Experiment 3 Fatty Acid Determination Using Gas Chromatography

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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).
2.      https://www.ncbi.nlm.nih.gov/pubmed/85206893.    http://www.doiserbia.nb.rs/img/doi/0352-5139/2015/0352-51391400073W.pdf

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