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Fatty
Acid
Determination
Using
Gas
Chromatography
OBJECTIVE
This experiment introduces a procedure that is used routinely for fat
analysis in which nonvolatile fatty acids are chemically converted to
the corresponding volatile methyl esters. The resulting volatile
mixture can be analyzed by gas chromatography.
INTRODUCTION
Fats consist of glycerol esters and long chain aliphatic acids
(fatty acids). The backbone of these compounds contains from 4 to
more than 20 carbon atoms. Most natural sources of these
compounds have an even number of carbon atoms because the
biosynthetic pathway builds the backbone two carbons at a time.
Fatty acid chains may contain one or more double bonds at specific
positions (unsaturated and polyunsaturated), or they may be fully
saturated. The physical and chemical properties of a fat depend on
the composition of the fatty acid mixture. Animal fats tend to have a
larger proportion of long chain-saturated acids and are solids at
room temperature. Fats from plant sources contain a higher
proportion of unsaturated acids and are often liquids at room
temperature due to hydrogen bonding. Polyunsaturated fats are
usually of vegetable origin. Crisco is an example of a vegetablederived, unsaturated fatty acid that has been hydrogenated to form
a solid material. Fats are used in cooking because they are very high
boiling compounds. Their high boiling points therefore make this
class of compounds ill suited for analysis by gas chromatography.
EXPERIMENTAL PROCEDURE
a.
samples
1.
2.
3.
5mL of 0.5 M methanolic solution was added into the flask and
was refluxed for about 3 to 4 minutes until the mixture turned
to golden brown colour.
4.
5.
6.
7.
The organic layer was transferred into a screw cap vial and be
made sure that only the organic layer was transferred.
b.
c.
1.
2.
3.
Peaks
Retention
Time of
Peaks
(min)
Base Peak
Width of
Peaks
(min)
Area of
Peaks
(pA*s)
Peak 2
Peak 3
2.084
2.765
0.0160
0.0217
107.40754
117.88110
Resolutio
n of 2
peaks
(peak 3 &
peak 4)
Response
Factor (Rf)
of Peaks
0.93103
0.8483
Peak 4
Peak 5
Peak 6
3.921
5.899
6.865
0.0299
0.0496
0.0432
1022.17914
698.57605
276.47214
45
0.09783
0.1431
0.3617
= 45 #
Peak 2
Response Factor (RF) = amount
peak area
= 100 (ppm)
107.40754
2.
= 0.93103 #
Peak 3
Response Factor (RF) = amount
peak area
= 100 (ppm)
117.88110
3.
Peak 4
= 0.8483 #
= 0.09783 #
Peak 5
Response Factor (RF) = 100 (ppm)
698.57605
1.
= 0.1431 #
Peak 6
Response Factor (RF) = amount
peak area
= 100 (ppm)
276.47214
B.
Peak 2
Peak 3
Peak 4
= 0.3617 #
Retention
Retention
Retention
Time, Rt for
Time, Rt for
Time, Rt for
Time, Rt for
Standard
Sample 1
Sample 2
Sample 3
(min)
2.084
(min)
= 2.105 +
(min)
= 2.106 +
(min)
= 2.106 +
2.106
2.106
2.106
= 2.106
= 2.811 +
= 2.106
= 2.814 +
= 2.106
= 2.812 +
2.812
2.812
2.813
= 2.812
= 4.004 +
= 2.813
= 4.003 +
= 2.813
= 4.006 +
4.004
4.004
4.005
2.765
3.921
Peak 5
Peak 6
C.
5.899
6.865
= 4.004
= 6.360 +
= 4.004
= 6.363 +
= 4.006
= 6.371 +
6.369
6.365
6.365
= 6.365
= 7.005 +
= 6.364
= 6.368
= 7.023 =
7.004
7.015
= 7.005
= 7.019
Base Width of
Resolution of
Peak 3 & 4
Peak 3 & 4
Sample 1
5 (min)
2.812, 4.004
(min)
0.0494,
18
Sample 2
Sample 3
2.813, 4.004
2.813. 4.006
0.08495
0.0493, 0.0857
0.0493, 0.0838
18
18
Sample 1
Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 4.004 2.812 )
(0.0494 + 0.08495)
= 18 #
2.
Sample 2
Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 4.004 2.813)
(0.0493 + 0.0857)
= 18 #
3.
Sample 3
Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 4.006 2.813 )
(0.0493 + 0.0838)
= 18 #
D.
Peak Area
(pA*s)
Correspond
Sample
1
Sample
2
Sample
3
Amount of
FAME (Fatty
Acid),
Peak
Peak
Peak
Peak
Peak
2
3
4
5
6
ing peak
0.93103
0.8483
0.09783
0.1431
0.3617
35.89295
16.54252
234.95041
18.673855
6.16273
(ppm)
33.417
14.033
22.958
2.6729
2.2291
Peak
Peak
Peak
Peak
Peak
2
3
4
5
6
0.93103
0.8483
0.09783
0.1431
-
11.16741
3.72386
44.85535
33.72593
-
10.397
3.159
4.388
4.8261
-
Peak
Peak
Peak
Peak
Peak
2
3
4
5
6
0.93103
0.8483
0.09783
0.1431
0.3617
14.295845
5.26132
68.67184
44.42255
10.71028
13.310
4.4632
6.718
6.357
3.874
DISCUSSION
Fatty acid is an important component of lipids (fat-soluble
components of living cells) in plants, animals, and microorganisms.
Generally, a fatty acid consists of a straight chain of an even
number of carbon atoms, with hydrogen atoms along the length of
the chain and at one end of the chain and a carboxyl group
(COOH) at the other end. It is that carboxyl group that makes it an
acid (carboxylic acid). If the carbon-to-carbon bonds are all single,
the acid is saturated; if any of the bonds is double or triple, the acid
is unsaturated and is more reactive.
GC can be used to analyze fatty acids either as free fatty acids
or as fatty acid methyl esters. The primary reasons to analyze fatty
acids as fatty acid methyl esters include; in their free, underivatized
form, fatty acids may be difficult to analyze because these highly
polar compounds tend to form hydrogen bonds, leading to
adsorption issues. Reducing their polarity may make them more
amenable for analysis and to distinguish between the very slight
differences exhibited by unsaturated fatty acids, the polar carboxyl
functional groups must first be neutralized. This then allows column
chemistry to perform separations by boiling point elution, and also
by degree of unsaturation, position of unsaturation, and even the cis
vs. trans configuration of unsaturation.
The esterification of fatty acids to fatty acid methyl esters is
performed using an alkylation derivatization reagent. Methyl esters
GC
analysis.
The
esterification
reaction
involves
the
retention time are almost the same, which means that the 5
compounds of samples elute out at almost the same time as the
standard. Methyl laurate is represented by peak 2, and at the
standard it eluted out at 2.084th minute and the samples it eluted
out at 2.1 minute. Peak 3 is methyl myristate, and in the standard
the compound elute out at 2.765th minute and the samples at 2.8th
minute. The 4th peak is methyl palmitate, and in the standard it
elute out at 3.921 minute and the in the samples methyl palmitate
elute out at the 4th minute. Methyl stearate is represented by peak 5
and in the standard it elute out at the 5.899 th minute but in the
samples peak 5 elute out at 6.3 rd minute, which differs slightly. And
lastly at peak 6, is methyl linoleate and it elute out at 6.865 th minute
and in the sample it elute out at 7th minute which also differs
slightly.
By the response factor calculated in the standard, we can tell
the amount of each methyl esters in the sample. And the amount of
fatty acids in each sample are calculated and stated in the results
and data section.
CONCLUSION
The derivatization technique used in this experiment is esterification
to convert non-volatile fatty acids to more volatile fatty acid methyl
ester (FAME). There are 5 components in the standard mixture that
is the methyl esters. The concentration of each component is
calculated by using the response factor of the standard.
REFERENCES
1.
http://global.britannica.com/science/fatty-acid
2.
http://www.sigmaaldrich.com/analyticalchromatography/analytical-products.html?
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