FACULTY OF APPLIED

SCIENCES LABORATORY
REPORT

INSTRUMENTAL ANALYSIS OF FOOD

FST 606

TITLE OF DETERMINATION OF FATTY ACIDS IN COOKING

EXPERIMENT OIL BY GAS CHROMATOGRAPHY

NO. EXPERIMENT 3

NUR SYAZA HANI BINTI NICK HAMASHOLDIN

STUDENT’S NAME (2018653554)

GROUP AS246 5B

DATE OF
EXPERMENT 02/11/2020

SUBMISSION DATE 09/11/2020

LECTURER’S NAME DR SO’BAH AHMAD

INTRODUCTION

According to Abidin et al. (2013), there are several analytical methods that can be used
for the identification and quantification of a component in fats and oil. Gas chromatography is
one of the popular techniques to allow the identification and quantification of individual
components in fats and oils. Theoretically, it is possible to analyse some foods by direct
injection of the food into a gas chromatography. Direct injection is sometimes used to
determine oxidation in vegetable oils. Since vegetable oils are reasonably thermally stable and
free of water, this method is particularly well suited to oil analysis (Qian et al., 2017). Besides,
this well-established method is an integral tool in the characterization of authenticity and
nutritional value of food. One of the most common detectors for gas chromatography is the
Flame Ionization Detector (FID).

In terms of its operating principles, the compounds eluted from the analytical column
will burned in a hydrogen flame where it carries a current across the potential which is
proportional to the organic ions present in the flame from the burning of an organic compound.
The current flowing across the flame is amplified and recorded. The FID responds to organics
on a weight basis and gives the best response with compounds containing C-C or C-H bonds.
The output of the detector will be recorded as a series of peaks where each one representing a
compound in the mixture passing through the detector. The retention times obtained can be
used to identify the compounds present in a sample and the areas under the peaks are
proportional to the amount of each compound which has passed the detector, and these areas
can be calculated automatically by the computer linked to the display (Clark, 2016).

As for applications, most food analyst often working with organic compounds, to which
this detector responds well. This detector is a great choice for most food work due to its very
good sensitivity, fast, easy to conduct, wide linear range in response, and dependability make.
Thus, this detector is used for virtually all food analyses for which a specific detector is not
desired. This includes flavour studies, fatty acid analysis, carbohydrate analysis, sterols,
contaminants in foods, and antioxidants (Qian et al., 2017).
OBJECTIVES
1. To determine the type and concentration of fatty acids presence in cooking oils using
gas chromatography.
2. To understand the principles of gas chromatography for chemical separation in food
samples.
APPARATUS AND REAGENTS

• Gas chromatography • Methanol

(with flame ionisation detector) • Pipette
• Capillary column • Vortex mixer
• Petroleum ether (GC grade) • Glass vials
• Standard fatty acids (oleic acid, palmitic acid)) • Sample: Cooking oil of different
• Sodium hydroxide (NaOH) brands

PROCEDURE

1. Preparation of Sodium Methoxide Reagent:

a) 1.15 g of sodium hydroxide had dissolved in 50 mL methanol and mixed well by
inverting the flask.
2. Preparation of the Standard:
a) 8000 ppm concentration of each fatty acids provided were prepared by dissolving
0.08 g (solid) or 80 µL (liquid) standards in 10 mL petroleum ether.
b) The solution was shaken well using the vortex mixer.
c) 0.4 µL of the sample was then injected into the GC injector port. When injecting a
sample, a thumb was positioned over the syringe plunger to prevent a blowback of
the sample by the carrier gas pressure in the injection port.
3. Preparation of the Sample:
a) 0.5 g of sample had weighed into a 20 mL volume glass vial.
b) 9.5 mL petroleum ether was added using a graduated pipette. The vial had closed
and shaken well to dissolve the sample.
c) 0.5 mL sodium methoxide reagent was added to the vial using a glass pipette. The
vial had immediately closed and was shaken vigorously for 10 seconds using the
vortex mixer.
d) The mixture was left for 20 minutes until a clear upper layer is formed.
e) The clear upper layer had pipetted out into a clean and dry vial for analysis.
f) 0.4 µL of the sample was injected into the GC injector port. The previous oil sample
had completely made sure it was eluted before second sample is injected.
PROCEDURE

1. Preparation of Sodium Methoxide Reagent

2. Preparation of the Standard

3. Preparation of the Sample
RESULTS

Table 1: Retention time for standards and samples

Retention Time (tR)

Standard/Sample tR
tR 1 tR 2
average ± std dev
Palmitic acid 1.419 1.395 1.407±0.017
Oleic acid 1.669 1.659 1.664±0.007

Palmitic acid 1.439 1.394 1.417± 0.032

Palm oil
Oleic acid 1.666 1.634 1.650±0.023

Palmitic acid 1.432 1.428 1.430±0.003

Olive oil
Oleic acid 1.618 1.625 1.622±0.005

Table 2: Peak area for standards and samples

Peak Area (PA)

Standard/Sample
PA
PA 1 PA 2
average ± std dev
Palmitic acid 5159.9087 6053.4644 5606.6866± 631.8393

Oleic acid 4077.4163 3842.1492 3959.7828 ± 166.3590

Palmitic acid 3114.2244 6558.0728 4836.1486 ± 2435.1686

Palm oil
Oleic acid 1990.0280 6867.3818 4428.7049 ± 3448.8099

Palmitic acid 2613.6567 2642.5564 2628.1066 ± 20.4352

Olive oil
Oleic acid 14015.1000 12937.8000 13476.4500 ± 761.7661
CALCULATIONS

A) Preparation of Standard (Palmitic Acid and Oleic Acid)

0.08 𝑔 1000 𝑚𝑔 1000 𝑚𝐿

Concentration (solid) = × ×
10 𝑚𝐿 1𝑔 1𝐿

= 8000 mg/mL
= 8000 ppm

80 µ𝐿 1 𝑚𝑔 1000 𝑚𝐿
Concentration (liquid) = × ×
10 µ𝐿 1𝑔 1𝐿

= 8000 mg/mL
= 8000 ppm

B) Average and Standard Deviation Retention Time (tR) for Standards and Samples.

Average retention time (tR average) Standard deviation

t R1 + t R 2
=
2 n -1
1.608 + 1.626
=
2
= 1.617Acid
Standard Palmitic
1.419 + 1.395
Average retention time (tR average) = = 1.407
2

(1.419−1.407)2 + (1.395−1.407)2
Standard deviation = √
2−1

= 0.017
Average±SD = 1.407±0.017
Standard Oleic Acid
1.669 + 1.659
Average retention time (tR average) = = 1.664
2

(1.669−1.664)2 + (1.659−1.664)2
Standard deviation = √
2−1

= 0.007
Average±SD = 1.664±0.007

Sample: Palm Oil

(Palmitic Acid)
1.439 + 1.394
Average retention time (tR average) = = 1.417
2

(1.439−1.417)2 + (1.394−1.417)2
Standard deviation = √
2−1

= 0.032
Average±SD = 1.417± 0.032

(Oleic Acid)
1.666 + 1.635
Average retention time (tR average) = = 1.650
2

(1.666−1.650)2 + (1.635−1.650)2
Standard deviation = √
2−1

= 0.023
Average±SD = 1.650±0.023
Sample: Olive Oil
(Palmitic Acid)
1.432 + 1.428
Average retention time (tR average) = = 1.430
2

(1.432−1.430)2 + (1.428−1.430)2
Standard deviation = √
2−1

= 0.003
Average±SD = 1.430±0.003

(Oleic Acid)
1.618 + 1.625
Average retention time (tR average) = = 1.622
2

(1.618−1.622)2 + (1.625−1.622)2
Standard deviation = √
2−1

= 0.005
Average±SD = 1.622±0.005
C) Average and Standard Deviation Peak Area (PA) for Standards and Samples.

Average Peak Area (PA average) Standard deviation

PA1 + PA2
=
2
n -1
3441.75415 + 3627.74756
=
2
Standard Palmitic Acid
= 3534.750855 5159.9087 + 6053.4644
Average Peak Area (PA average) = = 5606.6866
2

(5159.9087−5606.6866)2 + (6053.4644−5606.6866)2
Standard deviation = √
2−1

= 631.8393
Average±SD = 5606.6866± 631.8393

Standard Oleic Acid

4077.4163 + 3842.1492
Average Peak Area (PA average) = = 3959.7828
2

(4077.4163−3959.7828)2 + (3842.1492− 3959.7828)2

Standard deviation = √
2−1

= 166.3590
Average±SD = 3959.7828 ± 166.3590

Sample: Palm Oil

(Palmitic Acid)
3114.2244 + 6558.0728
Average Peak Area (PA average) = = 4836.1486
2

(3114.2244−4836.1486)2 + (6558.0728−4836.1486)2
Standard deviation = √
2−1

= 2435.1686
Average±SD = 4836.1486 ± 2435.1686
(Oleic Acid)
1990.0280 +6867.3818
Average Peak Area (PA average) = = 4428.7049
2

(1990.0280−4428.7049 )2 + (6867.3818−4428.7049 )2
Standard deviation = √
2−1

= 3448.8099
Average±SD = 4428.7049 ± 3448.8099

Sample: Olive Oil

(Palmitic Acid)
2613.6567 + 2642.5564
Average Peak Area (PA average) = = 2628.1066
2

(2613.6567−2628.1066 )2 + (2642.5564−2628.1066 )2
Standard deviation = √
2−1

= 20.4352
Average±SD = 2628.1066 ± 20.4352

(Oleic Acid)
14015.1000 + 12937.8000
Average Peak Area (PA average) = = 13476.4500
2

(14015.1000−13476.4500 )2 + (12937.8000−13476.4500 )2
Standard deviation = √
2−1

= 761.7661
Average±SD =13476.4500 ± 761.7661
D) Response factor for each of the standard fatty acids:

Response Factor (Rf) = Concentration / Peak Area

Standard Palmitic Acid

8000 ppm
Rf =
5606.6866
= 1.4269
Standard Oleic Acid
8000 ppm
Rf =
3959.7828
= 2.0203

E) Concentration of fatty acids in the samples:

Concentration = Response Factor (Rf) × Peak Area

Sample: Palm Oil

Concentration Palmitic Acid = 1.4269 × 4836.1486

= 6900.7004 ppm

Concentration Oleic Acid = 2.0203 × 4428.7049

= 8947.3125 ppm

Sample: Olive Oil

Concentration Palmitic Acid = 1.4269 × 2628.1066

= 3750.0453 ppm

Concentration Oleic Acid = 2.0203 × 13476.4500

= 27226.4719 ppm
DISCUSSION

In this experiment, two standard fatty acids which are palmitic acid and oleic acid were
used with their known concentration of 8000 ppm. In this experiment, two samples of different
cooking oils (palm oil and olive oil) were tested for determining their unknown concentration
and the type of fatty acids presence inside of them. According to the results tabulated in Table
1, the average retention time (tR) for standard palmitic acid is 1.407±0.017 whereas the average
retention time (tR) for standard oleic acid is 1.664±0.007. As for palm oil sample, the average
retention time (tR) obtained for both palmitic acid and oleic acid are 1.417± 0.032 and
1.650±0.023 respectively. Meanwhile for olive oil sample, the average retention time (tR)
obtained for palmitic acid and oleic acid are 1.430±0.003 and 1.622±0.005 respectively. From
here, it can be said that oleic acid has higher retention time compared to palmitic acid in both
standards and cooking oil samples.

According to Clark (2017), retention time is the time taken for a particular compound
to travel through the column to the detector. He mentioned that different compounds have
different retention times and for a particular compound, the retention time will vary depending
on the boiling point of the compound, the solubility in the liquid phase and the temperature of
the column. A compound with high boiling point and high solubility in liquid tends to have
higher retention time and a high column temperature will shortens retention times. From the
results in Table 1, oleic acid has higher retention time compared to palmitic acid in standards
and samples which agrees to the findings since oleic acid has higher boiling point at 360°C
compared to palmitic acid with boiling point of 351°C (National Center for Biotechnology
Information, 2020). Furthermore, the FID responses are proportional to the number of carbon
atoms or molecular weight where in this case, oleic acid has higher number of carbons that is
18 compared to palmitic acid which has 16 carbon atoms. In order to determine which type of
fatty acids presence in the cooking oils, the average retention time (tR) of fatty acids from the
samples were compared to the ones obtained from the standards. Based on the results obtained,
there were presence of palmitic acid and oleic acid in both palm oil and olive oil since the
retention time in both samples were similar and insignificant to the standard fatty acids.

Besides, the peak area (PA) was also recorded for each standards and samples in Table
2. From this table, the average peak area (PA) obtained from standard palmitic acid and oleic
acid were 5606.6866±631.8393 and 3959.7828±166.3590 which shows that palmitic acid has
higher peak area compared to oleic acid. As for palm oil sample, the average peak area obtained
for both palmitic acid and oleic acid are 4836.1486±2435.1686 and 4428.7049±3448.8099
respectively. In olive oil sample, the average peak area obtained for palmitic acid and oleic
acid were 2628.1066±20.4352 and 13476.4500±761.7661 respectively. Then, the response
factor (Rf) for each of the standard fatty acids have been calculated by using their known
concentration which is 8000ppm divided by their respective peak area obtained earlier. It was
calculated that the response factor, Rf for standard palmitic acid and oleic acid were 1.4269 and
2.0203 respectively. The response factor obtained was then used to calculate the unknown
concentration for each fatty acid in palm oil and olive oil. In palm oil, the concentration of
palmitic acid was 6900.7004 ppm whereas the concentration oleic acid was 8947.3125 ppm.
In olive oil, the concentration of palmitic acid was 3750.0453 ppm whereas the concentration
oleic acid was 27226.4719 ppm.

Based on that calculation, the concentration of oleic acid in palm oil is higher than the
concentration of palmitic acid. Same goes to olive oil, the concentration of oleic acid obtained
was significantly higher compared to the concentration of palmitic acid. The concentration of
oleic acid in olive oil is same to what is expected, however, the concentration of palmitic acid
in palm oil somehow lower than the expectation. This is because based on a finding by Ali and
Abdurrhman (2013), palmitic acid is the major saturated fatty acid in palm oil and this balanced
by almost 39% monounsaturated oleic acid and 11% polyunsaturated linoleic acid.

From this experiment, there are errors that could possibly contributes in the experiment
such as contamination of the column and injection port by non-volatile materials, some aqueous
layer was still presence in the organic compound or the occurrence of thermal degradation of
non-volatile food constituents in the injector. Therefore, some precautionary steps are needed
to take into consideration when conducting the experiment. First, since gas chromatography
(GC) involves the use of high temperature, it is very important to make sure that there are no
leaks in the hydrogen gas line every time when the instrument is being used to avoid
unnecessary accident. Next, it is necessary to discard little organic layer to make sure that there
is no aqueous layer to be injected into the gas chromatography to increase the efficiency
separation of the compounds. In addition, during the manual injection, it is better to inject the
samples and pressing the start button simultaneously since it can affect the running time.
CONCLUSION

In conclusion, this study has successfully developed a simple, fast and reliable method
for determining the type and concentration of fatty acids in cooking oils. There were two
components present in both palm oil and olive oil which are palmitic acid and oleic acid. The
average retention time, tR and peak area, PA for each standards and samples were recorded in
Table 1 and Table 2 respectively. The response factor, Rf obtained for standard palmitic acid
and standard oleic acid were 1.4269 and 2.0203 respectively. Besides, the concentration of both
samples was also determined by calculating the response factor and peak area obtained.
Therefore, the concentration of palmitic acid and oleic acid in palm oil were 6900.7004 ppm
and 8947.3125 ppm respectively whereas, the concentration of palmitic acid and oleic acid in
olive oil were 3750.0453 ppm and 27226.4719 ppm.
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doi: 10.1002/cjce.21848

2. Ali, A. S. M., & Abdurrhman, A. M. (2013). Determination of free fatty acids in palm
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