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EXPERIMENT 3: DETERMINATION OF FATTY ACID USING

GAS CHROMATOGRAPHY
NAME: MUHAMMAD TARMIZIE BIN MOHD SABERI
STUDENT ID: 2018695406
LECTURER: PUAN HALIZA KASSIM
GROUP MEMBERS:
1. MUHAMMAD HUZAIRE BIN ASNAN (2018272592)
2. DIANA SUDA ANAK ALAU (2019702461)
3. MOHAMAD NORSYHAMI BIN MOHD AZMI (2018264508)
DATE OF EXPERIMENT:23/10/2019

CONTENT
1) INTRODUCTION

2) METHODOLOGY

3) RESULTS AND DISCUSSION

4) CONCLUSION

5) REFERENCE

Abstract:
Gas chromatography is an instrumental method for separation and identification of
compounds. In GC, a liquid or gas sample is introduced into the injection port through a
rubber septum into a stream of inert gas, which is called the mobile phase or carrier gas. The
gas then passes through a chromatographic column where the partition between the mobile
phase and stationary phase occur. Analytes with the lower affinity for the stationary phase
spend less time in the column and pass through quickly while analytes with higher affinity for
the stationary phase spend more time bound to the column and pass through more slowly. In
this experiment, it introduces a derivatization procedure routinely used for fat analysis in
which nonvolatile fatty acid that chemically converted to the corresponding volatile methyl
esters (FAME). The resulting volatile mixture can then be analysed by GC. There are several
of fame such as methyl laurate, methyl myristate, and methyl palmitate. The objective of this
experiment is to produce methyl esters and determined it using gas chromatography and
determine the amount of FAME in the sample using the response factor formula.

Method:

a. Preparation of fatty acid methyl ester sample from fat sample.

i.2g of fat was approximately weighed and the exact weight recorded.

ii. The sample was transfer into 5ml flask equipped with air condenser.

iii.5ml of 0.5M methalonic acid was added and it refluxed for three to four minutes.

iv.15ml of esterification reagent was added and reflux for three minutes.

v. The mixture was transfer into a separatory flask.50ml of saturated NaCl and 25ml of
diethyl ether was added. The mixture was vigorously shake for two minutes and the aqueous
layer discarded.

b. Quantitative analysis of FAME

i. 0.4µL standard esters was injected onto the column. The injection was repeated to get
reproducible peak areas

ii. 0.4µL of the derivatized sample was injected. By using the data from the standard esters,
the amount of each fatty acid in the sample was calculated.

Result and Calculation:


a. Response factor (RF) for analytes in standard FAME.

Amount of sample ( standard)


Response factor =
Peak area

Amount of FAME in Peak area (pA*s) Response Factor


-1
standard (mg mL ) (RF)

Peak 2 0.10 846.10657 1.1819 x 10-4


Peak 3 0.10 1375.05994 1.0908 x 10-3
Peak 4 1.50 1725.53064 4.0567 x 10-4
Peak 5 0.70 573.69727 3.4861 x 10-4
Peak 6 0.20 812.15619 2.4626 x 10-4

b. Comparison of retention time for standard and samples.

Retention Retention Average Retention time of


time 1st (min) time 2nd (min) (min) standard
Sample 1 Peak 2 1.512 1.516 1.514 1.489
Peak 3 1.972 1.975 1.974 1.937
Peak 4 2.870 2.873 2.8715 2.810
Peak 5 3.923 3.926 3.9245 3.828
Peak 6 4.058 4.060 4.058 4.002
Sample 2 Peak 2 1.519 1.513 1.516 1.489
Peak 3 1.976 1.973 1.975 1.937
Peak 4 2.872 2.870 2.871 2.810
Peak 5 3.923 3.922 3.9225 3.828
Peak 6 4.061 4.060 4.0605 4.002
Sample 3 Peak 2 1.517 1.512 1.515 1.489
Peak 3 1.978 1.974 1.976 1.937
Peak 4 2.883 2.882 2.8825 2.810
Peak 5 3.939 3.937 3.938 3.828
Peak 6 4.069 4.067 4.083 4.002

c. Peak area sample

Peak Area (pA*s) Peak Area (pA*s) Average Peak


1st injection 2nd injection Area (pA*s)

Sample 1 Peak 2 32.4894 31.30498 31.8972


Peak 3 97.8832 92.14003 95.0116
Peak 4 3407.940 3247.907 3327.923
Peak 5 3234.034 3130.697 3182.865
Peak 6 862.315 725.524 793.919
Sample 2 Peak 2 17.490 18.164 17.827
Peak 3 58.440 55.251 56.845
Peak 4 2314.468 2358.385 2336.427
Peak 5 2226.209 2301.579 2263.894
Peak 6 585.623 607.988 596.805
Sample 3 Peak 2 44.892 47.234 46.063
Peak 3 163.049 172.173 167.611
Peak 4 5834.212 6082.991 5958.612
Peak 5 5812.727 6041.184 5926.955
Peak 6 1562.537 1697.149 1629.843

d. Amount of FAME in samples.

RF of Peak Area (pA*s) Amount of


corresponding FAME
peak
Sample 1 Peak 2 1.1819 x 10-4 31.8972 3.76x10-3
Peak 3 1.0908 x 10-3 95.0116 0.1036
Peak 4 4.0567 x 10-4 3327.923 1.350
Peak 5 3.4861 x 10-4 3182.865 1.1096
Peak 6 2.4626 x 10-4 793.919 0.1955
Sample 2 Peak 2 1.1819 x 10-4 17.827 2.10x10-3
Peak 3 1.0908 x 10-3 56.845 0.0620
Peak 4 4.0567 x 10-4 2336.427 0.9478
Peak 5 3.4861 x 10-4 2263.894 0.7892
Peak 6 2.4626 x 10-4 596.805 0.1469
Sample 3 Peak 2 1.1819 x 10-4 46.063 5.44x10-3
Peak 3 1.0908 x 10-3 167.611 0.1828
Peak 4 4.0567 x 10-4 5958.612 2.4172
Peak 5 3.4861 x 10-4 5926.955 2.0662
Peak 6 2.4626 x 10-4 1629.843 0.4014

e. Sample calculation:
0.10
Response factor peak 3 in standard = = 1.0908 x 10-3
1375.05994
Amount FAME in peak 3 (sample 1) = 1.0908 x 10-3× 95.0116
= 0.1036
Amount of FAME in peak 3 (sample2) = 1.0908 x 10-3×56.845
= 0.0620
Amount of FAME in peak 2 (sample 3) = 1.0908 x 10-3×167.611
=0.1828

Discussion:

The components in the samples are compared with the standard components by the
retention time. In standard mixture, five peaks are shown as a signal. Therefore, there is five
analytes in the mixture. The amount of FAME in standard is different based on it given . The
response factor for the analytes in standard mixture is calculated. The response factor (RF)
for peak 2 is 1.1819 x 10-4, peak 3 is 1.0908 x 10-3 and for 4 is 4.0567 x 10-4. For peak 5 and
peak 6, the value of RF is 3.4861 x 10-4 and 2.4626 x 10-4 respectively. The Rf of standard is
used to calculate the amount of fame in the samples.

For each of the samples, the average retention time and peak areas are calculated. It will
be more easier to compared the samples with the standard mixture. This is because each
samples have two injection. The peak is chosen by comparing the retention time of sample
with the retention time of standard.

The average retention time for sample 1 is 1.514 min, 1.974 min, 2.871 min, 3.924 min
and 4.058 min while. The average retention time for sample 2 is 1.516 min, 1.975 min, 2.871
min, 3.923 min and 4.061min. The average retention time is 1.515min, 1.976 min, 2.883 min,
3.938 min and4.083 min for the sample 3.

The amount of fame in the sample 1 is 3.76x10-3 ,0.1036 ,1.350, 1.1096 and 0.1955
respectively to the peak. In the sample 2 the amount fame respectively to the peak is 2.10x10-
3
, 0.0620, 0.9478, 0.7892 and 0.1469 and for sample 3 the amount of fame is 5.44x10-3,
0.1828, 2.4172, 2.0662 and 0.4014.

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 indicate all 5 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

References:

1. Nor’Ashikin Saim, Ruziyanti Tajuddin,Mardiana Saaid.(2017) Analytical


Separation Methods Laboratory Guide. 2nd edition. Shah Alam,Selangor:
UiTM Press

2. A., Azhar. (n.d.). FATTY ACID DETERMINATION USING GAS


CHROMATOGRAPHY (GC). Retrieved November 25, 2018, from
https://www.scribd.com/document/249342201/FATTY-ACID-
DETERMINATION-USING-GAS-CHROMATOGRAPHY-GC