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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Borhade
World Journal of Pharmacy and Pharmaceutical
SJIF Impact Factor 2.786
Volume 3, Issue 7, 977-989.
Research Article
ISSN 2278 – 4357
SYNTHESIS AND CHARACTERIZATION OF FATTY ACID METHYL
ESTER FROM LINUM USITATISSIMUM (LINSEED) SEED OIL
Shobha Borhade*
Department of Drug Chemistry, S.M.B.S.T.College, Sangamner, DistAhmednagar,University of
Pune, Maharashtra, INDIA-422605
Article Received on
17 April 2014,
Revised on 16 May
2014,
Accepted on 09 Jun 2014
ABSTRACT
Linum usitatissimum (Linseed) seed is popularly used in various food
products, because of its nutritional values and potential health benefits.
Natural seed of of Linum usitatissimum (Linseed) contains 35 % of
oil. Linum usitatissimum (Linseed) seed oil contains 53.21 % alpha-
*Author for Correspondence
linolenic
Shobha Borhade
acid ). Seed oil was extracted by n-hexane & fatty acids present in the
Department of Drug
Chemistry, S.M.B.S.T.College,
( omega-3 fatty acid ) & 17.25 % linoleic ( omega-6 fatty
seed oil is necessary to apply for transesterification to convert fatty
Sangamner, DistAhmednagar,
acid methyl ester. Fatty acid methyl ester (FAME) is created through
University of Pune,
an alkali catalyzed reaction between fats or fatty acids and methanol.
Maharashtra, INDIA
In the transesterification of fatty acids, the carboxyl group of an acid
and the hydroxyl group of an alcohol are condensed. This makes the
fatty acids methyl ester Now FAME excellent for stability as samples for analysis.
Transesterification is a general term used to indicate the direct conversion of triacylglycerols
lipids by alcohols to alkyl esters without first isolating the free fatty acids (FFA). The alkaline
catalyst, sodium hydroxide (NaOH),was used in the methanolysis of Linum usitatissimum
(Linseed) seed oil Physico-chemical properties of FAME of of Linum usitatissimum
(Linseed) seed oil was studied .Fatty acid methyl ester was characterized by UV, IR &
LCMS.
INTRODUCTION
Linum usitatissimum (Linseed) seed oil is extracted from the seed by extraction with nhexane. Linum usitatissimum (Linseed) is believed to have originated inEgypt. Now, this
plant grows through out Canada and the north western United States.
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Linum usitatissimum (Linseed)
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Malpighiales
Family: Linaceae
Genus: Linum
Species: L. usitatissimum
Linum usitatissimum (Linseed) seed is popularly used in various food products, because of its
nutritional values and potential health benefits. Linseed is rich in alpha linolenic acid (ALA),
a plant-based omega-3 fatty acid, as well as fiber and lignans (phytoestrogens), making it a
possible functional food for reducing cardiovascular risk factors linseed positively affected
lipoprotein A (Lp(a)) and insulin sensitivity. They also found a modest but short-lived
lowering effect in participants' LDL ("bad") cholesterol levels.Linseed oil is derived from the
hard, tiny seeds of the linseed plant. Like fish oil, linseed oil contains an omega-3 fatty acid =
alpha-linolenic acid (ALA). In addition to alpha-linolenic acid, linseed oil also contains
omega-6 fatty acids. Both acids are essential to health.Linseed have long been used in human
and animal diets and in industry as a source of oil and as the basic component or additive of
various paints or polymers. Recently there has been a growing interest in the probiotic
properties of linseed and in its beneficial effects on coronary heart disease, some kinds of
cancer and neurological and hormonal disorders1. The beneficial effects are mostly due to
linseed lipids. linseed oil is the richest plant source of linoleic (omega-6) and linolenic
(omega-3) polyunsaturated fatty acids (PUFA), which are essential for humans since they
cannot be synthesized in the organism and must be ingested in food. Linseed oil is
qualitatively different from the more common vegetable oils with high PUFA proportions,
such as soya oil, sunflower oil, rape oil, olive oil, etc. Linseed oil is a rich source of the
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following unsaturated fatty acids: oleic (C18, 16–24 %),linoleic (C18, 18–24 %), and
linolenic acid (C18, 36–50 %)2 and it has a relatively low glucosinolate content3.The protein
and fiber content in the seed are also important nutritional parameters: the crude protein
content in the seed ranges from 25 % to 45 %, while the crude fiber content is about10%4
.The
results
of
the
analyses
of
linseed
oil
have
been
reported5-6
Unsaponifiable lipid constituents of seed oils naturally contain hydrocarbons, terpene
alcohols, sterols, tocopherols and other phenolic compounds which may act as oxidation
inhibitors under a range of conditions7. The effectiveness of lipid unsaponifiable matters in
retarding oil deterioration has been demonstrated by many investigators8-9. In linseed grains,
lipids are protected against oxidation by various mechanisms, for example, the presence of
antioxidants such as lignans, phenols, tocopherols-(vitamin E) and
flavonoids10- 11
.In addition to
preventing fat rancidity, these antioxidants could increase commercial value of food products
and have beneficial effects on human health. When consumed together with essential
unsaturated fatty acids, they can reduce the risk of various diseases
12
The antioxidant ability
of phenols, tocopherols (vitamin E) and flavonoids is related to the presence of OH groups
which may directly bind to free radicals and chelate metals13 .Linseed improves the quality of
hair, nails, and skin, helps to regulate body weight, lowers cholesterol and blood pressure,
and prevents arthritis and cancers. The omega 3 fatty acids of linseed oil lower high blood
cholesterol and triglycerides. They also decrease the probability of clots in the arteries which
may lead to stroke, heart attack, pulmonary embolism, or peripheral vascular disease
occurring. They can also lower high blood pressure.It can kill human cancer cells on the
same culture.omega 3 fatty acids have been shown to be helpful in the treatment and
prevention of arthritis. Linseed oil can relieve asthma noticeably by decreasing inflammation
and improving lung function.omega 3 fatty acids can relieve or even eliminate PMS
symptoms.omega 3 fatty acids help to decrease allergic response. Linseed oil helps the
kidneys to remove sodium and water.Linseed oil is renowned for its ability to improve the
texture and quality of the skin, and will also alleviate skin conditions that are caused by the
lack of omega 3 fatty acids in the diet. the use of linseed oil can result in increased vitality
and more energy. Stamina is improved omega 3 fatty acids prevent excess toxic
biochemicals that the body produces under stress.Linseed oil has been shown benefits on
lowering heart diseases by reducing cholesterol levels, high blood pressure and slowing down
atherosclerosis. Linseed oil may also benefit people suffered from bipolar disorder, cancer,
acne and rheumatoid arthritis. In this section, we are going to review how linseed oil may
benefit our health, based on recent research findings. Oral linseed oil reduces the
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inflammation in rheumatoid arthritis14 .Linseed oil in inflammatory disorders like
rheumatoid arthritis15. Dietary level of alpha-linolenic acid (10% linseed oil) results in
modest improvements in some bone linseed oil may benefit people on bone health16. ALA
significantly decreased hepatic cholesterol but no effect was observed on heart and kidney
cholesterol levels17. Further, ALA is capable to alter platelet function and inflammation18
.Linseed oil are probably effective to decrease the risk factors associated with cardiovascular
disease19 & did not increase the effect of dietary therapy in correction of glycemia,
hypertension and atherogenic lipid levels20.Oil supplements are also believed to benefit
patients suffered from different types of cancers21.High doses of linseed oil could also delay
in the growth of mammary cancers 22 and is effective in preventing colon tumor development
23
.
Fatty acid methyl ester prepared by transesterification.In the transesterification of oils, a
triglyceride reacts with an alcohol in the presence of a strong acid or base, producing a
mixture of fatty acids alkyl esters and glycerol 24-25 . Transesterification is the general term
used to describe the important class of organic reactions where an ester is transformed into
another through interchange of the alkoxy moiety. When the original ester is reacted with an
alcohol, The transesterification process is called alcoholysis (Scheme 1) 26 .
A
In the transesterification of vegetable oils, a triglyceride reacts with an alcohol in the
presence of a strong acid or base, producing a mixture of fatty acids alkyl esters and glycerol
27-28
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The transesterification process is catalyzed by BrØnsted acids, preferably by sulfonic
29
and
sulfuric acids These catalysts give very high yields in alkyl esters, but the reactions are slow,
requiring, tipically, temperatures above 100 °C and more than 3 h to reach complete
conversion. The base-catalyzed transesterification of vegetable oils proceeds faster than the
acid-catalyzed reaction30-31.Dueto this reason, together with the fact that the alkaline catalysts
are less corrosives than acidic compounds, industrial processes usually favor base catalysts,
such as alkaline metal alkoxides
32-33
and hydroxides
34-38
as well as sodium or potassium
carbonates 39-40
The overall process is a sequence of three consecutive and reversible reactions, in which diand monoglycerides are formed as intermediates
41
. The stoichiometric reaction requires 1
mol of a triglyceride and 3 mol of the alcohol. However, an excess of the alcohol is used to
increase the yields of the alkyl esters and to allow its phase separation from the glycerol
formed. Fatty acid methyl esters can be transformed into a lot of useful chemicals, and raw
materials for further synthesis
41
.The alkanolamides, whose production consumes the major
part of the methyl esters produced in the world, have a direct application as non-ionic
surfactants, emulsifying, thickening and plastifying agents, etc . The fatty alcohols are
applied as pharmaceutical and cosmetics additives (C16-C18) as well as lubricants and
plastifying agents (C6-C12), depending on the length of their carbon chain
42
. The isopropyl
esters are also applied as plastifying agents and emollients. However, they can not be
produced in a convenient way by esterification of fatty acids, as an azeotrope formed by
water and isopropanol avoids the recycling of the alcohol. The fatty acid methyl esters are
further used in the manufacture of carbohydrate fatty acid esters (sucrose polyesters), which
can be applied as non-ionic surfactants or edible non-calorific oils 43-46 and can be used as an
alternative fuel substitute for diesel engines (biodiesel) 47-52 . The glycerol also has important
applications, in cosmetics, toothpastes, pharmaceuticals, food, lacquers, plastics, alkyl resins,
tobacco, explosives, cellulose processing, etc
53
.
Fatty acid methyl ester obtained from natural seed oil. Fatty acid methyl ester are
conventionally produced by the transesterification of Linum usitatissimum (Linseed) seed
using methanol in presence of suitable catalyst.
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MATERIAL AND METHODS
Collection of materials
The dried Linum usitatissimum (Linseed) seeds were obtained from local market in
Ahmednagar, Dist Ahmednagar, Maharashtra, India. They are dried in room, clean and stored
in a sealed vessel wrapped with polyethylene bag at 40C. Linum usitatissimum (Linseed)
seed oil , Methanol, NaOH, High purity water , Sulphuric acid.The instrument used were UV
spectrophotometer with wavelength range 200-450 nm. IR spectra of FAME was taken in the
range of 4000cm-1 to 600 cm-1 on perkin Elmer 221 IR spectrophotometer using KBR pellet
techniques & LCMS chromatogram.
Extraction of oil
After cleaning and removal of the sand and foreign material , the dried Linum usitatissimum
(Linseed) seeds were ground to a fine powder using a grinde. The oil was extracted with nhexane ( 1:4 w/v ) by continous extraction in a soxhlet apparatus for 12 hours.
Experimental set up
The experimental set up as a 1000 ml three necked round bottom flask was used as a reactor.
The flask was placed in heating mantle whose temperature about 55-600C. Two side necked
was equipped with a condenser and other was used as a thermometer for temperature
measurement. Stirrer placed at the centre for stirring the reaction mixture.( fig.1 )
Pretreatment of oil
Linum usitatissimum (Linseed) seed oil is first filtered to remove solid material then it is
preheated at 10500C for 25 min to remove moisture ( Presence of moisture responsible for
saponification in the reaction )
Transesterification – Based catalysed reaction : Mixing of alcohol and Catalyst
Weight 12 kg of Linum usitatissimum (Linseed) seed oil and pour it into the reactor for
preliminary heating to temperature of about 60-700C. In beaker dissolve 45.6 grams of
NaOH ( 3.8 grams per liter of oil , got by 3.5 grams stoichiometric equivqlent and 0.3 grams
for neutralizing FFA in 2.4 L methanol, 200 ml per lit of oil ). Add the NaOH slowly. This
combined mixture makes sodium methoxide. Adding sodium methoxide in Linum
usitatissimum (Linseed) seed oil provide rigorous mixing with the use of stirrer. The cloudy
looking of free fatty acids called glycerine, will sink to the bottom and the methyl ester a
translucent liquid will remain on top. When the separation appears not to be advancing any
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more, stop mixing. Let the mixture settle overnight. Meanwhile another batch can be started
as the reactor is not being used. The liquid on the top is fatty acid methyl ester, but before
using it any remaining soaps or salt which have to be removed. The glycerin which has sunk
to the bottom can be used in the production of cosmetics.
B
KOH
Methanol & Ethanol
Fatty acid methyl ester
Reactor Flash
Extract
of seed powder
Glycerol & Seed powder
Block Diagram for Production of Fatty Acid Methyl Ester ( FAME )
Rinsing Fatty Acid Methyl Ester
Fatty acid methyl ester was poured off into a separate clean container, where it was washed
free of any remaining soaps and salts. Warm water was added to the methyl ester. It was
stirred lightly and then allowed to settle. The warm water was heated in the main reactor
itself. The water was drained out from the bottom. The process was repeated until the
discarded rinse water reached pH level of 6-7 and no soap bubbles appeared in it. If the liquid
remaining is cloudy, there is water being retained in the methyl ester and it will need to be
reheated slowly to evaporate out the water. Any white substances forming at the bottom or
any bubbles forming at the surface is a sign of soaps and should be removed or the liquid
should be re-washed. The cleaned methyl ester is characterizwd by UV, IR and LCMS.
Physico-chemical Properties of FAME
Physico-chemical Properties of FAMS of Linum usitatissimum (Linseed) seed
oil was
studied. Density was determined picnometrically, where as refractive index was determined
at 400C with Abbey Refractometer equipped with a thermostated circulator. Viscosity was
determined by Ostwald method .( Table 1.)
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Absorption spectra of FAME
The absorption spectra of FAME of Linum usitatissimum (Linseed) seed oil was recorded
against a blank solution ans shown in Fig. 2 The absorption spectra was recorded in the
wavelength range 220-520 nm. FAME of Linum usitatissimum (Linseed) seed oil shows the
absorption maximum
at 208.20 nm shows absorption 2.130.
Infrared Spectra of FAME
The infrared spectra of fatty acid methyl ester of Linum usitatissimum (Linseed) seed oil was
taken in the range of 4000 cm-1 to 600 cm-1 on perkin Elmer 221 IR Spectrophotometer using
KBR pellet techniques. The characteristic bands observed are in Table 2. Fig 3. Shows IR
spectra of FAME of Linum usitatissimum (Linseed) seed oil
LCMS of FAME
HPLC was applied for testing the presence of number of organic components available of
methanolic extract of FAME of Linum usitatissimum (Linseed) seed oil.
Method
Column : YMC ,ODS, 50x4.6 mm, 3µ, Column ID : E-AC-1 / 12 / COL-03
Mobile phase : A : 0.05 % TFA in water & B : 0.05 % TFA in Acetonitrile
Inj , Volume : 5.0 µL, Flow Rate : 1.2 mL / minute, Gradient program : 20 % B to 100 % B
in 3.0 minute, At 4.50 min B conc is 20 % hold till 5.0 min Mass parameters : Scan mode
+ve, CDL temperature 250 0C , Heat block temperature 200 0C Interface temperature 250 0C
( Table 3. Peak Table , Fig 4)
RESULT AND DISCUSSION
The extraction of oil from Linum usitatissimum (Linseed) seeds were done by Soxlet method
using n-hexane as a solvent at 400C. yield obtained 46 % of oil.Extracted Linum
usitatissimum (Linseed) seed oil consisted of 94 % pure triglyceride esters and the rest were
free fatty acids. The physico-chemical properties were determined. Fatty acid methyl ester of
Linum usitatissimum (Linseed) seed oil shows moisture 0.001 % and ash content 0.02 Wt
%.FAME has density 0.7989 gm/cc and Refractive index at 400C 1.2894. Viscosity at 400C is
1.6231. Acid value, Iodine value and Saponification value are 0.34 mg KOH / gm, 91 g / 100
g of oil and 142 mg KOH / g of oil. UV spectra of FAME of Linum usitatissimum (Linseed)
seed oil was performed at 208.20 nm wavelength. At 208 nm absorbance was 2.130.. IR
spectra was done in the range of 4000cm-1 to 600cm-1 shows P-O Stretch at 1016.42, C=C
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Stretch at 1643.24, Alkynes ( RHC =CH2) at 2104.19, O-CH3 at 2842.88, Alkanes ( -CH3 ) at
2954.74, N-H Stretch at 3398.34, Primary free N-H at 3411.84 & 3436.91, N-H Stretch at
3454.27. LCMS is suitable analytical method for determining FAME of Linum usitatissimum
(Linseed) seed oil. It shows 31.96 % at 0.525 retention time, 03.72 % at 0.596, 34.31 % at
0.668 , 03.38 % at 0.798 , 06.48 % at 0.967, 06.48 % at 0.967, 02.39 % at 1.063, 06.49 % at
2.464 and 00.94 % at 2.648.
Table 1. Physico-chemical properties of FAMS of Linum usitatissimum (Linseed) seed oil
Sr.No.
1)
2)
3)
4)
5)
6)
7)
8)
Table 2.
Properties
Moisture
Ash content
Density
Refractive index at 400C
Viscosity at 400C
Acid value
Iodine value
Saponification value
Cst
mg KOH / gm
g / 100 g of oil
mg KOH / g of oil
Values
0.001
0.02
0.7989
1.2894
1.6231
0.34
91
142
IR of FAMS of Linum usitatissimum (Linseed) seed oil
Sr.No.
Frequency
Wavenumber
1016.42
1643.24
2104.19
2842.88
2954.74
3398.34
3411.84
3436.91
3454.27
1)
2)
3)
4)
5)
6)
7)
8)
9)
Table 3.
Unit
%
Wt %
gm / cc
Expected Elements
P-0 Stretch
C=C Stretch
Alkynes ( RHC =CH2)
O-CH3
Alkanes ( -CH3 )
N-H Stretch
Primary free N-H
Primary free N-H
N-H Stretch
LCMS of FAMS of Linum usitatissimum (Linseed) seed oil
Sr.No.
1)
2)
3)
4)
5)
6)
7)
8)
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Ret. Time
0.525
0.596
0.668
0.798
0.967
1.063
2.464
2.648
Area
60856
7087
65321
6428
12339
4567
12363
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Area %
31.96
03.72
34.31
03.38
06.48
02.39
06.49
00.94
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Figure 1. Experimental set for preparation of Linum usitatissimum (Linseed) methyl
ester
A
B
C
S
T
TC
-------------------------
Speed controller
Stirrer
Condensed ( Water condenser )
Sample inlet
Temp. indicator
Temp. controlled
Figure 2 . UV Spectra of FAMS of Linum usitatissimum (Linseed) seed oil
Figure 3 . IR of FAMS of Linum usitatissimum (Linseed) seed oil
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Figure 4 . LCMS of FAMS of Linum usitatissimum (Linseed) seed oil
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