Experiment 3: Fatty Acid Determination using Gas

Chromatography (GC)

NAME: NADIRAH BINTI ZAKARIA

ID: 2016586557

PARTNERS’ NAME:

1. NUR SYAZWANI BINTI MUHAMMAD YAZID (2016596153)

2. SITI ZAHARIAH BINTI ZAHARUDIN (2016552077)
3. WAN NURUL IHSAN BINTI WAN ZAINUDDIN (2016552007)

DATE EXPERIMENT CONDUCTED: 7 JUNE 2017

DATE EXPERIMENT SUBMITTED: 19 JUNE 2017

LECTURER: MADAM ROHAIZA MOHAMAD

Experiment 3: Fatty Acid Determination using Gas Chromatography (GC)

Objective:

1. To introduce a derivatization procedure routinely used for fat analysis in which non-volatile fatty

acids are chemically converted to the corresponding volatile methyl ester (FAME)

2. To determine the amount of FAME in the derivative samples.

Introduction:

In this experiment, we used Gas Chromatography (GC) to separate the analytes that is volatile

and chemically stable. The principle of GC is the sample solution injected into the instrument enters

a gas stream which transports the sample into a separation tube known as the "column." (Helium or

nitrogen is used as the so-called carrier gas.) The various components are separated inside the column.

The column is kept at a control temperature inside an oven, so that the mixture remains in vapor form

to be eluted. The detector is maintaining at a higher temperature than the column so that all analytes

will be gaseous. From the time the mixtures are injected until they reach the detector, they are being

retained by the liquid in the column (the stationary phase).

As the fatty acids not sufficiently volatile for GC analysis, it needs to modify chemically to

produce new compound that has properties that are suitable for analysis. If the unstable and unsuitable

compound is injected into GC analysis, it tends to cause peak tailing due to adsorption and non – specific

interaction with the column. In this experiment, the fatty acid is changed to fatty acid methyl ester

(FAME) that is more volatile and suitable for GC analysis by using esterification reagent.

Apparatus:

Beaker, volumetric flask, pipette and vial.

Reagents and solutions:

a. Individual FAME in the following concentration:

FAME Concentration
1. Methyl laurate C12:0 0.10 mg mL-1
2. Methyl myristate C14:0 0.10 mg mL-1
3. Methyl palmitate C16:0 1.50 mg mL-1
4. Methyl stearate C18:0 0.70 mg mL-1
5. Methyl linoleate C18:2 0.35 mg mL-1

b. Methanolic solution (0.5 M): NaOH in methanol.

c. Saturated NaCl.

d. Analytical grade diethyl ether.

e. Esterification reagent: 7.5 mL H2SO4 and 5 g of NH4Cl is added in a 500 mL flask and refluxed for

15 minutes.

Sample:

Oil or fat samples (margarine or butter) – 10 g

Procedure:

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

1. 2 g of oil or fat is approximately weighed out and the exact weight is recorded.

2. The sample is transferred into a 50 mL flask equipped with air condenser.

3. 5 mL of 0.5 M methanolic solution is added and refluxed for 3 – 4 minutes.

4. 15 mL of esterification reagent is added and then refluxed for 3 minutes.

 5. The mixture is transferred into a separatory flask and then 50 mL of saturated NaCl and 25

mL of diethyl ether is added. Vigorously shake the mixture for 2 minutes and the aqueous

layer is discarded.

6. Step 5 is repeated with another 25 mL of saturated NaCl and the aqueous layer is once again

discarded.

7. The organic layer is transferred into a screw vial cap. Make sure that only the organic layer is

injected into the GC as water can ruin the GC column.

NOTE: You may want to run your calibration standards while the esterification reaction is in progress.

b. Instruments set – up (may vary depending on instrument):

Injection port : Split (40:1)

Injection port temperature : 250˚c

Column temperature : 100˚c to 290˚c at 40˚c min-1

Carrier gas flow rate : 30 mL s-1

Detector temperature : 250˚c

c. Qualitative analysis of FAME:

1. 0.4 µL of standard esters is injected into the column. The injection is repeated to get

reproducible peak areas.

2. 0.4 µL of the derivatized sample is injected. The injection is repeated to get reproducible

peak areas.

3. The amount of each fatty acid in the sample is calculated using the data from standard

esters.
Results:

A. Retention time for standard:

Compound Retention time (tR) Width (Wb)

Methyl Laurate 3.994 0.0239
Methyl Myristate 4.543 0.0258
Methyl Stearate 4.222 0.0705
Methyl Linoleate 4.543 0.0222

B. Resolution:

1. Methyl Laurate and Methyl Myristate

2. Methyl Myristate and Methyl Stearate

3. Methyl Stearate and Methyl Linoleate

C. Retention time for compound in the sample of Fatty Acid:

Peak Compound Retention time (tR) Resolution

1 Methyl Laurate 3.750 Peak 1 & 2 = 22.0926
Peak of Methyl
2 Methyl Myristate 4.545 Myristate and Methyl
Stearate = 6.6667
Methyl Stearate +
3 4.206 Peak 3 = 6.9256
Methyl Linoleate
Discussion:

In order to obtain required accuracy and precision, each of these steps has to be optimized.

Esterification of lipids can be carried out with several reagents based on acid-catalysed or base-catalysed

reactions. To prepare the sample solution of fatty acid Methyl Esters, 2.1200 g of oil is weighed and the

exact weight is recorded. The sample is transferred into 50mL flask equipped with air condenser. 5mL of

0.5M methanolic solution is added and refluxed for 4 minutes. Next, 15mL of esterification reagent is

added and again refluxed for 3 minutes. The mixture transferred into separatory flask. 50mL of

saturated NaCl and 25mL of diethyl ether are added. The mixture is shaken vigorously for 2 minutes and

the aqueous layer is discarded. The organic layer that is formed will be transferred into a small screw

cap vial.

The fatty acids were identified by lining up the solvent peak of standard to the chromatogram

that is trying to identify. The retention times of standard for peaks of Methyl Laurate, Methyl Myristate,

Methyl Stearate and Methyl Linoleate are 3.994, 4.543, 4.222 and 4.543 respectively. Meanwhile, the

retention time for sample of Methyl Laurate, Methyl Myristate and Methyl Stearate + Methyl Linoleate

are 3.750, 4.545 and 4.206 respectively. The retention times observed for sample was not quite match

the peak of standard due to technique of sample preparation.

The peaks obtained may be good and poor based on the preparation techniques. In the results,

the overlapping peaks are appeared due to poor resolution and split peaks. This is because of poor

efficiency, sample overload, incomplete vaporization and so on. To overcome these problems,

appropriate stationary phase and column dimension must be choose, optimize the temperature

program set up and also adjust the sample concentration or amount injected on column.
Conclusion:

The retention times of standard for peaks of Methyl Laurate, Methyl Myristate, Methyl Stearate

and Methyl Linoleate are 3.994, 4.543, 4.222 and 4.543 respectively. Meanwhile, the retention time for

sample of Methyl Laurate, Methyl Myristate and Methyl Stearate + Methyl Linoleate are 3.750, 4.545

and 4.206 respectively. In conclusion, fatty acids (FAME) can be detected using GC if it undergoes

alternation through derivatization.

References:

1. Analytical separation methods laboratory guide: 2nd edition. Nor’ashikin Saim, Ruziyati Tajuddin

and Mardiana Saaid. (2016)

2. Derivatization of Fatty Acids to FAMEs, retrieved on May 25, 2017 from

http://www.sigmaaldrich.com/analytical-chromatography/analytical-

products.html?TablePage=105120181,

3. High resolution GC Analyses of Fatty Acid Methyl Esters. (2006). Restek Corporation. Retrieved

on May 25, 2017 from http://www.restek.com/pdfs/59584B.pdf