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%.