Extracting Lipid and Carotenoids from Microalgae with ... · ii Extracting Lipid and Carotenoids from Microalgae with Lecithin-Linker Microemulsions Johanna Chan Master of Applied

Extracting Lipid and Carotenoids from Microalgae with Lecithin-Linker Microemulsions

By

Johanna Chan

A thesis submitted in conformity with the requirements

for the degree of Master of Applied Science

Department of Chemical Engineering and Applied Chemistry

University of Toronto

copy Copyright by Johanna Chan 2012

ii

Extracting Lipid and Carotenoids from Microalgae with Lecithin-Linker

Microemulsions

Johanna Chan

Master of Applied Science



2012

ABSTRACT

This study investigated the extraction of lipids and β-carotene from microalgae using

microemulsions as an alternative to current solvents Type I and type IV microemulsions

composed of 4 lecithin sorbitan monooleate PEG-6-caprylic glycerides and ethyl caprate

were able extract lipids from lyophilized microalgae better than hexane and ethyl caprate

HPLC quantified the extracted β-carotene with type IV microemulsions extracting the most

β-carotene at 0137plusmn0074 (ww) of the total microalgae biomass after an hour The

growth recovery of the microalgae after extraction was observed over 2 weeks Variability in

the data prevented definite conclusions about the ability of algae to grow after extraction

The type IV extractions consistently showed some signs of survival After two weeks a pale-

green colour was observed in the 15min and 1h extractions This study showed that

microemulsions can successfully extract lipids from microalgae future work would apply

microemulsion formulations to live algal cells for in-situ extraction

iii

ACKNOWLEDGEMENTS

I would like to thank both my supervisors Professor Edgar Acosta and Professor Levente

Diosady for their advice and support throughout the course of this research Professor

Acostarsquos knowledge of microemulsions has been very helpful and inspiring and I am

grateful for his guidance As well Professor Diosadyrsquos constructive criticisms and insight

were fundamental to the completion of this work

I would also like to acknowledge Pond Biofuels for providing various materials and support

throughout this project I appreciated the financial support provided by the McLean

Scholarship Fund and the Alexander Graham Bell Canada Graduate Scholarship which

allowed me to focus on my research

Additionally I would like to thank both the members of the Food Engineering Group and the

Laboratory of Colloid and Formulation Engineering (LCFE) for their support and friendship

Specifically I am thankful for the advice and discussions from the Algae Group at the


Finally I appreciate the encouragement and guidance of my family throughout my life

iv

Table of Contents

1 Introduction 1

11 Objective 4

2 Background 4

21 Microalgae 4

22 Extracted Products and their Uses 6

221 Fuel Industry 6

222 Pharmaceutical and Food Industry 7

223 Other Commercial Applications 8

23 Algae Bioreactors 8

24 Extracting OilCarotenoids from Algae 10

241 Current Lipid Extraction Techniques 10

242 Current Carotenoid Extraction Techniques 13

243 Microalgae Milking with Two-Phase Bioreactors 14

25 Microemulsion and Aqueous Oil Extraction Processes 16

251 Microemulsions 16

252 Vegetable Oil Extraction and Extended-Surfactants 18

253 Linker-Based Biocompatible Microemulsions 19

3 Extracting Lipid and Carotenoids from Microalgae with Lecithin-Linker Microemulsions 20

31 Materials and Methods 20

311 Materials 20

312 Characterization of Microalgae 21

313 Microemulsion Formulation and Characterization 22

314 Lipid Extraction and Quantification 24

315 Carotenoid Extraction and Characterization 26

316 Biocompatibility Regrowth Study 27

32 Results 29




v


325 Biocompatibility Regrowth Testing 37

33 Discussion 42



333 Biocompatibility Testing 48

4 Conclusions 49

5 Recommendations 50

6 References 52

Appendix A BBM Solution 56

Appendix B Folch Method Validation 57

Appendix C FAMEs Procedure 59

Appendix D GC Conditions 60

Appendix E HPLC Conditions and Procedure 62

Appendix F GC Calibration 63

Appendix G Lipid Extraction Data 64

Appendix H HPLC Calibration 66

Appendix J Extended Surfactant Extraction 67

vi

List of Tables

Table 1 Fatty Acid Composition of Various Microalgae Species () 5

Table 2 General Composition of Microalgae compared to Food Sources 7

Table 3 Current Methods of Oil Extraction from Algae 11

Table 4 Current Methods of Carotenoid Extraction from Algae 13

Table 5 4 Lecithin Microemulsion Series 23

Table 6 Hansen Solubility Parameters 24

Table 7 Microemulsion Formulations for Subsequent Extractions 32

Table 8 Microemulsion Characterization 32

Table 9 Micrographs of C Vulgaris After Initial Extraction 41

Table 10 ANOVA Two-Factor with Replication 1h Extraction 44

Table 11 ANOVA - Two-Factor With Replication 120 extraction 45

Table 12 Microemulsion Composition based on Phase Scan Results 46

Table 13 HPLC Quantification of β-Carotene Extraction 47

vii

List of Figures

Figure 1 Microalgal Biorefinary 2

Figure 2 Growth Curve of Algal Cells 6

Figure 3 ndash Transesterification of Triglycerides 7

Figure 4 Two-Phase Bioreactor 15

Figure 5 Microemulsion Types 17

Figure 6 GC Chromatograms of Standards Olive Oil and Algae Oil 30

Figure 7 GC Chromatograms comparing Residual Lipids after Microemulsion Extraction 31

Figure 8 Phase Scan of 4 Lecithin Series with Varying Ratios of PEG-6-Caprylic Lecithin 32

Figure 9 Extraction Efficiency at 10h 33

Figure 10 Extraction Efficiency of 120 (MassSolvent) Ratio 34

Figure 11 Absorbance Spectrum of CarotenoidChlorophyll Extraction at 30min 160 dry

biomass(g)solvent (mL) 35

Figure 12 Absorbance Kinetics at 445nm with UV-Visible Spectrophotometry 35

Figure 13 HPLC Chromatogram of Algae Extract by Various SolventsMicroemulsions 36

Figure 14 Extracted Beta-Carotene Quantified with HPLC 37

Figure 15 Algae Recovery after 1h SolventMicroemulsion Extraction 38

Figure 16 Algae Growth Recovery 2 weeks after MicroemulsionSolvent Exposure 39

Figure 17 Algae Recovery after 15 minute SolventMicroemulsion Extraction 40

Figure 18 Micrograph of C Vulgaris 40

Figure 19 FAME Lipids Extracted vs Ethyl Caprate Concentration 46

1

1 Introduction

As the demand for energy increases there is a growing interest in biofuels as a sustainable

alternative Traditionally biofuels are produced from rapeseed soybean sunflower and

palm crops (Luisa Gouveia amp Oliveira 2009) However these oil crops require a large

cropping area compared to microalgae (Chisti 2007) Other benefits of microalgae as a

biofuel source include a high oil content quick growth rate use of non-arable land and non-

potable water use of less water and it does not displace food crops (Luisa Gouveia amp

Oliveira 2009) Ideally the production of biodiesel from microalgae would be carbon

neutral as the photosynthesis required to produce the biomass would consume greenhouse

gases (Mercer amp Armenta 2011) This carbon could be sourced from existing power plants

and smokestacks at minimal costs (Chisti 2007)

In addition microalgae are rich in high-value compounds and specialty lipids including

astaxanthin neurotoxins ω3-long-chain polyunsaturated fatty acids (PUFAs) and β-carotene

There is interest in extracting PUFAs such as docosahexaenoic acid (DHA) and

eicosapentaenoic acid (EPA) since there is an insufficient fish source that is also expensive

(Cohen 1994) Microalgae also contain a range of carotenoids which are antioxidants Beta-

carotene is pro-vitamin A and is converted to vitamin A in the body (Baker amp Guumlnther

2004) Microalgae have the potential to provide products in the food cosmetic

pharmaceutical and fuel industries (Mercer amp Armenta 2011)

A major limitation in using microalgal species in industry is the high cost of production

(EW Becker 1994) Currently large-scale production of microalgae involves raceway

ponds or tubular photobioreactors to produce an algal broth The productivity of algae culture

is a bottleneck in biomass and product formation (M A Hejazi amp Wijffels 2004) In

addition the biomass must be recovered via filtration or centrifugation prior to the extraction

of the microalgal oil and then dried (Chisti 2007) which are energy-intensive processes

(Mercer amp Armenta 2011)

2

The need for microalgae milking fits into the proposed microalgae biorefinary (Figure 1)

where lipids are extracted from a fixed algae biomass to produce lipid fractions for biofuels

and nutraceuticals The current lipid extraction techniques are energy intensive because the

water must be separated from the biomass Microalgae milking would involve in-situ

extraction of the lipids Furthermore the lag experienced by growing the algae is eliminated

since the live microalgae can continuously produce more lipids for future extractions

Figure 1 Microalgal Biorefinary (Allen 2010)

Ideally lipids could be lsquomilkedrsquo from the microalgae in a continuous process

Microemulsions would remove the lipids and value-added products from the microalgae

while leaving the cells intact The cell would then regenerate new lipids and carotenoids for

future extraction This would overcome the current bottleneck of regrowing algae for each

extraction Since the lipids are removed in-situ the cells would not require drying or lysing

thus decreasing the energy consumption of the extraction process Current extraction

methods include mechanical disruption solvent extraction supercritical fluid extraction

ultrasound or sonnication (Mercer amp Armenta 2011) Many of these extraction methods are

energy intensive Furthermore the use of microemulsions as biocompatible solvents would

improve extraction safety

3

The concept of lsquomicroalgae milkingrsquo has been demonstrated by Hejazi and Wijffels for the

continuous extraction of β-carotene with an organic solvent (M A Hejazi amp Wijffels 2004)

A production yield of 245mgm-2day

-1 was achieved by recirculating dodecane at

200mLmin-1

in a two-phase bioreactor The algae remained viable for more than 47 days but

cell growth was slow (M A Hejazi Holwerda amp Wijffels 2004) However the interface

between the solvent and biomass limited this extraction efficiency The use of

microemulsions as a solvent would overcome this limitation since microemulsions have a

greater surface area to volume ratio Theoretically other biocompatible solvents such as

microemulsions can be used to milk other components from the algae

Microemulsions have been shown to successfully extract oil from other sources with high

yields The application of microemulsions to extract peanut oil has been reported by Nguyen

et al The peanut oil is extracted directly into the oil phase of the microemulsion and almost

95 extraction efficiency was achieved at room temperature with a 10 minute extraction

time (Nguyen Do amp Sabatini 2010) However a biocompatible microemulsion would be

desirable to continuously extract by-products without killing the microalgae As such

lecithin-linker microemulsion formulations have been reported to be biocompatible and could

be used in this application (E J Acosta Chung amp Xuan 2011)

The hypothesis is that by combining the concept of lsquomilking microalgaersquo with the

microemulsion as a solvent there is potential to improve lipid and carotenoid extraction

yields The purpose of this study is to prove that microemulsions can extract more lipids and

carotenoids from microalgae than hexane In-situ extraction has potential benefits over

solvent extraction

To determine the effectiveness of lsquomicroalgae milkingrsquo the ability of microemulsions to

extract lipids and carotenoids from microalgae must be assessed first Initially lipids and β-

carotene were extracted from dried microalgae and quantified with gas chromatography and

high-performance liquid chromatography The effect of time and dry biomass to solvent ratio

was examined The biocompatibility between the microemulsion and microalgae was

4

observed by monitoring the growth recovery of the algae after an initial extraction with the

microemulsion

11 Objective

The objective is to investigate the extraction of lipids and β-carotene from microalgae

using microemulsions In particular this work will examine the extraction efficiency and

biocompatibility of various microemulsion formulations compared to current lipid extraction

techniques The focus of this report will be applying lecithin-linker microemulsions to extract

lipids from dried algae Finally the β-carotene extraction will also be quantified

This thesis is organized into four chapters Chapter 1 provides an introduction to the benefits

of extracting lipids and carotenoids from microalgae and the objective of this thesis Chapter

2 presents an overview of the literature including information regarding microalgae

applications for extracted products current extraction techniques and microemulsions

Chapter 3 focuses on the use of lecithin-linker microemulsions for lipid and carotenoid

extraction Finally Chapters 4 and 5 presents the conclusions and recommendations for

future work respectively

As described in section 251 there are two main microemulsion extraction mechanisms The

focus of this report and Chapter 3 has been on the supersolubilization method and the use of

lecithin-linker microemulsions In the alternative microemulsion extraction mechanism

known as capillary displacement low interfacial tension is achieved with very low surfactant

concentrations The preliminary results of this study can be found in the appendix

2 Background

21 Microalgae

Microalgae consist of lipids proteins carotenoids pigments vitamins sterols and

polysaccharides that could be extracted for applications in the fuel pharmaceutical food and

5

cosmetic industry (EW Becker 1994) The composition of the fatty acids depends on the

microalgae species nutritional factors and environmental factors (Spolaore Joannis-Cassan

Duran amp Isambert 2006) There is variability in the oil content and composition of algae

reported in literature due to varying environmental conditions

Table 1 summarizes the fatty acid composition of several common microalgae species

Depending on the application the species can be selected to produce certain fatty acids For

example nutraceutical applications would focus on algal species with more polyunsaturated

fatty acids (PUFAs) Approximately 80 of algal lipids are stored as triglycerides Other

storage molecules include sulfoquinovosyl diglyceride mono- and digalactosyl diglyceride

lecithin phosphatidyl-glycerol and γ-inositol (EW Becker 1994)

Table 1 Fatty Acid Composition of Various Microalgae Species ()

fatty Chlorella Dunaliella Scenedesmus Spirulina Spirulina

acid vulgaris bardawil obliquus maxima platensis

120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02

150 16 - - traces traces

160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -

Reference Spoehr amp Fried Kenyon Hudson amp Hudson amp

Milner 1949 et al 1982 et al 1972 Karis 1974 Karis 1974

(Adapted from Becker 1994 References as cited in Becker 1994)

After an algae culture is inoculated into new growth media a typical growth curve can be

observed as shown in Figure 2 For 2-3 days the algae cells acclimatize to the new environment

and conditions and growth is constant this is the lag phase Then the exponential growth phase

6

lasts for 4-6 days The cells grow exponentially since the optimal amount of light and nutrients

are present When the nutrients begin to deplete and light is limited due to crowding of the cells

then the cell division slows down and the stationary phase is observed Eventually the lack of

nutrients and light prevents further cell division resulting in the death phase

Figure 2 Growth Curve of Algal Cells

22 Extracted Products and their Uses

221 Fuel Industry

Vegetable oils are often used to produce biodiesel Lipids extracted from microalgae are

different from most vegetable oils since they contain higher amounts of PUFAs Commonly

eicosapenatenoic acid (EPA C205n-3) and docosahexaenoic acid (DHA C226n-3) are

found in algae These fatty acids with four or more double bonds have an increased

susceptibility to oxidation which reduces their acceptability for biodiesel use (Belarbi

Molina amp Chisti 2000) Some vegetable oils have similar problems with oxidative stability

and limit the presence of linolenic acid methyl esters in vehicle biodiesels The unsaturation

of microalgal oil can be reduced by catalytic hydrogenation (Chisti 2007)

The extracted oil consists of triglycerides three fatty acid molecules esterified with a

glycerol molecule To produce biodiesel the triglycerides are converted into methyl esters

through transesterification as shown in Figure 3 Transesterification typically occurs by

7

adding methanol to the triglycerides in the presence of a catalyst such as sodium hydroxide

or sodium methoxide

Figure 3 ndash Transesterification of Triglyceride

222 Pharmaceutical and Food Industry

The alga cell is composed of lipids carbohydrates proteins and carotenoidschlorophylls

For reference the general composition of several microalgae species is compared to various

food sources (Spolaore et al 2006) and is presented in Table 2

Table 2 General Composition of Microalgae compared to Food Sources

Source of dry matter

Protein Carbohydrate Lipid

Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20

Chlorella vulgaris 51-58 12-17 14-22

Dunaliella salina 57 32 6

Scenedesmus obliquus 50-56 10-17 12-14

Spirulina maxima 60-71 13-16 6-7

The lipid content ranges between 1-70 of the algal cell and consists of many

polyunsaturated fatty acids in the ω3 and ω6 families that are desirable for nutraceutical

applications (Spolaore et al 2006) Specifically DHA is required for the development and

functioning of the brain retina and reproductive tissues Also DHA has been used to treat

cardiovascular diseases cancer and inflammatory diseases (Jiang amp Chen 2000) The fatty

acid formation and composition depends on the microalgae growth conditions such as the

medium and environment and some algae sources can have up to 50 DHA Lipid

8

accumulation is observed when the microalgae are nutrient-limited (M A Hejazi amp Wijffels

2004)

Microalgae have high protein content and the amino acids present are comparable to other

food proteins The carbohydrates consist of starch glucose sugars and other polysaccharides

(Spolaore et al 2006)

Carotenoids present in microalgae include astaxanthin β-carotene lutein zeaxanthin

lycopene and bixin (Spolaore et al 2006) Astaxanthin is known to scavenge free-radicals

which is desirable for immunomodulation and cancer prevention (M A Hejazi amp Wijffels

2004) This carotenoid has a market value of at least $2000USkg (J Li Zhu Niu Shen amp

Wang 2011) Beta-carotene is another carotenoid demanded by the pharma and agrofood

industry due to its pro-vitamin A role (Baker amp Guumlnther 2004) Its market value is

approximately $300-3000USkg Carotenoids typically make-up 01-02 of dry weight but

can be as high as 14 dry weight in the case of Dunaliella Microalgae is also a source of

many essential vitamins including vitamins A B1 B1 B6 B12 C E nicotinate biotin folic

acid and panthothenic acid However the quantities present in the microalgae will fluctuate

depending on the environmental conditions (Spolaore et al 2006)

223 Other Commercial Applications

Microalgae extracts have also been found in face and skin care products sun protection

products and hair care products Chlorella vulgaris and Arthrospira are the two most

commonly used species for cosmetic applications Extracts have been used to support tissue

regeneration and wrinkle reduction (Spolaore et al 2006)

23 Algae Bioreactors

A current limitation is microalgal biomass production which can be expensive and time-

consuming Large-scale production of microalgae involves a continuous feed of synthetic

culture media containing the necessary nutrients and pH levels for optimal algae growth

(Grima Acie amp Chisti 1999) The microalgal broth is removed simultaneously followed by

9

processing There are two main methods of large-scale production raceway ponds and

photobioreactors (Chisti 2007)

In a raceway pond the microalgal broth is circulated through a closed loop channel with

constant mixing from a paddlewheel The algae culture is fed continuously in front of the

paddlewheel and harvested after the culture has flowed through the whole loop Raceway

ponds are low-cost but produce less biomass compared to photobioreactors per unit area

(Chisti 2007)

In a tubular photobioreactor an array of transparent tubes known as the solar collector

collects sunlight The algae broth is flowed with a mechanical or airlift pump to ensure light

penetration and allow for maximum algal growth The photobioreactor requires cooling

during the day and temperature control during the night with heat exchangers (Chisti 2007)

Photobioreactors can be designed to provide control over culture conditions and growth

parameters including temperature pH mixing CO2 and O2 levels This prevents

contamination of the algal culture and invasion by competing microorganisms (Mata

Martins amp Caetano 2010)

When comparing raceway ponds and photobioreactors both methods can produce 100t of

biomass each year with the same carbon dioxide consumption However photobioreactors

have yielded more oil per hectare compared to raceway ponds due to a 13-fold increase in

volumetric biomass productivity Both methods have been used in commercial production

facilities (Grima et al 1999) (Spolaore et al 2006)

After the microalgal biomass is recovered from the broth various oils and carotenoids can be

extracted from the algae The biomass recovery itself is an expensive process The biomass

from the photobioreactors costs less than the recovery of biomass from the raceway ponds

due to a higher biomass concentration (Chisti 2007)

10

24 Extracting OilCarotenoids from Algae

241 Current Lipid Extraction Techniques

Prior to extraction the microalgal biomass is typically recovered with a solid-liquid

separation step such as flocculation filtration flotation andor centrifugation These

processes are energy intensive (Y Li Horsman Wu Lan amp Dubois-calero 2008) and

contribute to approximately 20-30 of the total production costs (Grima Acie Medina amp

Chisti 2003) The resulting biomass slurry (5-15 dry solid content) is then dried for higher

extraction efficiency (Brennan amp Owende 2010) Dehydration of the algal biomass increases

its shelf-life and common methods include spray-drying drum-drying freeze-drying and

sun-drying (Richmond 2004) Purification of the crude lipids is also required (Grima et al

2003) The overall processing cost is dependent on the desired product but it is currently an

economic limitation (Mata et al 2010)

Current methods of oil extraction from algae include mechanical pressing homogenization

milking solvent extraction supercritical fluid extraction enzymatic extraction ultrasonic-

assisted extraction and osmotic shock Hexane is commonly used for solvent extraction due

to its low cost and high extraction efficiency Pressing homogenization and milling are often

used in combination with solvents presenting potential safety hazards (Mercer amp Armenta

2011) Mechanical disruption in addition to the solvent extraction improved the oil recovery

for Scenedesmus dimorphos and Chlorella protothecoides Wet milling of S dimorphos

combined with a hexane extraction yielded 253 oil recovery compared to only 63 in a

soxhlet extraction Similarly bead-beating of C protothecoides produced 188 oil versus

56 oil from a soxhlet extraction (Y Shen Pei Yuan amp Mao 2009) A summary of several

current extraction methods can be found in Table 3 The required extraction time and fraction

of oil recovery is dependent on the algae species

11

Table 3 Current Methods of Oil Extraction from Algae

Method Description Advantages Disadvantages Ref

Mechanical

disruption - Rupturing the cell

wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization

(forcing the biomass

through an orifice)

- Maintains chemical

integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent

extraction - Organic solvents

(ex hexane

acetone

chloroform) degrade

the cell wall The oil

is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform

methanol) is used as

a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)

- Non-toxic and non-

flammable

- Simple and quick

- Good for high-

value products free

of solvent residues

- Safe for thermally

sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound

sonnication - Recover oils

through cavitation

(form bubbles at

location where

pressure of liquid is

lower than vapor

pressure)

- The collapse of the

bubble can damage

cell wall nearby and

release cell contents

- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs

- Negligible toxicity

- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)

Typical lipid extraction methods such as those used for food may not be applicable to

microalgae Microalgae are single cells with individual cell walls Additionally the fatty

acids are unique from most higher animal and plant organisms Organic solvents are

commonly used to extract lipids from tissues The solvent should be adequately polar to

remove the lipid from the cell membrane but not so polar that non-polar lipids (such as

triacylglycerols) are not readily solubilised (Iverson Lang amp Cooper 2001)

The Folch method is commonly used to extract lipids from microalgae with gt95 recovery

of total lipids A ratio of 843 of chloroformmethanolwater is used to extract the total lipids

from the tissue sample with a ratio of 120 (gmL)of sample to chloroformmethanol (vv)

This method typically involves three sequential extractions (Iverson et al 2001) An

alternative solvent extraction technique is the Bligh and Dyer method which was originally

developed for marine tissues This method is similar to the Folch method except the ratio of

solvents is 1208 of chloroformmethanolwater The ratio of solvent to sample is 13 In

this method the lipid content was underestimated by up to 50 for samples with higher lipid

content in literature However this method is often chosen for its reduced solvent to sample

ratio For samples with less than 2 lipid content the Bligh and Dyer method is comparable

to the Folch method in terms of reliability and effectiveness (Iverson et al 2001)

The microalgae should be lyophilized to remove excess water in the cell and cell-disruption

was unnecessary when a minimum of two extractions were performed (Ryckebosch

Muylaert amp Foubert 2011)

13

242 Current Carotenoid Extraction Techniques

Similar to lipid extraction carotenoids are typically extracted by cell disruption followed by

solvent extraction Typically a yield of 25 carotenoid extract can be achieved with solvent

extraction but further purification is needed (Fernaacutendez-Sevilla Acieacuten Fernaacutendez amp Molina

Grima 2010) Table 4 summarizes common methods for carotenoid extraction

Table 4 Current Methods of Carotenoid Extraction from Algae

Method Description Advantages Disadvantages Reference

Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common

vegetable oils - Up to 939

recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-

stage system with an

organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1

- Ability to extract

from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)

243 Microalgae Milking with Two-Phase Bioreactors

Hejazi and Wijffels have harvested β-carotene from Dunaliella salina in a two-phase

bioreactor They proposed the possibility of extracting these secondary metabolites through a

lsquomilkingrsquo process Milking involves the continuous production and removal of metabolites

from the cell (M A Hejazi amp Wijffels 2004) After the lipid and high-value compounds are

extracted the microalgae would be able to regenerate new compounds for future extractions

thus bypassing the bottleneck of algae production

Hejazi and Wijffels grew algal cells under normal conditions and then stressed them with

light to produce extra β-carotene A biocompatible organic solvent was circulated throughout

the media containing algal cells to extract the β-carotene as shown in Figure 4 At the

interface between the cell and solvent the desired component was extracted Meanwhile the

algal cells were able to produce new β-carotene for future extraction in its own media (M A

Hejazi amp Wijffels 2004)

15

Figure 4 Two-Phase Bioreactor (M A Hejazi amp Wijffels 2004)

In the proposed extraction mechanism the solvent is taken up by the cell where it alters the

cell membrane The β-carotene accumulates in oil globules inside the chloroplast for D

Salina As the organic solvent dissolves into the cell membrane the globules are extracted

from the chloroplast to the space between the chloroplast and cell membrane Eventually the

globules are extracted to outside the cell In this mechanism direct contact between the

hydrophobic parts of the cell and the lipophilic solvent are required (M A Hejazi amp

Wijffels 2004) Another possible mechanism is the ability of the cell to make small vesicles

to take-up components of the solvent in the process of endo- and exo-cytosis (M A Hejazi amp

Wijffels 2004) More recently it was suggested that the extract was due to phase toxicity

where the dodecane enters the cell and alters the cell membrane As a result the cell

membrane is weakened and the cell is fragmented which allows the release of the lipid

globules (Kleinegris van Es Janssen Brandenburg amp Wijffels 2011)

Further understanding at the cellular level is required to describe the mechanism of the

milking process The molecular structure of the solvent will determine how the cell

membrane will react and what components of the cell will be extracted For example

16

extraction of chlorophyll can be difficult since it is located in the thylakoid membrane and is

bound strongly to other cell components (M A Hejazi amp Wijffels 2004)

There are three hypothesized extraction mechanisms product excretion cell

permeabilization or induced cell death Ideally product excretion would occur where the

lipids are transported out of the cell by exocytosis However it is hypothesized that the lipids

undergo a permeabilization mechanism and the lipids leak through the cell wall (Kleinegris

Janssen Brandenburg amp Wijffels 2011)

25 Microemulsion and Aqueous Oil Extraction Processes

251 Microemulsions

Microemulsions are composed of both an aqueous and oil domain coexisting in

thermodynamic equilibrium due to an emulsifying agent They have many unique properties

such as ultralow interfacial tension and ultrahigh solubilisation As a result they have the

capacity for oil-soluble and water-soluble solutes As well they are less than 100nm in size

so they are often transparent They have been used in a range of applications including

cosmetics drug delivery cleaning technologies and soil remediation (L D Do

Withayyapayanon Harwell amp Sabatini 2008)

There are three types of microemulsions that can be formed at low to moderate surfactant

concentrations The phase behaviour of microemulsions can be investigated by scanning

certain conditions (eg electrolyte concentration temperature etc) to affect the morphology

of the microemulsion Typically a phase scan involves the control of certain variables such

as temperature and surfactant concentration all variables are kept constant except for the

scanning variable In a Winsor Type I (OW) microemulsion a normal oil-swollen micelle is

formed in equilibrium with an excess oil phase In Winsor Type II a reverse micelle is

formed (water in oil WO) Finally a Winsor Type III consists of a bicontinuous phase A

fourth type of microemulsion Winsor Type IV is formed when the surfactant concentration

increases in a Type III system In a Type IV system a single middle phase forms Type I II

17

and IV microemulsions are illustrated in Figure 5 The transition between the phase types can

be controlled by electrolyte concentration for ionic surfactants or temperature for non-ionic

surfactants (L D Do et al 2008) When salinity is increased the electrical double layer

around the surfactant is reduced which causes the electrostatic repulsion to decrease too As

a result the surfactant-water interaction increases and the microemulsion shifts from Type I

(OW) to Type III to Type II (WO) (E Acosta Uchiyama amp Sabatini 2002) In Figure 5 the

water and oil volume ratios are fixed at 11 while the hydrophilic linker is increased along the

scan

Figure 5 Microemulsion Types

Triglycerides such as those found in algal oil are very hydrophobic due to long and bulky

alkyl chains and high polarity ester regions As a result they are difficult to solubilise and

form ldquospongerdquo phases when mixed with the aqueous and emulsifying components To

overcome this obstacle co-oils or linkers combined with extended-surfactants are used to

form the microemulsions Linkers are amphiphiles that improve the surfactant-oil and

surfactant-water interactions at the interface (L D Do et al 2008)

Microemulsions have been used to extract or solubilise oils in previous literature The use of

microemulsions to extract oils can be based on two proposed schemes In the first scheme

extended-surfactants are used to decrease the interfacial tension (IFT) This proposed

Increasing hydrophilic linker concentration

18

mechanism has been used to extract vegetable oil from ground oil seeds where the oil is

released from the cell membrane when it overcomes the IFT (L D Do et al 2008) In the

second scheme supersolubilization is achieved with a high surfactant concentration and

various linkers (Edgar J Acosta Nguyen Witthayapanyanon Harwell amp Sabatini 2005)

The solubilisation can be controlled through temperature to release excess oil Application

and examples of these two mechanisms are given in the proceeding sub-sections

252 Vegetable Oil Extraction and Extended-Surfactants

In previous studies reverse-micellar microemulsions were used to extract oil from ground

oilseeds (Leser Luisi amp Paimieri 1989) Vegetable oils could be extracted at high surfactant

concentrations between 2-4wt(L D Do et al 2008) The oil extraction efficiency

depended on the type of solvent structure of the oil contact time between the oilseed and the

solvent the shaking speed the temperature and the solid-to-solvent ratio In one study the

solid-to-solvent ratio was optimized to 15 (wv) when the shaking speed was set to 200rpm

for 40min (Nguyen et al 2010)

However the separation of the microemulsion and oil can be an obstacle Ideally an aqueous

surfactant-based process would be desirable where the surfactant remains separate from the

extracted oil More recently a new class of surfactants known as extended surfactants have

been able to produce ultralow interfacial tension (IFT) with vegetable oils (L D Do et al

2008) In typical surfactants the alkyl chain length increases proportionally with

hydrophobicity However there is a trade-off with water solubility In extended surfactants

there are propoxylate (PO) andor ethoxylate (EO) groups between the hydrophilic head and

hydrophobic alkyl chain thus increasing the hydrophobicity of the surfactant without

compromising water solubility As a result ultralow IFT much less than 01mNm can be

achieved (L D Do et al 2008)

Using extended surfactants 93-95 extraction efficiency was achieved for peanut and canola

oils at 25degC In addition the oil quality was better than that obtained from hexane extraction

The extraction mechanism is hypothesized to involve the lowering of the IFT at the oilwater

19

interface facilitating the oil droplet within the oil seed to break-up and diffuse out of the

cellular matrix (L D Do et al 2008)

253 Linker-Based Biocompatible Microemulsions

The type of microemulsion that is formed depends on the hydrophiliclipophilic balance

(HLB) of the surfactant and the oilwater interface Typically hydrotropes cosurfactants

cosolvents and electrolytes can be used to control the HLB Another option is the use of

linkers to extend the surfactant effect either in the oil or water phase Hydrophilic linkers

improve the surfactant-water interaction and lipophilic linkers improve the surfactant-oil

interaction These linkers can be used to complement each other and improve the

solubilisation results (E Acosta et al 2002)

Acosta et al(2005) have formulated microemulsions using lecithin as the surfactant in

addition to bio-compatible linker molecules for dry-cleaning applications Microemulsions

were formulated under isotonic conditions (09 NaCl) by adjusting the linker proportions

The solvency of the lecithinlinker formulation was compared with tetrachloroethylene

(PCE) a typical dry-cleaning solvent In both cases the solvent was used to remove

hexadecane from cotton fabrics At low loading ratios (lt1 oil volume fraction) the

lecithinlinker formulation and PCE removed the same amount of oil At higher loading

ratios the lecithinlinker formulation maintained its efficiency unlike PCE This study

showed that bio-compatible linker-based lecithin formulations have the potential to replace

organic solvents (Acosta et al 2005)

20

3 Extracting Lipid and Carotenoids from Microalgae with

Lecithin-Linker Microemulsions

31 Materials and Methods

311 Materials

The following chemicals were purchased from Sigma-Aldrich (Oakville ON Canada) ethyl

hexanoate (gt98 ethyl caprate) sodium chloride (99+ Fluka brand) and sorbitan

monooleate (SMO Spanreg 80 995) PEG-6-capryliccapric glycerides (Softigen 767) was

donated by Sasol North America (Houston TX USA) Olive oil was purchased from the

local market (Toronto ON Canada) Laboratory grade soybean lecithin was purchased from

Fisher Scientific (Fairlawn NJ USA)

The microalga studied was Chlorella vulgaris and was obtained from Pond Biofuels

(Toronto ON Canada) in paste form The original seed was Chlorella vulgaris (CPCC 90)

from the Canadian Phycological Culture Centre at the University of Waterloo The algae

were grown in Pond Biofuelrsquos 1000L bioreactor under flashing LED lights at 08wattslitre

flashing at 10Hz The algae were also continuously agitated with air spargers and fed with

bottled carbon dioxide Prior to extraction the alga was lyophilized to remove the excess

water and the algae were stored at 4degC

The lipids extracted from the algae were converted into fatty acid methyl esters (FAMEs)

with NaOH (reagent grade ge98) HCl (ACS reagent 37) methanol (ACS reagent

ge998) and MTBE (HPLC grade 998) from Sigma-Aldrich The FAMEs were then

quantified using gas chromatography (GC) and FAME standards from Sigma Aldrich

(FAME Mix GLC-10 1891-1AMP Oakville ON Canada) and olive oil (Bertolli) from the

local grocery store

The extracted carotenoids were quantified with UV-Visible spectrophotometry and high

performance liquid chromatography (HPLC) β-carotene (Type I synthetic ge93 (U )

powder) was obtained from Sigma-Aldrich The solvents required for HPLC such as

21

methanol and acetonitrile were provided by the ANALEST facility at the University of

Toronto

For the biocompatibility studies the algal cultures were grown in Boldrsquos basal medium The

composition of this stock solution can be found in Appendix A The algae was incubated at

room temperature in 150mL flasks plugged with foam stoppers and shaken under two 26W

full spectrum compact fluorescent bulbs (Exo-Terra Repti Glo China)

312 Characterization of Microalgae

An algae paste was obtained from Pond Biofuels (Toronto ON Canada) for lipid and

carotenoid extraction Prior to extracting lipids and carotenoids with microemulsions the

microalgae paste was characterized for water and lipid content To determine the water

content of the microalgae a known amount of microalgae paste was frozen at -80degC and

lyophilized overnight in a Labconco Freezone 12 Plus Freeze Dry System (Kansas City

Missouri) at 0018mBar and -40degC The change in mass indicated the water content of the

algae The lipid content could then be determined using a Folch extraction (Iverson et al

2001) followed by transesterification of the crude lipids and quantification via GC

3121 Lipid Extraction via Folch Method

As mentioned in section 241 the Folch method is the most common and accurate method of

lipid extraction from microalgae with more than 2 lipid content After lyophilisation and

grinding of the algae tissue the procedure found in Appendix B was followed

The final lipid mass measured was assumed to be the crude lipid content of the microalgae

sample The fatty acid composition of the extracted crude lipids was quantified using GC

following transesterification of the crude lipids

A baseline was established to determine the total lipid content of the algae sample prior to

extraction with microemulsions The Folch method was chosen to extract lipids from the

lyophilized algae due to its extensive application to microalgae samples in literature This

method was validated and optimized for C Vulgaris in Appendix B

22

The Folch method involves the addition of dry algae biomass to a chloroformmethanol (21

vv) mixture in a ratio of 120 (mv gmL) The mixture is agitated for 15 minutes then

centrifuged for 20 minutes The solvent is removed from the tube and new solvent is added

A 09 NaCl solution is then added to the solvent to separate the polar and non-polar phases

of the solvent mixture The upper methanol phase contains the non-polar lipids This method

was optimized for maximum lipid extraction three sequential extractions were determined to

be best

3122 Transesterification of Lipids for GC

Due to the small sample size the crude lipids that were extracted from the microalgae were

converted into fatty acid methyl esters (FAMEs) for more precise measurements with GC

This method was adapted from a Microbial ID System used to identify FAMEs present in

bacteria (Haack et al 1994) Typically a broth culture is used instead of crude algal lipids

As well the saponification and esterification steps occur at 87degC The full procedure can be

found in Appendix C To summarize the FAMEs were prepared in four steps in a process

involving saponification methylation extraction and sample clean-up During

saponification the triglycerides were treated with a strong base for 30 minutes The ester

bond was cleaved to release the fatty acid salt and glycerol molecules this step took 10

minutes Methylation involved the addition of a methyl group with methanol and a strong

acid to reduce polarity and this took 10 minutes In the third step the FAMEs were extracted

into the desired solvent and used as the mobile phase in GC Finally the solvents were rinsed

with a strong base to remove any impurities

The GC settings are found in Appendix D The prepared FAMEs were injected in the GC and

measured using a calibration curve and known standards

313 Microemulsion Formulation and Characterization

Lecithin was chosen as the surfactant due to its biocompatibility and food safety (E J Acosta

et al 2011) A starting point of 4 lecithin was based on previous work (X-Y Xuan

23

Cheng amp Acosta 2012) A phase scan was performed by varying the ratio of hydrophilic

linker to surfactant as found in Table 5

Table 5 4 Lecithin Microemulsion Series

Sorbitan monooleate Lecithin Softigen 20 NaCl dH2O Ethyl Caprate

g g g g g g

040 040 060 065

07010

0225 Balance to 10g 420

The chemicals were mixed in a 15mL tube until a uniform solution was observed The tubes

were left at room temperature to reach equilibrium this took 1-2 days When the temperature

of the microemulsion system changes the solubility will be affected The effect of

temperature was observed in this study since it is easily reversible The microemulsion series

was placed at room temperature 4degC and 37degC until equilibrium was reached The ratio of

the excess oil to microemulsion to excess water was measured and plotted against the

Softigenlecithin ratio as in Figure 8

A type I microemulsion can be identified by the presence of an excess oil phase at the top of

the vial Conversely a type II microemulsion will have an excess water phase on the bottom

A type III microemulsion will have both an excess oil and water phase while a type IV

microemulsion will have neither This shift in phase can be controlled with the amount of

surfactant hydrophilic linkers or lipophilic linkers After a type I type II and type IIIIV

microemulsion was identified these formulations were used in subsequent lipid and

carotenoid extractions

The conductivity of the microemulsions was measured with a VWR conductivity meter and

MI-905MI-915 conductivity probes (Microelectrodes Inc Bedford USA) The viscosity was

measured with a CV-2200 falling ball viscometer (Gilmont Instruments Barrington IL

USA) The drop size was measured by dynamic light scattering (DLS) with a 90Plus Particle

Size Analyzer from Brookhaven Instruments Corporation with a photomultiplier detector at

90deg (BI-200SM)

24

314 Lipid Extraction and Quantification

3141 Lipid Extraction

To determine the amount of lipids extracted from the algae using microemulsions an indirect

method was developed

First the algae paste received from Pond Biofuels was freeze-dried overnight and ground

using a coffee grinder to break-up the cell walls and maximize extraction Meanwhile the

microemulsions were prepared and characterized separately Once both components were

prepared the dried algae was added to either hexane ethyl caprate or the microemulsion and

agitated for 05-55 hours The mixture was centrifuged for 15 minutes to separate the solvent

or microemulsion from the algae biomass The upper solventmicroemulsion phase was

analyzed using HPLC or UV-Vis to observe the kinetics of carotenoid extraction

Hexane was chosen as a control because it is a common solvent used in extraction of lipids

from tissues (Hara amp Radin 1978) Since the microemulsion consists of water and ethyl

caprate these two solvents were included as controls too As well the Hansen solubility

parameters obtained from the HSIP software (Abbott and Hansen Charles M Yamamoto

2010) indicate that ethyl caprate could be a good match to the triglycerides as shown in

Table 6 Since the Hansen solubility parameter is much closer between ethyl caprate and the

various methyl esters and triglycerides it should solubilise more of these components than

hexane theoretically

Table 6 Hansen Solubility Parameters (Abbott and Hansen Charles M Yamamoto 2010)

Dispersion (MPa12

) Polar (MPa12

) Hydrogen Bonding (MPa12

)

Ethyl Caprate 158 205 55

Ethyl Oleate 162 32 40

Glycerol Trioleate 16 38 32

Methyl Oleate 162 38 45

25

Methyl Caproate 160 37 57

Methyl Caprylate 160 42 48

Tricaprylin 163 39 49

Tripalmitin 163 31 38

Hexane 149 0 0

Since the microemulsion itself contains fatty acids similar to the algal lipids it would be

difficult to differentiate the source of the lipids in this upper solventmicroemulsion phase

The lipid source could be either the microemulsion or the algae Therefore this indirect lipid

quantification method was used

3142 Lipid Quantification with GC

After the initial extraction with either solvent or microemulsions the residual lipids were

extracted and quantified The bottom phase contained the algal biomass which included any

remaining lipids not extracted by the microemulsion or solvent Any residual microemulsion

was rinsed away by adding DI water to the remaining biomass After agitating and

centrifuging this mixture again the excess microemulsion rose to the top of the vial since it

had a lower density than the water This algal biomass then underwent a second extraction

using the Folch method as described in Appendix B In an ideal extraction where 100 of

the lipids were extracted by the microemulsions no residual lipids would be extracted These

residual lipids were analyzed by GC Prior to GC the crude lipids underwent

transesterification (3122) to become fatty acid methyl esters

After the FAMEs were quantified using GC this value represents the ldquo esidual Lipidsrdquo The

ldquoBaselinerdquo was previously determined with the Folch method and represents the total lipids

initially present in the microalgae The ldquoLipids Extractedrdquo can be calculated indirectly as the

difference between these two values

Lipid Extracted (g) = Baseline (g) ndash Residual Lipids (g) (1)

26

Where the baseline was determined by the Folch extraction and GC analysis on the initial

dried algal biomass and the residual lipids was determined by the Folch extraction and GC

analysis on the algal that was pre-treated with the microemulsion (an initial extraction)

It should be noted that the calculated ldquoLipids Extractedrdquo is a conservative value that may be

lower than the actual value since the ldquo esidual Lipidsrdquo may include some components of the

microemulsion that would inflate this value

The extraction efficiency was calculated as

Extraction Efficiency = Lipid ExtractedBaseline (2)

315 Carotenoid Extraction and Characterization

3151 UV-Visible Spectrophotometry

A 661 (ww) ratio of algae biomass to solvent or microemulsion was mixed for 55h in a

vial Water hexane and ethyl caprate were used as controls

At 05h 15h 25h and 55h samples were analyzed using a Cary 50 Ultraviolet-Visible

Spectrophotometer At each time point the vial was centrifuged and 05mL of the

solventmicroemulsion was removed and diluted with either 35mL of the same solvent or

isopropanolethyl caprate (21 vv) for the microemulsions The sample was placed in the

UV-Vis spectrophotometer and the absorbance spectrum between 190nm to 1100nm was

obtained A baseline obtained as the absorbance of the pure solvent was subtracted Since β-

carotene has a characteristic peak at 450nm (Hart amp Scott 1995) the absorbance at this

wavelength was plotted against time However there is the potential for the chlorophyll

peaks and other carotenoid peaks to overlap at this wavelength Therefore the absorbance

kinetics is indicative of the overall pigment extraction

3152 HPLC

A 201 (ww) ratio of algae biomass to solvent or microemulsion was mixed for 1hour The

mixture was then centrifuged for 15 minutes to separate the algae and

27

solventmicroemulsion The extracted carotenoids that were solubilised in the solvent or

microemulsion were quantified using HPLC

A calibration curve was produced to relate known concentrations of β-carotene with the peak

area A protocol from Current Protocols in Food Analytical Chemistry (2001) F231-F2315

was adapted and can be found in Appendix E An isocratic C18 reversed-phase protocol with

a C18 column from Supelco (Supelcosil LC-18 25cm 46mm 5μm) and a Phenomenex

Guard cartridge (C18 5μm 4 30mm Phenomenex Corp) was used The mobile phase

consisted of 900mL methanol 100mL acetonitrile (ACN) 1mL triethylamine (TEA) and was

passed through the column at a flow rate of 1mLmin Since β-carotene has a maximum

absorbance at 452nm the UVVis detector was set to monitor absorbance at this wavelength

The β-carotene peak (retention time) was observed at 11-12minutes Samples of pure

microemulsion or solvent were also injected into the HPLC to ensure that nothing else eluted

at the β-carotene retention time

316 Biocompatibility Regrowth Study

To test for biocompatibility between the algae cells and the microemulsions algae paste was

added to water hexane ethyl caprate type I microemulsion type II microemulsion or type

IV microemulsion in a 12 (gmL) ratio The water was a negative control as it should not

kill the algae any cell death in this scenario was primarily due to the stress caused by the

mixing and centrifugation of cells

The algae and solventmicroemulsion were mixed for 1h on the multi-tube vortexer (VWR

VX-2500 USA) then centrifuged for 15 minutes to separate the algae from the solution The

solventmicroemulsion was removed from the vial using a pipette Distilled water was added

to the remaining algae and the vial was shaken and centrifuged again this was to remove

excess solventmicroemulsion that would rise to the top of the vial The remaining algae at

the bottom of the vial were added to a BBM synthetic media A sample of this alga was also

observed with a light microscope

28

The algae solution was diluted by BBM media at a ratio of 199 (vv) and placed in 150mL

shaker flasks under cool white light and constant agitation Samples of the algae solution

were taken at 1 3 7 and 13 days to observe cell growth In a healthy algal suspension the

algae are inoculated again after 2 weeks to ensure sufficient nutrients in the media A two

week observation of the growth curve would ideally include the lag phase exponential

growth phase and stationary phase

The viability of the algae was also tested in a less intensive extraction the algae paste was

mixed with the solventmicroemulsion at a 12 ratio (wv) in a glass tube for 15minutes

Every five minutes the tube was inverted several times to ensure gentle mixing After the

initial extraction the excess solventmicroemulsion was removed in the same manner as in

the previous biocompatibility test The remaining algae were inoculated with BBM media

and allowed to re-grow as before over a two week period with periodic measurements of

growth Small 5mL samples of the algae were dried on filter disks and quantified as Total

Suspended Solids (TSS) Any increase or decrease in biomass concentration was quantified

according to the American Public Health Association (APHA) standards 2540D and 2540E

1 Glass-fiber filter disks were ignited at 550degC for 15 minutes then weighed

2 Vacuum filtered 5mL of algal solution through the 15microm Whatman 934-AH glass-

fiber filter disk

3 Dried the filter disk and collected algae at 103-105degC for one hour or until a constant

mass was observed

The increase or decrease in mass or TSS may have represented the cell growth or death over

two weeks However the flasks were not homogenized prior to taking the sample Flocks at

the bottom of the flask were avoided during sampling which could contribute to a false

decrease in TSS This test was not a definite indicator for cell death The cells were also

observed with an Olympus BX-51 light microscope (Richmond Hill ON Canada)

29

32 Results


Prior to lipid and carotenoid extraction the microalgal paste obtained from Pond Biofuels

was characterized First the water content of the Chlorella vulgaris paste was determined to

be 635plusmn19 after lyophilisation

The viscosities at room temperature were determined with a FallingBall Viscometer The

Type I II and IV microemulsions had viscosities of 4537plusmn71 mPas 1737plusmn207 mPas

and 1481plusmn63 mPas respectively These values are lower than conventional medium-chain

alcohol lecithin microemulsions which are approximately 1000mPas (D A Sabatini

Acosta amp Harwell 2003) However the viscosity is higher than those reported by JS Yuan

et al (2008) for a similar microemulsion formulation The microemulsions consisted of

lecithin sorbitan monooleate sodium caprylate caprylic acid isopropyl myristate and

sodium chloride and produced a viscosity of 10-40mPas (J S Yuan Ansari Samaan amp

Acosta 2008) Comparatively the microemulsions formulated in this work includes PEG-6-

capryliccapric glycerides which increases the viscosity

Using DLS the drop size of the microemulsions was determined to be between 1-2nm This

small drop size is due to the high viscosity determined by the viscometer However the

microscale viscosity of the microemulsions could be much lower which would increase the

measured drop size

The total lipid content and fatty acid profile of the C vulgaris samples were determined as

described in section 3141 and 3142 A sample of olive oil was also converted into

FAMEs with the procedure in Appendix C A standard purchased from Sigma-Aldrich

(FAME Mix GLC-10 1891-100mg) was already in methyl ester form and was diluted using

the same solvent (hexaneMTBE) These three samples were injected into the GC and the

chromatograms are shown in Figure 6 In all samples an internal standard (IS) of hexadecane

was added

30

Diluted samples of the calibration standards were also injected into the GC to produce a

calibration curve for the following methyl esters methyl palmitate (C160) methyl oleate

(C181) methyl linoleate (C182) and methyl linolenate (C183) Each methyl ester has a

specific retention time The resulting calibration curves can be found in Appendix F

Olive oil was observed to have a very similar fatty acid profile to the oil extracted from C

Vulgaris A calibration curve was produced based on varying amounts of olive oil to

determine the total lipid content of the algae Again the calibration curves are in Appendix F

The R2 of these calibration curves were between 095-099

Figure 6 GC Chromatograms of Standards Olive Oil and Algae Oil

After the microalgae were extracted with the microemulsions the remaining lipids were mainly

C160 C180 C181 and C182 as shown in Figure 7 The longer fatty acids are not detected in

the residual lipids indicating that the microemulsions are effective in extracting various

polyunsaturated fatty acids and longer chained fatty acids

31

Figure 7 GC Chromatograms comparing Residual Lipids after Microemulsion Extraction


After the microemulsions were formulated with an increasing ratio of Softigen to lecithin a

clear phase transition could be observed As more hydrophilic linker was added to the

formulation the microemulsion shifted from Winsor Type II to Type III to Type I

A decrease in temperature decreased the solubility of the microemulsion and the excess oil

phase increased This indicated a shift towards the Type I microemulsion Conversely an

increase in temperature shifted the phase scan towards the Type II microemulsion Ideally

after the oil is extracted from the algae at room temperature or warmer where the

microemulsion is able to solubilise more oil the temperature could be lowered At a low

temperature the oil is less soluble in the microemulsion and the excess oil phase increases

This excess oil could be easily separated

32

Figure 8 Phase Scan of 4 Lecithin Series with Varying Ratios of PEG-6-Caprylic Lecithin

At room temperature three vials were chosen to represent the desired microemulsion types

This specific formulation was used for all subsequent experiments The chosen formulations

are presented in Table 7

Table 7 Microemulsion Formulations for Subsequent Extractions

Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420

The conductivity and viscosity of these microemulsions are found in Table 8 Typically the

droplet size increases as the formulation approaches the Winsor Type IV biocontinuous

system (J S Yuan et al 2008)

Table 8 Microemulsion Characterization

Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621

Type II Type IV Type I

33



Dried algae were used to demonstrate the extraction capability of the microemulsion

formulation In the following results the extraction efficiency of type I II and IV

microemulsions are compared to hexane and ethyl caprate In the Folch method a 120 ratio

of dry biomass to solvent was repeated three times a total mass (g)solvent (mL) ratio of

160 was required The biomass was exposed to the solvent for a total of 105min

3232 Effect of Mass Solvent Volume Ratio

The effect of mass solvent volume ratios at 1h are shown in Figure 9 In general increasing

the solventmass ratio improved the extraction efficiency of the system This is best

illustrated by Figure 9 where the extraction efficiency at 1h is quantified for different

masssolvent ratios Specific values and additional plots at 05h and 3h can be found in

Appendix G However the lipid extraction results at 05h and 3h were not as consistent

Figure 9 Extraction Efficiency at 10h

0

20

40

60

80

100

hexane ethyl caprate

type I type II type IV Extr

acti

on

Eff

icie

ncy

SolventMicroemulsion

1h Extraction Efficiency

16

120

160

n=3 pge005 pge001 pge0001

34

3233 Effect of Time

In a typical extraction the initial rate of extraction is high and slowly decreases as the

concentration gradient between the cell and environment reaches equilibrium Figure 10

compares the extraction efficiency of 120 (biomasssolvent) systems at 05h 1h and 3h

Specific values and data for the 16 and 160 systems can be found in Appendix G

Figure 10 Extraction Efficiency of 120 (MassSolvent) Ratio



UV-Visible spectrophotometry was used to monitor the extraction capability of the

microemulsions and compared to water hexane and ethyl caprate An increase in absorbance

indicated that more carotenoids and chlorophylls were being extracted into the

microemulsionsolvent Figure 11 shows one sample of a scan between 350-1000nm For

reference a β-carotene standard was dissolved in hexane and the absorbance was recorded

Beta-carotene has a characteristic peak at 452nm as observed The local maximum can shift

depending on the solvent

0

20

40

60

80

100

120


type I type II type IV

Extr

acti

on

Eff

icie

ncy


120 Extraction Efficiency

05h

1h

3h

n=3 pge005

35


biomass(g)solvent (mL)

The absorbance scan shown in Figure 11 was repeated at 15h 25h and 55h The

absorbance at 445nm a local maximum was recorded The overall extraction kinetics is

shown in Figure 12 All extractions involving the microemulsions showed a higher

absorbance than extractions with ethyl caprate or hexane Therefore microemulsions are

effective for pigment extraction

Figure 12 Absorbance Kinetics at 445nm with UV-Visible Spectrophotometry

160 dry biomass (g) solvent (mL)

-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)

Absorbance (30 min extraction) type I

type II

type IV

ethyl caprate

hexane

water

standard (10ugmL beta carotene)

0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)

Absorbance Kinetics (445nm) Type I

Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC

Although UV-visible spectrophotometry is a good indicator of the overall carotenoid and

chlorophyll extraction kinetics the amount of extracted specific carotenoids is unknown

HPLC was used to quantify the extracted β-carotene using the calibration curve with an R2 of

098 (Appendix H) Extraction conditions were chosen to be 1 hour contact time and 120

biomass to solventmicroemulsion ratio (gmL) since these conditions gave more consistent

results in the lipid extraction

Figure 13 shows the HPLC chromatograms of the algae extracts Between 11-12 minutes a

β-carotene peak was eluted The peak was much larger when a microemulsion was used to

extract the β-carotene Appendix H

Figure 13 HPLC Chromatogram of Algae Extract by Various SolventsMicroemulsions

β-carotene

37

Figure 14 shows that extraction with the microemulsion yielded more β-carotene than both

hexane and ethyl caprate The type I microemulsion extracted the most β-carotene

Figure 14 Extracted Beta-Carotene Quantified with HPLC

325 Biocompatibility Regrowth Testing

After the microalgae were mixed with water ethyl caprate hexane or a microemulsion

formulation for 1h the excess solventmicroemulsion was removed The remaining algae

were allowed to recover in BBM and the growth was monitored As a blank control one

sample was treated with water As expected this sample had the best recovery as

represented by the increase in total suspended solids (TSS) in Figure 15

All samples demonstrated an increase in cell growth up to 6 days (Figure 15) However at 13

days the majority of the samples appeared clear The samples involving type I

microemulsions and ethyl caprate were the exceptions where the algae remained a pale green

compared to the clear water present in the other flasks However the growth was still very

weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)

Extracted Beta-Carotene (1h 120)

38

In most cases except the water control the growth was not statistically significant based on a

t-test between TSS values The only significant increase in biomass was after the microalgae

were allowed to recover for 6 days after the Type IV extraction (Figure 15)

Figure 15 Algae Recovery after 1h SolventMicroemulsion Extraction

Figure 16 shows the flasks after 2 weeks Initially all flasks were inoculated with the same

amount of C Vulgaris and the colour of the flasks was visually similar Based on visual

inspection after 2 weeks the microalgae exposed to hexane type I and type II

microemulsions appear very weak the flasks are clear In comparison a pale green is

observed the microalgae samples exposed to ethyl caprate and type IV microemulsions As

expected the microalgae exposed to water had the best growth

Another biocompatibility test was performed at milder conditions the algae paste was

exposed to the solvent or microemulsion for 15 minutes only Instead of vigorous shaking

the tube was inverted several times at 5 minute intervals As expected the microalgae

remained greener with the gentler 15 minute extraction

0

005

01

015

02

025

03

water hexane ethyl caprate


TSS

(gL

)

Solvent Used in Initial Extraction

Recovery of Microalgae in BBM after 1h Initial Extraction

Day 1

Day 3

Day 6

Day 13


39

Figure 16 Algae Growth Recovery 2 weeks after MicroemulsionSolvent Exposure

Similar to the previous biocompatibility test following the 1h extraction of the microalgae

paste with solvent or microemulsions the algae growth recovery was measured by TSS

values In Figure 17 the TSS of each recovering algae sample was measured over 2 weeks

Triplicate values were taken

40

Figure 17 Algae Recovery after 15 minute SolventMicroemulsion Extraction

Figure 18 is a micrograph of the diluted microalgae paste provided by Pond Biofuels The cells

are green and round and appear healthy A small percentage of the cells are fragmented For

comparison the microalgae cells were also observed under the light microscope after the initial

extraction with either solvent or microemulsions These micrographs are found in Figure 18

Images of the cell immediately after the extraction are compared with the cells 14 days after

extraction In all cases the cells appeared green and round There was no noticeable difference in

the amount of lysed cells It should also be noted that the cell density of the micrographs are not

indicative of the cell growth or healthiness of the culture

Figure 18 Micrograph of C Vulgaris

0000

0050

0100

0150

0200

0250

0300

0350


type I type II type IV water

TSS

(gL

)


Recovery of Microalgae in BBM after 15min Initial Extraction

Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41

Table 9 Micrographs of C Vulgaris After Initial Extraction

Solvent used in

Initial Extraction

Day 0 After Extraction Day14 After Extraction

Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion


In the lipid extraction experiments reported above dried algae was used to prove that

microemulsions could be used for extraction The ultimate goal is to use live algae in

lsquomicroalgae milkingrsquo The extracted lipids would likely be lower in yield since the cell wall

would be intact in the live algae Typically drying the algae helps to disrupt the cell wall and

release the lipids

For each set of results a new baseline was established to take changes and variation in the

algae sample into consideration To obtain reliable results and account for changes in the GC

column an olive oil calibration curve was prepared for each set of new data The olive oil

and algae oil were converted into FAMEs and quantified on the same days to prevent

discrepancies due to degradation in FAMEs or changes in the GC column The olive oil

calibration was sufficient because the main concern was quantifying total lipids and not

specific fatty acids Also the olive oil underwent the same transesterification process as the

algae oil and takes incomplete transesterification into consideration

Chlorella vulgaris has been well studied in literature it is approximately 14-22 lipids 12-

17 carbohydrates and 51-58 protein (E W Becker 2007) though the composition can

vary based on growing conditions The algae samples in these experiments were between 6-

11 lipids which is slightly lower than expected Also the fatty acid composition of these

samples are comparable to those found in literature (Table 1) As shown in Figure 6 the

microalgae analyzed in this report consist of mainly C160 C181 and C182 fatty acids In

comparison literature reported C Vulgaris to be primarily made up of C160 C180 and

C203 (EW Becker 1994) The lack of polyunsaturated fatty acids such as C203 in the

experimental samples could be due to the growing conditions (M A Hejazi amp Wijffels

2004)

43


Theoretically additional solvent will improve the extraction efficiency until the maximum is

reached However smaller amounts of microemulsion or solvent are desirable for scale-up

When less solventmicroemulsion is used the extracted lipids are more concentrated and

easier to purify and costs for materials are minimized

During extraction the difference in concentration gradient between the inside and outside of

the algae cell increases as more solvent is added to the system As a result equilibrium is

reached sooner when the solvent mass ratio is greater Between 05-3h a plateau does not

seem to be reached as extraction efficiency continues to improve

It should be noted that an assumed 100 extraction efficiency was determined by the Folch

method previously described In the results presented 100 extraction efficiency was only

achieved in two situations at 3h 120 masssolvent ratio with the type I and type IV

microemulsions Even with hexane an organic solvent known to have good extractability

complete extraction efficiency was not achieved A possible explanation is that the baseline

determined by the Folch extraction was inflated due to the inclusion of phospholipids or

other non-polar components of the algae cell The chloroform may have dissolved the cell

membrane in addition to the lipid pools inside the cell since it is a very strong organic

solvent Theoretically polar lipids such as phospholipids extracted by the Folch method

should be solubilised in the methanol phase In future work this could be confirmed

Also hydrated lipids are difficult to extract even with hexane Theoretically the

microemulsions should still be able to extract the hydrated lipids due to its hydrophilic

region In this perspective the microemulsions would extract even better than the hexane in

liquid systems such as in lsquomicroalgae milkingrsquo

In Figure 9 only data from the 1h extractions is shown Appendix G includes the extraction

efficiencies at 05h and 3h Also the measured extraction efficiency at 3h opposes theory in

general Typically a higher biomass to solvent ratio will extract more unless equilibrium has

44

been reached However the 120 system appears to extract more lipids than the 160 system

on average at 3h This discrepancy could be explained by experimental error since the

sample size is small so errors are magnified All algae samples were taken from a well-mixed

stock but variability still exists between samples Also the solubility of the microemulsion

can be affected by temperature fluctuations This would randomly affect the results

An ANOVA test for two-factors with replication can be found in Table 10 In this case the

two factors were (1) type of solventmicroemulsion and (2) ratio of dry biomasssolvent

When comparing the effect of the solventmicroemulsion on the extraction efficiency it was

determined that FgtFcrit this implies that the solventmicroemulsion type significantly affects

the extraction efficiency Similarly FgtFcrit when comparing the effect of dry biomasssolvent

ratios

Table 10 ANOVA Two-Factor with Replication 1h Extraction

ANOVA

Source of

Variation

SS df MS F P-value F crit

Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44

Samples hexane ethyl caprate type I type II type IV

Columns 16 120 160

3312 Effect of Time

When observing the effect of time on the extraction the extraction efficiency did not reach a

plateau When the biomass to solvent ratio was 120 as in Figure 10 the improvement in

extraction efficiency was proportional to time as expected When the biomass was in contact

with the solvent for longer periods of time more lipids were able to diffuse into the

solventmicroemulsion

45

In general the type I microemulsion had the highest extraction efficiency since a plateau was

not reached An extraction time beyond 3h could potentially extract more lipids The time

needs to be determined for maximum extraction However there is a trade-off between

extraction time and extraction rate If each batch of microalgae undergoes multiple

extractions and is circulated with fresh microemulsions then a short contact time could be

acceptable

The statistical analysis can be found in Table 11 When comparing the effect of time and type

of solventmicroemulsion both were statistically significant factors for determining

extraction efficiency

Table 11 ANOVA - Two-Factor With Replication 120 extraction

ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h

3313 Extraction Efficiency vs Microemulsion Composition

Using the phase scan to determine the amount of excess water andor oil in each formulation

the composition of the microemulsion can be determined Ideally one would want to

minimize the use of ethyl caprate in the formulation Therefore the amount of ethyl caprate

used in the formulation is compared to the amount of lipids extracted from the microalgae

This data is plotted in Figure 19 and shows that type I microemulsions are the most effective

for lipid extraction in terms of ethyl caprate used

46

Table 12 Microemulsion Composition based on Phase Scan Results

of Microemulsion

Vial Phase

Behaviour

Ethyl

caprate Water Lecithin

Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139

A possible explanation for the improved lipid extraction observed with type I microemulsions is

the presence of more linkers Of the three formulations tested the type I microemulsion

contained the highest percentage of sorbitan monooleate and PEG-6-caprylic glycerides Linkers

are believed to reduce surfactant rigidity and affect the dynamic properties of the microemulsion

When the microemulsion is less rigid less energy is required to modify the structure and

solubilisation increases (Edgar J Acosta Le Harwell amp Sabatini 2003)

Figure 19 FAME Lipids Extracted vs Ethyl Caprate Concentration

00

02

04

06

08

10

12

14

ethyl caprate type I type II type IV

mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47



UV- isible spectrophotometry was used to monitor the change in absorbance Typically β-

carotene has an absorbance maximum at 452nm but the solventmicroemulsion has shifted

the local maximum to 445nm Several carotenoids including β-carotene absorb at this same

wavelength Therefore UV-visible spectrophotometry is only useful to observe the overall

extraction kinetics and is unable to quantify specific carotenoids The extraction kinetics of

the β-carotene would be a good indicator of the extraction kinetics of the lipids since both

are non-polar molecules In contrast chlorophyll is a water-soluble molecule

3322 HPLC

Other known analytes with the same wavelength maxima at 452nm include β-cryptoxanthin

and zeaxanthin The pigments within C Vulgaris will vary based on the growing conditions

but studies have a found that 04 of algae is made up of carotenoid pigments Of this 03

was identified as lutein 12 as β-carotene 362 as canthaxanthin 550 as astaxanthin

and the remaining 73 was other pigments (L Gouveia et al 2002) Based on these

reported results β-carotene should make up approximately 00048 (ww) of the dry algae

biomass The extracted levels in these solvent and microemulsion extractions are small but

comparable to the amount of β-carotene recovered in other algae species The specific values

can be found in Table 13

Table 13 HPLC Quantification of β-Carotene Extraction

ww stdev

hexane 0002 0001

ethyl caprate 0015 0000

type I 0037 0023

type II 0055 0005

type IV 0137 0074

48

For reference blue-green algae such as Phormidium Persicinum had 003 (ww) β-carotene

of dry biomass Similarly P Faveolarum P Luridum and Anabaenea flos-aquae had

0021 0038 and 0023 β-carotene of the total dry algae biomass respectively In these

cases the carotenoids were extracted with acetone and identified using IR and mass spectra

and by co-chromatography with known samples (Hertzberg amp Liaaen-Jensen 1971)

333 Biocompatibility Testing

Two extraction times 15min and 1h were used to assess the level of stress on the cells

associated with the extraction time Overall the shorter extraction time produced less stress

on the cells Due to the variability in the data it was difficult to differentiate between the re-

growth potential obtained between different solvents It was clear that regardless of the

extraction solvent the algae were not able to reach the growth levels of the blank control In

the blank control water was used instead of a solvent However the type IV microemulsion

extractions consistently retained a green or pale-green colour after 2 weeks in both the 1h and

15 min extractions

It should also be noted that the microalgae paste used in these tests had been stored in the

fridge for 3 months and were assumed to be dormant Even in the best scenario the algae

culture had a concentration of less than 02gL compared to the concentration of 15-20gL

usually observed The cells may have been slow to recover due to this prolonged storage

The lack of statistically significant data could also be due to sampling error As the algae

were allowed to recover some of the biomass flocculated as a brown mass at the bottom of

the flask Although the flasks were gently shaken prior to the TSS measurement care was

taken to avoid sampling from this flocculated mass since it was assumed that this mass was

either bacteria or dead biomass By excluding this biomass from the sampling the TSS

values would be deflated thus explaining the decrease in TSS observed at the two week

point

49

4 Conclusions

Overall this work showed that microemulsions can extract lipids better than common

solvents such as hexane This is the first time that microemulsions have been applied to algal

lipids Microemulsions have an advantage over conventional solvent technology because

they can work with wet biomass The potential for in-situ extractions on biofilms would

eliminate the energy intensive process required to dry and grind the algae the cells thus

reducing VOC emissions

This study has shown that type I and type IV microemulsions composed of 4 lecithin PEG-

6-caprylic glycerides sorbitan monooleate water 09 NaCl and ethyl caprate are able to

extract lipids from lyophilized microalgae better than hexane and ethyl caprate

UV-visible spectrophotometry was used to monitor the overall extraction kinetics of the

microalgae It was observed that most carotenoids and chlorophylls were extracted after 4h

HPLC was used to quantify the extraction of β-carotene after 1h of extraction with a 120

lyophilized algae solvent ratio (gmL) The type I microemulsion extracted the most β-

carotene at 0137plusmn0074 (ww) of the total microalgae biomass which was nine times more

than the amount extracted by hexane

Variability in the data associated with the viability study prevented any definite conclusions

about the ability of algae to grow after an extraction However among the microemulsion

extractions the type IV extractions consistently showed some signs of survival After two

weeks a green or pale-green colour was observed in both the 15min and 1h extractions

Overall type I and type IV microemulsions were the most effective in extracting lipids All

microemulsions extracted β-carotene from the lyophilized microalgae better than hexane and

ethyl caprate with type IV microemulsions extracting the most Work relating to the

extended-surfactant microemulsions can be found in Appendix J

50

5 Recommendations

Even if the microemulsion kills some of the algae and only extracts small quantities of lipids

the process could be repeated by cycling the microemulsion through the biofilm Between

cycles the biofilm could be exposed to growth media for recovery Another possibility is that

the microemulsion could kill a lot of the algae but also extract large quantities of lipids In

this scenario the microemulsion extraction is still advantageous over conventional solvent

extractions since the biomass does not need to be dried

The following recommendations are made in order of importance

1 For microalgae milking to occur the lipids must be extracted from live algae The type I

and type IV formulations that were applied to lyophilized algae should be applied to live

algae paste to extract for carotenoids and lipids The extraction efficiency and extraction

kinetics can be measured using the same methods presented in this report

2 Scale-up of the current lipid extractions from lyophilized algae should be completed to

extract larger amounts of oil The oil should be separated from the microemulsion and

analyzed

3 The biocompatibility between the microemulsions and microalgae should be tested with

healthier algae samples Other viability studies should be considered such as cell staining

or monitoring oxygen evolution

4 HPLC-MS should be used to confirm the peak at 452nm at 11-12min of the current

procedure is β-carotene Other carotenoids such as zeazanthin and lutein could also be

quantified using HPLC

5 Quantifying the amount of β-carotene extracted at 05 1 3 and 6h with HPLC would

provide a more complete understanding of the extraction kinetics The effect of mass

solvent ratio could also be studied using HPLC to quantify the carotenoid extraction at

ratios of 16 120 and 160

51

6 Since the type I and type IV microemulsions extracted the highest lipid yield other

microemulsion formulations that are type I or type IV can be tested as well The lipid

extraction at more time points would provide a better representation of the lipid

extraction kinetics including extraction less than 05h and more than 3h

52

6 References

Abbott S amp Hansen Charles M Yamamoto H (nd) HSPiP Hansen Solubility Parameters in Practice

Acosta E J Chung O amp Xuan X Y (2011) Lecithin-linker microemulsions in transdermal delivery

J Drug Delivery Sci Technol 21(1) 77-87

Acosta E Uchiyama H amp Sabatini D (2002) The role of hydrophilic linkers J Surfactants Deterg

5(2) 151-157

Acosta Edgar J Le M A Harwell J H amp Sabatini D A (2003) Coalescence and Solubilization

Kinetics in Linker-Modified Microemulsions and Related Systems Langmuir (7) 566-574

Acosta Edgar J Nguyen T Witthayapanyanon A Harwell J H amp Sabatini D a (2005) Linker-

based bio-compatible microemulsions Environ Sci Tech 39(5) 1275-82

Allen DG (2010) NSERC Strategic Grant University of Toronto

Baker R amp Guumlnther C (2004) The role of carotenoids in consumer choice and the likely benefits from

their inclusion into products for human consumption Trends Food Sci Technol 15(10) 484-488

Becker E W (2007) Micro-algae as a source of protein Biotechnol Adv 25(2) 207-10

doi101016jbiotechadv200611002

Becker EW (1994) Microalgae biotechnology and microbiology Cambridge United Kingdom

Cambridge University Press

Belarbi E H Molina E amp Chisti Y (2000) A process for high yield and scaleable recovery of high

purity eicosapentaenoic acid esters from microalgae and fish oil Enzyme and Microb Technol

26(7) 516-529

Brennan L amp Owende P (2010) Biofuels from microalgaemdashA review of technologies for production

processing and extractions of biofuels and co-products Renewable Sustainable Energy Rev 14(2)

557-577

Chisti Y (2007) Biodiesel from microalgae Biotechnol Adv 25(3) 294-306

Chisti Y amp Moo-Young M (1986) Disruption of microbial cells for intracellular products Enzyme

Microb Technol 8(4) 194-204

Cohen Z (1994) Production Potential of Eicosapentaenoic Acid by Monodus subterraneus J Am Oil

Chem Soc 71(9) 941-945

Do L D Withayyapayanon A Harwell J H amp Sabatini D a (2008) Environmentally Friendly

Vegetable Oil Microemulsions Using Extended Surfactants and Linkers J Surfactants Deterg

12(2) 91-99

53

Fernaacutendez-Sevilla J M Acieacuten Fernaacutendez F G amp Molina Grima E (2010) Biotechnological

production of lutein and its applications ApplMicrobiol Biotechnol 86(1) 27-40

Gouveia L Choubert G Pereira N Santinha J Empis J amp Gomes E (2002) Pigmentation of

gilthead seabream Sparus aurata (L 1875 ) using Chlorella vulgaris (Chlorophyta Volvocales )

microalga Aquaculture (Goodwin 1984) 987-993

Gouveia Luisa amp Oliveira A C (2009) Microalgae as a raw material for biofuels production J Ind

Microbiol Biotechnol 36(2) 269-74

Greenwell H C Laurens L M L Shields R J Lovitt R W amp Flynn K J (2010) Placing

microalgae on the biofuels priority list a review of the technological challenges J R Soc Interface

7(46) 703-26

Grima E M Acie F G amp Chisti Y (1999) Photobioreactors light regime mass transfer and

scaleup J Biotechnol 70 231-247

Grima E M Acie F G Medina A R amp Chisti Y (2003) Recovery of microalgal biomass and

metabolites  process options and economics Biotechnol Adv 20 491-515

Haack S K Garchow H Odelson D A Larry J Klug M J Haack S K Garchow I H et al

(1994) Accuracy Reproducibility and Interpretation of Fatty Acid Methyl Ester Profiles of Model

Bacterial Communitiest Appl Environ Microbiol 2483-2493

Hara a amp Radin N S (1978) Lipid extraction of tissues with a low-toxicity solvent Anal Biochem

90(1) 420-6

Hart D J amp Scott K J (1995) Development and evaluation of an HPLC method for the analysis of

carotenoids in foods and the measurement of the carotenoid content of vegetables and fruits

commonly consumed in the UK Food Chem 101-111 Food Chemistry

Hejazi M a Holwerda E amp Wijffels R H (2004) Milking microalga Dunaliella salina for beta-

carotene production in two-phase bioreactors Biotechnol Bioeng 85(5) 475-81

Hejazi M A amp Wijffels R H (2004) Milking of microalgae Trends Biotechnol 22(4) 189-94

Hertzberg S amp Liaaen-Jensen S (1966) The Carotenoids of Blue-Green Algae --I The carotenoids of

Oscillatoria rubescens and an Athrospira sp Phytochem 5(4) 557-563

Iverson S J Lang S L amp Cooper M H (2001) Comparison of the Bligh and Dyer and Folch

methods for total lipid determination in a broad range of marine tissue Lipids 36(11) 1283-7

Jiang Y amp Chen F (2000) Effects of medium glucose concentration and pH on docosahexaenoic acid

content of heterotrophic Crypthecodinium cohnii Process Biochem 35 1205-1209

Kang C D amp Sim S J (2008) Direct extraction of astaxanthin from Haematococcus culture using

vegetable oils Biotechnol Lett 30(3) 441-4

54

Kleinegris D M M Janssen M Brandenburg W a amp Wijffels R H (2011) Continuous production

of carotenoids from Dunaliella salina Enzyme Microb Technol 48(3) 253-9

Kleinegris D M M van Es M a Janssen M Brandenburg W a amp Wijffels R H (2011) Phase

toxicity of dodecane on the microalga Dunaliella salina J Appl Phycol 23(6) 949-958

Leser M E Luisi P L amp Paimieri S (1989) The use of reverse micelles for the simultaneous

extraction of oil and proteins from vegetable meal Biotechnol Bioeng 34(9) 1140-6

Li J Zhu D Niu J Shen S amp Wang G (2011) An economic assessment of astaxanthin production

by large scale cultivation of Haematococcus pluvialis Biotechnol Adv 29(6) 568-74

Li Y Horsman M Wu N Lan C Q amp Dubois-calero N (2008) Biofuels from Microalgae

Biotechnol Prog (1) 815-820

Mac M D amp Mart E (2005) Supercritical fluid extraction of carotenoids and chlorophyll a from

Nannochloropsis gaditana J Food Eng 66 245-251

Mata T M Martins A a amp Caetano N S (2010) Microalgae for biodiesel production and other

applications A review Renewable Sustainable Energy Rev 14(1) 217-232

Mercer P amp Armenta R E (2011) Developments in oil extraction from microalgae Eur J Lipid Sci

Technol 113(5) 539-547

Nguyen T Do L amp Sabatini D a (2010) Biodiesel production via peanut oil extraction using diesel-

based reverse-micellar microemulsions Fuel 89(9) 2285-2291

Popoola T O S amp Yangomodou O D (2006) Extraction properties and utilization potentials of

cassava seed oil Biotechnol 5(1) 38-41

Richmond A (2004) Handbook of microalgal culture biotechnology and applied phycology Blackwell

Science Ltd

Ryckebosch E Muylaert K amp Foubert I (2011) Optimization of an Analytical Procedure for

Extraction of Lipids from Microalgae J Am Oil Chem Soc 89(2) 189-198

Sabatini D A Acosta E amp Harwell J H (2003) Linker molecules in surfactant mixtures Curr Opin

Colloid Interface Sci 8(4-5) 316-326

Shen Y Pei Z Yuan W amp Mao E (2009) Effect of nitrogen and extraction method on algae lipid

yield Int J Agric amp Biol Eng 2(1) 51-57

Spolaore P Joannis-Cassan C Duran E amp Isambert A (2006) Commercial applications of

microalgae JBioscience Bioeng 101(2) 87-96

Xuan X-Y Cheng Y-L amp Acosta E (2012) Lecithin-Linker Microemulsion Gelatin Gels for

Extended Drug Delivery Pharmaceutics 4(1) 104-129

55

Yuan J S Ansari M Samaan M amp Acosta E J (2008) Linker-based lecithin microemulsions for

transdermal delivery of lidocaine Int J Pharm 349(1-2) 130-43

ougagh M alc rcel M os a (2004) Supercritical fluid extraction a critical review of its

analytical usefulness TrAC Trends Anal Chem 23(5) 399-405

56

Appendix A BBM Solution

The following chemicals were added to a 1L flask DI water made up the remaining volume and NaOH or

HCl were added to obtain a pH of 68 The final solution was autoclaved

BBM Stock Solution

Stock Stock Solution mLL

KH2PO4 875g500mL 10

CaCl22H2O 125g500mL 10

MgSO47H2O 372g500mL 10

NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10

Na2EDTA KOH 10gL 62gL 10

FeSO47H2O H2SO4 498gL 1mLL 1

Trace Metal Solution See below 1

H3BO3 575g500mL 07

Trace Metal Solution

Chemical Concentration (gL)

H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57

Appendix B Folch Method Validation

1 Added dry algae biomass to chloroformmethanol (21 vv) mixture in a ratio of 120 (mv)

in a 15mL tube

2 Agitated mixture for 15 minutes

3 Centrifuged mixture for 20 minutes Removed solvent from the top of the tube and placed

into a separate vial

4 Repeated steps 1-3 twice

5 Added 02 volume of 09 NaCl solution to the solvent and vortexed for 20 seconds

6 Centrifuged the solution for 5 minutes The top phase consists of the methanolwater and

polar lipids The bottom phase consists of chloroform and non-polar lipids

7 The two phases were separated using a pipette

8 The interface was washed with a methanolwater (11) solution

9 The chloroform phase containing the non-polar lipids was evaporated under a stream of

nitrogen The remaining lipids were massed When the mass stopped decreasing then all the

chloroform was assumed to be evaporated

To ensure that the Folch method was suitable for the Chlorella vulgaris samples involved in this study

the accuracy of the Folch method was tested Each time steps 1-3 is applied it is considered to be one

wash a 120 ratio of sample to solvent is used In literature three washes are standard to remove the

majority of the lipids in the tissue (160 ratio of sample to solvent) To ensure that all lipids were removed

from the algal tissue the effect of additional washes was quantified and presented in Figure Since each

additional wash requires more solvent and time the optimal method will involve three washes

Figure F-1 Lipids Extracted from Folch Method

To further validate the Folch procedure algae samples were also extracted using a Soxhlet apparatus and

a Swedish Tube extraction In the Soxhlet extraction three samples of lyophilized algae (3g each) were

placed in the Soxhlet apparatus and hexane was circulated The solvent is heated to reflux so that the

solvent vapor travels to a distillation arm connected to a thimble containing the sample A condenser then

cools the vapor and the solvent drips into the chamber with holding the sample where the lipids are

extracted out and solubilised into the warm solvent Once the chamber is full it is emptied and the cycle

repeats itself An average of 77 lipids was extracted from the algae using this method Another algae

0

5

10

15

20

1wash 3wash 4wash 5wash

L

ipid

s Ex

trac

ted

of Washes

Lipids Extracted from Folch Method

Gravimetric Analysis

GC Analysis

58

sample was tested by an external source using a Swedish Tube extraction These samples indicated 926

lipid content Compared to the lipid content determined by the optimized Folch method the 825plusmn005

lipid content was considered acceptable In the following experiments the Folch method was chosen for

any required lipid extractions

59

Appendix C FAMEs Procedure i Dishwashing and reagent preparation

Reagent SolventCompound Amount

R1 NaOH 75g

Distilled water 50mL

Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g


- Add 15mL or 2mL analyte or 02mL standard to vial

- Start 87degC water bath

ii Saponification

- Add 70mL of R1 to each vial and heat for 5 minutes

- Vortex for 10s

- Continue heating for 30 min

iii Methylation

- Cool the tubes

- Add 140mL of R2 to each vial and heat for 10 min

iv Extraction

- Add 30mL of R3 to each tube and tumble for 10 min

- Extract the lower (aqueous) phase and discard

v Base Wash

- Add 72mL of R4 into each tube and tumble for 5 min

- Pipette 23 of the organic phase for GC test

- Transfer final products into clean tubes

60

Appendix D GC Conditions Gas Chromatography Setup Conditions

Instrument Conditions

Capillary GC

Instrument GC5

Column Simplicity 5

Column Length 30m x 053mm (15um)

Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250

Detector 1 fid 200 (range 1)(att-3)

Detector 2 -

Instrument Control Method

Instrument Name GC5FPDNPD

Instrument Type PE AutoSystem GC with built-in autosampler

Channel Parameters

Data will be collected from channel A

Delay Time 000min

Run Time 2800 min

Sampling rate 125000 ptss

Channel A Channel B

Signal source Det A DetB

Analog Output INT Rec

Attenuation -4 0

Offset 50mV 50mV

Autosample Method

Syringe Capacity 50uL

Injection speed Normal

Viscosity delay 0

Pre-injection solvent washes 2

Post injection solvent washes (A) 2

Injection volume 10uL

Sample pumps 6

Washwaste via set 1

Pre-injection sample 2

Carriers Parameters

Carrier A control PFlow-H2 Diameter 250um

Column A length 3000m

Vacuum compensation OFF

Split ratio 201

Initial setpoint 10cms Initial hold 99900min

61

Auxiliary pnematics

Number Type Setpoint

1 press-PSIG 00 PSIG

Valve configuration and settings

Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters

Detector A Detector B

Detector FID None

Range 1 1

Time Constant 200 200

Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI

Initial Setpoint 250C initial hold 999 min

Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min

Ramp 1 50 0min to 270C hold for 0 min


Total run time 28min

Max temp 300C

Equilibrium time 0min

Real Time Plot Parameters

Pages Offset (mV) Setup (mV)

Channel A 1 5 200

62

Appendix E HPLC Conditions and Procedure

Adapted Basic Protocol 1 from

NE Craft ldquoChromatographic Techniques for Carotenoid Separationrdquo Current Protocols in

Food Analytical Chemistry (2001) F231-F2315

Materials

HPLC grade methanol

HPLC grade acetonitrile

HPLC grade triethylamine (TEA)

Calibration standards

Sample of interest

HPLC System

Column Supelcosil LC-18 25cm 46mm 5μm Phenomenex Guard cartridge C18

5μm 4 30mm

Data recorder computer data system

Detector UV-Vis detector

Injector automatic

Pump isocratic

Prepare mobile phase

1 Prepare mobile phase by mixing 900mL methanol 100mL acetonitrile and 1mL of

triethylamine (TEA)

2 Degas the mobile phase via inline vacuum degasser

Set HPLC Conditions

1 Set the pump flow rate at 10mLmin

2 Set UV-Vis detector at 452nm

3 Inject individual standards and the standard mixtures to generate a standard curve

4 Inject 20μL of sample 5 Calculate the final concentration of carotenoids in samples by multiplying the peak areas

of analytes by the calibration response factors Apply sample weight and dilution factors

to arrive at the concentration of carotenoids in the original sample

63

Appendix F GC Calibration

Figure F-1 C160 Methyl Palmitate Figure F-2 C183 Methyl Linolenate

Figure F-3 C180 Methyl Oleate Calibration Figure F-4 C182 Methyl Linoleate

Figure F-5 Olive Oil Calibration

y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt

Olive Oil Calibration Curve

64

Appendix G Lipid Extraction Data

Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60

Figure G-1 Extraction Efficiency at 05h

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65


Figure G-3 Extraction Efficiency of 16 (MassSolvent) Ratio


-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66

Appendix H HPLC Calibration

Correlation between HPLC Peak area and analyte concentration

Peak Area = 115615[β-carotene] (H-1)

y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea

Concentration (ugmL)

67

Appendix J Extended Surfactant Extraction

J1 Introduction

The use of microemulsions to extract oils can be based on two proposed schemes In the first scheme

extended-surfactants are used to decrease the interfacial tension (IFT) This proposed mechanism has

been used to extract vegetable oil from ground oil seeds where the oil is released from the cell membrane

when it overcomes the IFT (L D Do et al 2008) In the second scheme supersolubilization is achieved

with a high surfactant concentration and various linkers (Edgar J Acosta et al 2005) The solubilisation

can be controlled through temperature to release excess oil

J2 Materials

Extended surfactants were donated by Sasol specifically 123-4S and 145-8S were studied Other

components of these microemulsions were purchased from Sigma-Aldrich including NaCl oleic acid

and hexadecane DI water was obtained from the lab

J3 Methods

J31 Phase Scan

Similar to the super-solubilzation study a phase scan was performed to identify the phase transition and

formation of various types of microemulsions Phase scans were also used to identify the effect of

salinity on extended-surfactant formulations The formulations for these microemulsions can be found in

Table J-1

Table J-1 14 145-8S Series

Vial

Surfactant (g) Oil component (g) Aqueous Component (g) Aqueous Component (g)

NaCl 145-8S

Triglycerides (olive

oil) Water (20 NaCl) DI Water

1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80

The aqueous phase consisted of the surfactant NaCl and DI water The oil phase consisted of olive oil

and hexadecane Since it is difficult to produce a stable and clear microemulsions with triglycerides

initial formulations used 100 hexadecane As the NaCl concentration was optimized a larger proportion

of olive oil to hexadecane was used (2575 5050 7525 1000) NaCl concentrations were varied

between 4-8 at 05

68

J32 Interfacial Tension (IFT)

A model 500 spinning drop interfacial tensiometer from Temco Inc was used to measure the interfacial

tension between two phases Olive oil was used as the lighter phase and was injected into a glass tube

filled with a heavier phase The heavy phase consisted of water extended surfactant and various

concentrations of NaCl After the oil drop was injected the tube was spun at 2300-3600 rpm The

centrifugal force causes the heavier liquid to move outwards while the lighter fluid drop centers in the

middle of the tube and beings to spread out The spread of this drop is measured with a microscope The

width of the drop rotational velocity and difference in fluid densities can be used to calculate IFT The

effect of salinity on the IFT was observed over time Time (t=0) begins when the length of the drop is 4

times greater than the width The following equation can be used to calculate IFT

Equation M-1 γ=

Where

γ ndash interfacial tension (dynescm)

Δd ndash difference in fluid density (gmL)

ω ndash rotational velocity (rads)

y0 ndash radius of the drop (cm)

Converting the above equation to fit the units used by the model 500 Spinning Drop Tensiometer

Equation 1 γ= 342694 times 10-7

(Δh-Δd) ω2(D Cr)

3

Where


Δh ndash density of heavy (outer) phase (gmL)

Δl ndash density of lighter (drop) phase (gmL)

ω ndash rotational velocity (rpm)

D ndash measured drop width (diameter)(mm)

Cr ndash correction factor for refraction

A phase scan of 14 145-8S (of the aqueous component) with 4-8 NaCl (05 increments) was

observed to identify a type I II and IV microemulsion At these known concentrations of NaCl a

heavy phase was prepared (consisting of this known NaCl concentration and 1 145-8S) Olive oil

was used to represent the algae oil as the light phase The spinning drop tensiometer was set to 2300-

3600rpm The width of the drop was measured starting at t=0 once the length of the drop was four

times greater than the width Measurements of the width were taken every 3 minutes until the width

stabilized IFT was calculated with Equation 1

Table J-2 Heavy phase Composition

Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan

The effect of increasing the NaCl content is observed in Figure J-1 As NaCl increases the microemulsion

shifts from Type II to Type I The transition between these two phases Type IIIIV microemulsion is

between 7-75 NaCl

Figure J-1 Salinity Phase Scan of 14 145-8S with Olive Oil

J42 IFT Measurements

An ultralow IFT is desirable for the capillary displacement extraction mechanism to work When the IFT

is low a small shear force can be applied to the system to release the oil from the capillary

Hypothetically ultralow IFT would be achieved with a type IIIIV microemulsion Based on the phase

scan completed this transition is near 75 NaCl The IFT of a type I and type II microemulsion was also

measured with a spinning drop interfacial tensiometer for comparison purposes Figure J-2 shows the IFT

measurements of microemulsion systems with 4 75 and 8 NaCl The system contains a dilute

amount of extended surfactant at 1 145-8S Olive oil is used since it is similar to algal oil At

equilibrium 8 NaCl is required to achieve ultralow IFT of 00018 dynescm

Figure J-2 IFT Measurements of Extended Surfactant and Olive Oil at 4 75 and 8 NaCl

J4 Conclusions and Recommendations

NaCl concentrations between 75-8 are required to produce ultralow IFT Type I II and IV

microemulsions were synthesized with 14 extended surfactant (145-8S) The lipid and β-carotene

extractions can be applied to systems using these extended surfactant microemulsions

0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)

1 145-8S Solution amp Olive Oil

4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

ii

Extracting Lipid and Carotenoids from Microalgae with Lecithin-Linker

Microemulsions

Johanna Chan

Master of Applied Science



2012

ABSTRACT

This study investigated the extraction of lipids and β-carotene from microalgae using

microemulsions as an alternative to current solvents Type I and type IV microemulsions

composed of 4 lecithin sorbitan monooleate PEG-6-caprylic glycerides and ethyl caprate

were able extract lipids from lyophilized microalgae better than hexane and ethyl caprate

HPLC quantified the extracted β-carotene with type IV microemulsions extracting the most

β-carotene at 0137plusmn0074 (ww) of the total microalgae biomass after an hour The

growth recovery of the microalgae after extraction was observed over 2 weeks Variability in

the data prevented definite conclusions about the ability of algae to grow after extraction

The type IV extractions consistently showed some signs of survival After two weeks a pale-

green colour was observed in the 15min and 1h extractions This study showed that

microemulsions can successfully extract lipids from microalgae future work would apply

microemulsion formulations to live algal cells for in-situ extraction

iii

ACKNOWLEDGEMENTS















iv

Table of Contents

1 Introduction 1

11 Objective 4

2 Background 4

21 Microalgae 4


221 Fuel Industry 6














311 Materials 20






32 Results 29




v



33 Discussion 42




4 Conclusions 49


6 References 52










vi

List of Tables














vii

List of Figures





















1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

iii

ACKNOWLEDGEMENTS















iv

Table of Contents

1 Introduction 1

11 Objective 4

2 Background 4

21 Microalgae 4


221 Fuel Industry 6














311 Materials 20






32 Results 29




v



33 Discussion 42




4 Conclusions 49


6 References 52










vi

List of Tables














vii

List of Figures





















1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

iv

Table of Contents

1 Introduction 1

11 Objective 4

2 Background 4

21 Microalgae 4


221 Fuel Industry 6














311 Materials 20






32 Results 29




v



33 Discussion 42




4 Conclusions 49


6 References 52










vi

List of Tables














vii

List of Figures





















1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

v



33 Discussion 42




4 Conclusions 49


6 References 52










vi

List of Tables














vii

List of Figures





















1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

vi

List of Tables














vii

List of Figures





















1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

vii

List of Figures





















1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

1

1 Introduction


























2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

2


















3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

3





200mLmin-1



















solvent extraction






4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

4


microemulsion

11 Objective


















2 Background

21 Microalgae



5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

5














120 - - 03 traces 04

140 09 - 06 03 07

141 20 - 01 01 02


160 204 417 160 451 455

161 58 73 80 68 96

162 17 - 10 traces 12

164 - 37 260 - -

170 25 - - 02 03

180 153 29 03 14 13

181 66 88 80 19 38

182 15 151 60 146 145

183 - 205 280 03 03

184 - - - 203 211

202 15 - - - -

203 208 - - 08 04

others 196 - 25 - -







6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

6







221 Fuel Industry












7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

7


or sodium methoxide









Meat 43 1 34

Milk 26 38 28

Rice 8 77 2

Soybean 37 30 20












8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

8


2004)

























9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

9







(Chisti 2007)


















10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

10

























11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

11



Mechanical


wall by either

pressing (high

pressure) bead

milling (agitation

with beads) or

homogenization


through an orifice)


integrity of

extracted substance

- Simple

- No solvents

required (in

pressing)

- Slow

- Requires large

amount of

sample

- Cell breakage

is more

effective at

higher

concentrations

(100-200gL)

- Pretreatment

may be

required to

weaken the cell

wall

(Popoola

amp

Yangomo

dou

2006)

(Chisti amp

Moo-

Young

1986)

(Greenwe

ll

Laurens

Shields

Lovitt amp

Flynn

2010)

Solvent


(ex hexane

acetone

chloroform) degrade


is extracted due to

the high solubility

of the solvent The

solvent polarity

should match the

target compound

Bligh and Dyer

method

(chloroform


a benchmark

- Can be combined

with Soxhlet

extractor

- Possibility of

keeping cell intact

with biocompatible

solvents (ie

decane) and short

contact time

- Waste of

solvents at

large scale and

safety concern

- More effective

for dry

biomass

- Limited

applications in

food

processing

(Mercer

amp

Armenta

2011)

(Brennan

amp

Owende

2010)

Supercritical

fluid

extraction

- Some chemicals

behave as both

liquid and gas and

their solvating

power increases

above a critical

temperature and

pressure (ex CO2)


flammable

- Simple and quick

- Good for high-

value products free

of solvent residues


sensitive products

- Energy

intensive

- Expensive to

scale-up

- Limited by

moisture in the

sample (barrier

against

diffusion)

(Mercer

amp

Armenta

2011)(M

ac amp

Mart

2005)

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

12


Ultrasound


through cavitation

(form bubbles at

location where


lower than vapor

pressure)


bubble can damage



- Ultrasound and

microwave assisted

methods improve

efficiency

extraction time

and yields

- Low to moderate

costs


- High power

consumption

- Difficult to

scale-up

(Mercer

amp

Armenta

2011)





















13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

13








Organic

Solvent

Typically hexane is

used - Better than

alkaline

treatments since

lutein and

zeaxanthin are

converted to free

form and

carboxylic acids

and chlorophylls

are in the

aqueous phase

- Up to 95

recovery of

lutein with 6

hexane

extraction steps

- Easy solvent

removal

- Multiple

extraction steps

required for high

recovery time-

consuming

- Require drying of

microalgal

biomass

- If high

temperatures are

used (ie

accelerated

solvent extraction

methodology)

pheophorbides

can form from the

chlorophyll and is

a toxicological

concern

(Fernaacutendez-

Sevilla et al

2010)

Green

Solvents

Use of common


recovery of

astaxanthin

achieved with

olive oil and

75 yield of

lutein

- Application to

other carotenoids

are unknown

(Kang amp Sim

2008)

(Fernaacutendez-

Sevilla et al

2010)

Supercritical

Fluid-

Mediated

Extraction

- Relatively quick

and efficient

- Extraction

selectivity can

be controlled

with solvent

density

- Tends to recover

chlorophylls

better than

carotenoids

- More expensive

than solvent

extraction

- Requires dry

biomass

(Fernaacutendez-

Sevilla et al

2010)

( ougagh

alc rcel

os 2004)

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

14


In-Situ

Extraction

Flat-panel

photobioreactor

operating as a

turbidostat A two-


organic phase of

dodecane to extract

β-carotene

continuously

- Achieved

275mg β-carotene

Ldodecane-1

d-1


from wet algae

paste would save

energy from

drying

- Poor efficiency

(110th of that

removed from

reactor via

biomass overflow)

(Kleinegris

Janssen

Brandenburg amp

Wijffels 2011)














15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

15

















16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

16









251 Microemulsions


















17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

17








scan












18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

18




























19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

19





















20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

20




311 Materials



















local grocery store




21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

21


Toronto

























22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

22







be best




















23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

23





g g g g g g

040 040 060 065

07010





















90deg (BI-200SM)

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

24





















Dispersion (MPa12

) Polar (MPa12


)





25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

25





Hexane 149 0 0





















26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

26























3152 HPLC



27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

27



























28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

28


















fiber filter disk


mass was observed






29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

29

32 Results


















measured drop size







was added

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

30














31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

31













32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

32






Micro-

emulsio

n

Type

Sorbitan

monooleate

Lecithin PEG-6-

capric

glycerides

20

NaCl

dH2O Ethyl

Caprate

G g g g g g

I 040 040 100 0225 3775 420

II 040 040 060 0225 4175 420

IV 040 040 070 0225 4075 420





Viscosity

(cP)

Conductivity

(μScm)

Type I 453 1185

Type II 148 lt05

Type IV 174 1621


33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

33















0

20

40

60

80

100



acti

on

Eff

icie

ncy



16

120

160


34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

34

3233 Effect of Time















0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

n=3 pge005

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

35










-1

0

1

2

3

4

5

350 550 750 950

Ab

sorb

ance

Wavelength (nm)


type II

type IV

ethyl caprate

hexane

water


0

1

2

3

0 2 4 6

Ab

sorb

ance

Time (h)


Type IV

Type II

Ethyl Caprate

Hexane

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

36

3242 HPLC











β-carotene

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

37














weak in these cases

000

005

010

015

020

025



be

ta-c

aro

ten

e e

xtra

cte

d (

g)

dry

bio

mas

s (g

)


38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

38















0

005

01

015

02

025

03



TSS

(gL

)



Day 1

Day 3

Day 6

Day 13


39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

39






40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

40











0000

0050

0100

0150

0200

0250

0300

0350



TSS

(gL

)



Day 1

Day 3

Day 7

Day 14

n=3 pge005 pge001

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

41


Solvent used in

Initial Extraction


Water

Hexane

Ethyl Caprate

Type I

Type II

Type IV

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

42

33 Discussion






release the lipids


















2004)

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

43



























44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

44











ratios


ANOVA

Source of

Variation


Sample 04168 4 01042 9378 485E-05 2690

Columns 09239 2 04619 4158 225E-09 3316

Interaction 04767 8 00596 5363 00003 2266

Within 03333 30 00111

Total 2151 44


Columns 16 120 160

3312 Effect of Time






45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

45






acceptable





ANOVA

Source of

Variation


Sample 06873 4 01718 1214 551E-06 2689

Columns 01982 2 00991 6998 00032 3316

Interaction 05902 8 00738 5211 00004 2266

Within 04247 30 00142

Total 1900 44


Columns 05h 1h 3h








46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

46


of Microemulsion

Vial Phase

Behaviour

Ethyl


Sorbitan

monooleate

PEG-6-caprylic

glycerides

1 Type II 576 232 55 55 lt82

3 Type IV 420 430 40 40 70

9 Type I 192 557 56 lt56 139








00

02

04

06

08

10

12

14


mass lip

ids e

xtr

acte

d (

g)

mass e

thyl

cap

rate

(g

)

05h

1h

3h

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

47










3322 HPLC











ww stdev

hexane 0002 0001


type I 0037 0023

type II 0055 0005

type IV 0137 0074

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

48














15 min extractions











point

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

49

4 Conclusions
























50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

50

5 Recommendations














analyzed











51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

51





52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

52

6 References





5(2) 151-157














26(7) 516-529



557-577





Chem Soc 71(9) 941-945



12(2) 91-99

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

53










7(46) 703-26









90(1) 420-6















54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

54
















Technol 113(5) 539-547






Science Ltd











55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

55





56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

56




BBM Stock Solution


KH2PO4 875g500mL 10



NaNO3 125g500mL 10

K2HPO4 375g500mL 10

NaCl 125g500mL 10




H3BO3 575g500mL 07



H3BO3 286

MnCl24H2O 181

ZnSO47H2O 0222

Na2MoO22H2O 0390

CuSO25H2O 0079

Co(NO3)26H2O 00494

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

57



in a 15mL tube



























0

5

10

15

20


L

ipid

s Ex

trac

ted

of Washes



GC Analysis

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

58





59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

59



R1 NaOH 75g


Methanol 50mL

R2 HCl 14mL

Methanol 23mL

R3 Hexane 200mL

Methyl tert-butyl

ether

200mL

hexadecane 06mL

R4 NaOH 24g




ii Saponification


- Vortex for 10s


iii Methylation

- Cool the tubes


iv Extraction



v Base Wash




60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

60



Capillary GC

Instrument GC5

Column Simplicity 5


Carrier Gas H2

Flow rate 1mLmin

Split ratio 25mLmin

Temperature 55

Injection Temp 250


Detector 2 -




Channel Parameters


Delay Time 000min

Run Time 2800 min


Channel A Channel B



Attenuation -4 0

Offset 50mV 50mV

Autosample Method



Viscosity delay 0




Sample pumps 6

Washwaste via set 1


Carriers Parameters




Split ratio 201


61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

61

Auxiliary pnematics




Valve 1 SPLIT ON

Valve 2-6 NONE

Detector Parameters


Detector FID None

Range 1 1


Autozero ON ON

Polarity - -

Heated Zones

Injector A PSSI


Injector B NONE

Setpoint Off

Detector A 250C

Detector B 0

Auxiliary 0

Oven Program

Cryogenics off

Initial temp 170C

Initial Hold 0min




Max temp 300C




Channel A 1 5 200

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

62





Materials

HPLC grade methanol




Sample of interest

HPLC System


5μm 4 30mm



Injector automatic

Pump isocratic



triethylamine (TEA)


Set HPLC Conditions







63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

63





y = 10441x - 00554 Rsup2 = 09968

0 05

1 15

2 25

3 35

4

0 1 2 3 4

Rsp

(A

pe

akA

IS)

Amt (CpeakCIS)

y = 11998x + 00356 Rsup2 = 09997

0

05

1

15

2

25

0 05 1 15 2

Rsp

Amt

y = 04113x + 01697 Rsup2 = 09751

0

02

04

06

08

1

0 05 1 15 2

Rsp

Amt

y = 20412x - 01785 Rsup2 = 09463

0

05

1

15

2

25

3

35

4

0 05 1 15 2

Rsp

Amt

y = 01873x + 00294 Rsup2 = 09874

0

05

1

15

2

25

0 2 4 6 8 10 12 14

Rsp

Amt


64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

64


Extraction

Efficiency

Time 05h 1h 3h

extraction

efficiency stdev

extraction

efficiency stdev

extraction

efficiency stdev

Ratio

16 hexane 5997 25 5101 66 7775 32

ethyl caprate 3349 26 5204 47 6812 98

type I 3436 250 7215 40 2250 262

type II 059 164 2418 202 4848 33

type IV 3754 560 1094 125 3947 27

120 hexane 5322 145 5903 108 6255 184

ethyl caprate 5738 69 6102 42 8731 09

type I 7461 120 7931 62 10770 12

type II 1212 201 6934 144 6737 55

type IV 7420 187 5103 172 10057 39

160 hexane 0674521 128 2624 249

ethyl caprate 0769721 42 1429 26

type I 080899 127 5635 100

type II 075777 23 4373 137

type IV 0827567 07 4820 60


-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

nc

y



16

120

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

65




-20

0

20

40

60

80

100

120



Extr

acti

on

Eff

icie

ncy



16

120

166

-40

-20

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



05h

1h

3h

0

20

40

60

80

100



Extr

acti

on

Eff

icie

ncy



1h

3h

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

66




y = 164965x Rsup2 = 09849

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3

Peak A

rea


67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

67


J1 Introduction







J2 Materials




J3 Methods

J31 Phase Scan




Table J-1


Vial


NaCl 145-8S



1 08642 2000 0400 07358 40

2 08642 2000 0450 06858 45

3 08642 2000 0500 06358 50

4 08642 2000 0600 05358 60

5 08642 2000 0650 04858 65

6 08642 2000 0700 04358 70

7 08642 2000 0750 03858 75

8 08642 2000 0800 03358 80





between 4-8 at 05

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

68











Equation M-1 γ=

Where







(Δh-Δd) ω2(D Cr)

3

Where















Vial

Surfactant

(g)

20

NaCl

DI

water NaCl

1 01236 08525 31053 4

2 01236 15173 23839 75

3 01236 16209 23379 8

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

69

J4 Results

J41 Phase Scan



between 7-75 NaCl
















0

002

004

006

008

01

0 20 40 60

IFT

(dyn

es

cm)

Time (min)


4NaCl

4 NaCl (2)

75 NaCl

75 NaCl (2)

8NaCl

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