CHM510
ANALYTICAL SEPARATION METHOD
EXPERIMENT 3:
FATTY ACID
DETERMINATION USING GAS CHROMATOGRAPHY (GC)
INTRODUCTION
Gas chromatography separates the analytes that is volatile
and chemically stable. Fatty acids are not sufficiently volatile for GC
analysis, so that it needs to be modified chemically to produce a new compound
which has properties that are suitable for analysis. If the unsuitable sample
is introduced into GC analysis, it tends to cause peak tailing due to the
adsorption and non-specific interaction with the column. In this experiment,
the fatty acid is changed to fatty acid methyl ester (FAME) that is more volatile,
suitable for GC analysis by using esterification reaction that used methanolic
solution with catalyst of esterification reagent. The objective for this
experiment is to introduce a derivatization procedure routinely used for fat
analysis in which non-volatile fatty acids are chemically converted to the
corresponding volatile methyl ester (FAME) and to determine the amount of FAME
in the derivatives samples. The amount of FAME is determined by using the
response factor calculation:
PROCEDURE
a)
Preparation of fatty acid methyl
ester samples from fat samples.
2 g of oil or fat was
weighed out and the exact weight was recorded. The sample was transferred into
a 50 mL flask equipped with air condenser. Then, 5 mL of 0.5 M methanolic
solution was added and refluxed for 3-4 minutes. 15 mL of esterification
reagent was added and refluxed about 3 minutes. After that, the mixture was
transferred into a separatory flask. 50 mL of saturated sodium chloride and 25
mL of diethyl ether was added. The mixture was shaking vigorously for 2 minutes
and the aqueous layer was discarded. In addition, 25 mL of saturated sodium
chloride was added and the aqueous layer was discarded. The organic layer was
transferred into a screw cap vial. The samples were sealed by parafilm for the
quantitative analysis.
b)
Instrument set-up
Injection Port: Split (40:1)
Injection Port
Temperature: 250oC
Column Temperature: 100oC
to 290oC at 40oC min-1
Carrier Gas Flow Rate: 30
mL s-1
Detector Temperature: 250oC
c)
Quantitative analysis of FAME
0.4 µL of standard esters was injected
onto the column. Injection was repeated to get reproducible peak areas. 0.4 µL of derivative sample was injected. Injection was repeated
to get reproducible peak areas. The amount of each fatty acid in the sample was
calculated using the data from the standard esters.
RESULTS
A.
Response Factor (RF) for analytes in
standard FAME:
Peak
|
Amount of FAME in standard (ppm)
|
Peak Area(pA*s)
|
Response Factor
|
2
|
100
|
69.50613
|
1.4387
|
3
|
100
|
321.35806
|
0.31112
|
4
|
100
|
560.65057
|
0.17836
|
5
|
100
|
61.21711
|
1.6335
|
6
|
100
|
487.67694
|
0.2051
|
Comparison of retention time for
standard and samples:
Peak
|
Retention time for standard (min)
|
Retention time for sample 1 (min)
|
Retention time for sample 2 (min)
|
Retention time for sample 3 (min)
|
2
|
1.981
|
2.014
|
2.016
|
2.015
|
3
|
2.578
|
2.660
|
2.652
|
2.653
|
4
|
3.605
|
3.751
|
3.756
|
3.755
|
5
|
5.314
|
5.669
|
5.702
|
5.673
|
6
|
5.902
|
6.137
|
6.144
|
6.140
|
C.
Amount of FAME in samples:
Sample
|
Peak
|
Response factor of corresponding peak
|
Peak area (pA*s)
|
Amount of Fame (ppm)
|
1
|
2
|
1.4387
|
30.92022
|
44.48
|
3
|
0.31112
|
2.56153
|
0.80
|
|
4
|
0.17836
|
54.58322
|
9.74
|
|
5
|
1.6335
|
34.99018
|
57.16
|
|
6
|
0.2051
|
9.04965
|
1.86
|
|
2
|
2
|
1.4387
|
17.52389
|
25.21
|
3
|
0.31112
|
33.56306
|
10.44
|
|
4
|
0.17836
|
1261.23462
|
224.95
|
|
5
|
1.6335
|
1068.38159
|
1745.20
|
|
6
|
0.2051
|
263.29333
|
54.00
|
|
3
|
2
|
1.4387
|
5.08862
|
7.32
|
3
|
0.31112
|
21.01284
|
6.54
|
|
4
|
0.17836
|
848.58557
|
151.35
|
|
5
|
1.6335
|
1.15695
|
1.89
|
|
6
|
0.2051
|
180.49922
|
37.02
|
DISCUSSION
The components in the samples are compared with the standard
components by the retention time. From the retention time of standard and
samples, it is proven that component 5 (peak 6) is not present in all 3 samples
because of the difference of the retention time between standard and samples
are too big. The amount of each component is different in each samples may due
to the different mass of the fat initially. Peak 5 in each sample give very
large different in the amount of FAME, this may be due to the un-complete
separation process during shaking process or the discarding process. It is
necessary to discard little organic layer to make sure that there is no aqueous
layer anymore to be injected onto GC. That condition also affected by the
contaminants in the flask that is not clean before using. The other peaks that
correspond to specific component show small difference that assumed to be
correct. There is another way to derivatize or modified the low volatility
fatty acid to more volatile compound called silylation that BSTFA to yield
trimethylsilyl (TMS) ester that is more suitable to be analysed in GC.
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 while the 3
samples only indicate 4 components as shown in the standard mixture by comparison
of the retention time. The concentration of each component is calculated by
using the response factor of the standard.
REFERENCE
1. Nor’ashikin S., Ruziyati T., Mardiana
S. (2012), Analytical Separation Methods Laboratory Guide (2nd edition).
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