2.2.4 Determination of Free Fatty Acid Composition
Fatty acids were quantified by gas chromatography using the 789 GC
system (Agilent Technology, USA) and 5975C MSD with triple-axis detector
(Agilent Technology, USA). HP-5MS capillary was used as column in GC-MS
(5% - diphenyl, 95% - dimethylpolysiloxane; 30m length \(\times\)0.25mm ID \(\times\) 0.25\(\mu\)m film thickness).
The temperature program was set up from 50 to 250 with
4/min, both the injector and detector temperature were 280, and
helium gas was used as carrier gas. The injecting volume was
2μl. Ionization energy EI of 70eV was used for mass
spectroscopy detection with a source temperature of 150.
For the determination of FFA contained in the sample by gas
chromatography, the PFAD samples were prepared by esterification
according to the equation:
\begin{equation}
FAD+\ \text{CH}_{3}\text{OH\ }\ Methylester+\ C_{3}H_{8}O_{3}\nonumber \\
\end{equation}For GC-MS analysis, the sample containing fatty acids were derivatized
by esterification to the more volatile methyl esters by the addition of
methanol. 3g of PFAD was melted and heated to 55 in a water bath a
mixture of \(\text{CH}_{3}OH/NaOH\) was added (according to
stoichiometry) and stirred for 10mins. After reaction, solution was
centrifuged to separate layers. The top layer which contained the FAME
sample was collected and diluted with cyclohexane (5ml) and prepared for
GC analyses.
Fatty acid methyl ester (FAME) standards were prepared with analytical
grade cyclohexane in concentration of solution
(4\(\mu\)g\(\text{ml}^{-1}\)). For quantification of FAME in the
sample, external standard was analyzed in triplicate. The method was
subject to validation according to guidelines for validation of
chromatographic methods. In this regard, response linearity of the FAs,
sensitivity, limit of detection and quantification, recovery and
precision of the analytical procedure were all calculated. Calibration
curves were made as concentration dependence on the peak area of FAME.
For the calculation of the peak area, the ChemStation Integrator
software was used. The amount of each FAME was calculated from the
concerned calibration curve and mathematically recalculated to wt%.