EXPERIMENT 3

FATTY ACID DETERMINATION BY USING GAS

CHROMATOGRAPHY (GC)

NAME

: MOHD IQBAL BIN NORAZMAN

STUDENT ID

: 2015840192

GROUP

: AS2452s

PARTNERS NAME

: 1. AKMAL ARSYAD BIN MOHD. RAFFI

2. MUHAMMAD RAHIMI BIN ZAHURI
3. SYARAFUDDIN BIN MOHD SHAMSIBY

DATE OF EXPERIMENT :
DATE OF SUBMISSION

Introduction
Gas chromatography (GC) is an instrumental method for the separation and identification of
compounds. In GC, a liquid or gas sample is introduced into the injection port through the rubber
septum into a stream of inert gas which is called the mobile phase or carrier gas. The gas then
passes through a column which is kept in an oven that controls the column temperature. The
column is packed with stationary phase and when the analytes pass through the column, they will
be separated between the mobile phase and the stationary phase. The analytes which has higher
affinity toward the stationary phase will stay longer in the column which will elute later. While
the analyte which has lower affinity towards the stationaru phase and high affinity toward the
mobile phase will spend less time in the column and tend to pass quickly through the column. At
the end of the column, there is a detector which will detect the analytes quantitatively. The output
is displayed as a chromatogram which is a plot of detector response versus time. In this
experiment, the sample used is margarine or butter which is also known as fat samples. Fats
consist of glycerol esters and long chain aliphatic acids (fatty acids). The back bone of these
compounds contain 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 polysaturated), or they may be fully saturated. The physical or
chemical properties of a fat depend on the composition of the fatty acid mixture. Animal fats
tend to have larger proportion of long chain saturated acids and are solids at room temperature.
Fats from plant sources contain higher proportion of unsaturated acids and are often liquids at
room temperature due to hydrogen bonding. Figure below shows the general glycerol ester
structure. The main objective of this experiment is to introduce a derivatization procedure
routinely used for fat analysis at which non volatile fatty acids are chemically converted into the
corresponding volatile fatty acids methyl ester (FAME) and to determine the amount of FAME in
the derivatized sample. Fatty acids are not sufficiently volatile for the GC analysis, so it needs to
be modified chemically in order to produce new compound which has properties that are suitable
for the analysis. If the unsuitable sample is introduced to the GC analysis, it will causes peak
tailing due to the adsorption of and non specific interaction in the column. In this experiment, the
fatty acids are converted into fatty acid methyl ester (FAME) which is more volatile and suitable
for the GC analysis. The process of conversion of the fats into FAME is conducted by using the

esterification reaction which uses metholic solution catalyst of esterification reagent. The
purpose of this experiment is to study the procedure that is used in the fatty acid analysis at
which nonvolatile fatty acids are chemically converted to the volatile methyl ester and determine
the amount of fatty acid contained in a given sample of fats.

Method
Instrument used: Gas chromatograph (Agilent Technologies 6890N) equipped with flame
ionization detector (FID) and a 30m x 250m x 0.25m HP5-MS capillary column. Procedure to
prepare the fatty acid methyl ester samples from fat samples can be started from three fat
samples that weighed the mass approximately 2 g of fat and the exact weight was recorded. The
samples were transferred into 50mL flask equipped with air condenser. 5mL of 0.5M methanolic
solution was added into the flask and was reflux for about 3 to 4 minutes until the mixture turned
to dissolve. 15mL of esterification reagent was then added into the mixture and it was continued
to reflux for another 3 minutes. The mixture was then transferred into a separatory funnel flask
and 50mL of saturated sodium chloride (NaCl) and 25mL of diethyl ether were added into the
flask. The mixture was the shaken vigorously and the pressure formed was released. The
resulting aqueous layer was discarded. The steps were repeated with another 25mL of saturated
NaCl and the aqueous layer was discarded too. The organic layer was transferred into a screw
cap vial to be analyzed. Instrument was set up by injector port temperature was in split condition
(40:1) while its injector port temperature was 250C. Its carrier gas flow rate was 30mL s -1 and
the detector temperature was 250C. Quantitative analysis of FAME can be started by 0.4L of
standard esters was injected onto the column. The injection was repeated to get the reproducible
peak areas. 0.4L of the derivatized sample was injected onto the column. The injection was
repeated to get the reproducible peak areas. The amount of each fatty acid in the sample was
calculated using the data from the standard ester.

Results and Calculations

A. Response factor (RF) for the analytes in standard FAME:

Amount of FAME in
standard (ppm)

Peak Area

Response Factor (RF)

Peak 2

100

360.38354

0.2775

Peak 3

100

376.79562

0.2654

Peak 4

100

3656.82666

0.0273

Peak 5

100

563.72278

0.1774

Peak 6

100

3131.05029

0.0319

B. Comparison of retention time for standard and samples:

Peak 2
Peak 3
Peak 4
Peak 5
Peak 6

Retention time

Retention time

Retention time

Retention time

for standard

for sample 1

for sample 2

for sample 3

(min)
1.046
1.314
2.032
3.396
3.578

(min)
1.046
1.314
2.036
3.416
-

(min)
1.047
1.315
2.026
3.425
-

(min)
1.048
1.316
2.036
3.432
-

C. Amount of FAME in samples:

RF

Peak Area

Amount of

corresponding

Sample 1

Sample 2

Sample 3

Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6

peak
0.2775
0.2654
0.0273
0.1774
0.0319
0.2775
0.2654
0.0273
0.1774
0.0319
0.2775
0.2654
0.0273
0.1774
0.0319

D. Sample Calculation

Response Factor of peak 2 in standard =

= 0.2775

100
360.38354

FAME (ppm)
158.59912
75.79299
1285.79834
1342.02344
162.59578
68.37989
1016.37018
1030.79163
99.34875
38.72720
501.94928
523.87634
-

44.01
20.12
35.10
238.07
45.12
18.14
27.75
182.86
27.57
10.29
13.70
92.94
-

Amount of FAME in peak 2 (sample 1) = 0.2775 x 158.59912

= 44.01ppm
Amount of FAME in peak 2 (sample 2) = 0.2775 x 162.59578
= 45.12ppm
Amount of FAME in peak 2 (sample 3) = 0.2775 x 99.34875
= 27.57ppm

Discussion
The components in the samples were compared with the standard components by the retention
times. From the retention time of the standard and samples, it was proven that the component 5
(peak 6) did not present in all of the 3 samples because of the difference of the retention time
between the standard and the samples are too big. The amount of each components was different
in each samples might due to the different mass of the fats initially. Peak 5 in each sample give

very large different in the amount of FAME, this may due to the incomplete separation process
during the shaking process or the discarding process. It is necessary to discard little organic layer
to make sure that there was no aqueous layer anymore to be injected into the GC. The condition
also affected by the contaminants in the flask that wasnt clean before using it. The other peaks
that correspond to specific components show small differences that assumed to be correct. There
was another way to derivatize or modified the low volatility fatty acid to more volatile
compound called silylation to yield trimethylsilyl (TMS) ester which more suitable to be
analyzed in GC. When analyzing FAME chromatograph, the ghost peak existed at time 0.911
min. The ghost peak was obtained because not enough sonnicate of sample thus give any
dissolved gas that may interrupt in the GC column and give false reading towards detector. In
recommendation, sonnincation process to remove air bubbles must be done in appropriate and
have enough time to remove completely the air bubbles that could foul the analytical column in
GC

Conclusion
The amount of fame in sample 1,2 and 3 was 44.01ppm, 45.12ppm and 27.57ppm respectively.

References:

Gas Chromatography. (n.d.). Retrieved from

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm
Ichihara, K., Kohsaka, C., Kiyono, T., Tomari, N., Wada, J., & Hirooka, K. (n.d.). Fatty Acid
analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas
chromatography. Retrieved from ResearchGate: http://www.researchgate.net
Saim, N., Tajuddin, R., & Saaid, M. (2014). Experiment 3 Fatty Acid Determination using Gas
Chromatography(GC). In ANALYTICAL SEPERATION METHODS LABORATORY
GUIDE 2nd edition (pp. 5-8). Selangor: UiTM Press.