Helsinki.

University of Technology
Faculty of Process Engineering and Materials Science
Department of Chemical Engineering
Laboratory of Biotechnology and Food Engineering
FIN-02150 Fspoo, Finland
Technical Biochemistry Report 2/1995
Environmentally Friendly Products Based
on Vegetable Oils
Kerja Lamsa
Dissertation for the degree Doctor of Philosophy
to be presented with the due permission
for pUblic examination and debate
in Aucfltorium Kem 2
at Helsinki University of Technology (Espoo,Finland)
on the 11th of December, 1995, at 12 o'clock noon.
ESpoo, November, 1995.
TO Jyrki, Inka and Riku
CONTENTS
Abstract
Preface
List of publications
Introduction
1.1. Historical background of chemical esters used in lubricants 1
1.2. Preparation of fatty acid esters 2
1.2.1.Monoesters offatty acids 2
1.2.2.Diesters of fatty acids 4
1.2.3.Polyol esters of fatty acids 6
1.3. Lipase catalysed fatty acid ester production 10
1.3.1.Definition of Iipases 10
1.3.2.Structure of Iipases 11
1.3.3.Properties of lipases 11
1.3.4.Specificity of Iipases 13
1.3.5.Production of lipases 16
1.3.6.Reactions of Iipases 18
1.3.7.Effect of water on lipases 21
1.3.8.lmmobilisation of lipases 21
1.4. Aims of the present work 23
2. Materials and methods 24
2.1. Vegetable oils 24
2.2. Alcohols 25
2.3. Chemical catalysts 25
2.4. Reagents 25
2.5. Solvents 25
2.6. Enzymes 26
2.7. Analytical methods 26
2.8. Immobilisation of lipases 28
2.9. Chemical preparation of 2-ethyl-1-hexylester of rapeseed oil 29
2.10.Enzymatic preparation of 2-ethyl-1-hexylester of rapeseed oil 29
2.11.Chemical preparation of rapeseed oil methylester 29
2.12.Chemical preparation of soybean ethylester 29
2.13. Chemical preparation of coconut oil methylester 30
2.14.Chemical preparation of trimethylolpropane ester from tallow oil fatty acids 30
2.15.Chemical preparation of rapeseed oil trimethylolpropane ester 31
2.16.Enzymatic preparation of rapeseed oil trimethylolpropane ester 31
3. Results and discussion 32
3.1. Chemical synthesis of 2-ethyf-1-hexylester of rapeseed oil 32
3.1.1.Choice of catalyst 32
3.1.2.Effect of molar ratio 33
3.1.3.Effect of temperature and pressure 34
3.2. Enzymatic synthesis of rapeseed oil 2-ethyf-1-hexylester (I) 38
3.2.1.Choice of lipase (I) 38
3.2.2.Effect of substrate molar ratio (I) 39
3.2.3.Effect of lipase quantity (I) 39
3.2.4.Effect of added water (I) 41
3.2.5.Effect of temperature (I) 42
3.3. Small pilot scale enzymatic preparation of 2-ethyl-1-hexylester of rapeseed
oil (II) 43
3.4. Chemical synthesis of rapeseed oil methylester 44
3.5. Chemical synthesis of soybean oil ethylester 47
3.6. Chemical synthesis of coconut oil methylester 47
3.7. Chemical synthesis of trimethylolpropane ester from fatty acids of tallow oil 48
3.7.1.Choice of catalyst 48
3.7.2.Effect of catalyst quantity on esterification of tallow oil fatty acids 49
3.7.3.Effect of substrate molar ratio on esterification of tallow oil fatty acids 50
3.7.4.Effect of temperature, time and pressure on esterification of tallow oil
fatty acids 51
3.8. Chemical synthesis of TMP-ester from rapeseed oil methylester and
trimethylolpropane (III) 54
3.8.1.Choice and quantity of catalyst (III) 54
3.8.2.Effect of substrate molar ratio on RME transesterification (III) 55
3.8.3.Effect of temperature and pressure on transesterification of RME (III) 56
3.9. Small pilot scale production of TMP-ester from rapeseed oil methylester
and TMP (III) 59
3.10. Enzymatic preparation of rapeseed oil TMP-ester (IV) 62
3.10.1.Choice of lipase (IV) 62
3.10.2.Effect of lipase quantity and its stepwise addition (IV) 63
3.1 0.3.Effect of substrate molar ratio on enzymatic transesterification of RME (IV) 64
3.1 0.4.Effect of added water on enzymatic transesterification of RME (IV) 66
3.1 0.5. Effect of temperature on enzymatic transesterification of RME (IV) 66
3.10.6. Effect of pressure on enzymatic transesterification of RME (IV) 67
3.11. Immobilization experiments for the preparation of TMP ester from rapeseed
oil methylester 68
3.12. Applications and uses of rapeseed oil esters 70
3.12.1.Methyl-, ethyl-, and 2-ethyl-1-hexylesters of rapeseed oil 70
3.12.2.Hydraulic fluids made from rapeseed oil esters (V,VI) 71
3.13. Characterization of rapeseed oil ester based hydraulic fluids (V,VI) 73
3.13.1 Viscosity (V,VI) 73
3.13.2.Filterability (V,VI) 74
3.13.3.Standards of purity and impurity particles (V,VI) 75
3.13.4.Foaming (V,VI) 75
3.13.5.Colour (V,VI) 76
3.13.6.Cold stability (V,vl) 76
3.13.7.Friction and wear (V,VI) 77
3.13.8. Oxidation stability (V,vl) 78
3.13.9.Corrosion stability (V,VI) 79
3.13. 1O.Biodegradability (V,VI) 80
3.14. Conclusion of laboratory tests (VVI) 80
4. Condusions 81
References 83
Uimsa, Merja. Environmentally Friendly Products Based on Vegetable Oils.
Espoo 1995. Helsinki University of Technology. Technical Biochemistry Report 2/1995.
87p+ app.65p.
UDC
ISBN
Keywords
ABSTRACT
579.66..663.1 .. 582.28
951-22-2770-3
Vegetable oils, transesterification,alcohols, Iipases, biosolvents,
biolubricants, biofuels.
The scope of this work was to study and develop new methods and processes for the
production of vegetable oil esters as raw materials in the manufacture of biodegradable
lubricants and solvents. Different esters were produced either chemically or
enzymatically. In the chemical process, catalysts were either bases such as alkalimetal
oxides or alcoholates or acidic such as phosphoric, sulphUric or p-toluenesulphonic acid.
In the enzymatic reactions lipases used were produced by Candida rugosa (formerly
cyJindracea), Micor miehei, Pseudomonas Buorescens or Micor sp. The vegetable oils
used were Finnish rapeseed oil, soybean oil, coconut oil and their esters. Alcohols
employed were mono-, di- and polyols. The produced esters were methyl-, ethyl-, 2-ethyl-
1-hexyl- and trimethylolpropane esters.
The mono esters were prepared to be used as solvents or fuels or as raw materials for
further synthesis, preparation of raw materials for hydraulic fluid use. Polyol esters were
prepared to be used as raw materials for lubricants. In the chemical methods
temperature varied from 50C to 140C and pressure from ambient to 1.3 MPa.ln the
enzymatic methods temperature range was from 30C to 68C and pressure limit from
atmospheric to 2.0 MPa. In both cases different new routes were found to produce
desired esters with very good yields. In the chemical synthesis the conversion varied
between 85 % and 99%, depending on the ester. In the enzymatic reactions conversion
was between 82 % and 100 %.
2-ethyl-1-hexyl and trimethylolpropane ester were synthetized for the first time from
rapeseed oil. In the enzymatic preparation of the 2-ethyl-1-hexyl ester of rapeseed oil
conversion was 99.8%. Chemically, the conversion of 2-ethyl-1-hexyl ester of rapeseed
oil was 97.6%. Chemically from rapeseed oil methyl ester was succesfully synthetized
with trimethylolpropane trimethylolpropane ester with a conversion of 99%. Enzymatically
the conversion was 98%.
All of the esters have already been tested for industrial use either on the laboratory scale
or in field tests. The results were encouraging in respect of lubrication or solvent
properties.
2-ethyl-1-hexyl ester of rapeseed oil from chemical and enzymatic preparation have been
in field tests e.g.by the detergent industry to replace traditional organic solvents. These
esters are used today for example in carshampoos. Trimethylolpropane esters of
rapeseed oil (TMP ester), chemically and enzymatically synthetized, have been used as
starting materials for preparing hydraulic fluids for laboratory tests.
TMP-ester based hydraulic fluid resisted oxidation better than rapeseed oil or commercial
synthetic ester based fluids. Also cold stability properties of TMP ester were better than
rapeseed oil based or commercial synthetic ester based fluids. The products are also
readily biodegradable, non-toxic and do not bioaccumulate in nature.
PREFACE
This work was carried out dUring 1991-1995 at the Laboratory of Oil Milling Division,
Raisio Group and at the Laboratory of Biotechnology and Food Engineering, Department
of Chemical Engineering, Helsinki University of Technology.
My warmest thanks are to Professor Pekka Linko and Docent Yu-Yen Linko, whose
enthusiasm and fine co-operation made this work possible. I am deeply grateful to them
for their valuable advice and the expertise they have put to my disposal.
I express my graditude to Anne Huhtala, Esa Uosukainen,Sari Kiviniemi and Tomi
Virtanen, all of whom have done excellent work during this long process.
I also want to thank with my whole heart Mari Heikkila, Hannele Sulonen and Birgit
Vainio, whose practical work was very important in this project.
I could not have succeeded with the English language without the help of M.Sc. Anneli
Karjalainen, to whom my sincere thanks are not enough.
To my employer Raisio Group, Oil Milling Division and to my superior, director Matti
Soupas, warm thanks for making it possible to carry out this project.
I would also like to give my deepest thanks to my husband Jyrki, and to my parents Ritva
and Pekka for their caring, love and support during these years. Without them I would not
have succeeded in all this. The sweetest and lovecaring thanks to my little sunshines
Riku and Inka, who have given me the strength to continue.
LIST OF PUBLICATIONS
This thesis is based on the following publications, referred to as I to VI in the text.
Linko Y.-Y., Uimsa M., Huhtala A. and Linko P.,Lipase catalyzed transesterification of
rapeseed oil and 2-ethyl-1-hexanol, J.Am.Oil Chem.Soc. 71 (1994) 1411-1414.
II Lamsa M., Huhtala A., Linko Y.-Y.and Linko P., 2-Ethyl-1-hexanol fatty acid ester from
rapeseed oil by transesterification, Biotechnol.Techn.8 (1994) 451-456.
III Lamsa M., Process for preparing a synthetic ester from a vegetable oil, Patent
number SF 95367/1995.
IV Lamsa M., Linko P., Linko Y.-Y. and Uosukainen E., An enzymatic process for
preparing a synthetic ester from a vegetable oil, Patent number SF 95 395/1995.
v Lamsa M., Ecologically acceptable synthetic hydraulic fluids based on vegetable oils,
Proceedings of the 4th Scandinavian International Conference on Fluid Power,
September, 1995, Tampere, Finland.
VI Lamsa M., Vegetable oil based lubricants, Finnish Tribol. 14 (1) 1995 39-45.
Additional new results are also presented.
1
II In tiny, tiny drop, You can fit the whole world"
Aaro Hellaakoski
1.1NTRODUCTION
1.1. HISTORICAL BACKGROUND OF CHEMICAL ESTERS USED IN
LUBRICANTS
The development of synthetic esters in lubricant use (1) started in the 1930s in the
United States and in Germany. In Germany this was mainly due to the second world
war and shortage of petroleum. Diesters and polyol esters were developed in
Germany and first used as aircraft turbine lubricants. Their benefits were good low
temperature properties and resistance to thermal oxidation. Since 1960 the aviation
industry has used neopentyl polyol esters as lubricants for jet engines (2). Their
important feature is the flexibility of physical and chemical properties achieved by
changing the chemical structure of starting materials such as chain length,
branching, number of carbon atoms and type of alcohol.
In the 1960s synthetic esters for lubricants were further developed for arctic
conditions, functioning as crankcase, transmission and gear oils, and hydraulic fluids
and greases. During the 1970s motor oils (3) came into the picture, first semi-
synthetic oils, including diesters. Their most important benefits were good low
temperature properties and low volatility. The first synthetic motor oil esters came
onto the market in 1977 being mostly polyalfaolefins (PAO). The first two-stroke
outboard engine oils based on synthetic esters came onto the market in 1982 in
Switzerland and southern Germany (4). Their main base fluid was neopentylpolyol
ester of branched fatty acids.
The first laws concerning biodegradable lubricants were published in Portugal in
1991 by the Ministry for Environment and Natural Resources (4). It obligated the use
in outboard two-stroke engines oils, which biodegradability is a minimum of 66 % by
CEC-L-33-T-82-test. This test is the first one developed for biodegradation. It was
developed in 80s and has been used quite a long time and is a well accepted
lubricant test. OECD-tests (301-serie) have also been used since the beginning of
90s (4).
The next step from two-stroke engine oils in biodegradable products were
biodegradable chainsaw oils. They were launched in the middle of 80s. The first
products were based on natural esters, triglycerides of Finnish rapeseed oil (5).
In the 80s and still at the beginning of the 90s the main raw material for
environmentally acceptable products was rapeseed oil. Users and researchers
have noticed however that applications for example in high temperatures, demand
2
more from their raw material. From this started the development of biodegradable
synthetic esters for lubrication use. Application areas are hydraulic fluids, metal
cutting fluids, roller oils, turbine oils, industrial gear oils and compressor oils. Other
application areas are cosmetics, textile and fiber industry, heat transfer liquids and
plastics industry (6). Fatty acids for the synthetic esters were from vegetable oils or
animal fats. Pure or purified fatty acids were obtained by splitting, fractionating,
distilling, dewaxing, hydrating, cracking etc. Alcohols can vary from short chain
alcanols to polyols (4,7). Mixtures of esters are used to improve the properties of
lubricants. Mono-, di- and polyesters can be combined to achieve for example the
right viscosity behavior. Non-hindered dimer esters, made from dicarboxylic acids,
can be combined with polycarboxylic acid esters, prepared with alcohols containing
side chains. These synthetic esters can be even combined with mineral oil or
mineral oil based products ( PAO) (1,8,9).
In 1989 the first biodegradable greases, which were based on neopentyl esters
came to the market (4). Also new gas turbine engine oils based on monoerytritol and
trimethylolpropane were invented in 1989 (10).
1.2. PREPARATION OF FATTY ACID ESTERS
1.2.1. Monoesters of fatty acids
In the production of simple esters from short chain alcohols such as methanol,
ethanol or butanol and triglycerides from natural sources, most widely used catalysts
are sodium or potassium hydroxide, metal alcoholates, - hydroxides, - carbonates or
- acetates and different acids (11,12,13,14). An economic production of monoesters
in industrial scale should fulfill at least the following criteria: good yield, few by-
products, favorable energy balance, little impact on the environment and simple
process without complicated technical devices. Only few of the many different
published processes actually match with these requirements.
Figure 1: General molecular scheme of monoester synthesis from
triglycerides and short chain alcohols.
CHz-O-gR
6H- 0-8-RII +
hHz-o-gR'
triglyceride
3 R""OH
short chain
alcohol
CHz-OH
l \ c a t a l ~ t 3 R"'"..o-8-RIIII+ hH- OH
~ I
CHz-OH
ester of glycerol
short chain alcohol
3
The least expensive catalysts are sodium and potassium hydroxides. The quantity
needed is around 0.6 % (wlw) of total amount of reactants. The amount of alcohol
should exceed the theoretical value to force the reaction toward the products.
Mittelbach et al (11) used in their method a 1.6-fold alcohol excess. In the literature
many researchers have used acids to neutralize the alkali, but Mittelbach et al (11)
found out that washing with 50-60C pure water several times was a better way to
avoid saponification problems.
Kusy (15) used in the transesterification reaction 1.75 equivalent of ethanol and 0.5
% (wlw) sodium methoxide in methanol. These figures were based on the patent of
Bradshaw and Meoly (16). Reaction catalyst was deactivated by adding sulfuric acid
(1:1) to reaction mixture, adjust the pH between 5-6. The acidification prevents,
according to the authors, the reverse reaction towards the starting materials and
helps to separate the glycerol phase.
If the catalyst was not deactivated by acid, Kusy found out that only half of the
expected glycerol was obtained. The product contained mono- and diglycerides,
which remained in an inseparable emulsion, if only water washing was done. This
emulsion was analysed by IR-spectrophotometry and proved to be mono- and di-
glycerides.
Consequently, Kusy used both neutralisation with acid and water washing with warm
water to remove the rest of the glycerides, salts of sulfuric acid and the rest of the
used catalyst.
Freedman et al (12) suggested that the optimal excess of alcohol is six times the
theoretical value. They pointed out that acoording to Bradshaw (16) 4.8-fold excess
was enough and jf alcohol was added in three or four subsequent portions, this
could be reduced from 4.8 to 3.3. Freedman et al (12) studied also the effect of the
molar ratio on ester yield, because different researchers had published so variable
results (Figure 2). When the molar ratio of short chain alcanol and triglyceride was
6:1, 98 % ester conversion was obtained. When the molar ratio was decreased to
the theoretical level of 3:1, the ester yield decreased to 82 %, also the amount of di-
and monoglycerides increased. They were at their lowest at the molar ratio 6:1 and
increased thereafter showing that the conversion to ester was incomplete. The
optimal temperature was a few degrees lower than the boiling point of the alcohol
used. The reaction time varied from a few minutes to few hours, depending on the
alcohol used. Effect of temperature could be compensated by a shorter reaction
time. If tile reaction temperature was lower than a few degrees below the boiling
point, an almost equal conversion of ester could obtained with a longer reaction
time. For example, in the methanolysis of soybean oil at 60, 45 and 32C after one
hour reaction time the conversion was identical to that at 60C, 45C and 32C after
fOur hour reaction time, respectively.
4
Figure 2: Effect of molar ratio on ester yield, natural triglycerides and short chain
alcohols, 0.5 % (wlw) catalyst, 60
D
e, 1h).
Ester (_); triglyceride (0); diglyceride (.); monoglyceride (0).
100 ,.
I
90 t
80 t
,
_ 70 +
c 60+
g I
. ~ 50!
1; 40
o I
o ~ ~ ~ = = = = ~ = = " " ' 1
o i 2 3
molar ",no ( short chain alcohol:vege18bJe oil)
Different catalysts have been also tested. Using 15 %(wlw) sodium hydroxide or 0.5
% (wlw) sodium metoxide with a molar ratio 6:1 (alcohol: triglyceride), the
conversions were identical after one hour reaction time. With alkaline catalysts, the
results were much better than with acidic catalysts. With acidic catalysts reaction
times were much longer, required temperatures were much higher, and more excess
of alcohol was needed. For example. in soybean oil ethanolysis with unsatisfactory
conversion of ester was achieved 1 % (wlw) sulphuric acid as catalyst, molar ratio
20:1 (alcohol: triglyceride), after three and eighteen hours (11).
The technical use of monoesters of fatty acids have lately, from the beginning of
90s, been mainly fuels or solvents.
1.2.2. Diesters of fatty acids
Diesters are produced in the chemical reaction of two monohydric alcohols with a
dicarboxylic acid or a diole and a monocarboxylic acid (1,17). Since water is the
other product in the equilibrium reaction, it is essential to remove the produced
water.
5
Figure 3: The general scheme of diesters
dicarboxylic acid diester
diol carboxylic
acid
diester
The ester synthesis from dicarboxylic acids is more commonly used than ester
production from diols.
For example azelaic, sebatic, oleic, and malic acids and malic anhydride have been
used as the diacids. Most widely employed alcohols are 2-ethylhexanol, i-tridecyl
alcohol and a mixture of C8-C10-alcohols (1,12,13). One of the most common
diesters used in lubrication is di-2-ethyl-hexylazelate. This ester is prepared from 2-
ethyl-hexanol and azelaic acid (1). Diacids have reacted with alcohols using diacid:
alcohol molar ratios of 1:1 or 1:3. Catalysts have been either alkaline, e.g. sodium
hydroxide, carbonate or -phosphate or alternatively acidic types. The amount of
catalyst varies from 0.1 to 0.25 % (wlw). The optimal reaction temperature is
between 180-200C, always near the boiling point of the used alcohol, and the
pressure atmospheric. Reaction time varies from half an hour to five hours. The
produced water is always removed to force the reaction toward the side of products
(1 ).
Diesters have been used in gasturbine engines, compressors, and both hydraulic
and two-stroke engine oils. The first lubricants used in gasturbine engines were
dioctylsebacates. Later these esters were replaced by azelates and adipates and
more recently by polyolesters (3,18).
Diesters have exceptionally good low temperature properties and high viscosity-
index, and they operate quite well as lubricants. As an example of diesters, 2-
ethylhexanol diesters and their main properties are d e ~ b e d in Table 1.
6
Table 1: 2-ethylhexanol diesters and their properties (1,2,17,18,19).
acid viscosity viscosity VI pourpoint
37.8C 98.9C
(mm
2
/s) (OC)
adipine 8.2 2.4 123 -56
2,2,2-tri-
methyladipine 12.0 2.8 89 -54
atselaine 11.8 3.1 144 -58
sebasine 12.5 3.3 155 -51
dodecane-
dicarb. 15.2 3.9 168 -46
nonadecane 23.5 4.8 141 -49
ftalic 30.2 4.4 20 -42
isoftalic 32.6 4.7 47 -43
tereftal 30.5 5.1 100 -44
It is obvious from Table 1 that the use of branched chain alcohols and acids lowers
the viscosity-index more than the straight chain compounds. The use of cyclic
substrates increase the viscosity markedly.
For two-stroke engine oils esters, such as non-hindered dimer-ester can be used. It
is prepared from diacids such as oleic acid dimers combined with linear
monoalcohols (20). Dibasic acid esters are made from diacids like sebatic acid and
alcohols (21). This type of esters has the advantage of uniform chemistry, uniform
properties like low temperature properties. The pourpoint can be as low as -55C.
1.2.3. Polyolesters of fatty acids
Po/yol esters are produced in the reaction between polyhydric alcohols and mono- or
dicarboxylic acids. They are also called hindered esters. This reflects the structure of
the polyols, where the p.carbon atom does not have anylXf-hydrogen in it. The most
widely used polyalcohols are trimethylol propane (TMP) (A)
7
and other trimethylol alkanes, neopentylglycol (NPG) (8) , pentaerythritol (PE) (C),
ditrimethylolpropane (diTMP) and dipentaerytritol (diPE) (22,23,24,25). Some of them
are shown in Figure 4.
Figure 4: Examples of nOiX<-hydrogen alcohols used in polyol ester synthesis.
<;:;H
2
-OH
Et-C-CH2-0H
CH
2
-OH
(A)
Me
HO-CH
2
--CH
2
-OH
Me
(8)
9
H
2-
0H
HO-CH
2
-9-CH
2
-OH
CH
2
-OH
(C)
Acid composition varies widely, depending on the applications of the final polyol
ester. The acids used in polyol ester synthesis can be short-, /ong-, saturated-,
unsaturated-, straight- or branched chainacids. Recently the tendency has been to
use mixtures of different types of acids. Some examples of acids used in these
reactions are shown in Figure 6. The following features of the starting compounds
affect the properties of the resulting ester: molecular weight, the size of the acyl
groups, the functionality of polyols, and the method of preparation of the ester or the
mixture of esters (2,3).
Figure 5 demonstrates how the functionality and po/yol chain length and branching
affects the properties of the ester (26,27).
Figure 5: The effect of polyol chain length and side chains on the properties of
polyol ester.
Influence of chain length
:>.
.lJ
''';
UJ
a
o
UJ
...;
:>
length
.lJ
I::
''';
a
0.
\-l
::l
a
0...

Influence of side chains
:>.
.lJ
...;
rn
a
o
rn
...;
:>
8
Figure 6: Different acid types used in the preparation of polyol esters.
R
R-y-COOH
R"
monocarboxylic acids
R
I
R-CH-COOH
a..branched acids
Me R
Me-Q-(CH
2
)n-CH-(CH
2
)n-COOH (OH)x-R-COOH
Me
isocarboxylic acids acids cont. hydroxylic groups
Aviation gas turbine engines work with a polyester composition made by reacting
polyalcohols with monocarboxylic acids as the lubricant (28). An example of such
products is the commercial pentaerytritol esterified with a mixture of C5 to C9
carboxylic acids (28).
Car engine lubricants can be prepared by total esterification of TMP by a mixture of
saturated aliphatic carboxylic acids, typically by using 134 g (1 mol), TMP, 36.5 g
(0.25 mol) adipic acid, 130 g (1 mol) heptanoic acid and 465 g (1.5 mol) isostearic
acid (29). The reaction temperature is close to the boiling point of TMP and the
reaction time 8 hours. Ethyl Corporation has obtained a patent for producing TMP-
ester with a mixture of aliphatic monocarboxylic acids containing 4 to 12 carbon
atoms (30). An example of the reaction procedure is as follows: 67 parts of TMP,
235 parts of an acid mixture containing 26 % hexanoic acid, 43 % octanoic acid
and 31 % decanoic acid are dissolved in 17900 parts of xylene, using sodium
bisulphate (6 parts) as the catalyst and with a total reaction time of 8 h. Water is
removed from the reaction mixture during the entire reaction, and the mixture is
finally washed with 10 % caustic soda and with water until it is neutral.
One possibility for the acid composition is to useo<-branched acids (31). This type of
esters have been used in automatic transmission fluids. The reaction could be
carried out as follows to obtain an ester yield of 96 %: 25 parts of TMP, 2,5 parts of
a,branched chain acid and 0,5 parts of an acid with a lower molecular weight are
mixed. The catalyst is tetra-n-butyltitanate (TNST), which decomposes in air, so
nitrogen atmosphere is necessary as a protection. Reaction temperature is varied
between 180 to 250C to reflux reaction mixture. The total reaction time is about 8 h.
Synthetic polyol esters can also be made by using a single purified acid together
9
with polyols (32). The acid can, for example, be a commercial pelargonic acid (4582
parts), the other components TMP (1206 parts) and toluene (260 parts) as solvent
and p-toluene sulfonic acid (8 parts) as catalyst. The mixture is refluxed for 18.5
hours with a maximum temperature of 216C and water is separated continuously.
The crude ester is obtained upon removing of the excess of alcohol and solvent by
distillation and purifying by calcium oxide water at 50-60C. Water is then distilled off
and, decolorising carbon added. The esterification can also be done stepwise. There
are at least two different types of acids, which can be added. One example is
described by Metro, Hoffman and Matuszak using TMP as the alcohol (33).
TMP (1 mol, 134 g) and neo-octanoic acid (1 mol, 150 g) are mixed with sodium
bisulfate (0.4 % (wlw)) as catalyst and heptane as the water-removing agent. The
reaction mixture is then refluxed at atmospheric pressure, until 1 mol of water has
been removed. Then pelargonic acid (12.2 mol,34 g) is added together with
additional catalyst ( 10.1 % (wlw)). The reaction mixture is heated to reflux so that 2
moles of water are separated.
An improved lubricant composition has been achieved by using a mixture of acids
including straight chain acids (C6 to C10) and iso-acids (C6 to C10) for example iso-
nonanoic acid (10). An example of a reaction procedure of this type is the following:
66.7 parts of TMP, 20.2 parts of straight chain acids and 13.1 parts of iso- acids is
used as reactants, tinoxidate as the catalyst at 238 to 243C at 3.3 kPa with nitrogen
atmosphere. Water has to be separated during the whole reaction time. The total
reaction time is controlled by the hydroxyl value and the reaction is stopped, when
the hydroxyl number is sufficiently low.
Table 2: Properties of different acid containing polyol esters
(19,28,29,34,35).
Type of aCId Alcohol Viscosity VI Pour-
40'C point
(mm'ts) ('Cl
non -hydrogen TMP 23.9-35.0 -40-59
-branched TMP 32.6 137 -37
straIght chain
monocartloxylic di-TMP 27.9-28.1 135-140 -57-59
straight chain
monocartloxyhc 70% TMP 24.8 144
30% di-PE
mixture of straight
-50
chain and isochain TMP 23.9
mixture of straight
-54
chain and isochain PE 23.8
mixture of mono-
and dicartloxylic TMP 135-152
-20-40
hydroxyl containing TMP 20.5-35-2 106-167
hydroxyl containing PE 43.7-142.3 85-186
unsatisfactory PE 22.0-46.0 180-200
-30-40
10
1.3. LIPASE CATALYZED FATTY ACID ESTER PRODUCTION
1.3.1. Definition of lipases
Lipases (triacylglycerol acylhydrolases EC 3.1.1.3 ) are esterases widely found in the
animals, micro-organisms and plants. Their main biological function is to catalyse the
hydrolysis of triacylglycerols to fatty acids, diacylglycerol, monoacylglycerol and
glycerol (Figure 7). The reaction is reversible, so under certain conditions the
enzymes can also catalyse ester synthesis such as the formation of acylglycerols
from glycerols and free fatty acids (36-39).
Figure 7: Lipase reaction
Triglyceride
H
Diglyceride + free fatty acid
H
Monoglyceride + free fatty acid
H
Glycerol + free fatty acid
The natural substrates of Iipases are triacylglycerols of long chain fatty acids. They
have a very low water solubility but the lipases can quickly catalyse the hydrolysis of
ester bonds at the interface between an insoluble substrate and water. The ability of
lipases to catalyse the hydrolysis of water inspluble fatty acid esters separates them
from other esterases, which catalyse the hydrolysis of water soluble esters. Lipases
catalyse a large number of the hydrolysis of water insoluble fatty acid esters,
although the best substrates are acylglycerols.
11
. 1.3.2. Structure of Iipases
Lipases studied consist of 270 to 641 amino acids and they have a certain degree of
similarity, regardless of their origin. Some researchers have proposed, that Iipases
have an especially high proportion of hydrophobic amino acids and this results in the
interaction of hydrophobic amino acids and substrates (36,37). However, the
research of pUblished amino acid compositions clearly shows that as a group Iipases
are no more hydrophobic than other enzymes. The strong interaction with
hydrophobic compounds is propably due to the hydrophobic patches on lipase
surface. In the lipase from pancreas and MiJcor mieheifor example, the active
centres contain a structurally analogous Asparagine- Histidine-Serine triad (41). In
the Geotrichum lipase a catalytic triad of a similar structure Serine- Histidine-Glutine
was found. For each of these three lipases, the catalytic site has been found to be
covered by a helical lid, which opens at the lipid-water interface or in an organic
solvent. The lipases thus undergo a conformational change before becoming
catalytically active. The molar mass can vary from 27 000 g/mol ( lipase of
Penicillium cyclopium) to 500 000 g/mol ( lipase in human saliva) (36,37).
Several Iipases from different sources have been pUrified and crystallized. Most
purified Iipases contain 2- 15 % carbohydrates with usually glucoside residue
mannose. Galactose, xylose, arabinose and hexoseamine have also been reported.
The role of the carbohydrate moiety has not yet been clarified and its importance to
the activity has been questioned (36,37,41).
1.3.3. Properties of Iipases
Lipases are generally quite stable in neutral water solutions at room temperature.
Microbial lipases are usually more thermostable than animal or plant Iipases (42).
Lipases produced by Aspergillus niger, Rhicopus japonicus and Chromobacterium
viscosum are stable even at 50C. Humicola lanuginosa produces a lipase, that is
stable at 60C. The Pseudomonas Buorescens enzyme used in heat-treated dairy
products is only partly inactivated in these processes (36).
Generally Iipases are active in a wide pH range with the highest activity at a pH 6 to
8. Extracellular Iipases produced by Aspergillus niger, Cromobacterium viscosumand
Phizopus arrhizus are extremely active at a low pH and Pseudomonas nitroreducans
lipase is active at pH 11 (36).
Microbial lipases have an exceptionally high surface activity at air-water interface
when compared to other proteins. This high surface-activity leads to a strong
adsorption of Iipases to hydrophobic surfaces and can explain the high activity of
Iipases with insoluble substrates (37,43). Table 3 lists some properties of
commercially available lipases (36,37,43).
12
Table 3: Some properties of commerciallipases (36,37,43)
Species Molecular Carbohydrate Specific Average
weight content activity+ hydrophob.
(0/0)
Aspergillus
niger
25000 10 2400 1 058
Candida
rugosH>
120000 4.2 1 140 1 150
Chromobacterium 30000 0.0 5780 986
viscosU11f'
Geotrichum 54000 7.0 447 1 135
candidwrf
Humicola 27500 0.0 1 490 1 079
lanuginosa
e
Afucor
21 000 2.6 1 322
javanicuS
Pseudomonas 32000 0.0 4200 1 001
Duorescen#
Rhizopus 43000 6.7 9300 1 097
arrhizu1'
Rhizopus 41 300 4000 1 270
delemarJ-
+= From purified lipase ( lipase units! mg protein)
a= M.P.Tombs, private communication
b= Tomizuka et ai, 1966
c= Isobe and Sugiura, 1977; Horiutu and Imamura, 1977
d= Tsujisaka et aI, 1973
e= Liu et aI, 1973
f= Ishihara et ai, 1975
g= Sugiura and Oikawa, 1977; Sugiura et aI, 1977
h= Semeriva et ai, 1969; Laboureur and Labrousse, 1968
j= Chiba et ai, 1973; Iwai and Tsujisaka, 1974
13
1.3.4. Specificity of lipases
The specificity of Iipases can be divided into five main groups: Iipid-, regio- and fatty
acid specificity, stereochemical specificity and a combination of these. The degree of
specificity varies widely from distinctly specific to weakly or even non-specific. Lipid
specific Iipases have a high activity in hydrolysis of mono-, di- and triglycerides. For
example Penicillium cyclopium lipase has the highest activity to monoglyceride
substitutes (37,38,44).
Regiospecificity can be devided into three groups: 2-specific, 1,3-specific and non-
specific (31,37). Figure 8 shows some regiospecific reactions. Non-specific lipases
catalyse the total decomposition of triglycerides to free fatty acids and glycerols
with di- and mono-glycerides as products. When a non-specific lipase is used, the
resultant fatty acid composition is similar to that obtained with chemical
transesterification. If there is a double bond or a bulky substituent in the fatty acid
near to the carbonyl group, there is a resistance to the attack by enzyme. Such
components are poor substrates for lipase for steric reasons. Extracellular Iipases
produced by Candida rugosa, Cromobacterium aenes, Propiobacterium aenes,
St3phyloroccus aureus and Geotrium cadicum show no marked regiospecificity
(36,37,44).
The second group of Iipases are regiospecific, releasing fatty acids from 1- and 3-
positions of triacylglycerols. 1,3- Specified lipases produce free fatty acids, 1,2(2,3)-
diglycerides and 2-monoglycerides.as reaction products. Because the last two are
chemically unstable, under longer reaction times a total migration of fatty acids take
place. Regiospecific Iipases are produced for example by Aspergillus niger, Mucor
javanicus and Phiedzopus arrhizus. Also lipase Mucor miehei has been claimed to be
1,3-specific (44,45).
14
Figure 8: Products formed by lipase catalyzed hydrolysis of triglycerides.
1) Non-specific lipase
R COO-
y
H
2
9H2-0H
+ R'COOH + R"COOH + CH- OH
R"COO-CH
2
CH
2
-OH
2) 1,3-specific lipase
RCOO- yH
2
yH
2
-OH GH
2
-OH
R'COo-CH;=R COO-CH + COO-CH + R"COOH
R"COo-CH
2
R"COO-CH
2
CH
2
-OH
3) Fatty acid specific lipase
RCOO- yH
2
R'COO-QH
2
HO-QH
2
R'COO-9H
2
R'COO-CH + RCOO- RCOOH + R'COO-CH + CH-OH
R"COO-CH
2
R"COO-CH
2
R"COO-CH
2

In Figure 9 the reactions of non-specific and 1,3-specific microbial Iipases have been
demonstrated.
15
Figure 9: Positional specificity of microbial lipases.
1.Reaction catalysed by non-specific Iipases
2. Reaction catalysed by 1,3-specific lipases
CH
2
-o-gR CH
2
-OH
o I 0 I 0 !
R-t-O-CH ~ R - ~ - O - C H + R C O O H ~ R - t - O - CH + 2RCOOH
I 9 ! q !
CH
2
-O-C-R CH
2
-O-C-R HO-CH
2
2-specificity is extremely rare. For example Geotrichum viscosum lipase is claimed to
hydrolyse the fatty acid in the 2- position in oleic and linoleic acids (44).
One group of Iipases also express specificity towards a certain group of substrates.
Specificity can be for the chain length of carbonatoms in substrate, for a quantity of
unsaturated bonds or for the place of a double bond in the carbon chain (45). For
example lipases of Candida rugosa, Aspergillus niger, Geotrichum candicum catalyse
well the release of fatty acids of 18 carbom atoms, which have a cis- double bond in
position nine, as in oleic and linolene acid (36,46,47,48). The lipase in wheat embryo
is selective for monosubstituted fatty acids, and pancreatic lipase hydrolyses most
actively C4 saturated substitues of fatty acid chains.
Stereochemical specificity of some lipases has been reported for straight chain
secondary alcohols, optically active esters of acetinide and butyric acid,
cyclohexanals, 2-benzyl and glyceral ethers, sugaralcohols and enantiomeric esters
of ibuprofen (44). The human and rat saliva Iipases hydrolyse enantiomeric esters
from the 3-position. Many lipases are also stereo specific, which has been utilized in
optical resolution and in chiral synthesis. For example Candida rugosa lipase exhibits
preference toward (L)(-)-menthol (49).
16
1.3.5. Production of lipases
Lipases are produced by plants, animals and micro-organisms (39,45). The majority
of commercially employed Iipases are of fungal origin, and at least 20 are available
in commercial quantities (50). The porcine pancreatic lipase has been widely
studied. Also milk contains Iipases (51) and according to Hills and M u k h e ~ e e
rapeseed is a promising source for Iipases (52).
The formation of lipase is usually supported by the presence of mono- or
disaccharides, glycerol and/or lipids in the growth medium (40).
Carbon sources used in the production of extracellular Iipases are mainly
polysaccharides, such as starch, triglycerides or fatty acids. Common nitrogen
sources are soybean meal, yeast extract or com steep liquor. The most widely used
assay of Iipases is based on emulsions of insoluble triglycerides like olive oil. An
example of microbial lipase producers is shown in Table 4 (39,42).
17
Table 4: An example of microbial lipase producers (39,42).
Manufacturer Country Organism
Amano Pharmaceu- Japan Aspergillus niger
tical CO,Ltd Rhizopus niveus
Pseudomonas Buorescens
Biocatalysts Ltd United Aspergillus niger
Kingdom Candida rugosa
Chromobacterium viscosum
MiJcor miehei
Pseudomonas Buorescens
Gist-Brocades NV Holland MiJcor miehei
Hughes and Hughes United Rhizopus arrhizus
(enz.) Ltd Kingdom
John &E Stuge Ltd United Aspergillus niger
Kingdom
Meito Sangyo CO,Ltd Japan Candida rugosa
Novo Industry AfS Denmark Aspergillus niger
lYfncor miehei
Osaka Saikin Japan Rhiropus japonicus
Kenkyusho
Sapporo Breweries Japan Pseudomonas fi-agi
Toyo Jozo Co, Ltd Japan Chromobacterium viscosum
18
1.3.6. Reactions of Iipases
In addition to the hydrolysis of fats and oils transesterification is one of the most
common reactions performed by Iipases. Different types of reactions are shown in
Figure 10. Transesterifications without solvent have several benefits. Substrates can
be used in high concentrations. If substrates are used in stoiciometry, the final
product can easily be separated, for example by filtration. There are also fewer
byproducts and waste treatment is easier. The process is safer, because reaction
temperature and pressure is relatively low (43,51,52,53).
Transesterifications have been used in oil and fat industry in order to modify the
composition of fats and oils and to change the physical properties of triglycerides to
the desired direction (38). One major difference between chemical and enzymatic
interesterification reactions is that one can obtain acyl migration with 1,3-specific
enzymes, which is impossible to achieve with chemical catalysts.
19
. Figure 10: General reactions by Iipases.
I Hydrolysis of ester
R-Ko-R + Hp -- R-8-0H + HO-R
II Synthesis of ester
R-KoH + HO-R R-Ko-R + Hp
III Transesterification
III A Acidolysis
R
1
-gO-R + Rf gOH --Rz-gO-R + R
1
-gOH
III B Alcoholysis
R-g.O-R
1
+ HO-R
z
---- R.gO.R
z
+ HO-R
1
III C Interesterification
R
1
-g-O-R
1
+ RzgO-R
z
---R
1
-go-R
z
+ R
z
-gO-R
1
1110 Aminolysis
R.g-O-R
1
+ HzN-R
z
--- R-8-NH-R
z
+ HO-R
1
20
Alcoholysis of long-chain fatty acids has been investigated by quite a few
researchers. One example is celite immobilized Pseudomonas Duorescens lipase as
catalyst in the alcoholysis of tripalmitine, trioleine and olive oil together with ethanol
and isopropanol (54,55). Lipozyme (a Milcor miehei lipase, immobilized on
phenolformaldehyde resin; Novo Nordisk, Denmark) has been used for example in
transesterification of trioleine and stearyl alcohol. Also di- and mono-oleins were
produced (43). Enzymatic ester hydrolysis is described in general in Figure 11 (55).
The acyl-enzyme intermediate is similar to that of serine protease.
The removal of water during the reaction clearly affects the final yield of ester. An
example of this is the reaction of oleic acid with oleyl alcohol. The optimum yield of
85 % reported, when no water was removed during the reaction. If the reaction was
carried out in vacuum and the produced water was continously eliminated, the
esterification was forced to completion (55).
Figure 11: Enzymatic ester hydrolysis.
HOH R'OH
o Enz 9 Enz
R C - O R ' ~ RC-Enz---:;;;;=:
R OH HOH
R'-OH> HOH
o Enz 9 Enz
RC - O R ' ~ RC-Enz---:.;;;;;:::
R OH HOH
q
RC-OH
R ~ - O H
Miyoshi Oil and Fat Co of Japan have used Candida rugosa lipase to hydrolyse oils
for soap production (56). Enzymes were used instead of the conventional chemical
process to achieve better odour and colour of the products. It has also been
claimed that the enzymatic process is overall more economic than the chemical
method (56).
An example of an esterification reaction with lipases is the production of geranyl and
menthyl esters from butyric acid and geraniol or lauric acid and menthol,
respectively. The final products are high-valued chemicals, where the chemical
process is expensive and complicated (49).
It has been discovered recently that lipases also catalyse the formation of
peroxycarboxylic acids (41). An example is the epoxidation of cyclohexene with
21
lipase, hydrogen peroxide and a catalytic amount of a long or medium chain
carboxylic acid.
1.3.7. Effect of water on Iipases
Water is essential for enzyme catalysed reactions. Usually a low water activity is
favourable for lipase catalysed ester synthesis, while excessive water favors
hydrolysis (57,58). Water is essential for lipase, which will become inactive due to
structural changes when all water is lost (42). It is assumed that in an environment
of very low water content, the conformational space of the enzyme is restricted. In
completely waterless environment, there is no space for comformational changes
necessary for substrate binding. Lipases from moulds (like Rhimpus and Penici11um
sp.) seem to be more tolerant to low water actiVity than bacterial Iipases for
example Pseudomonas sp. (44,59). The water content is also important, affecting
reaction rate, product yield, selectivity and operational stability. For example when
pig pancreas lipase is wet (3.6 % (wlw) , the reaction speed for transesterification is
clearly faster than for "dry" lipase (0.48 %(wlw (60). Water quantity affects also
substrate specificity. Many Iipases have an optimum range for water quantity. For
example commercial Candida rugosa lipase shows little or no activity without added
water, but by adding water reaction rate rises markedly until it reaches the
maximum. Recently the emphasis in lipase studies has been on the measuring and
control of water activity in lipase catalyzed reactions (61). One way is to continuously
adjust the water activity in the headspace above the reaction mixture. This can be
done by operating a reactor with a saturated water solution in contact with the
reaction mixture.
Another possibility is to use pairs of salt hydrates acting as buffers of water activity.
1.3.8. Immobilization of Iipases
Because Iipases are active at the interface between water and oil, the total surface
area has a pronounced effect on the reaction rate. The surface can be increased
either by emulsifying or by immobilising the enzyme to a proper carrier. The purpose
of immobilisation is also to bring the lipase into a form, in which it can be physically
separated from the substrate or product for reuse. Additionally, with this technique
reactions can be carried out continously or repeatedly, and the thermo and storage
stability of lipases can be improved.
The carrier should be chosen in such a way that the essential water in lipase is
retained. The carrier should also have sufficient mechanical strenght, microbiological
durability, chemical resistance and functionality, hydrophobic or hydrophilic character
and the ability to regenerate easily (44).
There are two main categories for immobilisation, the chemical methods, in which
22
covalent bonds are created between the lipase and carrier and the physical
methods, in which weaker interactions in lipase are used. The most common
immobilisation method is adsorption to a proper carrier, like Celite, cellulose
derivatives, porous glass or ceramic pearls, aluminum- or titanium oxide and
ionexchange resins. Lipases can also be immobilised by entrapment in agaros in
cellulosic matrix, calcium alginate or polyacrylamide. However, there is no general
immobilization technique available for every process and an optimal application
should always be developed for each specific case (62).
23
1.4. AIMS OF THE PRESENT WORK
The main purpose of this work was to find new biotechnical and chemical ways to
produce in high yields vegetable oil based esters. Another purpose was to invent
new uses of vegetable oil based esters. This was planned to do by laboratory- and
field tests.
The alcohols used were chosen by the end use of the desired ester. They were
short, straight chain alcohols such as methanol, ethanol or long, side chain alcohols
like 2-ethyl-1-hexanol or polyols like trimethylolpropane.
Rapeseed oil, soybean oil and coconut oil or their esters, and tallow oil fatty acids
were used as the acid donor.
The transesterification reaction was to prepare monoester from vegetable oil and
short, straight chain alcohol (11,12,13,14). Reaction conditions were tried to be
improved, so that, for example, industrial scale production of rapeseed oil methyl
ester (RME) could be started. RME was also planned to be used as a solvent.
Because RME caused swelling and brittling of some gum materials, a more suitable
alcohol (2-ethyl-1-hexanol), was succesfully found from literature (17,18,19).
The 2-ethyl-1-hexyl ester of rapeseed oil was planned to prepare both chemically
and enzymatically. It was ment to compare the preparation methods, their costs and
conversion and purity of the wanted ester. 2-ethyl-1-hexyl ester of rapeseed oil was
planned to be tested as solvent in for example cleaning printing ink rollers or as to
replace organic solvents in carshampoos.
Different polyolesters were considered as raw materials for lubricants, especially
hydraulic fluids. Their raw materials were as acid donor tallow oil fatty acids or RME
and as alcohol trimethylolpropane (TMP). These reactions were planned to do either
chemically or enzymatically for the same reasons as for esters mentioned above.
From obtained esters hydraulic fluids were meant to prepare with additives. These
esters were tested in laboratory scale by standardized methods.
24
2. MATERIALS AND METHODS
2.1. VEGETABLE OILS
Rapeseed oil
Rapeseed oil was distil/ed and raffinated by Raisio Group, Oil Milling Division and was
entirely based on Finnish rapeseed.
The medium fatty acid composition was:
oleic acid 57 %
Iinolic acid 22 %
linolenic acid 12 %
palmitinic acid 4 %
stearic acid 1 %
eicosenic acid 2 %
erucic acid 1 %
others 1 %
The medium molar mass of rapeseed oil was 880 g/mol.
Soybean oil
Soybean oil was also distilled and raffinated by Raisio Group, Oil Milling Division.
The medium fatty acid concentration was:
linoleic acid 55 %
oleic acid 22 %
palmitinic acid 11 %
linolenic acid 8 %
stearinic acid 4 %
The medium molar mass of soybean oil was 885 g/mol.
Coconut oil
Coconut oil was a commercial product imported by Raisio Margarine.
The medium fatty acid concentration was
lauric acid 46 %
myristic acid 18 %
palmitic acid 10 %
oleic acid 7 %
caprylic acid 6.5 %
isodecanoic acid 6.5 %
stearic acid 3 %
linoleic acid 2.5 %
caproic acid 0.5 %
The medium molar mass of coconut oil was 795 g/mol.
25
Tallow oil
Commercial tallow oil was purchased by Raisio Chemicals.
The medium fatty acid concentration was:
oleic acid 41.5%
stearinic acid 15 %
myristic acid 1.5 %
palmitoleic acid 1 %
eicosenic acid 1 %
lauric acid 1%
others 39 %
The medium molar mass was 275 g/mol.
2.2. ALCOHOLS
Methanol (in the laboratory p.a., J.T. Baker, The Netherlands; at the factory Neste Resins
Ltd., Finland)
Ethanol Aa (Alko Ltd., Finland)
2-Ethyl-1-Hexanol (J.T. Baker, The Netherlands or Fluka Chemie Ag, Switzerland)
Trimethylolpropane (J.T. Baker, The Netherlands)
2.3. CHEMICAL CATALYSTS
Potassium hydroxide (Merck, Germany)
Sodium hydroxide (Merck, Germany)
Sodium methoxide (J.T. Baker, The Netherlands or Merck-Schubert, Germany)
Sodium ethoxide (J.T. Baker, The Netherlands)
Phosphoric acid (Merck, Germany)
Sulphuric acid (Merck, Germany)
p-Toluenesulphonic acid (Merck, Germany)
2.4. REAGENTS
Hydrogen chloride (Merck, Germany)
Sulphuric acid (Merck, Germany)
Sodium sulphate (Merck, Germany)
2.5. SOLVENTS
Toluene (J.T. Baker, The Netherlands)
Heptane (J.T. Baker, The Netherlands)
Acetone (Merck, Germany)
Acetone p.a. (Riedel de Haen, Germany)
26
2.6. ENZYMES
Lipases from the following micro-organisms were used
Candid1l rugosa (formerly cylindracea) (Biocatalyst Ltd., Great Britain, batches 1911021,
292173, activity 42500 U/g, batch 9922238, activity 80000 U/g; Meito Sangyo Co Ltd.,
Japan, batch L2502, activity 360000 Ulg)
Chromobacterium viscosum (Biocatalyst Ltd, Great Britain, batch 21690, activity 13300
Ulg)
Mucor miehei (Biocatalyst Ltd, Great Britain, batch 200906, activity 7200 Ulg; Lipozyme
1M 20, Novo Nordisk, Denmark, immobilised to porous, weakly alkalic anion exchange
resin, activity 380 Ulg)
Pseudomonas fJuorescens (Biocatalyst LTd., Great Britain, batch 24090111, activity 11900
Ulg; batch 10922342, activity 7700 Ulg)
MUcor sp. (Lipase M-AP 10, LMK 10529, Amano, Japan, activity 1700 U/g)
2.7.ANALYTICAL METHODS
Thin-layer-chromatography (TLC)
Thinlayer Kieselgel 60 F 254 (Merck, Germany) plates were used.
Standards:
All standards made in Raisio Group, Laboratory of Oil Milling Division, were purified with
preparative TLC. The standards were:
rapeseed oil (Raisio Group)
trioleine (Raisio Margarine or Sigma, USA)
dioleine (Raisio Margarine or Sigma, USA)
mono-oleine (Raisio Margarine or Sigma, USA)
methylester of rapeseed oil (Raisio Group)
TMP-oleate (Unichema, The Netherlands)
TMP-ester of rapeseed oil (Raisio Group)
2-ethyl-1-hexylester of rapeseed oil (Raisio Group)
Eluents
Hexane (Merck, Germany)
Diethylether ( Merck, Germany)
A
4
1
ratio
B
9
1
C
96
4
D
Hexane (Merck, Germany) 40
Diethylether (Rathburn, Great-Britain) 10
Acetic acid (Riedel de Haen, Germany) 1
27
colouring solutions ratio
I Ethanol Aa ( Alko Ltd., Finland) 50
Sulphuric acid ( Merck, Germany) 50
II Acetic acid( Riedel- de Haen, Germany) 100
Sulphuric acid ( Riedel- de Haen, Germany) 2
Anisealdehyde ( Merck, Germany) 1
III 2-7-Dichlorofluorocene ( Aldrich-Chemie, Germany) 0.1 % in
ethanol Aa (Alko Ltd, Finland)
High pressure liquid chromatography (HPLC)
Method 1.
pump: Waters 501 HPLC
detector: Waters differential refractometer
integrator. Merck-Hitachi D 2000
column: Ultrastyregel 500 A(Waters-Millipore)
Ultrastyregel 100 A (Waters-Millipore)
eluent: Tedrahydrofuran HPLC-quality (Merck, Germany), flow rate 0.5 mm
3
/min.
standards: rapeseed oil (Raisio Group)
methyl ester of rapeseed oil (Raisio Group)
trioleine (Raisio Group)
dioleine (Raisio Group)
mono-oleine (Raisio Group)
Samples were diluted to tedrahydrofuran (HPLC-quality) and filtered through 0.451Jm
disposable filter.
Method 2.
pump: Beckman system gold 126
detector. CUNOW light scattering detector DNL 21
column: Spherisorb (C 18) (Phase separations) reversed phase, particle size 3lffn,
length 15 em, diameter 4.6 mm
gradient: 30 % A and 70 % B to 70 % A and 30 % B, time 0.5 h
(A: CI
2
CH
2
+CICH
2
CH
2
CI (50:50);B: CH
3
CN)
28
Method 3.
pump: Perkin-Elmer series 4 pump module
detector: Hewlett-Packard 1047 A-R-refractometer
integrator: Perkin-Elmer 316 or SI-316 satellite integrator
column: Nova-Pak C 18 (Nordion) or Ultrastyregel 500 Aand 100 A (Waters-Millipore)
eluent: Tedrahydrofuran (HPLC-quality)
standards: 2-ethyl-1-hexylester of rapeseed oil (Raisio Group)
rapeseed oil (Raisio Group)
methylester of rapeseed oil (Raisio Group)
TMP ester of rapeseed oil (Raisio Group)
trioleine (Raisio Group)
dioleine (Raisio Group)
mono-oleine (Raisio Group)
Samples were diluted to tedrahydrofuran (HPLC-quality) and filtered through 0.451Jm
disposable filter.
Gas chromatography
Samples were methylated by IUPAC 2, 301-method and analysed using a
Hewlett-Packard 5890 gas chromatograph.
column: NB 351 (Nordion), length 25 m, diameter O. 32J.lm, film strength 0.21Jm driving-
program: 1 min 70C, 10C/min to 240C.
Infrared spectrophotometry
Perkin-Elmer 883 spectrophotometer
Perkin-Elmer FTIR 16 PC spectrophotometer
cuvette: NaCII 0.025 mm.
tablet: KBr
2.8. IMMOBILlSATJON OF L1PASES
Reagents: Na
2
HP0
4
x2HP (Merck, Germany)
NaH
2
P0
4
xHP (Merck, Germany)
Carriers:
Carriers were washed with hot, deionised water. The lipase solution was made by
slurrying 6 g lipase in 100 cm
3
0.05 M sodium phosphate buffer (pH 5.8), mixed for 2
hours and filtered. Buffered carrier (40 g) and enzyme solution (60 cm
3
) were mixed for 6
hours at 26C. Total mixture was filtered and cold-dried (20C) for 30 hours to dry-solid
content 99 %.
29
2.9. CHEMICAL PREPARATION OF 2-ETHYL-1-HEXYL ESTER OF RAPESEED OIL
Reaction conditions were as follows: 3.5 9 (0.4 mol) rapeseed oil, 2.1 9 (1.6 mol) 2-ethyl-
1-hexanol and 0.5 % (wlw) catalyst. Rapeseed oil and alcohol were measured to a 50
cm
o
three neck flask, equipped with a thermometer, stirrer and condenser. Catalyst was
added and stirring was started. Mixture was heated to 60-70C and mixed until the
catalyst was melted into the reaction mixture. Reaction mixture was then heated to 190-
200C in oil bath, so that alcohol was refluxing. The progress of the reaction was
followed by TLC. Mixture was neutralized by HCI-water or NaOH-water (1: 1) depending
on the catalyst. The excess alcohol was distilled at 120C/2.6 MPa for two hours.
Glycerol was separated and this phase was discarded. The product was washed three
times with warm water (50C) and dried over sodium sulfate.
2.10. ENZYMATIC PREPARATION OF 2-ETHYL-1-HEXYL ESTER OF RAPESEED OIL
Reaction conditions were: Rapeseed oil 0.2 g (0.28 mol), 107 dm
o
(0.68 mol) of 2-ethyl-1-
hexanol (molar ratio 1:3) and 3.0 % (wlw) added water were mixed in capped 13 mm
o
test tubes equipped with a magnetic stirrer at 200 rev min,1 at 37C. The reaction time
varied from 12 to 72 hours. The lipase was then separated by centrifugation for 5 min at
2000 rev min-
1
. The supernatant was pipetted into Eppendorf tubes and stored at -20C
for later analyses.
2.11. CHEMICAL PREPARATION OF RAPESEED OIL METHYL ESTER
Rapeseed oil (0.3 mol) was weighed into a 100 cm
o
three neck flask, equipped with a
thermometer, condenser, stirrer and sample adapter. Stirring was started and methanol
(2.0 mol) was added. Reaction mixture was heated to 60C and the alkaline catalyst used
was added 0.5 % (wlw). After four hour reaction time all rapeseed oil had reacted by
TLC. Reaction mixture was washed by acidic water. Glycerol was separated and the
excess alcohol was distilled. The reaction mixture was analysed by HPLC. The RME-
content varied from 95 % to 99 %.
2.12. CHEMICAL PREPARATION OF SOYBEAN OIL ETHYLESTER
Soybean oil (0.25 mol) was weighed into a 100cm
o
three neck flask, equipped with a
thermometer, condenser, stirrer and sample adapter. Stirring was started and ethanol
(1.5 mol) was added. Reaction mixture was warmed to 80C and 0.5 %(wlw) of alkaline
catalyst was added. Stirring was continued for two hours. The reaction was monitored by
TLC. Upon completion the reaction mixture was neutralised by acid water and washed
with warm water. The excess alcohol was distilled and glycerol separated. Reaction
mixture was analysed by HPLC. A typical conversion was 89 %.
30
2.13. CHEMICAL PREPARATION OF COCONUT OIL METHYLESTER
Reaction conditions were similar to those of rapeseed oil esterification; amount of
substrates were coconut oil (0.34 mol), methanol (2.0 mol) and an alkaline catalyst
0.6 % (wlw). Reaction time was five hours. Conversion was typically 97 % as
determined by HPLC.
2.14. CHEMICAL PREPARATION OF TRIMETHYLOLPROPANE ESTER FROM
TALLOW OIL FATTY ACIDS
Two methods were used.
A. Reaction conditions were: Fatty acids (161 g, 0.59 mol) were melted, warmed to
60C and TMP (25 g, 0.19 mol) was added under mixing. TMP was allowed to melt
and mix properly. Solvent (45 mm
3
) and catalyst (0.5 % (wlw were then added and
mixed properly. Temperature was raised to 120C and kept there for seven hours.
The reaction progress was monitored by TLC. After the completion of the reaction
about 11 mm
3
of water was separated. Heptane was separated. Reaction mixture
was neutralized using sodium hydroxide water and washed twice using warm water
(50G).
B. Reaction conditions were: Fatty acids (161 g, 0.59 mol) were melted, warmed to
60C and TMP (25 g, 0.19 mol) was added under mixing. TMP was allowed to melt
and mix properly. Solvent (45 mm
3
) and catalyst (0.5 % (wlw were then added and
mixed properly.
Reaction temperature was elevated to 70-75C and a slight vacuum was introduced,
so that reaction mixture refluxed and water was separated. Reaction temperature
was kept around 70C during whole reaction, but the pressure was reduced
smoothly. The amount of water separated was 155 mm
3
. The reaction mixture was
neutralized using sodium hydroxide solution and washed with warm (50C) water.
HPLC was used for analyses. The highest conversion was 87.3 % and 6.0 % fatty
acids were left in the product.
31
2.15. CHEMICAL PREPARATION OF RAPESEED OIL TRIMETHYLOLPROPANE
ESTER
RME (205.8 g, 0.7 mol) was weighed into a 500 cm
3
three neck flask equipped with
a thermometer, condenser, stirrer and sample adapter. Methylester was heated to
60C and TMP (25 g, 0.2 mol) was added and efficient stirred. When TMP was
melted and well-stirred, 0.5 % (wlw) catalyst was added and mixed. Reaction was
continued under reduced pressure (3.3 MPa) and the mixture was heated up to
refluxing. Samples were taken hourly with a total reaction time of 8 hours. After
cooling, the mixture was neutralized with alkaline or acidic water, washed with warm
(50C) water, and dried over anhydrous sodium sulfate. Analyses were carried out
by TLC during the course of the reaction and by HPLC for the final products.
2.16. ENZYMATIC PREPARATION OF RAPESEED OIL TRIMETHYLOLPROPANE
ESTER
Reaction conditions were TMP 0.6 g (4.5mmol), 10 % (wlw) added water, RME 4.0 g
(13.6 mol) and 40 % (wlw) lipase. Distilled water was added to TMP and once TMP
was in solution, RME and lipase were added. The capped test tubes were kept in a
water bath (37C) and stirred magnetically 250 rev min-
1
. The supernatant was
pipetted into Eppendorf tubes and stored at -20C for later analyses.
32
3.RESULTS AND DISCUSSION
3.1. CHEMICAL SYNTHESIS OF 2-ETHYL-1-HEXYLESTER OF RAPESEED
OIL
3.1.1 Choice of catalyst
The effect of different catalysts on the degree of conversion in the chemical
synthesis of 2-ethyl-1-hexyl ester of rapeseed oil was studied at a temperature 182-
185C. The catalysts tested for the esterification reaction were selected on the basis
of previous experience (11,12,15,28,63). They were alkaline sodium hydroxide,
potassium hydroxide, sodium methoxide and sodium ethoxide or acidic sUlphuric
acid and phosphoric acid. Reaction conditions were as described in 2.9. (page 29).
A sample was taken for HPLC analysis. The reaction proceeded slowly and
conversions were quite low. Best results are shown in Table 3. The highest
conversion, 68 %, was obtained with sodium methoxide. Sodium hydroxide was the
next best catalyst, which is an advantage for industrial production, because sodium
hydroxide is quite a cheap catalyst. With acidic catalyst only a 30 % conversion was
achieved. According to other literature and patent search, this type of reaction had
not been done before and these results can only be compared to shorter alcohol
reactions. There the best catalyst is sodium hydroxide (11,13,14) and conversions
up to 100 % have been reached at temperatures near the boiling point of the
alcohol. The reaction scheme is shown in Figure 12.
Table 3: Different catalysts in rapeseed oil and 2-ethyl-1-hexanol reaction, (0,5 %
(wlw) catalyst, 195-200C, 3h).
Catalyst
Sodium hydroxide
Potassium hydroxide
Sodium methoxide
Sodium ethoxide
Sulphuric acid
Conversion
(%)
58
54
68
30
30
33
Figure 12: Chemical reaction between rapeseed oil and 2-ethyl-1-hexanol reaction.
CH
2
-0-8-R
6H -o-8-R
bH
2
-0-8-R
rapeseed oil 2-ethyl-1-hexanol
2-ethyl-1-hexylester of
rapeseed oil
9
H
2-
0H
+ yH- OH
CH
2
-OH
glycerol
3.1.2. Effect of molar ratio
The stoichiometry of this reaction requires a 1:3 molar ratio for rapeseed oil: 2-ethyl-
1-hexanol (Figure 12). Molar ratio was varied in following reactions from 1:3 to 1:6,
being 1:3.0, 1: 3.5, 1:4.0, 1:4.5, 1:5.0, 1:5.5, 1:6.0. Nitrogen atmosphere was used to
prevent oxidation of fatty acids due to the high reaction temperature of 190-200C.
Reaction was carried out as described in 2.9.. The progress of the reaction was
followed by TLC analyses. After one or one and half hours no rapeseed oil was
detected in TLC. Reaction mixture was cloudy in all cases. In table 4 the effect of
molar ratio on the total conversion is demonstrated. In all cases fatty acids could be
detected by HPLC analysis, the amount depended only a little or not at all on the
molar ratio. The main reason for the formation of fatty acids was the high reaction
temperature. Fatty acids in final products may cause problems with corrosion and
oxidation. Best molar ratio found was 1:5.0, which was used in subsequent
experiments.
34
Table 4: The effect of molar ratio (rapeseed oil: 2-ethyl-1-hexanol) on the conversion
of their ester, (0.5 % (wlw) catalyst, 4 h, nitrogen atmosphere).
Molar ratio Conversion
(%)
ester RO FA
70.0 22.0
74.0 18.0
81.6 9.8
87.0 3.7
88.2 7.3
85.5 3.5
82.0 9.3
Gly
RO= unreacted rapeseed oil
FA= fatty acids produced in reaction
Gly= glycerol left in product mixture
ester= the ester percentage in the product calculated from rapeseed oil (from
HPLC-gram, method 1, page 23 )
3.1.3 Effect of temperature and pressure
Due to the high temperature and in spite of the nitrogen atmosphere, rapeseed oil
oxidized during the process and free fatty acids were found in the reaction mixture.
This reduced the degree of conversion to the desired ester, and the presence of free
fatty acids lowered the quality of the final product.
To avoid this, reactions were made under reduced pressure. The molar ratio of
rapeseed oil: 2-ethyl-1-hexanol was in this case 1:5.0 and 0.5 % (wlw) of alkaline
catalyst. Reaction was carried out at 95-120C/10.6 MPa, 90-115C/8.0 MPa, 85-
110C/5.3 MPa, 80-105 C/2.7 MPa, 80-100C/2.0 MPa. The progress of the reaction
was followed by TLC. Total reaction time was four hours.
Table 5 demonstrates the total conversions versus temperature and pressure.
35
Table 5: Effect of temperature and pressure on the conversion of 2-ethyl-1-
hexylester of rapeseed oil,(molar ratio 1: 5.0, 0.5 % (wlw) catalyst, 4 h).
Temperature/Pressure
(OC/MPa)
95-120/10.6
90-115/8.0
85-110/5.3
80-105/2.7
80-100/2.0
Conversion
(%)
88.4
89.6
92.0
97.6
93.2
Conversion was much higher (90%-99 %) at reduced pressure than at normal
pressure and no oxidation of rapeseed oil was discovered. The highest conversion of
97.6 % was obtained when temperature was 80-105C and pressure was 2.7 MPa.
2-Ethyl-1-hexYlester of rapeseed oil was subsequently produced using a molar ratio
1:5.0 of rapeseed oil:alcohol, 0.5 % (wlw) alkaline catalyst,at a reaction temperature
of aoc to 105C at 2.7 MPa reduced pressure. Figure 13 shows the HPLC-gram
from a small pilot scale procedure of 2-ethyl-1-hexyl ester of rapeseed oil.
Conversion was 97.6%. Figure 14 the IR-speetrum and its interpretation. It can be
confirmed from the IR-speetrum, that the product synthetized was 2-ethyl-1-hexyl
ester of rapeseed oil.
36
Figure 13: HPLC-gram of rapeseed oil and 2-ethyl-1-hexanol reaction,(molar ratio
1:5.0,0.5% (wlw) catalyst, 90-105C, 2.7 MPa, 4 h).
CSl
...,
<1"
~
'" CIl
"-
'"
'""
"-
lfl
0&
...
01
(S>
...
<N
1Il
U.
U.
<:)
M
...
G:
I-
~
(S> c-J
""!
<"I'"
.... V1
,
Z
V ' ! ~
w
'" c.>
I I I I I I I I I I I I I " I I I I I I I I I I I I I I II I I I I I I I I
~ ~ ~ $ ~ ~ ~
~ ~ ~ N ~ ~
<.J
37
Figure 14: IR-spectrum of 2-ethyl-1-hexyl ester of rapeseed oil and its interpretation.
Interpretation (em -1): 3470, broad, water in trace; 3020, (m), C-H-streehing, C=C-H
in R-group; 2940&2870, (5), C-H-streehing,
-CH
2
,-CH
3
; 1750, (5), C-O-streching, R-C-O-; 1390, (m), C-H-symmetrical
deformation,-CH
3
; 1250&1185, (5), C-O-streehing, R-C=O; 1125, (m), C-Q-streching,
C-O-CH
2
; 785, (w-m), C-H-out of plane deformation, HC=CH in R-group; 740, (m),
CH
2
-roeking, -CH
2

38
3.2. ENZYMATIC SYNTHESIS OF RAPESEED OIL 2-ETHYL-1-HEXYL
ESTER (I)
3.2.1.Choice of lipase (I)
Preliminary esterification tests with rapeseed oil and 2-ethyl-1-hexanol were carried
out in capped test tubes and the results analysed by TLC. Lipases produced by
Candida rugosa (formerly cylindracea) , MJcor miehei, Pseudomonas Duorescens and
Chromobacterium v.iscosum were used as they were considered to be the most
suitable for the esterification of fatty acids (64). Reaction conditions were as
described in chapter 2.10. (page 29). According to TLC, lipase from c.rugosa,
produced most of the desired ester; all rapeseed oil had reacted and only a small
amount of alcohol was left. P. Duorescens and Ch. viscoseum also lipases catalyzed
the ester synthesis well, but more unreacted alcohol and by-products were found.
Poorest results were obtained with M miehei lipase: little ester was formed and a lot
of unreacted rapeseed oil and alcohol remained. Samples after 24 and 48 hours
were also analysed by HPLC and the results are presented in Table 6. Table 7
shows the characteristics and prices of the different Iipases.
The best lipase was from C. rugosa which also turned out to be the least expensive.
Table 6: Different lipases in rapeseed oil and 2-ethyl-1-hexanol reaction, (molar ratio
1:3,3.5 % (wlw) lipase, 3.0 % (wlw) added water, 12-72 h).
C.rugosa
MMiehei
P.Buorescens
Ch. Yiscosum
Lipase
24 h
98
45
96
96
Reaction time
48 h
Conversion
(%)
98
87
99
97
39
Table 7: Properties and prices of different Iipases used in reaction 3.2.1 ..
Lipase
C.rugosa
Cr.viscosum
M.miehei
PS.f1uorescens
Activity
(U/g)
42500
13300
7200
11900
Water
(%wlw)
5,0
5,9
7,4
3,1
Price
USDlkg USD/10U
292 7
2300 172
2250 313
2100 177
3.2.2 Effect of substrate molar ratio (I)
The theoretical molar ratio in rapeseed oil 2-ethyl-1- hexanol ester synthesis is 1:3
rapeseed oil:2-ethyl-1-hexanol. Other reaction conditions were as in (2.10., page 29).
Ester synthesis was investigated using the following molar ratios of rapeseed oil and
2-ethyl-1-hexanol: 1:1.0, 1:1.5,1:2.5,1:2.6,1;2.7,1:2.8,1:2.9,1:3.0,1:4.0,1:6.0
and 1:10.0. On the basis of both TLC and HPLC the best results were obtained with
little or no excess alcohol. All rapeseed oil had reacted with a 98 % conversion to
the desired product, with no residual 2- ethyl-1-hexanol and no by-products. The
relative ester yield decreased when an excess amount of alcohol was used.
Apparently, excess alcohol inhibited the lipase and the use of excess of alcohol was
not further studied. Figure 2/1 demonstrates the effect of the molar ratio on the
conversion of 2-ethyl-1-hexyl ester of rapeseed oil. Molar ratio 1:2.8 seemed to yield
a little higher conversion in a shorter time than molar ratio 1:2.9 or 1:3.0 (in 1 h 58 %
compared to 50 % and 40 %, respectively, or in 5 h 96 % compared to 95 % and 85
%, respectively).
3.2.3 Effect of lipase quantity (I)
Figures 15 and 16 demonstrate the effect of lipase quantity on the conversion of
rapeseed oil to the desired ester. With a lipase concentration of between 0.3 to 1.7
% (wlw) transesterification was slow after one hour. By increasing the enzyme
quantity to 2.3-6.4 % (wlw), conversion was increased to 60 % in one hour, and
with a lipase quantity of 14.6 % (wlw) to almost 100 %. However with 0.3 % (wlw)
lipase, the conversion reached nearly 90 % in 7 hours and with 1.0-1.7 % lipase, the
conversion was over 80 % already in five hours. Bearing in mind the cost of the
enzyme, it is important to observe that almost a 100 % conversion could be
obtained with less enzyme at an extended reaction time.
40
lipase quantity of 14.6 % (wlw) to almost 100 %. However with 0.3 % (wlw) lipase,
the conversion reached nearly 90 % in 7 hours and with 1.0-1.7 % lipase, the
conversion was over 80 % already in five hours. Bearing in mind the cost of the
enzyme it is of importance to observe that almost a 100 % conversion could be
obtained with less enzyme at an extended reaction time.
Figure 15: Effect of lipase quantity on the conversion of 2- ethyl-1-hexyl ester of
rapeseed oil, (molar ratio 1: 2.8, added water 3.0 % (wlw), 37C, lipase
0.3 % (wlw) (.),1.0 % (wlw) (0),1.7 % (wlw) (+) and 2.3 % (wlw)
( ~ .
100 T
90 t
80 +
I
~ 70 t
~
~ 60 +
g I
. ~ 50 + I
~ 4 O + ~ /
u I
30 t,
20 + /
I
10 T ",#
o
o 5
time (h)
11
41
Figure 16: Effect of lipase quantity on the conversion of 2- ethyl-1-hexyl ester of
rapeseed oil, (molar ratio 1:2.8, added water 3.0 % (wlw), 37C, lipase
quantity 3.3 % (wlw) (0),4.9 % (wlw) (.), 6.4 % (wlw) (4> ) 14.6 %
(wlw) (Y.

4
time (h)

100 T
I
90 t
80 +
70 1
~ I
!L. 60 +
g I
'0 50 I
Qj T I /,
i!: 40 + ,I
o : I I>
u 30 + .,1 j
20 +!i
10 til
o".'- - - - + - - - ~ - - - . . . - - - - _ + _ _ - - - _ - - _ j
1
3.2.4. Effect of added water (I)
The effect of added water on the transesterification reaction is demonstrated in
Figure 5/1. When 0.5 % (wlw) added water was used, there were no separable
phases in the reaction system. When water content was between 10.0 to 50.0 %
(wlw), a distinct water phase was formed, into which the lipase and glycerol
produced, were dissolved. Only the organic phase was used for the ester analysis.
The water content of lipase alone (about 5.0 % (wlw was not sufficient for the ester
synthesis. Conversion after seven hours was only 24 % without added water and an
increase in water concentration up to 0.5 % (wlw) did not improve the conversion.
With 3.0 % (wlw) added water a complete conversion was reached in five hours.
Additional water increases did not further improve conversion.
42
3.2.5. Effect of temperature (I)
The effect of temperature is demonstrated in Figure 17. There were no significant
differences between 37C to 55C in seven hours: The conversion was almost
complete. In 55C, the conversion was 90 % already after two hours. The same
conversion was reached at 37C in three hours. The temperature of 60C inhibited
lipase and the conversion was only around 60 %. The results support the
observations of Mittelbach (11) and Hirata (65). Mittelbach (11) found that the
temperature limit for Candida sp. lipase catalysed sunflower oil transesterification is
45 to 50C. Hirata et al (65) reported 50C to be optimal for transesterification of
tributyrin and 1- octanol with c.cylindracea lipase. For economic reasons the
temperature 37C was chosen for the future industrial application of 2-ethyl-1-
hexylester of rapeseed oil.
Figure 17: Conversion of 2-ethyl-1-hexyl ester of rapeseed oil versus temperature,
(molar ratio 1:2.8, lipase 3.3 % (wlw) , added water 3.0 % (wlw),7 h).
11
100 T
90 + b
80t
70 + //
60 + _------.
0: /
;;SOt ///
40+ j)' /. //
8 I / /
30 +.,.//4
20 t "
10.i-
o.'
o S 7.
time (h)
1-37C!
I ----0- 4SC I
!--- SO"C
I '
1-0- SS'C i
1--- 6O"C i
I :
43
3.3. SMALL PILOT SCALE OF ENZYMATIC PREPARATION OF 2-ETHYL-1-HEXYL
ESTER OF RAPESEED OIL (II)
Figure 1/11 demonstrates the effects of different mixing rates on rapeseed oil
conversion. All experiments were performed with a substrate ratio 1:2.8, 1.0 % (wlw)
added water and 3.4 % (wlw) lipase. Magnetic stirrer (300 rev min-
1
) was used in flat
bottom flasks. Conversion increased to 65 % in a few minutes and was up to 80 %
in one and half hours. After 30 minutes the solid lipase had collected to the walls
and the bottom of the flask. This seemed to be beneficial for the esterification
reaction. The solid lipase preparation also absorbed the glycerol produced in the
reaction. After five hours the conversion was 87 %.In another experiment the speed
of the magnetic stirrer was 700rev min-
1
, in this case conversion was only 46 % for
the first five hours because the high speed dispersed the lipase to reaction mixture
and resulted in high shear forces. Lowering the stirring speed to 300 rev min-
1
, did
not increase the conversion to higher than 61 %. Lee and Choo (66) have also
noticed that C. rugosa is easily denatured when the mixing speed is higher than 75-
150 rev min-
1
. Clearly the activity of lipase decreases as a function of mixing rate
and time. This is in accordance with the results of Goldberg et al (67) on C. rugosa
lipase catalysed heptyl octanoate synthesis. When mixing rate was increased
sufficiently, for complete suspending of lipase, the enzyme became inactivated.
A few experiments were also carried out in which the lipase was reused after
decanting from the reaction mixture. Conversion then varied from 3 % to 13 %.
Figure 18 shows an example of such experiments. Because preliminary experience
had suggested that the immobilization of the lipase might increase conversion,
different carriers were also tested.
Figure 18: Conversion of 2-ethyl-1-hexyl ester of rapeseed oil (molar ratio 1:2.8,3.4
% (wlw) lipase, added water 1.0 % (wlw) , 37C, motor stirrer 300 rev (
f!lin-
1
lipase reused (0), magnetic stirrer 300 rev min-
1
( +)Iipase reused
(~ .
7
44
However, no improvement could be seen with glass beads, polyether foam or
hydrophilic Amberlite XAD-2 resin immobilized lipase. Figure 4/11 shows how XAD-7-
resin with different amounts of lipases affects to 2-ethyl-1-hexylester of rapeseed oil
conversion. The hydrophobic Amberlite XAD-7 resin gave the best results as a
carrier. After five hours the conversion was 95 % with 50 g of rapeseed oil, 2.5 g
lipase, 25 cm
3
2-ethyl-1-hexanol and 3.0 % (wlw) added water.
Under the optimised conditions a 2 kg batch of rapeseed oil was then esterified, with
1 dm
3
, (829 g) 2-ethyl-1-hexanol, 3 % (wlw) added water, 100 g lipase with 300 g
XAD-7 resin, speed of rotation 170 rev min-\ 37'C. The obtained conversion about
90 % was nearly theoretical. Total reaction time increased, but the conversion was
about 20 % higher than in earlier experiments with different carriers.
3.4. CHEMICAL SYNTHESIS OF RAPESEED OIL METHYL ESTER
Methylester of rapeseed oil (RME) has been produced by Raisio Group, Oil Milling
Division, since summer 1992, the total production of RME being 150-200 tons.
Production in Europe is estimated at 900 000 tons per annum and the total world
production is 1 million tons per year (68).
The preparation of RME on a laboratory scale was done as described in chapter
2.11. (page 8). The reaction scheme is shown in Figure 20. As can be seen from the
HPLC-gram (Figure 19), the total conversion was 97 %, the rest being unreacted
rapeseed oil. This matches other laboratory results well (11,12,13,14,15). Table 8
shows the most important biodiesel fuel demands according to c>NORM 1190/1991
and the standards, that they are made with. It is important to notice that the cetane
number is about 50, being clearly the best in comparison with alternative fuels (69).
Cold stability properties for winter season can be improved by additives. Pourpoint
can be decreased from -15 to -36C. Thermal value is also at the same level as
common diesel oil (69), but the distilling figure is totally different. Summer diesel
distills at 180-360C while rapeseed oil methylester at 240-270C (70).
Figure 19: HPLC-gram of rapeseed oil methyl ester (method 1, page 27).
N
""
,.)
":
N
, ~
N
M
'" u..
u..
\
0 N
'"
,...,
"' In
f-
,
...
f-
'" ""
<S>
'"
r
'"
, ~
,
r
-
u..
"'
, .... ("'01
" ' ~
..... l . ~ ~ _.
.-. '0'
l ~ ~ ~
.,
z r
l,.'l .... .u
'"
f tIl' 1111111111111111' II fIlII 1111I 111I1 II 'II
't? ~ fI) IS' U"J CSJ If) ($)
.... "'" ... ~ M ""') ..po
45
Figure 20: Reaction scheme of methyl ester synthesis.
CH
2
-0-8-R
6H- 0-8-R
6H
2
-0-gR
CH
2
-OH
I
+CH-OH
I
CH
2
-OH
rapeseed oil methanol methylester
of rapeseed oil
glycerol
46
Table 8: Specifications of Raisios RME for fuel use as modified from Biodiesel-
project report (71).
Property unit limits Standard
Density g/cm
3
0.87-0.90 DIN 51757
Viscosity
40C mm
2
/s 4.5-5.0 DIN 51562
20C 8.5-9.0 DIN 51562
OC 15.5-16.0 ASTM 0445
-20C 39.5-40.0 ASTM 0445
Surface tension dyn/cm
25C 30.5
Flashpoint COC C 190 ASTM 092
Pourpoint C -15 ASTM 097
Winter C -36 ASTM 097
Cetane number min 49
Thermal value MJ/kg 37.2
Carbon residue %M <0.02 ISO 10370
(Conradson)
Oistilling/350C % >90
Corrosion Cu 1a ASTM 0130
Sulphur content mglkg <5 ICP
Phosphor content mg/kg <5 ICP
Element analysis %M
Carbon 75.0
Hydrogen 12.6
Oxygen 11.1
Nitrogen <0.1
Methanol content % <0.03 OIN 51413,1
Water content mglkg <100 DIN 51777,1
TAN mgKOH/g <0.40 DIN 53402
Colour <G 10 ASTM 01500
Biodegradability % >90 CEC-L-33-T-82
47
3.5. CHEMICAL SYNTHESIS OF SOYBEAN OIL ETHYLESTER
Soybean oil ethyl ester was prepared as in chapter 2.12. (page 25). The reaction
scheme is similar to that for rapeseed oil ester (Figure 20, page 39). As can be seen
from Figure 21, conversion was typically around 90 % and the only other peak was
unreacted soybean oil.
Figure 21: HPLC-gram of the ethyl ester of soybean oil.
..
co
<$>
"
11'\
--
.., ..
'"
'<" ~
,...
..
'" ...
'"
=
.....
I Z.
,
...
... uJ
...
'"
1'1 1IIIIflllll,IIIIIIII II 1
'
11111111"'1111
n ~ n ~ ~ . ~ ~ s
~ ~ ~ ~ ~ ~ ~
3.6. CHEMICAL SYNTHESIS OF COCONUT OIL METHYLESTER
Coconut oil methylester was prepared as in chapter 2.13.(page 30). Also in this case
reaction scheme is similar to that for rapeseed oil methylester (Figure 20, page 46).
As can be seen from Figure 22, a two hour reaction time was not suffient. The
conversion after two hours was only 72 % and almost total conversion of 92 % was
achieved after 5 hours. It was more difficult to esterify coconut oil than rapeseed oil
due to its fatty acid content. According to literature the most frequently used oils are
rapeseed oil, soybean oil and sunflower oil (15).
48
Figure 22: A thin layer chromatogram from coconut oil and methanol reaction, (molar -
ratio 1: 6.0, 0.6 % catalyst (wlw) , 80C, 5 h).
CO=coconut oil
1=1h reaction
2=2h reaction
3=3h reaction
t
t f t i

t
4=4h reaction
t t
~
CO+2=coconut
f ,

oil+ 2h reaction

5=5h reaction
co
3
CO CO+2
5
3.7. CHEMICAL SYNTHESIS OF TRIMETHYLOLPROPANE ESTER FROM FATTY
ACIDS
3.7.1. Choice of catalyst
The synthesis of trimethylolpropane fatty acid esters was carried out using three
different catalysts sulfuric acid, phoshoric acid and p-toluenesulfonic acid. These
catalysts have been used previously in similar esterification reactions (10,31,35).
Reaction stoichiometry requires one TMP mole for three moles of fatty acids (Figure
23).
49
Figure 23: The reaction scheme of the esterification of TMP with fatty acids of tallow
oil.
CH
2
-OH
q I
3 R-C-OH + CH
3
-CH
2
-C-CH
2
-OH
I
CH
2
-OH
CH
2
-0-8-R
A 1 9
cata.!lst CH
3
-CH
2
-CCH
2
-O-C-R + 3 Hp
6H
2
-0-8-R
fatty acids TMP TMP- ester
To ensure that the reaction would progress towards the right direction, an excess of
fatty acids was used. Tallow oil fatty acids were distilled so that only certain types of
fatty acids were included in the starting material. Fatty acid concentration is
described in chapter 2.1., page 25 and reaction conditions are shown in chapter
2.14.A., page 30.
According to qualitative TLC best results were achieved with p- toluenesulphonic
acid, yielding most of the desired ester. Almost all of the alcohol had reacted and
only a small amount of fatty acids were left. Sulphuric acid and phosphoric acid as
catalysts also aided the production of the desired ester, but there were more
unreacted fatty acids and more byproducts. With phosphoric acid a much longer
reaction time was needed or the reaction temperature had to be increased, which
resulted in the oxidation of fatty acids.
3.7.2. Effect of catalyst quantity on esterification of tallow oil fatty acids
Figure 24 demonstrates the effect of different amounts of catalysts on the conversion
of fatty acids into esters. The amount of catalyst was varied from 0.1 % (wlw) to 2.0
% (wlw). Best results with a conversion of about 65 % as determined by HPLC were
achieved by using 0.3-0.5 % (wlw) catalyst.
50
Figure 24: The effect of the amount of catalyst on the conversion of fatty acids to
TMP-ester, (molar ratio 1:3, 120C,7 h.)
70 T
60 .,.
20 t
"
10 J.
1.2
0.5 0.7 0.9
amount of catalyst (%)
0.3
o ~ , -------------------
0.1
3.7.3. Effect of substrate molar ratio on esterification of tallow oil fatty acids
The theoretical molar ratio for the TMP fatty acid ester synthesis is 1:3 TMP:fatty
acids. The reaction conditions were as desribed earlier (2.14.), but the molar ratio
was varied from 1:3 to 1:6, as follows: 1:3.0 1:3.5, 1:4.0, 1:4.5, 1:5.0, 1:5.5, 1:6.0.
Analyses were carried out by TLC and HPLC. The conversion could also be
calculated approximately from the water quantity formed in the reaction. The best
result with almost all of the TMP reacted, a 65 % conversion to the desired product
and a little residual fatty acids, was achieved by using little or no fatty acid excess.
This can also be seen in Figure 25, which shows a thin layer chromatogram of the
reactions with different molar ratios. In further studies (next chapters) stoichiometric
quantities of fatty acids were used.
51
Figure 25: TLC-pictures of different molar ratios in reaction of TMP with fatty acids
of tallow oil, (0.5 % (wlw) catalyst, 120C, 7 h).
:
.':'.
: ,
l'<
JAn
1 2 .3 .. 5
l=A 2= l:J 1:3.5 4= 1:4 5=1:4.5 6=1:5 7=1:5.5 8=1:6
3.7.4. Effect of temperature, time and pressure on esterification of tallow oil fatty
acids
Reaction time varied from 2 to 11 hours, reaction temperature from 120 to 180C
and pressure from 18.6 MPa to 11.6 MPa. The conversion varied from 65 % to 100
% as calculated on the basis of water formed. The progress of the reaction was
followed by TLC. Highest conversion was obtained in 7 hours at 120 to 130C.
Because some unreacted, and also some oxidised fatty acids were detected in the
reaction mixture, the following changes were made: The reaction temperature was
adjusted to 70 to 80C and the pressure was reduced to 11.9 to 18.6 MPa. Table 9
presents data of the different reactions.
52
Table 9: The effect of temperature and pressure on the conversion of fatty acids
TMP-ester controlled by the produced water quantity, (molar ratio 1:3.0,
0.5% (w/w)p-toluenesulphonic acid, 2-9h)
Tempe-
rature
(oC)
Pressure
(MPa)
Time
(h)
Water
quant.
(%)
Conver-
sion
(%)
120 3.0 65
130 5.5 75
140 4.0 85
130-140 3.5 65
150 2.0 90
150-160 2.0 100
160 3.5 100
160 5.5 100
170 3.0 90
180 3.5 90
70-80 18.6 3.0 68
17.2 4.0 74
17.2 6.0 76
15.9 6.0 82
13.2 4.0 85
13.2 6.0 86
11.9 6.0 88
--------------------------------------------------------------------------------------------
The optimal reaction conditions were then employed in a larger scale experiment as
described in chapter 2.14., B.(page 30). The conversion was at best 87.3 %, with 6.0
% fatty acids still left in the product. In Figure 26 a comparison is made between
tallow oil trimethylolpropane ester and a comparable commercial TMP-ester. The
commercial TMP-ester is prepared from tallow oil fatty acids, which have been
distilled and purified to single fatty acids before used in esterification with TMP (4).
This can clearly be seen in the HPLC-gram as a narrow line of peaks. In all of the
reactions, unreacted starting materials, if only in a small amount, were always left in
the final product. Considerable difficulties were encountered in the neutralising,
washing
53
and purification steps of the ester-product owing to soaping and foaming. Because of
these problems, the transesterification of small alcanol esters of vegetable oils was
investigated in place of the esterification.
Figure 26: HPLC-gram (method 2) of tallow oil TMP-ester (above) and comparable
commercial TMP-ester (below).
E
-
"
~
~
!
l
! ~
~
! ~
J
.; ~
I
1
1
-
L ~
~
~
..
f
~
~ ~
i e'
L
i
L
~ ~
L
l.
I
r
~ f :
~
L
I
!
..
54
3.8 CHEMICAL PREPARATION OF TMP ESTER FROM RAPESEED OIL
METHYLESTER AND TRIMETHYLOLPROPANE (III)
3.8.1. Choice and quantity of catalyst (III)
The most frequently used catalysts in this type of transesterification reactions are
sodium and potassium hydroxides and alcoholates or p-toluenesulphonic acid
(72,73,74).
The quantity of catalyst has been generally varied from 0.2 % (wlw) to 2.0 % (wlw).
In the present work preliminary trials were done under following reaction conditions:
substrate ratio TMP:RME 1:3.5 and catalyst 0.5 % (wlw). Reduced pressure was
already known to be essential (3.1.3 and 3.7.4). Consequently, the reaction
temperature was 80 to 120C and pressure 3.3 MPa. The reaction temperature and
pressure were chosen so that TMP would properly melt and the reaction mixture
could be refluxed. The molecular scheme is shown in Figure 27. The best catalyst
under these reaction conditions seemed to be sodium methylate, although also
sodium hydroxide and potassium methylate gave satisfactory results.
Figure 27: Molecular schedule of rapeseed oil methylester and trimethylolpropane.
RME TMP
of rapeseed oil
TMP-ester methanol
The amount of sodium methylate was then varied from 0.1 % (wlw) to 2.0 % (wlw) ,
being 0.1 %,0.3 %, 0.5 %, 0.7 %, 0.9 %,1.2 %,1.4 %,1.6 %,1.8 % and 2.0 %
(w/w). Other reaction conditions were as given in chapter 2.15., page 31. The results
are shown in Table 10. The best results were achieved by using 0.7 % (wlw) sodium
methylate.
55
Table 10: The effect of the catalyst amount on the conversion of rapeseed oil methyl
ester to TMP-ester, (molar ratio 1:3.5, 60-130C , 3.3 MPa, 8 h).
------------------------------------------------------------------------------------------------------
Catalyst
(% (wlw)
0.1
0.3
0.5
0.7
0.9
1.2
1.4
1.6
1.8
2.0
Conversion
(%)
70.6
76.6
80.2
85.2
76.5
74.4
72.6
72.3
70.3
69.8
---------------------------------------------------------------------------------------------------------
3.8.2. Effect of substrate molar ratio on RME transesterification (III)
The stoichiometry of the transesterification reaction requires per one mole TMP
three moles RME (Figure 31). To ensure that the reaction proceeds towards the right
direction, a small excess of RME was needed. Molar ratios studied varied from 1:3.0
to 1:4.0, being 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.8, and 1:4.0.
Other reaction conditions were as described previously (2.15., page 10). The
differences in conversions were small, as can be seen from Table 11. Nevertheless
a slight excess of RME gave the highest conversion of 86 %, and was later used in
this study.
56
Table 11: The effect of molar ratio (TMP:RME) on the conversion to the desired
ester, (0.7 % (w/w) catalyst, 80-120C, 3.3 MPa, 8 hours).
Molar ratio
(RME:TMP)
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.8
4.0
Conversion
(%)
82.2
84.0
86.0
80.2
76.5
76.0
74.4
73.0
72.6
-------------------------------------------------------------------------
3.8.3 Effect of temperature and pressure on RME transesterification (III)
Both temperature and reduced pressure had a strong effect on the conversion to the
desired ester. The following conditions were investigated using a slight excess of
RME, TMP:RME (1 :3.2), 0.7 % (wlw) sodium methylate catalyst, other conditions
being as described in chapter 2.15. (page 26). Preliminary experiments were done
maintaining reduced pressure at 3.3 MPa. Temperature varied from 70 to 130C, at
levels 70-90C, 80-11 OC, 100C, 100-130C and 130C.
57
Table 12: Effect of the reaction temperature on the conversion of TMP-ester, (3.3
MPa reduced pressure, TMP:RME 1:3.2, 0.7 % catalyst, 8 hours).
Temperature
(0C)
Conversion
(%)
RME, unreacted
(%)
---------_._--_._--------
70-90
80-110
100
100-130
86.0
90.5
89.0
87.3
14.0
9.5
11.0
12.7
/._-------.-->--_.
As can be seen from Table 12, the conversion was higher when the temperature
varied during the reaction procedure. It could be seen clearly from TLC- and HPLC-
analyses, that the fatty acids of RME reacted with TMP in two steps: First the
shorter, straight chain, less substituted fatty acids and then the bigger molecules.
The reaction profile resembled that shown in Figure 28. Therefore following reactions
were carried out at two different temperatures or temperature ranges. Reaction time
in each case was decided on the basis of TLC runs dUring the reaction procedure.
Results are shown in Figure 29.
Figure 28: Typical profile of RME and TMP reaction. (Molar ratio TMP:RME 1:3.2,
0.7 % ( wlw) sodium methylate, 3.3 MPa).
120 T
100 '
U ~
"or 80 .....------
~ 60 ~
Q;
~ 40 +
Q) ,
- 20 +
ol....:- - - - - - - - - - - - - - - - ~ : _ _ : .
10
o
time (h)
100;
58
Figure 29A: The effect of the time on the conversion of TMP and RME ester, (molar
ratio TMP: RME 1: 3.2, at lower temperature area, mean value 90C,
3.3 MPa).
- T35 -;-
..___________\ ( 30 e-
Rao
- /0"
60 .. ./' t 15
(;; 40 +10'".,
I
8 20 r 5 -
L 0
oi 2.5 3,5 4 4.5 5.5
time (h)
Figure 298: The effect of the time on the conversion of TMP and RME ester, (molar
ratio TMP:RME 1:3.2, at higher temperature area, mean value 110C,
3.3 MPa).
- a, 35
_
" .. -;--__::-=__ f 30
801 f251J.J
g 60!
I 15
40 \ 10-5
g 20r 15
oLi 0 -
2.5 3.5
time (h)
5.5
Conversion was at lowest with 66.5 % of ester, 33.5 % of residual RME, when
temperature was 105-120C for 2.5 hours. It improved a little, when two temperature
areas were chosen, 80-100C, 2.0 hours and 100-130C, 2.5 hours, being 83 %. It
was noticed that the progress of the reaction did not only require two reaction
59
temperature areas, but also longer reaction times for better conversion. The best
. conditions observed for the temperature and pressure were: first 85-110C for 2.5
hours and then 110-120C for 8 hours in 3.3 MPa. Conversion was then 98.9 % of
the TMP-ester.
3.9. SMALL PILOT SCALE PRODUCTION OF TMP-ESTER FROM RAPESEED OIL
METHYLESTER AND TMP (III)
All reaction conditions that had earlier been optimal for the transesterification to
TMP-ester (3.8.1. - 3.8.3.) were combined and a pilot scale production of TMP-ester
was carried out. Five kilos of RME (17.1 mol) was weighed to a reaction vessel
equipped with a thermometer, a condenser and a stirrer. The methylester was
heated to 60C and TMP 716 g (5.3 mol) was added in five portions with adequate
mixing. When all TMP had melted and the mixture was well-stirred, 40.0 g (0.7 %
(wlw))sodium methylate was added and properly mixed. Pressure was reduced to
3.3 MPa. Temperature was first raised to 85C and then gradually elevated to 110C
during a period of two hours under refluxing. Thereafter the reaction temperature
was kept between 110 and 120C for 8.0 hours. Samples were taken every hour and
analysed with TLC. No RME could be seen on the TLC-gram after 10.5 hours, after
which the reaction mixture was neutralized by hydrogen chloride water (1:1) and
subsequently washed three times with warm (50C) water followed by drying over
anhydrous sodium sulfate. The final product was analysed by both IR and HPLC
with two different columns.
Conversion to the TMP-ester of rapeseed oil was 99.0 % and no methylester of
rapeseed oil could be found in the final product. Figure 30 shows the HPLC-grams of
product, and Figure 31 the IR- spectrum and its interpretation. HPLC-gram of RME
and TMP ester is comparable to HPLC-gram of commercial TMP-ester. It can be
seen, that the commercial TMP-ester contained fewer fatty acids. The main peaks,
however, were in the same area. The molecular structure of the product combination
was confirmed by IR-spectrum of rapeseed oil TMP-ester.The TMP-ester of
rapeseed oil thus obtained was used for producing and testing hydraulic fluids.
60
Figure 30: HPLC-gram of rapeseed oil TMP-ester (left) and comparable commercial
TMP-ester (right) with method 2 (page 27).
.~ !
;.1.;
: ~ + ~ ..
::
i :
61
Figure 31: IR-spectrum of rapeseed oil TMP-ester (obtained)
and its interpretation.
Interpretation (em-
1
): 3020, (m), C-H-streehing, H-C=C-H in R group; 2950&2860, (s),
C-H-streehing, -CH
2
,-CH
3
; 1750, (s), C-O-streehing, R-C-O-; 1470, (s), C-H-
deformation, -CH
2
,-CH
3
; 1385&1320, (m),-H
2
C-C-CH
2
-; 1250&1170, (s), C-O-
streehing, R-C=O; 1020, (m), C-O-streehing, R-C-O-C-; 785, (w-m), C-H-out of plane
deformation; 740, (s), -CH-roeking, -CH
2
.
62
3.10. ENZYMATIC PREPARATION OF RAPESEED OIL TMP-ESTER (IV)
3.10.1 Choice of lipase (IV)
The preliminary tests were carried out in capped test tubes and analysed by TLC.
Lipases used were produced by Candida rugosa (ex. cylindracea) , JlJucor miehei,
JlJucor sp. and Pseudomonas fluorescens. They were selected to be the most
suitable for this esterification on the basis of prior experiments (3.2.1.).
The Candida rugosa lipase appeared to produce most of the wanted ester according
to qualitative TLC. With JlJucor miehei lipase hardly any TMP-esters were obtained as
can be seen in Figure 32. Because conversion in capped test tubes was small or
none, the balance of transesterification was tried to shift to the side of desired
products by elevating temperature to 58C and with open testtubes. Conversion was
not much improved so the next step was to carry out the reaction under reduced
pressure. Candida rugosa was chosen to be the best lipase for further experiments.
Figure 32: Conversion of TMP-esters from rapeseed oil methylester by lipase
produced by C. rugosa{Cr) and Mmiehei (Mm), (molar ratio 1:3, added
water 15 % (wlw) , lipase 40 % (wlw) , 3rC).
TMPE, Cr (_); RME, Cr (0); TMPE, Mm (+); RME, Mm ().
0.04 "\
0'035
T
\L
0.03.). \
\\ .)
2) i . : _---0--0--
a 0.02 + \b-- --
!
0.015 + /
8 . /
0.01 + ! - _ _..--.__...... -+
'! /
0.005 + / /
o
o 12 24 36 48 60 72 84 96 108
time (h)
63
3.10.2 Effect of lipase quantity and its stepwise addition (IV)
The effect of the lipase quantity was tested under two different reaction conditions:
(1) substrate molar ratio (1 :3), reaction temperature 3rC, reduced pressure 12.0
MPa and reaction time 24 hours, and (2) 4rC, 5.3 MPa and 68 h. Substrate ratios
were the same as above. Lipase quantities tested were 10 %, 20 %, 40 % and 80 %
(wlw). In the reaction (1) the highest conversion of about 80 % was obtained, when
lipase quantity was 40 %. At higher temperature and pressure some RME remained
and 20 % (wlw) lipase quantity, conversion was increased to 95 % in 68 hours.
Figure 33 shows the results.
Figure 33: Conversion of rapeseed oil methyl ester with TMP to TMP-ester, with
various quantities of lipase, (molar ratio 1:3, added water 15 % ( wlw)
1. 3rC, 12.0 MPa, 24 h (.) and 2. 4rC, 5.3 MPa, 68 h (0.
100
,
90
80
..
70
~
60
..
C
. ~
50
..
;;
>
40 ~ c
0
...
30
.,.
20
~
10
..
0
0 10 20
enzyme (0/0)
40 80
64
Enzyme was also added stepwise to the reaction mixture. Reaction conditions were
as in chapter 2.16. (page 26) with the total enzyme quantity of 40 % (wlw). The
reaction time was 46 hours with two and 68 hours with three enzyme
supplementations. Figure 34 shows the conversion obtained with a number of lipase
additions. The conversion was approximately the same with one or two additions
and slightly lower with three steps. So there seemed to be no benefit with stepwise
addition of lipase.
Figure 34: Conversion of rapeseed oil methyl ester with TMP to TMP-ester, (molar
ratio 1:3, total lipase quantity 40 % added stepwise, added water 15 %
(wlw) , 4rC,5.3 MPa, 46 (. ) or 68 (0) h).
III
"
enzyme additions
r ~ l I I - ' __...JlII'- ~ L
I
3.10.3 Effect of substrate molar ratio on enzymatic transesterification of RME (IV)
In the first experiments the substrate molar ratio TMP:RME was always the
theoretical 1:3. In the following experiments the substrate molar ratio was varied
from 1:1.8 to 1:9.0, actual ratios being of 1:1.8, 1:2.0, 1:2.2, 1:2.3, 1:2.6, 1:3.0, 1:3.5,
1:4.5 and 1:9.0. Other reaction conditions were: temperature 3rC, reduced pressure
5.3 MPa, 40 % (wlw) lipase and 15 % added water (wlw). With 12 hour reaction time
at substrate molar ratio 1:3.5 and 1:4.5 conversion was 95 % and 85 %, respective.
In both cases some unreacted RME remained in the mixture. With substrate molar
ratios smaller than 1:3.5 or higher than 1:4.5 a lot of unreacted RME remained and
mono- and di-substituted TMP-esters and by-products were found in the mixture. No
trisubstituted ester was obtained under these conditions. Consequently, the molar
ratios of 1:3.5 and 1:4.5 were chosen for further experiments. Figure 35 shows the
effect of the molar ratio on the total conversion.
65
Figure 35: Conversion of rapeseed oil methyl ester with TMP to TMP-ester, when
molar ratio was varied, (lipase 40 % (wlw),added water 15 % (wlw),
37C, 5.3 MPa, 24 h).
9 4.5 3.5 2.3 2.6 3
relative quantity (TMP:RME)
2.2
2 .
l:H
~ 70 i
~
-;; 60
o
. 50 t
I ; ~ j'
20
10
o.l---+---+----+--->---+-----+----+-----<
1.8
3.10.4. Effect of added water on enzymatic transesterification of RME (IV)
The amount of added water for the first reactions was chosen on the basis of the
earlier esterification experiments (3.2.4.).
The following reaction conditions were used: (1) substrate ratio 1:4.5, reaction
temperature 3rC, 12.0 MPa pressure and reaction time 24 hours, and (2) substrate
ratio 1:3.5, reaction temperature 47C, pressure 5.3 MPa and reaction time 68 hours.
Lipase quantity was 40 % (wlw) in both cases.
The examined added water quantities were 8 %, 10 %,15 %, 30 %, 45 % and 60 %
(wlw) of the total mixture. In the first reaction procedure the conversion improved,
when the water content was increased from 8 % to 30 % (wlw). In the second
procedure the conversion was about 90 % with 15 % (wlw) added water with a little
RME left and no by-products. The detailed results are given in Figure 36.
66
Figure 36: Effect of the added water quantity on the TMP-ester conversion;1) molar
ratio 1:4.5, lipase 40 % (wlw) , 3rC, 12.0 MPa, 24h (.) (2) molar ratio
1:3.5, lipase 40 % (wlw) , 4rC, 5.3 MPa, 68h, (0.
1 ~ ~ I
~ 80
C 70
. ~ ~ ~ t
!II 40 t
g 30 t
(J 20 +
10
o ~ - - -
10 15 30 50
added water (%)
3.10.5 Effect of temperature on enzymatic transesterification of RME (IV)
The original temperature of 37C was chosen on the basis of the experience gained
from the preliminary experiments (3.2.5.). In the following experiments the pressure
was 5.3 MPa, reaction time 72 h, substrate ratio 1:4.5, lipase quantity 40 % (wlw)
and added water 15 % (wlw). The temperatures used were 37,42,47, 52 and 58C.
In Figure 37 the results are shown. The total reaction time was 72 hours. The
highest conversion of 97.5 % was obtained at 42C in 72 hours.
67
Figure 37: The effect of temperature on the conversion of RME with TMP to
rapeseed oil TMP-ester, (molar ratio of 1:4.5, lipase 40 % (wlw), added
water 15 % (wlw), and 5.3 MPa, in 72 h).
37C (.), 42C (0), 47C (+) and 52C (0).
72
48
24
time ChI
12
'./
80
70
60
~ 50
"
~ 4 0
~
:>
8 30
20
10
0r:------------ ~
o
3.10.6 Effect of pressure on enzymatic transesterification of RME (IV)
As was observed already in the first experiments, the reaction needed reduced
pressure (3.8.3., 3.10.1) to obtain the desired ester. The experiments under varying
pressure were done at 37C, substrate ratio 1:4.5, 40 % (wlw) lipase, 15 % (wlw)
added water, and reaction time 24 hours. The conversion was calculated only for the
main product, trisubstituted ester. The pressures used in the test were ambient, 12
MPa, 5.3 MPa and 2.0 MPa. The highest conversion of 70 % to tri-TMP-ester was
obtained with 2.0 MPa (Figure 38).
68
Figure 38: Effect of the pressure on the TMP-ester conversion, (molar ratio 1:4.5, 40
% (wlw) lipase, 15 % (wlw) added water, 37C, 24 h).
Normal pressure (.); 12.0 MPa (0); 5.3 MPa (.);2.0 MPa (0).
/
4 5
time (h)
11 24 30
3.11. IMMOBILISATION EXPERIMENTS FOR PREPARATION OF TMP-ESTERS
FROM RAPESEED OIL METHYL ESTER (IV)
Because previous experiments with different esters had clearly suggested that
conversion of RME to desired ester might increase by immobilising the lipase,
different carriers for immobilization were tested. They were Celites R-630, R-640 and
R-626, Diatomite, Duolite ES-762 (neutral adsorbtion resin), GCC, GDC 220 (weak
alkalic anion exchange resin), HPA 25 (strongly alkalic anion exchange resin),
silicagel and WA 30 (weak anion exchange resin). The reaction conditions were
those found optimal in earlier experiments (3.10.1- 3.10.6.): temperature 47C,
pressure 5.3 MPa, lipase 40 % (wlw) , added water 15 % (wlw), substrate ratio 1:4.5,
and reaction time 42 hours. The results are shown in Figure 39. With WA-30 and
Diatomite immobilised lipase no reaction could be seen. With Duolite ES 762, GDC,
GCC and HPA-25 immobolised lipase no improvement in conversion could be seen.
With silicagel immobilised lipase reaction ceased to mono- and di-ester of TMP. The
use of Celite R-630 as a carrier gave the highest conversion of 95 % to the tri-ester
in 22 hours.
69
A comparison experiment was done with commercial, immobilized MIcor miehei
lipase (Lipozyme 1M 20) under the same reaction conditions, except that no water
was added. A conversion of 92.5 % was obtained. The results show that by
immobilizing the Candida rugosa lipase to Celite R-630 conversion to the desired
ester was at the same level as without immobilization (95%). The reaction time,
however, was shorter compared to nonimmobilized lipase reaction (22 h compared
to 42 h or 68 h).
Figure 39: Effect of the immobilised lipase on the TMP-ester conversion, (molar ratio
1:4.5, added water 15 % (wlw) , immobilised lipase 40 % (wlw) , 47"C, 5.3
MPa, 42 h).
-
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~ 70
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00; 50
~ 40
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8 30
20
10
o
100
90
80
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~
u
~
g a;
. ~
m
Ql
u
'"
E
m'" '6
'"
;, ;,
"
"iii
~ ~
"
Z
::
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u
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is
."
70
3.12. APPLICATIONS AND USES OF RAPESEED OIL ESTERS
3.12.1 Methyl-, ethyl- and 2- ethyl-1-hexyl esters of rapeseed oil
The methyl-, ethyl- and 2-ethyl-1-hexyl esters of rapeseed oil have been used for
example as pure solvents or as solvents to replace organic solvents in detergents.
Pure solvent use is mainly in printing ink industry, for example to clean printing ink
rollers. First detergents using vegetable oil esters have been on the market for about
two years. Table 13 demonstrates how these detergents remove a dirty, solid phase,
which results from biodegradable hydraulic fluids dried on metal surface under
sunshine at extended periods.
Methyl- and ethyl esters of rapeseed oil are also used as fuel, biodiesel (71-76).
They were developed for environmental reasons. The main purpose is to reduce
exhaust gas emissions (Table 14).
Table 13: Different detergents in test. Test conditions (77): Steel plates (washed
and dried by acetone), 100 mm
3
tested oil, 1 min. The plates are under
UV-Iamp, 45-47C (max), t varies, depending on the tested oil, until the
film formation takes place. Washing: with pure liquid, 5 %, 10 % and
20% solutions, measured effiency and time.
Detergent Type Effect Time
Company +/+++++ (min)
I Environmentally
recommended
Limo/Solmaster limonene +++ 15-20
Pro-Clean/Ab Ind. env.friendly +++ 10-15
tekniklSwed en
Ecoclean autol RME +++++ 5-10
Trans-Clean
II Traditional
MT-Exima 095/TK-
10-15 +++
TK-123/Noiro/
10-15
Orion
+++
Goldfain2000/
++++
10-15
KS-Chemitra Ltd.
71
Table 14: Exhaust gas emissions of normal summer diesel ( DIK), and from
rapeseed oil bio diesel (Raisio) (modified from 71).
Fuel Exhaust gas emissions
(gIkWh)
Carbon HC-part PM-30 CO HC NO
x
particles
DIK 0.18 0.36 0.54 4.57 0.58 12.15
RME 0.04 0.27 0.31 2.91 0.45 13.36
RME+ 0.04 0.14 0.18 0.24 0.11 13.10
catalysator
limit 11.2 2.4 14.4
3.12.2 Hydraulic fluids made from rapeseed oil esters (V,VI)
TMP-ester based lubricants made in this work were compared with pure comparable
vegetable oil based and commercial synthetic ester based hydraulic fluids, using
commercial mineral oil based hydraulic fluid as a reference.
Main purpose for a hydraulic fluid is to reduce friction and decrease wear. It has to
have stability against heat, cold, oxidation and corrosion. It also has to be able to
maintain these qualities for extended periods. All of these properties can of coarse
be improved with different additives, but the basic fluid is of crucial importance.
These types of synthetic esters such as pure vegetable oils have great advantage in
the reduction of wear and friction due to their molecular structure (Figure 40).
72
Figure 40: The molecular structures of vegetable oil, TMP-ester of rapeseed oil and
mineral oil (78,79,80).
CH
2
-0-gR
bH- O-gR
bH
2
-0-8-R
vegetable oil
92
H
S
C2Hs-CH2-CH-CH2-H-CH3
C
2
H
s
CH
2
-0-gR
CH
3
-CH
2
-6-CH- 0-8.R
6H
2
-0-g-R
TMP-ester of rapeseed oil
mineral oil components
All esters include oxygen in their structure. Because of this oxygen these fluids can
form a very stable, unimolecular film over a metal surface (78,81,82,83). A major
advantage is their biodegradability and non-toxicity. They are not bioaccumulating
either. In many countries these requirements are essential for lubricants (25). A
general composition of hydraulic fluids from rapeseed oil TMP-ester is described
below.
Component
TMP-ester of rapeseed oil
antioxidant
pour-point depresser
antiwear
antifoamer
Amount
(%(wlw
90- 98
0.5- 2.5
1.0- 5.0
0.4- 2.0
0.1- 0.5
With the same procedure also vegetable oil and commercial, synthetic ester based
hydraulic fluids were made.
73
The hydraulic fluids have to pass certain standard tests to fulfill necessary criteria to
be classified as lubricant. In the next chapters the main tests, their purpose and
limits will be discussed together with test results of the three different types of
hydraulic fluids.
3.13. Characterization of rapeseed oil ester based hydraulic fluids (V,VI)
The lubricants tested in the present work were commercial trimethylolpropane ester
based hydraulic fluids (RBS 46S (A) and 68S (B, commercial vegetable oil based
hydraulic fluid (RBS 32L (C, reference mineral oil based hydraulic fluid (0) and
hydraulic fluid based on trimethylolpropane ester developed in this thesis, viscosity
grade 32 (E). These fluids are mentioned and marked in following text by their
letters (A)-(E).
3.13.1. Viscosity (V,VI)
Viscosity indicates how f1uidy a lubricant is. It is very important that the viscosity of a
lubricant is as stable as possible with temperature, which means that viscosity value
does not change with time at certain temperatures. A capillary viscosity value is
based on Hagen-Poiseville"s law (84,85) according to (AA):
{};: K x t, where K;: constant (mm
2
/s
2
) depending on viscosimeters capillary
length and diameter
( AA)
t;: time (s) needed for fluid to run a certain length in the
capillary
For hydraulic fluids the most common viscosity grades are 32, 46 and 68 mm
2
/s. To
be within its grade, the viscosity of hydraulic fluids can maximally vary +/- 10 %.
According to ASTM D 445 viscosity is generally measured at 40C and 100C and a
viscosity index is calculated on the basis of the results. Viscosity index is a number
telling how stable the fluid is with temperature. The higher it is, more stable the fluid
is with temperature. Viscosity Table 1 in publication VI shows that viscosities are in
their grade, except RBS 46S, which is a little high, 50,4 mm
2
/s, when the highest
limit is 46,9 mm
2
/s. Viscosity index for vegetable oil and their ester based hydraulic
fluids are typically higher than comparable mineral oil based products. A typical
mineral oil value is around 180, here 187 and for vegetable oil or its esters, the
value is around 220.
74
3.13.2 Filterability (V,VI)
Good filterability is necessary for well functioning hydraulic systems. Particles in
hydraulic fluids naturally increase wear. The filterability is always measured both
from the raw material and the final prodUct: hydraulic fluid.
The method used is under standardization and under development at the Royal
Technical University of Stockholm together with users (86). It is called percentage-
method, because results are given in percents. The higher the value is (max 100 %),
the better. Figure 41 demonstrates the filterability equipment and its basic function
(85). Because the test is under development, there are no official limits for
filterability, but for the research and development period, a limit has been set at 80
%. This limit is well accepted by production and customers. Filterability results vary
quite a lot. Commercial synthetic ester based fluids have a filterability of only 63 %.
This figure can improve, when oil has been in use for a while, but it can cause
problems before this. The main reason for such a low value are particles in the raw
material, which has not been purified as well as should have done. An other reason
is that the additive package used was not the right one for this type of raw material.
The mineral oil based fluid had a little better result, 72 %, but needs also a further
study of its particles. Vegetable oil and its esters all passed the demanded 80 %,
being 80 % to 98 % as can be seen in Table 2NI.
Figure 41: The filterability equipment of the percentage-method and its basic
function.
75
3.13.3. Standard of purity and impurity particles (V,VI)
Standard of purity and impurity particles are very meaningful characteristics in
hydraulic use. Small particles can cause various problems in fine hydraulic systems,
for example extremely high wear. Small particles can already be present in the basic
fluids, in additives, or in both. Therefore, the amount of particles is calculated before
use. This is done according to ISO 4406-standard by microscopically calculating
particles larger than 5 m, but smaller than 15 m, and particles larger than 15 m
microscopically (87). The type of crystals affecting the filterability can also be
determined microscopically.
Requirements for the cleanclass of hydraulic fluid varies depending on its end use,
being around 16/13. As can be seen from Table 3M, the cleanclass 13/8 of
vegetable oil and its esters based hydraulic lubricants fulfill very well these demands.
3.13.4 Foaming (V,VI)
Foaming can cause problems, in the machine operating and instability from
beginning in the hydraulic fluid tank. The less foaming, the better it is for the user.
Foaming is measured by ASTM D892-standard (88). Results are given in time (s)
and height (mm),that is how high the foam was originally and how long time it takes
for foam to break down. In this method air is bubbled at a certain pressure into a
certain volume of tested oil for a certain period of time (87). The maximum values for
foaming are 600 mm and 240 seconds. The results are shown in Table 15.
Differences in the foaming of different vegetable oil and its ester based hydraulic
lubricants were not big, the time varies from 120 s to 175 s and the height from 20
mm to 33 mm. For RBS 688, 40s and 8 mm, exceptionally good results were
obtained.
76
Table 15: Foaming (ASTM 0 892) and colour (ASTM 0 1500) of some hydraulic
fluids.
Hydraulic fluid TMPE R8S 32L R8S 46S R8S 68S MO
(E) (C) (A) (8) (0)
Foaming
time (s) 175 120 151 40 150
height (mm) 33 20 28 8 30
Colour 4- 3+ 4 4
3.13.5 Colour (V,VI)
Colour is measured by the ASTM 0 1500-standard. In this method the yellowish
colour of the tested oil is compared to reference colours on a yellow-orange-brown-
scale. The result is given in a scale from 1 to 20. The lighter the oil is, the smaller
the number (88). The colour is light in a new hydraulic fluid, but darkens quite
quickly in use. This can be seen well in the field test results (Table 8, in V). This
darkening is mainly caused by antioxidants.
3.13.6. Cold stability (V,VI)
Cold stability of hydraulic fluid is very important for machines working out of doors
during winter time, such as forest machinery. According to the ASTM 0 97 standard,
pour point is one way to measure cold stability. It indicates the temperature, at which
the fluid is still f1uidy (88).
Cold stability can also be expressed as longterm cold stability by indicating how
many days the tested fluid stays f1uidy at a certain temperature (VTT-standard).
Further, viscosity can be measured at cold temperatures according to ASTM 0 445
standard (88). Viscosity should be and should remain at a certain level at a certain
temperature in order not to cause trouble e.g. in COld-starting, if hydraulic fluid is too
viscous. It is important that the pourpoint is lower than -30C for winter use out of
doors in Nordic countries. Machine producers recommend as a limit for cold starting
the viscosity of hydraulic fluid to be less than 5000 mm
2
/s. The results have been
77
collected in Table 16. As can be seen from Table 16, the viscosity of RBS 46S and
RBS 68 S at cold temperatures was lower than 5000 mm
2
/s. No problems have,
however, been noticed in field tests with these oils. With vegetable oil based
hydraulic fluids, the viscosity at cold temperatures is not a problem. It stays around
3000 mm
2
/s at least for a week.
Table 16: Pourpoint (ASTM D 97) and viscosity at cold temperatures (ASTM D 445)
of different hydraulic fluids.
Hydraulic fluids
Pourpoint (0C)
TMPE
(E)
-41
RBS 32L
(C)
-39
RBS 46S
(A)
-36
RBS 68S
(B)
-39
MO
(D)
-40
Viscosity (mm
2
/s)
Temperature (OC)
o
-10
-20
-30
Time (days)
1
3
7
428 372 573
1030 829 1430 2160
1540 1628 1480 3670
3114 2940 4060 7170
3050
3050
3050
3.13.7. Friction and wear (V,VI)
As explained in chapter 3.12.2, all of the basic fluids studied here have good
properties for reducing friction and wear, which is one of the main functions of
lubricants (78,79,82,83,89,90). There are several standardised methods to measure
these properties at the laboratory level (75). One of them is the fourball test
(standards ASTM D 2783, IP 239) (88,91), in which wear under loading and
maximum loading at which the lubrication fails and the lubricating film breaks are
measured. Limits for hydraulic fluids are <0,5 mm (damage is small) and >1,0 mm
(damage is large). The bigger the maximum loading is and the smaller the wear, the
better. The test results are shown in publication V, Table 4. In all vegetable oil and
its ester based hydraulic fluids wear was under 0,5 mm. Under maximum load the
differences, from 2000 N to 3000 N, are due to different additive packages.
78
3.13.8. Oxidation stability (V,VI)
Poor oxidation stability has been claimed to be one of the weaknesses of vegetable
oil based hydraulic fluids. With the right choice of raw material and additives this is
no longer a problem. With TMP-esters of rapeseed oil, oxidation stability can be
even improved slightly. Table 17 gives the test results from two different oxidation
tests with ASTM D 525, oxidation bomb test and DIN 51586 viscosity change test
(78,82,83,88,89,92). In Figure 42 is shown the apparatus of ASTM D 525. In the
oxidation bomb test oxygen is lead to a steel vessel, Which is filled with the tested
oil. The steel vessel is kept in a hot water bath (98 +/- 2C). Either the time for total
oxygen consumption or oxygen pressure drop within a certain time is measured. The
longer the time or the smaller the pressure drop is, the better. In DIN 51586 the
viscosity of the tested fluid is measured before the test and after 312 hours at 95C
temperature under air bubbling (10 dm
3
/h). The smaller the difference is, the better it
is. The latest oxidation test modified for vegetable oil based products is the Baader-
test, DIN standard 51 554 teil 3. The temperature is 95C and the time needed 7
days. The oil (60 mm
3
) is mixed with a copper catalyst. The total acid number and
viscosity at 40C are measured before and after the test. The changes are
calculated, the smaller they are, the better. The limit for acid number change is < 0,6
mg KOH Ig and for the viscosity < 20 %.
Figure 42: Oxidationbomb test (ASTM D 525-standard) equipment.
OXYGEN
9 b ~ r
TESTOIL .
79
Table 17: Oxidation stability measurements (ASTM D525, DIN 51586-, DIN 51 554
standards) from some hydraulic fluids.
Standard TMPE RBS 32L RBS 46S RBS 68S MO
(E) (C) (A) (B) (D)
ASTM D (psi) 42 30 39 29 39
DIN 51586 (0/0) 12.4 28.8 20.3 24.1 15.8
DIN 51554
TAN (mgKOH/g) 0.02 0.05 0.13 0
visco (0/0) 4.2 6.2 0.86 5.5
3.13.9. Corrosion stability (V,VI)
It is essential that the lubricant does not corrode the metal surfaces with which it is
in contact. This can also be measured at the laboratory level. Widely used
standardised test is the Cincinnati-Milacron test, which does not only measure
copper and steel corrosion, but also what happens to the lubricant in contact with
metals during heating: Does it oxidize? Does there begin chemical reactions
catalysed by metals?
In this test viscosity and total acid number of a 200 mm
3
oil sample with steel and
copper plates are measured before and after the test at 135C for 168 hours and the
difference is calculated on percent. Total sludge formation is measured after the test
(93).
As can be noticed from the results (table 7, VI) TMPE developed in this research
gave the best results. The difference in total acid number was significantly smaller,
0.17 mg KOH/g, as compared to vegetable oil and commercial esters, 1.01 mg
KOH/g to 1.74 mg KOH/g. Also the total sludge formation, 1.1.mg/100 mm
3
, is much
smaller in the TMPE developed in this research compared to others, 28.8 mg/100
mm
3
. Total sludge is generated mainly with additive package. Other properties such
as viscosity change and copper and steel plates weight changes were similar with all
tested fluids.
80
3.13.10. Biodegradability (V,VI)
Biodegradability can be tested with many different methods. Table 18 shows results
as tested by the CEC-L-33-T-82-standard (89,94). These figures are compared with
other test results with RB5 32L. Bioaccumulation and toxicity properties of lubricants
are equally important characteristics as biodegradability. Bioaccumulation measures
if the product or parts of it accumulates in nature in micro-organisms, animals, plants
etc. It can be measured for example by GECD standard tests. Toxicity should be
measured together with bioaccumulation by GECD standards, because the product
may be toxic when disposed in nature.
Table 18 : Biodegradability of some hydraulic fluids.
Testmethod
CEC-L-33-T-82
. TMPE
>90 %
RB5 32L RB5 465 RB5 685 limit
>90 % >90 % >90 % 70 %
DIN 38412
GECD 301F
5 days
>85 %
14 days
60 %
3.14. Conclusion of laboratory tests (V,vl)
TMP ester developed in this thesis seemed to meet its requirements as a basic fluid
for hydraulic fluids very well. Laboratory testing showed that especially at higher
temperatures TMP ester worked better than a vegetable oil based hydraulic fluid and
as well as comparable, commercial synthetic ester based hydraulic fluids. The
oxidation stability of TMP-esters was better than that of a vegetable oil based
hydraulic fluid and as good as, or even better than, a comparable commercial
synthetic ester based hydraulic fluid. This difference could be best observed at
higher temperatures. In decreasing friction and wear all of these three different
types of hydraulic fluids were excellent. Cold stability of the TMP ester was better
than that of vegetable oil or commercial synthetic ester. The pourpoint was lower
and viscosity stability slightly better than with references. The next step is to finalize
already started field tests, to ensure the suitability of TMPE based hydraulic fluids for
the market.
81
4. CONCLUSIONS
The aim of this work was to find new ways to produce and use of vegetable oil
based esters.
The alcohols used were chosen with the end use of the required ester in mind. They
were short, straight chain alcohols such as methanol, ethanol, etc. or branched chain
alcohols such as 2-ethyl-1-hexanol or polyols like trimethylolpropane. With short
chain alcohols esters were produced chemically with excellent conversions (95 -100
%). These esters were also made on an industrial scale. They are mainly used as
fuel or solvents. Customers have been very pleased with these environmental
acceptable products. However in use as solvent problems such as swelling or
brittling of gum- and seal materials arose.
The next step was to use longer, branched chain alcohols, like 2-ethyl-1-hexanol.
Such esters were developed for the first time from rapeseed oil both chemically and
enzymatically. Good conversions, 96-100 %, were obtained using both methods. In
chemical synthesis the best results were obtained under reduced pressure and
sufficiently long reaction time. Rapeseed oil was used in excess of stoichiometric
amount, and the catalyst was alkaline sodium methylate. The conversion was 97.6
%. In enzymatic synthesis best results were obtained by Candida rugosa lipase. The
molar ratio was near to stoichiometric, added water was needed, and the amount of
lipase was 7,4 % (wlw). On the laboratory scale the conversion was 99.8%. The
enzymatic reaction was also examined on a pilot scale. The conversion was 87 %.
The lipase was immobilized to different carriers, of which XAD-7-resin, gave in pilot
scale 90 % conversion. For the chemical reaction, higher temperatures and reduced
pressure was needed than for the enzymatic reaction. This partly compensates the
fact, that lipases are more expensive than chemical catalysts, when bearing in mind
the costs of the reactions. No differences could be seen with the preparation
methods in either the conversions or in the purity of the wanted esters.
Both esters synthetized by chemical catalyst or enzymatic biocatalyst have been in
field tests e.g. in detergent industry use to replace traditional organic solvents. The
test results have been promising and these esters are today used for example in
carshampoos.
The chemical synthesis was also applied in tallow oil fatty acids esterification with
polyols. The best result, the conversion of 87 % and 6 % of unreacted fatty acid was
obtained at reduced pressure and middle temperature at quite long reaction time, 70-
75C/2-9 h. The molar ratio was stoichiometric and the catalyst was acidic p-
toluenesulfonic acid. Because there were several serious difficulties with this
synthesis, the next step was to use transesterification instead of esterification.
Transesterification was done both chemically and enzymatically.
Instead of fatty acids, the raw material was the methylester of rapeseed oil.
Chemically from rapeseed oil methylester was succesfully synthetized with
82
trimethylolpropane trimethylolpropane ester of rapeseed oil with conversion of 99 %.
. This reaction demands reduced pressure and at least two different temperature
areas. The methyl ester of rapeseed oil was used a little over stoichiometric quantity
and alkalic sodium methylate was used as catalyst. The fatty acid composition of the
product is similar to that of comparable,commercial synthetic esters. The production
of ester by the method developed in this work, is economically much cheaper than
with the methods based on purified fatty acids. TMP-ester of rapeseed oil has also
been produced chemically in pilot scale. The yield was 99.0 %. Enzymatically TMP-
ester of rapeseed oil was produced by Candida rl/gosa:.Jipase at a temperature, which
this lipase could tolerate. The reaction was proceed at reduced pressure with long
timeschedule, 47C, 5.3 MPa, 42 h. The methylester of rapeseed oil was used in
excess of stoichiometric to obtain the conversion of 98 %. The immobilizaton of
lipase was also researched. The best carrier was Celite R-630 with a conversion of
95 %. As a comparison, a commercial immobilized lipase, MIcor miehei-lipase
(Lipozyme 1M 20), was used. The conversion was 92,5 %. In the chemical reaction
slightly better conversion was achieved than in the enzymatic reaction. The reaction
time was much shorter in the chemical preparation, but at the same time the
temperature was much lower in the enzymatic preparation. In the chemical
preparation also more triglyceride ester of TMP was achieved.
Both of these esters were used as starting materials for producing hydraulic fluids for
laboratory tests. The esters were compared to commercial synthetic ester based
hydraUlic oils, and to rapeseed and mineral oil based hydraulic fluids. As a
conclusion of the laboratory tests the trimethylolpropane ester of rapeseed oil based
hydraulic fluid resisted oxidation better than the rapeseed oil or mineral oil based
products, and equally well or even better than commercial synthetic ester based
fluids. This difference was even more noticeable at higher temperatures. The cold
stability properties of trimethylpropane ester of rapeseed oil based fluids were better
than those of the rapeseed oil,mineral oil and commercial ester based fluids, as was
evident by the viscosity stability at cold temperatures.
Friction and wear properties were good for all vegetable oil based products, better
than for mineral oil based fluids. The TMP-esters developed in this work are ready
for fieldtests, which are essential before commercialization.
In this work new methods to produce vegetable oil esters were explored. The
methods have been succesfully used either in industrial or pilot scale production
with good yields. The aims of this work were fulfilled extremely well.
83
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Appendix I
with the permission of the publisher,
Lipase-Catalyzed Transesterification of Rapeseed Oil
and 2-Ethyl-1-Hexanol
Y.Y. LinkoB,*, M. Uimsa
b
, A. Huhtala
a
and P. LinkoB
aHelsinki University of Technology, Laboratory of Biotechnology and Food Engineering, FIN-02150 Espoo, Finland and bRaisio
Group Oil Milling Industry, FIN-21200, Raisio, Finland
1411
1//
Lipase-catalyzed transesterification (alcoholysis) of low-
erucic acid rapeseed oil and 2-ethyllhexanol without an
additional organic solvent was studied in stirred batch
reactors. Of a number of commercially available enzymes
investigated, the best results were obtained with a Can-
dida rugosa lipase. The optimal transesterification condi-
tions were an oilIalcohol molar ratio of 1:2.8, a minimum
of 1.0% (wlw) added water, and with a temperature of
37-55C. Under the optimal conditions, a nearly complete
conversion was obtained in one hour with 14.6% (wlw)
lipase, whereas 0.3% (wlw) lipase required 10 h for similar
results. The enzyme was inactivated at 60C.
KEY WORDS: Alcoholysis, biocatalysis, enzyme, 2-ethyll-hexanol,
2-ethyl-l-hexyl ester, lipase, rapeseed oil, transesterification.
Interest in Iipase-catalyzed biosynthesis is rapidly increas-
ing (1,2). Lipases have great potential in food-related lipid
modifications (3,4), in the production of esters (5,6), biode-
gradable polyesters (7,8) and fatty acids (9,10). Lipases
(triacylglycerol acylhydrolase, EC 3.1.1.3) are esterases that
catalyze hydrolysis and synthesis of glycerol esters. In trans-
esterification, the acyl moiety is exchanged either between
an ester and acid (acidolysis), ester and alcohol (alcoholysis)
or two esters (acyl exchange) (10,11). Acyl exchange between
two molecules is also called interesterification, and between
two acyl groups within a molecule it is called intraesterifica-
tion. Ester synthesis is favored under restricted water
availability (low water activity) (12), although a minimum
quantity of water is necessary for enzyme catalysis to take
place (131.
Lipase-catalyzed alcoholysis in the absence of solvent is
important in industrial applications, especially for food uses.
Complete transesterification between one mole of triacyl-
glycerol and three moles of alcohol yields three moles of ester
and one mole of glycerol (14). Although it has been claimed
that the presence of additional organic solvent may be
useful for example, in controlling water activity and
microbial contaminations (15,16), the absence of solvent
allows higher substrate and product concentrations (17),
simplifies downstream processing (18), and improves safety
(19). Zaks and Klibanov (20) were the first to study the
transesterification of tributyrin with a number of primary
and secondary alcohols, catalyzed by porcine pancreatic
lipase, to yield an ester of butyric acid and dibutyrin. They
demonstrated the importance of the quantity of water pres-
ent, both to the activity and stability of the biocatalyst.
Macrae (12) and Halling (21), among others, have further
emphasized the importance of water relationships in lipase-
catalyzed synthetic reactions. Hirata et aL (22) later demon-
strated that water requirements in the alcoholysis of
tributyrin by different lipases may vary widely.
In spite of the great technical interest of fatty acid esters
(23,24), lipase-catalyzed transesterification involving high-
molecular weight fatty acids has only recently been in-
vestigated.. Mittelbach (25) has studied the alcoholysis of
*To whom all correspondence should be addressed.
Copyright 1994 by AOCS Press
sunflower oil both in petroleumether as solvent and without
additional solvent, to synthesize methyl and ethyl esters as
diesel oil substitute. Shaw et aL (26) used Celite-inunobilized
PseuOOmonas fluorescence lipase in the alcoholysis of olive
oil An excess of alcohol has been claimed to be beneficial
in alcoholysis by suppressing the hydrolytic side reaction
(17,18). Nevertheless, in such cases, disadvantages such as
the removal of excess alcohol from the product should also
be considered. Erueyl erucate, the main component of jo-
joba oil, has been produced by transesterification of high-
erucic acid rapeseed oil and erucyl alcohol (18). The aim of
the present work was to investigate Iipase-catalyzed trans-
esterification (alcoholysis) of rapeseed oil and 2-ethyl1-
hexano!, currently used in the chemical synthesis of a
number of important compounds (27), without the use of
an additional organic solvent and as a possible alternative
to acid or base catalysis.
MATERIALS AND METHODS
Materials. Refined, low-erucic acid rapeseed oil and syn-
thetic rapeseed oil2-ethyl-1-hexylester were obtained from
the Raisio Group (RaisiO, Finland). The approximate fatty
acid composition of the oil was 57% oleic acid, 22% linoleic
acid. 12% linolenic acid, 4% palmitic acid, 1% stearic acid,
2% eicosanoic acid <1% erucic acid and 1% others.
2-Ethyl-1-hexanol was obtained from Fluka Chemie AG
(Buchs, Switzerland). MonO', di- and triolein standards
were from Sigma (St. Louis, MO), and glycerol from May
& Baker (Dagenham, United Kingdom).
Enzymes. The following powdered microbial lipases
were obtained from Biocatalysts Ltd. (Pontypridd, United
Kingdom): Candida rugosa (ex. cylindracea) (42,500 Ulg;
water 5.0% w/w), Chromobacterium viscosum (13,300 DIg;
water 5.9% w/w), Mucor miehei (7,200 Ulg; water 7.4% wlw)
and P. fluoreseens (11,900 Ulg; water 3.1% w/w).
Transesterifieation. A preliminary study with those four
Iipases (10 mg; 3.3% wlw) was carried out with 0.277 mmol
(ea. 0.2 g) rapeseed oil and 0.680 mmol (107 ilL) of
2-ethyl-1-hexanol (molar ratio of 1:3) in capped 13-mL test
tubes under magnetic stirring at 200 rpm with 3.0%
added water. Transesterification was allowed to continue
for 72 h, after which lipase was separated by centrifuga-
tion for 5 min at 2000 rpm (Martin Christ '1YPe UJ3;
Osterode, Germany), and the supernatant was pipetted
into Eppendorf tubes for storage at -20C and later
analysis. Further transesterification reactions were car-
ried out for up to 72 h with varying substrate molar ratios
(rapeseed oillethyl hexanol from 1:1 to 1:10), C rugosa
lipase (from 0.3 to 14.6% w/w) and added water (from 0.25
to 50% w/w) quantities and temperatures (from 37-60C).
Lipase activity. Lipase activity was determined accord-
ing to the Biocatalysts Ltd. assay method "Lipase AssaY,'
which is based Q.tl the hydrolysis of 50% (vol/vol) olive oil
emulsion (Product No. 800-1; Sigma) as shbstrate at pH
7.7, and 37Cin one hour. The quantity of free fatty acids
formed was titrated with O.lM sodium hydroxide. One
unit of lipase activity was defined as the quantity of
JAOCS, Vol. 71, no. 12 (December 1994)
1412
Y.Y. LINKO ET AL.
LII
FIG. 1. Thin-layer chromatograms with different Iipases (rapeseed
oilJ2-ethyl-l-hexanol substrate molar ratio 1:3; 3.5%, wlw, lipase; 3.0%,
w/w, added water; reaction time 24 h). 1, Rapeseed oil; 2,
2-ethyll-hexanol; 3, triolein; 4, diolein; 5, monoolein; 6, Candida
rugosa lipase; 7, Mucor mkhei lipase; 8, Pseudomo1l48 floorescence
lipase; 9, Chromobacterium viscosum lipase; 10, blank.
Consequently, the use of alcohol excess was not further
investigated.
About 50% rapeseed oil conversion was reached in one
hour, with a nearly complete conversion in 10 h when the
substrate molar ratio was between 1:2.8 to 1:3.0, lipase
quantity was 3.3% (w/w) and the added water 3.0%. It
could be concluded from several replicate transesterifica-
tions that the highest ester yield with the least residual
alcohol was obtained with the substrate molar ratio of
1:2.8, although the differences with different substrate
molar ratios, down to 1:2.5, were small. Consequently, the
molar ratio of 1:2.8 was used in most of the subsequent
trials.
Lipase quantity. As could be expected, an increase in
lipase quantity markedly increased the rapeseed oil con
version during the first few hours, but after seven hours
the differences had almost leveled off. Figure 2 illustrates,
as an example, the rapeseed oil conversion as the function
of time with 0.3, 2.3 and 14.6% (w/w) lipase, substrate
molar ratio of 1:2.8 and 3.0% (w/w) added water, The reac-
tion was nearly complete (the maximum theoretical con
version under the conditions used is 93.3%) in one hour
with the highest lipase quantity used, whereas with only
0.3% (w/w) lipase, the conversion in one hour was only
about 20%. Nevertheless, a nearly complete conversion
was obtained in 10 h, even with the lowest lipase quantity
used which, in addition to the stability of the enzyme, is
important in considering costs. Interestingly, Goldberg
et aL (29) reported that an increase in the quantity of
powdered C rugosa lipase results in a decrease in the ap-
parent enzyme activity in the production of heptyl oleate,
owing to an increase in diffusion limitation, a problem
which may be minimized in large-scale experiments by op-
timal biocatalyst and reactor design.
enzyme that catalyzes the release of one ,.,mole of free
fatty acid from olive oil in one minute under those
conditions.
Analytical methods. Qualitative analyses were carried
out by thin-layer chromatography (TLC). Samples were
diluted 1:10 (vol/vol) with ethanol, and 0.01 mL of the
diluted samples were used for TLC analysis. Hexane/di-
ethyl ether/acetic acid (80:20:1) was used as solvent on
Kieselguhr 60 F
254
plates (E. Merck. Darmstadt.
Germany) with one hour running time. Slightly dried
plates were sprayed with 0.1% 2',7'-dichlorofluorescein
(Aldrich-Chemie, Steinheim, Germany) in 99.5% ethanol
(Alko Ltd., Rajamaki. Finland) for detecting the spots at
254 and 360 nm.
Rapeseed oil conversion (% rapeseed oil used) and ester
yield (% of theoretical) were determined by reversed-phase
high-performance liquid chromatography (HPLC), as
modified from EI-Hamdy and Perkins (28) and Forssell et
aL (3), with a Perkin-Elmer (Norwalk, CT) 4 pump module,
ISS-lOO sampler, and 101 oven, Novapack C18 3.9 X 150
mm column with 4,.,m silica particles. HP 1047A refrac-
tive index detector (Hewlett-Packard, Palo Alto, CAl, PE
316 integrator and PE 7500 professional computer.
Samples were diluted with acetone to 10-20 mg/mL,
filtered through a Millex-LCR
4
disposable filter with 0.5
,.,m porosity (Millipore, Bedford, United Kingdom), and
0.02 mL of the filtrate was used for the analysis. The run-
ning solvent was acetone/acetonitrile (1:1) at 1.0 mL/min,
37C, 30 min. Residual 2-ethyl-1-hexanol could not be
determined by the HPLC method because the alcohol
overlapped with the acetone used as the diluent. Conse-
quently, any excess 2-ethyl+hexanol was determined by
TLC as described above. Moisture content of the enzyme
preparations was determined by drying about 4-g samples
overnight at 105C.
RESULTS AND DISCUSSION
Lipase. Th identify the most suitable enzyme for subse-
quent transesterification trials, preliminary experiments
were carried out with the most promising commercial
lipases of a total of 25 previously screened for n-butyl
oleate biosynthesis (5). A substrate molar ratio of 1:3, 3.3%
(w/w) of lipase and 3.0% (w/w) of added water were used.
Figure 1 shows that in 24 h the use of C rugosa lipase
as biocatalyst resulted in the highest ester production,
with no detectable residual rapeseed oil and little by-
product. A 98% conversion of rapeseed oil was obtained
in 24 h. Also, a superior cost/benefit ratio has been re-
ported previously for this lipase in the direct biocatalytic
synthesis of n-butyl oleate (4). The use of P. fluorescens
and C viscosum lipases also resulted in relatively high
ester production, with 96% conversion in 24 h and 97%
conversion or higher in 48 h, although clearly more resid-
ual alcohol and by-products could also be seen. The poor-
est results were obtained with M miehei lipase. Conse-
quently, C rugosa lipase was chosen for further studies.
Substrate molar ratio. One of the aims was to obtain
a maximum rapeseed oil conversion with no or little
residual 2-ethyl+hexanol. When an alcohol excess was
used, rapeseed oil conversion was always low, and the pro-
duct mixture contained large quantities of residual alcohol
and, in some cases, residual oil. The relative ester yield
decreased with an increase in the alcohol molar excess.
JAOCS, Vol. 71, no. 12 (December 1994)
1 2 3 5 6 7 8 9 10
3/1
1413
LIPASE-CATALYZED TRANSESTERIFICATION
FIG. 2. Effect of Candida cylindracea lipase (.,0.3%; 1:0., 2.3%; v,
14.6%; w/wl quantity on transesterification (substrate molar ratio
1:2.8; 3.0%, wlw added water).
j
J
I
I
1
3 4 5 6
Time (h)
FIG. 4. Effect of temperature (., 37C; 0, 45C; ~ , 5 0 C ; 1:0., 55C;
., 60C) on tr&nsesterification (substrate molar ratio 1:2.8; 3.3%, wIw,
lipase; 3.0%, w/w added water).
100
I
1
80
I
""
J
:::
I
c 60
i
I
I
"
I
;> 40
:::
c
U
I
20
I
I
.---.J
6 2 4
Time (h)
o
Added water. The importance of the control of water
content (and of water activity) in lipase-catalyzed esterifi-
cations has been frequently emphasized. Figure 3 shows
the effect of added water on transesterification when the
substrate molar ratio was 1;2.8 and the lipase quantity
was 3.3% (wlw). No phase separation took place with up
to 5% (wfw) of added water. At higher water quantities,
the organic phase was used for HPLC analyses. The water
(ca. 5% wfw of lipase) present in the lipase preparation was
insufficient, and only about 25% conversion was reached
in seven hours without added water. With 0.25% (wfw) of
added water, about 60% conversion was reached in one
hour, but an increase in time did not bring about a fur-
ther increase in rapeseed oil conversion. With a minimum
of about 1.0% added water, about 50% conversion was
reached in one hour, and a nearly complete conversion in
five hours. Little difference was observed between 1.0 and
3.0% added water. Additional increases in added water did
not result in further improvements. Interestingly, the reac-
tion proceeded nearly identically with 50% (wlw) added
water as with 3.0%. Although direct lipase-catalyzedester
synthesis may not be directly compared with transesteri-
fication, it is of interest to note that 90% or higher butyl
oleate yields have been reported in the presence of excess
water (6,30).
Temperature. Figure 4 illustrates the effect of tempera-
ture on the time course of rapeseed oil conversion. There
was little difference within the temperature range of 37-
55C. with about 90% conversion reached in 2-3 h and
nearly a complete conversion in 7 h. However, at 60C
lipase was clearly inactivated under the experimental con-
ditions. The results agreed well with those of Mittelbach
(25), according to whom the optimal temperature for Can-
dida sp. lipase-catalyzed sunflower oil alcoholysis is
45-50C. Hirata et aL (22) also reported 50C as the op-
timal temperature for the transesterification of tributyrin
and l-octanol with C rugosa lipase.
80 -
::: 60
c
'" 40 -
c
U ':.V.-----.-----.J
067
Time (h)
FIG. 3. Effect of added water (.,0%; 0, 0.25%; .,3.0%; 1:0., 50%)
on transesterification (substrate molar ratio 1:2.8; 3.3%; wlw, Iipasel.
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1993, pp. 205-219.
25. Mittelbach, M., J. Am. OiL Chem. Soc. 67:168 (1990).
26. Shaw, J.-F., D..L. Wang and Y.J. Wang, Enzyme Microb. TechnoL
13:544 (1991).
27. Chemical Marketing &porter 243:5 (1994).
28. El-Hamdy, A.H., and E.G. Perkins,J. Am. Oil Chem. Soc. 58.867
(1981).
29. Goldberg, M.. D. Thomas and M.-D. Legoy, Enzyme Microb.
TechnoL 12:976 (1990).
30. Nishio, T., T. Chikano and M. Kamimura, Agric. BicL Chern.
52:1203 (1988).
[Received May 14, 1994; accepted September 13, 1994]
9'1
Appendix U .
with the permission of the publisher
BIOTECHNOLOGY TECHNIQUES
Volume 8 No.6 (June 1994) pp.451-456
Received 5th May
2-ETHYL-I-HEXANOL FATTY ACID ESTERS FROM
RAPESEED OIL BY TRANSESTERIFICATION
Merja Uimsa*, Anne Huhtala. Yu-Yen Linko*" and Pekka Linko
Laboratory of Biotechnology and Food Engineering,
Helsinki Universiry of Technology, FIN-02l50 Espoo. Finland
SUMMARY
FaIry acid eSlers of 2-elhyl-I-hexanol were produced in a small pilol scale from rapeseed oil by
Candida cylindracea lipase catalyzed traJlseslerijication (alcoholysis) wilhow added solven!. Up
1090% conversion of rapeseed oil (97% of theoretical) was obtained in 8 h in 2 kg scale QI 37'
C wilh 3.4% (w/w) lipase imnwbilized on an anion exchange resin Amberlite XAD-7. rapeseed
oil:2-ethyl-I-hexanol substrale molar ratio of 2.8, alld 3% (wlw) ofadded warer.
INTRODUCTION
There is an increasing interest in using vegetable oils to obtain biodegradable esters
valuable as lubricants, biodiesel, surface active agents, solvents, etc. (Meffen, 1984;
Harrington and D'Arcy-Evans, 1985; Chopineau et al., 1988; van der Waa1, 1989).
Special attention has been recently paid to lipase catalyzed ester biosynthesis and
transesterification (Macrae and Hammond, 1985; Yamane, 1987: Mukerhjee, 1990;
Bjorkling et af., 1991; Linko et al., 1992). Nevertheless, enzymic alcoholysis of
\'egetable oils without additional organic solvent has been little investigated. Lazar et af.
(1985) have described the methanolysis of tallow in a bufferlhexane 2-phase system, and
Choo et al. (1987) used Candida cylindracea lipase in the methanolysis of vegetable oils
in hexane with an ester yield of 98%. Miuelbach (1990) employed Pseudomonas
,r7uorescens lipase both in petroleum ether and without an additional solvent for the
biosynthesis of methyl and ethly esters as diesel oil substitutes. Shaw et aJ. (1991) used
P. fluorescens lipase immobilized on Celite as a biocatalyst for the alcoholysis of olive
oil. Trani et af. (1991) used Lipozyme IM-20 (Novo Nordisk, Bagsvaerd, Denmark), a
Mucor miehei lipase immobilized on a weak anion exchange resin, to produce stearyl
oleate by transesterification of triolein and stearylalcohol, and erucylcrucate, the main
component of jojoba oil, by transesterification of rapeseed oil and erucyl alcohol. The
aim of the present work was to produce a blend of 2-elhyl-l-hexanol fatty acid esters by
enzymic alcoholysis of rapeseed oil.
Z Present address: Raisio Group Oil Milling Industry, r.O.Box 101, FIN-21201, Raisio, Finland
z* To whom all correspondence should be addressed
451
1/11
MATERIALS AND METHODS
Materials. Refined low erucic acid rapeseed oil and synthetic rapeseed oil 2-ethyl-l-
hexylester were obtained from Rasio Group (Raisio. Finalnd). The approximate fatty acid
composition of the oil was 57% oleic acid, 22o/c linoleic acid, 12% linolenic acid, 4%
palmitic acid, 1% strearic acid, 2% eicosaenic acid. < I% erucic acid, I % others. 2-
Ethyl-l-hexanol (water solubility at 25C ca. 2.5%) was obtained from Fluka Chemie
AG (Buchs, Switzerland). Mono-, di- and triolein standards were from Sigma (St. Louis,
MO), glycerol from May & Baker (Dagemham, UK), and powdered Candida rugosa
lipase (42,500 Ulg; water 5.0% w/w) was obtained from Biocatalysts Ltd (Pontypridd,
UK).
Transesterijication. The reaction mixture contained 50 g to 2000 g of rapeseed oil. 25
cm
3
(20 g) to 1.0 dm
3
(829 g) 2-ethyl-l-hexanol ratio of 1:2.8), 3.4%
(w/w) lipase, and I % to 5% (w/w) added water. Tne reaction was carried out for up 10 5
hours at 37C in a stirred vessel of varying shape either with free powdered lipase or
with enzyme immobilized on various carriers. Glass beads (20 g per 100 g rapeseed oil
and 5 g lipase), polyurethane foam (0.8 g), and adsorption resins Amberlite XAD-2 (2.5
g) and Amberlite XAD-7 (2.5 to 15 g) were used carriers.
Lipase activity. Lipase activity was determined according 10 the Biocatalysts Ltd. assay
method Lipase Assay, which is based on the hydrolysis oi 50% (v/v) olive oil (Product
No. 800-1, Sigma Chemical Company, St. Louis, Missouri) as substrate at pH 7.7, 37C
in one hour. The quantity of free fatty acids formed was titrated with O.IM sopdium
hydroxyde. One unit of lipase activity was defined as the quantity of enzyme which
catalyses the release of one 11 mole of free fauy acid from olive oil in one minute at the
above conditions.
Analytical methods. Qualitative analyses were carried OUl by thin layer chromatography
(TLC). Samples were diluted 1: 10 (v/v) with ethanol, and 0.01 ml of lhe diluted samples
were used for TLC analysis. Hexane:diethylether:acelic acid (80:20: 1) was used as
solvent on Kieselguhr 60 F254 plates (E. l\'1erck, Germany) wilh one hour
running time. Slightly dried plates were sprayed Wilh 0.1 % 2',7'-dichlorofluorescein
(Aldrich-Chemie, Steinhein1, Germany) in 99.5'* ethanol (Alko Ltd, Finland) for
detecting of the spots at 254 and 360 nm.
Rape seed oil conversion (% rapeseed oil used) and ester yield (% of theoretical) were
determined using reversed phase high performance liquid chromatography (HPLC) as
modified from El-Hamdy and Perkins (22) and Forsell et a1. (1), employing a Perkin-
Elmer (Norwalk, Connecticut) 4 pump module, ISS-lOa sampler, and 101 oven,
Novapack C18 3.9 x 150 mm column with 4 11m silica pJ.nicles, HP 1047A refractive
index detector (WaterslMillipore, Milford. UK) PE 316 integratOr and PE 7500
professional computer. Samples were diluted Wilh acetone to 10 to 20 mgiml, filtered
through a Millex-LCR4 disposable iilter 0.5 l-lm porosity (Millipore, Bedford, UK), and
0.02 ml of the filtrate was used for the analysis. The running solvent was
acetone:acetonitrile (1:1) at 1.0 ml/min, 37C, 30 min. Residual 2-ethyl-l-hexanOl could
not be determined by the HPLC method inasmuch as the alcohol overlapped with
acetone used as the diluent. Consequently, any excess 2-ethyl-I-hexanol was determined
by TLC as described above.
Moisture content of the enzyme preparations was determined by drying about 4 g
samples over night at 105C.
452
2/11
3/11
RESULTS AND DISCUSSION
Figure I shows the effect of different type of mixing on rapeseed oil conversion in a
series of experiments with substrate molar ratio of 1:2.8, 1.0% (w/w) added water and
3.4% (w/w) (25 g) lipase. With 100 g of oil and 50 cm
3
(41 g) of 2-ethyl-I-hexanol in
500 em3 flat bottomed flask under 300 rev min-I magnetic stirring conversion increased
in a few minutes up to about 65% and in 1.5 h to 80%, with a final conversion in 5 h of
87%(94% of theoretical maximum). Within 30 min the solid lipase was 'immobilized' on
the walls, adsorbing the glycerol formed in the reaction. Ester production was 21.7 g per
g lipase and 108.4 g per 100 g rapeseed oil. In another experiment with 500 g of oil and
700 rev min-
l
mechanical stirring for the first 5 h, followed by 300 rev min-
l
for a further
5 h the final conversion was only 61 % (66% of theoretical). During the frrst 5 h lipase
remained evenly suspended, and the conversion was only 46% (50% of theoretical). The
decrease of mixing rate resulted in the aggregation of lipase at the bottom, with an
increase in conversion. This agrees well with the observation of Goldberg et af. (1990)
during studies on C. rugosa lipase catalyzed heptyl octanoate synthesis, that when the
mixing rate was increased sufficiently for complete lipase suspension, the enzyme was
inactivated. Also Lee and Chao (1989) have reponed that C. rugosa lipase is sensitive to
shear forces in a 0.1 % solution with the activity decreasong with an increase both in
mixing rate and time. The degree of lipase inactivation could he markedly reduced by
propylen glycol addition.
!
..!
10 6 4 2
100 r I
J ....------....._ 1
80 1/--....----.... i
r
. / ~
60 - Q - - ~ I
//?,---."./,/" I
40 - c . / ~ / 1
I /
20 1- /
! /
i I
1/
,/
o -L -'- '--__---'- ..J
o
>
Reaction lime (11)
Fig. I. Effect of mixing on transesterification {. I(}() g rapeseed oil,
300 rev minot magnetic stirring; 0 500 g rapeseed oil, 700 rev min-!
mechanical stirring for 5 h, followed by 300 rev min-
l
for 5
453
Figure 2 shows as a result of an experiment with 200 g rapeseed oil under otherwise
similar conditions that with mechanical stirring at a 300 rev min-I from the beginning,
conversion increased to about 73% (78% of theoretical) in 30 min, without a further .
increase. As much as 89% of the product could be recover by simple decanting. Similar
results were obtained when the batch quantity of rapeseed oil was increased to 550 g.
Only a slight increase in conversion was observed when the flask was frlled to minimized
the air/liquid boundary as suggested by Lee and Choo (1989). When the lipase was
reused afer decanting the product, conversion was only about 3%.
100 r-----,r-----,r---,---.,.----,
tf2. 80 !
~ - - ~
~ 60V ~
'" r !
'" 40 r ~ ~
~ i
o w J
u 0 . = = = = = r = : : = : - t ~ - - l
o J 2 3 5
Reaction time (h)
Fig. 2. Effect of reusing the biocatalyst (. 200 g rapeseed oil,
300 rev min-l mechanical stiffing, 1st use: II 2nd usc).
Because the preliminary experiments were suggested [hat lipase immobilization might be
of advantage in increasing rapeseed oil conversion. a numher of carriers were tested. No
improvement was obtained with glass beads, polyurethane foam or Amberlite XAD-2
resin immobilized lipase (Figure 3). Figure 4 shows the effeCt of increa<;ing the relative
quantity the Amberlite XAD-7 resin as the carrier on conversion, with the highest
rapeseed oil conversion (50 g of oil) of about 95'X (100<;1 of theoretical) obtained in 5 h
with 7.5 g of resin per 2.5 g of ]ipa<;e. Figure 5 ckarly illustrates the imponance of the
quantity of added water on rapeseed oil conversion using Amberlite XAD-7 immobilized
lipase, with best result obtained with 3% added water. Finally, the optimal conditions
found were tested with a 2 kg batch of rapeseed oil (I dm3, 829 g, of 2-cthyl-I-hexanol,
3% added water, 100 g lipase with 300 g XAD-7 resin. 170 rev min'], 37C)(Fig. 6).
The result was an about 90% conversion (97% of theoretical) in 8 h, equal to 22.4 g of
ester per g lipase or 112 g ester per 100 g rapeseed oil. Although the total reaction time
increased with the immobilized biocatalyst, final conversion could be increased at least by
about 20% to nearly theoretical.
454
4/11.
95
. ] ,':r
3"J ",; 1
0" O. I
o 1 2 0 1 2
Reaction time (lJ) Reaction time (lJ)
5/11
80
o 60
Fig. 3. Transesteriiication with (e) glass
beads, polyurethane. and (0)
Amberlite XAD-2 resin as camer.
100

/f
I
II i
, 1
.;

It.........
o
o J 2
Reaction time (Ii)
Fig. 5. Effect oi added Ie 1'i<:. 0
2%, 3% w/w) on lranSeslerillcalion
with Arnberlite XAD-7 resin as carrier.
455
Fig. 4. Transesteriiication with
Amber-lite XAD-7 immobilized lipase
(e 3.75 g, /). 5.0 g and 0 7.5 g lipase)
Reaction time (ll)
Fig. 6. Time course of lransesteriiiction
of 2 kg rapeseed oil with Amberlile
XAD-7 resin immobilized lipase (insen:
BPLC analysis of product as the iunc-
li,m of lime; l, 5 min; 2, 2 h; 2h: 3, 11:
4.611; 5, Rh)
CONCLUSION
It has been demonstaned that fatty acid esters of 2-ethyl-l-hexanol can be produced in a
small pilot scale from rapeseed oil in near 100% conversion by Canditk1 cylindracea
lipase catalyzed transesterification (alcoholysis) without added so]venL Best results were
obtained with about 3% (w/w) added water and a slight oil excess under relatively mild
mixing with lipase immobilized on hydrophobic anion exchange resin Amberlite XAD-7.
REFERENCES
BjorkIing, E, Godfredsen, S. E and Kirk, O. (1991). Trends Biotechnol. 3, 360-363.
Choo, Y. M., Ong, S. H., Goh, S. H. and Khor, H. T. (1987) Brit. UK Pat. Appl. GB 2.188,057.
Chopineau, J., McCaffeny, F. D., Therisod, M. and KJibanov, A. M. (1988). Biolechnol.
Bioeng. 31, 208-214.
Goldberg" M., Thomas, D. and Legoy, M.-D. (1990) Enzyme Microb. Teelmol. 12,976-98 I.
Harrington, K. J. and D'Arey-Evans, C. (1985). J. Am. Oil Chem. Soc. 62, 1009- 1013.
Lazar, G. (1985). Fel/e, Seifen, Anstrichm. 87: 394-400.
Lee, Y.-K. and Choo, c.-L. (1989) Biotedmo!. Bioeng. 33,183-190.
Unko, Y.-Y., Rantanen, 0., Yu, H.-C. and Unko, P. (1992). Factors affecting lipase catalyzed
n-butyl oleate synthesis. In Biocataiysis in Non-Conventionai Media, J. Tramper, M. H.
Vermiie, H. H. Beefink and U. von Stockar, eds. Elsevier, Amsterdam
Macrae, A. R. and Hammond, R. C. (1985). BiOlechnoL. Genetic eng. Rev. 3, 193-217.
Meffert, A. (1984).1. Am. Oil Chem. Soc. 61,255-258.
Mittelbaeh, m. (1990). J. Am. Oil Chem. Soc. 67,168-170.
Mukerhjee, K. D. (1990). Biocatalysis 3, 277-293.
Trani, M., Ergan, E and Andre, G. (1991) J. Am. Oil Chem. Soc. 68,20-22.
Yamane, T. (1987). J. AnL Oil Chem. Soc. (A, 1567-1662.
van der Waal, B. (1989). NLGI Spokesman 53(8), 359-368.
456
6/11
Appendix III
with the permission of the publisher
Process for preparing a synthetic ester from a vegetable oil
97
95395
1/111
The objects of the present invention are a process for preparing a synthetic ester
from a vegetable oil and lubricants which contain a synthetic ester prepared by
said process.
Natural fats and oils have been used as lubricants already for thousands of years.
With industrialization mineral based lubricants came also to the market. The app-
lications of lubricants and thus also the requirements set for them have changed
,
and developed with the advance of technology. Various types of synthetic esters
and lubricants containing the same have been developed to meet the new require-
ments.
The purpose of a lubricant is to minimize friction and wearing of metals. Lubri-
cants are developed according to the use and they consist of a base fluid and
additives improving the lubricative properties. With the development of technolo-
gy, lubricants are used under more and more severe conditions, such as at very
low or very high temperatures (e.g. the turbine engines of aeroplanes). At the
same time biodegradability, unburden to the environment, non-toxicity and the
use of renewable raw materials have emerged as new requirements. The use of
biodegradable lubricants is of particular importance in the machines and devices
used in the fields of agriculture, forestry and building, as the oil used may be left
in the environment.
By the synthetic esters developed as lubricants are meant esters prepared from
mono-, di- or trialcohols and mono- or dicarboxylic acids by known esterification
and transesterification methods. Usually the process comprises combining all the
reactants and letting the reaction happen in one stage. The reaction may be car-
ried out in the presence of catalysts, such as acids, bases or metal oxides.
The structure of the synthetic ester used has a profound effect on the stability of
the lubricant. Esters decompose by the effect of heat and/or oxygen. It is known
to increase the thermal stability of synthetic esters by using in the preparation no
95395
2
beta hydrogen alcohols. Oxidative properties on the other hand can be improved
by deuteration of esters.
Synthetic esters intended for a lubricative use are classified by structure as mono-
carboxylic acid, dicarboxylic acid, polyol and complex esters. Due to their low
viscosity and high volatility monoesters are poorly suitable as lubricants. Polyol
esters are chemically more stable than for example diesters due to the structure of
the polyols used in the preparation of said esters, wherein no hydrogen atom is at-
tached to the {3 carbon atom. Complex esters have promising lubricative proper-
ties but the manufacture thereof on an industrial scale is difficult because of the
severe conditions required by the reaction, especially if said esters are prepared -
from purified fatty acids and alcohols.
If polyol esters are prepared by using no alfa hydrogen acids, the stability proper-
ties of the esters can be further improved. Metro et al. (CA 859 771) have shown
that the no alfa hydrogen carboxylic acids increase the thermal and oxidative
stability of esters prepared from no beta hydrogen alcohols, as well as slow down
the hydrolysis of the esters.
As the low viscosity polyol esters are not suitable for traditional uses wherein
high viscosity is required, it has been aimed at preparing polyol esters of higher
viscosity from for example trimethylol propane (TMP). However, it has been
found that it is difficult to obtain simple TMP esters with both high viscosity and
a low pour point (cf. for example US 4,061,581).
Products based on vegetable oils are nowadays used more and more as lubricants
because of their safety to the environment. Natural vegetable and animal oils are
glyceride diesters, i.e. tri-, di- or monoesters of glycerol and straight chain satu-
rated and unsaturated fatty acids. The lubricant industry uses for instance ra-
peseed, rape, soybean, castor, olive, coconut, palm and pine oils.
21111
9f
95395
3
The advantageous properties of vegetable oils include user friendliness and non-
toxicity. In addition to this they degrade in the environment, do not accumulate in
the food chain of nature and are renewable raw materials. However, the use of
vegetable oils as lubricants has been limited by their poor stability properties. The
poor thermal and oxidative stability is due to unsaturated and polyunsaturated
fatty acids. On the other hand, the unsatisfactory behaviour of vegetable oils at
low temperatures is due to the saturated fraction of fatty acids. By using suitable
additives and by favouring in cultivation such varieties which do not have a too
high degree of saturation, it has been possible to somewhat improve the stability
properties. Also the purification of the oil for technical use is helpful.
Furthermore, attempts have been made to modify natural glyceride esters in order
to improve their stability properties. Known processes include catalytic hydroge-
nation, alcoholysis, geometrical isomerization and sulfurization. For example by
hydrogenation a certain amount of double bonds can be removed, from the unsa-
turated part of vegetable oils and by isomerization the amount of undesired iso-
mers can be decreased.
Van der Waal and Kenbeek have presented a process for the preparation of synt-
hetic esters from vegetable oils or animal fats (proceedings of the Tribology
2000, 8th International Colloqium, Technische Akademie Esslingen, Germany,
14-16 June 1992, Vol II, pp 13.3-1 - 13.3-8). The process comprises first decom-
posing the glyceride esters of the starting material into fatty acids and glycerol
and subsequently separating the fatty acid fraction into liquid and solid phases.
The fatty acids of the liquid phase are separated by distillation into single fatty
acids which can be further modified e.g. by hydrogenation or cracking to obtain
the desired raw material. Fractions containing a single fatty acid are esterified
with no beta hydrogen polyols for preparing a synthetic ester.
The fatty acids of the ester prepared according to the above described process
usually contain less unsaturated double bonds than the fatty acids of the starting
material, which improves the oxidative stability. However, the costs of the pro-
3nll
4
95395
4/111
cess are extremely high, due to the multistage separation and purification reac-
tions and the most severe conditions (high pressure and temperature) required by
the reaction. Moreover, it has been found that when fractions containing only a
single fatty acid are reacted with polyols, plenty of mono- and diglycerides are
formed, i.e. all the OH groups of the polyols do not react. This decreases the
triglyceride yield and the raw material has to be recycled several times if the
yield is to be improved. Furthermore, the reaction of a fatty acid and an alcohol
creates water which has to be removed during the reaction.
According to the invention it has now been found that it is possible to prepare
synthetic esters with good lubricative properties from vegetable oils by a process
which avoids the multistage reaction with several separations and recyclings and
by which good yields are obtained.
In the process according to the invention a vegetable oil is first transesterified by
reacting the vegetable oil with a lower alkanol to obtain a mixture of fatty acid
lower alkyl esters. The process is characterised in that the obtained mixture of
esters is further transesterified by reacting the said mixture with a no beta hydro-
gen polyol of the formula
CH
2
0H
/
R - C - CH
2
0H
\
CHzOH
wherein R is a C
1
-C
6
alkyl group, particularly a C
1
-C
4
alkyl group, or a -CH
2
0H
group, and the synthetic ester obtained is recovered.
Vegetable oils suitable as a starting material of the process are for example ra-
peseed, rape, soybean, castor, olive, coconut, palm, pine, maize, walnut, flax-
seed, cotton, sunflower, sesame and almond oils, especially rapeseed oil, rape oil,
pine oil and soybean oil, particularly rapeseed oil.
5
95395
snll.
The fIrst transesterifIcation reaction of the process according to the invention is
carried out by a process known per se, by reacting a refIned or alkalirefIned
vegetable oil with a lower alkanol to obtain a mixture of fatty acid lower alkyl
esters.
The lower alkanol used in the fIrst transesterifIcation reaction is preferably a C
I
-
C
4
alkanol, especially methanol or ethanol. The obtained mixture of lower alkyl
esters of the vegetable oil is thus preferably a mixture of C
I
-C
4
alkyl esters,
especially a mixture of methyl or ethyl esters. If desiIed, usual esterifIcation
catalysts may be used in the reaction, and the amounts of the reactants and the
reaction conditions (pressure, temperature, reaction time) are either commonly
known or easily chosen by a person skilled in the art.
The fIrst transesterification reaction may be illustrated by the following general
reaction scheme I:
H
2
C-O-C( =O)-R
I
I
H
r
-O-C(=O)-R
2
+ 3 <= 3 +
H
2
C-O-C(=O)-R
3
(I)
wherein R
I
, R
2
and R
3
are fatty acid residues, is an alkyl residue, especially a
C
I
-C
4
alkyl residue, and R
x
is R
I
, R
2
or R
3
Glycerol is formed as a by-product.
The fatty acid lower alkyl ester obtained from the fIrst transesterification reaction
is thus a mixture comprising various fatty acids of the vegetable oil used as the
starting material. It is typical of the invention that this mixture of fatty acid lower
alkyl esters may be used directly as the starting material of the second transesteri-
fication reaction without separation or purification of fatty acids.
In the second transesterifIcation reaction according to the invention the mixture of
fatty acid lower alkyl esters obtained from the first transesterification reaction is
reacted with a no beta hydrogen polyol, such as for example trimethylol ethane,
6 95395
6/111
trimethylol propane, trimethylol butane or pentaerythritol, especially with penta-
erythritol or trimethylolpropane. The conditions required by the reaction are not
so severe than those required by the process according to the prior art, and the
by-products formed may be present in the reaction.
The second transesterification reaction may be illustrated with the following gene-
ral reaction scheme II:
/CH20H
3 + R-C - CH
2
0H --+
x "
CHpH
+ 3
wherein R
I
, R
2
, R
3
, and R
x
have the same meanings as in the reaction scheme
I and R is a C
I
-C
6
alkyl group, especially a C
C
C
4
alkyl group, or a -CH
2
0H
group.
Consequently, it is the question of a totally different chemical reaction than in the
process of the prior art wherein a free fatty acid is esterified with an alcohol. In
the process according to the invention, an ester is reacted with an alcohol, and
thus it is the question of a transesterification reaction which reaction, as well as
the reaction conditions required by it and the by-products formed therein, is
totally different from the reaction used in the process of the prior art.
The synthetic ester obtained from the second transesterification reaction is reco-
vered and, if desired, purified by conventional methods. for example by neutrali-
zation and washing with an aqueous acid. No distillation or any other special
treatment is needed as the obtained ester is ready to use as such as a raw material
of lubricants.
When a polyol is reacted with a mixture of fatty acid lower alkyl esters, almost
all OH-groups of the potyol react into trigtycerides. Over 90 % of the theoretical
yield of the triglyceride is obtained, the proportion of mono- and diglycerides
7
/03
95395
7/111
being in total about 10 %. The product obtained does not contain any free fatty
acids which makes it an especially advantageous raw material for lubricants whe-
rein the oxygenation of free fatty acids would cause problems (corrosion, cliange
of viscosity). The process is well adapted for industrial scale and the synthetic
ester obtained has better stability properties than the vegetable oil used as the raw
material, while at the same time the advantageous properties of a vegetable oil
(biodegradability, non-toxicity, user friendliness) are maintained. By the process
it is thus possible to prepare synthetic esters from vegetable oils, for example
from rapeseed oil, in a yield of even over 90 %of the theoretical.
According to the invention the second transesterification reaction may preferably
be carried out in two stages, the reaction temperature of the first stage being from
about 50 to about 110 e and of the second from about 110 to about 160 e.
Preferably the reaction temperature of the first stage is from 85 to 100 C and of
the second stage from 110 to 140 e. Reaction time may vary for example from
two to twelve hours. Preferably the reaction time of the first stage is about 1 to 7
hours and of the second stage about 1 to 10 hours.
The no beta hydrogen polyol and the mixture of esters are preferably reacted with
each other in a molar ratio of about 1:2 to 1:5, especially in the molar ratio of
about 1:3,5.
The second transesterification reaction is preferably carried out under reduced
pressure, for example under negative pressure of 1.3 to 13 kPa, and optionally in
the presence of a catalyst. As catalysts known esterification catalysts, such as acid
and base catalysts, for example p-toluene sulfonic acid, phosphoric acid, sodium
hydroxide. sodium ethoxide and sodium methoxide, of which sodium hydroxide
and sodium methoxide are especially advantageous may be mentioned.
The synthetic ester prepared by the process according to the invention is an excel-
lent raw material for the preparation of lubricants. Lubricants, especially hyd-
raulic oils, which contain a synthetic ester prepared by the process of the inventi-
8
95395
8/111
on, optionally with one more additives, are also included in the scope of the in-
vention. As additives for example oxidation inhibitors, antiwear agents, antifoam
agents, corrosion inhibitors, dispersants, viscosity index improvers and/or pour
point depressers which are generally known in the art, may be used.
Oxidation inhibitors include for example amines and phenols. As antiwear agents
and corrosion inhibitors for example phosphates or sulfonates and as antifoam
agents for example metal sulfonates, metal phenates, polyesters or silicones may
be used. Viscosity index improvers include for example polyisobutenes, styrene-
butadiene and ethene-propene-eopolymers ~ h i c h all are thus suitable also as pour
point depressers.
In the following the invention is further described by means of examples, the pur-
pose of which is to illustrate but not to limit the invention.
Example 1. Preparation of a methyl ester of rapeseed oil
Rapeseed oil (0.3 moles) was weighed into a three-necked flask provided with a
thermometer, cooler and a stirring device. Stirring was started and methanol (2.0
moles) was added. The reaction mixture was heated to 60C and the alkali cata-
lyst used was added (0.5 % by weight). Stirring was continued for three hours.
The progress of the reaction was followed by thin layer chromatography. The
reaction mixture was washed with an aqueous acid. The glycerol created in the
reaction mixture which as a heavier component settles on the bottom of the vessel
was separated and the product mixture was analyzed. Rapeseed oil ester content
was 97 %.
Example 2. Preparation of a synthetic ester from rapeseed methyl ester
The methyl ester of rapeseed oil (0.65 moles) was weighed into a three-necked
flask provided with a thermometer, a cooler, a stirring device and a reduced pres-
sure generator. The weighed rapeseed oil ester was heated to 50-110 e, after
9
/a;;-
95395
9/11'
which trimethylol propane (TMP, 0.19 moles) was added in small proportions
with proper stirring. After the alcohol was well mixed, sodium hydroxide used as
a catalyst was added (0.1-1.0 % by weight of the reaction mixture). Then the
reaction mixture was heated under reduced pressure (about 8 kPa) until it started
boiling. The reduced pressure was maintained during the whole reaction. The
mixture was allowed to boil at the lower temperature (50-110C) for 1 to 7 hours
and at the higher temperature (110-160 0c) for 1 to 10 hours. The progress of the
reaction was followed by thin layer chromatography. and quantitative IR spectrum.
At the end of the reaction the product mixture was neutralized and washed with
an aqueous acid, filtrated and washed with water. Drying was performed with
anhydrous sodium sulfate. A liquid chromatogram and an IR spectrum were run
of the final product. The yield was 90.5 % of the theoretical.
Example 3. Preparation of an ethyl ester of soybean oil
Soybean oil (0.2 moles) was weighed into a three-necked flask provided with a
thermometer, a cooler and a stirring device. Stirring was started and ethanol (1.5
moles) was added. The reaction mixture was heated to 80C and the alkali cata-
lyst used (0.4 % by weight) was added. Stirring was continued for two hours.
The progress of the reaction was followed by thin layer chromatography. The
reaction mixture was washed with an aqueous acid. Glycerol was separated from
the reaction mixture and the product mixture was analyzed by liquid chromato-
graphy. Soybean oil ester content was 96 %.
Example 4. Preparation of a synthetic ester from soybean oil ethyl ester
Soybean oil ethyl ester (0.7 moles) was weighed into a three-necked flask provi-
ded with a thermometer, a cooler, a stirring device and a reduced pressure gene-
rator. After the weighed ester was heated to 50-110C, trimethyloI ethane (TME,
0.2 moles) was added in small proportions with proper stirring. When the alcohol
was well mixed, the catalyst used (sodium hydroxide, 0.1-1.0 % by weight of the
reaction mixture) was added. Then the reaction mixture was heated under reduced
10
95395
10/1/1.
pressure (about 8 kPa) until it started boiling. The reduced pressure was main-
tained during the whole reaction. The mixture was allowed to boil at the lower
temperature (50-110C for I to 7 hours and at the higher temperature (110-160
0c) for 1 to 10 hours. The progress of the reaction was followed by thin layer
chromatography and quantitative IR spectrophotometry. At the end of the reaction
the product mixture was neutralized and washed with an aqueous acid, fI1trated
and washed with water. Drying was performed with sodium sulphate. A liquid
chromatogram and an IR spectrum were run from the final product. The yield
was 92 %of the theoretical yield.
Example 5. Preparation of a methyl ester of pine oil
Pine oil (0.3 moles) was weighed into a three-necked flask provided with a ther-
mometer, water separator and a cooler and a stirring device. Stirring was started
and methanol (2.0 moles) was added. The reaction mixture was heated to 60C
and the acid catalyst used (0.3 % by weight) was added. Stirring was continued
for six hours. The progress of the reaction was followed by thin layer chromato-
graphyand by the amount of water created. The reaction mixture was washed
with alkaline water and dried with sodium sulphate. The mixture was fI1trated and
analyzed by liquid chromatography. Pine oil ester content was 97 %.
Example 6. Preparation of a hydraulic oil from a synthetic rapeseed oil ester and
comparison of hydraulic oils
The raw material used was the synthetic rapeseed oil ester obtained in Example 2.
Said ester was mixed at a certain temperature with additives to obtain a hydraulic
oil having the following composition:
The synthetic ester from Example 2
Oxidation inhibitor
Pour point depresser
Antiwear agent
90 - 98 % by weight
0.1 - 2.5 % by weight
o-5.0 % by weight
0.1 - 2.0 % by weight
Antifoam agent
11
/07
95395
o- 0.5 % by weight
11/111
The technical properties studied of this ester containing additives were wearing,
friction, oxidation, low temperature properties and corrosion.
Wearing and friction were studied with a four ball test (ASTMD 2783, IF 239)
wherein wearing with respect to loading or the extreme loading where the lubrica-
tion still works, are measured. Oxidative properties were studied with an oxygen
bomb test (ASTMD 925) and with the oxidation test DIN 51586 where the change
of viscosity at 40C was monitored. In a corrosion test (Cincinnati-Milacron test)
the aging of the oil as well as copper and steel corrosion were studied. In said
test, the change of the total acid number (TAN) and viscosity, the weight change
of the copper and steel rods used as oxidation catalysts in the test procedure and
the formation of a precipitate under the test conditions are measured. Furthermo-
re, the pour point which illustrates the low temperature properties of an oil was
analyzed, i.e. the temperature where the oil is still fluid.
The corresponding properties were examined also from hydraulic oils based on
rapeseed oil and hydraulic oils based on commercial synthetic esters. All the hyd-
raulic oils were supplemented with the same additives as the hydraulic oil based
on the ester prepared by the process of the invention. The results are shown in
Table 1.
12
95395
-
Table 1. Comparison of the properties of hydraulic oils. A =hydraulic oil with
the ester prepared by the process of the invention as raw material, viscosity grade
32; B1 and B2 = hydraulic oils with commercial synthetic ester as raw materials,
viscosity grades 46 and 68; C =commercial hydraulic oil based on rapeseed oil,
viscosity grade 32.
A B1 B2 C
Four ball test
- extreme loading, N 2000 3000 2500 2000
- wearing, mm 0,4 0,46 0,41 0,64
Oxygen bomb test 42 39 29 30
ASTDM D445, psi
Oxidation inhibition test
DIN 51586, viscosity change, 12,4 20,3 24,1 28,8
%
Cincinnati-Milacron test
- TAN mg KOH/g
before 1,39 1,39 1,40 1,72
after 1,56 3,71 2,41 0,61
TAN 0,17 2,32 1,01 I,ll
- viscosity change, % 19,1 16,9 6,2 8,2
- total precipitate, mg/ 100 ml 1,1 17,0 28,8 4,4
- weight change of Cu rod, mg 1,7 -16,9

-0,5
- weight change of steel rod, -0,3 0,4 1,2 -0,5
mg
Pour point, C -41 -36 -39 -39
From the results it can be seen that as regards oxidative and low temperature
properties, the hydraulic oil based on the ester prepared by the process according
to the invention is better than the commercial hydraulic oil based on rapeseed oil.
The ester prepared by the process according to the invention and the correspon-
ding commercial esters are equal with respect to oxidative and low temperature
properties. From the Cincinnati-Milacron test it can be seen that the change of
total acid number (TAN) is clearly lowest with the ester of the invention. The
increase in viscosity at 40C is almost of the same order with all, as well as the
12/11\
13
13/111
weight change of copper and steel rods. Thus no corrosion is observed with any
of the tested hydraulic oils under the test conditions used. The results of the
oxygen bomb test are equal, as well as the results of the test according to DIN
51586 and the four ball test. Thus the wearing and friction properties are equally
good.
14
Claims
95395
1. A process for preparing a synthetic ester from a vegetable oil, comprising
- transesterification of said vegetable oil by reacting it with a
lower alkanol to form a mixture of lower alkyl esters of fatty acids,
- a second transesterification reaction wherein the obtained
mixture of esters is reacted with a no beta hydrogen polyol of the formula
CHzOH
,/
R - C - CHzOH
"CHzOH
wherein R is a C
C
C
6
alkyl group, particularly a C
1
-C
4
alkyl group, or a -CHzOH
group, and
- recovering the synthetic ester obtained.
2. The process according to claim 1, wherein the vegetable oil is selected from
the group consisting of rapeseed oil, rape oil, pine oil and soybean oil.
3. The process according to claim 1, wherein the lower alkanol is a C
1
-C
4
al-
kanol, especially methanol or ethanol.
4. The process according to claim 1, wherein the fatty acid lower alkyl ester is a
methyl ester of a fatty acid.
5. The process according to claim I, wherein the no beta hydrogen alcohol is
selected from the group consisting of trimethylol ethane, trimethylol propane,
trimethylol butane and pentaerythritol.
6. The process according to claim I, wherein the second transesterification reac-
tion is carried out under reduced pressure in the presence of a catalyst.
14nll
15
III
95395
7. The process according to claim 1, wherein the second transesterification is
carried out in two stages, with a reaction temperature of from 50 to 110 c in the
first and with a reaction temperature of from 110 to 160 C in the second stage.
8. The process according to claim 1, wherein the no beta hydrogen polyo1 and the
mixture of esters are reacted with each other in a molar ratio of from about 1:2 to
about 1:5, and especially in the molar ratio of about 1:3,5.
9. The use of a synthetic ester obtained according to claim 1 for the preparation
of lubricants, especially for the preparation of a hydraUlic oil.
10. A lubricant which comprises a synthetic ester obtained according to anyone
of the claims 1 to 8, optionally with one or more additives.
11. The lubricant according to claim 10 which comprises about 90 to 98 % of a
synthetic ester and about 2 to 10 % of additives.
12. The lubricant according to claim 10 wherein the additive(s) is (are) an oxida-
tion inhibitor, an antiwear agent, an antifoam agent, a corrosion inhibitor, a
dispersant, a viscosity index improver and/or pour point depresser.
16
Abstract
95395
The object of the invention is a process for preparing a synthetic ester from a
vegetable oil by a two-stage transesterification process. Further objects of the in-
vention are lubricants containing a synthetic ester prepared by the process accor-
ding to the invention.
161111
1/3
Appendix IV
with the permission of the publisher
95367
An enzymatic process for preparing a synthetic ester from a vegetable oil
The objects of the present invention are a process for preparing a synthetic ester
from a vegetable oil by means of lipase enzymes, and lubricants which contain a
synthetic ester prepared by said process.
Natural fats and oils have been used as lubricants already for thousands of years.
With industrialization mineral based lubricants carne also to the market. The app-
lications of lubricants and thus also the requirements set for them have changed
and developed with the advance of technology. Various types of synthetic esters
and lubricants containing the same have been developed to meet the new require-
ments.
The purpose of a lubricant is to minimize friction and wearing of metals. Lubri-
cants are developed according to the use and they consist of a base fluid and
additives improving the lubricative properties. With the development of technolo-
gy, lubricants are used under more and more severe conditions, such as at very
low or very high temperatures (e.g. the turbine engines of aeroplanes). At the
same time biodegradability, unburden to the environment, non-toxicity and the
use of renewable raw materials have emerged as new requirements. The use of
biodegradable lubricants is of particular importance in the machines and devices
used in the fields of agriculture, forestry and building, as the oil used may be left
in the environment.
By the synthetic esters developed as lubricants are meant esters prepared from
mono-, di- or trialcohols and mono- or dicarboxylic acids by known esterification
and transesterification methods. The conventional chemical process comprises
combining all the reactants and letting them react in one stage. The reaction may
be carried out in the presence of catalysts, such as acids, bases or metal oxides.
In addition to chemical agents, also lipase enzymes can act as catalysts of transes-
terification reactions.
111V
2
95367
Lipases (triacylglycerol acylhydrolase; EC 3.1.1.3) belong to the esterase enzyme
group, and fats and oils are their natural substrates. Several microbes (yeasts,
molds, bacteria) secrete in their growth media lipases by means of which lipids
decompose into nutrients of the microbe. Lipases catalyze the hydrolysis reactions
of oils and fats but under suitable conditions they also catalyze the synthesis and
transesterification of tri-, di- and monoglyceride esters (Yamane et al., J. Am.
Oil Chern. Soc. 64, 1987, 1657-1662).
On the basis of their specificity, lipases are divided into three groups, nonspeci-
fic, 1,3-specific and fatty acid specific lipases. Nonspecific 1ipases are produced
by for instance the yeast Candida rugosa (ex. cylindracae) and the bacteria Cory-
nebacterium acnes and Staphylococcus aureus. Nonspecific lipases release fatty
acids from all three positions of a triglyceride. According to their name, 1,3-li-
pases release fatty acids from positions 1 and 3 of trig1ycerides. These lipases are
produced by for instance the molds Aspergillus niger, Mucor javanicus, Mucor
miehei and Rhizopus arrhizus as well as by the yeast Candida lipolytica. The fatty
acid specific lipases release only certain fatty acids from triglycerides. Mucor
miehei, for example, produces also a lipase which in addition to 1,3-specificity is
also specific to fatty acids with 12 carbon atoms. However, the specificity is not
absolute.
The structure of the synthetic ester used has a profound effect on the stability of
the lubricant. Esters decompose by the effect of heat and/or oxygen. It is known
to increase the thermal stability of synthetic esters by using in the preparation no
beta hydrogen alcohols. Oxidative properties on the other hand can be improved
by deuteration of esters.
Synthetic esters intended for a lubricative use are classified by structure as mono-
carboxylic acid, dicarboxylic acid, polyol and complex esters. Due to their low
viscosity and high volatility monoesters are poorly suitable as lubricants. Polyo1
esters are chemically more stable than for example diesters, due to the structure
of the polyols used in the preparation of said esters wherein no hydrogen atom is
211V
3
Il5'
95367
311V
attached to the fJ carbon atom. Complex esters have promising lubricative proper-
ties but the manufacture thereof on an industrial scale is difficult because of the
severe conditions required by the reaction, especially if said esters are prepared -
from (purified) fatty acids and alcohols.
If polyol esters are prepared by using no alfa hydrogen acids, the stability proper-
ties of the esters can be further improved. Metro et ai. (CA 859 771) have shown
that the no alfa hydrogen carboxylic acids increase the thermal and oxidative
stability of esters prepared from no beta hydrogen alcohols, as well as slow down
the hydrolysis of the esters.
As the low viscosity polyol esters are not suitable for traditional uses wherein
high viscosity is required, it has been aimed at preparing polyol esters of higher
viscosity from for example trimethylol propane (TMP). However, it has been
found that it is difficult to obtain simple TMP esters with both high viscosity and
a low pour point (cf. for example US 4,061,581).
Products based on vegetable oils are nowadays used more and more as lubri-
cants because of their safety to the environment. Natural vegetable and animal
oils are glyceride diesters, i.e. tri-, di- or monoesters of glycerol and straight
chain saturated and unsaturated fatty acids. The lubricant industry uses for instan-
ce rapeseed, rape, soybean, castor, olive, coconut, palm and pine oils.
The advantageous properties of vegetable oils include user friendliness and non-
toxicity. In addition, vegetable oils are renewable raw materials and degrade in
the environment without accumulating in the food chain of nature. However, the
use of vegetable oils as lubricants has been limited by their poor stability proper-
ties. The poor thermal and oxidative stability is due to unsaturated and polyun-
saturated fatty acids. On the other hand, the unsatisfactory behaviour of vegetable
oils at low temperatures is due to the saturated fraction of fatty acids. By using
suitable additives and by favouring in cultivation such varieties which do not have
4
95367
4/1V
a too high degree of saturation, it has been possible to somewhat improve the
stability properties. Also the purification of the oil for technical use is helpful.
Furthermore, attempts have been made to modify natural glyceride esters in order
to improve their stability properties. Known processes include catalytic hydro-
genation, alcoholysis, geometrical isomerization and sulfurization. For example
by hydrogenation a certain amount of double bonds from the unsaturated part of
vegetable oils can be removed, and by isomerization the amount of undesired iso-
mers can be decreased.
Van der Waal and Kenbeek have presented a process for the preparation of synt-
hetic esters from vegetable oils or animal fats (proceedings of the Tribology
2000, 8th International Colloqium, Technische Akademie Esslingen, Germany,
14-16 June 1992, Vol II, pp 13.3-1 - 13.3-8). The process comprises first decom-
posing the glyceride esters of the starting material into fatty acids and glycerol
and subsequently separating the fatty acid fraction into liquid and solid phases.
The fatty acids of the liquid phase are separated by distillation into single fatty
acids which can be further modified e.g. by hydrogenation or cracking to obtain
the desired raw material. Fractions containing a single fatty acid are esterified
with no beta hydrogen polyols for preparing a synthetic ester.
The .fatty acids of the ester prepared according to the above described process
usually contain less unsaturated double bonds than the fatty acids of the starting
material, which improves the oxidative stability. However, the costs of the pro-
cess are extremely high, due to the multistage separation and purification reac-
tions and the most severe conditions (high pressure and temperature) required by
the reaction. Moreover, it has been found that when fractions containing only a
single fatty acid are reacted with polyols, plenty of mono- and diglycerides are
formed, i.e. all the OH groups of the polyols do not react. This decreases the
triglyceride yield and the raw material has to be recycled several times if the
yield is to be improved. Furthermore, the reaction of a fatty acid and an alcohol
creates water which has to be removed during the reaction.
5
1/7
95367
5JlV
'4
Transesterification of fats by means of lipases is known as such. The literature in
the field discloses especially various systems for the immobilization of the lipases
used (cf. for example EP patent application 579 928 and US patents 4,798,793
and 4,818,695). The immobilization of lipases facilitates their application both in
continuous and batch processes. Patent publication GB 1 577 933 discloses a pro-
cess for modifying trig1ycerides with a lipase, especially with an immobilized
lipase. However, the literature in the art does not describe the use of lipases as a
catalyst in the process according to the present invention.
According to the invention it has now been found that it is possible to prepare
synthetic esters with good lubricative properties from vegetable oils by an enzy-
matic process which avoids the multistage reaction with several separations and
recyclings and by which good yields are obtained.
In the process according to the invention a vegetable oil is first transesterified by
reacting the vegetable oil with a lower alkanol to obtain a mixture of fatty acid
lower alkyl esters. The process is characterised in that the mixture of esters obta-
ined from the first reaction is further transesterified by reacting said mixture with
a no beta hydrogen polyol of the formula
wherein R is a C
1
-C
6
alkyl group, particularly a C
1
-C
4
alkyl group, or a -CHzOH
group, in the presence of a lipase enzyme, and the synthetic ester obtained is
recovered.
Vegetable oils suitable as a starting material in the process are for example ra-
peseed, rape, soybean, castor, olive, coconut, palm, pine, maize, walnut, flax-
seed, cotton, sunflower, sesame and almond oils, especially rapeseed oil, rape oil,
pine oil and soybean oil, particularly rapeseed oil or rape oil.
6
95367
611V
The first transesterification reaction of the process according to the invention is
carried out by a process known per se, by reacting a refined or alkalirefined
vegetable oil with a lower alkanol to obtain a mixture of fatty acid lower alkyl
esters.
The lower alkanol used in the first transesterification reaction is preferably a C
1
-
C
4
alkanol, especially methanol or ethanol. The obtained mixture of lower alkyl
esters of the vegetable oil is thus preferably a mixture of C
1
-C
4
alkyl esters,
especially a mixture of methyl or ethyl esters. If desired, usual esterification
catalysts may be used in the reaction, and the amounts of the reactants and the
reaction conditions (pressure, temperature, reaction time) are either commonly
known or easily chosen by.a person skilled in the art. The reaction may also be
carried out by using a suitable enzyme as a catalyst.
The first transesterification reaction may be illustrated by the following general
reaction scheme I:
H
2
C-O-C( =O)-R
1
I
HC-O-C( =O)-R
2
I
H
2
C-O-C( =O)-R
3
+ 3 ~ - O H ~ 3 ~ - O - C ( =O)-R
x
H
2
C-OH
1
+ HC-OH
I
H
2
C-OH
(I)
wherein R
1
, R
2
and R
3
are fatty acid residues, ~ is an alkyl residue, especially a
C
C
C
4
alkyl residue, and R
x
is R
b
R
z
or R
3
- Glycerol is formed as a by-product.
The fatty acid lower alkyl ester obtained from the first transesterification reaction
is thus a mixture comprising various fatty acids of the vegetable oil used as the
starting material. It is typical of the invention that this mixture of fatty acid lower
alkyl esters may be used directly as the starting material of the second transesteri-
fication reaction without separation or purification of fatty acids.
In the second transesterification reaction according to the invention, the mixture
of fatty acid lower alkyl esters obtained from the first transesterification reaction
7
Ilfj
95367
711V
is reacted with a no beta hydrogen polyol, such as for example trimethylol etha-
ne, trimethylol propane, trimethylol butane or pentaerythritol, especially with
pentaerythritol or trimethylolpropane, in the presence of a lipase.
The second transesterification reaction may be illustrated with the following gene-
ral reaction scheme II:
/CH20H
3 + R-C - CH
2
0H
"'CH
2
0H
+ 3
wherein R
j
, R
2
, R
3
, and R
x
have the same meanings as in the reaction scheme
I and R is a C\-C
6
alkyl group, especially a C
j
-C
4
alkyl group, or a -CH
2
0H
group.
Consequently, it is the question of a totally different chemical reaction than in the
process of the prior art wherein a free fatty acid is esterified with an alcohol. In
the process according to the invention, an ester is reacted with an alcohol, and
thus it is the question of a transesterification reaction which reaction, as well as
the reaction conditions required by it and the by-products formed therein, is
totally different from the reaction used in the process of the prior art.
The synthetic ester obtained from the second transesterification reaction is reco-
vered and, if desired, purified by conventional methods, for example by neutrali-
zation and washing with an aqueous acid. No distillation or any other special
treatment is needed as the obtained ester is ready to use as such as a raw material
of lubricants.
When a polyol is reacted with a mixture of fatty acid lower alkyl esters in the
presence of a suitable lipase, almost all OH-groups of the polyol react into trigly-
cerides. From 75 to 98 % of the theoretical yield of the triglyceride is obtained,
the proportion of mono- and diglycerides being in total from about 2 to 25 %.
8
95367
allv
The product obtained does not contain any free fatty acids which makes it an
especially advantageous raw material for lubricants wherein the oxygenation of
free fatty acids would cause problems (corrosion, change of viscosity). The pro-
cess is well adapted for industrial scale and the synthetic ester obtained has better
stability properties than the vegetable oil used as the raw material, while at the
same time the advantageous properties of a vegetable oil (biodegradability, non-
toxicity, user friendliness) are maintained.
By the process according to the invention it is thus possible to prepare synthetic
esters from vegetable oils, for example from rapeseed oil, in a yield of even over
95 % of the theoretical. In this case, the di- and monoglycerides of the product
are also calculated in the yield. During the tests carried out it has been observed
that the advantageous properties of the product are maintained in spite of the
moderate (up to 30 %) di- and monoglyceride content.
The no beta hydrogen polyol and the mixture of esters are preferably reacted with
each other in a molar ratio of about 1:2 to 1:6, especially in the molar ratio of
about 1:3 to 1:3,5.
The second transesterification reaction, being characteristic of the invention, is
preferably carried out in a reduced pressure generator provided with reflux, for
example under negative pressure of 2.0 to 12 MFa, preferably under negative
pressure of 5.3 MPa. The reaction is carried out at a temperature wherein the
lipase used is active, for example at a temperature between 37C and 69 c,
preferably at a temperature between 42C and 47 C. A suitable reaction time is
from 24 hours up to 72 hours, depending on the other conditions and the enzyme
used. It is preferred to add water to the reaction mixture, for example about 0.1 -
29 %, preferably 8 - 15 %, or to carry out at a higher temperature without ad-
ding water.
The amount of the enzyme is preferably from about 2 % up to about 50 % calcu-
lated (w/w) on the substrates. With a 68 hour reaction, a methyl ester of rapeseed
9
/2/
9536/
19/IV
oil is completely made to react into products only with an enzyme amount of 10
%. The amount of the enzyme needed may be decreased by immobilizing the
enzyme. In the process according to the invention, a lipase obtained for example
from Candida rugosa (ex. cylindraceae), Mucor miehei or Pseudomonas fluores-
cellS may be used. The lipase may also be produced by transforming a gene co-
ding for the desired enzyme into another host organism, by cultivating the host
thus obtained and by isolating the lipase produced by it. Commercially obtainable
immobilized lipases may be used, or the free lipase may be immobilized before
use for example on an ion exchange resin, adsorption resin, celites, diatomaceous
earth or silica gel according to the conventional immobilization methods.
The synthetic ester prepared by the process according to the invention is an excel-
lent raw material for the preparation of lubricants. Lubricants, especially hyd-
raulic oils, which contain a synthetic ester prepared by the process of the inventi-
on, optionally with one more additives, are also included in the scope of the in-
vention. As additives for example oxidation inhibitors, antiwear agents, antifoam
agents, corrosion inhibitors, dispersants, viscosity index improvers and/or pour
point depressers which are generally known in the art, may be used.
Oxidation inhibitors include for example amines and phenols. As antiwear agents
and corrosion inhibitors for example phosphates or sulfonates and as antifoam
agents for example metal sulfonates, metal phenates, polyesters or silicones may
be used. Viscosity index improvers include for example polyisobutenes, styrene-
butadiene and ethene-propene-eopolymers which all are thus suitable also as pour
point depressers.
In the following the invention is further described by means of examples, the pur-
pose of which is to illustrate but not to limit the invention.
10
Example 1
95367
A methyl ester of rapeseed oil was prepared as follows: Rapeseed oil (0.3 moles)
was weighed into a three-necked flask provided with a thermometer, cooler and a
stirring device. Stirring was started and methanol (2.0 moles) was added. The
reaction mixture was heated to 60C and the alkali catalyst used was added (0.5
%, w/w). Stirring was continued for three hours. The progress of the reaction
was followed by thin layer chromatography. The reaction mixture was washed
with an aqueous acid. The glycerol created in the reaction mixture was separated
and the product mixture was analyzed. Rapeseed oil ester content was 97 %.
Example 2
In a 25 cm
3
round pottom flask attached to a Liebig-refluxer of 20 cm with a cold
(about +6C) tap water circulating in the cooling jacket, was weighed 0.607 g
(4.52 mmoles) of solid trimethylol propane (Merck, Darmstadt, Germany), and
0.7 ml of destilled water was added. After dissolution, 4.00 g (13.56 mmoles) of
methylated rapeseed oil (Raision Yhtyma, Finland) was added and finally 1.79 g
of Candida rugosa lipase (Biocatalysts Ltd., Pontypridd, Great Britain) in powder
form. A negative pressure of 5.3 MPa was sucked into the device. For stirring a
magnetic stirrer was attached to the device. The reaction mixture was stirred with
the magnetic stirrer at a speed of 200 rpm. The starting point of the reaction was
counted from the moment the suction for the reduced pressure was connected to
the device. The reaction temperature was 42C and the total reaction time 72
hours. The amount of substituted TMP esters in the final product was over 98 %
in total.
Example 3
Example 2 was repeated with 1.84 g of a Mucor miehei lipase Lipozyme 1M
(Novo Nordisk A/S, Bagsvaerd, Denmark) bound to a solid support. Water was
not added to the reaction mixture. The reaction temperature was 58C. The
10llV
11
(23
95367
1111V
TMPE content of the final product was 75.0 % after 24 hours and 92.5 % after
66 hours. There were no starting materials left after 66 hours.
Example 4
Example 2 was repeated with 1.84 g of a Candida rugosa lipase bound to a solid
support. 0.7 ml water was added to the reaction mixture. The reaction temperatu-
re was 47C. The TMPE content of the fmal product was 62.7 % after 48 hours
and 72.9 % after 78 hours.
The enzyme bound to the solid support was prepared as follows: 3.33 g of lipase
was dissolved in 100 ml of 0.05 M sodium phosphate buffer, stirred for 2 hours
and filtrated. To an erlenmeyer flask of 250 ml 40 g of a buffered support (e.g.
MWA-1, Mitsubishi Chemical Company, Japan; 43.4 % dry matter) and 60 m1 of
enzyme solution (2 g lipase) was added, shaken for 3 hours at a speed of 130
rpm, filtrated and lyophilized for 30 hours to a dry solids content of 98.9 %.
Example 5 Preparation of a hydraulic oil from a rapeseed oil ester and compa-
rison of hydraulic oils
The raw material used was the synthetic rapeseed oil ester obtained in Example 2.
said ester was mixed at a certain temperature with additives to obtain a hydraulic
oil having the following composition:
The synthetic ester from Example 2
Oxidation inhibitor
Pour point depresser
Antiwear agent
Antifoam agent
90 - 98 % by weight
0.1 - 2.5 % by weight
o- 5.0 % by weight
0.1 - 2.0 % by weight
o - 0.5 % by weight
The technical properties studied of this ester containing additives were wearing,
friction, oxidation, low temperature properties and corrosion.
12
95367
12/1V
Wearing and friction were examined with a four ball test (ASTMD 2783, IP 239)
wherein wearing with respect to loading or the extreme loading where the lubrica-
tion still works, are measured. Oxidative properties were studied with an oxygen
bomb test (ASTMD 925) and with the oxidation test DIN 51586 where the change
of viscosity at 40C was monitored. In a corrosion test (Cincinnati-Milacron test)
the aging of the oil as well as copper and steel corrosion were studied. In said
test, the change of the total acid number (TAN) and viscosity, the weight change
of the copper and steel rods used as oxidation catalysts in the test procedure and
the formation .of a precipitate under the test conditions are measured. Furthermo-
re, the pour point which illustrates the low temperature properties of an oil was
analyzed, i.e. the temperature where the oil is still fluid.
The corresponding properties were examined also from hydraulic oils of the state
of the art containing the same additives and from hydraulic oils based directly on
rapeseed oil containing also the same additives. The results are shown in Table 1.
/2?
13
95367
-
Table 1. Comparison of the properties of hydraulic oils. A = hydraulic oil with
the ester prepared by the process of the invention as raw material, viscosity grade
32; B1 and B2 = hydraulic oils with commercial synthetic esters as raw mate-
rials, viscosity grades 46 and 68; C =commercial hydraulic oil based on rape-
seed oil, viscosity grade 32.
A B1 B2 C
Four ball test
- extreme loading, N 2500 3000 2500 2000
- wearing, mm 0.42 0.46 0.41 0.64
Oxygen bomb test 40 39 29 30
ASTDM D445, psi
Oxidation inhibition test
DIN 51586, viscosity change, 11.5 20.3 24.1 28.8
%
Cincinnati-Milacron test
- TAN mg KOH/g
before 1.38 1.39 1.40 1.72
after 1.58 3.71 2.41 0.61
TAN 0.20 2.32 1.01 1.11
- viscosity change, % 19.0 16.9 6.2 8.2
- total precipitate, mg/l00 m1 1.0 17.0 28.8 4.4
- weight change of Cu rod, mg 1.5 -16.9 0 -0.5
- weight change of steel rod, 0.2 0.4 1.2 -0.5
mg
Pour point,
C
-41 -36 -39 -39
From the results it can be seen that as regards low temperature properties, the
ester prepared by the process according to the invention is equal to the commer-
cial raw materials on the market and better than the commercial product based on
rapeseed oil. From the Cincinnati-Milacron test it can be seen that the change of
the total acid number (TAN) is clearly the lowest with the ester of the invention.
The increase in viscosity at 40C is of the same order with all, as well as the
weight change of copper and steel rods. The results of the oxygen bomb test are
equal, as well as the results of the test according to DIN 51586 and the four ball
test.
13/1V
14
Claims
95367
1. An enzymatic process for preparing a synthetic ester from a vegetable oil,
comprising
- transesterification of said vegetable oil by reacting it with a
lower alkanol to form a mixture of lower alkyl esters of fatty acids,
- a second transesterification reaction wherein the obtained
mixture of esters is reacted in the presence of a lipase (triacylglycerol acylhydro-
lase; EC 3.1.1.3) with a no beta hydrogen polyol of the formula
wherein R is a C
C
C
6
alkyl group, particularly a C\-C
4
alkyl group, or a -CH
2
0H
group, and
- recovering the synthetic ester obtained.
2. The process according to claim I, wherein the vegetable oil is rapeseed oil.
3. The process according to claim 1, wherein the lower alkanol is a C\-C
4
al-
kanol, especially methanol or ethanol.
4. The process according to claim l, wherein the fatty acid lower alkyl ester is a
methyl ester of a fatty acid.
5. The process according to claim I, wherein the no beta hydrogen alcohol is
selected from the group consisting of trimethylol ethane, trimethylol propane,
trimethylo1 butane and pentaerythritol.
14/1V
15
/21
95367
1511V
6. The process according to anyone of the claims 1 to 5, wherein the second
transesterification reaction is carried out in the presence of an immobilized lipase.
7. The process according to anyone of the claims 1 to 6, wherein the second
transesterification reaction is carried out in the presence of a Candida rugosa
lipase.
8. The process according to anyone of the claims 1 to 6, wherein the second
transesterification reaction is carried out in the presence of a Mucor miehei lipase.
9. The process according to anyone of the claims 1 to 8, wherein the enzyme is
separated after the reaction and recycled.
10. The process according to anyone of the claims 1 to 9, wherein the second
transesterification reaction is carried out with a lipase obtained by transforming a
gene coding for said enzyme into another host organism for producing the lipase.
11. The process according to anyone of the claims 1 to 10, wherein the reaction
mixture contains about 0.1 to 29 % water.
12. The process according to anyone of the claims 1 to 11, wherein the reaction
temperature in the second transesterification is between 37C and 69 c.
13. The process according to claim 1, wherein the no beta hydrogen polyol and
the mixture of esters are reacted with each other in a molar ratio of from about
1:2 to 1:6, especially in a molar ratio of about 1:3 to 1:3,5.
14. The use of a synthetic ester obtained according to claim 1 for the preparation
of lubricants, especially for the preparation of a hydraulic oil.
15. A lubricant which comprises a synthetic ester obtained according to anyone
of the claims 1 to 13, optionally with one or more additives.
16
95367
( 1611\
16. The lubricant according to claim 15 which comprises about 90 to 98 % of a
synthetic ester and about 2 to 10 %of additives.
17. The lubricant according to claim 15 wherein the additive(s) is (are) an oxida-
tion inhibitor, an antiwear agent, an antifoam agent, a corrosion inhibitor, a
dispersant, a viscosity index improver and/or a pour point depresser.
17
Abstract
95367
The object of the invention is a process for preparing a synthetic ester from a
vegetable oil by means of lipase enzymes. Further objects of the invention are
lubricants containing a synthetic ester prepared by the process according to the
invention.
17/1\
Appendix V 1N
with the permission of the publisher
Merja Lamsa
R&D Manager
Research and Development Laboratory
Oil Milling Division
Raisio Group
P.O.Box 101
21201 Raisio
Tel: 921- 434 2746
Fax: 921- 434 2911
ECOLOGICALLY ACCEPTABLE SYNTHETIC HYDRAULIC FLUIDS BASED
ON VEGETABLE OILS
1. Introduction
There is an increasing interest of v e q e t a n ~ e oils to be
used as raw material for synthetic escer based
ecologically acceptable fluid (1,2,3).
Products based on natural oils and fats are used widely
as lubricants, mainly chainsaw oils and hydraulic
fluids. vegetable oil based hydraulic fluids entered to
the market at the middle of 80s (4,5).
These products fullfill their function very well, but
when requirements are higher for example for
temperature, synthetic esters corne on picture.
First synthetic esters were made for gasturbine engines
(6). In the late sixties and seventies the advantages of
synthetic esters were recoqnized also for other
applications like gear oils, hydraulic fluids and
compressor lubricants (7,8).
The-main raw materials for esters are alcohols and fatty
acids. Fatty acids are originally from natural oils and
fats. Alcohols may vary from mono- to oolyalcohols. The
careful choice of-fatty acid and alcohol gives the
demanded properties :or the final product. In hydraulic
fluids it is important to have right viscosity, good
oxidation and hydrolytic stability, low pourpoint and
stability of viscosity at cold temperatures and
naturally good antiwear properties.
In Raisio Finnish raoeseed oil and its esters have been
used together with polyols to synthetize polyolesters,
starting materials for synthetic hydraulic fluids
(9,10) .
Laboratorytest results are demonstrated from raw
materials and final products, hydraulic fluids.
Fieldtest results are shown from different hydraulic
applications.
From these testresults can easily be seen that
vegetableoil based synthetic esters work very well as
raw material for hydraulic fluids.
814
/J/
2.Methods
Synthetic esters of vegetable oil can be prepared either
chemically or enzymatically (9,10). Raw material can be
rapeseed-, tallo, soyabean-, coconut- or tallow oil
fatty acids. Alcohols used can be short, straightchain
or longer, sidechain alcohol, dialcohols or polyols
(1,5)" Examples of different esterification reactions
are shown in Figure 1.
Figure 1: Examples of molecular schedule of synthetic
ester reaction (11,12,13).
-------------------------------------------------------
2N
CH. -OR
""
o
CH -O-C-R
I a 0
II
CH-O-C-R"
I ';I 0
-O-C-R"
+ 3 R" OR
.D. ,catalyst 0
3 R'''-O-C-R
o1
' + CH-OR
"
B. Shortchain alcohol and diacids
A, catalyst Q 0
2ROR .,. ROOC- -COOR, - J RO-C- (CR.:l,)" -C-OR + R" 0
C.Polyols and triglycerides
a
CH... -a-CoR
I a
3 CH-O-C-R'
I ,j
-a-c-R
CH"OH
I
OR
I
CH;:.OR
6, catalyst
- 'R
..,
a
-O-C-R
I 9
-C-CR.-O-C-R +
I "0
CH.l -O-C-R
CH.t -OR
I
3 CR-OR
I
CR.-OR
...
-------------------------------------------------------
815
Figure 2:
The chainlenght and sidechains of acid effect
onto the physical properties of the polyol
esters (14) .
3N
--------------------------------------------------------
Influence of chain length

Influence of side Chains

cnatn noU1

cour I
POint!
V.l.
pour I
POint I
side cnalna
--------------------------------------------------------
Figure 3: Effect of polyol functionality and chain
lenght on the viscosity of polyol ester(15J.
40
3!l
30 ""1
., I
..

9
o Iff

_-ACro CHAIN LENGTH
--------------------------------------------------------
As an example of how the properties of the final ester
can be affected by raw materials are shown in Figure 2
and 3. The following features of the starting compounds
affect to the properties of the resulting ester:
molecular weight, the size of the acyl group, the
functionality of polyols and the method of preparation
of the ester or mixture of esters (15,16).
816
qualities of synthetic esters for hydraulic fluids
use one can get from polyolesters or combination of
polyolesters with diesters. It is also very important
not to forget the environmental side of synthetic
esters. There exist for example diesters that are
biodegradable, but do not work technically as
they should or vice versa. For environmental side
biodegradability is not enough, also toxicity and
bioaccumulation properties should be tested.
Different type of additives were also tested. They have
changed enourmously during last two- three years.
Information of their environmental side is now available
and additive industry make more custom- made additives.
4N
Figure 4 Examples of environmentally acceptbale
additives for hydraulic fluids.
--------------------------------------------------------
Add:"::ive Purpose Biodegradability Toxicity Bioaccumulation
Phenolic Antioxidant > 80%'(OECD 301B) EC >100 mg/l
Amineohos- Antiwear and
por:Jussalt antifirction > 70%'(OECD 301B) LC >10 rng/l log
P""" 6
Ester of Pourooint
oleic acid depresser > 70%' (OECD 301D) LC >50 rng/l log
Po"" >6
--------------------------------------------------------
These new additives have been tested in both natural and
synthetic esters of veqetable oil based hYdraulic
fiuids. All tests are performed in independent, outside
laboratories with standardized methods.
3.Results
PhYsical and chemical properties of natural and
synthetic esters of oil based hydraulic fluids
have been measured. The hydraulic fluids in test were
vegetable oil based hydraulic fluid (RES 32L old and new
version), commercial synthetic ester based hydraulic
fluids ( RBS 465 and RB5 685), the latest
deveploment work ( SE) and comparable mineral oil based
hydraulic fluid (MO).
817
Table 1: Physical and chemical properties of some
hydraulic fluids (13).
--------------------------------------------------------
5N
HF SE RBS32L RBS46S RBS 68S MO
Viscosi-
ty (mm2/s) (19)
40" C 32,9 33,S 32,6
100
0
C 8,0 7,8 6,4
VI 220 220 187
Filtera-
blity (%) (20) 92 96 72
Cleanclass (21) 13/8 B/S 14/10
Colour (22) 4- 3+ 4 4+
--------------------------------------------------------
One of the most important basic properties is the cold
stability Pourpoint is-one way to measure it,
more important is the stability of viscosity at cold
temperatures. Pourpoint reflects the temperature where
the-fluid is still-fluidy. Viscosity measurement at cold
temperature show the stabolity of the fluid in cold
(17) .
Table 2: pourpoint ( ASTM D97) (17) and viscosity at
cold- temperatures ( ASTM D 445) (17) of- some
hydraulic fluids.
--------------------------------------------------------
HF SE RBS 32L RBS 468 RBS68S MO
Pour-
point ('C) -41
Viscosity
(mm2/s)
Temp.
o
-10
-20
-25
-30
-39
372
829
1628
2940
-36
573
1430
2350
-39
2160
3670
496
1221
2038
Time ( days)
1 3050
3 3050
7 3050
--------------------------------------------------------
818
~ 3 S - 6N
Oxidation stability has been claimed to be one of the
weaknesses of vegetable oil based hydraulic fluids. with
the right choice of raw material and additives this is
no longer a problem. Oxidation stability can be measured
by many different standardized test like oxidation bomb
test ( ASTM D 525) , Baader test ( DIN 51 554 teil 3)
, viscosity test (DIN 51586) (19,21). problematic is
that there does not exist norms, that everyone would use
the same method.
Table 3: Examples of oxidation stability tests of
environmentally acceptable hydraulic fluids.
--------------------------------------------------------
SE
Test-
method
ASTM D445
(psi) 42
DIN 51586
(1:) %) 12,4
DIN 51554
TAN
(mgKOH/g)
vise.
( ~ % )
RBS 32L
30
28,8
0,05
6,2
RBS 46S
39
20,3
RBS 68S
29
24,1
MO
39
--------------------------------------------------------
All vegetable oil based products have good properties
for wear and friction reduction. This is due to their
chemical structure. They form an unimolecular, strenght
film on the metal surface, which lowers friction and
reduces wear. The molecular structure of vegetable oil,
vegetable oil based ester and mineral oil is showed in
Figure 5 to compare their chemical structures. As can
be seen, vegetable oil and its esters contain oxygen
atoms at their molecular structure. This explains the
mentioned properties. -
819
Figure 5: Molecular structure of vegetable oil (A),
vegetable oil based ester (B) and mineral oil
(C) for hydraulic fluid use (13,22).
--------------------------------------------------------
7N
o
CH.. -O-C-R
I 0
CH-O-C-R'
o
.\
C H ~ -O-C-R"
R"' - 0 - ~ - R"" Et Me
Et-CH" -CH-CH.t, -CH-Me
(A) (B) (C)
--------------------------------------------------------
Friction and wear nronerties can be measured at
laboratory scale for example by FZG-test ( DIN 51 354
teil 2) (21), Vickers- pumntest ( DIN 51 389 teil 2)
(21), Mobil-test (23) and fourball test (ASTM D 2783, IP
239) (17).
Table 4: Fourball test- eauinment and some testresults
from hydraulic fluids ( ASTM D 2783, IP 239) .
--------------------------------------------------------
HF SE RBS32L RBS46S RBS8S MO limit
max
load (N) 2000 2000
wear (rom) 0,4 0,48
2000
0,46
2500
0,41
2200
0,54 < 0,5
--------------------------------------------------------
Corrosion does not cause problems with vegetable oil
based products, but of coarse resistance can be improved
by additives. One laboratorytest to measure it, is
Cincinnati- Milacron test (24). In the testresults can
clearly be seen the affect of additives, how much better
are the custom-made additives.
820
/37
Table 5: Cincinnati- Milacron test from vegetable oil,
synthetic ester of it based hydraulic fluids.
--------------------------------------------------------
8N
HF SE RES 32Lo RBS 32Ln RBS 46S RBS 68S
TAN
(mgKOH/g) 0,17
vise.
(%)/40 C 19,1
total
sludge 1,1
(mg/l00ml)
weight change (mg)
Cu- plates 1,7
Fe-plates -0,3
1,74
10,2
45,4
0,3
0,5
1,11
6,2
20,5
- 0,8
o
1,01
16,9
28,2
o
1,2
2,32
12,9
17,0
-1,6
0,4
--------------------------------------------------------
4.Environmentally ~ r i e n d l v properties
Biodegradability can be tested by many different
methods. They are all standardlized methods and none of
them can be said to be clearly better than others.
OECD-301- serie (A-F) (25) is the newest and most
reliable for todays knowledge. Others are like CEC-L-33-
A-93(26), DIN 38412 (21).
Table 6: Biodegradability of vegetable oil and synthetic
esters of it based hydraulic fluids.
--------------------------------------------------------
HF SE RBS 32L RBS46S RBS68S limit
Testmethod
CEC >90% >90% >90% >90% 70%
DIN 38412
OECD 301F
5 days
>85%
14 days
60%
--------------------------------------------------------
As important as biodegradability are bioaccumulation and
toxicity properties of lubricants. Bioaccumulation means
if product or parts of it accumulates in nature in
micro- organisms, plants, animals etc. It is usually
measured by OECD- standards ( 3U5 A-E) (27). Toxicity
properties are also measured by OECD- standards, OECD
201- 203 (28). As toxicity- meter are used fishes,
water-fleas or algaes.
821
Table 7: Examnle of bioaccumulation ( OECD SOlD) and
toxicity test ( OECD 201- 203) of vegetable oil
based hydraulic fluid.
--------------------------------------------------------
9N
HF
Eioaccumulation ( 305D)
Toxicity
fishes ( 201)
waterfleas (202)
algaes ( 203)
RES 32L
not bioaccumulating
LQo > 1000 mg/l
LC:., > 100% WSE
ETCs.. > 50% WSE
--------------------------------------------------------
5. Fieldtests
synthetic esters of vegetable oil based hydraulic fluids
have been in fieldtests over two years. In fieldtest a
reallife machinery is filled with tested oil and
oilsamnles are taken after certain usaqehours. These
samples are examined at laboratory. -
Table 8: Fieldtest results from Wille- multifunctional
tractor (30) and Timberjack- forestmachine (
type 1210) with RES 46S and RES 68S hydraulic
fluids.
--------------------------------------------------------
Machinery Hvdraulic Hours Viscosity VI Purity Colour TAN
fiuid in use 4 0 ~ C 1.00"C
(h) (rnm2/s) (mgKOH/g)
Wille 845 RES 68S 0 59,0 11,1 1.83 10/8 4 2,45
290 54,9 10,8 193 15/13 5 2,05
872 54,9 10,5 181. 15/18 6 2,00
1140 55,1 10,7 190 14/12 6 1,95
1515 55,4 10,7 184 15/10 8 1,80
Timberjack
121.0 RES 465 0 43,3 8,7 186 13/11 1,67
629 42,7 8,7 187 13/11 10 1,68
1213 42,7 8,6 184 15/12 11 1,63
1577 43,0 8,8 188 14/11 11
--------------------------------------------------------
From fieldtest results can be seen ( Table 8) that
viscosity and viscosity index are very stable with time.
Also oxidation and hydrolytic stability are very good
and friction and wear additives work perfectly.
822
After these fieldcests the manufacturer of machinery can
grant approval for the cested hydraulic fluid. Also
research and develooment acauires a lot of information
to develope even betcer lubricants.
6. Conclusions
Both natural and synthetic esters based on vegetable oil
hydraulic fluids seem to fullfill their job very well.
Based on laboraccry tests, oxidation stability
especially at higner temperatures of vegetableoil based
synthtetic esters is better than in natural esters. This
difference could not be seen in fieldtests.
Coldstability, especially viscosity at low temperatures,
is clearly becter in synthetic escers at temperature
area -10 to -20 C, but then at colder temperatures it
is vice versa, natural esters are better in temoeracure
area -25 to -40 C. At fieldtests these differences could
not be noticed. In friction and wear by laboratory tests
no differences could be seen. In additives one could
very clearly see that with careful chose of raw material
and custom- made additive- package, at labscale,
testresults were much better than older packages.
From fieldtest results could be noticed that synthetic
ester based lubricants maintain thewir chemical and
physical properties very well.
Results from laboratory and fieldtests have been so
promising, that synthetic ester of vegetable oil based
hydraulic fluids are coming into the market.
References:
1. Linko, Y.-Y., Larosa, M.,Huhtala, A and Rantanen, 0.,
J.Am.Oil Chern. Soc. soecial issue on Biocatalysis
based on the speech at 86th AOCS Anual Meeting, San
Antonio, Texas, USA 6.-15.5.1995.
2. Metro, E.M. and Matuszak, A.H., Canadian Patent 859
771 ( 1970).
3. Berens, G. and Milton, L.H., U.S. Patent 4 263 159
(1981) .
4. Vilenius,M., Proceedings of the Nordtrib 1984, Nordic
Symposium on Tribology, Tampere, Finland.
5. Carr,D.D. andDeGeorge, N. U.S. Patent 4 826 633
(1989) .
6. Flowerday, P. and Robson, R., G.B.Patent 1 307 7271
(1970) .
7. Leleu,G., Bedague,p. and Sillion, B., u.S.Patent 4
061 581 (1977).
8. van der Waal,G. and Kenbeek,D., proceedings of
Tribology 2000, 8. International Colloqium,Esslingen,
Germany, Band I,vol IV, 1992, 13.3.
9. Larosa,M., A method for the preparation of synthetic
ester from vegetable oil, Finnish patent apply number
944 118, 1994.
823
1Q.(.\{
10.Larosa, M., Linko Y,-Y., Linko P. and Uosukainen E.,
An enzymatic method for preparing synthetic ester
from vegetable oil, Finnish patent number 944 119,
1994.
11.Raport of Biodiesel project in Finland, Ministry of
Agriculture and Forestry, Finland ,1992.
12.Wildersohn,M., Tribologie+Schmierrungstechnik 32
(1985) 70-75.
13.Larosa,M.,Finnish Tribol., 14(1), 1995 39-45.
14.Niedzielski,E.L., Ind.Eng.Chem.prod.Res. Dev. 15(1)
1976 54-58.
15.van der Waal, G., Proceedings of NLGI 55th Annual
Meeting 53(1989), Florida, USA, 359-378.
16.Szydywar,J., J.Synth.Lubr. 1 (1984) 153-169.
17.Annual Book of ASTM Standards, volume 05.02.,
Petroleum products an Lubricants, ASTM, Philadelphia,
PA 19103, USA.
18.Filterability- project, Royal Institute of Tecnology,
Stockholm, Sweden, 1988-1994.
19.ISO 4406-standard, International Orqanization for
Standardization, 1980, lOp. -
20.Annual Book of ASTM Standards,vol.05.01,Petroleum
Products and Lubricants,ASTM, Philadelphia, USA.
21.DIN-Taschenbuch 192, Schmierstoffe, Beuth Verlag
GMbH, Berlin, Germany,1983.
22.Larosa,M., proceedings of the 2nd Tampere Intern.
Conf. on Fluid POwer 1991, Tampere, Finland, 73-89.
23.The Lubrication Engineers Manual, 1st edition, United
States Steel, combiled and edited by Bailey,C.A. and
Aarons,J.S., 1971, 146-147.
24.The Lubrication Engineers Manual, 1st edition, United
States Steel, conbiled and edited by Bailey,C.A. and
Aarons. J.S., 1971, 152-153.
25.0ECD-Guidelines for testing of Chemicals, advice 301,
Paris, France, 1981.
26.CEC-L-33-A-93, Biodegradability of Two Stroke cycle
Outboard Engine Oils in Water, London, United
Kinadom.
27.0EcD- Guidelines for Testing of Chemicals, advice
305, Paris, France, 1981.
28.0ECD-Guidelines for Testing of Chemicals, advice 201-
203, Paris, France, 1981.
29.Virtanen, T., Research and Tests of New Synthetic
Esters as Hvdraulic Fluids, Master of Science
work(chem.eng.) ,Turku, Finland, 1993.
824
11N
/tf/
Appendix 'VI
with the permission of the publisher
Merja Liimsii
Raisio Group
Oil Milling Division. R&D Laboratory
P.O.Box 101
21201 Raisio
Tel. 921- 434 2746
Fax. 921- 434 2911
VEGETABLE OIL BASED LUBRICANTS
INTRODUCTION
Vegetable oils have been used as lubricants since 1000 B.C. 0,2). Revolution of
mineral oils started early 1900 and is still continuing (3,4). So called green values have
disiurbed this hegemonia. The green values demand in lubricant section products, that
would biodegradme. not bioaccumulate and not to be toxic in nature (5). These demands
are totally fullfilled by natural esters or synthetic esters based on vegetable oils. These
products operate also technically well or even better than conventional mineral oil based
products (6.7.8). New development in vegetable oil based lubricant section are totally
environmental friendly additives and lubricants based on synthetic esters of vegetable
oils. Latest development results and test results of new products are discussed.
METHODS
Synthetic esters of vegetable oils have been prepared either chemically or enzymatically
(9.10). Raw material has been mainly Finnish rapeseed oil. although tall-, soyabean-.
coconut- and tallow oils have also been in tests. Esters are mainly polyolester types.
These new products have been in laboratory tests and some of them already in field
tests. Detailed test methods and results are shown later.
Additive industry has started to produce environmentally more friendly additives during
last three years. These new generation additives have been tested in both natural esters
and synthetic esters of vegetable oil based lubricants. Also detailed information of them
is shown later. All tests are performed in external. independent laboratories with
standardized methods.
39
1N1
RESULTS
Physical and chemical properties of natural and synthetic esters of vegetable oil based
lubricants
Viscosity is determined according to ASTM D445-standard (11). It is measured in 40
0C and 100 0C and from them the viscosity index can be calculated. Viscosity in cold
and the stability of it in time are also very important quantities. They are measured by
the same standard.
Table 1: Viscosity of natural and synthetic ester based lubricants from vegetable oils.
ASTM D 445- standard.
2NI
Temp. Viscosity
(0C) (mm2ls)
CSE RBS 32Lo RBS 32Ln RBS46S SE
40 29,3 33,8 34,0 50,4 32,9
100 7,5 7,9 7,8 9,8 8,0
VI 242 220 220 191 220
MO
32,6
6,4
187
CSE= comparable,commercial synthetic ester based hydraulic fluid
RBS 32Ln= Raisio Bio Safe 32, new, vegetable oil based hydraulic fluid
RBS 32Lo= Raisio Bio Safe 32, old
RBS 46S= Raisio Bio Safe 46, synthetic ester based hydraulic fluid
SE= synthetic ester of vegetable oil based hydraulic fluid. 32
MO= commercial, comparable mineral oil based hydraulic fluid
Filterability is important to be good enough not to cause troubles in hydraulic systems.
If hydraulic fluid contains many particles in it, wear rate increases and finally filters
block- up. Filterability is always measured from the raw material (= natural and
synthetic esters) and from the final lubricant.
Method used is under standardizing and is developed in Royal University of Stockholm
together with users (12). It is called %- method, because results are given in pro cents.
The biggerthe value is (max 100%) the better.
40
1t.f3
3M
Table 2: Filterability of vegetable oil, synthetic ester based and comparable mineral oil
and synthetic lubricants.
Filterablity (%)
SE CSE
92 63
RES 32Ln
96
RES 32Lo
98
RES 46S
80
MO
72
Small particles can cause serious problems in fine hydraulic systems, for example high
wear. These particles can exist already in basic fluids, in additives or in combining these
two. They are calculated from new and used oil by ISO 4406- standard.
Table 3: ISO 4406- cleanclass of vegetable oil, synthetic ester and comparable synthetic
and mineral oils.
SE
13/8
CSE
15/10
RBS 32Ln
13/8
RES 32Lo
13/8
RBS46S
13/8
MO
14/10
Cold stability of hydraulic t1uids is very important for machines working outside during
wintertime, such as forest machines. Pourpoint is one way to measure it. It tells at what
temperature the t1uid is still fluidy. Standard is ASTM D 97 (11). Cold stability can also
be expressed by long-term coldstability (11). How many days fluid stays fluidy in
certain temperature. Viscosity should also be and stay at certain level in cold
temperature.
Table 4: Pourpoint and viscosity at cold temperatures of some hydraulic fluids.
Pour-
point ( C)
SE
-41
CSE
-48
41
RBS 32Ln
-39
RBS46S
-39
MO
-40
Table 5: Example of viscosity stability of some hydraulic fluids at cold temperatures.
Temp. Visco
(C) (mm2/s)
RES 32Ln MO
0 322
-10 829 496
-20 1628 1221
-25 2038
-30 2940
I day 3050
3 days 3050
5 days 3050
All these base fluids examined here have good properties for reduce wear and lower
friction. This is due to their chemical structure. that is shown in figure I. All these
components contain oxygen, which fonn an unimolecular, strenght film over metal
surface. This film lowers friction and reduces wear.
4NI
o
[H of.R
I 2 0 1
CH -(){.R
I 2

rapeseed 0 i I
minerai 011

CH
3
SHs
Figure I: Molecular structure of vegetable oil, synthetic ester of it and mineral oil (6).
42
To measure friction and wear at laboratory level, quite many different standardized
methods exist, such as FZG- test, Vickerspump- test, Mobil- test. One of them is so-
called fourballtest (standards ASTM D 2783, IP 239)( II), where wear is measured
under loading and maximum loading until lubrication fails. Limits for hydraulic fluids
are <0.5 mm (damage is small) and> 1.0 mm (damage is big).
Table 6: Fourballtest- equipment and some test results from hydraulic fluids.
5NI
SE
max 10ad(N) 2000
wear<mm) 0,4
RBS 32Ln
2000
0,48
RES 32Lo
3000
0,45
RBS46S
3000
0,46
MO
2200
0.54
Corrosion does not cause problems with vegetable oil based products. Of course.
corrosion resistance can also be improved by additives. Example of how these new
generation products are better than lubricants earlier is shown in table 7. This test is
called Cincinnati- Milacron test and it is done for 200 ml of tested oil, kept in 135 0C
for 168 hours with steel- and copper plates in it (12).
43
Table 7: Cincinnati - Milacron test from vegetable oil, synthetic ester based hydraulic
fluids.
TAN mgKOHIg SE RBS 32Ln RBS 32Lo RBS46S
before 1,39 1,72 0,83 1,40
after 1,56 0,61 2.57 2,41
TAN 0,17 1,11 1,74 1,01
Total sludge 1,1 20,S 45,4 28,2
(mgllOOml)
change in vise. 19,1 6,2 10,2 16,9
40 C(mm2/s)
weight change 1,7 -0,8 0,3 0
in Cu-plates (mg)
weight change -0,3 0 0.5 1,2
in Fe-plates (mg)
ENVIRONMENTALLY FRIENDLY PROPERTIES
Biodegradability can be measured by many different methods. They are all standardized
and no one of them can be said to be better than other. Examples of methods: CEC-L-
33-T-82(14),DlN 38412(15), Sturm- test, MITI- test. In table 8 some examples are
presented. As important as biodegradability are bioaccumulation and toxicity properties
of lubricants. Bioaccumulation happens if product or part of it accumulates in nature:
micro-organisms, animals, plants etc.
It can be measured for example by OECD-standard tests (16). Toxicity must be
measured together with bioaccumulation. because product can be, when disposed, toxic
in nature. This value can also be measured by OECD- standards.
Table 8: Biodegradability.
6NI
Testmethod
CEC-L-33-T-82 (%)
DIN 38412
Sturm -test
RBS 32Ln
>90%
5days
>90%
44
limit
70%
14 days
60%
//7
CONCLUSIONS
Both natural and synthetic ester of vegetable oil based lubricants seem to fullfill their
function very well. Based on laboratory test results. it can be said that oxidation and
t>specially thermal stability of synthetic esters are better than those of natural esters.
Cold stability. especially viscosity at low temperatures. is clearly better in synthetic
esters at temperature area -10 0C to - 20 oC, but then the role change: Natural esters are
better in colder temperatures ( -25 0C - -40 0C). In friction and wear properties no
difference can be found. Results are. however, so promising, that field test are already
going on.
REFERENCES
I. Ihrig, H.. Mineraloltechnik 35 1990 1-9,12-19.
2:-Boylan. J.B.. Nat. Lubr. Grease Inst. Spokesman 1987 185-195.
3. Odi- Owei. S..1. Soc. Trib. Lubr. Eng. 45 1989685-690.
4. Van der Waal. G.B., Nat. Lubr. Grease Inst. 55th Annual Meeting 53 1989359-368.
5. Stempiel. E.M. and Schmid. L.A.. Proceedings of the Tribology 2000 8th Intern.
Colloquium 1992 voll.
6. Uimsa. M.. Proceedings of the 2Nd Tampere Intern. Conf. on Fluid Power 1991 73-
89.
7. Lamsa. M.. Proceedings of Tech. Acad. Esslingen, Biologisch abbaubare
Schmierstoffe und Arbeitstlussigkeiten 1992 13.7.
8. Lamsa. M.. Proceedings of the 9th Intern. colloquium in Tribology 1994 vol I 2.8.
9. Lamsa. M.. Menetelma synteettisen esterin valmistamiseksi kasvioljysta. Finnish
patent apply number 944118 1994.9. 9.
10. Lamsa. :vl.. Linko. Y.- Y. and Linko. P.. Entsymaattinen menetelma synteettisen
esterin valmistamiseksi kasvioljysta. Finnish patent apply number 944119 1994.
II. Annual Book of ASTM- standards. vol 05.01.. Petro Prod. and Lubr. ASTM,
Philadelphia. USA.
12. Filterability- project. Royal Institute of Technology, Stockholm. Sweden 1988-
1994.
13. The Lubr. Eng. Manual 1st edition. United Steel Corporation USA.
14. CEC-L-33-T-82. Biodegr.of two-stroke cycle outboard engine oils in water. London.
United Kingdom.
15. DIN-Taschenbuch 192. Schmierstoffe. Beuth Verlag GMbH, Berlin, Germany 1983.
16. OECD Guidelines for testing of Chemicals. Advice 301. Paris, France 1981.
45
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