DETERMINATION OF FATTY ACID PROFILE USING GC-FID

Lipids are a quantitatively minor, but significant component of wheat grain, amounting to 0.9–
3.3%. Content of these compounds in wheat grain is connected with its maturity, classification of
cultivars into quality classes and cultivation conditions. Lipids from wheat germ isolated on a
commercial scale are important oils for direct human consumption, containing polyenic fatty acids
and tocopherols of high nutritive value. To determine the fatty acid components, present in a lipid
sample the fatty acid is converted to a fatty acid methyl ester. It can then be analyzed by GC/FID
and the individual fatty acid methyl esters identified by comparison of the retention times to
reference standards. Fatty Acid Profile of wheat flour was determined by GC-FID according to
methods described by Dodds et al. (2005). The detailed procedure for that is given below:

1.1.1 Material Required

Pipettes Water bath

Volumetric flask Vortex

Test tubes Sodium methoxide

Soxhlet apparatus n-Hexane

Rotary evaporator NaCl

1.1.2 Extraction of Oil

Oil from the wheat flour was extracted by Soxhlet extraction method using hexane as extraction
solvent. The extracted oil was converted into fatty acids methyl esters.

1.1.3 Fatty Acid Methyl Esters (FAME) Preparation

By using Pasteur pipettes 100±5 mL of oil sample was transferred to the pyrex test tube having a
tight sealing cap. Afterward, 5 mL of hexane was added to the test tube and vortexed briefly to
dissolve oil/lipid. Then 250 µL sodium methoxide reagent was added and vortexed for one minute,
pausing every 10 seconds. After complete mixing, 5 mL of saturated NaCl was added in the test
tube, shaken vigorously for 15 seconds and left the tubes vertically for 10 minutes. After that,
hexane layer was removed and transferred to a vial containing a small volume of Sodium Sulphate
for 15 minutes. Hexane layer was then transferred to a vial for subsequent GC analysis.
 1.1.4 GC-FID Conditions

Fatty acid methyl esters were analyzed by using GC (Agilent 6890) equipped with Flame
Ionization Detector. GC analysis was performed on an Agilent 6890N Network GC system, under
the following conditions: column, DB-wax Capillary; 30.0m×0.25mm×0.25m; oven temperature
program: the column held initially at 60oC for 3 min after injection, then increased to 185oC with
10oC/m in heating ramp for 1 min and increased to 200oC with 5oC/min heating ramp for 10 min.
Then the final temperature was increased to 220oC with 5oC/m in heating ramp for 20 min; injector
temperature, 250oC; detector (FID) temperature, 275oC; carrier gas, Nitrogen; inlet pressure, 40.65
psi; linear gas velocity, 39 cm/s; column flow rate, 2.7 ml/m in; split ratio, 40:1; injected volume,
1µL. After that, area percent method was used to quantify fatty acids.