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 (Baš

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