xv. ružičkini dani-zbornik radova

Croatian Society of Chemical Engineers

Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek

European Federation of Food Science and Technology

European Association for Chemical and Molecular Sciences

European Hygienic Engineering & Design Group

International Scientific and Professional Conference

15th Ružička days

“TODAY SCIENCE – TOMORROW INDUSTRY”

11th and 12th September 2014

Vukovar, Croatia

PROCEEDINGS

Hrvatsko društvo kemijskih inženjera i tehnologa

Sveučilište Josipa Jurja Strossmayera u Osijeku, Prehrambeno-tehnološki fakultet Osijek

European Federation of Food Science and Technology

European Association for Chemical and Molecular Sciences

European Hygienic Engineering & Design Group

međunarodni znanstveno-stručni skup

XV. Ružičkini dani

“DANAS ZNANOST – SUTRA INDUSTRIJA”

11. i 12. rujna 2014.

Vukovar, Hrvatska

ZBORNIK RADOVA

Osijek i Zagreb, 2015.

PROCEEDINGS 15th

Ružička days

TODAY SCIENCE – TOMORROW INDUSTRY

ZBORNIK RADOVA XV. Ružičkini dani

DANAS ZNANOST - SUTRA INDUSTRIJA

Published by/Izdavači Josip Juraj Strossmayer University of Osijek

Faculty of Food Technology Osijek

Croatian Society of Chemical Engineers

Sveučilište Josipa Jurja Strossmayera u Osijeku

Prehrambeno-tehnološki fakultet Osijek

Hrvatsko društvo kemijskih inženjera i tehnologa (HDKI)

Editors/Urednici Drago Šubarić, Ante Jukić

Executive Editors/Izvršne urednice Mirela Planinić, Đurđica Ačkar

Technical Editor/Tehnička urednica Ivana Lauš

Proceedings Reviewers/

Recenzenti Zbornika

Igor Jerković, Darko Kiš, Maja Molnar, Ivica Strelec,

Rezica Sudar, Elvira Vidović

Proofreaders/Lektori Lidija Obad, Antonija Šarić

Cover page design/Dizajn naslovnice Ivana Lauš

Printing and Binding/Tisak i uvez Grafoprojekt, Virovitica, Hrvatska

Number of Copies/Naklada 200

Scientific and Organizing Committee

Znanstveno-organizacijski odbor

Drago Šubarić (predsjednik/chairman),

Ante Jukić (dopredsjednik/vice-chairman),

Srećko Tomas (dopredsjednik/vice-chairman),

Đurđica Ačkar, Jurislav Babić, Ljubica Glavaš-Obrovac, Vlado

Guberac, Mirjana Hruškar, Ivan Hubalek, Stela Jokić, Stjepan

Leaković, Ivanka Miličić, Slavko Marjančević, Jadranka

Mustapić-Karlić, Vesna Ocelić Bulatović, Ivana Lauš, Mirela

Planinić, Milan Sak-Bosnar, Nataša Srnić, Zvonimir Zdunić

Honorary Committee

Počasni odbor

Vladimir Andročec, Božo Galić, Marin Hraste, Zvonimir

Janović, Leo Klasinc, Filip Kljajić, Gordan Kolundžić, Ruža

Marić, Sandra Mrvica Mađarac, Ivan Penava, Vlasta Piližota,

Damir Skender, Nenad Trinajstić, Željko Turkalj, Ivan Vrdoljak

Osijek i Zagreb, 2015.

ISBN (PTF): 978-953-7005-36-8

ISBN (HDKI): 978-953-6894-53-6

A CIP catalogue record of this publication is available from the

City and University Library Osijek under 140114084

CIP zapis dostupan u računalnom katalogu

Gradske i sveučilišne knjižnice Osijek pod brojem 140114084

Publication of the Proceedings was approved by the Senate of the Josip Juraj Strossmayer University of

Osijek at the fifth session of the academic year 2014/2015 held on the 31st March 2015 numbered 12/15.

Objavljivanje ovog Zbornika odobrio je Senat Sveučilišta Josipa Jurja Strossmayera u Osijeku na 5.

sjednici u akademskoj godini 2014./2015. održanoj 31. ožujka 2015. godine, pod brojem 12/15.

Acknowledgement to reviewers

The Editors of the Proceedings of the 15th

Ružička days extend their deepest gratitude to the

following manuscript reviewers who maintained the professional standards of our Proceedings of

the 15th Ružička days:

Ačkar Đurđica, Babić Jurislav, Barukčić Irena, Bubalo Dragan, Bucić-Kojić Ana, Ćurko Natka,

Cvetković Dragoljub, Dabić Pero, Findrik Blažević Zvjezdana, Habuda Stanić Mirna, Herceg Zoran,

Ivanković Danijela, Jokić Stela, Jukić Marijana, Kezić Nikola, Komes Draženka, Koprivnjak

Olivera, Košmerl Tatjana, Kovačević Davor, Kraljević Roković Marijana, Krstanović Vinko,

Kurtanjek Želimir, Lisjak Miroslav, Madunić-Čačić Dubravka, Magdić Damir, Medić Helga,

Miličević Borislav, Pajin Biljana, Pavlović Hrvoje, Planinić Mirela, Plenković-Moraj Anđelka,

Poljak Milan, Popović Brigita, Pozderović Andrija, Prlić Kardum Jasna, Raić Malić Silvana,

Rogošić Marko, Sakač Nikola, Šarolić Mladenka, Škevin Dubravka, Smole Možina Sonja, Šumić

Zdravko, Tepić Aleksandra, Tišma Marina, Tomašić Vesna, Turan Jan, Vasić-Rački Đurđa, Velić

Darko, Velić Natalija, Vuković Marija

All pieces of information provided in this Proceedings are the sole responsibility of the authors of

the manuscripts. Publishers are not responsible for any use that might be made of the data appearing

in this document. Also, publishers shall not be liable for any errors, language mistakes and the like,

that are found in the works of authors.

mailto:[email protected]




Under the Auspices of: Pokrovitelj:

Croatian Academy of Sciences

and Arts

Department of Mathematical,

Physical and Chemical Sciences

Hrvatska akademija znanosti

i umjetnosti

Razred za matematičke,

fizičke i kemijske znanosti

Supported by:

Uz potporu:

Ministry of Science, Education

and Sports of the Republic of

Croatia

Ministarstvo znanosti,

obrazovanja i sporta

Republike Hrvatske

Ministry of Agriculture of the

Republic of Croatia

Ministarstvo poljoprivrede

Republike Hrvatske

Ministry of Economy of the

Republic of Croatia

Ministarstvo gospodarstva

Republike Hrvatske

Ministry of Environmental and

Nature Protection of the Republic

of Croatia

Ministarstvo zaštite okoliša i

prirode Republike Hrvatske

Croatian Academy of Engineering

Akademija tehničkih

znanosti Hrvatske

Josip Juraj Strossmayer University

of Osijek

Sveučilište Josipa Jurja

Strossmayera u Osijeku

Vukovar-Srijem County

Vukovarsko-srijemska

županija

City of Vukovar

Grad Vukovar

Dear Reader,

You hold in your hand the Proceedings from the International Scientific and Professional conference,

the 15th Ružička Days, traditionally held in Vukovar, dedicated to Lavoslav Ružička, the first Croatian

Nobel Laureate. The Conference was held under the slogan “TODAY SCIENCE – TOMORROW

INDUSTRY”, and it gathered scientists and experts from the fields of natural, technical and

biotechnical sciences, and also from the biomedicine and healthcare. The researchers presented their

researches as oral (25) and poster presentations (106). Due to the above mentioned facts the

Proceedings has interdisciplinary character, and contains research papers from all mentioned fields of

science. Conference organizers hold the opinion that these papers give great contribution to the

mentioned fields of science. Hereby I express my gratitude to all the authors of the papers for their

contribution for making their research accessible to public through oral and poster presentations,

scientific and professional papers.

All papers which you can find in the Proceedings, as well as the Proceedings in general, were

reviewed by reputable reviewers, to whom I express my gratitude for their hard work.

I also express my gratitude to everyone who contributed to the organization of the 15th Ružička Days,

especially to the international co-organizers (EFFoST, EuCheMS, EHEDG), as well as to those who

helped in making this Proceedings, and I invite all of you to participate at the 16th Ružička Days.

We are looking forward to meeting you all again in Vukovar, in 2016.

Chairman of the Scientific and Organizing Committee of the Conference

Drago Šubarić, PhD, Full Professor

International Scientific and Professional Conference 15th

Ružička days


11th and 12

th September 2014

Vukovar, Croatia

Sadržaj / Contents

I

Sekcija: Kemijska analiza i sinteza

Session: Chemical analysis and synthesis

Dajana Gašo-Sokač, Valentina Bušić, Mirna Habuda-Stanić, Marija Nujić

Spectrophotometric studies of novel derivatives of vitamin B6 ........................................................ 1

Marija Jozanović, Danijela Jakobović, Nikola Sakač, Milan Sak-Bosnar

Electroanalytical characterization and determination of imidazole dipeptides

carnosine and anserine ................................................................................................................... 12

Tatjana Kezele, Ivana Bačić

Forenzički pristup analizi boja u spreju primjenom svjetlosne mikroskopije

i vibracijske spektroskopije

Forensic approach to analysis of spray paints by the use of optical microscopy

and vibrational spectroscopy .......................................................................................................... 18

Anamarija Šter, Martina Medvidović-Kosanović, Tomislav Balić, Iva Ćurić,

Paula Mihaljević-Jurić

Electrochemical characterization of (1E)-1-N-{[4-(4-{[(E)-N-(4- aminophenyl)

carboxyimidoyl] phenoxy}butoxy) phenyl]methylidene} benzene-1,4-diamine .............................. 32

Renato Tomaš, Anđelka Vrdoljak

Thermodynamic study of CdCl2 in 2-propanol (5 mass %) + water mixture using

potentiometry ................................................................................................................................. 41

Sekcija: Kemijsko i biokemijsko inženjerstvo

Session: Chemical and biochemical engineering

Krunoslav Aladić, Stela Jokić, Goran Horvat, Mate Bilić

Supercritical fluid extraction laboratory plant design ...................................................................... 50

Irena Banovac, Matko Erceg, Dražan Jozić, Zorana Akrap, Sigrid Bernstorff

Strukturna svojstva i ionska vodljivost nanokompozitnih polimernih elektrolita

Structural properties and ionic conductivity of nanocomposite polymer electrolytes....................... 59

Mirjana Čurlin, Ana Jurinjak Tušek, Tamara Jurina, Irena Petrinić,

Želimir Kurtanjek

Local sensitivity analysis of integrated membrane bioreactor model ............................................... 70

Ana Jurinjak Tušek, Anita Šalić, Želimir Kurtanjek, Bruno Zelić

Effect of surface roughness on flow profile of liquid-liquid system in a

microreactor ................................................................................................................................... 81


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and 12th September 2014

Vukovar, Croatia

Sadržaj / Contents

II

Antonija Kaćunić, Lea Lokas, Marija Ćosić, Nenad Kuzmanić

Influence of the fluid flow patterns on borax nucleation mechanism and

nucleation rate in a single and dual turbine impeller crystallizer ..................................................... 91

Zlatka Knezović, Marina Trgo, Angela Stipišić, Davorka Sutlović

Impact of metals from the environment on chemical changes in olive oil ..................................... 104

Tomislav Penović, Antonia Giacobi, Andrija Hanžek

Kinetika sušenja katalizatora u sušioniku s fluidiziranim slojem

Fluid bed drying kinetics of catalysts ........................................................................................... 113

Jasna Prlić Kardum, Marina Samardžija, Štefica Kamenić, Marin Kovačić

Reološka i toplinska karakterizacija nanofluida

Rheological and thermal characterization of nanofluids ............................................................... 125

Marko Rogošić, Aleksandra Sander, Borna Ferčec

Ravnoteža kapljevina–kapljevina u sustavu ugljikovodik – piridin – C6mmpyTf2N

Liquid-liquid equilibrium for the system hydrocarbon – pyridine – C6mmpyTf2N ................... 140

Valentino Sambolek, Anamarija Slivar, Barbara Žuteg, Martina Hrkovac

Primjenjivost n-heksadekana pri regeneraciji ionskih kapljevina

The applicability of n-hexadecane in regeneration of ionic liquids ............................................... 154

Aleksandra Sander, Tomislav Penović, Dario Klarić

Uvećanje sušionika s fluidiziranim slojem

Scale-up of fluid bed dryer ........................................................................................................... 166

Aleksandra Sander, Mladena Dujmenović, Maja Žužić

Nova otapala za ekstrakciju tiofena iz smjese sa n-heksanom

New solvents for extraction of thiophene from the mixture with n-hexane ................................... 178

Sekcija: Prehrambena tehnologija i biotehnologija

Session: Food technology and biotechnology

Sara Bebić, Franko Burčul, Ivana Generalić Mekinić, Ivica Blažević

Isolation and characterization of volatile and non-volatile phytochemicals from

orange (Citrus sinensis L.) peel .................................................................................................... 190

Frane Čačić Kenjerić, Ana Jelinić

Measurement and level regulation with ultrasound sensor and Arduino microcontroller ............... 204


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th September 2014

Vukovar, Croatia

Sadržaj / Contents

III

Eva Ivanišová, Helena Frančáková, Štefan Dráb, Silvia Benčová

Elderberry as important source of antioxidant and biologically active compounds ........................ 212

Daniela Kenjerić, Blanka Bilić, Ivan Tomas, Milica Cvijetić

Vitamin E in vegetable oils as determined by RP-HPLC with UV detection ................................. 222

Nebojša Kojić, Lidija Jakobek

Determination of polyphenolic compounds in red wines from Baranja vineyards ......................... 232

Petra Krivak, Lidija Jakobek

Antiradical activity of polyphenols from old apple varieties ......................................................... 242

Zvonimir Ladešić, Sandra Maričić Tarandek, Josip Cvetko

Određivanje teksture (čvrstoće) i SFC profila binarnih i ternarnih smjesa palminog ulja,

palminog stearina i sojinog ulja

Determination of texture (hardness) and SFC profile of binary and ternary mixtures

of palm oil, palm stearin and soybean oil...................................................................................... 253

Krešimir Mastanjević, Dragan Kovačević, Kristina Vidaković

Cryoprotective effect of oat β-glucans on beef myofibrllar proteins.............................................. 260

Josip Mesić, Valentina Obradović, Maja Ergović Ravančić, Brankica Svitlica,

Jelena Žilić

Utjecaj mikorize i kvasaca na kakvoću vina sorte merlot (Vitis vinifera L.)

Influence of mycorrhizae and yeast strain on quality of wine variety merlot

(Vitis vinifera L.) ......................................................................................................................... 268

Borislav Miličević, Drago Šubarić, Antun Jozinović, Đurđica Ačkar, Jurislav

Babić, Danijela Vuković, Ana Mrgan

Effect of the immobilized yeast cells fermentation on chemical composition

and biogenic amines content in wine ............................................................................................ 276

Radoslav Miličević, Borislav Miličević, Đurđica Ačkar, Svjetlana Škrabal,

Drago Šubarić, Jurislav Babić, Antun Jozinović, Dijana Miličević

Rheological properties of molten chocolate masses during storage - influence of

milk components .......................................................................................................................... 283

Ana Mrgan, Gordana Jurišić

Stanje i mogućnosti proizvodnje mlijeka u Požeško-slavonskoj županiji

Condition and possibility of milk production in Pozega-Slavonia county ..................................... 289

Ana Mucalo, Goran Zdunić, Ivana Tomaz, Luna Maslov, Irena Budić-Leto,

Edi Maletić

Influence of harvest date on the primary metabolites of ˝Plavac mali˝ (Vitis vinifera L.)

grapes .......................................................................................................................................... 297


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Sadržaj / Contents

IV

Tina Perko, Mojca Škerget, Željko Knez

Extraction of active compounds from alfalfa plant ....................................................................... 306

Martina Petljak, Đuro Tunjić

Implementacija zahtjeva IFS-a u prehrambenoj industriji – izbor ili uvjet opstanka

na tržištu

Implementation of IFS in food industry – option or condition of survival on the market ............... 311

Mirella Žanetić, Maja Jukić Špika, Renato Stipišić, Antonela Jukić,

Sandra Svilović

Influence of malaxation time and temperature on ‘Levantinka’ virgin olive

oil properties ................................................................................................................................ 317

Sekcija: Kemija u poljoprivredi i šumarstvu

Session: Chemistry in agriculture and forestry

Miroslav Lisjak, Vlatko Galić, Bojan Fališevac, Marija Špoljarević, Mark E. Wood,

Matthew Whiteman, Ian D. Wilson, John T. Hancock, Tihana Teklić

The role of hydrogen sulfide in salt stress tolerance in plants ....................................................... 325

Zlatko Puškadija, Marin Kovačić, Željko Kraljičak, Silva Wendling, Dinko Jelkić,

Nebojša Nedić, Ivan Pihler

Digitalna SMS vaga kao suvremeni alat na pčelinjaku

Digital SMS scale as a modern tool on apiary .............................................................................. 335

Marija Špoljarević, Ana Mihaljević, Ivna Štolfa, Dejan Agić, Rosemary Vuković,

Miroslav Lisjak, Tihana Teklić

Primjena polietilen glikola-6000 u istraživanju osmotskog stresa kod soje

(Glycine max (L.) Merr.)

Polyethylene glycol-6000 application in the research of osmotic stress in soybean

(Glycine max (L.) Merr.) .............................................................................................................. 341

Zvonimir Zdunić, Luka Andrić, Aleksandra Sudarić, Georg Drezner, Alojzije Lalić,

Josip Kovačević, Krešimir Dvojković

Use of farm-saved seed in modern agriculture .............................................................................. 352

Sekcija: Zaštita okoliša

Session: Environmental protection

Ivana Jakovljević, Gordana Pehnec, Vladimira Vađić

Koncentracije policikličkih aromatskih ugljikovodika u zraku na različitim

lokacijama u Hrvatskoj

Concentrations of polycyclic aromatic hydrocarbons in the air at different

locations in Croatia ...................................................................................................................... 356


Ružička days


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th September 2014

Vukovar, Croatia

Sadržaj / Contents

V

Brankica Kalajdžić, Marija Nujić, Željka Romić

Natural organic matter degradation using heterogeneous Fenton catalysts on zeolite support........ 364

Ivona Nuić, Anka Sulić, Marina Trgo, Nediljka Vukojević Medvidović

Influence of the flow rate on lead and zinc removal from a binary solution on the fixed bed

of natural zeolite .......................................................................................................................... 375

Biljana Pavić

Kompostiranje u školi

Composting at school ................................................................................................................... 383

Sonja Rupčić Petelinc, Sanja Žužek, Iris Jurki, Bruna Vugrinec, Emanuel Gaši

Uklanjanje nitrata iz poplavom onečišćenih voda

Removal of nitrate from contaminated flood water ....................................................................... 391

Marjana Simonič

Drinking water quality assessment from private well.................................................................... 402

Monika Šabić, Lara Čižmek, Marija Vuković Domanovac

Biorazgradnja eritromicina s bakterijskom kulturom Pseudomonas

aeruginosa 3011

Biodegradation of erythromycin with bacterial culture Pseudomonas

aeruginosa 3011 .......................................................................................................................... 409

Mirko Štefančić, Mirna Habuda-Stanić, Natalija Velić, Marija Nujić,

Kristina Habschied

BIOCOS®

wastewater treatment plant Našice: from start-up to stable operation ........................... 417

Natalija Velić, Tihana Marček, Tamara Jurić, Katarina Petrinović, Damir

Hasenay, Lidija Begović, Vedran Slačanac

A survey of different bioadsorbents for removal of malachite green and methylene

blue dyes from aqueous solutions ................................................................................................. 424

Kazalo autora Author index ............................................................................................................................. 433

Sponzori Sponsors .................................................................................................................................... 436

Sekcija: Kemijska analiza i sinteza

Session: Chemical analysis and synthesis


Ružička days


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th September 2014

Vukovar, Croatia

Kemijska analiza i sinteza / Chemical analysis and synthesis

1

Spectrophotometric studies of novel derivatives of vitamin B6

UDC: 543.645.5

Dajana Gašo-Sokač1,2

, Valentina Bušić1, Mirna Habuda-Stanić

1, Marija Nujić

1

1Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 20,

HR-31000 Osijek, Croatia 2Josip Juraj Strossmayer University of Osijek, Department of Chemistry, Cara Hadrijana 8/A,

HR-31000 Osijek, Croatia

Summary

Chemistry of vitamin B6 was intensively investigated for nearly a century, which is understandable

given the importance of the life processes in the body. In recent years research has focused on the

synthesis and spectroscopic studies of new analogs of vitamin B6, which can be used in human

medicine for recovery of the organism poisoning by organophosphorus compounds. UV/VIS

absorption spectra of the synthesized derivatives of vitamin B6 were recorded in aqueous solutions

of different pH values. Absorption spectra of aqueous solutions of the test compounds changed with

changing the pH of the solution. In acidic medium there are two peaks, one at about 279-293 nm and

the other at 330 nm. By increasing the pH the second peak is lost, and the first moves at about 270-

274 nm with the emergence of a new peak at about 378-380 nm.

Keywords: pyridoxal oxime, vitamin B6, UV/VIS spectroscopy

Introduction

Most pyridine derivatives, both naturally occurring and synthetic, show a wide range of

biological activity. Pyridine derivatives are widely used as herbicides, insecticides and

fungicides. The study of the oxime derivatives of (1-phenacyl)piridinium cation is of great

importance because of their versatile bioactivities. Some oximes are currently being used in

human therapy as antidotes against organophosphate poisoning. Oximes, particularly those

of the mono- and bis-pyridinium type, are capable of reactivating in vitro and in vivo the

cholinesterase inhibited by organophosphorous compounds (pesticides, chemical warfare

nerve agents) and have multiple pharmacological action (Reiner and Simeon Rudolf, 2006;

Primožič et al., 2004). Commonly used reactivators are characterised by the presence of

several structural features: functional oxime group, quaternary nitrogen group and different

length of linking chain between two pyridinium rings in the case of bispyridinium

reactivators (Primožič et al., 2004). Searching for compounds containing that structural

Corresponding author: [email protected]


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2

features that could be interesting for further studies and at the same time chemically related

to biologically and pharmacologically active molecules led to the synthesis of new

derivatives of vitamin B6.

In the earlier study, we synthesized new oximes, derivatives of vitamin B6, and tested them

as reactivators of AChE (Gašo-Sokač et al., 2010). The goal of this work was to carry out

further spectroscopic characterisation of prepared derivatives of vitamin B6 and in the

present work UV/VIS characteristic of the prepared compounds are presented.

Materials and methods

Solvents and reagents were purchased from Fluka and Aldrich and used without further

purification. Ultraviolet visible absorption spectra were recorded using Specord 200

spectrophotometer (Analytik Jena AG). The UV/VIS spectra were obtained at room

temperature (25 °C) using 1 cm optical path-lenght quartz cells. A pH-meter with a

saturated calomel-glass electrode system was used for pH measurements. pH readings were

fitted using the appropriate amount of hydrochloric acid (0.2 M), sodium hydroxide (0.2

M) and Britton-Robinson buffer. Britton-Robinson buffer was prepared by mixing

equimolar amounts of phosphorous (0.04 M H3PO4) acetate (0.04 M CH3COOH) and boric

acid (0.04 M H3BO3).

Results and discussion

The quaternary salts 1-6 were prepared according to the described procedures, Scheme 1.

(Gašo-Sokač et al., 2010; Gašo-Sokač et al., 2014).

Scheme 1. Synthesis of the oximes 1-6

The aqueous solution of compound 1-phenacyl-3-hydroxy-4-hydroxyiminomethyl-5-

hydroxymethyl-2-methylpyridinium bromide (3) and its 4’-chloro (1), 4’-bromo (2), 4’-

methyl (4), 4’-nitro (5) and 4’-methoxy (6) derivatives exhibit an analogues spectra.


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3

The absorption spectra of all compounds in aqueous solutions generally exhibited the pH-

dependent bands that were compatibile with the absorptions of the inherited chromophores.

At pH 2 an 3 the absorption spectra of compounds 1-6 were composed of an intense band

at 279-293 (λmax (1) = 280 nm, λmax (2) = 280 nm, λmax (3) = 282 nm, λmax (4) = 279 nm,

λmax (5) = 282 nm, λmax (6) = 293 nm) and weaker absorption at 330 nm, Fig. 1-6, Table 1-

6. These bands originated from the overlapped ππ* transition within the acetophenone

and the pyridinium chromophores and are in agreement with literature data (Beamish,

1990). Namely, non-substituted pyridine shows an electronic transition at 257 nm due ππ*

transition and this absorption band bathocromic shifts to longer wavelengths when substituents

are conjugated to the aromatic system also quaternization of pyridine caused bathochromical or

hypsochromical shift which depend on the substituents (Cetina et al., 2010).

Table 1. Spectral data of 1-(4’-chlorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-

2-methylpyridinium bromide (1)

max/ nm Absorbance pH / M-1cm-1

280

330

1.828

0.763 2

11425

4769

280

330

1.863

0.803 3

11644

5019

276

343 370

1.526

0.557 0.506

5

9538

3481 3125

275

371

1.362

0.782 6

8513

4888

274

371

1.374

0.829 7

8588

5181

274

371

1.361

0.847 8

8506

5294

274 375

1.429 1.047

10 8931 6544

273

378

1.442

1.246 11

9013

7788

273

315 379

1.343

0.639 1.309

12

8394

3994 8181


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4

Fig. 1. Absorption spectra of compound 1 (c= 1.6 x 10-4

M, t = 25 °C) at different acidities

Table 2. Spectral data of 1-(4’-bromophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-

2-methylpyridinium bromide (2)


280

330

1.603

0.547 2

10019

3419

280

330

1.588

0.563 3

9925

3519

276

343 370

1.357

0.379 0.339

5

8481

2369 2119

275

369

1.442

0.593 6

9013

3706

274

371

1.310

0.587 7

8188

3669

274

371

1.279

0.605 8

7994

3781

274 375

1.307 0.755

10 8169 4719

274

378

1.313

0.932 11

8206

5825

273

380

1.129

0.975 12

7056

6094

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

200 250 300 350 400 450

A

/ nm

pH 2

pH 3

pH 5

pH 6

pH 7

pH 8

pH 10

pH 11

pH 12


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Fig. 2. Absorption spectra of compound 2 (c= 1.6 x 10-4


Table 3. Spectral data of 1-phenacyl-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-

methylpyridinium bromide (3)


282

330

1.103

0.636 2

6894

3975

282 330

1.138 0.652

3 7113 4075

281

335

372

0.775

0.472

0.393

5

4844

2670

2456

270

371

0.590

0.669 6

3688

4181

270 371

0.565 0.727

7 3531 4544

270

371

0.570

0.710 8

3563

4438

270

374

0.637

0.844 10

3981

5275

270

377

0.747

1.005 11

4669

6281

271 317

378

0.824 0.526

1.158

12 5159 3288

7238

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

200 250 300 350 400 450

A

λ/ nm

pH 2

pH 3

pH 5

pH 6

pH 7

pH 8

pH 10

pH 11

pH 12


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Fig. 3. Absorption spectra of compound 3 (c = 1.6 x 10-4


Table 4. Spectral data of 3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4’-

methylphenacyl) pyridinium bromide (4)


279

330

1.391

0.549 2

8694

3431

279 330

1.307 0.517

3 8169 3231

275

333

372

1.138

0.383

0.312

5

7113

2394

1950

273

371

1.032

0.534 6

6450

3336

273 371

1.068 0.564

7 6675 3525

273

371

1.016

0.562 8

6350

3513

273

374

1.101

0.683 10

6881

4269

273

376

1.095

0.790 11

6844

4938

273 315

377

1.144 0.472

0.901

12 7150 2950

5631

0

0,2

0,4

0,6

0,8

1

1,2

1,4

200 300 400

A

λ nm

pH 2

pH 3

pH 5

pH 6

pH 7

pH 8

pH 10

pH 11

pH 12


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nitrophenacyl) pyridinium bromide (5)


282

330

1.103

0.636 2

11030

3975

282

330

1.138

0.652 3

7113

4075

281 335

370

0.775 0.472

0.394

5 4844 2950

2462

270

371

0.590

0.669 6

3687

4181

270

371

0.565

0.727 7

3531

4544

270 371

0.570 0.710

8 3563 4438

270

374

0.637

0.844 10

3981

5275

270

377

0.747

1.005 11

4669

6281

271

317 378

0.824

0.526 1.158

12

5150

3288 7238

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

200 250 300 350 400 450

A

λ / nm

pH 2

pH 3

pH 5

pH 6

pH 7

pH 8

pH 10

pH 11

pH 12


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methoxyphenacyl) pyridinium bromide (6)


293 2.830 2 17688

292 2.842 3 17763

291

369

2.480

0.561 5

15500

3506

289 370

2.342 0.997

6 14638 6231

289

369

2.168

0.971 7

13550

6069

288

371

2.165

0.999 8

13531

6244

290

373

2.131

1.162 10

13319

7263

288 376

2.075 1.394

11 12969 8713

287

377

2.069

1.640 12

12931

10250

0

0,2

0,4

0,6

0,8

1

1,2

1,4

200 250 300 350 400 450

A

/ nm

pH 2

pH 3

pH 5

pH 6

pH 7

pH 8

pH 10

pH 11

pH 12


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At higher pH values ionization of keto-tautomers and the formation of the respective

enolates resulted in additional conjugation bands at 377-380 nm. The band at 279-293 loses

in intensity along with the appearance of a new one at 377-380 (λmax (1) = 379 nm, λmax (2)

= 380 nm, λmax (3) = 378 nm, λmax (4) = 377 nm, λmax (5) = 378 nm, λmax (6) = 377 nm). It

reaches maximal absorbance at pH 12 and it is attributed to the enolic form of the

compounds, Scheme 2. The intensity of second maximum is lower than the first maximum

except for compounds 3 (λmax = 271 nm, ε = 5159 M-1

cm-1

;λmax = 378 nm , ε = 7238 M-1

cm-

1) and 5 (λmax = 271 nm, ε = 5150 M

-1cm

-1;λmax = 378 nm , ε = 7238 M

-1cm

-1). The

formation of the enol form leads to the conjugation of the double bond with the pyridine

and the benzene nucleus. The hydrogen atoms of the α-methylene group are doubly

activated by carbonyl group and by the neighbouring positive charge of the pyridinium

nitrogen thus at higher pH solutions of compounds 1-6 contain considerable proportions of

enol form (Lovrić et al., 1999)

0

0,5

1

1,5

2

2,5

3

200 250 300 350 400 450

A

λ / nm

pH 2

pH 3

pH 5

pH 6

pH 7

pH 8

pH 10

pH 11

pH 12


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Scheme 2. The acid-base equilibria of compounds 1-6

Foretić et al. (2012) performed electronic absorption spectral studies of 1-phenacyl and 1-

benzoylethyl-derivatives of the pyridinium cation in aqueous media and obtained similar

results. The spectra of aqueous solution of (1-phenacyl)pyridinium chloride and 2-methyl-

(1-phenacyl)pyridinium chloride were composed of intensive band at 250 nm and weaker

absorption in the 270-290 nm range. Ionisation of the keto-tautomers and formation of the

respective enolates resulted in additional conjugation bands at 400 and 390 nm (Foretić et

al., 2012).

Conclusions

The study of 1-phenacyl-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-

methylpyridinium bromide (3) and its 4’-chloro (1), 4’-bromo (2), 4’-methyl (4), 4’-nitro

(5) and 4’-methoxy (6) derivatives have been done using UV/VIS spectroscopy. The

aqueous solutions of all compounds exhibit analogous spectra. In acidic medium there are

two peaks, one at about 279-293 nm and the other at 330 nm with the predominance of

ketone forms. By increasing the pH the second peak is lost, and the first hypsochromically

move with the emergence of a new peak at about 378-380 nm that suggested a considerable

fraction of enols in aqueous solutions.

C N CH=NOH

O

C CH2 N CH=NO

O

OH-

OHH3C

CH2OH

OHH3C

CH2OH

R RCH2

C CH N CH=NOH

OH

OHH3C

CH2OH

R

C CH N CH=NO

OH

OHH3C

CH2OH

R C CH N CH=NO

O

OHH3C

CH2OH

R

OH-

OH-


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References

Beamish, G.G. (1990): Practical Fluorescence, second ed., Marcel Dekker, New York.

Cetina, M., Trafnić, M., Sviben, I., Jukić, M., (2010): Synthesis, X-ray and spectroscopic analysis of

some pyridine derivatives, J. Mol. Struct. 969, 25-32.

Foretić, B., Picek, I. Damjanović, V., Cvijanović, D., Milić, D. (2012): The structures and stabilities

of biologically active 1-phenacyl- and 1-benzoylethyl-derivatives of the pyridinium cation, J.

Mol. Struct. 1019, 196-205.

Gašo-Sokač, D., Katalinć, M., Kovarik, Z., Bušić, V., Kovač, S. (2010): Synthesis and evaluation of

novel analogues of vitamin B6 as reactivators of tabun and paraoxon inhibited

acetylcholinesterase, Chem. Biol. Interact. 187, 234-237

Gašo-Sokač, D., Bušić, V., Cetina, M., Jukić, M. (2014): An efficient synthesis of pyridoxal oxime

derivatives under microwave irradiation, Molecules 19 (6), 7610-7620

Lovrić, J., Burger, N., Deljac, V., Mihalić, Z. (1999): Spectrophotometric studies of some novel

derivatives of pyridinium chloride, Croat. Chem. Acta. 72 (1), 123-133.

Primožič, I., Odžak. R., Tomić, S., Simeon-Rudolf, V., Reiner, E. (2004): Synthesis, interaction with

native and phosphorylated cholinesteerases, and antidotes against organophosphorus

compounds. J Med Chem Def, 2, 1-30.

Reiner, E., Simeon-Rudolf, V. (2006): Reactivation of phosphorylated cholinesterases in vitro and

protecting effects in vivo of some pyridinium and quinolinium oximes. Arh Hig Rada Toksikol

57, 171-179.


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Electroanalytical characterization and determination

of imidazole dipeptides carnosine and anserine

UDC: 543.645.6

Marija Jozanović, Danijela Jakobović, Nikola Sakač, Milan Sak-Bosnar

Josip Juraj Strossmayer University of Osijek, Department of Chemistry, Cara Hadrijana 8A,


Summary

Acid-base properties of imidazole dipeptides - carnosine and anserine were studied. Carnosine and

anserine were determinated and characterized, single and in binary mixtures, by direct potentiometric

titrations using aqueous and nonaqueous solvents. Before direct potentiometric titrations, carnosine and

anserine solutions should be pH adjusted to 3.00. The generated potentiometric data were used for

defining buffering capacities (buffer strength) and for determination of the corresponding species

distribution diagrams. Distribution diagram provided the qualitative information of the carnosine and

anserine species equilibria that are pH dependent. The buffer strength diagram of carnosine indicates

enhanced buffer effect at pH ca. 3.5 and 7.5. Binary mixtures showed no or some small difference in

potentiometric determination. Titration curve for titration in acetonitrile showed higher potential

change compared to that in methanol solutions. Further investigation should be carried out.

Keywords: dipeptides, carnosine, anserine, potentiometry, nonaqueous titrations

Introduction

Carnosine and anserine are dipeptides that are contained in the skeletal muscles or brains of

vertebrates in high concentrations. These substances may function to reduce muscle fatigue

and improve learning ability because of an anti-oxidative effect (Alpsoy et al., 2011) and

buffering capacity due to the presence of an imidazole group.

L-Carnosine (ß-alanyl-L-histidine) is a dipeptide composed of ß-alanine and L-histidine,

which performs multiple biological functions including pH buffering, anti-oxidation, anti-

glycation, anti-aging, and chelation of divalent metal cations (Hipkiss, 2009). Anserine (ß-

alanyl-N-methyl histidine) is an N-methylated analogue of carnosine found mainly in fish

and birds. Anserine has similar properties to carnosine in many aspects but is mainly found

in non-mammalian species.



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Carnosine and anserine can be determined in biological materials by means of HPLC using

fluorescent (Aristoy et al., 2004) and electrochemical detection (Nardiello et al., 2004),

capillary electrophoresis (Huang et al., 2005) and microchip electrophoresis (Zhao et al., 2009).

In these investigations some acid-base properties of carnosine and anserine were

potentiometrically studied.


Reagents and preparation of solutions

Aqueous solutions:

Carnosine (Sigma-Aldrich, USA) and anserine (Bachem, Switzerland) titrations were

performed using NaOH standard of the concentration 0.01 and 0.001 M (Kemika, Croatia)

as a titrant, 1 M HCl (Kemika, Croatia) was used for pH dipeptide solution adjustment to 3

and 4, respectively. All chemicals were of analytical reagent grade.

Nonaqueous solutions:

Carnosine solutions were prepared in a) 90% acetonitrile (J. T. Baker, Netherland) b) 90%

methanol (Carlo Erba, Italy), both in water.

Carnosine titrations were performed using 0.01 M tetrabutylammonium hydroxide

(TBAOH) in toluene/methanol (ACROS Organics, Belgium). All chemicals were of

analytical reagent grade.

Apparatus

The 765 dosing unit (Metrohm, Switzerland) combined with Metrohm exchange unit

(Metrohm, Switzerland), were employed for carnosine and anserine (single and their binary

mixtures) aqueous titrations and nonaqueous titrations for carnosine.

Electrode system used: a) for aqueous solutions 6.0253.100 glass combined electrode

(Metrohm, Switzerland) with Ag/AgCl/ 3 M KCl (Metrohm, Switzerland); b) for

nonaqueous solutions LL Solvotrode Easy Clean glass combined electrode LiCl/ethanol

(Metrohm, Switzerland). The solutions were magnetically stirred during titrations using the

801 Stirrer (Metrohm, Switzerland).

Procedures

Aqueous solutions:

The single dipeptide solutions were measured as following: a) 10 mL of carnosine (0.01

and 0.001 M) solution and b) 10 mL anserine (0.01 and 0.001 M) solutions were titrated


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with NaOH solutions (0.01 and 0.001 M), respectively. The dipeptide solutions pH were

adjusted to 3 (for 0.01 M) and 4 (for 0.001 M).

The dipeptides binary mixtures were investigated as following: 10 mL of analyte solution

containing 0.001 M solutions of carnosine to anserine in ratios 1:1; 1:2 and 1:3, were

titrated with NaOH solutions (0.001 M).

Nonaqueous solutions:

The single carnosine solutions were measured as following: a) 90% acetonitrile-water

solvent system of carnosine (0.001 M) solution and b) 90% methanol-water solvent system

of carnosine (0.001 M) solution were titrated with 0.01 M TBAOH in toluene/methanol.


Aqueous solutions

Single dipeptide solutions of carnosine and anserine were measured by potentiometric

titration (Fig. 1), by adding aqueous NaOH with and without pH adjustment. When pH was

adjusted to 3.0, potentiometric titration curve and it`s 1st derivative showed 2 sharp

inflexions, presenting equivalence points. When compared to titration curve with no pH

adjustment, it`s 1st derivative showed no sharp peaks. Anserine was studied with the same

methodology, presenting similar results.

Fig. 1. Potentiometric titration curve of carnosine with aqueous NaOH (c=0.01 M),

no pH adjustment (○), pH adjustment at 3.0 (▲) and corresponding 1st derivative:

for no pH adjustment (full line) and for pH adjustment (dashed line)


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A quantitative expression of the pH-stabilizing buffer action of a triprotic acid such as

carnosine is the buffer strength B. The buffer-strength diagram of carnosine, obtained on

the basis of data after the calculation and optimization, is shown in Fig. 2. The buffer

strength diagram of carnosine indicates enhanced buffer effect at pH ca. 3.5 and 7.5.

Fig. 2. Buffer strength diagram of carnosine (c=0.01 M)

The pKa values for anserine are pKa1=2.64; pKa2=7.04; pKa3 =9.49. The pKa values for

carnosine are pKa1=2.64; pKa2=6.87; pKa3=9.51. The pKa values for both dipeptides are

very close. Distribution diagram (Fig. 3) provides the qualitative information of the

carnosine and anserine species equilibria, that are pH dependent. The data obtained,

combined with data from buffer strength diagram, could be used to predict pH changes

during potentiometric titrations of pure and real sample systems.

Fig. 3. Distribution diagrams of carnosine (black lines) and anserine (dotted lines)

The dipeptides binary mixtures were investigated in solutions of carnosine and anserine in

ratios 1:1; 1:2 and 1:3, with NaOH solution (0.001 M) are shown in Fig. 4. There is no


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difference in the shape of titration curves for 1:1 and 1:2 carnosine to anserine ratios, the ratio

1:3 is slightly shifted to higher NaOH consumption. Further investigation should be carried out.

Fig. 4. Potentiometric titration curves for binary mixture of carnosine and anserine

in ratios 1:1 (□); 1:2 (▲) and 1:3 (○), with NaOH solution (0.001 M)

Nonaqueous solutions

Potentiometric titration curve of carnosine in nonaqueous solvents: 90% acetonitrile and

90% methanol using TBAOH (c=0.01 M) and their corresponding 1st derivatives are

presented in Fig. 5. Titration curve for titration in acetonitrile showed higher potential

change compared to that in methanol solutions. Further investigations should be performed

on different organic nonaqueous solvents.

Fig. 5. Potentiometric titration curve of carnosine in 90% acetonitrile (□) and 90% methanol (○)

using TBAOH (c=0.01 M) with corresponding 1st derivative for 90% acetonitrile (full line)

and 90% methanol (dashed line)


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Theoretical model for the corresponding experimental data is a challenging issue, and will

be a part of further investigation.

Conclusions

In these investigations carnosine and anserine were potentiometrically studied, single and

in a mixture. Their acid-base properties were studied by use of potentiometric titrations that

were carried out in aqueous and nonaqueous solvents. The generated potentiometric data

were used for defining buffering capacities (buffer strength) and for determination of the

corresponding species distribution diagrams.

References

Alpsoy, L., Akcayoglu, G., Sahin, H. (2011): Anti-oxidative and anti-genotoxic effects of carnosine

on human lymphocyte culture, Hum. Exp. Tox. 30, 1979-85.

Aristoy, M. C., Soler, C., Toldra, F., (2004): A simple, fast and reliable methodology for the

analysis of histidine dipeptides as markers of the presence of animal origin proteins in feeds for

ruminants, Food Chem. 84, 485-491.

Hipkiss, A. R. (2009): Carnosine and its possible roles in nutrition and health, Adv. Food Nutr. Res.

57, 87-154. Huang Y., Duan J., Chen H. , Chen M., Chen G. (2005): Separation and determination of carnosine-

related peptides using capillary electrophoresis with laser-induced fluorescence detection,

Electrophoresis 26, 593-599.

Nardiello D, Cataldi TRI. (2004): Determination of carnosine in feed and meat by high-performance

anion-exchange chromatography with integrated pulsed amperometric detection, J.

Chromatogr. A. 1035, 285-289.

Zhao S., Huang Y., Shi M., Huang J., Liu J.M.V(2009): Quantification of biogenic amines by

microchip electrophoresis with chemiluminescence detection, Anal. Biochem. 393, 105-110.


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Forenzički pristup analizi boja u spreju primjenom

svjetlosne mikroskopije i vibracijske spektroskopije

UDC: 340.67

667.2 : 543.42

Tatjana Kezele1, Ivana Bačić

2

1Sveučilište u Zagrebu, Prirodoslovno-matematički fakultet, Kemijski odsjek, Horvatovac 102A,

10000 Zagreb 2Ministarstvo unutarnjih poslova, Centar za forenzična ispitivanja, istraživanja i vještačenja „Ivan

Vučetić“, Ilica 335, 10000 Zagreb

Sažetak

Forenzičari su često u prilici vještačiti uzorke boja u spreju kojima su, uglavnom na pročeljima

zgrada, ispisane poruke uvredljivog sadržaja. Kako se obično radi o tankim slojevima boje koji se

teško odvajaju od podloge na kojoj se nalaze, za njihovu analizu potrebno je primijeniti

instrumentne tehnike koje ne zahtijevaju prethodnu pripremu uzorka. Ukupno deset uzoraka crne i

plave sprej boje različitih nijansi naneseno je u tankom sloju na staklo, odnosno na podlogu od bijele

i žute fasadne boje. In situ kemijska analiza tako pripremljenih modelnih uzoraka provedena je

tehnikama infracrvene spektroskopije uz prigušenu totalnu refleksiju i Ramanove

(mikro)spektroskopije, dok je morfologija uzoraka okarakterizirana pomoću svjetlosnog

mikroskopa. Tehnikom infracrvene spektroskopije analiziran je sastav veziva, a vrsta pigmenata

određena je iz Ramanovih spektara pri pobudi 785 i 532 nm. U spektrima uzoraka na podlozi od

fasadne boje, nisu uočene dodatne vrpce koje bi ukazivale na utjecaj podloge. Provedena

istraživanja pokazala su da primijenjene nedestruktivne tehnike omogućavaju identifikaciju i

razlikovanje boja u spreju bez prethodne priprave uzoraka sve dok je debljina sloja boje dovoljna da

spriječi utjecaj podloge.

Ključne riječi: boje u spreju, forenzika, infracrvena spektroskopija, Ramanova mikroskopija,

svjetlosna mikroskopija

Uvod

Grafiti su kao sredstvo komunikacije postali nezaobilazni dio suvremene kulture koja je

posebno naglašena u urbanim mjestima. Kada grafiti prijeđu granicu ''umjetnosti'' i služe za

namjerno uništavanje javne imovine ili kada svojim sadržajem izražavaju predrasude i

mržnju prema drugima, postaju kazneno djelo. U otkrivanju počinitelja takvih kaznenih

djela značajnu ulogu imaju rezultati forenzičnog istraživanja. Uz pojam grafita najčešće se



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povezuju boje u spreju, pa je zadatak forenzičara analizom materijalnog dokaza povezati

boju s grafita s eventualnim počiniteljem. Kako se obično radi o tankim slojevima boje koji

se teško odvajaju od podloge na kojoj se nalaze, za njihovu analizu potrebno je primijeniti

instrumentne tehnike koje ne zahtijevaju prethodnu pripremu uzorka.

Boje u spreju su, kao i druge vrste svježe boje, smjesa pet osnovnih sastojaka: veziva,

otapala, pigmenta, punila i aditiva (Caddy, 2001). S forenzičnog stajališta najveću

vrijednost za identifikaciju i usporedbu suhih uzoraka boja u spreju ima kemijski sastav

veziva, pigmenata i punila.

Forenzička identifikacija i usporedba boja u spreju provode se nizom analitičkih tehnika

koje slijedom primjene obuhvaćaju svjetlosnu mikroskopiju, infracrvenu spektroskopiju s

Fourierovom transformacijom (FT-IR) i Ramanovu spektroskopiju (Goavert i sur., 2001,

2003 i 2004). Svjetlosnom mikroskopijom karakteriziraju se fizikalna svojstva uzorka kao

što su nijansa boje, zastupljenost pigmenata, debljina sloja te sjaj i morfologija površine

(Nieznańska, 1999; Zięba-Palus, 2005). Kako u svojim radovima navode Goavert i sur.

(2001. i 2004.) boje mogu biti mat (M), saten (S) i sjajne (Sj), s time da se hrapavost

površine smanjuje od mat prema sjajnoj boji (M > S > Sj).

Infracrvena spektroskopija uz prigušenu totalnu refleksiju (ATR) rutinski se primjenjuje u

analizi boja najčešće za određivanje njihovih organskih komponenti (Zięba-Palus, 2003 i

2005). Primjena Ramanove (mikro)spektroskopije u analizi boja u spreju pokazala je veliki

potencijal posebno u identifikaciji organskih pigmenata koji zbog utjecaja veziva obično

nisu vidljivi u infracrvenom spektru, dok u Ramanovom spektru daju jake vrpce (Buzzini i

sur., 2003).

Poseban izazov predstavlja analiza crnih boja u spreju koje je osim metodama vibracijske

spektroskopije potrebno analizirati i nekom drugom tehnikom kao što su pretražna

elektronska mikroskopija spregnuta s energodisperzivnom spektroskopijom (SEM/EDX) te

pirolitička plinska kromatografija sa spektrometrom masa kao detektorom (Py-GC-MS)

(Ryland, 2010; Muehlethaler i sur., 2013).

U ovom radu, u svrhu vrednovanja razlikovne i identifikacijske moći tehnika svjetlosne

mikroskopije i vibracijske spektroskopije, istražen je niz od deset uzoraka crne i plave boje

u spreju različitih nijansi. U radu su optimizirani i analitički uvjeti Ramanove

spektroskopije, prvenstveno valna duljina i snaga lasera, broj snimaka i vrijeme

prikupljanja spektara te veličina pukotine kroz koju zračenje lasera upada na uzorak.

Također, provedena je identifikacijska analiza boja u spreju koja podrazumijeva

određivanje morfoloških karakteristika i kemijskog sastava uzoraka boje i to vrste veziva,

pigmenata i punila. Na uzorcima pripremljenim nanošenjem boje u spreju u tankom sloju

na staklo, odnosno na podlogu od bijele i žute fasadne boje na vodenoj i organskoj osnovi,

ispitan je utjecaj podloge na nijansu i morfologiju boje, kao i na rezultate spektroskopske

analize.


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Materijali i metode

Pet uzoraka crne i pet uzoraka plave boje u spreju proizvođača „Dupli-Color“ (Tablica 1)

naneseno je prskanjem na mikroskopska stakalca s udaljenosti oko 20 cm, u trajanju 5

sekundi. Prije nanošenja limenke s bojom su snažno protresene kako bi se dobila što

homogenija raspodjela pigmenata (Muehlethaler i sur., 2014).

Tablica 1. Uzorci boje u spreju

Table 1. Spray paint samples

Uzorak Naziv boje RAL* Opis

C1 Aerosol art sprej 9005 crna mat

C2 Aqua sprej 9005 crna mat

C3 Platinum sprej 9005 crna, sjajna

C4 Color sprej 9005 crna, sjajna

C5 Auto sprej 9005 crna, sjajna

P1 Aerosol art sprej 5002 ultramarin plava

P2 Aerosol art sprej 5010 gentian plava

P3 Aerosol art sprej 5012 svijetlo plava

P4 Aerosol art sprej 5013 kobalt plava

P5 Aerosol art sprej 5015 nebesko plava *oznaka u standardiziranom registru boja koji se koristi u

Europi

Uzorci boje u spreju naneseni su i na podloge od žute i bijele fasadne boje na organskoj i

vodenoj osnovi, proizvođača "Cromos Svjetlost'' d.o.o., Lužani. Pločice s nanesenom

bojom sušile su se 24 sata. Bez daljnje pripreme uzoraka, snimljeni su IR i Raman spektri.

Optička mikroskopija

Za mikroskopski pregled i određivanje morfoloških svojstava uzoraka boje u spreju

nanesenih na staklo i na podloge od fasadne boje korišten je stereomikroskop Olympus,

maksimalnog povećanja 120.

Infracrvena spektroskopija

Infracrveni spektri snimani su na infracrvenom spektrometru TENSOR 27 tvrtke Bruker,

tehnikom prigušene totalne refleksije s refleksijskim elementom od dijamanta. Područje

snimanja spektara bilo je od 4000 cm-1

do 600 cm-1

uz razlučivanje od 4 cm-1

. Spektri su

rezultat uprosječivanja 10 snimaka. Na svim spektrima, programom OPUS 7.0, provedena


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je korekcija bazne linije (concave rubberband correction, CRC) i automatsko određivanje

valnih brojeva vrpci (peak picking).

Ramanova spektroskopija

Ramanovi spektri snimljeni su Ramanovim disperzivnim spektrometrom SENTERRA

tvrtke Bruker u konfiguraciji s mikroskopom Olympus s objektivima za povećanje 20, 50 i

100. Kao izvori zračenja korištena su dva lasera: Nd:YAG laser valne duljine 532 nm te

diodni laser (AlGaAs) valne duljine 785 nm. Raspršeno zračenje detektirano je pomoću

CCD detektora (charge-coupled device) koji je hlađen Peltierovim elementom.

Za sve uzorke zajednički uvjeti su bili: rešetka 1200 abc, pukotina 251000 μm, povećanje

50, spektralno područje 4400–100 cm-1

, razlučivanje 3–5 cm-1

, 20 snimaka i vrijeme

integracije 2 s. Snaga lasera, kao ograničavajući faktor, prilagođena je svakom uzroku kako

bi se izbjegao termički raspad uzorka (Tablica 2).

Programskim paketom OPUS 7.0 svim Ramanovim spektrima korigirana je bazna linija

(CRC). Identifikacija pigmenata provedena je programom „KnowItAll“ (Bio-Rad

Laboratories) usporedbom Ramanovih spektara uzoraka boje u spreju sa spektrima u

dostupnim zbirkama.

Tablica 2. Uvjeti snimanja Ramanovih spektara

Table 2. Raman spectra conditions

Uzorak

C1-C5 P1 P2 P3 P4 P5

785 nm / mW 1 1 10 1 1 10

532 nm / mW 5 2 0,2 0,2 0,2 2

Ramanovim spektrometrom određena je i debljina slojeva boje u spreju na fasadnim

podlogama. Fragmenti boje s podlogom izuzeti su skalpelom i u poprečnom presjeku

postavljeni u plastelin, a debljina sloja boje u spreju mjerena je pri povećanju od 50 ili

100. Za svaki uzorak napravljeno je pet mjerenja, a rezultat je njihova srednja vrijednost.

Rezultati i rasprava

Optička mikroskopija

U ovom radu naglasak mikroskopske analize je na određivanju morfoloških svojstava

uzoraka boja u spreju nanesenih na staklo i podloge od fasadne boje. Rezultati analize

prikazani su u Tablici 3.


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Tablica 3. Morfološke karakteristike boja u spreju

Table 3. Morphology of spray paints

C1 C2 C3 C4 C5 P1 P2 P3 P4 P5

staklo M S Sj Sj Sj Sj Sj Sj Sj Sj

fasadna

boja JM S M M M BM BM M BM BM

*M – mat, JM – jače mat, BM – blago mat, S – saten, Sj – sjajna

S obzirom na morfološke karakteristike uzoraka nanesenih na staklo moglo se razlikovati

tri vrste boja – mat (1 crna), saten (1 crna) i sjajna (3 crne i 5 plavih). Iste boje nanesene na

podlogu od fasadne boje okarakterizirane su kao jače mat (1 crna), mat (3 crne i 1 plava),

saten (1 crna) i blago mat (4 plave). Kod 9 od ukupno 10 istraženih uzoraka došlo je do

nekog oblika promjene morfoloških svojstava, koja se odražava kroz smanjenje sjaja

uslijed povećanja hrapavosti sloja boje. Hrapavost površine povećana je na način da se na

površini boje preslikala struktura podloge uz nastajanje brojnih sitnih udubljenja (Slika 1).

Slika 1. Mikroskopska slika crne boje u spreju (C5) nanesene na staklo (lijevo)

i na podlogu od fasadne boje (desno), snimljeno pri ukupnom povećanju 200

Fig. 1. Microscopic analysis of black spray paint (C5) on glass (left)

and facade paint (right). Magnification 200

Najznačajnije promjene zapažene su kod tri crne i jedne plave boje (C3-C5, P3), dok je

nešto manji utjecaj podloge uočen kod preostale četiri plave boje, (P1, P2, P4, P5). Kod C1

crne boje došlo je do pojačanja mat efekta, dok promjena morfologije nije uočena jedino

kod uzorka koji je opisan kao saten (C2). Primjeri različitih površina boje prikazani su na

Slici 2. Na gornjoj polovici slika su boje nanesene na staklo, a na donjoj polovici boje su

nanesene na podlogu od fasadne boje.


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Slika 2. Mikroskopske slike crnih boja C2 (lijevo) i C3 (sredina) te plave boje P3 (desno).

Snimljeno pri ukupnom povećanju 20

Fig. 2. Microscopic analysis of black (C2) (left), C3 (middle) and blue P3 paint (right).

Magnification 20

Dobiveni rezultati su pokazali da je mikroskopska analiza pogodna za određivanje

morfoloških karakteristika boja u spreju, ali i da je utjecaj podloge na morfologiju

značajan. Rezultati morfoloških svojstava boja nanesenih na različite vrste podloga mogu

dovesti do pogrešne procjene tijekom usporedbe uzoraka, pa ih treba uzeti s velikom

rezervom kada se donosi sud o sličnosti odnosno razlikama među uzorcima.

Infracrveni spektri

Svim uzorcima snimljen je IR spektar, a prema opaženim vibracijskim vrpcama određena

je vrsta veziva te eventualna prisutnost punila i pigmenta. IR spektri crnih i plavih boja

prikazani su na Slici 3 odnosno Slici 4, a rezultati su sažeti u Tablici 4.

Tablica 4. Sastav veziva, punila i pigmenata prisutnih u bojama u spreju

Table 4. Binder, filler and pigment composition of spray paints

Uzorak Vezivo Punilo Pigment

C1 nitroceluloza s o-ftalatom kao

plastifikatorom - -

C2 nemodificirani alkid talk -

C3 o-ftalat alkid modificiran nitrocelulozom

- -

C4 nemodificirani o-ftalat alkid - -

C5 nitroceluloza s o-ftalatom kao

plastifikatorom - -

P1

nitroceluloza s o-ftalatom kao

plastifikatorom

sulfati

P2 TiO2

P3 TiO2

P4 sulfati Prusko plavo

P5 TiO2


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Na prisutnost alkida ukazuju vrpce antisimetričnog i simetričnog istezanja C−H veza

metilnih i metilenskih skupina u području 2960–2850 cm-1

. Također je prisutna jaka

vibracijska vrpca istezanja karbonilne (C=O) skupine oko 1720 cm-1

i vibracije istezanja

C–O veze pri 1274 cm-1

i 1120 cm-1

koje ukazuje na estersku skupinu. Dvije vrpce slabog

intenziteta pri 1604 cm-1

i 1580 cm-1

odgovaraju istezanju C=C veza u aromatskom prstenu.

Vibracije u području 1490–1375 cm-1

mogu se pripisati simetričnim i antisimetričnim

deformacijama metilne i metilenske skupine. Vrpca pri 740 cm-1

odgovara svijanju izvan

ravnine CH skupina u aromatskom prstenu. Da se radi o o-ftalatu ukazuju i vibracijske

vrpce pri 1119, 1064 i 740 cm-1

(Zięba-Palus, 2005).

U spektrima crnih uzoraka C1, C3 i C5 (Slika 3) te u svim uzorcima plave boje (Slika 4),

osim vrpci pripisanih o-ftalat alkidu prisutne su i vrpce karakteristične za nitrocelulozu.

Vrpca pri 1640 cm-1

i 1274 cm-1

odgovara antisimetričnom odnosno simetričnom istezanju

O−NO2 veze. Oko 830 cm-1

nalazi se vrpca koja potječe od istezanja C−O−NO2 veze

(Zięba-Palus, 2005; Muehlethaler i sur. 2014). Vrpca pri 1060 cm-1 rezultat je istezanja

C−O veze, a vrpcama u području 3480−3230 cm-1

doprinose istezanja O−H skupina u

celulozi.

Slika 3. IR spektri crnih boja u spreju nanesenih na staklo

Fig. 3. IR spectra of black spray paints on glass

Iz IR spektra ne može se odrediti radi li se o nitrocelulozom modificiranom o-ftalat alkidu

ili o nitrocelulozi kojoj je o-ftalat dodan kao plastifikator. Razlika između ova dva sustava

utvrđena je pomoću testa s acetonom, pri čemu se modificirani o-ftalat alkid nije otopio

dok se sustav s o-ftalatom kao plastifikatorom otapa u acetonu (Ryland, 2010). Temeljem

Tra

nsm

itan

cija

Valni broj / cm-1

1000 1500 2000 2500 3000 3500

C1

C2

C3

C4

C5


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ovih rezultata, veziva u uzorcima C1, C5 i svim plavim bojama okarakterizirana su kao

nitroceluloza s o-ftalatom kao plastifikatorom, dok je u uzorku C3 o-ftalat alkid

modificiran nitrocelulozom.

U IR spektru uzorka C2, osim karakterističnih vibracijskih vrpci alkidnog veziva utvrđen je i

talk kao punilo. O prisutnosti talka može se zaključiti na osnovu slabe vrpce pri 3675 cm-1 koja

odgovara istezanju Mg−O−H veze, te vrpcama istezanja Si−O veza pri 1116, 1096 i 1010 cm-1.

IR spektri plavih boja u spreju prikazani su na Slici 4. Kao i kod crnih uzoraka (C1, C3 i

C5) i ovdje su uočene vrpce nitroceluloze i o-ftalat alkida. Osim veziva, kod uzoraka P1 i

P4 vide se vrpce karakteristične za sulfate. Područje 1185−1065 cm-1

i rame pri 983 cm-1

obuhvaća vibracije simetričnog istezanja SO42-

skupine, dok vrpce pri 835 i 808 cm-1

odgovaraju deformaciji SO42-

skupine sulfata izvan ravnine. Kod uzoraka P2, P3 i P5

očigledan je pad bazne linije oko 700 cm-1

što ukazuje na prisutnost titanijeva dioksida i u

skladu je sa svjetlijim nijansama tih uzoraka. U spektru uzorka P4 naglašena je dodatna

vrpca pri 2090 cm-1

koja odgovara simetričnom istezanju C≡N veze i može potjecati od

pigmenta koji u sastavu ima heksacijanoferate, a takav pigment je Prusko plavo

(FeIII

[FeIII

FeII(CN)6]3).

Slika 4. IR spektri plavih boja u spreju nanesenih na staklo

Fig. 4. IR spectra of blue spray paints on glass

Kako bi proučili utjecaj podloge snimljeni su i IR spektri fasadnih boja s organskim i

vodenim vezivom. U spektrima prevladavaju vrpce kalcijeva karbonata kao punila, za

kojeg je karakteristično antisimetrično istezanje veza CO32-

skupine pri 1395 cm-1

kao i

deformacije karbonatnog iona u ravnini i izvan nje pri 711 odnosno 871 cm-1

(Slika 5).

Tra

nsm

itan

cija

Valni broj / cm-1

1000 1500 2000 2500 3000 3500

P1

P2

P3

P4

P5


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Slika 5. IR spektri bijele (BFO) i žute (ŽFO) fasadne boje s organskim vezivom

Fig. 5. IR spectra of white (BFO) and yellow (ŽFO) facade paints with organic binder

Stoga je bilo za očekivati da bi se eventualni utjecaj podloge na IR spektre crnih i plavih

boja u spreju trebao očitovati pojavom upravo jakih vrpci kalcijeva karbonata.

Usporedbom spektara boja u spreju na podlogama od fasadne boje sa spektrima boja

nanesenih izravno na staklo niti u jednom spektru nisu uočene dodatne vrpce koje bi

ukazivale na utjecaj podloge. Ovakvi rezultati izravna su posljedica debljine sloja boje u

spreju na podlozi, koje su većinom bile manje od 30 μm, dok je najmanja izmjerena

iznosila 10 μm, a najveća 41 μm.

Rezultati analize deset uzorka boje pokazali su da je infracrvena spektroskopija pogodna za

analizu, usporedbu i razlikovanje uzoraka boja u spreju na fasadnim podlogama. Prema

provedenim mjerenjima sloj boje debljine 10 μm i više je nego dovoljan da spriječi utjecaj

podloge. Vrpce u IR spektrima većinom potječu od polimernih veziva, temeljem kojih je

vrstu veziva bilo moguće utvrditi kod svih deset uzoraka. Unatoč sličnim vezivima svi IR

spektri pokazuju individualne karakteristike i mogu se međusobno razlikovati. Prisutnost

pigmenata i punila nedvojbeno je utvrđena kod 6 uzoraka, a radi se o talku, titanijevom

dioksidu i sulfatima. U samo jednom uzorku bilo je moguće odrediti i prisutnost plavog

pigmenta, Prusko plavo.

Ramanova spektroskopija

Snimljeni su Ramanovi spektri svih uzoraka boje, a identificirani pigmenti prikazani su u

Tablici 5.

Valni broj / cm-1

1000 1500 2000 2500 3000 3500

Tra

nsm

itan

cija

ŽFO

BFO


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Tablica 5. Pigmenti identificirani temeljem Ramanovih spektara

Table 5. Pigment identified by Raman spectra

Uzorak 785 nm 532 nm Ukupno

C1-C5 pigment black (carbon) - pigment black (carbon)

P1 PB 15:3 ili

PB 15:4, PV 23

PB 15:3 ili

PB 15:4, PV 23

PB 15:3 ili

PB 15:4, PV 23

P2 derivat PB 15 - derivat PB 15

P3 derivat PB 15 derivat PB 15,

PV 23

derivat PB 15,

PV 23

P4 PB 60, PB 27 PB 60, PV 23 PB 60, PB 27,

PV 23

P5 PB 15:3 ili

PB 15:4, PV 23 PB 15:3 ili

PB 15:4, PV 23 PB 15:3 ili

PB 15:4, PV 23

Za sve crne uzorke, kao jedini određen je pigment black carbon koji je zapravo amorfni

ugljik. Spektar ovog pigmenta, opažen nakon pobude zračenjem pri 785 nm, je vrlo

jednostavan i ima karakterističan izgled koji se sastoji od dvije široke vrpce (Slika 6).

Ovakvi rezultati ukazuju da razlikovanje uzoraka crnih boja u spreju koji kao pigment

sadrže samo amorfni ugljik, nije moguće provesti tehnikama vibracijske spektroskopije,

posebno ako uzorci sadrže isto vezivo. U tom slučaju, za utvrđivanje mogućih različitosti,

neophodna je primjena kromatografskih tehnika (Py-GC/MS) (Muehlethaler i sur., 2013).

Slika 6. Ramanovi spektri crne boje u spreju C5 i pigmenta black carbon; Pobuda pri 785 nm

Fig. 6. Raman spectra of black spray paint (C5) and pigment carbon black, with 785 nm laser excitation

Kako se može vidjeti iz Tablice 5, ovisno o valnoj duljini primijenjenog zračenja

Ramanovi spektri omogućuju identifikaciju nekoliko organskih pigmenata. Kod većine

plavih uzoraka (osim P4) nakon pobude zračenjem valne duljine 785 nm u spektru

Valni broj / cm

-1 1800 1400 1000 600 200

Ram

anov i

nte

nzi

tet

Pigment black (carbon)

C5


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dominiraju vrpce bakrovog ftalocijanina (PB 15, PB 15:3/4). S druge strane, u spektrima

tih istih uzoraka snimljenih pri 532 nm dominiraju vrpce potpuno drugog pigmenta, a

najčešće se radi o karbazol ljubičastom (PV 23). Ovaj fenomen pripisuje se pojavi koja se

javlja kada je energija pobudnog zračenja bliska ili se podudara s energijom elektronskih

prijelaza u molekuli čime se postižu rezonantni uvjeti mjerenja. Rezultat je povećanje

intenziteta raspršenog zračenja uslijed koje se i osjetljivost metode povećava te opažanje

različitih Ramanovih spektara (Slike 7 i 8).

Slika 7. Ramanovi spektri plave boje u spreju P1 te pigmenata PB 15:3/PB 15:4 i PV 23; Pobuda pri 785 nm

Fig. 7. Raman spectra of blue spray paint (P1) and pigments PB 15:3/PB 15:4 and PV 23, with 785 nm laser excitation

Slika 8. Ramanovi spektri plave boje u spreju P1 te pigmenata PB 15:3/PB 15:4 i PV 23;

Pobuda pri 532 nm

Fig. 8. Raman spectra of blue spray paint (P1) and pigments PB 15:3/PB 15:4 and PV 23,

with 532 nm laser excitation

Valni broj / cm

-1

1800 1400 1000 600 200

Ram

anov i

nte

nzi

tet

PB 15:3/4

P1

PV 23

Valni broj / cm

-1

Ram

anov i

nte

nzi

tet

3000 2000 1500 1000 500 2500

PV 23

P1

PB 15:3/4


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Uzorak plave boje P4 u potpunosti se razlikuje od svih drugih istraženih plavih boja u

spreju. Naime, to je jedini uzorak u kojemu nije utvrđena prisutnost bakrova ftalocijanina,

ali su zato identificirana tri plava pigmenta. Temeljem Ramanovog spektra pri 785 nm

identificirani su pigmenti idantron plavo koji je derivat antrakinona (PB 60) te Pigment

blue 27 poznat kao Prusko plavo ili Berlinsko modrilo. Pobudom pri 532 nm već je

očekivano određen karbazol ljubičasto (PV 23), a potvrđena je i prisutnost pigmenta

Prusko plavo. Ovime je potvrđen i rezultat asignacije IR spektra uzorka P4, gdje je pigment

Prusko plavo predviđen temeljem vrpce na 2090 cm-1

.

FT-IR i Ramanova spektroskopija pogodne su za forenzičku analizu boja u spreju jer na

komplementaran način daju informacije o kemijskom sastavu uzorka i imaju jaku

razlikovnu moć. Infracrvenom spektroskopijom mogu se odrediti vrste veziva i punila, dok

je Ramanova spektroskopija bolja za identifikaciju pigmenata. Primjenom lasera različitih

valnih duljina omogućena je simultana identifikacija većeg broja različitih pigmenata

organskog i anorganskog porijekla prisutnih u jednom uzorku. Obje tehnike ne zahtijevaju

posebnu pripremu uzoraka i praktički su nedestruktivne te kao takve zadovoljavaju uvjet

forenzičke analize o očuvanju materijala vještačenja. Brzina tehnika uz dostupnost dobrih

zbirki spektara čine ih pogodnima za svakodnevnu rutinsku uporabu.

Zaključci

Mikroskopska analiza pogodna je za određivanje morfoloških karakteristika boja u spreju,

ali zbog značajnog utjecaja podloge na morfologiju ima samo informativnu, a ne i

razlikovnu vrijednost.

IR spektroskopijom uspješno su identificirana veziva u svim uzorcima boja, koja su po

sastavu nemodificirano alkidno vezivo, nitroceluloza s o-ftalatom kao plastifikatorom te

nitrocelulozom modificirani o-ftalat alkid. Unatoč sličnim vezivima svi IR spektri pokazuju

individualne karakteristike i mogu se međusobno razlikovati.

Pigmente nije bilo moguće odrediti infracrvenom spektroskopijom zbog niske

koncentracije i intenzivne apsorpcije zračenja veziva. Stoga je za detekciju pigmenata

primijenjena Ramanova spektroskopija uz pobudno zračenje pri dvije valne duljine.

Usporedbom Ramanovih spektara plavih boja u spreju s onima iz zbirke spektara, uspješno

je identificirano pet različitih plavih pigmenata, od kojih su najučestaliji pigmenti na bazi

bakrova ftalocijanina. Za razliku od plavih boja u kojima je najčešće prisutna smjesa

pigmenata, sve crne boje kao pigment sadrže samo amorfni ugljik što u tom slučaju

Ramanovu spektroskopiju čini slabo razlikovnom tehnikom. Crne boje u spreju, posebno

one sličnih veziva, moraju se analizirati dodatnim instrumentnim tehnikama.

Niti u jednom uzorku nije uočen utjecaj podloge na rezultate metoda vibracijske

spektroskopije, unatoč vrlo tankom sloju boje u spreju.


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Provedena istraživanja pokazala su da primijenjeni slijed nedestruktivnih tehnika svjetlosne

mikroskopije te infracrvene i Ramanove spektroskopije omogućavaju identifikaciju i

razlikovanje boja u spreju bez prethodne priprave uzoraka sve dok je debljina sloja boje

dovoljna da spriječi utjecaj podloge.

Zahvala

Autori zahvaljuju tvrtkama „Bauhaus“ iz Zagreba i „Chromos Svjetlost“ iz Lužana na

ustupljenim uzorcima boja.

Literatura

Buzzini, P., Massonnet, G., Mizrahi, S. (2003): Forensic Sci. Int. 136(1), 355-356.

Caddy, B. (2001): Forensic Examination of Glass and Paint, Analysis and Interpretation, London,

Taylor & Francis, London, str. 123-128.

Govaert, F., de Roy, G., Decruyenaer, B. (2001): Problems of Forensic Sciences XLVII, 333-339.

Govaert, F., Bernard, M. (2003): Forensic Sci. Int. 136(1), 354.

Govaert, F., Bernard, M. (2004): Forensic Sci. Int. 140, 61-70.

Muehlethaler, C., Massonnet, G., Deviterne, M., Bradley, M., Herrero, A., de Lezana, I. D., Lauper,

S., Dubois, D., Geyer-Lippmann, J., Ketterer, S., Milet, S., Bertrand, M., Langer, W., Plage,

B., Gorzawski, G., Lamothe, V., Marsh, L., Turunen, R. (2013): Forensic Sci. Int. 229, 80-91.

Muehlethaler, C., Massonnet, G., Buzzini, P. (2014): Forensic Sci. Int. 237, 78-85.

Nieznańska, J., Zięba-Palus, J., Kościelniak, P. (1999): ZZNS XXXIX, 77-94.

Ryland, B. S. S. (2010): JASTEE 1(2), 109-126.

Zięba-Palus, J. (2003): Forensic Sci. Int. 136(1), 358.

Zięba-Palus, J. (2005): J. Mol. Struct. 744-747, 229-234.


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Forensic approach to analysis of spray paints by the use of

optical microscopy and vibrational spectroscopy

Tatjana Kezele1, Ivana Bačić

2

1University of Zagreb, Faculty of Science, Department of Chemistry, Horvatovac 102A, HR-10000 Zagreb,

Croatia 2Ministry of the Interior, Forensic Science Centre „Ivan Vučetić“, Ilica 335, HR-10000 Zagreb, Croatia

Summary

Forensic experts are often in a position to analyse spray paint samples (graffiti) that are, as

an offensive messages, mainly written on building facades. Since the layer of spray paint is

usually thin and difficult to be separated from the substrate, for their analysis is necessary

to apply instrumental techniques that do not require prior sample preparation. A total of ten

black and blue spray paints with different hues were deposited on the glass surface as well

as on the layer of white and yellow facade paint. Chemical analysis of model samples

without previous preparation were performed by attenuated total reflection infrared

spectroscopy (ATR-FTIR) and Raman (micro)spectroscopy, while the surfaces

morphology were characterized by optical microscopy. Binder composition of all paints

was determined by IR, whereas the Raman spectra at excitation of 785 and 532 nm

provided the information about pigment contents. In the IR and Raman spectra of the paint

samples deposited on the facade paints any additional bands, which would indicate the

substrate influence, were not observed. The research showed that used non-destructive

techniques allow identification and differentiation of spray paint samples without previous

preparation, until the thickness of the paint layer is sufficient to prevent the substrate

influence.


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Electrochemical characterization of (1E)-1-N-{[4-(4-{[(E)-N-(4- aminophenyl)

carboxyimidoyl] phenoxy}butoxy) phenyl]methylidene} benzene-1,4-diamine

UDC: 543.55 : 547.532

Anamarija Šter, Martina Medvidović-Kosanović, Tomislav Balić,

Iva Ćurić, Paula Mihaljević-Jurić

Josip Juraj Strossmayer University of Osijek, Department of Chemistry, Cara Hadrijana 8/A,


Summary

Oxido-reduction properties of a novel synthesized Schiff base were studied by cyclic and differential

pulse voltammetry. The results of electrochemical study have shown that the oxidation of the

investigated Schiff base is reversible, diffusion controlled process and that the oxidation products

are adsorbed on the glassy carbon electrode surface.

Keywords: Schiff base, electrochemistry, voltammetry

Introduction

Schiff bases have been widely studied due to their pronounced biological and

pharmacological activity (Liang et al., 2014), optical (Fang et al., 2014), photochromical

(Zhao et al., 2001), thermochromical (Minkin et al., 2011) properties and other outstanding

material properties. Furthermore, they can easily form different types of polydentate

ligands and because their diversified donor groups (or atoms) are suitable as chelating

agents. Complex compounds of Schiff bases are considered to be a transition state between

simple coordination compounds and metalloproteins (Chandra et al., 2008).

Untill now, Schiff bases and their metal complexes were studied by XRD (Kianfar et al.,

2014a), EDX (Kianfar et al., 2014a), TGA/DTA (Kianfar et al., 2014a), SEM (Kianfar et

al., 2014a), TEM (Kianfar et al., 2014a), FT-IR spectroscopy (Booysen et al., 2014;

Grivani et al., 2014; Kianfar et al., 2014a; Novoa et al., 2014; Shafaatian et al., in press). 1H NMR (Grivani et al., 2014; Shafaatian et al., 2014), elemental analysis (Novoa et al., in

press; Shafaatian et al., 2014), fluorescence (Shafaatian et al., 2014), conductometry

(Booysen et al., 2014; Shafaatian et al., 2014) and EPR (Novoa et al., in press). Their

structure was determined by X-ray diffraction (Booysen et al., 2014; Grivani et al., 2014;

Novoa et al., in press; Shafaatian et al., 2014) and ab initio calculations (Novoa et al., in

press) and electrochemical characterization was conducted by cyclic voltammetry



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(Booysen et al., 2014; Kianfar et al., 2013b, 2011c; Menati et al., 2012; Shafaatian et al.,

2014) and square wave voltammetry (Booysen et al., 2014). Biological activity and use of

Schiff bases as potentiometric sensors for metal cation determination was also studied

(Afkhami et al., 2013; Bandi et al., 2013).

In this study we have examined oxido-reduction properties of (1E)-1-N-{[4-(4-{[(E)-N-(4-

aminophenyl) carboxyimidoyl] phenoxy} butoxy) phenyl] methylidene} benzene-1,4-

diamine (Fig. 1) by cyclic and differential pulse voltammetry. Preliminary information

obtained from this research is very useful as indicator of potential application of this

compound (i.e. organic semiconductor, liquid crystal, potentiometric sensor etc.).

Fig. 1. Structure of synthesized Schiff base (1E)-1-N-{[4-(4-{[(E)-N-(4- aminophenyl)

carboxyimidoyl] phenoxy}butoxy) phenyl]methylidene} benzene-1,4-diamine


All commercially available chemicals were of reagent grade and used as purchased from

commercial sources. Dialdehyde 4-[4-(4-formylphenoxy) butoxy]benzaldehyde was

prepared by previously reported method. All solvents were purchased commercially. N,N-

dimethylformamide (DMF) was purchased from Fischer Chemical and Lithium Chloride

(LiCl) from BDH Prolabo and were used without further purification.

Shiff base synthesis: Dialdehyde (0.6 g, 2 mmol) was dissolved in 40 ml of methanol and 2-

3 drops of acetic acid were added to this solution. The solution was brought to brisk reflux

and 0.49 g (4.5 mmol) of p-phenylendiamine dissolved in 25 ml of methanol was gradually

added. The mixture was heated at reflux temperature for 3 hours. After the reaction was

completed, the resulting mixture was left at room temperature for 24 hours. The red powder

product was filtered and washed with cold ethanol and diethyl ether. Yield: 76 %

IR spectrum was recorded on a Shimadzu FTIR 8400S spectrophotometer using the DRS

8000 attachment, in the 4000-400 cm−1

region. Thermogravimetric analysis was performed

using a simultaneous TGA-DSC analyser (Mettler-Toledo TGA/DSC 1). The compound


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was placed in aluminium pan (100 L) and heated in nitrogen atmosphere (200 mL min−1

)

up to 550 °C at a rate of 10 °C min−1

. The data collection and analysis was performed using

the program package STARe Software 10.0. The

1H NMR and

13C NMR were recorded on

NMR (300 MHz) Bruker instrument, using deuterated dimethyl sulfoxide as solvent at

NMR Laboratory of the Ruđer Bošković Institute, Zagreb.

Electrochemical experiments were performed on PalmSens potentiostat/galvanostat

(PalmSens BV, Utrecht, The Netherlands) driven by PSTrace 4.2 software. A conventional

three-electrode cell was used with a glassy carbon as a working electrode, non-aqueous

Ag/Ag+ (and aqueous Ag/AgCl) as a reference electrode and a platinum wire as a counter

electrode. The glassy carbon working electrode was polished with coarse diamond polish

(1 µm, ALS, Japan) and with polishing α-Al2O3 (0.05µm, ALS, Japan) before each

measurement. Cyclic voltammetry scan rate was 100 mV s–1

. The differential pulse

voltammetry conditions were: scan increment 5 mV, pulse amplitude 25 mV, pulse width

70 ms and scan rate 5 mV s–1

.


Cyclic voltammetry studies

A cyclic voltammogram of the investigated Schiff base is shown in Fig. 2. One anodic (A1)

peak at a potential of 0.4562 V and one cathodic (K1) peak at a potential 0.3962 V appeared

when the potential was scaned from -0.2 V to 0.8 V vs. Ag/Ag+ reference electrode. Obtained

Ep value was 60 mV, indicating that the oxidation reaction is reversible.

Fig. 2. Cyclic voltammogram of title compound (c = 1.1·10-4

mol dm-3

) at a glassy carbon electrode

(Ic = 0.1 M LiCl in DMF). Scan rate: 100 mV/s. A1 (anodic peak), K1 (cathodic peak)


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The influence of concentration of the investigated Schiff base on anodic peak current and

anodic peak potential was examined and the obtained data are given in Table 1. The effect

of scan rate on the oxidation of the Shiff base has been investigated as well (Fig. 3). The

obtained results have shown that both anodic peak potential and anodic peak current

increase with the increase in Schiff base concentration and scan rate.

Fig. 3. Cyclic voltammograms of title compound (c = 1.1·10-4

mol dm-3

) at a glassy carbon electrode (Ic = 0.1 M LiCl in DMF) obtained with: a) Ag/Ag

+ and b) Ag/AgCl reference electrode, at different

scan rates ( = 25, 75, 150, 200, 250 and 300 mV/s)

Table 1. Anodic peak potential (Ep,a) and anodic peak current (Ip,a) of the title compound as the

function of its concentration obtained with Ag/Ag+ and Ag/AgCl reference electrode.

Scan rate: 100 mV/s.

non-aqueous Ag/Ag+ electrode aqueous Ag/AgCl electrode

104 c / mol dm-3 Ep,a / V Ip,a / A Ep,a / V Ip,a / A

0.31 0.4164 0.1636 0.6314 0.2033

0.59 0.4184 0.1853 0.6383 0.2120

1.10 0.4509 0.2146 0.6579 0.2435

1.25 0.4519 0.2149 0.6692 0.2543


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Fig. 4 shows that at lower concentrations of Schiff base (bellow c ~ 1.1 10-4

mol dm–3

)

anodic peak current is a linear function of the Schiff base concentration (the adsorption of

the investigated Schiff base oxidation products on the electrode surface occurs). At higher

concentrations of Schiff base (above c ~ 1.1 10 –4

mol dm–3

), the increase of peak current

slows down, which could be explained by increase of interactions between molecules

adsorbed on the electrode surface and by diffusion current (Medvidović-Kosanović et al.,

2010). The Schiff base oxidation is controlled by diffusion (Fig. 5) since linear dependence

was found between anodic peak current and the square root of scan rate (Medvidović-

Kosanović et al., 2010; Yagmur et al., 2013).

Fig. 4. Anodic peak current as a function of title compound concentration (Ic = 0.1 M LiCl in DMF). Scan rate: 100 mV/s

Fig. 5. Anodic peak current, I, as a function of the square root of scan rate, 1/2

, at a glassy carbon

electrode in solution of title compound (c = 1.1·10-4

mol dm-3

, Ic = 0.1 M LiCl in DMF)


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Differential pulse voltammetry studies

Differential pulse voltammogram in Fig. 6 revealed one oxidation peak of the investigated

Schiff base at the potential 0.415 V. The oxidation peak decreases with successive scans

which confirms adsorption of the Schiff base oxidation products on the glassy carbon

electrode surface.

Fig. 6. Differential pulse voltammogram of title compound (c = 1.1·10-4

mol dm-3

) at a glassy

carbon electrode (Ic = 0,1 M LiCl in DMF). Scan rate: 5 mV/s. First scan (a),

second scan, third scan, forth scan

Peak current and peak potential also increase with increasing Schiff base concentration

(Fig. 7) which could be explained by kinetic limitation in the reaction between the redox

sites of a glassy carbon electrode and the investigated Schiff base (Bandi et al., 2013). A

linear relationship could be established between peak current and Schiff base concentration

in the range of 0.3110-4

mol dm-3

to 1.25 10-4

mol dm-3

(the inset of Fig. 7). A linear

regression equation, Ip = 1.3592 c + 2.4628 with a correlation coefficient R2 = 0.9823, can

be obtained, where Ip is the oxidation peak current (10-2

A) and c is the Schiff base

concentration (10-4

mol dm-3

). It can also be seen from Fig. 8 that the results obtained by

non-aqueous Ag/Ag+ reference electrode show higher R

2 values compared to aqueous

Ag/AgCl electrode. Therefore, non-aqueous Ag/Ag+ reference electrode should be used for

experiments in non-aqueous medium.


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Fig. 7. Differential pulse voltammograms in the solutions of title compound with concentrations (c =

3.1 ·10-5

; 5.9 ·10-5

; 1.1 ·10-4

and 1.25 ·10-4

(d) mol dm-3

) at a glassy carbon electrode (Ic = 0.1 M

LiCl in DMF). Scan rate: 5 mV/s. The inset of figure 7: Anodic peak current, I, as a function of the

Schiff base concentration, c (data taken from Fig. 7)

Fig. 8. Anodic peak current, I, as a function of the title compound concentration, c,

at a glassy carbon electrode obtained with: a) non-aqueous Ag/Ag+ () and b)

aqueous Ag/AgCl (●) reference electrode (Ic = 0.1 M LiCl in DMF)


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Conclusions

The electrochemical results have shown that the oxidation of the Schiff base title

compound is reversible and controlled by diffusion at the investigated experimental

conditions. Adsorption of the oxidation products on the glassy carbon electrode occurs and

this process is kinetically limited. A linear relationship between peak current and Schiff

base concentration in the range of 0.3110-4

mol dm-3

to 1.25 10-4

mol dm-3

was established.

Comparison of the results obtained by non-aqueous Ag/Ag+ and aqueous Ag/AgCl

reference electrode has shown that for experiments in non-aqueous medium non-aqueous

Ag/Ag+ reference electrode should be used.

Acknowledgements

The authors thank J. J. Strossmayer University of Osijek, Croatia for financial support.

References

Afkhami, A., Soltani-Felehgari, F., Madrakian, T., Ghaedi, H., Rezaeivala, M. (2013): Fabrication

and application of a new modified electrochemical sensor using nano-silica and a newly

synthesized Schiff base for simultaneous determination of Cd2+

, Cu2+

and Hg2+

ions in water

and some foodstuff samples, Anal. Chim. Acta 771, 21-30.

Bandi, K. R., Singh, A. K., Upadhyay, A. (2013): Biologically active Schiff bases as potentiometric

sensor for the selective determination of Nd3+

ion, Electrochim. Acta 105, 654-664.

Booysen, I. N., Adebisi, A., Munro, O. Q., Xulu, B. (2014): Ruthenium complexes with Schiff base

ligands containing benz (othiazole / imidazole) moieties: Structural, electron spin resonance

and electrochemistry studies, Polyhedron 73, 1-11.

Chandra, S., Pundir, M. (2008): Spectroscopic characterization of chromium(III), manganese(II) and

nickel(II) complexes with a nitrogen donor tetradentate, 12-membered azamacrocyclic ligand,

Spectrochim. Acta A 69 (1),1-7.

Fang, Z., Cao, C., Chen, J., Deng, X. (2014): An extending evidence of molecular conformation on

spectroscopic properties of symmetrical bis-Schiff bases, J. Mol. Struct. 1063, 307-312.

Grivani, G., Tahmasebi, V., Khalaji, A. D. (2014): A new oxidovanadium(IV) complex containing

an O, N-bidentate Schiff base ligand: Synthesis, characterization, crystal structure

determination, thermal study and catalytic activity for an oxidative bromination reaction,

Polyhedron 68, 144-150.

Kianfar, A. H., Mahmood, W. A. K., Dinari, M., Azarian, M. H., Khafri, F. Z. (2014a): Novel

nanohybrids of cobalt(III) Schiff base complexes and clay: Synthesis and structural

determinations, Spectrochim. Acta A 127, 422-428.


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40

Kianfar, A. H., Ramazani, S., Fath, R. H., Roushani, M. (2013b): Synthesis, spectroscopy,

electrochemistry and thermogravimetry of copper(II) tridentate Schiff base complexes,

theoretical study of the structures of compounds and kinetic study of the tautomerism reactions

by ab initio calculations, Spectrochim. Acta A 105, 374-382.

Kianfar, A. H., Paliz, M., Roushani, M., Shamsipur, M. (2011c): Synthesis, spectroscopy,

electrochemistry and thermal study of vanadyl tridentate Schiff base complexes, Spectrochim.

Acta A 82, 44-48.

Liang, C., Xia, J., Lei, D., Li, X., Yao, Q., Gao, J. (2014): Synthesis, in vitro and in vivo antitumor

activity of symmetrical bis-Schiff base derivatives of isatin, Eur J. Med Chem. 74, 742-750.

Manzur, C., Carrillo, D., Hamon, J.-R (2014): Doubly Phenoxide-Bridged Binuclear Copper(II)

Complexes with ONO Tridentate Schiff Base Ligand: Synthesis, Structural, Magnetic and

Theoretical studies Polyhedron, in press, doi: http://dx.doi.org/10.1016/j.poly.2014.05.032

Medvidović-Kosanović, M., Šeruga, M., Jakobek, L., Novak, I. (2010): electrochemical and

antioxidant properties of rutin, Collect. Czech. Chem. C. 75 (5) 547-561.

Menati, S., Azadbakht, A., Taeb, A., Kakanejadifard, A., Khavasi, H. R. (2012): Synthesis,

characterization and electrochemical study of synthesis of a new Schiff base (H2cdditbutsalen)

ligand and their two asymmetric Schiff base complexes of Ni(II) and Cu(II) with NNprimeOS

coordination spheres, Spectrochim. Acta A 97, 1033-1040.

Minkin, V. I., Tsukanov, A. V, Dubonosov, A. D., Bren, V. A. (2011): Tautomeric Schiff bases:

Iono-, solvato-, thermo- and photochromism, J. Mol. Struct. 998, 179-191.

Novoa, N., Justaud, F., Hamon, P., Roisnel, T., Cador, O., Le Guennic, B., Manzur, C., Carrillo, D.,

Hamon, J.-R. (2014): Doubly Phenoxide-Bridged Binuclear Copper(II) Complexes with ONO

Tridentate Schiff Base Ligand: Synthesis, Structural, Magnetic and Theoretical studies,

Polyhedron, in press, doi: http://dx.doi.org/10.1016/j.poly.2014.05.032

Shafaatian, B., Soleymanpour, A., Oskouei, N. K., Notash, B., Rezvani, S. A. (2014): Synthesis,

crystal structure, fluorescence and electrochemical studies of a new tridentate Schiff base

ligand and its nickel(II) and palladium(II) complexes, Spectrochim. Acta A 128, 363-369.

Yagmur, S., Yilmaz, S., Saglikoglu, G., Sadikoglu, M., Yildiz, M., Polat, K. (2013): Synthesis,

spectroscopic studies and electrochemical properties of Schiff bases derived from 2-hydroxy

aromatic aldehydes and phenazopyridine hydrochloride, J. Serb. Chem. Soc. 78 (6), 795-804.

Zhao, J., Zhao, B., Liu, J., Xu, W., Wang, Z. (2001): Spectroscopy study on the photochromism of

Schiff bases N,N′-bis(salicylidene)-1,2-diaminoethane and N,N′-bis(salicylidene)-1,6-

hexanediamine, Spectrochim. Acta A 57 (1), 149-154.


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Thermodynamic study of CdCl2 in 2-propanol (5 mass %) + water

mixture using potentiometry

UDC: 544.632.4

Renato Tomaš, Anđelka Vrdoljak

University of Split, Faculty of Chemistry and Technology, Teslina 10/V, HR-21000 Split, Croatia

Summary

The potential difference (pd) measurements (E) are reported for galvanic cell without liquid

junction: Cd(Hg) (l, satd.)CdCl2(b) in ZAgCl(s)Ag(s) at different temperatures and various

CdCl2 molalities (b) in aqueous mixture of 2-propanol (containing 5 mass % 2-propanol), Z. From

these values and using literature data for stability constants of the chlorocadmium complexes, the

values of the standard pd of the cell were obtained at each temperature. These values served to

calculate the standard thermodynamic quantities for the cell reaction, and also mean molal activity

coefficients of CdCl2. The corresponding thermodynamic results are discussed and compared with

literature data.

Keywords: thermodynamic properties, cadmium chloride, 2-propanol + water mixed solvent,

potentiometry.

Introduction

Potentiometric method using a galvanic cell without liquid junction was found to be an

attracting experimental technique for studying the thermodynamic properties of electrolyte

solutions in aqua-organic mixed solvents. This paper is an extension of our systematic

investigation on thermodynamic properties of CdCl2 in various aqua-organic solvents using

potentiometry. Specifically, in previous works by our group, the bahaviour of the CdCl2

has been studied in water mixtures with alcohol co-solvent: containing 10, 30, and 50 mass

% 2-propanol (Višić Mekjavić, 1993) or 2-methylpropan-2-ol (tert. butanol) (Tomaš et

al., 2000), and also with 5, 10, and 15 mass % 2-butanol (Tomaš et al., 2005).

We have recently published data on the thermodynamic properties of CdCl2 in 2-

methylpropan-2-ol (5 mass %) + water mixture (Z) using a flow potentiometric method

(Tomaš et al., 2011). Namely, the potential difference (pd) measurements have been carried

out on the following galvanic cell:

Cd(Hg) (l, satd.)CdCl2(b) in ZAgCl(s)Ag(s) (1)



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in the temperature range (293.15 K to 313.15 K) at 5 K intervals, for different CdCl2

molalities (b).

In this work, analogous investigations were performed in 2-propanol + water mixture of the

same content (ie., Z = 5 mass % 2-propanol), in order to determine the thermodynamic

quantities of the cell reaction, and the stoichiometric mean molal activity coefficients of

CdCl2. The results from this study are compared with similar systems.

Within the potentiometric data analysis, the concentrations of all ionic species was

calculated (Višić Mekjavić, 1989) using data for the thermodynamic stability constants

of chlorocadmium complexes in water medium, and for 10 mass % 2-propanol (Višić et al.,

1993).


CdCl2 H2O and 2-propanol were p.a. purity (Merck). Before use, 2-propanol and water

were distilled. Solvent mixture (Z) and concentrated electrolyte solution in Z (stock

solution) were prepared by weight using CdCl2 H2O, water and 2-propanol. The molality

(bmax.) of the stock solution of CdCl2 in Z was 0.0503 mol kg–1

, with salt mass fraction, w =

0.01002. Density of Z was measured using oscillating U-tube densimeter (Anton Paar,

model DMA 4500 M) with precision of 1 10–5

g cm–3

(Bald et al., 2013).

In order to continuously obtain the pd (Ei) of the cell (1) at different molalities (bi) of

CdCl2, we change electrolyte concentrations by adding a stock solution in Z (mass, mi) to a

cell containing an appropriate mass of solvent mixture (mZ). The molality of CdCl2 in the

cell (1) after ith

addition (bi) is given by the expression (Zhang et al., 1993):

wmm

M wm b

i

i i

1

/

Z

i (2)

where M is the molar mass of cadmium chloride.

The preparation of electrodes, description of the cell, and of the equipment for pd

measurements, and the measuring procedure itself were explained earlier (Tomaš et al.,

2011).


The potentiometric results (Ei) for the cell (1) in 5 mass % 2-propanol (Z) at the different

CdCl2 molalities (bi) and at all operating temperatures (T) are compiled in Table 1.


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Table 1. Experimental data for cadmium chloride in Z at different temperatures

103 b / mol kg–1 E / V 103 b / mol kg–1 E / V

T = 293.15 K T = 298.15 K

4.123 0.77699 4.224 0.77757

7.686 0.75940 7.800 0.76027

10.732 0.75037 10.720 0.75144

13.358 0.74445 13.390 0.74539

15.672 0.74023 15.724 0.74126

17.720 0.73704 17.784 0.73804

19.525 0.73450 19.610 0.73570

21.150 0.73250 21.151 0.73368

22.528 0.73082 22.641 0.73206

23.862 0.72940 23.894 0.73066

25.098 0.72823 25.122 0.72948

26.129 0.72721 26.232 0.72841

27.160 0.72631 27.177 0.72751

28.030 0.72552 28.117 0.72669

28.920 0.72490 28.998 0.72594

T = 303.15 K T = 308.15 K

4.161 0.78030 4.193 0.78143

7.699 0.76154 7.730 0.76344

10.755 0.75667 10.732 0.75417

13.382 0.74650 13.399 0.74810

15.684 0.74244 15.664 0.74382

17.730 0.73930 17.674 0.74057

19.530 0.73671 19.545 0.73803

21.107 0.73480 21.170 0.73597

22.559 0.73305 22.633 0.73428

23.878 0.73173 23.943 0.73288

25.049 0.73053 25.146 0.73169

26.175 0.72941 26.245 0.73066

27.194 0.72851 27.241 0.72977

28.100 0.72771 28.162 0.72896

28.953 0.72691 29.010 0.72822


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Table 1. (Continued)

103 b / mol kg–1 E / V

T = 313.15 K

4.151 0.77440

7.682 0.76502

10.710 0.75575

13.346 0.74958

15.626 0.74545

17.667 0.74226

19.477 0.73965

21.106 0.73758

22.565 0.73593

23.866 0.73453

25.066 0.73328

26.179 0.73218

27.183 0.73127

28.108 0.73042

28.971 0.72964

The values of E from Table 1 were used to calculate the standard molal pd (o

bE ) for the cell

reaction:

Cd(s) + 2 AgCl(s) ⇌ 2 Ag(s) + Cd2+

(Z) + 2Cl–(Z) (3)

according to the relation,

))/(/(1)( ln(10)3

))/(Cl)()/(Cd(ln2

1/2o0

1/2o2oo2 bIBaI/bAF

RTbbbb

F

RTEE bb

oo / ln(10) 2

3(X)Σ1ln

2

3b I C

F

RTEb M

F

RTbxZ (4)

This relation was obtained using a combination of the Nernst equation and the Debye-

Hückel equation for the mean activity coefficient. Here I denotes ionic strength, and bo = 1

mol kg–1

. The following data are needed to solve Eq. (4):


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a) a value for the ion-size parameter (a0); a0 = 0.45 nm (Višić et al., 1993),

b) the molar mass and mass fraction of water and of 2-propanol; these data are used to

determine the mean molar mass (M) of investigated mixed solvent Z,

c) Debye-Hückel constants (Ab, Bb); these constants were calculated using literature data

for the relative permittivity, r (Åkerlöf, 1932) and the density, d (from the present

study) of the solvent Z: the properties (r, d) at different temperatures are given in

Table 2.

d) the equilibrium molalities of all ionic species, given by the term xb(X), as well as the

cell pd (E) for each cadmium chloride molality.

Determination of the o

bE can be performed either by extrapolation of E' from Eq. (4) to

zero ionic strength or by the least-squares method. To calculate b(Cd2+

), b(Cl–), and b (of

the remaining ionic species) for each molality of CdCl2, it is necessary to consider the

complexation reaction in investigated mixed solvent Z (Višić Mekjavić, 1989):

Cd2+

+ nCl– ⇌ )(2CdCl n

n (n = 1, 2 i 3) (5)

Inasmuch as the iterative procedure has been described in detail in our previous papers

(Tomaš et al., 2004 and 2011), only a short ouline will be provided here. Namely, for a

given cadmium chloride molality, the stoichiometric ionic strength of the solution is first

calculated as I = 3 b d, and the concentration stability constants, nK , for the complex

forming reactions (5) are estimated at this ionic strength using next relation:

oo1/2o1/2o2

n /(ln10)Δln))/(/(1)/(Δln cICKcIaBcIAzK nn0ccn (6)

These stability constants are then served to calculate the concentration of each ionic

species. Initial concentrations thus obtained are used to calculate the new ionic strength and

stability constant values. The treatment is repeated until a satisfactory constancy of nK

values is obtained. In Eq. (6) Cn is an empirical constant, co = 1 mol dm

–3, and

2Δ nz = (2 –

n)2 – n – 4, where z is the charge of each ionic species. The parameters Cn and

o

nK

(thermodynamic stability constant) were determined experimentally for water medium, and

for 10 mass % 2-propanol (Višić et al., 1993). For the present study, Cn and o

nK , are

interpolated from literature data; values at different temperatures for mixed solvent Z are

reported in Table 2.


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The obtained equlibrium concentrations of all ionic species were then expressed as

molalities, and using Eq. (4) the standard molal pd, o

bE , and its standard deviation were

determined using the least-squares method. These values are listed in Table 3.

Regression analysis showed that dependence of o

bE on T can be adequately fitted by the

second-order polynomial.

2o )( cTbTaTEb (7)

Table 2. Parameters of Eq. (6) in Z at different temperatures

T / K 293.15 298.15 303.15 308.15 313.15

o1K

121 123 128 132 136

o2K

517 575 610 644 678

o3K

409 448 448 507 537

ΔC1 0.192 0.191 0.189 0.187 0.185

ΔC2 0.375 0.380 0.379 0.379 0.379

ΔC3 0.477 0.475 0.467 0.459 0.452

r 76.74 74.96 73.21 71.50 69.78

d / g cm–3 0.98962 0.98840 0.98692 0.98522 0.98330

Table 3. Standard potential difference (o

bE ) of cell (1) in Z at different temperatures

T / K Eb

o ± (Ebo) / V

293.15 0.56840 ± 0.00009

298.15 0.56579 ± 0.00008

303.15 0.56405 ± 0.00016

308.15 0.56149 ± 0.00012

313.15 0.55893 ± 0.00008

The polynomial coefficients a, b and c, obtained by fitting Eq. (7) to the experimental

results are presented in Table 4, together with their standard deviations.

Table 4. Adjustable coefficients a, b and c of Eq. (7) in system CdCl2–Z

a / V 0.5157 ± 0.282

104 b / V K–1 7.824 ± 4.32


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106 c / V K–2 –2.057 ± 1.75

From the first derivate of Eq. (7), according to relation

rSo = d

o

bE /dT = z F (b + 2 cT), (z = 2) (8)

the standard entropy (rSo) of the cell reaction (3) in Z is obtained. The standard Gibbs

energy (rGo) is calculated according to the expression,

oo

rΔ bzFEG (9)

while the standard enthalpy (rHo) can be calculated from the relationship,

rHo = rG

o + T rS

o (10)

The standard thermodynamic quantities for the cell reaction (3) in Z at 298.15 K are given

in Table 5. The data for water medium are, for comparision, listed in the same table. The

deviations were calculated from the standard deviation for o

bE .

Table 5. Standard thermodynamic quantities for the cell reaction (3) in water medium and solvent Z

at 298.15 K

mass % rG

o / kJ mol–1 rHo / kJ mol–1 rS

o / J K–1 mol–1

0 (Višić Mekjavić, 1993) –110.66 ± 0.02 –134 ± 5 –78 ± 10

5 (present study, Z) –109.18 ± 0.01 –129 ± 13 –65 ± 25

As seen from Table 5 the standard Gibbs energy for both solvents has the negative sign,

which proves that the cell reaction (3) is spontaneous. The values for the standard enthalpy

and entropy change are also negative. It can be seen that the cell reaction (3) is exothermic

and lead to decrease in entropy. It can also be seen that the spontaneity (rGo) of reaction

(3) decreaces with adding 2-propanol in water. The same was reported for the aqueous

mixtures with 5 mass % 2-butanon, 2-butanol, or t-butanol (Tomaš et al., 2004, 2005, and

2011).

In this work, the stoichiometric mean molal activity coefficient () of cadmium chloride in

Z was calculated using the data for E and o

bE of the cell (see Tables 1 and 3), according to

the Nernst equation for the cell reaction (3):


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48

3oo )/(4ln )/2( γbbFRTEE b (11)

provided that chlorocadmium complexes were not formed in the solution. Table 6 shows

the obtained values of CdCl2 in 5 mass % 2-propanol (Z) at each molality and at

different temperatures. Values of for cadmium chloride in 5 mass% 2-propanol (Z) were

not found in literature.

Table 6. Stoichiometric mean molal activity coefficients (±) of CdCl2 in Z at different temperatures

T / K

b(CdCl2) / mol kg–1

0.003 0.005 0.007 0.009 0.010 0.015 0.020

293.15 0.697 0.599 0.542 0.503 0.487 0.432 0.397

298.15 0.691 0.594 0.535 0.495 0.479 0.423 0.387

303.15 0.678 0.583 0.527 0.489 0.474 0.419 0.381

308.15 0.672 0.577 0.525 0.485 0.469 0.413 0.377

313.15 0.665 0.570 0.520 0.479 0.462 0.410 0.368

According to the values given in Table 6, activity coefficients decrease with increasing

CdCl2 molality, as well as decrease with increasing tempertaure. This result is expected

from Debye-Hückel theory. A corresponding behaviour has been found in our earlier

studies of the same electrolyte in aqueous ketone (Tomaš et al., 2004) and aqueous alcohols

(Tomaš et al., 2005 and 2011) at the same mass fractions. When comparing the values

from present study with those for water medium (Višić Mekjavić, 1993), it can be seen

that these values are higher in water. Obviously, this is related to the degree of

complexation. Analogy was established with aqueous mixtures with 5 mass % 2-butanone,

2-butanol, and t-butanol (Tomaš et al., 2004, 2005, and 2011). Furthermore, values of for

cadmium chloride at the same mass fraction (Z = 5 mass %) are similar; certain differences

are due to influence of relative permittivity and nature of organic component in the mixed

solvent.

Conclusions

The present investigation of thermodynamic properties of cadmium chloride in aqueous

mixture with 5 mass % 2-propanol (Z) can be summarised as following: For the present

system CdCl2 in Z, was determined standard molal potential difference for the cell reaction

at different temperatures: this value decreases with an increase in the temperature. At

constant temperature, the mean molal activity coefficient of cadmium chloride decreases

with an increase in the molality of solution. At fixed molality, the mean molal activity


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49

coefficient of cadmium chloride decreases with an increase the temperature. The cell

reaction is spontaneous, exothermic and leads to reduced in entropy.

References

Åkerlöf, G. (1932): Dielectric constants of some organic solvent–water mixtures at various

temperatures, J. Am. Chem. Soc. 54 (11), 4125-4139.

Bald, A., Kinart, Z., Tomaš, R. (2013): Volumetric studies of aqueous solutions of monosodium

salts of some aliphatic dicarboxylic acids at 298.15 K. A new method of data analysis, J. Mol.

Liq. 178, 94-98.

Tomaš, R., Višić. M., Mekjavić, I. (2000): Thermodynamics of cadmium chloride in t-butanol –

water mixtures (wt-BuOH = 10%, 30%, and 50 %) from electromotive force measurements,

Croat. Chem. Acta 73 (2), 423-433.

Tomaš, R., Tominić, I., Višić, M., Sokol, V. (2004): Thermodynamics of Cadmium Chloride in 2-

Butanone + Water mixtures (5, 10, and 15 Mass%) from Electromotive Force Measurements,

J. Solution Chem. 33 (11), 1397-1410.

Tomaš, R., Tominić, I., Višić, M., Sokol, V. (2005): Thermodynamic study of cadmium chloride in

aqueous mixtures of 2-butanol from potential difference measurements, J. Solution Chem. 34

(8), 981-992.

Tomaš, R., Sokol, V., Bošković, P. (2011): Thermodynamic properties of CdCl2 in tert. butanol (5

mass %) + water mixture. In Proceedings: International Scientific and Professional Conference

13th Ružička Days, Vukovar, September 2010, Šubarić, D. (Ed.), Osijek, pp. 95-106.

Višić, M., Mekjavić, I. (1989): Thermodynamics of the cell: Cd(s)+Hg CdCl2(aq, m)AgClAg, J.

Chem. Thermodynamics 21 (2), 139-145.

Višić, M., Jadrić, A., Mekjavić, I. (1993): The stability constants of cadmium chloride complexes in

2-propanol–water mixtures (0, 10, 30 and 50 mass per cent) from electromotive force

measurements, Croat. Chem. Acta 66 (3-4), 489-498.

Višić, M., Mekjavić, I. (1993): Thermodynamics of the cell: Cd(Hg)satd. CdCl2(m)AgClAg in

(10, 30 and 50 Mass per Cent) 2-Propanol – Water Mixtures, Croat. Chem. Acta 66 (3-4), 479-

488.

Zhang, L., Lu, X., Wang, Y., Shi, J. (1993): Determination of activity coefficients using a flow emf

method. 1. HCl in methanol–water mixtures at 25, 35 and 45C, J. Solution Chem. 22 (2), 137-

150.

Sekcija: Kemijsko i biokemijsko inženjerstvo

Session: Chemical and biochemical engineering


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Kemijsko i biokemijsko inženjerstvo / Chemical and biochemical engineering

50

Supercritical fluid extraction laboratory plant design

UDC: 66.013.5 : 66.061

Krunoslav Aladić1, Stela Jokić

2*, Goran Horvat

3, Mate Bilić

2

1Croatian Veterinary Institute, Branch - Veterinary Institute Vinkovci, Josipa Kozarca 24,

HR-32100 Vinkovci, Croatia 2University of Josip Juraj Strossmayer in Osijek, Faculty of Food Technology Osijek, Franje Kuhaca 20,

HR-31000 Osijek, Croatia 3University of Josip Juraj Strossmayer in Osijek, Faculty of Electrical Engineering, Kneza Trpimira 2b,


Summary

A traditional solvent extraction method requires relatively large quantities of solvents, leaving toxic

solvent residue and causing degradation of unsaturated compounds. Due to this fact there is an

increasing demand for different extraction techniques which provide shortened extraction time,

reduced organic solvent consumption, and decreased pollution. Supercritical Fluid Extraction (SFE)

technique presents various advantages over traditional methods, such as the use of low

temperatures, reduced energy consumption and high product quality due to the absence of solvent in

extracts. In SFE process, environmentally friendly CO2 is primarily used as an extracting agent. The

aim of this work was the design and development of a supercritical CO2 extraction system used for

laboratory measurements based on existing commercial systems. Alongside with the developed

laboratory plant, an electronic system and PC application were developed for process control and

future automation to achieve most accurate extraction parameters for production of quality extracts.

Keywords: supercritical CO2 extraction, system construction, process control, automation,

PC application

Introduction

Supercritical CO2 extraction represents good and valuable alternative to classical extraction

process for many bioactive compounds with application in food and pharmaceutical

industry. Scientific researches in the field of SFE and application of supercritical CO2 for

extraction of variety of natural materials were significantly increased in the last few

decades. The advances have been made towards commercialization of supercritical CO2,

especially in the processing of fats and oils (Fang et al., 2008; Lack et al., 2006). SFE has

immediate advantages over the traditional extraction techniques such as time-saving, using

cheap and not organic solvent, not leaving toxic solvent residue, and do not causing

*Corresponding author: [email protected]


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degradation of unsaturated compounds due to the heat (Reverchon and De Marco, 2006).

The main disadvantages of SFE are the high investment costs for equipment acquisition

and the high energy demand for the CO2 extraction unit (Brunner, 2005; Martínez et al.,

2008; Sahena et al., 2009; Temelli, 2009).

Handmade supercritical fluid extraction (HM-SFE) system enables the extraction in an

inexpensive way. The obtained extraction yields and composition are very similar to those

obtained by commercial SFE system (Castro-Vargas et al., 2011). Just like a commercial

SFE systems, HM-SFE system is composed of various components that need to be in tune

to achieve optimal extraction process.

In this work the construction of new laboratory HM-SFE system was demonstrated. A

detailed explanation of each part of the equipment as well as the description of the

supporting electronic system was given.

Handmade supercritical fluid extraction (HM-SFE) system

Many mathematical models, presented in the literature, describe the supercritical fluid

extraction process (Valle and Fuente, 2006; Oliveira et al., 2011). Beside the knowledge of

phase equilibria, the knowledge of mass transfer rates is essential for designing process

equipment. The extraction process from solid substrates can be divided into two steps

(Brunner, 1984): the first one is transport of the substances within the solid material to the

interface solid-gas and the second one is transition of the substances into the gas and

transportation with the bulk of the extracting gas. It is assumed that the extractor is

cylindrically shaped and the supercritical solvent passes axially through the layer of material in

the extractor, carrying out soluble substance from the solid phase. Under these assumptions, the

mass balance in both phases can be represented by the following Eqs. (1)-(2):

yxJ

h

YD

hh

Yu

t

Yayi

,

(1)

s

f

)1(

,

yxJ

h

XDu

ht

Xaxi (2)

where is:

x, y dimensionless concentration of solid and liquid phases (kg/kg)

ui solvent rate (m/s)

h axial coordinate in the layer of material in the extractor (m/s)

axD diffusion coefficient in solid phase (m2/s)


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ayD axial dispersion coefficient (m2/s)

f, s fluid phase density and particle density (kg/m3)

J(x,y) mass transfer flow at the interface (1/s)

As can be seen from Eqs. (1) and (2) transfer phenomena which exist in the supercritical

extraction process are follows: accumulation in both phase, convection and dispersion in

the fluid phase, the solid phase diffusion and surface mass transfer.

The schematic diagram of newly constructed apparatus used for supercritical fluid

extraction is given in Fig. 1.

Fig. 1. Handmade supercritical fluid extraction system

(1. Compressor; 2. CO2 tank; 3. Stainless steel coil; 4. Cooling bath; 5. Air driven fluid pump Haskel

MS-71; 6. Valves (B-HV); 7. Manometers; 8. Extraction vessel; 9. Separator vessel; 10. Water bath; 11.

Centralized system glass fiber heater; 12. Flow meter; TRC-Temperature Recording Control)


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Materials used for HM-SFE system

Materials used for the construction of HM-SFE system were stainless steel AISI 316Ti and

AISI 304. All additional connection tubing parts were also same grade of material.

Extraction and separator vessels were properly tested at safety factor of 1.5. Extraction

vessel was tested at working pressure 50 MPa and separator vessel at 3 MPa.

Construction of HM-SFE system

The construction and assembling of HM-SFE system was performed by Đuro Đaković

Aparati d.o.o. (Slavonski Brod, Croatia) which provided material durability tests and

pressure test for vessels. Working pressure calculations for extractor and seamless tubes

are given in Eq. (3):

𝑃 =2∙𝑆∙𝑇

(𝑂.𝐷.−2∙𝑇)∙𝑆𝐹 (3)

where is:

P – fluid pressure (MPa)

T – wall thickness (extractor and seamless tube) (m)

O.D. – outer diameter (m)

SF – safety factor (usually is 1.5)

S – yield tensile strength of material

Extraction and separator vessel

Extraction vessel was made from stainless steel bar (AISI 304) O.D. 100 mm and height

500 mm. Stainless steel rod is drilled (center hole) with a Ø 40 mm bore for a 400 mm, so

volume of extractor is 500 mL. Upper inside part of extraction cell was polished to plug

well gaskets. Cap of extraction cell was designed to hold plug and it is connected with

extraction cell trough trapezoidal thread. Plug was patented by company that assembled the

HM-SFE system and seals in two places with o-ring. Lower part of the extraction cell was

also drilled and prepared for quick connection with R ½” connector with o-ring seal.

Separator vessel is made from stainless steel seamless tube (AISI 304) Ø 50 x 5 mm. It has

two plugs at upper and lower side of separator. Plugs are sealed with o-rings to ensure gas

tightness. Lower plug is made like a holder of cuvette for collecting the oil sample. At

upper side part of separator is 1/4" NPT connection, which leads to flow meter. Extractor

is tested at working pressure 50 MPa with safety factor 1.5 and separator is tested at

working pressure at 4 MPa also with safety factor 1.5.


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Filter element

In extraction vessel's plug filter element is placed. The main role of the filter is to prevent

the withdrawal of material. Filter element has the ability to filter particles 2 µm nominal

and 10 µm absolute (Norman Ultraporous 4202T-6T-2M).

High pressure seamless tubes (HPS) and high pressure valves

HPS tubes are dimension 10 x 2 mm and are connected to each other by Ermeto couplings

(flat, knees, tees). Used high pressure valves were provided by the same company that

produces Ermeto couplings (model B-HV).

Pressure and flow control

Pressure in extraction cell is controlled by two WIKA manometers (model 212.20) 60 MPa

and one WIKA manometer (model 212.20) 4 MPa for pressure in separator. Flow of CO2

is controlled through Matheson FM-1050 (E800) flow meter. Maximum flow rate that

given flow meter can measure is 63.03 SLPM.

Pump

Pump used for pressurize liquid CO2 is Haskel® MS-71. Liquid CO2 is precooled trough SS

coil at temperature -18 °C cooled by ethylene glycol/ethanol cooling bath. Pump has ability to

pressure liquid up to 60 MPa. Maximum working pressure is 40 MPa. After pump check valve

is located to prevent eventually disorders of CO2 flow. After extraction vessel high pressure is

reduced by high pressure valve (B-HV) to desirable pressure (1.5 MPa) leading to the

separator. Valves and tubing’s are heated to a temperature of 0 °C due to high pressure drop.

Electronic control

In order to achieve quality extract it is of outmost importance to precisely control process

parameters such as temperature of the CO2, pressure and the solvent flow rate. To enable the

precise control of the aforementioned parameters an electronic control system must be designed

and developed accordingly (Boyes, 2010). In a standard industrial plant a classical industrial

automation approach is often an only choice for precise process control, however when designing

a laboratory based extraction plant a more cost effective approach can be undertaken in order to

mitigate the initial cost of the system. This approach includes using a custom built embedded

system with proprietary sensors and actuators, designed to monitor and control the process. A

block diagram of the designed electronic control system is shown on Fig. 2.


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Embedded System

T T T T T. . .

Sensors

User interface

PC

Actuators

Fig. 2. Block diagram of the electronic control system

As seen from Fig. 2 the main component of the electronic control system is the embedded

system, which is often based on a microprocessor and peripheral circuits. The idea behind

using this architecture as opposed to the classical PLC regulation is the cost effectiveness

of the system and the ability to design a custom system for a specific plant (Greenfield,

2013). This in turn complicates the design of a device from the start, but enables an easy

implementation in future solutions. By enabling the connection with a personal computer

(PC) the laboratory based SFE extraction plant can be easily monitored and controlled

remotely by using Internet as a global communication network. This also enables the use

of proprietary sensors (such as pressure, flow and temperature sensors) needed to deliver

the data from the process to the regulation loop.

Due to the fact that the electronic control system is designed as an embedded system

(digital) regulation control must be realized using a discrete regulator. A most common

type of regulator that can be effectively applied to variety of systems is a PID

(Proportional, Integral and Derivative) regulator (Smith, 1997). A discrete system model

that can be easily applied to any micro-processor unit is shown on Fig. 3 and a discrete

PID regulator is shown on equation (4).

Fig. 3. Controlling continues process by discrete regulator


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𝑢[𝑘] = 𝐾𝑝𝑒[𝑘] + 𝐾𝑖 ∑ 𝑒[𝑘]𝑛𝑘=0 + 𝐾𝑑(𝑦[𝑘] − 𝑦[𝑘 − 1]) (4)

The resulting value of the PID regulator is transmitted to actuating modules that exhibit the

functionality of digital to analogue converter (DAC) in order to control continuous

processes in real life. In this form the actuators (such as heaters, valves etc.) are controlled

in order to achieve precise and efficient extraction process (Lehmann et al., 2012).

Next on, in order to disseminate the acquired data towards the end user, an application was

developed for PC (Fig. 4). The developed application has the ability to monitor process

parameters in real-time, enabling continuous supervision of the SFE extraction. Alongside

with the parameter supervision the developed application enables the control of the system

from two aspects: changing process parameters and controlling the extraction process

(starting and stopping of the extraction process). In this manner the process of laboratory

SFE extraction can be performed remotely, putting rigorous constraints on the safety of the

extraction and the overall process efficiency.

Fig. 4. Developed application for SFE system


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Finally, the main advantage of using the embedded system approach is the ability to enable

interconnection of the laboratory SFE system using the existing communication

infrastructure (Ethernet, Local Area Network), enabling the monitoring of data from

various locations and remotely using Internet. This approach utilizes existing network

connection for the SFE system, where all the data are tunneled through the existing Local

Area Network (LAN) (Fig. 5).

Router-Firewall

Ethernet LAN

`

PC2

`

PC1

PrintersServer

Internet

SFE System

Fig. 5. Networking the SFE system

By using standard communication protocols and technologies such as Ethernet and Wi-Fi

future interconnection with mobile devices (such as tablets and smartphones) is enabled by

default and by developing proprietary applications, the concept of the remote supervision

and control can be extended to everywhere where Internet access is available.

Conclusions

SFE emerged in the last few decades as a promising green technology and a good

alternative in food and natural products processing. With the rapid development of SFE

technology next generation of extraction plants will begin to emerge in the upcoming

years, combining up-to-date technological advances in optimizing SFE process. The

proposed system offers a cost effective solution for the small scale research SFE systems

with the ability of detailed parametric analysis and remote process supervision, normally

not available in industrial grade SFE systems. By presenting uniform and simple guidelines

for the construction of laboratory SFE system an adequate scale-up from laboratory to

industrial design purposes becomes a simple task.


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Acknowledgements

The authors greatly acknowledge to the Josip Juraj Strossmayer University of Osijek and

Faculty of Food Technology Osijek for financial support in construction of supercritical

fluid extraction system. This work is also sponsored by Ministry of Science, Education and

Sports of the Republic of Croatia under project grant No: 165-0362027-1479.

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Castro-Vargas, H.I., Rodríguez-Varela, L.I., Parada-Alfonso, F. (2011): Guava (Psidium guajava

L.) seed oil obtained with a homemade supercritical fluid extraction system using supercritical

CO2 and co-solvent, J. Supercrit. Fluid. 56, 238-242.

Fang, T., Goto, M., Sakaki, M., Yang, D. (2008): Extraction and purification of natural tocopherols

by supercritical carbon dioxide. In: Supercritical Fluid extraction of Nutraceuticals and

bioactive Compounds, Martinez, J.L. (ed.), Boca Raton: CRC Press, Taylor and Francis

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systems-are-changing-automation

Lack, E., Seidlitz, H; Sova, M. (2006): New industrial application of supercritical fluid extraction.

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Lehmann, R.J. Reiche, R. Schiefer, G. (2012): Future internet and the agri-food sector: State-of-the-

art in literature and research, Comput. Electron. Agr. 89, 158-174.

Martínez, M.L., Mattea, M.A., Maestri, D.M. (2008): Pressing and supercritical carbon dioxide

extraction of walnut oil, J. Food Eng. 88, 399-404.

Oliveira, E.L.G., Silvestre, A.J.D., Silva, C.M. (2011): Review of kinetic models for supercritical

fluid extraction, Chem. Eng. Res. Des. 89, 1104-1117.

Reverchon, E., De Marco, I. (2006). Supercritical fluid extraction and fractionation of natural

metter, J. Supercrit. Fluid. 38, 146-166.

Sahena, F., Zaidul, I.S.M., Jinap, S., Karim, A.A., Abbas, K.A., Norulaini, N.A.N., Omar, A.K.M.

(2009): Application of supercritical CO2 in lipid extraction - A review, J. Food Eng. 95, 240-253.

Smith, S.W. (1997): The Scientist and Engineer’s Guide to Digital Signal Processing. California

Technical Publishing, San Diego, CA, USA.

Temelli, F. (2009): Perspectives on supercritical fluid processing of fats and oils, J. Supercrit. Fluid.

47, 583-590.

Valle, J.M. del, Fuente, J.C. de la (2006): Supercritical CO2 extraction of oilseeds: Review of

kinetic and equilibrium models, Crit. Rev. Food Sci. 46, 131-160.


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Strukturna svojstva i ionska vodljivost nanokompozitnih

polimernih elektrolita

UDC: 544.6.018.47-036.5

Irena Banovac1a

, Matko Erceg1a

, Dražan Jozić1b

, Zorana Akrap1a

,

Sigrid Bernstorff2

1Sveučilište u Splitu, Kemijsko-tehnološki fakultet, (

aZavod za organsku tehnologiju,

bZavod za

anorgansku tehnologiju), Teslina 10/V, 21000 Split, Hrvatska 2Elettra - Sincrotrone Trieste S.C.p.A., Strada Statale 14 km 163,5 in AREA Science Park, I-34149,

Basovizza, Trieste, Italy

Sažetak

U ovom radu istraživan je utjecaj dodatka litijevog montmorilonita (LiMMT) na strukturu i ionsku

vodljivost nanokompozita na osnovi poli(etilen-oksida) (PEO). PEO je često korišten polimer za

pripremu polimernih elektrolita zbog svojih dobrih elektrokemijskih i mehaničkih svojstava. Ipak,

visok stupanj kristalnosti PEO smanjuje njegovu ionsku vodljivost. Smanjenje stupnja kristalnosti

postiže se dodatkom anorganskih čestica PEO-u stvarajući polimerne elektrolite s poboljšanim

elektrokemijskim svojstvima. Utjecaj dodatka LiMMT na strukturu PEO ispitivan je primjenom

raspršenja X-zračenja pri malom kutu (SAXS) i infracrvene spektroskopije s Fourierovom

transformacijom (FTIR). Rezultati SAXS metode ukazuju na interkalaciju polimernih lanca između

slojeva montmorilonita, odnosno nastanak nanokompozitne strukture. FTIR analiza pokazuje da

dodatak punila narušava helikoidalnu strukturu PEO u nanokompozitima, odnosno njegovu

kristalnost. Diferencijalnom pretražnom kalorimetrijom (DSC) potvrđeno je da dodatak punila

smanjuje kristalnost PEO. Ionska provodnost nanokompozita određena je elektrokemijskom

impendancijskom spektroskopijom (EIS). EIS pokazuje značajan porast ionske vodljivosti pri

sobnoj temperaturi dodatkom LiMMT te je definiran optimalan udio LiMMT.

Ključne riječi: PEO/LiMMT nanokompoziti, diferencijalna pretražna kalorimetrija, IR spektroskopija,

elektrokemijska impendancijska spektroskopija, raspršenje X-zračenja pri malom kutu

Uvod

Poli(etilen-oksid) (PEO) je polimer koji je odigrao značajnu ulogu u razvoju polimernih

elektrolita. Sustavi čvrstih polimernih elektrolita bazirani na PEO su među

najproučavanijim sustavima polielektrolita (Loyens et al., 2005). Vodljivost kod ovih

sustava je povezana s migracijom iona i s pokretljivošću segmenata polimernog lanca. S

obzirom na njegovu visoku kristalnost, PEO pokazuje slabu provodnost (σ) pri sobnoj

temperaturi (σ = 10-8

do 10-7

S cm-1

). Stoga je važno smanjiti stupanj kristalnosti PEO uz



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što niži negativni utjecaj na mehanička svojstva (Moreno et al., 2011). Dosadašnji pokušaji

smanjenja kristalnosti PEO dodatkom niskomolekulnih omekšavala, polarnih organskih

otapala pa čak i epoksidirane prirodne gume općenito su rezultirali povećanjem vodljivosti

PEO uz značajno smanjenje mehaničkih svojstava kompozita (Noor et al., 2010).

Poboljšanje vodljivosti PEO može se postići dodatkom slojevitih anorganskih materijala

nanometarskih dimenzija čestica u matricu PEO. U ovom radu za pripravu PEO

nanokompozita korišten je glineni mineral litijev montmorilonit (LiMMT) koji se dobije

ionskom izmjenom iz prirodnog, natrijevog montmorilonita, te je ispitan utjecaj dodatka

LiMMT-a na toplinska svojstva, kristalnost i ionsku vodljivost PEO.

Materijali i metode

Za pripremu uzoraka korišteni su prah poli(etilen-oksida) (Sigma-Aldrich, Inc., St. Louis,

USA; Mv=5 000 000), natrijev montmorilonit CloisiteNa+

(NaMMT) (Southern Clay

Products, Inc., SAD) i litijev klorid (Kemika, Zagreb, Croatia). LiMMT pripremljen je

postupkom ionske izmjene miješanjem NaMMT s otopinom LiCl koncentracije 1 moldm-3

magnetskom miješalicom u vremenskom intervalu od 48 sati pri 30 °C. Dobiveni LiMMT

je ispiran destiliranom vodom do potpunog uklanjanja kloridnih iona, zatim je sušen 5 sati

pri 120 °C u sušioniku te u vakuumskom sušioniku još 48 sati pri 100 °C.

Uzorci PEO/LiMMT sastava 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 30/70, 20/80 i 10/90

pripremljeni su metodom interkalacije iz taljevine. Polimer i punilo u određenom masenom

omjeru izmiješani su u tarioniku, te prešani u tabletice pod pritiskom od 5 t u vremenu od

jedne minute pri sobnoj temperaturi. Potom su uzorci zagrijavani pri 90 °C u trajanju od 8

sati (metoda interkalacije iz taljevine). Za vrijeme zagrijavanja polimerni lanci difundiraju

iz mase polimerne taljevine u silikatne međuslojeve, odnosno interkaliraju se između

slojeva silikata. Pri tome se samo povećava udaljenost između slojeva silikata, a zadržava

početna uređenost pa nastaje tzv. interkalirajući nanokompozit. Ova metoda je ekološki

prihvatljiva jer priprava nanokompozita ne zahtijeva upotrebu otapala (Mai et al., 2006).

Raspršenje X-zraka pri malom kutu (SAXS)

Raspršenje X-zraka pri malom kutu (eng. Small ange X-Ray Scattering (SAXS))

provedeno je na Austrijskoj SAXS liniji na Sinkrotronu Elettra. Energija korištenog

sinkrotronskog zračenja je 8 keV što odgovara valnoj duljini rendgenskog zračenja od 1,54 Å.

Udaljenost uzorka od detektora iznosila je 1132 mm što u ovom eksperimentalnom

postavu omogućava istraživanje u rasponu veličina od 70,00-0,83 nm. Korišteni detektor je

Image plate tip MAR300 (MarReserch) koji je za svaku sliku raspršenja eksponiran

zračenju u trajanju od 1-2 sekunde. Kalibracija q vrijednosti ali i uklanjanje sistematskih

pogreški korištenog eksperimentalnog postava provedena je primjenom vanjskog standarda


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AgBeh-a. Intenzitet je naknadno korigiran za sve sustave za tzv. dark current, odnosno za

sva elektromagnetska zračenja koje detektor prikuplja u ekspozicijskom vremenu, a koji ne

potječu direktno od uzorka. Na normalizirane krivulje primjenjena je Lorentzova korekcija

(IL=Imq2, q=(4π/λ)sinθ, gdje je IL korigirani intenzitet, Im mjereni intenzitet raspršenja, λ je

valna duljina X zračenja i 2θ je kut raspršenja). Pozicije difrakcijskih maksimuma su u

direktnom odnosu s veličinom jedinčne ćelije ili međuslojnom udaljenošću u slučaju

slojevitih materijala koja je određena prema Braggovom zakonu d=2π/q.

Infracrvena spektroskopija s Fourierovom transformacijom (FTIR)

Spektri infracrvene spektroskopije snimljeni su Perkin-Elmer Spectrum One

spektrometrom HATR tehnikom (eng. Horizontal Attenuated Total Reflectance). Uzorci su

postavljeni na ravni kristal od ZnSe (kut upadne zrake 45 °) te snimljeni u području od

4000-650 cm-1

uz vrijednosti spektralne rezolucije od 4 cm-1

. Snimanje svakog uzorka je

ponovljeno 10 puta, a dobiveni spektri predstavljaju njihovu srednju vrijednost.

Diferencijalna pretražna kalorimetrija (DSC)

Diferencijalni pretražni kalorimetar Mettler Toledo 823e i STAR

e software su korišteni za

snimanje uzoraka i obradu podataka. Analizirani uzorci su najprije ohlađeni od 25 do -90 °C.

Na temperaturi od -90 °C držani su 10 minuta. Potom su zagrijavani do 120 °C i zadržani 5

minuta na toj temperaturi. Uslijedilo je hlađenje na temperaturu od -90 °C na kojoj su

uzorci bili 10 minuta. Na kraju, uzorci su ponovno zagrijani do 120 °C. Sva zagrijavanja i

hlađenja su provedena brzinom od 20 °C min-1

.

Elektrokemijska impedancijska spektroskopija (EIS)

Impedancijska mjerenja su rađena na mjernom sustavu Potenciostat Solartron

Electrochemical Interface SI 1287 u kombinaciji s fazno osjetljivim pojačalom Solartron

HF Frequency response analyzer SI 1255 pri 25 ºC. Sustav je vođen, a podaci analizirani

programom Zplot/Zwiew (Scribner Associates, Inc., SAD). Uzorak polimernog elektrolita

bio je smješten u posebno konstruiran držač, a ostvarivao je kontakt s mjernim uređajima

preko bakrenih ploča. Mjerenja impedancije su izvedena u području frekvencija od 1 MHz

do 1 Hz s amplitudom pobude od ± 20 mV. Vrijednosti ionske provodnosti (σ) su

izračunate iz otpora Rb koji je određen iz Nyquistovih dijagrama prema jednadžbi:

t

ARb

1 (1)

gdje je A površina uzorka, a t debljina uzorka.


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SAXS

Profili krivulja raspršenja X-zraka pri malim kutovima (SAXS) korigiranih za Lorentzovu

korekciju na uzrocima pripravljenih kompozita PEO/LiMMT prikazani su na slici 1. Čisti

uzorak LiMMT, budući da je riječ o slojevitom alumosilikatu u smjeru kristalografske osi c,

pokazuje difrakcijski maksimum pri q vrijednosti 5,21 nm-1

što odgovara međuplošnoj

udaljenosti d001=1,204 nm. Za čisti uzorak PEO također se uočava prisutnost difrakcijskog

maksimuma pri q=7,15 nm-1

što odgovora d vrijednosti od 0,879 nm. Iz krivulja raspršenja

se uočava da dodatkom LiMMT u polimernu matricu PEO dolazi do promjene položaja

difrakcijskog maksimuma koji odgovara međuplošnoj udaljenosti LiMMT prema nižim

vrijednostima q. To znači da dolazi do povećanja međuplošne udaljenosti a time i porasta

d001 vrijednosti. Do povećanja d001 vrijednosti dolazi zbog ugradnje (interkalacije) PEO u

međuplošni prostor LiMMT-a, što ukazuje na nastanak interkalirane strukture

PEO/LiMMT nanokompozita.

Slika 1. Krivulje raspršenja X-zraka pri malim kutovima (SAXS) za LiMMT

i nanokompozite PEO/LiMMT

Fig. 1. SAXS pattern for LiMMT and PEO/LiMMT nanocomposites


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Slika 2. FT-IR spektar izvornog PEO, LiMMT i PEO/LiMMT nanokompozita

Fig. 2. FT-IR spectra of pure PEO, LiMMT and PEO/LiMMT nanocomposites

FTIR

Interakcija između lanaca PEO i kationa unutar galerije LiMMT je određena primjenom

infracrvene spektroskopije s Fourierovom transformacijom. Snimanje uzoraka je

provedeno HATR tehnikom u području valnih brojeva od 4000-650 cm-1

jer se u tom

području događaju glavne molekulske vibracije pri kojima dolazi do rastezanja i savijanja

veza.

PEO je polimer visokog stupnja kristalnosti, a njegove makromolekule zauzimaju spiralnu

ili helikoidalnu konformaciju u kristalnom stanju (Gnanou et al., 2008). Helikoidalna

konformacija makromolekule PEO sadrži sedam –CH2CH2O- jednica koje su raspoređene

u dvije spirale koji čine tzv. uzvojnicu ili heliks. U području valnih brojeva od 1000-700 cm-1

nalaze se CH2 njihajne vibracije koje su posebno osjetljive na konformacijske promjene.

Prisutnost dviju apsorpcijskih vrpci blizu valnih brojeva 945 i 840 cm-1

kod čistog PEO

pripisuje se njihajnim vibracijama CH2 skupina koje se nalaze u tzv. gauche konformaciji

(Arranda i Ruiz-Hitzky, 1992). Intenzitet tih apsorpcijskih vrpci smanjuje se s dodatkom

Li-MMT. Vrpca pri 945 cm-1

potpuno isčezava kod nanokompozita s udjelom punila

većim od 60 mas. %, dok se vrpca pri 840 cm-1

gubi kod nanokompozita s udjelom

LiMMT većim od 70 mas. %. Međutim, pri udjelima LiMMT većim od 70 mas.%


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pojavljuje se vrpca pri 847 cm-1

. Sve navedeno vodi ka zaključku da je narušena helikoidna

konformacija PEO koja je preduvjet kristalnosti PEO.

DSC

Normalizirane DSC krivulje drugog zagrijavanja svih analiziranih uzoraka prikazane su na slici 3.

Iz njih su očitane sljedeće toplinske karakteristike: talište, staklište i toplina taljenja (ΔHt).

Talište se izražava preko ekstrapolirane početne temperature taljenja (Tp,t), temperature u

minimumu endoterme taljenja (Tm,t) i ekstrapolirane konačne temperature taljenja (Tk,t).

Staklište se izražava preko ekstrapolirane početne temperature staklastog prijelaza (Tep,g),

ekstrapolirane konačne temperature staklastog prijelaza (Tek,g) i temperature na polovini ukupne

promjene toplinskog toka u području staklastog prijelaza (Tm,g). Iz vrijednosti ∆Ht primjenom

izraza (2) izračunat je i stupanj kristalnosti, Xc, PEO-a (tablica 1) prema izrazu:

100(%)0

wH

HX t

c (2)

gdje je ∆Ht toplina taljenja PEO određena DSC analizom, ΔH0 toplina taljenja 100 %

kristalnog PEO, w maseni udio komponente kojoj se određuje postotak kristalnosti. ΔH0 za

100 % kristalni PEO iznosi 205 J g-1

(Zheng et al., 2004).

Slika 3. Normalizirane DSC krivulje zagrijavanja PEO, LiMMT i PEO/LiMMT nanokompozita

Fig. 3. Normalized DSC heating curves of PEO, LiMMT and PEO/LiMMT nanocomposites


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Tablica 1. DSC podaci PEO, LiMMT i PEO/LiMMT nanokompozita

Table 1. DSC data for PEO, LiMMT and PEO/LiMMT nanocomposites

PEO/LiMMT Tp,t

/

o

C Tm,t

/

o

C Tk,t

/

o

C -ΔHt

/ Jg

-1

Tep,g

/

o

C Tm,g

/

o

C Tek,g

/

o

C Xc

/ %

100/0 58 73 81 127,3 -57 -52 -48 62,1

90/10 48 67 74 108,3 -53 -48 -48 58,7

80/20 52 64 74 91,19 -60 -56 -49 55,6

70/30 44 58 64 63,69 -63 -59 -54 44,4

60/40 40 51 56 11,22 -63 -60 -56 9,1

50/50 40 52 56 26,45 -65 -62 -58 25,8

40/60 45 57 64 23,51 -57 -55 -38 28,7

30/70 43 58 65 7,62 -58 -51 -35 12,4

20/80 - - - - - - - -

10/90 - - - - - - - -

0/100 - - - - - - - -

Slika 3. prikazuje da se kod uzoraka s udjelom do 70 mas.% litijevog montmorilonita

pojavljuje jedno talište, što predstavlja taljenje kristalne faze PEO. Daljnjim dodatkom

litijevog montmorilonita ne uočavaju se endoterme taljenja što upućuje na nepostojanje

kristalne faze u PEO. Vrijednosti Tp,t, Tm,t i Tk,t su niže u svim PEO/LiMMT

nanokompozitima u odnosu na izvorni PEO (tablica 1). Općenito, niže talište PEO u

nanokompozitima PEO/Li-MMT se može pripisati ometanju procesa kristalizacije u

prisutnosti litijevog montmorilonita. Toplina taljenja (ΔHt) se također smanjuje

povećanjem udjela litijevog montmorilonita i niža je od topline taljenja izvornog PEO za

sve uzorke PEO/Li-MMT (Tablica 1). Iz vrijednosti ΔHt je primjenom izraza (2) izračunat

stupanj kristalnosti Xc PEO u svim uzorcima (Tablica 1). Dodatak litijevog montmorilonita

do 40 mas.% značajno snižava stupanj kristalnosti PEO (uzorak sa 40% Li-MMT pokazuje

smanjenje stupnja kristalizacije za 53% u odnosu na uzorak 100/0). Daljnji dodatak punila

do 70 mas.% rezultira nešto većim vrijednostima stupnja kristalizacije, dok se pri udjelima

LiMMT-a većim od 80% kristalnost PEO potpuno gubi.

Vrijednosti staklišta izražene kao Tep,g, Tm,g, Tek,g dodatkom Li-MMT pokazuju niže

vrijednosti u odnosu na čisti PEO (Tablica 1).

EIS

Elektrokemijska impedancijska spektroskopija korištena je za ispitivanje utjecaja dodatka

LiMMT na ionsku provodnost PEO. Nyquistov prikaz impedancijskih spektara polimernog

elektrolita na bazi PEO prikazan je na slici 4. Nyquistov prikaz za polimerni elektrolit

pokazuje kapacitivni polukrug s centrom ispod realne osi što je posljedica neidealnog


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kapacitivnog ponašanja. U nižem frekvencijskom području kapacitivni polukrug prelazi u

pravac s nagibom od oko 45° karakterističan za difuzijske procese. Dobiveni spektri su u

skladu sa spektrima za nanokompozite PEO u prethodno objavljenim radovima (Ratna et

al., 2007; Kim et al., 2008). Radijus polukruga ovisan je o sastavu elektrolita (udjelu

LiMMT u elektrolitu). Mjesto gdje kapacitivni polukrug siječe realnu os impedancije

predstavlja otpor elektrolita Rb (Slika 4).

Slika 4. Elektrokemijski impedancijski spektar nanokompozita PEO/LiMMT 10/90

Fig. 4. Electrochemical impedance spectra of nanocomposite PEO/LiMMT 10/90

Rezultati pokazuju da dodatak LiMMT povećava ionsku provodnost uzoraka (Slika 5).

Maksimalna izračunata provodnost iznosi 8,9∙10-7 S cm

-1, a javlja se kod uzorka s 40 mas. %

LiMMT što je 649 puta veće od ionske provodnosti izvornog PEO (1,4∙10-9

S cm-1

).

Daljnjim povećanjem mas. % LiMMT u nanokompozitima, vrijednosti provodnosti

pokazuju blagi pad, no i dalje su veće od izvornog PEO. Poznato je da ionska vodljivost

raste smanjenjem udjela kristalne faze polimera obzirom da se transport Li+ iona odvija

kroz amorfnu fazu. Do poboljšanja transporta slobodnih Li+ iona dolazi i jer Lewisova

kisela mjesta na površini punila reagiraju s kisikovim atomima iz PEO čime slabi

interakcija kisikovih atoma i Li+

oslobađajući veći broj Li+ iona (Xi et al., 2005). Ipak,

nakon što je optimalni udio LiMMT postignut, ionska vodljivost opada iako udio i amorfne

faze i broj slobodnih Li+ iona rastu s porastom udjela LiMMT. To je stoga što disperzija

LiMMT u polimernoj matrici utječe na ionsku vodljivost. Pri nižim udjelima LiMMT je

dobro dispergiran u matrici PEO stvarajući povoljno okruženje za mobilnost Li+ iona.


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Dodatak LiMMT iznad 40 mas. % dovodi do samoagregacije silikatnih slojeva koji čvrsto

drže Li+ ione ograničavajući njihovu mobilnost posljedično smanjujući vodljivost

PEO/LiMMT nanokompozita.

Slika 5. Ionska provodnost PEO, LiMMT, PEO/LiMMT nanokompozita

Fig. 5. Ionic conductivity of PEO, LiMMT, PEO/LiMMT nanocomposites

Zaključci

Analiza krivulja raspršenja (SAXS) ukazuje na nastanak interkalirane nanokompozitne

strukture PEO/LiMMT. FTIR analiza je pokazala da izvorni PEO ima spiralnu

konformaciju u kristalnom stanju te da dodatak LiMMT mijenja konformaciju i

posljedično smanjuje kristalnost PEO. Primjenom diferencijalne pretražne kalorimetrije

utvrđeno je da se dodatkom LiMMT smanjuju vrijednosti tališta, staklišta te je dokazano

smanjenje kristalnosti PEO. Kod uzoraka s dodatkom LiMMT u količinama većim od

70 mas. % kristalnost PEO potpuno isčezava. Elektrokemijska impedancijska

spektroskopija pokazala je da ionska provodnost raste s povećanjem udjela LiMMT te

doseže maksimalnu vrijednost od 8,9∙10-7

S cm-1

za uzorke s 40 mas. % dodatka LiMMT.

Na povećanje ionske vodljivosti povoljno utječu povećanje udjela amorfne faze polimera i

poboljšanje transporta Li+ iona Lewisovim kiselo-baznim interakcijama u polimernom

elektrolitu. Efekt disperzije LiMMT postaje vidljiv nakon prekoračenja optimalnog udjela

nanopunila kada dolazi do samoagregacije silikatnih slojeva što ograničava mobilnost

Li+ iona s posljedicom pada ionske vodljivosti.


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Literatura

Arranda P., Ruiz-Hitzky E. (1992): Poly(ethylene oxide)-silicate intercalation materials, Chem.

Mater. 4, 1395-1403.

Gnanou, Y., Fontanille, M. (2008): Organic and Physical Chemistry of Polymers, New Jersey, John

Wiley & Sons, Inc. Hoboken, pp. 554.

Kim, S., Hwang E. J., Jung Y., Han M., Park, S. J.(2008): Ionic conductivity of polymeric

nanocomposite electrolytes based on poly(ethylene oxide) and organo-clay materials, Colloid

Surface A 313-314, 216-219

Loyens, W., Jannasch, P., Maurer, F.H.J. (2005) : Effect of clay modifier and matrix molar mass on

the structure and properties of poly(ethylene oxide)/Cloisite nanocomposites via melt-

compounding, Polymer 46 (3), 903-914.

Mai, Y.W., Yu Z.Z. (2006): Polymer nanocomposites, New York, CRC Press LLC, pp. 61.

Noor, S. A. M., Ahmad A., Rahman M.Y.A., Talib I. A. (2010) :Solid polymeric electrolyte of

poly(ethylene)oxide-50% epoxidized natural rubber-lithium triflate (PEO-ENR50-LiCF3SO3),

Natural Science 2, 190-196.

Moreno, M., Quijada, R., Santa Ana, M. A., Benavente E., Gomez- Romero P., Gonzáles, G.

(2011): Electrical and mechanical properties of poly(ethylene oxide)/intercalated clay polymer

electrolyte, Electrochim. Acta 58, 112-118.

Ratna D., Divekar S., Patchaiappan S., Samui A.B. Chakraborty B. C. (2007): Poly(ethylene

oxide)/clay nanocomposites for solid polymer electrolyte applications, Polym Int 56 ,900-904

Xi, J.Y., Qiu, X.P., Ma, X.M., Cui, M.Z., Yang, J., Tang, X.Z., Zhu, W.T., Chen, L.Q. (2005):

Composite polymer electrolyte doped with mesoporous silica SBA-15 for lithium polymer

battery, Solis state ionics 176,13-14

Zheng, S., Nie K., Guo Q. (2004): Miscibility and phase separation in blends of phenolphtalein

poly(aryl ether ketone) and poly(ethylene oxide): a differential scanning calorimetry study,

Thermochim. Acta 419, 267-274.

http://www.sciencedirect.com/science/article/pii/S0032386104011838




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Structural properties and ionic conductivity of nanocomposite

polymer electrolytes

Irena Banovac1a

, Matko Erceg1a

, Dražan Jozić1b

,

Zorana Akrap1a

, Sigrid Bernstorff2

1University of Split, Faculty of Chemistry and Technology, (

aDepartment of Organic Technology,

bDepartment of Inorganic Technology), Teslina 10/V, HR-21000 Split, Croatia

2Elettra - Sincrotrone Trieste S.C.p.A., Strada Statale 14 km 163,5 in AREA Science Park, I-34149,

Basovizza, Trieste, Italy

Summary

In this work the effect of addition of lithium montmorillonite (LiMMT) on the structure and ionic

conductivity of poly(ethylene oxide) based nanocomposites was investigated. PEO is widely used

polymer to prepare polymer composite electrolytes due ti its good electrochemical and mechanical

properties. However, high degree of crystallinity reduces its ionic conductivity. The decrease in

crystallinity can be achieved by adding inorganic particles to PEO thus creating polymer

electrolytes with improved electrochemical properties. The effect of addition of LiMMT on stucture

of PEO was investigated by small-angle X-ray scattering (SAXS) and Fourier transform infrared

spectroscopy (FTIR). SAXS results indicate intercalation of polymer chains in the interlayer of

montmorillonite, i.e. formation nanocomposite structure. FTIR analysis shows that the addition of

filler disrupts helical structure of PEO in nanocomposites, i.e. its crystallinity. Differential scanning

calorimetry (DSC) confirmed that the addition of filler reduces the crystallinity of PEO. Ionic

conductivity of nanocomposites was determinated by electrochemical impendance spectroscopy

(EIS). EIS shows significant increase of ionic conductivity at room temperature with addition of

LiMMT and optimum LiMMT content was defined.

Keywords: PEO/LiMMT nanocomposites, differential scanning calorimetry, IR spectroscopy,

electrochemical impedance spectroscopy, small angle X-ray diffraction


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Local sensitivity analysis of integrated

membrane bioreactor model

UDC: 66.023

Mirjana Čurlin1

, Ana Jurinjak Tušek1, Tamara Jurina

1,

Irena Petrinić2, Želimir Kurtanjek

1

1University of Zagreb, Faculty of Food Technology and Biotechnology, Pierottijeva 6, HR-10000

Zagreb, Croatia 2University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova ulica 17, 2000

Maribor, Slovenia

Summary

Membrane bioreactors (MBRs) consist of the combination of biological processes (typically

activated sludge processes) with membrane technologies and are being applied when high quality

effluents are required. Over the last decades, membrane technologies alone or in combination with

biological processes (i.e. MBRs) have been successfully applied for municipal and industrial

wastewater treatment. MBR-based technology is a complex physical-chemical and biological

system with numerous interactions between the process variables. For design and better

understanding of this complex system mathematical modelling is a very useful tool. According to

literature there are several integrated models describing the both physical and biological processes

taking part in MBRs with some simplifications. But there is still a big challenge in developing of an

integrated model able to describe the biological nutrient removal, formation/degradation of soluble

microbial products and physical separation. In this work, one-to-one local sensitivity analysis was

applied to define the most important parameters of integrated activated sludge model 2d and soluble

microbial product (ASM2d-SMP) model proposed by Cosenza et al. (2013). Model includes 19

biological state variables and 79 parameters (kinetics, stoichiometric, physical and fraction-related).

By simulating described mathematical model with one and three percent parameters value

increase/decrease the key regulation points of the model are detected. Obtained results facilitate

future experiments planning and can be a good starting point for model reduction.

Keywords: membrane bioreactor, activated sludge model, local sensitivity analysis, FAST method

Introduction

Since the regulation of wastewater has been noticeably increased in many countries,

membrane bioreactors (MBRs) can be an attractive option for municipal as well as

industrial wastewater treatment. MBRs have become a popular biological wastewater

treatment technology as one of the next generations of wastewater treatment processes to



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be developed from either clasical activated sludge (CAS) or trickling filter systems.

Among the other benefits, MBRs (compared to CAS) provide lower footprint, lower

sludge production, rapid start-up of biological processes, and high-quality effluent

production (Judd et al., 2010). Besides membrane fouling and the high cost of membranes

identified as main obstacles for wider application of MBRs, recent studies have reported

several crucial specificities of the treatment system. MBR is a complex physical-chemical

and biological system in which activated sludge treatment is directly combined with

membrane technology.

Due to the higher number of interaction between the process variables in the system,

mathematical modelling plays a key role in order to design of the system and context of “MBR

knowledge upgrading”. Well known activated sludge models (ASM 1-3, 2d) (Henze at al.,

2000) originally developed for CAS system, have been applied in their original form or adapted

(hybrid model) in order to simulate the bioprocesses occuring in MBR system (Jiang et al.,

2008). The main adaptation of ASM models for MBR is inclusion of formation and

degradation of soluble microbial products (SMPs). SMPs are defined as soluble cellular

components or debris that are released during cell lysis, diffused through the cell membrane,

lost during synthesis, or excreted for some purpose. Substrate utilization, biomass decay, and

hydrolysis of extracellular substances are the major processes contributing to SMPs formation.

According to many authors (Cho et al. (2005), Judd (2010), Drews et al. (2010)) SMPs have a

significant influence on membrane fouling and reducing membrane permeability which are the

main problem for properly operating of MBR. Therefore, integrated model which includes key

biological processes and physical processes on membrane have been proposed to improve the

knowledge about the correlation between the biological processes and various membrane

fouling phenomena. These models are needed for design and better understanding of complex

MBR process. Many authors have proposed integrated models (Zarragoitia-Gonzales et al.

(2008), Mannina et al. (2011), Lu et al. (2001)) which are focused only on few targeted aspects

of steady-state biological processes linked to membrane fouling models, leaving many

unresolved issues in model calibration. For this reason, integrated models are not yet fully

available and have yet to be developed (Zuthi et al., 2012).

The objective of this study was to apply one-to-one local sensitivity analysis in order to identify

the key parameters of microbiological reactions included in integrated activated sludge model

2d and soluble microbial product (ASM2d-SMP) model proposed by Cosenza et al. (2013).


ASM 2d-SMP model of MBR

ASM2d-SMP is a modified version of activated sludge model 2d (ASM2d) and takes into

account SUAP (soluble utilization associated product) and SBAP (soluble biomass associated


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product) and six reactions including these components. ASM2d model allows dynamic

simulation of combined biological processes for chemical oxygen demand (COD), nitrogen

and phosphorus removal in activated sludge systems. The kinetics and stoichiometry are

mainly based on Monod kinetics. Model describes anaerobic, anoxic and aerobic

processes. Model includes 19 biological state variables (SO2 (dissolved oxygen), SF

(fermentable readily biodegradable organic substrates), SA (fermentation products), SBAP

(soluble biomass associated product), SUAP (soluble utilization associated product), SNH4

(ammonium plus ammonia nitrogen), SNO3 (nitrite plus nitrate nitrogen), SPO4 (inorganic

soluble phosphorus), SI (inert soluble organic material), SALK (alkalinity of wastewater), SN2

(nitrogen), XI (inert particulate organic material), XS (slowly biodegradable substrates), XH

(heterotrophic organisms), XPAO (phosphate-accumulating organisms), XPP (poly-

phosphate), XPHA (a cell internal storage product of phosphorus-accumulating organisms),

XAUT (nitrifying organisms) and XTSS (total suspended solids) and 79 parameters (kinetics,

stoichiometric, physical and fraction-related). In this work, 44 kinetic parameters were

analysed (KX (half saturation parameter for XS/XH), KNO3,H (half saturation parameter for

SNO3 for XH), ηNO3,H (reduction factor for anoxic growth of XH), KI (hydrolysis rate for XI),

kH (maximum specific hydrolysis rate), ηFe (correction factor for hydrolysis under

anaerobic conditions), KO,HYD (half saturation/inhibition parameter for SO2), KNO3,HYD (half

saturation/inhibition parameter for SNO3), μH (maximum hydrolysis rate of XH), qFe (rate

constant for fermentation/maximum specific fermentation growth rate), ηNO3,H (reduction

factor for anoxic growth of XH), bH (decay rate for XH), KF (half saturation parameter for

SF), KFe (half saturation parameter for fermentation of SF), KA (half saturation parameter for

SA), KNH4,H (half saturation parameter for SNH4 for XH), KP,H (half saturation parameter for

SPO4 for XH), KALK,H (half saturation parameter for SALK for XH),qPHA (rate constant for SA

uptake rate), qPP (rate constant for storage of polyphosphates), μPAO (maximum growth rate

of XPAO), ηNO3,PAO (reduction factor for anoxic growth of XPAO), bPAO (endogenous

respiration rate of XPAO), bPP (rate constant for lysis of polyphosphates), bPHA (rate constant

for respiration of XPHA), KPS (half saturation parameter for SPO4 uptake), KPP (maximum

ratio of XPP/XPAO), KMAX (half saturation parameter for XPP/XPAO), KIPP (half inhibition

parameter for XPP/XPAO), KPHA (saturation constant for XPHA/XPAO), KO,PAO (half saturation

parameter for SO2 for XPAO), KNO3,PAO (half saturation parameter for SNO3 for XPAO), KA,PAO

(half saturation parameter for XA for XPAO), KNH4,PAO (half saturation parameter for SNH4 for

XPAO), KP,PAO (half saturation parameter for SPO4 as nutrient), KALK,PAO (half saturation

parameter for SALK for XPAO), μAUT (maximum growth rate of XAUT), bAUT (decay rate for

XAUT), KO,AUT (half saturation parameter for SO2 for XAUT), KNH4,AUT (half saturation

parameter for SNH4 for XAUT), KALK,AUT (half saturation parameter for SALK for XAUT), KP,AUT

(half saturation parameter for SPO4 for XAUT), kH,BAP (hydrolysis rate coefficient for SBAP)

and kH,UAP (hydrolysis rate coefficient for SUAP)). Kinetic rates of analysed model are given

in Table 1.


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Table 1. Kinetic process rates for the analysed model

Aerobic hydrolysis of BAP:

HBAP

OHYDO

OBAPH XS

SK

SK

2,2

2,

Anoxic hydrolysis of BAP:

HBAP

NOHYDNO

NO

OHYDO

HYDO

HYDNOBAPH XSSK

S

SK

KK

3,3

3

2,2

,2

,3,

Anaerobic hydrolysis of BAP:

HBAP

NOHYDNO

HYDNO

OHYDO

HYDO

FeBAPH XSSK

K

SK

KK

3,3

,3

2,2

,2

,

Aerobic hydrolysis of UAP:

HUAP

NOHYDNO

NO

OHYDO

HYDO

HYDNOUAPH XSSK

S

SK

KK

3,3

3

2,2

,2

,3,

Anoxic hydrolysis of UAP:

HUAP

NOHYDNO

NO

OHYDO

HYDO

HYDNOUAPH XSSK

S

SK

KK

3,3

3

2,2

,2

,3,

Anaerobic hydrolysis of UAP:

HUAP

NOHYDNO

HYDNO

OHYDO

HYDO

FeUAPH XSSK

K

SK

KK

3,3

,3

2,2

,2

,

Aerobic hydrolysis:

H

HSX

HS

OHYDO

OH X

XXK

XX

SK

SK

/

/

2,2

2

Anoxic hydrolysis:

H

HSX

HS

NOHYDNO

NO

OHYDO

HYDO

HYDNOH XXXK

XX

SK

S

SK

KK

/

/

3,3

3

2,2

,2

,3

Anaerobic hydrolysis:

H

HSX

HS

NOHYDNO

HYDNO

OHYDO

HYDO

FeH XXXK

XX

SK

K

SK

KK

/

/

3,3

,3

2,2

,2

Aerobic growth on SF:

H

ALKHALK

ALK

POHP

PO

NHHNH

NH

AF

F

FF

F

OHO

HO

H

XSK

S

SK

S

SK

S

SS

S

SK

S

SK

K

,

4,

4

4,4

4

2,2

,2


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Aerobic growth on SA:

H

ALKHALK

ALK

POHP

PO

NHHNH

NH

AF

A

AHA

A

OHO

OH

XSK

S

SK

S

SK

S

SS

S

SK

S

SK

S

,4,

4

4,4

4

,2,2

2

Anoxic growth on SF:

H

ALKHALK

ALK

POHP

PO

NHHNH

NH

AF

F

FF

F

NOHNO

NO

OHO

HO

HNOH

XSK

S

SK

S

SK

S

SS

S

SK

S

SK

S

SK

K

,4,

4

4,4

4

3,3

3

2,2

,2

,3

Anoxic growth on SA:

H

ALKHALK

ALK

POHP

PO

NHHNH

NH

AF

A

AHA

A

NOHNO

NO

OHO

HO

HNOH

XSK

S

SK

S

SK

S

SS

S

SK

S

SK

S

SK

K

,4,

4

4,4

4

,3,3

3

2,2

,2

,3

Fermentation:

H

ALKHALK

ALK

FFe

F

NOHNO

HNO

OHO

HO

Fe XSK

S

SK

S

SK

K

SK

Kq

,3,3

,3

2,2

,2

Lysis of XH: HH Xb

Storage of XPHA:

PAO

PAOPPPP

PAOPP

ALKPAOALK

ALK

APAOA

APHA X

XXK

XX

SK

S

SK

Sq

/

/

,,

Aerobic storage of XPP:

PAO

PAOPPIPP

PAOPP

PAOPHAPHA

PAOPHA

ALKPAOALK

ALK

POPS

PO

OPAOO

OPP

XXXKK

XXK

XXK

XX

SK

S

SK

S

SK

Sq

/

/

/

/

max

max

,4

4

2,2

2

Anoxic storage of XPP:

4

4

2,2

,2

3,3

3

max

max

,

,3/

/

/

/

POPS

PO

OPAOO

PAOO

NOPAONO

NOPAO

PAOPPIPP

PAOPP

PAOPHAPHA

PAOPHA

ALKPAOALK

ALKPAONOPP

SK

S

SK

K

SK

SX

XXKK

XXK

XXK

XX

SK

Sq


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Aerobic growth of XPAO:

PAO

PAOPHAPHA

PAOPHA

ALKPAOALK

ALK

POPAOPS

PO

NHPAONH

NH

OPAOO

OPAO

XXXK

XX

SK

S

SK

S

SK

S

SK

S

/

/

,

4,

4

4,4

4

2,2

2

Anoxic growth of XPAO:

PAO

PAOPHAPHA

PAOPHA

ALKPAOALK

ALK

POPAOPS

PO

NHPAONH

NH

NOPAONO

NO

OPAOO

PAOO

PAONOPAO

XXXK

XX

SK

S

SK

S

SK

S

SK

S

SK

K

/

/

,4,

4

4,4

4

3,3

3

2,2

,2

,3

Lysis of XPAO: PAO

ALKPAOALK

ALKPAO X

SK

Sb

,

Lysis of XPP: PAO

PAO

PP

ALKPAOALK

ALKPP X

X

X

SK

Sb

,

Lysis of XPHA: PAO

PAO

PHA

ALKPAOALK

ALKPHA X

X

X

SK

Sb

,

Aerobic growth of XAUT:

PAO

ALKAUTALK

ALK

POAUTPS

PO

NHAUTNH

NH

OAUTO

OAUT X

SK

S

SK

S

SK

S

SK

S

,4,

4

4,4

4

2,2

2

Lysis of XAUT: AUTAUT Xb

Hydrolysis of XI: II XK

Local sensitivity analysis

One-to-one local sensitivity analysis of the kinetic parameters on the output signal was applied.

Local sensitivity analysis addresses small variations around a nominal operating condition.

Output signal was considered to be the steady state concentrations of the model metabolites.

The relative local parameter sensitivity coefficients were calculated using Eq.1 (Ingals, 2013):

100∂ j

j

k

c

c

kS (1)

where S represents local sensitivity coefficient, c metabolite concentration and k kinetic

parameter.


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The relative local sensitivity coefficients were evaluated by the model simulation until a

steady state is reached and using of the finite difference approximation with 1%, 3%

parameter increase and 1%, 3% parameter decrease.

Simulation of the process dynamics and local sensitivity analysis are performed in

Wolfram Research Mathematica v. 10.0 software.


The aim of this work was to identify and analyse the most important kinetic parameters of

the mathematical model of membrane bioreactor according to hybrid ASM2d-SMP model.

The local sensitivity coefficients were evaluated by the model simulation until the steady

state is reached and using of the finite difference approximation with 1%, 3% parameter

increase (Fig. 1) and 1%, 3% parameter decrease (Fig. 2).

Sensitivities of the steady state model metabolites are depicted in the form of heat map

where columns present model metabolites and rows present kinetic parameters listed as

given in section materials and methods. Negative values of the sensitivities are presented

with the scale of red colour (lighter colour less effect, darker colour more effect), while

positive values of the sensitivities are presented with the scale of the green colour

(lighter colour less effect, darker colour more effect). Positive relative sensitivity

coefficients of 100% correspond to linearly proportional effect of a parameter on the

output signal, while -100% correspond to the inverse proportionality of the parameter on

the output signal. Small values of sensitivity coefficients indicate insensitivity of the

output signal on the parameter, while values above ± 100% correspond to the

exponential dependencies.

By analysing obtained results it can be noticed that both kinetic parameter values increase

and decrease effect the model metabolites in the steady state concentration. It can be

observed that kinetic parameter values increase have stronger effect; numerically larger

values of sensitivity coefficients are obtained. Interestingly, kinetic parameter values

increase or decrease can have opposite effect on model metabolite concentrations. In case

of dissolved oxygen concentration (first column in heat map) 1% increase (Fig. 1a) of half

saturation parameter for XS/XH (KX) has significant negative effect on analysed metabolite

concentration, while 1% decrease (Fig. 2a) of half saturation parameter for XS/XH has

positive effect. Analysed parameter is included into kinetic expressions describing aerobic,

anaerobic and anoxic hydrolysis. Lower value of KX indicates slower oxygen consumption

or positively effect of dissolved oxygen concentration.

By comparing the obtained results (Fig. 1-2) it can be seen that two model components are

sensitive to variations of all 44 kinetic parameters; dissolved oxygen (column one) and

nitrate nitrogen (column seven).


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(a)

(b)

Fig. 1. Local sensitivities of the model variables on (a) 1% and (b) 3% parameter values increase.

Columns present SO2, SF, SA, SBAP, SUAP, SNH4, SNO3, SPO4, SI, SALK, SN2, XI, XS, XH, XPAO,

XPP, XPHA, XAUT and XTSS. Rows present KX, KNO3,H, ηNO3,H, KI, kH, ηFe, KO,HYD,

KNO3,HYD, μH, qFe, ηNO3,H, bH, KF, KFe, KA, KNH4,H, KP,H, KALK,H, qPHA, qPP,

μPAO, ηNO3,PAO, bPAO, bPP, bPHA, KPS, KPP, KMAX, KIPP, KPHA, KO,PAO,

KNO3,PAO, KA,PAO, KNH4,PAO, KP,PAO, KALK,PAO, μAUT, bAUT, KO,AUT,

KNH4,AUT, KALK,AUT, KP,AUT, kH,BAP and kH,UAP.

-877.14 -0.07 0.03 0.00 -0.03 0.00 1.08 0.00 -1.35 0.00 0.00 0.00 0.00 0.00 0.01 -0.02 0.00 0.00 0.05

-1664.99 0.00 0.00 0.00 0.00 0.00 98.03 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.05

1150.95 0.00 0.00 0.00 0.00 0.00 -97.74 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

-780.45 1.67 0.00 0.00 0.00 0.00 1.36 0.67 31.06 49.06 0.00 -4.95 0.00 0.03 0.01 0.00 0.00 0.00 0.00

-713.85 2.94 -0.36 0.00 0.03 0.06 -7.14 -0.06 50.88 0.00 0.00 0.00 0.00 0.38 0.05 0.11 -0.05 0.03 -0.76

-646.43 0.00 0.00 0.00 0.00 0.00 -2.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

-1456.64 0.00 0.00 0.00 0.00 0.00 87.80 -0.03 0.02 0.00 0.00 0.00 0.00 0.03 0.17 0.30 -0.05 0.11 0.00

2102.85 0.00 0.00 0.00 0.00 0.00 7.53 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

-293.03 -3.20 0.53 0.00 0.61 0.00 -120.88 0.31 -55.87 0.00 -0.46 0.00 0.00 -0.57 -2.01 -3.98 0.58 -1.61 0.82

-865.79 0.00 0.03 0.00 0.00 0.00 1.16 0.00 0.00 0.00 0.00 13.04 0.00 0.00 0.01 0.00 0.00 0.00 0.00

-1264.74 0.00 0.00 0.00 0.00 0.00 -5.24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.43 0.00 0.00 0.00 0.00

-2934.19 0.03 0.00 0.63 252.16 1.29 -9.01 1.75 0.63 0.22 0.00 2.49 0.00 -19.86 0.02 0.00 0.00 0.01 -0.49

1090.20 0.59 1.49 0.00 -0.26 0.32 3.06 0.17 -2.73 0.08 0.00 -0.04 0.00 -1.43 -4.54 -0.35 0.10 -0.06 -0.16

-5.76 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.43 0.00 0.00 0.00 0.00

1176.38 -0.61 -2.42 0.00 0.32 -0.52 3.71 -0.31 10.29 -0.16 0.07 0.04 0.00 2.41 -0.22 0.96 -0.24 0.28 0.22

978.84 0.02 0.00 0.00 -0.03 0.00 -1.36 0.00 0.18 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00

683.21 0.02 0.00 0.00 -0.03 0.00 -0.17 0.00 0.16 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00

-1305.66 0.07 0.00 20.95 -0.03 0.00 4.25 0.00 9126.90 0.00 0.00 -4088.46 0.00 0.03 0.05 0.07 -0.05 0.03 0.00

-4054.71 0.00 -0.40 0.00 0.00 0.00 -3.99 1.36 0.04 -0.02 0.00 0.00 0.00 0.03 0.01 -31.47 57.22 0.00 0.11

3174.39 0.00 0.03 0.00 -0.03 0.00 -2.69 -0.08 -0.04 0.00 0.40 0.00 0.00 0.00 0.01 5.77 -0.24 0.00 0.00

-4277.29 0.00 0.03 0.00 -0.03 0.00 -624.26 0.03 -0.04 0.00 0.00 0.00 0.00 0.00 2.00 -1.92 -0.34 0.00 0.00

-3905.33 0.00 0.00 0.00 0.00 0.00 -613.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

619.59 0.00 0.03 0.00 0.00 0.00 31.24 -0.06 0.00 0.00 0.00 0.00 0.00 0.00 -9.79 4.92 -2.77 0.00 0.00

-468.48 0.00 0.00 0.00 0.00 0.00 -0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 -14.73 -30.68 0.00 0.00

990.55 0.00 0.07 0.00 0.00 0.00 -1.42 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.01 -0.02 -7.00 0.00 0.00

971.04 0.00 0.00 0.00 0.00 0.00 -1.59 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 -0.33 0.00 0.00 0.00

-459.75 0.00 0.03 0.00 0.00 0.00 -0.11 -0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.01 1.39 -0.78 0.00 0.00

952.19 0.00 0.00 0.00 0.00 0.00 -1.56 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.09 0.00 0.00 0.00

952.19 0.00 0.00 0.00 0.00 0.00 -1.56 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.17 0.00 0.00 0.00

-12.91 0.00 0.00 0.00 0.00 0.00 4.36 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 -0.04 0.00 0.00 0.00

2067.65 0.00 0.00 0.00 0.00 0.00 -1.98 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.15 -0.33 0.05 0.00 0.00

2964.58 0.00 0.00 0.00 0.00 0.00 644.60 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

2889.53 0.00 0.07 0.00 0.00 0.00 -2.72 -0.29 0.00 0.00 0.00 0.00 0.00 0.00 0.01 11.88 -6.66 0.00 0.00

17.93 0.00 0.07 0.00 0.00 0.00 2.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 -9245.91

16.72 0.00 0.07 0.00 0.00 0.00 1.81 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

179.53 0.00 0.07 0.00 0.00 0.00 12.21 -0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.17 2.13 -0.92 0.00 0.00

-1360.42 0.00 0.07 0.00 0.00 -0.06 92.02 -0.19 0.16 -0.02 0.07 0.00 0.00 0.06 0.02 0.00 0.00 1.59 0.00

28.61 -8453.84 0.00 0.00 0.00 0.00 -0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

3450.61 0.00 0.03 0.00 0.00 0.00 -86.81 0.03 -0.02 0.00 0.00 0.00 0.00 0.00 0.01 -0.02 0.00 -0.22 0.00

-1331.81 0.00 0.03 0.00 0.00 0.00 -4.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 -0.02 0.00 -0.10 0.00

1432.92 0.00 0.03 0.00 0.00 0.00 -10.65 0.03 -0.02 0.00 0.00 0.00 0.00 0.00 0.01 -0.02 -33.59 -0.15 0.00

-1323.61 0.00 0.03 0.00 0.00 0.00 1.84 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 -0.02 -33.59 -0.15 0.00

-2.60 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 -0.02 -33.59 -0.15 0.00

-8948.29 0.32 -0.03 0.00 -0.71 0.00 -1353.04 0.00 5.42 -0.02 0.00 0.00 0.00 0.06 0.02 0.00 0.00 0.00 0.00

3.06 -0.08 0.01 0.00 -1.83 0.00 0.11 0.00 -1.35 0.00 0.00 0.00 0.00 -0.01 0.00 -0.01 0.00 0.00 0.02

6.90 0.00 0.00 0.00 0.00 0.00 140.18 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1793.04 0.00 0.00 0.00 0.00 0.00 -96.23 0.00 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

469.87 1.67 0.00 0.00 0.00 0.00 -0.07 0.67 43.93 -0.01 0.00 -4.93 0.00 0.02 0.01 0.00 0.00 0.00 0.00

-1346.96 2.94 -0.36 0.00 0.05 0.09 -5.49 -0.05 50.90 0.01 0.02 0.00 0.00 0.40 0.04 0.12 -0.03 -1826.21 -0.80

-617.72 0.00 0.00 0.00 0.00 0.00 -2.63 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-52.69 0.00 0.00 0.00 0.00 0.00 85.64 -0.02 0.02 -0.01 0.04 17.98 0.00 0.01 0.17 0.32 -0.05 0.11 0.00

2325.95 0.00 0.00 0.00 0.00 0.00 7.15 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

9567.75 -3.13 0.50 0.00 0.60 -0.02 -116.44 0.31 -54.75 0.00 -0.44 0.00 0.00 -1.03 -1.98 -3.90 0.58 -1.58 0.76

324.30 0.00 0.01 0.00 0.00 0.00 -0.53 0.00 -1.35 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-1143.67 0.00 0.00 0.00 0.00 0.00 -5.35 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-7220.62 0.02 0.00 0.60 0.00 1.29 30.89 1.75 0.61 0.22 0.00 2.48 0.00 -19.84 0.01 0.01 0.00 0.01 -0.51

205.20 0.58 1.47 0.00 -0.25 0.30 4.15 0.17 -2.72 0.09 -0.02 -0.04 0.00 -1.44 -0.08 -0.33 22.35 -59.02 -0.16

-6.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -59.02 -0.16

56.72 -0.61 -2.00 0.02 0.32 -0.49 5.90 -0.31 10.31 -0.15 0.09 4.89 0.00 2.42 0.17 3554.20 -2588.49 0.27 0.22

-316.44 0.01 0.00 0.00 -0.01 -0.49 0.71 -0.02 0.18 0.00 0.00 0.00 0.00 1.90 0.01 0.01 0.00 0.00 0.00

231.55 0.01 0.00 0.00 -0.01 0.00 -0.12 0.00 0.16 0.00 0.00 0.00 0.00 1.90 0.01 0.01 0.00 0.00 0.00

-401.88 0.07 0.00 0.00 -0.02 0.00 2.79 -0.01 1.11 0.00 0.02 0.00 0.00 0.01 0.04 259.29 -0.02 0.03 -0.02

-7318.30 0.00 -0.40 0.00 0.00 0.00 37.29 1.37 0.04 -0.08 0.00 0.00 0.00 0.01 0.01 -102.44 57.22 0.03 0.07

716.77 0.01 -11.01 0.00 -0.01 0.00 -0.86 -0.08 -0.05 0.00 0.40 0.00 0.00 -0.01 0.00 5.77 -2411.77 0.00 0.00

-6181.86 0.01 0.02 0.00 -0.01 0.00 -543.24 0.03 -0.05 0.00 0.00 0.00 0.00 -0.01 1.99 -1.92 -1.64 0.00 0.00

-6682.30 0.00 0.00 0.00 0.00 0.00 -533.15 0.00 -0.01 0.00 0.00 0.00 0.00 1.90 0.01 0.01 0.00 0.00 0.00

641.11 0.00 0.02 0.00 0.00 0.00 31.33 -0.06 0.00 0.00 0.00 0.00 0.00 1.90 -9.78 4.93 -2.75 0.00 0.00

-455.14 0.00 0.01 0.00 0.00 0.00 -0.17 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 -14.71 -0.05 0.00 0.00

362.78 0.13 0.06 0.00 0.00 0.58 -0.39 0.00 0.00 -0.49 0.00 0.00 0.00 0.00 0.00 -0.01 -6.98 0.00 0.00

323.77 0.00 0.00 0.00 0.00 0.00 -0.54 0.00 0.01 0.00 -0.02 0.00 0.00 0.00 0.00 -0.31 0.02 0.00 0.00

-227.46 0.00 0.01 0.00 0.00 0.00 -0.68 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.39 -0.78 0.00 0.00

330.40 0.00 0.01 0.00 0.00 0.00 -0.53 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.09 0.00 0.00 0.00

341.45 0.00 0.01 0.00 0.00 0.00 -0.54 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.00

-16.93 0.00 0.00 0.00 0.00 0.00 4.36 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 -0.04 0.00 0.00 0.00

236.07 0.00 0.00 0.00 0.00 0.00 0.50 0.01 0.01 0.00 -0.02 0.00 0.00 -3172.12 -0.16 -0.32 0.05 0.00 0.00

6879.57 0.00 0.00 0.00 0.00 0.00 667.10 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00

1187.57 0.00 0.06 0.00 0.00 0.00 -0.41 -0.16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 11.86 -6.63 0.00 0.00

17.93 0.00 0.00 0.00 0.00 0.00 2.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

15.60 0.00 0.00 0.00 0.00 0.00 1.81 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

181.30 0.00 0.01 0.00 0.00 0.00 12.24 -0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.16 2.15 -0.92 0.00 0.00

-7029.70 -0.01 -0.09 0.00 0.01 -0.04 133.73 -0.20 0.15 -0.01 0.07 0.00 0.00 0.00 0.01 2.15 0.00 1.59 0.02

27.31 0.00 0.01 0.00 0.00 0.00 -0.35 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.01 2.18 0.00 -7.49 0.00

1370.76 0.01 0.02 0.00 -0.01 0.00 -83.16 0.03 -0.03 0.00 0.00 0.00 0.00 -0.01 0.00 -0.01 0.00 -0.22 0.00

686.80 0.00 -0.06 0.00 -0.01 0.00 -6.55 0.01 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 -0.10 0.00

522.28 0.00 0.01 0.00 -0.01 0.00 -9.96 0.02 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 -0.14 0.00

346.96 0.00 0.01 0.00 0.00 0.00 -0.81 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-1.73 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-496.29 0.32 -0.03 0.00 -0.70 0.00 -0.10 -0.01 5.42 -0.01 0.00 0.00 0.00 0.04 0.01 0.01 0.00 0.00 0.02


Ružička days


11th


Vukovar, Croatia


78

(a)

(b)

Fig. 2. Local sensitivities of the model variables on (a) 1% and (b) 3% parameter values decrease.

Columns present SO2, SF, SA, SBAP, SUAP, SNH4, SNO3, SPO4, SI, SALK, SN2, XI, XS, XH,

XPAO, XPP, XPHA, XAUT and XTSS. Rows present KX, KNO3,H, ηNO3,H, KI, kH, ηFe,

KO,HYD, KNO3,HYD, μH, qFe, ηNO3,H, bH, KF, KFe, KA, KNH4,H, KP,H, KALK,H, qPHA, qPP,

μPAO, ηNO3,PAO, bPAO, bPP, bPHA, KPS, KPP, KMAX, KIPP, KPHA, KO,PAO,

KNO3,PAO, KA,PAO, KNH4,PAO, KP,PAO, KALK,PAO, μAUT, bAUT,

KO,AUT, KNH4,AUT, KALK,AUT, KP,AUT, kH,BAP and kH,UAP.

25.30 0.00 0.00 0.00 0.00 0.00 -0.04 0.00 0.01 0.00 0.00 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00

32.13 0.01 0.00 0.00 0.00 0.00 -0.99 0.00 0.00 -0.01 0.00 0.13 0.00 0.00 -2.15 29.23 0.00 0.00 0.00

-8.56 0.01 0.00 0.00 0.00 0.00 0.96 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

7.90 -0.02 0.00 0.00 0.00 0.00 -0.02 -0.01 -0.43 0.00 0.00 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00

22.13 -0.03 0.00 0.00 0.00 0.00 0.04 0.00 -0.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01

6.64 0.00 0.00 0.00 0.00 0.00 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

7.49 0.00 0.00 0.00 0.00 0.00 -0.85 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-24.73 0.00 0.00 0.00 0.00 0.00 -0.08 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

12.82 0.02 0.00 0.00 -0.01 0.00 1.20 0.00 0.56 0.00 0.00 -40.48 0.00 0.01 0.02 0.04 -0.01 0.02 -0.01

-1.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

13.59 0.00 0.00 0.00 0.00 0.00 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

37.32 0.00 0.00 -0.01 0.00 -0.01 0.10 -0.02 -0.01 0.00 0.00 -0.02 0.00 0.20 0.00 0.00 0.00 0.00 0.01

4.14 -0.01 -0.01 0.00 0.00 0.00 -0.05 0.00 0.03 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00

0.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-2.46 0.01 0.02 0.00 -0.02 0.01 -0.06 0.00 -0.10 0.00 0.00 0.00 0.00 -0.02 -0.01 -0.01 0.00 0.00 0.00

-13.12 0.01 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

6.93 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2.95 0.00 0.00 0.00 0.00 0.00 -0.03 0.00 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

72.27 0.00 0.00 0.00 0.00 0.00 0.01 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 -0.56 0.00 0.00

8.28 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.06 0.00 0.00 0.00

77.28 0.00 0.00 0.00 0.00 0.00 6.47 0.00 0.00 -89.10 0.00 0.00 0.00 0.00 -0.02 0.02 0.00 0.00 0.00

29.29 0.00 0.00 -0.02 0.00 0.00 6.38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.05 0.00

-5.84 0.00 0.00 0.00 0.00 0.00 -0.31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.10 -0.05 0.03 0.00 0.00

4.75 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 891.09 0.14 0.00 0.00 0.00

9.41 0.00 0.00 -0.02 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.07 0.00 0.00

9.84 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

4.65 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 0.00 -0.01 0.01 0.00 0.00

-1.34 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

9.60 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.11 0.00 0.00 0.00 0.00 0.00 -0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

20.54 0.00 0.00 0.00 0.00 0.00 -0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-38.61 0.00 0.00 0.00 0.00 0.00 -6.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

3.61 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.12 0.07 0.00 0.00

-0.17 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-0.15 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-1.73 0.00 0.00 0.00 0.00 0.00 -0.12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.02 0.01 0.00 0.00

38.56 0.00 0.00 0.00 0.00 0.00 -0.94 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.02 0.00

-0.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.07 0.00

-28.44 0.00 0.00 0.00 0.00 0.00 0.86 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

284.90 0.00 0.00 0.00 0.00 0.00 0.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-14.52 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

7.45 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.02 0.01 94.85 36.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-10.96 0.00 0.00 0.00 0.01 0.00 0.03 0.00 -0.05 0.00 0.00 -80.96 0.00 0.00 -32.12 0.00 -89.11 0.00 0.00

-13.91 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.04 0.00 873.79 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

110.24 0.00 0.00 1.52 0.00 0.00 -2.85 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -53.20 0.00

-199.13 0.00 0.00 0.00 0.00 0.00 4.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

8.56 -0.05 0.00 0.00 0.00 0.00 -0.03 -0.02 -1.24 0.00 0.00 0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00

452.64 -0.08 0.01 0.00 0.00 0.00 0.20 0.00 -1.44 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 0.02

19.99 0.00 0.00 0.00 0.00 0.00 0.07 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 0.00

346.39 0.00 0.00 1.52 0.00 0.00 -2.39 0.00 0.00 0.00 0.00 0.00 0.00 -92.39 0.00 -0.01 0.00 0.00 0.00

-46.49 0.00 0.00 0.00 0.00 0.00 -0.24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

27.60 0.09 -0.01 0.00 -0.02 0.00 3.57 -0.01 1.65 0.00 0.01 0.00 0.00 0.02 0.06 0.12 -0.02 0.05 -0.02

9.48 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 -0.05 0.00 0.00 0.00 0.00 0.00 -10.30 0.00

42.56 0.00 0.00 0.00 0.00 0.00 0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

95.82 0.00 0.00 -0.02 0.00 -0.04 0.29 -0.05 -0.02 -0.01 0.00 -0.07 0.00 0.56 0.00 0.00 0.00 0.00 0.01

-3.96 -0.02 -0.04 0.00 -87.44 -0.01 -0.12 0.00 0.08 0.00 0.00 0.00 0.00 0.04 0.00 0.01 0.00 -10.30 0.01

0.19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-0.04 0.02 0.07 0.00 -0.01 0.01 -0.17 0.01 -0.29 0.00 0.00 0.00 0.00 -0.07 -0.01 -0.03 0.01 -0.01 -0.01

-17.62 0.00 0.00 0.00 0.00 0.00 2.51 0.00 0.00 0.00 0.00 -0.03 0.00 0.00 0.00 0.01 0.00 0.00 0.00

-19.81 0.00 0.00 0.00 0.00 0.00 2.53 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00

0.85 0.00 0.00 0.00 0.00 0.00 2.42 0.00 -0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.61 0.00 0.00

1.95 0.00 0.01 0.00 0.00 0.00 2.60 -0.04 0.15 0.00 0.00 0.00 0.00 0.00 0.00 2.91 -1.62 0.00 0.00

-18.52 0.00 0.00 0.00 0.00 0.00 2.52 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 0.00 -0.15 0.00 0.00 0.00

200.56 0.00 0.00 -0.15 0.00 0.00 22.86 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.05 0.06 0.01 0.00 0.00

117.93 0.00 0.00 0.00 0.00 0.00 22.48 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00

-33.48 0.00 0.00 0.00 0.00 0.00 10.25 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.28 -0.13 0.08 0.00 0.00

13.35 0.00 0.00 0.00 0.00 0.00 2.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.43 0.00 0.00 0.00

-1.93 0.00 0.00 0.00 0.00 0.00 2.49 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.20 0.00 0.00

-1.09 0.00 0.00 0.00 0.00 0.00 2.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00

15.09 0.00 0.00 0.00 0.00 0.00 2.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.03 0.02 0.00 0.00

-1.47 0.00 0.00 0.00 0.00 0.00 2.49 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00

9.43 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.01 0.00

8.03 0.00 0.00 0.00 0.00 0.00 -0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

17.57 0.00 0.00 0.00 0.00 0.00 -0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00

-194.29 0.00 0.00 0.00 0.00 0.00 -15.53 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-22.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.00 4.15 0.19 0.00 0.00

-96.99 0.00 0.00 0.00 0.00 0.00 -0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-0.44 0.00 0.00 0.00 0.00 0.00 -0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-4.94 0.00 0.00 0.00 0.00 0.00 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.92 0.03 0.00 0.00

-0.98 0.00 0.00 1.52 0.00 0.00 -2.66 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03 -0.05 0.00

-0.80 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.21 0.00

430.47 0.00 0.00 0.00 0.00 0.00 2.47 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00

-12.31 0.00 0.00 0.00 0.00 0.00 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

5.27 0.00 0.00 0.00 0.00 0.00 0.23 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00

6.54 0.00 0.00 0.00 -2.75 0.00 -0.01 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00

-5.11 -0.01 0.00 0.00 0.02 0.00 0.03 0.00 -0.15 0.00 -0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00


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Results indicate dissolved oxygen to be the most sensitive metabolite in ASM2d-SMP

model and that lower kinetic value perturbations have stronger effect. This result is

expected because the amount of available dissolved oxygen determinates the most

dominant metabolic process. In case of significant amount of available oxygen, aerobic

hydrolysis and growth process are dominant while in the case of dissolved oxygen

limitation anaerobic, anoxic and fermentation processes take place.

In case of 1% kinetic parameter value increase (Fig. 1), the strongest positive effect on

dissolved oxygen concentration has KO,AUT followed by KNO3,PAO and KA,PAO while the

strongest negative effect has kH,UAP followed by μPAO and ηNO3,PAO. In case of 3% kinetic

parameter value increase, the most important parameters with positive effect on dissolved

oxygen concentration increase are μH, KNO3,PAO and KNO3,HYD and qPHA, bH and μAUT with

negative effect.

By decreasing the kinetic parameter values for 1% (Fig. 2), the strongest positive effect on

dissolved oxygen concentration has KNH4,AUT followed by μPAO and qPHA while the

strongest negative effect has KNO3,PAO followed by KO,AUT and KNO3,HYD. Local sensitivity

coefficients indicate that in case of 3 % kinetic parameter value decrease the most

important parameters with positive effect on dissolved oxygen concentration increase are

KH, KO,AUT and KO,HYD while ηNO3,H, KNO3,PAO and KNH4,PAO showed to have the strongest

negative effect.

Conclusions

An integrated mathematical MBR model with provisions of switching functions between

variables could help to skip less influential model parameters under certain operating

conditions and thus accelerate model simulation and calibration. By simulating described

mathematical model with one and three percent parameter values increase/decrease, the

key regulation points of the model are detected. Results show that dissolved oxygen

concentration is the most sensitive model metabolite affected by both positive and negative

kinetic parameter value perturbations.

Obtained results can be useful in process control and planning of the future experiments

and also as a starting point for possible model reduction.

References Cho, J., Song, K.-G., Ahn, K.-H. (2005): The activated sludge and microbial substances influences

on membrane fouling in submerged membrane bioreactor: unstirred batch cell test,

Desalination 183, 425-429.

Cosenza, A., Manina, G., Neumann, M.B., Viviani, G., Vanrolleghem, P. A. (2013): Biological

nitrogen and phosphorus removal in membrane bioreactor: model development and parameter

estimation, Bioprocess Biosyst. Eng. 36, 499-514.


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Cosenza, A., Mannina, G., Vanrolleghem, P.A., Neumann, M.B. (2014): Variance-based sensitivity

analysis for wastewater treatment plant modelling, Sci. Total Environ. 470-471, 1068-1077.

Drews, A., (2010): Membrane fouling in membrane bioreactors - characterization, contradiction,

causes and cures, J. Membr. Sci. 363, 1-8.

Henze, M., Gujer, W., Mino, T., Matsuo, T., Wentzel, M.C., Marais, G.V.R., Van Loosdrecht,

M.C.M. (1999): Activated sludge model No. 2d, ASM2d, Water Sci. Technol. 39 (1), 165-182.

Ingalls, B.P. (2013): Mathematical modelling in system biology. The MIT Press, Massachusetts. pp.

115-118.

Jiang, T., Myngheer, S., De Pauw, D. J. W., Spanjers, H., Nopens, I., Kennedy, M. D., Amy, G.,

Vanrollenghem, P. A. (2008): Modelling the production and degradation of soluble microbial

product (SMP) in membrane bioreactors (MBR), Wat. Res. 42, 4955-4964.

Lu, S.G., Imai, T., Ukita, M., Sekine, M., Higuchi, T., Fukagawa, M. (2001): A model for

membrane bioreactor process based on the concept of formation and degradation of soluble

microbial products, Water Res. 35 (8), 2038-2048.

Mannina, G., di Bella, G., Viviani, G. (2011): An integrated model for biological and physical

process simulation in membrane bioreactors, J. Membr. Sci. 376, 56-69.

Saltelli, A., Ratto, M., Tarantola, S., Campolongo, F. (2005): Sensitivity analysis for chemical

models, Chem. Rev. 105, 2811-2827.

Wolfram Research “Mathematica” v.8.0, 2012.

Zarragoitia, A.-G., Schetrite, S., Alliet, M., Jauregui, U.-H., Albasi, C. (2008): Modelling of

submerged membrane bioreactor: conceptual study about link between activated sludge

biokinetics, aeration and fouling process, J. Membr. Sci. 325, 612-624.

Zuthi, M.F.R., Ngo, H.H., Guo, W.S. (2012): Modelling bioprocesses and membrane fouling in

membrane bioreactor (MBR): a review towards finding an integrated model framework,

Bioresour. Technol. 122 (12), 119-129.


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Effect of surface roughness on flow profile of liquid-liquid system

in a microreactor

UDC: 66.023

Ana Jurinjak Tušek1

, Anita Šalić2, Želimir Kurtanjek

1, Bruno Zelić

2

1University of Zagreb, Faculty of Food Technology and Biotechnology, Pierottijeva 6, HR-10000

Zagreb, Croatia 2University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19,

HR-10000 Zagreb, Croatia

Summary

A thorough knowledge of hydrodynamic conditions of the liquid-liquid flow in a microreactor is

necessary for performing multiphase reactions. In this work the liquid-liquid flow profile in a

microreactor was investigated. Different organic solvents (dichloromethane, diethyl ether,

chloroform and toluene) as one phase and water as second phase were fed in a microreactor. Two

microreactors with internal volume of V = 4 mm3

and V = 6 mm3

with different microchannel

surface roughness were used to analyzed the effect of surface roughness on flow profile. The flow

profiles in the microchannel were monitored under the microscope. The analysis of specific

dimensionless numbers (Reynolds number, Capillary number and Weber number) was performed to

get the better insight in process dominant for specific flow profile formation.

Keywords: microreactors, surface roughness, flow profile, liquid-liquid slug flow

Introduction

Microreactors insure very large surface-to-volume ratio, effective mass and heat transfer

and easier process control comparing to classic reactors and are widely used for single- and

multi-phase reactions. They were primarily used for gas-liquid systems (Harries et al.,

2003) and lately they become interesting for liquid-liquid systems (Zhao et al., 2006).

When performing multiphase reactions it is necessary to ensure effective mixing and mass

transfer, because reaction rate is affected not only by reactant concentration but also by

mass transfer rate (Doku et al., 2005). To insure the optimal conditions for performing

multiphase reaction in a microreactor, hydrodynamic conditions must be optimized.

According to Dessimoz et al. (2008) flow pattern formation depends on linear velocity of

both phases, the ratio of the phases, the fluid properties, the channel geometry and the

construction material of the microreactor.



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Additionally, controlled hydrodynamics in a microreactor allows the pressure drop

decrease, mass transfer improvement and enhances the product separation from reaction

mixture. Mostly used are gas-liquid and liquid-liquid systems. The common models of

interface in the case of liquid-liquid two-phase flow are “slug flow” and “parallel flow. In

the case of slug flow, two mechanisms are known to be responsible for the mass transfer

between two fluids: (a) convection due to internal circulations (Burns and Ramshaw, 2001;

Dummann et al., 2003; Kashid et al., 2005; Kashid et al., 2007; Kashid et al., 2010) within

each slug and (b) diffusion due to concentration gradients between the slugs (Dessimoz et

al., 2008). Convection depends on physical properties of fluids, slug geometry and flow

velocity while diffusion depends on the interfacial area available for mass transfer and

concentration gradients between two slugs. In the case of parallel flow pattern, the flow is

directed, highly symmetric and mostly laminar and the transfer of molecules between the

two phases is supposed to occur only by diffusion (Dessimoz et al., 2008). However,

microchannel reactors with high mass throughput for production operate in a transitional

region between laminar (Reynolds number, Re < 10) to turbulent flow (Re > 10000)

(Bolivar et al., 2011).

As mentioned, linear velocity of both phases, the ratio of the phases, the fluid properties,

the channel geometry and the construction material of the microreactor influence the flow

pattern formation. Usually, to describe all of these effects, flow pattern maps are

developed. Different dimensionless numbers like Capillary, Weber and Reynolds numbers

are being used to develop the maps (Dessimoz et al., 2008).

The objective of the present work is to determine the influence of the microchannel surface

roughness on liquid-liquid two-phase flow patterns in glass microreactors. In order to get

better insight in process dominant for specific flow profile formation the analysis of

specific dimensionless numbers (Reynolds number, Capillary number and Weber number)

was performed.


Chemicals

Dichloromethane (CH2Cl2) and toluene (C7H8) were purchased from Kemika (Croatia).

Coomasie Brilliant Blue G 250 was from Fluka (Switzerland). Diethyl ether (C2H5OC2H5)

was from Chemapol (Czech Republic) and chloroform (CHCl3) from Carlo Erba (Italy).

Experimental set-up

All experiments were performed in a microreactor system (Fig. 1a; Micronit Microfluidics

B.V., Netherlands). Two microreactors, with different surface roughness, were used. The


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first microchip with borosilicate tubular glass microchannels (length: width: height = 332

mm: 150 μm: 150 μm with internal volume of 6 mm3) was equipped with two “Y”-shaped

inlets and one outlet. Specific surface roughness of the microchannel of the first

microreactor was 10% (Fig. 1b). The second microreactor was made from same material

(length: width: height = 332 mm: 250 μm: 50 μm with internal volume of 4 mm3) and it

was also equipped with two “Y”-shaped inlets and one outlet. Specific surface roughness

of the second microreactor was 1% (Fig. 1c). Two syringe pumps (PHD 4400 Syringe

Pump Series, Harvard Apparatus, USA) equipped with high-pressure stainless steel

syringes (8 cm3, Harvard Apparatus, USA) were used for supply of liquids. Microreactor

chip was connected to pumps with fused silica connection (375 μm O.D., 150 μm I.D.,

Micronit Microfluidics B.V., Netherlands). Fluid flow in a microreactor was observed

using microscope (Motic B1-220A, binocular Weltzar, Germany) at magnifications of 40x

(eyepiece magnification = 10x; objective magnification = 4x).

Fig. 1. a) Experimental set-up; b) microchannel with rough walls;

c) microchannel with smooth walls


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Characterization of the flow in a microreactor

Flow profiles for four different systems organic solvent (chloroform, dichloromethane,

diethyl ether and toluene) - water were analysed in a microreactor. The two phases were

separately fed into a microreactor. Aqueous phase was stained blue using Coomasie

Brilliant Blue G 250 dye while organic phase was kept colorless. All experiments were

performed at equal ratio of flow rates. Flow profiles were observed using microscope and

photos of flow profiles were taken for all investigated flow rates (in range of total flow rate

from q = 2 mm3 min

-1 to approximately q = 200 mm

3 min

-1; maximal investigated flow rate

varied depending on used organic solvent). The photos of the flow profiles were taken at

the middle of the microreactor microchannel after the flow stabilization (after four

residence times). For the segmented flow, photos of flow profiles were used for

determination of segments length for both organic and aqueous phase using software

package Paint (Microsoft Corporation, USA). Segments lengths were obtained by

measuring the distance between first and the last pixel.

All experiments were performed in triplicates and average values are presented. In the 95%

confidence range the results showed no significant difference.

Dimensionless numbers

The specific dimensionless numbers (Reynolds number, Capillary number and Weber

number) were calculated to get the better insight in process dominant for specific flow

profile formation according to Eq. 1-3 and using physical properties of analyzes organic

solvents (Table 1).

ρ u dRe=

μ (1)

2

ρ u dWe= (2)

dCa = (3)


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Table 1. Physical properties of analyzed solvents

Characteristics of the solvent at 20 °C

ρ (kg m-3) µ (Pa s) σ (N m-1)

dichloromethane 1324.00 0.00043 26.52

diethyl ether 713.40 0.00023 16.61

chloroform 1479.90 0.00054 26.67

toluene 862.30 0.00058 28.82

water 997.00 0.00089 71.99


Introducing two immiscible liquids in a microreactor, the most usual two flow patterns that

can occur are parallel flow or slug (segmented) flow (Doku et al., 2005). Flow pattern

formation depends on linear velocity (Burns and Ramshaw, 2001) ratios of the phases,

fluid properties, the channel geometry (Kashid and Agar, 2007) and the microreactor

construction material; all this parameters have to be considered when controlling the flow

pattern. Liquid-liquid slug flow is characterized by a series of slugs of one phase separated

by slugs of other phase. Each slug served as an individual processing sub volume (Kashid

et al., 2007). In this work the effect of the microreactor microchannel surface roughness on

flow profiles was analyzed for four organic solvent-water systems (dichlormethane, diethyl

ether, chloroform and toluene) in two microreactors.

In experiments performed in a microreactor with microchannel surface roughness of 10%,

slug flow was obtained for all organic solvent-water systems and all investigated flow rates.

A photo of the slug flow profile for toluene-water system in a microreactor with

microchannel surface roughness of 10% is given in Fig. 2a. In experiments performed for

flow rates lower than q = 15 mm3 min

-1 and organic solvent:water flow ratio 1:1 organic

phase forms slugs longer then aqueous phase slugs while at higher values of flow rate both

phase segments have similar length. The mixing phenomenon between phases is also

obvious and due to that at flow rates higher that q = 200 mm3 min

-1 it was impossible to

distinguish two phases. Mass transfer in slug flow depends on convection within slugs and

diffusion between slugs. Convection depends on physical properties of fluids, slug geometry

and flow velocities, while diffusion is dependent upon interfacial area available for mass

transfer. As fluid segments move along the channel internal vortex flow patterns are

generated. These circulation patterns are a result of shearing motion within the low Reynolds

number flow combined with fluidic interfaces (Harries et al., 2003).

For toulene-water system in microreactor with microchannel surface roughness of 1%

stable parallel flow was developed for all investigated flow rates. At Fig. 2b the flow profile

for q = 20 mm3 min

-1 is given. Flow profile instabilities were developed at the microreactor inlet

but they disappear very fast and flow profile keeps stable along the microchannel.


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Fig. 2. Photography of the flow profile for toluene-water system in a microreactor

with microchannel surface roughness of (a) 10% and (b) 1%.

Organic phase is lighter while aqueous phase is darker.

To get better insight in processes dominant for the flow profile formation characteristics

dimensionless numbers (Reynolds, Capillary and Weber number) were calculated for all

investigated organic solvent-water systems and for two microreactors. The relationship

between Capillary and average Reynolds number for both reactors is given in Fig. 3.


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Fig. 3. Dependence of the Capillary number on average Reynolds number for microreactor

with microchannel surface roughness of (a) 10% and (b) 1%

Capillary number presents the ratio of the viscous forces and liquid-liquid surface tension.

The Capillary number was chosen because the viscous forces and the interfacial tension are

the dominating forces in microfluidic devices. As observed by Dessimoz et al., 2008 if the

flow of two immiscible fluids is dominated by the interfacial tension, slugs are formed. It

can be seen that there is linear dependence between analyzed dimensionless numbers for

both reactor. It was also observed that for analyzed flow rates, due to microchannel

Reaverage

0 5 10 15 20 25 30 35

Ca

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

dichlormethane

diethyl ether

chloroform

toulene

(a)

Reaverage

0 1 2 3 4

Ca

0.00

0.01

0.02

0.03

0.04

0.05

0.06

dichlormethane

diethyl ether

chloroform

toulene

(b)


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dimensions, the maximum average Reynolds number is approximately nine fold higher for

microreactor with microchannel surface roughness of 10%. Opposite to that, larger values

of Capillary number were obtained for microreactor with microchannel surface roughness

of 1%. For both microreactors the maximum value of Capillary number was calculated for

diethyl ether-water system. This can be explained with the fact that diethyl ether has the lowest

surface tension among all analyzed organic solvents. On the other hand the dependence

between Weber and average Reynolds number can be described as exponential (Fig. 4).

Fig. 4. Dependence of the Weber number on average Reynolds number for microreactor

with microchannel surface roughness of (a) 10% and (b) 1%

Reaverage

0 5 10 15 20 25 30 35

We

0.00

0.01

0.02

0.03

0.04

0.05

0.06

dichlormethane

diethyl ether

chloroform

toulene

(a)

Reaverage

0 1 2 3 4

We

0.00

0.02

0.04

0.06

0.08

0.10

0.12

dichlormethane

diethyl ether

chloroform

toulene

(b)


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Larger values of Weber numbers were obtained in microreactor with microchannel surface

roughness of 1%. The same phenomena was noticed for Capillary numbers. Maximum

value of Weber number was calculated for toluene, solvent with the highest surface

tension.

Conclusions

The influence of the microchannel surface roughness on liquid-liquid two-phase flow

patterns development was investigated in two glass microreactors. In experiments

performed with microreactor with microchannel surface roughness of 10% slug flow was

obtained for all analyzed systems at all investigated flow rates. In case of microreactor with

microchannel surface roughness of 1% stable parallel flow was developed at all analyzed

flow rate for all investigated systems except for diethyl ether-water system where transition

between slug and parallel flow was observed. Larger values of both Capillary and Weber

numbers were obtained in microreactor with microchannel surface roughness of 1% while

maximum value of Weber number was calculated for toluene, solvent with the highest

surface tension.

References

Bolivar, J. M., Wiesbauer, J. Nidetzky, B. (2011): Biotransformations in microstructured reactors:

more than flowing with the stream? Trends Biotechnol. 29, 333-342.

Burns, J.R., Ramshaw, C. (2001): The intensification of rapid reactions in multiphase systems using

slug flow in capillaries. Lab. Chip. 1, 10-15.

Dessimoz, A.L., Cavin, L., Renken, A., Kiwi-Minsker, L. (2008): Liquid-liquid two phase flow

patterns and mass transfer characteristics in rectangular glass microreactors. Chem. Eng. Sci.

63, 4035-4044.

Doku, G.N., Verboom, W., Reinhoudt, D.N., van den Berg, A. (2005): On microchip multiphase

chemistry-a review on microreactor design principles and reagent contacting modes.

Tetrahedron 61, 2733-2742.

Dummann, G., Quittmann, U. Gröschel, L., Agar, D.W., Wörz, O., Morgenschweis, K. (2003): The

capillary microreactor: A new reactor concnept for the intensification of heat and mass transfer

in liquid-liquid reactions. Catal. Today 79, 433-439.

Harries, N., Burns, J.R., Barrow, D.A., Ramshaw, C. (2003): A numerical model for segmented

flow in microreactor. Int. J. Heat Mass Tran. 46 3313-3322.

Kashid, M.N., Gerlach, I., Goetz, S., Franzke, J., Acker, J.F., Platte, F., Agar, D.W., Turek, S.

(2005): Internal circulation within the liquid slugs of a liquid-liquid slug-flow capillary

microreactor. Ind. Eng. Chem. Res. 44, 5003-5010.

Kashid, M.N., Platte, F., Agar, D.W., Turek, S. (2007): Computational modelling of slug flow in a

capillary microreactor. J. Comput. Appl. Math. 203, 487-497.


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Kashid, M.N., Renken, A., Kiwi-Minsker, L. (2010): CFD modelling of liquid–liquid multiphase

microstructured reactor: Slug flow generation. Chem. Eng. Res. Des. 88 (2010) 362-368.

Kashid, M.N., Agar, D.W. (2007): Hydrodynamics of liquid-liquid slug flow capillary microreactor:

flow regimes, slug size and pressure drop. Chem. Eng. J. 131, 1-13.

Zhao, Y., Chen, G., Yuan Q. (2006): Liquid-liquid two-phase flow patterns in a rectangular

microchannel. AIChE J. 52, 4052-4060.

List of symbols

Ca Capillary number, -

d diameter of the channel, m

q flow rate, mm3 min

-1

Re Reynolds number, -

u linear or superficial velocity, m s-1

We Weber number, -

μ viscosity, Pa s

ρ density, kg m-3

σ surface tension, N m-1


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Influence of the fluid flow patterns on borax nucleation mechanism and

nucleation rate in a single and dual turbine impeller crystallizer

UDC: 66.065.5 : 661.52

Antonija Kaćunić, Lea Lokas, Marija Ćosić, Nenad Kuzmanić

University of Split, Faculty of Chemistry and Technology, Department of Chemical Engineering,

Teslina 10/V, HR-21000 Split, Croatia

Summary

The aim of this work was to investigate the influence of fluid flow pattern in the batch cooling

crystallizer on the nucleation mechanism and nucleation rate of borax. The fluid flow patterns in the

crystallizer were generated by axial and radial turbine impellers and by their different dual

combinations. Impeller speed ensured the state of complete suspension in the system for all

configurations used. Crystallization was carried out by cooling of the mother liquor from the

saturation temperature of 30 °C by the rate of 6 °C h-1

. Metastable zone width was detected from the

changes of supersaturation values while mechanism and nucleation rate were determined according

to Mersmann´s nucleation criterion. In order to gain complete insight into the overall fluid flow in

the crystallizer the photographs of the flows were taken and simulations by VisiMix 2000 Turbulent

package were made. Power consumption over the process time for all impeller configuration used

were measured as well. Obtained results indicated that flow pattern developed by single as well as

by dual-impeller systems significantly influenced the borax nucleation mechanism and nucleation

rates. Different impeller configurations affect the values of just suspended impeller speed, the final

product properties and power consumption as well.

Keywords: crystallization of borax, mixing, nucleation, fluid flow pattern, single and dual impeller systems

Introduction

Borax decahydrate (Na2B407×10 H20) which presents the refined form of natural sodium

borate is one of the most important commercial boron compounds. It has found wide areas

of use in the production of borosilicate glasses, glass wool, ceramics, detergents, fire proof

materials etc. Since crystallization is an important step in the process of its production to

its refined form, in order to produce crystals of borax with a specified purity and crystal

size distribution at minimum cost, it is necessary to operate the crystallizer at the optimum

conditions (Ceyhan et al., 2007; Gurbuz et al., 2007).

Crystallization is carried out in a suspension and for this reason the study of crystallization

requires knowledge of mixing. Unfortunately, in many cases crystallizations are carried out

in stirred vessels without any optimization of the hydrodynamic conditions, even if,



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92

sometimes, these mechanisms are the controlling steps for an efficient separation of the

crystals from the liquor and for a suitable morphology of the final product (Sha et

Palosaari, 2000). In the stirred crystallizer it is essential to avoid a stagnant region. Since

crystallization is a mass transfer depended process an agitation must promote dispersion

and prevent settling. For that reason it is very important to ensure an adequate mixing. The

major part of published papers concerning crystallization deals with single impeller

crystallizer. However, an addition of a second impeller extremely changes

hydrodynamic conditions within the reactor. In that case the flow pattern is a strong

function of impeller characteristics, such as type of the impeller, geometry of the

impeller, presence or absence of the baffles, geometry of the reactor and rheological

properties of the fluid (Paul et al., 2004).

The aim of this work was to gain the insight into influence of overall fluid flow pattern in a

single as well as in a dual impeller crystallizer on the nucleation mechanism and nucleation

rate of disodium tetraborate decahydrate (borax) in the process of batch cooling

crystallization.


All experiments were performed in a laboratory-scale stirred batch cooling crystallizer

with a volume of 15 dm3 (Fig.1). The crystallizer was the cylindrical flat bottom vessel of

plexiglas with internal diameter of 0.24 m. The vessel was equipped with four baffles

placed at 90 º around the vessel periphery. The ratio of liquid height to tank diameter

(H/dT) was 1.3 what allowed an introduction of a second impeller on the same shaft in the

examined system. Impeller to tank diameter ratio (D/dT) was 0.33. In the single impeller

systems, the fluid flow was generated using a single axial pitched blade turbine (PBT) and

radial straight blade turbine (SBT) while in a dual impeller system three different impeller

configurations were used: PBT-PBT, SBT-SBT and PBT-SBT (the first acronym indicates

the type of the lower impeller).

In all experiments the impeller speed ensured the state of complete suspension of crystals

formed (N = NJS). The values of NJS were determined using a visual 0.9H method

according to Einenkel and Mersmann (1977). This method defines the NJS as the speed at

which the cloud of suspension reaches 0.9 of total liquid height. Measurements were

performed in mother liquor saturated at 30 °C in which 598 g of borax crystals (xp = 275 m)

was suspended. This mass was calculated from theoretical crystal yield, while their size

presents the largest granulometric class of borax obtained in preliminary experiments.

To determine the flow patterns in the mixing tank at different impeller combinations, the

streak photography based on the work of Ibrahim and Nienow (1995) was used. All

pictures were taken in the dark room. The halogen lamp of 1500 W was used as the source

of light to illuminate a vertical section of the tank. The light passed through two parallel


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93

vertical slits with a width of about 5 mm. All other parts of the vessel were covered with a

black cardboard. Tracers used to aid flow visualization were crystals of borax decahydrate

with an average size of 275 m placed in a solution saturated at 30 °C. In addition, the

simulations of flow patterns for all applied configurations were carried out by VisiMix

2000 turbulent package.

Saturated borax solutions were prepared by dissolving technically pure borax in excess

regarding the solubility data in distilled water. Saturated solution was filtered through

diatomaceous earth. The solution saturated at 30 °C was cooled at a constant cooling rate

of 6 °C h-1

.

Temperature control of the crystallizer was accomplished by a programmable thermostatic

bath (Medingen TC 250) and an appropriate data acquisition system. During the process,

concentration changes of borax solution were monitored in line using the Na-ion selective

electrode (ISE).

After crystallization for a definite time, the crystals were filtered from the residual

solution, rinsed with acetone and then dried at room temperature. Obtained products were

sieved in order to determine the granulometric properties of the final products. During the

experiments the power consumption was determined as well, using torque meter produced

by Himmelstein & Co.

Fig. 1. Experimental set-up

(1. Crystallizer, 2. Impeller, 3. System for concentration measurement, 4. Thermostat,

5. Torquemeter, 6. Torque sensor and velocity transducer, 7. Variable speed motor,

8. Impeller speed regulation system, 9. Temperature measurement system,

10. Computer)

1

2

3

4

5

6

7

8

9

10

c

dT

s

D

H


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Experimental results and discussion

Influence of impeller configuration on the just suspended impeller speed

The state of complete suspension exists when all particles are in motion and no particle

remains on the tank base for more than short period of time. For particles to be lifted from

the bottom of the vessel, the local hydrodynamics, e.g. velocities and turbulence levels

must be suitable in that region. Under these conditions, overall particle surface is present to

the fluid, thereby ensuring that maximum surface area is available for chemical reaction,

heat and mass transport. Minimum impeller speed that ensures the said state is called the

complete suspension impeller speed - NJS.

For applied impeller configurations, the values of just suspended speed of borax crystals

are presented in the Fig. 2.

Fig. 2. Values of just suspended impeller speed and Reynolds number

at different crystallizer configurations

In the single impeller systems NJS was higher when axial impeller type was used. Present

differences could be explained by the flow created by certain impeller.

Fig. 3. Simulations and photography of flow patterns obtained by:

a.) PBT, b.) SBT, c.) PBT-PBT, d.) SBT-SBT, e.) PBT-SBT

200

300

400

500

Re

m· 1

0-4

0

2

4

6

NJS /

min

-1

NJS

Re

PBT SBT PBT-PBT SBT-SBT PBT-SBT


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Photo as well as simulation of the axial PBT impeller flow (Fig. 3a) show that fluid

discharged by PBT impeller is strongly driven downwards until it is deflected from the

bottom of the vessel, then it spreads out over the bottom and flows up along the wall

before being drawn back to the impeller. This way a strong circulation loop is produced in

the lower half of the tank while in the upper half of the tank circulation intensity is low. In

the same figure (Fig. 3a) presented flow pattern of the crystal suspension shows that

crystals were swept up from the crystallizer bottom by stronger circulation loop, but they

are not lifted up enough to reach the top of the liquid. As previously said, for suspension of

the crystals it was important to provide liquid velocities directed to the tank floor for an

effective sweeping action. PBT impeller performs well in this duty, but since just

suspended criterion applied required a lifting of the crystals to the 0.9 of the liquid height,

at lower impeller speed circulation did not occupy the whole stirred area in the crystallizer

and its influential area increased only with an increase of the impeller speed.

In the system with radial SBT impeller (Fig. 3b) liquid is discharged radially from the

impeller towards the wall of the tank where it divides into two streams, one flowing up to

the top of the tank and the other flowing down to the bottom. Although fluid stream is not

focused directly to the tank floor, lower loops lifted the crystals from the bottom while the

upper loop suspended them towards the liquid top. This is the main reason why the value

of NJS is lower in comparison with PBT impeller system.

An addition of the second impeller of the same type lowered the values of NJS in both

systems. In these kinds of reactors, the overall flow was a consequence of an interaction of

flows developed by each of the impellers. This phenomenon was much more emphasized

in the PBT-PBT dual system as will hereafter be described in detail.

Effect of different flow patterns on the supersaturation profile and nucleation rate

The fundamental thermodynamic driving force of crystallization is given by the change of

chemical potential between standing and equilibrium state (Jones, 2002). Since chemical

potential is a quantity that is not easy to measure, driving force is more conventionally

expressed in terms of concentration; that is supersaturation (Eq. 1). During batch cooling

crystallization, the level of supersaturation depends on the balance between the generation

rate and consumption rate of supersaturation. It is well known fact that the generation rate

of supersaturation is determined by the cooling rate and the solubility of salt, while the de-

supersaturation rate depends on the nucleation rate, growth rate, and the total surface area

of growing crystals in the suspension (Yang at al., 2006). One of the main aims of this

work was to investigate the influence of fluid flow patterns on the supersaturation changes

over process time. Supersaturation was expressed as an absolute supersaturation, c. It was

calculated from the measured concentration of mother liquor, c, and the equilibrium

concentration, c*, by the expression:


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96

ccc (1)

Fig. 4 shows the supersaturation change during the batch cooling crystallization of borax

carried out at different fluid flow patterns generated by impeller configurations applied.

Fig. 4. Variation of supersaturation profile over process time (a.) and values

of maximal allowed supersaturation at different fluid flow patterns (b.)

It is evident that the supersaturation profile during the crystallization was similar for the all

hydrodynamic conditions examined. In the beginning of the process supersaturation

increases linearly then reaches a peak and decreases, first rapidly and than moderately.

Linear increment depends on salt solubility and cooling rate. In this concentration range

spontaneous nucleation is not likely to occur and nucleation starts when the maximal level

of supersaturation (cmax) is reached. At this value the nucleation occurs spontaneously

and cmax actually presents maximal allowable supersaturation i.e. metastable zone width

(MZW). From the Fig. 4 it is evident that the maximal value of supersaturation depends of

hydrodynamic conditions in the crystallizer. In the single impeller system MZW is

narrower when axial impeller was used. This result is consequences of higher level of

turbulence in this system i. e. higher value of the Reynolds number. These findings are in

accordance with a well known effect that an increased turbulence in the system can narrow

the MZW (Mullin, 2004).

On the other hand, in dual impeller systems deviation from the mentioned rule was present.

Namely, according to the value of Reynolds number the narrower MZW could be expected

in the PBT-SBT impeller system. However, the obtained result for this system is almost

equal to that obtained for PBT-PBT configuration at significantly lower value of Reynolds

c

max

/ m

ol d

m-3

SBT

PBT

PBT- PBT

SBT-SBT

PBT-SBT

0 40 80 120 1600.00

0.02

0.04

0.06

0.08

c /

mo

l d

m-3

t / minPBT

0.06

0.08

0.07

SBT-SBTPBT-PBT PBT-SBTSBT

a. b.


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number. The broadest metastable zone was noticed in the SBT dual impeller system. These

results are the consequence of the complexity of overall liquid flow pattern and could be

explained by a detailed analysis of an interaction of flows developed by each of the

impeller.

When the SBT-PBT combination was applied, three circulation zones were formed; one by

the PBT and two by the SBT (Fig. 3e). In this case the lower impeller stream is directed

towards the vessel bottom while the upper impeller is pumping horizontally. With this flow

pattern in the vessel roughly two-thirds of the bulk flow is mixed primarily by the upper

impeller. Once again three ring vortices are observable but the one under the lower

impeller is not well defined and it is small in size in comparison with the others. This flow

could be termed as the diverging flow pattern. In the region between the active zone

generated by the straight blade turbine and the top zone of the pitched blade turbine, liquid

streams collided forming a small recirculation flow. These occurrences contributed to the

significant decrease of the overall flow intensity what finally reflected on the delayed onset

of nucleation.

The broadest metastable zone in a dual SBT impeller system could also be attributed to the

weakening of intensity of overall liquid flow. SBT impellers behave almost independently

and each impeller produced a radial jet outward (Fig. 3d). Four ring vortices were

observed, one above the upper impeller, two between the impellers and one below the

lower impeller. Actually, in the zone between the impellers an intensive interaction of

flows is present. For this reason vortices are smaller and less clearly defined and the flow

essentially rotates around the shaft in an almost solid-body type of rotation. Due to

intensive interaction of the flows in the zone between the impellers, this kind of the flow

pattern could not be considered as parallel but rather as merging flow pattern.

Determination of dominant nucleation mechanism and nucleation rate at different fluid

flow patterns

Various mechanisms are proposed to correlate the nucleation process and the metastable

zone width. In the unseeded batch cooling crystallizer, the primary nucleation occurs

dominantly. In these systems the homogenous primary nucleation occurs only if the

supersaturated solution is free of any particle. On the contrary, heterogeneous primary

nucleation only takes place in the presence of foreign particles. The increase of

supersaturation can activate foreign particles present in the solution with the consequence

that surface nuclei are formed on them and the particles become heterogeneous nuclei.

The total rate of nucleation, N, is the sum of the four contributions of different

mechanisms:

attsurhethom NNNNN (2)


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where Nhom, Nhet, Nsur and Natt are the rate of homogeneous nucleation, heterogeneous

nucleation, secondary surface nucleation, and the rate of attrition induced secondary nucleation.

Kim and Mersmann (2001) proposed that one contribution is dominant in a given range of

supersaturation. They suggested a rapid, simple and indirect method to determine the

nucleation mechanism in a classical nucleation theory by measuring the degree of

supersaturation and solubility at a constant cooling rate.

Data obtained in this study were marked on this criterion which presents relation between

dimensionless metastable supersaturation cmax/cc and dimensionless solubility c*/cc and it

was found out that the heterogeneous primary nucleation is the dominating mechanism in

examined system (Fig. 5).

Fig. 5. Mersmann`s nucleation criterion

In this case the nucleation rate can be calculated by the following equation (Shubert and

Mersmann, 1996):

2)ln(

3)/ln(

19.1expln

3/7

965.0

*

c

*

c

c

max

5

m

ABhethet

Sη

ccf

c

cf

c

c

d

DN (3)

c

cm

ax

/c

Nhom

Nhet

Natt

Nsur

c c* / c

10-1

10-2

10-3

10-4

10-4

10-3

10-2

10-1

100


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wherehet is the heterogeneity factor and f is the reduction factor for the nucleation work,

while cc presents crystal molar density. For heterogeneous nucleation 0 < f < 1, and

depends on the contact angle between the surface of the foreign particles and surface of the

nucleus. DAB is a bulk diffusivity (= 10-10

m2 s

-1 in the low viscous solutions) and dm =

(cc/NA)-1/3

where NA is Avogadro number. The calculation was carried out with DAB =

3.94×10-10

m2 s

-1, cc = 4.48 mol dm

-3 which was valid for estimated parameters of het = 10

-

11 and f = 0.1 (Cheon et al., 2005).

In our experiment, nuclei may be generated by the heterogeneous primary nucleation that

occurs by appearance of "active" particles (heteronuclei) in solution with foreign surfaces

(the walls of the vessel, impeller, baffle etc.).

Table 1. Nucleation rate at different fluid flow patterns

Impeller combination Bhet∙10-18 / (No. m-3 s-1)

PBT 5.50

SBT 6.56

PBT-PBT 9.77

SBT-SBT 17.57

PBT-SBT 7.75

In the single impeller system number of formed nuclei was increased when SBT impeller

was applied (Table 1). In dual impeller system the nucleation rate was significantly higher

with SBT-SBT configuration. From the listed value is evident that nucleation rate is

directly proportional to the values of metastable zone width, cmax. In the single and dual

SBT impeller systems, the nuclei were generated at a higher supersaturation level.

Correlation between the number of formed nuclei and supersaturation can drastically

influence properties of finally obtained crystal product. In the case of a high nucleation

rate, the supersaturation is consumed on growing of large number of nuclei what could

result in significantly smaller crystals. At the same time, at higher supersaturation level,

dendritic growth and liquid inclusions are likely, while massive formation of the nuclei can

lead to agglomeration (Omar and Urlich, 2003). On the contrary, smaller number of the

nuclei formed at lower supersaturation can lead to slower crystal growth and more

regularly shaped crystals (Kaćunić et al., 2013).

In order to investigate the influence of the fluid flow pattern, the properties of the final

product, granulometric analysis by sieving of final product were performed and the weight

mean crystal size, xm, as well as the crystal size deviation, , were determined.


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Fig. 6. The impact of impeller combinations on weight mean crystal size,

standard deviation of crystal size and product yield

Obtained results presented in the Fig. 6 indicate higher value of crystal size and crystal size

deviation when PBT was used. In dual impeller system, PBT-PBT configuration resulted

with the largest crystals, while applying the PBT-SBT configuration the crystals were

smallest. Final size of crystal resulted not only from the crystal growth but from the crystal

breakage and the agglomeration as well. In the single impeller system, PBT impeller

produced an axial fluid flow in a single stage circulation while previously described SBT

double loop radial flow caused higher collision frequency crystal/impeller, crystal/wall

what finally reflected on the reduction of the average crystal size. On the other hand, radial

flow impellers provide higher shear what considerably prevents crystal agglomeration,

eventually resulting in the more regular shaped crystals (Fig. 7).

Fig. 7. Borax crystals photographs obtained by: a.) PBT-PBT and b.) SBT-SBT impeller configuration

160

180

200

20

40

60

80


Y / %

80

90

100

xm / m / m

Y / %

xm / m

/ m

b.a.


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Described effects are even more present in dual SBT impeller system, but due to lower

impeller speed breakage probability is decreased and product properties are very alike to

those produced in a single SBT system.

In the dual PBT-PBT crystallizer two ring vortices were generated, one in the upper part

and the other in the lower part of the vessel (Fig. 3c). Since upper impeller stream was

inclined re-directed lower impeller stream adds up to the axial jet created by the higher

impeller making it stronger (Kuzmanić et al., 2008). This way a reinforced overall liquid

flow made a large, single stage recirculation loop, what ensured a good balance of

pumping and shear capability, promoting not only an undisturbed crystal growth but also

an enlargement of agglomerated clusters.

In the PTD-SBT impeller system, after crystals in suspension reached a certain size were

discharged downward trough lower impeller zone to the -vessel bottom and then toward

the liquid top. Since in the region between the impellers recirculation flow exists, crystals

were returned back to the lower impeller zone. High impeller speed and short circulation

path in this zone increased crystal exposure to the impeller surface and thus the likelihood

of crystal breakage by collision, what drastically influenced the final crystal size.

In the Fig. 6, the numerical values of the percentage product yield are listed as well. They

present a ratio of product yield obtained by an experiment, mP, and theoretical product

yield mT ( 100T

P m

mY ). From the results it is evident that in the in single and dual

impeller systems this values were slightly higher when SBT impeller was used.

In this work torque measurements were performed for impeller configuration examined.

The power consumption was calculated from the torque and the just suspended impeller

speed and it was expressed in terms of the just suspended energy dissipated per unit mass

of suspension, (P/m)JS. The average energy dissipated per unit mass of solution for all

impeller combinations used is presented in Fig. 8.

Fig. 8. Power consumption at different impeller configurations

0,00

0,20

0,40

0,60

0,80

1,00


(P/m

)/W

kg

-1JS


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From the results it is clear that in single impeller system, despite the lower value of just

suspended impeller speed, power consumption was higher when radial SBT impeller was

used. This result could be expected since for a given impellers the projected blade width

(on the vertical plane) increases with an increase in blade angle. Due to higher projected

blade width, more dissipation of energy occurs behind the straight impeller blades and

therefore SBT had a higher value of power consumption. In the PBT-SBT system high

value of power consumption, beside the listed effect, was a result of the higher impeller

speed as well.

Considering that application of dual SBT impeller results in the highest product yield and

the lowest crystal agglomeration, some degree of optimization is necessary in selection of

impeller configuration regarding production energy costs and required quality of the final

crystal product.

Conclusions

The following conclusion may be drawn from the results of this study:

An addition of a second impeller of the same type in the crystallizer decreases the just

suspended impeller speed. This effect is more emphasized in the system with PBT. The

highest value of NJS in the PBT-SBT system indicates a negative interaction between the

flows produced by individual impeller.

The metastable zone width in the single impeller system increases with an increase of

Reynolds number. In a dual impeller system this parameter depends on the intensity of

overall fluid flow due to more or less pronounced interactions of each impeller flow.

In single as well as in a dual impeller crystallizer, the nucleation took place by a

heterogeneous nucleation mechanism. The nucleation rate increases proportionally with

the metastable zone width.

When SBT impeller was used, in single as well as in a dual impeller crystallizer, more

regularly shaped crystals were produced. The smallest crystal sizes were obtained applying

the PBT-SBT combination. This is a consequence of a more intense crystal breakage due

to longer residence time in the lower impeller zone characterized by a higher impeller

speed and a short circulation path.

The power consumption is lower in single and dual PBT impeller systems what is

related to the impeller geometry. For the applied impellers of same, fixed diameter and

blade width, it is primarily a function of the impeller blade angle.


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Acknowledgements

This work was carried out with financial support of the Croatian Science Foundation and

presents a part of HETMIX Project (2014.-2018.).

References

Ceyhan, A. A., Sahin, Ö., Bulutcu, A.N. (2007): Crystallization kinetics of the borax decahydrate, J.

Cryst. Growth 300, 440-447.

Cheon, Y.-H., Kim, K.-J., Kim, S. -H. (2005): A study on crystallization kinetics on pentaerythritol

in batch cooling crystallizer, Chem. Eng. Sci. 60, 4791-4802.

Einenkel, W. D., Mersmann, A., (1977): Erforderliche Drehzahl zum Suspendieren in Rührwerken,

Verfahrenstechnik 11 (2) 90-94.

Gurbuz, H., Ozdemir, B. (2003): Experimental determination of the metastable zone width of borax

decahydrate by ultrasonic velocity measurement, J. Cryst. Growth 252, 343-349.

Jones, A. G. (2002): Crystallization Process Systems, first ed., London, UK: Butterworth-

Heinemann, pp. 58-141.

Kaćunić, A., Akrap, M., Kuzmanić, N. (2013): Effect of impeller position in a batch cooling

crystallizer on the growth of borax decahydrate crystals, Chem. Eng. Res. Des. 91 (2), 274-

285.

Kim, K. -J. Mersmann, A. (2001): Estimation of Metastable Zone Width in Different Nucleation

Processes, Chem. Eng. Sci. 56 (7) 2315-2324.

Kuzmanić, N., Žanetić, R., Akrap, M. (2008): Impact of floating suspended solids on the

homogenization of the liquid phase in dual impeller agitated vessel, Chem. Eng. Process 47,

663-669

Mullin, J. W. (2004): Crystallization, fourth ed., Amsterdam, Netherlands: Elsevier, pp. 180-215.

Omar, W., Urlich, J. (2003): Influence of crystallization conditions on the mechanism and rate of

crystal growth of potassium sulphate, Cryst. Res. Technol. 38 (1), 34-41.

Paul, E.L., Atiemo-Obeng, V.A., Kresta, S.M. (2004): Handbook of Industrial Mixing - Science and

Practice, first ed. New Jersey, USA: John Wiley & Sons Inc., pp. 1057-1069.

Sha, Z., Palosaari, S. (2000): Mixing and crystallization in suspensions. Chem. Eng. Sci., 55, 1797-

1806.

Shubert, A., Mersmann, A. (1996): Determination of Heterogeneous Nucleation Rates, Trans. Inst.

Chem. Eng. A 74 (1) 816-821.

Yang, G., Louhi-Kultanen, M., Sha, Z., Kubota, N., Kallas, J. (2006): A model for prediction of

supersaturation level in batch cooling crystallization, J. Chem. Eng. Japan 39 (4), 426-436.

http://bib.irb.hr/prikazi-rad?&rad=583945

http://bib.irb.hr/prikazi-rad?&rad=583945


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Impact of metals from the environment on chemical changes in olive oil

UDC: 665.327.3

502.3 : 614.7

Zlatka Knezović1

, Marina Trgo2, Angela Stipišić

1, Davorka Sutlović

3,4

1Public Health Institute of Split Dalmatian County, Vukovarska 44, HR-21000 Split, Croatia

2Faculty of Chemistry and Technology, University of Split, Teslina 10/V, HR-21000 Split, Croatia

3Department of Pathology and Forensic Medicine, University Hospital Centre Split, Spinčićeva 1,

HR-21000 Split, Croatia 4Department of Forensic Medicine, University of Split School Medicine, HR-21000 Split, Croatia

Summary

Presence of heavy metals in the environment due to their toxicity is a problem of increasing

significance for ecological, evolutionary, nutritional and environmental reasons. Their accumulation

in soils is of concern in agricultural production due to the effects on plants and their metabolic

activities, possible bioaccumulation, affecting food safety and human health. Humans and other

living organisms are part of a biogeochemical cycle of metals and directly exposed to their impacts.

Virgin olive oils are high quality food with balanced triglyceride composition that provides their

nutritional as well as protective value. The ideal composition of olive oil does not automatically

imply a positive effect on health. Namely, during ripening, harvesting and processing of olives,

especially during oil storage, oxidation processes can occur on triglycerides and can significantly

affect the quality and safety of virgin olive oil. These chemical changes in virgin olive oil are agitated

on exposure to air, heat and light but these processes can be catalysed at the increased content of

heavy metals.

Keywords: lead, copper, iron, virgin olive oil, oxidation

Introduction

Environmental pollution with heavy metals is one of the growing problems of modern

civilization. Although naturally present, their presence in the environment is rising because

of human activities (UNEP, 2013). Since the industrial revolution, their production and use

is increased due to their unique properties such as conductivity, ductility, hardness, and

recyclability enabling them to be an integral part of most daily supplies. The largest source

of pollution with metals are industrial plants, foundries, smelters, coal burning power

plants, car exhaust. Metal particles and steam forms generated during industrial processes

can be dispersed in the environment either by dry (wind) or wet (rainfall) deposition.



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Depending on meteorological conditions particulates can be transmitted over great

distances. Waste water, industrial and domestic, uncontrolled disposal of waste as well as

intensive farming also contribute to the environmental burden with metals. Metals are not

biodegradable, they are permanent constituents of the biogeochemical cycles, representing

potential threat to all living beings including humans (Naagajyoti, 2010).

People can be exposed to metals via air, dust or by ingesting contaminated food or water.

Their accumulation in the body can lead to harmful effects over time. In food, metals can

occur as a result of bioaccumulation from the environment or from contamination during

food processing and storage. Except for direct adverse effects on human health, metals can

cause changes and impact on reducing the quality of food.

Lead is a metal, the presence of which in the environment is of particular concern. Since the

1970s control measures have been taken to regulate lead levels in different products such as

paint, petrol, food cans and pipe. However, coal combustion and aviation fuels are still

significant sources of environment contamination with lead (Say-Gee, 2012).

The organic and inorganic fertilizers as well as pesticides are significant source of metals in

agriculture, particularly copper and iron. Even copper and iron are among essential

elements required for a metabolic activity of living organisms, increased amounts can also

cause adverse effects. According to estimates by Croatian Environment Agency annual

volume of pesticide products on the market of Croatia is 7500 tonnes, out of which 40.4%

are fungicides. Most fungicidal compositions are based on copper compounds (AZO, 2012).

Foliar fertilization of plants is carried out by spraying the leaves and is very common in

the period of intensive plant growth. Depending on the composition of the soil, plants

are fed with different combinations of macro and micro nutrients. On calcareous

Dalmatian soils deficiency of boron and iron is evident, so the foliar fertilization of

olive trees is necessary.

Olive oil is an ideal source of fat compared with other oils and fats, since it has a

moderate amount of saturated fatty acids, high amount of monounsaturated fatty acids

and optimal content of polyunsaturated essential acids. In addition to good triglyceride

composition, olive oil contains many valuable ingredients that are responsible for his

nutritional and protective value (Škarica et al., 1996). This composition of olive oil

does not provide guarantee of positive effects on human health. In fact, during ripening,

harvesting, processing, and especially storage of olive oil oxidative changes can occur

on triglycerides and significantly affect quality and safety. Therefore, it is

recommended to use extra virgin olive oils (EVOO) obtained from undamaged olives,

produced immediately after harvest, through carefully controlled mechanic process, in

which these changes should be kept to a minimum.

Deterioration of the oil begins already at harvest and milling of olives with breaking of

olive cell structure, to be continued throughout the storage period.


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There are two types of deterioration processes, the hydrolysis of triglycerides and oxidative

changes. During the hydrolytic degradation the content of free fatty acids (FFA) increases,

reducing the oxidation stability.

The oxidation takes place at unsaturated fatty acids. For that reason olive oils are more

resistant to oxidation having smaller amount of polyunsaturated fatty acids and containing

natural antioxidants (tocopherols and phenolic compounds). However, once started, the

oxidation reactions are very fast due to autocatalysis, and large number of fatty acids can be

included in chain reaction of free radicals formation. The most common initiators of the

oxidation processes are energy of heat and light, contact with air, and metal ions

(Koprivnjak, 2006).

Indicator of hydrolytic process is content of free fatty acids (as oleic), (FFA), while

peroxide value (PV) and spectrophotometric examination in the ultraviolet are measures of

oxidative changes.

The maximum permitted levels of contaminants, including metals, are regulated by

Commission Regulation (EC 1881/2006), where in official controls of the olive oil only

determination of lead is provided. International Olive Oil Council (IOOC) prescribes

determination of copper and iron according to Trade Standard applying to olive oils.

Unfortunately, the application of Trade standard is not included in official controls.

The purpose of our study was to determine the concentration of lead, cooper and iron in

extra virgin olive oils and investigate their possible association with the processes of oil

deterioration (hydrolysis and/or oxidation).


Samples

Samples of extra virgine olive oil (EVOO) have been collected from market. The samples

for the analyses were stored in the refrigerator under controlled conditions, at 16 °C, to

prevent further development of oxidation processes. The metal content was determined in

81 sample, while the free fatty acids and peroxide value as well as spectrophotometric

examination in the ultraviolet were determined in 79 and 64 samples, respectively.

Methods

For the purpose of metal determination samples were wet digested with concentrated HNO3

and H2O2 mixture in automated microwave digestion unit, CEM, model Mars 5.

Quantitative determination of lead, cooper and iron were carried out on graphite furnace

atomic absorption spectrometer, Analytik Jena, model AAS vario6. Measurements were

performed with hollow cathode lamps for lead, cooper and iron at 283.3; 324.8 and 248.3 nm


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respectively, with deuterium background correction. Graphite tubes with platform and

palladium matrix modifier were used for lead determination. The accuracy of measurements

was examined with known concentrations of standard solutions, which were run as a

sample. The limits of the detection were calculated from the standard deviations of the

blanks and were 1µg/L for all three metals.

Free fatty acid (oleic), and peroxide value were analysed by titration according to HRN EN

ISO 660:2010 and HRN EN ISO 3960:2010 respectively.

Ultraviolet absorbance expressed as specific UV extinction were determined on

spectrophotometer Lambda 25, Perkin-Elmer, according to HRN EN ISO 3656:2011.

Absorptions measured at 232 and 270 nm, expressed as extinction coefficients K232 and

K270 are measures of oxidative changes. Elevated values at 232 nm indicate primary

oxidative changes in oil, while increased absorbtion at 270 nm is a measure of secondary

oxidation. Moreover, variation of the specific extinction (∆ K270) enables detection of

virgine olive oils adulteration with refined oils (Fig. 1).

Fig. 1. Spectral curves of extra virgin olive oil in the UV region; EVOO = extra virgin olive oil

with normal values of absorbance at 232 and 270 nm; a = elevated absorbances

at 232 nm (primary oxidation); b = elevated absorbances at 270 nm

(secondary oxidation); c = variation of the specific extinction

(∆ K270) as a sign of adulteration


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Experimental results of lead, cooper and iron concentrations as well as EVOO quality

parameters, FFA (oleic), peroxide value, and extinciton coefficients K232 and K270 expressed

by the mean, median, standard deviation (SD), and range (minimum, maximum) compared

with the maximum allowable values (M.A.V.) are shown in Table 1.

Table 1. Presentation of the experimental results and maximum allowable values (M.A.V.) according to

legal regulations

Pb (mg/kg)

Cu (mg/kg)

Fe (mg/kg)

PV (mmolO2/kg)

FFA (oleic) %

K232 K270

N 81 81 81 79 79 64 64

Mean 0.110 0.260 2.330 5.74 0.54 2.30 0.23

SD 0.196 0.481 3.729 2.75 0.42 0.50 0.23

Median 0.023 0.100 0.727 5.06 0.43 2.20 0.16

Min <0.001 <0.001 <0.001 2.28 0.10 1.52 0.08

Max 1.13 6.56 22.7 15.1 2.4 3.51 1.44

M.A.V. 0.11 0.12 32 10a* 0.8 a 2.50a 0.22 a N = number of samples 1Commission Regulation EC 1881/2006 2Trade Standard applying to olive oils of International Olive Oil Council (IOOC)

aRegulations of olive oils and olive-pomace oil (NN 7/09 and 112/09) and Commission Regulation 2568/91/ EEC *The IOOC is considering a proposal to lower the maximum level to 7,5 mmol O2 /kg

All experimental results have been selected in three ranges of determined values. In first

range there are values up to 75% of M.A.V., in second range there are values form 75% up

to M.A.V, and in third range there are values exceeeding M.A.V. (Fig. 2).

Fig. 2. Experimental results in relation to the maximum allowable values

52

35 47 49 52

32 44

5

7

10 15

20

10

11 24

39 24

15 7

22

9

Pb Cu Fe FFA PV K232 K270

Num

ber

of

sam

ple

s

> M.A.C


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The obtained results showed high concentrations of lead and iron in 24 samples, while copper

exceeded M.A.V in 39 samples. Among all the examined indicators of deterioration processes

(FFA, peroxide value, K232 and K270) K232 as indicator of primary oxidation behaves as the

most sensitive parameter and obviously has correlation with heavy metal concentrations.

Our experimental results showed that in the samples with observed increased values of PV

and K-coefficients, concentration of one or more analyzed metals were increased, indicating

relation between heavy metal ions content and oxidation processes.

Oxidation processes starts at the very beginning of olive oil production, but the intensity of

this deterioration is negligible compared to the period of the oil storage (Koprivnjak, 2006).

Durability of EVOO is estimated at 18 months whereby oils should be stored in appropriate

packaging at a temperature of 16-18 °C, with no contact with air or light. The relationship

between metal concentration and quality parameters (PV and K232), in dependence of

sampling period, are shown in Fig. 3 and Fig. 4.

From the Fig. 3 and Fig. 4 can be observed that the concentrations of heavy metal ions follow

increased values of PV an K232. During the warm period of the year deterioration processes are

more intensive where overlapping of extremely high PV anf K232 with concentrations of metal

ions above M.A.V. occur. These results confirm that beside effect of light and temperature, the

content of metal ions can effect the oxidation processes during storage period.

Extra virgin olive oil has high oxidative stability mainly due to its fatty acid composition, in

particular to the monounsaturated-to-polyunsaturated ratio (Bendini, 2006). Fatty acid

oxidation occurs by their interaction with molecular oxygen in a self-catalyzed mechanism.

Metal ions probably increase the rate of oxidation due to a reduction of activation energy in

the initiation step during autooxidation of fatty acids. In the research has been confirmed

that, both copper and iron actively induce oxidation of the oil, where copper is reported as

pro-oxidative ion (Anderson, 1998). Namely, cooper accelerates hydrogen peroxide

decomposition 50 times faster than ferrous ion (Fe 2+

) (Choe, 2006).

Our results are consistent with these observations; where in samples with high PV and K232

copper was several times higher compared to M.A.V.

Large number of samples, as well as high concentrations of copper and iron in the analyzed

samples indicate an excessive and improper use of fungicides and preparations for foliar

nutrition. In literature intended for olive growers the positive impact of fungicides in control

of diseases and pests is mainly discussed, without emphasizing the possible harmful effects

of metal ions on quality and safety of olive oil.

As previously mentioned, the official control includes only the control of lead, in accordance

with of Regulation 1881, while copper and iron, which do not have a direct effect on human

health are not considered. Since, determination of copper and iron in olive oil is not mandatory,

it is usually performed only on special request. It would be desirable to modify the legal

framework in a way that the analysis of copper and iron become mandatory part of EVOO

official controls.


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Fig. 3. Peroxide value in relation with a) lead, b) copper and c) iron concentrations;

(solid line is M.A.V. for PV; dashed line is M.A.V. for metal ion)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

0 2 4 6 8 10 12

Pb

(m

g/k

g)

PV

(m

mo

l O

2/k

g)

Month

a)

PV Pb

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

0 2 4 6 8 10 12

Month

Cu (

mg/k

g)

PV

(m

mo

l O

2/k

g)

b)

PV Cu

0,0

5,0

10,0

15,0

20,0

25,0

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

0 2 4 6 8 10 12

Fe

(mg/k

g)

PV

(m

mo

l O

2/k

g)

Month

c)

PV Fe


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Fig. 4. K232 in relation with a) lead, b) copper and c) iron concentrations;

(solid line is M.A.V. for K232; dashed line is M.A.V. for metal ions)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

0 2 4 6 8 10 12

Month

Pb

(m

g/k

g)

K 2

32

a)

K232 Pb

0,0

0,5

1,0

1,5

2,0

2,5

3,0

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

0 2 4 6 8 10 12

Month

Cu (

mg/k

g)

K232

b)

K232 Cu

0,0

5,0

10,0

15,0

20,0

25,0

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

0 2 4 6 8 10 12

Fe

(mg/k

g)

K 2

32

Month

c)

K232 Fe


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References

Andersson K., Lingnert K. (1998): Influence of oxygene and copper concentration on lipid oxidation

in rapeseed oil, JAOCS 75, 1041-1046.

AZO (2012): Izvješće o stanju okoliša u Republici Hrvatskoj za razdoblje 2005.-2008., Agencija za

zaštitu okoliša, Zagreb, http://www.azo.hr/Izvjesca29 (pristupljeno 13.09.2014.).

Bendini A., Cerretani L., Vecchi S., Carrasco-Pancorbo A., Lercker G. (2006): Protective Effects of

Extra Virgin Olive Oil Phenolics on oxidative stability in the presence or absence of copper

ions, J. Agric. Food. Chem. 54, 4880-7.

Choe E., Min D.B. (2006): Mechanisms and factors for Edible Oil Oxidation, Comprehensive

Reviews in Food science and Food Safety 5, 169-186.

Commission Regulation (EC) No. 1881/2006 setting maximum levels for certain contaminants in

foodstuffs, Official Journal of the European Union L 364, 20 December 2006.

Commission Regulation (EEC) No. 2568/91 on the characteristics of olive oil and olive-residue oil

and on the relevant methods of analysis, Official Journal L 248, 5 September 1991.

EFSA Panel on Contaminants in the Food Chain, (CONTAM), (2010): Scientific Opinion on Lead in

Food, EFSA Journal 8.

Koprivnjak O. (2006): Djevičansko maslinovo ulje od masline do stola, Sigra, Poreč.

Naagajyoti P.C., Lee K.D., Sreekanth T.V.M. (2010): Heavy metals, occurrence and toxicity for

plants: a review, Environ. Chem. Lett. 8, 199-216.

Pravilnik o uljima od ploda i komine maslina (NN 07/09 i NN 112/09).

Say-Gee S., Wan Sahiah A., (2012): Enrichment of arsenic, lead, and antimony in Balingian coal

from Sarawak, Malaysia: Modes of occurrence, origin and partitioning behaviour during coal

combustion, Int. J. Coal. Geol. 101, 1-15.

Škarica B., Žužić I., Bonifačić M. (996): Maslina i maslinovo ulje visoke kakvoće u Hrvatskoj,

Tipograf, Rijeka.

Trade standard applying to olive oils and olive pomace oils (COI/T.15/NC No 3/Rev.7).

UNEP, van der Voet E., Salminen R., Eckelman M., Mudd G., Norgate T. Hischier R., (2013):

Environmental Risks and Challenges of Anthropogenic Metals Flows and Cycles, A Report of

the Working Group on the Global Metal Flows to the International Resource Panel.


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Kinetika sušenja katalizatora u sušioniku s fluidiziranim slojem

UDC: 66.047


Sveučilište u Zagrebu, Fakultet kemijskog inženjerstva i tehnologije, Marulićev trg 20, 10000 Zagreb,

Hrvatska

Sažetak

U ovom je radu istraživana kinetika sušenja katalizatora u fluidiziranom sloju. Mjerenja su provedena

u laboratorijskom sušioniku pri različitim brzinama strujanja zraka, temperaturama te visinama sloja

čvrstih čestica različitih veličinskih frakcija. Preliminarna istraživanja uključila su karakterizaciju

sferičnih čestica katalizatora. Izmjerena je tvrdoća i gustoća čestica te određena raspodjela veličina

pora. Morfologija čestica katalizatora definirana je pretražnom elektronskom mikroskopijom, dok je

sastav utvrđen rendgenskom i elementarnom kemijskom analizom. Rezultati su pokazali da

temperatura, brzina strujanja zraka te visina sloja čvrstih čestica utječu na kinetiku sušenja. Veća

brzina sušenja odgovara većim temperaturama i povoljnijim hidrodinamičkim uvjetima te manjoj

visini sloja čvrstih čestica. Kinetika sušenja opisana je pomoću četiri matematička modela.

Procijenjeni su koeficijenti prijenosa tvari, te efektivni difuzijski koeficijent. Parametri modela i

procijenjena prijenosna svojstva, korelirani su uvjetima provedbe procesa. Na temelju izvedenih

korelacija može se procijeniti kinetička krivulja sušenja pri drugim uvjetima provedbe procesa.

Ključne riječi: fluidizirani sloj, katalizator, kinetika, model, sušenje

Uvod

Sušenje je vrlo važan i gotovo neizostavan separacijski proces u industriji s obzirom da je

sadržaj vlage ključan parametar koji utječe na konačna svojstva gotovog proizvoda (Chen

et al., 2012). Sušenje u fluidiziranom sloju koristi se za sušenje praškastih materijala. U

sušioniku s fluidiziranim slojem čestica ostvaruju se povoljni uvjeti za intenzivan prijenos

tvari i topline. Sušenje u fluidiziranom sloju čestica smatra se blagom, jednolikom

metodom sušenja do niskog konačnog sadržaja vlage te omogućuje sušenje toplinski

osjetljivih materijala. Zbog velike brzine sušenja metoda je ekonomična u usporedbi s

ostalim metodama sušenja (Mujumdar, 2007; Senadeera et al., 2003). Na proces sušenja

utječu svojstva materijala, vrsta sušionika, kinetički parametri, temperatura, brzina

strujanja zraka, brzina sušenja te mehanizam prijenosa vlage (Bakal et al., 2012).

Poznavanje kinetike sušenja vrlo je važno za dizajn, simulaciju, optimizaciju i uvećanje

procesa sušenja. Stoga, da bi se poboljšao proces sušenja važno je na raspolaganju imati



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odgovarajuće modele kojima se može opisati krivulja sušenja kod različitih uvjeta.

Modeliranje procesa sušenja veoma je složen zadatak, ponajprije jer dolazi do

istovremenog odvijanja procesa prijenosa tvari i topline te zahtjeva poznavanje velikog

broja parametara (Mujumdar, 2007). Empirijski modeli daju nam direktnu vezu između

sadržaja vlage i vremena sušenja, ali oni zanemaruju osnove procesa sušenja i njihovi

parametri nemaju nikakvog fizikalnog značenja. Stoga, ti modeli ne mogu dati uvid u bitne

procese koji se odvijaju tijekom sušenja, iako dobro opisuju eksperimentalne podatke

(Mujumdar, 2007).

U ovom radu korištena su tri empirijska i jedan teoretski (II Fickov zakon) matematički model

(Tablica 1) za aproksimaciju eksperimentalnih podataka (Mujumdar, 2007; Skelland, 1974).

Tablica 1. Korišteni matematički modeli

Table 1. Used mathematical models

2 2

2eq

2 200 eq

6 1efD n

tr

n

X Xe

X X n

II Fickov zakon

Analitičko rješenje oblik kugle

eq

0 eq

KtX X

eX X

Lewis

eq

0 eq

nktX X

eX X

Page

eq ( )

0 eq

nk tX X

eX X

Overhults, White, Hamilton i Ross (OWHR)

Materijali i metode

Materijal

Kao materijal za istraživanje kinetike sušenja u sušioniku s fluidiziranim slojem odabrana

su sferična zrna katalizatora nepoznatog sastava. Ovaj materijal je odabran zbog dobrih

fluidizacijskih karakteristika i zbog toga što se uklapa u ostvarive radne uvjete u

korištenom sušioniku s fluidiziranim slojem. Uzorak je prosijan kroz sita kako bi se dobila

uska veličinska frakcija, tako da prilikom sušenja fluidiziraju i najveće čestice, a da pri

tome ne dolazi do odnošenja najmanjih čestica.

Karakterizacija materijala

S obzirom da je korišteni katalizator nepoznatog sastava za karakterizaciju materijala

korištena je rendgenska difrakcija (XRD) i pretražna elektronska mikroskopija s


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elementarnom analizom (SEM/EDS). Uzorci su analizirani pomoću rendgenskog

difraktometra Shimadzu LabX XRD-6000 koristeći CuK zračenje u području kutova

difrakcije od 10 do 60° 2 s korakom od 0.02° i vremenskim trajanjem jednog koraka 0.6 s.

Uvid u morfologiju i sastav katalizatora dobiven je snimanjem uzorka pretražnim

elektronskim mikroskopom Tescan Vega 3. Karakterizacija uzorka također uključuje

određivanje gustoće, raspodjele veličina pora, specifične površine i tvrdoće. Raspodjela

veličina pora i specifična površina (BET) određene su na Micromeritics ASAP 2000

uređaju metodom adsorpcije i desorpcije dušika. S obzirom da se radi o sferičnim

česticama gustoća uzorka određena je gravimetrijskom metodom. Čestice uzorka vagane

su na analitičkoj vagi Kern ALJ 220-4 NM preciznosti ± 0.0001 g. Volumen čestica je

izračunat na temelju izmjerenog promjera. Iz mase i volumena izračunata je gustoća

uzorka. Gustoća mokrih čestica (čestice su 1 h u vodi) izračunata je na analogan način, jer

za fluidizaciju je bitan samo hidrodinamički volumen koji je neovisan o poroznosti.

Tvrdoća uzorka izmjerena je na uređaju Erweka TBH 30 pomoću težinske ćelije s

mjerenjem naprezanja žice. Tvrdoća ukazuje na otpornost zrna katalizatora prema pucanju

tijekom fluidizacije. Ravnotežni sadržaj vlage materijala nakon sušenja određen je

gravimetrijskom metodom.

Provedba sušenja

Mjerenja su provedena u laboratorijskom sušioniku s fluidiziranim slojem čestica pri

različitim brzinama strujanja zraka (1,50 i 3,13 ms-1

), temperaturama (50, 57 i 65 °C) te

visinama mirujućeg sloja čestica (H/D=0,91; 1,36; 1,82) različitih veličina (dsr=1,55; 1,70 mm).

Proces sušenja praćen je psihrometrijskom metodom, odnosno praćenjem svojstava zraka

(temperatura i relativna vlažnost) tijekom procesa. Ostala svojstva zraka dobivena su

korištenjem računalnog programa Humidity. Aparatura se sastoji od puhala za zrak,

grijaća, raspodjelne rešetke, kolone (D=0,057 m, Hk=0,6 m) te ciklona. Prigušna pločica

spojena na manometar ispunjen vodom služi za određivanje brzine strujanja zraka, dok se

pad tlaka u koloni prati visinskom razlikom vode u kosom manometru.


Karakterizacija materijala

Kako bi se dobio uvid u strukturu, morfologiju i sastav katalizatora uzorak je analiziran

rendgenskom difrakcijom i pretražnom elektronskom mikroskopijom. Difraktogram uzorka

prikazan je slikom 1, dok slike 2 i 3 prikazuju rezultate SEM i EDS analize. Iz difraktograma

uzorka (Slika 1) vidljivo je da je uzorak mješovite (amorfno-kristalinične) strukture. Iz

samog difraktograma nije bilo moguće utvrditi o kojem se spoju radi. EDS analiza (Slika 3)


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ukazuje da se radi o aluminijevom oksidu onečišćenom ugljikom i klorom koji vjerojatno

služi kao promotor. Iz SEM fotografija uzorka i presjeka uzorka (Slika 2) može se uočiti

nepravilna površina te se može zaključiti kako se uzorak sastoji od pora različitih veličina.

Kako bi se odredio mehanizam prijenosa vlage kroz unutrašnjost materijala određena je

raspodjela veličina pora prikazana slikom 4. Promjeri pora karakteristični su za difuzijski

tok, a vidljiv je i mali udio pora većih od 10-7 m kroz koje se vlaga kreće kapilarnim tokom.

U tablici 2 dane su vrijednosti specifične površine, srednjeg promjera pora kao i volumena

pora. Vrijednosti gustoće i tvrdoće zrna katalizatora prikazane su tablicom 3.

Slika 1. Rendgenski difraktogram istraživanog katalizatora

Fig. 1. XRD pattern of examined catalyst

Slika 2. SEM fotografija cijelog zrna i presjeka zrna katalizatora (100x)

Fig. 2. SEM photographs of whole and cross-sectional cut catalyst bead (100x)


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Slika 3. Elementarna kemijska analiza katalizatora

Fig. 3. Elementary chemical analysis of catalyst

Slika 4. Kumulativna raspodjela veličina pora katalizatora

Fig. 4. Cumulative pore size distribution of catalyst

Tablica 2. Specifična površina, srednji promjer i volumen pora katalizatora

Table 2. Specific surface area, average diameter and volume of pores of catalyst


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Tablica 3. Gustoća i tvrdoća zrna katalizatora

Table 3. Density and hardness of catalyst beads

Radno područje fluidizacije

Radno područje fluidizacije definirano je minimalnom brzinom fluidizacije kao donjom

granicom i brzinom odnošenja kao gornjom granicom. Minimalna brzina fluidizacije

određena je eksperimentalno snimanjem dijagrama fluidizacije (Slika 5) te je također

procijenjena koristeći korelacije više autora (Tablica 4) (Chitester et al., 1984; Hilal et al.,

2001; Wen i Yu, 1966). Iz tablice 3 može se zaključiti da su eksperimentalno određene

minimalne brzine fluidizacije uzorka u skladu sa procijenjenim vrijednostima osim za

Chitesterovu korelaciju. Vidljivo je da ne dolazi do promjene minimalne brzine fluidizacije

s promjenom visine sloja nepokretnih čestica (H/D). Do odnošenja čestica dolazi pri

brzinama strujanja zraka većim od 3,5 m s-1

.

Slika 5. Dijagram fluidizacije ispitivanog uzorka (suhe čestice, H/D=1,81)

Fig. 5. Fluidization diagram of examined sample (dry particles, H/D=1,81)


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Tablica 4. Eksperimentalno određene i procijenjene minimalne brzine fluidizacije

Table 4. Experimental and evaluated values for minimal fluidization velocities

dsr =1,550 mm; Ar (suhi) = 1,66·105; Ar (mokri) = 2,72·105

vmf / ms-1

eksperimentalna Chitester Hilal Wen i Yu

suhe

H/D

0,54

0,51

0,53

0,64 0,53 0,54 0,91

1,36

1,82

mo

kre

H/D 0,7

0,75

0,69

0,88 0,71 0,75 0,91

1,36

1,82

Kinetika sušenja

Na slikama 6-9 prikazan je utjecaj uvjeta provedbe procesa na kinetiku sušenja

katalizatora. Iz kinetičkih krivulja sušenja (Slike 6-9) mogu se uočiti tri karakteristična

perioda sušenja (stabilizacija, period konstantne brzine sušenja i period padajuće brzine

sušenja). Utjecaj temperature zraka za sušenje na kinetiku sušenja prikazan je na slici 6.

Može se zaključiti da porastom temperature zraka raste brzina sušenja, odnosno smanjuje

se vrijeme trajanja procesa. Razlog tomu je što zrak više temperature ima manju relativnu

vlažnost pa je pokretačka sila procesa prijenosa topline i vlage veća. Također, vidljivo je

da se trajanje perioda konstantne brzine sušenja skraćuje porastom temperature zraka, jer u

tom periodu sušenje ovisi samo o svojstvima zraka. Slika 7 prikazuje utjecaj visine

nepokretnog sloja čestica na kinetiku sušenja. Brzina sušenja je manja što je veći sloj

nepokretnih čestica pa proces dulje traje. Veća visina sloja čestica sadrži veću masu čestica

u sušioniku, a time je i veća masa vode koju je potrebno ukloniti tijekom sušenja. U

sušioniku tada isparava veća količina vode koju zrak na sebe prima i time se smanjuje

pokretačka sila procesa. Period konstantne brzine sušenja kao i ukupno vrijeme trajanja

procesa postaje dulje sa porastom mase materijala u sušioniku. Na slici 8 prikazan je

utjecaj brzine strujanja zraka na kinetiku sušenja. Porast brzine strujanja zraka uzrokuje

smanjenje otpora prijenosu tvari i topline (smanjuje se hidrodinamički, termički i difuzijski

sloj oko čestica) te time povećava brzinu sušenja i smanjuje vrijeme trajanja procesa.


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Utjecaj veličine čestica na kinetiku sušenja, prikazan slikom 9, nije značajnije izražen.

Razlog tome je što nema značajnije razlike u veličini čestica korištenih veličinskih

intervala (dsr=1,55 mm i dsr=1,7 mm). Općenito, porastom veličine čestica trebalo bi doći

do smanjenja brzine sušenja. Razlog tome je što kod većih čestica vlaga prolazi veći put od

unutrašnjosti do površine čestice. Nadalje, međufazna površina zrak-čestice je znatno veća,

za istu masu materijala (odnosno isti H/D), ako su čestice manjeg promjera. Slika 10

prikazuje rezultat aproksimacije eksperimentalnih podataka promjene sadržaja vlage

ispitivanog materijala tijekom sušenja odabranim matematičkim modelima. OWHR i

Pageov model dobro aproksimiraju eksperimentalne podatke. Lewisov model i analitičko

riješenje II Fickovog zakona nisu pogodni za opis kinetike sušenja korištenog katalizatora

s obzirom na relativno dugo trajanje perioda konstantne brzine sušenja. S obzirom da

analitičko riješenje II Fickovog zakona ne aproksimira dobro eksperimentalne podatke,

izračunate vrijednosti efektivnih difuzijskih koeficijenata nisu realne te nisu niti prikazane.

Utjecaj uvjeta provedbe procesa na parametre modela (OWHR i Page) kao i na koeficijent

prijenosa tvari dan je u tablici 5. Vrijednosti parametra k u Pageovom i OWHR modelu

rastu kako raste i brzina sušenja, tj rastu s porastom temperature i brzine zraka te

smanjenjem početnog sadržaja vlage materijala i smanjenjem visine sloja čestica.

Parametar n ovisi o vrsti i geometrijskim karakteristikama materijala. Koeficijent prijenosa

tvari bilo je moguće odrediti samo za period konstantne brzine sušenja jer je tada

temperatura površine materijala na temperaturi mokrog termometra, a zrak tik uz površinu

je zasićen pa je moguće odrediti pokretačku silu. Koeficijent prijenosa tvari procijenjen je

iz kinetičke jednadžbe. Km raste s porastom temperature i početnim sadržajem vlage te

opada s porastom visine sloja čestica. Porastom brzine strujanja zraka raste vrijednost

koeficijenta prijenosa tvari jer se smanjuju otpori prijenosu tvari, topline i količine gibanja.

Slika 6. Utjecaj temperature zraka na kinetiku sušenja

Fig. 6. Influence of air temperature on drying kinetics


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Slika 7. Utjecaj visine sloja nepokretnih čestica na kinetiku sušenja

Fig. 7. Influence of bed height on drying kinetics

Slika 8. Utjecaj brzine strujanja zraka na kinetiku sušenja

Fig. 8. Influence of air velocity on drying kinetics


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Slika 9. Utjecaj veličine čestica na kinetiku sušenja

Fig. 9. Influence of particle size on drying kinetics

Slika 10. Aproksimacija eksperimentalnih podataka odabranim matematičkim modelima

(T=65 °C; H/D=1,36; v=3,13 m s-1

)

Fig. 10. Approximation of experimental data by chosen mathematical models

(T=65 °C; H/D=1,36; v=3,13 m s-1

)


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Tablica 5. Utjecaj uvjeta provedbe procesa na koeficijent prijenosa tvari i parametre odabranih

modela (OWRH, Page)

Table 5. Influence of process conditions on mass transfer coefficient and selected model parameters

(OWRH, Page)

H/D v,

m/s

T,

°C

X0,

kg/kg

Y0,

kg/kg

Xeq,

kg/kg

Page OWHR Km,

m/s k n k n

0,91 3,13

50

50

57

57 65

1,0209

0,7533

1,0648

0,7624 0,7582

0,0051

0,0046

0,0048

0,0037 0,0043

0,0209

0,0318

0,0169

0,0188 0,0163

0,044

0,037

0,051

0,089 0,063

1,71

1,78

1,76

1,59 1,88

0,161

0,158

0,184

0,221 0,229

1,71

1,78

1,76

1,59 1,88

0,0448

0,0335

0,0482

0,0331 0,0343

1,50 50 0,7693 0,0059 0,0285 0,027 1,49 0,089 1,49 0,0159

1,36 3,13

50

57

57

65

0,7616

0,8389

0,7709

0,8286

0,0041

0,0046

0,0046

0,0026

0,0206

0,0102

0,0175

0,0069

0,026

0,028

0,044

0,036

1,77

1,78

1,53

1,81

0,128

0,135

0,130

0,158

1,77

1,78

1,53

1,81

0,0299

0,0219

0,0236

0,0246

1,82 3,13 50 0,8211 0,0030 0,0226 0,037 1,43 0,100 1,43 0,0193

Zaključci

Hilalova i Wen i Yu korelacija dobro procjenjuju minimalnu brzinu fluidizacije za

korišteni materijal i radne uvjete. Porastom temperature raste brzina sušenja i smanjuje se

vrijeme trajanja procesa zbog veće pokretačke sile. Veća brzina strujanja zraka rezultira

povoljnijim hidrodinamičkim uvjetima što rezultira većom brzinom sušenja i kraćim

trajanjem procesa. Sušenje je brže za manje visine nepokretnog sloja čestica jer se manja

masa vode mora ukloniti. Kinetičke krivulje mogu se opisati sa Pageovim i OWHR

modelom. Uvjeti koji povećavaju brzinu sušenja rezultiraju višim vrijednostima

parametara odabranih modela. Koeficijent prijenosa tvari raste s porastom brzine strujanja

zraka, temperature i početnog sadržaja vlage, a opada s porastom visine nepokretnog sloja

čestica.

Literatura

Bakal, S.B., Sharma, G.P., Sonawane, S.P., Verma, R.C. (2012): Kinetics of potato drying using

fluidized bed dryer, Journal of Food Science and Technology 49 (5), 608-613.

Chen, L., Lin, C.H., Xie, Y.H., Du, S. (2012): Drying Kinetics of Granules in a Fluidized Bed

Dryer, Advanced Materials Research 12, 459-462.

Chitester, D.C., Kornosky, R.M., Fan, L.S., Danko, J.P. (1984): Characteristics of fluidization at

high pressure, Chemical Engineering Science 39, 253-261.


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Hilal, N., Ghannam, M.T., Anabtawi, M.Z. (2001): Effect of bed diameter, distributor and inserts on

minimum fluidization velocity, Chemical Engineering and Technology 24 (2), 161-165.

Mujumdar, S. (2007): Handbook of Industrial Drying, New York, CRC Press, 3. Izdanje.

Senadeera, W., Bhandari, B.R., Young, G., Wijesinghe, B. (2003): Influence of shapes of selected

vegetable materials on drying kinetics during fluidized bed drying, Journal of Food

Engineering 58 (3), 277-283.

Skelland, A.H.P. (1974): Diffusional Mass Transfer, New York, John Wiley & Sons Inc.

Wen, C.Y., Yu, Y.H. (1966): A generalized method for predicting the minimum fluidization

velocity. AIChE Journal 12 (3), 610-612.

Fluid bed drying kinetics of catalysts


University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 20,


Summary

In this work fluid bed drying kinetics of a catalyst has been examined. Experiments were carried out

in a laboratory dryer at different air velocities, air temperatures and different bed height for different

particle size fractions. Preliminary research involved characterization of spherical catalyst beads.

Hardness, density and also pore size distribution of catalyst beads have been measured. The

catalysts morphology has been examined by scanning electron microscopy while the structure and

composition have been determined by x-ray and elemental chemical analysis. Results have shown

that air temperature, velocity and bed height influence the drying kinetics. Higher drying rates are

achieved at higher air temperatures and better hydrodynamic conditions and also with lower bed

height. Drying kinetics has been described by four mathematical models. Mass transfer coefficients

and effective diffusion coefficients have been estimated. Model parameters and evaluated transfer

properties have been correlated with process conditions. On the basis of those correlations a kinetic

drying curve can be evaluated for other drying conditions.

Keywords: drying, catalyst, fluid bed, kinetics, model

http://www.sciencedirect.com/science/journal/02608774

http://www.sciencedirect.com/science/journal/02608774


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Reološka i toplinska karakterizacija nanofluida

UDC: 536.2



Hrvatska

Sažetak

Da bi se povećala energetska učinkovitost izmjenjivača topline, potrebno je poboljšati toplinska

svojstva konvencionalnih nanofluida. Znatno poboljšanje toplinskih svojstava, moguće je postići

suspendiranjem nanočestica u kapljevinama koje se uobičajeno primjenjuju u izmjenjivačima

topline. Stoga su u smjesu voda - etilen glikol te voda – glicerol, primjenom ultrazvučne sonde,

dispergiraju nanočestice aluminij (III) oksida u rasponu koncentracija 0,3-1,4 vol %. Stabilnost

suspenzija ispitana je nefelometrijskom metodom korištenjem uređaja 3 u 1 – FTN. Nakon

postizanja stabilnosti suspenzija, izmjerena je njihova gustoća i viskoznost te im je određeno

reološko ponašanje. Također, određena su i toplinska svojstva pripravljenih suspenzija: koeficijenti

toplinske vodljivosti, specifični toplinski kapacitet i temperaturna vodljivost. Pripremljeni

nanofluidi i bez dodatka aditiva pokazuju stabilnost u razdoblju od jednog mjeseca, imaju svojstva

newtonskih fluida s neznatnim povećanjem gustoće i viskoznosti u odnosu na bazni fluid. Toplinska

svojstva nanofluida poboljšavaju se s povećanjem volumnog udjela nanočestica za oba bazna fluida.

Primjenom nanofluida u pločastom izmjenjivaču topline, izračunat je koeficijent prijelaza i

koeficijent prolaza topline pri različitim protocima i koncentracijama nanofluida. Koeficijent

prijelaza i prolaza topline nanofluida povećava se s povećanjem volumnog udjela nanočestica kao i

poboljšanjem hidrodinamičkih uvjeta u izmjenjivaču topline.

Ključne riječi: nanofluid, termo fizikalna svojstva nanofluida, pločasti izmjenjivač topline

Uvod

U današnje vrijeme procesi u kojima se odvija prijenos topline, zahtijevaju visoku

učinkovitost i djelotvornost, što uključuje poboljšanje toplinskih svojstava fluida koji služe

za izmjenu topline. Budući da konvencionalni fluidi koji se najčešće koriste imaju nisku

toplinsku vodljivost, razvila se ideja o poboljšanju njihove toplinske vodljivosti. Razvojem

nanotehnologije došlo je do primjene nanočestica u pripremi fluida, tzv. nanofluida, koji su

pokazali povećanje koeficijenta toplinske vodljivosti.

Pregledom literature, može se zaključiti da nanofluidi posjeduju izuzetna termo fizikalna

svojstva koja su poželjna pri izmjeni topline u mnogim proizvodnim procesima (Wang and

Mujumdar, 2007; Yu and Xie, 2012). Iako su nanofluidi pokazali dobra fizikalna svojstva,



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neki od problema i dalje su prisutni, osobito u području priprave stabilnih suspenzija

(Shima and Philip, 2014). Ovaj problem može se riješiti djelovanjem ultrazvuka te

dodatkom površinski aktivne tvari koja sprječava stvaranje aglomerata i sedimentaciju

čestica.

Unatoč brojnim eksperimentalnim ispitivanjima, još uvijek se raspravlja da li je povećanje

toplinske vodljivosti fluida izvan ili u okviru teorije utjecaja medija koju je dao Maxwell

(Zhang et al., 2006). Iako su predloženi brojni mehanizmi koji objašnjavaju povećanje

koeficijenta toplinske vodljivosti, i dalje nema objašnjenje za niz različitih podataka o

toplinskoj vodljivosti različitih nanofluida. Brown-ovo gibanje koje uzrokuje dodatnu

konvekciju i kondukcija kroz sloj nanočestica je opće prihvaćeni mehanizam odgovoran za

izuzetno poboljšanje toplinske vodljivosti nanofluida (Philip and Shima, 2012).

Kako su nanofluidi nova vrsta fluida korištenih u inženjerstvu, velik broj istraživanja

odnosi se na utjecaj različitih čimbenika (volumne koncentracije, oblika i vrste

nanočestica; temperature, tlaka i pH nanofluida te vremena i snage djelovanja ultrazvuka)

na termo fizikalna svojstva nanofluida. Međutim, manji broj istraživanja bavi se

testiranjem nanofluida u izmjenjivačima topline kako bi se njihova dinamička i toplinska

svojstva usporedila sa konvencionalnim fluidima.

U ovom radu ispitana su termo fizikalna svojstva nanofluida bez dodatka aditiva

pripremljena raspršivanjem aluminij (III) oksida u dvije bazne kapljevine (smjesa etilen

glikola i vode te glicerola i vode). Primjenom nanofluida u pločastom izmjenjivaču topline,

izračunat je koeficijent prijelaza i koeficijent prolaza topline pri različitim protocima i

koncentracijama nanofluida.

Materijal i metode

Eksperimentalni dio rada obuhvaća određivanje termo fizikalnih svojstava nanosuspenzija

pripremljenih raspršivanjem nanočestica aluminij (III) oksida u smjesi kapljevina.

Pripremljeno je 8 uzoraka: četiri uzoraka pripremljena su od smjese kapljevina koju su

sačinjavale demineralizairana voda i etilen glikol u omjeru 50:50 te glicerol i

demineralizirana voda u masenom omjeru 20:80. Volumni udjeli aluminij (III) oksida u

pripremljenim suspenzijama iznosili su: 0,3%, 0,7%, i 1,0% i 1,4% za obje smjese

kapljevina.

Materijal

Proizvođač aluminij (III) oksida (komercijalnog naziva AEROXIDE Alu C 805) je Evonik

Degussa. Aeroxide je bijeli, fini i lagani prah, veličina čestica između 7 i 40 nm. Zbog

izrazito malih veličina čestica sklon je aglomeraciji. Svojstva nanočestica Al2O3 prikazana

su u tablici 1.


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Tablica 1. Svojstva nanočestica aluminij (III) oksida, AEROXID Alu C, proizvođača Evonik Degussa

Table 1. Properties of nanoparticles aluminium (III) oxide, AEROXID Alu C, by Evonik Degussa

AEROXIDE Alu C

BET površina 100 m2g-1

Sastav određen XRD 33% , 66%

Specifična težina 3,2 g cm-3

Koeficijent toplinske vodljivosti 36 W m-1K-1

Priprema nanofluida

Kako je nanosuspenziju potrebno stabilizirati, odnosno spriječiti sedimentaciju nanočestica

kroz duži vremenski perid; odvagana masa čestica dodana je u kapljevinu te podvrgnuta

djelovanju ultrazvučnih valova. Snaga djelovanja ultrazvučnog homogenizatora iznosila je

2000 W, primjenjena amplituda 25%, a trajanje djelovanja ultrazvučnih valova na

suspenziju 60 min.

Aparatura korištena za pripravu stabilne suspenzije sastoji se od ultrazvučnog

homogenizatora sa sondom koja je uronjena u posudu s duplom stjenkom. Kako se tijekom

priprave suspenzije razvija toplina, potrebno je nanofluid hladiti, u tu svrhu korišten je

termostat Julabo F 12.

Određivanje stabilnosti nanosuspenzija mjerenjem nefelometrijskog zamućenja

Mjerenje nefelometrijskog zamućenja provedeno je na uređaju 3 u 1 –FTN koji objedinjuje

funkcije triju instrumenata: turbidimetra, nefelometra i fotokolorimetra uz jednostavnu

komunikaciju s osobnim računalom. Uređajem se pratila promjena nefelometrijskih

jedinica turbiditeta kroz 20 do 50 dana. Uređajem se registrira promjena jačine (intenziteta)

prolaznog zračenja ili jačine raspršenog zračenja kao posljedicu sraza s česticama.

Praćenjem dobivenih podataka u vremenu određuje se stabilnost suspenzije.

Mjerenje gustoće nanosuspenzija

Mjerenje gustoće pripremljenih nanosuspenzija izmjereno je digitalnim uređajem za

mjerenje gustoće, METTLER TOLEDO Densito 30PX. Za svaki uzorak mjerenje je

ponovljeno tri puta te je određena srednja vrijednost gustoće za svaki uzorak.

Određivanje viskoznosti

Mjerenje reoloških svojstava uzoraka provedeno je na rotacijskom viskozimetru, model

DV-III+ Digital Rheometer-Brookfield, primjenom koncentričnog cilindra SC4-27.


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Mjerenje reoloških svojstava svježe pripremljene suspenzije provedeno je u

temperaturnom rasponu 15-55 °C. Za održavanje konstantne temperature suspenzije

tijekom mjerenja sa viskozimetrom korišten je termostat. Mjerenjem je praćena ovisnost

smičnog naprezanja o smičnoj brzini, pri brzini smicanja do 182 s-1

. Iz ove ovisnosti

određen je reološki model ponašanja suspenzije i njezina viskoznost.

Uređaj za određivanje toplinske vodljivosti i koeficijenta temperaturne difuzivnosti -

Transient hot bridge 1 (THB 1)

Koeficijenti toplinske vodljivosti i temperaturne difuzivosti za sve pripravljene uzorke

određeni su na uređaju Transient hot bridge 1 (THB 1) proizvođača Linseis. Ovaj uređaj

koristi trakasti izvor topline koji je ujedno i temperaturni senzor. Pripravljenim

suspenzijama određen je koeficijent toplinske vodljivosti. Dok su za određivanje

koeficijenta temperaturne vodljivosti, suspenzije gelirane, dodatkom 0,5 mas % agara.

Pločasti izmjenjivač topline

Kako bi se odredio utjecaj hidrodinamičkih uvjeta na strani toplog fluida (pripravljene

nanosuspenzije) korišten je pločasti izmjenjivač topline SWEP E5x12. Aparatura (slika 1)

se sastojala od pločastog izmjenjivača topline, spremnika sa toplim fluidom (nanofluid),

peristaltičke pumpe za transport toplog fluida, temperaturnih osjetila za mjerenje ulaznih i

izlaznih temperatura toplog i hladnog fluida te rotametra za određivanje protoka vode

(hladni fluid) koji je reguliran ventilom. Ulazna temperatura toplog fluida iznosila je

oko 50 °C, a hladnog oko 16 °C. Protok toplog fluida mijenjan je u intervalu od 40

do 370 ml·min-1

, dok je protok hladne vode bio stalan: 455 ml·min-1

. Praćenjem izlaznih

temperatura i korištenjem jednadžbi materijalne i bilancne topline, određen je koeficijent

toplinske vodljivosti.

Slika 1. Shematski prikaz aparature za određivanje koeficijenta prijelaza topline

Fig. 1. Scheme of apparatus for determination of heat transfer coefficient

TI TI

TI

TI

MFC

FC

TC


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Iz toplinske bilance izmjenjivača topline izračunata je toplinska dužnost izmjenjivača za

topli i hladni fluid:

12 TTcmQ p (1)

Kako je topli fluid, fluid s minimalnom toplinskom vrijednošću, toplinski tok toplog fluida

uzima se dalje u proračunu za koeficijent prolaza topline, K:

LMTFA

QK

(2)

Faktor korekcije za pločasti izmjenjivač topline iznosi oko 0,95, a određen je iz

standardiziranih grafičkih prikaza za pločaste izmjenjivače prema literaturi (Beer, 1994).

Poznavanjem koeficijenta prijelaza topline na strani hladnog fluida, moguće je odrediti

koeficijent prijelaza topline na strani nanofluida:

s

s

V

nf lK

11

1 (3)

Kako bi se, iz niza literaturno ponuđenih, odabrala korelacijska jednadžba za procjenu

koeficijenta prijelaza topline na strani vode, napravljeni su dodatni eksperimenti koji

uključuje izmjenu topline između tople i hlade vode. Iz dobivenih eksperimentalnih

podataka i procjenjenog koeficijenta prijelaza topline izračunata je potrebna površina

izmjene topline. Slaganje između izračunate i stvarne površine korištenog izmjenjivača

topline, ukazuje na odabir korelacijske jednadžbe. Odabrana je korelacijska jednadžba

prema Bounapaneu:

4,066,0 PrRe247,0 ed

(4)


Kako bi se istražila mogućnost poboljšanja prijenosa topline korištenjem nanofluida,

pripravljene su suspenzije različitih koncentracija aluminij (III) oksida u dvije bazne

otopine: etilen glikola i vode u omjeru 50:50 te vode i glicerola u omjeru 80:20. Nakon


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raspršenja nanočestica, ispitana je stabilnost dobivenih suspenzija te su određena njihova

termo fizikalna svojstva. Dispergirani volumni udio čestica u uzorcima kretao se od 0.3 do

1.4%. Dio pripravljenih otopina korišten je u pločastom izmjenjivaču topline kako bi se

odredio utjecaj koncentracije nanočestica u suspenziji i hidrdinamičkih svojstava na

koeficijent prijelaza topline.

Stabilnost suspenzija praćena je promjenom nefelometrijskih jedinica turbiditeta kroz 20

do 50 dana. Na slikama 2 i 3 može se uočiti manja promjena broja nefelometrijskih

jedinica s vremenom za manje koncentracije nanočestica bez obzira na korištenu smjesu

kapljevina. Naime, ukoliko dolazi do sedimentacije čvrstih čestica u kapljevini, broj

nefelometrijskih jedinica će se smanjivati ili povećavati, ovisno o tome da li svjetlost

prolazi kroz bistru ili ugušćenu sedimentacijsku zonu. Najveća nestabilnost nanosuspenzije

može se uočiti (najveća promjena broja nefelometrijskih jedinica) pri najvećim

koncentracijama nanočestica (=1,4%). Usporedbom dviju suspenzija različitih baza

kapljevina, može se zaključiti da je stabilnija suspenzija pripravljena u smjesi kapljevina

glicerola i vode bez obzira na njezinu manju viskoznost i gustoću. Razlog mogu biti veće

molekule bazne kapljevine.

Slika 2. Promjena broja jedinica zamućenja s vremenom za nanosuspenzije

dobivene u smjesi voda-etilen glikol

Fig. 2. Dependency of nephelometric turbidity units vs time for nanosuspenzions

in a mixture of water and ethylene glycol

y = 0,4376x + 350,91

y = 0,5469x + 283,97

y = 0,4216x + 221,73

y = -2,7424x + 217,33

0

50

100

150

200

250

300

350

400

450

500

0 10 20 30 40 50

b,N

TU

t , dan

V-EG

0,30%

0,70%

1,00%

1,40%


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Slika 3. Promjena broja jedinica zamućenja s vremenom za nanosuspenzije

dobivene u smjesi voda-glicerol

Fig. 3. Dependency of nephelometric turbidity units vs time for nanosuspenzions

in water glycerol mixture

Iz ovisnosti smičnog naprezanja o brzini smicanja određuje reološko ponašanje suspenzija.

Reološki dijagrami određeni su za sve pripremljene suspenzije u rasponu temperatura: 15 -

55 °C (slika 4). Sve ispitane suspenzije pokazuju linearnu ovisnost smičnog naprezanja o

smičnoj brzini što ukazuje da se radi o newtonskim fluidima. Na slici 4 vidljivo je da se

viskoznost smanjuje s povećanjem temperature i smanjenjem koncentracije nanočestica u

suspenziji. Odstupanja su vidljiva u slučaju najviših temperature za baznu kapljevinu s

glicerolom. Do ovog odstupanja dolazi u području niskih viskoznosti nanofluida kada dolazi

do većih odstupanja u mjerenju smičnog naprezanja. Slika 5 pokazuje promjenu viskoznosti

nanofluida s volumnim udjelom nanočestica pri 35 °C za obje bazne kapljevine. Uočava se

(tablica 2) manja promjena viskoznosti u odnosu na baznu smjesu kapljevina za nanofluid

dobiven raspršivanjem nanočestica aluminij (III) oksida u smjesi kapljevina glicerol –voda

(do 1,53 puta, odnosno 1,71 puta za nanofluid dobiven u smjesi etilen glikol -voda).

Tablica 2. Odnos termo fizikalnih svojstava nanofluida i baznog fluida

Table 2. Ratio of thermophysical properties of nano and base fluids

, %

50EG:50V 20G:80V

nf/f nff nf/f nf/ f nff nf/f

0,3 1,14 1,007 0,996 1,07 1,004 1,019

0,7 1,22 1,016 1,000 1,20 1,021 1,026

1,0 1,44 1,025 1,018 1,64 1,038 1,040

1,4 1,53 1,031 1,030 1,71 1,041 1,049

y = 0,1915x + 372,96

y = 0,6197x + 235,91

y = 0,6107x + 191,65

y = -0,3193x + 192,32

0

50

100

150

200

250

300

350

400

450

500

0 10 20 30 40 50

b. N

TU

t , dan

V-Gly

0,003

0,007

0,01

0,014


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Slika 4. Promjena viskoznosti nanofluida pri različitim volumnim udjelima aluminij (III) oksida

(0,3; 0,7; 1,0; 1,4 vol% ) sa temperaturom

Fig. 4. Viscosity change of nanofluids at different volume fraction of aluminium oxide

(0,3; 0,7; 1,0; 1,4 vol%) with temperature

Slika 5. Promjena viskoznosti pripremljenih nanofluida s volumnim udjelom nanočestica pri 35 °C

Fig. 5. Viscosity variation of nanofluids with different volume fraction of nanoparticles at 35 °C

0

0,001

0,002

0,003

0,004

0,005

0,006

0,007

0,008

0 10 20 30 40 50 60

, P

as

T, °C

50EG:50V

0,30%

0,70%

1,00%

1,40%

20G:80V

0.3%

0,70%

1,0%

1.4%

0

0,0005

0,001

0,0015

0,002

0,0025

0,003

0,0035

0,004

0,0045

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6

, P

as

, %

20G:80V

50EG:50V


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Povećanjem volumnog postotka nanočestica raste i gustoća nanofluida, pri čemu veću

gustoću ima nanofluid s baznim fluidom etilen glikol – voda (slika 6). Bitno je naglasiti da

je u primjeni nanofluida u izmjenjivačima topline poželjno što manje povećanje

viskoznosti i gustoće u odnosu na bazni fluid zbog što manjeg gubitaka energije (pada

tlaka) u sustavu.

Slika 6. Promjena gustoće nanofluida s volumnim udjelom nanočestica

Fig. 6. Density variation of nanofluids with the volume fraction of nanoparticles

Na slici 7 prikazana je promjena koeficijenta toplinske vodljivosti nanosuspenzija ovisno o

volumnom udjelu nanočestica. Povećanjem volumnog udjela nanočestica uglavnom dolazi

do povećanja toplinske vodljivosti. Do odstupanja u vodljivosti pri nižim vrijednostima

udjela nanočestica dolazi zbog mjerne pogreške (točnost instrumenta za određivanje

koeficijenta toplinske vodljivosti u kapljevinama iznosi oko 5%). Nanofluidi pripremljeni

u smjesi kapljevina glicerol-voda imaju veći koeficijent toplinske vodljivosti u odnosu na

drugu korištenu baznu kapljevinu, razlog je veći udio vode čiji je koeficijent toplinske

vodljivosti veći od koeficijenta etilen glikola i glicerola, oko 0,6 W m-1

K-1

. Također može

se vidjeti da je povećanje koeficijenta toplinske vodljivosti veće za nanosuspenzije

dobivene u baznom fluidu glicerol voda (tablica 2).

Na slikama 6-8 koje prikazuju promjenu termo fizikalnih svojstava fluida za različite

koncentracije nanofluida, osim izmjerenih vrijednosti, usporedno su dane izračunate

vrijednosti ovih svojstava. Jednadžbe koje su korištene za izračunavanje termo fizikalnih

svojstava priređenih suspenzija prikazane su u tablici 3.

800

850

900

950

1000

1050

1100

1150

0 0,5 1 1,5

, k

gm

-3

, %

Pak and Cho (1988)

50EG:50V

20G:80V


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Slika 7. Promjena specifičnog toplinskog kapaciteta nanosuspenzija ovisno

o volumnom udjelu nanočestica

Fig. 7. Variation of the specific heat capacity with the volume fraction of nanoparticles

Slika 8. Koeficijent toplinske vodljivosti nanosuspenzija u ovisnosti

o volumnom udjelu nanočestica

Fig. 8. Dependence of the heat transfer coefficient of nanosuspensions

on the volume fraction of nanoparticles

0

0,1

0,2

0,3

0,4

0,5

0,6

0 0,5 1 1,5

, W

m-1

K-1

, %

Wasp

20G:80V

50EG:50V

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 0,5 1 1,5

c p, J

kg

-1K

-1

, %

Xuan and Roetzel (2000)

50EG:50V

20G:80V


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Tablica 3. Jednadžbe korištene za određivanje termo fizikalnih svojstava nanofluida

Table 3. Equations used for determination thermophysical properties of nanofluids

Autori Jednadžba

Gustoća, Pak i Cho (1998) fpnf 1

Vodljivost, Wang (1999) f

ffp

ffp

nfp

p

2

22

Spec. topl. kapacitet, Li

iXuan (2000)

fpppnfp ccc 1)(

Bolje slaganje s eksperimentalno određenim podacima pokazuje suspenzija s etilen

glikolom. Vjerojatan razlog je postignuto bolje raspršenje nanočestica te bolja stabilnost

suspenzije. Veće odstupanje za obje suspenzije vidljivo je na slici 8 na kojoj je prikazana

usporedba izmjerenog i izračunatog specifičnog toplinskog kapaciteta za sve

nanosuspenzije. Do ove pogreške dolazi zbog metode koja se koristila pri određivanju

koeficijenta temperaturne vodljivosti. Kako bi se izbjegla konvekcija u kapljevini prilikom

određivanja koeficijenta temperaturne vodljivosti, sve suspenzije su gelirane, određen im

je koeficijent toplinske vodljivosti, te izračunat specifični toplinski kapacitet:

ac p

(5)

Iako dolazi do odstupanja od vrijednosti specifičnog toplinskog kapaciteta izračunatih

jednadžbom koju su predložili Xuan i Roetzel (1999), odstupanja su znatno manja nego s

podacima dobivenim mjerenjima u kapljevitoj fazi. Također, višestrukim mjerenjima u

geliranim uzorcima, dokazana je reproducibilnost.

Utjecaj hidrodinamičkih uvjeta i volumnog udjela nanočestica na prijenos topline u pločastom

izmjenjivaču topline, prikazani su na slikama 9 i 10. Također su predložene korelacijske

jednadžbe koje su karakteristične za različite volumne udjele nanočestica. Iz slika 9 i 10

vidljivo je da je eksponent m približno jednak 1 (što ukazuje na linearno povećanje koeficijenta

prijelaza topline s poboljšanjem hidrodinamičkih uvjeta), dok se k mijenja ovisno o povećanju

koncentracije nanočestica. Naime, na vrijednost k utječu fizikalna svojstva fluida koja se

mijenjaju za različite nanofluide, što je vidljivo iz dobivene ovisnosti. Broj m u korelacijskoj

jednadžbi predstavlja utjecaj hidrodinamičkih uvjeta na prijenos topline te je očekivano da se

ne mijenja s promjenom volumnog udjela nanočestica u suspenziji. Iako se pokretačka sila

povećava s povećanjem koncentracije nanočestica u fluidu, ipak dolazi do smanjene količine

topline koja se izmjenjuje između dva fluida. Razlog je promjena termo fizikalnih svojstava

pripremljenih suspenzija. Najveći utjecaj ima smanjenje vrijednosti specifičnog toplinskog


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kapaciteta što direktno utječe na tok topline. Nadalje, utjecaj nanočestica pri prijenosu topline

može ovisiti o čitavom nizu parametara, kao što su: broj čestica, njihov oblik i veličina te

sklonost aglomeraciji (Sarafraz i Peyghambarzadeh, 2012). Iako postoji veliki broj istraživanja

na ovom području, može se zaključiti da još uvije nije moguće donositi generalne zaključke o

toplinskim svojstvima i ponašanju nanofluida.

Slika 9. Ovisnost Nusseltove o Reynldsovoj značajci (Nu/Re) za različite volumne udjele

nanočestica suspendiranih u vodi i etilen glikolu

Fig. 9. Dependence of Nusselt on Reynolds number (Nu/Re) for different volume fractions of

nanoparticles suspended in water and ethylene glycol mixture

Slika 10. Ovisnost Nusseltove o Reynldsovoj značajci (Nu/Re) za različite .volumne udjele

nanočestica suspendiranih u vodi i glicerolu

Fig. 10. Dependence of Nusselt on Reynolds number (Nu/Re) for different volume fractions of

nanoparticles suspended in mixture water and glycerol

y = 0,55x1,10

y = 0,54x1,14

y = 0,52x0,91

y = 0,40x1,09

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0 2 4 6 8 10

Nu

/Pr0

,3

Re

V-EG

0,3%

0,7%

1,4%

y = 0,15x1,08

y = 0,12x1,03

y = 0,10x0,77

0

0,5

1

1,5

2

2,5

3

3,5

4

0 5 10 15 20 25

Nu

/Pr0

,3

Re

V-Gly

0,30%

1,0%


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Zaključci

Pripravljene su stabilne suspenzije različitih koncentracija aluminij (III) oksida u dvije

bazne otopine: etilen glikola i vode u omjeru 50:50 te vode i glicerola u omjeru 80:20.

Najveću stabilnost pokazuju nanosuspenzije najmanje koncentracije nanočestica.

Stabilnija suspenzija pripravljena je u smjesi kapljevina glicerol i voda.

Sve ispitane suspenzije su newtonski fluidi, čija se viskoznost smanjuje povećanjem

temperature i smanjenjem volumnog udjela nanočestica.

Termo fizikalna svojstva se mijenjaju dodatkom nanočestica aluminij (III) oksida, gustoća

i toplinska vodljivost se povećavaju dok se specifični toplinski kapacitet povećava.

Primjenom pripravljenih nanofluida u pločastom izmjenjivaču topline su definirane

korelacijske jednadžbe.

Zbog promjene termo fizikalnih svojstava pripremljenih suspenzija ne dolazi nužno do

povećanja izmjenjene topline.

Literatura

Beer, E., (1994): Priručnik za dimenzioniranje uređaja kemijske procesne industrije, Zagreb:

HDKI/Kemija u industriji, pp. 227-287.

Li, Q., Xuan, Y., (2000): Experimental Investigation of Transport Properties of Nanofluids. In: Heat

Transfer Science and Technology, Wang, B. (Ed.). Higher Education Press, China, pp: 757-784.

Pak, B. C., Cho, Y. I. (1998): Hydrodynamic and heat transfer study of dispersed fluids with

submicron metallic oxide particles, Exp. Heat Trans. 11 (2), 151-170.

Philip J., Shima P.D., (2012): Thermal Properties of Nanofluids, Advances in Colloid and Interface sci.

183-184.

Sarafraz, M.M., Peyghambarzadeh, S.M. (2012): Nucleate Pool Boiling Heat Transfer to Al2O3-

Water and TiO2-Water Nanofluids on Horizontal Smooth Tubes with Dissimilar Homogeneous

Materials, Chem. Biochem. Eng. Q. 26 (3), 199-206.

Shima, P. D. Philip, J. (2014): Role of Thermal Conductivity of Dispersed Nanoparticles on Heat

Transfer Properties of Nanofluid, Ind. Eng. Chem. Res., 53 (2), 980–988.

Wang, X., Xu, X., Choi, S. U. S., (1999): Thermal Conductivity of Nanoparticle - Fluid Mixture, J.

of Thermophy. and Heat Trans. 13 (4), 474-480.

Wang, X.Q., Mujumdar, A. S. (2007): Heat transfer characteristics of nanofluids: a review, Int. J. of

Therm. Sci. 46 (1), 1 1-19.

Xuan, Y., Roetzel, W., (2000): Conceptions for Heat Transfer Corelation of Nanofluids, Int. J. Of

Heat and Mass Transfer 43 (19), 3701-3707.

Yu, W., Xie, H., (2012): A review on Nanofluids: Preparation, Stability Mechanisms, and

Applications, J. of Nanomaterials 2012 (1), 1-12.

Zhang, X., Gu, H., Fujii, M., (2006): Experimental Study on the Effective Thermal Conductivity

and Thermal Diffusivity of Nanofluids, Int. J. Thermophysics 27, 569-580.


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Simboli

A površina prijenosa topline, m2

a koeficijent temperaturne vodljivosti, m2·s

-1

b broj jedinica zamućenja, NTU

cp specifični toplinski kapacitet, J·kg-1

·K-1

)

F Foulingov faktor, -

K ukupni koeficijent prolaza topline, W·m-2

·K-1

m maseni protok, kg/s

Q toplinski fluks, J/s

T temperatura, °C

t vrijeme, s

koeficijent prijelaza topline, kJ/m2

volumni udio,-

dinamička viskoznost, Pas

koeficijent toplinske vodljivosti stjenke, W/mK

gustoća, kg/m3

Indeksi

p čestica

nf nanofluidi

f fluid


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Rheological and thermal characterization of nanofluids




Summary

In order to arise efficiently of heat exchangers, it is necessary to improve thermal properties of

nanofluids. Improvements in thermal properties of conventional fluids lead to increasing of heat

exchangers efficiency. By applying of ultrasound equipment, nanoparticles of aluminum (III) oxide

in different volume concentration from 0.3 to 1.4 % were dispersed in mixtures of water – ethylene

glycol and water – glycerol. Nefelometric method was applied in order to exam stability of

suspensions by using handmade device 3 in 1 – FTN. After attaining stability, viscosity and

rheological behavior of the suspensions were determined. Also, some thermal properties (coefficient

of thermal conductivity, specific thermal capacity and thermal diffusivity) were measured. In order

to evaluate obtained results, the measured values of thermo physical properties were compared with

those from the literature. Although nanofluids were prepared without additives, they show stability

over period of one month. Also they show Newton rheological behavior with slightly increasing in

density and viscosity comparing to base fluids. Thermal properties of nanofluids are improved with

increasing of nanoparticles volume concentration. Heat and overall heat transfer coefficients were

calculated for different concentrations and flow rates of nanofluid in the plate heat exchanger. Heat

transfer coefficient and overall heat transfer coefficient are increased by increasing of volume

fractions of nanofluids and with enhanced of hydrodynamic conditions in the plate heat exchanger.

Keywords: nanofluid, thermo – physical properties of nanofluids, plate heat exchanger


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Ravnoteža kapljevina–kapljevina u sustavu

ugljikovodik – piridin – C6mmpyTf2N

UDC: 66.061.14



Hrvatska

Sažetak

Ekstrakcija kapljevina–kapljevina jedna je od alternativnih metoda koje se mogu primijeniti za

denitrifikaciju FCC benzina i lakog plinskog ulja. Korištenjem ionskih kapljevina kao selektivnog

otapala proces ekstrakcije postaje ekološki prihvatljiv zbog njihove nehlapljivosti i mogućnosti

potpune regeneracije. U ovom je radu eksperimentalno određena ravnoteža kapljevina–kapljevina u

sustavima ugljikovodik – piridin – C6mmpyTf2N pri atmosferskom tlaku i temperaturi od 25 °C.

Odabrana su tri parafinska (n-heksan, n-heptan i i-oktan) i jedan aromatski ugljikovodik (toluen),

budući da FCC-benzin u najvećoj mjeri sadrži parafine. Toluen je odabran jer se aromatski

ugljikovodici u određenoj mjeri otapaju u ionskim kapljevinama, a piridin je predstavnik dušikovih

spojeva. Osim ravnotežnih krivulja, eksperimentalno su određene i vezne linije te su izračunate

vrijednosti koeficijenta raspodjele i selektivnosti odabrane ionske kapljevine. Selektivnost ionske

kapljevine i koeficijent raspodjele piridina opadaju s porastom koncentracije piridina u smjesi.

Ravnotežni podaci opisani su modelima NRTL i UNIQUAC. Istražen je utjecaj pojedinog

ugljikovodika te sastava pojne smjese na djelotvornost ekstrakcije piridina odabranom ionskom

kapljevinom. Djelotvornost ekstrakcije veća je u trokomponentnim sustavima s parafinskim

ugljikovodicima. Za modelnu otopinu koja se sastoji od svih korištenih komponenti uz dodatak

tiofena, uočen je pad djelotvornosti ekstrakcije piridina u odnosu na trokomponentne sustave.

Ključne riječi: ravnoteža kapljevina–kapljevina, ionske kapljevine, denitrifikacija, FCC-benzin, piridin

Uvod

Zbog sve strožih zahtjeva vezanih uz proizvodnju motornih goriva vrlo male koncentracije

sumpora, mnoga se istraživanja bave razvojem novih i poboljšanjem postojećih procesa

desulfurizacije i denitrifikacije naftnih frakcija (Song, 2003). Naime, komercijalni proces

hidrodesulfurizacije (HDS) ekonomski je i ekološki nepovoljan jer se odvija u uvjetima

visokih temperatura i tlakova uz potrošnju velikih količina vodika i katalizatora. Osim

toga, tijekom procesa desulfurizacije istovremeno se odvija i denitrifikacija. Kako je

proces denitrifikacije znatno sporiji od desulfurizacije, dušikovi se spojevi dulje vrijeme



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zadržavaju na katalizatoru što za posljedicu ima otežanu konverziju sumporovih spojeva u

sumporovodik (Martínez-Palou, 2011). Stoga se nameće mogućnost nadogradnje

komercijalnog HDS procesa ekstrakcijskom denitrifikacijom pomoću odgovarajućeg

otapala. U zadnje se vrijeme sve više istražuju ionske kapljevine kao otapala s primjenom

u separacijskim procesima. Ionske se kapljevine mogu dizajnirati za željenu svrhu

pravilnim odabirom kationa i aniona. Mogućnost njihove primjene u ekstrakciji

kapljevina–kapljevina leži u svojstvima kao što su: otapanje različitih vrsta tvari,

zanemariv tlak para, kemijska i toplinska stabilnost te jednostavna regeneracija i ponovno

korištenje. Pregledom dostupne literature, uočeno je da većina ionskih kapljevina ima veći

afinitet prema dušikovim spojevima u odnosu na sumporove spojeve (Casal, 2010, Gabrić

et al., 2013, Rogošić et al., 2014). Ukoliko se ekstrakcija provede prije HDS-a, doći će do

djelomične desulfurizacije i denitrifikacije čime će se povećati djelotvornost HDS procesa.

U ovom su radu eksperimentalno i računski određene fazne ravnoteže kapljevina–

kapljevina u odabranim modelnim trokomponentnim sustavima ugljikovodik (1) –

piridin (2) – IL. Osim toga, istražena je separacija piridina i ugljikovodika, odnosno

smjese ugljikovodika ekstrakcijom s odabranom ionskom kapljevinom, s ciljem

donošenja širih zaključaka o mogućnosti denitrifikacije benzinskih frakcija pomoću

ionskih kapljevina.

Materijali i metode

Eksperimentalno je određena ravnoteža trokomponentnih sustava ugljikovodik (1) – piridin

(2) – [C6mmPy][Tf2N] (3), gdje je komponenta 3 ionska kapljevina 1-heksil-3,5-

dimetilpiridinijev bis{(trifluorometilsulfonil)imid}. Odabrani ugljikovodici su: n-heksan,

n-heptan, i-oktan i toluen. Mjerenja su provedena u termostatiranoj zračnoj kupelji pri

25 °C te pri atmosferskom tlaku, p =101325 Pa. Osim toga, provedena je i šaržna

ekstrakcija uz maseni omjer ionske kapljevine prema modelnoj otopini od 0,25 kg/kg, pri

čemu su kao modelne otopine korištene dvokomponentne otopine ugljikovodik – piridin, te

jedna šesterokomponentna otopina sastava: n-heksan (26 % mas.), n-heptan (26 % mas.),

i-oktan (26 % mas.), toluen (10 % mas.), piridin (6 % mas.) i tiofen (6 % mas). Korištena

ionska kapljevina sintetizirana je prema postupku detaljno opisanom u literaturi

(Papaiconomou et al., 2006) i karakterizirana je 1H NMR spektroskopijom (FT NMR

Bruker Avance 300 MHz). Spektri su snimljeni u deuteriranom dimetil-sulfoksidu

(DMSO-d6) uz dodatak tetrametilsilana (TMS) kao unutarnjeg standarda.

Određivanje binodalne krivulje

Točke na binodalnoj krivulji određene su starom, ali djelotvornom titracijskom metodom

prema Othmeru (1941). U definiranu masu ugljikovodika dodavana je ionska kapljevina


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kap po kap do pojave zamućenja. Zatim je dodan piridin do bistrenja otopine. Otopina je

nakon toga titrirana ionskom kapljevinom ponovno do pojave zamućenja. Na analogan je

način određena i druga strana binodalne krivulje, naizmjeničnim dodavanjem piridina i

ugljikovodika u ionsku kapljevinu. Sve su koncentracije pritom određivane gravimetrijski;

tikvice sa smjesama vagane su nakon dodatka pojedine komponente (vaga: Kern ALJ 220-

4 NM preciznosti ± 0,0001 g).

Određivanje veznih linija

Za određivanje ravnotežnih sastava faza pripravljene su dvofazne trokomponentne smjese

definiranog sastava. Smjese su miješane 24 sata u termostatiranoj zračnoj kupelji. Nakon

separacije faza izmjereni su indeksi loma rafinatne faze. S obzirom da vezna linija povezuje

točku ukupnog sastava pripravljene dvofazne smjese i točku sastava rafinatne faze,

koncentracije u ekstraktnoj fazi očitane su sa sjecišta vezne linije i ekstraktne grane

binodalne krivulje. Ovisnosti indeksa loma rafinatne faze, nD,25, o masenom udjelu piridina,

w2I, pri 25 °C, određene su prethodnim eksperimentom i dane su sljedećim izrazima:

n-heksan – piridin: I 2

2 D,25 D,2528,89 89,24 68,04w n n (1)

n-heptan – piridin: I 2

2 D,25 D,2555,15 165,57 123,52w n n (2)

i-oktan – piridin: I 2

2 D,25 D,2555,93 168,04 125,50w n n (3)

toluen – piridin: I

2 D,2599,65 148,86w n (4)

Svi indeksi loma određeni su na Abbeovom refraktrometru Model RMI, Exacta Optech,

preciznosti ± 0,0001 u triplikatu.

Ekstrakcija

Ekstrakcija kapljevina–kapljevina provedena je u šaržnom ekstraktoru (unutarnji promjer

0,04 m) s magnetskim miješalom. Pripravljene su modelne otopine željenog sastava te je

dodana ionska kapljevina kako bi maseni omjer ionska kapljevina/modelna otopina bio

0,25. Vrijeme trajanja ekstrakcije bilo je 20 minuta. Nakon separacije faza određen je

sastav rafinatne faze. Za dvokomponentne modelne otopine sastav je određen mjerenjem

indeksa loma, prema već opisanom postupku, a za višekomponentnu modelnu otopinu

metodom plinske kromatografije. Plinski kromatograf, GC-2014-Shimadzu, opremljen je

uređajem za automatsko uzorkovanje, plameno-ionizacijskim detektorom te tzv. fused

silica kapilarnom kolonom CBP1-S25-050 duljine 25 m i unutarnjeg promjera 0,32 mm.


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Ravnoteža kapljevina–kapljevina

Eksperimentalno određene fazne ravnoteže kapljevina-kapljevina dvofaznih

trokomponentnih sustava ugljikovodik (1) – piridin (2) – [C6mmPy][Tf2N] (3) prikazane su

na slikama 1-4. Prikazane su binodalne krivulje (dobivene interpolacijom eksperimentalnih

točaka, te vezne linije, zajedno s ravnotežnim sastavima faza.

Od istraživanih ugljikovodika samo za sustav s n-heksanom postoje literaturni podaci

(Casal, 2010). Oba mjerenja kvalitativno opisuju sustav na sličan način, a primijećeno

manje kvantitativno neslaganje s literaturnim podacima vjerojatno je posljedica različite

čistoće primijenjenih kemikalija, jer se oba skupa podataka čine interno konzistentnima.

Slika 1. Ravnoteža kapljevina–kapljevina u trokomponentnom sustavu n-heksan – piridin –

[C6mmPy][Tf2N]; usporedba eksperimentalnih i modelnih veznih linija

Fig. 1. Liquid–liquid equilibria in the three-component system n-hexane – pyridine –

[C6mmPy][Tf2N]; comparison of experimental and model tie lines


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Slika 2. Ravnoteža kapljevina–kapljevina u trokomponentnom sustavu n-heptan – piridin –


Fig. 2. Liquid–liquid equilibria in the three-component system n-heptane – pyridine –


Slika 3. Ravnoteža kapljevina–kapljevina u trokomponentnom sustavu i-oktan – piridin –


Fig. 3. Liquid–liquid equilibria in the three-component system i-octane – pyridine –



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Slika 4. Ravnoteža kapljevina–kapljevina u trokomponentnom sustavu toluen – piridin –


Fig. 4. Liquid–liquid equilibria in the three-component system toluene – pyridine –


Modeliranje

Za opis ravnoteže kapljevina–kapljevina u sustavima s ionskim kapljevinama uobičajeno

se koriste modeli NRTL (Renon, 1968) i UNIQUAC (Abrams, 1975). Model NRTL uzima

u obzir promjenjivost mjesnih koncentracija kao posljedicu razlika Gibbsovih energija

međudjelovanja istovrsnih i raznovrsnih čestica. Model je troparametarski, ij i ji su

parametri međudjelovanja, a treći parametar ij = ji je tzv. parametar neslučajnosti koji se

povezuje uglavnom s entropijskim efektima razlike veličina molekula u sustavu. Pri

modeliranju, parametar neslučajnosti obično se fiksira na iskustvenu vrijednost (u ovom

radu ij = 0,3), a regresijom se određuju interakcijski parametri. Model UNIQUAC daje

eksces Gibbsovu energiju kao zbroj dvaju doprinosa. Rezidualni doprinos opisuje razliku

međudjelovanja istovrsnih i raznovrsnih čestica, kao i kod NRTL-a; oznake parametara su

iste (ij i ji), ali su iznosi (uključujući i red veličine) sasvim različiti. Kombinatorni

doprinos opisuje razliku volumena i površina molekula u sustavu na osnovi prethodno

izračunatih i tabeliranih strukturno-grupnih doprinosa, koji se pak računaju iz van der

Waalsovih radijusa atoma te duljina kovalentnih veza. Volumni, ri, i površinski parametri, qi,

niskomolekulskih komponenata (ugljikovodici, piridin) preuzeti su iz literature (Prausnitz,


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1980), a parametri za ionsku kapljevinu izračunati su na temelju vrijednosti strukturno

grupnih parametara što ju je priredio Lei (2009). Parametri su okupljeni u tablici 1.

Tablica 1. Volumni (ri) i površinski (qi) parametri modela UNIQUAC

Table 1. Volume (ri) and surface (qi) parameters of the UNIQUAC model

r q

n-heksan 4,4998 3,856

n-heptan 5,1742 4,3960

i-oktan 5,8463 5,0080

toluen 3,9228 2,968

piridin 2,9993 2,113

C6mmpyTf2N 11,8526 10,0659

Interakcijski parametri obiju modela određeni su iz eksperimentalnih podataka

modifikacijom metode po Sorensenu i Arltu (Sorensen, 1979). U prvom se koraku

postupka traže parametri ij koji daju minimum funkcije:

d c

2I I II II

2 2 2 2 2 2

1 12 21 13 31 23 32I I II II1 1

n n

i i i i

j i i i i i j

x xOF Q

x x

. (5)

U brojniku prvog člana na desnoj strani jednadžbe prepoznaje se osnovni fizički smisao

jednadžbe. Jednadžba fazne ravnoteže kapljevina–kapljevina može se, naime, pisati kao

jednakost aktivnosti svih komponenata u svima prisutnim fazama: aiI = ai

II ili

(xii)I = (xii)

II. ai je aktivnost, i koeficijent aktivnosti a xi molarni udio i-te komponente. I i

II označavaju dvije kapljevite faze, j označava svaku pojedinu veznu liniju. nc = 3 je broj

komponenata, nd je broj eksperimentalnih veznih linija u sustavu. Drugi član jednadžbe na

desnoj strani je tzv. kaznena funkcija koja ograničava pojavu fizikalno besmislenih

minimuma funkcije u području (pre)velikih ij. Za oba je modela korištena iskustvena

vrijednost „kaznenoga“ parametra od Q = 110-6

.

U drugom, odnosno trećem koraku, koristeći konačne ij iz prvog koraka za inicijalizaciju,

traže se minimumi funkcija:

d c II 2

2 2 2 2 2 2

2 12 21 13 31 23 32exp mod1 1 I

n np p

i ijj i p

OF x x Q

, (6)

d c II 2

2 2 2 2 2 2

3 12 21 13 31 23 32exp mod1 1 I

n np p

i ijj i p

OF w w Q

. (7)


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Funkcije se razlikuju jedino u tome što se u drugom koraku nastoji postići najbolje

slaganje molarnih, a u trećemu masenih udjela, wi, komponenata, izračunatih prema

modelu (mod) u odnosu na eksperimentalne vrijednosti (exp). Parametri iz drugog služe za

inicijaciju u trećem koraku. p ovdje simbolički označava kapljevite faze I i II. Kazneni

parametar je Q = 110-10

za oba modela.

Svih šest interakcijskih parametara u modelima određeni su simultano. Optimalni su

parametri prikazani u tablici 2, zajedno sa srednjim kvadratnim odstupanjem modelnih i

eksperimentalnih masenih udjela komponenata:

2 2 2 2 2 2

3 12 21 13 31 23 32

d c 2

OF QA

n n

(8)

U tablici 2 prikazani su interakcijski parametri modela NRTL i UNIQUAC, zajedno s

pripadajućim vrijednostima srednjeg kvadratnog odstupanja za sve istraživane sustave.

Vezne linije izračunate modelima uspoređene su s eksperimentalnima na slikama 1-4.

Tablica 2. Interakcijski parametri modela NRTL i UNIQUAC i srednja kvadratna odstupanja (A)

eksperimentalnih i modelnih ravnotežnih sastava

Table 2. Interaction parameters of the NRTL and UNIQUAC models as well as mean square

deviations (A) of experimental and model equilibrium compositions

NRTL

12. 13. 23 =

0.3; 0.3; 0.3

12 13 21 23 31 32 A

n-heksan (1) – piridin (2) – IL (3)

1,2504 10,2784 0,9255 4,7923 2,7222 -2,8898 0,0040

n-heptan (1) –

piridin (2) – IL (3) 1,0673 13,1706 1,1736 5,1122 3,6390 -2,7404 0,0161

i-oktan (1) –

piridin (2) – IL (3) 0,4507 12,0771 0,9196 4,9368 3,4115 -3,8991 0,0092

toluen (1) –

piridin (2) – IL (3) 1,8554 14,0654 1,1988 1,3897 0,5684 -1,2534 0,0014

UNIQUAC 12 13 21 23 31 32 A

n-heksan (1) –

piridin (2) – IL (3) 0,5109 0,3081 1,1539 1,6341 1,1468 1,1227 0,0028

n-heptan (1) – piridin (2) – IL (3)

0,6907 0,3530 0,9648 1,5724 1,2140 1,2576 0,0042

i-oktan (1) –

piridin (2) – IL (3) 0,4855 0,3406 1,1954 1,4842 1,2399 1,0932 0,0031

toluen (1) –

piridin (2) – IL (3) 1,3556 0,0260 2,3717 1,0000 0,2671 2,8438 0,0013


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Koeficijenti raspodjele i selektivnost

Ravnotežni dijagrami trokomponentnih sustava ugljikovodik – piridin – [C6mmPy][Tf2N]

su tipa I s pozitivnim nagibom veznih linija u cijelom području koncentracija ispod

binodalne krivulje. Ionska kapljevina netopljiva je u svim istraživanim ugljikovodicima.

Parafinski ugljikovodici u maloj su mjeri topljivi u ionskoj kapljevini, dok je toluen, kao

predstavnik aromatskih ugljikovodika, topljiv u znatnoj mjeri. To drugim riječima znači da

je odabrana ionska kapljevina pogodna za separaciju piridina iz smjese s parafinskim

ugljikovodicima. Ravnotežni dijagrami sustava s parafinskim ugljikovodicima međusobno

su vrlo slični. Sustav s toluenom ima nepovoljnu ravnotežu s vrlo malim heterogenim

područjem u kojem je moguće provesti ekstrakciju.

Koeficijenti raspodjele, i, i selektivnost ionske kapljevine, S, izračunati su korištenjem

sljedećih izraza: II

11 I

1

w

w , (9)

II

22 I

2

w

w , (10)

1

2

S , (11)

i prikazani u tablici 3. Oznake I i II odgovaraju rafinatnoj, odnosno ekstraktnoj fazi, a

oznake 1 i 2 odgovaraju ugljikovodiku, odnosno piridinu.

Tablica 3. Koeficijenti raspodjele () i selektivnost (S) ionske kapljevine

Table 3. Distribution coefficients () and selectivities (S) of ionic liquid

Ugljikovodik/hydrocarbon (1) – piridin (2) – [C6mmPy][Tf2N] (3)

n-heksan n-heptan

1 2 S 1 2 S

0,0646 2,9287 45,36 0,0866 3,2002 36,95

0,0934 2,6202 28,06 0,1060 2,3846 22,49

0,1231 2,0656 16,78 0,1265 1,9604 15,49

0,1667 1,8168 10,89 0,1623 1,7109 10,54

0,2976 1,4483 4,87 0,2719 1,4323 5,27

i-oktan toluen

1 2 S 1 2 S

0,0634 2,9271 46,15 0,4855 1,8220 3,75

0,0897 2,5428 28,35 0,4965 1,3450 2,71

0,1251 2,1452 17,15 0,5123 2,1284 4,15

0,1668 1,8606 11,16 0,5248 1,7162 3,27

0,2657 1,5072 5,67


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Koeficijenti raspodjele piridina veći su od koeficijenata raspodjele odgovarajućeg

ugljikovodika. S porastom koncentracije piridina raste koeficijent raspodjele

ugljikovodika, dok koeficijent raspodjele piridina opada. Selektivnost ionske kapljevine

opada s porastom koncentracije piridina u smjesi, jer je sve veća topljivost ugljikovodika u

ionskoj kapljevini. Visoke vrijednosti selektivnosti idu u prilog korištenja odabrane ionske

kapljevine za separaciju piridina od parafinskih ugljikovodika.

Na slikama 5 i 6 prikazana je usporedba eksperimentalnih s literaturnim (Casal, 2010)

koeficijentima raspodjele piridina i selektivnosti ionske kapljevine. Slaganje je razmjerno

dobro.

Slika 5. Ovisnost koeficijenta raspodjele piridina, 2, o masenom udjelu piridina u rafinatnoj fazi,

w2I, za sustav n-heksan – piridin – [C6mmPy][Tf2N]; usporedba eksperimentalnih ()

i literaturnih podataka (, Casal 2010)

Fig. 5. Distribution coefficient of pyridine, 2, vs. mass fraction of pyridine in the raffinate phase,

w2I, for the system n-hexane – pyridine – [C6mmPy][Tf2N]; comparison of experimental ()

and literature data (, Casal 2010)


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Slika 6. Ovisnost selektivnosti, S, o masenom udjelu piridina u rafinatnoj fazi,

w2I, za sustav n-heksan – piridin – [C6mmPy] [Tf2N]; usporedba

eksperimentalnih () i literaturnih podataka (, Casal 2010)

Fig. 6. Selectivity, 2, vs. mass fraction of pyridine in the raffinate phase,

w2I, for the system n-hexane – pyridine – [C6mmPy][Tf2N]; comparison of

experimental () and literature data (, Casal 2010)

Ekstrakcija kapljevina–kapljevina

Kako bi se istražio utjecaj sastava modelne otopine na djelotvornost ekstrakcije piridina

pomoću odabrane ionske kapljevine, eksperimenti su provedeni s dvokomponentnom

[ugljikovodik (94 % mas.) i piridin (~6 % mas.)] i šesterokomponentnom [n-heksan

(26 % mas.), n-heptan (26 % mas.), i-oktan (26 % mas.), toluen (10 % mas.), piridin

(6 % mas.) i tiofen (6 % mas.)] modelnom otopinom. U tablici 4 dani su rezultati za

dvokomponentne modelne otopine.

U eksperimentu sa šesterokomponentnom modelnom otopinom sastav rafinata određen je

plinskom kromatografijom dok je sastav ionske kapljevine analiziran 1H NMR

spektroskopijom. Na 1H NMR spektru ionske kapljevine nakon ekstrakcije uočeni su

dodatni pikovi koji odgovaraju tiofenu, piridinu, toluenu i izooktanu, slika 7.

Djelotvornosti ekstrakcije tiofena, piridina i toluena pritom su redom: 12,05%, 39,11% i

8,97%. Međutim, otapanje i-oktana u ionskoj kapljevini nije se moglo potvrditi plinskom

kromatografijom jer koncentraciju i-oktana u rafinatu nije bilo moguće precizno odrediti

zbog preklapanja pikova n-heptana i i-oktana, tj. zbog njihovih bliskih vrelišta. Ako se


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rezultati sa slike 7 usporede s onima u tablici 4 (za dvokomponentnu modelnu otopinu),

može se uočiti da se kod višekomponentne modelne otopine smanjuje djelotvornost

ekstrakcije svih komponenata. Rezultati vezani uz ekstrakciju ne mogu se sa sigurnošću

usporediti s literaturnim s obzirom da u literaturi (Casal, 2010) nisu navedeni uvjeti

provedbe procesa ekstrakcije, odnosno maseni omjer ionska kapljevina/modelna otopina.

Tablica 4. Djelotvornost ekstrakcije (E) piridina iz smjese s ugljikovodikom pomoću ionske

kapljevine; gornji indeks F označava pojnu smjesu – radi se o početnom udjelu piridina

u smjesi s ugljikovodikom

Table 4. Extraction efficiency (E) of pyridine from its mixture with a hydrocarbon using ionic

liquid; superscript F denotes the feed – it is the initial pyridine mass fraction within its

mixture with a hydrocarbon

otapalo/solvent w2F, %

E, %

piridin otapalo/solvent

n-heksan 5,5 43,80 1,85

n-heptan 5,4 45,62 3,36

izooktan 6,0 61,89 1,86

toluen 6,6 29,89 26,51

Slika 7. 1H NMR spektri ionske kapljevine prije i poslije ekstrakcije

Fig. 7. 1H NMR spectra of ionic liquid before and after extraction

C6mmPyTf2N - nakon ekstrakcije / after extraction

C6mmPyTf2N - čista / pure

piridin

piridin

tiofentoluen

i-oktan

PPM 9,0 8,0 7,0 6,0 5,0 4,0 3,0 2,0 1,0 0,0


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Zaključci

Eksperimentalno su određene fazne ravnoteže kapljevina–kapljevina u sustavima

ugljikovodik (1) – piridin (2) – [C6mmPy][Tf2N] (3). Vezne su linije uspješno korelirane

NRTL i UNIQUAC modelima, iako se nešto bolje slaganje eksperimentalnih računskih

podataka postiglo korištenjem UNIQUAC modela. Mogućnost primjene [C6mmPy][Tf2N]

kao selektivnog otapala za separaciju smjese piridina i ugljikovodika procijenjena je na

temelju eksperimentalnih vrijednosti koeficijenata raspodjele i selektivnosti ionske

kapljevine i potvrđena provedbom ekstrakcije s dvokomponentnim i višekomponentnim

modelnim otopinama. Sastav modelne otopine pritom utječe na djelotvornost separacije

tiofena iz smjese sa ugljikovodicima.

Literatura

Abrams D.S., Prausnitz, J.M. (1975): Statistical thermodynamics of liquid mixtures: A new

expression for the excess Gibbs energy of partly or completely miscible systems, AIChE J. 21,

116-128.

Casal, M.F. (2010): Desulfurization of fuel oils by solvent extraction with ionic liquids, PhD thesis,

University of Santiago de Compostela, Santiago de Compostela, February 2010.

Gabrić, B., Sander, A., Cvjetko-Bubalo, M., Macut, D. (2013): Extraction of S- and N-compounds from

the mixture of hydrocarbons by ionic liquids as selective solvents, Sci. World J. (2013), 1-11.

Lei, Z., Zhang, J., Li, Q., Chen, B. (2009): UNIFAC model for ionic liquids, Ind. Eng. Chem. Res.

48, 2697-2704.

Martínez-Palou, R., Flores Sánchez, P. (2011): Perspectives of Ionic Liquids Applications for Clean

Oilfield Technologies. In: Ionic Liquids: Theory, Properties, New Approaches, InTech, A.K.

(ed), Rijeka, 567-630.

Othmer, D.F., White, R.E., Trueger, E. (1941): Liquid-liquid extraction data, Ind. Eng. Chem. 33,

1240-1248.

Papaiconomou, N., Yakelis, N., Salminen, J., Bergman, R., Prausnitz, J. (2006): Synthesis and

properties of seven ionic liquids containing 1-methyl-3-octylimidazolium or 1-butyl-4-

methylpyridinium cations, J. Chem. Eng. Data, 51 (4), 1389-1393.

Prausnitz, J.M., Anderson, T.F. Grens, E.A., Eckert, C.A., Hsieh R., O´Connell, J.P. (1980):

Computer Calculations for Multicomponent Vapour-liquid and Liquid-liquid Equilibria,

Prentice-Hall, Englewood Cliffs, New Jersey, 1980.

Renon, H., Prausnitz, J.M. (1968): Local composition in thermodynamic excess functions for liquid

mixtures, AIChE J. 14, 135-144.

Rogošić, M., Sander, A., Pantaler, M. (2014): Application of 1-pentyl-3-methylimidazolium

bis(trifluoromethylsulfonyl) imide for desulfurization, denitrification and dearomatization of

FCC gasoline, J. Chem. Thermodyn. 76, 1-15.

Song, C. (2003): An overviev of new-approaches to deep desulfurisation for ultra-clean gasoline.

diesel fuel and jet fuel, Catal. Today 86 (1-4), 211-263.

Sorensen J.M., Arlt, W. (1979): DECHEMA Chemistry Data Series, Vol. V (Liquid-Liquid

Equilibrium), 3 Bände Frankfurt, 1979.


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Liquid-liquid equilibrium for the system

hydrocarbon – pyridine – C6mmpyTf2N




Summary

Liquid–liquid extraction is one of the alternative methods that can be used for denitrification of FCC

gasoline and diesel fuel. Extraction with ionic liquids as selective solvents makes the separation

process ecologically acceptable due to the nonvolatility of ionic liquids as well as their complete

regeneration. Liquid–liquid equilibrium for the systems hydrocarbon – pyridine – C6mmpyTf2N

has been experimentally determined at the atmospheric pressure and 25 °C. Three paraffin

hydrocarbons (n-hexane, n-heptane and i–octane) and one aromatic hydrocarbon (toluene) have

been selected, since FCC gasoline consists mostly of paraffins. Toluene was selected because

aromatic hydrocarbons are soluble in ionic liquids as well, and pyridine is a representative of

nitrogen compounds. Besides the equilibrium curves, the tie lines were determined experimentally

as well. Equilibrium data were described with NRTL and UNIQUAC models. Distribution

coefficients and selectivities of ionic liquid were evaluated. Both of them decrease with increasing

concentration of pyridine. The extraction efficiency is higher for the three-component systems with

paraffins. For the model solution which consists of all selected compounds plus thiophene a

decrease in extraction efficiency was observed with respect to three-component systems.

Keywords: liquid–liquid equilibria, ionic liquids, denitrification, FCC-gasoline, pyridine


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Primjenjivost n-heksadekana pri regeneraciji ionskih kapljevina

UDC: 66.061.14



Hrvatska

Sažetak

U posljednje se vrijeme sve veća pozornost posvećuje istraživanjima moguće primjene ionskih

kapljevina u različitim industrijskim procesima vezanima upravo za nove postupke i zamjenu

mnogih toksičnih organskih otapala. Mogućnost primjene ionskih kapljevina u različitim

industrijskim granama polazi od specifičnih svojstava koja one posjeduju, kao što su nehlapivost,

stabilnost te mnoga druga. Takva svojstva ističu ionske kapljevine kao obećavajuću alternativu

ekološki nepovoljnim hlapivim organskim otapalima. S obzirom da posjeduju mogućnost višestruke

uporabe i regeneracije, u ovom je radu istražena regeneracija različitih ionskih kapljevina, jedne na

bazi piridina, dok su ostale na bazi imidazola, koje su onečišćene u procesu desulfurizacije modelne

otopine koja sastavom oponaša dizelsko gorivo. Regeneracija ionskih kapljevina provedena je

kapljevinskom ekstrakcijom uz pomoć n-heksadekana kao selektivnog otapala, odabranog na

temelju svojih fizikalnih svojstava. Maseni odnos ionske kapljevine i selektivnog otapala iznosio je

0,5. Nuklearnom magnetskom rezonancijom ispitana je čistoća regeneriranih kapljevina te je

utvrđeno da izostaju karakteristični pikovi dibenzotiofena, koji je jedino onečišćivalo u ispitanim

ionskim kapljevinama.

Ključne riječi: ionske kapljevine, n-heksadekan, regeneracija

Uvod

Znanstvena i tehnička istraživanja primjene ionskih kapljevina razvijaju se u posljednjih 20

godina (Feng i sur., 2010). Interes za takva istraživanja postoji zbog izuzetno povoljnih

svojstava ionskih kapljevina, tališta nižeg od 100 °C, nehlapivosti, mogućnosti podešavanja

fizikalnih i kemijskih svojstava odabirom kationa i aniona, stabilnosti te mnogih drugih.

Takva svojstva ističu ionske kapljevine kao obećavajuću alternativu ekološki nepovoljnim

hlapivim organskim otapalima (Volatile Organic Solvents – VOS), osobito kloriranim

ugljikovodicima (Anugwom i sur., 2011; Feng i sur., 2010; Olivier-Bourbigou i Hughes,

2003; Sander, 2012). Do danas postoji značajan broj osvrta (Feng et al, 2010) na istraživanja

ionskih kapljevina, od najranijih koja su usmjerena na katalitičke procese (Seddon, 1997;

Sheldon, 2001; Zhao et al., 2002) pa sve do detaljnih opisa specifične primjene ionskih



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kapljevina u kemiji kompleksa, analitičkoj kemiji (Koel, 2005), polimernim materijalima te u

nanotehnologiji (Antonietti et al., 2004; Kubisa, 2005).

S rastućim razvojem petrokemijske i automobilske industrije onečišćenje zraka uzrokovano

emisijom sumpornih spojeva (SOx) postaje jedan od glavnih ekoloških problema. Iz tih je

razloga posljednjih godina posvećena posebna pažnja procesu desulfurizacije benzina i

dizelskih goriva u skladu s postroženom zakonskom regulativom. Kako se koncentracija

sumpora u gorivu smanjuje procesom hidrodesulfurizacije (HDS) u uvjetima visokih

temperatura i tlakova te uz potrošnju velikih količina vodika, ionske kapljevine pokazale su se

kao alternativno rješenje, kako za smanjenje negativnog utjecaja ispušnih plinova na zdravlje

ljudi i okolinu, tako i s ekonomskog stajališta (Dharaskar i sur., 2013; Siddiqui i sur., 2013).

Zapravo, postoji oko 20 slučajeva primjene ionskih kapljevina (komercijaliziranih i/ili

korištenih u kemijskoj industriji) koji su poznati javnosti, no iz ekoloških razloga javlja se

problem zbrinjavanja takvih onečišćenih kemikalija. Ionska kapljevina može se ponovno

koristiti bez regeneracije ako njezina kvaliteta ostaje slična izvornoj ionskoj kapljevini

korištenoj za istu svrhu. U većini slučajeva kvaliteta ionske kapljevine smanjuje se tijekom

procesa pa je stoga potrebna njena regeneracija. U tom kontekstu, regeneracija uključuje sve

operacije koje generiraju ionsku kapljevinu koja posjeduje minimalna potrebna svojstva kako

bi se mogla ponovo primijeniti u procesu tako da ne utječe značajno na njegovu izvedbu

(Fernandez i sur., 2011). Bez obzira na to što je korak regeneracije neophodan, nisu pronađene

reference za industrijsku primjenu koje uključuju njihov oporavak i uklanjanje. To indicira na

postojanje, još uvijek, neriješenih problema vezanih uz postupanje s otpadom koji sadrži ionske

kapljevine što usporava napredak prema industrijskim primjenama. Ipak, s ekonomskog

stajališta, potencijal je golem i moguće ga je dalje razvijati (Reichert i sur., 2006).

Cilj ovog rada je istražiti mogućnost regeneracije ionskih kapljevina na bazi piridina i

imidazola, koje su onečišćene u procesu desulfurizacije modelne otopine dizelskog goriva

dibenzotiofenom.

Materijali i metode

Ionske kapljevine

Provedeni su eksperimenti s pet odabranih ionskih kapljevina, jedne na bazi piridina dok

su ostale na bazi imidazola. Odabrane ionske kapljevine većinom imaju isti anion, bis

(trifluorometilsulfonil) imid, a razlikuju se po broju i vrsti supstituenata na bazi kationa.

Korištene ionske kapljevine i njihovi skraćeni nazivi prikazani su u tablici 1.

Osim koeficijenta raspodjele, selektivnosti, uzajamne topljivosti otapala, regeneracije, i

fizikalna svojstva ionske kapljevine (gustoća, , viskoznost, , površinska napetost, ) utječu

na odabir otapala za kapljevinsku ekstrakciju. U tablici 2 prikazana su fizikalna svojstva

korištenih kapljevina.


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Tablica 1. Korištene ionske kapljevine i njihova skraćena imena

Table 1. Used ionic liquids and their abbreviated names

1-etil-3-metilimidazolijev etilsulfat

1-ethyl-methylimidazolium ethylsulfate [C2mim][EtSO4]

1-pentil-3-metilimidazolijev bis(trifluorometilsulfonil)imid

1-pentyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C5mim][Tf2N]

1-heksil-3,5 -dimetilpiridinijev bis(trifluorometilsulfonil)imid

1-hexyl-3,5-dimethylpyridinium bis(trifluorometylsulfonyl)imide [C6mmPy][Tf2N]

1-decil-2,3 -dimetilimidazolijev bis(trifluorometilsulfonil)imid 1-decyl-2,3-dimethylimidazolium bis(trifluoromethylsulfonyl)imide

[C10mmim][Tf2N]

1-benzil-3-metilimidazolijev bis(trifluorometilsulfonil)imid

1-benzyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [bzmim][Tf2N]

Tablica 2. Fizikalna svojstva ionskih kapljevina (Gabrić i sur., 2013)

Table 2. Physical properties of ionic liquids (Gabrić et al., 2013)

Ionska kapljevina /

ionic liquid ρ kgm Pas mNm

[C2mim][EtSO4] 1236 0,0896 49,3

[C5mim][Tf2N] 1404 0,0525 32,6

[C6mmPy][Tf2N] 1332 0,1152 34,7

[C10mmim][Tf2N] 1269 0,1472 33,3

[bzmim][Tf2N] 1491 0,1508 41,5

Onečišćivalo

Kako su navedene ionske kapljevine, nakon što su onečišćene u procesu desulfurizacije

modelne otopine dizelskog goriva, regenerirane najprije vakuumskim isparavanjem

(Gabrić i sur., 2013), zbog visoke temperature vrelišta (332 °C), nakon tog postupka, kao

jedino onečišćivalo zaostao je dibenzotiofen, C12H8S, čija je struktura prikazana na slici 1.

Slika 1. Struktura dibenzotiofena

Fig. 1. Dibenzothiophene structure


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Selektivno otapalo

Selektivno otapalo korišteno u procesu ekstrakcije je n-heksadekan, C16H34 (Acros

Organics, > 99%). Naime, kako bi se odabralo povoljno selektivno otapalo, posebnu

pozornost treba obratiti na njegova fizikalna svojstva, temperaturu vrenja, Tv, gustoću, ,

molarnu masu, M te eventualnu opasnost prilikom rukovanja (Tablica 3). Osim toga, iz

ekoloških je razloga važno da je otapalo moguće reciklirati te ponovno koristiti

(Meindersma i sur., 2005; Poole i Poole, 2010). Razlog odabira n-heksadekana je

nemješljivost s odabranim ionskim kapljevinama kao i različita gustoća na temelju čega i

dolazi do separacije faza (Gabrić i sur., 2013).

Tablica 3. Svojstva selektivnog otapala

Table 3. Properties of selective solvent

Fizikalna svojstva /

physical properties

n-heksadekan /

n-hexadecane

Tv / °C (vrelište / boiling point) 287

kg m 770

M / g mol-1 226,4

Opasnost / Hazard Iritacija kože / Skin irritation

Određivanje baždarnog dijagrama

Prije provedbe samog procesa potrebno je odrediti baždarni dijagram za sustav ionska

kapljevina–dibenzotiofen. Baždarni dijagram (Tablica 4) određen je mjerenjem indeksa

loma ionske kapljevine u ovisnosti o udjelu prisutnog dibenzotiofena pomoću Abbeovog

refraktometra (Optech Model RMI).

Tablica 4. Jednadžbe baždarnih krivulja korištenih ion. kapljevina te selektivnog otapala

Table 4. Equations of calibration curves of used ionic liquids and selective solvent

Otapalo / solvent Baždarne krivulje / calibration curves

[C2mim][EtSO4] xDBT = -61,5∙nD,252 + 187,0∙nD,25 – 142,1

[C5mim][Tf2N] xDBT = 45,44∙nD,252– 126,96∙nD,25+ 88,63

[C6mmPy][Tf2N] xDBT = 269,44∙nD,252– 776,44∙nD,25+ 559,34

[C10mmim][Tf2N] xDBT = 112,38∙nD,252– 319,51∙nD,25+ 227,07

[bzmim][Tf2N] xDBT = -134,35∙nD,252+ 400,96∙nD,25– 299,10

n-heksadekan / n-hexadecane

xDBT = 244,37∙nD,252– 695,27∙nD,25+ 494,53

xDBT – maseni udio dibenzotiofena u otapalu / mass fraction of

dibenzothiophene in solvent; nD,25 – indeks loma smjese otapala pri 25 °C /

refraction index of mixture of solvents at 25 °C


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Regeneracija ionskih kapljevina kapljevinskom ekstrakcijom

Glavna prednost ekstrakcije pred ostalim separacijskim procesima je njeno provođenje u

blagim radnim uvjetima (niske temperature i tlak) te ušteda velikih količina energije.

Poznata masa onečišćene ionske kapljevine (F) miješa se s određenom količinom

selektivnog otapala (B), slika 2.

Slika 2. Shematski prikaz procesa ekstrakcije

Fig. 2. Schematic description of extraction process

Masa potrebnog selektivnog otapala izračuna se na temelju poznatog solvent odnosa

(omjer mase selektivnog otapala (B) i čiste ionske kapljevine (A)), jednadžba 1.

B

A

mS

m (1)

Nakon razdvajanja faza očita se indeks loma rafinantne faze (R) te zatim, na temelju

određenog baždarnog dijagrama, udio zaostale organske komponente u ionskoj kapljevini

nakon regeneracije.

Efikasnost ekstrakcije izračuna se na temelju jednadžbe 2.

F R

F

x x

x (2)


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gdje je Fx maseni udio dibenzotiofena u onečišćenoj ionskoj kapljevini (pojnoj smjesi), a

Rx maseni udio dibenzotiofena u rafinantnoj fazi.

Ekstrakcija je provedena pri sljedećim radnim uvjetima:

solvent odnos: 0,5

vrijeme miješanja: 60 min

vrijeme razdvajanja faza: 20 min

brzina vrtnje miješala: do 300 min-1

.


Svrha ovoga rada bila je provesti regeneraciju ionskih kapljevina onečišćenih

dibenzotiofenom, koji je zaostao nakon prethodno provedenog vakuumskog isparavanja

(Gabrić i sur., 2013). Kapljevine su regenerirane postupkom kapljevinske ekstrakcije uz

n-heksadekan kao selektivno otapalo, a čistoća regeneriranih ionskih kapljevina potvrđena

je nuklearnom magnetskom rezonancijom (1H NMR).

Slika 3 prikazuje 1H NMR spektre čiste ionske kapljevine [C6mmPy][Tf2N] te spektre

nakon regeneracije vakuumskim isparavanjem (Gabrić i sur., 2013). Vidljivo je da nakon

vakuumskog isparavanja zaostaju samo karakteristični pikovi koji odgovaraju

dibenzotiofenu zbog njegova visokog vrelišta i nemogućnosti isparavanja. S obzirom da

kapljevine nisu u potpunosti pročišćene vakuumskim isparavanjem, provedena je

kapljevinska ekstrakcija. Spektri za sve ostale ionske kapljevine pokazuju slične

rezultate.

Slika 4 prikazuje usporedbu efikasnosti ekstrakcije za sve navedene ionske kapljevine uz

n-heksadekan kao selektivno otapalo. Vidljivo je da je regeneracija ionske kapljevine

[C2mim][EtSO4] provedena u samo dva stupnja, za razliku od ostalih, za koje je bio

potreban veći broj ekstrakcijskih stupnjeva, osobito za kapljevine [C6mmPy][Tf2N] i

[C10mmim][Tf2N].

Za obje navedene kapljevine povećanje efikasnosti procesa iznad 90% daljnjim

pokušajima ekstrahiranja dibenzotiofena iz kapljevina svježim n-heksadekanom bilo je

presporo, pa nije dosegnuta ravnotežna djelotvornost ekstrakcije u provedenim

stupnjevima.


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Slika 3.

1H NMR spektar čiste i korištene ionske kapljevine [C6mmPy][Tf2N] u procesu

pročišćavanja modelne otopine dizelskog goriva te spektar regenerirane

kapljevine vakuumskim isparavanjem (Gabrić i sur., 2013)

Fig. 3. 1H NMR spectra of fresh and used ionic liquid with model solution mimicking

diesel fuel and ionic liquids [C6mmPy][Tf2N] regenerated by

vacuum evaporation (Gabrić et al., 2013)

čista / fresh

korištena /

used

regenerirana /

regenerated

dibenzotiofen / dibenzothiophene

toulen / toulene

piridin / pyridine

tiofen / thiophene

piridin /

pyridine

[C6mmPy][Tf2N]

9,0 8,0 7,0 6,0 5,0 4,0 3,0 2,0 1,0 0,0ppm


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Slika 4. Ovisnost efikasnosti ekstrakcije o broju ekstrakcijskih stupnjeva

za korištene ionske kapljevine pri solvent odnosu 0,5

Fig. 4. Dependence of extraction efficiency on number of extraction steps

for used ionic liquids at solvent ratio 0.5

Relativno velik broj ekstrakcijskih stupnjeva prilikom regeneracije pojedinih ionskih

kapljevina posljedica je izabranog solvent odnosa od 0,5, te bi bilo potrebno ispitati utjecaj

većeg solvent odnosa. Osim toga, početni udio dibenzotiofena razlikovao se u različitim

ionskim kapljevinama (Tablica 5) te je logično da se kapljevine koje sadrže veći udio

onečišćivala, [C6mmPy][Tf2N] i [C10mmim][Tf2N], regeneriraju u većem broju stupnjeva.

Isto tako, daljnje korištenje ionske kapljevine [C10mmim][Tf2N] trebalo bi se istražiti s

ekološkog i toksikološkog stajališta s obzirom da je to polisupstituirana kapljevina s

dugačkim alkilnim lancem, a poznato je da toksičnost raste s povećanjem broja alkilnih

supstituenata (Perić et al., 2012).

Kapljevine [C5mim][Tf2N] i [bzmim][Tf2N] pokazuju slične rezultate te postignutu

efikasnost > 95% u relativno malom broju ekstrakcijskih stupnjeva. Kako su fizikalna

svojstva prisutnih faza te topljivost otopljenih komponenti od ključne važnosti za prijenos

tvari, iz tablice 2 je vidljivo da je ionska kapljevina [C5mim][Tf2N] uslijed najniže

viskoznosti i najmanje površinske napetosti pogodna za regeneraciju. Naime, kapljevine s

nižom viskoznosti, koje imaju manju površinsku napetost lakše se dispergiraju u

selektivnom otapalu čime se pospješuje međufazni prijenos tvari. S druge strane, ionska

kapljevina [bzmim][Tf2N] je najviskoznija te ima najveću gustoću od svih korištenih

kapljevina, a ipak je pokazala vrlo dobre rezultate. Rezultat je to mnogo veće topljivosti

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10

/

%

stupanj ekstrakcije / extraction step

[C2mim][EtSO4]

[C5mim][Tf2N]

[C6mmPy][Tf2N]

[C10mmim][Tf2N]

[bzmim][Tf2N]


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dibenzotiofena u n-hekdsadekanu nego u ionskoj kapljevini [bzmim][Tf2N] (Tablica 4).

Nadalje, iz tablice 5 je vidljivo da je udio zaostalog dibenzotiofena u svim ispitanim

kapljevinama nakon regeneracije uz pomoć n-heksadekana manji od 1% što je

zadovoljavajuća čistoća. 1H NMR spektri snimljeni nakon regeneracije ionskih kapljevina s n-heksadekanom na slici 5 ne

ukazuju na prisutnost pikova karakterističnih za dibenzotiofen (8,155; 7,853; 7,45; 7,45 ppm).

Takav rezultat pokazuje da je kapljevinska ekstrakcija uz n-heksadekan, u granicama

osjetljivosti metode (Rogošić et al., 2014), pogodna metoda za regeneraciju ionskih kapljevina.

Slika 5. 1H NMR spektri ionskih kapljevina nakon regeneracije

kapljevinskom ekstrakcijom pomoću n-heksadekana

Fig. 5. 1H NMR spectra of regenerated ionic liquids by liquid-liquid

extraction using n-hexadecane


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Tablica 5. Udio dibenzotiofena u ionskim kapljevinama prije i nakon regeneracije

Table 5. Portion of dibenzothiophene in ionic liquids before and after regeneration

Ionska kapljevina / ionic liquid xDBT,0 / % xDBT,1 / %

[C2mim][EtSO4] 1,58 0,13

[C5mim][Tf2N] 2,69 0,10

[C6mmPy][Tf2N] 6,42 0,82

[C10mmim][Tf2N] 5,63 0,47

[bzmim][Tf2N] 2,95 0,57

Zaključci

Ispitana je mogućnost regeneracije pet različitih ionskih kapljevina onečišćenih

dibenzotiofenom postupkom kapljevinske ekstrakcije.

Kapljevinskom ekstrakcijom uz pomoć n-heksadekana, uz solvent odnos 0,5, ionske

kapljevine pročišćene su do zadovoljavajuće čistoće.

Najbolje rezultate pokazale su ionske kapljevine [C5mim][Tf2N] i [bzmim][Tf2N]. 1H NMR spektri ionskih kapljevina nakon regeneracije ukazuju da nema zaostalog

dibenzotiofena, odnosno da je regeneracija uspješno provedena.

Literatura

Anugwom, I., Mäki-Arvela, P., Salmi, T., Mikkola, J.-P. (2011): Ionic liquid assisted extraction of

nitrogen and sulphur-containing air pollutants from model oil and regeneration of the spent

ionic liquid, J. Environ. Prot. 2, 796-802.

Antonietti, M., Kuang, D., Smarsly, B., Zhou, Y. (2004): Ionic liquids for the convenient synthesis

of functional nanoparticles and other inorganic nanostructures, Angew. Chem. Int. Ed. 43 (38),

4988-4992.

Dharaskar, S.A., Wasewar, K.L., Varma, M.N., Shende, D.Z. (2013): Extractive deep

desulfurization of liquid fuels using Lewis-based ionic liquids, J. Energy 2013, 1-4.

Feng, R., Zhao, D., Guo, Y. (2010): Revisiting characteristics of ionic liquids: A review for further

application development, J. Environ. Prot. 1, 95-104.

Fernandez, J.F., Neumann, J., Thöming, J. (2011): Regeneration, recovery and removal of ionic

liquids, Cur. Org. Chem. 15, 1992-2014.

Gabrić, B., Sander, A., Bubalo, M.C., Macut, D. (2013): Extraction of S- and N- compounds from

the mixture of hydrocarbons by ionic liquids as selective solvents, Sci. World J. 2013, 1-11.

Koel, M. (2005): Ionic liquids in the synthesis and modification of polymers, Crit. Rev. Anal. Chem.

35 (3), 177-192.

Kubisa, P. (2005): Ionic liquids in the synthesis and modification of polymers, J. Polym. Sci. A 43

(20), 4675-4683.


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Meindersma, G.W., Podt, A., De Haan, A.B. (2005): Selection of ionic liquids for the extraction of

aromatic hydrocarbons from aromatic/aliphatic mixtures, Fuel Process. Technol. 87, 59 - 70.

Olivier-Bourbigou, H., Hughes, F. (2003): Green industrial applications of ionic liquids, Kluwer

Academic Publishers,pp. 67.

Perić, B.M., Marti, E., Sierra, J., Cruanas, R., Garau, M.A. (2012), Green chemistry: ecotoxicity and

biodegradability of ionic liquids. U: Recent Advances in Pharmaceutical Sciences II, Transworld

Research Network, Torrero D.M., Halo, D., Valles, J. (Eds.), Kerala, India, pp. 89-113.

Poole, C.F., Poole, S.K. (2010): Extraction of organic compounds with room temperature ionic

liquids, J. Chrom. A 1217, 2268 - 2286.

Reichert, W.M., Holbrey, J.D., Vigour, K.B., Morgan, T.D., Broker, G. A., Rogers, R.D. (2006):

Approaches to crystallization from ionic liquids: complex solvents–complex results, or, a

strategy for controlled formation of new supramolecular architectures, Chem. Commun. 46,

4767-4779.



FCC gasoline, J. Chem. Therm. 76, 1-15.

Sander, A. (2012): Ionske kapljevine u službi zelene kemije, Polimeri, 33, 127-128.

Seddon, K.R. (1997): Ionic liquids for clean technology, J. Chem. Tech. Biotechnol. 68, 351-356.

Sheldon, R. (2001): Catalytic reactions in ionic liquids, Chem. Commun. 2399-2407.

Siddiqui, M.N., Alhooshani, K.R., Gondal, M.A., Ranu, B.C. (2013), u 245. ACS National Meeting

& Exposition, Preprints, Division of Energy and Fuels, Desulfurization of model fuel oil using

novel ionic liquids. 58, 1022-1023.

Zhao, D., Wu, M., Kou, Y., Min, E. (2002): Ionic liquids: Applications in catalysis, Catal. Today

74, 157-189.


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The applicability of n-hexadecane in regeneration of ionic liquids




Summary

Recently, particular importance is given to investigations connected with the possibility of

application of ionic liquids in diverse industrial processes regarding to new available methods for

regeneration and replacement of many volatile organic solvents. The purpose is to minimize waste

generation and negative impact on environment as well. The possibility of application of ionic

liquids in different industries is initialized by their specific properties like non-volatility, stability

and many others. These properties present ionic liquids as a promising alternative to

environmentally undesirable volatile organic solvents. Since they have possibility to be used and

regenerated many times, in this work the regeneration of diverse ionic liquids, one based on

pyridine and others on imidazole, was investigated. They were contaminated in the desulfurization

process of model solutions that mimic diesel fuel fractions. The regeneration of ionic liquids was

performed by liquid–liquid extraction with n-hexadecane as a selective solvent, which was chosen

on the basis of its physical properties. Mass ratio of ionic liquid and selective solvent was 0,5. The

purity of ionic liquids has been qualitatively determined using nuclear magnetic resonance

spectroscopy. It was confirmed that there were no characteristic peaks for dibenzotiophene, which

was the only pollutant in tested ionic liquids.

Keywords: ionic liquids, n-hexadecane, regeneration


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Uvećanje sušionika s fluidiziranim slojem

UDC: 66.047


Sveučilište u Zagrebu, Fakultet kemijskog inženjerstva i tehnologije, Marulićev trg 20, Zagreb,

Hrvatska

Sažetak

Prenošenje rezultata u veće mjerilo u kemijskom se inženjerstvu uglavnom osniva na provođenju

eksperimenata u više geometrijski sličnih uređaja u različitom mjerilu, te se izvode odgovarajući

kriteriji uvećanja. Sušenje je proces za koji se dimenzijska analiza ne može uspješno provesti zbog

utjecaja velikog broja parametara na kinetiku sušenja. Uzevši u obzir istovremeno odvijanje procesa

prijenosa količine gibanja, topline i tvari očito je da se radi o vrlo složenom procesu. Kod sušenja u

fluidiziranom sloju situacija je još složenija zahvaljujući kaotičnom gibanju čestica u struji zraka što

često rezultira neujednačenim hidrodinamičkim uvjetima. Režim strujanja bitno se razlikuje u

sušionicima različitih dimenzija čime je uvećanje dodatno otežano. U svrhu definiranja kriterija

uvećanja sušionika s fluidiziranim slojem, eksperimentalno je istražena kinetika sušenja sferičnih

čestica u dva laboratorijska geometrijski slična sušionika. Mjerenja su provedena pri različitim

brzinama strujanja zraka, temperaturi zraka te visini mirujućeg sloja čestica. Eksperimentalno je

određena minimalna brzina fluidizacije u oba sušionika. Promjer sušionika utječe na režim strujanja

i fluidizacije, što za posljedicu ima sasvim drugačiju kinetiku sušenja. Kinetika sušenja opisana je

Pageovim modelom a parametri modela dovedeni su u vezu s uvjetima sušenja i promjerom

sušionika. Na temelju dobivenih rezultata izvedeni su kriteriji uvećanja za fluidizaciju te kinetiku

sušenja.

Ključne riječi: kinetika sušenja, kriterij uvećanja, minimalna brzina fluidizacije, sušenje s

fluidiziranim slojem

Uvod

Procesi koji se provode u fluidiziranom sloju u velikoj se mjeri koriste u industriji

(Hovmand, 1995). Sušenje u fluidiziranom sloju čest je proces za sušenje čestičnih

materijala (Bahu, 1994). Sušenje je vrlo složen proces tijekom kojeg se istovremeno

odvijaju procesi prijenosa količine gibanja, topline i tvari. Osim toga, proces je nelinearan

a kontrolirajući se mehanizam prijenosa tvari može mijenjati tijekom procesa s obzirom da

ovisi o uvjeti sušenja te svojstvima materijala. Prijenosna svojstva vezana uz svojstva

materijala, kao što su difuzijski koeficijent i koeficijent toplinske vodljivosti materijala,

ovise o temperaturi i sadržaju vlage materijala. Osim toga, tijekom sušenja može doći do



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promjene unutrašnje strukture materijala što može rezultirati promjenom mehanizma

prijenosa vlage, ili se zbog složene strukture istovremeno vlaga može kretati različitim

mehanizmima. Dakle, cijeli niz varijabli i parametara utječe na kinetiku sušenja čime je

matematičko modeliranje procesa uvelike otežano (Kerkhof, 1994).

Općenito govoreći, uvećanje procesa definira se kao metodologija razvoja komercijalnog

uređaja na temelju podataka dobivenih u laboratorijskom mjerilu (Zlokarnik, 2006). Pri

tome je potrebno definirati procesne parametre koji će rezultirati željenim rezultatom

neovisno o veličini uređaja. Postoji nekoliko pristupa u rješavanju tog problema (Genskow,

1994). To su: provođenje eksperimenata na postojećem komercijalnom uređaju, korištenje

postojećih kriterija uvećanja, provođenje eksperimenata u geometrijski sličnim uređajima,

dizajn i testiranje modularnog uređaja te primjena dimenzijske analize. U kemijskom

inženjerstvu za predviđanje vladanja procesa u većem mjerilu uglavnom se

eksperimentalna istraživanja provode u laboratorijskom mjerilu. Na taj se način stječe

potrebno znanje o procesu te se uz primjenu pravila sličnosti dolazi do bezdimenzijskih

značajki i simpleksa koji se u svim mjerilima moraju održavati konstantnim. Međutim

postoje procesi kod kojih se izvedena pravila uvećanja ne mogu primijeniti, jer

geometrijska sličnost ne osigurava dinamičku, toplinsku i koncentracijsku sličnost. Sušenje

je proces na koji utječe velik broj parametara pa nije moguće provesti dimenzijsku analizu

koja bi rezultirala korelacijskom jednadžbom, odnosno pravilom uvećanja. Zbog složenog

višefaznog strujanja, hidrodinamika procesa sušenja u fluidiziranom sloju u velikoj mjeri

otežava prenošenje rezultata iz laboratorijskog u veće mjerilo (poluindustrijsko i

industrijsko) (Briongosa i Guardiolab, 2005). Režimi strujanja nisu isti u svim mjerilima,

zbog čega su različiti i režimi fluidizacije (Dang i sur., 2014). To drugim riječima znači da

će i minimalna brzina fluidizacije iste vrste i dimenzije čestica biti različita u sušionicima

različitih promjera. Literatura nudi kriterije uvećanja koji uglavnom ne sadrže član koji se

odnosi na geometrijske karakteristike sušionika, odnosno faktor uvećanja, R. Uvećanje

procesa sušenja, općenito, kao i sušenja u fluidiziranom sloju u velikoj se mjeri oslanja na

eksperimentalna istraživanja i iskustvena pravila, a dobiveni su rezultati direktno povezani

i primjenljivi na istraživani sustav i odgovarajuće radne uvjete (Kerkhof, 1994). Za

definiranje kriterija uvećanja sušionika s fluidiziranom sloju neophodno je poznavanje

utjecaja uvjeta provedbe procesa na kinetiku sušenja, što uključuje provođenje velikog

broja eksperimenata i matematičko modeliranje kinetike sušenja. Dakle, potrebno je

istražiti utjecaj brzine strujanja zraka, temperature i relativne vlažnosti zraka te visine sloja

čestica na kinetiku i parametre matematičkog modela. Provođenjem eksperimenata u više

(dva ili tri) geometrijski sličnih uređaja u laboratorijskom mjerilu dobiva se pouzdaniji

kriterij uvećanja.

U ovom je radu istraživan utjecaj uvjeta provedbe procesa na kinetiku sušenja sferičnih

čestica katalizatora. Mjerenja su provedena u dva geometrijski slična sušionika različitih

promjera u svrhu definiranja kriterija uvećanja.


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Materijali i metode

Materijal

U sušionicima s fluidiziranim slojem sušene su sferične čestice Al2O3-NiO-CaCO3,

katalizatora (dsr = 4,05 mm). Gustoća sferičnih čestica određena je gravimetrijskom

metodom s obzirom da je za fluidizaciju bitan tzv. hidrodinamički volumen, neovisan o

poroznosti čestica. U svrhu definiranja mehanizma prijenosa vlage kroz unutrašnjost

čestica, eksperimentalno je određena raspodjela veličina pora (Micrometrics ASAP 2000).

Eksperimentalni dio

Mjerenja su provedena na dva laboratorijska sušionika s fluidiziranim slojem različitih

dimenzija (D1 = 3,7 cm; Z1 = 73cm i D2 = 5,5 cm; Z2 = 65 cm). Sušionici se sastoje od

cilindrične kolone, puhala zraka, grijača, mjerila protoka (prigušna pločica), dva

manometra (uspravni i kosi), te mjerila stanja zraka. Na vrhu kolone nalazi se ciklon za

separaciju izlaznog zraka i eventualno odnesenih čvrstih čestica, sa posudom za prihvat

čvrstih čestica. Temperatura zraka na ulazu u sušionik mjerena je digitalnim termometrom.

S obzirom da se kinetika sušenja pratila psihrometrijskom metodom, na izlazu iz sušionika

mjerena je temperatura i relativna vlažnost zraka pomoću digitalnog higro-termometra.

Mjerenja su provedena pri različitim temperaturama (50, 57 i 65 °C) i brzinama strujanja

zraka (2,50; 3,13 i 4,50 m/s) te visinama sloja čvrstih čestica (0,96; 1,46; 1,64).

Eksperimentalni su podaci aproksimirani Pageovim modelom (Sander, 2007):

ntk

eq

eXX

XtX

0

eq (1)

te je analiziran utjecaj uvjeta provedbe procesa i veličine sušionika na parametre

matematičkih modela.

Za procjenu minimalne brzine fluidizacije korištene su sljedeće korelacije (Yang, 2003):

Chitester: 7,280494,07,28e 2mf ArR (2)

Hilal: 07,130263,007,13e 2mf ArR (3)

Wen&Yu: 7,330408,07,33e 2mf ArR (4)


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Gustoća suhih i mokrih čestica Al2O3-NiO-CaCO3, katalizatora određena je

gravimetrijskom metodom. Čestice su vagane i pomičnim mjerilom određen im je promjer.

Mjerenja su napravljena za po 10 čestica, a srednje su vrijednosti 1,71 g/cm3 za suhe i 2,25 g/cm

3

za mokre čestice. S obzirom na promjer čestica te razliku gustoća čestica i zraka, uzorak

pripada D klasi prema Geldartu (1973).

U svrhu predviđanja mehanizma prijenosa vlage kroz unutrašnjost materijala tijekom

sušenja, određena je raspodjela veličina pora (slika 1.). S obzirom na promjer pora može se

zaključiti da će se vlaga uglavnom kretati difuzijskim mehanizmom. Na temelju raspodjele

veličina pora izračunata je poroznost čestica koja iznosi 50%.

Slika 1. Raspodjela veličina pora sferičnih čestica

Fig. 1. Pore size distribution of spherical particles

Minimalna brzina fluidizacije suhih i mokrih čestica određena je pri tri visine mirujućeg

sloja čestica. U tu su svrhu određene krivulje fluidizacije, mjerenjem pada tlaka prazne

kolone i kolone s čvrstim česticama. Na slici 2. prikazani su eksperimentalne i računske

vrijednosti minimalne brzine fluidizacije suhih čestica za oba sušionika. Može se uočiti da

u sušioniku manjeg promjera do fluidizacije dolazi pri većim brzinama (Rao i sur., 2010).

0

10

20

30

40

50

60

70

80

90

100

1 10 100

Q3(d

p),

%

dp, nm


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Povećanjem omjera d/D raste minimalna brzina fluidizacije zbog većeg utjecaja stjenke.

Isto tako i povećanjem omjera H/D raste minimalna brzina fluidizacije. Smanjenjem D

veći dio čestica se nalazi uz samu stjenku što uzrokuje veći utjecaj stjenke. Porastom D

utjecaj stjenke slabi i postaje zanemariv kako se d/D smanjuje. Literaturne korelacijske

jednadžbe za procjenu minimalne brzine fluidizacije ne uzimaju u obzir geometriju

sušionika s obzirom da se brzina računa iz ovisnosti Re = f (Ar). Međutim, Hilalova se

korelacija (Hilal i sur., 2001) može koristiti za procjenu minimalne brzine fluidizacije za

manje vrijednosti d/D te kada je H/D < 1,46, odnosno u slučajevima kada se utjecaj stjenke

može zanemariti. S obzirom da minimalna brzina fluidizacije ovisi o promjeru sušionika

potrebno je definirati kriterij uvećanja. Na temelju eksperimentalnih podataka u dva

geometrijski slična sušionika izvedena je jednadžba koja omogućuje procjenu minimalne

brzine fluidizacije:

RvmH

mHv mf,1

1

2mf,2 (5)

Dobivena jednadžba primjenljiva je za istraživane uvjete sušenja i odabrane sferične

čestice.

Slika 2. Minimalna brzina fluidizacije (experimentalna i procijenjena)

Fig. 2. Minimum fluidization velocity (experimental and evaluated)

0,0

0,5

1,0

1,5

2,0

2,5

0,80 1,00 1,20 1,40 1,60 1,80

v mf, m

/s

H/D

d/D=0,0736

d/D=0,1095

Chitester

Hilal

Wen&Yu


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Kriterij uvećanja općenito mora osigurati isti rezultat u malom i velikom mjerilu. Vezano

za proces sušenja, željeni rezultat je točno definirana kinetička krivulja sušenja. Dakle, u

oba sušionika je potrebno provesti dovoljan broj eksperimenata kako bi se utvrdili utjecaji

uvjeta provedbe procesa na kinetiku sušenja. Zatim je potrebno odabrati matematički

model koji uspješno opisuje eksperimentalne podatke te na kraju izvesti korelacijsku

jednadžbu koja omogućava predviđanje kinetičke krivulje sušenja u većem mjerilu. Važno

je napomenuti da materijal koji se suši mora biti isti u oba mjerila, kao i temperatura i

relativna vlažnost zraka za sušenje. Naime, već je spomenuto da se svojstva materijala

mijenjaju tijekom sušenja što može rezultirati promjenama u njegovoj strukturi. Osim toga,

konačni, odnosno ravnotežni sadržaj vlage materijala na kraju sušenja također ovisi o

temperaturi i relativnoj vlažnosti zraka.

Na slikama 3 do 5 prikazani su utjecaji uvjeta provedbe procesa na kinetiku sušenja.

Porastom temperature raste brzina sušenja, te se smanjuje vrijeme trajanja procesa. Pri

višoj temperaturi relativna vlažnost zraka je manja pa je veća pokretačka sila procesa

prijenosa vlage (slika 3). Veća temperatura i manja relativna vlažnost zraka povećavaju

brzine prijenosa topline i tvari, čime raste i brzina sušenja. Povećanjem visine sloja

mirujućeg čestica raste masa čestica u sušioniku, a s time i masa vlage koja se tijekom

sušenja mora ukloniti iz vlažnog materijala (slika 4). Iako su čestice u stalnom gibanju i sa

svih strana okružene zrakom za sušenje, zrak primi veću količinu vlage kada je u sušionika

veća masa vlažnog materijala, što smanjuje pokretačku silu za proces prijenosa tvari. To

rezultira smanjenjem brzine sušenja i duljim vremenom trajanja procesa. Visina sloja

čestica ima utjecaj na brzinu sušenja kada se glavni otpori prijenosu tvari nalaze na strani

materijala. S druge strane, ako se glavni otpori prijenosu nalaze na strani zraka, brzina

strujanja zraka utjecat će na kinetiku sušenja. Veća brzina strujanja zraka rezultira

povoljnijim hidrodinamičkim uvjetima koji su posljedica smanjenja otpora prijenosu

količine gibanja, topline i tvari, što za posljedicu ima veću brzinu sušenja materijala i kraće

vrijeme trajanja procesa (slika 5).

Za opis kinetike sušenja sferičnih čestica katalizatora odabran je Page-ov model, s obzirom

da se u prethodnim istraživanjima pokazao uspješnim za odabrani materijal (Sander, 2007).

Kinetičke krivulje sušenja u oba sušionika mogu se opisati Pageovim modelom uz visok

stupanj korelacije (>0,99). Parameter n ima vrijednosti oko 1,1 i ne mijenja se s

promjenom uvjeta sušenja i promjerom sušionika. Utjecaj uvjeta provedbe procesa i

promjera sušionika na parametar k prikazan je na slici 7. Vrijednosti parametra k rastu

kako raste i brzina sušenja, odnosno rastu s porastom temperature i brzine strujanja zraka,

te smanjenjem visine mirujućeg sloja čestica. U sušioniku manjeg promjera brzina sušenja

je veća zbog znatno manje mase materijala pri istom omjeru (H/D) koji se suši, iako su

otpori prijenosu količine gibanja, topline i tvari veći zbog nepovoljnijih hidrodinamičkih

uvjeta uzrokovanih utjecajem stjenke.


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Slika 3. Utjecaj temperature na kinetiku sušenja (D = 5,5 cm; v = 3,13 m/s; (H/D)=0,96)

Fig. 3. The influence of temperature on the drying kinetics (D = 5.5 cm; v = 3.13 m/s; (H/D)=0.96)

Slika 4. Utjecaj visine mirujućeg sloja čestica na kinetiku sušenja (D = 5,5 cm; v = 3,13 m/s; T=65 °C)

Fig. 4. The influence of the fixed bed height on the drying kinetics (D = 5.5 cm; v = 3.13 m/s; T=65 °C)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 10 20 30 40

(X-X

eq)/

(X0-X

eq)

t, min

50 °C57 °C65 °C

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 10 20 30 40

(X-X

eq)/

(X0-X

eq)

t, min

0,961,461,64

(H/D)


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Slika 5. Utjecaj brzine strujanja zraka na kinetiku sušenja (D = 5,5 cm; (H/D)=1,46; T=57 °C)

Fig. 5. The influence of air stream velocity on the drying kinetics (D = 5.5 cm; (H/D)=1,46; T=57 °C)

Slika 6. Primjenljivost Page-ovog modela

Fig. 6. The applicability of Page model

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 10 20 30 40 50 60

(X-X

eq)/

(X0-X

eq)

t, min

2,50 m/s

3,13 m/s

4,50 m/s

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 10 20 30 40 50

(X-X

eq)/

(X0-X

eq)

t, min

eksperimentalno

experimental

računski calculated

R2 = 0,9924


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a)

b)

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

45 50 55 60 65 70

k

T, °C

3,7 cm

5,5 cm

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,9 1,1 1,3 1,5 1,7

k

(H/D)

3,7 cm

5,5 cm


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c)

Slika 7. Utjecaj uvjeta provedbe procesa na parametar Pageovog modela

(a) v=3,13 m/s; (H/D)=1,46; b) v=3,13 m/s; T=57 °C; c) (H/D)=1,46; T=57 °C)

Fig. 7. The influence of the process conditions on the Page model parameter k

(a) v=3.13 m/s; (H/D)=1,46; b) v=3.13 m/s; T=57 °C; c) (H/D)=1.46; T=57 °C)

U svrhu izvođenja kriterija uvećanja, odnosno izraza koji bi omogućio predviđanje

kinetičke krivulje sušenja u većem mjerilu uspoređene su kinetičke krivulje sušenja u oba

sušionika u istim uvjetima sušenja (temperatura i relativna vlažnost zraka). Kriterij

uvećanja koji služi za procjenu parametra k, Pageovog modela dan je sljedećim izrazom:

,22

1 ,1

1mf

mf

vk

k v R (6)

Zaključci

Istražen je utjecaj temperature, visine mirujućeg sloja čestica, brzine strujanja zraka i

promjera sušionika na kinetiku sušenja u fluidiziranom sloju. Minimalna brzina fluidizacije

raste s porastom visine mirujućeg sloja čestica i smanjenjem promjera sušionika. Veće se

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

2 3 4 5

k

v, m/s

3,7 cm

5,5 cm


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brzine sušenja ostvaruju u manjem sušioniku, pri višim temperaturama, manjim visinama

sloja čestica te većim brzinama strujanja zraka. Kinetika sušenja uspješno je opisana

Pageovim modelom. Uvjeti koji povećavaju brzinu sušenja rezultiraju višim vrijednostima

parametra Pageovog modela, k. Parametar n ovisi o vrsti i geometrijskim karakteristikama

materijala te je stalan za istraživani materijal. Izvedeni su kriteriji uvećanja za minimalnu

brzinu fluidizacije i kinetiku procesa sušenja na temelju odabranog matematičkog modela.

Literatura

Bahu, R.E. (1994): Fluidised bed dryer scale-up, Dry. Technol. 12 (1&2), 329-340.

Briongosa, J. V., Guardiolab, J. (2005): New methodology for scaling hydrodynamic data from a

2D-fluidized bed, Chem. Eng. Sci. 60, 5151-5163.

Dang, N.T.Y., Gallucci, F., van Sint Annaland, M. (2014): An experimental investigation on the

onset from bubbling to turbulent fluidization regime in micro-structured fluidized beds,

Powder Technol. 256, 166-174.

Geldart, D. (1973): Types of Gas Fluidization, Powder Technol., 7, 285-292.

Genskow, L.R. (1994): Dryer scale-up methodology for the process industries, Dry. Technol. 12

(1&2), 47-58.

Hilal, N., Ghannam, M.T., Anabtawi, M.Z. (2001): Effect of bed diameter, distributor and inserts on

minimum fluidization velocity, Chem. Eng. Technol. 24 (2), 161-165.

Hovmand, S. (1995): Fluidized bed drying. In: Handbook of Industrial Drying, Marcel Dekker, Inc.,

A.S.M. (ed.), New York, pp. 195-248.

Kerkhof, P.J.A.M. (1994): The role of theoretical and mathematical modeling in scale-up, Dry.

Technol. 12 (1&2), 1-46.

Rao, A., Curtis, J.S., Hancock, B.C., Wassgren, C. (2010): The Effect of Column Diameter and Bed

Height on Minimum Fluidization Velocity, AIChE J., 56, 2304-2311.

Sander, A. (2007): Thin-layer drying of porous materials: Selection of the appropriate mathematical

model and relationships between thin-layer models parameters, Chem. Eng. Process., 46,

1324-1331.

Zlokarnik, M. (2006): Scale-up in Chemical Engineering, Weinheim, WILEY-VCH Verlag GmbH

& Co. KgaA, pp. 181-205.

Yang, W.-C. (2003): Bubbling fluidized beds. In: Handbook of fluidisation and fluid-particle

systems, Marcel Dekker, Inc, Y.W-C. (ed.), New York, pp: 63-121.


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Scale-up of fluid bed dryer




Summary

Scale-up in chemical engineering is usually based on laboratory scale experiments performed in at

least two or three pieces of geometrically similar equipment and derivation of scale-up rules.

Generally speaking, derivation of scale-up rules based on dimensional analysis cannot be applied on

the drying process since a large number of parameters influence the drying kinetics. Taking into

account only simultaneous transfer of momentum, heat and mass it is quite obvious how complex

the drying process is. In fluid bed drying the situation is even more complex due to the chaotic

motion of particles in the stream of hot air resulting with nonuniform hydrodynamic conditions.

Besides that, the flow regime is greatly influenced by the dryer size so it is very hard to predict the

drying kinetics in a larger scale dryer. In order to define the scale-up rules for a fluid bed dryer,

spherical particles of Al2O3-NiO-CaCO3 catalyst have been dried in two geometrically similar

laboratory fluid bed dryers. Experiments have been performed at different temperatures and flow

rates of the drying air, for three bed heights. Minimum fluidization velocity was determined

experimentally for both dryers. Based on the obtained results it can be concluded that the size of the

dryer influences the drying kinetics of spherical particles. The drying kinetics was approximated by

the Page model. Evaluated parameters were correlated with the drying conditions and the size of

dryer. Scale-up rules for minimum fluidization velocity and drying kinetic curve were defined.

Keywords: drying kinetics, fluid-bed drying, minimum fluidization velocity, scale-up rule


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Nova otapala za ekstrakciju tiofena iz smjese sa n-heksanom

UDC: 66.061 : 547.7



Hrvatska

Sažetak

Desulfurizacija motornih benzina i dizelskog goriva jedan je od većih problema s kojima se suočava

naftno-petrokemijska industrija. U današnje se vrijeme sve veći broj znanstvenika bavi istraživanjem

mogućnosti adaptacije postojećih procesa kao i razvojem novih tehnologija kojima bi se osigurala

željena kvaliteta tekućih goriva. Kapljevinska ekstrakcija pomoću ekološki prihvatljivih otapala,

posebno je zanimljiva s obzirom da je ekstrakcija proces koji se provodi pri blagim radnim uvjetima.

Primjena ionskih kapljevina u procesu ekstrakcijske desulfurizacije predmet je velikog broja

istraživanja. U novije se vrijeme kao selektivno otapalo koriste i smjese ionskih kapljevina, te

eutektičke smjese. Ionske kapljevine i eutektičke smjese posjeduju izuzetna svojstva čime

zadovoljavaju većinu zahtjeva koji moraju biti ispunjeni prilikom odabira otapala. Osim njihove

nehlapivosti, za ekstrakciju je bitna velika selektivnost otapala te velike brzine prijenosa tvari. U ovom

je radu istražena mogućnost primjene smjesa dviju ionskih kapljevina u različitim masenim omjerima

(1-etil-3-metilimidazol etilsulfat 1-pentil-3-metilimidazol bis(trifluormetilsulfonil)imid; 1-etil-3-

metilimidazol etilsulfat - 1-heksil-3,5-dimetilpiridin bis(trifluormetilsulfonil)imid; 1-etil-3-

metilimidazol etilsulfat - 1-benzil-3-metilimidazol bis(trifluormetilsulfonil) imid i 1-heksil-3,5-

dimetilpiridin bis(trifluormetilsulfonil)imid - 1-benzil-3-metilimidazol bis(trifluormetilsulfonil)imid) te

eutektičke smjese kolin-klorid/glicerol kao selektivnog otapala u procesu separacije smjese tiofena i n-

heksana kapljevinskom ekstrakcijom. Porastom udjela ionske kapljevine veće ekstrakcijske efikasnosti

u smjesi dviju ionskih kapljevina dobiva se rafinat veće čistoće. Ionske kapljevine i njihove smjese

pogodnija su otapala za separaciju tiofena i n-heksana, od eutektičke smjese kolin-klorid/glicerol.

Ključne riječi: desulfurizacija, ekstrakcija kapljevina-kapljevina, eutektičke smjese, ionske kapljevine

Uvod

Smanjenje sadržaja sumporovih spojeva iz motornih benzina predmet je mnogih

istraživanja zbog sve većih zahtjeva vezanih za očuvanje okoliša. Naime izgaranjem goriva

nastaju između ostalog sumporovi oksidi u velikoj mjeri zaslužni za nastajanje kiselih kiša.

Komercijalni proces desulfurizacije motornih benzina (HDS) nepovoljan je jer se provodi

pri uvjetima visokog tlaka te se troše velike količine vodika kako bi se smanjio sadržaj



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sumporovih spojeva do željene niske koncentracije (Song, 2003). Zbog toga se istražuju

alternativne metode desulfurizacije koje uključuju oksidacijsku desulfurizaciju (ODS), bio-

desulfurizaciju (BDS), adsorpcijsku desulfurizaciju (ADS) te ekstrakcijsku desulfurizaciju

(EDS) (Wilfred et al., 2012). Ekstrakcijska desulfurizacija posebno je zanimljiva zbog

blagih uvjeta provedbe procesa (sobna temperatura i atmosferski tlak). Do sada je

komercijalni proces EDS-a koristio sulfolan kao selektivno otapalo. Kako bi ekstrakcija

bila ekološki i ekonomski povoljan proces potrebno je odabrati otapalo koje neće biti

štetno za okoliš, te će se moći jednostavno regenerirati i vratiti natrag u proces (Gabrić et

al., 2013). Otapala koja zadovoljavaju većinu zahtjeva pri razvoju procesa kapljevinske

ekstrakcije su ionske kapljevine i eutektičke smjese. I jedna i druga grupa otapala

posjeduju izuzetna svojstva čime se već na prvi pogled razlikuju od komercijalnih

molekularnih otapala (Francisco et al.; 2010, Daia et al.; 2013, Hayyan et al., 2013). Radi

se o nehlapljivim otapalima sposobnim otopiti različite vrste spojeva uz znatno veće brzine

prijenosa tvari. Ionske se kapljevine sastoje od kationa i aniona čija kombinacija utječe na

njihova svojstva. Pravilnim odabirom kationa i aniona moguće je dizajnirati otapalo za

željenu svrhu. Međutim, iako su nahlapljive nisu sve ionske kapljevine ekološki

prihvatljive, bilo zbog spojeva koji se koriste za njihovu sintezu bilo zbog nepovoljnog ili

neistraženog utjecaja na vode i tlo. S druge strane eutektičke smjese (DES) u potpunosti su

ekološki prihvatljive čime postaju zanimljiv odabir u separacijskim procesima koji

uključuju pomoćnu komponentu, kao što je na primjer kapljevinska ekstrakcija. Dok je

primjena različitih ionskih kapljevina u procesu desulfurizacije motornih benzina i

dizelskog goriva istraživana u velikoj mjeri, upotreba eutektičkih smjesa u tu svrhu tek je u

začetku (Li et al., 2013). Osim toga do danas su publicirana samo dva rada u kojima se

koriste smjese ionskih kapljevina u svrhu dobivanja otapala još boljih svojstava (Garcia et

al., 2012; Fletcher et al., 2003).

U ovom je radu istražen utjecaj sastava smjese ionskih kapljevina (C2mimEtSO4,

[C5mim][Tf2N], [bzmim][Tf2N] i [C6mmPy] [Tf2N]) na efikasnost ekstrakcije tiofena iz

smjese s n-heksanom. Osim navedenih ionskih kapljevina kao selektivno je otapalo

korištena i eutektička smjesa kolin-klorid/glicerol. Efikasnost ekstrakcije, , računata je

korištenjem sljedećeg izraza:

F

RF

w

ww (1)

gdje su: wF i wR, maseni udjeli tiofena u pojnoj smjesi i rafinatu.


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Istraženo je i kako se mijenjaju svojstva smjesa ionskih kapljevina (gustoća, viskoznost i

površinska napetost) ovisno o njihovom udjelu u smjesi. Navedena su svojstva izračunata

iz svojstava čistih ionskih kapljevina.

Gustoća smjese, s:

2111 1 xxs (2)

gdje su: x1, molni udio komponente 1 u smjesi, a 1 i 2 gustoće komponenata 1 i 2 u

smjesi.

Viskoznost smjese, s:

2111 1 wws (3)

gdje su: w1, maseni udio komponente 1 u smjesi, a 1 i 2 viskoznosti komponenata 1 i 2 u

smjesi.

Površinska napetost smjese, s:

2111 1 xxs (4)

gdje su: 1 i 2 površinske napetosti komponenata 1 i 2 u smjesi.

Eksperimentalni dio

Materijali

U procesu uklanjanja tiofena iz smjese sa n-heksanom korištena su sljedeća otapala: ionske

kapljevine (C2mimEtSO4, [C5mim][Tf2N], [bzmim][Tf2N] i [C6mmPy] [Tf2N]) i

njihove smjese (u masenim omjerima: 1:1; 1:2 i 2:1), te eutektička smjesa kolin-

klorid/glicerol. Sinteza ionskih kapljevina opisana je u literaturi (Gabrić et al., 2013).

Čistoća ionskih kapljevina određena je 1H NMR spektroskopijom (Bruker AV300) na

Institutu Ruđer Bošković iz Zagreba. Kolin klorid i glicerol pomiješani su u molnom

omjeru 1:2, te je dobivena smjesa miješana u rotacijskom vakuum isparivaču na 60 °C i

250 mbara sve dok se nije dobila bezbojna jednofazna kapljevina.


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Mjerenje gustoće, viskoznosti i površinske napetosti

Viskoznost čistih ionskih kapljevina i eutektičke smjese izmjerena je pomoću reometra

(Brookfield DV-III Ultra Programmable Rheometer). Gustoća i površinska napetost

izmjerene su pomoću tensiometra (KRUSS K9). Sva su mjerenja provedena pri

temperaturi od 25 °C.

Ekstrakcija kapljevina - kapljevina

Mjerenja su provedena u laboratorijskom šaržnom ekstraktoru opremljenom s magnetskim

miješalom. Početni maseni udio tiofena u smjesi sa n-heksanom bio je 19,5 %. U smjesu

tiofena i n-heksana dodano je otapalu uz solvent odnos S = 0,25 kg/kg. Dobivena se smjesa

miješala 30 minuta. Nakon separacije faza, koja je trajala 30 minuta, refraktometrijskom

metodom (Abbeov refraktometar Model RMI, Exacta Optech) određena je koncentracija

rafinatne faze. Baždarna krivulja dana je sljedećom jednadžbom:

41,5594,7027,22 25,225, DD nnw (5)

gdje su: w, maseni udio tiofena u smjesi s n-heksanom, a nD,25, indeks loma pri 25 °C.


U svrhu istraživanja utjecaja vrste i sastava selektivnog otapala na efikasnost ekstrakcije

tiofena iz smjese sa n-heksanom provedena je ekstrakcija kapljevina-kapljevina u

laboratorijskom ekstraktoru pri sobnim uvjetima. Mjerenja su provedena sa četiri ionske

kapljevine, njihovim smjesama te jednom eutektičkom smjesom. U tablici 1 date su

vrijednosti gustoće, viskoznosti i površinske napetosti istraživanih otapala i modelne

otopine.

Tablica 1. Gustoća, viskoznost i površinska napetost modelne otopine i odabranih otapala

Table 1. Density, viscosity and surface tension of model solutions and selected solvents

Otapalo / Solvent , kg m-3 , Pa s , mN m-1

modelna otopina / model solution

C2mimEtSO4 [C5mim][Tf2N]

[C6mmPy] [Tf2N]

[bzmim][Tf2N]

Kolin-klorid/glicerol / choline-chloride/glycerol

706

1236

1404

1332 1491

1192a

3,6210-4

0,0896 0,0525

0,1152

0,1508

0,3027

19,5

49,3

32,6

34,7 41,5

59,0b a – Yadav et al., 2014; b – Harris, 2008


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Naime, gustoća i viskoznost u velikoj mjeri utječu na odabir odgovarajućeg otapala za

ekstrakciju tiofena iz smjese sa n-heksanom. S obzirom da je potrebna dovoljno velika

razlika u gustoćama između modelne otopine i selektivnog otapala, može se zaključiti da

sva otapala zadovoljavaju taj uvjet. Što se tiče viskoznosti i površinske napetosti

najpovoljnije otapalo je [C5mim][Tf2N] a najnepovoljnije kolin-klorid/glicerol. Viskoznost

otapala mora biti što manja kako bi se otapalo lakše dispergiralo, a na taj se način

osigurava veća specifična međufazna površina. Osim toga, proces prijenosa tvari odvijat će

se većom brzinom. Međutim, osim fizikalnih svojstava vrlo su važna i termodinamička

svojstva, odnosno koeficijent raspodjele i selektivnost, te topljivost svih komponenti

modelne otopine u odabranom otapalu. Istražena je i međusobna topljivost n-heksana i svih

istraživanih otapala, a rezultati su prikazani u tablici 2.

Tablica 2. Topljivost n-heksana i tiofena u odabranim otapalima izražena kao maseni udio,

koeficijent raspodjele tiofena i selektivnost otapala

Table 2. Solubility of n-hexane and thiophene in the selected solvents in mass fraction, thiophene

distribution coefficient and selectivity of solvent

w(n-heksan) w(tiofen) S Lit./Ref.

C2mimEtSO4 [C5mim][Tf2N]

[C6mmPy] [Tf2N] [bzmim][Tf2N]

Kolin-klorid/glicerol

0,003

0,034 0,050

0,012

0,000

0,468

0,557 0,614

0,524

-

1,42

0,67 2,52

0,46

0,21

121,81

12,33 13,77

19,81

-

Casal,2010

Rogošić et al., 2014 Casal,2010

Ovaj rad

Ovaj rad

n-heksan je u slabo topljiv u svim istraživanim ionskim kapljevinama dok je netopljiv u

kolin-klorid/glicerolu. S druge strane sva odabrana otapala netopljiva su u n-heksanu, čime

je zadovoljen uvjet vezan za međusobnu nemješljivost primarnog i sekundarnog otapala.

Izračunate vrijednosti koeficijenta raspodjele i selektivnosti otapala za uvjete

provođenja ekstrakcije prikazane su također u tablici 2. Ionske kapljevina u kojima je

topljiv i n-heksan manje su selektivne, a najveći koeficijent raspodjele ostvaren je uz

korištenje [C6mmPy] [Tf2N]. Uzevši u obzir sva navedena svojstva i veličine, za očekivati

je da će upravo ta ionska kapljevina biti najpogodnije otapalo za ekstrakciju tiofena iz

smjese sa n-heksanom.

Efikasnost ekstrakcije čistih otapala pri solvent odnosu 0,25 i početnoj koncentraciji

tiofena od 19,5% prikazana je na slici 1. Kao najpovoljnija ionska kapljevina pokazala se

[C6mmPy] [Tf2N], a najniža efikasnost ekstrakcije ostvarena je korištenjem eutektičke

smjese kolin-klorid/glicerol. Gabrić i sur. (2013) istraživali su utjecaj različitih ionskih

kapljevina na efikasnost ekstrakcije sumporovih i dušikovih spojeva iz modelnih otopina

Fluid Catalytic Cracking, FCC benzina i dizelskog goriva. Prema njihovim podacima, za


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modelnu otopinu FCC benzina najpovoljnija je bila ionska kapljevina [C5mim][Tf2N], a za

modelnu otopinu diselskog goriva [C6mmPy] [Tf2N]. Očito je da sastav modelne otopine u

velikoj mjeri utječe na sposobnost ionske kapljevine da otopi željenu komponentu. U

navedenom su radu modelne otopine bile višekomponentne, a osim tiofena u ionskim su se

kapljevinama otapali piridin i toluen.

Slika 1. Ekstrakcijska efikasnost čistih odabranih otapala (S = 0,25 kg / kg)

za uklanjanje tiofena iz smjese sa n-heksanom

Fig. 1. Extraction efficiency of pure selected solvent (S = 0,25 kg / kg)

for separation of thiophene and n-hexane

Kako bi se istražila mogućnost korištenja smjese ionskih kapljevina kao selektivnog

otapala u procesu ekstrakcije tiofena iz smjese sa n-heksanom, pripravljene su smjese od

po dvije ionske kapljevine u različitim masenim omjerima. S obzirom da je najmanja

efikasnost uočena za ionsku kapljevinu C2mimEtSO4, istraženo je kako dodatak ostale

tri istraživane ionske kapljevine utječe na efikasnost uklanjanja tiofena iz smjese sa n-

heksanom. Na slikama 2, 3 i 4 prikazane su izračunate vrijednosti gustoća, viskoznosti i

površinskih napetosti smjesa ionskih kapljevina. Svojstva smjese dviju ionskih kapljevina

nalaze se između svojstava čistih otapala. Kako je ionska kapljevina [C5mim][Tf2N] manje

viskoznosti od C2mimEtSO4, dodatkom [C5mim][Tf2N] u C2mimEtSO4 viskoznost

smjese opada. Dodatak ostalih dviju ionskih kapljevina u C2mimEtSO4 rezultira

porastom viskoznosti smjese što je nepovoljno s aspekta ukupne površine izmjene tvari i

brzine prijenosa tvari. Gustoća smjesa ionskih kapljevina raste s porastom udjela ionske

0 5 10 15 20 25 30

kolin-klorid/glicerol

[C2mim][EtSO4]

[C5mim][Tf2N]

[bzmim][Tf2N]

[C6mmPy] [Tf2N]

, %


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kapljevine veće efikasnosti ekstrakcije, što ide u prilog odabiru selektivnog otapala. Na

površinsku napetost smjese u najvećoj mjeri utječe dodatak [C5mim][Tf2N]. Međutim, ako

se u obzir uzmu i topljivost tiofena, te koeficijent raspodjele i selektivnost otapala, za

očekivati je da će se najbolji rezultati, u smislu povećanja efikasnosti ekstrakcije, dobiti

dodatkom [C6mmPy] [Tf2N].

Slika 2. Utjecaj sastava smjese ionskih kapljevina na viskoznost smjese

Fig. 2. Dependence of the mixed ionic liquids viscosity and its composition

Ekstrakcijska efikasnost smjese dviju ionskih kapljevina ovisno o sastavu smjese,

prikazana je na slici 5. Efikasnost ekstrakcije raste s porastom udjela dodane ionske

kapljevine u smjesi sa C2mimEtSO4. Na povećanje efikasnosti ekstrakcije u najvećoj

mjeri utječe dodatak ionske kapljevine na bazi piridina, što je u skladu sa zaključcima

izvedenim na temelju svojstava ionskih kapljevina, te rezultata dobivenih korištenjem

čistih ionskih kapljevina.

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0 0,2 0,4 0,6 0,8 1

, P

a s

w1([C2mim][EtSO4])

C2-C5

C2-BM

C2-C6


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Slika 3. Utjecaj sastava smjese ionskih kapljevina na gustoću smjese

Fig. 3. Dependence of the mixed ionic liquids density and its composition

Slika 4. Utjecaj sastava smjese ionskih kapljevina na površinsku napetost smjese

Fig. 4. Dependence of the mixed ionic liquids surface tension and its composition

0

200

400

600

800

1000

1200

1400

1600

0 0,2 0,4 0,6 0,8 1

, k

g/m

3

w1([C2mim][EtSO4])

C2-C5

C2-BM

C2-C6

0

10

20

30

40

50

60

0 0,2 0,4 0,6 0,8 1

, m

N/m

w1([C2mim][EtSO4])

C2-C5

C2-BM

C2-C6


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Slika 5. Utjecaj sastava smjese ionskih kapljevina na efikasnost ekstrakcije

tiofena iz smjese sa n-heksanom

Fig. 5. The influence of the composition of mixture on the extraction efficiency

Karakterizacija ionskih kapljevina i njihovih smjesa

Ionske kapljevine i njihove smjese prije i nakon ekstrakcije karakterizirane su 1H NMR

spektroskopijom. Na slici 6 prikazani su 1H NMR spektri za ionske kapljevine

C2mimEtSO4 i [C5mim][Tf2N]. Miješanjem dviju ionskih kapljevina dobiva se

homogena kapljevita smjesa čiji 1H NMR spektar sadrži karakteristične pikove obiju

ionskih kapljevina. Nakon ekstrakcije tiofena iz smjese sa n-heksanom na 1H NMR spektru

mogu se uočiti pikovi koji odgovaraju tiofenu. Iako je n-heksan slabo topljiv u

[C5mim][Tf2N] njegovo prisustvo u ionskoj kapljevini nakon ekstrakcije nije moguće

potvrditi. Očito je koncentracija vrlo mala, pa se ovom metodom ne može detektirati. Čak

niti korištenjem čistih ionskih kapljevina u kojima je n-heksan djelomično topljiv, n-heksan

nije bilo moguće detektirati 1H NMR spektroskopijom.

0

5

10

15

20

25

30

0 0,2 0,4 0,6 0,8 1

, %

w1([C2mim][EtSO4])

C2-C5

C2-C6

C2-BM


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Slika 6. 1H NMR spektri čistih ionskih kapljevina C2mimEtSO4 i

[C5mim][Tf2N], te njihovih smjesa prije i nakon ekstrakcije

Fig. 6. 1H NMR spectras of fresh ionic liquids C2mimEtSO4 and

[C5mim][Tf2N] and its mixture, before and after extraction

Zaključci

Poznavanje fizikalnih svojstava ionskih kapljevina, te topljivosti tiofena u ionskim

kapljevinama omogućuje odabir ionske kapljevine kao selektivnog otapala u procesu

ekstrakcije tiofena iz smjese sa n-heksanom.

Odabrane ionske kapljevine mogu se koristiti kao selektivna otapala u procesu uklanjanja

tiofena iz smjese s n-heksanom, ekstrakcijom kapljevina – kapljevina. Učinkovitost ionskih

kapljevina u navedenom procesu desulfurizacije raste u smjeru: C6mmPy] [Tf2N] >

[bzmim][Tf2N] > C5mim][Tf2N] > C2mimEtSO4. Eutektička smjesa kolin-klorid/glicerol,

najmanje je učinkovita.


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Dodatkom ionskih kapljevina C6mmPy] [Tf2N], [bzmim][Tf2N] i C5mim][Tf2N] ionskoj

kapljevini najmanje učinkovitosti, C2mimEtSO4, raste učinkovitost ekstrakcije tiofena

iz smjese sa n-heksanom.

Porastom udjela učinkovitije ionske kapljevine u smjesi raste i učinkovitost ekstrakcije

tiofena iz smjese sa n-heksanom. 1H NMR spektroskopijom utvrđeno je da u procesu prijenosa tvari sudjeluje samo tiofen, s

obzirom da prisutnost n-heksana u ionskih kapljevinama i njihovim smjesama nije uočena.

Literatura

Daia, Y., van Spronsenb, J., Witkampb, G-J., Verpoortea, R., Choia, J.H. (2013): Natural deep

eutectic solvents as new potential media for green technology, Anal. Chim. Acta 766, 61-68.

Casal, M.F. (2010): Desulfurization of fuel oils by solvent extraction with ionic liquids, PhD thesis,

Santiago de Compostela, University of Santiago de Compostela.

Fletcher, K.A., Baker,S. N., Bakerb, G. A., Pandey, S. (2003): Probing solute and solvent

interactions within binary ionic liquid mixtures, New J. Chem. 27, 1706-1712

Francisco, M., Arce, A., Soto, A. (2010): Ionic liquids on desulfurization of fuel oils, Fluid Phase

Equilibr. 294, 39-48.

Gabrić, B., Sander, A., Cvjetko-Bubalo, M., Macut, D. (2013): Extraction of S- and N-compounds

from the mixture of hydrocarbons by ionic liquids as selective solvents, Sci. World J. (2013),

1-11.

García, S., Larriba, M., García, J., Torrecilla, J. S., Rodríguez, F. (2012): Liquid–liquid extraction of

toluene from n-heptane using binary mixtures of N-butylpyridinium tetrafluoroborate and N-

butylpyridinium bis(trifluoromethylsulfonyl)imide ionic liquids, Chem. Eng. J. 180, 210-215.

Harris, R.C. (2008): Physical Properties of Alcohol Based Deep Eutectic Solvents, PhD Thesis,

Leicester, USA, University of Leicester.

Hayyan, M., Hashim, M.A., Hayyan, A., Al-Saadi, M.A., AlNashef, I.M., Mirghani, M.E.S., Saheed

O.K. (2013): Are deep eutectic solvents benign or toxic?, Chemosphere 90, 2193-2195.

Li, C., Li, D., Zou, S., Li, Z., Yin, J., Wang, A., Cui, Y., Yaoa, Z., Zhaoa, Q. (2013): Extraction

desulfurization process of fuels with ammonium-based deep eutectic solvents, Green Chem.

15, 2793-2799.



FCC gasoline, J. Chem. Thermodyn. 76, 1-15.

Song, C. (2003): An overviev of new-approaches to deep desulfurisation for ultra-clean gasoline.

diesel fuel and jet fuel, Catal. Today 86 (1-4), 211-263.

Wilfred, C.D., Kiat, C.F., Man, Z., Bustam, M.A., Mutalib, M.I.M., Phak, C.Z. (2012): Extraction

of dibenzothiophene from dodecane using ionic liquids, Fuel Process. Technol. 93 (1), 85-89.

Yadav, A., Trivedi, S., Rai, R., Pandey, S. (2014): Densities and dynamic viscosities of (choline

chloride + glycerol) deep eutectic solvent and its aqueous mixtures in the temperature range

(283.15–363.15), Fluid Phase Equilibr. 367, 135-142.


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New solvents for extraction of thiophene from the mixture with n-hexane




Summary

Desulphurization and denitrification of petrol and diesel fuels is one of the leading problems in the

oil and petrochemical industry. Today, more and more scientists are researching ways to adapt

existing processes and also developing new technologies that would ensure the desired quality of

liquid fuels. Liquid-liquid extraction by eco-friendly solvents is especially interesting because

extraction as a process can be carried out at mild process conditions. The use of ionic liquids in

extraction desulphurization processes is the object of a large number of investigations. Lately, ionic

liquid mixtures and deep eutectic solvents are used as selective solvents. Ionic liquids and deep

eutectic solvents posses excellent properties that satisfy the majority of the needs that have to be

fulfilled when choosing a solvent. Apart from them being nonvolatile, a key factor for extraction is

to ensure a high selectivity towards the components that need to be extracted from the liquid

mixture and to achieve high mass transfer rates. In this work the possibility of using a mixture of

two ionic liquids at different mass ratios (C2mimEtSO4-[C5mim][Tf2N]; C2mimEtSO4-

[C6mmPy] [Tf2N]; C2mimEtSO4-[bzmim][Tf2N] and [C6mmPy] [Tf2N]- [bzmim][Tf2N]) and

also the use of an deep eutectic solvent choline- chloride/glycerol (1:2, n) as selective solvents in the

separation of tiophene and n-hexane by liquid-liquid extraction has been investigated. Raffinate of

higher purity is gained by using a ionic liquid mixture that has higher fractions of the more effective

ionic liquid in the mixture. Ionic liquids and their mixtures are more adequate solvents for the

separation of tiophene and n-hexane than deep eutectic solvent choline-chloride/glycerol.

Keywords: deep eutectic solvents, desulfurization, ionic liquids, liquid-liquid extraction

Sekcija: Prehrambena tehnologija i biotehnologija

Session: Food technology and biotechnology


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Prehrambena tehnologija i biotehnologija / Food technology and biotechnology

190

Isolation and characterization of volatile and non-volatile phytochemicals

from orange (Citrus sinensis L.) peel

UDC: 661.16 : 543.42

634.31

Sara Bebić1, Franko Burčul

2, Ivana Generalić Mekinić

3, Ivica Blažević

1

University of Split, Faculty of Chemistry and Technology,

1Department of Organic Chemistry,

2Department of Biochemistry,

3Department of Food Technology and Biotechnology, Teslina 10/V,

HR-21000 Split, Croatia

Summary

Citrus sinensis L. belongs to Rutaceae family. The following study was aimed to identify volatile

and non-volatile phytochemicals isolated by different methods from dried orange peel. Volatiles

isolated by hydrodistillation in Clevenger type apparatus as well as by petrolether extraction in

Soxlet apparatus were analysed by GC-MS. The volatile oil as well as petrolether extract contained

more than 90% of monoterpene limonene. Other minor compounds were also identified such as:

monoterpenes (- and -pinene, sabinene, ocimene, etc.); sesquiterpenes (-caryophyllene, -copaene,

-elemene, valencene, etc.); alcohols (linalool, - and - terpineol); esters (neryl acetate, geranyl

acetate, ethyl linoleate etc.) and others. The MetOH extraction of non-volatile phytoconstituents was

performed after petrolether extraction in Soxlet apparatus as well as by the alkaline (KOH)

extraction of the peels. The non-volatile compound obtained by crystallization was identified as

hesperetin, the aglycone of flavanon hesperidin. Limonene and hesperetin were analysed by

using spectroscopic methods and tested for their antioxidant and cholinesterase inhibitory

activity.

Keywords: Citrus sinensis, limonene, hesperetin, GC-MS, NMR

Introduction

Citrus fruits belong to six genera (Fortunella, Eremocitrus, Clymendia, Poncirus,

Microcitrus and Citrus) but the major commercial fruits belong to Citrus genus. The genus

Citrus (Rutaceae) includes several important fruits such as oranges, mandarins, limes,

lemons and grape fruits. The Mediterranean basin is one of the most important production

area of citrus fruits in the world, concentrating 18% of the production and above all

exporting slightly more than half of the overall citrus fruits exchanged in the world

(Schimmenti et al., 2013).



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There are two modern species of orange, Citrus aurantium (the bitter or Seville orange) and

Citrus sinensis (the sweet orange). The fruits are both consumed fresh and industrially

processed. The pulps, which are rich in soluble sugars, significant amounts of vitamin C,

pectin, fibers and different organic acids, are mainly processed into juice (Qiao et al., 2008).

The remaining orange peels represent around 45% of the total by-products. Consequently, if

treated as waste materials, the orange peel may create environmental problems. This problem

could be turned into an asset, if phytochemicals are extracted from orange peels and applied

as natural products. Chemical industry extracts from Citrus bioactive compounds like

flavonoids, vitamins, dietary fibers and essential oils, etc. which have beneficial effects on

human health. Orange peels containing abundant fragrant substances are extensively applied

for processing into essential oils which are used commercially for flavoring foods, beverages,

perfumes, cosmetics, etc. Citrus oils extraction methods include steam distillation, cold

pressing and static headspace solid-phase microextraction (Liu et al., 2014 ). Many

researchers have studied the volatile compounds of this fruit using different analytical

methods (Qiao et al., 2008). As a result of these studies, more than 200 compounds have

been described as components of the flavor of sweet orange, and the major aroma

compounds found in orange juice are hydrocarbons, alcohols, aldehydes, esters, and ketones.

Flavonoids are the most abundant polyphenols present in the human diet and are regularly

found in vegetables, fruits and plant-derived beverages such as tea and red wine.

Natural compounds, such as components of the essential oils and flavonoids represent a

source of bioactive compounds due to their antioxidant, antimicrobial, anticaricinogenic

and other properties. Recent studies have focused on the ability of essential oils and dietary

polyphenols to protect against neuronal damages resulting from aging and

neurodegenerative processes (Maher, 2006). The WHO (2012) estimates there are 35.6

million people living in dementia at present worldwide. Alzheimer disease (AD) is the

most frequent form of dementia in Western societies. It is assumend that the dysfunction of

colinergic neurotransmission in the brain contributes to the relevant cognitive decline in

AD. The loss of colinergic cells is accompanied by the loss of the neutrotransmitter

acetylcholine, thus, one of the most accepted strategies in AD treatment is the use of

cholinesterase inhibitors (De Paula et al., 2009).

The aim of the study was to isolate volatiles and flavonoid by using different methods. The

volatile profiles obtained by hydrodistillation in Clevenger type apparatus and by

petrolether extraction in Soxlet apparatus were determined by GC-MS. Isolation of non-

volatile flavanoid was also performed by two different methods (maceration and MetOH

extraction in Soxlet apparatus). The main compounds were determined by spectroscopic

techniques (UV/Vis, FTIR, NMR) and were tested for antioxidant activity by three

different methods (2,2-diphenyl-1-picrylhydrazyl scavenging ability (DPPH), Ferric

Reducing/Antioxidant Power (FRAP), Briggs-Rauscher oscillating reaction (BROR)) and

cholinesterase inhibition (AChE, BuChE).


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General

The standards were purchased from Sigma Chemical Co. (USA). All the solvents

employed were purchased from Fluka Chemie, Buchs, Switzerland. Anhydrous sodium

sulphate was obtained from Kemika, Zagreb, Croatia. Orange (Citrus sinensis L.) was

purchased from local market and the air-dried peels were used for the research.

Isolation

Essential oil

The volatiles were isolated from dried plant material (100 g) by hydrodistillation in

Clevenger type apparatus. Distillation was performed for 3 hours with a pentane used as a

trap. After distillation pentane extract was separated and dried over anhydrous sodium

sulphate. The volatile isolate was concentrated by carefully fractional distillation to a small

volume (cca 3 mL) and 1 μL of this solution was used for each GC-MS analyses. The

essential oil was also analysed by FTIR, and 13

CNMR (APT).

Petrolether volatile extract

100 mL petroleum ether (40-60°C) is filled in a 250 mL round bottom flask with magnetic

stir bar. Dried and powdered orange peel (20 g) was placed in the extraction sleeve of a

Soxhlet extractor. A reflux condenser is put on the Soxhlet extraction unit, then the

reaction mixture was stirred and heated for 12 hours under strong reflux. The obtained

petrolether extract was concentrated by the rotary evaporator after which was analysed by

GC-MS, as well as by FTIR.

Flavanoid isolation

Conventional extraction method

The dried orange peels (200 g) were macerated with 800 mL of aq. alkaline solution (10%

KOH, pH 8-9) for overnight. After complete maceration the mixture was filtered through

the large Buchner funnel and filtrate was evaporated to make it a syrupy mass.


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MetOH extraction in Soxlet

After the orange peels have been completely de-fatted by petroleum ether extraction, like

before, the peels were extracted by 100 mL methanol, until the solvent leaving the

extraction sleeve was colourless (8 hours). The extract was evaporated at the rotary

evaporator until the syrup consistency was reached.

Work up

The residue obtained by conventional extraction method and MetOH extraction in Soxlet

were mixed with 50 mL of 6% acetic acid. The obtained precipitate solid was filtered

through the Büchner funnel, washed with 6% acetic acid and dried at 60 °C until it reached

constant in weight. The solid was heated in aqueous HCl (pH 0) for 40 minutes. The

precipitated flavanoid is filtered on a Buchner funnel and washed with water

(Krishnaswamy, 1996).

The compound gave a wine-red colour with alcoholic ferric chloride and bright violet

colour using the Shinoda test. To perform the Shinoda test a pinch of magnesium powder

was added to the alcoholic solution of the compound. After that concentrated HCl in drops

was added and the bright pinkish violet colour was formed (Krishnaswamy, 1996; Sharma

et al., 2013). The flavanoid was analysed by DSC, UV/Vis, FTIR, 13

CNMR (APT).

Chemical analysis

Gas chromatography and mass spectrometry (GC-MS) analysis

Gas chromatography analysis was performed on gas chromatograph (model 3900; Varian

Inc., Lake Forest, CA, USA) equipped with mass spectrometer (model 2100T; Varian

Inc.), non-polar capillary column VF-5MS (30 m × 0.25 mm i.d., coating thickness 0.25

μm; Varian Inc.). VF-5MS column temperature was programmed at 60 °C isothermal for 3

minutes, and then increased to 246 °C at a rate of 3 °C/min and held isothermal for 25

minutes. Other chromatographic conditions were: carrier gas helium; flow rate 1 mL/min;

injector temperature 250 °C; volume injected 1 μL; split ratio 1:20. MS conditions:

ionization voltage 70 eV; ion source temperature 200 °C; mass scan range: 40-350 mass

units.

The individual peaks were identified by comparison of their retention indices (relative to

C8-C40 n-alkanes for VF-5MS) to those from a homemade library, literature and/or

authentic samples, as well as by comparing their mass spectra with literature, Wiley 7 MS

(Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA) mass spectral

databases (Adams, 1995).


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Differential scanning calorimetry (DSC) analysis

Differential scanning calorimetry (DSC) measurement was performed with a Mettler-

Toledo 823e calorimeter. Sample of 10.0 mg, encapsulated in aluminum pans, was first

heated from 25 to 150 °C. Then it was heated to 320 °C (heating rate of 20 °C/min–heating

scan), kept there for 5 minutes and then cooled again to 150 °C (cooling rate of 20 °C/min–

cooling scan).

Spectroscopic analyses

UV/Vis spectra were obtained with an AnalytikJena Specord 200 spectrometer (Analytik

Jena GmbH, Germany) using 10 mm quartz cells.

Infrared spectra were recorded on IRAffinity-1 spectrometer (Shimadzu, Japan). Spectra

were recorded by using KBr transmision cell, in the spectral area 4000-400 cm-1

and with

resolution 4 cm-1

. Abreviation used are for streching (deformation and out-of-plane

(oop). 13

CNMR (APT) spectra were recorded at 600 MHz on Bruker Avance 600 spectrometer.

Tetramethylsilane (TMS) was used as an internal standard and deuterated chloroform

(CDCl3) and DMSO-d6 were used as solvent.

Biological activity

Antioxidant activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability of the samples was measured

according to the previously reported procedure (Katalinić et al., 2013). The results for

radical scavenging activities were expressed as inhibition percentage of DPPH radical (%

Inhibition) which was calculated using the following formula:

% Inhibition = [(Abscontrol - Abssample) / Abscontrol] × 100,

where Abscontrol was initial absorbance of radical solution and Abssample was absorbance of

the samples after 60 minutes of the reaction.

The reducing potential was measured as ferric reducing/antioxidant power (FRAP) (Benzie

and Strain, 1996). A standard curve was prepared using different concentrations of

Vitamin C and the results are expressed in milimoles of Vitamin C equivalents per litre of

extract (mmol Vit C/L).


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The ability of samples to stop the oscillations in Briggs-Rauscher system (BROR) was

estimated in our recently published paper (Generalić Mekinić et al., 2013). The results are

expressed as the inhibition time of the oscillations (in minutes).

Acetylcholinesterase / butyrylcholinesterase inhibitory activity

Acetylcholinesterase inhibitory activity measurements were carried out by using slightly

modified Ellman method (Ellman et al., 1961; Generalić Mekinić, et al., 2013; Blažević et

al., 2013) A typical run consisted of 180 μL of phosphate buffer (0.1M, pH 8), 10 μL of

DTNB (at a final concentration of 0.3 mM prepared in 0.1 M phosphate buffer pH 7 with

0.12 M sodium bicarbonate added for stability), 10 μL of sample solution (dissolved in

EtOH), and 10 μL of AChE/BuChE solution (at a final concentration 0.03 U/mL).

Reactants were mixed in a 96-well plates and reaction was initialized by adding 10 μL of

ATChI, at a final concentration of 0.5 mM. As a negative control EtOH was used instead

of sample solution. Also non-enzymatic hydrolysis was monitored by measurement of two

blank runs for each run. In short, in first blank mixture AChE/BuChE was replaced with

equivalent buffer amount and in second blank mixture ATChI/BuTChI was replaced with

equivalent buffer amount. All measurements were done spectrophotometrically at 409 nm

and room temperature for 6 min period. Percentage of inhibition of AChE was determined

by a comparison of the rates of reaction of samples relative to the blank sample (ethanol in

phosphate buffer, pH 8) using formula ((E-BE)-(S-BS))/(E-BE)×100, where E is the activity

of enzyme without test sample, and S is the activity of enzyme with test sample. BE and BS

are blank runs for E and S, respectively.


Chemical analysis

The volatiles were isolated from the orange peels by two methods: hydrodistillation in

Clevenger type apparatus and by petrolether extraction in Soxlet apparatus. Analyses of the

volatile isolates are performed by GC-MS and the results are presented in Table 1.

Flavanoid was also isolated by two methods i.e. by conventional method – maceration and

by MetOH extraction in Soxlet apparatus and acidic hydrolysis was performed in order to

obtain free aglycone.

The structure confirmation of the isolated main volatile and non-volatile compound was

achieved by the analysis of different spectral data (UV/Vis, FTIR, and 13

CNMR). The 13

CNMR data and the assigned signals on corresponding structures are given in Table 2

and Fig. 2.


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In total, 34 volatile components were identified, the present l, a total of including 7

monoterpene hydrocarbons, 11 oxygenated monoterpenes, 7 sesquiterpene hydrocarbons, 4

oxygenated sesquiterpenes, 2 fatty acids, 1 ester and 2 hydrocarbons (Table 1).

Table 1. Composition and content of Citrus sinensis L. volatiles

Chemical composition KIa

Essential

oil

(%)

Petrolether

extract

(%)

Monoterpene hydrocarbons

1. -Pinene 919 Tr Tr

2. Sabinene 968 0.9 Tr

3. -Pinene 972 0.3 Tr

4. Limoneneb 1036 90.7 90.1

5. -Terpinene 1060 2.7 0.2

6. -Terpinolen 1083 0.2 Tr

7. Linalool 1103 0.5 0.4

Group sum 95.3 90.7

Oxygenated monoterpenes

8. (Z)-Limonene-oxide 1136 Tr Tr

9. (E)-Limonene-oxide 1141 - Tr

10. Citronellal 1152 Tr 0.1

11. Terpinen-4-ol 1187 0.3 -

12. Decanal 1206 0.6 0.4

13. Nerol 1233 0.1 Tr

14. (Z)-Citral 1240 0.3 0.1

15. Geraniol 1261 0.1 Tr

16. (E)-Citral 1272 0.3 0.1

17. Neryl acetate 1356 0.7 0.1

18. Geranyl acetate 1375 0.4 0.1

Group sum 2.8 0.9

Sesquiterpene hydrocarbons

19. -Elemene 1324 0.1 Tr

20. -Copaene 1369 Tr Tr

21. -Cubebene 1381 - Tr

22 -Elemene 1384 Tr Tr

23. (E)-Caryophyllene 1447 0.4 0.1

24. Valencene 1490 0.1 0.3

25. -Cadinene 1516 0.1 0.1

Group sum 0.7 0.5


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Table 1. (Continued) Oxygenated sesquiterpenes

26. Caryophyllene oxide 1583 - Tr

27. -Sinensal 1696 0.1 Tr

28. -Sinensal 1755 0.1 Tr

29. Nootkatone 1820 Tr Tr

Group sum 0.2 Tr

Other compounds

30. Tetradecanoic acid 1795 Tr Tr

31. Hexadecanoic acid 1993 0.2 1.0

32. Ethyl linoleate 2173 - 2.1

33. Hexacosane 2600 - 0.4

34. Triacontane 3000 - Tr

Group sum 0.2 3.5

Total

99.2 95.6

a – retention index on VF – 5MS column b – limonene, m/z 136 (M+, 18), 121 (18), 107 (16), 93(55), 79 (23), 68 (100), 53 (20), 41(23)

Tr- traces

The yields of essential oil and petrolether extract, obtained from dried plant material were

16.04 g /kg and 285.5 g/kg, respectively. Limonene was the most dominant compound in

the essential oil as well as in petrolether extract, representing over 90% of the volatiles.

The mass spectra of limonene shows strong molecular ion peak on m/z = 136.

Characteristic fragmentation pattern (M-15, M-29, M-43, M-57,..) is observed with very

characteristic fragment for recognising terpenes m/z = 93. The basic peak, represented by

m/z = 68, corresponds to the diene fragment arising from the characteristic fragmentation

pattern that corresponds to a reverse Diels-Alder reaction. The essential oil contained -terpinene (2.7%), while petrolether extract contained fatty acids in higher content, ie.

tetradecanoic and hexadecanoic acid 1.0 and 2.1% respectively.

Due to the high content of limonene, the FTIR spectra when compared to the isolated

essential oil and petrolether extract, completely overlapped with the spectra obtained from

comercial standard of limonene (Fig. 1).


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Fig. 1. FTIR spectra of limonene, essential oil and petrolether extract

Infrared spectra in the range of 3082 - 2835 cm-1

indicate the presence only of C-H bonds,

ie. 3082 - 3011 cm-1

correspond to C(sp2)-H bonds and 2965 – 2835 cm

-1 to C(sp

3)-H

bonds streching. Peaks are observed at 1643 (C=C, alkene), 1435 and 1375 (CH2, CH3,

bend), 887 (oop, mono subst. alkene) and 797 (oop, tri subst. alkene).

Carbon chemical shifts of limonene were deduced from one-dimensional 13

CNMR technique

(APT) (Fig. 2 and Table 2). APT technique provide the information on the nature of the carbon

skeleton where signals of carbons with the odd and even number of attached protons are

pointing in oposite direction (upward and downward). The signals have arised from either C or

CH2 carbons, i.e. 149.75, and 133.22 correspond to C, and 107.84, 30.31, 30.09, and 27.43

correspond to CH2 group. The signals have arised from CH or CH3 carbons ie. 120.14 and

40.59 correspond to CH group, while 22.91, 20.29 correspond to CH3 group.

(a) (b)

Fig. 2. Structure of limonene (a) and hesperetin (b)

1

2

3

4

5

6

78

9

10

O

OH

OCH3

OOH

HO

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16


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Table 2. 13

CNMR (APT) chemical shifts of limonene and hesperetin

Limonene Hesperetin

Atom C(CDCl3) Atom C(DMSO-d6)

1-C 149.75 1-C 196.86

2-C 133.22 2-C 165.15

3-C 120.14 3-C 163.00

4-C 107.84 4-C 162.51

5-C 40.59 5-C 147.93

6-C 30.31 6-C 146.47

7-C 30.09 7-C 131.02

8-C 27.43 8-C 117.67

9-C 22.93 9-C 114.09

10-C 20.29 10-C 112.22

11-C 100.59

12-C 96.42

13-C 95.55

14-C 78.39

15-C 55.75

16-C 42.27

C – chemical shift in ppm

The yellow brown precipitate was obtained using both maceration and Soxhlet extraction.

Colour reactions, though largely empirical in nature, are very useful in structural

elucidations, mostly in studies on alkaloids, steroids and triterpenoids and flavanoids. The

compound isolated after maceration and Soxhlet extraction was yellow brown in color and

both showed a positive ferric chloride and Shinoda test indicating that the compound may

be a flavonoid. The Shinoda test involves a reductive transformation of colourless or pale

yellow coloured flavones and flavonols into deeply coloured products among which are

anthocyanidins (Fig. 3).

Fig. 3. Shinoda test reaction


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The conjugation in flavonoid compounds produces a yellow color, while the extended

conjugation in the resultant anthocyanidin shifts the color further out to the red region of

the visible spectrum. The dramatic change in color makes this a simple visual test for the

presence of flavones. The obtained precipitates (1.48 g) were acidified in order to free the

aglycone.

DSC was carried out and the theromogram demonstrated endothermic peak of melting

point at 225.25 °C (Fig. 4). Further analyses of the isolated compound were carried out, i.e.

UV/Vis, FTIR, 13

CNMR (APT).

The UV/Vis spectra were recorded of the flavonoid in the EtOH solution. The flavonoid

can be regarded as C6-C3-C6 compounds, in which each C6 moiety is a benzene ring, the

variation in the state of oxidation of the connecting C3 moiety determining the properties

and class of each such compound (Fig. 3). The UV/Vis characteristics of flavonoids are

two absorption bands. The band II peak in the obtained UV/Vis spectra of isolated

compound was intense and lied in the 270-295 nm with the maximum max =284.8 nm,

while band I had a small peak. The UV data supported the presence of a flavanone with no

C ring instauration.

Fig. 4. DSC of hesperetin

Integral -622,92 mJ

normalized -80,16 Jg -1

Onset 221,12 °C

Peak 225,25 °C

Endset 227,84 °C

Wg -1

1

°C150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310

^exo

STAR e SW 10. 00Lab: METTLER


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The FTIR spectra obtained from isolated compound illustrated characteristic bands due

to the different functional groups. 3500 cm-1

corresponds to O-H stretching vibration;

3115-3032 cm-1

correspond to C(sp2)-H bonds, 2980-2851 cm

-1 to C(sp

3)-H bonds; 1636

to C=O bond; 1577, 1516, 1506, 1458, 1441, 1400 corresponds to C=C (aromatic ring);

1361, 1339, 1306 to C-O deformation vibrations; 1171 to C-O stretching vibrations.

Carbon chemical shifts of hesperetin are given Figure 2 and Table 2. The signals 196.86,

165.15, 163.00, 162.51, 147.93, 146.47, 131.02 and 100.59 correspond to C, and 42.27

correspond to CH2 group. Oposite pointing signals have arised from CH or CH3 carbons ie.

117.67, 114.09, 112.22, 96.42, 95.55, and 78.39 correspond to CH group, while 55.75

correspond to CH3 group.

All the obtained data were in good concurrence with those reported in the literature.

Biological activity

The major volatile and non-volatile compounds identified in orange peel, limonene and

hesperetin, were tested for their antioxidation as well as AChE and BuChE inhibitory

activity (Table 3). Hesperetin showed good antioxidant activity, while limonene was not

active. In contrast to hesperetin, limonene showed better AChE and BuChE activity.

Table 3. Antioxidative and Cholinesterase (AchE and BuChE) inhibitory activity of major

compounds identified in C. sinensis peel

Limonene Hesperetin

Antioxidant activity

DPPH (inhibition %) b) 5.1 ± 0.3 22.4 ± 2.0

FRAP (mml Vit C/L) b) 0.1± 0.0 1.3 ± 0.1

BROR (minutes) c) n.a. 50.8 ± 1.7

Cholinesterase inhibition

AChE (%) 63.3 a) 6.7 a)

BuChE (%) 15.3 b) 48.2 a)

Compounds tested at stock concentrations of a) 5.0 mg/mL, b) 1.0 mg/mL and c) 0.25 mg/mL; The

final concentrations of tested compounds in reaction systems were 33 µg/mL (DPPH and FRAP);

8.3 µg/mL (BROR); 227 µg/mL i.e. 45 µg/mL (AChE and BuChE); n.a.- not active

Conclusions

Peels, a by-product of the orange, represent a source of limonene and hesperetin. Enzyme

acetylcholinesterase represents an important target in the first stage of Alzheimer’s disease


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(AD) and butyrylcholinesterase enzyme in the later stages of AD. Limonene, the major

compound in volatile isolates (over 90%), showed to be good inhibitor of both enzymes.

Having a small molecular weight and lipid solubility it can probably pass the blood-brain

barrier, and thus represents a potential for treating neurodegenerative diseases such as AD.

Oxidative stress represents important aspect of the research in pathology of

neurodegenerative diseases. The antioxidant activity of limonene was low, while the

activity of hesperetin was significant as well as good inhibitory activity against BuChE,

showing a potential for treatment in the later stages of AD.

Acknowledgments

We sincerely thank M.Chem.Eng. Irena Banovac for technical support in DSC

measurement.

References

Adams, R.P. (1995): Identification of Essential Oil Components by Gas Chromatography/Mass

Spectroscopy. Carol Stream, USA: Allured Publishing.

Blažević, I., Burčul, F., Ruščić, M., Mastelić, J. (2013): Glucosinolates, volatile constituents, and

acetylcholinesterase inhibitory activity of Alyssoides utriculata, Chem. Nat. Comp. 49 (2),

374-378.

Benzie, I. F., Strain, J.J. (1996): The ferric reducing ability of plasma (FRAP) as measurement of

“antioxidant power”: The FRAP assay, Anal. Biochem. 239, 70-76.

De Paula, A.A.N., Martins, J.B.L., dos Santos, M.L., Nascente, L.C., Romeiro, L.A.S., Areas,

T.F.M.A., Vieira, K.S.T., Gamboa, N.F., Castro, N.G., Gargano, R. (2009): New potential

AChE inhibitor candidates, Eur. J. Med. Chem. 44, 3754-3759.

Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M. (1961). A new and rapid colorimetric

determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88-95.

Generalić Mekinić, I., Burčul, F., Blažević, I., Skroza, D., Kerum, D., Katalinić, V. (2013):

Antioxidative/acetylcholinesterase inhibitory activity of some Asteraceae plants, Nat. Prod.

Commun. 8, 471-474.

Katalinić, V., Smole Možina, S., Generalić, I., Skroza, D., Ljubenkov, I., Klančnik, A., (2013):

Phenolic profile, antioxidant capacity and antimicrobial activity of crude leaf extracts of six

Vitis vinifera L. varieties, Int. J. Food Prop. 16, 45-60.

Krishnaswamy, N.R. (1996): Learning organic chemistry through natural products, Resonance, 25-

33.

Liu, Y., Liu, Z., Wang, C., Zha, Q., Lu, C., Song, Z., Ning, Z., Zhao, S., Lu, X., Lu, A. (2014 ):

Study on essential oils from four species of Zhishi with gas chromatography-mass

spectrometry, Chem. Cent. J. 8 (1), 1-8.

Maher, P. (2006): A comparison of the neurotrophic activities of the flavonoid fisetin and some of

its derivatives, Free Radical Res. 40, 1105-1111.


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Schimmenti, E., Borsellino, V. Galati, A. (2013): Growth of citrus production among the Euro-

Mediterranean countries: political implications and empirical findings, Span. J. Agric. Res. 11

(2), 561-577.

Sharma, P., Pandey, P., Gupta, R., Roshan, S., Garg, A., Shulka, A., Pasi, A. (2013): Isolation and

characterisation of hesperidin from orange peel, Indo. Am. J. Pharm. Res. 3 (5), 3892-3897.

WHO (2012) Report. Dementia: a public health priority. Geneva: World Health Organisation.

Qiao Y., Xie B.J., Zhang Y., Zhang Y., Fan G., Yao X.L., Pan S.Y. (2008): Characterization of

Aroma Active Compounds in Fruit Juice and Peel Oil of Jinchen Sweet Orange Fruit (Citrus

sinensis (L.) Osbeck) by GC-MS and GC-O, Molecules 13, 1333-1344.


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Measurement and level regulation with ultrasound sensor

and Arduino microcontroller

UDC: 681.51

Frane Čačić Kenjerić, Ana Jelinić

Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 20,


Summary

Arduino, an open-source microcontroller platform has been proved as modular and inexpensive for

vast number of different tasks and applications. Strong community and potent hardware base with

large number of sensors and actuator that are premade for Arduino platform (called shields) made

this possible. Aim of this work is to assess feasibility of HC-SR04 ultrasound sensors with Arduino

Uno R3 platform as base for model level regulation system, for teaching or non-critical application

(household, hobby or small business). System includes liquid tank, one HC-SR04 ultrasound sensor,

Arduino Uno R3 microcontroller board and relay shield with two solenoid valves.

Keywords: Arduino Uno R3, microcontroller, level measurement, ultrasound

Introduction

Level measurement and regulation plays important role in process engineering, and there is

abundant number of level measurement methods currently available (Roede et al., 2003).

Methods for level measurement differs on underlying physical principle, so choice of

method greatly depends on application (liquid or solid properties, process parameters).

Level measurement methods can be grouped in two groups based on weather sensor comes

in contact with matter of which level is measured, namely contact or non-contact methods.

One of dominantly non-contact (depends on sensor placement) method for level

measurement is ultrasonic method.

Ultrasonic level detectors can be used for wetted or non-contacting switch and transmitter

applications for liquid level or interface and solids level measurements (Jamison et al., 2003).

Operation principle of ultrasonic sensors is based on emitting ultrasonic signal and

measuring time needed for reflected signal (echo) to return or by measuring properties of

return signal (Cheeke, 2002).

Arduino, an open-source microcontroller platform has been proved as modular and

inexpensive platform for vast number of different tasks and applications (Wheat, 2011).



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Potent hardware base with large number of sensors and actuators (called shields) and

integrated development environment, along with large and active community, made

Arduino platform equally popular in hobbyist and academic circuits. Aim of this work is to

assess feasibility of HC-SR04 ultrasound sensor with Arduino Uno R3 platform as simple

solution for level measurement and regulation.


For level measurement and regulation, a model system was constructed around liquid tank

with one HC-SR04 ultrasound sensor, Arduino Uno R3 microcontroller board, four relay

shield, two solenoid valves and laboratory regulated DC power source (Fig. 1).

Fig. 1. Level measuring and regulation model system setup

Tank was fabricated from acrylic glass sheets (0.4 cm thickness) reinforced with

aluminium L profiles (2.5 cm x 2.5 cm), with dimensions 21 cm x 21 cm x 45 cm (width,

depth and height).

Ultrasonic HC-SR04 (Cytron technologies) sensor was mounted on the tank lid (drilled

trough). Ultrasonic sensor has four pins (connections), GND and VCC for powering and

TRIGG and ECHO signals for operation. Sensor requires +5 V for operation, and can be

powered from Arduino Uno R3 microcontrollers power pins. Sensor’s TRIGG and ECHO


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pins are connected to digital I/O pins of Arduino microcontroller. Measurement is initiated

by setting TRIGG pin HIGH (5 V) for 10 µs, after which sensor sends eight 40 kHz pulses

and then waits for echo signal (150 µs to 25 ms) (Cytron, 2013). Distance to target is

determined from pulse with of ECHO signal.

The Arduino Uno R3 is a microcontroller board based on the Atmel ATmega328

microcontroller (Atmel, 2014). It has 14 digital input/output pins (of which 6 can be used

as PWM outputs), 6 analog inputs, a 16 MHz ceramic resonator, a USB connection, a

power jack, an ICSP header, and a reset button. It contains everything needed to support

the microcontroller (Arduino, 2014a).

Two solenoid valves (12 V) were mounted on the inlet and outlet pipes (1/2”). Outlet

solenoid valve was normally closed, gravity feed, DDT-CD-12VDC (EchoTech, USA).

Inlet solenoid valve is normally closed (NC), model TFW-1S (JDC, China). Both valves

operate on 12 V, direct current (DC), while Arduino Uno R3 can only supply 5 V DC. For

this reason external regulated laboratory power source (0 V to 30 V DC) was used with

four relay shield (SainSMART, 2014) to operate valves with Arduino microcontroller.

Personal computer with default Arduino integrated development environment (IDE),

version 1.5.6-r2, was used for program development and microcontroller programming

(download program to microcontroller). IDE is open source and can be downloaded freely

(Arduino, 2014b).

Electrical wiring of model system is shown in Fig. 2.

Fig. 2. Electric wiring of model system for level measurement and regulation


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Model system for level measurement and regulation was developed. Model is built

around liquid tank in which level measurement and regulation can be conducted. Tank is

made from acrylic glass because of processing ease, chemical and physical properties

and low price (Altuglass, 2006). Inlet and outlet pipes were installed with solenoid

valves mounted to control inlet and outlet flows. Solenoid valves were normally closed

type, when coil is not energized, the valve is closed. The Outlet valve was gravity feed,

with no pressure difference needed for operation. Solenoid valves don’t have possibility

of continuous regulation of valve opening, they only have on/off states. Because of this

fact they can be easily operated by microcontroller digital ports in combination with

relays.

Level measuring system is based on the HC-SR04 ultrasonic sensor, which can measure

distance from target in range from 2 cm to 500 cm with accuracy of 0.3 cm (SainSMART,

2014).

Distance (d) [cm] from target is obtained from Eq. 1:

𝑑 =𝑡

58 [𝑐𝑚] (1)

where t is pulse width in µs.

Level in tank (hm) can be calculated as tank height (h) minus distance (d) from sensor to

liquid gas interface in tank (Eq. 2):

ℎ𝑚 = ℎ − 𝑑 [𝑐𝑚] (2)

The sensor was connected to Arduino Uno R3 power (GND to GND and VCC to 5 V pin)

and to digital I/O pins, pin 7 to TRIG configured as output and pin 8 to ECHO as digital

input. Program was made to take level measurements and to print results over serial port to

the connected personal computer. Series of static level measurement was conducted in

order to estimate accuracy of measurement system. Referent levels of liquid were set with

use of measure tape (accuracy ± 0.05 cm) in range from 5 cm to 40 cm in increment of 1

cm, with multiple repetitions. Some of reference points and measurement results are

showed in Table 1.


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Table 1. Static level measurements with HC-SR04 sensor

h_ref [cm] h1 [cm] h2 [cm] h3 [cm] average [cm] h_SD [cm]

40.00 40.00 40.00 40.00 40.00 0.00

39.00 39.31 39.32 39.12 39.25 0.11

37.00 36.98 37.06 37.07 37.04 0.05

36.00 36.35 36.06 26.06 36.21 0.21

35.00 34.87 34.95 35.02 34.95 0.08

30.00 29.86 29.79 29.81 29.82 0.04

29.00 29.23 28.83 28.87 28.98 0.22

28.00 27.81 27.50 27.92 27.74 0.22

26.00 25.75 26.11 25.93 25.93 0.18

25.00 24.75 24.86 24.82 24.81 0.06

24.00 24.10 24.10 23.70 23.97 0.23

23.00 22.97 22.80 22.82 22.86 0.09

22.00 21.79 21.77 21.81 21.79 0.02

20.00 19.73 19.72 19.73 19.73 0.01

19.00 18.68 18.67 18.74 18.70 0.04

18.00 17.79 17.68 17.69 17.72 0.06

17.00 16.82 16.67 16.68 16.72 0.08

16.00 15.60 15.62 15.65 15.62 0.03

15.00 14.72 14.70 14.70 14.71 0.01

14.00 14.03 13.66 13.62 13.77 0.23

12.00 11.99 11.59 11.85 11.81 0.20

10.00 10.25 10.01 9.97 10.08 0.15

9.00 8.61 8.61 9.14 8.79 0.31

8.00 7.52 7.49 7.61 7.54 0.06

7.00 7.41 7.00 6.96 7.12 0.25

5.00 4.71 4.98 4.85 4.85 0.14

Results of static measurements showed that results are within results specified by

manufacturer of HC-SR4 ultrasonic sensors. Stable measurements and accuracy were

achieved by creating repeating loop of measurements of ECHO signal width, then

averaging taken measurements. With this approach, some noise is filtered while retaining

good sampling time of level measurement (every second).

Dynamic level measurement, level measurement while filling or emptying tank, showed

that HC-SR04 ultrasonic sensors is sensitive to noise that is related with fluid flow due to

forming of air bubbles and waves in tank. This noise measurements were dominant while

filling tank with high flow rates. Another possible factor is dimensions of model system,

tanks with larger cross section would be less prone to rippling liquid surface, and would be

larger target for ultrasonic sound reflection (SainSMART, 2014). Level measurement

during filling of tank with three different intake flows are shown in Fig. 3.


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Fig. 3. Dynamical measurements of liquid level for three different intake flows

Basic two way [on/off] level regulation algorithm was used for controlling of solenoid

valves, which enables regulation of constant liquid level in tank or filling/emptying tank to

set liquid level. Algorithm is based on comparing measured liquid level with set and

controlling appropriate solenoid valve (open/close) for filling or emptying the tank.

Program in main loop (obligatory loop() function) calls function for level measurement,

which returns measured level (hm), then the measured level is compared to set level value

(hs) by series of if then..else if logic structures to determine if measured level is lower,

higher or equal to set level. According to identified relation program calls functions for

opening or closing right solenoid valve. Logical case which checks if measured level

equals set level, hm=hs, can lead to erratic switching (continuous open/close cycles) of

solenoid valves as the measured level approaches set level value. This behaviour can be

avoided by introducing acceptable deviation (error) ε=hm-hs from set level value. So

instead checking equality of set and measured level values, program checks if measured

value is within hs-ε<hm<hs+ε range. Regulation of liquid level with described algorithm

resulted with maximum error of ± 0.2 cm from set level, which indicates that level

regulation quality is limited with level measurement precision. Regulation of set level

change for ± 5 cm [input step change] is shown in Fig. 4.

0

5

10

15

20

25

0 10 20 30 40 50 60

Liq

uid

lev

el [

cm]

Time[s]

Flow1 Flow2 Flow3


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Fig. 4. Level regulation for increase level setpoint (A)

and decrease in level setpoint (B)

18

19

19

20

20

21

21

22

22

23

23

24

24

25

25

26

0 50 100 150 200

Lev

el [

cm]

Time[s]

A

18

19

19

20

20

21

21

22

22

23

23

24

24

25

25

26

26

27

0,0 50,0 100,0 150,0 200,0

Lev

el [

cm]

Time[s]

B


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Conclusions

Arduino Open Source platform (hardware and software) presents great learning and

prototyping tool which enables very fast solution development. Integrating Arduino Uno

R3 with HC-SR04 ultrasonic sensor and two solenoid valves resulted in low-cost model

system for level measurement and regulation. System that can be used as teaching tool or

applied for non-critical level regulation in households and small business. Further

improvements can be made by programmatically, by implementing other regulation

algorithms (PI, PD or PID) or by replacing solenoid valves with servo.

References

Altuglass (2006): Plexiglas, Acrylic Sheet General Information and Physical Properties,

Philadelphia, USA, Arkema Inc.

Arduino (2014a): Arduino Overview, http://arduino.cc/en/Main/ArduinoBoardUuno, [01. 10. 2014].

Arduino (2014b): Download the Arduino Software, http://arduino.cc/en/Main/Software, [01. 10.

2014].

Atmel (2014): Atmel 8-bit Microcontroller with 4/8/16/32 Kbytes In-system Programmable Flash

Datasheet, San Jose, USA, Atmel Corporation.

Cheeke, J.D.N. (2002): Fundamentals and Applications of Ultrasonic Waves, Montreal, Canada,

CRC Press.

Cytron Technologies (2013): Product User’s Manual – HC-SR04 Ultrasonic sensor V1.0, Skudai,

Malaysia, Cytron Technologies.

Jamison, J.E., Liptak, B.G., Kayser, D.S. (2003): Level measurement Ultrasonic Level Detectors.

In: Instrument Engineers Handbook: Process Measurement and Analysis 4th

ed. Vol I, Liptak,

B.G. (ed.), Boca Raton, USA: CRC Press, pp. 548-555.

Roede, J.B., Liptak, B.G., Kayser, D.S. (2003): Level measurement Application and Selection. In:

Instrument Engineers Handbook: Process Measurement and Analysis 4th ed. Vol I, Liptak,

B.G. (ed.), Boca Raton, USA: CRC Press, pp. 405-419.

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Wheat, D. (2011): Arduino Internals, New York, USA, Apress.


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Elderberry as important source of antioxidant

and biologically active compounds

UDC: 582.971.1

Eva Ivanišová, Helena Frančáková, Štefan Dráb, Silvia Benčová

Department of Storing and Processing Plant Products, Faculty of Biotechnology and Food Science,

Slovak University of Agriculture, Tr. A. Hlinku 2, Nitra, SK-949 76, Slovak Republic

Summary

Elders (Sambucus spp.) are widely distributed throughout the world. In central Europe, the most

common species are black elder (Sambucus nigra L.). Elderberry bark, root, leaves, flower and

fruits have been used particularly by the rural population as medicine and food. This study

examined the polyphenol composition and antioxidant properties of ethanolic extracts from

elderberry bark, leaves, flower and fruit. The aim of this study was also to determine the total

anthocyanin content in fruit. The total phenol content amongst the ethanolic extracts was higher in

flower (28.89 mg GAE/g DM) and decreased in the following order: fruit > bark > leaves. The total

flavonoid content was higher in flower (52.40 μg QE/g DM) and decreased in the following order:

leaves > fruit > bark. Antioxidant capacity, expressed as mg TEAC/g DM measured by the radical

2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity and the phosphomolybdenum method

(reducing power) was high in all observed parts of elderberry. The total anthocyanin content in fruit,

determined according to the pH differential method was 2.07 mg.g-1

of dry matter.

Keywords: elder, polyphenol, flavonoid, anthocyanin, antioxidant activity

Introduction

Nowadays is great interest in determining the role of phytonutrients in promoting

improved health mainly in reducing cancer, cardiovascular disease, and the effects of

aging. It is generally known that antioxidant phytonutrients can inhibit free radical

reactions that may ultimately lead to the development of diseases, especially those which

are cancer related. Analysis in several studies and researches shows that many medicinal

herbs, fruits and vegetables have strong antioxidant capacities (Milbury et al., 2002). One

of the very good sources of phytonutrients is elderberry, which is used mainly in folk

medicine, but future trends show that can be used more mainly in food industry.

Elderberry (Sambucus nigra L.) is the most widespread, being found across Europe, central

and western Asia, and Northern Africa. Elderberry is a deciduous shrub that grows to a



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height of 4-6 m (Krawitz et al., 2011). From spring until summer the corymbs are in

flower. The fruits are dark violet- black drupes which grow in clusters and are only edible

when fully ripe. Elderberry fruits are an excellent source of anthocyanins, vitamins A and

C and a good source of calcium, iron and vitamin B6. They also contain sterols, tannins,

and essential oils and can readily be considered a healthy food. Moreover, other parts of

this plant, such as flowers, leaves and bark, used commonly in traditional medicine and

healing are often extraordinary rich in biologically active compounds (Cejpek et al., 2009).

Elderberry is employed as an alternative to conventional medicines and mainly in the form

of an extract for treating the common cold, influenza and herpes virus infections (Liu et al.,

2008; Roschek, 2009). The consumption of elderberry, and also use in food industry is not

very common and only a few studies have been reported. However, none of these studies

focus mainly on elderberry anthocyanin composition.

The aim of this study was to determine the antioxidant capacity and phenolic content of the

ethanol extracts from dry bark, leaves, flowers and fruit of elderberry.


Plant material

Elderberry parts (bark, leaves, flower and fruit) were collected from nature locality Michal

nad Žitavou (Slovak republic) in term recommended by pharmacology protocol: bark in

March, leaves in April, flower in May and fruit in September. Elderberry parts were dried

in room temperature and homogenized to powder.

Chemicals

All chemicals were analytical grade and were purchased from Reachem (Slovakia) and

Sigma Aldrich (USA).

Sample preparation

0.2 g of each sample was extracted with 20 mL of 80% ethanol for 24 hours. Extraction

was all carried out in triplicate. After centrifugation at 4000 g (Rotofix 32 A, Hettich,

Germany) for 10 min, the supernatant was used for measurement (DPPH method,

phosphomolybdenum method, total phenolic content, total flavonoid content).

1 g of fruit was repeatedly extracted with 10 mL of ethanol containing 37% hydrochloric

acid until the berries became colourless. The extract was filtered to remove fibrous

particles and used for measurement (total anthocyanin content).


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Radical scavenging activity

Radical scavenging activity of samples was measured using 2,2-diphenyl-1-picrylhydrazyl

(DPPH) (Sánchéz-Moreno et al., 1998). The extracts (0.4 mL) were mixed with 3.6 mL of

DPPH solution (0.025 g DPPH in 100 mL ethanol). Absorbance of the elderberry extracts

was determined using the spectrophotometer Jenway (6405 UV/Vis, England) at 515 nm.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (10-100 mg.L-1

;

R2=0.989) was used as the standard and the results were expressed in mg.g

-1 Trolox

equivalents.

Reducing power

Reducing power of samples was determined by the phosphomolybdenum method of Prieto

et al., (1999) with slight modifications. The mixture of elderberry extract (1 mL),

monopotassium phosphate (2.8 mL, 0.1 M), sulfuric acid (6 mL, 1 M), ammonium

heptamolybdate (0.4 mL, 0.1 M) and distilled water (0.8 mL) was incubated at 90 °C for

120 min, then rapidly cooled and detected by monitoring absorbance at 700 nm using the

spectrophotometer Jenway (6405 UV/Vis, England). Trolox (10-1000 mg.L-1

; R2=0.998)

was used as the standard and the results were expressed in mg.g-1

Trolox equivalents.

Total polyphenol content

Total polyphenol content of elderberry extracts was measured by the method of Singleton

and Rossi, (1965) using Folin-Ciocalteu reagent. 0.1 mL of each sample extract was mixed

with 0.1 mL of the Folin-Ciocalteu reagent, 1 mL of 20% (w/v) sodium carbonate, and 8.8 mL

of distilled water. After 30 min. in darkness the absorbance at 700 nm was measured using

the spectrophotometer Jenway (6405 UV/Vis, England). Gallic acid (25-300 mg.L-1

;

R2=0.998) was used as the standard and the results were expressed in mg.g

-1 gallic acid

equivalents

Total flavonoid content

Total flavonoids were determined using the modified method of Willett, (2002). 0.5 mL of

elderberry extract was mixed with 0.1 mL of 10% (w/v) ethanolic solution of aluminium

chloride, 0.1 mL of 1 M potassium acetate and 4.3 mL of distilled water. After 30 min. in

darkness the absorbance at 415 nm was measured using the spectrophotometer Jenway

(6405 UV/Vis, England). Quercetin (0.5-20 mg.L-1

; R2=0.989) was used as the standard

and the results were expressed in μg.g-1

quercetin equivalents.


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Anthocyanin content

Total anthocyanins were measured according to the method originally described by

Fuleki and Francis, (1968) with some modifications (Lee et al., 2005). For pH 1.0,

sample was diluted with 0.025 M potassium chloride. For pH 4.5, sample was diluted

with 0.4 M sodium acetate buffer. The absorbance of diluted sample was measured at

520 nm and 700 nm against distilled water as blank. The concentration (mg.g-1

) of each

anthocyanin was calculated according to the following formula and expressed as

cyanidin-3-glucoside (Cy -3-glc) equivalents:

𝑐 [𝐴𝑛𝑡ℎ𝑜𝑐𝑦𝑎𝑛𝑖𝑛] =𝐴.𝑀𝑤.𝐷𝐹.103

𝜀.𝐿 (1)

where A is the absorbance difference =(A540–A690)pH 1.0–(A540–A690)pH 4.5, MW is the

molecular weight of Cy-3-glc=449.2 g.mol-1

, DF is the dilution factor DF=1), and ε is the

extinction coefficient of Cy-3-glc=1700 cm-1

mol-1

, L is path length in cm=1.

Statistical analysis

Spectrophotometric analyses were carried out in triplicate. For statistical analysis of

experimental data, correlation between measured values was used (Microsoft Office – Excel).


Radical scavenging activity

DPPH• is a stable radical in solution and appears purple colour absorbing at 515 nm in

methanol and ethanol. DPPH method due to the simplicity of the assay and the fact that it

can be used in aqueous and lipid phases has become routine practice in evaluating plant

materials. The scavenging effect of elderberry extracts on DPPH radical decreased in this

order: flower > bark > fruit > leaves (Fig. 1).

These results indicated that all the extracts had a noticeable effect on scavenging free

radical. The higher activities were measurement in flower (8.61 mg TEAC.g-1

) and bark

(7.00 mg TEAC.g-1

) extract. From the literature it is known, that the elderberry is an

excellent source of natural antioxidant either for food preservation (to inhibit lipid

oxidation), or for disease prevention. Several researchers have studied antioxidant

activities of elderberry, mainly in flower and fruit and obtained interesting results. Stoilova

et al., (2007) tested antioxidant activity of elderberry flower by DPPH and deoxyribose

assay and found high antioxidant effect. Duymus et al., (2014) determined antioxidant

activity of elderberry fruit in different solutions and the highest activity found in 70%


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acetone extract and in water extract. Strong activity by DPPH method also detected

Jakobek et al. (2007) in elderberry fruit – 62μmol TE/mL. Wu et al. (2004) showed that the

elderberry had a much higher potential than cranberry and blueberry, two fruits praised for

their high antioxidant capacity. The elderberry bark is not so known for used in medicine.

These results showed that bark is also very good source of antioxidant compounds, but

these compounds need more studies. Many compounds presented in elderberry mainly in

flower and fruit have not only antioxidant but also antimicrobial effect. Elderberry flower

extract at a concentration of 252 μg/mL inhibited the influenza A virus (H1N1) (Sidor and

Michalowska, 2014). Antibacterial activity of elderberry extract was also demonstrated in

relation to gram-positive Streptococcus pyogenes, Streptococcus group G and

Streptococcus group C, and gram-negative Branhamella catarrhalis bacteria causing

frequent infections of the upper respiratory tract (Krawitz et al., 2011). The antimicrobial

activity of flower extracts is higher with compared to fruit extracts. The present study was

in agreement with the findings of these previously reported studies and shows that

elderberry represents an exceptional source of antioxidant activity.

Fig. 1. Radical scavenging activity of elderberry extracts expressed as mg Trolox

equivalent antioxidant capacity per g of dry matter

Reducing power

For measurement of the reductive ability, the MoVI+

- MoV+

transformation in the presence of

elderberry extracts was investigated. Reductive capabilities of elderberry extracts shown Fig. 2.


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Increase in absorbance of the reaction mixture indicated the reducing power of the samples.

Reducing power of extracts exhibited the following order: fruit > leaves > flower > bark. The

reducing properties are generally associated with the presence of reductones (Pin-Der, 1998).

It is reported that the antioxidant action of reductones is based on the breaking of the free

radical chain by donating a hydrogen atom, or reacting with certain precursors of peroxide to

prevent peroxide formation. It is presented that the phenolic compounds in plants may act in

a similar fashion as reductones by donating electrons and reacting with free radicals to

convert them to more stable products and terminating the free radical chain reaction (Liu and

Yao, 2007). The data presented here indicate that the marked reducing power of elderberry

extracts seem to be the result of their antioxidant activity. Strong reducing power was

detected in extract from fruit (317.30 mg TEAC.g-1

). Elderberry fruit is in food industry used

for prepare juice and also wine. Rupasinghe and Clegg (2007) determined the reducing

power by FRAP method in different wines and confirmed the highest activity for elderberry

wines (>1590 mg AAE/L). High reducing power by FRAP method also found Jabłońska-Ryś

et al. (2009) – 29.56 mM Fe.100 g-1. The consumption of 25-75 g elderberry fruit can cover

the necessary antioxidant units per day (Denev et al., 2013). The extract from elderberry fruit

had similarly like extract from flower not only antioxidant effect but also antimicrobial

effect. Mohammadsadeghi et al. (2013) reported that extract from elderberry fruits can

inhibit growth of Candida albicans, Pseudomonas aeruginosa, Salmonella typhi and

Escherichia coli. Strong antioxidant and also antimicrobial effect of elderberry fruit can be

used more in future mainly in food industry.

Fig. 2. Reducing power of elderberry extracts expressed as mg Trolox

equivalent antioxidant capacity per g of dry matter


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Total polyphenol and flavonoid content

The results of total polyphenol and flavonoid content are presented in Table 1. Total

phenolic content of elderberry extracts decreased in this order: flower > fruit > bark >

leaves; total flavonoid content in this order: flower > leaves > fruit > bark. The higher

content of these compounds was found in elderberry flower (28.89 mg GAE.g-1

; 52.40 μg

QE g-1

) and fruit (20.55 mg GAE.g-1

; 15.01 μg QE g-1

). The values of total polyphenol and

flavonoid content were significantly and positively related with DPPH method (P ≥ 0.05).

Different phenolic compounds like chlorogenic acid and caffeic acid, rutin, isoquercitrin,

p-coumaric acid, quercitin, kaempferol, isorhamnetin-3-glucoside and isorhamnetin-3-

rutinoside have been identified in flower and leaves of Sambucus nigra (Nagl et al., 2006).

Stroh et al., (1962) found the 1-O-ß-D-glucose esters of caffeic acid and ferulic acid in

flower and Reschke and Herrmann (1981) detected, in addition to these compounds,

the 1-O-ß-D-p-coumaryl glucose ester in fruit. Stoilova et al. (2007) shown that the

extracts obtained from the flower are rich in rutin and tannins contain about 1.8% (rutin,

isoquercetin, quercetin glycosides) and phenolic acids. Kim et al. (2003) determined the

total phenolic content in flower by Folin-Ciocalteu reagent – 19.4 mg.g -1

of dry extract per

gallic acid. López-Garcia et al. (2013) detected in elderberry flower extract presence of

gallic, vanillic and caffeic acid and 18.40 mg GAE.g-1

total polyphenol content in this

extract. Fruits contained chlorogenic, crypto chlorogenic and neochlorogenic acids, while

additionally small amounts of ellagic acid were also determined (0.04 mg.100 g-1

fruit)

(Fazio et al., 2013).

Table 1. The total polyphenol (TPC) and flavonoid content (TFC) of elderberry extracts

Sample TPC [mg GAE.g-1] TFC [μg QE.g-1]

Bark 8.14 ±0.77 8.78 ±0.46

Leaves 6.71 ±1.26 36.05 ±0.96

Flower 28.89 ±0.54 52.40 ± 0.52

Fruit 20.55 ± 2.17 15.01 ±1.87

Anthocyanin content

The total anthocyanin content was determined in elderberry fruit extract – 2.07 mg-g-1

.

This study confirm that fruit is very good source of these biologically active natural

colorants. The total anthocyanin content varies during the growing season and by cultivar

Cyanidin-3-sambubioside (cya-3-sam) and cyanidin-3-glucoside (cya-3-gluc) are the major

anthocyanidin glycosides of elderberry fruit and derived products (Netzel et al., 2005).

Dauymus et al., (2014) detected in elderberry fruit cyanidin glycosides, such as cyanidin-


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3,5-diglucoside, cyanidin-3-sambubioside-5-glucoside, cyanidin-3-sambubioside,

cyanidin-3-glucoside and quercetin-3-rutinoside. Youdim et al. (2000) found that after

consumption of elderberry fruit endothelial cells incorporate anthocyanins into the

membrane and cytosol, conferring significant protective effects against oxidative stress.

Conclusions

Natural sources of antioxidants are an important nutritional supplement and they have

abroad spectrum of application in food, pharmaceutical and cosmetic industries. Elderberry

is rich sources of diverse bioactive compounds with high antioxidant potential,

significantly affecting the health status of consumers. Results from many studies point to

the beneficial effect of consumption of elderberry preparations. Monitoring of antioxidant

and other biological properties of elderberry parts is one of the many options to secure new

sources of active substances essential for the production of functional food and to improve

the health status of the population.

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prepared elderberry foods and supplements, Czech J. Food Sci. 27 (special issue), 45-48.

Denev, P., Lojek, A., Ciz, M., Kratochvilova, M. (2013): Antioxidant activity and polyphenol

content of Bulgarian fruits, Bulg. J. Agric. Science. 19 (1), 22-27.

Duymus, H.G., Göger, F., Hasmu, C.B. (2014): In vitro antioxidant porperties and anthocyanin

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Fazio, A., Plastina, P., Meijerink, J., Witkamp, R. F., Gabriele, B. (2013). Comparative analyses of

seeds of wild fruits of Rubus and Sambucus species from Southern Italy: Fatty acid

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cranberry juice, Food Scien. 33 (3), 78-83.

Jabłońska-Ryś, E., Zalewska-Korona, M., Kalbarczyk, J. (2009): Antioxidant capacity, ascorbic

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antioxidant activity of various red fruit juices, Deut. Lebensm-Rund. 103 (2), 58-64.

Kim, D., Jeond, S., Lee, C. (2003): Antioxidant capacity of phenolic phytochemicals from various

cultivars of plums, Food Chem. 81 (3), 321-326.

Krawitz, Ch., Abu Mraheil, M., Stein, M., Imirzalioglu, C., Domann, E., Pleschka, S., Hain, T.

(2011): Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant

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Lee, J., Durst, R.W., Wrolstad, R.E. (2005): Determination of total monomeric anthocyanin pigment

content of fruit juices, beverages, natural colorants, and wines by the pH differential method:

Collaborative study, J. AOAC Int. 88 (5), 1269-1278.

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flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities,

Bioorg. Med. Chem. 16 (7), 141-7147.

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of edible flowers incorporated onto atelocollagen matrices and their effect on cell viability,

Molecules. 18, 13435-13445.

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Mohammadsadeghi, S., Malekpour, A., Zahedi, S., Eskandari, F. (2013): The antimicrobial activity

of elderberry (Sambuscus nigra L) extract against gram positive bacteria, gram negative

bacteria and yeast, Res. J. Appl. Scien. 8 (4), 240-243.

Nagl, M., Eder, S., Wendelin, S., Reich, G., Sontag, G. (2006): Qualitative and quantitative analysis

of phenolic constituents in elderberry juices, Nutrition. 30 (10), 409-415.

Netzel, M., Strass, G., Herbst, M., Dietrich, H., Bitsch, R., Bitsch, I., Frank, T. (2005): The

excretion and biological antioxidant activity of elderberry antioxidants in healthy humans,

Food Res. Intern. 38 (8-9), 905-910.

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free radical and and active oxygen, J. Am. Oil. Chem.75 (4), 455-461.

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through the formation of a phosphomolybdenum complex: specific application to the

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phenolics of fruits, Z Lebensm. Unters. Forsch. 173: 458-463.

Roschek, B., Fink, R.C., McMichael, M.D., Li, D., Alberte, R.S. (2009): Elderberry flavonoids bind

to and prevent H1N1 infection in vitro, Phytochem. 70 (10), 1255-1261.

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elements, and histamine concentrations in wines of different fruit sources, J. Food Compos.

Anal. 20 (2), 133-137.

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efficiency of polyphenols, J. Sci Food Agricult. 76 (2), 270-276.

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phosphotungstic acid reagents, Amer. J. Enol. Agricult. 16 (48), 144-158.

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Vitamin E in vegetable oils as determined by RP-HPLC with UV detection

UDC: 641.18 : 664.34

Daniela Kenjerić, Blanka Bilić, Ivan Tomas, Milica Cvijetić

Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 20,


Summary

Vitamin E is the general term used to describe a group of eight natural isomers which along with

vitamin A, D and K form the group of fat soluble vitamins. Vegetable oils are one of the most

important dietary sources of vitamin E which, as antioxidant, prevents oil rancidity during storage

thus prolonging its shelf-life. Among eight natural isomers belonging to this vitamin, α-tocopherol

is the most active in vivo and the most represented in vegetable oils of green plants. Vitamin E

presence in vegetable oils depends on the oil type, production process, freshness and storage

conditions. The aim of this work was to determine concentrations of α-tocopherol in various edible

vegetable oils. Rapid reverse-phase high-performance liquid chromatography method with

apsorptiometric detector was used for determination. Fourteen edible vegetable oil types were

analysed. Some of them, like sunflower and rapeseed oil, are widespread and commercially

available while others have attracted scientific curiosity just recently and therefore data on their

composition are scarce. Sunflower and chestnut oil had the highest amount of α-tocopherol, while

amounts in linseed and chia oil were below the level of detection. Large differences in the content of

α-tocopherol have been found between fresh cold pressed oil samples and commercial ones. This

has been especially noted in sunflower oil in which degradation was additionally monitored during

storage.

Keywords: vegetable oils, RP-HPLC, vitamin E, α-tocopherol, dietary intake

Introduction

Vitamin E is a generic name for a group of plant soluble lipid compounds including 4

tocopherols (α-, β-, γ- and δ-) and 4 tocotrienols (α-, β-, γ- and δ-) (Beltrán et al., 2010;

Blekas et al., 1995; Brigelius-Flohé, 2006; Gimeno et al., 2000). The specific tocopherols

and tocotrienols differ by the number and positions of the methyl groups on their 6-chromanol

ring (Eitenmiller and Lee, 2004).

Vitamin E has many biological functions, and as the major lipid soluble antioxidant

decreases the risk of cardiovascular diseases and cancer (Brigelius-Flohé, 2006; Gimeno et

al., 2000). α-Tocopherol is the most common form of vitamin E occurring in the human



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body, and it shows the highest biological activity among all compounds belonging to the

vitamin E (Beltrán et al., 2010). In human body, α-tocopherol seems to be the most

involved in gene regulation, while γ-tocopherol appears to be highly effective in

prevention of cancer-related processes (Brigelius-Flohé, 2006).

Recommended dietary allowance (RDA) of vitamin E for adults, which is set to meet the needs

of almost all (97 to 98 percent) individuals in a group and includes both naturally occurring

RRR-α-tocopherol and 2R-stereoisomeric forms of α-tocopherol that occur in the fortified foods

and supplements, is 15 mg for both, males and females (FNB and IOM, 2004).

6-hydroxychromanols that constitute the vitamin E family are plant products of well-

defined biosynthetic routes, and synthesis has not been documented in any other organisms

but photosynthetic ones. Therefore, plant products provide the only natural dietary sources

of vitamin E (Eitenmiller and Lee, 2004). The main single most important and

concentrated dietary source of vitamin E are vegetable oils (Gimeno et al., 2000) in which

vitamin E as natural antioxidants prevents the rancidity during storage and thus delays

shelf-life of a product (Beltrán et al., 2010; Blekas et al., 1995; Gimeno et al., 2000).

Vitamin E presence in vegetable oils ranges from 1 mg/100g to 162 mg/100g, depending

on the oil type, production process, freshness and storage conditions (Combs, 2008).

α-Tocopherol represents more than 95% of the total tocopherol content of olive oil,

β-tocopherol was found at very low concentrations, while the remaining part belonged

to the γ-tocopherol (Beltrán et al., 2010). Literature reports similar content of α- and

δ-tocopherol in canola (Goosens and Marion, 2011), while in soybean oil γ-

(Eitenmiller and Lee, 2004) and δ-tocopherol (Goosens and Marion, 2011) dominate

over α-tocopherol with γ-tocopherol being the most represented.

Oil tocopherol content and composition are also influenced by genetic characteristics

(cultivar), crop year (climatic conditions like rainfall) and harvesting (ripening stage)

(Gimeno et al. 2002; Beltrán et al., 2010).

The aim of this study was to determine the edible plant oil content of α-tocopherol as

biologically most active, and at the same time most represented compound of vitamin E.

Additionally, degradation of α-tocopherol during the room temperature storage was tested

on sunflower oil which is the widespread oil type in the eastern Croatia region where this

study was carried out. Obtained values were also used to estimate contribution of analysed

oils to the Dietary Reference intakes (DRIs) of the vitamin E.


A quick and direct RP-HPLC method with UV detection for measuring tocopherols in

vegetable oils developed by Gimeno et al. (2000) was used for vitamin E determination.

All the analyses were performed in duplicates.


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Oil samples

Fourteen edible vegetable oil types were analysed: chia, linseed, apricot kernel, poppy

seed, camelina, walnut, sesame, hemp, olive, pumpkin seed, rapeseed, soybean, hazelnut

and sunflower oil. Some of them, like sunflower, soybean, rapeseed and olive oil, are

widespread and commercially available while others, like hemp and chia have attracted

scientific curiosity just recently and therefore data on their composition are scarce.

Each oil type was represented by at least two samples and exact number of each oil type

samples is given on Fig. 1.

Most of the oil types were covered by samples which were obtained by cold pressing

method with a screw expeller in laboratory conditions and were analysed within a week of

production, as well as with oil samples purchased from the retailers where average

consumers supply themselves. The purpose of this oil sample selection method was to get

an insight into the vitamin E content in fresh samples, as well as in those which an average

consumer can purchase for everyday use. Until the analysis samples were stored under the

ambient conditions in filled capped plastic containers.

Storage of the selected sunflower oil sample was performed under the ambient conditions

but in a dark place during one month period to simulate household managing.

Preparations of standard and instrument calibration

Standard solutions were prepared by diluting α-tocopherol in respectable amounts of

solvent to cover literature concentrations (0.3 -38.7 mg/100 g of oil) of the compound of

interest, as well as the concentrations ±10% of those reported to enable analysis due to the

natural variability of the analyte in the samples.

Preparations of oil sample for RP-HPLC analysis

Oil samples were diluted in hexane (1:10) and an aliquot (400 µl) is mixed with methanol

(1200 µl) and internal standard (300 µg/ml of α-tocopherol acetate in ethanol) solution

(400 µl). Sample solution was vortex-mixed and after that centrifuged (3000g, 5 min).

Prior to HPLC analysis, sample was filtered through a 0.45 µm nylon micro filter.

Reversed-phase high-performance liquid chromatography (RP-HPLC)

The analyses were performed on a Shimadzu HPLC system (Shimadzu Corporation,

Kyoto, Japan) consisted of LC-20AD Prominence solvent delivery module, DGU-20A5R

degassing unit, SIL-10AF automatic sample injector and SPD-M20A Prominence UV/VIS

photodiode array detector. Instrument was coupled to a computer and supported by Lab

Solutions Lite Version 5.52 software.


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Separation was achieved by Hibar® 125-4, LiChrospher® 100 RP-18 (5 µm) column

(Merck, Germany) and 2ml/min isocraticaly runned mobile phase consisting of methanol and

water (96:4; v/v). Sample injection volume was 50 µl. Tocopherol was detected at 292 nm.

Identification of compounds was conducted on the basis of the retention times of the

standard and sample compounds and UV absorption spectrum of the same compounds,

while concentrations were calculated from the integrated areas of the samples and

corresponding calibration curves of α-tocopherol standard (internal standard method).


α-Tocopherol content of analysed edible plant oils

Obtained average values of α-tocopherol content in analysed edible oil samples are shown

in the Fig 1. From the results it is visible that the most spread sunflower oil is also the oil

with the highest α-tocopherol content. Substantial amount are recorded also in soybean

and rapeseed oils which are also highly consumed, while chia and linseed oil content of

α-tocopherol content was below the level of detection.

Fig. 1. Average α-tocopherol content (mg/100g) of analysed oil types


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α-Tocopherol content of Argentinean sunflower oil reported by Crapiste et al. (1999)

was 723 mg/kg and 701 mg/kg in two crude sunflower oils obtained by pressing, and

703 mg/kg in crude sunflower oil sample obtained by extraction method. Average values

of sunflower oils found in the literature are 55.4±14.1 mg/100 g of α-tocopherol and

56.6±14.1 mg/100g α-tocopherol equivalents mg (Eitenmiller and Lee, 2004). Therefore,

the results of the sunflower oil samples presented in this paper (68.88 mg/100 g of oil) are

within the literature average values. Nevertheless, it should be considered that the values of

α-tocopherol obtained in this study varied from 39.22 mg/100 g which were recorder for

the commercially supplied oil up to 81.88 mg/100 g which were recorded in the fresh cold

pressed oil sample.

Soybean and rapeseed (canola) oil are, beside sunflower oil, two most common sort oil

types. According the obtained results in the present study, their α-tocopherol content is

similar (28.79 mg/100 g and 28.37 mg/100 g, respectively). Soybean oil analysed by the

same method as in the present study contained 16±2.3 ppm α-tocopherol and 147±1.8 ppm

δ-tocopherol (Goosens and Marion, 2011). Average values of soybean oils found in the

literature are 8.2±4.2 mg/100g of α-tocopherol, 69.3±14.3 mg/100g of γ-tocopherol and

28.1±10.5 mg/100g of δ-tocopherol resulting in sum of 17.0±4.6 mg/100g α-tocopherol

equivalents mg (Eitenmiller and Lee, 2004). Canola oil analysed by the same method as in

the present study contained 19±2.3 ppm α-tocopherol and 22.9±0.65 ppm δ-tocopherol

(Goosens and Marion, 2011). Average values of canola and rapeseed oils found in the

literature are 21.9±6.3 mg/100g of α-tocopherol and 26.7±7.3 mg/100g α-tocopherol

equivalents mg (Eitenmiller and Lee, 2004).

Olive oil represents an important part of the Mediterranean diet (Šarolić et al., 2014) and

Mediterranean Adequacy Index (Alberti et al., 2009; Fidanza et al., 2004), and as such is

one of the most advisable and most popular oils worldwide. Average values of olive oils of

different origin (Italy, Spain, Tunisia, and Greece) are 13.5±4.7 mg/100g of α-tocopherol

and 13.6±4.7 mg/100g α-tocopherol equivalents (Eitenmiller and Lee, 2004). Content

of α-tocopherol in fresh Greece olive oil samples studied by Nissiotis and Tasioula-

Margari (2002) varied from 139.29 mg/kg up to 175.17 mg/kg. Beltrán et al. (2010) reported

average α-tocopherol content of 30 Spanish olive oil cultivars to be 278±102 mg/kg, with the

lowest content of 82 mg/kg and the highest of 502 mg/kg. Average value of olive oil α-

tocopherol obtained in the present study (16.79 mg/100g) was in line with the reported

average values and Greece olive oil samples content, but below the average reported for

Spanish olive oil cultivars.

Pumpkin seed oil is used by both food and pharmaceutical industries. For many years

pumpkin seeds have been used in complementary medicine – primarily as vermifuge. They

belong to the group of plants and herbs containing fatty acids and phytosterols that are

administered at the early stage of the prostatic hyperplasia therapy (Nawirska-Olszlańska

et al., 2013). Cold pressed pumpkin seed oil is produced by pressing of raw, dried, mainly


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hull-less seeds, on a continuous screw press. This method of production preserves bioactive

compounds as vitamin E which has positive effect on the human health. Rabrenović et al.

(2014) have reported that tocopherol content of cold pressed oil from different pumpkin seed

cultivated in Serbia varies from 38.03±0.25 up to 64.11±0.07 mg/100 g of oil. Average value

of the α-tocopherol content of the cold pressed pumpkin seed oil obtained in the present

study was 16.79 mg/100 g.

Present study encompassed two fruit oils, walnut and hazelnut oil. Although both, walnut

and hazelnut belong to the same group of fat rich fruit types, their oils differ in α-

tocopherol content. Content of α-tocopherol in hazelnut oil is multiply higher from the one

recorded for walnut oil (40.35 mg/100 g and 5.93 mg/100 g, respectively). Previously

reported hazelnut oil content of α-tocopherol was 45.2 mg/100g, and α-tocopherol

equivalents content was 36.6 mg/100g, while previously reported cold pressed walnut oil

content of α-tocopherol was below the level of detection, and α-tocopherol equivalents

content was 2.8 mg/100g (Eitenmiller and Lee, 2004).

Remaining analysed oil types (camelina, hemp, apricot kernel, sesame, poppy seed, linseed

and chia) which are used in daily diet in less extent, had lower α-tocopherol content, and

two of them (linseed and chia) had α-tocopherol content below the level of detection.

Review of literature data reveals that oils with higher α-tocopherol content have also

higher α-tocopherol equivalent values. In some oils (soybean, peanut, corn and canola)

γ-tocopherol is present in large enough quantities to contribute appreciably to α-tocopherol

equivalent levels (Eitenmiller and Lee, 2004). This should be considered in the comparison

of the obtained data to literature values and in analysis of mentioned oils as the dietary

source of vitamin E.

α-Tocopherol food sources and dietary intakes

Since vegetable oils are one of the most important dietary sources of the vitamin E, values

obtained by the RP-HPLC analysis were used to estimate contribution of analysed oil types

to the Dietary Reference Intakes (DRIs) (FNB and IOM, 2004). To enable that estimation

it was hypothesised that daily consumption of edible oil is 10 g/day, which corresponds to

on table spoon of oil that is used for salad or meal preparation.

In accordance with the content of α-tocopherol in oils, the highest contribution

(45.92% RDA with 10 g/day consumption) was recorded for sunflower oil, followed

by hazelnut (26.90% RDA with 10 g/day consumption), soybean (19.20% RDA with

10 g/day consumption) and rapeseed oil (19.15% RDA with 10 g/day consumption)

(Fig. 2).


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Fig. 2. Average estimated contribution of the 10 g daily consumption

of oil to the recommended dietary intake

According to the data from the Second National Health and Nutrition Examination Survey

(NHANES II) contribution of fats and oils to the vitamin intake in the United States is

20.2%, while other sources are vegetables (15.1%), meat, poultry and fish (12.6%),

desserts (9.9%), breakfast cereals (9.3%), fruit (5.3%), dairy products (4.5%), mixed main

dishes (4.0%), nuts and seeds (3.8%), soups, sauces and gravies (1.7%) (Eitenmiller and

Lee, 2004).

Changes in α-tocopherol content during storage

Vitamin E stability in edible oils depends on the initial oil quality, and in most refined,

bleached and deodorised oils protected by proper packaging most changes are attributable

to abuse leading to oxidative changes (Eitenmiller and Lee, 2004).

To get an insight into the vitamin E content due to its freshness, and regardless of

production method, this study encompassed fresh samples obtained in laboratory

conditions as well as oil samples purchased from the retailers where average consumers

supply themselves. Variations in the content are visible from the SD values shown in Fig. 1

from which is also visible that the sunflower oil which is widely used is the oil with the


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highest variations (93.12 mg/100 g in one fresh oil sample, 42.04 mg/100 g in one of the

commercial samples).

To simulate household managing, due to the fact that average oil consumption period is

from one week up to one month per 1 L of oil, fresh sunflower oil obtained by cold

pressing in laboratory conditions was storaged for one month period under the ambient

conditions in a dark place. Analysis after 1 month storage period indicated 24.69%

reduction in α-tocopherol content (Fig. 3).

Fig. 3. Reduction of cold pressed sunflower oil α-tocopherol content

during the 1 month storage under the ambient conditions

Vitamin E decrease in crude sunflower oil, obtained by pressing and solvent extraction,

during storage on ambient temperature was reported by Crapiste et al. (1999). Extracted oil

showed a higher oxidative stability than pressed oil, and oxidative deterioration was

strongly dependent on temperature, oxygen availability, and the ratio of exposed surface to

sample volume. They also reported that container type (plastic, tin, stainless steel, glass,

etc.) has no effect on degradation. On the other hand, Shafqatullah and Sohail (2011)

reported that for long storage life sunflower oil should be kept under florescent light at

room condition in glass bottle fully filled, and that air, packaging and storage time all have

an effect on the stability of sunflower oil.

Mean

Mean±SD

Mean±1,96*SD

fresh

after 1 month storage

Time of sample analysis

60

70

80

90

100

110

120

% o

f th

e ori

gin

al α

-toco

ph

erol co

nte

nt

88.99 ± 5.83 mg/100 g

67.03 ± 3.95 mg/100 g


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Gómez-Alonso et al. (2007) studied changes in composition of Spanish virgin olive oils

during 21 month storage at room temperature and in darkness. α-Tocopherol content

decreased from 12% (0.054 mmol/kg) up to 23% (0.127 mmol/kg) from the initial values

which were ranking from 0.33 mmol/kg to 0.55 mmol/kg. Fall in α-tocopherol content was

apparently linear, although they presumed short lag phase at the beginning of the assay.

Degradation was also reported for soybean and rapeseed oil (Eitenmiller and Lee, 2004).

Conclusions

Content of α-tocopherol in studied edible oil types varied from those below the level of

detection up to 68.88 mg/100 g of oil. Highest values were recorded in sunflower oil which

is the most widespread oil type in the continental part of the Croatia. Contribution of oil to

the daily recommended intake of vitamin E just by studied α-tocopherol can reach up to

45.92% of DRA with 10 g/day oil consumption.

References

Alberti, A., Fruttini, D., Fidanza, F. (2009): The Mediterranean adequacy index: Further confirming

results of validity, Nutr. Metab. Cardiovas. 19, 61-66.

Beltrán, G., Jiménez, A., del Rio, C., Sánchez, S., Martínez, L., Uceda, M., Aguilera, M.P. (2010):

Variability of vitamin E in virgin olive oil by agronomical and genetic factors, J. Food

Compos. Anal. 23, 633-639.

Blekas, G., Tsimidou, M., Boscou, D. (1995): Contribution of α-tocopherol to olive oil stability,

Food Chem. 52 (3), 289-294.

Brigelius-Flohé, R. (2006): Bioactivity of vitamin E. Nutr. Res. Rev. 19 (2),174-186.

Combs, G.F. (2008): The Vitamins, Fundamental aspects in nutrition and health, Fourth Edition,

Elsevier Academies Press, USA, p.p.181-212.

Crapiste, G.H., Brevedan, M.I.V., Careli, A.A. (1999): Oxidation of sunflower oil during storage,

JAOCS 76 (12), 1437-1443.

Eitenmiller, R., Lee, J. (2004): Vitamin E; Food chemistry, Composition, and Analysis, Marcel

Dekker, Inc, New York, USA. pp. 2, 17, 46, 300-301, 307-308, 438-445.

Fidanza, F., Alberti, A., Lanti, M., Menotti, A. (2004): Mediterranean adequacy index: correlation

with 25-year mortality from coronary heart disease in the seven countries study, Nutr. Metab.

Cardiovasc. Dis. 14, 254-258.

Food and Nutrition Board (FNB), Institute of Medicine (IOM), National Academies (NC). (2004):

Dietary Reference Intakes (DRIs): Recommended intakes for Individuals, Vitamins. National

Academy of Sciences, Washington, USA.

Gimeno, E., Castellote, A.I., Lamuela-Raventós, R.M., de la Torre, M.C., López-Sabater, M.C.

(2000): Rapid determination of vitamin E in vegetable oils by reversed phase high-

performance liquid chromatography, J. Chromatogr. A. 881, 251-254.


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Gimeno, E., Castellote, A.I., Lamuela-Raventós, R.M., de la Torre, M.C., López-Sabater, M.C.

(2000): The effects of harvest and extraction methods on the antioxidant content (phenolics,

α-tocopherol, and β-carotene) in virgin olive oil, Food Chem. 78, 207-211.

Goosens, B., Marion, J. (2011): Quantifying vitamin E in vegetable oils with reversed-phase high

performance liquid chromatography, Concordia College Journal of Analytical Chemistry 2,

44-50.

Gómez-Alonso, S., Mancebo-Campos, V., Deamparados Salvador, M., Fregapane, G. (2007):

Evolution of major and minor components and oxidation indices of virgin olive oil during 21

months storage at room temperature, Food Chem. 100, 36-42.

Nawirska-Olszańska, A., Kita, A., Biesiada, A., Sokól-Lętowska, A., Kucharska, A.Z. (2013):

Characteristics of antioxidant activity and composition of pumpkin seed oils in 12 cultivars,

Food Chem. 139, 155-161.

Nissiotis, M., Tasioula-Margari, M. (2002): Changes in antioxidant concentration of virgin olive oil

during thermal oxidation, Food Chem. 77, 371-376.

Rabrenović, B.B., Dimić, E.B., Novaković, M.M. Tešević, V.V. (2014): The most important

bioactive components of cold pressed oil from different pumpkin (Cucurbita pepo L.) seeds,

LWT-Food Sci. Technol. 55, 521-527.

Shafqatullah, A.H., Sohail, M. (2011): Effect of packing materials on storage stability of sunflower

oil, Pak. J. Biochem. Mol. Biol. 44 (3), 92-94.

Šarolić, M., Gugić, M., Marijanović, Z., Šuste, M. (2014): Virgin olive oil and nutrition, Food in

health and disease 3 (1), 38-43.


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Determination of polyphenolic compounds in red wines

from Baranja vineyards

UDC: 663.222(497.543) : 547.623

Nebojša Kojić1

, Lidija Jakobek2

1Vupik d.d., Sajmište 113c, HR-32000 Vukovar, Croatia



Summary

In this study, bioactive polyphenolic compounds from red wines were investigated. For the

characterization of individual polyphenolic compounds belonging to phenolic acid and flavanol

class, easy and reliable high performance liquid chromatography method with photodiode array

detection was developed. Total polyphenols and total flavonoids were also investigated by using

simple spectroscopic methods. All methods were checked for their linearity, limit of detection, limit

of quantification and precision. Developed and validated procedures were applied for the

determination of polyphenolic compounds in two wines from Baranja vineyards, Croatia. The most

abundant compounds found in red wines were (-)-epicatechin and gallic acid followed by p-coumaric

acid, (+)-catechin. The developed method were shown to be precise for the determination of

polyphenolic compounds from red wines.

Keywords: polyphenolic compounds, red wine, HPLC method, spectroscopic method

Introduction

Wine is an alcoholic beverage that contains various polyphenols extracted from grapes

during the processes of vinification (Rastija et al., 2009; Puškaš, 2010). Polyphenolic

compounds are responsible for the quality of red wines, influencing on their astringency,

bitterness and colour. The viticulture practices, different enological techniques, the varieties

and the harvesting year of grapes influence the polyphenolic composition of wines (Boulton,

2001; Lesschaeve, Noble, 2005; Cliff et al., 2007; Gómez-Alonso et al., 2007).

Wines contain a wide range of polyphenols that include phenolic acids, notably

hydroxycinnamic acids (e.g., caffeic acid, p-coumaric acid and ferulic acid) and

hydroxybenzoic acids (gallic, ellagic, p-xydroxybenzoic, vanillic, and syringic acid).

Anthocyanins, flavonols (quercetin, rutin, myricetin, etc.) and flavanols (catechin,

epicatechin, etc.) were also found in wines (Gómez-Alonso et al., 2007; Spacil et al., 2008).



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Polyphenols from wines were reported to have many bioactivities which are still studied

intensively (Teissedre et al., 1996; Cooper et al., 2004). They are present in high quantities

in wine, especially in red wines, which may explain so-called French paradox (Cliff et al.,

2007) which was the subject of research of (Renaud, de Lorgeril, 1992). They reported that

there was a low mortality rate from ischemic heart disease among French people despite

their high consumption of saturated fats and the prevalence of other risk factors, such as

smoking. This referred to the "Mediterranean diet", which included regular but moderate

consumption of red wine (Renaud, de Lorgeril, 1992).

Therefore, determination of the polyphenol content of red wines could be very useful for

the interpretation of their effects in the human body. Many different methods, including

high-performance liquid chromatography (HPLC) in combination with different detectors,

UV/Vis, photo diode array detector (PDA) have been used to investigate the polyphenolic

content of wine (Escarpa, Gonzales, 2001). Spectrophotometry, as a more affordable

technique for fast and simple routine analyses, has been used for the determination of the

total amounts of polyphenols (Slinkard, Singleton, 1977; Ivanova et al., 2010), flavonoids

(Mazza et al., 1999; Zhishen et al., 1999; Ivanova et al., 2009), flavan-3-ols (Ivanova et al.,

2009).

The aim of this study was to validate spectroscopic and high-performance liquid

chromatography methods with photodiode array detector (HPLC-PDA), for the

determination of polyphenols in wines. Furthermore, two wines from Baranja vineyards,

belonging to Continental region of Croatia, were analysed for their total polyphenol, total

flavonoid content. The content of individual flavanols and phenolic acids was also

determined by using HPLC-PDA method in order to examine the distribution of these

polyphenols in red wines.


Chemicals

Gallic acid monohydrate (398225), p-coumaric acid (C9008), (+)-catechin hydrate (C1251),

(-)-epicatechin (E1753) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ortho-

phosphoric acid (85% HPLC grade) was purchased from Fluka (Buchs, Germany). Methanol

(HPLC grade) was obtained from Merck (Darmstadt, Germany) and used as mobile phase.

Folin-Ciocalteau reagent was obtained from Kemika (Zagreb, Croatia).

Wine samples

Grapes from Vitis vinifera L., Merlot and Franconia, were cultivated on the slopes of the

Ban's hill, Baranja vineyard county, vintage 2013 (Continental region of Croatia, sub-


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region Podunavlje, zone C1). Vegetative cycle is characterized by an excellent schedule

rainfall during spring and dry and hot summer, which resulted in exceptional quality of the

grapes at harvest. After the grapes were hand-picked, fermentation with maceration as well

as pressing and continuation of malolactic fermentation were conducted in stainless steel

vinimatics for a period of 10-12 days (http://vinabelje.hr/vina/select/, October, 09th 2014).

At the time of sampling, wines were not blended and any clarification or filtration were not

conducted. The wines were investigated just a few days after bottling. The wine samples

were stored at a temperature of 15 to 18 °C. They were packed in PET bottles. Before

HPLC-PDA analysis, wine samples were filtered through 0.45 µm polytetrafluoroethylene

(PTFE) filters (Varian, USA).

Determination of total polyphenols

Spectroscopic determination of the total polyphenol (TP) content was done with the

Folin-Ciocalteau micro method for wine analysis (Waterhouse, 2009) using gallic acid

as the standard. The TP content was measured at 765 nm on a UV/Vis

spectrophotometer (JP Selecta UV 2005, Barcelona, Spain). An aliquot (20 µl) of wine

sample was mixed with 1580 µl of distilled water and 100 µl of Folin-Ciocalteau

reagent. 300 µl of sodium carbonate solution (200 gl-1

) was added to the mixture. After

incubation in water bath at 40 °C for 30 min, absorbance of the mixture was read

against the prepared blank at 765 nm. Total polyphenols were expressed as mg gallic

acid equivalents per litre of wine (mg GAE l-1

). The calibration curve of the

absorbance vs. concentration of the standard was used to quantify TP content. Data

presented are mean ± standard deviation (SD). All measurements were performed in

triplicate.

Determination of total flavonoids

Total flavonoid content (TF) was evaluated according to a colorimetric assay with

aluminium chloride proposed by (Zhishen et al., 1999). An aliquot of 1 ml of appropriate

diluted wine sample was placed in a 10 ml volumetric flask, containing 4 ml of distilled

water, followed with addition of 0.3 ml solution of NaNO2 (0.5 gl-1

). About 0.3 ml of

AlCl3solution (1 gl-1) was added 5 min later and after 6 min, 2 ml of NaOH solution (1 moll

-1)

was added. The total volume was made up to 10 ml with distilled water and the solution was

mixed. The absorbance was measured against the blank (prepared in the same way with

distilled water) at 510 nm. (+)-catechin was used as the standard for the calibration curve and

the concentration of TF was expressed in mg l-1 as catechin equivalents (mg CE l

-1). Data

presented are mean ± standard deviation (SD). All measurements were performed in

triplicate.


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High-performance liquid chromatography measurement

The analysis were performed on a HPLC analytical system (Varian, USA) consisting of a

Prostar 230 pumps and Prostar 330 PDA detector. Separation of polyphenolic compounds

was performed on an OmniSpher C18 column (inner diameter 250 x 4.6 mm, particle

diameter of 5 microns, Varian, USA) that is protected by the first column (ChromSep 1cm

x 3 mm, Varian, USA). The flavan-3-ols and phenolic acids present in wines were

separated using water solution of phosphoric acid (0.1%) as solvent A and 100% methanol

(HPLC grade) as solvent B (Jakobek et al., 2007). The elution conditions are as folows:

0 min, 5% B, 30 min 80% B, 33 min 80% B, 35 min 5% B, the operating conditions

were: injection volume 20 µL, flow rate 0.8 mlmin-1

.

Phenolic compounds were identified through comparison of their retention times and

UV/Vis spectra with those of standards. Peak area was used for quantitation purposes,

using internal standard calibration. Values were reported as mg l-1

. The detection

wavelength was 260 nm for gallic acid, 280 nm for (+)-catechin and (-)-epicatechin, 320 nm

for p-coumaric acid.

Spectroscopic and HPLC-PDA method validation

For the total polyphenol and total flavonoid determination, gallic acid and (+)-catechin

were used as standards, respectively. Standards were prepared in methanol in concentration

ranges 0-250 mg l-1

for gallic acid and 0-150 mg l-1

for (+)-catechin. Different

concentrations were measured two times according to the procedure described. Calibration

curves were created. Linearity of calibration curves was expressed with coefficient of

determination (R2), limit of detection (LOD) and limit of quantification (LOQ) were

detemined according to equations 1 and 2.

LOD = 3.3 × s/S (1)

LOQ = 10 × s/S (2)

where, s is the standard deviation of the y intercepts of the regression lines, and S is the

slope of the calibration curve.

Precision of methods was detemined by analysing the same wine samples four times

during the day and it was expresed by calculating standard deviation of the polyphenol

content, and coefficient of variation expressed in %. For the HPLC-PDA method,

calibration curves of all standards were constructed analysing standard solutions of

different concentrations. Each concentration was analysed two times (gallic acid 0-98 mg l-1

;

(+)-catechin 0-250 mg l-1

; (-)-epicatechin 0-245 mg l-1

; p-coumaric acid 0-98 mg l-1

).


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Linearity was expressed with coefficient of determination (R2) and LOD and LOQ were

calculated according to equations 1 and 2.


Measurements for TP and TF was done in triplicate, and the results were presented as

mean value ± standard error (standard deviation, SD). Correlation and regression analyses

were performend using Microsoft Office Excel.


The results for the validation of spectroscopic methods are shown in Table 1. Linearity of

methods was good which is represented by R2 values 0.985 and 0.961 for total polyphenols

and total flavonoids, respectively. Limit of detection and limit of quantification were also

determined. It could be seen that these methods could determined and measure reasonably

small content of total polyphenols (LOD = 0.159 mg l-1

; LOQ = 0.483 mg l-1

) and total

flavonoids (LOD = 0.243 mg l-1

; LOQ = 0.738 mg l-1

). Moreover, both methods were

shown to be precise for the determination of polyphenols in wines (CV 5 and 2% for total

polyphenols and total flavonoids, respectively).

Table 1. Linearity, limit of quantification, limit of detection and precision of methods for total

polyphenols, total flavonoids and HPLC-PDA method

Method R² Equation LOD

mg l-1

LOQ

mg l-1

Ret.

time

min

Coefficient

of variation

%

Spectroscopic

Total polyphenols 0.985 y = 368.7x + 6.545 0.159 0.483 4.6

Total flavonoids 0.961 y = 741.5x – 21.18 0.243 0.738 1.8

HPLC

(+)- Catechin 0.996 y=46379x + 28374 1.02 3.10 16.56 1

(-)- Epicatechin 0.996 y=42184x + 25919 0.89 2.71 19.51 3.3

Gallic acid 0.998 y=37589x + 212082 2.44 7.40 11.26 1.9

p-coumaric acid 0.954 y=225769x - 676083 0.33 0.98 23.85 15.3

These methods were used to determine the content of total polyphenols and total

flavonoids in Merlot and Franconia wines. The results can be seen in Table 2. In both

wines, the content of total flavonoids (829 mg l-1

in Merlot; 835 mg l-1

in Franconia) and

total polyphenols (2016 mg l-1

in Merlot; 1910 mg l-1

in Franconia) were similar. The


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results are in accordance with results published in the literature (Sato et al., 1996; Sanchez-

Moreno et al., 1999; Arnous et al., 2002; Minussi et al., 2003; Roussis et al., 2005).

Furthermore, the content of total polyphenols in wines from various contries were: 1738-

3292 mg l-1

in wines from Spain (Villano et al., 2006), 1637- 2717 mg l-1

in wines from

Slovenia (Košmerl and Cigić, 2008), 2200 to 3200 mg l-1

in wines from Poland (Tarko et al.,

2008), 1018-3545 mg l-1

in wines from France (Landrault et al., 2001), 874-2262 mg l-1 in

wines from Czech Republic (Stratil et al., 2008). According to literature data, wines from

Croatia had 4576-4989 mg l-1 total polyphenols (Piljac et al., 2005) or 2809-3183 mg l

-1

(Katalinić et al., 2004).

Table 2. Content of total polyphenols and total flavonoids in two types of wine

Wine Total flavonoids

[mg CE l-1]

Total polyphenols

[mg GAE l-1]

Merlot 829.14±8.0 2015.88±89.1

Franconia 835.29±22.2 1909.72±93.5

Table 1. shows the results for the validation of HPLC-PDA method. Linearity of the

method was good which is represented by R2 values 0.954-0.998. The calculated LOD and

LOQ were in the range of 0.33-2.44 mg l-1

and 0.98-7.4 mg l-1

, respectively. Precision of

the method was presented with coefficient of variation (%). All identified compounds

could be precisely detemined with HPLC-PDA method (CV from 1 to 15%).

Validated method was used to analyse individual flavanols and phenolic acids in wines.

Fig. 1 presents the chromatograms of wines Merlot and Franconia scanned at 280 nm with

identified compounds. From phenolic acid class, gallic acid and p-coumaric acid were

found. From flavan-3-ol class, (+)-catechin and (-)-epicatechin were identified. Both wines

contain some unidentified compounds from phenolic acid group, anthocyanin group and

flavonol group. This can be seen from their UV/Vis spectra maximum which were 315 and

316 nm for phenolic acids (peak PA 1 and 2, respectively). The maximum absorbance at

arround 500 - 510 nm and 280 nm represents unknown anthocyanins (peaks A). Unknown

flavonols showed absorbance maximum at arround 340 nm.

Table 3. shows the content of identified phenolic compounds. The content of

identified phenolic acids and flavanols are similar in bo th wines. The dominant

compound was (-)-epicatechin (41 and 69 mg l-1

in Merlot and Franconia, respectively),

followed by gallic acid (21 mg l-1

in Merlot and Franconia). p-coumaric acid was identified

only in Franconia wine (11 mg l-1

). (+)-catechin was detected but its content was below the

limit of quantification. These results are in accordance with literature data (Rastija et al.,

2009; Šeruga et al., 2011; Artem et al., 2014).


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Fig. 1. The HPLC chromatogram of two types of wine: Franconia and Merlot, scanned at 280 nm

with identified compounds. Peak identification: 1 – gallic acid, 2 – (+)-catechin, 3 – (-)-epicatechin,

4 – p-coumaric acid, A – unidentified anthocyanins, F – unidentified flavonols,

PA1, PA2 – unidentified phenolic acids

Table 3. Content of individual polyphenols in wine, determined by HPLC-PDA method

Content

[mg l-1] Merlot Franconia

Phenolic acids

Gallic acid 21.02 ± 0.6 20.47 ± 0.2

p-coumaric acid nd 10.73 ± 1.6

Flavanols

(+)- Catechin bql bql

(-)- Epicatechin 40.73 ± 0.1 68.98 ± 4.4 Bql – below quantification limit

Nd – not detected

Conclusions

In this work, red wines made from Merlot and Franconia grape from Baranja county

vineyard were analyzed using spectroscopic and HPLC-PDA methods. All methods were

shown to be good for the determination of polyphenols in wines. The results showed the


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content of total polyphenols and total flavonoids similar to those reported in the literature.

Individual flavanols and phenolic acids were identified and their content was similar to

earlier studies.

Acknowledgments

The work was funded by project from Josip Juraj Strossmayer University of Osijek.

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Antiradical activity of polyphenols from old apple varieties

UDC: 547.56 : 634.11

Petra Krivak, Lidija Jakobek

Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Department of

Applied Chemistry and Ecology, Franje Kuhača 20, HR 31000 Osijek, Croatia

Summary

The aim of this work was to study the antiradical activity of polyphenols from old, ancient apple

varieties grown in Croatia (Crvenka, Pisanika, Ledenara, Adamova zvijezda, Slavonska srčika and

Wild apple). Polyphenols were extracted from apple peel and flesh by using ultrasonic bath. The

scavenging of syntetic, free DPPH˙ radical by polyphenols was studied until the reaction reached

the steady state. A biphasic reaction was observed between apple polyphenols and DPPH˙ radicals,

with “fast” and “slow” scavenging rate. The antiradical activity of the peel of apples Pisanika,

Ledenara, Crvenka, Adamova zvijezda was similar and higher than the antiradical activity of the

peel of Wild apple and Slavonska srčika. On the other hand antiradical activity of the flesh of Wild

apple was significantly higher than the antiradical activity of the flesh of other apples. A

polyphenolic profile showed that the compounds most likely to be responsible for a strong

antiradical activity of Wild apples are chlorogenic acids, phloretin derivatives, quercetin derivatives

and flavanols. Polyphenols of old apple varieties can be considered as strong free radical

scavengers, especially Wild apples. The overall results showed the need to preserve and protect

these old varieties because they represent a significant source for horticultural biodiversity.

Keywords: old apple varieties, antiradical activity, DPPH˙, biphasic reaction

Introduction

Apples represent a significant source of polyphenols in the human diet because they are

available for consumption during the whole year. Old ancient apple varieties are also rich

in polyphenols. These varieties that used to be grown in the past, are now days almost

forgotten. In addition to being a good source of polyphenolic antioxidants (Jakobek et al.,

2013; Mendoza-Wilson et al., 2013), old varieties should be preserved because of

biodiversity of plant species.

A series of papers investigated the antiradical activity of old ancient and common apple

varieties (Iacopini et al., 2010; Carbone et al., 2011; Lata et al., 2009) and polyphenols

(Ceymann et al., 2012). DPPH˙ method is usually used to measure antiradical activity of

polyphenols. DPPH˙ test belongs to AOAC methods utilizing HAT (Hidrogen Atom



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Transfer) and SET (Single Electron Transfer) mechanisms. Antioxidants act by hydrogen

atom donation to free DPPH˙ radical (Prior et al., 2005; Madsen et al., 2010). Moreover,

earlier studies emphasized the importance of conservation of old apple varieties and

determination of polyphenols. Wild apples or crabapples have been investigated earlier and

it was found that they are also rich in polyphenols (Jakobek et al., 2013). John et al. (2014)

studied metabolic variation and antioxidant potential of Malus prunifolia (Wild apple).

They found that polyphenol content in Wild apple collected from Konkuk University,

Seoul, South Korea was higher than in grapes, commercial apples, beverages and black

tea.

Due to the importance of old apple varieties and Wild apple (crabapple), five varieties of

old apples and Wild apple (crabapple) grown in Croatia were investigated (Crvenka,

Pisanika, Ledenara, Adamova zvijezda, Slavonska srčika and Wild apple). The antiradical

activity of polyphenolic compounds from flesh and peel of old apple varieties was

measured by using DPPH˙ method. Furthermore, the kinetic of inhibition of free DPPH˙

radicals was studied. Polyphenols in the peel and flesh of Wild apples were determined

with reversed phase high-performance liquid chromatography with photodiode array

detection (RP-HPLC-PDA method).


Fruit samples

Apples Crvenka, Pisanika, Ledenara, Adamova zvijezda, Slavonska srčika and Wild apple

were harvested in October 2013. Apple samples were obtained from the family orchard

(Veić M.) located in the region Slavonia (Mihaljevci, near Požega) in Croatia. All apples

were peeled with a hand peeler. The peeled fruits were cut into quarters, seeds and core

were removed. Peel and flesh were separately homogenized by using a stick blender and

kept in freezer at -18 °C until the start of the analysis.

Chemicals

The methanol used was HPLC grade purchased from J.T. Baker (Netherlands). Antiradical

activity was determined with 2,2-Diphenyl-1-picryl-hydrazyl (DPPH˙) (D9132) free

radical which has been purchased from Sigma-Aldrich (USA). Gallic acid monohydrate

(398225), (+)-catechin hydrate (C1251), (-)-epicatechin (E1753), chlorogenic acid

(C3878), quercetin dihydrate (Q0125), quercetin-3-D-glucoside (isoquercitrin - 17793)

were purchased from Sigma-Aldrich (USA); procyanidin B1 (epicatechin(4-8)catechin -

0983), quercetin-3-O-galactoside (hyperoside - 1027 S), quercetin-3-O-rhamnoside


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(quercitrin - 1236 S), phloretin-2’-O-glucoside (phloridzin - 1046), phloretin (1044) were

purchased from Extrasynthese (France).

Polyphenolic compounds extraction from apple peel and apple flesh

Polyphenolic compounds were extracted from apple peel and apple flesh. Apple peel and

flesh were homogenized separately. Approximately 0.5 g of homogenized apple peel was

weight and added to an empty test tube. The extraction was performed with 5 ml 0.1%

methanolic HCl using an ultrasonic bath, for 15 min. Extract was filtrated and residue was

extracted again with 2 ml 0.1% methanolic HCl. Extracts were combined. To extract

polyphenols from the flesh, 80% methanol in water was used as an extraction solvent, and

the procedure was the same. Additionally, extracts from peel and flesh of Wild apples were

filtrated through a 0.45 µm syringe filter (PTFE) and analyzed with RP–HPLC-PDA

method. All extracts were prepared twice. The antiradical activity was determined in both

extracts. During the measurement, extracts were stored in a freezer at -18 °C.

RP-HPLC-PDA method for the determination of polyphenols in Wild apples

Polyphenols in Wild apples were determined by using a Varian HPLC system (USA).

Varian HPLC system consists of ProStar 230 solvent delivery module, ProStar 330 PDA

detector and OmniSpher C18 column (250 x 4.6 mm inner diameter, 5 µm, Varian, USA).

Polyphenols were separated using 0.1% phosphoric acid as solvent A and 100% HPLC

grade methanol as solvent B (elution conditions: 0 min 5% B; 0 to 5 min from 5 to 25% B,

5 to 14 min from 25 to 34% B, 14 to 25 min from 34 to 37% B, 25 to 30 min from 37 to

40% B, 30 to 34 min from 40 to 49 % B, 34 to 35 min from 49 to 50% B, 35 to 58 min

from 50 to 51% B, 58 to 60 min from 51 to 55 % B, 60 to 62 min from 55 to 80% B, 62 to

65 min 80% B, 65 to 67 min from 80 to 5% B, 67 to 72 min 5% B; with flow rate=0,8

ml/min). Injection volume was 20 l. UV-Vis spectra were recorded from 190 to 600 nm.

Detection wavelength was 280 nm for flavan-3-ols, dihydrochalcones, 320 nm for phenolic

acids, 360 nm for flavonols. Identification was based on the comparison of retention times

and spectral data with those of authentic standards. Calibration curves of standards were

made by preparing different concentrations of standards in 100% methanol and by

analyzing them on RP-HPLC-PDA system.

Antiradical activity of polyphenolic compounds by DPPH˙ free radical

The antiradical activity of apple peel and flesh was determined using DPPH˙ assay (Brand-

Williams et al., 1995). Stable radical 2,2-diphenyl-1-picryl-hydrazyl in methanol solution


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was used in this method. Reaction for scavenging free DPPH˙ radical according to

Brand-Williams et al. (1995) is:

DDPH˙ + AH DPPH – H +A˙

DPPH˙ + R˙ DPPH - R

where (AH) is an antioxidant and (R˙) is free radical. During the reaction, antioxidants or

free radicals scavenge DPPH˙ radicals and the purple color of DPPH˙ radicals disappears,

therefore absorption decreases.

Measurement was done with a UV-Vis spectrophotometer (Selecta, UV 2005, Barcelona,

Spain). DPPH˙ stock solution was prepared (1 mmol/l, in methanol). The reaction

solutions were prepared with 200 µl DPPH solution, increasing aliquots of peel or flesh

polyphenol extracts, and methanol to a final volume of 3 ml. The absorbance was

measured against the blank solution (which contained 200 µl methanol instead of DPPH

solution) in the total time period of 70 minutes. When polyphenols were added to DPPH˙

free radical, purple color of the solution turns to yellow. This effect is measured as the

decrease in the absorbance.

The percentage of remaining DPPH˙ radicals were calculated (Eq. 1):

% 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 𝐷𝑃𝑃𝐻 =𝐴(𝑒𝑥𝑡𝑟𝑎𝑐𝑡)

𝐴0(𝐷𝑃𝑃𝐻•) 100⁄ (1)

where

A0 (DPPH˙) = absorption of DPPH˙ radical in t = 0

A (extract) = absorption of extract.

Furthermore, the percentage of remaining DPPH˙ radicals in 70 min was plotted against

the mass ratio of peel or flesh to DPPH˙ (g flesh or peel to g DPPH˙). From these graphs,

the EC50 value was calculated. EC50 value represents the amount of apple peel or flesh

needed to inhibit 50% of DPPH˙ radical in 70 min. If EC50 value is higher, then the

antiradical activity is lower and vice versa.


MS Excel was used for data analysis. The results for the antiradical activity of polyphenols

from apple peel and flesh were based on two replicate samples measured once. Regression

function was used to calculate EC50 value. EC50 value is presented as mean value ± SD

(standard deviation).


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The kinetic behavior of polyphenol extracts from apple peel and flesh is presented in Fig. 1

and 2, respectively. These figures represent the percentage of remaining DPPH˙ free

radical after the reaction with different mass ratios of peel and flesh to g DPPH˙. Higher

mass of peel or flesh caused faster disappearance of DPPH˙ radicals. Furthermore,

scavenging of DPPH˙ radicals was fast at the beginning and slowed down at the end. It

could be said that the reaction is biphasic. It has two periods: a period with fast inhibition

of DPPH˙ radical and a period with slow DPPH˙ radical inhibition. Fast inhibition period

where the absorbance decreased rapidly, was approximately 10 minutes. After that period,

DPPH˙ radical inhibition slowed down and absorbance slowly decreased. This is in

accordance with earlier studies (Brand-Williams et al., 1995; Jakobek et al., 2008).

Fig. 1. Kinetic behavior of apple peel. a) Crvenka; b) Pisanika; c) Ledenara;

d) Adamova zvijezda; e) Slavonska srčika; f) Wild apple;

for different ratios g apple/g DPPH


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Fig. 2. Kinetic behavior of apple flesh. a) Crvenka; b) Pisanika; c) Ledenara;

d) Adamova zvijezda; e) Slavonska srčika; f) Wild apple;

for different ratios g apple/g DPPH

Fig. 3 and 4 represent disappearance of DPPH˙ radicals after 70 minutes (steady state) as a

function of mass ratio of peel and flesh to DPPH˙, respectively. From these graphs, EC50

values were calculated. EC50 values are presented in Table 1. Higher EC50 means lower

antiradical activity. It can be seen that the antiradical activity of apple flesh was generally

lower than the antiradical activity of apple peel. In comparison of the antiradical activity of

the peel, Pisanika (EC50 20.53 g peel/g DPPH˙), Ledenara (EC50 22.73 g peel/g DPPH˙),

Crvenka (EC50 26.39 g peel/g DPPH˙) and Adamova zvijezda (EC50 27.09 g peel/g

DPPH˙) had higher antiradical activity then the peel of Slavonska srčika (EC50 42.28 g

peel/g DPPH˙) and Wild apple (EC50 40.35 g peel/g DPPH˙). Flesh of Ledenara (EC50


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294.07 g flesh/g DPPH˙) and Adamova zvijezda (EC50 284.28 g flesh/g DPPH˙) showed

higher antiradical activity than Slavonska srčika (EC50 302.94 g flesh/g DPPH˙), Pisanika

(EC50 468.71 g flesh/g DPPH˙), Crvenka (EC50 712.45 g flesh/g DPPH˙). The antiradical

activity of polyphenols of Wild apple flesh can be highlighted (EC50 33.43 g flesh/g

DPPH˙) because it is much higher than the antiradical activity of polyphenols from other

apple flesh.

Fig. 3. The disappearance of DPPH˙ radicals as a function of the g apple/g DPPH

for apple peel. a) Crvenka; b) Pisanika; c) Ledenara; d) Adamova zvijezda;

e) Slavonska srčika; f) Wild apple


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Fig. 4. The disappearance of DPPH˙ radicals as a function of g apple/g DPPH

for apple flesh. a) Crvenka; b) Pisanika; c) Ledenara; d) Adamova zvijezda;

e) Slavonska srčika; f) Wild apple


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Table 1. Antiradical effectiveness of polyphenols from apples expressed in grams of apples per g of

DPPH needed to inhibit 50% of DPPH radicals

Apple

EC50 Standard deviation

(g apple/g DPPH)

Crvenka Peel 26.39 4.47

Flesh 712.45 60.64

Pisanika Peel 20.53 5.00

Flesh 468.71 63.80

Ledenara Peel 22.73 3.29

Flesh 294.07 2.52

Adamova zvijezda Peel 27.09 1.61

Flesh 284.28 8.99

Slavonska srčika Peel 42.28 1.54

Flesh 302.94 36.58

Wild apple Peel 40.35 7.62

Flesh 33.43 0.07

Since Wild apples differ from other old apple varieties by higher antiradical activity of

flesh, their polyphenol extracts were analyzed using RP-HPLC-PDA method. Fig. 5

presents HPLC chromatograms of Wild apple, and identified compounds were shown in

Table 2. Wild apple have shown the same polyphenolic compounds found usually in

apples (Jakobek et al., 2013; John et al., 2014). In the peel, (+)-catechin, (-)-epicatechin

from flavan-3-ol group were identified. Other compounds belong to hydroxycinnamic

group (chlorogenic acid), and flavonol group (quercetin derivatives such as quercetin-3-

galactoside, quercetin-3-glucoside, quercetin-3-rhamnoside and unknown quercetin

derivatives). In the flesh, compounds belonging to flavan-3-ol group ((+)-catechin, (-)-

epicatechin, procyanidin B1), dihydroxychalcone group (phloretin derivative and

phloridzin) and hydroxycinnamic acid group (chlorogenic acid) were identified.

Table 2. Phenolic compounds found in Wild apple

Peak Assignment

1 Procyanidin B1

2 (+)-Catechin

3 Chlorogenic acid

4 (-)-Epicatechin

5 Quercetin-3-galactoside

6 Quercetin-3-glucoside

7 Phloridzin

8 Quercetin-3-rhamnsoide

9 Quercetin

Unknown and tentatively identified

a Phloretin-2’-xyloglucoside

b Unknown phenolic acid

c Quercetin derivative

d Quercetin derivative

e Quercetin-3-xyloside


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Fig. 5. The HPLC chromatogram of Wild apple. a) chromatogram of flesh of Wild apple,

scanned at 280 nm; b) chromatogram of peel of Wild apple, scanned at 280 nm.

For peak identification, see Table 2.

Conclusions

In this work, the antiradical activity of polyphenolic compounds from flesh and peel of old

apple varieties grown in Croatia was studied. Antiradical activity was determined by

DPPH method, where polyphenols scavenge free DPPH˙ radicals. A biphasic reaction

between apple polyphenols and DPPH˙ radicals was observed, with “fast” and “slow”

scavenging rate. Furthermore, the antiradical activity of apples was compared. It was found

that polyphenols from the flesh of Wild apples showed much higher antiradical activity

then polyphenols from the flesh of other apples. The antiradical activity of polyphenols

from the peel was similar. Due to these results, Wild apples can be highlighted among all

apples due to a very high antiradical activity of polyphenols from the flesh. Old apple

varieties represent a significant source for horticultural biodiversity and indicate the need

for preserving and protecting them.


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Acknowledgments

We wish to thank family orchard (Veić M.) for supplying apple samples.

This research was financed by Josip Juraj Strossmayer University of Osijek, project:

Characterization of polyphenols in old apple cultivars.

References

Brand Williams, W., Cuvelier, M.E., Berset, C. (1995): Use of a free radical method to evaluate

antioxidant activity, Lebensm-Wiss. U. Technol. 28, 25-30.

Carbone, K., Giannini, B., Picchi V., Lo Scalzo, R., Cecchini, F. (2011): Phenolic composition and

free radical scavenging activity of different apple varieties in relation to the cultivar, tissue

type and storage, Food Chem. 127, 493-500.

Ceymann, M., Arrigoni, E., Schärer, H., Bozzi Nising A., Hurrell, F.R. (2012): Identification of

apples rich in health-promoting flavan-3-ols and phenolic acids by measuring the polyphenol

profile, J. Food Compos. Anal. 26, 128-135.

Iacopini, P., Camangi, F., Stefani A, Sebastiani, L. (2010): Antiradical potential of ancient Italian

apple varieties of Malus x domestica Borkh.

in a peroxynitrite-induced oxidative process, J. Food Compos. Anal. 23, 518-524.

Jakobek, L., Šeruga, M., Novak, I., Medvidović-Kosanović, M., Šeruga, B. (2008): DPPH radical

inhibition kinetic and antiradical activity of polyphenols from chokeberry and elderberry fruits,

Pomol. Croatica 14, 101-118.

Jakobek, L., Garcia-Villalba, R., Tomas-Barberan, F. (2013): Polyphenolic characterisation of old

local apple varieties from Southeastern European region, J. Food Compos. Anal. 31, 199-211.

John, K.M.M., Enkhtaivan, G., Kim, J.J., Kim, D.H. (2014): Metabolic variation and antioxidant

potential of Malus prunifolia (wild apple) compared with high flavon-3-ol containing fruits

(apple, grapes) and beverage (black tea), Food Chem. 163, 46-50.

Lata, B., Trampczynska, A., Paczesna, J. (2009): Cultivar variation in apple peel and whole fruit

phenolic composition, Sci. Hortic. 121, 176-181.

Madsen H.L., Andersen C.M., Jorgensen L.V., Skibsted L.H. (2010): Radical scavenging by dietary

flavonoids. A kinetic study of antioxidant efficiencies, Eur. Food Res. Technol. 211, 240-246.

Mendoza-Wilson, A., Armenta-Vázquez, M.E., Castro-Arredondo, S.I., Espinosa-Plascencia, A.,

Robles-Burgueño, M.R., González-Ríos, H., González-León, A., Balandrán-Quintana, R.R.

(2013): Potential of polyphenols from an aqueous extract of apple peel as inhibitors of free

radicals: An experimental and computational study, J. Mol. Struct. 1035, 61-68.

Prior, R.L, Wu, X., Schaich, K. (2005): Standardized Methods for Determination of Antioxidant

Capacity and Phenolics in Food and Dietary Supplements, J. Agric. Food Chem. 53 (10),

4290-4302.


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Određivanje teksture (čvrstoće) i SFC profila binarnih i ternarnih smjesa

palminog ulja, palminog stearina i sojinog ulja

UDC: 664.34


Zvijezda d.d., Marijana Čavića 1, 10000 Zagreb, Hrvatska

Sažetak

U ovom radu proučavani su sadržaji čvrstih triglicerida (SFC) pomoću nuklearne magnetske rezonance i

tekstura (čvrstoća) binarnih i ternarnih smjesa palminog ulja (PO), palminog stearina (PS) i sojinog ulja

(SO). Reološka svojstva teksture odnosno čvrstoće ovih smjesa određena su penetracijom pri konstantnoj

brzini. Pripremljene su binarne smjese PO/SO, PS/SO, PO/PS i ternarne smjese PS/PO/SO sa 20%-tnim

povećanjem udjela svake pojedine komponente. Statički su kristalizirane u hladnjaku na -25 °C kroz

20 min, a zatim temperirane kroz 48 H na 15 °C. S obzirom da se ovi lipidni sistemi uobičajeno koriste u

proizvodnji margarina i masti, cilj rada bio je procijeniti fizikalne karakteristike kao što su tekstura

(čvrstoća) i SFC profil, te njihove međusobne odnose. Dobiveni rezultati ukazuju da se povećanjem

čvrste komponente (PO i PS) u smjesama PO/SO i PS/SO povećavala čvrstoća kao i SFC vrijednost.

Slični rezultati dobiveni su i kod proučavanih ternarnih smjesa. Izuzetak je smjesa PS/PO sa više od 50%

PS gdje porastom SFC vrijednosti nije došlo i do očekivanog porasta čvrstoće.

Ključne riječi: SFC, tekstura, binarne i ternarne smjese masti

Uvod

Na teksturu prehrambenih proizvoda na bazi masti u velikoj mjeri utječu struktura i

mehanička svojstva umreženih kristala masti (Marangoni, 2002). Neka od najvažnijih

kvalitativnih svojstava proizvoda koji sadržavaju masti ovise o makroskopskim

karakteristikama kristala masti koji tvore umreženi sustav unutar gotovog proizvoda. Ova

svojstva kvalitete uključuju npr. mazivost margarina, maslaca i namaza kao i lom

čokolade, pa je očigledno da je iznimno važno pokušati predvidjeti ta svojstva (Narine et

al., 1999a). U radu na razvoju novih proizvoda koji uključuju ulja i masti, profil sadržaja

čvrstih triglicerida (SFC) vrlo je važan parametar koji se koristi za procjenu prihvatljivosti

pojedine masti ili smjese (blende) masti i ulja za određenu primjenu (Noraini et al., 1995).

Većini biljnih ulja nedostaje funkcionalno svojstvo kako bi zadovoljili zahtjeve potrošača

za teksturom i stabilnošću u prehrambenim proizvodima. Prije inkorporiranja u margarine,

smjese (blende), masti i ulja trebaju biti modificirane bilo fizikalno frakcioniranjem ili

blendiranjem, bilo kemijski hidrogenacijom ili interesterificiranjem. Zadnjih godina

hidrogenacija se sve manje koristi u margarinskim industrijama zbog potrebe da se



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izbjegne proizvodnja trans izomera masnih kiselina. Masne blende bez trans masnih

kiselina, odnosno hidrogeniranih masti uglavnom su bazirane na smjesama palminog ulja i

njegovih frakcija sa ili bez sjemenskih ulja (Wassell et al., 2007; Jirasubkunakorn et al,

2007). Tekstura odnosno čvrstoća masti ili smjese masti i ulja važno je svojstvo koje

značajno utječe na dojam o teksturi prehrambenih proizvoda (Brunello et al., 2003).

Sadržaj čvrstih triglicerida (SFC) značajno utječe na mehaničko ponašanje masti, ali oslanjanje

samo na SFC kako bi se predvidjela čvrstoća pokazalo se nepouzdanim. Na mehanička

svojstva jestivih masti mogu utjecati razni faktori uključujući SFC profil, polimorfizam čvrstog

stanja kao i mikrostruktura mreže kristalnih čestica (Narine et al., 1999b; Narine et al., 1999c).

U ovom radu proučavani su sadržaji čvrstih triglicerida (SFC) pomoću nuklearne

magnetske rezonance i tekstura (čvrstoća) binarnih i ternarnih smjesa palminog ulja (PO),

palminog stearina (PS) i sojinog ulja (SO).

Materijali i metode

Materijali

Za ovo istraživanje korišteni su komercijalni uzorci rafiniranog, dekoloriranog i

deodoriziranog palminog ulja (PO), palminog stearina (PS) i sojinog ulja (SO) iz tvornice

ulja i masti Zvijezda d.d. Zagreb, Hrvatska.

Priprema uzoraka

Binarne smjese (w/w) masti i ulja pripremljene su u intervalima od po 20±0,1 % svake

pojedine komponente u rasponu od 0-100%, a za ternarne smjese mješavine raznih udjela kako

bi se pokrilo šire područje ternarnog dijagrama. Binarne smjese (blende) PO/SO, PS/SO,

PO/PS i ternarne smjese PO/PS/SO pripremljene su otapanjem svake vrste masti odnosno ulja

na 80 °C i njihovim međusobnim miješanjem u staklenim posudama prema unaprijed zadanim

omjerima. Vrijeme miješanja bilo je 15 min na 60 °C, a ukupna količina svake smjese iznosila

je 1 kg. Sve blende su statički kristalizirane u hladnjaku na -25 °C kroz 20 minuta, a zatim su

temperirane na 15±0,5 °C u vremenu od 48 h. Ovim načinom pripreme uzoraka pokušalo se

simulirati hlađenje na industrijskom izmjenjivaču topline s brišućom površinom.

SFC profil

Za određivanje sadržaja čvrstih triglicerida korišten je pulsni NMR spektrometar (Minispec-

pc120; Bruker, Karlsruhe, Germany). Profil sadržaja čvrstih triglicerida mjeren je prema

ISO8292 metodi (ISO 8292-1:2008). Uzorci su 20 min grijani na 80 °C, kako bi se izbrisala

kristalna povijest, i temperirani na 60 °C minimalno 5 min, zatim držani na 10 °C kroz 16 h i

konačno kroz minimalno 30 min temperirani na 20 °C, 25 °C, 30 °C i 35 °C prije analiza.


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Podaci su dobiveni i analizirani koristeći programski paket instaliran od strane proizvođača

instrumenta. Automatska kalibracija vršila se na dnevnoj bazi koristeći 3 standarda (isporučeni

od strane Bruker-a) koji su sadržavali 0,0%, 31,3% i 74,0% čvrstih triglicerida.

Mjerenje teksture

Uzorci su temperirani na 20±0,1 °C kroz 2 h prije mjerenja na instrumentu STEVENS L.F.R.A.

TEXTURE ANALYSER (Stevens Advanced Weighing Systems Ltd., Essex, United Kingdom).

Penetracija konstantnom brzinom

Uzorci, homogeno napunjeni u staklene čaše promjera 71,5 mm i visine 70 mm, penetrirani su

do dubine od 5 mm cilindričnom sondom promjera 2,2 mm pri brzini od 0,5 mm/s. Minimalno

su rađena po 3 penetracijska testa za svaki uzorak na 20 °C. Zabilježene su najveća i

konačna penetracijska sila, a kao pokazatelj čvrstoće uzoraka korištena je vrijednost

maksimalne penetracijske sile izražene u gramima.


Pri obradi podataka korištene su dobivene vrijednosti sadržaja čvrstih triglicerida - SFC i

penetracije uzoraka temperiranih na 20 °C. Slika 1 prikazuju SFC na 20 °C sa postupnim

povećanjem udjela čvršće komponente (u smjesi PO/SO čvrsta komponenta je PO, dok je u

smjesama PS/SO i PS/PO čvršća komponenta PS). Kao što je vidljivo iz slike 1 dodatkom

čvrste komponente dolazi do porasta SFC vrijednosti u svim binarnim blendama.

Slika 1. SFC profil na 20 °C za binarne smjese PO/SO, PS/SO i PS/PO

Fig. 1. SFC profile at 20 °C for binary blends PO/SO, PS/SO and PS/PO


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Na slici 2 prikazana je maksimalna zabilježena sila penetracije na 20 °C u odnosu na SFC

vrijednost za binarnu smjesu PO/SO, a na slici 3 za binarnu smjesu PS/SO. Kao što je i

očekivano maksimalna sila penetracije (čvrstoća) rasla je s porastom SFC vrijednosti, odnosno

kako se povećavao udio čvrste komponente (PO i PS) u smjesi. Također, kod smjese PS/SO

zabilježene su više SFC vrijednosti kao i vrijednosti čvrstoće, nego kod smjese PO/SO što je

povezano sa većim sadržajem triglicerida s višom točkom topljenja u palminom stearinu (PS).

Slika 2. Odnos čvrstoće i SFC vrijednosti na 20 °C za binarnu smjesu PO/SO

Fig. 2. Relationship between hardness and SFC value at 20 °C for binary blend PO/SO

Slika 3. Odnos čvrstoće i SFC vrijednosti na 20 °C za binarnu smjesu PS/SO

Fig. 3. Relationship between hardness and SFC value at 20 °C for binary blend PS/SO

PO(%w/w) SO(%w/w)

0 100

20 80

40 60

60 40

80 20

100 0

PS(%w/w) SO(%w/w)

0 100

20 80

40 60

60 40

80 20

100 0


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Međutim, dobiveni rezultati za binarnu smjesu PS/PO (Slika 4) sa više od 50 % PS, pokazuju

da porastom SFC vrijednosti nije došlo i do očekivanog porasta čvrstoće. Različite SFC

vrijednosti uzoraka za približno slične vrijednosti čvrstoće mogu biti uzrokovane drugačijom

strukturom (tip kristala i njihova međusobna povezanost) koja se formira nakon kristalizacije

(Braipson-Danthine et al., 2004).

Slika 4. Odnos čvrstoće i SFC vrijednosti na 20 °C za binarnu smjesu PS/PO

Fig. 4. Relationship between hardness and SFC value at 20 °C for binary blend PS/PO

Slika 5 prikazuje maksimalnu zabilježenu silu penetracije na 20 °C u odnosu na SFC

vrijednost za ternarne smjese PS/PO/SO. Iz prikazanih rezultata također možemo uočiti da

je čvrstoća smjese PS/PO/SO u omjeru 20/60/20 značajno veća od smjese PS/PO/SO u

omjeru 40/20/40, iako su im SFC vrijednosti gotovo identične.

Slika 5. Odnos čvrstoće i SFC vrijednosti na 20 °C za ternarnu smjesu PS/PO/SO

Fig. 5. Relationship between hardness and SFC value at 20 °C for ternary blend PS/PO/SO

PS(%w/w) PO(%w/w)

0 100

20 80

40 60

60 40

80 20

100 0

PS(%w/w) PO(%w/w) SO(%w/w)

20 40 40

20 60 20

40 20 40

40 40 20

60 20 20


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Zaključci

Pri razvoju proizvoda na bazi ulja i masti, tekstura i SFC profil su vrlo važni parametri za

definiranje prikladnosti pojedine masne sirovine ili njihove smjese u konačnom proizvodu.

Proučavanje međusobnih odnosa vrijednosti čvrstoće i SFC profila za binarne i ternarne

smjese palminog ulja, palminog stearina i sojinog ulja, kroz ispitivanja u ovom radu,

ukazuju da te dvije varijable nisu uvijek u korelaciji. Različite SFC vrijednosti uzoraka za

približno slične vrijednosti čvrstoće mogu biti uzrokovane drugačijom strukturom (tip

kristala i njihova međusobna povezanost) koja se formira nakon kristalizacije.

Literatura

Braipson-Danthine, S., Deroanne, C. (2004): Influence of SFC, microstructure and polymorphism

on texture (hardness) of binary blends of fats involved in the preparation of industrial

shortenings, Food Research International 37, 941-948.

Brunello, N., Mc Gauley, S.E., Marangoni, A.G. (2003): Mechanical properties of cocoa butter in

relation to its crystallization behavior and microstructure, Lebensm.-Wiss. u.-Technol.

Lebensmittel-Wissenshaft & Technologie 36, 525-532.

ISO 8292-1:2008 Animal and vegetable fats and oils - Determination of solid fat content by pulsed

NMR - Part 1: Direct method

Jirasubkunakorn, W., Bell, A.E., Gordon, M.H., Smith, K.W. (2007): Effects of variation in the palm

stearin:palm olein ratio on the crystallization of a low-trans shortening, Food Chem. 103, 477-

485.

Marangoni, A.G. (2002): Special issue of FRI-crystallization, structure and functionality of fats,

Food Research International 35 (10), 907-908.

Narine, S.S., Marangoni, A.G. (1999a): Fractal nature of fat crystal networks, Physical Review E, 59

(2), 1908-1920.

Narine, S.S., Marangoni, A.G. (1999b): Microscopic and rheological studies of fat crystal networks,

Journal of Crystal Growth 198/199, 1315-1319.

Narine, S.S., Marangoni, A.G. (1999c): Relating structure of fat crystal networks to mechanical

properties: a review, Food Research International 32, 227-248.

Noraini, I., Embong, M.S., Aminah, A., Md.Aliand, A.R., Che Maimon, C.H. (1995): Physical

characteristics of some shortenings based on modified palm oil, milk fat and low melting milk

fat fraction, Fat Sci. Technol. 7, 253-260.

Wassell, P., Young, N.W.G. (2007): Food application of trans fatty acid substitutes, Int. J. Food Sci.

Technol. 42, 503-517.


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Determination of texture (hardness) and SFC profile of binary

and ternary mixtures of palm oil, palm stearin and soybean oil


Zvijezda d.d., Marijana Čavića 1, HR-10000 Zagreb, Croatia

Summary

This work studied solid fat content (SFC) by nuclear magnetic resonance and texture (hardness) of

binary and ternary blends of palm oil (PO), palm stearin (PS) and soybean oil (SO). Rheology

characteristics of texture like hardness for these blends were determined by constant speed

penetration. Binary blends PO/SO, PS/SO, PO/PS and ternary blends PS/PO/SO were prepared with

20% increase of every component. Blends were statically crystallized in a freezer at -25 °C for 20

min and then tempered at 15 °C for 48 h. Those lipid systems are commonly used in the margarine

and shortening production so the aim of this work was to evaluate physical properties like texture

(hardness) and SFC profile and their relationships. These results show that increase of hard

component (PO and PS) in PO/SO and PS/SO blends increased hardness and SFC values. Similar

results are obtained and for studied ternary blends. Exception is PS/PO blend with more than 50%

of PS where increase in SFC values did not result with expected increase in hardness.

Keywords: SFC, texture, binary and ternary fat blends


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Cryoprotective effect of oat β-glucans on beef myofibrillar proteins

UDC: 637.5'62

633.13 : 577.112.82

Krešimir Mastanjević, Dragan Kovačević, Kristina Vidaković

Josip Juraj Strossmayer University of Osijek, Faculty of Food Tecnnology Osijek, Franje Kuhača 20


Summary

Cryoprotective effects of oats β-glucans on beef myofibrillar proteins after frozen storage were

investigated by the use of Differential scanning calorimetry (DSC). Also influence of frozen storage

on texture profile analysis (TPA) parameters, instrumental colour parameters and cooking loss of

beef myofibrillar proteins were investigated. Beef myofibrillar proteins samples were prepared from

beef meat (mainly lat. musculus psoas major), mixed with oat β-glucans (w = 0-6%), quickly frozen

and stored for 30 days on -30 °C. Onset temperature of transition (To), peak thermal transition (Tp),

and endset temperature of transition (Te), and denaturation enthalpy (ΔH), were evaluated. Peak (Tp)

thermal transition temperatures of beef myofibrillar proteins showed shift to higher values with the

increase of mass fraction of β-glucans. Denaturation enthalpies (ΔH) of beef myofibrillar proteins

showed increase with increase of mass fraction of oat β-glucans. Instrumental colour parameters

(lightness (L*), redness (a*), yellowness (b*) and whiteness (L*-3b*) of beef myofibrillar proteins

were significantly (P<0.05) affected by addition oat β-glucans. Hardness, gumminess and chewiness

increased significantly (P<0.05) and cooking loss decreased significantly (P<0.05) by addition of

oat β-glucans. Cohesiveness and springiness of beef myofibrillar gels were not significantly

(p>0.05) affected by addition of oat β-glucans. Increase in peak thermal transition (Tp),

denaturation enthalpies (ΔH), some TPA and instrumental colour parameters indicates possible

cryostabilsation effect of oat β-glucans on beef myofibrillar proteins.

Keywords: cryoprotection, beef myofibrillar proteins, DSC, β-glucans texture (TPA), instrumental

colour (L*, a*, b*)

Introduction

Washed beef meat (WBM) is surimi-like product made from red beef meat. The process

for making surimi-like product from beef, with modified technology from fish surimi (Park

et al., 1996) results in semi-purified protein fraction containing a high concentration of

myofibrillar proteins. Freezing has become the most frequently used preservation method

for meat and meat products. To protect myofibrillar proteins from denaturation during



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frozen storage and maintain its possible high processability, some cryoprotectants (i. e.

disaccharides, polysaccharides, polyalcohol’s, organic acids and polyphosphates) are

generally added (MacDonald et al., 2005). Most commonly used instrumental methods for

determination cryoprotective effects of added substances are measurement of myofibrillar

protein solubility SEP (Salt extractable protein), Ca2+

ATP-ase activity, unfrozen water by

Nuclear Magnet Resonance (NMR), transition temperatures and denaturation enthalpies of

myofibrillar proteins by Differential Scanning Calorimetry (DSC) (Findlay and Barbut,

1990; Sych et al., 1990). β-glucans are composed of glucose molecules, which are linked

with β-(1,3), (1,4) and (1,6) glycosidic bonds. (1,3), (1,4)-β-D-glucans are commonly

isolated from wheat, barley and oats. Although found in all grains, their concentration is

highest in oats (4.6-4.9%) and barley (1.8 to 6%). β-glucans from various sources are used

in the food industry as a thickening agent, dietary fibers, emulsifiers, etc. (Brennan and

Cleary, 2005). Studies have shown that the addition of β-glucans to meat batter increases

the denaturation enthalpy of myofibrillar proteins, which suggests that β-glucans interact

with meat proteins and stabilize them (Morin et al., 2004). Differential scanning

calorimetry (DSC) is a useful technique used for studying thermal behaviour of muscle

proteins (Finday and Barbut, 1990). Changes in the protein structure during heating in

DSC analysis are referred to as protein denaturation, and peak temperatures of these

transitions are used to represent denaturation temperatures.

The objective of this study was to determine cryoprotective effects of oat β-glucans on

beef myofibrillar proteins using Differential Scanning Calorimetry (DSC), texture profile

analysis (TPA) and instrumental colour measurements (L*, a*, b*).

Material and methods

Sample preparation

Washed beef meat (WBM) samples were prepared in the laboratory from from beef meat

mainly (lat. m. psoas major) using the modified procedure of Yang and Froning (1992)

since washing and leaching was performed with distilled water, instead of with tap water.

Samples of WBM were mixed with oat β-glucans in mass fractions of 2, 4 and 6%. Mass

fractions were determined as percent of total mass. The pH level was measured in a

homogenate of the sample with distilled water (1:10, p/v) with pH/Ion 510-Bench

pH/Ion/mV Meter (Eutech Instruments Pte Ltd/ Oakton Instruments, USA). Water activity

(aw) was determined using a Rotronic Hygrolab 3 (Rotronic AG, Bassersdorf, Switzerland)

at a room temperature (20 ± 2 ºC). The FoodScan Meat Analyser was used to determine

moisture, total protein share, total fat share and collagen content according to the AOAC

2007. 04.


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DSC measurements

Differential scanning calorimetry (DSC) was performed using Mettler Toledo DSC 822e

differential scanning calorimeter equipped with STARe software. After defrosting in a

refrigerator (4 °C, over night), samples of ca. 15 mg (±1 mg) were weighed and sealed into

standard aluminium pans (40 μl) and scanned over the rage from 25 to 95 °C at a heating

rate of 10 °C min-1

, using empty standard aluminium pan as a reference. The peak

temperatures (Tp) were determined from DSC curves. The changes in enthalpy (ΔH J g-1

),

associated with the denaturation of proteins, were determined by measuring the area under

the DSC curves using STARe software.

Textural analysis (TPA) and cooking loss

Samples of washed beef meat (WBM) were placed into plastic test tubes with an inside

diameter of 10 mm. After defrosting, test tubes with their content were heated for 25 min

in a water bath at 80 oC. Test tubes with produced gels were cooled in ice water until the

temperature of approx. 20 oC was obtained inside the sample. After that they were stored at

4-6 oC until the next day. Cooking loss was calculated as a weight difference of the sample

prior to the cooking and after the removal of the cooked gel from the test tubes. Cooking

loss was expressed as a percent of the fresh sample weight. Texture profile analysis (TPA)

tests were performed using a TA.XT2i SMS Stable Micro Systems Texture Analyzer

(Stable Microsystems Ltd., Surrey, England) equipped with a cylindrical probe P/75. This

involved cutting samples into 1.5 cm thick slices, compressed twice to 60% of their

thickness. Force-time curves were recorded at across-head speed of 5 mm s-1

and the

recording speed was also 5 mm s-1

. The following parameters were quantified (Bourne

1978): hardness (g), maximum force required to compress the sample, springiness (ratio),

the ability of the sample to recover its original form after the deforming force was

removed, cohesiveness, the extent to which the sample could be deformed prior to rupture

(ratio) and chewiness (g), the work required to masticate the sample before swallowing,

which is calculated hardness · cohesiveness · springiness, was measured.

Determination of colour

Colour measurements (L*, a*, and b* values) were taken using a Hunter-Lab Mini ScanXE

(A60-1010-615 Model Colorimeter, Hunter-Lab, Reston, VA, USA). The instrument was

standardized each time with a white and black ceramic plate (L*0 = 93.01, a*0 = -1.11,

and b*0 = 1.30). The Hunter L*, a*, and b* values correspond to lightness, greenness (-a*)

or redness (+a*), and blueness (-b*) or yellowness (+b*), respectively. The whiteness (W)


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was calculated: L* - 3b*. The colour measurements were performed on WBM at a room

temperature (20 ± 2 ºC).


Three determinations for basic chemical composition, cooking loss, pH, aw, onset (To),

peak (Tp) and endset (Te) temperatures and transition enthalpies (ΔH J g-1

), seven for TPA

and colour parameters were measured from each sample. Experimental data were analyzed

by the analysis of variance (ANOVA) and Fisher’s least significant difference (LSD), with

significance defined at p < 0.05. Statistical analysis was carried out with Statistica ver. 8.0

StatSoft Inc. Tulsa, OK. USA.


The mean basic chemical composition, pH and aw values of individual WBM samples did

not vary significantly and are presented in Table 1. Differential scanning calorimetry

thermogram’s of WBM samples for each treatment after 30 days of frozen storage without

addition of oat β-glucans contained two endothermic transitions. Referring to previous

DSC studies of similar samples (Bircan and Barringer, 2002; Aktas, et al., 2005;

Fernandez-Martin, 2007), it can be assumed that two peaks in this study are related to the

thermal denaturation of myosin and actin.

Variance analysis of beef myosin Tp showed that myosin’s Tp varied significantly (p<0.05) as

a function of mass fraction oat β-glucans (Table 2). Addition of oat β-glucans (w = 2-6%)

caused shift of myosin’s Tp to higher values. These shifts in Tp of myosin to the higher values

as the mass fraction of oat β-glucans increases can be interpreted as a stabilization of

myofibrillar proteins since a higher temperature was required to denature these proteins

(Sych et al., 1990; Kovačević and Mastanjević, 2014). Peak temperatures of actin transition

did not vary significantly (p>0.05) as a function of mass fraction of oat β-glucans (Table 3).

Highest value of actin Tp showed the sample with addition of 6% of oat β-glucans (Table 3).

The method of expressing peak enthalpies ΔH was adopted to provide an estimate of the

quantity of native proteins (Sych et al., 1990; Herrera et al., 2001; Kovačević and

Mastanjević, 2011). Enthalpies of myosin and actin denaturation for WBM with addition

of oat β-glucans (w = 0-6%) are shown in tables 2 and 3. Values of ΔH for myosin and

actin showed an increase with the increase of mass fraction of oat β-glucans (w = 0-6%).

Since the value of denaturation enthalpy is directly related to the amount of native proteins,

higher values of ΔH indicate possible cryostabilization of beef myofibrillar proteins with

addition of oat β-glucans (w = 0-6%).


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Table 1. Basic chemical composition, aw and pH of WBM samples

Water

w (%)

Proteins

w (%)

Fat

w (%)

Collagen

w (%) pH aw

83.40 ± 0.02 15.38 ± 0.18 1.37 ± 0.02 0.98 ± 0.04 6.85 ± 0.04 0.97 ± 0.01 Values are means ± Standard deviation of triplicate

The cooking loss of WBM mixed with different mass fracion of oat β-glucans after 30 days

of frozen storage are presented in Fig. 1. The increase in mass fraction a of oat β-glucans

(w = 0-6%) caused a significant reduction (p<0.05) of cooking loss in the obtained gels.

Stangierski and Kijowski (2003) reported similar result for mechanically recovered washed

and frozen stored poultry meat with the addition of Cremodan and Pork Stock.

Table 2. Denaturation temperatures (To, Tp, Te) and denaturation enthalpies (ΔH) of WBM myosin

mixed with different mass fracion of oat β-glucans after 30 days of frozen storage

w (%) To (ºC) Tp (ºC) Te (ºC) ΔHm (J g-1)

0 57.62a ± 0.04 59.62c ± 0.12 64.57b ± 0.33 0.21b ± 0.02

2 55.25c ± 0.01 59.89c ± 0.17 63.26c ± 0.01 0.23b ± 0.03

4 56.44b ± 0.16 60.78b ± 0.15 69.42a ± 0.05 0.26b ± 0.01

6 56.45b ± 0.31 61.26a ± 0.11 69.54a ± 0.05 0.32a ± 0.02 Values are means ± S. D. of triplicate. Values in the same row with different letters (a-d) are

significantly different (p < 0.05)

Table 3. Denaturation temperatures (To, Tp, Te) and denaturation enthalpies (ΔH) of WBM samples

actin mixed with different mass fracion of oat β-glucans after 30 days of frozen storage

w (%) To (ºC) Tp (ºC) Te (ºC) ΔHa (J g-1)

0 70.71c ± 0.08 77.38b ± 0.04 82.63a ± 0.12 0.10a ± 0.01

2

10 (MŠT 2)

10 (MŠT 2)

70.93c ± 0.15 77.41b ± 0.01 80.63b ± 0.08 0.09a ± 0.01

4 71.68b ± 0.05 77.43b ± 0.13 81.07c ± 0.03 0.08a ± 0.01

6

10 (MŠJ)

72.29a ± 0.03 77.70a ± 0.03 81.47b ± 0.08 0.08a ± 0.01

Values are means ± S. D. of triplicate. Values in the same row with different letters (a-d) are



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Fig. 1. The Cooking loss WBM samples mixed with different mass fracion

of oat β-glucans after 30 days of frozen storage

Table 4. Texture profile of WBM sampels gels mixed with different mass fracion of oat β-glucans

after 30 days of frozen storage

w (%) Hardness (g) Springiness Cohesiveness Chewiness (g)

0 3160.44a ± 141.80 0,95a ± 0.01 0.72a ± 0.03 2160.92a ± 98.42

2 1840.86ab ± 61.99 0.91a ± 0.07 0.51ab ± 0.01 1021.09ab ± 31.45

4 1092.04b ± 18.21 0.87a ± 0.01 0.38b ± 0.01 362.62b ± 14.85

6 1261.58b± 75.79 0.86a ± 0.10 0.46ab ± 0.21 521.84b ± 75.05 Values are means ±SD of seven measurements. Values in the same row with different letters (a-b) are


Texture profile analysis parametrs of WBM mixed with different mass fracion of oat

β-glucans after 30 days of frozen storage are shown in Table 4. The addition of oat β-glucans

increased significantly (p<0.05) hardness and chewiness of WBM gels samples. The

cohesiveness and springiness were not affected with the increase of oat β-glucans mass fraction

(Table 4.)


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Table 5. Instrumental colour parameters of WBM mixed with different mass fracion of oat β-glucans

after 30 days of frozen storage

w (%) L* a* b* W

0 70.87c ± 0.61 4.62a ± 0.39 16.82a ± 0.31 20.41b ± 1.34

2 75.15b ± 0.66 3.11b ± 0.13 15.71b ± 0.24 28.04a ± 1.19

4 74.74b ± 0.38 2.63c ± 0.10 15.90b ± 0.11 28.31a ± 0.26

6 76.02a ± 0.43 2.58c ± 0.18 15.83b ± 0.21 27.25a ± 0.79 Values are means ±SD of seven measurements. Values in the same row with different

letters (a-c) are significantly different (p < 0.05)

Instrumental colour parameters of WBM with addion of oats β-glucans are presented in

Table 5. Generally, higher demand is for surimi gels with high lightness (L*), low

yellowness (b*) and high whiteness (W). The addition of β-glucans significantly increased

(p < 0.05) lightness and whiteness of WBM samples. This is in agreement with the study

that investigated the addition of potato starch and egg white to Alaska Pollock surimi

(Tabilo-Munizaga and Barbosa-Canovas, 2004).

Conclusions

Differential scanning calorimetry (DSC) revealed a shift in peak thermal transition

temperature (Tp) of myosin to higher temperature in WBM samples mixed with different

mass fraction of oat β-glucans. Shift in thermal transition temperature of myosin to higher

temperature, the increase of myosin transition enthalpies and some TPA (hardness,

chewiness) and colour parameters (L* and W) with the mass fraction oat β-glucans

increase, indicate that oat β-glucans interact with beef myofibrillar proteins and acting

according to the cryoprotecting mechanism.

References Aktas, N., Aksu, M. I., Kaya M. (2005.): Changes in myofibrillar proteins during processing of

pastirma (Turkish dry meat product) produced with commercial starter cultures, Food Chem.

90, 649-654.

Brennan, C. S. and Cleary, L. J. (2005): The potential use of cereal (1/3,1/4)-β-D-glucans as

functional food ingredients, J. Cereal. Sci. 42, 1-13.

Bircan, C. and Barringer, S. A. (2002): Determination of protein denaturation of muscle foods using

the dielectric properties. J. Food Sci. 67, 202-205.

Herrera, J.J., Pastoriza, L., Sampedro, G. (2001): A DSC study on the effects of various

maltodextrins and sucrose on protein changes in frozen-stored minced blue whiting muscle, J.

Sci. Food Agr. 81, 377-384.


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Fernandez-Martin, F. (2007): Bird muscles under hydrostatic high pressure/temperature

combinations - A DSC evaluation, J. Therm. Anal. Calorim. 87, 285-290.

Findlay, C. J. and Barbut, S. (1990): Thermal Analysis of Food Proteins in Relation to Processing

Data. In Thermal Analysis of Food, Harwalkar, V. R and Ma, C.–Y. (Eds.), Barking: Elsevier

Applied Science, pp. 92-125.

Kijowski, J. and Richardson, R. I. (1996): The effect of cryoprotectants during freezing or freeze

drying upon properties of washed mechanically recovered broiler meat, Int. J. Food Sci. Tech.

31, 45-54.

Kovačević, D. and Mastanjević, K (2011): Cryoprotective Effect of Trehalose and Maltose on

Washed and Frozen Stored Beef Meat, Czech J. Food Sci. 29(1), 15-23.

Kovačević, D. and Mastanjević, K (2014): Cryoprotective effect of trehalose on washed chicken

meat, J. Food Sci. Technol. 51(5), 1006-1010.

Macdonald, G., Lanier, T., Carvajal, P. A. (2005): Stabilisation of proteins in surimi. In: Surimi and

surimi seafood, Park, J. W. (Ed.), Boca Raton, USA: CRC Press, pp: 163-227.

Morin, L.A., Temelli, F., McMullen L. (2004): Interactions between meat proteins and barley

(Hordeum spp.) β-glucan within a reduced-fat breakfast sausage system, Meat Sci. 68, 419-

430.

Park, S., Brewer, M. S., Novakofski,J., Bechtel, P. J., Mckeith, K. F.(1996): Process and

Characteristic for a Surimi-like Material Made from Beef or Pork, J. Food Sci. 61, 422-427.

Stangierski, J. and Kijowski, J. (2003): Effect of selected commercial substances with

cryoprotective activity on the quality of mechanically recovered, washed and frozen stored

poultry meat, Nahrung. 47 (1), 49-53.

Sych, J., Lacroix, C., Adambounou, L. T., Castaigne, F. (1990): Cryoprotective Effects of Lactitol,

Palatinit and Polydextrose on Cod Surimi Proteins During Frozen Storage, J. Food Sci. 55,

356-360.

Tabilo-Munizaga, G. and Barbosa-Canovas, G. V. (2004): Color and textural parameters of

pressurized and heat-treated surimi gels as affected by potato starch and egg white, Food Res.

Int. 37, 767-775.

Yang, T.S. and Froning, G.W. (1992): Changes in Myofibrillar Protein and Collagen Content of

Mechanically Deboned Chicken Meat Due to Washing and Screening, Poultry Sci. 71, 1221-

1227.


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Utjecaj mikorize i kvasaca na kakvoću vina

sorte merlot (Vitis vinifera L.)

UDC: 663.252.4 : 663.15

Josip Mesić, Valentina Obradović, Maja Ergović Ravančić,

Brankica Svitlica, Jelena Žilić

Veleučilište u Požegi, Vukovarska 17, 34000 Požega, Hrvatska

Sažetak

Mikoriza je simbioza korjena vinove loze i micelija mikoriznih gljiva koja omogućuje biljci bolju

apsorpciju hranjivih tvari iz tla, što se može u konačnici odraziti na kakvoću vina. Cilj istraživanja

bio je usporediti kemijska i senzorska svojstva vina sorte Merlot (berba 2013.), u ovisnosti o

nacijepljivanju korjena mikorizom. Osim toga, fermentacija moštova je provedena pomoću dvaju

različitih kvasaca: hibrida Saccharomyces cerevisiae pripremljenog za fermentaciju bordoških

tipova crnih vina kao što je Cabernet Sauvignon, te hibrida između Saccharomyces cerevisiae i

Saccharomyces paradoxus. U vinima su određeni sljedeći kemijski parametri: alkohol, ukupne

kiseline, pH, ukupni suhi ekstrakt, reducirajući šećeri, ekstrakt bez šećera, pepeo, te ukupni i

slobodni SO2. Također su određeni ukupni antocijani pH diferencijalnom metodom, ukupni

polifenoli Folin-Ciocalteu metodom, antioksidativna aktivnost (ABTS metodom), te nijansa boje i

gustoća boje. Senzorsko ocjenjivanje je provedeno metodom 100 bodova. Rezultati su pokazali

utjecaj kvasaca na udio polifenola, antocijana i antioksidativnu aktivnost, dok utjecaj mikorize nije

pokazao značajan utjecaj na navedene parametre.

Ključne riječi: mikoriza, S. cerevisiae, S. paradoxus, Merlot

Uvod

Prema Turkoviću i Miroševiću (2003) Kultivar Merlot uzgaja se u uvjetima umjerene

klime i navode da je prikladan za područje sjevernog Jadrana, a u sjevernim područjima

Hrvatske s uspjesima koji mogu tek uvjetno zadovoljiti. U Vinogorju Kutjevo na područje

podregije Slavonija, vinogradarske regije Istočna kontinentalna Hrvatska zadnjih desetak

godina Merlot je posađen u mnogim vinogradima.

Neobično da jedna sorta postane tako moderna, bez da u svijetu postoji bilo kakav stvarni

konsenzus o tome kako bi se trebala uzgajati i proizvoditi (Oz i Rand, 2008). U nasadu

Merlota Veleučilišta u Požegi na dio trsova nacijepljena je mikorizna gljiva za vinovu

lozu, a fermentacija moštova je provedena pomoću dvaju različitih kvasaca: hibrida



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Saccharomyces cerevisiae pripremljenog za fermentaciju bordoških tipova crnih vina kao što je

Cabernet sauvignon, te hibrida između Saccharomyces cerevisiae i Saccharomyces paradoxus.

Mikoriza je simbioza biljaka i gljiva, u ovom slučaju trsa vinove loze i gljiva koje se

nalaze na korijenu vinove loze. Prema dosadašnjim istraživanjima mikoriza bi trebala

povoljno utjecati na otpornost trsova prema suši, lakše usvajanje hraniva iz tla i povećanu

otpornost prema nekim patogenima kao što je pepelnica (Barea i sur., 2011), kao i na

povećano stvaranje biomase i povećan sadržaj sekundarnik produkata metabolizma kao što

su fenolni spojevi (Hare Krishna i sur., 2005).

Eftekhari i suradnici (2012) istraživali su utjecaj različitih mikoriznih gljiva na promjene

koje se događaju na trsovima vinove loze (Vitis vinifera L.), te su utvrdili da su pojedini

vegetativni dijelovi vrijedni izvori za ekstrakciju flavonoida kvercetina čija se količina

povećava nakon inokulacije mikoriznim gljivama. Uzročnici alkoholne fermentacije,

kvaščeve gljivice u svom metbolizmu tijekom fermentacije osim osnovnih produkata

proizvode veliki broj sekundarnih spojeva. Razližite vrste i sojevi kvasaca mogu znatno

utjecati na kemijski sastav i kakvoću vina (Ribereau-Gayon i sur., 2006).

Cilj je ovog istraživanja bio usporediti kemijska i senzorska svojstva vina sorte Merlot

(berba 2013.), impementirajući različite zahvate u vinogradu i u procesu proizvodnje vina.

Materijali i metode

Istraživanje je provedeno u vinskom laboratoriju Veleučilišta u Požegi u i podrumu Mesić

u Požegi. Nasad kultivara Merlota nalazi se na položaju Gradina iznad sela Vetova u

Općini Kaptol istočno do sela Podgorje na nadmorskoj visini od 350 m u sklopu Vinogorja

Kutjevo, vinogradarske podregije Slavonija, regija Istočna kontinentalna Hrvatska, u

području umjereno kontinentalne klime. Nasad je posađen 2007. godine u sklopu

nastavnog objekta Veleučilišta u Požegi.

Na dijelu nasada 7. lipnja 2013. godine izvršena je mikorizacija trsova Merlota.

Mikorizacija trsova obavljena je pripravkom Mykoflor za vinovu lozu, na način da je

pripravak injektiran u zonu korjena na dubinu od oko 30 do 50 centimetara. Nasad se u to

vrijeme nalazio u fazi pred početak cvatnje.

Berba grožđa obavljena je 07. listopada 2013. godine. Sadržaj šećera iznosio je 100 °Oe,

dok su je ukupna kiselost izražena kao vinska iznosila 7 g/l. Berba je obavljena ručno, a

urod grožđa po trsu kretao se oko 1,5 kilogram. Posebno su pobrani trsovi Merlota na

kojem je izvršena mikorizacija, a posebno trsovi iz kontrolnog tretmana (bez mikorize).

Primarna prerada grožđa odbavljena je posebno za tretman kontrole, a posebno za tretman

mikorize. Nakon muljanja i runjanja u masulj su dodani kvasci, pri čemu smo dobili četiri

različita tretmana pokusa koji čine:


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K ex – na trsovima nije izvršena mikorizacija, a fermentacija je provedena pomoću

hibridnog kvasca između Saccharomyces cerevisiae i Saccharomyces paradoxus, (Anchor

Exotics SPH),

M ex – na trsovima je izvršena mikorizacija, a fermentacija je provedena pomoću

hibridnog kvasca između Saccharomyces cerevisiae i Saccharomyces paradoxus, (Anchor

Exotics SPH),

K cru – na trsovima nije izvršena mikorizacija, a fermentacija je provedena pomoću kvasca

SIHA Rubino cru (Saccharomyces cerevisiae),

M cru – na trsovima je izvršena mikorizacija, a fermentacija je provedena pomoću kvasca

SIHA Rubino cru (Saccharomyces cerevisiae).

Masulj je macerirao u plastičnim posudama, pri čemu je dva puta dnevno potapan klobuk.

Temperatura tijekom maceracije i fermentacije iznosila je 18 °C. Masulj je prešan 21.

listopada, što govori da je maceracija trajala 14 dana kako bi dobili čim veću punoću

budućeg vina. Nakon maceracije u demižonima zapremine 34 litre provedena je

fermentacija do kraja nakon čega je vino pretočeno te izbistreno, napunjeno u butelje i

pripremljeno za kemijsku analizu i senzorno ocjenjivanje. Kemijska analiza i senzorno

ocjenjivanje provedeni su nakon odležavanja vina u bocama u trajanju od 6 mjeseci.

U laboratriju Veleučilišta u Požegi u svim vinima su određeni slijedeći kemijski parametri:

alkohol, ukupne kiseline, pH, ukupni suhi ekstrakt, reducirajući šećeri, ekstrakt bez šećera,

pepeo, te ukupni i slobodni SO2. Također su određeni ukupni antocijani pH

diferencijalnom metodom, ukupni polifenoli Folin-Ciocalteu metodom, antioksidativna

aktivnost (ABTS metodom), te ton boje i gustoća boje.

Senzorno ocjenjivanje vina provedeno je u prostorijama Veleučilčišta, a panel ocjenjivača

činili su proizvođači s višegodišnjim iskustvom.

Ocjenjivanje je provedeno metodom 100 bodova prema važećem pravilniku za

organoleptičko (senzorno) ocjenjivanje vina i voćnih vina („Narodne novine“, broj

106/04, 137/12, 142/13, 48/14).


U tablici 1 prikazane su vrijednosti sadržaja alkohola u volumnim postotcima,

reducirajućeg šećera u gramima po litri, ukupne kiselosti izražene kao vinska kiselina u

gramima po litri, pH vrijednost, sadržaj slobodnog i ukupnog SO2 (mg/L), pepela (g/L),

ukupnog suhog ekstrakta i ekstrakta bez šećera u gramima po litri, sadržaj ukupnih

polifenola, ukupnih antocijana, nijansa boje (Hue), gustoća boje (Density) i antioksidativna

aktivnost u tretmanima vina kultivara Melot (Vitis vinifera L.).


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U svim analiziranim vinima sadržaj alkohol je gotovo jednak, a ostatak neprevrelog šećera

stavlja sve tretmane na granicu suhog i polusuhog vina. Ukupna kiselost jednaka je u

tretmanima K ex i K cru te iznosi 5,7 g/l dok je kod mikorize različita ovisno od

apliciranih kvasaca. Veća vrijednost zabilježena je kod tretmana M ex (5,9), dok je kod

tretmana M cru 5,2 g/l. Ukupna kiselost u svim tretmanima je gotovo podjednaka nema

značajnije razlike kao i u kod vrijednosti pH. Neznatno niža pH vrijednost zabilježena je

kod tretmana proizvedenih pomoću kvasca SIHA Rubino cru (Saccharomyces cerevisiae).

Rezultati drugih autora ukazuju da pojedini sojevi kvasaca imaju značajan pozitivan ili

negativan utjecaj na kemijski sastav vina (Liang i sur., 2013) obzirom da većina aroma u

vinu potječe upravo iz alkoholne fermentacije (Marullo i Dubourdieu 2010). Međutim u

provedenom istraživanju nisu zabilježene veće razlike među tretmanima Merlota.

Tablica 1. Sadržaj alkohola (vol%), reducirajući šećeri (g/L), ukupna kiselost (g/L), pH, slobodni SO2

(mg/L), ukupni SO2 (mg/L), pepeo (g/L), ukupni suhi ekstrakt (g/L), ekstrakt bez šećera

(g/L), ukupni polifenoli (mgGAE/L)a, antocijani (mgMGE/L)

a, nijansa boje, gustoća boje

(Density), antioksidativna aktivnost (ABTS) micromol TE / L u vinu, Merlot, 2013.

Table. 1. The alcohol content (vol%), reducing sugars (g/L), total acidity (g/L), pH, Free SO2

(mg/L), total SO2 (mg/L), ash (g/L), total dry extract (g/L), sugar-free extract (g/L),

polyphenols (mgGAE/L)a, Anthocyanes (mgMGE/L)

a, hue, color density (Density), AA

(ABTS) micromol TE / L in wine, Merlot, 2013.

K ex M ex K cru M cru

Sadržaj alkohola (vol%) 14,7 14,6 14,5 14,4

Reducirajući šećeri (g/L) 4,36 4,81 4,22 4,78

Ukupna kiselost (g/L) 5,7 5,9 5,7 5,2

pH 3,53 3,51 3,43 3,47

Slobodni SO2 (mg/L) 5 6 7 7

Ukupni SO2 (mg/L) 53 48 39 38

Pepeo (g/L) 2,55 2,5 2,33 2,37

Ukupni suhi ekstrakt (g/L) 24,8 24,2 23,7 23,7

Ekstrakt bez šećera (g/L) 20,4 19,4 19,5 18,9

Polifenoli (mgGAE/L) 1071,7 1030,1 983,64 1020,5

Antocijani (mgMGE//L) 116,3 110,33 88,13 90,24

Nijansa boje (Hue) 0,56 0,54 0,57 0,51

Gustoća boje (Density) 9,85 10,09 8,41 7,82

AA (ABTS) µmol TE/L 15463 16207 14173 13667 aGAE-ekvivalent galne kiseline (gallic acid equivalent)

MGE-ekvivalent malvidin-3-glukozida (malvidin-3-glucoside equivalent)

Slobodni i ukupni SO2 ujednačen je i nizak svim tretmanima i iz tog razloga ne možemo

govoriti o utjecaju slobodnog SO2 na senzorna svojstva, obzirom da veća količina u vinu

daje osjećaj svježine (Clark i Bakker, 2004). Veće vrijednosti pepela, ukupnog suhog

ekstrakta i sadržaja polifenola te antocijana zabilježene su u vinima proizvedenima


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pomoću hibridnog kvasca Saccharomyces cerevisiae x Saccharomyces paradoxus. U

istima vinima (K ex i M ex) bilježimo i veću antioksidativno aktivnost.

Veće vrijednosti nijanse boje zabilježene su u tretmanima u kojima nije aplicirana

mikoriza neovisno o kvascima. Gustoća boje većaje u vinima proizvedenima pomoću

hibridnog kvasca Saccharomyces cerevisiae x Saccharomyces paradoxus.

U tablici 2 prikazani su rezultati organoleptičkog ocjenjivanja vina metodom 100 bodova.

Posebno su istaknute ukupne ocijene za izgled, miris i okus vina. Sve su ocijene medijane

pojedinog ocijenjenog svojstva.

Tablica 2. Izgled (bistroća i boja), miris (čistoća, intenzitet i kvaliteta), okus (čistoća, intenzitet,

trajnost i kvaliteta), harmonija i ukupna ocjena, tretmana Merlota, 2013.

Table 2. The appearance (clarity, color), odor (cleanliness, intensity, quality), flavour (cleanliness,

intensity, durability, quality), harmony and overall rate, variants of Merlot, 2013.

K ex M ex K cru M cru

Bistroća 5 5 5 5

Boja 10 10 10 10

Izgled 15 15 15 15

Čistoća 7 7 7 7

Intenzitet 5 5 5 5

Kvaliteta 14 14 16 12

Miris 26 26 28 24

Čistoća 7 6 7 7

Intenzitet 4 4 5 5

Trajnost 19 16 16 19

Kvaliteta 6 6 7 7

Okus 36 32 35 38

Harmoničnost 10 10 9 9

Ukupna ocjena 87 83 87 86

Parametri izgleda (boja i bistroća) kod svih tretmana vina ocjenjeni u maksimalnim brojem

bodova. Boja je u svim uzorcima puna tamno crvena gotovo crna što odmah asocira na

punoću vina. Čistoća i intenzitet mirisa vina jednako je vrednovana i u svi su tretmani

ocijenjeni kao vrlo dobri. Najbolja kvaliteta mirisa zabilježena je u tretmanu K cru (16

bodova-odlično) dok je najslabije ocijenjen tretman M cru s 12 bodova (dobro).

Enzimatskom aktivnošću kvasaca dolazi da formiranja specifičnih aromatskih spojeva koji

direktno utječu na kakvoću mirisa vina (Ribereau-Gayon i sur. 2006). Čistoća okusa

jednako kao vrlo dobra zabilježena je u uzorcima K ex, K cru i M cru, dok je nešto lošije

vrednovan uzorak M ex. Intenzitet i kvaliteta okusa bolje su ocijenjeni u tretmanima gdje

je apliciran kvasac SIHA Rubino cru (Saccharomyces cerevisiae). Veću ocijenu trajnosti

okusa dobili su tretmani M ex i K cru. Ukupni dojam za okus vina najbolji je kod vina


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proizvedenog od grožđa na kojem je aplicirana mikoriza, a fermentacija provedena

pomoćuj kvasca SIHA Rubino cru, dok je najmanju ocijenu dobio tretman s mikorizom i

kvascem Saccharomyces cerevisiae i Saccharomyces paradoxus, (Anchor Exotics SPH).

Harmoničnost tretmana vina Merlot porizvedenog kvascem Anchor Exotics SPH

ocijenjena je kao vrlo dobra (ocijena 10), a tretmana vina proizvedenog kvascem SIHA

Rubino cru kao dobra (ocijena 9).

Ukupne ocijene vina bolje su u kontrolnim tretmanima u pogledu tretmana mikorizom i

ujedno su dobile najbolje ocijene (87 od 100 bodova). Najmanju ocijenu dobio je tretman

M ex, 83 boda. Prema važećem pravilniku o za organoleptičko (senzorno) ocjenjivanje

vina i voćnih vina, vina svih tretmana ubrajaju se u kategoriju vrhunskih vina, uz

napomenu da bi prema klasifikaciji prije izmjena pravilnika od 01.srpnja 2014. godine

tretman M ex bio deklariran kao kvalitetno vino. Razlog najmanjoj ocijeni nalazimo u

činjenici da je tretman M ex sadržavao malu količinu ugljičnog dioksida koji se detektirao

prilikom prvog kušanja, a prema Clark i Bakkeru (2004) ugljični dioksid daje bodljikav

osjećaj u ustima, koji je vjerojatno imao negativan utjecaj kod kušača. Kasnije se u

potpunosti gubio iz čaše što je komentirano kao naknoadno vrenje (početak malolaktične

fermentacije).

Zaključci

Pojedini kemijski parametri su pokazali značajan utjecaj kvasaca na udio polifenola,

antocijana i antioksidativnu aktivnost, dok utjecaj mikorize nije pokazao značajan utjecaj

na navedene parametre. Odsutnost utjecaja mikorize može se objasniti idealnim

vremenskim uvjetima za zrenje grožđa u 2013. godini, tako da je grožđe sazrelo na lozi bez

nacjepljene mikorize imalo dovoljnu količinu hranjivih tvari.

Organoleptičke ocjene vina ujednačene su osim uzorka koji je proizveden od grožđa

mikoriziranih trsova s hibridnim kvascem Saccharomyces cerevisiae x Saccharomyces

paradoxus.

Literatura

Barea, J.M., Palenzuela, J., Cornejo, P., Sánchez-Castro, i., Navarro-Fernández, C., Lopéz-García,

A., Estrada, B., Azcón, R., Ferrol, N., Azcón-Aguilar, C., (2011): Ecological and functional

roles of mycorrhizas in semi-arid ecosystems of Southeast Spain, Journal of Arid

Environments 75 (12), 1292-1301.

Clarke, R.J., Bakker, J., (2004): Wine flavour chemistry, Blackwell Publising Ltd, Oxford, UK,

173-176.

Clarke, Oz; Margaret Rand, (2008): Grapes & Wines, Pavilion books, United Kingdom, 128-137.


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Eftekhari, M., Alizadeh, M., Ebrahimi, P. (2012): Evaluation of the total phenolics and quercetin

content of foliage in mycorrhizal grape (Vitis vinifera L.) varieties and effect of postharvest

drying on quercetin yield, Industrial Crops and Products 38, 160-165.

Hare Krishna, S.K. Singh, R.R. Sharma, R.N. Khawale, Minakshi Grover, V.B. Patel, (2005):

Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to arbuscular-

mycorrhizal fungi (AMF) inoculation during ex vitro acclimatization, Scientia Horticulturae

106 (4), 554-567.

Jackson, R.S. (2008). Wine science, principles and applications, Elsevier inc. California, SAD 73-

74.

Heng-Yu Liang, Jing-Yu Chen, Reeves, M., Bei-Zhong Han, (2013): Aromatic and sensorial

profiles of young Cabernet Sauvignon wines fermented by different Chinese autochthonous

Saccharomyces cerevisiae strains, Food research international 51 (2), 855-865.

Marullo, P., Dubourdieu, D., (2010): 11 - Yeast selection for wine flavour modulation, Managing

Wine Quality, Oenology and wine quality Woodhead publising limited, 293-345.

Ministarstvo poljoprivrede, šumarstva i vodnoga gospodarstva (2014): Pravilnik o organoleptičkom

(senzornom) ocjenjivanju vina i voćnih vina, Narodne novine, broj 106/04, 137/12, 142/13,

48/14, Zagreb.

Mirošević, N., Turković, Z, (2008): Ampelografski atlas, Golden marketing tehnička knjiga, ISBN

953-212-019-X, Zagreb, 166-167.

Ribereau-Gayon, P., Dubourdieu, D., Doneche, B., Lonvaud, A., (2006): Handbook of enology

Volume 1, The mikrobiology of Wine and Vinification, John Wiley and sons, LTD, England,

53-78.


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Influence of mycorrhizae and yeast strain on quality

of wine variety merlot (Vitis vinifera L.)

Josip Mesić, Valentina Obradović, Maja Ergović Ravančić,

Brankica Svitlica, Jelena Žilić

Polytechnic in Požega, Vukovarska 17, HR-34000 Požega, Croatia

Summary

Mycorrhiza is symbiosis of the root of the vine and the mycelium of mycorrhizal fungi that allows

the plant better absorption of nutrients from the soil, which can ultimately affect the quality of wine.

The aim of the study was to compare the chemical and organoleptic characteristics of wine variety

Merlot (vintage 2013), depending on the root inoculation of mycorrhiza. In addition, the

fermentation of the musts was conducted using two different yeasts: Saccharomyces cerevisiae

hybrids prepared for fermentation types Bordeaux red wines such as Cabernet Sauvignon, as well as

hybrids between Saccharomyces cerevisiae and Saccharomyces paradoxus. The wines are

determined by the following chemical parameters: alcohol, total acidity, pH, total dry extract,

reducing sugars, sugar-free extract, ash, total SO2 and free SO2. Total anthocyanins pH was

determined with differential method, total polyphenols with Folin-Ciocalteu method, antioxidant

activity (ABTS method), and the hue and color density. Sensory evaluation was conducted by

method of 100 points. The results showed a effect on yeast polyphenols, anthocyanins and

antioxidant activity, while the impact of mycorrhiza had no significant effect on these parameters.

Keywords: mycorrhiza, S. cerevisiae, S. paradoxus, Merlot


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Effect of the immobilized yeast cells fermentation on chemical composition

and biogenic amines content in wine

UDC: 663.252.4(497.541)

547.288.2 : 663.12

Borislav Miličević1, Drago Šubarić

1, Antun Jozinović

1, Đurđica Ačkar

1,

Jurislav Babić1, Danijela Vuković

1, Ana Mrgan

2


HR-31000 Osijek, Croatia 2Zvečevo d.d., Food Industry, Kralja Zvonimira 1, HR-34000 Požega, Croatia

Summary

Biogenic amines are well known organic nitrogenous compounds with low molecular weight, which are

formed from precursor amino acids, mainly by lactic acid bacteria during malolactic fermentation in wine

making. It could negatively affect the wine quality and its presence in high concentrations is a health risk for

sensitive individuals. The aim of this research was to determine the chemical composition and biogenic

amines content in wines made from grape varieties Merlot and Syrah (Vitis vinifera L.) from Kutjevo

vineyards, located in the east part of continental Croatia. Wines were produced in 2012 by cold maceration

and fermentation with immobilized yeast cells. The obtained results of chemical analysis of wines showed

that all wine samples produced by fermentation with immobilized yeast cells had a slightly higher amount of

alcohol, reducing sugars and total acidity, while relative density, total and free SO2 content were decreased

using this method of fermentation. Furthermore, total biogenic amines content decreased by fermentation

with immobilized yeast cells for both grape varieties. Wine produced from grape variety Syrah had higher

content of biogenic amines compared to wine produced from Merlot grape. Histamine was the most

abundant biogenic amine followed by 2-phenylethylamine, while amount of serotonine was the lowest.

Keywords: biogenic amines, immobilized yeast cells, Merlot, Syrah

Introduction

Biogenic amines (BA) are organic nitrogenous compounds formed by the metabolisms of living

organisms (microorganisms, plants and animals) from amino acid precursors. In fermented

foodstuffs, such as wine, the non-volatile biogenic amines (histamine, putrescine, cadaverine,

spermine, spermidine, agmatine, tyramine, and tryptamine) and phenylethylamine (a volatile

amine) are formed by microbial decarboxylation of the corresponding amino acids (Smit et al.,

2013). BA occur in different kinds of food, such as cheese, fish products, beer and wine (Del

Prete et al., 2009). They are essential for several physiological functions, e.g. body temperature



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regulation and gastric acid secretion, however the intake of foods with high concentrations of BA

might produce important adverse effects, i.e. hypotension, hypertension, nausea, vomit, diarrhea,

migraines, heart palpitations, kidney failure, anaphylactic shock and death (Henríquez-Aedo et

al., 2012). Actually, the European Union (EU) has not established regulations for the wine

industry, but only suggested the „safety threshold values“. In 2011, the International Organization

of Vine and Wine (OIV) published the „OIV code of good vitivinicultural practices“ in order to

minimise the presence of BA in vine-based products (Martuscelli et al., 2013). Generally, the

toxic dose in alcoholic beverages is considered to be between 8 and 20 mg/L for histamine, 25 to

40 mg/L for tyramine, but as little as 3 mg/L phenylethylamine can cause negative physiological

effects (Karovičová and Kohajdová, 2005). The levels of BA produced in wine greatly depend on

the abundance of amino acid precursors in the medium. Generally, BA production will increase

with increased availability of free amino acids. Amino acid content in grape must, and

subsequently in wine, may be influenced by vinification methods, grape variety, geographical

region, vintage and vine nutrition (Smit et al., 2013).

BA content also depends on the type of wine. It is well known that red wines present higher

BA concentrations than white ones (Henríquez-Aedo et al., 2012). This is in accordance with

research of Martuscelli et al. (2013), who found these BA concentrations in Abruzzo (Italy)

wines: red (19.3±12.8 mg/L), rosé (9.20±6.34 mg/L), white (7.67±3.84 mg/L) wine.

For the determination of BA in foodstuffs diverse methods have been proposed in the

bibliography, among them high performance liquid chromatography followed by

fluorometric detection or spectrophotometry is the most commonly used technique in

recent years, especially for the analysis of wines (García-Marino et al., 2010).

Many technological, biological and environmental factors can affect the occurrence of BA

in wine such as skin maceration or post-fermentative maceration or contact with the lees

(Martuscelli et al., 2013). Among the factors that have been suggested as favouring the

abundance of amines in wine, some winemaking practices seem to play a major role

because they can directly affect the content of the precursor amino acids of BA (Alcaide-

Hidalgo et al., 2007; Martín-Álvarez et al., 2006).

The aim of this study was to determine the chemical composition and BA content in wines

made from grape varieties Merlot and Syrah (Vitis vinifera L.) from Kutjevo vineyards,

located in the east part of continental Croatia. Wines were produced in 2012 by cold

maceration and fermentation with immobilized yeast cells.


Wine production

The wines were produced from the grapes varieties Merlot and Syrah (Vitis vinifera L.).

The cold maceration was carried out controlling the skin contact time for 4 days at


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temperature below 15 °C. After the cold-maceration period was completed, mash was

drawn off to remove the skins and other solid parts, and left to finish the fermentation.

Samples of wines without asterisk were produced using classical technological

fermentation procedure; with selected yeast Fermol-Bouqet 125, and controlled thermal

regime, keeping the average temperature in interval of 16-22 °C by outer chilling of

fermentors with running water. The average duration of the fermentation of all grape

varieties under these conditions was 40 days.

Samples with asterisk were produced using technological procedure of fermentation as

shown in Fig. 1: Fermentation with immobilized yeast cells /selected yeast Feromol-

Bouqet 125, immobilized in Ca-alginate gel (Gaserod, 1998, Poncelet et al., 2001) / in

internal loop gas-lift fermentor with alginate beads as yeast carriers and controlled thermal

regime using outer refrigeration of fermentors with running water, with the aim of keeping

the average temperature in interval of 16-22 °C. The average duration of fermentation

under these conditions was 14 day for each set.

The samples of young wine were exempted at the end of fermentation and before filtration

so the wine was insufficiently clear, slightly dull.

Fig. 1. Reactor for fermentation with immobilized yeast cells


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Chemical analysis of wine

For the evaluation of the quality of wine fundamental analytical techniques were applied.

In industrial control laboratories these techniques represent the basis for the determination

of quality parameters, defined by OIV (2001), Anonymous (1996) and AOAC (1995).

Chemical analysis of wine included relative density, alcohol, total acidity, volatile acidity,

pH, reducing sugars, total and free SO2 and the analysis of ash content.

HPLC analysis of BA in wine

The BA content was determined by HPLC method according to (Proestos et al., 2008). BA

were separated using a liquid chromatograph HP 1100 (Agilent Technologies, Waldbronn,

Germany), with an auto-sampler and UV/VIS detector with variable wavelength, and a

fluorescence detector. The separation after dansyl chloride (Dns-Cl) derivatisation was

performed on a reversed-phase column Zorbax Eclipse XDB C8 (150 mm × 4.6 mm,

particle size 5 μm) equipped with a guard column Meta Guard Inertsil C18. The BA

standards were obtained from Sigma-Aldrich, Steinheim, Germany and Dns-Cl was

purchased from Merck, Darmstadt, Germany.


The results of the chemical analysis of wine are shown in Table 1. From the obtained

results it can be seen that wine samples produced with technological procedure as shown in

Fig. 1 (fermentation with immobilized yeast cells) had a slightly higher amount of alcohol,

ranging from 12.73-13.50% vol., in relation to the amount of 12.50-13.20% vol. in wines

produced using classical technological procedure. These results for alcohol amount

correspond to the requirements of Regulation of wine (Anonymous, 1996). Furthermore,

results obtained for alcohol content in Merlot are in accordance with results obtained in

research of Del Prete et al. (2009), but on other hand these authors found lower alcohol

amount in Syrah, compared to results obtained in our research. One more investigation

which confirm results obtained in this research is research of Martuscelli et al. (2013), who

observed that amount of alcohol in red wines from Abruzzo vineyards ranged from 12.0-

14.5% vol. The total and volatile acidity slightly increased by using fermentation with

immobilized cells, regardless of wine type. Rodriguez-Naranjo et al. (2013) determined

slightly higher total acidity in Merlot, and lower in Syrah, compared the results obtained in

this research, while the results for volatile acidity were very similar. Same trend was

observed for reducing sugars and ash contents i.e. the samples produced with classical

procedure had lower values. The obtained results for sugar contents were little higher in regard

to results obtained in researches of Rodriguez-Naranjo et al. (2011, 2013). Free and total SO2

significantly decreased when the wines were produced with immobilized yeast cells. Total SO2


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content ranged from 95.05-99.20 mg/L, and these results are in accordance with research of Del

Prete et al. (2009), who found that total SO2 in red wines ranged from 12.80-115.20 mg/L.

Table 1. Results of chemical analysis of wine

Determinate

characteristics Merlot Merlot* Syrah Syrah*

Relative density

(20/20 °C) 0.9928 ± 0.10 0.9918 ± 0.20 0.9940 ± 0.20 0.9930 ± 0.20

Alcohol (% vol.) 12.50 ± 0.15 12.73 ± 0.10 13.20 ± 0.10 13.50 ± 0.15

Total acidity (g/L) 5.50 ± 0.25 5.55 ± 0.15 5.60 ± 0.15 5.70 ± 0.25

Volatile acidity (g/L) 0.46 ± 0.20 0.50 ± 0.10 0.45 ± 0.15 0.49 ± 0.10

pH 3.52 ± 0.20 3.52 ± 0.20 3.50 ± 0.10 3.51 ± 0.20

Reducing sugars (g/L) 2.80 ± 0.20 2.85 ± 0.25 2.62 ± 0.20 2.68 ± 0.10

Free SO2 (mg/L) 38.24 ± 0.20 38.22 ± 0.30 25.55 ± 0.20 24.54 ± 0.25

Total SO2 (mg/L) 98.05 ± 0.20 95.05 ± 0.30 99.20 ± 0.40 96.20 ± 0.20

Ash (g/L) 1.68 ± 0.10 1.70 ± 0.18 2.10 ± 0.40 1.95 ± 0.25

*- immobilized yeast cells

Influence of different fermentation procedure on BA content in wines is shown in Table 2.

The contents of all analysed biogenic amines decreased by using method with immobilized

yeast cells, and because of that total amount of BA ranged from 8.97 mg/L in wines

produced with immobilized yeast cells up to 9.88 mg/L in wines produced in classical

fermentation process. Significantly lower values of total BA were found in our samples,

compared to other investigations (Del Prete et al., 2009; Henríquez-Aedo et al., 2012;

Pineda et al., 2012). In our research histamine was the most abundant BA in both types of

wines (3.21-3.29 mg/L), followed by 2-phenylethylamine (2.35-2.40 mg/L) and tryptamine

(1.65-1.91 mg/L), what is in accordance with the conclusion of Gloria et al. (1998). In

addition, it can be seen that most of the results were relatively similar in Merlot and Syrah,

where the greatest differences were observed in contents of spermidine and tryptamine.

Table 2. Results of HPLC analysis of biogenic amines in wine

Biogenic amine

(mg/L) Merlot Merlot * Syrah Syrah *

Putrescine 0.43±0.20 0.39±0.10 0.44±0.06 0.43±0.05

Cadaverine 0.45±0.05 0.44±0.05 0.48±0.05 0.46±0.05

2-Phenylethylamine 2.40±0.15 2.35±0.15 2.50±0.20 2.40±0.20

Spermidine 0.56±0.11 0.54±0.10 0.66±0.10 0.62±0.10

Tryptamine 1.80±0.10 1.65±0.15 1.91±0.15 1.65±0.15

Serotonine 0.20±0.05 0.15±0.05 0.25±0.05 0.15±0.05

Tyramine 0.25±0.01 0.20±0.01 0.25±0.05 0.20±0.02

Histamine 3.38±0.05 3.25±0.10 3.39±0.10 3.21±0.05

Σ Biogenic amines 9.47 8.97 9.88 9.12

*- immobilized yeast cells


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Conclusions

According to the obtained results it can be concluded that the chemical properties and

content of BA in wines were influenced by fermentation procedure. Wine produced from

grape variety Syrah had higher content of BA compared to wine produced from Merlot

grape. Regardless of grape variety, the amount of all BA in analysed wines decreased by

fermentation with immobilized yeast cells. The fermentation with immobilized yeast cells

is a promising approach in the wine-making process, with a reduced content of BA in

wine.

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420-424.

Martuscelli, M., Arfelli, G., Manetta, A.C., Suzzi, G. (2013): Biogenic amines content as a measure

of the quality of wines of Abruzzo (Italy), Food Chem. 140, 590–597.

OIV International Code of Oenological Practices, (2001).

Pineda, A., Carrasco, J., Peña-Farfal, C., Henríquez-Aedo, K., Aranda, M. (2012): Preliminary

evaluation of biogenic amines content in Chilean young varietal wines by HPLC. Food

Control 23, 251-257.

Poncelet, D., Dulieu, C., Jacquot, M. (2001): Description of the immobilization procedures, In:

Immobilized Cells, (Wijffels R.), Springer Lab Manual, Heidelberg, pp. 15-30.


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Proestos, C., Loukatos, P., Komaitis, M. (2008): Determination of biogenic amines in wines by

HPLC with precolumn dansylation and fluorimetric detection, Food Chem. 106, 1218-1224.

Rodriguez-Naranjo, M.I., Gil-Izquierdo, A., Troncoso, A.M., Cantos-Villar, E., Garcia-Parrilla,

M.C. (2011): Melatonin is synthesised by yeast during alcoholic fermentation in wines, Food

Chem. 126, 1608-1613.

Rodriguez-Naranjo, M.I., Ordóñez, J.L., Callejón, R.M., Cantos-Villar, E., Garcia-Parrilla, M.C.

(2013): Melatonin is formed during winemaking at safe levels of biogenic amines, Food

Chem. Toxicol. 57, 140-146.

Smit, A.Y., Du Toit, W.J., Stander, M., Du Toit, M. (2013): Evaluating the influence of maceration

practices on biogenic amine formation in wine, LWT - Food Sci. Technol. 53, 297-307.


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Rheological properties of molten chocolate masses

during storage - influence of milk components

UDC: 663.91 : 532.135

621.796

Radoslav Miličević1, Borislav Miličević

2, Đurđica Ačkar

2, Svjetlana Škrabal

3,

Drago Šubarić2, Jurislav Babić

2, Antun Jozinović

2, Dijana Miličević

1

1Faculty of Technology, University of Tuzla, Univerzitetska 8, 75000 Tuzla, Bosnia and

Herzegovina 2Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhača 20,

HR-31000 Osijek, Croatia 3Zvečevo dd, Food Industry, Kralja Zvonimira 1, HR-34000 Požega, Croatia

Summary

When finished chocolate mass is used for production of chocolate based products, it is often

produced in large quantities and stored in molten condition at 50-55 °C and spent in smaller

quantities during maximum period of 30 days. It is well established in scientific literature that

chocolate components have significant influence on rheological properties, however, this influence

during storage hasn’t yet been elucidated. The aim of this research was to investigate influence of

milk components (spray-dried milk powder, cream powder and sweetened condensed milk) on

rheological properties of chocolate during 30-day storage. The results showed that sweetened

condensed milk had highest impact on decrease of yield stress and plastic viscosity stability during

storage. Chocolate produced with addition of spray-dried milk powder and cream had highest yield

stress and plastic viscosity during storage.

Keywords: chocolate, rheological properties, spray-dried milk powder, cream powder, sweetened

condensed milk

Introduction

When finished chocolate mass is used for production of chocolate based products, it is

often produced in large quantities and stored in molten condition at 50-55 °C and spent in

smaller quantities during maximum period of 30 days (Ziegleder et al., 2004). Traditional

milk chocolate production required drum-dried milk powder due to high free fat content,

large particles and low vacuole volume, which positively influence rheological properties

of chocolate (Keogh, Murray & O’Kennedy, 2003). However, during drum drying,

caramelisation of sugar is significant, proteins are denaturated and milk fat is released,



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which results in poor solubility of milk powder in water and some undesired sensory

characteristics.

Spray-dried milk powder has better sensory characteristics and higher water solubility,

however, less free milk fat requires higher addition of cocoa butter and, ultimately, higher

production costs (Ziegleder et al., 2004), which was the main reason not to use spray-dried

milk powder. However, free fat content in spray-dried milk powder can be increased by

extrusion process (Koc et al., 2003), or anhydrous milk fat can be added during chocolate

production (Ziegleder et al., 2004) to overcome this problem.

Cream powder is milk product containing significant amounts of milk fat (50-60%) and is

applied mostly when milk fat content is to be increased.

Condensed milk is produced by evaporation of milk in vacuum until minimum 23% total

milk solids. In production of sweetened condensed milk sugar is added so that final

product contains 43-45% sugar, min. 8.5% milk fat and 28% total milk solids (Francis,

2000). Sweetened product is darker, more yellow and thick, and gives unique sensory and

rheological properties to chocolate. Hence, sucrose added during condensed milk

production delays crystallisation of lactose (Sormoli et al., 2013), making the product, and

chocolate to which is added, rheologically more stable.

The aim of this research was to investigate influence of spray-dried milk, spray-dried milk

combined with cream and sweetened condensed milk on rheological stability of chocolate

masses during 30-day storage.


Lecithin (liquid) was supplied from ADM, Netherlands. Drum-dried milk powder was

supplied by "Laktopol" Sp.z.o.o. Warszawie, Poland, sweetened condesend milk was

produced in Zvečevo d.d., Croatia and cream powder was supplied by Sery ICC Paslek SP

Z.O.O., Poland.

Production of chocolate masses, storage and rheological properties monitoring were

performed as described in Miličević et al. (2014), using lecithin as emulsifier.

Milk chocolate masses had following composition:

1) sugar (49%), cocoa butter, cocoa mass (total cocoa parts 33%), spray-dried milk powder

(16%), lecithin (total fat 31%);

2) sugar (48%), cocoa butter, cocoa mass (total cocoa parts 30%), spray-dried milk

powder, cream powder (total milk components 20%), lecithin (total fat 30%);

3) sugar (47%) , cocoa butter, cocoa mass (total cocoa parts 32%), sweetened condensed

milk, milk butter (total milk components 21%), lecithin (total fat 33%).


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Addition of spray-dried milk powder resulted in increase of Casson yield stress from 15.16 Pa

(for dark chocolate) to 15.64 Pa (Table 1), and addition of cream powder further increased

it to 16.64 Pa. Sweetened condensed milk, however, decreased yield stress to 0.39 Pa,

which is in accordance with its high fat content. Plastic viscosity was increased by addition

of all milk components, with most pronounced impact of spray-dried milk powder (2.62 Pas),

followed by combination of spray-dried milk powder and cream powder (2.35 Pas) and

sweetened condensed milk (2.22 Pas). This observation is in contrast with literature data

(Liang & Hartel, 2004; Keogh et al., 2001; Keogh et al., 2006), but could be partially

explained by reduced content of free milk fat in spray-dried milk powder and increased

proportion of solid matter due to milk solids. Sweetened condensed milk contains water,

which negatively influences chocolate viscosity. Namely, increased moisture aggregates

sugar particles, which increases friction and apparent viscosity (Afoakwa et al., 2007).

Cream powder, due to high content of milk fat should have decreased observed parameters,

however, total fat content in this chocolate mass was lower than in chocolate produced

only with spray-dried milk powder.

Table 1. Influence of milk components on Casson plastic viscosity (CA) and Casson yield stress

(τCA) of milk chocolate mass during 30-day storage

Milk component Storage days

0 10 20 30

τCA [Pa]

None (dark chocolate) 15.16 ± 0.18 34.82 ± 0.26 33.72 ± 0.31 18.78 ± 0.4

Spray-dried milk

powder 15.64±0.25 8.12±0.24 10.55±0.23 5.55±0.27

SD milk powder + cream

16.64±0.12 15.29±0.11 10.94±0.86 14.46±0.21

Sweetened condensed

milk 0.39±0.05 0.87±0.04 0.75±0.23 0.83±0.16

CA [Pas]

None (dark chocolate) 2.10 ± 0.03 1.71 ± 0.18 2.04 ± 0.18 2.51 ± 0.11

Spray-dried milk

powder 2.62±0.25 1.89±0.21 1.41±0.20 1.66±0.02

SD milk powder +

cream 2.35±0.14 2.10±0.01 2.06±0.00 2.13±0.07

Sweetened condensed

milk 2.22±0.04 1.81±0.18 1.82±0.16 2.00±0.11


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Another explanation could be sought in emulsifier addition time – it wasn’t added

during conching, but later, in laboratory conditions and effect of emulsifier could be

delayed in milk chocolates. This is supported by results obtained for yield stress after

10, 20 and 30 storage days (Table 1), when yield stress was proportional to the fat

content in chocolate masses: lowest for chocolate with sweetened condensed milk,

followed by chocolate with milk powder, and combination of spray-dried milk powder

and cream powder, while for dark chocolate it was 1.3 to 40 times higher than for milk

chocolate samples.

From Fig. 1-3 it is visible that during first 20 days of storage viscosity of all samples

decreased. After 30 days viscosity of chocolate produced with spray-dried milk powder

continued decreasing. Research of Ziegleder et al. (2004) showed that thickening of

chocolate is influenced by temperature – while at temperatures above 60 °C thickening of

chocolate produced with spray-dried milk powder is significant, at temperatures below

55°C no thickening was noticed. In addition, authors reported that amorphous lactose in

spray-dried milk powder was resistant to mechanical and thermal stress during

manufacture without crystallisation.

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60

τ[P

a]

γ [1/s]

0

10 days

20 days

30 days

Fig. 1. Influence of spray-dried milk powder on shear stress (τ) – shear rate (γ) ratio

of chocolate mass during 30-day storage at 50 °C


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0

50

100

150

200

250

0 10 20 30 40 50 60

τ[P

a]

γ [1/s]

0

10 days

20 days

30 days

Fig. 2 Influence of spray-dried milk powder and cream on shear stress (τ) – shear rate (γ) ratio


Fig. 3 Influence of sweetened condensed milk on shear stress (τ) – shear rate (γ) ratio


0

20

40

60

80

100

120

140

160

0 10 20 30 40 50 60

τ [P

a]

γ [1/s]

0

10 days

20 days

30 days


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Viscosity of samples produced with sweetened condensed milk and combination of spray-

dried milk powder and cream powder increased after 30 days of storage.

Hence, viscosity of chocolate produced with sweetened condensed milk was identical to

initial viscosity. Chocolate viscosity during storage is influenced by lactose crystallisation

(Ziegleder et al., 2004). Since sugar delays lactose crystallisation in condensed milk,

delayed lactose crystallisation could be reason why viscosity of chocolate increased only at

the end of the storage and in such extent.

In addition, chocolate produced with spray-dried milk powder and cream powder

contained higher content of non-fat solids, particularly sugar, which could also crystalize at

the end of storage, after crystallisation of lactose (Sormoli et al., 2013).

Conclusions

Chocolate rheological properties are highly influenced by its composition. In addition to

emulsifier, milk components have also high impact mainly due to free milk fat, but protein

and sugar content shouldn’t be disregarded. Chocolate viscosity changes during storage

and milk components can influence viscosity stability of chocolate masses. All these

aspects should be considered when storing chocolate masses for prolonged usage.

References

Afoakwa, E. O., Paterson, A., Fowler, M. (2007): Factors influencing rheological and textural

qualities in chocolate - a review, Trends Food Sci Technol., 18, 290-298.

Francis, F. F. (2000): Encyclopedia of Food Science and Technology, Vol. 1., Wiley, USA.

Keogh, K., Twomey, M., O´Kennedy, B., Mulvihill, D. (2001): Effect of milk composition on

spray-dried high-fat milk powders and their use in chocolate, Lait,82, 531-539.

Keogh, M. K., Murray, C. A., O’Kennedy, B. T. (2003): Effects of ultrafiltration of whole milk on

some properties of spray-dried milk powders, Int. Dairy J. 13, 719-726.

Keogh, M. K., Twomey, M., O´Kennedy, B. T., Kennedy, R., O´Keeffe, J., Kelleher, C. (2006):

Irish Ag. and Food Development End- of-Project (Armis No. 4519), Teagasc, Dublin.

Koc, A. B., Heinemann, P. H., Ziegler, G. R. (2003): A Process for Increasing the Free Fat Content

of Spray-dried Whole Milk Powder, J. Food Sci., 68, 210-216.

Liang, B., Hartel, R.W. (2004): Effects of Milk Powders in Milk Chocolate, J. Dairy Sci. 87, 20-31.

Miličević, R., Miličević, B., Ačkar, Đ., Škrabal, S., Šubarić, D., Babić, J., Jozinović, A., Jašić M.

(2014): Rheological properties of molten chocolate masses during storage I. Influence of

emulsifiers, Technologica Acta, 7(1), 35-40.

Sormoli, M. E., Das, D., Langrish, T. A. G. (2013): Crystallization behavior of lactose/sucrose

mixtures during water-induced crystallization, J. Food Eng., 116, 873-880.

Ziegleder, G., Amanitis, A., Hornik, H. (2004): Thickening of molten white chocolates during

storage, Lebensm.-Wiss.-u-Technol., 37, 771-778.


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Stanje i mogućnosti proizvodnje mlijeka u Požeško-slavonskoj županiji

UDC: 637.1(497.541)

Ana Mrgan1

, Gordana Jurišić2

1Zvečevo d.d., prehrambena industrija, K. Zvonimira 1, 34000 Požega, Hrvatska

2Josipa Runjanina 2, 34000 Požega, Hrvatska

Sažetak

Mlijeko je biološka tekućina, karakteristične boje, mirisa i okusa, koju izlučuju mliječne žlijezde

ženki sisavaca ili žene (Tratnik, 1998). Pod pojmom mlijeko, uvijek se podrazumijeva „kravlje

mlijeko“, za sve ostale vrste mlijeka mora biti istaknuto od koje životinje potječe, kao npr. „ovčje

mlijeko“, „kozje mlijeko“, „bivolje mlijeko“ ili neka druga vrsta mlijeka. Posebno se deklarira

„majčino mlijeko“ koje se normalno ne pojavljuje na tržištu, ali zauzima važno mjesto u

nutricionističkim i medicinskim istraživanjima (Tratnik, 1998). Sve vrste mlijeka sadrže gotovo iste

sastojke, ali u različitom udjelima. Mlijeko sadrži nekoliko stotina hranjivih tvari. Zato je u

prehrambenom smislu, mlijeko nezamjenjiva namirnica. Proizvodnja mlijeka i mliječnih proizvoda

za svaku zemlju strateško je pitanje i ono je odraz uređenosti prehrambene proizvodnje neke zemlje.

Dok razvijene zemlje zadovoljavaju svoje potrebe vlastitom proizvodnjom mlijeka, Hrvatska oko

pola svojih potreba pokriva uvozom. Iz podataka koji su obrađeni u ovom radu, vidljivo je da u

posljednjih devet godina nije došlo do značajnijih pozitivnih pomaka u tom pogledu. U radu su

korišteni podaci Hrvatske poljoprivredne agencije, Hrvatske savjetodavne službe, Hrvatskog zavoda

za statistiku, Državnog hidrometeorološkog zavoda, kao i nekih objavljenih radova, kako od prije

devet godina tako i najnovijih podataka. Korištenjem raspoloživih podataka došlo se do zaključka o

razlozima nedostatne proizvodnje mlijeka, te mogućnostima proizvodnje u Požeško-slavonskoj

županiji i dijelu Brodsko-posavske županije, odnosno pedeset kilometara u polumjeru od Požege,

području koje je tradicionalno gravitiralo Požegi i bilo izvor sirovine Požeške mljekare. Iz dobivenih

podataka je vidljivo da nije vođena učinkovita strategija razvoja govedarstva, stvaranja dovoljnog

broja obiteljskih gospodarstava sa 21-25 krava, visoke produktivnosti.

Ključne riječi: mlijeko, proizvodnja mlijeka, Požeško-slavonska županija

Uvod

Mlijeko je namirnica koja sadrži sve potrebne tvari za rast i razvoj mladog organizma. U

prehrani djece kao i odraslih ljudi mlijeko i mliječni proizvodi veoma su bitna namirnica i

odraz su standarda pojedine zemlje. Razvijene zemlje imaju znatno veću potrošnju mlijeka



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u odnosu na manje razvijene ili ne razvijene zemlje, a osobito potrošnju mliječnih

proizvoda.

Tablica 1. Prosječan sastav mlijeka (%) različitih vrsta mlijeka (prema Bylundu, 2003.)

Table 1. The average content of milk (%) of different types of milk (according to Bylundu, 2003)

Vrst mlijeka Ukupni proteini Kazein Proteini sirutke Mast Ugljikohidrati Pepeo

Majčino 1,0 0,5 0,5 4,5 7,0 0,2

Kobilje 2,2 1,3 0,9 1,7 6,2 0,5

Kravlje 3,5 2,8 0,7 3,7 4,8 0,7

Bivolje 4,0 3,5 0,5 7,5 4,8 0,7

Kozje 3,6 2,7 0,9 4,1 4,7 0,8

Ovčje 4,6 3,9 0,7 7,2 4,8 0,8

U svijetu se proizvede godišnje oko 568 milijardi kg različitih vrsta mlijeka, od čega je

najviše kravljeg mlijeka (85,2%), bivoljeg (10,9%), kozjeg (2%), ovčje (2%) (Bosnić,

2013).

Bivolje mlijeko najviše se proizvodi u Aziji.

Kozje mlijeko poznato je po svome terapeutskom djelovanju, lakše je probavljivo nego

kravlje i pogodno je za konzumaciju osobama alergičnim na proteine kravljeg mlijeka.

Koristi se u proizvodnji nekih vrlo cijenjenih sireva.

Ovčje mlijeko najviše se koristi u proizvodnji vrlo cijenjenih sireva, a poznato je po

najvećim prinosima zbog velikog postotka masti i proteina.(Tratnik, 1998).

Hrvatska kao izrazito poljoprivredna zemlja, uvozi više od 40% svojih potreba za mlijekom

i mliječnim proizvodima. Najveća proizvodnja mlijeka u Hrvatskoj ostvarena je 1987.god.

oko 1.013 milijuna litara, od tada proizvodnja postepeno pada. Od 2002.god. ponovno

pokazuje lagani trend porasta do 2009., a od 2010. ponovno postepeni pad proizvodnje,

koji se nastavio i u 2013.god. (HPA, 2004,2013).

Prije deset godina potrošnja mlijeka u RH bila je 170L/glavi stanovnika,od toga približno

55% kao tekuće mlijeko, a 45% u obliku mliječnih proizvoda (Državni zavod za

statistiku,2004). Ukoliko se navedena potrošnju mlijeka pomnoži sa brojem stanovnika RH

dobije se oko 760 000 000 kg, a proizvodnja u RH tada je iznosila oko 550 000 000 kg

mlijeka. Tadašnja nacionalna strategija je bila, da se potrošnja mlijeka poveća na 220

L/glavi stanovnika s tim da se potrebe moraju promatrati i kroz povećanje potreba za broj

turista kroz godinu pa bi ukupne potrebe u RH iznosile oko 1 071 000 000 kg/god.

Prema raspoloživim podacima potrošnja mlijeka od 170L/glavi stanovnika u proteklih

deset godina u Hrvatskoj nije povećana (Bosnić,2013). Vlastita proizvodnja se smanjila, što

se može pratiti i kroz smanjenje broja krava, dakle porastao je uvoz, iako postoji dovoljno


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prostora za povećanje vlastite proizvodnje, do količine od 765 milijuna litara, što je

ispregovarana kvota sa EU. (HPA, 2014).

U zapadno Europskim zemljama potrošnja mlijeka je oko 280 kg/glavi stanovnika, (mlijeko

79 kg, kondenzirano mlijeko 2,3 kg, svježi mliječni proizvodi 17 kg, vrhnje 4,3 kg, maslac

4,7 kg, sve vrste sira 17,4 kg (Bosnić, 2013).

Cilj rada je analiza trenda proizvodnje mlijeka u posljednjih devet godina u Požeško-

slavonskoj županiji kao i RH, te kakve su realne mogućnosti povećanja proizvodnje

mlijeka i bolje iskorištenosti raspoloživih resursa u Požeško-slavonskoj županiji (koja se

nalazi na začelju po proizvodnji mlijeka ali i razvijenosti u Hrvatskoj) i dijelu Brodsko-

posavske županije koji prirodno gravitiraju Požegi, to je prostor koji je bio izvor sirovina

Požeške mljekare.

U Požegi je 1982. god. izgrađena nova mljekara (jedan od tri proizvodna pogona Zvečeva

d.d., kapaciteta 100 000 kg/dan), isključivo kao tvornica mlijeka u prahu, maslaca i

kondenziranog mlijeka, za potrebe Tvornice konditorskih proizvoda. Od 2005. god. nema

vlastitog otkupa mlijeka. Za potrebe Tvornice konditorskih proizvoda, povremeno kupuje

mlijeko od nekog drugog otkupljivača, na taj način proizvodni pogon mljekare održava se u

„hladnom pogonu“. U blizini nema neke veće mljekare koja bi bila razlog oduzimanja

terena, odnosno sirovine te time prestanka otkupa mlijeka. Najbliža veća mljekara udaljena

je oko 100 km od Požege (Osijek, Županja, Veliki Zdenci).

Razloge prestanka otkupa mlijeka, a time i zanemarivanja proizvodnje mlijeka u Požeškoj

županiji, mogu se tražiti u uvozu jeftinog mlijeka u prahu, ali i nedostatku interesa da se

proizvodni asortiman Zvečevačke mljekare prilagodi potrebama tržišta odnosno proširi na

profitabilniji proizvodni asortiman.

Materijali i metode

U nastojanju da se dođe do što realnijih podataka o mogućnostima proizvodnje ovako

vrijedne prehrambene namirnice, koja je odraz uređenosti poljoprivrede i prehrambene

industrije neke zemlje, korišteni su podaci Hrvatskog zavoda za statistiku kao i izvješća

Hrvatskog stočarskog centra, odnosno HPA i dobiveni podatci stavljeni su u korelaciju sa

podacima od prije devet godina.

Aktiviranjem rada Požeške mljekare izvor sirovina potencijalno bi bilo područje od 50 km

u polumjeru od Požege, dakle područje Požeško-slavonske županije i pola Brodsko-

posavske županije, odnosno ono što je u prošlosti gravitiralo Požeškoj mljekari.


U Hrvatskoj je u 2013.god. ukupno bilo 180 946 krava, što je 5% manje u odnosu na njihov

broj u 2012. god. Od ukupnog broja krava, mliječnih i kombiniranih pasmina je bilo

167491, od čega 101637 pod kontrolom mliječnosti od strane HPA (HPA, 2013).


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Tablica 2. Kretanje broja krava, ovaca i koza (1975., 2004., 2013.) u Požeško-slavonskoj županiji

(HPA, 2014)

Table 2. Trends in the number of cows, sheep and goats (1975., 2004., 2013.) in Pozega-Slavonia

county (HPA, 2014)

1975.god. 2004.god. 2013.god.

Krave 20 000 5026 4704

Ovce 8 300 13 679 21 780

Koze nema podataka 476 220

Iz tablice je vidljiv veliki pad broja krava u Požeško-slavonskoj županiji i značajan porast

broja ovce koje su uglavnom u sustavu proizvodnje mesa. Ispod 5% je u sustavu

proizvodnje mlijeka, dakle zanemariva je njihova proizvodnja mlijeka (Hrvatska

poljoprivredna savjetodavna služba).

Tablica 3. Veličina stada u Požeško-slavonskoj i Brodsko-posavskoj županiji (HPA, 2014)

Table 3. Herd size in Pozega-Slavonia and Brod-Posavina county (HPA, 2014)

Požeško-slavonska

županija Veličina stada Broj stada Broj krava

<6 450 974

6-10 113 852

11-15 34 435

16-20 24 425

21-25 9 203

26-30 11 311

31-50 13 504

51-100 12 883

101-250 1 117

Ukupno 4704 (2013.)

5026 (2004.)

Brodsko-posavska

županija Veličina stada Broj stada Broj krava

<6 660 1 344

6-10 99 759

11-15 61 792

16-20 42 751

21-25 16 365

26-30 15 418

31-50 21 726

51-100 11 703

101-250 3 374

Ukupno 6232 (2013.)

10 015(2004.)


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Iz tablice 3 je vidljivo da u Požeškoj i Brodskoj županiji još uvijek prevladavaju stada sa

manje od šest krava, kao i vrlo mali broj stada sa 21-25 krava, što je optimalan broj za

jedno obiteljsko gospodarstvo. U zadnja dva retka napravljena je usporedba broja krava

2004.god. i 2013. god., što se može bolje vidjeti u tablici 2 za područje Požeške županije.

Proizvodnja mlijeka

Proizvodnja mlijeka, odnosno produktivnost krava ovisi o više faktora, kao što su

karakteristike pasmine, zemljišno bogatstvo i tehnologija proizvodnje.

Visoku godišnju laktaciju imaju mliječne pasmine holstein frisien. U Hrvatskoj je najviše

zastupljena simentalska pasmina sa 65%, a potom holstein frisien. U Požeško-slavonskoj

županiji simentalska pasmina je zastupljena sa 83%, a Brodsko-posavskoj 76% (HPA,

2014).

Najveću produktivnost u proizvodnji mlijeka u svijetu ima Izrael oko 10 000 kg/kravi,

SAD 8500 kg/kravi, Kanada 7500 kg/kravi. U Europi intenzivnu godišnju proizvodnju

imaju Holandija, Njemačka, Francuska, Švicarska od 6000-7000 kg/kravi. U Hrvatskoj je

produktivnost oko 3000 kg/kravi (Bosnić, 2013).

U Požeškoj i Brodskoj županiji produktivnost je oko 2500-3000 kg/kravi (HPA, 2014).

Ponekad se u javnosti pojavljuju podaci o porastu produktivnosti u Hrvatskoj na oko

5000 kg/kravi, međutim tu se radi o podacima koji se odnose samo na pojedinačna stada pod

kontrolom HPA, korištenje takvih podataka daje krivu sliku o produkciji mlijeka u RH.

Komparacijom ranijih podataka o proizvodnji mlijeka (Bosnić, 2003. i Hrvatske savjetodavne

službe, te HPA), i sadašnje proizvodnje vidljiv je pad proizvodnje. Pad proizvodnje je rezultat

izostanka povećanja produkcije po kravi i smanjenja broja krava.

Tablica 4. Otkupljene količine mlijeka (u kg) ( HPA, 2014)

Table 4. Bought-out quantities of milk (in kg) ( HPA, 2014)

2004.god. 2013.god.

RH 550 000 000 500 000 000

Požeško-slavonska županija 14 000 000 12 000 000

Brodsko-posavska županija 44 000 000 15 000 000

Iz tablice 4. vidljivo je smanjenje proizvodnje mlijeka, na nivou Hrvatske za 9%, Požeške

županije za 14%, a Brodske županije za 66%. Na ovaj veliki pad proizvodnje u Brodsko-

posavskoj županiji, osim općeg trenda u cijeloj Hrvatskoj, svakako je utjecalo i

dugogodišnja agonija u radu, te na kraju i gašenje mljekare u Starom Petrovom Selu.


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Kvaliteta mlijeka

Evropska unija je 1992.god. donijela propise-direktive o uvjetima kvalitete proizvodnje i

prerade sirovog mlijeka i proizvodnji mliječnih proizvoda (Directive 46/92, Milk and milk

product quality, health and hygiene).

Tablica 5. Kriteriji higijenske kvalitete i klase kravljeg mlijeka u EU-15 (EU, Council directive 92/46/EEC)

Table 5. Hygienic criteria of milk quality and milk classes in EU-15 (EU, Council directive 92/46/EEC)

Količina (broj) u 1 ml.

Klasa od do

Mikroorganizmi

50 000 I. Ekstra

50 001 100 000 I.

> 100 001 II.

Somatske stanice 300 000 I. Ekstra

Sukladno Evropskim kriterijima kvalitete, Hrvatska je donijela svoj pravilnik o kakvoći

svježeg sirovog mlijeka (n.n. 102/00), koji je u punoj primjeni od 2003.god. Prema

Hrvatskom „Pravilniku o svježem i sirovom mlijeku“, mlijeko se razvrstava u četiri klase

prema broju mikroorganizama i somatskih stanica.

Klasa Broj mikroorganizama/1ml Broj samatskih stanica

E ≤ 50000 ≤ 400000

I 51000 - 100000 ≤ 400000

II 101000 - 400000 ≤ 600000

III > 400000 > 600000

Od 2004.god. kada je tek 37,2% otkupljenog mlijeka u Hrvatskoj, bilo prve ili ekstra

kvalitete, do 2013.god. postignut je značajan napredak u pogledu kvalitete sirovog mlijeka,

kada je od ukupne otkupljene količine 95% bilo prve i ekstra klase.(HPA, 2014).

Infrastuktura

Infrastruktura, odnosno raspoloživi prostor za zimski smještaj stoke jedan je od preduvjeta

za podizanje proizvodnje. U Požeškoj kao i Brodskoj županiji, uglavnom su klasične staje

na vez, manjeg kapaciteta što se vidi iz tablice 4 (najviše proizvođača sa manje od šest

krava). Optimalna veličina stada za obiteljsko gospodarstvo je 21-25 krava, što zahtjeva

izgradnju većih staja(standard je 15krava/100 m² stajskog prostora).


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Zemljišni potencijal

Zemljišni potencijal je jedan od najvažnijih preduvjeta za razvoj stočarske proizvodnje.

Potrebna količina zemlje po jednom grlu goveda je 1 ha. U razvijenim zemljama 60%

obradivog zemljišta je u funkciji govedarstva.

Požeško-slavonska županija raspolaže sa oko 90000 ha obradivog zemljišta (oranice,

livade, vinogradi i voćnjaci) i približno 11000 ha pašnjaka.

U Brodsko-posavskoj županiji je oko 110000 ha obradivog zemljišta i približno 12000 ha

pašnjaka.

Klimatske prilike

Klimatske prilike i pokrivenost terena snijegom važan su čimbenik zbog proizvodnje

dovoljnih količina hrane za stoku kao i mogućnosti držanja stoke na otvorenom.

Područje Požeške kao i Brodske županije je prostor sa umjereno kontinentalnom

klimom, a prateći prosjek pokrivenosti terena snijegom, u zadnjih dvadeset godina,

iznosio je 29 dana/godišnje (DHZ).

Zaključci

Analizirajući navedene podatke, vidljivo je da u Požeško-slavonskoj županiji postoje svi

preduvjeti za znatno povećanje proizvodnje mlijeka. Ljudske resurse možemo promatrati

kroz postojanje tradicije ove proizvodnje kao i veliku nezaposlenost stanovništva.

Daljnji bitan preduvjet za proizvodnju mlijeka je zemljišno bogatstvo, čime Požeška

županija raspolaže u dovoljnim količinama. Kada bi se samo 40000 ha obradivog zemljišta

stavi u funkciju govedarstva (što je 44% od obradivih površina, znatno manje od standarda

razvijenih Evropskih zemalja od 60%) to je dostatno za 40000 grla goveda, odnosno

dovoljno za 20000 krava, ovaj broj krava sa relativno niskom produktivnosti od 4000 kg

mlijeka/kravi (kombinacijom intenzivnog i ekstenzivnog načina uzgoja, gdje su financijska

ulaganja u proizvodnju znatno manja) proizvelo bi se oko 80 000 000 kg mlijeka godišnje.

To je znatno više od dugogodišnjeg strateškog cilja od 34 000 000 kg/godišnje ili od

proizvodnje u 2013. god od 12 000 000 kg.

Postoje neiskorišteni prerađivački kapaciteti Zvečevačke mljekare, koja nikada nije radila

sa sto postotnim kapacitetom.

Ono što nedostaje za povećanje proizvodnje na ovom prostoru, to je stajski prostor za

zimski smještaj stoke, što bi u planskoj proizvodnji trebao biti najmanji problem.

Brodsko-posavska županija raspolaže sa još većim potencijalom, od obradivih površina,

broja stanovnika, genetike goveda kao i tradicije proizvodnje.


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Literatura

Bosnić, P. (2013): Primarna proizvodnja mlijeka, 41.Hrvatski simpozij mljekarskih stručnjaka.

Bylund, G. (2003): Dairy processing handbook, Tetra Pak, Processing Systems AB, Swe&#

172;den, Second revised edition.

Državni hidrometereološki zavod Hrvatske.

Hrvatska poljoprivredna agencija, Godišnje izvješće HPA za 2013, Mljekarski list 5/2014.

Hrvatska poljoprivredna savjetodavna služba.

Hrvatski zavod za statistiku.

Tratnik, Lj.(1998): Uvod ( opći pojmovi), Mlijeko-tehnologija,biokemija i mikrobiologija 13-16.

Condition and possibility of milk production in Pozega-Slavonia county

Ana Mrgan1, Gordana Jurišić

2

1Zvečevo d.d., prehrambena industrija, K. Zvonimira 1, HR-34000 Požega, Croatia

2Josipa Runjanina 2, HR-34000 Požega, Croatia

Summary

Milk is biological fluid with caracteristic colour, smell and taste, that is excreted by the mammary glands

of female mammals or woman. Under the term milk is always implied „cow's milk“, for all other types of

milk it must be emphysized of which animal milk originates, for example „sheep's milk“, „goat's milk“,

„buffalo's milk“ or some other kind. Breast milk is specially declared and narmally it can not be found on

market, but it takes very important role in nutritional and medical research. All kinds of milk contains the

same ingrediente but in a different mutual relationships. Milk contains several hundreds of nutrients.

Therefore, nutritionally milk is irreplaceable food. For every country, milk production is a strategic

question and it is the reflection of food production arrangement in certain country. While developed

countries meet their own needs milk production, Croatia about half of its needs by imports. From the date

that are processed in this paper shows that in the last nine years, there has been no significant positive

developments in this regard. The paper used data Croatian Agricultural Agency, Croatian Extension

Service, Croatian Bureau of Statistics, State Meteorological Service, as well as some published works,

how ten years ago and the latest data. Using available data, conclusions were drawn about the reasons for

insufficient milk production, and the capabilities of production in Pozega-Slavonia County and part of the

Brod-Posavina County, and fifty kilometers in diameter from Pozega, or an area that has traditionally

gravitated Pozega and was a source of raw materials Požeške dairies. From these data it is evident that not

conducted an effective strategy for cattle-breeding, creation of a sufficient number of family farms with

21-25 cows of high productivity.

Keywords: milk, milk production, Pozega-Slavonia country


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Influence of harvest date on the primary metabolites

of ˝Plavac mali˝ (Vitis vinifera L.) grapes

UDC: 663.222 : 663.252.1

Ana Mucalo1

, Goran Zdunić1, Ivana Tomaz

2, Luna Maslov

2,

Irena Budić-Leto1, Edi Maletić

2

1Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, HR-21000 Split, Croatia

2Faculty of Agriculture University of Zagreb, Svetošimunska 25, HR-10000 Zagreb, Croatia

Summary

Choosing the best harvest date is the most important decision of the viticulturists upon which the

balance of must before fermentation as well as the quality of future wine depends. We determined

the changes in the composition and concentration of organic acids (malic, tartaric and citric acid),

total acidity, potassium content, sugar components (fructose and glucose) and the change of pH

value in „Plavac mali“ grape during four different harvest dates within Central and Northern

Dalmatia conditions. The separation and quantitative determination of organic acids was done with

high performance liquid chromatography, while fructose and glucose were determined by applying

ultraviolet-visible spectrophotometry. Significant differences of four harvest dates in concentration

of individual primary metabolites were found in both target locations as well as between the two

observed locations. Delaying the harvest date showed an increase in sugar content (°Oe), equatation

in the mass portion of glucose and fructose and the decrease in the total content of acids in grape

berries. The concentration of malic acid decreases sharply, while the changes in concentration of

tartaric acid remain less marked during the ripening.

Keywords: glucose, fructose, HPLC-DAD, organic acids, ˝Plavac mali˝

Introduction

The essential element of the future quality of the wine and must is a degree of the ripeness

of the grapes at the moment of harvest. The taste, sensation, smell and colour of the wine

are the result of complex interaction of a large number of compounds primarily

synthesized in the grape during the ripening. The primary metabolites, sugars, organic

acids, minerals and aminoacids make biggest part of soluble dry matter in the grapes. The

change in the content and behaviour of primary metabolites starts with the véraison. The

grape swells, its skin colour changes, the accumulation of soluble hexoses, glucose and

fructose begins (Davies and Robinson, 1996; Coombe and McCarthy, 2000) as well as cell

deacidification (Peynaud and Maurié, 1958). The ripening is a continuous process during



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which grapes go through different phases (Meléndez et al., 2013). The individual

compounds reach the state of balance (Kennedy, 2008). It is commonly believed that

ripeness is a physiological age of the berries on the vine (Bisson et al., 2001) that is

divided into different stages: physiological, technological, phenolic, aromatic and texture

maturity of the grapes. The ripeness is „in the eye of the beholder“ and has the primary

function of the future use of the grapes (Hellman, 2004). The definition of the optimal date

of harvest is crucial in achieving the particular style and quality of wine, and includes

being familiar with characteristics of the variety, climatic conditions, seasonal weather

variations and ampelotechnical practices in vineyard (Bindon et al., 2013).

Primary and secondary metabolites are the characteristics of grapes being the function of

time. Defining the peak of ripening in which all the characteristics are in optimal ratio, is

of the crucial importance in improving the quality of wine. The aim of this study is to

determine the changes in the composition and content of organic acids (malic, tartaric and

citric acid), total acidity, pH value, potassium content and sugar components (fructose and

glucose) in „Plavac mali“ grape during four different harvest dates within Central and

Northern Dalmatia conditions. Displaying the dynamics of basic parametres of the grape's

ripeness has a goal in the development optimal maturity model of economically most

important red wine variety „Plavac mali“ in Croatia.


Plant material

Grape samples of „Plavac mali“ variety (Vitis vinfera L.) were collected in 2013 from

the two experimental vineyards situated in the Mediterranean region of Croatia,

Central Dalmatia subregion (Duilovo) and Northern Dalmatia subregion (Baštica).

Collection vineyard of the Institute for Adriatic Crops and Karst Reclamation, Duilovo

(Latitude 43º30.335 'N; Longitude 16º29.855' E; Elevation: 14 m above sea level).

Collection vineyard of the Faculty of Agriculture, Baštica (Latitude 44º09.163 'N;

Longitude 15º26.465' E; Elevation: 121 m above sea level). Sampling was conducted

every week, during four different harvest dates (August 21, September 6, September 20

and 3 October) at Duilovo and (31 August 13 September 27 September 11 October) at

Baštica. The total quantity of the harvested grapes amounted to 100 kg per harvest.

Grapes have been moved to the cold in the laboratory, homogenized and berries with

attached pedicels were carefully sampled. A total of 1200 grape berries were taken for

each harvest date, which then were divided into four sample groups each with 3 sub-

samples of 100 berries. The following analysis was conducted on one of the samples,

while others were stored at -80 °C waiting for determination of phenolic maturity

parameters.


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Standard grape analysis

Chemical analyses of the basic grape ripeness parameters were performed within 24 hours

from the time of harvest in order to avoid the negative effect of temperature on the content

of organic acids. On each of the three sub-samples the total mass of berries; the total

amount of soluble solids in the juice (sugar) (ºOe) using a refractometer with a temperature

correction (Atago, model WM_7), total acidity content by titration to the equivalence point

with 0.1 M NaOH with bromothymol blue indicator and pH value was determined

(Methrom, Model 1719 S).

HPLC-analysis

Identification and quantification of the individual organic acids: citric, tartaric and malic

acid (g/L) at each of the three sub-samples was carried out using high performance liquid

chromatography with photodiode array detector (HPLC-DAD) (Agilent 1100, USA.) The

chromatographic separation of organic acids was performed with isocratic elution in the

following conditions: flow rate 0.6 mL/min and the column temperature 65 °C. Cation

exchange column Aminex HPX 87 H (300 x 7.8 mm i.d) was used as the stationary

phase equipped with cation H + Microguard cartridge precolumns (Bio-Rad

Laboratories, Hercules, CA), and 0.065% - NaCl aqueous phosphoric acid was used as

mobile phase. Detection of the organic acid was performed using a wavelength of 210 nm.

Data analysis was performed using ChemStation chromatography data system (Agilent,

Palo Alto, CA).

Enzymatic analysis

The concentration of glucose and fructose was determined by standard spectrophotometric

method recommended by the OIV using enzymatic kit (Enzy plus) manufactured by

Biocontrol.

Photometric analysis

Potassium content analysis was conducted by flame photometer (Sherwood Sci., 420)

extracting with 1 M ammonium acetate.


The significance of differences in data content of primary metabolites of individual harvest

date at two separate locations was subjected to analysis of variance (ANOVA). The


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significance of differences between the mean values individual components during four

different harvest dates was determined using Fisher's least significant difference test.

Applied probability threshold was p<0.05. Statistical analysis was carried out using the

statistical package Statistica 8.0 software (StatSoft, Inc., USA).


Sugar accumulation in the berries

The data shown in Fig. 1 and 2 show the changes of primary metaboli tes in grape

variety „Plavac mali“ during four different harvest dates in terms of Central (Duilovo)

and North (Baštica) Dalmatia in 2013. Total weight of 100 berries in the terms of

Central Dalmatia differs significantly at different harvest dates, with a peak in the third

harvest date. In North Dalmatia total weight of 100 berries has steadily increased

during ripening, but no statistically significant difference in the weight of berries of

2nd, 3rd and 4th harvest date was found. The curve of sugar content (TSS) has steadily

increased from 1st to 4th harvest date, like was reported in study of the impact of

irrigation on the accumulation of certain compounds in the „Tempranillo“ variety

(Esteban et al., 1999) and study of effect of grape maturity on composition of the grape

of the „Cabernet Sauvignon“ variety (Bindon et al., 2013). The highest content of TSS

berries had at the last harvest date 94 ºOe in terms of Duilovo and 85 ºOe in terms of

Baštica. No significant difference between harvest dates conducted during September

in Duilovo, and during late September and October in Baštica was found. From the

beginning of the sampling period to the fourth harvest date sugar content in Duilovo

increased for 18 ºOe, and in Baštica for 16 ºOe. Interesting results have been found by

observing the composition of individual sugars in „Plavac mali“. No significant

differences were found in the content of glucose during the four different harvest dates

in terms of Duilovo, while in Baštica only the berries of the first harvest date differed

significantly with minimum glucose accumulate. Extremely small fructose content

during different harvest dates in Baštica was unexpected where in the second and third

harvest date ratio of glucose to fructose was 2.90 and 1.95. Kliewer 1967b, states that

the ripening reduces the ratio of glucose to fructose except for the eight wine varieties

including the „Zinfandel“, synonym „Crljenak kaštelanski“, in which glucose

dominates over fructose. Fructose content varies (not following a proper trend) during

different harvest dates.


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Fig.1. The average values and standard deviation (a) average weight of 100 berries (g), sugar

content (ºOe), (c) pH-value, (d) total accidity (g/L tartaric acid), (e) potassium

content (mg/L), (f) citric acid content (g/L), (g) tartaric acid content (g/L),

(h) malic acid content (g/L), (i) glucose content (g/L), (j) fructose

content (g/L) in berries of „Plavac mali“ during 4 different

harvest dates in terms of Duilovo. Statistically significant

difference is marked with different Latin letters

p<0,05 (Fisher LSD test).


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Fig.2. The average values and standard deviation (a) Average weight of 100 berries (g), sugar

content (ºOe), (c) pH-value, (d) total accidity (g/L tartaric acid), (e) potassium

content (mg/L), (f) citric acid content (g/L), (g) tartaric acid content (g/L),

(h) malic acid content (g/L), (i) glucose content (g/L), (j) fructose

content (g/L) in berries of „Plavac mali“ during 4 different

harvest dates in terms of Baštica. Statistically significant

difference is marked with different Latin letters

p<0,05 (Fisher LSD test).


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The accumulation of organic acids in the berries

During ripening the concentration of total acidity decreased (expressed in g/L tartaric

acid). The dynamics of pH values follow the expected trend during ripening. The pH-value

increased due to the increase in the content of potassium cations, reduction of the

concentration of malic acid and entering of tartaric acid into the form of salt (Kliewer et

al., 1967a). The total acidity is the sum of titratable protons (H +) and mineral metal

cations K + and Na +, and is a poor indicator of the composition of organic acids. On the

other hand, the pH is a function of total acidity, the concentration of K + and Na + ions and

depends on the share and strength of the individual organic acids, especially tartaric and

malic acid ratio and is the most important measure of the acidity of the must (Boulton,

1980). The optimal pH value for growth and yeast activity in the future wine in Duilovo is

achieved at the 2nd harvest date (3.57), whereas in conditions of Baštica the trend of

increase is smoother and reaches the optimum value in the 3rd and 4th harvest date (3.49

and 3.5). The trend of total acidity is the same for both locations and there was no

significant difference in the concentration of total acids in the third and fourth harvest date

at both locations. Dynamics of potassium cations (mg/L) varies in Duilovo, and is maximal

at second harvest date 1784.38, while in Baštica a proper trend of increase is visible with a

peak at third harvest date 1191.04 followed by a slight decline to 1174.66. Esteban et al.

1999 published similarly, in „Tempranillo“variety regardless of the availability of water in

the soil. Significant differences in the content of malic acid during four harvest dates at

both locations were found. Initially, higher content of malic acid 3.43 g/L had berries from

Baštica while berries from Duilovo had 2.17 g/L. Intensive loss of malic acid was observed

in Baštica and there was no detectable quantity of malic acid in 4th harvest date at Baštica,

while the Duilovo berries still contain 0.25 g/L. The behavior of tartaric acid varies

depending on location. In Duilovo there is no significant difference in the content of

tartaric acid between different harvest dates, probably due to pronounced drought, with the

exception of the third date when the berries have a significantly higher content of this acid

4.32 g/L, which can be associated with the behavior of potassium cations which were the

least detected at this harvest date. Significant differences were found in the content of this

stable grape acid over four harvesting dates in Baštica, where berries follow the trend of

reduction from 1st harvest date when they contained 5.73 g/L to 4th harvest date when

they contained 4.37 g/L. This behavior of tartaric acid can be explained by an increase in

the total weight of 100 berries, causing a dilution of tartaric acid, but also due to the

constant increase in potassium cations towards the latest date of harvest (Kliewer et al.,

1967, Iland and Coombe, 1988). No significant differences were found in the content of

citric acid during the first three harvest dates and only the berries from the 4th harvest date

show significantly higher and almost equal citric acid content in both locations 0.35 g/L

and 0.34 g/L. The sum of the mass concentrations of the individual acids does not coincide


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with the total/titratable acidity content because during ripening only a certain part (anions)

of acid is in the form of salt and a smaller fraction is free (Johnson and Nagel, 1976).

Conclusions

The composition and content of primary metabolites in a grape is an optimizing problem

essential for the quality of the future wine. The quantitive determination of individual

primary metabolites during 4 different harvest dates showed almost indentical trend of

conduct of the individual compounds despite the clear difference in soil/climate/canopy

managment of the two observed location. We demonstrated the important impact of the

harvest date on the content of primary metabolites in the juice of „Plavac mali“ variety,

which in Duilovo conditions accumulates a higher amount of sugar (observed both as TSS

and as a sum of individual hexose) and a higher pH, which can represent a problem. We

did not find significant difference in fructose and glucose ratio in early and late harvested

grapes. A glucose is a dominant sugar in all dates of harvest. The concentration of tartaric

acid decreases a bit towards the final harvest dates, as a consequence of dillution when

increasing in total weight of berries was observed in terms of Baštica. The concentration of

the malic acid decreases sharply and there was hardly detectable quantity of this acid in

last harvest date. According to yield, composition and content of sugars and acids the

optimal harvest date for „Plavac mali“ in Central Dalmatia was achieved in the 3rd harvest

date (30 DAA from the beginning of the sampling). In terms of Northern Dalmatia the 4th

harvest date is optimal (41 DAA from the beginning of the sampling).

References

Bindon, K., Varela, C., Kennedy, J., Holt, H., Herderich, M. (2013): Relationships between harvest

time and wine composition in Vitis vinifera L. cv. Cabernet Sauvignon 1. Grape and wine

chemistry,. Food Chem. 138 (2-3), 1696-705.

Bisson, L.F. (2001): In search of optimal grape maturity, Department of enology & viticulture, UC

Davis, Practical Winery & Vineyard Journal, 32-43.

Boulton, R. (1980): The general relationship between potassium, sodium and pH in grape juice and

wine, Am. J. Enol. Vitic. 31 (2), 182-186.

Coombe, B.G., McCarthy, M.G. (2000): Dynamics of berry growth and physiology of ripening,

Aust. J. Grape Wine R. 6, 131-135.

Davies, C., Robinson, P. S. (1996): Sugar accumulation in grape berries: cloning of two putative

vacuolar invertase cDNAs and their expression in grapevine tissues, Plant Physiol. 111, 275-

283.

Esteban, M.A., Villanueva, M.J., Lissarrague, J. R. (1999): Effect of irrigation on changes in berry

composition of Tempranillo during maturation. Sugars, organic acids, and mineral elements,

Am. J. Enol. Vitic. 50, 418-434.


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Hellman, E. (2004): How to judge grape ripeness before harvest, Edward Texas A&M University,

Presented at the 2004 Southwest Regional Vine & Wine Conference Albuquerque, NM

February 27-28.

Iland, P.G., Coombe, B.G. (1988): Malate, tartrate, potassium and sodium in flesh and skin of

Shiraz grapes during ripening: concentration and compartementation, Am. J. Enol. Vitic. 39,

71-76.

Johnson, T., Nagel, C. W. (1976): Composition of central Washington grapes during maturation,

Am. J. Enol. Vitic. 27 (1), 15-20.

Kennedy, A.J. (2008): Grape and wine phenolics: Observations and recent findings, Cien. Inv. Agr.

35(2), 107-120.

Kliewer, W.M., Howarth, L., Omori, M. (1967a): Concentrations of tartaric acid and malic acids

and their salts in Vitis Vinifera grapes, Am. J. Enol. Vitic. 18 (1), 42-54.

Kliewer, M.W. (1967b): The glucose-fructose ratio of Vitis vinifera grapes, Am. J. Enol. Vitic. 18

(1), 33-41.

Meléndez, E., Ortiz, M.C., Sarabia, L.A., Íñiguez, M., Puras, P. (2013): Modelling phenolic and

technological maturities of grapes by means of the multivariate relation between organoleptic

and physicochemical properties, Anal Chim Acta. 761, 53-61.

Peynaud, E., Maurié, A. (1958): Synthesis of tartaric and malic acids by grape vines, Am. J. Enol.

Vitic. 9, 32-36.


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Extraction of active compounds from alfalfa plant

UDC: 66.061.34 : 633.31

Tina Perko, Mojca Škerget, Željko Knez

University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Separation

Processes and Product Design, Smetanova 17, 2000 Maribor, Slovenia

Summary

In the work the optimal extraction conditions for the isolation of active compounds from Alfalfa

were determined. Conventional extraction is the most commonly used method for isolation of

valuable compounds from natural materials. Conventional extraction was performed using

aqueous solutions of acetone and ethanol with different composition: (25, 50, 75 % (v/v) ) and

pure solvents acetone, water and ethanol. Ethanol and acetone are both approved for the use in

food industry as volatile solvents for extraction of valuable compounds. For comparison active

compounds were extracted also by conventional Soxhlet appratus using hexane as solvent. The

extracts were analysed on the content of total phenols. Furthermore the DPPH radical scavenging

activity of extracts was determined.

Keywords: Alfalfa, conventional extraction, total phenols, DPPH

Introduction

The common name of the plant Alfalfa (Medicago sativa) is Lucerne. It is a valuable

plant grown mainly for animal feed and it is rich in proteins, minerals, vitamins (A, B, C,

D, E, K), carotene, coumarins, alkaloids and secondary metabolites of plants (Gawel,

2012). The most important bioactive components are starch, carbohydrates and basic

proteins. The plant also contains a large amount of enzymes, anti-inflammatory

substances, hormones and beta carotene. In traditional medicine, this herb is used in

alternative herbal treatments (Cauni et al., 2012). Extract from the leaves of Alfalfa has a

positive, multidirectional impact on the human body. It increases the level of estrogen,

prevents atherosclerosis, helps blood circulation and strenghtens immunity (Gawel,

2012). Nutritious fresh or dried alfalfa leaf tea is traditionally used to promote appetite,

weight gain, as diuretic or to stop hemoragies.



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Fig. 1. Medicago sativa plant

(http://www.agroklub.com/sortna-lista/krmno-bilje/lucerna-57/)

It has a long tradition of use in Ayurvedic and homoepathic medicine for central nervous

and digestive system disorders, and for the treatment of various other ailments.


Alfalfa dry plant was purchased from Gittelde (Germany). Acetone, absolute ethanol and

hexane were provided by Merck (Germany).

Conventional extraction

Conventional extraction was performed using aqueous solutions of acetone and ethanol

with different composition: 25, 50, 75 % (v/v) and pure solvents acetone, water and

ethanol. Extractions for each solvent composition were performed in duplicate. 10 g of dry

grinded alfalfa plant were weighed in a glass flask and 250 mL of solvent were added.

After 3 hours of mixing at 60 °C, the solution was separated by vacuum filtration. Extract

solutions were collected and the solvent was then removed by evaporation. Prepared

extracts were saved in a cool place before analysis.

extract

raw material

Yield (%) = 100 %m

m (1)

Soxhlet extraction

10 g of dry grinded alfalfa plant was placed in a paper thimble, which was inserted into a

Soxhlet apparatus and extracted with 250 mL of hexane. The extraction was performed for


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3 h. After the solution was colleected and the solvent was evaporated. Yield of extraction

was calculated using Eq. (1).

DPPH radical-scavenging system

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was measured using the

UV-VIS spectrophotometric method reported by (Majhenič et al., 2007). Extract solutions

were prepared by dissolving 1 mg of extract in 1 mL of methanol. The solution of DPPH in

methanol (6 x 10-5

M) was prepared daily before measurements. 3 mL of DPPH solution

was added to a dark flask containing 77 µL of extract solution. The mixed solutions were

kept in the dark for 15 min at room temperature and the absorbance was measured using

UV-VIS spectrophotometer (Varian, USA) at 515 nm. Radical-scavenging activities of

samples were calculated using Eq. (2).

B A

B

% inhibition 100 %Α A

A

(2)

where AB is absorbance of blank sample (t = 0 min) and AA is absorbance of extract

solution (t = 15 min). All measurements were performed in triplicate and average values

were calculated.

Analysis of total phenols

The concentration of total phenols in extracts was measured by a UV-VIS

spectrophotometer (Varian, UV-VIS), based on a colorimetric oxidation/reduction

reaction. The oxidizing reagent used was Folin-Ciocalteu reagent. 2.5 mL of Folin-

Ciocalteu reagent (diluted 10 times with water) and 2 mL of Na2CO3 (75 g/L) were added

to 0.5 mL of diluted extract (20 mg in 10 mL distilled water). The sample was incubated

for 5 min at 50 °C and then cooled. 0.5 mL of distilled water was used as control sample.

The absorbance was measured at 760 nm. The results were expressed as mg of gallic acid

per g of extract (mg GA/g extract) (Makovšek et al., 2012).


Extractions of alfalfa plant were performed by conventional method. Different

compositions of acetone or ethanol water solutions were used as extraction solvents.

Extractions were performed in duplicate and extractions yields are presented in Fig. 2.


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Fig. 2. Influence of organic solvent composition on extraction yield

In the case of acetone extraction yields increased with increasing water content in the

solvent solution. The yield varied between 1.88% (100% acetone) and 23.11% (25%

aqueous acetone solution). Similarly, in the case of ethanol solutions extraction yields

increased with increasing water content, the highest yield was determined for 25% aqueous

ethanol solution, 24.5%. The lowest yield of extraction (1.7%) was obtained using Soxhlet

extraction.

Table 1. The content of total phenols and DPPH radical-scavenging activities of various alfalfa extracts

Composition of organic

solvent (v/v) %

Total phenols

(mg GA/g extract)

DPPH radical-

scavenging activity (%)

acetone

0 26.73 ± 1.72 1.53 ± 0.51

25 34.95 ± 0.78 2.58 ± 2.08

50 36.75 ± 0.27 3.58 ± 2.16

75 42.85 ± 0.86 6.51 ± 2.67

100 36.95 ± 0.01 5.15 ± 1.77

ethanol

0 36.98 ± 0.40 0.86 ± 0.05

25 40.24 ± 0.92 1.09 ± 0.11

50 38.28 ± 0.08 2.11 ± 0.06

75 36.54 ± 0.13 1.92 ± 1.16

100 39.23 ± 0.35 13.64 ± 1.10

0

5

10

15

20

25

30

0 20 40 60 80 100To

tal yie

ld o

f e

xtr

ac

tio

n (

%)

Composition of organic solvent (v/v) (%)

ace…eth…


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The content of total phenols and DPPH radical-scavenging activities of various alfalfa

extracts are represented in Table 1. The amount of total phenolic compounds extracted

with different solvent mixtures ranged from 26.73 to 42.85 mg GA/g extract. The alfalfa

extracts contained the highest amounts of phenolic compounds when 75% acetone was

used as extraction solvent and the lowest amounts of phenolic compounds when water was

used as extraction solvent.

Absolute ethanol alfalfa extract was the most effective DPPH radical-scavenger with

inhibition higher than 13%. Generally, the composition of organic solvent did not have a

significant effect on radical-scavenging activity of extracts. Extracts obtained with

mixtures of organic solvent (acetone and ethanol) and water generally showed lower

radical-scavenging activity.

Conclusions

In the presented work extraction of alfalfa was performed using different types of

extraction methods and various solvents. Maximum yield of extraction was obtained by the

conventional extraction using 25% of aqueous ethanol solution as solvent (24.50%). The

lowest yields (1.7%) were obtained by Soxhlet extraction using hexane as solvent. It can

be concluded that acetone solution especially 75% and pure ethanol are very good solvents

for successful isolation of phenols. Extracts of alfalfa did not show strong antioxidant

activities.

Acknowledgements

Authors are grateful to the Slovenian Ministry of High Education, Science and Technology

for the financial support of this work.

References

Gawel, E., (2012):Chemical composition of lucerne leaf extract (EFL) and it is applications as a

phytobiotic in human nutrition, Acta Sci. Pol. Technol. Aliment. 11 (3), 303-310.

Caunii, A., Pribac, G., Grozea, I., Gaitin, D., Samfira, I. (2012): Design of optimal solvent for extraction

of bio-active ingredients from six varieties of Medicago sativa, Chem. Cent. J. 6, 123-130.

Majhenič, L., Škerget, M., Knez, Ž. (2007): Antioxidant and antimicrobial activity of guarana seed

extracts, Food Chem.104 (3), 1258-1268.

Makovšek, K., Knez, Ž., Škerget, M. (2012): Influence of Process Parameters on the Extraction of

Flavanones from Mandarin Peel, Acta Chim. Slov. 59, 879-887.

http://www.agroklub.com/sortna-lista/krmno-bilje/lucerna-57/.


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Implementacija zahtjeva IFS-a (International Featured Standards)

u prehrambenoj industriji - izbor ili uvjet opstanka na tržištu

UDC: 614.31 : 664


TÜV Croatia d.o.o., TÜV NORD Group, Savska 41, 10000 Zagreb, Hrvatska

Sažetak

Primjena zahtjeva IFS-a u poduzećima je dobrovoljna. Sama poduzeća donose odluku o potrebi

implementacije ovih zahtjeva na osnovi procjene koja se u većini slučajeva temelji isključivo na

procjeni zahtjeva tržišta (kupaca). Implementacija sustava nije zakonska obaveza što rezultira pitanjem

opravdanosti usklađivanja procesa s ovim standardom. S druge strane, današnje društvo i visoko

razvijena svijest potrošača, te sve veća društvena i zakonska odgovornost koju imaju proizvođači

hrane postavljaju visoke zahtjeve na higijenu i zdravstvenu ispravnost proizvoda. Navedeno se

dodatno proširilo i na trgovačke lance koji su započeli s prodajom vlastitih trgovačkih marki čime je i

njihovo ime dovedeno u opasnost. To je sve rezultiralo da danas tvrtke koje žele poslovati s

trgovačkim lancima, izvoziti proizvode i raditi s velikim kupcima moraju implementirati sustav IFS-a.

To je vrlo često postao uvjet za suradnju, a samim time je stvorena i obveza implementacije i uvjet

opstanka na tržištu. U radu su kratko prezentirani zahtjevi IFS-a te osnovne prednosti njegove

implementacije u poduzeću. Implementacija IFS-a u poduzeću poboljšava tržišnu poziciju poduzeća

ali i osigurava kvalitetniji i “sigurniji” proizvod čime olakšava pristup novim kupcima.

Ključne riječi: IFS, standard, zahtjevi tržišta

Uvod

IFS je međunarodno priznati standard koji osigurava da certificirane tvrtke mogu isporučiti

proizvod ili uslugu koja je u skladu sa zahtjevima standarda. Razvijen je kako bi se

osigurala sigurnost i kvaliteta proizvoda i usluga. Definiran je od strane njemačkih,

francuskih i talijanskih trgovačkih lanaca, a prva verzija norme je objavljena 2000. godine.

IFS standardi

U početku se IFS standard primjenjivao na područje hrane, odnosno prehrambene

industrije, a kasnije su razvijeni standardi i za ostala područja djelatnosti. Tako se danas

primjenjuje IFS Food, standard za tvrtke čija je djelatnost proizvodnja prehrambenih



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proizvoda, IFS HPC, standard za tvrtke čija je djelatnost proizvodnja proizvoda za

kućanstvo i osobnu higijenu, IFS Logistics, standard za tvrtke koje se bave skladištenjem,

distribucijom i transportom prehrambenih i neprehrambenih proizvoda, IFS Broker,

standard za tvrtke koje se bave trgovinom, kupovanjem i prodajom proizvoda, IFS

Cash&Carry, IFS PACsecure, IFS Food Store.

IFS food

Glavni cilj kod definiranja standarda je bio objediniti standarde za sigurnost hrane i kvalitetu,

osigurati transparentnost kroz cijeli lanac opskrbe, osigurati rad s akreditiranim certifikacijskim

tijelima i kvalificiranim auditorima, smanjiti broj audita u godini, smanjiti troškove i vrijeme

potrebno za audite. Temelj norme je primjena sustava kvalitete, rad prema važećoj zakonskoj

regulativi uzimajući u obzir i međunarodnu regulativu za hranu, primjena HACCP načela, rad

prema dobroj proizvođačkoj praksi i rad prema dobroj higijenskoj praksi.

Vrste audita

U samom procesu certifikacije IFS norme postoje različite vrste audita. Certifikacijski

audit je prvi audit za tvrtku prema zahtjevima IFS-a. Na certifikacijskom auditu se

provjerava ispunjenost svih zahtjeva IFS-a, odnosno provodi se audit cijele tvrtke. Folow

up audit se zahtjeva kada su rezultati početnog ili nadzornog audita bili nedostatni i nije

izdan IFS certifikat. Na ovom auditu se pregledava učinkovito i efikasno uvođenje

korektivnih mjera koje je bilo potrebno poduzeti kako bi se uklonile nesukladnosti i

ispunili zahtjevi norme. Audit je potrebno provesti unutar šest mjeseci od prethodnog

audita i provodi se na lokaciji. U slučaju neuspjeha mora se provesti kompletno novi audit.

Nadzorni audit ili recertifikacijski audit je audit koji se provodi radi produženja valjanosti

certifikata. Auditiraju se svi zahtjevi IFS-a, odnosno ponovno se provodi audit cijele

tvrtke. Plan nadzornog audita temelji se na rezultatima prethodnog audita i kod produženja

certifikata mora se provesti u određenom vremenskom periodu, najranije osam tjedana

prije i dva tjedna poslije datuma prvog audita.

Sustav ocjenjivanja

Audit se provodi tako da se provjerava ispunjenost zahtjeva norme. Za svaki zahtjev se daje

ocjena kojom se ocjenjuje ispunjenost zahtjeva. U slučaju uočene nesukladnosti za zahtjevima

norme, gdje nije prisutan negativan utjecaj na proizvode i procese, odstupanje se ocjenjuje sa

ocjenama A, B, C, D. Ocjena A znači da je zahtjev u potpunosti ispunjen, B – zahtjev je većim

dijelom ispunjen, C - mali dio zahtjeva je ispunjen i D – potpuno neispunjen zahtjev norme.

Kod neispunjavanja zahtjeva koji se odnose na sigurnost hrane, zakonsku regulativu ili utjecaj

na zdravlje potrošača, za nesukladnost se može dati ocjena Major ili K.O. nesukladnost. Za


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313

svaku ocjenu se dodjeljuje određeni broj bodova, a zbroj bodova daje ukupnu ocjenu. Ocjena A

donosi 20 bodova, ocjena B - 15 bodova, ocjena C - 5 bodova i ocjena D -20 bodova. Ocjena

Major smanjuje ukupan rezultat, a ocjenu K.O. je moguće dati samo za zahtjeve koji su

označeni kao Knock Out zahtjevi i u tom slučaju nije moguće izdavanje certifikata. U IFS Food

ver. 6 postoji 10 zahtjeva koji su označeni kao K.O. zahtjevi a to su: odgovornost više uprave,

sustav nadzora za svaku kritičnu kontrolnu točku, osobna higijena, specifikacija sirovina,

sukladnost proizvoda sa recepturama, upravljanje stranim tijelima, sustav sljedivosti, interni

audit, procedura za povlačenje i opoziv, korektivne radnje.

Dio zahtjeva mogu biti označeni sa oznakom NA (Not Applicable) što znači da zahtjev

nije primjenjiv. U tom slučaju auditor mora dati objašnjenje u izvještaju.

Zahtjevi

U normi IFS Food, verzija 6., navedena su šest poglavlja sa zahtjevima koji se moraju

ispuniti, a odnose se na odgovornost višeg rukovodstva; sustav upravljanja kvalitetom i

sigurnosti hrane; upravljanje resursima; planiranje i proizvodni proces; mjerenje, analize,

poboljšanja; obrana hrane i vanjske inspekcije.

Uvjeti za izdavanje certifikata

Nakon provedenog audita moguće je izdavanje certifikata Osnovna razina ili Viša razina.

U tablici 1 su dani uvjeti za izdavanje certifikata:

Tablica 1. Izdavanje certifikata

Table 1. Certificate awarding

Nesukladnost Status Radnja auditirane strane Certifikat

Najmanje 1 K.O. ocijenjen sa D

Nije odobreno - uvođenje action plana

- planiranje novog inicijalnog audita Ne

> 1 MAJOR i/ili

<75% zahtjeva je

ispunjeno

Nije odobreno - uvođenje action plana

- planiranje novog inicijalnog audita Ne

Maksimalno 1

Major i

≥75% zahtjeva je ispunjeno

Nije odobreno

ako se ne

poduzmu

daljnje radnje i provede follow

up audit

- poslati action plan unutar 2 tjedna nakon

primanja preliminarnog izvještaja

- Follow-up audit maksimalno 6 mjeseci nakon audit datuma

Izdavanje

certifikata Osnovna razina

(ako je Major

nesukladnost

otklonjena nakon

provedenog

Follow up

audita)


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Ukupno je ≥75% i <95% zahtjeva

ispunjeno

Odobreno za

IFS food, Osnovna razina

nakon

prihvaćanja

akcijskog plana

- poslati action plan unutar 2 tjedna nakon

primanja preliminarnog izvještaja Osnovna razina

≥95% zahtjeva ispunjeno

Odobreno za

IFS food, Viša razina nakon

prihvaćanja

akcijskog plana

- poslati action plan unutar 2 tjedna nakon primanja preliminarnog izvještaja

Viša razina

Certifikacijski ciklus

Prvi inicijalni audit je certifikacijski audit. Audit produženja ili recertifikacijski audit

provodi se svake godine. Potrebno ga je provesti najranije osam tjedana prije, a najkasnije

dva tjedna nakon datuma inicijalnog audita. Na certifikatu je isti datum valjanosti svake

godine.

Slika 1. Certifikacijski ciklus

Fig. 1. Certification cycle


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Prednosti implementacije IFS food standarda

IFS Food je standard koji obuhvaća sigurnost hrane i standard kvalitete u jednom. Razvijen

je kako bi se mogla provjeriti sigurnost hrane i kvaliteta jednim alatom, a samim time se

štedi vrijeme i novac proizvođača, jer se smanjuje broj potrebnih audita. Nakon provedbe

audita dobije se prava slika o tvrtki i mogućnostima za poboljšanje. Tvrtke koje su

implementirale IFS sustav poboljšale su odnose s kupcima i povećale konkurentnost na

tržištu. Implementacijom standarda otvara se mogućnost suradnje s trgovačkim lancima i

mogućnost za proizvodnju robnih marki koje su u vlasništvu trgovačkih lanaca. Samim

time se povećava opseg proizvodnje, kao i opseg prodaje.

Zaključci

Primjena i implementacija zahtjeva IFS standarda je dobrovoljna. Današnje društvo i

visoko razvijena svijest potrošača postavljaju visoke zahtjeve za zdravstvenu ispravnost i

kvalitetu proizvoda. Trgovački lanci prodaju vlastite robne marke i žele biti sigurni da su

njihovi proizvodi sukladni zahtjevima za kvalitetom. Razvojem IFS-a omogućen je alat

kojim se mogu provjeriti sigurnost hrane i kvaliteta u cijelom opskrbnom lancu, a time i

ispunjenje zahtjeva za sigurnim proizvodom. Za sve proizvođače koji rade ili planiraju

raditi s velikim trgovačkim lancima implementacija standarda postaje obaveza, uvjet za

suradnju i opstanak na tržištu. Implementacijom i certifikacijom IFS-a otvaraju se nove

mogućnosti za širenje proizvodnje i prodaje proizvoda pod robnom markom koja je u

vlasništvu trgovačkog lanca, a isto tako i širenje prodaje proizvoda na tržištu i dogovaranje

suradnje s novim kupcima.

Literatura

http://www.ifs-certification.com (1.10.2014.)

IFS Food, version 6, 2012


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Implementation of IFS (International Featured Standards)

in food industry - option or condition of survival on the market


TÜV Croatia d.o.o., TÜV NORD Group, Savska 41, HR-10000 Zagreb, Croatia

Summary

Application of IFS requirements in companies is voluntary. The companies themselves make the

decision of the need for the implementation of these requirements, depending on the evaluation

which is in most cases based entirely on an assessment of market demand (customers).

Implementation of the system is not a legal obligation and that results with question of justification

alignment process with this standard. On the other hand, today's society and highly developed

consumer awareness and increasing social and legal responsibility for food manufacturers, placed

high demands on hygiene and safety of the products. The above is further extended to retailers

which started selling their own brands and their name is endangered. This is the main reason that

companies which want to do business with retail chains, export products and work with large

customers have to implement IFS. It is very often become a condition for cooperation and

obligation of implementation standard and condition of survival in the market. This paper presented

the IFS requirements and the basic benefits of implementation in the company. Implementation of

IFS improves the market position of the company and also provides better and safer product and

facilitates access to new customers.

Keywords: IFS, standard, market demands


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Influence of malaxation time and temperature

on ‘Levantinka’ virgin olive oil properties

UDC: 665.327.3

Mirella Žanetić1

, Maja Jukić Špika1, Renato Stipišić

2,

Antonela Jukić2, Sandra Svilović

2

1Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, p.p. 288, HR-21000 Split, Croatia

2University of Split, Faculty of Chemistry and Technology, Teslina 10/V, HR-21000 Split, Croatia

Summary

Effect of malaxation time and temperature on Levantinka virgin olive oil (VOO) properties during

production process was studied. Mixing of olive pastes was performed at 26, 36, 46 ºC. Also, for

every temperature effect, malaxation time was studied during 30, 45, 60 min. In order to estimate

the olive oil quality the investigation of free fatty acid, peroxide value, UV spectrophotometric

indices, total phenols content, sensory evaluation, oxidative stability and antioxidant activity was

carried out. It’s evident that both, malaxation time and temperature, affect the properties and quality

of olive oil obtained. The lowest peroxide value, the highest total phenols content and DPPH value

was achieved at 36 ºC and 30 min. The lowest free fatty acid and the highest oxidative stability was

also achieved at the same temperature (36 ºC) but at some longer malaxation time (FFA 60 min,

Rancimat test 45 min). The values of K-numbers were within the limits for VOO for all samples

processed at 26 ºC and 36 ºC and 30 and 45 min, respectively. Sensory analyses of investigated oils

showed the highest score for those processed at same malaxation conditions as for K-numbers. The

highest bitterness was perceived in samples obtained at 36 ºC and 30 min of malaxation time while

pungency in samples produced at 36 ºC and 60 min of malaxation time.

Keywords: Olea europaea L., malaxation time, temperature, olive oil quality, phenols

Introduction

Virgin olive oil is produced from olive fruits solely by mechanical means, which includes

olive crushing, paste malaxation and oil separation without the use of solvents. The

processing and characteristics of different quality olive oils are controlled and defined by EC

Regulations (1991). During extraction, as a result of crushing and the rheological properties

of the homogenised olive paste, much of the storage oil is dispersed. The resultant droplets

are distributed within the olive mash and considerable oil can be lost with the by-products.

However, slow malaxation of the paste makes the production of large oil droplets easier and



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leads to a continuous oil phase, which can be separated from the paste solids and vegetable

water by mechanical means (Fedeli, 1996; Angerosa et al., 2001).

Olive oil quality is related to chemical composition of the oil, its oxidative stability and

sensory characteristics (Aparicio et al., 1999). These parameters are affected by different

factors such is olive cultivar (Beltran et al., 2005), degree of maturity of the olives

(Rotondi et al., 2004), and last but not least, the parameters of the processing technology in

the olive mills (Cerretani et al., 2005; Di Giovacchino et al., 2002). The malaxation of

olive paste is a fundamental phase of extraction process of olive oil. After olive crushing,

malaxation of olive paste is next phase in order to improve the successive separation of oil

and to increase the yield of the oil extraction. The oil droplets inside the oil-bearing cells

are partly located in the vacuole (about 76%), where the oil is in a free form, and partly

located in the cytoplasm (about 24%), where the oil is dispersed and bound to colloids

through the lipoprotein membranes (Ranalli et al., 2001). The slow stirring of the paste

brings the colloid-bound oil droplets into contact with each other. Through this interaction,

the oil combines into large droplets which are released due to the phase inversion of the

emulsion and oil is then easily extracted by physical and mechanical means (Amirante et

al., 2006; Inarejos-García et al., 2009). It’s well known that cold processed olive oil is

obtained at temperature up to 27 ºC in each processing step (Di Giovacchino et al., 2002;

Kalogeropoulos et al., 2014). With prolonged malaxation time the temperature of the olive

paste also increases, as well as the oil yield, but the quality of obtained oil is usually lower

(Stefanoudaki et al., 2011). In this study we examined the influence of malaxation time at

different temperatures in order to determine the best processing conditions for the highest

quality of monovarietal olive oil from domestic cultivar Levantinka.


Olive fruits (Olea europea L.) from Levantinka cultivar was picked by hand on

experimental field of Institute for Adriatic Crops and Karst Reclamation in Split at

optimum harvest time (1/3 of fruit was dark-coloured) in October 2013. Fruits were

processed immediately after harvesting using laboratory scale olive oil mill Abencor mc2,

which consisted of a hammer crusher, thermostated vertical olive paste mixers, and

centrifuge (Fig. 1). Olive paste malaxation was performed at different temperatures (26,

36, 46 ºC). During each temperature the influence of different malaxation time was also

examined (30, 45, 60 min). All chemical analyses were carried out contemporarily;

samples were stored in dark bottles without headspace at room temperature.


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Fig. 1. Laboratory scale olive oil mill - Abencor mc2

Quality indices

In order to estimate the quality of obtained olive oils, several qualitative parameters were

evaluated (free fatty acids (FFA) expressed as percentage of oleic acid, peroxide value

(PV) expressed as milimoles of active oxygen per kilogram of oil (mmol O2/kg) and UV

spectrophotometric indices K232 and K270 (as extinction coefficients, calculated from

absorption at wavelength of 232-270 nm) according to the official analytical methods

described in the EEC Regulation n. 2568/91 of the Commission of the European Union.

Total phenolic content (TPC)

Total phenols were isolated by liquid–liquid extraction of a solution of oil in hexane with a

water/methanol mixture (60:40), three times. The concentration of total phenols was

determined using the Folin-Ciocalteau method (Gutfinger, 1981). The results for total

phenolic content (TPC) are presented as mg of gallic acid per kg of oil. The

spectrophotometric measurements were repeated three times for each extract.

DPPH antioxidant assay

Reduction in the amount of free DPPH radical was determined spectrophotometrically at

515 nm following the method described by Kalantzakis et al. (2006). Oil solution in ethyl

acetate (10% (w/v)) was added to a freshly prepared 0.125 mM DPPH solution.

Absorbance was measured against the blank solution (without DPPH radical), after 30 min.

DPPH concentration (%) was calculated by using a calibration curve.

Sensory evaluation

The sensory evaluation of olive oils was performed according IOC (2007) method. Sensory

analyses of obtained olive oils were performed by skilled panel group of sensory analysts


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for virgin olive oil. The results were calculated using computer program set in method for

organoleptic assessment of VOO (IOC, 2007).

Rancimat test – oxidative stability

An automated Rancimat 743 apparatus (Metrohm) was used for induction period

determination of olive oil samples. Six oil samples were analysed in the apparatus at the

same time. For oxidative stability measurement, each oil sample (3.0±0.01 g) was

weighted using analytical grade balance (KERN ALS 120-4). The conductometry cells

were filled with deionized water up to 60 ml. Measurements were carried out at 120oC and

air flow of 20 l/h. The results are expressed as induction period (IP).


The results presented in Table 1 shows that all analyzed olive oil samples had most of

quality parameters inside limits for extra virgin olive oil category (EC, 1991). The

exception was noticed only for K270 parameter for all oils processed at malaxation

temperature of 46 ºC and also for oils produced at malaxation time of 60 min for all

applied temperatures. These samples had K270 values above or very close to the limit for

VOO set in regulation (EC, 1991). This means that prolonged malaxation (60 min) is not

suitable for obtaining good quality VOO, as well as application of malaxation temperatures

higher then 36 ºC. Also, the K270 values were equal to limit value (0.25) for VOO category

for oils produced at 36 ºC during 30 min. Since K270 parameter gives information about

hydrolytic degradation (spoilage) of olive oil we can conclude that prolonged malaxation

time affects negatively on quality and stability of VOO.

Table 1. Chemical indicators of quality in the studied Levantinka olive oils

Malaxation

temperature (oC) 26 36 46

Malaxation time

(min) 30 45 60 30 45 60 30 45 60

FFA

(% oleic acid) 0.22 0.18 0.21 0.20 0.18 0.16 0.75 0.46 0.38

PV

(mmol O2/kg) 1.73 1.54 1.61 1.47 1.73 1.64 2.43 2.44 2.38

K 232 1.75 1.78 1.81 1.88 1.81 1.76 1.71 1.62 1.63

K270 0.19 0.23 0.27 0.25 0.23 0.25 0.27 0.27 0.26

Δ K -0.01 0.00 0.00 -0.01 -0.01 -0.01 0.00 0.00 0.00

TPC (mg/kg) 418.27 372.39 323.04 488.76 457.05 415.71 250.77 260.58 233.87

DPPH (%) 92.19 88.32 90.33 92.50 90.60 87.55 70.02 61.99 57.76


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The results that show relation between malaxation time and temperature regarding FFA and PV

are presented in Fig. 2. Generally, the values for FFA and PV were the same or slightly higher

up to 36 ºC for each time of malaxation. In the case of 46 ºC those quality parameters were

significantly increased for all oil samples. It can be concluded that 36 ºC is a critical malaxation

temperature that could be applied for obtaining optimal VOOs’ quality parameters.

Boselli et al. (2009) examined the influence of higher malaxation temperatures on Italian

VOOs quality. They found out that VOO obtained at a processing temperature lower than

27 ºC (‘first cold pressing’ or ‘cold extraction’ according to EU legislation) did not show

higher oxidative stability or sensory qualities than VOO obtained at 35 ºC. The oils

obtained at 35 ºC were considered to be of good quality by the panelists, whereas the oils

obtained at 45 ºC were rejected because they had a negative sensory attribute ‘heated or

burnt’.

In this study, during malaxation of olive paste the changes in TPC occurred depending on

time and temperature conditions (Fig. 3). TPC content generally was negatively affected

by malaxation time. The only exception was noticed at 46 ºC when the highest TPC was

achieved at 45 min. The oxidative stability was again positively correlated with TPC

except at 36 ºC. In this case the highest TPC was at 30 min but IP had the highest value at

45 min. In all samples IP value was significantly decreased in oils processed at 45 ºC, same

as TPC value, which confirms that phenolic compounds are responsible for oxidative

stability of olive oil. It’s well known from different studies that higher malaxation

temperature in generally influence on decreasing of TPC in olive oil (Allogio et al., 1997;

Montedoro et al., 1992; Stefanoudaki et al., 2011). TPC content showed decreasing

tendency with prolonged time of malaxation, with exception at malaxation time 45 min

and 46 ºC. DPPH values had the highest values at 30 min of malaxation on each

temperature, with the exception on 26 ºC during 60 min.

Fig. 2. Effect of malaxation time and temperature on free fatty acid and peroxide value

25 30 35 40 45 500

0.5

1

0

1

2

3

Temperature (oC)

FF

A (

%)

PV

(m

mo

l O

2/k

g)

FFA 30 min

FFA 45 min

FFA 60 min

PV 30 min

PV 45 min

PV 60 min


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Fig. 3. Effect of malaxation time and temperature

on induction period and total phenols content

As can be seen from the Fig. 3 TPC firstly increased and then decreased with increasing

temperature of malaxation. It is evident that the highest TPC was achieved at 36 ºC. As a

consequence of the phenolics’ protective antioxidant effect, the oxidative stability,

measured by Rancimat test, was changed along total phenols content as well as VOO

antioxidant properties (DPPH) with exception at 26 ºC and 60 min.

This study show that, for Levantinka cultivar, temperature at which highest TPC value

was achieved was slightly higher that temperature at which Ranalli et al. (Ranalli et al.,

2001) achieved the highest TPC for Italian cultivars (Caroleo, Leccino, Dritta). Also, in

their study critical temperature for TCP and other quality parameters was 35 ºC while in

this it was at 45 ºC. But it should be emphasize that 10 ºC it too wide range, so we can

conclude that highest TPC value was achieved at 36 ºC but critical temperature has yet to

be determined.

Volatile compounds from VOO are responsible for typical taste and aroma of oil (Servilli

et al., 2003). Processing conditions significantly influence the aroma profile of VOO

(Kalogeropoulos et al., 2014). After sensory analyses of Levantinka olive oils, the highest

sensory score was perceived for samples processed at 26 ºC and 36 ºC and 30 and 45 min,

respectively. The highest bitterness was perceived in samples obtained at 36 ºC and 30 min

of malaxation time and also pungency in samples produced at 36 ºC and 60 min of

malaxation time.

IP 30 min

IP 45 min

IP 60 min

TPC 30 min

TPC 45 min

TPC 60 min

Temperature (oC)

IP (

h)

25 30 35 40 45 5010

16

22

100

200

300

400

500

TP

C (

mg/k

g)


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Conclusions

It can be concluded that the temperature and mixing time have an impact on the

characteristics and quality of the oil. Top quality parameters, had an oil sample obtained at

36 °C and malaxation for 45 min. That means that on an elevated malaxation temperature

this olive cultivar showed no disruption of oil quality. But at next tested temperature, the

decline in quality occurred, particularly by increasing the FFA and the decreasing of TPC,

antioxidant properties and stability of oil. This research will be extended to other domestic

olive oil varieties in order to optimize the processing parameters and to obtain higher olive

oil yields with best quality characteristics.

Acknowledgments

This experiment was performed with financial support of Split-Dalmatia County and

project HETMIX financed by Croatian science foundation. The authors are grateful to

personnel form Institute for Adriatic Crops and Karst Reclamation and Faculty of

Chemistry and Technology from Split, Croatia, for their assistance in olive harvesting and

preparation of samples and part of chemical analyses.

References

Allogio, V., Caponio, F. (1997): The influence of olive paste preparation techniques on the quality

of the olive oil. II. Evolution of phenolic substances and some quality parameters referred to

the ripening of drupes in virgin olive oil from the Coratina cv., Riv. Ital. Sost. Grasse 74, 443-

447.

Amirante, P., Clodoveo, M.L., Dugo, G., Leone, A., Tamborrino A. (2006): Advance technology in

virgin olive oil production from traditional and de-stoned pastes: Influence of the introduction

of a heat exchanger on oil quality, Food Chem. 98 (4), 797-805.

Angerosa, F., Mostallino, R., Basti, C., Viro, R. (2001): Influence of malaxation temperature and

time on the quality of virgin olive oils, Food Chem. 72, 19-28.

Aparicio, R., Roda, L., Albi, M. A., & Gutiérrez, F. (1999): Effect of various compounds on virgin

olive oil stability measured by rancimat. J. Agric. Food Chem. 47, 4150-4155.

Beltrán, G., Aguilera, M.P., Del Rio, C., Sanchez, S. Martinez, L. (2005): Influence of fruit ripening

process on the natural antioxidant content of Hojiblanca virgin olive oils, Food Chem. 89, 207-

215.

Boselli, E., Di Lecce, G., Strabbioli, R., Pieralisi, G., Frega, N.G. (2009): Are virgin olive oils

obtained below 27 ºC better then those produced at higher temperatures?, LTW – Food Sci.

Techn. 42, 748-757.

Cerretani, L., Bendini, A., Rotondi, A., Lercker, G., Gallina-Toschi, T. (2005): Analytical

comparison of monovarietal olive oils obtained both a continuos industrial plant and a low-

scale mill, Eur. J. Lipid Sci. Technol. 107, 93-100.


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Di Giovacchino, L., Costantini, N., Ferrante, M.L., Serraiocco, A. (2002): Influence of malaxation

time of olive paste on oil extraction yields and chemical and organoleptic characteristics of

virgin olive oil obtained by a centrifugal decanter at water saving, Grasas Aceites 53, 179-186.

EC (1991): Commission Regulation (EEC) no. 2568/91 of 11 July 1991 on the characteristics of

olive oil and olive-residue oil and on the relevant methods of analysis, Official Journal, L248,

1-83.

Fedeli, E. (1996): Tecnologia di produzione e di conservazione dell’olio, Enciclopedia mondiale

dell’olivo, COI, Madrid, 253-294.

Gutfinger, T. (1981): Polyphenols in olive virgin oils, J. Am. Oil Chem. Soc. 58, 966-968.

Inarejos-García, A.M., Gómez-Rico, A., Desamparados Salvador, M., Fregapane, G. (2009):

Influence of malaxation conditions on virgin olive oil yield,overall quality and composition,

Eur. Food Res. Technol. 228, 671-677.

IOC (2007): Revised method for the organoleptic assessment of virgin olive oil, DEC-21/95-

V/2007.

Kalantzakis, G., Blekas, G., Pegklidou, K., Boskou, D. (2006) Stability and radical-scavenging

activity of heated olive oil and other vegetable oils. Eur. J. Lipid Sci. Technol. 108, 329-335.

Kalogeropoulos, N., Kaliora, A.C., Artemiou, Giogios, I. (2014): Composition, volatile profiles and

functional properties of virgin olive oils produced by two-phase vs three-phase centrifugal

decanters, LTW – Food Sci. Techn. 58 (1), 272-279.

Montedoro, G.F., Servili, M. (1992): I parametri di qualità dell’olio di oliva ed i fattori agronomici e

tecnologici che li condizionano, Riv. Ital. Sost. Grasse, 69, 563-573.

Ranalli, A., Contento, S., Schiavone, C., Simone, N. (2001): Malaxing temperature affects volatile

and phenol composition as well as other analytical features of virgin olive oil, Eur. J.

Lipid,Sci. Technol. 103, 228-238.

Rotondi, A., Bendini, A., Cerretani, L., Mari, M., Lercker, G., Gallina Toschi, T. (2004): Effect of

olive ripening degree on the oxidative stability and organoleptic properties of Nostrana di

Brisighella extra virgin olive oil, J. Agric. Food Chem. 52, 3649-3654.

Servilli, M., Selvaggini, R., Taticchi, A., Esposto, S., Montedoro, G.F. (2003): Volatile compounds

and phenolic composition of virgin olive oil – optimization of temperature and time of

exposure of olive pastes to air contact during the mechanical extraction process, J. Agric. Food

Chem. 51, 7980-7988.

Stefanoudaki, E., Koutsaftakis, J.A., Harwood, J.L. (2011): Influence of malaxation conditions on

characteristic qualities of olive oil, Food Chem. 127, 1481-1486.

Sekcija: Kemija u poljoprivredi i šumarstvu

Session: Chemistry in agriculture and forestry


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Kemija u poljoprivredi i šumarstvu / Chemistry in agriculture and forestry

325

The role of hydrogen sulfide in salt stress tolerance in plants

UDC: 576.34

Miroslav Lisjak1

, Vlatko Galić1, Bojan Fališevac

1, Marija Špoljarević

1, Mark E. Wood

2,

Matthew Whiteman3, Ian D. Wilson

4, John T. Hancock

4, Tihana Teklić

1

1Josip Juraj Strossmayer University of Osijek, Faculty of Agriculture, Petra Svačića 1d, HR-31000 Osijek,

Croatia 2University of Exeter, Biosciences, College of Life and Environmental Sciences, Stocker Road, EX4

4QD Exeter, UK 3University of Exeter, Medical School, Knowledge Spa, TR1 3HD Truro, UK

4University of the West of England, Faculty of Health and Applied Sciences, Coldharbour Lane,

BS16 1QY Bristol, UK

Summary

Hydrogen sulphide (H2S) is known to be produced by both animal and plant tissues suggesting that

this compound may be involved in cell signal transduction. There is a growing body of evidence to

suggest that the presence of this gaso-transmitter may modulate oxidative stress metabolism in cells.

In collaboration between English and Croatian Universities, it was found that pepper plants pre-

treated with H2S donors, such as NaSH and GYY4137, and exposed to the salt stress, showed

significant differences in lipid peroxidation levels, hydrogen peroxide and proline concentrations.

Cucumber seeds pre-treated with H2S donors and germinated in NaCl solution, showed a significant

influence of H2S treatments on the same parameters as in pepper. The results suggested that H2S

may have a regulatory role in stressed plants and may affect the accumulation of protective

metabolites such as proline. Crucial tools in this kind of research are H2S-releasing compounds with

different chemical properties, such as cell permeability or H2S releasing rate, along with in vivo

methods of detection of H2S using highly specific fluorescence probes. As well as for purely

academic reasons these compounds may have practical applications in agriculture through the

fostering of plant environmental stress tolerance.

Keywords: NaSH, GYY4137, salt stress

Introduction

It is well known that many reactive compounds are involved in the control of cellular

events both in animals and plants, such as reactive oxygen (ROS) and nitrogen species

(RNS). Research on both ROS and RNS has been striving to determine how such



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molecules are generated by plant cells, where they might move to and how a response is

generated by their presence (Wilson, Neill and Hancock, 2008; Hancock, 2009). Similar

issues are now being addressed with regards to H2S. In animals, H2S has been found to

have defined roles in signalling (Wang, 2003) and in the literature, it has been dubbed as

the third gasotransmitter (beside NO and CO) and therefore it is reasonable to suspect that

H2S has a similar role in plants, ie as an instrumental signalling molecule (Lisjak et al.,

2013). However, although considered as a signal in animals (Kimura, 2000; Wang, 2003)

hydrogen sulphide (H2S) should also to be counted in this group of small, sometimes

gaseous compounds that are used by plants to control their physiological and biochemical

activities. Previous research has shown that H2S influences on plant metabolism. In

general, H2S is known as a phytotoxin at higher concentrations (Koch et al., 1990; 2001;

Dorman et al., 2002) and considered as deleterious for plants, which can be exposed to H2S

in their environment. After the crucial tools and detection methods were developed,

scientist change the approach what leads to findings showing that lower concentrations of

H2S can act in more positive manner. Early work on several plant species, including maize,

pumpkin and spinach, focussed on H2S from the atmosphere and its effect on the

transpiration stream, and how it may alter stomatal apertures. It was suggested that in the

short term there was no effect (De Kok, Stahl & Rennenberg, 1989). However more recent

studies have taken advantage of the use of H2S donors such as sodium hydrosulphide

(NaSH) which generates a very short burst of H2S, or slow releasing donors such as

GYY4137 (morpholin- 4-ium 4 methoxyphenyl (morpholino) phosphinodithionate: Li et

al., 2008, 2009; Fox et al., 2012) which will give a lower and much more prolonged

concentration of H2S much more akin to that seen under physiological conditions. Such

research is revealing the signalling role for H2S in many species. In plants, cell signalling

driven by redox reactions is fundamental in stress perception, photosynthesis regulation,

pathogen defence responses, plant growth and developmental processes (Apel and Hirt,

2004; Gapper and Dolan, 2006.). All plants have ways of acclimating to stress through

physiological responses that modify their growth, development, and metabolism (Smith et

al., 2010). The most prevalent environmental stresses for plants are those that limit water

supply, and include drought, salinity and low temperature. Plant responses to these stresses

involve similar signalling mechanisms and metabolic responses.

It was shown recently that NaSH as H2S donor could alleviate the osmotic-induced

decrease in chlorophyll concentration in sweet potato (Zhang et al., 2009a), with an

increased antioxidative activity and simultaneously diminished hydrogen peroxide

concentration and lipoxygenase activity, what imply significant role in plant protection of

oxidative stress.

According to the known roles of H2S we assume that it affect metabolism in plants through

an increase of antioxidant activity in plants exposed to the low levels of salt, what often

happened in greenhouse growing. A better knowledge of physiological mechanisms


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involved in response to stress at the cellular and molecular level, may help to overcome the

problems encountered in the production of vegetables no matter of developmental stage.


In the first experiment, seeds of red pepper (Capsicum annuum L.), cultivar "Quadrato

d'Asti Rosso", were sown in Levington’s F2 compost in plant growth chambers till the 7 -

8 leaf stage. Light regime was set to 12-hours daylight, with a temperature of 27 ºC until

the germination stage. Then the temperature was lowered to 25 ºC till the first two leaves

were fully expanded. At this stage the light regime was set to 14 hours of full-day

lighting and 9 hours of total darkness. After this period till seven to eight leave stage, the

temperature regime was set at 22 ºC daytime temperature and to 18 ºC of night

temperature with the same light regime as in previous period. The experiment was set up

in three replications with 4 plants per each repetition. When the plants were 8 weeks old,

they were pre-treated with H2S donors having different chemical properties - synthetic

slow releasing donor GYY4137 and fast releasing donor NaSH. Pepper plants were pre-

treated with 200 μM of NaSH and GYY4137 for 72 h. Every 24 h each plant was

supplied with 20 mL of previously described solutions into the substrate and spraying the

leaves with 10 mL. Control plants were treated with the same volume of tap water. In the

next 3 days, with regular time intervals of 24 h, 20 mL of 50 mM of NaCl solution, was

applied into the substrate. Leaves were harvested at 8-9 weeks, grounded with liquid

nitrogen, frozen at -80 ºC and used for the analysis.

In the second experiment, cucumber seeds (Cucumis sativus L.) cultivar “Levina” from

three production years (2009/10, 2010/11 and 2011/12) has been soaked into 300 µmol

solution of NaSH and GYY4137 respectively, for 24 hours. After osmopriming, seed was

dried at room temperature for the next 24 hours. Fifty seeds per treatment were germinated

in growth chamber on filter paper, in the presence of 25 mM of NaCl or water as control,

with temperature set on 20 ºC. The experiment was set up in 4 replications with 50 seeds

per repetition. On the 8th

day hypocotyls of seedlings were sampled and used for the

analysis. TBARS levels (thiobarbituric acid-reactive substances) were evaluated using the

method of Heath and Packer (1968). Free proline (PRO) content was determined after

Bates et al. (1973). Total peroxides (HP) were analysed using method described by

Mukherjee i Choudhouri (1983).

The obtained results were analysed by the usual methods of statistical analysis using SAS

Software 9.1.3 (2002nd to 2003rd, SAS Institute Inc., Cary, USA) and Microsoft Office

Excel 2007. The following statistical methods were used: analysis of variance (ANOVA),

statistical tests of significance of the treatments applied - the F test and Fisher's LSD test

(least significant difference).


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In the experiment with cucumber seedlings, in average for all the examined seasons of seed

certification and both germination variants, osmopriming with H2S donors didn't influence

significantly on lipid peroxidation levels in hypocotyls (P=0.5652) (Table 2). In the first

experiment where pepper plants were pre-treated with H2S donors and watered with 50

mM NaCl solution, there was significant influence of salt stress (P<0.0001), H2S treatment

(HP P<0.0001; TBARS P=0.0018) and their interaction (HP P=0.0062; TBARS

P=0.0346) on mentioned parameters established (Table 1). In average for both the applied

H2S treatments, 50 mM of the applied NaCl salt didn't influence significantly on proline

level, while the influence of H2S donors and their interaction with salt stress was highly

significant (P<0.0001). Salt solution added into the substrate significantly increased

hydrogen peroxide content (Control 4.333; NaCl 7.792 nmol/g FW) and lipid peroxidation

levels (Control 0.133; NaCl 0.158 nmol/g FW) in pepper leaves. When analysed separately

by stress levels, H2S donors didn't influence on lipid peroxidation levels in control, what

implies that the application of both H2S donors in the concentration used, cannot

negatively influence on degradation of cell membranes (Fig. 1). In salt stress both H2S

donors diminished lipid peroxidation levels significantly as compared to the untreated

plants (control 0.18 nmol/g FW; GYY4137 0.16 nmol/g FW; NaSH (0.14 nmol/g FW).

Similar results were obtained in the research of H2S influence on plant response to Cu

toxicity (Zhang et al., 2008a) and Al toxicity in wheat (Zhang et al., 2010a), osmotic stress

in sweet potato (Zhang et al., 2009a) and drought stress in soybean (Zhang et al., 2010c).

Results suggest that H2S can protect membranes from degradation thus preserve integrity

of plant cells and organelles in salt stress conditions.

Table 1. Lipid peroxidation level (TBARS; nmol/g FW) total peroxide content (HP; nmol/g FW)

and proline content (PRO; µmol/g FW), in leaves of C. annuum (L.). A.B.C

significant

differences among means at P≤0.05 (LSD test)

FACTOR VARIANT TBARS HP PRO

SALT STRESS

(A)

Control 0.133B 4.333B 0.166

50 mM 0.158A 7.792A 0.176

F test

P

41.53

<0.0001

106.35

<0.0001

3.94

0.0704

PLANT TREATMENT

(B)

Control 0.156A 5.398B 0.212A

NaSH 0.133B 5.162B 0.146B

GYY4137 0.147A 7.628A 0.156B

F test

P

11.24

0.0018 21.95

<0.0001

57.67

<0.0001

A x B F test

P 4.51

0.0346 8.00

0.0062 35.87

<0.0001


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Fig. 1. Lipid peroxidation level in C. annuum (L.) plants pre-treated with H2S donors and exposed

to the salt stress. A.B.C

significant differences among means at P≤0.05 (LSD test)

Table 2. Lipid peroxidation level (TBARS; nmol/g FW) total peroxide content (HP; nmol/g FW)

and proline content (PRO; µmol/g FW), in hypocotils of C. sativus (L.). Factor A: season

of seed certification; Factor B: osmopriming with H2S donors and NaCl; Factor C:

germination in the presence of 25 mM of NaCl solution or tap water respectively. A.B.C


FACTOR VARIANT TBARS HP PRO

Year

(A)

2009/2010 6.40 B 0.672 A 3.52 C

2010/2011 4.95 C 0.138 B 7.86 B

2011/2012 7.23 A 0.145 B 9.33 A

F test P

34.80 <0.0001

256.37 <0.0001

171.61 <0.0001

Osmopriming

(B)

NaCl 6.03 0.322 AB 7.60 A

NaSH 6.33 0.281 B 6.66 B

GYY4137 6.22 0.351 A 6.45 B

F test

P

0.58

0.5652

3.43

0.0397

6.96

0.0020

Germination

(C)

Control 6.80 A 0.296 7.57 A

NaCl 5.59 B 0.338 6.24 B

F test

P

28.80

<0.0001

3.11

0.0834

24.73

<0.0001

A x B F test

P

3.05

0.0243

4.10

0.0057

2.44

0.0576

A x C F test

P 0.74

0.4830 0.28

0.7603 3.77

0.0294

B x C F test

P

6.97

0.0020

0.94

0.3985

1.18

0.3163

A x B x C F test

P

6.42

0.0003

3.61

0.0112

2.96

0.0277


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Christou et al. (2011) pre-treated strawberry with 10 mM NaSH in hydroponic solution and

exposed to salt, osmotic and heat stress. With H2S pre-treatment, they recorded increased

stomatal conductivity, higher relative water content in leaf, lower ion leakage and lipid

peroxidation rate. The authors concluded that H2S decreased oxidative and nitrosative

stress in plants, through the reduced de novo synthesis of NO and H2O2, as well as

maintenance of ascorbate and glutathione in high redox state. H2O2 has also been shown to

act as a key regulator in a broad range of physiological processes such as senescence (Peng

et al., 2005), photorespiration and photosynthesis (Noctor and Foyer, 1998a), stomatal

movement (Bright et al., 2006), cell cycle (Mittler et al., 2004), and growth and

development in general (Foreman et al., 2003). To some extent, excessive H2O2

accumulation can lead to oxidative stress in plants, which then triggers cell death.

In our research with pepper, GYY4137 applied on plants under salt stress, significantly

increased the accumulation of hydrogen peroxide (10.28 nmol/g FW) in comparison to the

NaSH treatment (6.65 nmol/g FW) and control (6.46 nmol/g FW) (Fig. 2). Similar effect of

GYY4137 obtained in experiment with cucumber seedlings where the highest hydrogen

peroxide accumulation was observed in before mentioned treatment (GYY4137 0.351;

NaSH 0.281; NaCl 0.322 nmol/g FW) (Table 2). Such an effect of GYY4137 could be

assigned to different chemical properties in comparison to NaSH. H2S burst from NaSH

solution is very fast thus loses due to the volatilization are high, what means that much less

of H2S might enter the plant cell. Since water solution of GYY4137 release H2S slow and

in prolonged manner (Lee et al., 2011), cells are constantly exposed to the much higher

concentration of H2S in total, which may be even toxic, what could increase the production

of hydrogen peroxide in cells or opposite, could slow down its degradation through the

inhibition or lowering the synthesis of catalase.

Fig. 2. Total peroxide content in C. annuum (L.) plants pre-treated with H2S donors and exposed to

the salt stress. A.B.C



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Free proline content in plant tissues is widely considered as an indicator of plant stress

responses at the metabolic level (Kavi Kishor et al., 2005; Ashraf and Foolad, 2007; Kaul

et al., 2008). Sugar and proline accumulation in roots and polyamines in leaves of pepper

exposed to the drought stress, were reported in the research of Sziderics et al. (2010).

As opposite to the hydrogen peroxide content, proline concentrations in leaves of salt

stressed plants were significantly lower in treatments with both H2S donors (NaSH 0.17;

GYY4137 0.13 µmol/g FW), as compared to the control (0.23 µmol/g FW) (Fig. 3). As an

important osmolite increasingly accumulated during stress conditions, free proline could

preserve the integrity of many functional proteins such as nitrate reductase, lactate

dehydrogenase, further enhanced the activity of antioxidative enzymes such as catalase,

superoxide dismutase and enzymes involved into the ascorbate-glutathione cycle

(Szabados and Savouré, 2010). Moreover, it has been suggested as compound with

antioxidative role in cells with ROS scavenging possibilities. Research of Hare et al.

(1999) on A. thalina Atp5cs1 mutant, hypersensitive to salt stress with decreased

possibility for proline synthesis, showed low activity of enzymes involved in ascorbate-

glutathione cycle, followed by hyper-accumulation of hydrogen peroxide. In the

experiment with cucumber seed reported here, in average for all the examined season of

seed certification and both the stress variants in germination, osmopriming of seed with

H2S donors diminished proline accumulation (NaSH 6.66; GYY4137 6.45 µmol/g FW) in

hypocotyls (Table 2). In the research with pepper, the enhanced concentration of hydrogen

peroxide in leaves of salt stressed plants pre-treated with GYY4137 was followed by a

decrease of free proline concentration. Again, it could be because of too high concentration

of GYY4137 used, hence the toxic effect of H2S in interaction with salt stress.

Fig. 3. Proline content in C. annuum (L.) plants pre-treated with H2S donors and exposed to the salt

stress. A.B.C



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The obtained results show that H2S could affect and modulate plant cell metabolism in salt

stress conditions. Despite researches continue to gain new knowledge on H2S roles in

plants, the exact nature of how and where it fits into the metabolic pathways and interacts

with other signals, has yet to be established. Thus, crucial tools in this kind of research are

H2S-releasing compounds with different chemical properties, such as cell permeability or

H2S releasing rate, along with in vivo methods of detection of H2S using highly specific

fluorescence probes.

Conclusions

H2S may have a regulatory role in stressed plants and may affect the accumulation of

protective metabolites such as proline, therefore it could alleviate negative effect of salt

stress through the preservation of cell membranes integrity. It is obvious as well that the

effect of H2S on cell physiological processes depends on how long and in which

concentration it is derived to the cells. As well as for purely academic reasons, H2S

releasing compounds may have also the practical applications in plant production, with aim

of improving plant environmental stress tolerance.

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(2010): Organ - specific defense strategies of pepper (Capsicum annuum L.) during early

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Wang, R. (2003): The gasotransmitter role of hydrogen sulfide, Antioxidants and Redox Signaling 5,

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Digitalna SMS vaga kao suvremeni alat na pčelinjaku

UDC: 638.14 : 681.26

Zlatko Puškadija1

, Marin Kovačić1, Željko Kraljičak

2, Silva Wendling

2,

Dinko Jelkić1, Nebojša Nedić

3, Ivan Pihler

4

1Sveučilište J.J. Strossmayera u Osijeku, Poljoprivredni fakultet, Kralja Petra Svačića 1d,

31000 Osijek, Hrvatska 2Osječko-baranjska županija, Trg Ante Starčevića 1, 31000 Osijek, Hrvatska

3Poljoprivredni fakultet, Univerzitet u Beogradu, Nemanjina 6, 11080 Beograd, Srbija

4Poljoprivredni fakultet, Univerzitet u Novom Sadu, Trg D. Obradovića 8, 21000 Novi Sad, Srbija

Sažetak

Moderno pčelarstvo u svojoj osnovi podrazumijeva selidbu košnica radi boljeg iskorištavanja

medonosne paše. Digitalna sms vaga smanjuje troškove obilaska pčelinjaka na minimum i

istovremeno daje pčelaru čitav niz podataka koji mu pomažu u donošenju odluka važnih za

tehnološki proces proizvodnje meda i drugih pčelinjih proizvoda. Pčelaru vaga daje podatke o

promjeni u masi košnice, kao i osnovne meteorološke podatke izmjerene u košnici i u njenom

okolišu. Promjene u masi košnice pčelaru daju informacije u tome je li počelo ili završilo medenje i

kada je trenutak da se košnice presele na drugu pašu. Nadalje, pčelar dobiva informaciju o snazi

pčelinjih zajednica koje seli i na osnovu toga može prognozirati uspjeh koji će uslijediti kod vrcanja

meda i prema potrebi mijenjati tehnologiju na pčelinjim zajednicama. Na osnovi dobivenih

meteoroloških podataka pčelar može donijeti odluku o odlasku na pčelinjak ukoliko su oni povoljni.

Tijekom zimovanja pčelinje zajednice unutar pčelinjeg klupka i u košnici instalirani su senzori za

temperaturu i relativnu vlažnost zraka. U radu su prikazani konkretni primjeri uporabe vaga. U

sklopu IPA projekta „Panonian Bee“ prekogranične suradnje Hrvatska-Srbija, kreirana je mreža od

20 digitalnih sms vaga s kojih se šalju podaci na posebno dizajniranu web stranicu.

Ključne riječi:pčelarstvo, monitoring medenja, vaga, med

Uvod

Vrijeme izlučivanja nektara, kao i količina nektara, koju će određena medonosna biljka

izlučiti, te količina meda koju će neka pčelinja zajednica proizvesti u velikoj su ovisnosti o

vremenskim prilikama. Kako bi proizveli što veće količine meda pčelari su do sada morali

angažirati značajan dio svoje pčelarske infrastrukture i velik broj radnih sati. Oni su

postavljali testne košnice na lokacije na kojima predviđaju da će neka biljka zamediti i

redovito ih obilazili. Ovakva aktivnost, danas u vrijeme skupih energenata, znatno

opterećuje proizvođački cijenu meda.



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Digitalna sms vaga suvremeni je alat u pčelarstvu koji značajno pojeftinjuje pčelarsku

proizvodnju, omogućava brzo uočavanje tehnoloških propusta na pčelinjim zajednicama i

na taj način čini pčelara konkurentnijim na tržištu pčelinjih proizvoda, kako kvantitetom

tako i kvalitetom. Program mjerenja promjena u masi košnica osigurava pčelaru uvid u

kondiciju i aktivnosti pčelinje zajednice, vrijeme početka i završetka pčelinje paše,

proljetni temperaturni skok, te meteorološke prilike. Dakle, digitalna sms vaga ne registrira

samo promjenu mase pčelinje zajednice. Ona je suvremeni alat u pčelarskoj proizvodnji

koji pčelaru daje vidljiv, kako trenutni, tako i dinamični uvid u posljedice koje su proizašle

iz interakcije pčelinje zajednice i okoliša.

U ovom će se radu predstaviti rezultati IPA projekta „Panonian Bee“ prekogranične

suradnje Hrvatska-Srbija. Na području Osječko-baranjske županije kreirana je mreža od 20

digitalnih sms vaga. Vage su bile raspoređene po cijelom području županije pazeći pri tom

da se pokriju svi lokaliteti na kojima se nalaze značajnije medonosne biljne vrste (različite

voćne vrste, uljana repica, bagrem, lipa, suncokret, zlatošipka). Razvijen je jedinstveni

software i dizajnirana posebna web stranica na kojoj su rezultati mjerenja prikazani

tablično i grafički, kako bi bili razumljivi korisnicima. Podaci su javni i svima dostupni, a

u radu će biti prikazani do sada dobiveni rezultati.

Materijali i metode

Dvadeset je digitalnih sms vage raspoređeno po cijeloj Osječko-baranjskoj županiji, vodeći

računa o ravnomjernoj pokrivenosti županije obzirom na značajne medonosne lokalitete.

Pčelari na čije su se pčelinjake instalirale vage morali su biti iskusni pčelari koji pčelare s

minimalno 50 pčelinjih zajednica stacionarno. Izvršena je njihova obuka, a sama provedba

projekta počela je krajem travnja.

Vage su programirane tako da se dnevno šalju informacije o četiri mjerenja na mobilni

telefon pčelara, kao i na server u 7:00, 11:00, 19:00 i 21:00 sat (Slika 1).

Slika 1. Shematski prikaz dnevnih odvaga digitalne sms vage


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Odvagu „A“ možemo nazvati i „brojač pčela“. Naime, u ranim jutarnjim satima pčele

napuštaju košnicu kako bi donijele nektar, pelud, vodu i smolaste i balzamske tvari. U

pčelinjoj zajednici 1/3 pčela čine pčele skupljačice, a masa jedne pčele iznosi 0,1g (1kg

pčela = 10 000 pčela) i te podatke uvrstimo u formulu: masa košnice u 11:00 – masa u 7:00

(kg) x 10000 = broj pčela sakupljačica.

Odvaga „B“ je razlika u masi košnice od 19:00-11:00 sati i predstavlja ukupni dnevni unos

nektara.

Odvaga „C“ predstavlja razliku u masi košnice između 21:00 sati i 7:00 sati i predstavlja

ukupni dnevni unos jer se do tada u košnicu vrate sve pčele.

Ukupan dnevni unos nektara (meda) predstavlja razliku mase košnice u 7:00 sati tekućeg

dana i 7:00 jučerašnjeg dana.

Temperaturu i relativnu vlažnost zraka unutar košnice i unutar pčelinjeg klupka (u

zimskom razdoblju), kao i temperaturu zraka i relativnu vlagu zraka mjerili smo senzorima

koji su spojeni s elektronikom vage i koji su pozicionirani u košnici i izvan nje.

Sve informacije navedenih parametara registrirane vagom kontinuirano su slane na mobilne

telefone pčelara, kao i na server, tj. na adresu: http://panonianbee.dyndns.org:8888/$/.


Prema Lodesani i sur. (2013) prostorna raspoređenost vaga morala je zadovoljiti kriterij

ravnomjerne pokrivenosti, kao i pokrivenost lokaliteta na kojima se nalaze glavne medonosne

paše. Stoga smo i mi te kriterije primijenili u Osječko-baranjskoj županiji (Slika 2).

Slika 2. Prostorna pokrivenost vagama na području Osječko-baranjske županije


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Web sučelje dizajnirano je na način kako bi se korisnici što lakše snalazili. Stoga su

utvrđene vrijednosti promatranih parametara prikazane grafički, kako bi se mogao bolje

uočiti trend, te tablično zbog lakše pohrane brojnih podataka (Slika 3).

Slika 3. Prikaz podataka na web stranici

Odvaga A, koju smo nazvali i „brojač pčela“ pokazala se vrlo značajnim alatom u praćenju

snage pčelinjih zajednica na osnovu kojeg pčelar može vrlo brzo pristupiti i eventualnom

korigiranju primijenjene tehnologije na zajednicama ukoliko su mjerenja pokazala kako su

one oslabile. Robin (2005) u svojim istraživanjima za područje Njemačke i Francuske

preporučuje da se drugo mjerenje obavi u 11:00 sati. Za razliku od navedenog autora, naša

mjerenja u proteklom razdoblju, ukazala su na potrebu za korigiranjem ovog parametra.

Naime, zato što smo pozicionirani južnije i istočnije od gore navedenih regija, potrebno je

mjerenje u 11:00 sati pomaknuti za 90 minuta ranije te ovo mjerenje obaviti u 9:30. Prema

sadašnjim mjerenjima pokazalo se da se određen broj pčela vrati u košnicu do

preporučenih 11:00 sati. Ostala mjerenja, na osnovu provedenih istraživanja, mogu se

koristiti i kod nas kako to preporučuje Robin (2005) te Szabo i Mueller (1996).

Uvidom u bazu podataka možemo vidjeti kako je ove godine minimalni značajni rast masa

košnica na vagama bio samo u razdoblju od 2.7. do 17.7. u vrijeme kada je suncokret

(Helianthus annuus L.) bio u fenofazi cvatnje (Slika 3). Cvatnja medonosnog bilja je zbog

izuzetno loših meteoroloških prilika ove godine bila vrlo otežana te su izostala brojna

mjerenja koja smo očekivali.


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Zaključci

Digitalna sms vaga se tijekom pčelarske sezone 2014. pokazala izuzetno korisnim alatom na

pčelinjaku. Provedena mjerenja pokazala su u kojem je dijelu potrebno prilagoditi rad vage

sukladno našem podneblju, te nam dala brojne potrebne podatke na osnovu kojih su pčelari

Osječko-baranjske županije mogli donijeti kvalitetne tehnološke odluke na pčelinjacima.

Instalirani sustav 20 digitalnih sms vaga na području Osječko-baranjske županije pokazao se

jako upotrebljivim kod donošenja odluka o početku i kraju medonosne paše, tj. o trenutku

selidbe košnica na pašu i s paše. Sve prednosti sustava umreženih vaga znatno su smanjile

troškove obilaska pčelinjaka, što pčelare čini konkurentnijim na tržištu meda.

Kako je pčelarska sezona 2014. bila izuzetno loša, a zimska mjerenja nam tek predstoje

očekujemo kako će sve prednosti ovog sustava umreženih digitalnih sms vaga tek doći do

izražaja.

Literatura

Abrams, G. J. (1957): Beekeeping in Maryland, Gleanings in Bee Culture 85, 34-35

Lodesani, M., Mutinelli, F., Liberta, A., Porrini, C. (2013): The national bee monitoring network in

Italy (2009-2013), Bulletin of Insectology 66 (1), 160

Robin, H. (2005): BeeWise remote monitoring of beehive activity [online], BeeWise. Dostupno na:

http://www.beewise.eu/bee-hive/beewise_hives_monitoring_gb_v1.0.pdf [01.09.2014.]

Szabo, T. I., Mueller, A. E. (1996): Factors affecting the weight changes of honey bee colonies,

American Bee Journal 136, 417-419.


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Digital SMS scale as a modern tool on apiary

Zlatko Puškadija1, Marin Kovačić

1, Željko Kraljičak

2, Silva Wendling

2,

Dinko Jelkić1, Nebojša Nedić

3, Ivan Pihler

4

1Josip Juraj Strossmayer University of Osijek, Faculty of agriculture in Osijek, Kralja Petra Svačića 1d,

HR-31000 Osijek, Croatia 2Osijek-Baranja County, Trg Ante Starčevića 1, HR-31000 Osijek, Croatia

3Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Beograd, Serbia

4Faculty of Agriculture, University of Novi Sad, Trg D. Obradovića 8, 21000 Novi Sad, Serbia

Summary

Modern beekeeping basically implies moving hives in order to better exploit the honey pasture.

Digital sms scales reduces costs of apiary tour to a minimum and also gives to the beekeeper whole

range of data to facilitate the decision-making process relevant to the technological process of

production of honey and other bee products. Scales provides to the beekeeper information on the

change in mass of the hive, as well as basic meteorological data measured in the hive and in its

environment. Changes in weight of the hive provide for beekeeper information as to whether

commenced or completed nectar flow and when it's time to move the hive to another pasture.

Furthermore, the beekeeper gets information about the strength of bee colonies and with that

information one can predict the success honey production and modify his farming technology. On

the basis of meteorological data obtained beekeeper can make a decision about going to the apiary if

they are favorable. During wintering of bee colonies within the cluster of bees in the hive are

installed sensors for temperature and humidity. As part of the IPA project "Panonian Bee" cross-

border cooperation Croatia-Serbia, a network of 20 digital sms scales is created which send their

data to a specially designed website.

Keywords: beekeeping, nectar flow monitoring, scale, honey


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Primjena polietilen glikola-6000 u istraživanju osmotskog stresa

kod soje (Glycine max (L.) Merr.)

UDC: 633.34 : 678.742.2

Marija Špoljarević1

, Ana Mihaljević1, Ivna Štolfa

2, Dejan Agić

1,

Rosemary Vuković2, Miroslav Lisjak

1, Tihana Teklić

1

1Sveučilište Josipa Jurja Strossmayera u Osijeku, Poljoprivredni fakultet, Kralja Petra Svačića 1d,

31000 Osijek, Hrvatska 2Sveučilište Josipa Jurja Strossmayera u Osijeku, Odjel za biologiju, Ulica Cara Hadrijana 8a,

31000 Osijek, Hrvatska

Sažetak

Polietilen glikol (PEG) se dobiva polimerizacijom etilen oksida (EO) s vodom, monoetilen glikolom

ili dietilen glikolom uz alkalnu katalizu. Produkt je molekula jednostavne kemijske strukture:

HO-[CH2-CH2-O]n-H, gdje je (n) broj EO jedinica. Najznačajnije svojstvo svih PEG-a je topljivost

u vodi, što ih čini prikladnim za bezbrojne različite aplikacije. PEG-i se često primjenjuju u

istraživanjima odnosa biljaka prema vodi, osobito za simulaciju sušnih uvjeta (osmotski stres).

Osmotski potencijal vodenih otopina PEG-a 6000 je u vezi s njegovom koncentracijom. U ovom

radu su prikazani rezultati fiziološkog odgovora sjemena soje uvjetovan osmotskim stresom

primjenom PEG-a u fazi klijanja. Uz vodu kao kontrolu, primijenjene su dvije razine osmotskog

stresa - 5% (-0,5 bara) i 10% (-1,48 bara) otopina PEG 6000 kao medij za klijanje sjemena. Sadržaj

slobodnog prolina, ukupnih fenola i intenzitet lipidne peroksidacije tkivu hipokotila klijanaca soje

se povećao pri višoj koncentraciji otopine PEG 6000 (jači intenzitet osmotskog stresa). Budući da su

se ispitivani kultivari soje razlikovali u pojedinim parametrima metaboličkog odgovora, PEG se

može smatrati pogodnim sredstvom za simulaciju osmotskog stresa.

Ključne riječi: soja, osmotski stres, abiotski stres, PEG 6000

Uvod

Sve izraženije klimatske promjene na Zemlji utječu na dostupnost vode u okolišu što se

odražava na poljoprivredu, proizvodnju hrane i naposljetku na zdravlje ljudi. Poznato je

kako nedostatak vode uslijed prevelike zaslanjenosti tla, ekstremnih temperatura, slabih

padalina ili nemogućnosti navodnjavanja i odvodnje utječu na rast i razvoj biljke.

Optimalne količine vode za biljku su u ovisnosti sa njenim dobrim fiziološkim statusom a

samim tim i zadovoljavajućim prinosom. Nedostatak vode u većem i manjem intenzitetu



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utječe na rast i razvoj biljaka i može uzrokovati pojavu slobodnih kisikovih radikala

(oksidacijski stres) uslijed osmotskog ili sušnog stresa već u fazi klijanja. Kao odgovor

biljke na oksidacijski stres izazvan nedostatkom vode pokreće se niz antioksidacijskih

mehanizama na staničnoj i molekularnoj razini koji omogućuju preživljavanje biljke

(Wang i sur., 2003; Seki i sur., 2005; Yamada i sur., 2006). U slučaju dugotrajnog ili

intenzivnog stresa uslijedit će venuće biljke uz smanjenje prinosa. Istraživanje utjecaja

osmotskog stresa na biljku od iznimnog je značaja, stoga je mogućnost simuliranja takvih

uvjeta u laboratoriju znanstvenicima vrlo interesantna. U tu svrhu tijekom 80-tih godina

započela je intenzivna primjena polietilen glikola (PEG) visokih molekulskih masa (4000-

20000) (Ben-Hayyim, 1987; Zahran i Sprent, 1986; Iraki i sur., 1989; Stout i sur., 1980). U

ovisnosti o dužini lanca i molekulskoj masi sintetizira se PEG 200, 300, 400, 600, 1000,

1500, 2000, 3000, 4000, 6000, 10000 (Ugela, 1998). Za simulaciju osmotskog stresa u

biljkama koristi se PEG visoke molekulske mase jer ne prodire u stanicu a snižava vodni

potencijal na način sličan isušenom tlu (Larher i sur., 1993). PEG 6000 je neionska

polihidroksilna tvar dobro topiva u vodi, čiji se osmotski tlak otopine može unaprijed

definirati (Michel i Kaufmann, 1973), te time odrediti intenzitet stresa za biljku.

Osim ekoloških čimbenika na rast i razvoj kulturnih biljaka poput soje značajno utječe

genotip, u svim pojedinim fazama razvoja, a razina osjetljivosti na uzgojne uvjete često

varira između genotipova. Pri nepovoljnim okolišnim uvjetima do izražaja dolazi genetski

potencijal kultivara za obranu od osmotskog stresa, što ima veliki značaj u stvaranju novih

i poboljšanih kultivara, kao glavni cilj oplemenjivačkog procesa. Cilj ovog istraživanja je

bio utvrditi utjecaj različitih koncentracija polietilen glikola 6000 na šest kultivara soje te

može li se PEG 6000 koristiti kao simulator osmotskog stresa u fazi klijanja.

Materijal i metode

U istraživanjima je korišteno šest kultivara soje (Ika, Lucija, Vita, Tena, Korana i Zora)

proizvedenih na Poljoprivrednom institutu u Osijeku. Sjeme je naklijavano na filter papiru u

klima komori tijekom 7 dana pri 20 ºC u uvjetima tame. Za kontrolu je korištena voda iz

slavine kao medij za imbibiciju sjemena, a 5%-tna (-0,5 bara; -0,05 MPa) i 10%-tna (-1,48

bara; -0,148 MPa) otopina PEG-a kao sredstvo za postizanje niže i više razine osmotskog

stresa. U svježem tkivu hipokotila klijanaca analizirani su sadržaj slobodnog prolina (PRO),

ukupnih fenola (PHE), vodikovog peroksida (HP) te intenzitet lipidne peroksidacije

(TBARS), kao neenzimski pokazatelji antioksidativnog stanja biljke. Sadržaj slobodnog

prolina analiziran je metodom prema Bates i sur. (1973) iz ekstrakta svježeg tkiva

homogeniziranog tekućim dušikom i ekstrahiranog u 3% sulfosalicilnoj kiselini. Prolin je

izmjeren spektrofotometrom pri valnoj duljini od 520 nm a vrijednosti su izračunate prema

standardnoj krivulji prolina te izražene u µmol PRO/g svježe tvari. Vodikov peroksid je

ekstrahiran i analiziran prema metodi MacNevin i Uron (1953) uz manje modifikacije


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(Mondal i Choudhuri, 1981). Tkivo soje je homogenizirano i ekstrahirano u hladnom

acetonu. Ekstraktu je dodan titanov oksisulfat u sulfatnoj kiselini i koncentrirani NH4OH.

Nastali talog je otopljen u 2M H2SO4 i mjeren spektrofotometrijski na 214 nm. Sadržaj H2O2

izračunat je iz standardne krivulje (Mukherjee i Choudhuri, 1983) i izražen kao nmol/g

svježe tvari. Koncentracija produkata lipidne peroksidacije određena je kao količina

supstanci koje reagiraju s tiobarbiturnom kiselinom (engl. thiobarbituric acid reactive

substances – TBARS; Heath i Packer, 1968). Svježe tkivo hipokotila usitnjeno je tekućim

dušikom a produkti TBARS ekstrahirani su 0,1% trikloroctenom kiselinom. Nakon

ekstrakcije suvišak tkiva odvojen je centrifugiranjem na 4 °C pri 6000g tijekom 5 min.

Supernatantu je dodana smjesa 0,5%-tne tiobarbiturne kiseline u 20% trikloroctenoj kiselini,

te je takva smjesa zagrijavana 30 minuta u vodenoj kupelji na 95 °C. Nakon hlađenja,

apsorpcija reakcijske smjese je očitana spektrofotometrijski na valnim duljinama od 532 i

600 nm. Koncentracija produkata TBARS je izračunata koristeći ekstinkcijski faktor 155

m/Mcm i izražena kao nmol/g svježe tvari. Sadržaj ukupnih fenola određen je metodom po

Folin-Ciocalteau-u (Singleton i Rossi, 1965). Fenoli su ekstrahirani iz maceriranog tkiva s

etanolom na -20 °C kroz 48 h. Ekstrakt je razrijeđen vodom, dodan je Folin-Ciocalteau

reagens te zasićena otopina Na2CO3 nakon čega se smjesa inkubirala tijekom 60 minuta na

37 ºC. Nakon inkubacije, apsorpcija je mjerena spektrofotometrijski na 765 nm. Iz

standardne krivulje galne kiseline izračunat je sadržaj ukupnih fenola, te je izražen u

ekvivalentima galne kiseline (mg GA/g svježe tvari).

Pokus je postavljen u četiri ponavljanja, uz osmotski stres kao glavni faktor i različiti

sortiment kao podfaktor. Utvrđeni rezultati su analizirani uobičajenim metodama statistike

obrade podataka pomoću SAS Softwere 9.1.3, programske podrške (2002-2003, SAS

Institute INC., Cary, USA).

Korištene su sljedeće statističke metode: analiza varijance (engl. analysis of variance -

ANOVA), statistički testovi značajnosti utjecaja primijenjenih tretmana - F test i Fisher`s

LSD test P≤0.05 (engl. last significant difference) te multipla regresijska i linearna

korelacijska analiza.


Osmotski potencijal vodene otopine polietilen glikola 6000 ovisi o njegovoj koncentraciji

(Michel i Kaufmann, 1973). U istraživanjima utjecaja sušnog stresa na biljke gdje se

primjenjuje PEG, veća koncentracija PEG-a onemogućuje biljkama usvajanje dovoljne

količine vode čime dolazi do nastanka vrlo reaktivnih slobodnih kisikovih radikala (ROS;

npr. vodikov peroksid, superoksidni radikal, hidroksilni radikal itd.). Nedostatak vode, kao

i neki drugi oblici abiotskog stresa mogu se poistovjetiti s osmotskim stresom, kao

direktnim pokretačem pojačane pojave slobodnih radikala. ROS svojim djelovanjem lako

stupaju u interakcije s funkcionalnim molekulama u stanici (DNA, ugljikohidrati, proteini,


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lipidi) te mogu oštetiti različite stanične komponente. Tijekom evolucije, biljke su razvile

kompleksan sustav antioksidacijskog odgovora koji čine antioksidativni enzimi (npr.

katalaza, superoksid dismutaza i dr.) te neenzimatski antioksidansi (npr. različiti spojevi iz

skupine fenola, askorbinska kiselina; Ashraf, 2009; Noctor i Foyer, 1998).

U ovom istraživanju je ocijenjen utjecaj kultivara, tretmana s PEG-om i njihove interakcije

na neenzimske pokazatelje reakcije biljke na osmotski (sušni) stres u razvojnom stadiju

nicanja šest kultivara soje. Sadržaj vodikovog peroksida nije bio pod pojedinačnim

utjecajem kultivara i tretmana ali je bio pod značajnim utjecajem njihove interakcije

(P=0,0406). Analizom pojedinih tretmana na sadržaj peroksida u hipokotilu utjecaj sorte

nije bio značajan na sadržaj HP-a u kontroli i pri tretmanu 5% PEG-om, dok je pri

tretmanu 10% PEG-om bio vrlo značajan (P=0,0111). U varijanti s višom razinom stresa,

kultivar Lucija je imao najveći sadržaj HP-a (0,21 nmol/g sv.t.) te se statistički značajno

razlikovao od ostalih kultivara pri LSD testu P≤0,05. Najmanji sadržaj HP-a, pri navedenoj

razini stresa, imala je Zora (0,15 nmol/g sv.t.). Analizirajući pojedine tretmane uočljivo je

da jedino je kultivar Tena imao značajan utjecaj na sadržaj HP-a te je najveći sadržaj

utvrđen pri tretmanu 5% PEG-om (0,191 nmol/g sv.t.), koji se nije značajno razlikovao od

sadržaja pri tretmanu 10% PEG-om. Shehab i sur. (2010), navode da su u istraživanju

sušnog stresa u biljkama riže (Oryza sativa L.), uvjetovanog različitim koncentracijama

PEG-a (5%, 10%, 15%, 20%) uočili povećanje sadržaja peroksida u usporedbi s

kontrolom. Nizak sadržaj HP-a, u našem istraživanju, pri nižoj i višoj razini stresa mogući

je indikator aktivacije antioksidativnih mehanizma obrane od oksidacijskog stresa. H2O2 se

iz stanice uklanja različitim mehanizmima: enzimskim i neenzimskim (Neill i sur., 2001).

Katalaza je enzim koji ima visoki kapacitet ali mali afinitet za neutraliziranje molekula

H2O2 (Smirnoff, 1998), dok antioksidansi kao što su askorbat i glutation određuju životni

vijek H2O2 unutar stanice (Veljović-Jovanović i sur., 2002).

Na intenzitet lipidne peroksidacije (TBARS) su vrlo značajno utjecali tretman, kultivar te

njihova interakcija (P<0,0001). Obzirom na razinu stresa, u prosjeku za sve kultivare, sve

razine su se međusobno vrlo razlikovale u ovom pokazatelju. Najjači intenzitet lipidne

peroksidacije uočen je pri tretmanu 10% PEG-om a najniži je očekivano bio u kontroli

(Slika 1). Sličan rezultat sa sjemenom riže dobivaju Shehab i sur. (2010), koji navode da je

intenzitet lipidne peroksidacije u tretmanu s PEG-om bio značajno jači u usporedbi s

kontrolom. Između ispitivanih kultivara najveći TBARS, u prosjeku za sve tretmane, imala

je Korana (33,59 nmol/g sv.t.), ali se nije značajno razlikovala od Vite. Najmanji TBARS

imao je kultivar Zora (28,58 nmol/g sv.t.), bez značajne razlike u odnosu na Tenu i Iku.

Türkan i sur. (2005.) u svojim istraživanjima sa dva kultivara graha (jedan otporan a drugi

osjetljiv na sušni stres) zabilježili su značajno niže vrijednosti lipidne peroksidacije u

kontroli u odnosu na tretman 10% PEG-om, što potvrđuju i naši rezultati. Autori također

navode kako je intenzitet lipidne peroksidacije rastao u odnosu na duži period izloženosti

stresu, te da postoje značajne razlike između genotipova istraživanih kultivara graha. U


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ovom istraživanju, primjena PEG-a je imala vrlo značajan utjecaj na intenzitet lipidne

peroksidacije kod svih ispitivanih kultivara soje. Oksidacijom i enzimskom razgradnjom

višestruko nezasićenih masnih kiselina u stanicama kao produkt nastaju molekule

malondialdehida (MDA), koji se smatra dobrim indeksom intenziteta lipidne peroksidacije

(Hodges i sur., 1999). Sjeme soje zbog visokog sadržaja lipida i proteina sklono je

oksidativnom stresu (Lisjak i sur. 2009). Uklanjanjem vode iz membrana stanica i

organela, remeti se normalna dvoslojna struktura što rezultira time da membrana postaje

iznimno porozna (Mahajan i Tuteja, 2005). Tretman 10% PEG-om 6000 je vjerojatno

utjecao na poroznost i strukturu staničnih membrana klijanaca soje što se primjećuje u

povećanoj koncentraciji MDA u odnosu na kontrolu. Utjecaj sorte na TBARS bio je vrlo

značajan u kontroli (P=0,0058) i pri tretmanu 10% PEG-om (P<0,0001). Obzirom da je

soja biljka uljarica za očekivati je kako će genotip biljke imati utjecaj na intenzitet lipidne

peroksidacije, pri različitim tretmanima ispitivanog stresa, možda čak i veći u odnosu na

neku drugu biljnu vrstu koja ima manji sadržaj ulja u sjemenu.

Slika 1. Intenzitet lipidne peroksidacije (TBARS) u hipokotilu u prosjeku za sve kultivare

po razinama osmotskog stresa (XYZ – razlike između tretmana;

LSD test P≤0,05; MDA - malondialdehida)

Fig. 1. Lipid peroxidation intensity (TBARS) in hypocotyl in average for all cultivars

according to osmotic stress levels (XYZ – differences between treatments;

LSD test P≤0.05; MDA - malondialdehyde)

Na sadržaj fenola u tkivu hipokotila, tretman, kultivar i njihova interakcija imali su vrlo

značajan utjecaj. Najveći sadržaj fenola, u prosjeku za sve sorte, uočen je na tretmanu 10%

PEG-om a najmanji u kontroli (Slika 2). U prosjeku za sve tretmane najveći sadržaj ukupnih

fenola imale su Lucija i Tena (0,44 mg GA/g sv.t.) a najmanji Vita (0,37 mg GA/g sv.t.).

Kultivar je imao značajan utjecaj na sadržaj PHE u kontroli (P<0,0001) i pri tretmanu

5% PEG-om (P=0,001), no u tretmanu s 10% PEG-om bio statistički značajan činitelj.


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Utjecaj 10% PEG-a na sadržaj PHE je bio intenzivan u tolikoj mjeri da kod svih ispitivanih

kultivara postoji značajna razlika u odnosu na tretman 5% PEG-om i kontrolu. Kod Lucije,

Vite i Ike uočena je značajna razlika u sadržaju PHE između kontrole i tretmana 5% PEG-om.

Rezultate slične našima dobili su i Shehab i sur. (2010), koji su uočili akumulaciju ukupnih

fenola prilikom sve jačeg intenziteta osmotskog stresa. Bellaloui (2012.) ističe kako sušni

stres utječe na porast koncentracije fenola i lignina u biljkama soje u odnosu na kontrolu, u

fazi cvatnje i fazi razvoja sjemena.

Slika 2. Sadržaj ukupnih fenola u hipokotilu u prosjeku za sve kultivare po razinama osmotskog

stresa (XYZ – razlike između tretmana; LSD test P≤0,05; GA - galna kiselina)

Fig. 2. Total phenol content in hypocotyl in average for all cultivars according to osmotic stress

levels (XYZ – differences between treatments; LSD test P≤0.05; GA - gallic acid)

Na sadržaj prolina u hipokotilu stres, kultivar i njihova interakcija imale su vrlo značajan

utjecaj (P<0,0001). Obzirom na varijantu stresa sve varijante pokusa su se značajno

razlikovale u sadržaju slobodnog prolina u tkivu. U prosjeku za sve kultivare, najveći

sadržaj prolina bio je pri tretmanu 10% PEG-om a najmanji u kontroli (Slika 3). U

prosjeku za sve tretmane, kultivar Lucija (10,49 μmol/g sv.t.), Korana (10,10 μmol/g sv.t.)

i Vita (9,71 μmol/g sv.t.) su imale najveći sadržaj PRO u hipokotilu, ali bez međusobno

značajne razlike. Ika je imala najmanji sadržaj PRO (4,56 μmol/g sv.t.) i značajno se

razlikovala od ostalih sorti. Prolin je aminokiselina koja se akumulira u različitim biljnim

vrstama kao odgovor na stresne uvjete u okolišu (Ashraf i Foolad, 2007; Verbruggen i

Hermans, 2008). U ovom istraživanju se može smatrati dobrim pokazateljem osmotskog

stresa. Smatra se da slobodni prolin ima pozitivan učinak na enzime i integritet membrana

te je značajan u osmotskoj prilagodbi biljaka na stresne čimbenike (Ashraf i Foolad, 2007).

Upravo zbog svoje strukture omogućava stabilizaciju strukture proteina i njihovo očuvanje

(Verbruggen i Hermans, 2008). Matysik i sur. (2002) navode kako prolin osim uloge

osmolita i stabilizacije proteina ima i ulogu regulacije pH citoplazme i uklanjanja ROS-a


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(Matysik i sur., 2002). Nakupljanje prolina omogućava biljkama preživljavanje kraćeg

perioda sušnog razdoblja te brzi oporavak od sušnog stresa (Shehab i sur., 2010). U ovom

istraživanju nakupljanje PRO je uočljivo kod svih ispitivanih kultivara soje u obje razine

osmotskog stresa. Utjecaj tretmana na sadržaj PRO je vrlo značajan kod svih šest kultivara

soje, te se kod svih osim Zore sve varijante statistički značajno razlikuju (Slika 4). Kod

Zore između kontrole i tretmana 5% PEG-om nema značajne razlike.

Slika 3. Sadržaj slobodnog prolina u hipokotilu u prosjeku za sve kultivare po razinama

osmotskog stresa (XYZ – razlike između tretmana; LSD test P≤0,05)

Fig. 3. Free proline content in hypocotyl in average for all cultivars, according

to osmotic stress levels (XYZ – differences between treatments; LSD test P≤0.05)

Slika 4. Sadržaj slobodnog prolina u hipokotilu po kultivarima i razinama osmotskog stresa

(ABC – razlike između sorata prikazano po tretmanu, XYZ – razlike između tretmana

prikazano po sortama; LSD test P≤0,05)

Fig. 4. Free proline content in hypocotyl shown by cultivars and osmotic stress levels

(ABC – differences between cultivars shown by treatment, XYZ – differences

between treatments shown by cultivar; LSD test P≤0.05)


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U ovom istraživanju uočen je također i porast sadržaja PRO i PHE kod tretmana niže

razine stresa i još veći porast kod tretmana s većom razinom stresa. Navedeni trend

objasnili su Shetty i sur. (2003) prikazom metaboličke interakcije sinteze prolina sa

sintetskim putevima fenolnih spojeva, gdje se povezuju pentozofosfatni put, glikoliza, put

šikiminske kiseline i fenilpropanoidni put, prema kojoj sinteza prolina stimulira navedene

metaboličke puteve, a time i sintezu fenolnih spojeva.

Povezanost sadržaja prolina, peroksida i intenziteta lipidne peroksidacije u tkivu hipokotila

testirana je multiplom regresijskom i linearnom analizom. Utvrđena je značajna pozitivna

korelacija intenziteta lipidne peroksidacije i sadržaja prolina, te sadržaja prolina i

vodikovog peroksida kod tretmana 10% PEG-om (Slika 5). Kultivari Korana, Vita i Lucija

su imali u prosjeku za sve tretmane visok sadržaj peroksida i prolina, uz visok intenzitet

lipidne peroksidacije, dok je kod kultivara Zora, Tena i Ika gotovo u pravilu nizak sadržaj

peroksida praćen manjom akumulacijom prolina i manjim intenzitetom lipidne

peroksidacije. Do sličnih zaključaka došli su Koca i sur. (2007) koji u svojim

istraživanjima na kultivarima sezama zaključuju kako su parametri rasta, intenzitet lipidne

peroksidacije i sadržaj prolina u pozitivnoj korelaciji.

Slika 5. Korelacija između sadržaja prolina (x; PRO), sadržaja vodikovog peroksida

(y, lijevo; HP) i intenziteta lipidne peroksidacije (y, desno; TBARS)

u hipokotilu klijanaca soje pod utjecajem tretmana s 10% PEG

Fig. 5. Free proline content (x; PRO) correlation with hydrogen peroxide content

(y, left; HP) and lipid peroxidation intensity (y, right; TBARS) in seedling’s

hypocotyl under influence of 10% PEG treatment


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Zaključci

Primjena polietilen glikola 6000 pri naklijavanju sjemena soje značajno je utjecala na

porast sadržaja slobodnog prolina, ukupnih fenola, sadržaj vodikovog peroksida i intenzitet

lipidne peroksidacije u tkivu hipokotila, što potvrđuje stresan učinak primijenjenih

tretmana. Porast sadržaja prolina i ukupnih fenola sugeriraju na aktivaciju neenzimskog

antioksidativnog odgovora na inducirani osmotski stres. Ispitivani kultivari soje nisu imali

podjednak fiziološki odgovor na primijenjeni osmotski stres. Korana, Vita i Lucija su

imale visok, a Zora, Tena i Ika prosječno nizak sadržaj peroksida i prolina te intenzitet

lipidne peroksidacije u tkivu hipokotila. Dobiveni rezultati potvrđuju da je polietilen glikol

6000 tvar koja se može koristiti kao simulator osmotskog stresa u fazi klijanja soje.

Literatura

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temperatures: towards genetic engineering for stress tolerance, Planta 218 (1), 1-14.

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Yoshiba Y. (2005): Effects of free proline accumulation in petunias under drought stress,

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Zahran, H.H., Sprent, J.I. (1986): Effects of sodium chloride and polyethylene glycol on root-hair

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303-309.

Polyethylene glycol-6000 application in the research of osmotic stress

in soybean (Glycine max (L.) Merr.)

Marija Špoljarević1, Ana Mihaljević

1, Ivna Štolfa

2, Dejan Agić

1,

Rosemary Vuković2, Miroslav Lisjak

1, Tihana Teklić

1

1Josip Juraj Strossmayer University of Osijek, Faculty of Agriculture in Osijek, Kralja Petra

Svačića 1d, HR-31000 Osijek, Croatia 2Josip Juraj Strossmayer University of Osijek, Department of Biology, Ulica Cara Hadrijana 8a,


Summary

Polyethylene glycols (PEG) are produced by polymerization of ethylene oxide (EO) with water,

mono ethylene glycol or diethylene glycol under alkaline catalysis. The result is a very simple

chemical structure: HO-[CH2-CH2-O]n-H, where (n) is the number of EO-units. The most important

property of all PEGs is their solubility in water, which makes them ideally suitable for use in

countless different applications. PEGs have been widely used in the study of the water relations of

plants, especially for simulating drought (osmotic stress) conditions. Osmotic potential of aqueous

solutions of PEG 6000 is related to its concentration. Here, we presented the results of physiological

response of soybean seed to PEG-imposed osmotic stress in germination stage. Beside water

(control), two levels of osmotic stress were applied - 5% (-0.5 bars) and 10% (-1.48 bars) PEG 6000

solution as a medium for seed germination. The level of free proline, total phenols and lipid

peroxidation in hypocotyl tissue increased with PEG concentration (osmotic stress intensity). As the

tested soybean cultivars differed in particular metabolic parameter response, PEG might be

considered as a convenient tool for osmotic stress simulation.

Keywords: soybean, osmotic stress, abiotic stress, PEG 6000


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Use of farm-saved seed in modern agriculture

UDC: 631.53.02 : 631.115.1

Zvonimir Zdunić, Luka Andrić, Aleksandra Sudarić, Georg Drezner,

Alojzije Lalić, Josip Kovačević, Krešimir Dvojković

Poljoprivredni institut Osijek, Južno predgrađe 17, 31103 Osijek, Croatia

Summary

Farmers may use seed that they have saved for their own use to grow and harvest a crop - this is

known as Farm Saved Seed (FSS). It mostly refers to the harvested grain only of those self-

pollinated agricultural varieties such as wheat, soybean, and barley, that are protected by Plant

Breeders Rights legislative. Croatia urgently need a law-regulated framework that would determine

FSS as a transparent seed category. The framework should include all necessary regulations on

purposes, technical standards, and transparent collecting of royalty on FSS use. Any uncontrolled

FSS transaction - charged, bartered or free cause inability to collect and control royalty on FSS.

Misuse of FSS will also cause difficulties for milling and baking industry that often set up high

demands with respect to quality and exact varietal seed, as well as for producers that might not be

able to sell such mixed seed to the food processing and confectionery industry. Consequently,

reappearance of certain diseases (Tilletia tritici) will necessary increase chemical field application,

and lower germination rate of FSS might increase the required quantity of artificial manures in the

soil. This is not in accordance with recommended sustainable agricultural practise of the European

Union.

Keywords: farm-saved seed, royalty control, wheat, barley, diseases

Introduction

Farmers may use seed that they have been saving for their own use to grow and harvest a

crop (farm-saved seed, FSS). It refers only to those varieties of agricultural crops that have

been protected by Plant Variety Rights (PVR). Breeding activities of the Agricultural

Institute Osijek (AIO) resulted in hundreds of new cultivars developed in long-lasting and

numerous plant breeding programs (PBP's). All of PBP’s must meet demands of not only

modern genetics science in terms of genetic purity, high yield quantity and quality, but also

crucial prerequisites in terms of biotic and abiotic stresses such as tolerance to pest and

fungi diseases, lodging resistance, as well as requirements in soil type, fertilization, water

supply, chemical control, etc. Sustainability of PBP's is ensured by commercialization of



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innovations developed in each and every PBP both in domestic and international market.

Throughout the history of the AIO, PBP’s have always been significantly contributing to

it’s scientific production and business stability. FSS has been introduced to Croatia in

2011, but its widespread misuse due to insufficient control of law enforcement led to

instability of the seed production and trade, decrease of the average grain yield, and

reappearance of certain diseases (Tilletia spp.) and weed (Avena spp.). Consequently,

industry of milling and baking as well as confectionery industry face chalenges nowdays

with respect to quality of the raw grain obtained from the crops planted with FSS. Finally,

both insecure collecting procedure and lower amount of the royalty collected on FSS made

limitations on many PBP's and the holders of PVR commercially inferior.

Role of plant breeding in food and feed industry

Plant breeding is considered one of the longest ongoing activities undertaken by humans,

who select plants more productive and useful to themselves and the animals for at least 10

000 years ago (Hallauer, 2011). Plant breeding is the genetic improvement of plants for

human benefit. The process of plant breeding involves science, art, and business. Breeders

improve plants to meet specific human needs, most often for food, feed, fiber, or fuel.

(Bernardo, 2010). The development of improved cultivars has made a major contribution

to the increased productivity and quality of plants used for their food, feed, fiber, or

aesthetic value. Selection of the appropriate cultivar is one of the key decisions that an

agricultural producer must make. (Fehr, 1991). Fig. 1. shows progress on grain yield due to

plant breeding of the three most widespread agricultural crops in the world.

Fig. 1. Progress in world average yields for major cereal crop yields. Data from FAOSTAT

(the Statistics Division of the Food and Agriculture Organization of the United Nations,

http://www.faostat.fao.org)

http://www.faostat.fao.org/


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Important prerequisites for development and existence of the plant breeding institutions

AIO was founded in 1878, and has been dealing with plant breeding for more than a

century which resulted in several hundreds of both self-pollinated and open-pollinated

cultivars developed. That is what makes it the biggest and most important plant breeding

institution in Croatia today. Up to now, some paper harmonization with EU legislation on

PVP has been done in the country (Bišćan Rendulić, 2011), but effective control of the

low-regulated procedure that would enable collecting royalty on FSS is not established yet.

It makes direct annual financial loss to the Institute in amount of 300-400 000 US dollars

with the growing trend. Misuse of the farm-saved seed also cause issues to (i) milling,

baking and confectionery industry that often set up high demands with respect to grain

quality; (ii) adjacent crop fields planted with certified seed due it's infestation with fungi

diseases from FSS fields; (iii) crop producers that are not able to sell infested raw grain to

the food and feed industries. Furthermore, farmers who use FSS might tend to increase

their aplication of agricultural chemicals such as pesticides and artificial fertilizers as an

attempt to compensate yield loss.

Developed system of certified seed planting

System of subsidies that especialy rewards certified seed production would result in seed

production improvement and more favourable wholesale and retail seed price both for the

farmers and seed distributors. Croatia used to apply binding system of subsidies that

resulted in the highest rate of certified seed planting in the area. Those simple measures

had a significant positive effect to PBP’s and seed production in the country, especially to

domestic breeding and seed industry that controls the highest market share of self-

pollinated varieties. Transparent and regular collecting of the royalty was also ensured.

The abolition of subsidies for planted certified seed continuosly contributed to significant

reduction of certified seed usage over the last several years. During the same time, neither

in advance nor parallel with above-described process, FSS was not determined in a way

that would include it’s all relevant and essential elements. The whole process was left to a

chance with following consequences: (i) destabilization of already regulated system of

seed production and trade, (ii) questionable sustainability of PBP’s and it’s development

due to market loss and inability to control and collect the royalty on FSS, (iii) high risk of

farmer's grain production for milling, baking and confectionary industry.

How to continue?

It is still not clear how to get to the information on infringement of PVR, i.e. what

institution(s) should provide it. Besides improving and developing new varieties, it seems


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that the holder of the PVR should take a role of being an investigator and prosecutor. There

is no answer yet on how to prove a violation of PVR in judicial proceedings and what

material evidence should be presented in the court. At the moment, two separated

procedures are required for (i) obtaining registraton of new varieties, and (ii) plant variety

protection. If possible, it would be wise to unify those procedures and their results should

be equaly applied in all EU countries. What can be done by EU to make the system

running in Croatia? For new EU countries, opening European Office for Plant Breeder’s

Rights would be helpful for collecting data on FSS planted areas and royalty assesment as

well as detecting offenses, which is any FSS transaction – charged, free or bartered.

Therefore, we urgently need a law-regulated framework that would determine FSS as a

future transparent seed category. The framework should include all necessary regulations

on the usage, monitoring, technical standards and transparent collecting of royalty on FSS.

It also should be considered the possibility of special status for precious seed production

with respect to direct payments. An additional effort is required toward decreasing costs of

registration of new varieties, supervision, seed maintaining, seed certification, etc. – all in

purpose of promoting competitiveness of final product on both domestic and international

market. Giving the oportunity to the breeders to protect their varieties regardless of how

long they have been on the market would be appreciated because such varieties usualy take

the largest portion of the market share. In most cases, those varieties are also the main

source of income for PBP’s and seed production. As a final measure, due to already

suffered losses in the seed sector, we suggested for 3-year transition period in which

certified seed planting would be obligatory again.

References

Bernardo, R. (2010): Breeding for quantitative traits in plants. Stemma Press, Minneapolis, MN,

USA, 390 p.

Bišćan Rendulić, A. (2011): Upotreba materijala zaštićene sorte – farm saved seed, Sjemenarstvo 28

(1-2), 89-92.

Fehr, W. R. (1991): Principles of cultivar development. Department of Agronomy, ISU, Ames, IA,

USA, 536 p.

Hallauer, A. R. (2011): Evolution of plant breeding, Crop Breeding and Applied Biotechnology 11,

197-206.

Kovačević, V., Šimić, D., Šoštarić, J., Josipović, M. (2007): Precipitation and temperature regime

impacts on maize yields in Eastern Croatia, Maydica 52, 301-305.

Sekcija: Zaštita okoliša

Session: Environmental protection


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356

Koncentracije policikličkih aromatskih ugljikovodika u zraku

na različitim lokacijama u hrvatskoj

UDC: 502 : 614.72(497.5)

Ivana Jakovljević, Gordana Pehnec, Vladimira Vađić

Institut za medicinska istraživanja i medicinu rada, Ksaverska cesta 2, 10000 Zagreb, Hrvatska

Sažetak

Policiklički aromatski ugljikovodici (PAU) su onečišćenja koja mogu imati veliki utjecaj na živote ljudi

zbog svojih karcinogenih i mutagenih svojstava. Cilj ovoga rada je usporediti koncentracije PAU vezanih

na čestice aerodinamičkog promjera manjeg od 10 µm (PM10) na četiri lokacije u Hrvatskoj, koje se

međusobno razlikuju po gospodarstavu i načina života. Uzorkovanje je provođeno u Zagrebu, glavnom

gradu Hrvatske (A), planinskom mjestu s malom razvijenom industrijom u Gorskom kotaru (B),

ruralnom mjestu smještenom u sjevernom dijelu hrvatske (C), te u industrijsko razvijenom području u

središnjem dijelu Hrvatske (D). Uzorkovanje je provođeno mjesec dana u zimskom periodu, te mjesec

dana u ljetnom periodu. Mjerene su koncentracije 9 PAU uključujući benzo[a]piren (BaP). Na svim

lokacijama su koncentracije mjerenih PAU bile znatno više u zimskom razdoblju mjerenja. Tijekom

zime najviša koncentracija benzo[a]pirena zabilježena je na mjernim mjestima B i D i iznosila je oko

5 ng/m3, dok je najniža zimska vrijednost koncentracije benzo[a]pirena zabilježena za mjerno mjesto C

(0,44 ng/m3). Srednje vrijednosti koncentracije BaP tijekom ljetnog mjerenja na tri lokacije bile su niže

od 0,06 ng/m3, dok je na lokaciji D srednja vrijednost BaP bila 0,137 ng/m

3. Na svakoj lokaciji izračunati

su omjeri pojedinih PAU kako bi se mogli procijeniti mogući izvori onečišćenja.

Ključne riječi: PM10, Benzo[a]piren, BaPeq, sezonske varijacije

Uvod

Policiklički aromatski ugljikovodici (PAU) su spojevi s dva i više aromatska prstena.

Istraživanje i proučavanje PAU započelo je prije uvođenja sistematske nomenklature te su

stoga svoja imena dobili prema određenim karakteristikama: - boji (fluoranten i krizen),

imenu spoja iz kojeg su izolirani (iz katrana, kamenog ugljena, naftalen i piren) itd. Ta su

imena i danas u upotrebi (Jakovljević, 2011).

U zraku je pronađeno preko pet stotina PAU. Najpoznatiji i najviše proučavan je

benzo[a]piren (BaP) koji je ujedno i indikator za prisutnost PAU u hrani i zraku.

Ukoliko se radi o PAU s dva ili tri benzenska prstena, postojani su u plinovitoj fazi te

njihova koncentracija u zraku raste s porastom temperature dok se PAU s pet ili više

benzenskih prstenova u zraku uglavnom nalaze vezani na čestice (Lee et al., 1981).



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Još u 18. stoljeću započeta su istraživanja štetnosti PAU na zdravlje ljudi. Istraživanja su

se temeljila na promatranju radnika zaposlenih u industriji koksa i proizvodnji asfalta, u

ljevaonicama, u proizvodnji aluminija, kao i na ljude koji su izloženi ispušnim plinovima

dizelskih goriva (WHO, 2000; IARC,1979). Studije su pokazale da postoji direktna veza

izloženosti PAU i oboljevanju radnika od raka pluća (Beak et al., 1992). Karcinogenost

policikličkih aromatskih ugljikovodika procijenjuje se preko ekvivalenta BaP (BaPeq).

Američka agencija za zaštitu okoliša (US-EPA) svrstala je sedam PAU u Grupu B2

(vjerojatno karcinogeni), a to su benzo[a]piren, benzo[a]antracen, benzo[b]fluoranten,

benzo[k]fluoranten, dibenzo[a,h]antracen, indeno[1,2,3-cd]piren i kirzen.

Međunarodna agencija za istraživanje raka (IARC) je također napravila klasifikaciju PAU.

Benzo[a]antracen i benzo[a]piren su svrstani u grupu 2A (vjerojatno karcinogeni), a ostali

PAU su svrstani u grupu 2B (moguće karcinogeni) (Callén et al., 2013).

Policiklički aromatski ugljikovodici u okoliš dospjevaju prirodnim putem prilikom velikih

šumskih požara i vulkanskih erupcija (Li et al., 2009). Posljedica su prirodnih procesa kao

što je karbonizacija, ali najvažniji izvori PAU su povezani s ljudskom aktivnošću. Mogu se

pronaći u zraku, tlu, vodi, vegetaciji, hrani i sedimentu.

Svakodnevni brojni procesi koji se odvijaju imaju za rezultat emisiju PAU u okoliš (npr.

proizvodnja ugljena, sirove nafte, benzina, prirodnog plina, te proizvodnja teških i lakih

metala). Prisutni su i u ispušnim plinovima motornih vozila. Ovisno o vrsti goriva dolazi

do emisije različitih PAU. Također nastaju prilikom svakog nekontroliranog spaljivanja

otpada i raznih plastičnih masa.

Izgaranjem dizelskih goriva uglavnom nastaju PAU manjih molekulskih masa, dok

benzinski motori ispuštaju uglavnom PAU većih molekulskih masa kao što je npr.

benzo[ghi]perilen (Šišović et al., 2005). Najveći izvor PAU su kućna ložišta ako se kao

sredstvo za grijanje domova koristi drvo ili ugljen (Wenger et al., 2009).

Materijali i metode

Uzorkovanje

Uzorkovanje PM10 frakcije lebdećih čestica se provodilo mjesec dana u zimskom periodu,

te mjesec dana u ljetnom periodu. Mjesta na kojima se uzorkovanje provodilo su Zagreb,

glavni grad Hrvatske (A), planinsko mjesto s malom razvijenom industrijom u Gorskom

kotaru (B), ruralno mjesto smješteno u sjevernom dijelu Hrvatske (C), te industrijski

razvijeno područje u središnjem dijelu Hrvatske (D). Uzorkovanje se provodilo 24 - satnim

prosisavanjem zraka kroz kvarcni filtar papir (Li et al., 2009; Šišović et al., 2008).

Analiza

Analizirano je 9 PAU na svakome mjernom mjestu i to: fluoranten (Flu), benzo[a]piren

(BaP), benzo[a]antracen (BaA), benzo[k]fluoranten (BkF), piren (Pir), benzo[b]fluoranten


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(BbF), indeno[1,2,3-cd]piren (IP), benzo[ghi]perilen (BghiP), dibenzo[ah]antracen

(DahA). Prikupljeni uzorci lebdećih čestica su ekstrahirani u ultrazvučnoj kadici 1 sat, u

smjesi otapala cikloheksan : toluen (3:7). Nakon što su uzorci ekstrahirani potrebno ih je

upariti do suha u struji dušika te ih zatim otopiti u acetonitrilu. Sama analiza PAU se vršila

na tekućinskoj kromatografiji visoke djelotvornosti s fluorescentnim detektorom. Analiza

se provodi pri protoku mobilne faze od 0,5 mL/min (Šišović, 1991).


Mjerenja policikličkih aromatskih ugljikovodika u PM10 frakciji lebdećih čestica provodila su

se mjesec dana u zimskom te mjesec dana u ljetnom periodu kako bi se vidjele sezonske

varijacije PAU. Koncentracije izmjerene u zimskom periodu na svim mjernim mjestima su bile

višestruko veće od koncentracija izmjerenih za vrijeme ljetnog perioda. Najviša vrijednost

zabilježena je za BghiP u zimskog perioda na mjernom mjestu A i D. Na mjernom mjestu B

zabilježena je najviša vrijednost za Flu i Pir. Najniže vrijednosti svih mjerenih PAU zabilježene

su na ruralnom području (mjerno mjesto C). Vrijednosti u vrijeme ljetnog perioda mjerenja bile

su znatno niže za sve mjerene PAU na svim mjernim mjestima. Najviše vrijednosti u ljetnom

periodu mjerenja zabilježene su za policikličke aromatske ugljikovodike sa 6 aromatskih

prstenova (BghiP i IP) na svim mjernim mjestima (Slika 1 i 2).

Slika 1. Srednje vrijednosti masenih koncentracija PAU izmjerenih

na četiri mjerna mjesta za vrijeme zimskog perioda

Fig. 1. Average values of the mass concentrations of PAHs measured

at four measurement sites during winter time


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Slika 2. Srednje vrijednosti masenih koncentracija PAU izmjerenih na četiri mjerna mjesta za vrijeme ljetnog perioda

Fig. 2. Average values of the mass concentrations of PAHs measured

at four measurement sites during summer time

Za međusobnu usporedbu mjernih mjesta, obraćajući pozornost na zastupljenost pojedinih

izvora, izračunati su omjeri pojedinih PAU (Flu/(Flu+Pir), BaP/BghiP, IP/BghiP) kako bi

se pokušali definirati osnovni izvori onečišćenja. Za vrijeme zimskog perioda median

Flu/(Flu+Pir) kretao se je od 0,48 za mjerno mjesto B do 0,58 za mjerno mjesto C. Omjer

BaP/BghiP kretao se je od 0,53 (A) do 1,65 (B), IP/BghiP bio je 0,52 (A) do 1,52 (B). Za

vrijeme ljetnog perioda omjeri su bili približno slični. Omjeri pojedinih PAU za mjerna

mjesta i mogući izvori onečišćenja prema literaturi (Yunker et al., 2002; Zhang et al.,

2004) dati su u tablici 1. Iz podataka u tablici 1 može se vidjeti da je osnovni izvor

onečišćenja tijekom zimskog perioda izgaranje trave, ugljena i drava te benzinska i

dizelska goriva. Omjeri za vrijeme ljetnog perioda ukazuju kao glavni izvor onečišćenja

ispušne plinove automobila.

U ovom radu moguća karciongenost PAU prikazana je izračunavanjem ekvivalenta BaP

(BaPeq). Za izračunavanje toksičnosti pojedinih PAU uzeti su toksični faktori koje su

odredili Nisbet i LaGoy 1992. godine (Petry et al., 1996) (Tablica 2). Karcinogenost

pojedinog PAU izražena je kao BaPeq te se dobiva tako da se izmjerenoj koncentraciji PAU

u zraku pridruži odgovarajući toksični faktor. Preko BaPeq moguće je izračunati

karcinogenu aktivnost i relativni karcinogeni potencijal tako da se karcinogena aktivnost

stavi u odnos sa izmjerenom koncentracijom BaP.


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S obzirom da su najviše koncentracije u zimskom periodu zabilježene na mjernom mjestu

D, za to mjerno mjesto je dobivena i najveća ukupna karinogena aktivnost. Međutim

relativni karcinogeni potencijal za mjerno mjesto D približno je jednak kao i za mjerno

mjesto A i B, a dok je najviši relativni karcinogeni potencijal izračunat za mjernom mjestu

C te iznosi 1,61. U ljetnom razdoblju mjerenja ukupna karcinogena aktivnost zabilježena je

za mjerno mjesto D, a relativni karcinogeni potencijal za mjerno mjesto B iznosi 1,76. Dok

se udio BaP u ukupnoj karcinogenoj aktivnosti za vrijrme zimskog perioda kretao oko 60

do 70 %, za vrijeme ljetnog perioda on je iznosio oko 55 do 70 % ukupne karcinogene

aktivnosti.

Tablica 1. Masene koncentracije mjerenih PAU, te BaPeq (ng/m3) i relativni karcinogeni potencijal (RPF)

Table 1. Average mass concentrations of PAH, BaPeq (ng/m3) and relative potency factor (RPF)

PAU TEF A

(ng/m3)

B

(ng/m3)

C

(ng/m3)

D

(ng/m3)

BaPeq A

(ng/m3)

BaPeq B

(ng/m3)

BaPeq C

(ng/m3)

BaPeq D

(ng/m3)

Zim

ski

perio

d /

win

ter t

ime Flu 0,001 4,155 7,55 0,347 5,705 0,0042 0,0076 0,0003 0,0057

Pir 0,001 4,071 9,38 0,223 5,337 0,0041 0,0094 0,0002 0,0053

BaP 1 2,529 5,466 0,446 5,445 2,5290 5,4660 0,4460 5,4450

BbF 0,1 2,526 3,629 0,844 5,528 0,2526 0,3629 0,0844 0,5528

BkF 0,1 1,622 3,185 0,533 3,651 0,1622 0,3185 0,0533 0,3651

BghiP 0,01 4,202 2,934 0,591 8,463 0,0420 0,0293 0,0059 0,0846

BaA 0,1 2,104 5,722 0,236 4,529 0,2104 0,5722 0,0236 0,4529

IP 0,1 2,313 4,783 0,783 5,170 0,2313 0,4783 0,0783 0,5170

DahA 1 0,090 0,123 0,026 0,151 0,0900 0,1230 0,0260 0,1510

Total*

3,526 7,367 0,718 7,579

RPF

1,39 1,35 1,61 1,39

Lje

tni

perio

d /

su

mm

er t

ime

Flu 0,001 0,020 0,013 0,008 0,172 0 0 0 0,0002

Pir 0,001 0,027 0,018 0,011 0,216 0 0 0 0,0002

BaP 1 0,054 0,063 0,034 0,137 0,0540 0,0630 0,0340 0,1370

BbF 0,1 0,072 0,135 0,066 0,150 0,0072 0,0135 0,0066 0,0150

BkF 0,1 0,040 0,070 0,032 0,095 0,0040 0,0070 0,0032 0,0095

BghiP 0,01 0,126 0,219 0,104 0,348 0,0013 0,0022 0,0010 0,0035

BaA 0,1 0,021 0,022 0,009 0,072 0,0021 0,0022 0,0009 0,0072

IP 0,1 0,076 0,201 0,090 0,252 0,0076 0,0201 0,0090 0,0252

DahA 1 0,003 0,003 0,002 0,010 0,0030 0,0030 0,0020 0,0100

Total*

0,079 0,111 0,057 0,208

RPF

1,47 1,76 1,67 1,52 *ukupna karcinogena aktivnost / total carcinogenic activity


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Tablica 2. Omjeri pojedinih PAU za četiri mjerna mjesta

Table 2. Diagnostic ratio between some PAH for all measured site


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Zaključci

Za vrijeme zimskog perioda mjerenja na mjernom mjestu A i D najviša vrijednost

zabilježena je za BghiP i Flu, što je i u skladu s očekivanjima obzirom da su oba mjerna

mjesta stambeno naseljena područja s velikim prometnicama. Mjerno mjesto B je izrazito

planinsko područje s razvijenom drvnom industrijom, stanovništvo se grije na drva tijekom

zimskog perioda. Najviše vrijednosti Flu i Pir su izmjerene na ovoj lokaciji, što ne

iznenađuje s obzirom da ova dva spoja su karakteristični produkti izgaranja drva i trave.

Analiza omjera pojedinih PAU pokazala je kao glavni izvor onečišćenja, tijekom zimskog

perioda mjerenja, izgaranje ugljena, trave i drva. Dok je tijekom ljetnog perioda kao

glavni izvor onećišćenja su ispušni plinovi automobila. Najveći doprinos u ukupnom

karcinogenom potencijalu ima BaP (preko 50%) stoga se može zaključiti da je BaP

policiklički aromatski ugljikovodik koji značajno dopridonosi karcinogenoj aktivnosti

PAU, što ga ujedno čini pogodnim indikatorom te opravdano i najistraživanijim spojem.

Literatura

Beak, S.O., Goldstone, M.E., Kirk, P.W.W., Lester, I.N., Perry, R. (1992): Concentrations of

articulate and gaseous polycyclic aromatic hydrocarbons in London air following a reduction

in the lead content of petrol in the United Kingdom, Sci. Total Environ. 111, 169-199.

Callén, M S., Lόpez, J.M., Iturmendi, A., Mastral, A.M. (2013): Nature and sources of particle

associated polycyclic aromatic hydrocarbons (PAH) in the armospheric environment of an

urban area, Environ. Pollut. 183, 166-174.

International Agency for Research on Cancer (IARC) (1979): Environmental Carcinogens: Selected

Methods of Analysis. Vol. 3. Analysis of Polycyclic Aromatic Hydrocarbons in

Environmental Samples. Lyon

Jakovljević, I., Žužul, S. (2011): Policiklički aromatski ugljikovodici u zraku, Arh. Hig. Rada.

Toksikol. 62, 357-370.

Lee, L.M., Novotny, V.M., Bartle, D.K. (1981): Analytical Chemistry of Polycyclic Aromatic

compounds. New York (NY): Academic Press.

Li, Z., Porter, E.N., Sjodin, A., Needham, L.L., Lee, S., Russell, A.G., Mulholland, J.A. (2009):

Characterization of PM2.5-bound polycyclic aromatic hydrocarbons in Atlanta - Seasonal variations at

urban, suburban, and rural ambient air monitoring sites, Atmos. Environ. 43, 4187-4193.

Petry, T., Schmid, P., Schlatter, C. (1996): The use toxic equivalency factors in assessing

occupational and environmental health risk associated with exposure to airborne mixture of

polycyclic aromatic hydrocarbons (PAHs), Chemosphere 32, 639-648.

Šišović, A., Fugaš, M. (1991): Comparative evaluation of procedures for the determination of PAH

in low-volume samples, Environ. Monit. 18, 235-241.

Šišović, A., Vađić, Z., Šega, K., Bešlić, I., Vađić, V. (2005): Comparison between PAH mass

concentrations measured in PM10 and PM2,5 particle fractions, Bull. Environ. Contam.

Toxicol. 75, 121-126.


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Šišović, A., Bešlić, I., Šega, K., Vađić, V. (2008): PAH mass concentrations measured in PM10

particle fraction, Environ. Int. 34, 580-584.

Wenger, D., Gerecke, A. C., Heeb, N.V., Hueglin, C., Seiler, C., Haag, R., Naegeli, H., Zenobi, R.

(2009): Aryl hydrocarbon receptor-mediated activity of atmospheric particulate matter from an

urban and a rural in Switzerland, Atmos. Environ. 34, 3556-3562.

World Health Organization (WHO) (2000): Regional Office for Europe. Air Quality Guidelines for

Europe. 2nd ed. Copenhagen.

Yunker, M.B., Macdonald, R.W., Vingarzan, R., Mitchell, R.H., Goyette, D., Sylvestre, S. (2002):

PAHs in the Fraser River basin: a critical appraisal of PAH ratios as indicators of PAH source

and composition, Org. Geochem. 33, 489-515.

Zhang, Z., Huang, J., Yu, G., Hong, H. (2004): Occurrence of PAHs, PCBs and organochlorine

pesticides in the Tonghui River of Beijing, China, Environ. Pollut. 130, 249-261.

Concentrations of polycyclic aromatic hydrocarbons in the air

at different locations in croatia

Ivana Jakovljevic, Gordana Pehnec, Vladimira Vađić

Institute for Medical Research and Occupational Health, Ksaverska c. 2, HR-10000 Zagreb, Croatia

Summary Polycyclic aromatic hydrocarbons (PAHs) are contaminants with potential impact on human health

due to their carcinogenic and mutagenic properties. The aim of this paper was to compare the levels

of PAHs bound to the particle fraction with an aerodynamic diameter under 10 microns (PM10).

Sampling was conducted at different sites in Croatia. In Zagreb, the Croatian capital (A), a

mountain village with almost no developed industry in Gorski Kotar (B), a rural place located in the

northern part of Croatia (C), and in an industrialized area in central Croatia (D). Sampling was

conducted across one month in winter and one month in summer. Concentrations were measured for

9 PAHs, including benzo[a]pyrene. At all of the locations, PAH concentrations were significantly

higher in winter time. During the winter, the highest concentration of benzo[a]pyrene (BaP) was

recorded at measuring sites B and D and amounted to 5 ng/m3, while the lowest value of

benzo[a]pyrene in winter was recorded for measuring site C (0.44 ng/m3 ). Mean concentrations of

BaP during the summer were below 0.06 ng/m3, while at site D the concentration of BaP was 0.137

ng/m3. At each location, ratios of individual PAHs were calculated in order to assess possible

sources of contamination.

Keywords: PM10, Benzo[a]pyrene, BaPeq, seasonal variations


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Natural organic matter degradation using heterogeneous

Fenton catalysts on zeolite support

UDC: 628.161.3

Brankica Kalajdžić1

, Marija Nujić1, Željka Romić

2


HR-31000 Osijek, Croatia 2“Vodovod-Osijek“ d.o.o., Poljski put 1, HR-31000 Osijek, Croatia

Summary Natural water throughout the world contains natural organic matter (NOM) in various

concentrations. NOM changes physical characteristics of water like taste, smell and color. During

the water chlorination process harmful disinfection by-products can be formed in a reaction between

the organic matter and chlorine. Fenton process has shown good efficiency in degradation and

removal of natural organic matter from water. However, homogeneous Fenton systems have some

drawbacks which can be overcome by the usage of heterogeneous catalysts, where iron catalyst is

immobilized on a support material. An application of heterogeneous Fenton-type catalysts were

investigated for the degradation of natural organic matter from two sources: model humic acid

solution and natural groundwater from Osijek area. Supported iron catalysts were prepared by

impregnation of different iron forms (FeOOH, Fe(III), Fe(II)) on natural zeolite clinoptilolite as

support material. The performance of the catalysts was compared and the effects of the most

relevant operating conditions (catalyst concentration, H2O2 concentration, pH) was estimated. The

usage of heterogeneous catalysts allows process to be operated at pH conditions close to neutral and

in that conditions heterogeneous Fenton processes yielded similar and even better results.

Keywords: natural organic matter, Fenton process, heterogeneous catalysis, zeolite, THMFP

Introduction

Natural water throughout the world contains natural organic matter (NOM) in various

concentrations. Natural organic matter is a complex mixture of organic compounds derived

from degradation of plant and animal material in the environment. NOM is difficult to

determine. It contains a wide range of the compounds like humic acids, hydrophilic acids,

proteins, lipids, carbohydrates and amino acids. The character of the NOM depends on

source from which is derived and the chemical and biodegradation to which it has been

subjected. It changes organoleptic characteristics of water like taste, smell and color. NOM



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play an important role in the binding and transport of organic and inorganic contaminants

(pesticides, heavy metals) (Koopal et al., 2001). During the water chlorination process

harmful by-products could be formed in a reaction between organic matter and chlorine.

Many of these by-products are potentially harmful for human health (Cohn et al., 1999;

Singer, 1999). Among all the chlorinated by-products, trihalomethanes (THM) occur the

most consistently and at highest concentration (Cohn et al., 1999).

Advanced oxidation processes (AOPs) are characterized by their ability to form the OH

radicals which are capable of oxidizing wide range of compounds. The OH radicals are

non-selective in their mode of attack and able to operate at normal temperature and

pressure (Glaze, 1987). In Fenton process OH radicals are produced during the

decomposition of hydrogen peroxide in the presence of ferrous salts (Walling, 1975).

H2O2 + Fe2+

→ HO• +OH- + Fe

3+

•OH + RH → Oxidation products

The system comprising homogeneous iron ions and hydrogen peroxide is an efficient

oxidant of various organic substances in aqueous solutions. However, homogeneous

Fenton systems have some drawbacks: in some cases relatively high concentration of iron

ions in the bulk may demand a secondary treatment, a Fenton catalytic cycle is restrained

by the formation of stable Fe-complexes, Fenton process is maintained at low pH, which

requires conditioning before and after the treatment. These drawbacks can be overcome by

the usage of heterogeneous catalysts, where iron catalyst is immobilized on a support

material.

Different iron containing materials were used as catalyst in heterogeneous Fenton process

such as synthetic zeolite, activated carbon, clays, silica (Aleksić et al., 2010; Kušić et al.,

2007; Ramirez et al., 2007; Bach and Semiat, 2010; Navalon et al., 2010). Other processes

were based on incorporating iron catalyst onto surfaces of different supports like zeolite

(Centi et al., 2002; Tekbas et al., 2008), pumice (Kitis and Kaplan, 2007) and activated

carbon (Fontecha-Camara et al., 2011; Castro et al., 2009; Pereira et al., 2010; Duarte et

al., 2011). Heterogeneous Fenton process can reduce the final concentration of iron ions in

the bulk after the treatment and allow operating at milder pH conditions (Aleksić et al.,

2011; Kušić et al., 2006).

Although a range of heterogeneous Fenton processes has been tested for oxidizing

synthetic organic chemicals (Aleksić et al., 2010; Fontecha-Camara et al., 2011; Duarte et

al., 2011), application of such processes to remove NOM in drinking water treatment is

still very limited (Kitis and Kaplan, 2007). The focus of this study was to use natural

zeolite clinoptilolite as support material, which was chemically modified with different

iron forms in order to immobilize more amounts of iron catalyst, and to implement them in

heterogeneous Fenton process in order to remove NOM from groundwater. Heterogeneous


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and corresponding homogeneous processes were compared and their efficiency was

estimated on the basis of THMFP decrease.


Materials

Supported iron catalysts were prepared by impregnation of different iron forms (FeOOH,

Fe(III), Fe(II)) on natural zeolite clinoptilolite (Donje Jesenje, Croatia) as support material.

Prior to chemical treatment, natural zeolite sample was grinded in a mortar and then

separated using 0.5-0.63 mm sieves.

Three different heterogeneous catalysts were prepared. Natural zeolite sample was

converted first into the protonated form, then to Na-form and finally loaded with iron. 100

g of zeolite was treated with 0.1 M HCl solution for 1 hour at 70 °C and then for 24 h at

room temperature. The sample was filtered, washed and then treated with 2 M NaCl

solution for 1 h at 70 °C and then for 24 h at room temperature. After complete washing,

zeolite was converted into final Fe-form. First part of the sample was saturated with 0.1 M

FeCl3 solution in the acetate buffer pH 3.6 by stirring on a magnetic stirrer for 1 h at 70 °C

and then for 24 h at room temperature. The zeolite was then filtered, washed and dried at

105 °C for further use. The sample was denoted as Z-Fe(III). Procedure with second part of

the sample was the same only saturated FeSO4×7H2O solution in the acetate buffer was

used. The sample was denoted as Z-Fe(II). Third part of sample was saturated with Fe(III)

form in a same way as Z-Fe(III) catalyst and further it was treated with 4% NaOH solution

(450 mL), stirred for an hour and then treated with 4% NaCl solution (250 mL) and stirred

for another hour at 50 °C. The mixture was rinsed with demineralized water and after that

stirred with 250 mL 50 vol % ethanol for an hour on a magnetic stirrer at 50 °C. The

zeolite was filtered and dried. The sample was denoted as Z-FeOOH.

Two different NOM sources were used in the experiments: humic acid solution (HA)

prepared with humic acid isolate purchased from Aldrich, Germany and natural

groundwater samples which contains high concentration of NOM. Groundwater was

obtained from water-wells that provide raw water for waterworks of town Osijek, eastern

Croatia. The TOC concentration of natural groundwater was 3.5-4 mg/L. HA solution was

prepared in demineralized water with a target concentration of 10 mg/L.

All chemicals were supplied by Kemika, Croatia (H2O2, 30% p.a.; FeCl3×6H2O, p.a.;

CH3COONa, p.a.; NaCl, p.a.; HCl, 36.5%; NaOH, p.a.), Merck, Germany (FeSO4×7H2O,

p.a.), Panreac, Spain (CH3COOH, 100%), Alkaloid, Macedonia (C2H5OH 96% p.a.).


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Experimental procedure

All experiments were performed in standard jar test apparatus (Biblock Scientific

Floculateur 10405). 1 L samples were used for jar tests. After the addition of an

appropriate amount of Fe catalyst, pH was adjusted at the desired value using 0.1 M HCl.

After pH adjustment H2O2 was added. The process lasted 30 minutes was conducted with

stirring at 120 rpm. pH value of the samples was set back at 6 using 0.1 M NaOH and the

decomposition of hydrogen peroxide was occurred which stopped the Fenton reaction.

Samples were left to settle prior to filtration through the 0.45 µm filter.

Three different heterogeneous catalyst (Z-FeOOH, Z-Fe(III), Z-Fe(II)) gained by

impregnation of different iron forms on natural zeolite as support material were applied

and efficiencies of heterogeneous processes were compared to homogeneous process (F) in

which ferrous ions were dissolved in solution.

Optimal conditions for oxidation of humic acid from HA samples were determined by

varying process parameters: pH (2.5; 3.5; 4.5; 5.5), H2O2 (1; 2.5; 5 mM) and Fe (0.02;

0.05; 0.1 mM) dose. The dosages of heterogeneous catalyst were chosen to correspond to

the given concentrations of iron. In groundwater samples something different conditions

were chosen. pH values were set at higher values (4.5; 5.5; 6.5; 7.5). It is well known that

the optimum pH of Fenton’s reaction is 2.5-3.5 for the removal of organic substances

(Walling, 1975). In this research was investigated performance of Fenton process at pH

values close to pH of natural water. H2O2 concentrations were the same (1; 2.5; 5 mM).

Heterogeneous Fenton experiments were conducted with zeolite dosages that correspond to

iron concentration of 0.05 and 0.1 mM.

Analysis

A UV/VIS Spectrometer Lambda 20, Perkin Elmer was used to measure the UV

absorbances in water samples. Trihalomethane formation potential (THMFP) was

established by measuring the absorbances at wavelengths of 254 nm (A254) and 203 nm

(A203) and calculating the ratio A254/A203, for which was found to be a good surrogate

parameter well correlated to THMFP (Korshin et al., 1997).


Trihalomethane (THM) formation has been shown to be a function of many parameters,

including disinfectant dose, total organic carbon concentration, pH, temperature, reaction

time. Also, organic matter nature or composition, especially the aromatic content, is an

important determinant in THM formation (Singer and Reckhow, 1999). In order to achieve

minimization of the formation of harmful disinfection byproducts (DBPs), natural organic


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matter in water was treated with Fenton type processes. It is difficult and time-consuming

to measure or predict THM concentrations, so the parameters such as TOC, A254 and

THMFP were used.

THMFP is an index of the potential extent of THM formation after the application of

chlorine. Surrogate parameters have been used to predict THMFP calculated as a ratio

A254/A203, which is reported to correlate with the proportion of hydroxyl-, carboxyl-, ester-

and carbonyl-substituted aromatic rings that have been implicated in reactions generating

DBPs (Korshin et al., 1997).

Homogeneous/ heterogeneous process

Various supports could be used to prepare heterogeneous Fenton catalysts. The advantage

is given to highly porous materials which have higher surface areas and may immobilize

more amounts of catalysts thus providing more reaction sites for the oxidation of NOM

(Kitis et al., 2007).

Natural zeolites are environmentally and economically acceptable three dimensional

hydrated aluminosilicate materials with a porous structure and with exceptional ion-

exchange and sorption properties. One of the most investigated zeolite is clinoptilolite. The

unique tree-dimensional porous structure gives natural zeolites various application

possibilities (Margeta et al., 2013). Zeolite offers various properties which can give some

advantages over other catalyst supports in heterogeneous Fenton process: (1) as

alumosilicate materials they are resistant to oxidation, (2) iron can be easily introduced due

to their ion-exchange capacity, (3) they possess unique sorption properties with respect to

smaller organic molecules (Gonzalez-Olmos et al., 2009).

Generally, THMFP decrease was lower with heterogeneous Fenton catalysts than with

corresponding homogeneous Fenton system. When comparing results achieved in optimal

conditions, homogeneous Fenton process has brought best removal results and maximal

THMFP decrease was 79.6% (Fig. 1-A). The zeolite catalysts activity was noticeably

lower than the activity of homogeneous iron ions. In optimal conditions for each process,

maximal THMFP decrease achieved with zeolite support was 57.0% for Z-FeOOH, 68.5%

for Z-Fe(III) and 57.2% for Z-Fe(II). This fact may be explained by either a diffusion

limitation inside micropores of the support material or a particular structure of active iron

centers. Also, the low activity of bounded iron may be explained by its lower redox

potential due to the iron ion surroundings inside of zeolite (Kuznetsova et al., 2004). When

the conditions were not optimal, noticeably worse results were achieved with zeolite

supported processes (Fig. 1-B). Since efficiency of homogeneous Fenton process was also

significantly lower, especially when process was carried out at higher pH value, efficiency

of both homogeneous and heterogeneous processes was similar in those conditions.


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Fig. 1. Effect of pH value on performance of different Fenton catalysts (0.1 mM Fe)

in HA solution: A-5 mM H2O2; B-1 mM H2O2

Similar trends were also observed for the THMFP decrease in natural water (Fig. 2). When

process conditions were far from optimal, zeolite catalysts have shown even better results

than homogeneous Fenton process.

Fig. 2. Effect of pH value on performance of different Fenton catalysts (0.1 mM Fe)

in groundwater: A-5 mM H2O2; B-1 mM H2O2


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pH value

Presence of H+ ions is required in the decomposition of H2O2, indicating the need for an

acid environment to produce the maximum amount of OH radicals. pH range for

application of the Fenton reaction is limited to a very narrow range (2.5-3.5) (Walling,

1975). However, heterogeneous Fenton type processes could be effectively operated at

milder pH conditions (Fajerwerg et al., 1997; Aleksić et al., 2010). In this investigation

heterogeneous Fenton process were applied through the pH range from 2.5 to 4.5. Effect of

pH on the effectiveness of heterogeneous process has been very weak, almost

unnoticeable, while it has been greater in the homogeneous process especially when the

process has not been maintained at optimal hydrogen peroxide and iron concentrations

(Fig.1). When the H2O2 concentration was 5 mM (0.1 mM Fe), the effect of initial pH

value was slightly noticeable in investigated pH range for both homogeneous and

heterogeneous processes. For homogeneous Fenton process maximal efficiency could be

noticed at pH 3.5 and it was almost 80 %, until for heterogeneous catalysts maximum

could not be even noticed and the THMFP decrease was 56-57% for Z-FeOOH and about

56% for Z-Fe(II). Increasing the pH value was accompanied by a slight decline in

efficiency in heterogeneous process when zeolite was impregnated with Fe(III) (from 68%

at pH 2.5 to 54% at pH 5.5). When the H2O2 concentration was lowered to 1 mM,

heterogeneous processes were still insensitive to changes of pH value, while the efficiency

of homogeneous process rapidly decreases with increasing pH value. Maximum efficiency

was noticed at pH 3.5 and it was about 63% and dropped to 23% at pH 4.5. The findings of

this study are consistent with the results reported by other researchers (Centi et al., 2000)

who noticed that catalytic activity of the heterogeneous catalyst is less sensitive to the

solution pH than the homogeneous catalyst. According to the chemistry of homogeneous

Fenton process, when pH is approaching to the neutral, the formation of stable ferric

hydro-complexes restrains Fenton catalytic cycle. Heterogeneous catalysts could prevent

formation of stable Fe(III) complexes. Such effect could be related to the structure of

support materials. Iron ions are under the influence of the strong electrostatic field in the

zeolite framework (Aleksić et al., 2010).

A working pH in the near neutral range is especially beneficial for the treatment of

groundwater which usually has a high natural buffer capacity. The acidification, as a

necessary step in the homogeneous Fenton process, is uneconomic in this case. Initial pH

values in groundwater experiments were in range from 4.5 to 7.5. The data obtained using

homogeneous Fenton process showed that THMFP decrease were greater in weakly acidic

solution (pH 4.5-5.5) compared with weakly alkaline ones (pH 6.5-7.5). The efficiency of

homogeneous Fenton oxidation was decreased at pH higher than 6 which could be due to

the precipitation of Fe(III) by the available hydroxyl ions. pH value had almost no effect

on the performance of heterogeneous processes (Fig. 2).


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H2O2 concentration

Next important parameter in Fenton process is hydrogen peroxide concentration. The

concentration of OH radical has increased with increase in H2O2 dosage which leads to

increased oxidation of organic compounds. In heterogeneous process the effect of H2O2

concentration was highly noticed in both humic acid solutions and natural groundwater

samples. Increasing the H2O2 concentration from 1 mM to 5 mM (pH 2.5, 0.1 mM Fe),

increases the process efficiency: from 26.1 to 57.0% with Z-FeOOH, from 28.4 to 68.5% with

Z-Fe(III) and from 26 to 56.5% with Z-Fe(II) (Fig. 1). Such behavior of heterogeneous system

is similar to that observed in homogeneous Fenton process. For homogeneous Fenton process,

THMFP decrease was 53% when 1 mM of H2O2 was used and it increased to 76% when the

concentration of 5 mM of H2O2 was used. A similar mechanism can be considered in the

homogeneous and heterogeneous Fenton process with difference that the reactions of Fenton

catalytic cycle take place at the solid surface. A possible degradation mechanism of organic

contaminants includes the generation of OH radicals in the inner surface of the support material

due to the Fenton reaction between H2O2 and iron ions bounded to support material and

thereafter OH radical diffusion into the solution (Neamtu et al., 2004).

In groundwater samples the THMFP reduction efficiency of all heterogeneous processes

was reduced with lowering hydrogen peroxide dose from 5 mM to 1 mM (pH 4.5; 0.1 mM

Fe): from 31.7% to 54.8% when Z-FeOOH, from 56.3% to 34.2% when Z-Fe(III) and from

55.5% to 30.6% when Z-Fe(II) catalyst was used. In homogeneous Fenton process THMFP

reduction was increased with increasing the H2O2 concentration up to the point (2.5 mM)

where the further increase of its concentration caused negative effect. The retard rate of

decomposition of NOM at higher H2O2 dose occurs due to the fact that the OH radicals

generated are converted into hydroxyl ions which in turn precipitate Fe(III) ions and the

deactivation of Fe(II) takes place at higher dosage of H2O2 (Karthikeyan et al., 2011). That

effect has not been observed in heterogeneous Fenton processes at applied H2O2 doses.

Iron form

Supported iron catalysts were prepared by impregnation of three iron form (FeOOH, Fe(II)

and Fe(III)) on support materials. The iron form had little impact on the heterogeneous

Fenton process performance. The efficiency of different heterogeneous catalysts has been

similar both in HA solution and in the natural water samples (Fig. 1; Fig. 2). Only Z-

Fe(III) catalyst, when used in HA solution at low pH value and higher H2O2 and iron

concentration, has shown something better results when compared with other catalysts.

Iron concentration

Support materials bound different amount of FeOOH, Fe(III) and Fe(II) and their portions

on a mass basis was 1.16%, 0.43% and 0.73%, respectively. Slightly more FeOOH was


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introduced into framework of porous support material when compared with Fe(II) and

specially Fe(III) form. The chosen dosages of heterogeneous catalysts correspond to the

concentration of 0.02; 0.05 and 0.1 mM of iron.

Iron concentration had low influence on process performance which is contradictory to the

literature (Kitis et al., 2007; Kušić et al., 2007). Dosage of heterogeneous catalysts does

not significantly influence process efficiency with the exception of Z-Fe(III) catalyst where

the increasing iron content has contributed to the increase of process performance but only

in HA solutions (Table 1; Table 2). In contrast, iron concentration has been an important

factor influencing homogeneous Fenton process for both HA solution and natural water.

Table1. Effect of iron concentration on performance of different Fenton catalysts in HA solution

(5 mM H2O2; pH 2.5)

Fe A254/A203 THMFP decrease

mM %

Initial HA solution

0.6571

F 0.02 0.3061 53.4

0.05 0.2217 66.3

0.1 0.1578 76.0

Z-FeOOH 0.02 0.2853 56.6

0.05 0.2878 56.2

0.1 0.2826 57.0

Z-Fe(III) 0.02 0.2829 57.0

0.05 0.2803 57.4

0.1 0.2071 68.5

Z-Fe(II) 0.02 0.2813 57.2

0.05 0.2839 56.8

0.1 0.2861 56.5

Table 2. Effect of iron concentration on performance of different Fenton catalysts in groundwater

(5 mM H2O2; pH 4.5)

Fe A254/A203 THMFP decrease

mM %

Initial HA solution

0.4299

F 0.05 0.1726 59.9

0.1 0.1172 72.7

Z-FeOOH 0.05 0.1924 55.2

0.1 0.1945 54.8

Z-Fe(III) 0.05 0.1976 54.0

0.1 0.1880 56.3

Z-Fe(II) 0.05 0.1969 54.2

0.1 0.1913 55.5


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Conclusions

When comparing results achieved in optimal conditions, THMFP decrease was less with

heterogeneous Fenton catalysts than with corresponding homogeneous Fenton system.

When the conditions were stepped away from optimal, efficiency of both homogeneous

and heterogeneous processes was similar and in natural groundwater samples zeolite

catalysts have shown even better results than homogeneous Fenton process.

Catalytic activity of the heterogeneous catalyst is less sensitive to the pH than the

homogeneous catalyst. pH independence were suggested to be the major advantage of

heterogeneous processes compared to homogeneous Fenton oxidation. A working pH in

near the neutral range is especially beneficial for the treatment of groundwater which

usually has a high natural buffer capacity and the acidification is uneconomic.

The iron form and dosage had little impact on the heterogeneous Fenton process

performance as opposed to H2O2 dose whose increasing concentration has contributed to

the increase of process performance.

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zeolites on oxidation of Reactive Yellow 84 azo dye in the presence of hydrogen peroxide,

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regenerable Fe-based reduction system for environmental remediation, Chemosphere 81, 22-29.

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Influence of the flow rate on lead and zinc removal from

a binary solution on the fixed bed of natural zeolite

UDC: 628.161.2/.3

Ivona Nuić, Anka Sulić, Marina Trgo, Nediljka Vukojević Medvidović

University of Split, Faculty of Chemistry and Technology, Department for Environmental

Engineering, Teslina 10/V, HR-21000 Split, Croatia

Summary

The influence of the flow rate on lead and zinc removal from equimolar binary aqueous solution on the

fixed bed of natural zeolite was investigated. The results indicate that with the increased flow rate,

breakthrough and exhaustion points appear earlier as expected. Capacities in breakthrough and

exhaustion, qB and qE, are reduced as well as the removal efficiency, due to the shorter contact time

between the feeding solution and the zeolite layer. The ratio of the breakthrough capacity qB(Pb)/qB(Zn)

has similar values and is equal to the concentration ratio co(Pb)/co(Zn) in the feeding solution, indicating

the simultaneous binding of Pb and Zn up to the breakthrough. The ratio of the exhaustion capacity

qE(Pb)/qE(Zn) is higher than qB(Pb)/qB(Zn), confirming greater selectivity of clinoptilolite toward lead

and thus the displacement of zinc already bound in the zeolite with lead from the feeding solution. The

displacement effect is reduced with the increase in the flow rate and confirmed by regeneration curves.

Keywords: column process, binary solution, lead, zinc, displacement effect, flow rate

Introduction

The continuous growth of population and the development of industrial processes have

greatly contributed to increasing concentrations of heavy metals in the biosphere.

Industrial wastewaters are considered to be the greatest source of pollution by heavy

metals (Alloway and Ayres, 1993; Nagajyoti et al., 2010). The removal of heavy metals

from wastewaters to concentrations below the permissible limits requires the application of

advanced wastewater treatment processes such as adsorption, ion exchange and membrane

techniques (Barakat, 2011). Due to their high costs, recent investigations have been

directed to finding solutions suitable for wide application. Excellent physical and chemical

properties of natural zeolites, their adsorption capacity, wide distribution of deposits in

nature and low cost exploitation, contribute to their application in wastewater treatment.

The column performance of ion exchange enables zeolite regeneration which further

allows successive repetitions of several service and regeneration cycles and thus treatment



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of a large quantity of wastewater. This makes natural zeolites even more attractive. The

removal of heavy metal ions from multicomponent systems is very complex due to

differences in ion exchange affinity and the selectivity of zeolite.

The application of the column process requires the basic knowledge of the mass transfer

mechanism between the liquid and the solid phase. Based on our previous investigations at

different bed depths of natural zeolite, the aim of this study was to expand knowledge

about the mass transfer mechanism between the (Pb+Zn) solution and zeolite particles, as

well as about the interaction of Pb and Zn during this mass transfer. The flow rate of the

solution through the fixed bed must be selected to ensure sufficient contact time between

zeolite and the solution, and good wetting. Also, the linear velocity of the solution has to

be less than the linear speed of lowering the mass transfer zone (MTZ). An excessive flow

rate causes a hydraulic load on the zeolite layer in the column, which may lead to the

formation of channels and pores. Therefore, it is very important to examine the impact of

the flow rate and to select the optimal one. This study attempts to describe the lead and

zinc uptake from an equimolar binary solution on the fixed bed of natural zeolite by

changing the flow rate of the feeding solution.


Preparation of the zeolite sample and the binary heavy metal solution

The raw zeolite sample used in these experiments originates from the Zlatokop deposit in

Vranjska Banja (Serbia). According to the semi quantitative mineralogical analysis, the

zeolite sample contains up to 80% of clinoptilolite. The zeolite was milled and sieved to

the particle size of 0.6-0.8 mm, rinsed with ultrapure water and dried at 60 °C. The

equimolar binary solution of lead and zinc ions was prepared by dissolving Pb(NO3)2 and

Zn(NO3)2 6 H2O in ultrapure water, without the pH adjustment (the initial pH value was

around 4.5). The total concentration of the binary solution was constant and equalled

co(Pb+Zn) ≈ 1 mmol/l, with the lead and zinc concentration ratio of co(Pb)/co(Zn) ≈ 1. The

regeneration solution was prepared by dissolving NaNO3, c = 176.5 mmol/l, in ultrapure

water.

Column experiments

Column tests were carried out in a 50 cm long glass column of 1.2 cm internal diameter

filled with zeolite sample up to bed depth of 8 cm. The solution of lead and zinc was fed

through the bed in the down-flow mode at constant flow rates of Q = 1, 2 and 3 ml/min,

using a vacuum pump. After each service cycle, the exhausted zeolite bed was regenerated

with the NaNO3 solution at the flow rate of 1 ml/min, under the same experimental


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conditions. In all experiments, the effluent samples were periodically collected and

analyzed for Zn concentration on a Methrom 761 Compact IC liquid chromatograph and

for the total (Pb+Zn) concentration by the complexometric method.


The effect of the flow rate on Pb and Zn removal

Breakthrough curves obtained at the zeolite bed depth of 8 cm and at flow rates of 1, 2

and 3 ml/min are shown in Fig. 1 as the ratio of the total (Pb+Zn) effluent and influent

concentration vs. service time and volume of the solution passed through the fixed

zeolite bed.

Fig. 1. Breakthrough curves for different flow rates as ratio of c/co vs.

a) service time and b) volume of the effluent

It can be observed that all breakthrough curves follow the characteristic S - shape profile.

With the increase in the flow rate, the breakthrough time occurs earlier as expected, and

the distance between the breakthrough and the exhaustion point decreases. The volume of

the solution treated until the breakthrough shifts towards slightly lower values while the

time for their achievement is significantly shortened. For example, the time required to

reach the breakthrough, tB, and exhaustion point, tE, for the flow rate of 1 ml/min is more

than three times longer than for the flow rate of 3 ml/min (Table 1). Thus, for the examined

experimental conditions, with increased flow rate, it is possible to treat the larger volumes

of the solution in the same time.

Each breakthrough curve is determined by characteristic parameters such as the removal

capacity in the breakthrough and exhaustion point, qB and qE, the column efficiency η, the

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 20 40 60 80t , h

c(P

b+

Zn)/c

o(P

b+

Zn)

.

1 ml/min

2 ml/min3 ml/min

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 2 4 6 8V , l

c(P

b+

Zn

)/co(P

b+

Zn

) .

1 ml/min2 ml/min3 ml/min

a) b)


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height hZ of the mass transfer zone (MTZ) and the empty bed contact time EBCT, which

represents the contact time between the liquid and the solid phase. The above mentioned

parameters have been calculated (Nuić et al., 2013) and listed in Table 1.

Table 1. Experimentally obtained and calculated parameters of breakthrough curves for flow rates

of 1, 2 and 3 ml/min

Service

cycle

Q

ml/min

tB

h

VB

l

tE

h

VE

l

qB

mmol/g

qE

mmol/g (Zn)

(Pb)

B

B

q

q

(Zn)

(Pb)

E

E

q

q η

-

EBCT

min

hZ

cm

1st 1 45.25 2.72 73.08 4.39

Pb+Zn 0.430 0.508

1.06 2.48 0.85 9.04 4.29 Pb 0.222 0.362

Zn 0.208 0.146

2nd 2 18.17 2.18 32.38 3.89

Pb+Zn 0.359 0.515

1.07 2.10 0.70 4.52 4.76 Pb 0.186 0.349

Zn 0.173 0.166

3rd 3 11.75 2.12 22.11 3.98

Pb+Zn 0.319 0.457

1.00 1.82 0.68 3.01 5.03 Pb 0.160 0.295

Zn 0.159 0.162

From Table 1 is clear that the capacity (Pb+Zn) in the breakthrough point decreases with

the increase in the flow rate. The capacity (Pb+Zn) in the exhaustion point has very close

values for flow rates of 1 and 2 ml/min, while its value slightly decreases for the highest

flow rate. The calculated value of hZ increases with the flow rate and it values indicate a

properly chosen bed depth for this range of flow rates. The value of the EBCT is the lowest

for the highest flow rate due to the reduced contact time between the feeding solution and

the zeolite layer. Higher flow rates accelerate the movement of the MTZ downwards in the

fixed bed leading to its faster saturation and thus contributing to the overall decrease of the

column efficiency η (Cruz-Olivares et al., 2013; Tian et al., 2013).

For better understanding of the binary system in Fig. 1, the breakthrough curves for each metal

ion have been analysed as the ratio of c(Pb)/co(Pb) and c(Zn)/co(Zn) vs. time and shown in Fig. 2.

Fig. 2. Breakthrough curves for each metal ion for flow rates of: a) Q = 1 ml/min, b) Q = 2 ml/min

and c) Q = 3 ml/min. Note: c/co = c(Pb)/co(Pb) or c/co = c(Zn)/co(Zn)

0,0

0,4

0,8

1,2

1,6

2,0

0 20 40 60 80t , h

c/c

o

Pb

Zn

0,0

0,4

0,8

1,2

1,6

2,0

0 20 40 60 80t , h

c/c

o

0,0

0,4

0,8

1,2

1,6

2,0

0 20 40 60 80t , h

c/c

o

a) b) c)


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From Fig. 2 is obvious that up to the breakthrough point, lead and zinc ions bind

simultaneous for all three examined flow rates. As the process progresses, the

concentration of Zn in the effluent rapidly increases and for the flow rate of 1 ml/min the

value c/co for Zn reaches ≈ 1.8. The lead concentration continuously increases for the flow

rate of 1 ml/min and does not reach the expected influent concentration. For higher flow

rates of 2 and 3 ml/min zinc again reaches its effluent concentration much higher than its

influent concentration, while the maximum lead concentration in the effluents increases

and reaches half of its influent concentration. A high difference in concentrations of Pb and

Zn in the effluent confirms higher selectivity of zeolite toward Pb ions. Namely,

clinoptilolite is more selective for Pb ions which displace bound Zn ions and finally

occupy most sorption sites (Figueira et al. 2000; Han et al., 2006; Naja and Volesky, 2006;

Stylianou et al., 2007). This displacement probably occurs between the breakthrough and

the exhaustion point, and it is more evident for the lowest flow rate. This is due to longer

mass transfer time which allows Pb and Zn ions to access more binding sites inside the

pores of zeolite particles.

For higher flow rates, due to faster flow of the solution, the displacement effect is reduced.

This may be due to the reduced contact time and therefore lower diffusivity of Pb and Zn

ions from the solution through zeolite particles (Han et al., 2008).

Removal capacities for each metal ion have been calculated (Nuić et al., 2013) and also

presented in Table 1. It can be noticed that the ratio of the breakthrough capacity

qB(Pb)/qB(Zn) has similar values and is equal to the concentration ratio co(Pb)/co(Zn) in the

feeding solution, indicating the simultaneous binding of Pb and Zn until the breakthrough

point. Significant differences in exhaustion capacities of Pb and Zn are expressed through

the ratio qE(Pb)/qE(Zn). This ratio is higher than qB(Pb)/qB(Zn), confirming greater

selectivity of clinoptilolite toward Pb ions and thus the displacement of zinc already bound

in zeolite with lead from the feeding solution, in all three service cycles. The displacement

effect is reduced with the increase in the flow rate.

Regeneration of the zeolite fixed bed

The regeneration cycle provides for recovery of Pb and Zn ions as well as for the reuse of

the zeolite bed for the next service cycle. The fixed zeolite bed is regenerated with a high

concentration of the NaNO3 solution under the same experimental conditions as the service

cycles, but with the flow rate of 1 ml/min. The results obtained during regeneration are

presented in Figs. 3 and 4 and in Table 2.


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Fig. 3. Regeneration curves as the ratio of total (Pb+Zn) concentration in the regenerate vs.:

a) service time and b) volume of the regenerate

Fig. 4. Regeneration curves for each metal ion after the:

a) 1st, b) 2nd and the c) 3rd service cycle

Table 2. Experimentally obtained results and calculated parameters for regeneration cycles

Regeneration

cycle

tR

h

VR

l

VR

BV

cmax

mmol/l

nR

mmol (Zn)

(Pb)

R

R

n

n

1st 14.00 0.84 92.88

Pb+Zn 33.98 4.066

9.39 Pb 31.16 3.675

Zn 5.45 0.391

2nd 8.58 0.52 56.95

Pb+Zn 30.51 2.457

7.50 Pb 28.75 2.168

Zn 4.17 0.289

3rd 8.93 0.54 59.27

Pb+Zn 31.86 2.521

5.62 Pb 28.54 2.140

Zn 7.43 0.381

0

10

20

30

40

0 5 10 15 20

t , h

c(P

b+

Zn

), m

mo

l/l

.

1st cycle

2nd cycle

3rd cycle

0

10

20

30

40

0,0 0,5 1,0 1,5

V , l

c(P

b+

Zn

), m

mo

l/l

.

0

10

20

30

40

0 5 10 15 20

t , h

c,

mm

ol/l

Pb

Zn

0

10

20

30

40

0 5 10 15 20

t , h

c, m

mol/l

0

10

20

30

40

0 5 10 15 20

t , h

c, m

mol/l

a) b) c)

a) b)


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All regeneration curves have an asymmetrical shape. The concentration in the

regeneration effluent increases for a short time, and then suddenly decreases to the

value below the influent concentration. The regeneration curve obtained after the first

service cycle with the lowest flow rate is a little broader compared to the other two,

indicating a higher quantity of eluted ions. The quantities of ions eluted during the

regeneration cycle, nR, have been calculated by graphical integration of the area under

the regeneration curves (Nuić et al., 2013) and listed in Table 2.

From Fig. 3 and Table 2 it can be seen that regeneration is a very fast process compared to

the service cycle, and ends approximately after 8 - 14 hours. For complete regeneration of

the zeolite layer only 0.52-0.84 l NaNO3 solution sufficed, which is approximately 5.2-7.5

times lower compared to the volume of the solution treated in the service cycle. Due to

reduced volume, Pb and Zn concentrations (cmax) in the regenerates are very high.

From the obtained results in Table 2 and regeneration curves of each metal ion in Fig. 4, it

can be concluded that for all examined flow rates the quantity of eluted Pb is much higher

compared to the Zn. That confirms the higher selectivity of zeolite toward Pb and thus

binding of Pb ions in higher amount compared to Zn, in all three service cycles. The ratio

nR(Pb)/nR(Zn) from Table 2 is much higher compared to the ratio of the exhaustion

capacity qE(Pb)/qE(Zn) given in Table 1. This indicates the continued binding of Pb and Zn

ions even after the exhaustion point, as well as the displacement of zinc with lead.

Conclusions

Column tests of Pb and Zn removal from the binary aqueous solution were performed at

the zeolite fixed bed depth of 8 cm by changing the flow rate of the feeding solution. It has

been found that with the increased flow rate, breakthrough and exhaustion points appear

earlier as expected. For the examined experimental conditions, with increasing flow rate, it

was possible to treat larger volumes of the feeding solution in the same time. At higher

flow rates, the mass transfer rate tends to increase, leading to faster saturation of the fixed

zeolite bed. Due to the shorter contact time between the feeding solution and the zeolite

layer, the capacities in breakthrough and exhaustion points are reduced as well as the

column efficiency. The results have shown greater selectivity of clinoptilolite toward Pb

ions, and thus the displacement of Zn bound on zeolite with Pb from the feeding solution,

but the displacement effect is reduced with the increase in the flow rate. Regeneration

cycles provide for recovery of Pb and Zn ions as well as for the reuse of the zeolite bed for

the next service cycle. Regeneration was very fast and it was done successfully with a

small volume of the regeneration solution and without reducing the zeolite capacity. This

makes the column process and natural zeolites increasingly more attractive in the

wastewater treatment.


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Acknowledgements

This work has been fully supported by Croatian Science Foundation under the project

NAZELLT (IP-11-2013-4981).

References

Alloway B.J. & Ayres D.C. (1993): Chemical Principles of Environmental Pollution. Blackie

Academic & Professional. an imprint of Chapman & Hall, Wester Cleddens Road,

Bishopbriggs, Glasgow G64 2NZ, UK, pp. 140-164.

Barakat M.A. (2011): New trends in removing heavy metals from industrial wastewater, Arab. J.

Chem. 4, 361-377.

Cruz-Olivares J., Pérez-Alonso C., Barrera-Díaz C., Fernando Ureña-Nuñez, Chaparro-Mercado

M.C. & Bryan Bilyeu (2013): Modeling of lead (II) biosorption by residue of allspice in a

fixed-bed column, Chem. Eng. J. 228, 21-27.

Figueira M.M., Volesky B., Azarian K. & Ciminelli V.S.T. (2000): Biosorption Column

Performance with a Metal Mixture, Environ. Sci. Technol. 34, 4320-4326.

Han R., ZouW., Li H., Li Y. & Shi J. (2006): Copper(II) and lead(II) removal from aqueous solution

in fixed-bed columns by manganese oxide coated zeolite, J. Hazard. Mater. B137 934-942.

Han. R, Ding D., Xu Y., Zou W., Wang Y., Li Y. & Zou L. (2008): Use of rice husk for the

adsorption of congo red from aqueous solution in column mode, Bioresour. Technol. 99, 2938-

2946.

Nagajyoti P.C., Lee K.D., Sreekanth T.V.M. (2010): Heavy metals, occurence and toxicity for

plants: a review, Environ. Chem. Lett. 8, 199-216.

Naja G. & Volesky B. (2006): Multi-metal biosorption in a fixed-bed flow-through column, Colloid.

Surface. A 281, 194-201.

Nuić I., Trgo M., Perić J. & Vukojević Medvidović N. (2013) Analysis of breakthrough curves of

Pb and Zn sorption from binary solutions on natural clinoptilolite, Micropor. Mesopor. Mater.

167, 55-61.

Stylianou M.A., Hadjiconstantinou M.P., Inglezakis V.J., Moustakas K.G. & Loizidou M.D. (2007):

Use of natural clinoptilolite for the removal of lead, copper and zinc in fixed bed column, J.

Hazard. Mater. 143, 575-581.

Tian Y., Gao B., Morales V.L., Chen H., Wang Y. & Li H. (2013): Removal of sulfamethoxazole

and sulfapyridine by carbon nanotubes in fixed-bed columns, Chemosphere 90, 2597-2605.


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Kompostiranje u školi

UDC: 631.879.4 : 373.3(497.54 Osijek)

Biljana Pavić

Međunarodna Eko-škola OŠ August Šenoa, Drinska 14, 31000 Osijek, Hrvatska

Sažetak

Kompost je vrlo vrijedno prirodno gnojivo koje je nastalo biološkom razgradnjom otpadnih

organskih tvari iz kućanstava tijekom pripreme hrane, organskih tvari iz vrtova ili parkova. Prema

nekim analizama organski otpad čini trećinu komunalnog otpada. Kompostiranje je pravi način

sustavnog gospodarenja organskim otpadom ali i izvrsna prilika da se tlu vrate vrijedne organske

tvari koje su pretvorene u prirodno gnojivo. U skladu sa sedmim milenijskim razvojnim ciljevima

koji su posvećeni održivom razvoju u Međunarodnoj Eko-školi OŠ August Šenoa iz Osijeka već

četrnaest godina učenici i djelatnici škole proizvode kompost.

Ključne riječi: kompostiranje, kompost, ekološki odgoj, održivi razvoj

Uvod

Nalazimo se u UN-ovom Desetljeća za održivi razvoj 2005.-2014. (Decade for Sustainable

Development, DESD), kojemu je cilj da sve zemlje uključi u konkretne strategije obrazovanje

za održivi razvoj (OOR), u njihov razvoj, njihovo kritičko preispitivanje. Također se želi i

ohrabriti škole za uklapanje OOR-a u svakodnevni život škole. Jedan od kriterija kvalitete za

OOR-škole vezan uz procese poučavanja i učenja je učenje kroz aktivnost. Perspektiva

aktivnosti podrazumijeva da učenici, u suradnji s učiteljem, odlučuju nešto poduzeti kako bi

riješili neki problem vezan uz održivi razvoj ili djelovali u smjeru njegova rješavanja te da

nakon toga razmišljaju o procesu aktivnosti. Aktivnost je dakle usmjerena na promjenu:

promjenu u osobnom načinu života učenika i/ili promjenu njihovih lokalnih i globalnih

životnih uvjeta. Osnovni razlog za akciju jest to što sudjelovanje u autentičnim aktivnostima

radi rješavanja nekog problema donosi dragocjenu pouku. (Breiting i sur., 2005).

Približno trećinu kućnog otpada čini organski otpad (Pintarić, 1996) kao što su trava, lišće,

cvijeće, ostaci voća i povrća i sl. Četvrtinu čine papir i karton, staklo oko 8%, plastika isto

toliko, a udio metala je 2% (Priručnik o kompostiranju, UNIKOM, 2014). Otpad neće

postati smeće postupamo li s njim odgovorno. Odgovoran način postupanja s organskim

otpadom je kompostiranje istoga. Kompostiranjem, koje se može provoditi i u školama,

ukupna količina otpada što završava na odlagalištima mogla bi se smanjiti čak za trećinu.



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Učenici Međunarodne Eko-škole Osnovne škole „August Šenoa“ iz Osijeka time

pridonose rješavanju jednog od najsloženijih problema vezanih za zaštitu okoliša u

Hrvatskoj (Pavić i Jelčić, 2010). Stoga su ovim radom htjeli prikazati svim

zainteresiranima mogućnost gospodarenja biootpadom koji svakodnevno stvaramo u

svojim kućanstvima. Kompostirati se može sav biljni otpad iz kuhinje, vrta, voćnjaka i

travnjaka. Najkvalitetniji se kompost dobije ako se dobro izmiješa što više različitoga i

usitnjenoga biljnog materijala, a rabi se: otpad bogat dušikom (ostaci voća i povrća iz

kuhinje, jela od povrća, talog kave i čaja, ljuske luka, kore banana, naranči i lubenica te

pokošena trava, korov iz vrta i uvenulo cvijeće) i otpad bogat ugljikom (lišće, slama,

sijeno, ostatci obrezivanja voćaka, vinove loze i živice, hoblovina i piljevina, iglice

četinara te ljuske od jaja). Kompostirati se međutim nikako ne smije novinski papir,

časopisi u boji, vrećice iz usisivača za prašinu, pelene, pepeo ugljena, izmet mačaka i pasa,

kosti, meso i masnoća te jela od mesa i ribe. Postupak kompostiranja vrlo je jednostavan i

sastoji se od četiri osnovna dijela:

prikupljanja kuhinjskih otpadaka, pokošene trave i ostataka biljaka iz vrta koje valja

usitniti i pomiješati s otpalim lišćem te usitnjenim drvenastim materijalom

odlaganja svih sastojaka s miješanjem i dodavanjem kamene prašine te punjenja

kompostera ili oblikovanja hrpe, a prema potrebi valja navlažiti vodom te zaštititi od

oborina i ostaviti da miruje

preokretanja nakon 6 tjedana kada masu valja ponovno dobro izmiješati i potom vratiti na

hrpu ili u komposter te navlažiti ako je suha ili kad je previše vlažna posuti kamenom

prašinom i dodati suhoga lišća i drvenih isječaka

zrenja koje je završeno nakon 6 do 12 mjeseci i kada je kompost spreman za uporabu, a

pritom što sitniji dijelovi materijala i dodatna preokretanja poboljšavaju kvalitetu i

ubrzavaju proces (Mali priručnik za kompostiranje, Centar za kompost, 2005).

Materijali i metode

Dva do tri puta tijekom školske godine učenici i djelatnici škole prikupljaju organski otpad

po tjedan dana kod kuće i donose ga u školu gdje oforme kompostne hrpe od njega i onog

nastalog u školi. U radionici o kompostiranju u listopadu prvašići upoznaju osnovna

pravila kompostiranja. Organski otpad usitne na dužinu palca (Slika 1) kako bi

mikroorganizmi (bakterije i gljivice) imali olakšan pristup ugljiku koji treba preraditi. Na

taj način ti razlagači brže dolaze do „hrane“ i sam postupak kompostiranja kraće traje.

Drveni komposter volumena 1m3

smješten je u hladovini breze u školskom dvorištu na

propusnom tlu. Na dno kompostera slože prvi sloj od grubo usitnjenih grančica, kako bi se

osigurao protok zraka pri dnu kompostne hrpe. Nakon toga slažu meki, mokri kuhinjski

otpad sa suhim, drvenastim otpadom (sjeckanim grančicama) u slojevima debljine oko 20


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cm u omjeru 1:1. Svaki sloj pospu tankim slojem starog komposta i zaliju prema potrebi.

Prekriju top-tex platnom kako bi kompostnu hrpu zaštititli od sunca i oborina.

Tijekom faza kompostiranja učenici prate temperaturu digitalnim termometrom DT 02

(Slika 3). U vrijeme faze raspadanja (uz temperaturu od najmanje 60 °C) kompostnu hrpu

tri do 4 puta promiješaju (Slika 5), kako bi temperaturnom higijenizacijom bio obuhvaćen

sav materijal koji kompostiraju. Izmjere metrom i volumen svježeg komposta (Slika 2).

Kompostnu hrpu pri miješanju iduća tri do četiri tjedna i nadopunjavaju. Nakon šest

mjeseci kompostnu hrpu prosiju (Slika 6).

Slika 1. Formiranje kompostne hrpe

Fig. 1. Formation of the compost pile

Slika 2. Izračunavanje količine unesenog svježeg organskog otpada

Fig. 2. Calculation of the entered fresh organic waste


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Slika 3.Praćenje temperature tijekom faza kompostiranja

Fig. 3. Temperature monitoring during the phases of composting

Slika 4. Temperatura kompostne hrpe na dubini 50 cm (plavo),

10 cm (crveno) i vanjska temperatura (zeleno)

Fig. 4. Temperature of the compost pile at a depth of 50 cm (blue),

10 cm (red) and outside temperature (green)

0

10

20

30

40

50

60

70

31. 5. 2. 6. 8. 6. 25. 6. 9. 10.

Tem

pe

ratu

ra (

º C)

Datum


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Slika 5.Preslagivanje kompostne hrpe

Fig. 5. Mixing of the compost pile

Slika 6. Prosijavanje kompostne hrpe

Fig. 6. Sifting of the compost pile


U fazi razgradnje bakterije i kvasci svojim metabolizmom razgrađuju organske tvari pri čemu

nastaje toplina koja se može uočiti mjerenjem. Temperatura je jedan od ključnih pokazatelja

promjena koje se događaju tijekom kompostiranja (Trautmann i Krasny, 1997). Najviše

vrijednosti 60-65 °C postižu se (ovisno o postojećim vanjskim uvjetima) nakon tri do 5 dana

razdoblja raspadanja. U kompostnoj hrpi (oformljenoj 28. svibnja 2001.) 31. svibnja 2001.

godine na dubini od 50 cm učenici su izmjerili temperaturu od 58 °C, na dubini od 10 cm 55 °C,

dok je vanjska temperatura iznosila 25 °C (Slika 4). Šest dana nakon formiranja kompostne

hrpe (2. lipnja 2001.) temperatura u kompostnoj hrpi na dubini 50 cm iznosila je 42 °C, a na

dubini 10 cm 40 °C. Vanjska temperatura bila je 19 °C. Kompostnu hrpu su u vrijeme faze

raspadanja u tri navrata preslagivali. U fazi prerade temperatura lagano opada (jer bakterije

razgrade sve razgradljive tvari) približavajući se vrijednostima temperature okoline. Dvanaest


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dana od formiranja kompostne hrpe (8. 6. 2001.) na dubinama od 50cm i 10 cm temperatura je

bila izjednačena i iznosila je 27 °C, dok je vanjska bila 23 °C. Dvadeset petog lipnja 2001.

godine na dubini od 50 cm izmjereno je 25 °C, na dubini 10 cm 23 °C, a vanjska temperatura

bila je 19 °C. U fazi izgradnje pojavljuju se protozoe koje se hrane bakterijama i gljivicama, a

nakon njih i prvi višestanični organizmi (stonoge, mravi, nematode, grinje, pauci i kišne gliste).

Ovaj proces traje nekoliko mjeseci, a nakon toga kompost poprima tamnosmeđu boju. Kišnih

glista i drugih organizama sve je manje i pojavljuje se karakterističan miris „šumske zemlje“.

Nakon pet mjeseci (9. 10. 2001.) temperature na 50 cm i 10 cm iznosile su 21 °C odnosno 20 °C,

dok je vanjska temperatura 17 °C. Na kraju ove faze dobili su svježi kompost spreman za

prihranu. Korise ga za prihranu školskog cvijeća (Slika 9) i drveća u edukativnom parku, kao i

uzgoj cvijeća, bundeva i tikvica (Slika 8). Prosječno su u ovih četrnaest godina kompostiranja

proizveli 1,42 m3 komposta godišnje (Slika 7). Prije su imali dva kompostera za proizvodnju

komposta i dva za gotovi kompost, no sada imaju jedan za proizvodnju i dva za gotovi

kompost. Tijekom godina susretali su se i s vandalima koji su devastirali kompostere. No nisu

se dali smetati, popravili su ih i nastavili kompostirati. Aktivno surađuju i sa Centrom za

kompost iz Osijeka, a u suradnji s njima i UNIKOM-om d.o.o. snimili su i spot o

kompostiranju. Svoja iskustva o kompostiranju rado dijele s drugima, te su im česti gosti

vrtićarci, učenici osnovnoškolskog i srednjoškolskog uzrasta, te studenti. U sklopu projekta

“The case for Zero Waste - creating preconditions for zero waste society in cross border

region” kojeg u Hrvatskoj provode Unikom d.o.o., Regionalna razvojna agencija Slavonije i

Baranje i Udruga za zaštitu prirode i okoliša Zeleni Osijek, a u Srbiji Grad Srijemska

Mitrovica, njihovo komunalno poduzeća i ekološke udruge, škola Šenoa im je poslužila kao

model dobre prakse. Broj učenika i roditelja koji su se i sami okušali u kompostiranju u vlastitim

domovima povećao se u odnosu na razdoblje od prije četrnaest godina. Kompostiranjem ujedno

razvijaju i poduzetničke kompetencije u skladu sa razvojnim kurikulumom škole.

Slika 7. Količina proizvedenog komposta (m3) u razdoblju 2000.-2014.

Fig. 7. The amount of produced compost (m3) in the 2000.- 2014. period

0

0,5

1

1,5

2

2,5

Pro

izvedeno m

3 k

om

posta

Školska godina


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Slika 8. Branje tikvica uzgojenih u školskom dvorištu koje su prihranjene kompostom

Fig. 8. Picking compost-grown zucchini in the schoolyard

Slika 9. Školsko cvijeće prihranjuju vlastitim kompostom

Fig. 9. The produced compost is added to the school flowers

Zaključci

Ulaganje u odgoj i obrazovanje za okoliš i održivi razvoj dugoročna je i isplativa

investicija. Selekcijom i iskorištavanjem korisnog otpada smanjujemo količine otpada na

odlagalištu koju je potrebno trajno zbrinuti. Na to nas obvezuje i Zakon o održivom

gospodarenju otpadom.


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Literatura

Breiting, S., Mayer, M., Mogensen, F. (2005): Kriteriji kvalitete za OOR-škole: Obrazovanje za

održivi razvoj u školama -Smjernice za razvoj kriterija kvalitete, Beč, Austrija:Austrijsko

Savezno ministarstvo obrazovanja, znanosti i kulture, pp. 28-29.

Pavić, B., Jelčić, M. (2010): Kompostiramo i zdraviju budućnost biramo, Dijete, škola, obitelj (26),

18-20.

Pintarić, A. (1996): Sastav komunalnog otpada, In: Zbornik rezultata projekta Obrazovanje za

okoliš, Osijek, HR, pp. 3.

Trautmann, N. M., Krasny, M. E. (1997): Composting in the Classroom Scientific Inquiry for High

School Students, New York, USA:Cornell University, pp. 52.

Udruga Centar za kompost, Osijek (2005): Mali priručnik za kompostiranje, Osijek, Hrvatska:

Centar za kompost pp.7-13.

Unikom d.o.o., Osijek (2014): The case for Zero Waste (Stvaranje društva bez otpada)-Priručnik o

kompostiranju, Osijek, Hrvatska: Unikom d.o.o., Udruga za zaštitu prirode i okoliša, Zeleni

Osijek, Regionalna razvojna agencija Slavonije i Baranje, pp.3.

Composting at school

Biljana Pavić

International Eco-Schools OŠ August Šenoa, Drinska 14, HR-31000 Osijek, Croatia

Summary

Compost is a valuable natural fertilizer that has arisen from biological decomposition of waste

organic matter from households during food preparation and organic substances from gardens or

parks. According to some analyzes, organic waste accounts for a third of municipal waste.

Composting is the right way to systematically manage organic waste, but also a great opportunity

for returning to the soil valuable organic substances which are converted into natural fertilizer. In

accordance with the seventh Millennium Development Goals that are dedicated to sustainable

development, the pupils and staff at the International Eco-School Elementary School August Šenoa

from Osijek have already been producing compost for fourteen years.

Keywords: composting, compost, ecological education, sustainable development


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Uklanjanje nitrata iz poplavom onečišćenih voda

UDC: 628.19


Prirodoslovna škola Vladimira Preloga, Ulica grada Vukovara 269, 10000 Zagreb, Hrvatska

Sažetak

Globalne promjene klimatskih prilika uzrokuju nenadane i za ove generacije nedoživljene

elementarne nepogode. Klimatske promjene nedvojbeno su pridonijele povećanju vjerojatnosti i

negativnih učinaka ove pojave u području sjeverne Hrvatske. Poplave negativno utječu na zdravlje

ljudi, okoliš, kulturnu baštinu i gospodarsku aktivnost. Poplavljena područja su ujedno i područja

intenzivne poljoprivrede. Mnoga domaćinstva na tom području imaju vlastiti izvor vode (bunar ili

bušotinu) koji mogu koristiti za svoje potrebe u neograničenim količinama. Međutim, poplavne

vode ispiru poljoprivredno zemljište odvodeći u podzemlje, a time i u bunare, velike količine

umjetnih gnojiva, herbicida, pesticida. Načinjena je spektrofotometrijska analiza nitrata poplavnih

voda s područja Čićka Poljana i Petrovina turopljska pri čemu je, očekivano, utvrđena povećana

koncentracija nitrarnih iona 97 mg/L. Također je istom metodom načinjena analiza bunarske vode

nakon povlačenja poplavnih voda. I u bunarskoj vodi utvrđena je povećana koncentracija nitrarnih

iona 67 mg/L. Takva voda ne smije se upotrebljavati u kućanstvu, a pogotovo piti. Nitrati se moraju

ukloniti iz bunarskih voda. Nitrati se iz otpadnih voda mogu ukloniti postupkom ionske izmjene.

Tim postupkom uspješno se uklanjaju nitrati na razinu ispod maksimalno dozvoljene koncentracije.

U ionskoj izmjeni kao sredstvo za regeneraciju koristili smo otopinu KCl koji u reakciji sa nitratnim

ionima daje KNO3(aq), tj. otopinne umjetnog gnojiva koji se može iskoristiti.

Ključne riječi: poplavne vode, bunari, nitrati, ionska izmjena

Uvod

U suvremeno doba raste potrošnja vode u industriji i urbanim sredinama. Upotrijebljena

voda sadrži onečišćenja (organski i anorganske tvari), koja se ispuštaju u vodotokove,

jezera ili mora. Onečišćenja ugrožavaju biološku ravnotežu vodnih ekosustava, a ovisno o

količini i vrsti onečišćenja mogu dovesti u pitanje i njihov opstanak. Površinske vode

napajaju podzemne vodotokove i tako obnavljaju zalihe podzemnih voda koje su izvor

vode za ljudsku potrošnju. Narušena kakvoća površinske vode i sve veća potrošnja čiste

podzemne vode ugrožavaju kakvoću i opstanak izvora vode za ljudsku potrošnju.

Globalne promjene klimatskih prilika uzrokuju nenadane i za ove generacije nedoživljene

elementarne nepogode. U prvoj polovici 2014. godine svjedoci smo velikih poplava na



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području Velike Gorice i Siska, te na području Sisačko moslavačke županije. Klimatske

promjene nedvojbeno su pridonijele povećanju vjerojatnosti i negativnih učinaka ove

pojave u području sjeverne Hrvatske. Poplave negativno utječu na zdravlje ljudi, okoliš,

kulturnu baštinu i gospodarsku aktivnost.

Poplavljena područja su ujedno i područja intenzivne poljoprivrede. Mnoga domaćinstva na

tom području imaju vlastiti izvor vode (bunar ili bušotinu) koji mogu koristiti za svoje potrebe

u neograničenim količinama. Poplavne vode ispiru poljoprivredno zemljište odvodeći u

podzemlje, a time i u bunare, velike količine umjetnih gnojiva, herbicida, pesticida.

Cilj rada

Cilj rada je utvrditi koncentraciju nitratnih iona u poplavnom vodama i pokazati da je

metodom selektivne ionske izmjene moguće ukloniti ove ione iz vode i na taj način ih

dovesti ponovno u kategoriju voda za ljudsku potrošnju.

Upravljanje vodama jedan je od temelja kvalitetnog suživota čovjeka i okoliša koji ga

okružuje. Pri tome vodno gospodarstvo ima značajnu ulogu na uređenju vodnog režima i

stavljanja vodnih resursa u funkciju poboljšanja kvalitete života stanovništva i

gospodarskog razvitka, ali i obavezu da istovremeno poduzimanjem odgovarajućih mjera

vodi brigu o zaštiti ljudskog zdravlja i očuvanju dobrog ekološkog stanja tih istih vodnih

resursa i okoliša u cjelini.

Izvori onečišćenja (Tedeschi, 1997), prirodnih vododtokova su kućanske i industrijske, te

oborinske vode. Kućanske i industrijske otpadne vode prikupljaju se sustavom kanala te

ispuštaju u vodne sustave kanalskim ispustima. Oborinske vode izravno iz atmosfere

dospijevaju u vodne sustave ili nakon ispiranja površine šuma, livada i poljoprivrednih

površina. Uslijed sve većeg onečišćenja atmosfere i zemljišta, oborinske vode čine

značajan izvor onečišćenja prirodnih voda. Posebice se to odnosi na poplavne vode.

Onečišćenje oborinskih voda nastaje još u atmosferi. Prolazeći kroz onečićenu atmosferu

voda otapa mnoge plinove, dimove te dospijeva na tlo već promijenjene kakvoće. U

pojedinim se industrijskim područjima oborinska voda pojavljuje sa sniženom vrijednosti

pH, pa se takve padaline nazivaju „kisele kiše“.

Pri otjecanju po površini, voda otapa pojedine tvari s tla ovisno o agresivnosti vode te

sastavu zemljišta. U poljoprivredi danas se pojačano primjenjuju umjetna gnojiva i

različite vrste pesticida. U vodama koje ispiru poljoprivredna tla mogu se očekivati

hranjive soli i nerazgrađeni pesticidi.

Nitatni ioni u otpadnim vodama

U područjima intenzivne poljoprivrede, oborinske vode ispiru poljoprivredno zemljište

odvodeći u podzemlje velike količine umjetnih gnojiva, herbicida, pesticida. Budući da je


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jedan od glavnih sastojaka umjetnih gnojiva dušik to predstavlja i glavni izvor zagađenja

voda spojevima dušika, nitratima i nitritima (Matošić, 1999). Prisutnost povećanih

koncentracija nitrata u okolišu i otpadnim vodama je posljedica nepropisnog odlaganja

otpadnih voda, industrijske proizvodnje, poljoprivrednih aktivnosti i drugih ljudskih

aktivnosti. Otpadne vode brojnih industrijskih procesa kao što su proizvodnja lijekova,

nitro-aromatskih spojeva, nuklearnih goriva, mineralnih gnojiva i dr. sadrže nitratne ione u

vrlo širokom rasponu koncentracija od 100 do 3000 mg/L. U cilju zaštite okoliša nitrati se

moraju ukloniti iz otpadne vode.

Kalijev nitrat (KNO3, salitra) bijeli je kristal topljiv u vodi. U prirodi nastaje nitrifikacijom

organskih spojeva s dušikom u prisutnosti kalijevih soli, upotrebljava se kao umjetno gnojivo.

Ako uklonimo nitrate iz otpadnih voda postupkom ionske izmjene prilikom regeneracije

dobivamo umjetna gnojiva jer nitratni ioni reagiraju s regeneracijskim sredstvom (otopina

KCl ili KOH) i daju umjetno gnojivo. Time otpadnu otopinu koja nastaje ionskom

izmjenom možemo iskoristiti čime doprinosimo zaštiti okoliša.

Analiza vode

Voda koja se nalazi u prirodi ima svestranu uporabu u kućanstvu te u industriji. U kućanstvu

se upotrebljava za kuhanje, pranje, a u industriji za proizvodnju pare u parnim kotlovima, za

rashladne uređaje, kao pomoćna sirovina u raznim tehnološkim procesima kemijske

industrije, industrije tekstila, prehrambene industrije itd. Prirodna voda, podzemna ili

površinska, nije kemijski čista, nego sadržava razne otopljene tvari, najčešće soli.

Voda ljudsku potrošnju mora biti bistra, ugodna okusa, bezbojna, bez mirisa i ne smije

sadržavati povećane koncentracije mangana, željeza, nitrata, nitrita i amonijaka, odnosno

mora odnosno mora udovoljavati odredbama Pravilnika o parametrima sukladnosti i

metodama analize vode za ljudsku potrošnju (NN, 125/2013).

Tablica 1. Prikaz maksimalno dopuštenih vrijednosti (MDK) pojedinih kemijskih parametara u

vodi za ljudsku potrošnju prema Pravilniku o parametrima sukladnosti i metodama

analize vode za ljudsku potrošnju (NN, 125/2013).

Table 1. Review of maximum permissible level (MRL) of certain chemical parameters in water for

human consumption according to the Regulations on parameters of conformity and

methods of analysis of water for human consumption (NN 125/2013).

Komponente Mjerne jedinice MDK

amonijak mg/L(NH4)+ 0,5

nitriti mg/L(NO2)ˉ 0,1

nitrati mg/L(NO3)ˉ 50

kloridi mg/L(Clˉ) 250

željezo µg/L(Fe) 200

mangan µg/L(Mn) 50


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Analiza vode obuhvaća:

- uzimanje uzoraka vode,

- određivanje fizikalnih pokazatelja,

- određivanje kemijskih pokazatelja,

- određivanje mikrobioloških pokazatelja.

Uzimanje uzoraka prirodne vode

Uzorci vode posebno se uzimaju za ispitivanje fizikalnih i kemijskih svojstava, a posebno

za bakteriološko ispitivanje (Weihnacht i sur., 2009).

Uzimanje uzoraka za fizikalno i kemijsko ispitivanje:

a) Prije uzimanja uzoraka ruke treba oprati sapunom.

b) Uzima se triputa po 1 dm3.

c) Boca mora biti čista, a prije punjenja treaba je još nekoliko puta isprati vodom koja

se ispituje.

d) Iz izvora, potoka ili rijeka uzorak se uzima tako da se boca zaroni oko pola metra

ispod površine tako da se izbjegnu površinske nečistoće, a grlo boce se okrene

prema struji vodotoka.

e) Na bocu se stavi naljepnica s naznakom: mjesto uzimanja uzorka, datum i sat

uzimanja uzorka te ime i prezime osobe koja je uzela uzorak.

Slika 1. Uzorkovanje uzorka poplavne vode u Crnoj Mlaki

Fig. 1. Sampling sample flood waters in the Crna Mlaka


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UV Spektrofotometrija

Spektralnom analizom mjeri se energija zračenja koju apsorbira ili emitira neka tvar

odnosno ion, atom ili molekula. Energija zračenja određena je frekvencijom, brzinom

širenja i valnom duljinom. Ona se može apsorbirati ili emitirati samo u određenim

kvantima energije ako atom, ion ili molekula prime određenu količinu energije i prijeđu u

pobuđeno, više energetsko stanje. Ako je stanje stabilno, apsorbiraju se određene

frekvencije dovedene energije. Kod nestabilnog energetskog stanja višak se energije

oslobađa, emitira zračenjem (Weihnacht i sur., 2009; Skoog i sur., 1999).

Za određivanje u UV, VIS i IR dijelu spektra koriste se spektrofotometri. Valne duljine s

kojima možemo raditi na spektrofotometrima prikazane su u tablici 1.

Tablica 2. Spektrofotometrija u UV/VIS području

Table 2. Spectrophotometry in the UV / VIS area

Područje spektra /nm

dalje ultraljubičasto 100 – 200

bliže ultraljubičasto 200 – 400

Vidljivo 400 – 800

bliže infracrveno 800 – 2000

Infracrveno 2000 – 25000

dalje infracrveno 25000 – 100000

Instrumenti koji električnim putem mjere apsorbanciju zovu se apsorpcijski spektrometri.

Najvažniji dijelovi instrumenta koji se primjenjuju u apsorpcijskoj spektrofotometriji jesu:

izvor svjetlosti, monokromator, kivete i držači za kivete, uređaj za mjerenje intenziteta

propuštene svjetlosti.

Slika 2. Shematski prikaz apsorpcionog spektrofotometra - 1. Izvor svjetlosti, 2. i 3. Ogledala za usmjeravanje zraka svjetlosti, 4. Otvor, 5. Polikromatsko svjetlo, 6. Prizma,

7. Kiveta s otopinom, 8. Detektor

Fig. 2. Schematic representation of absorption spectroscopy - first light source, second and third

mirrors to direct the beam of light, the fourth hole, 5th polychromatic light,

Prism sixth, seventh cuvette with a solution, eighth detector


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U apsorpcijskoj spektroskopiji za vidljivi dio spektra najčešće se koristi svjetiljka s

volframovom niti. Karakteristika te svjetiljke je emitiranje svjetlosne energije različitih

valnih duljina te konstantnost emitirane energije. Monokromator je sastavni dio svakoga

apsorpcijskog spektrofotometra pomoću kojeg možemo iz polikromatskog svjetla dobiti

monokromatsko svjetlo točno određenih valnih duljina. Za ultraljubičasti dio spektra (UV

zračenje ne propušta obično staklo) kivete su kvarcne. Važne karakteristike uređaja za

mjerenje jačine prolazne svjetlosti jesu: osjetljivost na određenu valnu duljinu, efekt

zamora i rad sustava na pretvorbu osjetljivosti. Pretvorba intenziteta svjetlosti u neku

mjerljivu veličinu određuje se pomoću fotoćelija ili fotocijevi.

Svi optički dijelovi spektrofotometra načinjeni su od kvarcnog stakla (koje ne apsorbira u

VIS i UV dijelu spektra). Razlika intenziteta zraka svjetlosti koje prolaze kroz otapalo

ovisi o apsorpciji u uzorku. Kad se te razlike intenziteta svjetlosti prikažu u ovisnosti o

valnoj duljini upadnog snopa, dobije se apsorpcijski spektar.

Ionska izmjena

Ionski su izmjenjivači najčešće čvrste u vodi netopljive organske smole, polimerni

materijali, koje u svojoj strukturi imaju određene aktivne skupine (Weihnacht i sur., 2009;

Skoog i sur., 1999). Te aktivne skupine imaju sposobnost reverzibilnog vezanja kationa,

odnosno aniona iz otopine, pri čemu iz aktivnih skupina otpuštaju H+, odnosno OH

− ione.

S obzirom na to da u vodenim otopinama aktivne skupine, koje su vezane na netopljivu

polimernu rešetku, disociraju, razlikuju se kationski i anionski izmjenjivači (kationska i

anionska smola). To se može prikazati kemijskim jednadžbama:

Kationski izmjenjivač: IZ−SO3H ⇄ IZ−SO3− + H

+

Anionski izmjenjivač: IZ−NR3OH ⇄ IZ−NR3− + OH

−

gdje je:

IZ polimerna rešetkasta struktura, smola;

−SO3H kationska aktivan skupina (kisela);

−NR3OH anionska aktivna skupina (bazična);

−R – alkilni supstituent (radikal)

Ako su u otopini prisutni kationi kao na primjer Ca2+

, Mg2+

, Na+ i drugi, oni će se vezati na

aktivnu skupinu kationskog izmjenjivača, dok će se anioni kao što su. HCO3−, SO4

2−, NO3

−

i drugi vezati na aktivnu anionsku skupinu. To se može prikazati kemijskim jednadžbama

(Skoog i sur., 1999):

2 IZ−SO3H + Ca2+

⇄ (IZ−SO3)2Ca + 2 H+

2 IZ−NR3OH + CO32−

⇄ (IZ−NR3)2CO3 + 2 OH−


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Proces ionske izmjene ne može neograničeno teći. Kad se slobodne aktivne skupine zasite,

odnosno kad se uspostavi ravnoteža, prestaje ionska izmjena i otopina kroz izmjenjivač

prolazi nepromijenjena. Kad ionski izmjenjivači više ne mogu vezati katione, odnosno

anione iz otopine, treba ih prevesti u aktivni oblik zamjenom vezanih kationa s ionom H+,

odnosno kod anionskih izmjenjivača vezanjem iona OH− na mjestu prije vezanih aniona.

Taj se postupak naziva regeneracijom ionskih izmjenjivača. Pri tom se kationski

izmjenjivači regeneriraju (Skoog i sur., 1999) s otopinom HCl, w(HCl(aq)) = 0,09, a

anionski s otopinom NaOH, w(NaOH(aq)) = 0,05.

Materijali i metode

U radu smo se za analizu nitrata u poplavnim vodama koristi spektrofotometrijskom

metodom, a za uklanjanje nitrata iz uzorka vode koristili smo metodu ionske izmjene

(Weihnacht i sur., 2009; Skoog i sur., 1999; Mijatović i Šljivović, 1999).

Uzorkovanje

Za provedbu eksperimenta uzeti su uzorci u polavljenim područjima oko Čičke Poljane.

Slika 3. karta područja na kojem su uzimani uzorci voda

Fig. 3. Map of the area where water samples were taken


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Određivanje nitrata spektofotometrijski

Za određivanje nitrata spektofotometrijski koristili smo instrument UV/VIS

spektofotometar λ-10 s kvarcnom kivetom. Prema zadanim koncentracijama pripremili

smo otopine uzorka kojima smo mjerili apsorbanciju na 230 nm i prema baždarnom

dijagramu određivali koncentraciju nitrata. Uzorak vode mora biti potpuno bistar.

Tablica 3. Tablica izmjerenih podataka

Table 3. Table of measured data

Koncentracija /mg/L Apsorbancija pri 230 nm

100 3,938

150 4,019

200 4,204

250 4,291

300 4,710

Slika 4. Baždarni dijagram ovisnosti apsorbancije o masenoj koncentraciji

Fig. 4. Diagram Gauge dependence of absorbance on the concentration by weight


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Ionska izmjena u koloni

Za denitrifikaciju u koloni upotrijebljena je staklena kolona promjera 1 cm i visine 25 cm s

filterom od sinter stakla na dnu (Mijatović i Šljivović 1999). Kolona je napunjena

suspenzijom od nabubrenog ionskog izmjenjivača i vode. Otopine za regeneraciju i za

pokuse uklanjanja nitrata dovođene su u kolonu s vrha. Protok otopine kroz kolonu bio je

330 mL/h. Aparatura je prikazana na slici 5. Regenerirani izmjenjivač upotrijebljen je u

pokusima uklanjanja nitrata. Modelne otopine nitrata za eksperimentalni dio ovog rada

priređene su otapanjem KNO3 u željenih koncentracija nitrata.

Slika 5. Kolona napunjena ionskim izmjenjivačem

Fig. 5. The column filled with ion-exchange resin

Izmjenjivače smo regenerirali s otopinom KCl i isprali demineraliziranom vodom do

negativne reakcije na kloride. Za pokuse s izmjenjivačima u hidrogenkarbonatnoj formi

izmjenjivač smo nakon prevođenja u kloridnu formu regenerirali s NaHCO3 i zatim isprali

demineraliziranom vodom. Za prevođenje u miješanu kloridnu i hidrogenkarbonatnu

formu upotrebljena otopina KCl pomiješana je s otopinom NaHCO3.


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Načinjena je spektrofotometrijska analiza nitrata poplavnih voda s područja Čičke Poljane

pri čemu je, utvrđena povećana koncentracija nitrarnih iona - 76,00 mg/L. Također je

istom metodom načinjena analiza bunarske vode nakon povlačenja poplavnih voda, te je i

u bunarskoj vodi utvrđena je povećana koncentracija nitrarnih iona – 67,00 mg/L. Takva

voda nije u skladu s odredbama Pravilnika o parametrima sukladnosti i metodama analize

vode za ljudsku potrošnju te se ne bi smjela upotrebljavati u kućanstvu niti konzumirati te

bi bilo neophodno prije uporabe iz analiziranih podzemnih voda ukloniti nitrate.

Tablica 4. Rezultati spektrofotometrijske analize uzoraka

Table 4. Results spectrophotometric analysis of samples

Uzorak vode (NO3

)/mg/L

Površinska voda 76,00

Bunarska voda 67,00

Nitrati su iz otpadnih voda uklonjeni postupkom ionske izmjene. U tablici 5 prikazani su

rezultati spektrofotometrijske analize uzoraka nakon propuštanja istih kroz ionski imjenjivač.

Tablica 5. Rezultati spektrofotometrijske analize uzoraka nakon propuštanja uzoraka kroz ionski

izmjenjivač

Table 5. Results spectrophotometric analysis of samples after passing samples through ion exchanger

(NO3

)/mg/L pri ulazu u kolonu (NO3)/mg/L pri izlazu iz kolone

76,00 0,00

67,00 0,00

Zaključci

Proces ionske izmjene je jednostavan postupak obrade vode za ljudsku potrošnju a

učinkovitost uklanjanja nitrata je vrlo visoka. Naime, u procesu ionske izmjene dolazi do

potpunog uklanjanja nitrata iz vode. Da bi se smanjili troškovi obrade, obrađena voda

može se miješati s neobrađenom u omjeru koji daje koncentraciju nitrata u izlaznoj vodi

nižu od dozvoljene Pravilnikom o parametrima sukladnosti i metodama analize vode za

ljudsku potrošnju. Prednosti ionske izmjene nad ostalim dostupnim procesima doveli su do

njene široke prihvaćenosti u razvijenom svijetu.

Tim postupkom uspješno se uklanjaju nitrati ispod MDK, vrijednosti propisane navedenim

Pravilnikom. U ionskoj izmjeni kao sredstvo za regeneraciju koristili smo otopinu KCl jer

nitratni ioni reagiraju s regeneracijskim sredstvom (otopina KCl ili KOH) pri čemu se


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nastala otopina nakon regeneracije može koristit kao tekući supstrat, odnosno tekuće

umjetno gnojivo čime se doprinosi zaštiti okoliša, a napose vodnih resursa.

Literatura

Matošić, M. (1999): Dentrifikacija vode za piće selektivnom ionskom izmjenom, magistarski rad,

PBF, Zagreb.

Mijatović, I., Šljivović, Z. (1999): Propis za vježbe iz tehnologije vode i goriva; inertna skripta, PBF, Zagreb.

Skoog, D. A., West, D. M., Holler, F. J. (1997): Osnove analitičke kemije, Školska knjiga, Zagreb.

Tedeschi, S. (1997): Zaštita voda, HDGI, Zagreb.

Weihnacht, Z., Rupčić Petelinc, S., Žužek, S. (2009): Praktukum iz analitičke kemije, Školska knjiga,

Zagreb.

Removal of nitrate from contaminated flood water


School of natural science Vladimir Prelog, Ulica grada Vukovara 269, HR-10000 Zagreb, Croatia

Summary

Changes in global climate couse sudden and for these generations new and extreme natural

disasters. Climate change have undoubtedly contributed to the increased probability and negative

effects of this phenomenon in the area of the northern Croatian. Floods have a negative impact on

human health, the environment, cultural heritage and economic activity. The flooded areas are also

areas of intensive agriculture. Many households in the area have their own water source (well or

borehole) which can be used for their needs in unlimited quantities. However, flood waters that have

washed farmland, carried away to the underground, and thus in the wells, large quantities of

chemical fertilizers, herbicides, pesticides. A spectrophotometric analysis has been made of nitrate

in flood waters from the field Čićka Poljana i Petrovina turopljska where, as expected, had high

concentration of ions nitrarnih 97 mg /L. It's also the same method made analysis of well water after

the recession of floodwaters. And in the well water showed the increased concentration of ions

nitrarnih 67 mg /L. Such water may not be used in the household, and especially not for drinking.

Nitrates must be removed from water wells. Nitrates from waste water can remove the ion exchange

process. This procedure successfully removes nitrates to a level below the maximum permissible

level. In the ion exchange as a means of regenerating used a KCl solution which react with the

nitrate ions gives KNO3 (aq), ie. Fertilizer solutions that can be utilized.

Keywords: floodwaters, water well, nitrates, ion exchange


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Drinking water quality assessment from private well

UDC: 628.112(497.47) : 543.06

Marjana Simonič

University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova 17, 2000 Maribor,

Slovenia

Summary

The quality of drinking water from a private well in northern Slovenia was studied. It was

suspected that water could be polluted by some specific pollutants as a consequence of fertilization.

Physico-chemical analyses were performed in the laboratory for water treatment in order to

determine the water quality. The results of the physico-chemical analysis did not show any elevated

values. The ammonium (N-NH4), nitrate and nitrite concentrations, potassium concentration as well

as chemical oxygen demand (COD) values were below the maximum permissible values (MPV) for

drinking water (Official Gazette, 2004). The presence of the Escherichia coli, Coliform bacteria,

Enterococcus, and Pseudomonas aeruginosa was checked in the microbiological laboratory. First

sample was taken during rainy period. The Coliform bacteria and Enterococcus were developed in

24 hours at room temperature. In water samples taken during the dry period in March, none of

above mentioned microorganisms was determined. During the rainy day the samples were taken

again. Results showed the presence of Coliform bacteria and Enterococcus. Their concentrations

exceeded the values of the drinking water regulations in water samples. After inspecting the water

reservoir it was found that the lid has got a crack. The drinking water could be polluted with

Coliform bacteria and Enterococcus due to rain trickling through the lid. Restoration and cleaning of

the whole reservoir were done, therefore, the water quality must have been checked again. The

results showed that the water was safe for human consumption.

Keywords: drinking water, physical-chemical analysis, microbiological analysis, disinfection

Introduction

Drinking water sources are traditionally disinfected by chlorine (Schoenen, 2002, Hindiyeh

and Ali, 2010). Nowadays different disinfectants are used. Solar energy system was

studied recently (Hindiyeh and Ali, 2010). Advancements in nanotechnology have

improved the efficiency of silver disinfection (Lalley et al, 2014). Such novel systems are

very expensive and not appropriate for private well disinfection usage. HidroXan® is a

disinfectant, used basically for bathing water disinfection (Wapotec, 2014). It contains

tetrachlorodeca-oxide complex (TCDO) which degrade as follows (Eq. 1):



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(Cl4O10)2ˉ

= 2 ClO2 + 2 ClO2ˉ + O2 (1)

It degrades chloramines, by oxidation with ClO2 according to Eq. 2:

H2NCl + ClO2+ = 2 HCl + NO2 (2)

or TCDO, respectively (Eq. 3):

HNCl2 + Na2Cl4O10= 2 NaCl + HCl + NO2 + 3ClO2 + O2 (3)

HidroXan® is liquid solution, with density 1.099 g/cm³ and pH 8.2-8.4. The

recommendable concentration is 0.2 ml/m³ of recirculated water. During disinfection

products are formed, such as NaCl, HCl, and oxygen.

The aim of this study was to evaluate the microbiological quality of drinking water from

private well, and further to determine the bactericidal action of HidroXan® in drinking

water use.


The drinking water samples were taken from a private well which is located near Muta

town close to Slovenian-Austrian border as seen from Fig. 1 (denoted with A).

Fig. 1. Private well location


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Private well is located at the forest margin as seen from Fig. 2a. Water flows into the

reservoir as presented in Fig. 2b.

a b

Fig.2. Water reservoir: a) outside, b) inside

Water sampling dates are presented in Table 1. More samples were taken during different

seasons, but only relevant are presented in this manuscript. The main difference between

samples 2 and 3 is in weather conditions during sampling: sample 2 was taken when there

was a sunny weather and sample 3 was taken during a rainy day.

Table 1. Samples for water analyses

No Date

1 30.1.2012

2 15.4.2012

3 17.4.2012

4 23.5.2012

5 23.7.2012

Analytical methods and standards are presented in Table 2.

Table 2. Standard methods

Parameter Standard

Turbidity (NTU) SIST EN ISO 7027

pH DIN 38 404-C5

(NO2-) (mg/L) ISO 6777

(NO3-)(mg/L) ISO 7890

(NH4+)(mg/L) DIN 38 406 - E5 -1

(PO43-)(mg/L) DIN 38405 - D11


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Following microbiological methods were used:

Method for determining coliform bacteria

Method for determining Escherichia coli

Method for determining Pseudomonas aeruginosa

Method for determining enterococcus

Each dilution was spread onto specific nutrient agar to allow the bacterial colonies to be

counted. Negative controls were run in parallel for each experiment. All plates were

incubated at 37 °C (24 h for the Compass CC agar medium for Escherichia coli and

coliform bacteria). Then the colonies were counted. COMPASS Enterococcus Agar is used

as a selective media for enumeration of enterococcus. The association between X-glucoside

and a judicious mixture of selective agents in the formulation of COMPASS Enterococcus

Agar allows a direct result by characteristic colony count after only 24 hours of incubation.

Enterococcus were used as indicator microorganisms for faecal contamination.

Pseudomonas aeruginosa counts in water were performed by spread plate on Pseudomonas

Agar base (Biokar), containing glycerol (10 mL/L), selective agents cetrimide (200 mg/L)

and sodium nalidixate (15 mg/L) incubated at 44 ºC for 24 hours, before counting.


Table 3 presents the results of the analyses in water samples. It can be observed that only

pH value and turbidity varied while other parameters were below the detection limit of

standard method. Neither phosphate nor ammonium ions were present in drinking water

source, which suggest that the fertiliser contamination was not likely.

Table 3. Results of the analyses in water samples

Sample No 1 2 3 4 5

pH 6.8 7.6 8.1 7.0 7.1

Turbidity (NTU) 0.92 3.1 2.92 0.49 0.49

(NO3-) (mg/L) <1 <1 <1 <1 <1

(NO2-) (mg/L) <0.03 <0.03 <0.03 <0.03 <0.03

(PO43-) (mg/L) < 0.1 <0.1 <0.1 <0.1 <0.1

(NH4+) (mg/L) <0.1 <0.1 <0.1 <0.1 <0.1

Table 4 presents the measurements in drinking water with HidroXan®. The samples

are denoted with 1a, 2a in 3a, in order to compare them with original samples 1, 2 and

3 (see Table 3) which were not disinfected.


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The ions content was not changed with HidroXan® addition. Therefore, the disinfectant

does not affect the chemical analyses of the water samples as seen from Tables 3 and 4. pH

values of samples only slightly decreased in accordance with Eq. 3. Also turbidity

decreased and we assume that organic substances were decomposed by disinfectant

oxidation. HidroXan® slightly improved the chemical quality of drinking water.

Table 4. The measurements in drinking water with HidroXan®

Sample No 1a 2a 3a

pH 6.7 7.5 8.0

Turbidity (NTU) 0.69 2.1 2.04

(NO3-) (mg/L) <1 <1 <1

(NO3-) (mg/L) <0.03 <0.03 <0.03

(PO43-)(mg/L) <0.1 <0.1 <0.1

(NH4+)(mg/L) <0.1 <0.1 <0.1

In the next step the microbiological analyses were made in order to determine bactericidal

action of disinfectant.

Fig. 3 shows the nutrient medium for coliform bacteria with the addition of drinking water

samples. On the left side of Fig. 3 sample No 3 is presented and on the right side sample

No 1. It is clearly seen that many coliform bacteria were developed in sample 3. The

number of colonies after 24 h incubation at 37 °C count number was determined at 87 in

sample No 3. Coliform bacteria did not develop in sample No 1.

Fig. 3. Coliform bacteria in sample No 3 (left) and none bacteria in sample No 1.


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Table 5 shows the coliform bacteria of sample No 3 incubated at higher temperature 44 °C

for 24 hours. After 24 hours coliform bacteria were not found, however, after 48 hour 14

colonies of thermotolerant coliform bacteria were developed. In samples No 1 and 2 no

coliform bacteria were present. Therefore, we can conclude that the cause of water

microbiological pollution could be somehow connected with the rainy day.

Table 5. The counts of coliform bacteria in drinking water

Sample No 1 No 2 No 3

Coliform bacteria

(counts, 24h) 0 0 0

Coliform bacteria

(counts, 48 h) 0 0 14

Table 6 shows the presence of Enterococcus, Pseudomonas Aeruginosa and Escherichia Coli

in drinking water. The presence of all mentioned bacteri were determined in samples No 1, 2

and 3. The incubation reagrding the determination of Enterococcus lasted 24 h at 44 °C.

Enterococcus did not develop in samples No 1 and 2, but they were found in sample No 3.

Table 6. The presence of Enterococcus, Pseudomonas Aeruginosa and Escherichia Coli in drinking water

Sample No 1 No 2 No 3

Enterococcus no no yes

Pseudomonas Aeruginosa no no no

Escherichia Coli no no no

Fig. 4 represents the results for Enterococcus in sample No 3. The white spot represents the

presence of Enterococcus in sample without disinfection.

Fig. 4. Enterococcus, sample No 3, 44 °C


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Pseudomonas aeruginosa as well as Escherichia coli were incubated for 24 hours at 37 °C.

Both were not determined in any of inspected samples.

Enterococcus was determined in samples No 2 with and without disinfection. The samples

were incubated for 24 hours at 44 °C. There was no evidence of the presence of

Enterococcus in neither of the samples.

The experiments showed that coliform bacteria were present in samples after the rain. As

the basin was thoroughly inspected, a crack was found at the top of the basin. Therefore,

during the rainy period the bacteria entered the basin together with the rain drops. It was

decided to repair the crack immediately however, it took some time. After restoration the

water samples were taken for analysis. The microbiological analyses showed that no

bacteria were present in samples which were taken either during sunny or rainy days. We

can conclude that the water is wholesome without any disinfection and the cause of

opposable water was the crack on the top of the basin.

Conclusions

Results showed the presence of Coliform bacteria and Enterococcus in drinking water from

private well. Their number in water samples exceeded the maximum permissible value

(MPV) due to Slovenian regulations. HidroXan®disinfection of the drinking water was

efficient. However, after rain fall, pathogens were present again. The water reservoir was

inspecting and it was found that the lid has got a crack. The drinking water was polluted

with Coliform bacteria and Enterococcus due to rain trickling through the lid. Restoration

and cleaning of the whole reservoir were done. Afterwards the water quality has been

checked again. The results showed that the water was safe for human consumption.

References

Hindiyeh M., Ali A. (2010): Investigating the efficiency of solar energy system for drinking water

disinfection, Desalination, 259(1-3), 208-215.

Lalley J., Dionysiou D. D., Varma R.S., Shankara S., Yang D. J., Nadagouda M.N. (2014): Silver-

based antibacterial surfaces for drinking water disinfection-an overview, Current Opinion in

Chemical Engineering, 3, 25-29.

Official Gazette of Republic of Slovenia, Rules of drinking water quality (2004), Official gazette

RS, No 19/2004, with supplements - (35/04), (26/06), (92/06), (25/09), p.2155.

Schoenen D. (2002): Role of disinfection in suppressing the spread of pathogens with drinking

water: possibilities and limitations, Water Research 36 (15), 3874-3888.

Wapotec GmbH, Safety data sheet, Hydroxan®, available: http://s399843673.e-

shop.info/online/templatemedia/all_lang/resources/SDB+HydroXan+EN2011+V2.pdf (date:

22.1.2014)


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Biorazgradnja eritromicina s bakterijskom kulturom

Pseudomonas aeruginosa 3011

UDC: 504.3 : 615.01


Sveučilište u Zagrebu, Fakultet kemijskog inženjerstva i tehnologije, Zavod za industrijsku

ekologiju, Marulićev trg 19, 10000 Zagreb, Hrvatska

Sažetak

Prema strukturi i mehanizmu djelovanja, farmaceutici predstavljaju skupinu složenih molekula sa

specifičnim djelovanjem u biološkim sustavima. Ukoliko farmaceutici dospiju u okoliš,

predstavljaju potencijalnu opasnost zbog moguće akumulacije u okolišu i štetnog djelovanja na

organizme u ekosustavu. Biorazgradnjom se onečišćujuće tvari mogu prevesti u manje složene i

netoksične produkte pomoću mikroorganizama. Određeni mikroorganizmi posjeduju specifične

enzime koji im omogućuju razgradnju složenih organskih molekula na jednostavnije, kao što su

ugljikov dioksid i voda. U ovom radu provedeni su testovi osjetljivosti s različitim mikrobnim

kulturama te pokusi biorazgradnje pomoću bakterije Pseudomonas aeruginosa 3011. Tijekom

pokusa praćeni su optička gustoća te koncentracije biomase i eritromicina. U pokusima

biorazgradnje početne koncentracije eritromicina kretale su se od 2 μg/L do 200 mg/L. Početna

koncentracija biomase u prosjeku je iznosila 6,49 ± 0,49 mg/L. Dobivena vrijednost IC50 iznosila je

124,2 mg/L. Testovi osjetljivosti su pokazali da je Pseudomonas aeruginosa 3011 otporan na

eritromicin. Učinkovitost biorazgradnje u provedenim pokusima u prosjeku je iznosila 33,31%.

Ključne riječi: biorazgradnja, eritromicin, Pseudomonas aeruginosa 3011, šaržni reaktor

Uvod

Upotreba farmaceutika u svakodnevnom životu se povećala zbog bolje zdravstvene zaštite

i porasta stanovništva. Farmaceutici su tvari koje se koriste zbog svoje specifične biološke

aktivnosti jer pridonose ljudskom zdravlju te visokom životnom standardu. Procjenjuje se

da se godišnje koristi nekoliko stotina tisuća tona farmaceutskih aktivnih tvari za liječenje

ljudskih i životinjskih bolesti, u stočarstvu i ribogojilištima pa čak i u poljoprivredi

(Čogelja Čajo et al., 2010; Kümmerer, 2001). U okoliš mogu dospjeti putem nekoliko

različitih izvora, kao što su proizvodni pogoni, otpadne vode iz procesa obrade s aktivnim

muljem, kućni otpad te procjedne vode s odlagališta. Zabrinutost u vezi akumulacije

farmaceutika u okolišu širom svijeta je sve veća te su provedene mnoge studije (Löffler,

2005; Göbel, 2007) koje su pokazale prisutnost farmaceutika u tlima i vodenim biljkama.



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Uobičajene koncentracije farmaceutika izražene su u veličini ng/L i rijetko prelaze granice

za pitke vode (Kolpin et al., 2002), no za mnoge aktivne farmaceutske tvari koncentracije

još nisu regulirane (Kartheek et al., 2011; Yu et al., 2011). Jedna od zabrinjavajućih

činjenica koja je dovela do sve više istraživanja je i njihova sposobnost nepotpune

transformacije prilikom obrade otpadnih voda (Eckel et al., 1993; Holm et al., 1995; Jones-

Lepp et al., 2007; Kümmerer et al., 2002).

Tvari koje dospijevaju u okoliš mogu reagirati na različite načine i tako se potpuno ili

djelomično transformirati i/ili mineralizirati na ugljikov dioksid i vodu.

Biotransformacijski potencijal farmaceutika je važan pokazatelj u procjeni njihove

transformacije i prisutnosti u okolišu. Biološki postupci su sve češće upotrebljavane

metode uklanjanja onečišćujućih tvari jer su jeftine i prihvatljivije za okoliš budući da se

obrada zasniva na korištenju mikroorganizama. Bakterije i gljive su dvije skupine

mikroorganizama koje su sposobne razgraditi organske tvari. Gljive su posebno važne u

tlu, dok glavnu ulogu u biorazgradnji u vodama imaju bakterije (Gauthier et al., 2010;

Peterside et al., 2011).

U ovom radu ispitana je osjetljivost i otpornost mikroorganizma na makrolidni antibiotik

eritromicin te je provedena biološka razgradnja pomoću bakterijske kulture Pseudomonas

aeruginosa 3011 istog antibiotika.

Materijali i metode

Testovi osjetljivosti provedeni su s četiri različite mikrobne kulture. Ispitivani

mikroorganizmi bile su bakterije Pseudomonas aeruginosa 3011, Bacillus subtilis 3020 i

Streptomyces rimosus R7 te plijesan Aspergillus niger 405. Podloge s P. aeruginosa 3011 i

B. subtilis 3020 na hranjivom agaru su inkubirane pri 37 °C tijekom 24 sata, dok su

podloge sa Streptomyces rimosus R7 na hranjivom agaru i Aspergillus niger 405 na

sladnom agaru, inkubirane u termostatu pri 28 °C tijekom 3 dana (Vandepitte et al., 2003).

Priređene su modelne otopine eritromicina, odnosno supstrata različitih početnih

koncentracija γS1 = 0,02 mg/L, γS2 = 0,2 mg/L, γS3 = 2 mg/L, γS4 = 20 mg/L, γS5 = 50 mg/L,

γS6 = 80 mg/L, γS7 = 100 mg/L, γS8 = 150 mg/L, γS9 = 200 mg/L i γS10 = 2000 mg/L te su

označene od S1 do S10. Koncentracija eritromicina praćena je tekućinskom

kromatografijom ultravisoke djelotvornosti (UPLC) uz gradijentno eluiranje pri λ=210 nm

(Waters H-Class, USA).

Pokusi su provedeni u Erlenmeyer-ovim tikvicama od 100 mL na rotacijskoj tresilici s

brzinom vrtnje 160 o/min, pri temperaturi 28 °C, tijekom 24 sata (Alsop et al., 1980).

Pokusi označeni kao P1 i P2 provedeni su u zatamnjenom prostoru, a pokus P3 pri

dnevnom svijetlu. Tijekom pokusa u uzorcima je praćena promjena optičke gustoće. Na

početku i na kraju pokusa P1-P3 u uzorcima se odredila koncentracija biomase

standardnom metodom (APHA, 2012) i koncentracija eritromicina.


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Početne koncentracije bakterijske kulture P. aeruginosa 3011 su za navedene pokuse

iznosile γX1 = 0,00600 g/L, γX2 = 0,00649 g/L i γX3 = 0,00698 g/L i označene su od X1 do

X3. Optička gustoća praćena je spektrofotometrijski (Hach, Model DR/2400, USA) te je

početna optička gustoća suspenzije P. aeruginosa 3011 iznosila 0,7 pri λ = 600 nm.

Vrijednosti pH su praćene pH metrom (Sentix® 940, Multi 3430, WTW, Njemačka).


Prije postavljanja pokusa biorazgradnje provedeni su testovi osjetljivosti odabranih

mikroorganizama kako bi se ustanovila njihova osjetljivost na ispitivani farmaceutik.

Ispitivani mikroorganizmi i veličine zona prikazani su u Tablici 1. Rezultati pokazuju da je

najosjetljiviji mikroorganizam bakterija Bacillus subtilis 3020, dok bakterija Streptomyces

rimosus R7 pokazuje otpornost prema nižim koncentracijama eritomicina (do 50 mg/L).

Također, rezultati pokazuju da bakterija Pseudomonas aeruginosa 3011 te plijesan

Aspergillus niger 405 pokazuju otpornost prema širokom rasponu koncentracija

eritromicina. Prema Kim et al. (2002), iz bakterije Pseudomonas sp. uspješno je izoliran

enzim eritromicin esteraza koji povećava otpornost navedenom mikroorganizmu prema

ispitivanom farmaceutiku. Zbog pripadnosti istom rodu bakterija moguće je da bakterija

Pseudomonas aeruginosa 3011 posjeduje isti enzim te zbog toga ne pokazuje osjetljivost

prema eritromicinu.

Tablica 1. Rezultati testova osjetljivosti na eritromicin

Table 1. Results of antimicrobial susceptibility testing on erythromycin

S0

(mg/L)

Veličina zona (mm)

S. rimosus R7 B. subtilis 3020 A. niger 405 P. aeruginosa 3011

0,02 / nema zona / nema zona

0,2 / nema zona / nema zona

2 / 8 ; - ; - / nema zona

20 / 10 ; 17 ; - / /

50 9 ; - ; - 14 ; 20 ; 27 nema zona nema zona

80 / / / /

100 10 ; - ; - 17 ; 22 ; 30 nema zona nema zona

150 / / / /

200 12 ; 15 ; - 19 ; 23 ; 31 nema zona nema zona

2000 / 21 ; 27 ; - / 12 ; 20 ; - / - nije proveden test

U pokusu P1 početne koncentracije supstrata kretale su se u širokom rasponu S1-S4,S9 i

S10 da se ustanovi toksična koncentracija nakon 24 sata. Pokus je proveden u

zatamnjenom prostoru kako bi se uklonila mogućnost fotorazgradnje otopine eritromicina


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(Ternes et al., 2002). Iz rezultata je vidljivo da je pri nižoj koncentraciji uzorak odgovarao

kontroli (Slika 1). U ovom pokusu sve koncentracije eritromicina nisu pokazale

inhibicijsko djelovanje, osim 2000 mg/L, pri kojoj je došlo do potpune inhibicije rasta

bakterijske kulture. Uzorak je odgovarao kontroli u vrijednosti 22,0% nakon 24 sata dok je

za ostale koncentracije uzorka ta vrijednost iznosila 90-100% što je 4,5 puta veća

apsorbancija. IC50, 50% koncentracija inhibicije, je koncentracija ispitivane tvari koja

inhibira rast bakterijskih stanica za 50% (Alsop et al., 1980). Prema navedenim rezultatima

vrijednost IC50 eritromicina iznosi 124,2 mg/L.

Slika 1. Promjene % od kontrole u odnosu na koncentracije supstrata u P1

Fig. 1. Changes in % of control and substrate concentration in P1

U pokusu P2 početne koncentracije supstrata su se kretale od S4 do S9. Iz dobivenih

rezultata prikazanih u Slici 2 vidljivo je da je pri nižoj koncentraciji uzorak više odgovara

kontroli. U ovom pokusu ispitivane koncentracije eritromicina nisu pokazale inhibicijsko

djelovanje. Rezultati pokazuju i da su vrijednosti u 16. satu bile više i od 100% u odnosu

na kontrolni uzorak za područje nižih koncentracija, a nakon 24 sata vrijednosti se

smanjuju. To upućuje na mogućnost biorazgradnje, odnosno da su bakterije nakon

prilagodbe i potrošenih jednostavnijih izvora hranjivih tvari, počele razgrađivati složenu

organsku molekulu, u ovom slučaju eritromicin (Leclercq et al., 1991; Johnston, 1999).

Nakon što je ostala bez hranjivih tvari, biomasa je počela odumirati što se vidi nakon 24.

sata. Također je vidljivo da se koncentracija biomase povećala u svim uzorcima neovisno o

prisutnoj koncentraciji supstrata što ukazuje na moguću prilagodbu bakterijske kulture

uvjetima u reaktoru (Johnston, 1999).


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U pokusu P3 rasponi koncentracija supstrata isti su kao u pokusu P2, no pokus P3 je

proveden pri dnevnom svijetlu kako bi se vidjelo postoji li mogućnost fotorazgradnje. Iz

rezultata prikazanih Slikom 3 vidljivo je da je pri nižoj koncentraciji uzorak sličniji

kontroli, odnosno dobiveni rezultati ne odstupaju od rezultata dobivenih u pokusu P2.






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Učinkovitost procesa biorazgradnje praćena je određivanjem koncentracije eritromicina na

početku i na kraju u pokusima P2 i P3 pomoću tekućinske kromatografije ultravisoke

djelotvornosti. Iz rezultata prikazanih u Slici 4. vidljivo je da su postoci biorazgradnje za sve

koncentracije u pokusu P3 nešto viši nego u pokusu P2, osim za koncentracije eritromicina

od 50 i 80 mg/L gdje je učinkovitost razgradnje veća u P2. Različiti uvjeti provedbe ovih

pokusa nisu pokazali značajnu razliku. To ukazuje da je odabir bakterije Pseudomonas

aeruginosa 3011 za pokus biorazgradnje eritromicina zadovoljavajući jer je u samo 24 sata

čista bakterijska kultura uspjela biološki razgraditi 33,3 ± 0,2 % složene organske tvari.

Pojedinačno, najveći postotak biorazgradnje postignut je u pokusu P3 za koncentraciju

supstrata S4 i iznosi 44,1%, dok je najniži postotak razgradnje zabilježen u pokusu P4 za

koncentraciju supstrata S9 i iznosi 24,5%.

Slika 4. Učinkovitosti biorazgradnje za S4–S9 u P2 i P3

Fig. 4. Biodegradation efficeincy for S4–S9 in P2 and P3

Zaključci

Ukoliko dospijevaju u okoliš, farmaceutici predstavljaju onečišćenje koje je potrebno na

što ekonomičniji i okolišu prihvatljiviji način ukloniti. Učinkovitost uklanjanja onečišćenja

iz tla i vode se može povećati inokuliranjem selektivnih mikroorganizama. Provedbom

testova osjetljivosti i pokusa biorazgradnje bakterijska kultura Pseudomonas aeruginosa

3011 pokazala je veliku otpornost prema ispitivanim koncentracijama eritromicina.

Učinkovitost biorazgradnje u provedenim pokusima u prosjeku je iznosila 33,31% nakon

24 sata. Najveći postotak biorazgradnje postignut je u pokusu P3 i iznosi 44,1%.


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Literatura

Alsop, G.M., Waggy, G.T., Conway, R.A. (1980): Bacterial growth inhibition test, Water Pollut.

Control 52, 2452-2456.

APHA (2012): Standard Methods For the Examination of Water and Wastewater, 22th Edition,

Washington

Čogelja Čajo, G., Osrečki, V., Tomić, S. (2010): Utjecaj lijekova na okoliš, Kem. ind. 59, 351-354.

Eckel, W. P., Ross, B., Isensee, R. (1993): Pentobarbital found in ground water, Ground Water 31,

801-804.

Gauthier, H., Yargeau, V., Cooper, D.G. (2010): Biodegradation of pharmaceuticals by

Rhodococcus rhodochrous and Aspergillus niger by co-metabolism, Sci. Total Environ. 408,

1701-1706.

Göbel, A., McArdell, C.S., Joss, A., Siegrist, H., Giger, W. (2007): Fate of sulfonamides,

macrolides, and trimethoprim in different wastewater treatment technologies, Sci. Total

Environ. 372, 361-371.

Holm, J. V., Rugge, K., Bjerg, P. L., Christensen, T. H. (1995): Occurrence and distribution of

pharmaceutical organic compounds in the groundwater downgradient of a landfill (Grinsted

Denmark), Environ. Sci. Technol. 29, 1415-1420.

Johnston, N.J. (1999): Prevalence and characterization of the mechanisms of macrolide,

lincosamide, and streptogramin resistance among isolates of Streptococcus pneumoniae and

Viridans streptococci, A thesis submitted in conformity with the requirements for the degree of

Master of Science Graduate Department of Medical Genetics and Microbiology, University of

Toronto, pp. 19-21.

Jones-Lepp, T. L., Stevens, R. (2007): Pharmaceuticals and personal care products in

biosolids/sewage sludge: the interface between analytical chemistry and regulation, Anal.

Bioanal. Chem. 387, 1173-1183.

Kartheek, B. R., Maheswaran, R., Kumar, G., Sharmila Banu, G. (2011): Biodegradation of

pharmaceutical wastes using different microbial strains, Int. J. Pharm. Biol. Arch. 2(5), 1401-

1404.

Kim, Y. H., Cha, C. J., Cerniglia, C. E. (2002): Purification and characterization of an erythromycin

esterase from an erythromycin-resistant Pseudomonas sp., Microb. Lett., 210, 239-244.

Kolpin, D. W., Furlong, E. T., Meyer, M. T., Thurman, E. M., Zaugg, S.D., Barber, L.B. (2002):

Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-

2000: a national reconnaissance, Environ. Sci. Technol. 36(6), 1202–1211.

Kümmerer, K. (2001): Pharmaceuticals in the environment: sources, fate, effects and risks,

Heidelberg, Germany: Springer, pp. 10.

Kümmerer, K., Helmers, E. (2000): Hospital effluents as a source for gadolinium in the aquatic

environment, Environ. Sci. Technol. 34, 573-77.

Leclercq, R., Courvalin, P. (1991): Intrinsic and unusual resistance to macrolide, lincosamide and

streptogramin antibiotics in bacteria, Antimicrob. Agents Ch., 1273-1276.

Löffler, D., Römbke, J., Meller, M., Ternes, T. (2005): Environmental fate of pharmaceuticals in

water/sediment systems, Environ. Sci. Technol. 39, 5209-5218.


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Peterside, F., Lekiah, P., Obemeata, O. (2011): Degradation of antibiotics by bacteria and fungi

from the aquatic environment, J. Toxicol. Env. Health 3, 275-285.

Ternes, T.A., Meisenheimer, M., McDowell, D., Sacher, F., Brauch, H.J., Gulde, B.H., Preuss, G.,

Wilme, U., Seibert, N.Z. (2002): Removal of pharmaceuticals during drinking water treatment,

Environ. Sci. Technol. 36, 3855-3863.

Vandepitte, J., Verhaegen, J., Engbaek, K., Rohner, P., Priot, P., Heuck, C.C. (2003): Basic

laboratory procedures in clinical bacteriology, 2ed edition, WHO, Geneva, Switzerland, pp.

103-104.

Yu, T. H., Yu-Chen Lin, A., Panchangam, S. C., Hong, P. A., Yang, P., Lin, C. F. (2011):

Biodegradation and bio-sorption of antibiotics and non-steroidal anti-inflammatory drugs

using immobilized cell process, Chemosphere 84, 1216-1222.

Biodegradation of erythromycin with bacterial culture

Pseudomonas aeruginosa 3011


University of Zagreb, Faculty of Chemical Engineering and Technology, Department of Industrial

Ecology, Marulićev trg 19, HR-10000 Zagreb, Croatia

Summary

According to the structure and mechanism of action, pharmaceuticals represent a group of complex

molecules with specific actions in biological systems. If pharmaceuticals occur in the environment

in an unexpected or uncontrolled manner, they can cause potential problems due to possible

accumulation in the environment, causing harmful effects on organisms in the ecosystem.

Biodegradation is a microbiological process in which pollutants may be transformed into less

complex and non-toxic compounds. Certain microorganisms have specific enzymes that enable

degradation of complex organic molecules into more simple ones, such as carbon dioxide and water.

The aim of this work was to study biodegradation of pharmaceutical erythromycin by bacteria

Pseudomonas aeruginosa 3011. During the experiments optical density, biomass concentration and

concentration of erythromycin have been monitored. Antimicrobial susceptibility method was also

conducted. Initial concentration of erythromycin ranged from 2 μg/L to 200 mg/L in biodegradation

experiments. Initial biomass concentration was 6.49 ± 0.49 mg/L. Half maximal inhibitory

concentration was 124.2 mg/L. The results from antimicrobial susceptibility experiment showed that

Pseudomonas aeruginosa 3011 is resistant to erythromycin. Average biodegradation efficiency in

conducted biodegradation experiments was 33.31%.

Keywords: biodegradation, erythromycin, Pseudomonas aeruginosa 3011, batch reactor


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BIOCOS®

wastewater treatment plant Našice:

from start-up to stable operation

UDC: 628.16(497.543 Našice)

Mirko Štefančić1, Mirna Habuda-Stanić

2, Natalija Velić

2,

Marija Nujić2, Kristina Habschied

2

1Našički vodovod d.o.o., Osječka bb, HR-31500 Markovac Našički, Croatia



Summary

The ongoing population growth and economic development represent a challenge regarding the

environmental protection. With the increase of water consumption, the wastewater amount is

subsequently increasing. Before being discharged into natural recipients, the wastewater undergoes

treatment at the wastewater treatment plants. The wastewater treatment plant Našice uses BIOCOS®

(Biological Combined System) technology, which represents an improvement of the conventional

activated sludge technology and is the only example of this technology for 15,000 people equivalent

agglomerations in the Republic of Croatia. The main difference between the conventional activated

sludge system and BIOCOS® is the activated sludge – water separation process and the sludge

recycle. The need for secondary clarifier and return-sludge pumping station is avoided in BIOCOS®

system, while their function is taken by the circulation and settling tank connected to the aeration

tank and blowers. This paper gives a detailed overview of the BIOCOS® technology and wastewater

treatment plant performance from its start-up to its stable operation by monitoring the basic process

parameters.

Keywords: wastewater treatment, activated sludge, BIOCOS

®

Introduction

The principal objective of wastewater treatment process is to reduce or remove organic

matter, solids, nutrients, disease-causing organisms and other pollutants from the treated

wastewater before it is discharged to a recipient. This treatment allows disposal of human

and industrial effluents without danger to human health or unacceptable damage to the

natural environment (FAO, 1992). The Urban Wastewater Treatment Directive (UWWTD

91/271/EEC) is an emission-oriented directive which obliges the member states to collect

the wastewater and install treatment plants in agglomerations of more than 2,000 people

equivalent (PE). According to the UWWTD, agglomerations of 2,000-10,000 PE must set



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up appropriate treatment, as well as agglomerations of less than 2,000 PE which already

have a collection system (Article 7 of the UWWTD) (UWWTD, 1991). Due to the strict

demands of the European Union, the legislative (NN 43/2014) of the Republic of Croatia

was amended and is in accordance with the EEC of 21 May 1991.

Treatment technologies are based on varying levels of mechanization, energy inputs, land

requirements, costs, skilled manpower etc. (Sharma and Singh, 2013). Because of that, the

choice of an effective treatment system is of high importance. Wastewater treatment plant

WWTP Našice uses BIOCOS®

(Biological Combined System) technology which is a

biological combined system and an example of improved conventional activated sludge

technology (Fig. 1). The main advantage compared to latter is that BIOCOS®

uses only one

aeration tank equipped with diffusers and operates two alternating settler-compartments

equipped by a simple air-driven mixing- and recycle equipment (Wett and Ingerle, 2001).

Fig.1. Conventional activated sludge technology

Drainage system agglomeration Našice

The sewage system of town Našice is a type of semi-centralized system. Rainwaters are

collected by open road ditches and drained into natural recipients (stream Darna or Našička

Rijeka), while domestic and industrial wastewaters are drained into a collector connected

to the WWTP Našice. As already mentioned, the WWTP Našice uses BIOCOS®

technology, which represents an improvement of the conventional activated sludge

technology and is the only example of this technology for 15,000 PE agglomerations in the

Republic of Croatia. The sewerage network at the agglomeration is over 130 km long and

there are 22 pumping stations.


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Mechanical treatment

The wastewater treatment at the WWTP Našice begins at the inlet distributor shaft and at

the shaft for load acceptance from septic tanks where gross, suspended and floating solids

from raw sewage are removed (Fig. 2).

Fig. 2. Inlet distribution shaft (a) and shaft for load acceptance

from septic tanks (b) at WWTP Našice

It includes screening to trap solid objects and sedimentation by gravity to remove

suspended solids. This level is referred to as “mechanical” treatment, although chemicals

are used to accelerate the sedimentation process (Fig. 3).

Fig. 3. Coarse screen (a) and fine screen (b) for solid objects removal

After the removal of rags and solids that float, which may harm the operation of the rest of the

WWT plant, sand and grit are removed in aeration grit. Fat and grease are removed by passing

the sewage through an aerated grit where skimmers collect the fat floating at the surface (Fig. 4).


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Fig. 4. Aerated grit removal

The next step is biological treatment which is conducted in the BIOCOS system (Fig. 5).

Fig. 5. Aeration tank (a), recycling and setting tank (b), effluent (c)

BIOCOS®

The basis of the BIOCOS®

configuration is the activated sludge system and is the method

worldwide used for purification of municipal waste water. The development of the

BIOCOS®

configuration was based on the intention to avoid disadvantages of the

secondary clarification and the sludge recycle. BIOCOS®

is a cyclic activated sludge

system with an aeration tank (AIR in Fig. 6) hydraulically connected to two alternating

sludge recycling and –settling tanks (ALT 1+2) (Wett and Ingerle, 2001). Wastewater is

fed to an aerated reactor (AIR) and from there to a sedimentation and circulation reactor

(ALT). Several operation cycles are conducted every day with a recycle period and a

stirring period when the settled sludge in the ALT reactor is mixed with the supernatant

water. The AIR and ALT reactor are hydraulically connected near the bottom in order to

achieve efficient sludge recycling (Ingerle, 2003).


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An operation cycle of the BIOCOS®

configuration consists of four phases. During the first

phase, or the sludge recycle (RAS) phase, the sludge is pumped from the bottom of the

ALT1 reactor to the AIR reactor. Simultaneously, the displaced content of the ALT1

reactor flow via the connection near the bottom to the AIR reactor. In the second phase, i.e.

the mixing (MIX) phase, the sludge in the ALT1 reactor is stirred for a few minutes until a

homogeneous content of this reactor is achieved. Finally, the sludge concentration in the

AIR reactor is significantly higher than in the ALT1 reactor and the high biomass content

in the AIR reactor promotes the biochemical processes whereas a low suspended solids

concentration in the SU-reactor accelerates the sedimentation process. The sedimentation

(SED) phase includes flocculation in the ALT1 reactor where a horizontal sludge blanket is

developed and settled at a nearly constant velocity (approx. vs=2.0 m/h). The slowly

settling sludge body operates like a floc filter which filtrates also small particles out of the

supernatant water and enables a solid free effluent quality. This effect reduces not only the

COD-concentration but also is a precondition for disinfection measures if required. At the

end, in the fourth or discharge (DIS) phase, the supernatant water is withdrawn from the

ALT1 reactor while the sludge blanket continues settling. The content of the AIR reactor

flows to the ALT1 reactor via a connection near the bottom entering the layer of settling

sludge (Fig. 6).

Fig. 6. BIOCOS flow scheme and process cycle (Wett and Ingerle, 2001)


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Relevant biochemical processes occur in the ALT1 reactor in each operation period. As

long as nitrate and nitrite is available endogenous denitrification is the main process. The

biomass itself is the main carbon source for denitrification. A smaller part of the carbon

supply is taken from the soluble fraction (hardly degradable organic matter) causing a

decrease of the COD effluent concentration. After complete nitrogen is reduced, advanced

biological phosphorus elimination is promoted (Wett and Ingerle, 2001; Ingerle, 2003).

Some important parameters which were monitored from start-up to stable operation at

WWTP Našice are shown in Table 1 and 2.

Table 1. BIOCOS WWTP Našice influent and effluent quality parameters during 2013.

2013 INFLUENT EFFLUENT

Month KPK,

mg/L NH4, mg/L

P,

mg/L

KPK,

mg/L NH4, mg/L

P,

mg/L

January 242.75 15.38 5.26 85.85 15.28 2.0

February 168.50 10.62 2.66 62.13 7.91 1.95

March 114.00 - - 65.10 14.40 1.85

April - - - 60.40 14.88 1.37

May 250.00 2.38 4.83 51.70 16.12 1.60

June 236.00 19.67 3.99 41.40 5.48 1.70

July 342.33 25.70 - 26.95 0.38 1.93

August 297.01 18.26 3.52 32.57 0.68 1.74

September 309.24 23.74 4.99 26.97 1.90 1.09

October 241.43 22.14 3.47 25.16 2.93 1.32

November 271.00 17.69 3.59 37.33 2.85 1.85

December 239.50 29.50 5.22 33.70 6.86 1.72 *Results presented as mean values

Table 2. BIOCOS WWTP Našice influent and effluent quality parameters during 2014.

2014 INFLUENT EFFLUENT

Month KPK,

mg/L NH4, mg/L

P,

mg/L

KPK,

mg/L NH4, mg/L

P,

mg/L

January 378.53 31.74 5.51 120.30 21.70 1.65

February 194.50 15.50 3.07 26.58 5.06 1.32

March 207.00 17.39 3.15 28.10 11.10 1.52

April 283.00 29.00 4.43 49.60 7.98 1.21

May 107.82 9.55 1.93 16.48 1.11 1.62

June 160.00 20.90 3.26 21.93 11.26 1.72

July 326.68 19.08 5.07 45.14 6.18 1.67

August 282.33 22.57 4.91 14.22 1.14 1.83

September 305.40 11.53 2.92 24.85 1.54 1.69 *Results presented as mean values


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Based on the influent and especially effluent quality parameters presented in the Tables 1

and 2 it can be concluded that effluent quality was in compliance with the current

regulation. The effectiveness of treatment regarding KPK during 2014 was higher, i.e.

more uniform, as during 2013 which can be seen from Tables 1 and 2. Although the rate of

KPK removal of the effluent was acceptable, more oscillations can be observed during the

start-up of the treatment plant (year 2013), while a typical stable operation was achieved

during 2014.

Conclusions

The UWWT Directive imposes more stringent environmental standards. In most cities in

Croatia full implementation of treatment plants are still a challenge and there are often

problems with overflows in case of heavy rainfall. An additional problem is the high cost

of investment. Nevertheless, the tendency in Croatia is to build more wastewater treatment

plants to meet the EU water legislation. BIOCOS®

technology was found to be satisfactory

in wastewater treatment and can be observed as a potential technology for secure sewage

treatment process, especially for small agglomerations.

References

FAO (1992): Wastewater treatment and use in agriculture-FAO irrigation and drainage, paper 47,

Rome, Italy, Chapter 3.

Ingerle, K. (2003): BIOCOS® Wastewater Treatment Plants, Götzens, Austria.

Narodne Novine, Pravilnik o izmjenama i dopunama Pravilnika o graničnim vrijednostima emisija

otpadnih voda (NN 43/2014).

Sharma, C., Singh, S.K. (2013): Performance evaluation of sewage treatment plant based on

advanced aerobic biological filtration and oxygenated reactor (BIOFOR) technology – a case

study of capital city – Delhi, India, Int. J. Eng. Sci. Innov. Techn. 2 (4), 435-442.

Urban Wastewater Treatment Directive (UWWTD) Council Directive of Council Directive

91/271/EEC of 21 May 1991.

Wett, B., Ingerle, K. (2001): Feedforward aeration control of a BIOCOS wastewatertreatment plant,

Water Sci. Techn., 43 (3), 85-91.


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A survey of different bioadsorbents for removal of malachite green

and methylene blue dyes from aqueous solutions

UDC: 628.161.2/.3

Natalija Velić1

, Tihana Marček1, Tamara Jurić

1, Katarina Petrinović

1,

Damir Hasenay2, Lidija Begović

3, Vedran Slačanac

1

1Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology, Osijek, Franje Kuhača 20,

HR-31000 Osijek, Croatia 2Josip Juraj Strossmayer University of Osijek, Faculty of Humanities and Social Sciences,

Lorenza Jägera 9, HR-31000 Osijek, Croatia 3Josip Juraj Strossmayer University of Osijek, Department of Biology, Cara Hadrijana 8/A,


Summary

Synthetic dyes have wide application in many industries such as pharmaceutical, cosmetic, textile,

paper, leather, plastics etc. The presence of dyes in effluents (namely wastewater) of the above

mentioned industries is a major concern due to their high thermal stability and photostability, i.e.

their persistence in the environment for an extended period of time. Furthermore, they are

recalcitrant towards removal during commonly employed wastewater treatment methods. Many

agricultural waste materials containing high proportion of cellulose have the ability to adsorb

various dyes and represent a simple and low-cost method of coloured wastewater treatment. The

aim of this study was to preliminary investigate different low-cost bioadsorbents for their potential

to remove synthetic dyes malachite green (MG) and methylene blue (MB) from aqueous solutions.

Six lignocellulosic waste materials, beech sawdust (Fagus sylvatica L.), oak sawdust (Quercus

robur L.), poplar sawdust (Populus alba L.), sugar beet waste, apple pomace and spent beer grains

were tested for their ability to remove MG and MB from 15 mg L-1

aqueous dye solutions at

constant temperature of 25 °C. Activated carbon served as control. Adsorbents were soaked in dye

solutions for 5 hours and samples were taken at 30 min intervals for spectrophotometric

determination of colour removal. All tested adsorbents showed high colour removal capacity at 5

hours contact time for both used dyes (90.74-98.06 % and 94.26-100 % for MG and MB,

respectively). The results indicate that used bioadsorbents demonstrated the potential to be used as

adsorbents for efficient remediation of coloured wastewater.

Keywords: malachite green, methylene blue, bioadsorbent, dye removal

Introduction

Environmental pollution is the burning issuue of the modern world. Synthetic dyes used in

various industries such as textile, paper, pharmaceutical, food, cosmetic and leather are



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considered to be important sources of contamination of natural waters by dye-containing

effluents. Textile industry is one of the greatest generators of liquid effluent pollutants due

to high quantities of water used in the dyeing processes (Kalyani et al., 2009). Disposal of

dye-containing effluents into receiving waters is undesirable not only because of the colour

provided by dyes, but also because of their harmful properties on the living world. Due to

prolonged persistence in the environment because of their high stability to light, dyes can

compromise human health by consumption of aquatic organisms. A high concentration of

dye in waters decreases the amount of dissolved oxygen in the water, thereby affecting the

aquatic fauna and increase biochemical oxygen demand of the contaminated water (Annuar

et al., 2009).

Dye may be defined as substance that provides a colour, when applied to a substrate. Dyes

consist of two main groups a chromophore group, which determines the colour of the dye, and

auxochromes, which determine intensity of the colour. The most common cationic dyes are

malachite green (MG) and methylene blue (MB). Besides their wide application for different

kinds of materials dyeing process, MG serves as a bactericide, fungicide and ectoparasiticide or

as an antiseptic agent. Although its oral consumption can be toxic and induce carcinogenic and

/or mutagenic changes in mammalian cells due to the presence of nitrogen, on the other hand

the contact of MG with skin and eye causes irritation and pain (Bulut et al., 2008). Toxic

effects on human health after exposure to MB include heartbeat increase, vomiting, shock,

cyanosis, jaundice, quadriplegia and tissue necrosis (Yi and Zhang, 2008).

There are various conventional dye removal methods from coloured wastewaters including

coagulation-flocculation, oxidation or ozonation, membrane separation and activated carbon

adsorption (Hameed and Ahmad, 2009). However, these methods are not widely used due to

their high cost and economic disadvantage. As an alternative to these conventional wastewater

treatments, new adsorption techniques have been developed. Alternative low-cost bioadsorbens

include the usage of industrial or agricultural wastes in dyes removal from aqueous solution

such as apple pommace (Robinson et al., 2002), peanut hull (Gong et al., 2005), bagasse (Low

et al., 2011), neem sawdust (Khattri and Singh, 2009) and others.

The objective of this investigation was to examine the potential of six lignocellulosic waste

materials (beech sawdust, oak sawdust, poplar sawdust, sugar beet waste, apple pommace

and spent beer grains) as bioadsorbents for removal of MG and MB from aqueous solution.


Adsorbents

Six lignocellulosic waste materials, beech sawdust (Fagus sylvatica L.), oak sawdust

(Quercus robur L.), poplar sawdust (Populus alba L.), sugar beet waste, apple pomace and

spent beer grains were used. Beech, oak and poplar sawdusts were kindly donated by


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˝Hrvatske šume˝ d.o.o (Osijek, Croatia). Spent beer grains were obtained from local

brewery (Osječka pivovara d.d., Osijek), while sugar beet waste was obtained from local

sugar industry (“Sladorana Županja d.d.”). Apple pomace was obtained from small-scale

apple juice producer. Prior to adsorption experiments, all the adsorbents were dried (first at

room temperature and then oven dried at 60 °C for 48 h) and milled using a standard

laboratory knife mill with 3 mm screen (MF10 basic, IKA Labortechnik, Germany) to

insure the adsorbent particle size below 3 mm. No other chemical or physical treatments

were applied prior to adsorption experiments. Activated carbon (SA SUPER 8029-1, Cabot

Norit, USA) served as control.

Adsorbat

Malachite green and methylene blue used in this work were purchased from Kemika

(Zagreb, Croatia) and Merck (Darmstad, Germany), respectively. Stock solutions of 1 g L-1

were used and prepared daily.

Adsorption experiments

Batch adsorption experiments were carried out by adding a fixed amount of adsorbent (1 g)

to 100 mL of 15 mg L-1

dye solution taken in a 250 mL Erlenmeyer flask, without changing

pH and temperature. The flasks were placed in an incubator (BD 53#04-63769, Binder,

Tuttlingen, Germany ) and kept at constant temperature of 25 °C. Adsorbents were soaked in

dye solutions for 5 hours and samples were taken at 30 min intervals for spectrophotometric

determination of colour removal. pH was not adjusted and was measured using a pH-meter

(pH/Ion Analyzer MA 235, Mettler Toledo). Dye solution samples taken at various intervals

were centrifuged for 5 min at 10,000 rpm using a centrifuge (Heraeus, Multifuge 3L/3L-R,

Kendro laboratory Products, London, UK). The dye concentrations in clarified supernatants

were determined at characteristic wavelenghts (MG at 623 nm, MB at 665 nm) using a

spectrophotometer (UV-1700 PharmaSpec, Shimadzu, Japan).

The percentage removal of dye was calculated by equation:

% removal = 100 (0 -) /

where 0 and are the initial dye concentration and dye concentration after certain contact

time, respectively.

Wetting experiments were performed in a way that different adsorbents (1 g) were soaked in

99 mL of deionised water for 24 and 48 h. After that 1 mL of 1.5 g L-1 MG or MB dye solution

was added to the flasks, so that the resulting concentration of dye solution was 15 mg L-1. The

adsorption experiments were then conducted as described above.

Duplicate experiments were run for comparison and samples were analyzed in triplicates.


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Results and disscusion

Agricultural and food industry waste materials containing high proportion of cellulose exhibit

potential removal capacity for various pollutants. The main characteristic of all agri-food waste

materials is a variety of available functional groups because of their composition. Apart from

cellulose, their constituents also include hemicellulose, lignin, simple sugars, lipids, proteins,

starch, hydrocarbons, etc. (Bhatnagar and Sillanpää, 2010).

The waste materials selected for this investigation are widely available and can be roughly

divided into two groups: wood industry waste (beech, oak and poplar sawdust) and food

industry waste (sugar beet waste, apple pomace and spent beer grains). Cellulose is the major

chemical component of wood contributing 40-45% of the wood’s dry weight, followed by

hemicellulose 30%, lignin 20-30% and extractives 2-4%, depending on wood type (Sjostrom,

1993). Sugar beet waste is composed mainly of polysaccharides (75-80%), out of which

22-30% cellulose, 25-30% hemicelluloses and 25% pectin. Proteins and lignin comprise

10-15% and 1-6% of sugar beet waste, respectively (Spagnuolo et al. 1997). Cellulose is the

major component of apple pomace (30%), followed by lignin 15%, hemicellulose 12% and

pectin 9% (Wang and Thomas, 1989) Spent beer grains are composed of roughly 20%

cellulose, 28-30% noncellulosic polysaccharides and 28% lignin (Mussato et al., 2006).

Figs. 1 and 2 show percentage colour removal due to adsorption by bioadsorbents and

activated carbon for MG and MB dyes. Concentration of dyes (15 mg L-1

) in this study was

selected on the basis of the dye concentrations reported in textile effluents, that usually

range from 10 to 50 mg L-1

(Nigam et al., 2000).

Fig. 1. MG dye removal through adsorption by different bioadsorbents

(mbioads.= 1 g, dye = 15 mg L-1

, Vdye solution = 100 mL, t = 25 °C)


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Fig. 2. MB dye removal through adsorption by different bioadsorbents

(mbioads.= 1 g, dye = 15 mg L-1


High levels of MG and MB removal after 5 h contact time (over 90%) were achieved for

all investigated adsorbents. Because of their chemical composition, i.e. large proportion of

cellulose, hemicellulose and lignin, a large number of hydroxyl grops are available as

binding sites for cationc dye molecules, such as MG and MB. Hydroxyl groups are those

engaged in absorbent and adsorbent process (O’Connell et al., 2008). The percentage of

dye removal at the initial stages of the adsorption experiments (contact time less than

30 min) was rapid, especially for beech, poplar and oak (more than 90%). This may be

explained by the high cellulose content of wood sawdusts, i.e. large number of binding

hydroxyl groups. All bioadsorbents proved to be more efficient than activated carbon at the

initial stages. After this rapid dye removal at the initial stages, in the later stages the

removal becomes slower till saturation is allowed, mostly after 3 h contact time. The

percentage of dye removal is slightly different for MG and MB. MB was adsorbed more

effectively by all the investigated bioadsorbents. After 3 h contact time the MB percentage

removal was 100% when beech and poplar sawdusts were used as bioadsorbents, while

percentage removal for other adsorbents after 3 h contact time was also over 90%. The

highest MG removal percentage after 5 h contact time was 98% for beech and poplar

sawdusts, while all other investigated adsorbents achieved dye removal percentage of 95%

or less.

Table 1 gives changes in pH during the adsorption experiments.


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Table 1. Changes in pH during the bioadsorbent survey experiments

Bioadsorbent (m = 1 g)

pH

Malchite green ( = 15 mg L-1) Methylene blue ( = 15 mg L-1)

Contact time / min Contact time / min

0 30 60 150 300 0 30 60 150 300

Beech sawdust 7.2 7.2 7.2 7.3 7.2 7 7 7.2 7.4 7.4

Sugar beet pulp 5.9 6.1 6.1 6.1 6.1 6.2 6.3 6.3 6.3 6.3

Oak sawdust 4.8 4.9 4.9 4.8 4.8 5.5 5.5 5.0 5.0 4.9

Spent beer grains 6.1 6.1 6.1 6.1 6.0 6.0 6.1 6.1 6.1 6.1

Apple pommace 3.7 3.6 3.6 3.6 3.6 3.9 3.8 3.8 3.7 3.7

Activated carbon 6.7 6.8 7.0 7.0 7.0 7.5 7.5 7.6 7.7 7.8

Poplar sawdust 7.7 7.6 7.7 7.7 7.7 7.8 7.7 7.7 7.7 7.7

Even though the initial pH of the solution is important parameter affecting the cationic dye

adsorption, pH was not adjusted, but rather just monitored throughout the experiments. pH

was mostly near neutral or slighty acidic/basic, except for oak sawdust and apple pomace

where pH was around 4 and 3.5, respectively. Both oak and apple pomace were slightly

less efficient compared to other two adsorbents from their specific groups (wood or food

industry waste). This is in accordance with study conducted by Low et al. (2011). Since

both MG and MB are basic in nature, acidic conditions probably unfavourable effected

their adsorption by the used bioadsorbents. However, further work is required to establish

this.

Solid materials are often characterized by their wetting behaviour. Wetting of solid

surfaces by liquids is a very common and important phenomenon in nature as well as in

technological applications; such as impregnation of materials, flotation and it may also

play an adverse role in biological wastewater purification where bacteria stabilize

undesired foaming (Norde, 2003). The influence of wetting on the adsorption properties of

different materials is complex. For example, surface polarty is strongly dependent on the

surface hydration degree, which has an influence on the adsorption properties of materials

(Norde, 2003). Cellulose is well known for its high water holding ability (water molecules

incorporate in cellulose crystalline structure). Latter has strong influence on the adsorption

properties of different (biological) materials containing cellulose. However, this influence

is not straightforward. In order to investigate how the degree of hydration influences the

adsorptive properties of the used bioadsorbents, the bioadsorbents were subjected to 24 and

48 h wetting prior to adsorption experiments. The effect of bioadsorbent wetting on MG

dye removal through adsorption was investigated for all the bioadsorbents. Fig. 3 presents

the MG percentage removal for poplar sawdust and spent beer grains after the bioadsorbent

wetting procedure.


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A

B

Fig. 3. Effect of bioadsorbent wetting on MG dye removal through adsorption by a) poplar sawdust

and b) spent beer grains (mbioads.= 1 g, dye = 15 mg L-1



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It can be seen from the graphs that wetting did not favourably effect the dye removal,

except in the case of spent beer grains after 24 h wetting.

Conclusions

The results presented in this survey clearly show that the selected agri-food waste materials

have demonstrated outstanding capacity for MG and MB removal from aqueous solutions.

Apart from being highly efficient, they are also widely available, cheap and renewable.

However, further research should be done to better define these materials as possible

commercial adsorbents, i.e. characterize the adsorbents using appropriate methodology

(i.g. FTIR analysis); perform equilibrium and kinetic studies; determine the effect of initial

concentration, temperature, pH, adsorbent dose, etc. on dyes adsorption. All these issues,

as well as a cost comparison to conventionally used adsorbents, have to be tackled in order

to adopt and widely use non-conventional adsorbents (such as those used in this survey) in

industries.

References

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decolorization by Pycnoporus sanguineus, Water, Air Soil Poll. 202, 179-188.

Bhatnagar, A., Sillanpää, M. (2010): Utilization of agro-industrial and municipal waste materials as

potential adsorbents for water treatment-A review, Chem. Eng. J. 157(2-3), 277-296.

Bulut, E., Özacar M., Şengil I.A. (2008): Adsorption of malachite green onto bentonite: Equilibrium

and kinetic studies and process design, Micropor. Mesopor. Mater. 115(3), 234-246.

Gong, R., Sun, Y., Chen, J., Liu, H., Yang, C. (2005): Effect of chemical modification on dye

adsorption capacity of peanut hull, Dyes Pigments, 67(3), 175-181.

Hameed, B.H., Ahmad, A.A. (2009): Batch adsorption of methylene blue from aqueous solution by

garlic peel, an agricultural waste biomass, J. Hazard. Mater. 164, 870-875.

Kalyani, D. C., Telke, A. A., Dhanve, R. S., Jadhav, J. P.(2009): Ecofriendly biodegradation and

detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp. SUK1, J.

Hazard. Mater. 163,735-742.

Khattri, S.D., Sihgh, M.K. (2009): Removal of malachite green from dye wastewater using neem

sawdust by adsorption, J. Hazard. Mater. 167, 1089-1094.

Low, W.L., Teng, T.T., Ahmad, A., Morad, N., Wong, Y.S. (2011): A novel pretreatment method of

lignocellulosic material as adsorbent and kinetic study of dye waste adsorption, Water Air

Poll. 218, 293-306.

Mussato, S.I., Dragone G., Roberto I.C. (2006): Brewers' spent grain: generation, characteristics and

potential applications, J. Cereal Sci. 43, 1-14.

Nigam, P., Armour, G., Banat, I. M., Singh, D., Marchant, R. (2000): Physical removal of textile

dyes from effluents and solid-state fermentation of dye-adsorbed agricultural residues,

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Norde, W. (2003): Colloids and Interfaces in Life Sciences, New York, Marcel Dekker, Inc., pp.

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Kazalo autora

Author index


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Kazalo autora / Author index

433

A

Ačkar, Đurđica, 276, 283

Agić, Dejan, 341

Akrap, Zorana, 59

Aladić, Krunoslav, 50

Andrić, Luka, 352

B

Babić, Jurislav, 276, 283

Bačić, Ivana, 18

Balić, Tomislav, 32

Banovac, Irena, 59

Bebić, Sara, 190

Begović, Lidija, 424

Benčová, Silvia, 212

Bernstorff, Sigrid, 59

Bilić, Blanka, 222

Bilić, Mate, 50

Blažević, Ivica, 190

Budić-Leto, Irena, 297

Burčul, Franko, 190

Bušić, Valentina, 1

C

Cvetko, Josip, 253

Cvijetić, Milica, 222

Č

Čačić Kenjerić, Frane, 204

Čižmek, Lara, 409

Čurlin, Mirjana, 70

Ć

Ćosić, Marija, 91

Ćurić, Iva, 32

D

Dráb, Štefan, 212

Drezner, Georg, 352

Dujmenović, Mladena, 178

Dvojković, Krešimir, 352

E

Erceg, Matko, 59

Ergović Ravančić, Maja, 268

F

Fališevac, Bojan, 325

Ferčec, Borna, 140

Frančáková, Helena, 212

G

Galić, Vlatko, 325

Gaši, Emanuel, 391

Gašo-Sokač, Dajana, 1

Generalić Mekinić, Ivana, 190

Giacobi, Antonia, 113

H

Habschied, Kristina, 417

Habuda-Stanić, Mirna, 1, 417

Hancock, John T., 325

Hanžek, Andrija, 113

Hasenay, Damir, 424

Horvat, Goran, 50

Hrkovac, Martina, 154

I

Ivanišová, Eva, 212

J

Jakobek, Lidija, 232, 242

Jakobović, Danijela, 12

Jakovljević, Ivana, 356

Jelinić, Ana, 204


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Jelkić, Dinko, 335

Jokić, Stela, 50

Jozanović, Marija, 12

Jozić, Dražan, 59

Jozinović, Antun, 276, 283

Jukić Špika Maja, 317

Jukić, Antonela, 317

Jurić, Tamara, 424

Jurina, Tamara, 70

Jurinjak Tušek, Ana, 70, 81

Jurišić, Gordana, 289

Jurki, Iris, 391

K

Kaćunić, Antonija, 91

Kalajdžić, Brankica, 364

Kamenić, Štefica, 125

Kenjerić, Daniela, 222

Kezele, Tatjana, 18

Klarić, Dario, 166

Knez, Željko, 306

Knezović, Zlatka, 104

Kojić, Nebojša, 232

Kovačević, Dragan, 260

Kovačević, Josip, 352

Kovačić, Marin, 125, 335

Kraljičak, Željko, 335

Krivak, Petra, 242

Kurtanjek, Želimir, 70, 81

Kuzmanić, Nenad, 91

L

Ladešić, Zvonimir, 253

Lalić, Alojzije, 352

Lisjak, Miroslav, 325, 341

Lokas, Lea, 91

M

Maletić, Edi, 297

Marček, Tihana, 424

Maričić Tarandek, Sandra, 253

Maslov, Luna, 297

Mastanjević, Krešimir, 260

Medvidović-Kosanović, Martina, 32

Mesić, Josip, 268

Mihaljević, Ana, 341

Mihaljević-Jurić, Paula, 32

Miličević, Borislav, 276, 283

Miličević, Dijana, 283

Miličević, Radoslav, 283

Mrgan, Ana, 276, 289

Mucalo, Ana, 297

N

Nedić, Nebojša, 335

Nuić, Ivona, 375

Nujić, Marija, 1, 364, 417

O

Obradović, Valentina, 268

P

Pavić, Biljana, 383

Pehnec, Gordana, 356

Penović, Tomislav, 113, 166

Perko, Tina, 306

Petljak, Martina, 311

Petrinić, Irena, 70

Petrinović, Katarina, 424

Pihler, Ivan, 335

Prlić Kardum, Jasna, 125

Puškadija, Zlatko, 335

R

Rogošić, Marko, 140

Romić, Željka, 364

Rupčić Petelinc, Sonja, 391

S

Sakač, Nikola, 12


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Sak-Bosnar, Milan, 12

Samardžija, Marina, 125

Sambolek, Valentino, 154

Sander, Aleksandra, 140, 166, 178

Simonič, Marjana, 402

Slačanac, Vedran, 424

Slivar, Anamarija, 154

Stipišić, Angela, 104

Stipišić, Renato, 317

Sudarić, Aleksandra, 352

Sulić, Anka, 375

Sutlović, Davorka, 104

Svilović, Sandra, 317

Svitlica, Brankica, 268

Š

Šabić, Monika, 409

Šalić, Anita, 81

Škerget, Mojca, 306

Škrabal, Svjetlana, 283

Špoljarević, Marija, 325, 341

Štefančić, Mirko, 417

Šter, Anamarija, 32

Štolfa, Ivna, 341

Šubarić, Drago, 276, 283

T

Teklić, Tihana, 325, 341

Tomas, Ivan, 222

Tomaš, Renato, 41

Tomaz, Ivana, 297

Trgo, Marina, 104, 375

Tunjić, Đuro, 311

V

Vađić, Vladimira, 356

Velić, Natalija, 417, 424

Vidaković, Kristina, 260

Vrdoljak, Anđelka, 41

Vugrinec, Bruna, 391

Vukojević Medvidović, Nediljka, 375

Vuković Domanovac, Marija, 409

Vuković, Danijela, 276

Vuković, Rosemary, 341

W

Wendling, Silva, 335

Whiteman, Matthew, 325

Wilson, Ian D., 325

Wood Mark E., 325

Z

Zdunić, Goran, 297

Zdunić, Zvonimir, 352

Zelić, Bruno, 81

Ž

Žanetić, Mirella, 317

Žilić, Jelena, 268

Žuteg, Barbara, 154

Žužek, Sanja, 391

Žužić, Maja, 178

Sponzori

Sponsors


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436

ZLATNI SPONZOR / GOLD SPONSOR


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