BACHELOR OF SCIENCE (HONOR) APPLIED CHEMISTRY (AS245)

ANALYTICAL SEPARATION METHOD

CHM510

EXPERIMENT 3: Fatty Acid Determination using Gas

Chromatography (GC)

NAME NURANISAH NADIAH BINTI MOHD NIZAM

STUDENT ID 2022855638

GROUP MEMBERS
1) NUR AMIRAH BALQIS BINTI MOHD KOSIM
(2022800388)
2) AINUL SYARAFANA BINTI SHAMSUL HAYAT
(20224942820)
3) NURATHIRAH NABIHAH BINTI SAIRI (20224942820)
4) NUR HIDAYAH MOHD NAJIB (2022626786)

CLASS AS2452S1

NAME OF LECTURER DR WAN NAZIHAH WAN IBRAHIM

a. Introduction
Both free fatty acids and those found in complex lipids play crucial roles in metabolism as main
metabolic fuel (energy storage and transit), necessary elements of all membranes, and gene
regulators. Additionally, dietary lipids contain polyunsaturated fatty acids (PUFAs), which are
the basic building block for the potent locally acting metabolites known as eicosanoids. Fatty
acids are crucial for mechanical protection as well as thermal and electrical insulation because
they are a component of complex lipids. Furthermore, due to their amphipathic characteristics
and the creation of micelles, free fatty acids and their salts may serve as detergents and soaps.

Most of these fatty acids are liquid at room temperature in terms of physical state. The presence
of saturation or unsaturation is largely correlated with the various qualities. In general, liquid
oils show a high concentration of unsaturated fatty acids, while solid fats are indicated by a
predominance of saturated fatty acids.

Esterification

When an alcohol and a carboxylic acid react with the help of a mineral acid catalyst like sulfuric
acid, it is known as an esterification. These reactions produce an equilibrium mixture of both
products and reactants, hence changing the reaction conditions is necessary to get a good yield.

b. Procedure

• Preparation of fatty acid methyl ester samples from fat samples

1. 2 g of oil or fat was weighed out approximately and the exact weight was recorded.
2. The sample was transferred into a 50 mL flask equipped with air condenser.
3. 5 mL of 0.5 M methanolic solution was added and refluxed for 3-4 minutes.
4. 15 mL of esterification reagent was added and refluxed for 3 minutes.
5. The mixture was transferred into a separatory flask. 50 mL of saturated NaCl and 25
mL of diethyl ether were added. The mixture was shaken vigorously for 2 minutes and
the aqueous layer was discarded.
6. Steps 1-5 were repeated with another 25 mL of saturated NaCl and again the aqueous
layer was discarded.
7. The organic layer was transferred into a screw cap vial. Make sure that only the
organic layer is injected into the GC as water can ruin the GC column.

• Instrument set-up (may vary depending on instrument):

Injection port: Split (40:1)

Injection port temperature: 250˚C
Column temperature: Ram:190oC (1min) to 210˚C (5min) at 30˚C min-1; 210˚C to
230˚C at 30oC min-1
Carrier gas flow rate: 70 cm sec-1
Detector temperature: 250˚C

• Quantitative analysis of FAME

1. 0.4 μL of standard esters was injected onto the column. The injection was
repeated to get reproducible peak areas.
2. 0.4 μL of the derivatized sample was injected. The injection was repeated to
get reproducible peak areas.
3. The amount of each fatty acid in the sample was calculated using the data from
the standard esters,

• Alternate Method for Derivatization of Fatty Acids

1. 500 mg of fatty acid sample was combined with 5 mL of boron trifluoride-

methanol (BF3-methanol) in a 50 ml volumetric flask.
2. The mixture was heated on a steam bath for 5-10 minutes.
3. Enough saturated NaCl solution was added to raise the fluid level in the flask
well into the neck.
4. The flask was capped and mixed well by repeatedly inverting the flask.
5. The organic phase was allowed to collect in the neck of the flask, and remove it
from the top of the aqueous phase (bottom layer).
6. The hexane extracts were dried over anhydrous sodium sulphate and transferred
to a clean, dry container.
 7. The hexane layer was analysed by gas chromatography directly or, if
concentration is desirable, evaporate the hexane and analyse the residue.

c. Data and results

1. Response factor (RF) for analyte in standard FAME

Peak No. of Amount of Peak Area Average Response
injection FAME in (pA*s) Peak Area Factor (RF)
standard (pA*s)
(ppm)

2 1 100 331.87665 328.5025 3.285025

2 100 325.12839
3 1 100 331.41095 327.0778 3.270778
2 100 322.74472
4 1 100 1714.39587 1685.2022 16.85202
2 100 1656.00854
5 1 100 929.86871 924.0413 9.240413
2 100 918.21381
6 1 100 1225.56799 1218.7965 12.187965
2 100 1212.02502

2. Comparison of retention time for standard and samples

Peak No. of Retention Average Retention Average Retention Average Retention Average
injection time of Retention time of Retention time of Retention time of Retention
standard time of sample 1 time of sample 2 time of sample 3 time of
(min) standard (min) sample (min) sample 2 (min) sample 3
(min) (min) (min) (min)
2 1 1.197 1.197 1.197 1.196 1.234 1.234 1.195 1.195
2 1.197 1.195 1.233 1.195
3 1 1.936 1.937 1.683 1.680 1.680 1.679 1.676 1.677
2 1.937 1.677 1.677 1.677
4 1 2.677 2.677 2.666 2.663 2.666 2.663 2.660 2.662
2 2.677 2.660 2.659 2.663
5 1 3.964 3.965 3.993 3.987 3.986 3.985 3.981 3.982
2 3.965 3.981 3.983 3.983
6 1 4.170 4.172 4.155 4.153 4.155 4.151 4.150 4.152
2 4.173 4.150 4.147 4.154
3. Amount of FAME in sample 1
Sample Peak No of RF of Peak Area Average Amount
injection corresponding (pA*s) peak area of
peak (pA*s) FAME
(ppm)

1 2 1 3.285025 5.55587 5.44751 1.66

2 5.33915
3 1 3.270778 19.16391 18.30820 5.60
2 17.45249
4 1 16.85202 845.83264 800.79236 47.52
2 755.75208
5 1 9.240413 1086.95886 1039.54443 112.50
2 992.13000
6 1 12.18797 192.38061 176.95332 14.52
2 161.52602

Amount of FAME in sample 2

Sample Peak No of RF of Peak Area Average Amount
injection corresponding (pA*s) peak area of
peak (pA*s) FAME
(ppm)
2 2 1 3.285025 11.78556 11.63817 3.54
2 11.49078
3 1 3.270778 14.4723 14.15005 4.33
2 13.82779
4 1 16.85202 663.16113 652.11139 38.70
2 641.06165
5 1 9.240413 1128.0763 1097.47833 118.77
2 1066.8804
6 1 12.18797 125.31517 129.53254 10.63
2 133.74991

Amount of FAME in sample 3

Sample Peak No of RF of Peak Area Average Amount
injection corresponding (pA*s) peak area of
peak (pA*s) FAME
(ppm)
3 2 1 3.285025 7.38562 7.62273 2.32
2 7.85984
3 1 3.270778 29.18389 29.00590 8.87
2 28.8279
4 1 16.85202 1356.1748 1365.62183 81.04
 2 1375.0689
5 1 9.240413 1817.5253 1828.20862 197.85
2 1838.892
6 1 12.18797 219.18767 219.81935 18.04
2 220.45102

Calculation

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴

Sample calculation for response factor = 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑜𝑜𝑜𝑜 𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝑖𝑖𝑖𝑖 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠

328.5025
• Response factors peak 2 in standard mixture = 100

= 3.285025

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴

Sample calculation for amount of FAME in sample = 𝑅𝑅𝑅𝑅

11.63817
• Amount of FAME in sample 1, peak 2 = 3.285025

= 3.54ppm
d. Discussion

Comparing the components in the samples to the standard components is done using retention
time. Depending on whether the fat was originally one mass or several, each sample contains
a different amount of each component. Peak 5 in each sample varies greatly in terms of the
amount of FAME; this may be because the separation process was not fully completed during
the shaking operation or the discarding process. A little portion of the organic layer must be
removed to make sure that the GC does not need an additional aqueous layer to be injected on
top of it. Also contributing to such condition were the contaminants in the flask, which had not
been fully cleaned before use.

e. Conclusion

In this experiment, the derivatization technique of esterification was used to convert non-
volatile fatty acids into the more volatile fatty acid methyl ester (FAME). It can be observed
from comparing the retention durations that the usual combination comprises 5 components,
but the 3 samples only show 3 components. The concentration of each component is calculated
using the response factor of the standard.

f. Reference

• Gas Chromatography Mass Spectrometry (GCMS). (n.d.)

.https://www.shimadzu.eu.com/sites/shimadzu.seg/files/SEG/GCMSBASIC.pdf

• Chromatography - LABORATORY REPORT - Studocu. (n.d.). Studocu. Retrieved

January 26, 2023, from https://www.studocu.com/my/document/universiti-teknologi-
mara/food-analysis/practical/lab-5-identification-and-determination-of-fatty-acids-
using-gas-chromatography/10574485/view