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EXPERIMENT 1

DETERMINATION OF FATTY ACIDS IN COOKING OIL BY USING GAS


CHROMATOGHRAPHY
OBJECTIVE:
1. To determine the fatty acids contain in cooking oil (corn oil and palm oil)
by using gas

chromatography

2. To learn on how to operate gas chromatography instrument


INTRODUCTION
Gas chromatography is a term used to describe the separation
techniques used to analyses volatile substances in the gas phase. In gas
chromatography, the components of a sample are dissolved in a solvent
and vaporized in order to separate the analytes by distributing the sample
between two phases which are stationary phase and mobile phase such as
helium or hydrogen. The mobile phase is a chemically inert gas that
serves to carry the molecules of the analyte through the heated column.
The liquid stationary phase is adsorbed onto a solid inert packing or
immobilized on the capillary tubing walls in a thin layer. A typical gas
chromatograph consists of an injection port, a column, carrier gas flow
control equipment, ovens and heaters for maintaining temperatures of the
injection port and the column, an integrator chart recorder and a detector.
Fatty Acids are aliphatic carboxylic acid with varying hydrocarbon
lengths at one end of the chain joined to terminal carboxyl (-COOH) group
at the other end. The general formula is R-(CH 2)n-COOH. Fatty acids are
predominantly unbranched and those with even numbers of carbon atoms
between 12 and 22 carbons long react with glycerol to form lipids which is
fat-soluble

components

of

living

cells

in

plants,

animals,

and

microorganisms. The objectives of this experiment are to determine the


fatty acids that contain cooking oil by using gas chromatography
instrument and to learn the operation of gas chromatography itself.

MATERIALS
1. Gas chromatography

6. Methanol

2. Capillary column

7. Pipette

3. Petroleum ether (GC grade)

8. Vortex mixer

4. Standard fatty acids (palmitic acid, stearic acid


5. Sodium hydroxide (NaOH)

9. Glass vial

10. Sample (Corn oil and

Palm oil)
METHODS
A.

Preparation of Sodium Methoxide Reagent


1.15 g of sodium hydroxide was dissolved in 50 mL methanol

B.

Preparation of the Standard


1. 8000 ppm concentration of each fatty acids was prepared by
dissolving 0.08 g in 10 mL petroleum ether
2. The standard prepared was then shake well by using the vortex
mixture
3. 0.4 uL of the sample was injected into the GC injector port. The
present of bubbles was first removed during drawing in the sample
into the syringe and the thumb was positioned over the syringe
plunger to prevents a blowback of the sample by the carrier gas
pressure in the injection port

C.

Preparation of the Sample


1. 0.5 g of sample was weighed into a 20 mL volume glass vial
2. 9.5 mL petroleum ether was added by using a graduated pipette
and the vial was closed and shake well to make sure the sample
was dissolved
3. 0.5 mL of sodium methoxide was added by using glass pipette and
the vial was closed immediately. The vial was then shake vigorously
for 10 seconds by using vortex mixture

4. The mixture was leaved for 20 minutes and the present of clear
upper layer that formed was observed
5. The clear upper layer was then pipette out into a clean and dry vial
for analysis
6. 0.4 uL sample was injected into the GC injector port carefully

RESULTS
A.

Record of retention time and peak area data


Peak Area (cm3)

Retention time (tR)


Standard

TR
TR 1

TR 2

(averag

PA 1

PA 2

1.5635

1057.19

1357.60

1.4000

3715.66

3471.52

e)
Stearic Acid
Palmitic Acid

1.549

1.578

0
1.386

0
1.414

Sample

TR
TR 2

(averag

Acid

Corn Oil
Palm Oil

Palmiti
c Acid

Corn Oil
Palm Oil

1.623

1.622

0
1.583

0
1.619

0
1.419

0
1.416

0
1.371

0
1.414

1.6225
1.6010
1.4175
1.3925

5
3593.59

PA 1

PA 2

PA
(averag
e)

1287.08

2026.48

1656.78

84
5554.63

80
7928.75

82
6741.69

18

64
1189.68

41
973.178

3744.61

74
6158.25

1
4951.43

21

68

45

e)
Stearic

1207.39

Peak Area (cm3)

Retention time (tR)


TR 1

PA
(average
)

3040.95

CALCULATION
Response Factor
Response Factor (Rf) = Concentration / Peak Area
1. Stearic acid methyl ester
Response Factor (Rf)

= 8000 ppm / 1207.395 cm2

= 6.6258
2. Palmitic aid methyl ester
Response Factor (Rf)

= 8000 ppm / 3593.59 cm2

= 2.2262
Concentration
Concentration (ppm) = Retention Factor (Rf) x Peak Area (cm2)
1. Palm Oil
a) Stearic acid methyl ester
Concentration (ppm) = 6.6258 x 6741.6941
= 44669.12 ppm
b) Palmitic acid methyl ester
Concentration (ppm) = 2.2262 x 4951.4345
= 11022.88 ppm

2. Corn Oil
a) Stearic acid methyl ester
Concentration (ppm) = 6.6258 x 1656.78821
= 10977.55 ppm
b) Palmitic acid methyl ester
Concentration (ppm) = 2.2262 x 973.1781
= 2166.49 ppm

DISCUSSION
Through this experiment, the fatty acids contained in cooking oils
which are palm oil and corn oil was determined by using gas
chromatography. The standard (palmitic acid methyl ester and stearic acid
methyl ester) was prepared first into desired concentration which is 8000
ppm before it was injected to GC injector port. The samples was then
prepared appropriately and leaved for 20 minutes until clear upper layer
was formed before it was also injected to GC injector port. The retention
time and peak area data was then recorded and the result was analysed.
From the results, the retention time of standard fatty acids was
compared with unknown samples (corn oil and palm oil) to identify types
of fatty acid present. Retention time plays big role in analysing the sample
in the gas chromatography. According to Wirasnita et. al, (2013) retention
time are differentiated according to the number of carbon, molecular
weight and the boiling point of the samples. The retention time will
increase when the molecular weight and boiling point of the compound
increased.
Based on the retention times (t R) data, the average tR for stearic
acid is 1.5635 meanwhile palmitic acids average t R is 1.4000. Meanwhile,
the average tR value of corn oil and palm oil is 1.6225 and 1.6010

respectively. This result shows the nearest tR value to the stearic acid
methyl ester standard (1.5635). In contrast, the others average t R value
for corn oil and palm oil are 1.4175 and 1.3925 respectively. This data
shows the nearest tR value to palmitic acid methyl ester standard
(1.4000). Both data shows that samples contain both standard which are
stearic acid methyl ester (C18) and palmitic acid methyl ester (C16).
Meanwhile, response factor (Rf) for each standard was calculated
which are 6.6258 for stearic acid methyl ester and 2.2262 for palmitic acid
methyl ester. The response factors were calculated by using this formula:Response Factor (Rf) = Concentration / Peak Area
The response factor for each standard was then used to calculate the
concentration of the appropriate fatty acids in the samples (corn oil and
palm oil) by using this formula:Concentration (ppm) = Retention Factor (Rf) x Peak Area (cm2)
From the results obtained, the concentration of palmitic acid in corn
oil and palm oil are 2166.49 ppm and 11022.88 ppm respectively.
Meanwhile, the concentration of stearic acid in corn oil and palm oil are
10977.55 ppm and 44669.12 ppm respectively. These results show that
palm oil and corn oil contain higher number of stearic acid than palmitic
acid. In addition, corn oil also show that it contain higher number of
stearic acid contain than palm oil. These results were not corresponding
with the actual result that was based on studies. This was maybe due to
several errors during preparation. According to Zambiaki et. al (2010),
palm oil should contain a lot of palmitic acid (C16) than stearic acid (C18)
as the peak area average data of palmitic acid in palm oil has the highest
value than in corn oil which results in higher concentration of fatty acid
through the concentrations calculation.
During the experiment, they are some sources of error that occur
which may affect the accuracy of the result. For example, there are
parallax error that happen when the eyes does not directly proportional to

the reading of the syringes measurement. The present of bubbles during


drawing in the sample into the syringe might also interrupt the GC
readings. The preparation of standard might be contaminated. Besides,
three replications of readings by using GC instrument were not taken as
only two data was used which may contribute to the error of the handler.
To minimize these errors various precautions should be taken such
as, make sure our eyes are directly proportional during reading the
measurement in the syringe, the syringe was rinsed and cleaned by using
pet ether before and after each sample was used to prevent any
contaminant from others solution that will affect the data obtained and
the formation of bubbles was avoided from affecting the reading in the
column. To add on, the standard should be freshly prepared. Lastly, three
consecutive of measuring need to be carried out to eliminate the errors of
the handlers.
CONCLUSION
In conclusion, the fatty acids contained in cooking oil (corn oil and palm
oil) were determined which are stearic acid and palmitic acid by using gas
chromatography. Palm oil and corn oil also was found to contain a lot of
stearic acid than palmitic acid.
REFERENCE
R. Wirasnita, T. Hadibarata, Y. Maria Novelina, A.Y. Rahim, and Z. Yusop,
(2013), A Modified

Methylation

Content by Gas Chromatography,

Method
Retrieved

to

Determine

April

6,

Fatty

2013

Acid

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file:///C:/Users/user/Downloads/Modified%20Methylation%20Process
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etermination%20of%20Fatty%20Accid.pdf

R. C. Zambiazi, R. Przybylski, M. W. Zambiazi and C. B. Mendonca, (2010),


Fatty Acid Composition of

Vegetable Oils and Fats, Retrieved Jun 25,

2010 from http://www.nononsensecosmethic.org/wpcontent/uploads/2015/01/fatty-acid-oil-

composition.pdf

Vesna K. , S. Memeti and B. Bauer, (2009), Fatty Acid Composition of


Edible Oils and Fats, Journal of Hygienic Engineering and Design, Retrieved
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http://www.jhed.mk/filemanager/JHED

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