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The Production of a Potential Feedstock for Biodiesel using
Water and Isopropyl Alcohol to Extract Yellow Mustard Oil
by
Veronica Maria Ataya Pulido
A thesis submitted in conformity with the requirements for the degree of Master of Applied Science (M.A.Sc.)
Graduate Department of Chemical Engineering and Applied Chemistry
University of Toronto
© Copyright by Veronica Maria Ataya Pulido (2010)
ii
The Production of a Potential Feedstock for Biodiesel using Water and
Isopropyl Alcohol to Extract Yellow Mustard Oil
Veronica Maria Ataya Pulido
Master of Applied Science
Graduate Department of Chemical Engineering and Applied Chemistry
University of Toronto
2010
Abstract
The aim of this project was to produce a potential feedstock for biodiesel by developing a
process to extract oil from yellow mustard seeds using aqueous and isopropyl alcohol extraction.
The aqueous extraction of yellow mustard flour was performed at pH 11 using 4:1 water to flour
ratio and constant stirring at room temperature for 30min, with a second washing stage. Oil was
separated as oil-in-water emulsion with 37% oil recovery from the flour. The oil in the emulsion
was then extracted with isopropyl alcohol. Single and multiple stage extractions were evaluated
and the optimal conditions were four-stage extraction at 2:1 IPA:Oil weight ratio, with 96.3% oil
recovery from the emulsion. A preliminary evaluation of the final solution of isopropyl alcohol,
water and yellow mustard oil concluded that it is indeed a potential feedstock for biodiesel,
however it needs to be further processed to meet optimal conditions for transesterification.
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank Professor Diosady for giving me this amazing
opportunity and for being a great supervisor. His guidance and support made possible the
completion of this project. Thanks for believing in me and for introducing me to the wonderful
world of food.
To Prof. Boocock and Prof. Acosta for taking the time to read about my work and for the
valuable feedback.
Being part of the Food Engineering Group was a pleasure and I am very grateful for all the
things I have learned and all the people I have meet. They were a very important part of my life
at the University of Toronto and in Canada. Olive, Kristen, Karina, Divya, Solmaz, Dan, Xu Lei
and Bih-King, thanks for your friendship and great advice.
To my friends and family for giving me love and encouragement, thanks for always being there
for me!
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TABLE OF CONTENTS
Abstract ..................................................................................................................................... ii
ACKNOWLEDGEMENTS....................................................................................................... iii
LIST OF TABLES .................................................................................................................. viii
LIST OF FIGURES .................................................................................................................. ix
1. INTRODUCTION ...............................................................................................................1
2. LITERATURE REVIEW .....................................................................................................3
2.1 Mustard seed......................................................................................................................3
2.1.1 Structure of Mustard Seeds ..........................................................................................4
2.1.2 Classification of Yellow Mustard Seeds in Canada ......................................................6
2.1.3 Composition of Yellow Mustard Seeds ........................................................................6
2.1.3.1 Yellow Mustard Protein ........................................................................................7
2.1.3.1.1 Oleosins..........................................................................................................8
2.1.3.2 Oil ........................................................................................................................9
2.1.3.2.1 Erucic Acid .................................................................................................. 11
2.1.3.3 Mucilage ............................................................................................................. 13
2.1.3.4 Anti-Nutrients ..................................................................................................... 13
2.1.3.4.1 Glucosinolates .............................................................................................. 13
2.1.3.4.2 Phenolic Compounds .................................................................................... 14
2.1.3.4.3 Phytic Acid ................................................................................................... 15
2.2 Uses of Yellow Mustard Products .................................................................................... 16
2.3 Oil extraction ................................................................................................................... 17
v
2.3.1 Traditional Methods .................................................................................................. 18
2.3.1.1 Hard press and Pre-press Solvent Extraction ....................................................... 18
2.3.2 Solvent Extraction ..................................................................................................... 19
2.3.3 Alternative Solvents .................................................................................................. 21
2.3.3.1 Isopropyl Alcohol as a Solvent ............................................................................ 22
2.3.4 Aqueous Extraction ................................................................................................... 24
2.4 Food Emulsions ............................................................................................................... 28
2.5 Biodiesel Production ........................................................................................................ 31
2.5.1 Water Adsorption ...................................................................................................... 35
3. PROJECT OBJECTIVES................................................................................................... 37
4. EXPERIMENTAL METHODS ......................................................................................... 38
4.1 Starting Materials ............................................................................................................ 38
4.2 Reagents and Materials .................................................................................................... 39
4.3 Equipment ....................................................................................................................... 40
4.4 Experimental Methods ..................................................................................................... 40
4.4.1 Aqueous Extraction of Oil and Protein ...................................................................... 40
4.4.2 Emulsion Destabilization ........................................................................................... 43
4.4.3 Oil Extraction from the Emulsion using Isopropyl Alcohol as a Solvent .................... 43
4.4.3.1 Single Extraction................................................................................................. 43
4.4.3.2 Multiple Stage Extraction .................................................................................... 44
4.4.4 Water Adsorption using Molecular Sieves 4A ........................................................... 44
4.4.5 Analytical Methods ................................................................................................... 45
5. RESULTS AND DISCUSSION ......................................................................................... 46
5.1 Starting Material Analysis................................................................................................ 46
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5.2 Aqueous Extraction of Oil and Protein from Yellow Mustard Flour ................................. 47
5.3 Emulsion Analysis ........................................................................................................... 50
5.4 Destabilization of the Yellow Mustard Oil Emulsion ....................................................... 51
5.5 Determination of the Molecular Weight of Yellow Mustard Oil ....................................... 52
5.6 Oil Extraction from the Emulsion Using Isopropyl Alcohol as a Solvent .......................... 54
5.6.1 Single Stage Extraction ............................................................................................. 54
5.6.2 Multiple Stage Extraction .......................................................................................... 61
5.6.2.1 Three-Stage Oil Extraction using IPA ................................................................. 62
5.6.2.2 Four-Stage Oil Extraction using IPA ................................................................... 63
5.6.3 Characterization of the Miscellas from the Four-Stage Oil Extraction ........................ 68
5.6.4 Characterization of the Final Miscella ....................................................................... 70
5.7 Preliminary Evaluation of the Final Miscella as Feedstock for Biodiesel Production ........ 72
6. CONCLUSIONS ............................................................................................................... 75
7. RECOMMENDATIONS ................................................................................................... 76
8. REFERENCES .................................................................................................................. 77
APPENDICES .......................................................................................................................... 87
APPENDIX A ....................................................................................................................... 88
Analytical and Experimental Methods ................................................................................... 88
A1. Determination of oil content using Mojonnier Method ................................................. 89
A.2 Oil Content in Miscella................................................................................................ 92
A.3 Determination of protein content using Kjeldahl Method (AOCS Method Ba4d-90) .... 93
A.4 Determination of Moisture Content (AACC Method 44-15A)...................................... 96
APPENDIX B ....................................................................................................................... 97
Results ................................................................................................................................... 97
vii
B1. Yellow Mustard Flour Analyses................................................................................... 98
B2. Aqueous Extraction of Oil and Protein from Yellow Mustard Flour ........................... 100
B3. Emulsion Analysis ..................................................................................................... 103
B3. Oil Extraction from the Emulsion Using Isopropyl Alcohol as a Solvent ................... 108
B.3.1 Single Stage Extraction ....................................................................................... 108
B.3.2 Multiple Stage Extraction .................................................................................... 119
B.4 Characterization of the Miscellas from the Four-Stage Oil Extraction ........................ 133
B.5 Final Miscella Analyses ............................................................................................. 136
viii
LIST OF TABLES
Table 2-1. Quality of 2007 Yellow Mustard Seeds .....................................................................6
Table 2-2. Essential amino acid content of mustard compared to recommended intakes and soy (mg amino acid/ g protein) ..........................................................................................................7
Table 2-3. Fatty Acid Composition (%) of Canadian Yellow Mustard Seeds (Harvest 2007) ..... 10
Table 2-4. Characteristics of n-Hexane and Isopropyl Alcohol .................................................. 22
Table 2-5. Optimal Conditions for Aqueous Extraction of Yellow Mustard Seeds ..................... 26
Table 2-6. Comparison of the cold flow properties of isopropyl and methyl esters of soybean oil and yellow grease ...................................................................................................................... 33
Table 4-1. Solvents used in the experiments .............................................................................. 38
Table 5-1. Composition of yellow mustard flour ....................................................................... 46
Table 5-2. Composition of Yellow Mustard Oil Emulsion ......................................................... 50
Table 5-3. Fatty Acid Composition of Canadian No. 1 Yellow Mustard Seeds (Harvest 2007) .. 53
Table 5-4. Composition of the solids after four-stage extraction ................................................ 64
Table 5-5. Protein balance for the four-stage extraction process ................................................ 65
Table 5-6. Summary of the results for the four-stage oil extraction ............................................ 66
Table 5-7. Oil yields for each stage in the multistage extraction ................................................ 68
Table 5-8. Composition of the miscellas for the four-stage extraction ........................................ 69
Table 5-9. Water content in miscellas for different stages .......................................................... 70
Table 5-10. Composition of the final miscella ........................................................................... 71
Table 5-11. Water adsorption using 4A molecular sieves........................................................... 73
ix
LIST OF FIGURES
Figure 2-1. Structure of a maize oil body (Tzen and Huang 1992) ...............................................5
Figure 2-2. Triacylglycerol molecule (Palmitic acid, oleic acid and α-linolenic acid) ...................9
Figure 2-3. Oil extraction combining solvent extraction and pressing (Rosenthal et al. 1996) .... 19
Figure 2-4. Solubility of cottonseed oil in absolute ethanol and IPA, and their azeotropes ......... 21
Figure 2-5. Structure of the yellow mustard oil-in-water emulsion............................................. 29
Figure 2-6. Transesterification of a triglyceride molecule with isopropyl alcohol ...................... 31
Figure 4-1. Experimental setting for the aqueous extraction process .......................................... 41
Figure 4-2. Experimental procedure for the aqueous extraction of oil from yellow mustard flour ................................................................................................................................................. 42
Figure 4-3. Experimental procedure for oil extraction using IPA ............................................... 43
Figure 5-1. Yellow mustard flour. ............................................................................................. 46
Figure 5-2. Aqueous extraction of yellow mustard flour ............................................................ 47
Figure 5-3. Oil and protein distribution during aqueous extraction of yellow mustard flour ....... 48
Figure 5-4. Yellow mustard oil emulsion ................................................................................... 51
Figure 5-5. Triglyceride molecule ............................................................................................. 54
Figure 5-6. Oil extraction from emulsion using isopropyl alcohol .............................................. 55
Figure 5-7. Single stage oil extraction from emulsion at different IPA:Oil weight ratios ............ 56
Figure 5-8. Solvent holdup for different IPA:Oil weight ratios .................................................. 57
Figure 5-9. Composition of miscella (w/w) for different IPA:Oil weight ratios .......................... 58
Figure 5-10. Relationship between solvent holdup and residual oil content ................................ 60
Figure 5-11. Oil extraction from emulsion at low weight ratios ................................................. 61
Figure 5-12. Three-stage oil extraction using IPA...................................................................... 62
x
Figure 5-13. Four-stage oil extraction using IPA ....................................................................... 63
Figure 5-14. Oil and protein extraction from the emulsion for 2:1 IPA: Oil weight ratio and four-stage extraction ......................................................................................................................... 67
Figure 5-15. Miscellas from the four-stage oil extraction using IPA at 2:1 IPA:Oil weight ratio 68
Figure 5-16. Final Miscella from 2:1 IPA:Oil weight ratio, four-stage extraction ....................... 71
1
1. INTRODUCTION
Canada is largest exporter of mustard seeds in the world, controlling over 50% of the total
export market in 2007 and standing in second place in world production with 160,000 metric
tonnes, with yellow mustard representing over 40% of the total production.
Yellow mustard seed (sinapis alba) is traditionally used as a condiment, to prepare “hotdog”
mustard in North America; however, there are several other important applications of
mustard products such as emulsifiers, binding agents, and food preservatives. The seed is
high in oil and protein, with a well balanced amino acid profile and recent research has been
focused on the production of yellow mustard protein isolates for use in different products
such as protein bars, meat and soy substitutes, and beverages. Yellow mustard oil is high in
erucic acid, a monounsaturated fatty acid associated with certain heart conditions and
banned from food use in North America and Europe. However, erucic acid is recognized for
its excellent lubricating properties and it has many potential applications in different
markets.
Oil extraction from oilseeds is usually performed by using hexane as a solvent at elevated
temperatures, achieving high extraction yields but causing significant damage to the protein
in the seed, compromising its use. Furthermore, there are several environmental and safety
implications limiting the use of hexane and driving scientists to find alternative solvents for
oil and protein extraction. Aqueous extraction represents a very good option because it
allows simultaneous recovery of oil and protein. A process developed by the Food
Engineering Group at the University of Toronto involves the use of alkaline pH to extract
protein and oil from yellow mustard seeds with yields of over 85% and 55-80%,
respectively.
Due to the structure of mustard seeds and the presence of oleosin proteins, the oil is
extracted in form of an oil-in-water emulsion and further processing is necessary in order to
obtain free oil. Techniques previously studied to break this type of emulsions include
2
heating and centrifuging, freezing and thawing, pH variations, enzymatic treatment, and the
use of different solvents. The use of isopropyl alcohol (IPA) to recover the oil in the
emulsion is evaluated in this investigation. Once the oil is extracted with the IPA, the
solvent can be distilled and the oil purified, however, the use of this mixture could find an
application in the production of biodiesel.
Biodiesel is a renewable fuel, environmentally friendlier than petroleum diesel and with
comparable energy content. It is produced by the transesterification of vegetable oils or
animal fats in the presence of alcohol and a catalyst. Methanol and ethanol are the preferred
types of alcohol, however they produce fuels with poor cold flow properties limiting their
use in cold climates. The use of isopropyl alcohol significantly improves the cold flow
properties of biodiesel and represents a promising alternative. In addition, using IPA could
enhance the conditions for the transesterification reaction. When other alcohols are used, the
reaction is slow, limited to the interface. The use of IPA eliminates mass transfer limitations
due to the complete miscibility of oil in IPA at high concentrations. The use of the IPA and
yellow mustard oil solution in the production of biodiesel seems very promising, since it
would combine the excellent lubricity of yellow mustard oil with the superior cold flow
properties of isopropyl esters.
The aim of this project is to develop an integrated process involving aqueous and isopropyl
alcohol extraction of yellow mustard oil and to evaluate the potential use of this mixture in
the production of biodiesel. The use of isopropyl alcohol to extract oil from the yellow
mustard oil emulsion is discussed and optimal conditions are selected. The final solution is
characterized and a preliminary assessment of its use as feedstock for biodiesel production is
performed.
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2. LITERATURE REVIEW
2.1 Mustard seed
Mustard is a member of the Brassica family of plants and it is an annual crop very tolerant to
drought, heat and frost. The name mustard is derived from the Latin mustum ardens, which
means burning must, in reference to the old French practice of mixing ground mustard seeds
with must (unfermented juice of wine grapes) and the spicy heat of such mixture.
There are several types of mustard plants but only three are used for culinary purposes:
yellow or white mustard (sinapis alba), black (brassica nigra) and brown (brassica juncea).
In Canada, three types of mustard derived from two different species are produced: yellow
(Sinapis alba), brown and oriental (Brassica juncea).
Yellow mustard is the mildest of the three types and it is the main ingredient of traditional
hotdog mustard. Other applications include dry milling for flour, wet milling for mustard
pastes, and whole ground seed for spice mixes, meat processing, and other food products.
The oil content ranges between 25-35% (Agriculture and Agri-Food Canada 2009).
Brown and oriental mustard seeds are hot and spicy. Brown mustard seed is ground into flour
and it is used in the manufacturing of European products such as Dijon and English mustards.
Oriental mustard seed is often used to produce spicy cooking oils consumed in some parts of
Asia. The oil content of brown mustard seeds is around 36% and 39% for oriental, although
oil contents of up to 50% have been found in some varieties of oriental mustard grown in
Canada (Agriculture and Agri-Food Canada 2009).
Mustard production began in Alberta in 1936 with 40 hectares. At this time, mustard
production was concentrated in the states of California and Montana, but the high quality of
Canadian mustard seed led to an increase in Canadian production during the 1950s and
1960s. The hot and dry weather conditions in the Prairies help produce a mustard seed with
lower oil concentration and higher protein content. At the same time, seed quality is
4
preserved due to the cold and dry winters conditions (Agriculture and Agri-Food Canada
2008). Today, Saskatchewan accounts for over 80% of total mustard production.
Canada is the largest exporter of mustard seed in the world with nearly 160,000 tonnes in
2008, accounting for over 50% of all mustard seed exports worldwide. In 2007, over 40% of
total exports were destined to the United States; other important markets are Belgium,
Germany, Netherlands and Japan (Statistics Canada 2009). Canada ranks second in world
production with 35 % world production (Agri-Food Trade Service 2009). In 2008, Mustard
production increased 41%, from the previous year, to 161,000 metric tonnes as a result of
increased planted area and higher than average yields. Yellow mustard represented 44% of
total production, while brown and oriental accounted for 42% and 14%, respectively
(Canadian Grain Commission 2008).
2.1.1 Structure of Mustard Seeds
Mustard plants typically grow 1m in height and have yellow flowers. Seeds grow in pods of
2.0-4.2 cm with 2-4 seed in each of them. Seeds are approximately 2 mm in diameter and
contain a thick seed coat that accounts for 20% of the weight (Balke 2006). In oilseeds,
cotyledon cells contain oil and protein stored in discrete cellular organelles called lipid and
protein bodies (Rosenthal et al. 1996). Oil is stored by the seed as triacylglycerol (TAG) as
food reserve for germination and postgermination growth. The TAG are present in spherical
particles surrounded by a layer of phospholipids embedded with proteins called oleosins,
which are alkaline proteins of small molecular mass, with most of the oleosins present on the
surface of the phospholipid layer (Tzen et al. 1993; Murphy and Cummins 1989).
Oil-bodies have a diameter ranging from 0.5 to 2 µm depending on the type of seed. For
mustard seed, oil-bodies are 0.73 µm in diameter with a composition of 94.64% TAG, 3.25
% proteins, 1.97 % phospholipids and 0.17% Free Fatty Acids (FFA) (Tzen et al. 1993). Oil-
bodies have very small sizes for rapid mobilization of the reserved TAG during
postgerminative growth since they provide large surface areas for the attachment of lipase to
the organelles. (Tzen and Huang 1992).
5
Figure 2.1 is a representation of a maize oil body based on results from Tzen and Huang
(1992). TAG are represented in blue, phospholipids in red and oleosins in yellow. About 2/5
of the oleosin is hydrophobic and it is embedded in the phospholipids layer and TAG matrix.
The remaining 3/5 covers the outside of the oil-body. The head of the oleosin is the
hydrophilic portion and the tail the hydrophobic. The dotted lines in the phospholipid
represent the hydrophobic portion attached to the TAG.
Figure 2-1. Structure of a maize oil body (Tzen and Huang 1992)
The size of oil-bodies will have a direct effect on oil extraction. In traditional solvent-based
processes, seeds are ground or flaked to disrupt the cells and expose the oil. The difference in
size of oil-bodies for different seeds or fruits explains why oil extraction is easier for some
oleaginous materials than for others. The diameter of oil-bodies in mustard seeds is less than
1 µm, while for avocado, olive and palm oil-bodies is often from 10-20 µm (Murphy 1993).
Due to the small size of oil-bodies in mustard seeds, even after grinding and flaking, oil-
bodies will remain unaffected complicating oil extraction.
6
2.1.2 Classification of Yellow Mustard Seeds in Canada
Depending on the characteristics of the seeds harvested, yellow mustard seeds are classified
by the Canadian Grain Commission into 4 categories: No. 1, No. 2, No. 3 and No. 4.
Canada No. 1 yellow mustard seeds are well matured, sweet and of good natural colour.
Canada No. 2 yellow mustard seeds are fairly well matured, sweet and of reasonable good
colour. Canada No. 3 and Canada No. 4 yellow mustard seeds may have the natural odour
associated with low-quality seed, but not any odour that would indicate serious deterioration.
2.1.3 Composition of Yellow Mustard Seeds
Yellow mustard seeds contain 28-40% of protein, 24-36% of oil, 20-25% carbohydrates, 4 %
minerals, 2% fiber and antinutritional factors such as glucosinolates, phenolic compounds
and phytic acid (Agriculture and Agri-Food Canada 2009).
The composition of yellow mustard seeds is very dependent on the variety and environmental
conditions during seed growth and will vary slightly every year depending on the area of
cultivation. The protein and oil contents of yellow mustard seeds harvested in western
Canada in 2007 are shown in the following table.
Table 2-1. Quality of 2007 Yellow Mustard Seeds Grade % Protein 1 % Oil 2
Canada No. 1 27.1 33.7
Canada No. 2 27.0 34.0
Canada No. 3 28.0 32.4
Canada No. 4 28.2 32.7
Data from Canadian Grain Commission report on Quality of Western Canadian Mustard 2007 1 % N x 6.25; dry matter basis 2 Dry Matter basis
7
2.1.3.1 Yellow Mustard Protein
Proteins are organic compounds made of amino acids arranged in a linear chain. They are
formed by series of up to 20 amino acids and they are essential components of organisms and
participate in almost every process within cells. The main role of proteins is as enzymes to
catalyze reactions involved in metabolism and DNA replication, repair and transcription.
Table 2-2 presents the amino acid profile of mustard seeds compared to soy and the
recommended intakes (FAO/WHO; MIT).
Table 2-2. Essential amino acid content of mustard compared to recommended intakes and soy (mg amino acid/ g protein)
Essential
Amino Acids
Recommended Intakes Mustard Soy
FAO/WHO
(UNU 1996)
MIT
(Young &
Borgonha 2000)
(Bhattacharyya
& Sen 2000)
(Sadeghi et al.
2006)
(Solae 2004)
Isoleucine 38 35 38 37 49
Leucine 65 65 81 78 82
Lysine 50 50 54 49 63
Methionine +
Cysteine
25 25 ----- 56 26
Phenylalanine
+ Tyrosine
65 65 92 68 90
Threonine 25 25 45 43 38
Tryptophan 10 10 17 16 13
Valine 35 35 35 52 50
8
Humans cannot synthesize all 20 amino acids, in fact, they can only synthesize half of the
necessary amino acids and the rest must be consumed regularly in the diet (Whitaker and
Tannerbaum 1977; Voet and Voet 2004). As it is shown in Table 2-2, mustard seeds contain
a well-balanced amino acid profile relative to the recommended intakes for proper nutrition
(FAO/WHO; MIT) and to soy protein. The proteins in yellow mustard seeds could provide
more than the necessary requirements for protein consumption.
Some individuals can develop allergic reactions to mustard products such as anaphylaxis
(Panconesi et al. 1980; Caballero et al. 2002). Sin A1 and Sin A2 are two allergens identified
in mustard protein isolates (Palomares et al. 2005; Mustorp et al. 2007), nevertheless, only
low levels of the allergens identified in soy are present in yellow mustard seeds (Bell and
Rakow 1996), making it a suitable substitute for people with allergies to soy.
2.1.3.1.1 Oleosins
Most of the protein present in yellow mustard seeds are storage proteins, in addition to
oleosins. Oleosins protect and stabilize oil-bodies after environmental stress such as
desiccation, rehydration, heating and cooling; they maximize the surface area of oil-bodies in
order to accelerate oil mobilization by lipase after germination, and they act as receptors for
lipase binding and/or activation. (Murphy and Cummins 1989; Murphy 1993; Tzen et al.
1993).
Oleosins consist of three structural domains, including an amphipathic NH2-terminal domain,
a central hydrophobic domain, and an amphipathic α-helical domain at or near the COOH-
terminus. The hydrophobic domain is in direct contact with the triglycerides, while the
amphipathic N and C terminals are exposed to the aqueous phase (Tzen et al. 1993; Murphy
1993; Nikiforidis et al. 2009). The oleosin provides an amphipathic surface and steric
hindrance to prevent coalescence.
The behaviour of artificially prepared particles of TAG surrounded by either phospholipids
or oleosin, and their combination has been studied (Tzen and Huang 1992). It was found that
9
when only the phospholipids or the oleosin layer was present, oil-bodies tended to coalesce
and were less stable than native oil-bodies. However, when both components were added,
oil-bodies became as stable as native oil-bodies and did not coalesce even when they were
pressed against each other. Therefore the presence of both phospholipids and oleosins is
necessary to preserve the TAG for use in later stages of seed growth.
2.1.3.2 Oil
Oilseeds contain oil stored as triacylglycerol or triglycerides, which are composed of three
fatty acids esterified with a glycerol molecule. Fatty acids are formed by carbon, hydrogen
and oxygen arranged as a carbon chain skeleton with a carboxyl group (-COOH) at one end.
When the fatty acid has zero double bonds is it referred to as a saturated fatty acid. When
only one double bond exists then it is a monounsaturated fatty acid; and when there are two
or more double bonds it is known as polyunsaturated fatty acids.
Figure 2-2. Triacylglycerol molecule (Palmitic acid, oleic acid and α-linolenic acid)
Yellow mustard seeds generally contain 24-35% oil, characteristically lower than oil contents
for oriental and brown mustard seeds, which can reach values of up to 50%. The fatty acid
composition is shown in Table 2-3. According to the Canadian Grain Commission, for the
harvest of 2007, oil content for Canada No. 1 seeds ranged from 23.7% to 35.3%, with an
average value of 30.1%.
10
Table 2-3. Fatty Acid Composition (%) of Canadian Yellow Mustard Seeds (Harvest 2007)
Fatty Acid Canada No. 1 Canada No. 2 Canada No. 3 Canada No. 4
Palmitic (C16:0) 2.90 2.90 2.90 2.90
Palmitoleic (C16:1) 0.20 0.20 0.20 0.20
Stearic (C18:0) 1.00 1.00 1.00 1.00
Oleic (C18:1) 26.50 25.80 25.50 25.50
Linoleic (C18:2) 10.50 10.40 10.30 10.30
α-Linolenic (C18:3) 8.73 9.30 9.40 9.80
Arachidic (C20:0) 0.70 0.70 0.70 0.60
Eicosenoic (C20:1) 11.03 10.90 11.10 11.00
Eicosadienoic (C20:2) 0.30 0.30 0.30 0.30
Behenic (C22:0) 0.60 0.60 0.60 0.50
Erucic (C22:1) 34.10 34.30 34.80 34.40
Docosadienoic (C22:2) 0.30 0.30 0.30 0.30
Lignoceric (C24:0) 0.30 0.30 0.30 0.30
Nervonic (C24:1) 2.30 2.30 2.20 2.30
Saturated Fatty Acids 1 5.50 5.40 5.40 5.40
Iodine Value 100.00 101.00 101.00 101.00
Data from Canadian Grain Commission report on Quality of Western Canadian Mustard 2007 1 Saturated Fatty Acids are defined as the sum of C16:0, C18:0, C20:0, C22:0, and C24:0
11
Yellow mustard oil is low in saturated fatty acids and it is a good source of omega-3 fatty
acids such as α-linolenic acid, which helps prevent heart disease. However, yellow mustard
oil has a high content of erucic acid, a fatty acid that has been associated with certain heart
conditions.
2.1.3.2.1 Erucic Acid
Erucic acid (13-docosenoic acid) is a monounsaturated fatty acid with 22-carbon chain length
and a double bond in the omega-9 position. It is present in several oilseeds such as rapeseed,
wallflower and mustard. It is believed to contribute to certain heart conditions such as
myocardial lipidosis, which is accumulation of triglycerides in the heart. In response to
health concerns, canola was developed as a variety of rapeseed with very low erucic acid
content (less than 2%).
The first report on the possible toxics effects of erucic acid in rats being fed rapeseed oil was
presented 1960 (Roine et al.). Cardiac lesions were studied in rats fed rapeseed oil with
different erucic acid contents (1.6%, 4.3% and 23 %) and it was found accumulation of fat in
myocardial fibers when more erucic acid was given to the rats, and that female rats tended to
have fewer lesions than males (Charlton et al. 1975). Hulan et al. (1976), studied the
relationship between erucic acid and myocardial changes in male rats and found that the
myocardial lesions associated with feeding rapeseed oil with 20% erucic acid to rats was not
linked to the consumption of erucic acid, but to poor metabolism of rats when digesting
triglycerides. It was established that rats have a poor ability to metabolize fats (Kramer et al.
1979) and are more susceptible to myocardial lipidosis (Grice and Heggtveit 1983).
Furthermore, their metabolism is very different from that of humans and they should not be
used as a model to predict adverse effects of erucic acid in humans (Kramer and Sauer,
1983).
In a different study, a group of newborn pigs were fed erucic acid in different amounts and it
was found that the cause of myocardial lipidosis was their low capacity to oxidize fatty acids
and it was not attributed to any specific fatty acid (Werner et al. 1983). Several other studies
12
were performed to evaluate the incidence of heart lesions in adult pigs being fed erucic acid
in their diet and no relationship was found between heart lesions and any particular kind of
fat in the diet (Aherne et al. 1976; Friend et al. 1976). It was concluded that adult pigs are
more able to tolerate erucic acid in their diets than newborns. Experiments with monkeys
suggested that in general they tolerate high levels of erucic acid in their diets without
presenting cardiac lesions or any other adverse effect (Kramer and Sauer, 1983).
A risk analysis report presented in Australia and New Zealand in 2003 (FSANZ 2003)
established a tolerable level of human exposure and considering that the intake of erucic acid
comes generally from canola oil with a low concentration of erucic acid (well below the
tolerable intake), and it was concluded that the consumption of erucic acid does not represent
a risk to humans.
The effect of erucic acid in human health was studied in some parts of India were edible oil
with high levels of erucic acid was consumed and it was found that significant amounts of
erucic acid was accumulated in the heart muscles but there was no association with
myocardial lesions (Grice and Heggtveit 1983). Although it has not been proven that erucic
acid has negative effects on the health of humans, it has been banned in the European Union
and North America and it is considered unsuitable for human consumption. However, erucic
acid has been widely studied for industrial applications. Its durability, chemical and water
resistance, dimensional stability, and electrical properties are valuable characteristics for
numerous applications and it is extensively recognized for its excellent lubricating properties
and high resistance to hydrolysis and oxidation (Fobert et al. 2008).
A very important advantage of erucic acid-based products is their biodegradability compared
to petroleum-based products, making them environmentally friendlier. There are more than
200 potential applications of erucic acid and more than 1000 patents have been issued
(Leonard, 1994; Mietkiewska et al., 2004). Erucamide is a very important chemical
derivative used in the plastic film industry and to produce, among other things, bread
wrappers and garbage bags (Leonard, 1993). Erucic acid can also be used in many
applications such as a plasticizer for vinylchloride resins, in the manufacture of polymers and
13
liquid wax esters, as an additive in lubricants and solvents, softener in textiles, to produce hot
water detergents, high temperature fluidity lubricants, surfactants, surface coating materials
(since it is a water-repelling agent and helps prevent corrosion), pharmaceuticals, concrete
mould-release agents (Leonard, 1994; Ohlson, 1983; Van Dyne and Blase, 1990).
2.1.3.3 Mucilage
Mucilage is a polysaccharide that contains of 80-94% carbohydrates, 1.7-15.0 % ash and 2.2-
4.4% protein. The carbohydrates are mainly composed of glucose (22–35%), galactose (11–
15%), mannose (6.0–6.4%), rhamnose (1.6–4.0%), arabinose (2.8–3.2%) and xylose (1.8–
2.0%), (Cui et al. 1993). It is present in the outer surface of the hulls and it represents 25% of
the hull weight and 5% of the dry seed weight, much higher than the <1% found in other
types of mustard (Cui et al. 2006). It plays an important role in protecting the seed during
germination and it is responsible for its resistance to drought (Wu et al. 2009a; Balke and
Diosady 2000). It is also a good emulsifier and this creates problems during aqueous oil
extraction (Balke 2006).
Mucilage could be an excellent gum with thickening, stabilizing and texturizing properties. It
can exhibit interactive properties with other polysaccharides, such as starches and
galactomannans to produce gels at very low concentrations, which could find many
applications in the food industry (Cui et al. 2006). It is very good for increasing viscosity of
solutions at very low concentrations 0.5-1.0% w/w and its properties are very similar to
xanthan gum, implying that yellow mustard mucilage could be as a substitute for xantham
gum in food products (Balke and Diosady 2000; Cui et al. 1993).
2.1.3.4 Anti-Nutrients
2.1.3.4.1 Glucosinolates
Glucosinolates are sulphur-containing organic compounds present in plants of the Brassica
family and they act as insecticides generating a very unpleasant taste when they get
hydrolyzed by the enzyme myrosinase in the presence of water. The reaction of myrosinase
14
and glucosinolates produces isothiocyanates and other derivatives, responsible for the
pungent and bitter flavour of mustard. Moisture must be kept bellow 10% to prevent reaction
between myrosinase and glucosinolates (Balke 2006).
Mustard seeds have a very high concentration of glucosinolates (140 µmol/g) (Mathaus
1997), with 90% being p-hydroxybenzyl glucosinolates, also known as sinalbin (Hopkins et
al. 2009; Hemingway 1995, Mithen et al. 2000). Myrosinase is stored in special
compartments within the plant and when the compartments are damaged, for example by the
action of chewing, they come in contact with the glucosinolates. The products of the reaction
are toxic and pungent products such as isothiocyanates, thiocyanates and nitriles (Bones and
Rossiter 2006; Mithen et al. 2000).
The hot flavour of mustard is associated with allyl isothiocyanate. This compound is soluble
in oil and it is therefore extracted with the oil, giving it heat and flavour (Fenwick et al.
1983). Isothiocyanates are toxic in high concentrations and they can interfere with thyroid
function and are linked to depression, liver lesions, poor palatability, hypertrophy and
hyperplasia of the thyroid (goiter) (Hopkins et al. 2009; Fenwick et al. 1983; Anilakumar et
al. 2006; Heany and Fenwick 1995). Luckily, due to the unpleasant flavour of
isothiocyanates, consumption in excess is not likely.
Glucosinolates may have important anticarcinogenic effects (Verhoeven et al. 1997; Hecht
1999), especially in colon and rectum cancers (World Cancer Research Fund, 1997), and they
could also help reduce the risk of developing mammary and lung tumors (Wattenberg 1977;
Guo et al. 1992; Lugasi and Varga 2006). Their role as pesticides has also been considered
(Haramoto and Gallandt 2005).
2.1.3.4.2 Phenolic Compounds
Phenolic compounds are substances that contain an aromatic ring with one of more hydroxyl
groups. They are essential during plant growth and reproductive stages and also help fight
pathogens (Butler 1992). Some examples of these compounds are phenol, phenylpropanoids,
benzoic acid derivatives, flavonoids, stilbenes, tannins, lignans and lignins.
15
Esterified phenolic esters constitute about 80 % of the phenolic compounds present in
mustard and rapeseed (Shahidi and Naczk 2004), being sinapine the major component of this
group in mustard seeds. The hydrolysis of sinapine produces sinapic acid, the main phenolic
compound present in mustard flour (Sharma 2007). Sinapine is partially responsible for the
bitter taste, while tannins are responsible for the astringency of mustard (Shahidi and Naczk
2004).
The content of sinapine in yellow mustard seeds is around 13 mg/g (Matthaus 1997),
compromising the uses of mustard products. Hens being fed a diet containing rapeseed with
high levels of sinapin produced eggs with a fishy flavour (Pearson et al. 1980; 1982) and it is
expected that using mustard products will generate the same effect.
Phenolics lower the nutritional value of protein isolates because they may contribute to their
dark colour, astringency and bitter taste, and also because they remain bound to proteins and
carbohydrates after extraction (Shahidi and Naczk 1995; Nackz et al. 1998). Tannins are
antinutritional and they bind to peptides and proteins and prevent their hydrolysis and
digestion in the stomach (Sosulski 1979; Shahidi and Naczk 2004).
Even though phenolic compounds are considered antinutritional, they posses antimicrobial,
antiviral and antioxidant properties and can be used as nutraceuticals, antioxidants,
antibiotics, pesticides, insulating materials, protecting UV agents (Shahidi and Naczk 2004).
2.1.3.4.3 Phytic Acid
Phytic acid (inositolhexaphosphate) is a compound with 6 groups of phosphates attached to
the inositol group. It accounts for 2-3% of the seed weight and represent 60-90% of total
phosphorous (Graf et al, 1984). In yellow mustard seed, the content of phytic acid is 14 mg/g
(Matthaus 1997). It accumulates during seed development until the seed reaches maturity and
serves as storage of phosphates as energy source and antioxidants during germination (Raboy
2003).
Due to the chelating effect of the phosphate groups, phytic acid binds to mineral cations such
as Cu+2, Zn+2, Ni+2, Co+2, Mn+2, Fe+3 and Ca+2 (Persson et al. 1998; Vohra et al. 1965).
16
A diet high in phytic acid is linked to malnutrition and mineral deficiency because the
solubility of phytic acid-metals complexes is low at the pH of most part of the intestines
(Cheryan 1980). Animals with diets high in phytic acid tend to excrete large quantities of
phosphorous, leading to eutrophication of fresh water and lakes (Vats et al. 2005).
In spite of all the antinutritional characteristics of phytic acid, it is a very good antioxidant
and it tends to bind and inactivate Fe ions in solution, avoiding the formation of hydroxyl
radicals when Fe+2 is oxidized. It works better than EDTA (Graf et al. 1984; Wong and Kitts
2001) and the inactivation of these radicals could help in the prevention of Alzheimer’s,
Parkinson’s, arthritis and cancer, among other conditions (Benzie 2003). It also prevents
oxidation of cellulose, increasing the half-life of documents (Neevel 1995), prevents
calcification and kidney stones (Grases et al. 1998; 2004), helps lower cholesterol and
triglycerides (Onomi et al. 2004) and it has been recently tested for the storage of meat
(Stodolak et al. 2007). It has been considered in cancer therapy for its ability to inhibit iron
mediated oxidative reactions, enhancing immunity and preventing tumour metastasis
formation (Vucenik and Shamsuddin 2006; Bohn et al. 2008).
2.2 Uses of Yellow Mustard Products
Mustard is generally used in the production of North American “yellow” mustard or Dijon
mustard, but it is a vital ingredient in mayonnaise, ketchup, pickles, relishes, sauces and salad
dressings and because mustard has the same emulsifying properties as egg yolks, it can be
used as a replacement in a variety of products.
Mustard has been used as a food preservative since some components of mustard seeds
(phenolic compounds, glusocinolates and isothiocyanates) have good antiseptic and
antifungal properties and may inhibit growth of some types of bacteria, yeasts and moulds
(Agriculture and Agri-Food Canada 2008). Mustard oil is a very important cooking oil in
some parts of Asia such as India and Bangladesh, but because of its high level of erucic acid
it is not considered edible in North America or Europe; for this reason, mustard oil prices are
lower than canola oil prices, making it suitable for industrial applications such as lubricants,
17
solvents or in the production of biodiesel. As it was previously mentioned, there are more
than 200 potential applications of erucic acid (Leonard 1994) and this provides mustard oil
with important characteristics to compete in the industrial market.
Deheated Mustard flour (previously heated to inactivate the enzyme myrosinase and prevent
the formation of pungent and hot flavours) is used as a binding agent in the meat industry and
it also helps improve the flavour of some products and provide a smooth texture
(Saskatchewan Mustard Development Commission 2009). Mustard protein has a well-
balanced amino acid profile and the allergens identified in mustard are different to the ones
found in soy, therefore mustard protein could be used as a substitute for soy protein products
such as vegetarian meat products, infant formulations, protein bars, protein-enhanced
beverages and soups (Lorenzo 2008).
It is important to remove the undesirable compounds such as glucosinolates, phenolic
compounds and phytic acid, which give the seed unfavourable properties. The
anticarcinogenic and antioxidant properties of phytic acid and glucosinolates could be
applied in food products or nutraceuticals and phenolic compounds can be used as pesticides.
One important aspect of mustard products is their biodegradability and environmentally
friendly label compared to petroleum based products. Some Canadian companies such as
Nematrol in Ontario and Peacock Industries in Saskatchewan have developed bio-pesticides
derived from mustard seeds with good performance. Bio-green Technologies in
Saskatchewan has introduced a new product called Mustard Organic Soil Stabilizer (MOSS)
which is ideal for restoring damaged or poor soils (Agriculture and Agri-Food Canada 2008).
2.3 Oil extraction
Each oilseed has specific conditions for extraction depending on its characteristics. The seed
is first analyzed for moisture content and dried to 10-12% moisture before storage. Seeds
must be kept at low moisture content to retard the development of free fatty acids (Lusas
1983). The next step is to clean the material and remove undesirable particles such as sticks,
18
leaves and other plant materials, before the dehulling process starts. Dehulling involves two
operations: cracking of the seeds and separation of the kernels from the hulls by gravity
tables or sieving. Complete removal of the hulls is not desired because their presence
enhances percolation during extraction (Lusas 1983), unless they can be used for other
purposes.
Once the seeds have been cleaned and dehulled, the next step involves techniques designed
to free the oil by rupturing the seed cells using thermal or mechanical stress. Some of these
techniques are: hard press, direct solvent extraction, prepress-solvent extraction, enzymatic
extraction and aqueous extraction.
The final step in oil extraction is to refine the oil. The refining process involves several
operations: degumming for removing phosphatides and mucilaginous gum, alkali-refining
treatment to remove free fatty acids, colour bodies and metallic pro-oxidants, bleaching to
remove pigments and residual soaps and improve the taste of the oil, and at last, a
deodorization step by high-vacuum steam distillation (Brekke 1980).
2.3.1 Traditional Methods
2.3.1.1 Hard press and Pre-press Solvent Extraction
Hard press is the oldest method used and its principle is to press the seeds in order to squeeze
the oil from the rest of the solids. Seeds are usually cooked to reduce their moisture content
and then they are continuously pressed. A screw press is designed to accept the feed and
subject it to gradually increasing pressure as it is conveyed through a barrel cage. The friction
caused by the material moving in the barrel generates excess heat that can damage the cake
and the protein. Usually there is a residual oil content of 6-12%, although some operations
produce meals with 3-4% residual oil content (Lusas 1983).
Pre-press solvent extraction is a method usually used before solvent extraction to remove oil
from oilseeds with over 30% oil content. In this case, the seeds are pressed with less intensity
19
than in hard-pressing and the oil content is reduced to 14-18%, before solvents are used to
remove the remaining oil (Lusas 1983).
2.3.2 Solvent Extraction
Solvent extraction is the most widely used method for oil extraction due to its ability to
remove the oil from the seeds almost completely. Residual oil can be as low as 0.5-1% (Wan
and Wakelyn, 1997; Lusas, 1983). In this process (shown in Figure 2-3), the seeds are
cooked or conditioned and flaked to about 0.010 inch in thickness and then extracted with
solvents in a continuous extractor. The heating step reduces the viscosity of the oil, partially
coagulates proteins, improves permeability of cells due to cell rupture and helps agglomerate
oil droplets (Lajara 1990). Flaking is very important because reducing the size of particles
will increase the contact surface between seeds and solvent.
Figure 2-3. Oil extraction combining solvent extraction and pressing (Rosenthal et al. 1996)
20
The mixture of solvent and oil is called “miscella”, and the components need to be separated
by distillation to recover solvent for reuse. The extracted solids are called the “marc” and are
usually subject to solvent removal processes to minimize solvent losses. Solvent removal
occurs at high temperatures (100-115 ºC) and causes denaturation of the meal compromising
its use in other products.
The efficiency of the extraction will depend on three factors: the solubility of the oil in the
solvent, the degree of penetration of the solvent in the solid matrix (percolation), and the
degree of final drainage once the oil has been extracted (Lajara 1990). If percolation is high
there will be more contact between the solvent and solids, but it will be more difficult to
separate the solvent from the marc after extraction. Retained miscella represents additional
oil remaining in the marcs, which along with undissolved oil, determines the total residual oil
after extraction.
The solvent selected will determine the quantity and quality of oil extracted, since different
solvents have different affinities for triglycerides and for undesirable components such as
phosphatides and pigments. Various solvents have been used for commercial extraction but
the preferred solvent for commercial oil extraction over the past 60 years has been n-hexane
or mixtures of hexanes, rich in n-hexane. Yields for oil recovery can be as high as 95%
(Rosenthal et al. 1996).
Hexane is a flammable organic solvent considered a greenhouse gas and it is a volatile
organic compound (VOC), which can react in the atmosphere with other chemicals to
produce ozone and other photochemicals which affect human health and the environment.
Worker exposure to hexane raises concerns about its effects on the central nervous system
and motor neuropathy (Lusas et al. 1990) and handling hexane presents a potential risk of
explosion. The Environmental Protection Agency regulates n-hexane under the U.S. Clean
Air Act and has designated n-hexane as a hazardous air pollutant. It has been listed in the
National Pollutants Release Inventory since 1999 for industrial release reporting but it is still
permitted for use as extraction solvent by Health Canada. Hexane losses range from 0.2-0.5
gal/ton (Wan and Wakelyn 1997) and releases into the atmosphere have increased in Canada
21
in the last 10 years (NPRI 2008). Given its environmental effects and potential risks, in
addition to fluctuating prices, new solvent alternatives need to be found.
2.3.3 Alternative Solvents
An ideal solvent should be highly soluble or completely miscible with triglycerides.
Furthermore, the ideal solvent should be neither toxic nor teratogenic to workers, humans or
animals; it should shave selectivity for certain components such as triglycerides but not for
free fatty acids, waxes, pigments and phosphatides in the meal; it should be low in cost, non-
flammable, non-corrosive and stable for repeated recycling. Unfortunately the perfect solvent
does not exist and compromises must be made to find the best possible alternative. Research
in alternative solvents has been concentrated on ethanol, isopropyl alcohol and water.
Figure 2-4. Solubility of cottonseed oil in absolute ethanol and IPA, and their azeotropes
(Johnson and Lusas 1983)
Alcohols represent a very good option because they have high oil solubility at elevated
temperature and low oil solubility at ambient temperature, making separation of oil from
22
solvent without evaporation possible. The solubility of oil in IPA and ethanol varies with
water content and temperature (Wan and Wakelyn 1997, Lusas 1983). Ethanol and isopropyl
alcohol (IPA) form azeotropes with water at 96 and 87.7 wt%, respectively. These azeotropic
mixtures have lower oil solubility than pure solvents and have limited oil solubility at low
temperature as shown in figure 2-4.
Ethanol can be produced by fermentation and it is inexpensive, however it is tightly regulated
and it is subject to taxes. Isopropyl alcohol is made from petroleum refinery by-products and
it is effective as a solvent for vegetable oils.
2.3.3.1 Isopropyl Alcohol as a Solvent
Isopropyl alcohol (IPA) is safer than hexane because it has a higher flash point, upper
explosive limits and autoignition temperature. The major disadvantages of using IPA is that
more energy is required to recover the solvent that in the traditional hexane process, and that
oil solubility is lower than hexane. Since isopropyl alcohol forms an azeotrope with water at
87.7 wt% IPA (IPAWA), oil extraction was limited to the use of IPAWA solutions,
restricting oil solubility and extraction. With the development of pervaporation, absolute IPA
could be obtained and oil extraction at IPA concentrations of 93% or higher could be tested.
Recovering IPA by distillation is more energy-intensive than recovering hexane, however
chill separation is possible for IPA-oil mixtures. Chill separation is a process of cooling IPA
miscella to reduce oil solubility resulting in 2 phases, a solvent-rich upper phase and an oil-
rich lower phase. Some properties of IPA and hexane are presented in Table 2-4.
Table 2-4. Characteristics of n-Hexane and Isopropyl Alcohol
Characteristic n-Hexane Isopropyl Alcohol
Molecular Weight (g/mol) 85.17 60.11
Density (g/ml at 25ºC) 0.655 0.786
23
Water Azeotrope (wt%) 5.6 12.3 IPAWA
Boiling Point (ºC) 68.7 100% IPA= 82.3
IPAWA = 80.2
Vapour Pressure (kPa@20 ºC)
17.3 4.4
Flash Point (ºC) -22.8 11.7
Viscosity (cP at 65 ºC) 0.22 0.65
Autoignition Temperature (ºC)
224 339
Oil Solubility Completely miscible Varies with water content
100% IPA = completely miscible at 20 ºC
95% IPA= 100% at 50 ºC
Water Solubility Immiscible Completely miscible
Flammable Limits Lower 1.15%,Upper 7.5% Lower 2%, Upper 12.7%
Latent Heat of Vaporization 80 kcal/kg 100% IPA= 160 kcal/kg
95% IPA= 179 kcal/kg
IPAWA= 206 kcal/kg
Energy for Chill Separation Not possible IPAWA= 9 kcal/kg
24
IPA is completely miscible with water and it may dehydrate the seed during extraction,
therefore moisture content in the seeds must be kept low, bellow 7% (Lusas et al. 1996).
Lusas et al. (1996) reported solubilities for IPAWA of 15-18% for cottonseed oil and 25-28%
for soybean oil, and infinite miscibility of cottonseed oil at IPA concentrations of 93% or
higher and 91% or higher for soybean oil. It was concluded that when the IPA concentration
increases, even though oil solubility improves, it is more difficult to separate oil by chilling
from the upper phase due to the solvent effect, reducing the effectiveness of chill-separation.
95% IPA was selected as the most practical solvent because it guarantees full miscibility and
does not restrict chill separation. Moreover, the refined cottonseed and soybean oils extracted
with IPAWA and 95% IPA exhibited similar characteristics and lower Free Fatty Acid (FFA)
content than hexane-extracted oils (Lusas et al. 1996; Ghandi et al. 2003; Seth et al. 2007).
IPA is less aggressive than hexane as an extraction solvent and more stages are needed to
remove oil from seeds because diffusion is lower and requires more time (Lusas et al. 1996;
Zhang et al. 2002). Solvent holdup in the marcs is higher for IPA than for hexane, because
hexane is less viscous than IPA and drains more easily. The difference in polarities also
contributes to the higher affinity of IPA towards the protein and carbohydrates in the meal
(Zhang et al. 2002). Higher solvent holdup means more energy is necessary to recover the
solvent from the marc. A recent study has found IPAWA to recover more oil than hexane for
soybean flakes, with oil yields up to 95% compared to 93% with n-hexane (Seth et al. 2007);
however these results are contradicting previous observations. More research is still
necessary to improve IPA extraction and make it more competitive with hexane extraction,
especially in reducing operating costs.
2.3.4 Aqueous Extraction
Aqueous extraction is an emerging alternative to hexane extraction because it represents a
cleaner and safer process in terms of environmental effect and worker exposure, since it
improves safety due to low risk of fire and explosion. The extracted oil requires less refining
due to its lower phospholipids content. Water is used as the extraction solvent in order to
separate different components of the seed. It involves a different approach to hexane
25
extraction: instead of using a solvent with high affinity for triglycerides, a solvent with low
affinity for oil is used in order to allow separation of oil in a distinct phase. Separation is
based more on the insolubility of oil in water than on the dissolution of oil (Johnson and
Lusas 1983). This process is suitable for small-scale operations and it offers the advantage of
recovering both oil and protein simultaneously without damaging any of the products. Some
disadvantages of aqueous extraction are reduced yields, demulsification requirements and
treatment needs of the effluents.
In the traditional solvent extraction process the seed is flaked in order to break the cell wall
and expose the oil, which will then diffuse into the solvent while the protein remains in the
meal. In the aqueous process, the soluble components diffuse into the water and the oil forms
a separate phase, in many cases an emulsion with water due to the presence of oleosins. As it
was previously mentioned, mustard oil bodies are very small (< 1µm) and they are not
completely disrupted during flaking, therefore separation of free oil is very difficult.
Aqueous extraction has been studied for several oleaginous materials such as coconut, palm,
peanut, rapeseed, soy and sunflower, with oil yields ranging from 60-90% (Cater et al. 1974;
Kim 1989; Hagenmaier 1974; Embong and Jalen 1977, Rosenthal et al. 1996, Chen and
Diosady 2003); however oil yields lower than 80 % are generally obtained. Some of the
products made from aqueous processing of various oleaginous materials include fresh and
preserved coconut milk, corn oil, starch, cream, cheese, butter, palm oil, soy mild and tofu.
In the Food Engineering Group at the University of Toronto, dehulled or partially dehulled
yellow mustard flour was used as starting material instead of yellow mustard seeds. The flour
is mixed with water at a specific water to seed ratio, then extracted at the desirable pH and
centrifuged. After centrifugation, separation of 3 phases is possible: a solid phase called the
residue which is rich in fiber and carbohydrates, a protein-rich phase called the aqueous
extract, and an oil-rich phase in form of oil-in-water emulsion. The aqueous extract is further
processed using membranes to concentrate the solution and isoeletric precipitation to recover
the protein products. Two types of protein isolates are obtained, a precipitated protein isolate
and a soluble protein isolate, both with more than 85% protein concentration and high protein
26
recovery (over 85%) (Xu et al. 2003). Oil recovery ranges from 55-80% in form of an oil-in-
water emulsion (Balke 2006).
Extraction involves stirring the solution to promote oil release through disintegration of the
cells. Factors affecting aqueous extraction are water to seed ratio, pH, extraction time,
temperature and number of extraction stages (Lusas et al. 1982; Balke 2006). Table 2-5
presents a summary of the optimal conditions for aqueous extraction of protein and oil from
yellow mustard seeds.
In general, the extraction conditions that produce the highest protein yield will also produce
the highest oil yield; therefore, the highest oil extraction yield occurs at pH values
corresponding to maximum protein yield (Rosenthal et al. 1996). Optimal pH conditions for
sunflower and rapeseed are 10.0 and 6.6, respectively. (Hagenmaier 1974; Embong and Jalen
1977). For yellow mustard seeds, the optimal pH for protein extraction is 11 (Xu et al. 2003;
Balke 2006).
Table 2-5. Optimal Conditions for Aqueous Extraction of Yellow Mustard Seeds
Parameter Optimal Value
pH 11
Water to Seed Ratio 4:1
Temperature Room Temperature
Extraction Time 20-30 min
Number of Extraction Stages
2
Blending Time 180 s
Data Compiled from Balke, 2006.
27
Temperature is considered to have a more significant effect on oil than on protein extraction
(Lusas et al. 1982). Temperatures used in some processes are: room temperature for
sunflower oil, 60-65 ºC for peanut oil, 70 ºC for rapeseed oil (Hagenmaier 1974; Rhee et al
1972; Embong and Jalen 1977). Balke (2006) studied the effect of temperature on oil and
protein extraction from yellow mustard seeds and found that room temperature extraction
was successful at high pHs.
Water to seed ratio is an important parameter in aqueous processes. Lower ratios generate
less emulsion and less effluents, however, higher yields are obtained when large quantities of
water are used (Rosenthal et al. 1996). Recommended values for different materials are: 2.5:1
and 3.5:1 for rapeseed, 10:1 for sunflower and 12:1 for soybeans (Embong and Jelen 1977;
Hagenmaier 1974; Lusas et al 1982). In the case of yellow mustard, low water to flour ratios,
such as 4:1, subject the seed material to higher stresses and produce more disruption of the
cells, increasing oil and protein extraction (Balke 2006).
The longer the extraction time, the more stable emulsion is likely to be. Optimal time for
soybean oil extraction is 40 min (Lusas et al 1982) and 20-30 min for yellow mustard, with
two 20-30 min extraction stages needed to achieve maximum extraction (Xu et al. 2003;
Balke 2006).
Blending time was another factor studied in our group. A blending time of 180s helped
increase oil and protein extraction because it reduced mean size of particles compared to
experiments where the blending stage was eliminated (Balke 2006).
One of the problems of aqueous extraction is the low efficiency of the process: oil yields are
lower than in hexane extraction. A solution to this problem is the use of enzymes in order to
break the structure of the cell walls and release more oil and protein during the aqueous
extraction process. Some of the enzymes used are cellulases, hemicellulases, proteases and
pectinases. Oil yields of up to 90% from soybeans have been obtained with proteases,
however most of it remains in the form of a cream or emulsion fraction, with no free oil
separated (Lamsal and Johnson 2007; Lamsal et al. 2006) and the valuable protein is
28
destroyed. The choice of enzyme will depend on the nature of the oilseed, and in most cases,
enzyme mixtures with combined activity give better results than individual enzymes
(Rosenthal et al. 1996).
Enzyme aided aqueous extraction is a very good alternative to hexane extraction and its
application in oil and protein extraction from yellow mustard seeds needs to be further
studied. However, it is beyond the scope of this investigation.
2.4 Food Emulsions
An emulsion is a mixture of two immiscible liquids in which one of the liquids is dispersed
in the other. One of the phases exists as discrete droplets, known as discrete phase, suspended
in the second phase, called continuous phase, due to the presence of a surfactant material at
the interface.
Generally, emulsions occur in mixtures of water and oil. The surfactant (surface acting
agent) is a charged particle that lowers the interfacial tension and creates a repulsive force
between drops which prevents coalescence. Without the presence of the surfactant, the drops
would tend to minimize the interfacial area by coalescing and creating two separate layers of
oil and water. Surfactant molecules are amphiphilic: they contain both hydrophobic and
hydrophilic domains, therefore, one part of the surfactant dissolves in the fat or oil and the
other part in the aqueous phase. Examples of surfactants are egg yolk (where the active
ingredient is lecithin), proteins, honey, mustard (due to the presence of mucilage), cetearyl
alcohol and polysorbates (Tween 20 and 80), among others.
There are two types of emulsions which are important in foods: oil-in-water (o/w), water-in-
oil (w/o). Oil-in-water emulsions are the most common and versatile type of emulsion and
can be found in milk, mayonnaise, vinaigrettes, creamers, whippable toppings, cream
liqueurs and ice creams mixes. Their properties can be controlled by varying the surfactants
used and the components present in the aqueous phase. Water-in-oil emulsions are widely
used in the production of margarine, butter and fat-based spreads. They are more difficult to
29
control because their properties depend more on the properties of the fat and surfactant and
less on the properties of the aqueous phase. (Friberg et al. 2004).
Proteins absorb to the oil-water interface, their hydrophobic regions dissolve in the oil phase.
Once the protein is adsorbed, its structure will prevent close packing of the points of contact
with the interface and thus reduce interfacial tension. Although some proteins are excellent
emulsifiers, in some cases they cannot adsorb to the oil-water interface because of their rigid
structure and strongly hydrophilic side chains, such as in the case of gelatin and lyzosome,
which tend to be poor emulsifiers (Dickinson et al. 1985; Kato et al. 1981). Phospholipids are
also important in defining the properties of an emulsion. They are usually present in the
interfacial layer and help stabilize the emulsion, even though they are not as efficient as
proteins or large molecular weight emulsifiers (Friberg et al. 2004; Wu et al. 2009b).
Figure 2-5. Structure of the yellow mustard oil-in-water emulsion
In the case of mustard, the surfactant is mainly the oleosin protein, although the presence of
phospholipids enhances its stability. Figure 2-5 is a representation of the yellow mustard oil-
in-water emulsion. As it was previously mentioned, oleosins are composed of three sections,
a hydrophobic middle section in direct contact with the triglyceride molecules and embedded
30
in the phospholipid layer, and two amphipathic N and C terminals exposed to the aqueous
phase. The stability associated with oil bodies is due both to the negative charge of the
droplet surface and the steric forces originated from the hydrophilic parts of the oleosin
molecule. Furthermore, the properties of the mixed phospholipid-oleosin layer provide more
stabilization and prevent coalescence (Nikiforidis and Kiosseoglou 2009).
The existence of oil-in-water emulsions after the aqueous extraction process limits its
application because a new demulsification step is necessary to fully recover the oil.
Heating and centrifuging has not been effective in breaking emulsions (Lamsal and Johnson
2007). Freezing and thawing resulted in oil yields of up to 25 % in the form of free oil
(Morales et al. 2008). pH adjustment using hydrochloric acid has been studied to destabilize
emulsions by promoting precipitation of interfacial protein. Lowering pH of soy emulsion to
near the isoelectric point (4.5) resulted in recovery of 83-100% free oil (Wu et al. 2009b;
Morales Chabrand and Glatz 2009; Jung et al. 2009). Nevertheless, the addition of salts to
the process limits the ability to recycle the aqueous fraction and generates undesired waste.
Li et al. (1976) developed a process to break emulsions by adding a mixture of two or more
comiscible volatile solvents, at least once miscible with oil and one miscible with water,
causing the separation of the emulsion in two separate layers. The solvents used were
isopropyl alcohol and cyclohexane mixed in equal volume with emulsions for a time
sufficient to separate the oil and water in two phases, after which the solvents were distilled
for reuse.
Current research is focused on the use of enzymes to hydrolyze the interfacial proteins
reducing the rigidity of the oil droplet interface or hydrolyzing phospholipids to eliminate
their surface activity and permit coalescence of the oil droplets. Endopeptidases have been
used and resulted in oil recoveries of 95% (Morales Chabrand and Glatz 2009). Proteases and
phospholipases have been studied to break almost completely or completely soybean oil
emulsions, either separately or in combination (Wu et al. 2009; Jung et al. 2009). It was
found that proteases act at lower concentration than phospholipases indicating than proteins
31
are the major contributors to emulsion stability. With the implementation of enzyme
recycling, the use of an enzyme mixture of proteases and phospholipases during the
extraction and demulsification steps could help achieve efficiencies similar to those of
hexane extraction.
2.5 Biodiesel Production
Biodiesel is produced by a transesterification reaction (Figure 2-6) in which a vegetable oil or
animal fat reacts with an alcohol in the presence of a catalyst to produce alkyl esters.
Glycerol is formed as a by-product and it separates from the ester phase by gravity. Biodiesel
can be produced from a variety of feedstocks such as vegetable oils from soybean,
cottonseed, sunflower, palm, peanut, canola, or from animal fats (usually tallow) and waste
oils. Methanol is normally used because it is the least expensive alcohol; however ethanol,
isopropyl alcohol and butanol are good alternatives. The catalyst is usually a base such as
NaOH or KOH.
Figure 2-6. Transesterification of a triglyceride molecule with isopropyl alcohol
Biodiesel can be used as a fuel in pure form (B100) or mixed with petrodiesel at different
concentrations (B20, B30). The use of biodiesel compared to petrodiesel offers great
32
advantages. Biodiesel is produced from renewable sources and it is biodegradable; it has a
higher flash point which makes it safer to handle and store; it is low in sulphur and reduces
most exhaust emissions with the exception of nitrogen oxides (NOx); and it offers excellent
lubricity. On the other hand, biodiesel has lower energy content (37.2 MJ/kg for soy
biodiesel) than No. 2 diesel fuel (42.6 MJ/kg), it does not offer good cold flow properties and
its price is higher than petrodiesel, making it less competitive in the market. This is due to the
fact that the feedstocks used to produce biodiesel are generally employed in the food industry
and edible oil market, such as in the case of soybean and canola oils. In the United States,
soybean oil is the prime feedstock while in Europe it is canola oil, and in tropical countries
palm oil (Knothe et al. 2005). When petroleum prices are low, the price of biodiesel can be as
high as four times the price of petroleum diesel. Feedstocks account for 70-85% of the
overall production cost, therefore an alternative feedstock with lower costs is desired.
Mustard oil represents a great option because it does not have an edible oil market and its
high erucic acid content would provide biodiesel with even better lubricating properties.
Three types of catalyst can be used in the transesterification reaction: alkaline, acid and
enzymes. When alkaline catalysts are used, less catalyst is required and shorter reaction times
are observed. NaOH is the most widely used catalyst. Potassium hydroxide could be used as
a catalyst and the advantage would be the potential economic value of the waste stream as a
fertilizer. However, the cost of KOH is much higher than sodium catalysts, limiting its use.
Methyl alkoxides such as sodium or potassium methylate could also be used and even though
they are more expensive than hydroxides, they are safer to handle and produce purer
glycerol. Acid catalysts are used when the oil has a large concentration of free fatty acids
(FFA) and reaction times are slower than when alkaline catalysts are used. FFA neutralize
alkaline catalysts and form soaps, which will increase the viscosity of the solution and
interfere with the separation of glycerol (Chi 1999). The use of enzymes to catalyze
transesterification reactions is an emerging approach which offers great advantages such as
mild conditions, easy recovery of products and an environmentally friendlier process,
however the cost of enzymes limits its application in industrial settings (Ranganathan et al.
2008).
33
Diesel and biodiesel fuels contain small amounts of long-chain hydrocarbons which
crystallize at low temperatures. Once they crystallize, these wax crystals will agglomerate
and plug fuel filters, disrupting engine operation. The temperature at which crystals become
visible is defined as Cloud Point (CP), and at temperatures below the CP crystals start to
agglomerate until free pouring of fluid is prevented, this temperature is known as Pour Point
(PP). Biodiesel made from feedstocks containing higher concentrations of high-melting point
saturated long-chain fatty acids tends to have poor cold properties (Knothe et al. 2005).
Methyl esters will start to crystallize at ~0ºC for soybean oil and at 13-15ºC for animal fats
and frying oils (Lee et al. 1995). Mustard oil is low in saturated fatty acids and the CP and PP
for ethyl esters from mustard seed were found to be 1ºC and -15ºC, respectively (Dunn and
Bagby 1996).
The use of longer-chain alcohols such as isopropyl alcohol or butanol can lower the freezing
point of biodiesel and improve its cold flow properties. Crystallization is more difficult due
to the branched structure of isopropyl alcohol. Lee et al. (1995) compared the properties of
isopropyl and methyl esters of soybean oil and tallow and found that the crystallization
temperature for isopropyl esters was 7-11ºC lower than for the methyl esters. Wang et al.
(2005) compared the cold flow properties of isopropyl and methyl esters of soybean oil and
yellow grease. As it is shown in Table 2-6, the CP and PP temperatures were reduced by over
7ºC when isopropyl alcohol was used.
Table 2-6. Comparison of the cold flow properties of isopropyl and methyl esters of soybean oil and yellow grease
Property Diesel No. 2 Soybean Oil Methyl Ester
Yellow Grease Methyl Esters
Soybean Oil
Isopropyl Esters
Yellow Grease
Isopropyl Esters
CP (ºC) -18 -2 8 -9 0
PP (ºC) -30 -6 6 -12 -3
Data compiled from Wang et al., 2005.The production of Fatty Acid Isopropyl Esters and Their Use as a Diesel Engine Fuel. Nov 2005.
34
In order to obtain the maximum yield in the transesterification reaction, the water and free
fatty acids (FFA) content should be low. FFA react with the catalyst to produce soap and
their level should be below 2% (Sharma et al. 2008). It is very important to maintain a low
moisture level because water can hydrolyze alkyl esters to produce soaps instead of biodiesel,
and it can consume the catalyst reducing its effectiveness. Sharma et al. (2008) concluded
that acid catalysts are more affected by the presence of water, followed by alkaline catalysts
and little effect had been observed when using lipase as a catalyst. The limit when using
alkaline catalysts was set to 1-1.5%, and less than 0.5% for acid catalysts.
The molar ratio of alcohol to oil is a very important parameter. The stoichoimetric amount is
3:1, however excess alcohol is always used to drive the reaction to completion. Base-
catalyzed transesterification reactions are generally conducted near the boiling point of the
alcohol in order to improve the rate of the reaction. The standard conditions for methanol
transesterification are 6:1 alcohol:oil molar ratio, 1% NaOH and 60ºC, with conversion of
99% in 1 h at ambient pressure and vigorous mixing, however if lower temperatures are used
(32ºC), the reaction could be completed only in 4h (Freedman et al. 1984). Other alcohols
require higher temperatures for optimum conversion, such as in the case of ethanol and
butanol, with temperatures of 75 and 114ºC, respectively (Lee et al. 1995). For conversion of
soybean oil to isopropyl esters, a 20:1 isopropanol:oil molar ratio and 1% sodium metal
catalyst (based on TAG amount) is required at a temperature of 80 ºC (Wang et al. 2005).
Special care must be taking when using large quantities of excess alcohol because it
complicates separation of glycerol and esters (Miao and Wu 2006)
One problem in biodiesel production is the long reaction times observed at ambient
temperature. The solubility of oil in methanol or ethanol is low and the reaction presents
mass transfer limitations, taking place only at the interface. Several studies have considered
the use of cosolvents such as tetrahydrofuran (THF) or methyl tert-butyl ether (MTBE) in
order to decrease the reaction time. The use of a cosolvent creates a single phase and
accelerates the reaction. 99.4% conversion can be achieved in 7 min using THF and a
methanol:oil molar ratio of 27:1. (Boocock et al. 1998; Zhou et al. 2003).
35
Isopropyl alcohol and oil are completely miscible, therefore there are no mass transfer
limitations and cosolvents are not necessary. Taking into consideration that isopropyl esters
have significantly better cold flow properties than methyl esters as it was shown in Table 2-6,
and that mustard oil ethyl esters already have low Cloud Point and Pour Point temperatures,
it is expected that isopropyl esters produced from mustard oil will have even lower CP and
PP temperatures, making it a suitable fuel for cold climates.
Oil extraction with ethanol followed by the transesterification of the vegetable oil has been
previously investigated (Lago et al. 1985). Oilseeds where extracted with ethanol and the
resulting miscella was used in the production of ethyl esters by adding 0.8% NaOH at 50ºC
and stirring for 1 h with conversion yields close to 96%. Shi et al. (2008) used ethanol-
rapeseed oil mixtures with methanol to produce methyl esters with 1.1% NaOH as a catalyst,
9:1 methanol:oil ratio and 50-60ºC with yields of 98%. The idea of using an integrated
process involving isopropyl alcohol extraction of yellow mustard oil and reaction of the
miscella to produce biodiesel represents a good alternative which needs to be evaluated.
2.5.1 Water Adsorption
The presence of water represents a major disadvantage in the transesterification reaction. If
water is present, there a chance of soap formation due to the presence of oil in a basic
medium. Adsorption represents an approach to remove the water from the system and it is
usually performed in columns with packed sorbent particles. The separation is achieved by
one of three mechanisms: steric, kinetic, or equilibrium effect (Yang 2003). The steric effect
refers to the use of molecular sieves to separate molecules based on a difference in size.
Some small molecules will diffuse into the adsorbent while bigger ones will be excluded.
Kinetic separation is based on the difference in diffusion rates of different molecules. Many
processes operate through the equilibrium adsorption of mixture and are called equilibrium
separation processes.
Three types of sorbents are used for water adsorption: zeolites, silica gel and activated
alumina. Silica gel and activated alumina have a large capacity for water (40% by weight)
and ease of regeneration (150 ºC compared to 350 ºC for zeolites). They have higher
36
capacities for water vapour than zeolites do, however zeolites perform better at low
concentrations. Some of their important applications are purification of gas streams in the
petroleum industry, dehydration of gases and alcohols, and separation of hydrocarbons.
Zeolites are used due to their unique surface chemistries and crystalline pore structures. They
are selective ion exchangers, selective and strong adsorbers and they can be used as catalysts.
They are crystalline aluminosolicates of alkali or alkali earth metals such as sodium,
potassium and calcium. When Al and/or Si are substituted by other elements, the term
molecular sieve is used. There is some confusion between the terms molecular sieves and
zeolites and they are often used interchangeably. A molecular sieve is defined as a material
capable of separating molecules based on the basis of molecular size and shape. Therefore
zeolites are one type of molecular sieves along with other microporous materials.
There are at least 40 different types of naturally occurring zeolites and more than 150 have
been synthesized and are designated by a letter or group of letters, such as Type A, Type Y,
Type X, Type ZSM, among others (Yang 2003). Their pore size ranges from 3-12Å and the
regularity of the pores differentiates them from other molecular sieves.
Molecular sieves 4A and 3A have been previously studied in the adsorptive drying of
ethanol, isopropyl alcohol and non-polar organic solvents (Teo and Ruthven 1986; Joshi and
Fair 1988, 1991; Jain and Gupta 1994, 1998). In the case of water adsorption, while both the
solvent and the water will adsorb strongly to the molecular sieve surfaces, the large surface
area within the pores is only accessible to the smaller water molecules (1.93Å in size), so
they are effectively removed from the solvent.
Molecular sieves 4A have a pore size of 3.8Å and they are usually used to absorb water,
methanol, ethanol, carbon dioxide, ethylene, propylene, among other compounds with
molecular size below 4Å. Jain and Gupta (1994) studied the adsorptive drying of isopropyl
alcohol on 4A molecular sieves zeolites. They used cylindrical pellets and demonstrated the
feasibility of the process. The adsorption capacity was defined as 0.25g of water/ g of
molecular sieve.
37
3. PROJECT OBJECTIVES
The main objective of this project is to investigate the use of isopropyl alcohol in the recovery of
oil from the emulsion obtained by aqueous extraction of yellow mustard flour and to evaluate the
potential use of the resulting solution as feedstock for biodiesel production.
In order to develop an integrated process, the following detailed objectives were pursued:
Recovery of oil in the form of an emulsion from mustard flour using an aqueous process.
Extraction of the oil from the emulsion using isopropyl alcohol as a solvent.
Evaluation of the suitability of the isopropyl alcohol–oil solution for direct conversion to
biodiesel in the form of isopropyl esters, and recommendations for further processing.
38
4. EXPERIMENTAL METHODS
4.1 Starting Materials
Yellow Mustard Flour
The yellow mustard flour used in this project was a powder milled from Canadian No. 1
yellow mustard seed produced by Mustard Capital Inc. in Gravelbourg, SK and supplied
by Hermann Laue Spice Company Inc. in Uxbridge, ON. The material had a pale yellow
colour and was free of hard lumps or foreign materials, with 98% of the material passing
though US Mesh 45. The maximum oil content was specified by the producer as 35%,
minimum protein content 27%, maximum moisture 8% and 4-6% ash content with traces
of allyl isothiocyanate. The product was packed in a 20kg multi-wall poly lined bag and
stored at room temperature.
Solvents
The following table lists the solvents used in the experiments of oil extraction. Water was
used in the aqueous extraction of oil and protein from yellow mustard flour and Isopropyl
Alcohol was used in the oil extraction from the emulsion.
Table 4-1. Solvents used in the experiments
Solvent Grade Supplier
Water Reverse Osmosis Wallberg Building General Supply
Isopropyl Alcohol 100% Caledon Laboratories LTD. Georgetown, ON.
39
4.2 Reagents and Materials
Sodium Hydroxide (50% w/w, analytical grade): VWR, West Chester, PA, USA. Sodium Hydroxide 10% w/w was used to control the pH.
Ethanol 100%: University of Toronto Stores, Toronto, ON, Canada.
Diethyl Ether, ACS Reagent >99.0%. Sigma Aldrich, St Louis, MO, USA.
Petroleum Ether, ACS Reagent >99.0%. Boiling range 35-60ºC. BDH Chemicals Ltd., Poole, England
Ammonium Hydroxide 28-30%. BDH Chemicals Ltd., Poole, England
Hydrochloric Acid 36.5-38.0%, ACS Reagent. Fisher Scientific, Nepean, ON.
Hydrochloric Acid (25 + 11) was prepared as described in Appendix A.
Phenolphthalein Powder, ACS Reagent, J. T. Baker, Phillipsburg, NJ, USA. Phenolphthalein 0.5% (w/v) in Ethanol was prepared as described in Appendix A.
Water, Reverse Osmosis. University of Toronto General Supply. Toronto, ON, Canada.
Kelmate MT-37 Kjeldahl digestion mixture (3.5g K2SO4/0.175g HgO per tablet) Product
No. KX0015A-1: EMD, Gibbstown, NJ, USA.
Sulphuric Acid (concentrated 95-98%): EMD, Gibbstown, NJ, USA.
Boric Acid (powder, analytical grade): BDH Chemicals Ltd., Poole, England.
N-Point Indicator (Product No. NX0847/3, contains methanol, water, bromocresol green,
and methyl red): EMD, Gibbstown, NJ, USA.
Glass wool (low in lead): BDH Chemicals Ltd., Poole, England.
Sodium Thiosulfate (analytical grade): Fisher Scientific, Fair Lawm, NJ, USA.
Sulfuric Acid (0.1000 N, analytical grade): VWR, West Chester, PA, USA.
Stock Solutions (preparation instruction available in appendix A):
- Boric Acid 4% w/v
- Sodium Thiosulfate 8% w/v
- Sodium Hydroxide 32% w/w
40
Molecular sieves 4A, beads, 8-12 mesh (1.68-2.38mm), activated: J. T. Baker Chemical
Co., Phillipsburg, NJ, USA.
4.3 Equipment
Centrifuge (Beckman Coulter J20XP High Speed Refrigerated): Beckman Instruments,
Fullerton, CA, USA.
Balance (Mettler PC 4400-DeltaRange): Mettler Instruments A.G., Zurich, Switzerland.
Microscale (Model No. 2001 MP2): Sartorius, Germany.
Convection Oven (Blue M-Constant Temperature Cabinet Model No. 0V-490A-2): Electric
Company, Blue Island, IL, USA.
Refrigerator Samsung (Model No. RB1855SW): (1-4 ºC)/ Freezer (-20 ºC).
pH meter (model 8000): VWR Scientific, West Chester, PA, USA.
Blender (2-speed commercial): Waring, Torrington, CT, USA.
Overhead Mixer (RZR50 Stirrer type mixer with variable speed 45-2000 RPM transmission):
Caframo, Wiarton, ON, Canada.
Hand Blender (MR 400): Braun, Spain.
Freeze dryer (Freezeone12 Plus No.7960044): Labconco, Kansas City, MO, USA.
Centrifuge bottles (50mL, 250mL, 500mL): Beckman Instruments, Fullertong, CA, USA.
Wrist Action Shaker (Model 75): Burrel, Pittsburg, PA, USA.
Kjeldahl Digester (Model No. 425): Buchi, Switzerland.
Kjeldahl Digestion Tubes (300mL), holder, manifold and clamps: Buchi, Switzerland.
Kjeldahl Distillation Unit (Model No. K-350): Buchi, Switzerland.
4.4 Experimental Methods
4.4.1 Aqueous Extraction of Oil and Protein
Oil was extracted from yellow mustard flour via an aqueous process. The conditions for this
process were:
41
- 700g of yellow mustard flour and 2800g of water, 4:1 Water to flour ratio.
- Blending Time 180s
- pH 11.00 ± 0.05
- Extraction time 30min
- Room Temperature
- Re-extraction/ Washing step under the same conditions
The procedure is illustrated in Figure 4-1 and it is based on the experimental procedures
followed by Balke (2006). The yellow mustard flour was mixed with water in a blender in a 1L
glass jar for 3 min in three different portions. The mixture was then transferred to a large beaker
(4000mL). The native pH of the mixture was around 5.00 ± 0.05 and it was adjusted to 11.00 ±
0.05. Approximately 220 mL of sodium hydroxide (10% w/w) were added to increase the pH to
11. Afterwards, NaOH was added as needed to maintain the pH within the target range.
Figure 4-1. Experimental setting for the aqueous extraction process
42
The extraction was performed for a period of 30 min and the mixture was then transferred to 4
1000mL centrifuge bottles in order to separate the different fractions. The centrifuge was
operated at 6,500 RPM (~9000 g) for 20 min. After centrifugation, three fractions were
separated: a solid residue at the bottom of the bottles, a liquid extract, and an emulsion phase at
the top of the liquid and on one side of the centrifuge bottle. The fractions were recovered in
separate beakers. The weight of the solid residue was recorder and enough water was added to
reach the weight of the original mixture (3,500mg). The residue was dispersed in the water using
a hand blender and re-extracted for additional 30 min, making sure the pH was maintained at 11
by adding NaOH when necessary. The same procedure described above was used to separate the
fractions. After centrifugation, the second extract was mixed with the initial extract and the
second emulsion was mixed with the initial emulsion. The weights of the extract, emulsion and
final residue were recorded. Oil content was determined in freeze-dried samples of extract and
residue, for all further experiments, wet samples were used.
Figure 4-2. Experimental procedure for the aqueous extraction of oil from yellow mustard flour
43
4.4.2 Emulsion Destabilization
Three techniques were evaluated in order to destabilize the emulsion: heating and centrifuging,
freezing and thawing, and pH adjustments. For heating and centrifuging, the emulsion was
heated at 90ºC for 1 h. It was then centrifuged at 3,500 rpm for 20 min. For freezing and
thawing, the emulsion was placed in the freezer at -20ºC for 24h. It was then thawed at room
temperature. For pH adjustments, the pH of the emulsion was reduced to 7.
4.4.3 Oil Extraction from the Emulsion using Isopropyl Alcohol as a Solvent
4.4.3.1 Single Extraction
Before doing any extraction experiments, the oil content of the emulsion obtained from the
aqueous process had to be determined using the Mojonnier method. A known amount of
emulsion (15-25g) was weighted in a centrifuge bottle and enough isopropyl alcohol was added
to satisfy the required IPA:Oil ratio. The mixture was shaken for 30 min in a wrist action shaker
to allow enough time for the extraction to proceed, and it was then centrifuged for 20 min at
4,500 RPM. After centrifugation, two phases were separated: a solid phase composed of protein,
residual oil, IPA and water, and a liquid phase “miscella” composed of IPA, oil and water.
Figure 4-3. Experimental procedure for oil extraction using IPA
44
The weight of the miscella and solids was recorded. For most of the experiments, oil extraction
was calculated by determining oil content in the miscella, however, for low ratios only the
residual oil content was determined because it provided more reproducible results. Oil content in
the miscella was determined by weight difference after the solvent was evaporated in the fume
hood and the sample dried in the oven for 1.5 h at 110 ºC. For weight ratios below 3:1, residual
oil content in the solids was determined using the Mojonnier method (AOAC 922.06). Solvent
holdup was determined gravimetrically by calculating the weight loss after the sample was
heated in the oven at 110 ºC for 24h (AACC Method 44-15A). The detailed procedures for all
methods can be found Appendix A.
4.4.3.2 Multiple Stage Extraction
The procedure previously described for the single extraction was followed. However, after
centrifugation, the solids were re-extracted with the same volume of IPA used for the first
extraction step, then mixed for 30 min and centrifuged. For subsequent stages, the procedure was
repeated.
Oil extraction was calculated by determining oil content in the miscella. After the last stage, the
solids were dried in the oven to determine solvent holdup. For residual oil content determinations
wet solid samples were used, while for protein analysis samples were first dried.
For the final four-stage 2:1 IPA to oil weight ratio experiments, water content was determined by
Karl Fischer Titration. The samples were sent to the analysis laboratory of the BIOX Group in
Hamilton, ON.
4.4.4 Water Adsorption using Molecular Sieves 4A
A known amount of the miscella was placed in a glass beaker with enough molecular sieves 4A
to adsorb the water, according to the theoretical adsorption capacity. After 24h, the molecular
sieves were removed and water content determined using the Karl Fischer method.
45
4.4.5 Analytical Methods
Oil was determined by the Mojonnier method. AOAC Method 922.06 was used for fat in flour or
solid samples, AOAC Method 995.19 was used for fat in emulsions, and AOAC Method 989.05
was used for fat in the extract. Protein content was determined by Kjeldahl method AOCS Ba4d-
90 and reported as Nx6.25%. Moisture content was determined by AACC method 44-155A.
Water content in miscellas was determined by coulometric Karl Fischer titration ASTM standard
method D6304. For details refer to Appendix A.
46
5. RESULTS AND DISCUSSION
5.1 Starting Material Analysis
The same batch of yellow mustard ground seed, referred to as yellow mustard flour, was used in
all experiments presented in the following sections. Protein and oil analyses were performed two
times during the course of the experiments in February and May 2009. Moisture analyses were
performed monthly from February to June. The quality of the yellow mustard flour is presented
in Table 5-1. Protein and oil content did not vary significantly and the moisture content remained
in the 5-7.5% range. The values are consistent with protein and oil contents for yellow mustard
seeds harvested in 2007.
Table 5-1. Composition of yellow mustard flour Protein Content
(wt%, as is)
Oil Content
(wt%, as is)
Moisture
(wt %)
30.4 ± 0.3 26.6 ± 1.1 6.0 ± 1.0
The flour used in this project had been only partially dehulled and therefore had a lower protein
and oil content than completely dehulled flour reported by Balke in 2006, for which oil and
protein contents were in the range of 33-38%.
Figure 5-1. Yellow mustard flour.
47
5.2 Aqueous Extraction of Oil and Protein from Yellow Mustard Flour
Aqueous extraction of yellow mustard flour was performed following the procedure described in
Section 4.4.1. In a previous project carried out in our food engineering group (Balke 2006),
many variables where considered to have an influence in the aqueous extraction of yellow
mustard seed. It was concluded that pH had a very significant effect. Maximum oil and protein
extraction was observed at high pH values. Water to flour ratio, extraction time, temperature and
blending time were also studied and it was concluded that pH 11, 4:1 water to flour ratio, 3 min
blending time, room temperature and 30 min of extraction, with a second extraction/washing
stage of the solid residue under the same conditions, represented the best set of conditions for
optimal oil and protein recovery.
For all extractions, 700g of yellow mustard flour were mixed with 2800g of water, and
approximately 240mL of NaOH were added in order to maintain the pH at 11.0 ± 0.5. After
centrifugation, as shown in Figure 5-2, three phases were produced: a solid residue phase, a
liquid extract phase and an emulsion phase. Each batch produced 120-130g of emulsion. The
liquid extract represented 73 % of the total weight of the three phases, the solid residue 25% and
the emulsion phase less than 3%.
Figure 5-2. Aqueous extraction of yellow mustard flour
48
The distribution of oil and protein for the aqueous extraction process is presented in Figure 5-3.
The extract is rich in protein, with 91.7% of the total protein present in the original flour, while
the emulsion and residue only contain 1.6% and 4.7% of the original protein, respectively.
Mustard protein is highly soluble at high pH and similar results with protein extraction yields of
over 90% have been previously reported (Balke 2006). The low protein content in the emulsion
phase indicates that most of the protein present is oleosin protein, which is not soluble at alkaline
pH, while most of the non-oleosin protein is dissolved in the solution and recovered in the
extract.
Figure 5-3. Oil and protein distribution during aqueous extraction of yellow mustard flour
49
Most of the protein can be found in the extract but its concentration is low, ~ 4%. Further
processing is necessary to recover protein isolates with high purity. A process involving
isoelectric precipitation followed by ultrafiltration and diafiltration before drying of the final
products has been developed in our group to recover mustard precipitated and soluble protein
isolates with protein contents higher than 85% (Xu et al. 2003). Given the well-balanced amino
acid profile of yellow mustard seed, the production of protein isolates could yield protein isolates
comparable to those of soy for use in different products such as protein bars, protein enhanced
beverages, soups, or in the meat industry as meat substitutes for example.
Recovering mustard protein via an aqueous process is a feasible process; however, recovering oil
represents a challenge because the oil is extracted in the form of a very stable oil-in-water
emulsion and separation of free oil is very difficult. The stability of the emulsion is due mainly to
the presence of oleosin proteins surrounding the oil bodies, as it was described in section 2.4,
however, phospholipids help enhance its stability. The oleosin proteins act as surfactants
stabilizing the oil drops and preventing their coalescence. This represents a major disadvantage
in the aqueous process because demulsification steps are necessary to recover the oil. Several
techniques used to destabilize the emulsion are discussed in sections 5.5 and 5.6.
Only 39% of the oil present in the original flour is extracted in the emulsion, while 33% remains
in the solid residue after extraction and 29% in the extract. In a previous study using fully
dehulled yellow mustard flour, oil recovery in the emulsion was as high as 75% at alkaline pH
(Balke 2006). In the current project, the mustard flour has been partially dehulled and that could
explain the low oil yields observed. About 25% of the seed coat is composed of mucilage, a
polysaccharide with very good emulsifying properties which complicates oil recovery.
According to observations made by Balke (2006), when the mucilage gets hydrated, it creates a
gelatinous layer on the surface of the solids after centrifugation. When mucilage was completely
removed from the seeds, the gelatinous layer was no longer observed and multi-phase separation
was improved. During the current investigation, the gelatinous layer was observed in all
extractions and oil could be seen in the solids after separation. The mucilage prevented oil from
separating from the solids by creating a barrier. A solution to this problem would be to use
50
completely dehulled mustard flour or to remove the mucilage before performing aqueous
extraction. A rapid method for mucilage removal has been proposed by Balke and Diosady
(2003).
Another approach to the low oil recovery problem would be to use enzymes during the aqueous
extraction process. The use of enzymes has been proven to improve oil recovery. They can help
achieve higher extraction yields by breaking the cell walls thus releasing more oil.
Overall, the aqueous process is very good for protein extraction but not effective for oil recovery.
Improving the latter will make aqueous extraction a more competitive technique considering the
different products that could be obtained from such process.
5.3 Emulsion Analysis
Emulsions obtained from the aqueous extraction of yellow mustard flour were analyzed for oil,
moisture and protein. All measurements were performed in triplicate. In the course of the project,
15 emulsions were prepared and characterized. 121.4 ± 8.6g of emulsion were generated in each
extraction. Emulsions were kept refrigerated at 4ºC for a maximum period of one month, after
that time, they became very compact and were not used in any experiment. Oil extraction yields
based on oil recovered in the form of an emulsion were determined for all extractions. The mean
oil recovery was 37.0 ± 1.9 and the values were in the range of 35-40%. The average protein, oil
and moisture contents are presented in Table 5-2. Oil content was in the 55-60% range, moisture
content between 38-44% and protein content between 2.4-2.9%. For complete results refer to
Appendix B.
Table 5-2. Composition of Yellow Mustard Oil Emulsion
Oil Content (wt %) Protein Content (wt %) Moisture (wt %)
57.0 ± 3.3 2.6 ± 0.1 40.5 ± 2.2
51
Figure 5-4. Yellow mustard oil emulsion
5.4 Destabilization of the Yellow Mustard Oil Emulsion
In the aqueous process, the presence of the oleosin proteins makes the recovery of free oil very
difficult and contributes to the emulsification of the oil. The yellow mustard oil emulsion
produced in this investigation contained less than 3% protein, however, this amount is sufficient
to create a very stable system. Protein contents of 0.5-1% in soy emulsions have been previously
reported (Wu et al. 2009b).
Heating and centrifuging: The emulsion was heated at 90 ºC for 1 h and centrifuged but there
was no separation of oil. Lamsal and Johnson (2007) reported similar observations.
Freezing and thawing: Freezing the emulsion at -20 ºC for 24h and thawing resulted in some
separation of the oil in a clear upper phase and an oily solid phase. Similar results were reported
by Morales et al. (2008). This method was not further studied because the energy requirements
would make it unfeasible in industrial applications.
pH adjustments: separation of 83-100% of the oil has been previously reported by lowering the
pH of soy emulsions to 4.5, which is the isoelectric point of soy protein (Morales Chabrand and
Glatz 2009; Jung et al. 2009). The pH of the yellow mustard oil emulsion is 10.4 and the
52
isoelectric point of yellow mustard protein is pH 7 (Balke 2006). The pH of the emulsion was
lowered to 7 but the emulsion became very thick, similar to a paste, and some of the protein
coagulated but still there was no visible separation of oil.
Isopropyl alcohol has been studied as an extraction solvent for other triglyceride oils due to its
high miscibility with oil at high concentrations (Lusas et al. 1990, Zhang et al. 2002) and its
performance is discussed in section 2.3.3.1. Using isopropyl alcohol for breaking the yellow
mustard oil emulsion represented a very promising alternative and the following sections present
and discuss the results obtained using this approach.
5.5 Determination of the Molecular Weight of Yellow Mustard Oil
The use of isopropyl alcohol for breaking the yellow mustard oil emulsion involves mixing the
solvent with the emulsion at different isopropyl alcohol to oil ratios. The first approach was to
use molar ratios. The moles of oil in the sample were calculated by determining the grams of oil
in the sample and dividing them by the molecular weight of yellow mustard oil. The average
molecular weight of yellow mustard oil can be calculated from the fatty acid profile of the oil.
Data from the Canadian Grain Commission for the harvest of 2007, which is the year of
production of the seeds used in the experiments, was used.
In Canada, yellow mustard seeds are classified in 4 categories: No 1, No. 2, No. 3 and No. 4. The
yellow mustard flour used in this project was obtained from ground Canada No. 1 yellow
mustard seeds. The fatty acid profile for Canadian No. 1 yellow mustard seed is presented in
Table 5-3.
As it was mentioned in section 2.1.3.2, oil is stored in the seed as triacylglycerol, which is
composed of a glycerol molecule combined with three fatty acids on each of the OH groups as
shown in Figure 5-5. The red box represents the fatty acids and the blue box the glycerol.
53
Table 5-3. Fatty Acid Composition of Canadian No. 1 Yellow Mustard Seeds (Harvest 2007)
Fatty Acid Composition
(%)
Molecular
Weight (g/mol)
Composition
(g/mol)
Palmitic (C16:0) 2.90 256.42 7.44
Palmitoleic (C16:1) 0.20 254.41 0.51
Stearic (C18:0) 1.00 284.48 2.84
Oleic (C18:1) 26.50 282.50 74.86
Linoleic (C18:2) 10.50 280.50 29.45
α-Linolenic (C18:3) 8.73 278.43 24.31
Arachidic (C20:0) 0.70 312.52 2.19
Eicosenoic (C20:1) 11.03 310.51 34.25
Eicosadienoic (C20:2) 0.30 308.00 0.92
Behenic (C22:0) 0.60 340.58 2.04
Erucic (C22:1) 34.10 338.57 115.45
Docosadienoic (C22:2) 0.30 336.55 1.01
Lignoceric (C24:0) 0.30 368.63 1.11
Nervonic (C24:1) 2.30 366.62 8.43
TOTAL 304.82
Data from Canadian Grain Commission report on Quality of Western Canadian Mustard 2007
54
Figure 5-5. Triglyceride molecule
The general formula of a fatty acid is: The total molecular mass of the fatty
acid radicals in yellow mustard oil is 304.82 g/mol. Considering the structure of the triglyceride
molecule, in order to determine the molecular weight of the oil, the molecular mass of the fatty
acid radicals has to be multiplied by 3 and added to the molecular mass of glycerol minus the
mass of 3 H2O. Therefore the molecular mass of yellow mustard oil was calculated to be 952.45
g/mol.
5.6 Oil Extraction from the Emulsion Using Isopropyl Alcohol as a Solvent
5.6.1 Single Stage Extraction
As it was described in section 4.4.3.1, the emulsion was mixed with isopropyl alcohol at
different IPA to oil ratios in order to find the optimal conditions for oil extraction. Molar ratios
were used for the single stage extraction, however the results are presented for both molar and
weight ratios. The results are discussed in terms of weight ratios. Oil content was determined for
each emulsion before calculating the corresponding amount of IPA required in order to ensure
exact ratios, since the oil content of the emulsions had small variations. 15-25g of emulsion were
used in all experiments. After centrifugation, a solid and a liquid phase were obtained as shown
in Figure 5-6.
55
Figure 5-6. Oil extraction from emulsion using isopropyl alcohol
The solid phase was composed of protein, water, residual oil and IPA, while the liquid phase was
composed of IPA, extracted oil and water. Oil extraction was calculated either based on the
determination of residual oil content in the solids or by measuring the oil content in the miscella.
The first approach was to determine residual oil in solids and it was used in calculating most of
the extraction yields. However, for the last three ratios, determination of oil in miscella was
implemented because it involved less time. In order to verify and compare results obtained by
using the two methods, oil extraction yields were determined using oil content in both solids and
miscella for 3:1 and 25:1 weight ratios. For 3:1 the results for oil extraction were 59.96% and
58.82% for oil in solids and in miscella, while for 25:1 the results were 90.57% and 90.39%,
respectively. These results indicate that the two techniques can be used interchangeably. For
ratios higher than 3:1, the use of either of these methods guaranteed accurate results, however,
for ratios below 3:1 determination of residual oil in solids was the only method generating
reproducible results.
56
Figure 5-7. Single stage oil extraction from emulsion at different IPA:Oil weight ratios
Figure 5-7 presents the results for single stage oil extraction from yellow mustard oil emulsion
using IPA. For 1:1 weight ratio there was very little separation of oil from the emulsion due to
the fact that there was not enough solvent to break the emulsion. The expected trend would be
that as IPA volume increases, extraction should also increase as a result of more solvent being
available to dissolve the oil. Nevertheless, results show that oil extraction exhibits values of 59-
62% between 1.5:1 - 3:1 weight ratio, it then decreases to nearly 20% at 6:1 weight ratio before
increasing continuously until most of the oil is extracted. 31:1 is the ratio, at which 94% of the
oil can be recovered in the IPA phase. Higher ratios were not examined because the potential
increase in oil recovery would not be significant enough to justify the use of larger quantities of
IPA.
There are three important factors affecting oil extraction: the solubility of the oil in IPA, the
degree of percolation and the degree of final drainage. As it was shown in figure 2-4, the
57
solubility of oil in IPA varies with temperature and water content, being oil completely miscible
at very high concentrations, even at room temperature. The high affinity of IPA towards water
represents a major disadvantage because at low weight ratios the water content is higher and the
solubility of oil in IPA is limited. For weight ratios below 6:1, oil drops were observed in the
miscella suggesting low solubility of oil in IPA, at greater ratios the miscella formed a
homogeneous phase indicating full miscibility of oil and IPA.
The permeability of the solid matrix also affects extraction. When more solvent percolates
through the solid matrix, contact area increases and extraction should also increase, however, it
is more difficult to drain the solids and this retention of miscella represents an increase in
residual oil in the marc. The residual oil would represent not only the undissolved oil but also the
oil present in the retained miscella.
The degree of final drainage was measured as solvent holdup in the marc after extraction and it is
shown in Figure 5-8 for different IPA:Oil weight ratios. Solvent holdup represents the amount of
IPA and water being retained in the solid matrix after extraction. When high solvent holdup
values are observed, drainage is considered poor due to high retention of the miscella.
Figure 5-8. Solvent holdup for different IPA:Oil weight ratios
58
Solvent holdup values in the range of 26-29% were found for ratios of 1.25:1 – 2:1. For 3:1, 5:1
and 6:1 the values were 31.5%, 32.2% and 34.4%, respectively. Above 6:1, it increased
significantly until a maximum holdup of 94.5% at 31:1 weight ratio. When more oil was
extracted, the retention of miscella in the solid matrix was higher.
In order to fully understand the behaviour of the extraction process, it was necessary to
determine the composition of the miscella for each weight ratio. Oil, water and IPA content were
calculated and the results are presented in Figure 5-9. The estimation of water content was based
on the assumption that the total amount of water in the emulsion would be transferred to the
miscella due to the high affinity of IPA towards water.
Figure 5-9. Composition of miscella (w/w) for different IPA:Oil weight ratios
Concentrations of IPA higher than 91% and 93% were previously reported for infinite miscibility
of cottonseed and soy oil, respectively (Lusas et al. 1996). For weight ratios below 6:1, the
59
concentration of water is higher than 10% and this explains the presence of oil drops in the
miscella, since there is limited solubility of oil in IPA. It is at weight ratios above 6:1 when no
more oil drops are observed in the miscella and this corresponds to concentrations of water
below 10%, indicating that yellow mustard oil is completely miscible in IPA at concentrations
above 90%
For low weight ratios (1.5:1 – 3:1), water content is in the range of 15-25% and low extraction
yields would be expected, however, yields around 60% are observed. Even though the solubility
of oil is limited by high water content, the degree of percolation is low and there is good
drainage, with 26-29% solvent holdup, making the separation of miscella, oil and solids easier
and facilitating oil extraction. The amount of IPA is enough to disrupt the emulsion and separate
the oil, even when the oil is not being completely dissolved in the solvent.
As the amount of IPA increases, solubility improves and more solvent permeates through the
solids. Extraction yields are lower because oil is being retained in the solids due to poor
drainage, as it is shown in Figure 5-8, with solvent holdup values increasing with higher volumes
of IPA.
Oil drops were observed for ratios below or equal to 6:1. The decrease in oil extraction between
3:1 and 6:1 weight ratios can be attributed to the presence of oil drops and to higher holdup
values. Mustard oil is denser than IPA and tends to remain with the solids, reducing oil recovery.
When oil agglomerates, separation is more difficult.
Oil extraction starts increasing for ratios above 6:1 when no more oil drops are observed and
completely miscibility has been reached at concentrations of IPA higher than 90%. Oil content in
the miscella increases between 6:1 and 19:1 weight ratios with a maximum oil content of 3.8% at
19:1. The maximum in oil content indicates the point at which sufficient IPA has been used to
completely dissolve the oil in the emulsion. The low extraction yield observed (75%) is
attributed to miscella being retained in the solid matrix as a result of poor drainage. For ratios
above 19:1, oil concentration decreases with higher volumes of IPA, and miscella retention does
60
not represent a big contribution to residual oil content. Oil extraction yields up to 94% can be
achieved.
As it was shown in Figure 5-8, solvent holdup increased with higher IPA:Oil weight ratios and
higher extraction yields. The relationship between solvent holdup and residual oil content was
studied and it is presented in Figure 5-10. There is an inverse relationship between solvent
holdup and residual oil content which corresponds to observations made by Zhang et al. (2002)
with isopropyl alcohol extraction of cottonseed. When more oil is extracted, there is more space
available to entrain solvent in the solids and solvent holdup increases. For low extraction yields,
more oil is present in the solids, there is less space available and less solvent is retained.
Figure 5-10. Relationship between solvent holdup and residual oil content
61
5.6.2 Multiple Stage Extraction
In the single stage process described above, 94% of the oil could be extracted at a 31:1 weight
ratio; however, extraction yields around 60% were observed at low weight ratios (1.5:1 – 3:1).
Figure 5-11 presents oil extraction yields at low weight ratios for a single extraction step. There
was a maximum at 2:1 weight ratio with 62.10% oil extraction, while for 1.5:1, 1.75:1 and 3:1 oil
yields were 59.52%, 60.48% and 59.96%, respectively. Taking into account that high oil
recovery yields are observed, multiple stage extraction was considered at these low weight ratios
in order to find the optimal conditions for oil extraction. The reasoning behind this consideration
was that if it could be possible to extract as much oil as with high weight ratios but using only a
fraction of the IPA, the process could be more economically feasible.
Figure 5-11. Oil extraction from emulsion at low weight ratios
The emulsion was mixed with IPA at the desired ratio and centrifuged. The solids separated after
centrifugation were then mixed with the same volume of IPA used in the first stage and
centrifuged once more. The process was repeated for every stage of the extraction process.
62
5.6.2.1 Three-Stage Oil Extraction using IPA
Results of three-stage oil extraction at different weight ratios are presented in Figure 5-12. 94%
of the oil in the emulsion was extracted at 3:1 weight ratio. 2:1 is less effective with 87%
extraction yield, while 1.75:1 and 1.5:1 only extract 80% and 78% of the oil, respectively.
Figure 5-12. Three-stage oil extraction using IPA
The observed trend indicates that at these low ratios, oil extraction is directly affected by the
amount of isopropyl alcohol used. When the proportion of IPA increases, oil extraction responds
in the same way. For all ratios, the highest extraction yield is observed in the first stage with
values in the range of 57-62%. Drops of oil can be seen in the miscella and the colour is dark
yellow due to the high oil content. Solids are very compact and separation is easy indicating low
solvent holdup as observed in the single stage extraction. For the second stage, extraction is
63
around 17% for 3:1 and 10% or less for lower ratios. The miscella is lighter in colour and no oil
drops are observed, however, after centrifugation the solids tend to mix with the miscella and
separation is a little more difficult because solids are easily disturbed. For the third stage, oil
extraction is close to 20% for 3:1 weight ratio, 15% for 2:1, and 11% for 1.75:1 and 1.5:1.
Special care must be taken when separating solids and miscella. When more oil is removed,
solvent holdup increases complicating separation after extraction.
Oil extraction was not complete with the three-stage process, however, if the same trend
continues, complete oil recovery could be obtained for 3:1 and 2:1 weight ratio with one
additional stage. Lower ratios could require more than four stages to fully extract the oil.
5.6.2.2 Four-Stage Oil Extraction using IPA
To confirm and better quantify the trends observed with three stages, four-stage extraction was
evaluated for 1.5:1, 2:1 and 3:1 solvent to oil weight ratios. The results are shown in Figure 5-13.
Figure 5-13. Four-stage oil extraction using IPA
64
For the four-stage process, 99.40% of the oil was extracted using a 3:1 IPA:Oil weight ratio,
96.29 % using a 2:1 ratio, and 90.77% with 1.5:1. In order to confirm the results, residual oil
content was determined in the solids after the four stages for 3:1 and 2:1. Residual oil content in
the solids for 3:1 ratio was 1.01%, representing less than 0.93% of the original oil. For the 2:1
ratio, residual oil content in the solids was 4.2%, representing 4% of the original oil. Both results
satisfy the mass balance and validate the results obtained by determining oil content in miscellas.
For 1.5:1 weight ratio, more than four stages will be required to fully extract the oil.
The yellow mustard oil emulsion is composed of oil, water and protein. The oil is extracted in
the IPA phase and it is assumed that the protein would be in the solid phase. However, protein
analyses were carried out in the extracted solids for 2:1 and 3:1 weight ratios. Solvent holdup
was also determined for the same ratios and the results are presented in Table 5-4.
Table 5-4. Composition of the solids after four-stage extraction
IPA:Oil Weight Ratio 2:1 3:1
Oil Content (%) 4.2 ± 0.07 1.01 ± 0.09
Protein Content (%) 3.85 ± 0.04 3.59 ± 0.08
Solvent Holdup Content (%) 89.27 ± 1.94 93.22 ± 0.40
TOTAL 97.32 97.82
The mass balance could account for 98% of the solid phase, the missing is 2-3% could be
attributed to experimental error or more likely, loss of sample during transfers.
65
Solvent holdup values are higher for 3:1 weight ratio than for 2:1, which could be expected,
since more oil is extracted at 3:1 and hence there is more space available for the solvent in the
solid matrix.
Protein content is very similar in both solids. A protein mass balance was performed to
determine the amount of the original protein in the solids after extraction and to estimate how
much of the protein could be in the miscella. The protein in the starting material, yellow mustard
flour, was set to 100%.
Table 5-5. Protein balance for the four-stage extraction process
IPA:Oil Weight Ratio Protein in solids from
original flour (%)
Protein in miscella from
original flour (%)
2:1 0.43 1.17
3:1 0.43 1.17
From the aqueous process mass balance discussed in section 5.2, 92% of the protein was
recovered in the extract, 4.7% in the residue and 1.6% in the emulsion. After extracting the oil in
the emulsion with IPA, the same protein balance was obtained for both weight ratios, with 1.17%
of the original protein present in the extracted solids after the four-stage extraction process and
only 0.43% in the miscella. Considering that 700g of flour were used in the aqueous extraction
experiments, 2.49g of protein can be found in the extracted solids and 0.92 g in the miscella.
The amount of protein in the miscella could be attributed to the presence of solid particles after
the separation. As it was previously mentioned, after the second stage, it was very difficult to
separate the miscella from the solids and some solid particles were transferred to the miscella. It
is believed that if separation is done perfectly, there should not be any protein in the miscella,
only oil, water and IPA.
66
Table 5-6 summarizes the results obtained for the four-stage oil extraction process from the
emulsion using IPA as a solvent. 99.4% of the oil in the emulsion is extracted using a four-stage
extraction process with 3:1 weight ratio and 96.3% with 2:1 weight ratio; however, the difference
between oil extraction from the original oil in the flour is only 1%.
Table 5-6. Summary of the results for the four-stage oil extraction
IPA:Oil Weight Ratio 2:1 3:1
Total IPA:Oil weight ratio 8 12
Oil extraction from emulsion
(%)
96.3 99.4
Oil extraction from original
flour (%)
35.7 36.8
The four-stage process at 2:1 weight ratio was chosen as the optimal process for oil extraction,
since the requirements of isopropyl alcohol are lower than with 3:1 weight ratio but the reduction
in oil recovery is not significant, with 35.7% oil extraction from the original oil in the flour. The
small improvement in oil recovery does not justify the use of larger quantities of IPA.
Figure 5-14 illustrates the overall oil extraction for 2:1 weight ratio, starting with aqueous
extraction of oil from yellow mustard flour in form of emulsion, and ending with multistage
extraction of the oil in the emulsion. Results for protein content in the emulsion, in the final
solids and in the miscella are also presented.
67
Oil Balance
Protein Balance
700 g Flour
2800 g Water
Emulsion
37.0 % Extraction
68.9g Oil 186.2g Oil
IPA 2:1 Weight Ratio
4-stage Extraction
Emulsion 66.4g Oil
35.7 % from Original Oil in the Flour
212.8g Protein in
Flour
1.6% in Emulsion (3.4g Protein)
1.2% in Final Solids (2.5g Protein)
0.4% in Miscella (0.9g Protein)
After 4-stage extraction
Figure 5-14. Oil and protein extraction from the emulsion for 2:1 IPA: Oil weight ratio and four-stage extraction
68
5.6.3 Characterization of the Miscellas from the Four-Stage Oil Extraction
The optimal conditions for oil recovery from the emulsion were chosen as 2:1 weight ratio and
four-stage extraction of the emulsion with IPA. The results presented and discussed below refer
to these conditions.
Figure 5-15. Miscellas from the four-stage oil extraction using IPA at 2:1 IPA:Oil weight ratio
The miscellas for each stage of the process are shown in Figure 5-15 and the oil extraction yields
for each stage are presented in Table 5-7. The colour of the miscella for the first stage is darker
because of the higher oil concentration compared to the other stages, with 61% of the oil
extracted in that stage. As oil extraction increases, separation of solids and miscella is more
difficult and even though it was handled with extreme care, some solid particles were transferred
to the miscella, especially in the last stage, which explains the cloudiness observed in that
miscella.
Table 5-7. Oil yields for each stage in the multistage extraction
Stage 1 2 3 4
Oil Extraction (%) 61.23 ± 1.24 11.50 ± 0.50 15.17 ± 1.26 8.40 ± 0.43
69
The composition of the miscellas for the four stages was determined and it is presented in Table
5-8. Oil content was determined as oil content in miscella following the procedure described in
Appendix A. Water content was determined by Karl Fischer titration.
The water content in the first stage is higher than 20%, which explains the presence of oil drops
due to the limited solubility of oil at concentrations of IPA below 90%. Oil drops are not seen in
later stages because water content is well below 5%, indicating full miscibility of oil and IPA.
Table 5-8. Composition of the miscellas for the four-stage extraction
Stage Water Content
(%)
Oil Content
(%)
IPA Content
(%)
1 20.14* 19.78 ± 0.14 60.08 ± 0.14
2 3.29 ± 0.03 6.98 ± 0.21 89.73 ± 0.18
3 0.92 ± 0.01 8.23 ± 0.64 90.85 ± 0.63
4 0.43 ± 0.01 4.43 ± 0.30 95.14 ± 0.30
* Only one measurement was possible
The first stage presented large amounts of oil separated in a denser phase and it was very
difficult to determine its water content. Coulometric Karl Fischer titration was used in all
determinations but this method is more appropriate for low water contents. The value of 20.14%
may not be very accurate due to the presence of oil drops and the high water content. It is
recommended to use volumetric Karl Fischer for high water content; however, the presence of
two separate phases would still complicate any determination of water in the sample.
70
Based on these results, the amount of water extracted from the emulsion was calculated for each
stage as shown in Table 5-9. 78.23% of the water in the emulsion was extracted in the first stage,
7.02% in the second, 2.20% in the third and 1.06% in the fourth stage, accounting for 88.51% of
the water in the emulsion. The problems associated with determining water content in the first
stage miscella might be responsible for the remaining 11%, since most of the water is expected
to be transferred to the miscella in the first stage due to the high affinity of IPA towards water.
Considering that there is more confidence on the results from the second, third and fourth stage
miscellas, the amount of water extracted in the first stage must be close to 90%.
Table 5-9. Water content in miscellas for different stages
Stage Water in miscella
(%)
Water extracted
from emulsion (%)
1 20.14 78.23
2 3.29 7.02
3 0.92 2.20
4 0.43 1.06
5.6.4 Characterization of the Final Miscella
The composition of the final miscella is presented in Table 5-10. The results are theoretical,
based on an oil extraction yield of 96.3%, and based on the assumption that all the water and IPA
are in the miscella after separation.
71
Table 5-10. Composition of the final miscella
Water Content (%) Oil Content (%) IPA Content (%)
7.47 ± 0.68 10.02 ± 0.28 82.51 ± 0.40
Figure 5-16. Final Miscella from 2:1 IPA:Oil weight ratio, four-stage extraction
The final miscella composition indicates that the water content is 7.47%. Coulometric Karl
Fischer determination resulted in 8.91%, which is 1.4% higher than the theoretical result. Water
content higher than 8% would represent a contradiction in the mass balance, since it would
indicate there is more water in the final miscella than the amount of original water in the
emulsion. IPA is very volatile and the samples were sent for analysis to a different facility. It is
believed that from the moment when the samples were taken until the moment water content was
determined, some of the IPA might have evaporated and this loss would be reflected in a higher
72
concentration of water in the sample, nevertheless it represents a good estimation of the actual
value. It can be concluded that the theoretical assumptions used to determine the composition of
the final miscella are correct.
5.7 Preliminary Evaluation of the Final Miscella as Feedstock for Biodiesel
Production
The complete process for oil extraction from yellow mustard seeds will consist of aqueous
extraction of the yellow mustard flour followed by four-stage extraction of the emulsion using
2:1 IPA:Oil weight ratio. The final miscella will be the feedstock for the transesterification
reaction. Important variables in the production of biodiesel are alcohol:oil molar ratio,
temperature, catalyst, reaction time, water content and free fatty acid content.
The free fatty acid content in the final miscella was not determined, however, it is an important
factor affecting the transesterification reaction and it needs to be evaluated in future
investigations.
The molar ratio of alcohol to oil is a very important factor. Excess alcohol must be ensured to
drive the reaction to completion. In the final miscella, the IPA content is 82.51% and the oil
content 10.02%. This represents an IPA:Oil weight ratio of 8.2:1. The theoretical value is 8:1
assuming all the oil is extracted, however oil recovery yield was 96.3% and the weight ratio
resulted in a higher value than the theoretical estimation because less oil was recovered. The
IPA:Oil molar ratio is 130.5:1, representing more than enough alcohol to shift the equilibrium
towards production of isopropyl esters. Wang et al. (2005) reported conversion of soybean oil to
isopropyl esters using a 20:1 isopropanol:oil molar ratio and 1% sodium metal catalyst at 80 ºC.
An IPA:Oil molar ratio around 20-25:1 is expected to be optimal for the transesterification of
mustard oil using IPA, however, it must be studied in further detail.
73
Large quantities of excess alcohol can have a negative effect in biodiesel production. Wu et al.
(2006) reported that addition of large quantities of methanol in the transesterification reaction
affected separation of the esters and glycerol. Nevertheless, the goal is to use a process where the
IPA will be separated by distillation before phase separation of glycerol and esters in a
continuous process as patented by BIOX Corporation (US Patent 6712867).
It is very important to maintain a low moisture level, below 1.5% for alkaline catalysts (Sharma
et al. 2008) because water can consume the catalyst and reduce its efficiency. Moreover, water
causes the triglycerides to hydrolyze and produce soaps instead of undergoing transesterification
to produce biodiesel. The water content in the final miscella is 7.47%. Water adsorption
represents a good alternative for the removal of water from the miscella. Water adsorption from
isopropyl alcohol using 4A molecular sieve pellets (1.8mm diameter) has been previously
investigated (Jain and Gupta, 1994; 1998) and the adsorption capacity of 4A molecular sieves
was determined to be 0.25g of water per gram of dry adsorbent, except at water concentrations
below 1%.
In order to remove the water from the final miscella, 4A molecular sieve beads (8-12 mesh) were
used as an adsorbent for the preliminary evaluation of their adsorption capacity. The results are
presented in Table 5-11.
Table 5-11. Water adsorption using 4A molecular sieves
Experiment Theoretical Water
Adsorption
Water Content
Before (%)
Water Content
After (%)
Water Adsorption
(%)
Water Adsorption
(g)
1 0.25g water/ g molecular
sieve
8.91 3.82 57.14 2.6
2 0.12g water/ g molecular
sieve
8.90 1.70 80.90 3.6
Initial mass of water in samples: 4.45g
74
In the first experiment, the theoretical adsorption of 0.25gwater/g of molecular sieves was
presumed; therefore the mass of 4A molecular sieves added was 4 times the mass of water in the
sample. Adsorption was performed during a period of 24h. Only 57.14% of the water was
adsorbed.
For the second experiment, twice the amount of molecular sieves required for the theoretical
capacity of 0.25g water was used. The mass of molecular sieves was 8 times the mass of water,
representing a theoretical adsorption capacity of 0.12g water/ g molecular sieves. Water
adsorption was 80.90%.
Using the theoretical adsorption capacity did not guarantee complete water removal, with 80% of
the water being removed when twice the theoretical amount of molecular sieves was used. The
theoretical capacity was determined for cylindrical molecular sieves and the adsorbents used in
these experiments were beads of larger size, which could represent a source of overestimation in
the adsorption capacity. However, even if the capacity of the molecular sieves is overestimated,
there is no doubt that large quantities of molecular sieves will need to be used in order to bring
the water content of the miscella below 1.5% or less, probably more than 8 times the weight of
water. It is necessary to study the adsorption phenomenon in detail in order to find the best
possible adsorbent for water removal in the miscella.
The solution of isopropyl alcohol and yellow mustard oil is definitely a potential feedstock for
biodiesel production but the preparation of this mixture to undergo transesterification needs to be
investigated in more detail. Free fatty acid content needs to be determined and water needs to be
adsorbed to acceptable levels. The biodiesel produced from this mixture will exhibit the great
cold flow properties of isopropyl esters combined with the excellent lubricity of mustard oil due
to the high erucic acid content.
75
6. CONCLUSIONS
Aqueous extraction represents a good approach to simultaneous recovery of oil and
protein from yellow mustard flour. In the aqueous extraction process, 37.0% of the oil
was recovered as an oil-in-water emulsion, composed of 57.0% oil, 40.5% water and
2.6% protein. Higher oil extraction yields must be achieved to make it competitive with
hexane extraction.
Isopropyl alcohol can be used to effectively extract the oil in the yellow mustard oil
emulsion. For single stage extraction, 94% of the oil was extracted using a 31:1 IPA:Oil
weight ratio.
Multiple stage extraction of oil from the emulsion was proven to be more efficient for oil
recovery than single stage extraction, with lower volumes of IPA required to achieve high
extraction yields. The optimal conditions for multiple stage extraction were four stages
and 2:1 IPA:Oil weight ratio, with 96.3% oil recovery yield from the emulsion.
A process based on aqueous extraction of yellow mustard flour followed by multiple
stage isopropyl alcohol extraction of the emulsion and subsequent water removal will
produce a solution suitable for biodiesel production. The potential feedstock is expected
to produce fuel with great lubricity and excellent cold flow properties, suitable for cold
climates. Further processing of the solution is required to meet the optimal conditions for
transesterification.
76
7. RECOMMENDATIONS
Higher oil recovery yields must be achieved in the aqueous extraction process. The use of
completely dehulled yellow mustard flour is recommended to eliminate the presence of
mucilage and facilitate oil separation. The use of enzymes is also recommended since oil
extraction yields up to 90% have been reported. Cellulases, hemicellulases, and
pectinases are good alternatives but they need to be evaluated in the aqueous extraction of
yellow mustard seeds.
The separation of solids and miscella should be improved for the last stages of multiple
stage extraction using IPA. The use of filters represents a good approach to solving this
problem.
Karl Fischer determinations should be carried out in a way to minimize losses of IPA. For
miscellas with water content, higher than 10%, volumetric Karl Fisher titration must be
used.
A complete evaluation of the variables affecting the transesterification reaction is
required. Optimal conditions for the production of biodiesel using the mixture of IPA and
yellow mustard oil must be determined.
The free fatty acid content of the final miscella must be calculated to better evaluate its
potential use in biodiesel production.
The optimal molar ratio of IPA to oil for the transesterification reaction must be studied
and a process to distil and recycle some of the IPA must be evaluated.
The use of different adsorbents to reduce water content in the miscella should be
investigated in further detail. A preliminary evaluation on the use of molecular sieves 4A
was performed, however, deeper understanding of the process is necessary.
77
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Zhou, W., S. K. Konar, and D. G. B. Boocock (2003). Ethyl Esters from the Single-Phase Base-Catalyzed Ethanolysis of Vegetable Oils. J. Am. Oil. Chem. Soc. 80: 367-371.
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APPENDICES
88
APPENDIX A
Analytical and Experimental Methods
89
A1. Determination of oil content using Mojonnier Method All measurements were performed in triplicate. For all analysis, corks were soaked in water for at least 1 h to improve seal.
For solid samples: AOAC Method 922.06. Fat in Flour (acid hydrolysis)
1. Place 2g of sample (weigh to nearest 0.1mg) in 50mL beaker. 2. Add 2mL ethanol and stir to moisten particles to prevent lumping on addition of
acid. 3. Add 10mL HCl (25+11), mix well, set beaker in water bath held at 70-80ºC and
stir in frequent intervals during 30-40min. 4. Add 10mL ethanol and cool. 5. Weigh 4 150mL beakers to nearest 0.1mg. 6. Transfer mixture to Mojonnier flask. Rinse beaker into extraction flask with
25mL diethyl ether added in 3 portions, stopper flask (cork stopper) and shake vigorously for 1min.
7. Add 25mL petroleum ether and shake for 1min. 8. Let stand until upper liquid is practically clear. Draw off as much possible of
ether-fat solution through filter consisting of cotton ball packed just firmly enough in funnel stem to let ether pass freely into weighted 150mL containing boiling chips.
9. Re-extract liquid remaining in flask twice, each time with only 15mL of each ether. Shake well on addition of each ether and draw off ether solution into same beaker. Wash tip of funnel and end of funnel with few mL of mixture of ethers in equal volumes.
10. Evaporate ether on steam bath, then dry fat in over al 100ºC for 90min. 11. Remove beaker from over, let stand and weight. 12. Run a blank using only reagents for each set of experiments. 13. Calculation:
Oil (%) = [(weight beaker +fat) – (weight beaker)] – weight blank * 100 weight sample
For emulsions: AOAC Method 995.19. Fat in cream
1. Place test sample in water bath at 38 ± 1ºC. Mix thoroughly and weight aliquot immediately. Do not let samples remain in water bath more than 15min after reaching 38ºC.
2. Weight empty flask. 3. Pipet into flask enough cream to yield 0.3-0.6g of extracted fat (0.8g 60%
emulsion, 1g 40% emulsion, 2g 20% emulsion) and weight to the nearest 0.1mg.
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4. Dilute test portion with 10mL distilled water at room temperature. 5. Weigh 4 150mL beakers to the nearest 0.1mg. 6. To test portions in flask add 1.5mL NH4OH and mix thoroughly. It neutralizes
any acid present in dissolves casein. 7. Add 3 drops of phenolphthalein indicator to sharpen visual appearance of
interface. 8. Add 10mL ethanol, stopper with water-soaked cork and shake vigorously 15s. 9. For first extraction add 25mL diethyl ether, stopper with cork and shake for 1min.
Hold body of flask horizontally with lower bulb and stopper up. 10. Loosen cork gently to release built-up pressure. Add 25mL petroleum ether, shake
for 1min. 11. Let stand until ether phase and the pink aqueous phase are separated and transfer
ether-fat solution to weighted beaker. 12. For second extraction add 5mL ethanol, stopper with same cork used for first
extraction and shake 15s. 13. Add 15mL diethyl ether, replace cork and shake 1min. 14. Add 15mL petroleum ether, stopper with same cork and shake 1min. 15. Let phases separate. If interface is below neck of flask, add water to bring level
half way up neck. Add water slowly to cause minimum disturbance of separation. Decant ether solution into same beaker used for first extraction.
16. For third extraction omit addition of ethanol and repeat procedure for second extraction.
17. Evaporate solvents in the fumehood on hot plate. Dry extracted fat at 100ºC for 90min.
18. Remove beaker from oven and place them in a dessicator. Cool down and weigh. 19. Run 1 blank with reagents and substitute emulsion with 10mL distilled water.
Reagent blank should be < 0.0020g residue. 20. Calculation:
Oil (%) = [(weight beaker +fat) – (weight beaker)] – weight blank * 10 (weight flask +sample) – (weight flask)
For skim fraction: AOAC Method 989.05. Fat in Milk
1. Place test sample in water bath at 38 ± 1ºC. Mix thoroughly and weight aliquot immediately.
2. Weight empty flask. 3. Pipet into flask ca 10g of milk and weight to the nearest 0.1mg. 4. Weigh 4 150mL beakers to the nearest 0.1mg. 5. To test portions in flask add 1.5mL NH4OH and mix thoroughly. It neutralizes
any acid present in dissolves casein.
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6. Add 3 drops of phenolphthalein indicator to sharpen visual appearance of interface.
7. Add 10mL ethanol, stopper with water-soaked cork and shake vigorously 15s. 8. For first extraction add 25mL diethyl ether, stopper with cork and shake for 1min.
Hold body of flask horizontally with lower bulb and stopper up. 9. Loosen cork gently to release built-up pressure. Add 25mL petroleum ether, shake
for 1min. 10. Let stand until ether phase and the pink aqueous phase are separated and transfer
ether-fat solution to weighted beaker. 11. For second extraction add 5mL ethanol, stopper with same cork used for first
extraction and shake 15s. 12. Add 15mL diethyl ether, replace cork and shake 1min. 13. Add 15mL petroleum ether, stopper with same cork and shake 1min. 14. Let phases separate. If interface is below neck of flask, add water to bring level
half way up neck. Add water slowly to cause minimum disturbance of separation. Decant ether solution into same beaker used for first extraction.
15. For third extraction omit addition of ethanol and repeat procedure for second extraction.
16. Evaporate solvents in the fumehood on hot plate. Dry extracted fat at 100ºC for 90min.
17. Remove beaker from oven and place them in a desiccator. Cool down and weigh. 18. Run 1 blank with reagents and substitute milk with 10mL distilled water. Reagent
blank should be < 0.0020g residue. 19. Calculation:
Oil (%) = [(weight beaker +fat) – (weight beaker)] – weight blank * 100 (weight flask +sample) – (weight flask)
Sample calculation. Fat in emulsion
Weight flask = 75.7784 Weight flask + sample = 76.7430 Weight beaker = 68.9179 Weigh beaker + fat = 69.4843 Weight blank = 0.0007
Oil (%) = [(69.4843) – (68.9179)] – 0.0007 * 100 = 58.65 (76.7430) – (75.7784)
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Reagents for Mojonnier Method
HCl (25 + 11): Mix 100mL of HCl (36.5-38.0%) and 44mL of distilled water and shake
well before using.
Phenolphthalein 0.5% (w/v) in ethanol: Weigh 0.5g of phenolphthalein power and
transfer to volumetric flask. Fill with 100% ethanol until the mark. Shake well.
A.2 Oil Content in Miscella All measurements performed in triplicate for single stage extraction, taking samples from well mixed miscella. For multiple stage extraction, all miscella was used in calculation.
1. Weigh 50mL beaker. 2. Make sure miscella is well mixed and take sample ca 20-25mL. Weigh to nearest
0.1mg. 3. Evaporate solvents in fume hood on hot plate. Dry fat in oven at 100ºC for 1h. 4. Cool samples and record weight. 5. Oil (%) = [(weight beaker + fat) – weight beaker] * 100
(weight sample + beaker) – weight beaker
Sample calculation:
Weight beaker + fat = 34.8734
Weight beaker = 28.1402
Weight beaker + sample = 52.3143
Oil (%) = [(34.8734) – 28.1402] * 100 = 27.85 (52.3141 – 28.1402)
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A.3 Determination of protein content using Kjeldahl Method (AOCS Method Ba4d-90)
Kjeldahl Digestion:
1. Preheat the Digestion Unit to setting 4.
2. Place sample in the Buchi tubes. All determinations should be done in triplicate and one tube for the blank. The weight of the sample should be enough to guarantee a reading of 10mL in the titration. If the sample is 100% protein, around 0.1g of sample should be used.
- For solids samples: For mustard flour (30% protein) weight around 0.3g. For emulsion around 2g. Too much sample for emulsion can make the digestion very long and with too much foaming. Blank: weighing paper.
- For liquid samples: For extract (4% protein) weigh 2-2.5g. Blank: distilled water.
3. Add 4 Kjeldahl tablets to each tube.
4. Add 25mL concentrated sulphuric acid to each tube.
5. Place the glass manifold on the tubes and make sure there is glass fiber on the side of the blank. Use the clamp wires to tighten the tubes and the manifold.
6. Connect the suction tube to the glass manifold and turn on the tap water.
7. Place the tubes on the digestion unit and run on setting 4 for 20min. Then move to setting 6 for 10min and setting 10 for 35min. The walls of the tubes must be clean and the solution should be colourless for the last 10 min of digestion. This is standard time but sometimes samples can take longer. If there is foam formation please remove the samples from the digestion unit and cool down, then place the tubes back on the digestion unit.
8. Remove the tubes form the digester and place in a rack with the water running for 20min until the solution is cool. The tubes can be then removed and the rack placed in the fumehood. When the tubes cool, a white precipitate will appear. Distillation can be performed later on the day or on a different day. If solution is too viscous because digestion took too long, distillation should be done on the same day.
Kjeldahl Distillation:
1. Remove the glass manifold and be careful not to spill any condensed acid. Rinse with distilled water.
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2. Swirl the sample and make sure the white precipitate mixes with the clear liquid. Add 50mL distilled water and mix very well. The solution should be completely clear at this point.
3. Add 25mL sodium thiosulfate (8%), swirl the samples and cover. The solution will turn dark gray. Swirl the samples every 5 min for the next 15 min to increase the extent of the reaction in the tubes.
4. Check the distillation unit and make sure the distilled water compartment is ¾ full and the 32% NaOH solution 1/3 full.
5. Turn on the tap water and turn on the distillation unit and wait until it beeps, indicating it is ready for use.
6. Place 60mL boric acid (4%) into Erlenmeyer flasks and add 3 drops of indicator.
7. After hearing the beep, open the glass compartment and remove tube. Replace water in the tube with fresh distilled water and fill it halfway. Replace water in the Erlenmeyer with distilled water as well.
8. Set distillation time to 2 min by pressing the time button and then press start. The machine will beep when it is finished. Wait until there is no more noise and remove the tube using the tongs.
9. Replace water tube with blank tube and the Erlenmeyer flask with one with boric acid solution. Ensure the tip of the tube is fully submerged in the solution. Place paper towels underneath if necessary.
10. Add 90mL NaOH solution by pressing the reagent button until the 180mL mark. The button should be pressed twice to ensure it is supplied manually, otherwise the machine will do it automatically. Enough NaOH should be added until there are no more bubbles in the solution indicating it has been neutralized.
11. Set time to 5min and press start to run distillation.
12. The machine will beep when it is finished. Wait until there are no more noises and use the tongs to remove the tube and put it back on the rack.
13. Remove the Erlenmeyer flask and rinse the tip with distilled water. The pink solution should be light pink or clear.
14. Repeat steps 9 to 13 by placing a new sample tube and a new Erlenmeyer flask with boric acid solution. The solution in the Erlenmeyer will turn green indicating the presence nitrogen in the sample.
15. Once all the samples have been distilled, place a tube with fresh distilled water (half way) and an Erlenmeyer with fresh distilled water. Run distillation for 5min.
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16. Repeat the cleaning procedure as many times as necessary.
17. When the machine is clean, replace tube and Erlenmeyer with fresh distilled water, turn off the unit and turn off the tap water.
18. Sing the logbook.
Titration:
1. Pump 0.1N sulphuric acid slowly until the burette is set to 0mL.
2. Titrate the sample until the pink endpoint is reached. The sample will transition from green to pink. The endpoint should be the same as the blank.
3. Record the volume of the sulphuric acid used to reach the endpoint
Calculation:
N (%) = [H2SO4 ml used for sample - H2SO4 ml used for blank] * 0.1N * 1.4 Weight of sample Protein (%) = N (%) * 6.25
Sample Calculation:
H2SO4 ml used for sample = 10.70 H2SO4 ml used for blank = 0.40 Weight of sample = 0.2974 N (%) = [10.70 – 0.4] * 0.1N * 1.4 = 4.85 0.2974
Protein (%) = 4.85 * 6.25 = 30.30
Reagent Solutions:
32 % (w/w) NaOH (Preparation for 3.5L)
1. Weigh 3,000g NaOH 50% (w/w) solution into a 4L beaker. 2. Place beaker in the fumehood on a stir plate. 3. Add large spin bar into the NaOH solution. 4. Weigh 1.69g distilled water in a separate beaker. 5. Place ice in a bucket around the NaOH beaker. The reaction between NaOH and
water is very exothermic and the ice is used to manage the generated heat. 6. Add the water slowly to the NaOH. 7. Stir the solution for at least 2 hours until it is no longer cloudy.
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4 % (w/v) Boric Acid
1. Weigh 80g of boric acid into a 2L beaker. 2. Fill the beaker up to 1,800mL. 3. Add a large spin bar and stir solution until clear (over 2.5h). Cover the beaker
with aluminum foil to prevent contamination of the solution. 4. Transfer solution to a 2L volumetric flask and add distilled water up to the mark,
shake well.
8% (w/v) Sodium Thiosulfate
1. Weigh 160g of sodium thiosulfate into a 2L volumetric flask. 2. Fill the flask with distilled water up to the mark and shake well.
A.4 Determination of Moisture Content (AACC Method 44-15A) Always perform in duplicate or triplicate.
1. Weigh an aluminum tray and record weight.
2. Weight sample in aluminum tray and record weight.
3. Put sample in oven at 105 ºC for at least 20h, preferably 24h.
4. Remove samples from oven, cool in a dessicator and record weight.
5. Calculation:
Moisture (%) = (weight sample + tray)wet – (weight sample + tray)dry * 100 (weight sample + tray)wet – weight tray 6. Sample Calculation: Weight tray = 1.0965 Weight sample + tray wet = 3.2641 Weight sample + tray dry = 3.0869 Moisture (%) = 3.2641– 3.0869 * 100 = 8.17 3.2641– 1.0965
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APPENDIX B
Results
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B1. Yellow Mustard Flour Analyses Protein Content
Date Sample Sample (g) V Titration
(mL) % N %
Protein Mean STDV Blank 0.3
Feb 17, 2009 1 0.0992 3.72 4.8266 30.17
2 0.1005 3.82 4.9035 30.65 30.61 0.43 3 0.0987 3.80 4.9645 31.03 Blank 0.45 1 0.2974 10.70 4.8252 30.16
May 19, 2009 2 0.2981 10.80 4.8608 30.38 30.26 0.11
3 0.2981 10.75 4.8373 30.23
Overall Mean (%) 30.44 Overall STDV (%) 0.34
Oil Content
Date Sample Sample
(g) Fat (g) % Oil Mean STDV Blank -0.0041
Feb 11, 2009 1 1.9960 0.4922 24.87
2 2.0055 0.5365 26.96 26.63 1.62 3 2.0125 0.5607 28.06 Blank 0.0005
May 1, 2009 1 1.9960 0.5407 27.06
2 1.9969 0.5255 26.29 26.50 0.49 3 2.0093 0.5259 26.15
Overall Mean (%) 26.57 Overall STDV (%) 1.07
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Moisture Content
Date 1 2 3 Mean STDV Beaker (g) 1.8075 1.8013 1.8032
7-Feb B + Wet (g) 2.2231 2.2156 2.2345 6.50 0.19 B + Dry (g) 2.1954 2.1895 2.2063 Moisture % 6.67 6.30 6.54 Beaker (g) 1.8022 1.8031 1.8015
15-Mar B + Wet (g) 2.2035 2.1946 2.2027 5.33 0.06 B + Dry (g) 2.1823 2.1735 2.1814 Moisture % 5.28 5.39 5.31 Beaker (g) 1.8006 1.8026 0.0000
6-Apr B + Wet (g) 2.2164 2.2935 0.0000 7.38 0.91 B + Dry (g) 2.1884 2.2541 0.0000 Moisture % 6.73 8.03 0.00 Beaker (g) 1.1260 1.1268 0.0000
19-May B + Wet (g) 2.4311 2.5199 0.0000 6.52 0.05 B + Dry (g) 2.3455 2.4295 0.0000 Moisture % 6.56 6.49 0.00 Beaker (g) 1.8199 1.8012 1.8182
7-Jun B + Wet (g) 4.0118 4.7308 4.2809 4.87 0.09 B + Dry (g) 3.9047 4.5912 4.1590 Moisture % 4.89 4.77 4.95
Overall Mean 5.99 Overall STDV 0.97
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B2. Aqueous Extraction of Oil and Protein from Yellow Mustard Flour W Flour = 700g W Water = 2800g W Oil in flour (26.63%) = 186.41g Oil Distribution. May 07, 2009
Extract Emulsion Residue Total Weight (g) 4158.8 128.73 1318.80
% Oil (as is) 1.03 ± 0.04 56.36 ± 0.62 5.45 ± 0.02 % Oil (freeze-dried) 12.67 ± 0.52 ------ 27.35 ± 0.12
Total Weight (freeze-dried) (g)
262.7 ------- 339.2
Weight Oil (g) 42.98 72.55 71.85 % Oil Recovery 23.06 38.92 38.54
Protein Distribution. Aug 25, 2009 W Protein in flour (30.4%) = 212.80g
Extract Emulsion Residue Total Weight (g) 4033.7 + 1100.8 130.77 1373.10 – 1100.80
% Protein 3.80 ± 0.04 2.58 ± 0.01 3.72 ± 0.04 Weight Protein (g) 195.11 3.37 10.07
% Protein Recovery 91.69 1.59 4.73 Oil and Protein Yield Calculations The residue contains some of the extract, therefore moisture content for the residue was determined and the mass of protein and oil equivalent to that mass of extract in the residue was subtracted from the total protein content in residue. Moisture Residue = 80.17% ± 0.53 Protein Balance: Protein in Flour = 700g * (30.4%) = 212.8g Protein in Extract = 4033.7g * (3.8%) = 153.28g Protein in Residue = 1373.1g * (3.78%) = 51.90g Protein in Emulsion = 130.77g * (2.58%) = 3.37g 1.59% Yield Extract in Residue = 1373.1g * (80.17%) = 1100.8g with 3.8% protein = 41.83g protein
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Total Protein in Residue = 51.90g – 41.83g = 10.07g 4.73% Yield Total Protein in Extract = 153.28g + 41.83g = 195.11g 91.69% Oil Balance: Oil in Flour = 700g * (26.63%) = 186.41g Oil in Extract = 4158.8g * (1.03%) = 42.98g Oil in Residue = 1318.8g * (5.45%) = 71.85g Oil in Emulsion = 128.73 * (56.36%) = 72.55g 38.92% Yield Extract in Residue = 1318.8g * (80.17%) = 1057.28g with 1.03% oil = 10.89g oil Total Oil in Residue = 71.85g – 10.89g = 60.96g 32.70% Yield Total Oil in Extract = 42.98g + 10.89g = 53.87g 28.86% Oil Content in fractions May 4, 2009. Emulsion May 1
B 1 2 W Flask + stopper 0.0000 76.4530 77.0450
W sample 0.0000 77.2345 78.1978 W Beaker 66.5981 77.5026 66.7096
W Beaker + Fat 66.5963 77.9378 67.3625 W Sample 0.0000 0.7815 1.1528
W Fat -0.0018 0.4352 0.6529
% Fat 55.92 56.79 56.36 0.62
May 4, 2009. Residue extraction May 1 (Freeze-dried) B 1 2 3
W Flask + stopper 0.0000 0.0000 0.0000 W sample 0.0000 1.9996 2.0034 W Beaker 68.1456 66.2451 69.7181
W Beaker + Fat 68.1458 66.7938 70.2645 W Sample 0.0000 1.9996 2.0034
W Fat 0.0002 0.5487 0.5464
% Fat 27.43 27.26 27.35 0.12
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May 4, 2009. Extract from May 1 extraction (Freeze-dried) B 1 2
W Flask + stopper 0.0000 0.0000 0.0000 W sample 0.0000 2.0111 1.9946 W Beaker 66.7449 82.2545 69.3012
W Beaker + Fat 66.7507 82.5077 69.5670 W Sample 0.0000 2.0111 1.9946
W Fat 0.0058 0.2532 0.2658
% Fat 12.30 13.04 12.67 0.52 Protein Content in Fractions August 25, 2009. Emulsion Aug 25
Sample Weight
(g) V Titration (mL) % N %
Protein Blank 0.2
1 1.5500 4.80 0.4155 2.60 2 1.5500 4.80 0.4155 2.60 3 1.5800 4.85 0.4120 2.58
2.58 0.01 Aug 26, 2009. Residue extraction Aug 25
Sample Weight
(g) V Titration (mL) % N %
Protein Blank 0.3
1 2.0500 9.25 0.6112 3.82 2 2.1100 9.40 0.6038 3.77 3 2.1100 9.30 0.5972 3.73
3.78 0.04 Aug 26, 2009. Extract extraction aug 25
Sample Weight (g) V Titration (mL) % N %
Protein Blank 0.3
1 3.0400 13.55 0.6102 3.81 2 3.0000 13.45 0.6137 3.84 3 3.0100 13.25 0.6023 3.76
3.80 0.04
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B3. Emulsion Analysis Oil Recovery from Yellow Mustard Flour
W Oil in flour (26.57%) = 185.99g
Date W Emulsion % Oil Emulsion % Oil Extracted 19-Jan 117.56 59.89 37.86 10-Feb 118.44 57.93 36.89 20-Feb 133.99 56.86 40.96 2-Mar 109.96 58.06 34.33 5-Mar 117.65 57.61 36.44 6-Apr 109.36 58.40 34.34 28-Apr 137.41 49.51 36.58 1-May 128.73 56.36 39.01
12-May 121.39 59.87 39.08 17-Jun 120.92 56.33 36.62 22-Jun 122.17 55.82 36.67 8-Jul 120.46 57.53 37.26 15-Jul 113.01 57.24 34.78 25-Aug 130.77 55.38 38.94 2-Oct 115.56 58.11 36.11
Mean Oil Extraction 37.0 STDV Oil Extraction 1.9
Mean Weight Emulsion 121.4 STDV Weight Emulsion 8.6
Oil Content in Emulsion
Date Blank 1 2 3 Mean STDV Sample (g) 0 1.7941 2.7445 2.3097
19-Jan Oil (g) 0.0017 1.0727 1.6809 1.3595 59.89 1.21 % Oil 59.70 61.18 58.79 Sample (g) 0 0.7161 1.3912
Feb 10 Oil (g) 0.0111 0.4372 0.7952 57.93 2.22 % Oil 59.50 56.36
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Sample (g) 0 0.9119 1.2121 0 Feb 20 Oil (g) 0.0006 0.5206 0.6877 0 56.86 0.24
% Oil 57.02 56.69 Sample (g) 0 1.8998 2.2772 2.386
2-Mar Oil (g) -0.0002 1.1172 1.3177 1.3717 58.06 0.68 % Oil 58.82 57.87 57.50 Sample (g) 0 1.483 1.5603 1.3888
5-Mar Oil (g) 0.0004 0.8509 1.0025 0.7121 57.61 6.49 % Oil 57.35 64.22 51.25 Sample (g) 0 1.057 1.19 1.6999
6-Apr Oil (g) 0.0007 0.6131 0.715 0.9736 58.40 1.45 % Oil 57.94 60.03 57.23 Sample (g) 0 0.931 0.8162 1.2407
28-Apr Oil (g) 0.0007 0.4626 0.401 0.6196 49.51 0.43 % Oil 49.61 49.04 49.88 Sample (g) 0 0.7815 1.1528 0
1-May Oil (g) -0.0018 0.4352 0.6529 0 56.36 0.62 % Oil 55.92 56.79 Sample (g) 0 0.84 1.18 1.04
12-May Oil (g) 0.0006 0.4994 0.7151 0.6211 59.87 0.61 % Oil 59.38 60.55 59.66 Sample (g) 0 0.98 0.99 0.86
17-Jun Oil (g) 0.0005 0.5416 0.5535 0.4986 56.33 1.41 % Oil 55.21 55.86 57.92 Sample (g) 0 0.73 0.95 0.89
22-Jun Oil (g) -0.0007 0.3918 0.4998 0.5422 55.82 4.52 % Oil 53.77 52.68 61.00 Sample (g) 0 0.7 0.8 1
08 jul Oil (g) -0.0007 0.421 0.4586 0.5485 57.53 2.7 % Oil 60.24 57.41 54.92 Sample (g) 0 0.7 0.9646 1.0021
15 jul Oil (g) -0.0007 0.3856 0.5664 0.5779 57.24 1.85 % Oil 55.19 58.79 57.74 Sample (g) 0 0.8 0 0.7
25-Aug Oil (g) 0.0014 0.4337 0 0.3984 55.38 1.89 % Oil 54.04 56.71 Sample (g) 0 0 0 1.00
2-Oct Oil (g) 0.0009 0 0 0.5820 58.11 % Oil 58.11
Overall Mean 56.97 Overall STDV 3.29
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Protein Content in Emulsion
Date Sample Sample
(g) V Titration
(mL) % N %
Protein Mean STDV Blank 0.35
Feb 10, 2009 1 0.1300 0.70 0.3769 2.36
2 0.1200 0.70 0.4083 2.55 2.45 0.14 3 Blank 0.4 1 1.1000 3.80 0.4327 2.70
Feb 20, 2009 2 1.0000 3.60 0.4480 2.80 2.83 0.14
3 1.0000 3.80 0.4760 2.98 Blank 0.1 1 2.0100 6.00 0.4109 2.57
Mar 02, 2009 2 2.0200 6.08 0.4145 2.59 2.58 0.01
3 2.0200 6.05 0.4124 2.58 Blank 0.2 1 2.0000 6.00 0.4060 2.54
Mar 05, 2009 2 2.0000 5.80 0.3920 2.45 2.49 0.04
3 2.0000 5.90 0.3990 2.49 Blank 0.1 1 2.0000 6.00 0.4130 2.58
May 12, 2009 2 2.1000 6.15 0.4033 2.52 2.58 0.05
3 2.0000 6.10 0.4200 2.63 Blank 0.2 1 1.5500 4.80 0.4155 2.60
July 15, 2009 2 1.5500 4.80 0.4155 2.60 2.59 0.01
3 1.5800 4.85 0.4120 2.58
Overall Mean 2.59 Overall STDV 0.14
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Moisture Content in Emulsion
Date 1 2 3 Mean STDV Beaker (g) 1.8134 1.7923 1.8132
Feb 10, 2009 B + Wet (g) 3.1080 2.6815 3.1925 42.97 0.64
B + Dry (g) 2.5534 2.2932 2.6075 Moisture % 42.84 43.67 42.41 Beaker (g) 1.8094 1.8012 1.7920
Feb 20, 2009 B + Wet (g) 2.7197 2.7895 2.7001 43.67 0.18
B + Dry (g) 2.3208 2.3599 2.3030 Moisture % 43.82 43.47 43.73 Beaker (g) 1.8027 1.8068 1.8045
Mar 02, 2009 B + Wet (g) 2.5287 2.5906 2.7757 39.61 0.21
B + Dry (g) 2.2406 2.2788 2.3933 Moisture % 39.68 39.78 39.37 Beaker (g) 1.7979 1.7962 1.7881
Mar 05, 2009 B + Wet (g) 2.5659 2.6025 2.4706 40.71 0.31
B + Dry (g) 2.2507 2.2746 2.1947 Moisture % 41.04 40.67 40.42 Beaker (g) 1.8055 1.8122 1.8002
Apr 06, 2009 B + Wet (g) 2.3870 2.4060 2.4969 38.75 0.18
B + Dry (g) 2.1620 2.1767 2.2255 Moisture % 38.69 38.62 38.96 Beaker (g) 1.1281 1.1179 1.1139
Apr 28, 2009 B + Wet (g) 2.0598 2.1882 2.3838 45.44 0.40
B + Dry (g) 1.6322 1.7037 1.8105 Moisture % 45.89 45.27 45.15 Beaker (g) 1.8191 1.8085 1.8059
May 1, 2009 B + Wet (g) 2.9612 3.0702 3.2057 38.54 0.18
B + Dry (g) 2.5214 2.5859 2.6635 Moisture % 38.51 38.38 38.73 Beaker (g) 1.1321 1.1170 1.1300
May 12, 2009 B + Wet (g) 2.1539 2.1638 2.3830 37.87 0.04
B + Dry (g) 1.7665 1.7676 1.9089 Moisture % 37.91 37.85 37.84
107
Date 1 2 3 Mean STDV Beaker (g) 1.1208 1.1405 1.1301
Jun 17, 2009 B + Wet (g) 2.0795 2.0148 1.9765 39.95 0.14
B + Dry (g) 1.6953 1.6654 1.6396 Moisture % 40.08 39.96 39.80 Beaker (g) 1.1074 1.1429 1.1345
Jun 22, 2009 B + Wet (g) 2.1518 2.1566 2.2610 40.22 0.06
B + Dry (g) 1.7311 1.7494 1.8080 Moisture % 40.28 40.17 40.21 Beaker (g) 1.1449 1.1466 1.1479
July 08, 2009 B + Wet (g) 2.0873 2.0045 1.8523 38.83 0.14
B + Dry (g) 1.7213 1.6703 1.5798 Moisture % 38.84 38.96 38.69 Beaker (g) 1.1116 1.1311 1.1461
July 15, 2009 B + Wet (g) 1.9866 1.9029 2.0197 38.72 0.10
B + Dry (g) 1.6470 1.6040 1.6823 Moisture % 38.81 38.73 38.62 Beaker (g) 1.1478 1.1345 1.1098
Aug 25, 2009 B + Wet (g) 2.1945 2.1740 2.2118 42.84 0.59
B + Dry (g) 1.7451 1.7232 1.7467 Moisture % 42.93 43.37 42.21 Beaker (g) 1.1076 1.1197 1.1185
Oct 02, 2009 B + Wet (g) 2.0145 2.2803 2.3770 41.23 0.33
B + Dry (g) 1.6373 1.8026 1.8617 Moisture % 41.59 41.16 40.95
Overall Mean 40.54 Overall STDV 2.23
108
B3. Oil Extraction from the Emulsion Using Isopropyl Alcohol as a Solvent
B.3.1 Single Stage Extraction
Oil Extraction
IPA: Oil Weight Ratio
IPA:Oil Molar Ratio
W Emulsion (g) W Oil (g)
W water (g)
V IPA (mL)
% Oil Extracted
W Oil Extracted (g) W IPA (g)
1.00 16 15.00 7.43 6.82 9.46 5.61 0.42 7.43 1.25 20 15.00 8.63 5.82 13.74 45.12 3.89 10.79 1.50 24 15.00 8.63 5.82 16.49 59.52 5.14 12.95 1.75 28 15.00 8.63 5.82 19.24 60.48 5.22 15.10 2.00 32 15.00 8.63 5.82 21.98 62.10 5.36 17.26 3.14 50 20.00 11.61 7.92 46.38 59.96 6.96 36.41 4.70 75 30.00 17.97 12.65 107.65 31.73 5.70 84.51 6.27 100 25.00 14.48 10.74 115.70 19.93 2.89 90.83 9.41 150 15.00 8.76 5.81 104.97 30.60 2.68 82.41 12.54 200 25.00 14.48 10.74 231.39 41.53 6.01 181.65 15.68 250 25.00 14.22 10.92 283.90 58.25 8.28 222.88 18.81 300 25.00 14.22 10.92 340.68 76.51 10.88 267.45 21.95 350 25.00 14.22 10.92 397.46 81.94 11.65 312.03 25.09 400 25.00 14.52 9.90 463.82 90.57 13.15 364.12 28.22 450 25.00 14.52 9.90 521.80 92.20 13.38 409.64 31.36 500 15.00 8.64 6.11 345.17 94.04 8.13 270.98
109
Miscella Composition
Weight Ratio % Oil % Water % IPA % Oil
Extracted 1.00 2.8 46.5 50.7 5.6 1.25 19.0 28.4 52.6 45.1 1.50 21.5 24.4 54.2 59.5 1.75 20.0 22.3 57.8 60.5 2.00 18.8 20.5 60.7 62.1
3 13.6 15.4 71.0 60.0 5 5.5 12.3 82.2 31.7 6 2.8 10.3 87.0 19.9 9 2.9 6.4 90.7 30.6
13 3.0 5.4 91.6 41.5 16 3.4 4.5 92.1 58.3 19 3.8 3.8 92.5 76.5 22 3.5 3.3 93.3 81.9 25 3.4 2.6 94.0 90.6 28 3.1 2.3 94.6 92.2 31 2.8 2.1 95.0 94.0
Results for each weight ratio For 1:1 Weight Oil in Miscella
W emulsion (g) 15 % Oil 49.51 W Total (g) 22.4265
W Oil (g) 7.4265 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0078 W beaker liquid 29.8395 W beaker + liquid 39.22 W Liquid 9.38
Weight Ratio 1 W IPA (g) 7.4265
ρ IPA (g/mL) 0.78505 % Oil liquid 4.44 MW IPA (g/mole) 60.1 W Oil Liquid 0.42
V IPA (mL) 9.46 Moles IPA 0.1236 W Oil Extracted (g) 0.42
Molar Ratio 16 % Oil Extracted 5.61
110
For 1.25:1 Oil in Solids W emulsion (g) 15
% Oil 57.53 W Total (g) 25.7869 W Oil (g) 8.6295
MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0091
W beaker solids 10.10 W beaker + solids 17.40 W Solids 7.30
Weight Ratio 1.25 W IPA (g) 10.7869
ρ IPA (g/mL) 0.78505 % Oil Solids 64.88 MW IPA (g/mole) 60.1 W Oil Solids 4.74
V IPA (mL) 13.74
Moles IPA 0.1795 W Oil Extracted
(g) 3.89 Molar Ratio 20 % Oil Extracted 45.12
For 1.5:1 Oil in Solids W emulsion (g) 15
% Oil 57.53 W Total (g) 27.9443 W Oil (g) 8.6295
MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0091
W beaker solids 10.30 W beaker + solids 15.90 W Solids 5.60
Weight Ratio 1.5 W IPA (g) 12.9443
ρ IPA (g/mL) 0.78505 % Oil Solids 62.38 MW IPA (g/mole) 60.1 W Oil Solids 3.49
V IPA (mL) 16.49
Moles IPA 0.2154 W Oil Extracted
(g) 5.14 Molar Ratio 24 % Oil Extracted 59.52
111
For 1.75:1 Oil in Solids W emulsion (g) 15
% Oil 57.53 W Total (g) 30.1016 W Oil (g) 8.6295
MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0091
W beaker solids 10.40 W beaker + solids 15.80 W Solids 5.40 Weight Ratio 1.75
W IPA (g) 15.1016 ρ IPA (g/mL) 0.78505 % Oil Solids 63.16
MW IPA (g/mole) 60.1 W Oil Solids 3.41 V IPA (mL) 19.24
Moles IPA 0.2513 W Oil Extracted
(g) 5.22 Molar Ratio 28 % Oil Extracted 60.48
For 2:1 Oil in Solids W emulsion (g) 15
% Oil 57.53 W Total (g) 32.2590 W Oil (g) 8.6295
MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0091
W beaker solids 10.30 W beaker + solids 15.60 W Solids 5.30 Weight Ratio 2
W IPA (g) 17.2590 ρ IPA (g/mL) 0.78505 % Oil Solids 61.71
MW IPA (g/mole) 60.1 W Oil Solids 3.27 V IPA (mL) 21.98
Moles IPA 0.2872 W Oil Extracted
(g) 5.36 Molar Ratio 32 % Oil Extracted 62.10
112
For 3:1 weight ratio, 50:1 molar ratio Oil in solids
W emulsion (g) 25 % Oil 56.86 W Total (g) 69.5746
W Oil (g) 14.2150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0148 W beaker solids 43.6 W beaker + solids 53.20 W Solids 9.60
Ratio (Molar) 50 Moles IPA 0.7417
ρ IPA (g/mL) 0.78505 % Oil Solids 57.58 MW IPA (g/mole) 60.1 W Oil Solids 5.52768
V IPA (mL) 56.78 W IPA (g) 44.5746 W Oil Extracted (g) 8.6873
% Oil Extracted 59.96 Oil in miscella
W emulsion (g) 20 % Oil 58.06 W Total (g) 56.4123
W Oil (g) 11.6120 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0121 W beaker liquid 98.5 W beaker + liquid 146.30 W Liquid 47.80 Ratio (molar) 50
Moles IPA 0.6059 ρ IPA (g/mL) 0.78505 % Oil liquid 14.29
MW IPA (g/mole) 60.1 W Oil Liquid 6.83062 V IPA (mL) 46.38
W IPA (g) 36.4123 W Oil Extracted
(g) 6.8306 % Oil Extracted 58.82
113
For 5:1 weight ratio 75:1 molar ratio Oil in solids
W emulsion (g) 30 % Oil 59.89 W Total (g) 115.0294
W Oil (g) 17.9670 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0189 W beaker solids 43.5 W beaker + solids 63.30 W Solids 19.80
Ratio (Molar) 75 Moles IPA 1.4148
ρ IPA (g/mL) 0.78505 % Oil Solids 61.95 MW IPA (g/mole) 60.1 W Oil Solids 12.2661
V IPA (mL) 108.31 W IPA (g) 85.0294 W Oil Extracted (g) 5.7009
% Oil Extracted 31.73 For 6:1 weight ratio 100:1 Molar ratio Oil in solids
W emulsion (g) 25 % Oil 57.93 W Total (g) 115.8269
W Oil (g) 14.4825 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0151 W beaker solids 43.6 W beaker + solids 62.10 W Solids 18.50
Ratio (Molar) 100 Moles IPA 1.5113
ρ IPA (g/mL) 0.78505 % Oil Solids (dry) 62.68 MW IPA (g/mole) 60.1 W Oil Solids 11.5958
V IPA (mL) 115.70
W IPA (g) 90.8269 W Oil Extracted
(g) 2.8867 Weight ratio 6 % Oil Extracted 19.93
114
For 9:1 weight ratio 150:1 molar ratio Oil in Miscella
W emulsion (g) 15 % Oil 58.40 W Total (g) 97.9140
W Oil (g) 8.7600 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0092 W beaker liquid 119.4 W beaker + liquid 203.70 W Liquid 84.30
Ratio (Molar) 150 Moles IPA 1.3796
ρ IPA (g/mL) 0.78505 % Oil liquid 3.18 MW IPA (g/mole) 60.1 W Oil Liquid 2.68
V IPA (mL) 105.62 W IPA (g) 82.9140 W Oil Extracted (g) 2.68
Weight ratio 9 % Oil Extracted 30.60 For 13:1 weight ratio 200:1 molar ratio Oil in solids
W emulsion (g) 25 % Oil 57.93 W Total (g) 207.7704
W Oil (g) 14.4825 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0152 W beaker solids 67.9 W beaker + solids 90.90 W Solids 23.00
Ratio (molar) 200 Moles IPA 3.0411
ρ IPA (g/mL) 0.78505 % Oil Solids 36.82 MW IPA (g/mole) 60.1 W Oil Solids 8.4686
V IPA (mL) 232.81 W IPA (g) 182.7704 W Oil Extracted (g) 6.0139
Weight ratio 13 % Oil Extracted 41.53
115
For 16:1 weight ratio 250:1 molar ratio Oil in Solids
W emulsion (g) 25 % Oil 56.86 W Total (g) 249.2431
W Oil (g) 14.2150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0149 W beaker solids 67.9 W beaker + solids 95.40 W Solids 27.50
Ratio (molar) 250 Moles IPA 3.7312
ρ IPA (g/mL) 0.78505 % Oil Solids 21.58 MW IPA (g/mole) 60.1 W Oil Solids 5.9345
V IPA (mL) 285.64 W IPA (g) 224.2431 W Oil Extracted (g) 8.2805
Weight ratio 16 % Oil Extracted 58.25 For 19:1 molar ratio 300:1 weight ratio Oil in Solids
W emulsion (g) 25 % Oil 56.86 W Total (g) 294.0918
W Oil (g) 14.2150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0149 W beaker solids 67.3 W beaker + solids 99.40 W Solids 32.10
Ratio (molar) 300 Moles IPA 4.4774
ρ IPA (g/mL) 0.78505 % Oil Solids 10.40 MW IPA (g/mole) 60.1 W Oil Solids 3.3384
V IPA (mL) 342.77 W IPA (g) 269.0918 W Oil Extracted (g) 10.8766
Weight ratio 19 % Oil Extracted 76.51
116
For 22:1 weight ratio 350:1 molar ratio Oil in solids
W emulsion (g) 25 % Oil 56.86 W Total (g) 338.9404
W Oil (g) 14.2150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0149 W beaker solids 67.8 W beaker + solids 101.40 W Solids 33.60
Ratio (molar) 350 Moles IPA 5.2236
ρ IPA (g/mL) 0.78505 % Oil Solids (dry) 7.64 MW IPA (g/mole) 60.1 W Oil Solids 2.56704
V IPA (mL) 399.90 W IPA (g) 313.9404 W Oil Extracted (g) 11.6480
Weight ratio 22 % Oil Extracted 81.94 For 25:1 weight ratio 400:1 molar ratio Oil in solids
W emulsion (g) 25 % Oil 58.06 W Total (g) 391.3611
W Oil (g) 14.5150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0152 W beaker solids 105.9 W beaker + solids 139.30 W Solids 33.40
Ratio (molar) 400 Moles IPA 6.0959
ρ IPA (g/mL) 0.78505 % Oil Solids (dry) 4.10 MW IPA (g/mole) 60.1 W Oil Solids 1.3694
V IPA (mL) 466.67 W IPA (g) 366.3611 W Oil Extracted (g) 13.1456
Weight ratio 25 % Oil Extracted 90.57
117
Oil in miscella
W emulsion (g) 25 % Oil 58.06 W Total (g) 391.3611
W Oil (g) 14.5150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0152 W beaker liquid 208 W beaker + liquid 558.80 W Liquid 350.80 Ratio (molar) 400
Moles IPA 6.0959 ρ IPA (g/mL) 0.78505 % Oil liquid 3.74
MW IPA (g/mole) 60.1 W Oil Liquid 13.12 V IPA (mL) 466.67
W IPA (g) 366.3611 W Oil Extracted
(g) 13.12 Weight Ratio 25 % Oil Extracted 90.39
For 28:1 weight ratio 450:1 molar ratio Oil in miscella
W emulsion (g) 25 % Oil 58.06 W Total (g) 437.1562
W Oil (g) 14.5150 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0152 W beaker liquid 297.4 W beaker + liquid 688.70 W Liquid 391.30 Ratio (molar) 450
Moles IPA 6.8578 ρ IPA (g/mL) 0.78505 % Oil liquid 3.42
MW IPA (g/mole) 60.1 W Oil Liquid 13.38 V IPA (mL) 525.01
W IPA (g) 412.1562 W Oil Extracted
(g) 13.38 Weight ratio 28 % Oil Extracted 92.20
118
For 31:1 weight ratio 500:1 molar ratio
Oil in miscella
W emulsion (g) 15 % Oil 57.61 W Total (g) 287.6412
W Oil (g) 8.6415 MW Oil (g/mole) 952.45 After Centrifugation:
Moles Oil 0.0091 W beaker liquid 52.2 W beaker + liquid 311.00 W Liquid 258.80 Ratio (molar) 500
Moles IPA 4.5365 ρ IPA (g/mL) 0.78505 % Oil liquid 3.14
MW IPA (g/mole) 60.1 W Oil Liquid 8.13 V IPA (mL) 347.29
W IPA (g) 272.6412 W Oil Extracted
(g) 8.13 Weight ratio 32 % Oil Extracted 94.04
Solvent Holdup and Residual Oil in Solids
Weight Ratio
% Oil in Solids % Holdup
% Oil Extracted
1.25 64.88 26.24 45.12 1.50 62.38 28.79 59.52 1.75 63.16 27.67 60.48 2.00 62.71 27.52 62.10
3 57.58 31.50 59.96 5 61.95 32.18 31.73 6 62.68 34.37 19.93 9 46.38 37.51 30.60 13 36.82 61.20 41.53 16 21.58 74.11 58.25 19 10.40 85.83 76.51 22 7.64 89.51 81.94 25 4.10 92.88 90.57 28 2.61 93.57 92.20 31 1.90 94.46 94.04
119
Oil Distribution
B.3.2 Multiple Stage Extraction For the first stage, oil extraction was determined from residual oil in solids, for subsequent
stages, oil in miscellas was measured.
Three stage extraction
Triple Extraction. Run 1. April 09 Molar Ratio 50 : 1
Weight Ratio 3 : 1
% Oil Extracted First Stage 58.59 58.59
Second Stage 17.32 75.91
Third Stage 18.28 94.19
120
Triple Extraction. Run 2. April 29 Molar Ratio 50 : 1
Weight Ratio 3 : 1
% Oil Extracted First Stage 57.88 57.88
Second Stage 15.77 73.65
Third Stage 19.87 93.52
Triple Extraction. May 05, 2009 Triple Extraction. May 08, 2009 Run 1 Run 2
Molar Ratio 32 : 1 Molar Ratio 32 : 1 Weight Ratio 2 : 1 Weight Ratio 2 : 1
% Oil Extracted % Oil Extracted
First Stage 62.10 62.10 First Stage 61.50 61.50 Second Stage 9.70 71.80 Second Stage 10.12 71.62
Third Stage 14.82 86.62 Third Stage 15.88 87.50 Triple Extraction. May 05, 2009 Molar Ratio 16 : 1
Weight Ratio 1 : 1
% Oil Extracted First Stage 5.61 5.61
Second Stage 44.68 50.29
Third Stage 4.44 54.73 Triple Extraction. June 26, 2009 Molar Ratio 24 : 1
Weight Ratio 1.5 : 1
% Oil Extracted First Stage 59.52 59.52
Second Stage 7.03 66.55
Third Stage 11.16 77.71
121
Triple Extraction. June 30, 2009 Molar Ratio 28
Weight Ratio 1.75 : 1
% Oil Extracted First Stage 59.13 59.13
Second Stage 10 69.13
Third Stage 11.16 80.29 Four-stage Extraction 4-stage Extraction. July 16, 2009 4-stage Extraction. Aug 27, 2009
Molar Ratio 32
Molar Ratio 32
Weight Ratio 2 : 1
Weight Ratio 2 : 1
% Oil Extracted % Oil Extracted
First Stage 60.35 60.35
First Stage 62.1 62.10
Second Stage 11.85 72.20
Second Stage 11.14 73.24
Third Stage 16.06 88.26
Third Stage 14.28 87.52
Fourth Stage 8.77 97.03
Fourth Stage 8.02 95.54
4-stage Extraction. Average Jul 16 and Aug 27 Molar Ratio 32
Weight Ratio 2 : 1
% Oil Extracted First Stage 61.23 61.23
Second Stage 11.50 72.72
Third Stage 15.17 87.89 Fourth Stage 8.40 96.29
122
4-stage Extraction. July 20, 2009 Molar Ratio 50
Weight Ratio 3 : 1
% Oil Extracted First Stage 59.96 59.96
Second Stage 19.8 79.76
Third Stage 15.29 95.05 Fourth Stage 4.35 99.40
4-stage Extraction. July 23, 2009 Molar Ratio 24
Weight Ratio 1.5 : 1
% Oil Extracted First Stage 59.52 59.52
Second Stage 7.74 67.26
Third Stage 11.35 78.61 Fourth Stage 12.16 90.77
4-stage Titration
16-Jul-09 For 2:1 Weight First Oil in Solids W emulsion (g) 15 % Oil 57.53 W Total (g) 32.2590 W Oil (g) 8.6295 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0090 % Extn 60.35 Weight Ratio 2 W IPA (g) 17.2590 ρ IPA (g/mL) 0.78505 MW IPA (g/mole) 60.1 V IPA (mL) 21.98 Moles IPA 0.2872 W Oil Extracted (g) 5.21 Molar Ratio 31.89 % Oil Extracted 60.35
123
16-Jul-09 For 2:1 Weight Second
Oil in Miscella W solids (g) 9.79 W Total (g) 27.0511 W Oil (g) 3.4216 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0036 W beaker liquid 77.5 W beaker + liquid 91.84 W Liquid 14.34 Ratio 2 Moles IPA 0.0071 ρ IPA (g/mL) 0.78505 % Oil liquid 7.13 MW IPA (g/mole) 60.1 W Oil Liquid 1.02 V IPA (mL) 21.98 W IPA (g) 17.2590 W Oil Extracted (g) 1.02 % Oil Extracted 29.87
% Oil from original 11.85
16-Jul-09 For 2:1 Weight Third Oil in Miscella W Total (g) 17.2590 W Oil (g) 2.3994 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0025 W beaker liquid 66.7039 W beaker + liquid 82.67 W Liquid 15.97 Ratio 50 Moles IPA 0.1252 ρ IPA (g/mL) 0.78505 % Oil liquid 8.68 MW IPA (g/mole) 60.1 W Oil Liquid 1.39 V IPA (mL) 21.98
W IPA (g) 17.2590 W Oil Extracted (g) 1.39
% Oil Extracted 57.75
% Oil from original 16.06
124
16-Jul-09 For 2:1 Weight Fourth Oil in Miscella W Total (g) 13.5492 W Oil (g) 1.0137 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0011 W beaker liquid 65.253 W beaker + liquid 81.56 W Liquid 16.31 Ratio 50 Moles IPA 0.0529 ρ IPA (g/mL) 0.78505 % Oil liquid 4.64 MW IPA (g/mole) 60.1 W Oil Liquid 0.76 V IPA (mL) 17.26
W IPA (g) 13.5492 W Oil Extracted (g) 0.76
% Oil Extracted 74.66
% Oil from original 8.77
Total Extn 60.35 60.35 11.85 72.20 16.06 88.25 8.77 97.02
4-stage Titration
27-Aug-09 For 2:1 Weight First
Oil in Solids W emulsion (g) 20 % Oil 55.38 W Total (g) 42.1520 W Oil (g) 11.0760 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0116 % Extn 62.10 Weight Ratio 2 W IPA (g) 22.1520 ρ IPA (g/mL) 0.78505 MW IPA (g/mole) 60.1 V IPA (mL) 28.22 Moles IPA 0.3686 W Oil Extracted (g) 6.88 Molar Ratio 31.89 % Oil Extracted 62.10
125
27-Aug-09 For 2:1 Weight Second
Oil in Miscella W solids (g) 13.12 W Total (g) 35.2738 W Oil (g) 4.1978 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0044 W beaker liquid 28.1218 W beaker + liquid 46.18 W Liquid 18.06 Ratio 2 Moles IPA 0.0088 ρ IPA (g/mL) 0.78505 % Oil liquid 6.83 MW IPA (g/mole) 60.1 W Oil Liquid 1.23 V IPA (mL) 28.22 W IPA (g) 22.1520 W Oil Extracted (g) 1.23 % Oil Extracted 29.39
% Oil from original 11.14
27-Aug-09 For 2:1 Weight Third
Oil in Miscella W Total (g) 22.1520 W Oil (g) 2.9642 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0031 W beaker liquid 29.5854 W beaker + liquid 49.92 W Liquid 20.33 Ratio 2 Moles IPA 0.0062 ρ IPA (g/mL) 0.78505 % Oil liquid 7.78 MW IPA (g/mole) 60.1 W Oil Liquid 1.58 V IPA (mL) 28.22
W IPA (g) 22.1520 W Oil Extracted (g) 1.58
% Oil Extracted 53.36
% Oil from original 14.28
126
27-Aug-09 For 2:1 Weight Fourth Oil in Miscella W Total (g) 17.3904 W Oil (g) 1.3825 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0014 W beaker liquid 29.4437 W beaker + liquid 50.50 W Liquid 21.05 Ratio 50 Moles IPA 0.0721 ρ IPA (g/mL) 0.78505 % Oil liquid 4.22 MW IPA (g/mole) 60.1 W Oil Liquid 0.89 V IPA (mL) 22.15
W IPA (g) 17.3904 W Oil Extracted (g) 0.89
% Oil Extracted 64.27
% Oil from original 8.02
Total Extn 62.10 62.10 11.14 73.24 14.28 87.52 8.02 95.54
4-stage Titration
20-Jul-09 For 3:1 Weight First
Oil in Solids W emulsion (g) 30 % Oil 57.24 W Total (g) 81.5160 W Oil (g) 17.1720 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0179 % Extn 59.96 Weight Ratio 3 W IPA (g) 51.5160 ρ IPA (g/mL) 0.78505 MW IPA (g/mole) 60.1 V IPA (mL) 65.62 Moles IPA 0.8572 W Oil Extracted (g) 10.30 Molar Ratio 47.84 % Oil Extracted 59.96
127
20-Jul-09 For 3:1 Weight Second
Oil in Miscella W solids (g) 19.70 W Total (g) 71.2197 W Oil (g) 6.8757 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0072 W beaker liquid 77.5 W beaker + liquid 122.53 W Liquid 45.03 Ratio 3 Moles IPA 0.0215 ρ IPA (g/mL) 0.78505 % Oil liquid 7.55 MW IPA (g/mole) 60.1 W Oil Liquid 3.40 V IPA (mL) 65.62 W IPA (g) 51.5160 W Oil Extracted (g) 3.40 % Oil Extracted 49.45
% Oil from original 19.80
20-Jul-09 For 3:1 Weight Third Oil in Miscella W Total (g) 51.5160 W Oil (g) 3.4758 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0036 W beaker liquid 68.9194 W beaker + liquid 116.24 W Liquid 47.32 Ratio 3 Moles IPA 0.0109 ρ IPA (g/mL) 0.78505 % Oil liquid 5.55 MW IPA (g/mole) 60.1 W Oil Liquid 2.63 V IPA (mL) 65.62
W IPA (g) 51.5160 W Oil Extracted (g) 2.63
% Oil Extracted 75.55
% Oil from original 15.29
128
20-Jul-09 For 3:1 Weight Fourth Oil in Miscella W Total (g) 51.5160 W Oil (g) 0.8498 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0009 W beaker liquid 77.5713 W beaker + liquid 128.68 W Liquid 51.11 Ratio 3 Moles IPA 0.0027 ρ IPA (g/mL) 0.78505 % Oil liquid 1.46 MW IPA (g/mole) 60.1 W Oil Liquid 0.75 V IPA (mL) 65.62
W IPA (g) 51.5160 W Oil Extracted (g) 0.75
% Oil Extracted 87.81
% Oil from original 4.35
Total Extn 59.96 59.96 19.80 79.76 15.29 95.05 4.35 99.40
4-stage Titration
23-Jul-09 For 1.5:1 Weight First
Oil in Solids W emulsion (g) 30 % Oil 57.24 W Total (g) 55.7580 W Oil (g) 17.1720 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0179 % Extn 59.52 Weight Ratio 1.5 W IPA (g) 25.7580 ρ IPA (g/mL) 0.78505 MW IPA (g/mole) 60.1 V IPA (mL) 32.81 Moles IPA 0.4286 W Oil Extracted (g) 10.22 Molar Ratio 23.92 % Oil Extracted 59.52
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23-Jul-09 For 1.5:1 Weight Second Oil in Miscella W solids (g) 19.78 W Total (g) 45.5372 W Oil (g) 6.9512 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0073 W beaker liquid 77.5006 W beaker + liquid 100.38 W Liquid 22.87 Ratio 3 Moles IPA 0.0218 ρ IPA (g/mL) 0.78505 % Oil liquid 5.81 MW IPA (g/mole) 60.1 W Oil Liquid 1.33 V IPA (mL) 32.81 W IPA (g) 25.7580 W Oil Extracted (g) 1.33 % Oil Extracted 19.12
% Oil from original 7.74
23-Jul-09 For 1.5:1 Weight Third Oil in Miscella W Total (g) 25.7580 W Oil (g) 5.6222 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0059 W beaker liquid 65.2534 W beaker + liquid 89.53 W Liquid 24.28 Ratio 3 Moles IPA 0.0176 ρ IPA (g/mL) 0.78505 % Oil liquid 8.03 MW IPA (g/mole) 60.1 W Oil Liquid 1.95 V IPA (mL) 32.81
W IPA (g) 25.7580 W Oil Extracted (g) 1.95
% Oil Extracted 34.67
% Oil from original 11.35
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23-Jul-09 For 1.5:1 Weight Fourth Oil in Miscella W Total (g) 25.7580 W Oil (g) 3.6728 MW Oil (g/mole) 952.45 After Centrifugation: Moles Oil 0.0038 W beaker liquid 66.8754 W beaker + liquid 92.15 W Liquid 25.27 Ratio 3 Moles IPA 0.0115 ρ IPA (g/mL) 0.78505 % Oil liquid 8.26 MW IPA (g/mole) 60.1 W Oil Liquid 2.09 V IPA (mL) 32.81
W IPA (g) 25.7580 W Oil Extracted (g) 2.09
% Oil Extracted 56.83
% Oil from original 12.16
Residual Oil Content for 2:1 and 3:1 after Four-stage Extraction
July 16, 2009. Solids Exp July 16. 2:1, 4-stage extraction B 1 2 3
W Flask + stopper 0.0000 0.0000 0.0000 0.0000 W sample 0.0000 1.9409 2.1815 0.0000 W Beaker 66.2450 67.0538 68.5657 0.0000
W Beaker + Fat 66.2450 67.1429 68.6694 0.0000
W Sample 0.0000 1.9409 2.1815 0.0000 W Fat 0.0000 0.0891 0.1037 0.0000
% Fat 4.59 4.75 0.00 4.67 0.12
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Aug 27, 2009. Solids Exp Aug 27. 2:1, 4-stage extraction B 1 2 3
W Flask + stopper 0.0000 0.0000 0.0000 0.0000 W sample 0.0000 3.1255 2.2945 0.0000 W Beaker 66.2450 66.7042 68.5651 0.0000
W Beaker + Fat 66.2450 66.8204 68.6511 0.0000
W Sample 0.0000 3.1255 2.2945 0.0000 W Fat 0.0000 0.1162 0.0860 0.0000
% Fat 3.72 3.75 0.00 3.73 0.02
July 20, 2009. Solids Exp July 20. 3:1, 4-stage extraction B 1 2 3
W Flask + stopper 0.0000 0.0000 0.0000 0.0000 W sample 0.0000 1.7974 1.7054 0.0000 W Beaker 66.2450 66.7019 66.8814 0.0000
W Beaker + Fat 66.2450 66.7212 66.8975 0.0000
W Sample 0.0000 1.7974 1.7054 0.0000 W Fat 0.0000 0.0193 0.0161 0.0000
% Fat 1.07 0.94 0.00 1.01 0.09
Protein and Holdup Analysis for 2:1 and 3:1 after Four-stage Extraction
2:1 Weight Ratio Holdup July 16 Wbeaker = 29.4428 Wb + wet = 38.3246 Wb + dry = 30.5173 Holdup = 87.90 %
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Aug 27 Wbeaker = 28.7537 Wb + wet = 33.8362 Wb + dry = 29.2293 Holdup = 90.64 % Average Holdup = 89.27 ±1.94 Protein Aug 27, 2009. Solids (dried) after 2:1 4-stage
Sample Weight
(g) V Titration (mL) % N %
Protein Blank 0.4
1 0.2009 7.80 5.1568 32.23 2 0.2019 7.70 5.0619 31.64 3 0.2020 7.70 5.0594 31.62
31.83 0.35
W solids dry= 1.075 %Protein= 31.83 gProtein= 0.3422 W solids wet= 8.8818 %Protein wet= 3.85 ±0.04 gProtein emulsion= (18g emulsion * 2.59% protein) = 0.4662 %Protein in solids= 73.40% %Protein in miscella= 26.60% gProtein in total emulsion from flour= 3.4 ----------------- 1.6% of protein in flour gProtein in solids after extn= 2.5 ----------------------------- 1.17% of protein in flour gProtein in miscella after extn= 0.92 ------------------------ 0.43% of protein in flour
3:1 Weight Ratio Holdup July 20 Wbeaker = 28.1428 Wb + wet = 37.8612 Wb + dry = 28.8286 Holdup = 92.94 %
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Protein July 20, 2009. Solids (dried) after 3:1 on July 20. 4-stage Sample Weight (g) V Titration (mL) % N % Protein Blank 0.3
1 0.2028 12.00 8.0769 50.48 2 0.2016 12.30 8.3333 52.08 3 0.2002 11.70 7.9720 49.83
50.80 1.16 W solids dry= 1.1155 %Protein= 50.80 gProtein= 0.5667 W solids wet= 15.8 %Protein wet= 3.59 ±0.08 gProtein emulsion= (30g emulsion * 2.59% protein) = 0.777 %Protein in solids= 72.93% %Protein in miscella= 27.07% gProtein in total emulsion from flour= 3.4 ----------------- 1.6% of protein in flour gProtein in solids after extn= 2.5 ----------------------------- 1.17% of protein in flour gProtein in miscella after extn= 0.92 ------------------------ 0.43% of protein in flour
B.4 Characterization of the Miscellas from the Four-Stage Oil Extraction Coulometric Karl Fischer Titration
Stage Weight Sample
% Water 1
% Water 2
Ave Water %
sd % Water g Water
% water from
emulsion 1 49.92 20.140 20.140 10.054 78.23 2 27.38 3.274 3.314 3.294 0.028 0.902 7.02 3 30.68 0.918 0.928 0.923 0.007 0.283 2.20 4 31.40 0.439 0.428 0.434 0.008 0.136 1.06
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Miscellas for 4-stage 2:1 Weight Ratio Experiment July 16 Experiment Aug 27 Stage 1 Stage 1
% component g sample g component % in sample %
component g sample g component % in sample Water 20.14 26.21 5.28 20.14 Water 20.14 34.94 7.04 20.14
Oil 60.35 oil extn 8.6295 5.21 19.88 Oil 62.10 oil
extn 11.076 6.88 19.69 IPA 15.72 59.98 IPA 21.03 60.17
26.21 34.94 Stage 2 Stage 2
% component g sample g component % in sample %
component g sample g component % in sample Water 3.274 14.34 0.47 3.27 Water 3.314 18.06 0.60 3.31
Oil 7.13 14.34 1.02 7.13 Oil 6.83 18.06 1.23 6.83 IPA 12.85 89.60 IPA 16.23 89.86
14.34 18.06 Stage 3 Stage 3
% component g sample g component % in sample %
component g sample g component % in sample Water 0.918 15.97 0.15 0.92 Water 0.928 20.33 0.19 0.93
Oil 8.68 15.97 1.39 8.68 Oil 7.78 20.33 1.58 7.78 IPA 14.44 90.40 IPA 18.56 91.29
15.97 20.33
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Stage 4 Stage 4
% component g sample g component % in sample %
component g sample g component % in sample Water 0.439 16.31 0.07 0.44 Water 0.428 21.05 0.09 0.43
Oil 4.64 16.31 0.76 4.64 Oil 4.22 21.05 0.89 4.22 IPA 15.48 94.92 IPA 20.07 95.35
16.31 21.05
Experiment July 16 Experiment Aug 27
Stage % Water % Oil % IPA % Oil extn Stage % Water % Oil % IPA % Oil extn
1 20.14 19.88 59.98 60.35 1 20.14 19.69 60.17 62.10 2 3.27 7.13 89.60 11.85 2 3.31 6.83 89.86 11.14 3 0.92 8.68 90.40 16.06 3 0.93 7.78 91.29 14.28 4 0.44 4.64 94.92 8.77 4 0.43 4.22 95.35 8.02 97.03 95.54
Final result with both experiments together Stage Water (%) sd water Oil (%) sd Oil IPA (%) sd IPA Oil extn sd Oil extn 1 20.14 0.00 19.78 0.14 60.08 0.14 61.23 1.24 2 3.29 0.03 6.98 0.21 89.73 0.18 11.50 0.50 3 0.92 0.01 8.23 0.64 90.85 0.63 15.17 1.26 4 0.43 0.01 4.43 0.30 95.14 0.30 8.40 0.53 96.29 1.05
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B.5 Final Miscella Analyses
Water Oil IPA 17-Jul 6.99 10.22 82.79
27-Aug 7.95 9.82 82.23
ave 7.47 10.02 82.51 sd 0.68 0.28 0.40
Water Adsorption with Molecular Sieves 4A
Coulometric Karl Fischer Exp Sep 08 g sample 50 % Water Miscella 8.912
Exp W water before
% Water after
W Water after
W water adsorbed
% water adsorbed
1 4.456 3.793 1.897 2.559 57.437 2 4.456 3.846 1.923 2.533 56.848 average 2.546 57.142 sd 0.019 0.417
Exp Oct 07 g sample 50
% water miscella 8.9
W
water before
% Water After
W Water after
W water adsorbed
% water adsorbed
4.450 1.7 0.850 3.600 80.899