Documente Academic
Documente Profesional
Documente Cultură
Chromatography (GC)
ID: 2016586557
PARTNERS’ NAME:
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)
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
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:
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
c. Saturated NaCl.
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:
Procedure:
1. 2 g of oil or fat is approximately weighed out and the exact weight is recorded.
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
NOTE: You may want to run your calibration standards while the esterification reaction is in progress.
1. 0.4 µL of standard esters is injected into the column. The injection is repeated to get
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:
B. Resolution:
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 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
References:
1. Analytical separation methods laboratory guide: 2nd edition. Nor’ashikin Saim, Ruziyati Tajuddin
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