Gas Chromatography (GC)
Gas chromatography is an analytic technique used in many laboratories for identification and quantification of compounds in a mixture. It is frequently used technique because it allows detection of very small quantities.
The mobile phase and stationary phase is required for this technique. The mobile phase is a carrier gas that is made of inert gas and the stationary phase consists of a packed column. This packed or solid support itself act as stationary phase or is coated with liquid stationary phase. Mostly used analytical GC is capillary column immobilized with liquid phase. The separation of compounds is based on its interaction with stationary phase. (Gas Chromatography Theory)
Polarity of the liquid stationary phase is the major factor influencing separation process in GC (Ulberth, 2003). The sample solution injected into the instrument enters a gas stream which transports the sample into column. Column is a separation tube. Detector measure the quantity of components that exit the column. A standard sample with known concentration is injected into the GC instrument. This helps to measure the quantity of components in an unknown sample. The peak produced by the standard sample is compared to that of the test sample (Shimadzu).
Preparation of sample
GC requires the lipids to be capable of being volatized inside the instrument. Not being volatile, intact triacylglycerols and free fatty acids are therefore difficult to analyze using GC. To solve this problem, lipids are usually derivitized prior to analysis, which increases their volatility. Triacylglycerols have to be first saponified which breaks them down to glycerol and free fatty acids, and are then methylated (Analytical Techniques in Aquaculture Research).
Triacylglycerol ——> Fatty acid methyl esters (FAMEs) + methylated glycerol
The sample (0.1 to 1ul) is drawn up into the syringe barrel with the help of the needle and leaving it empty. The needle is inserted firmly into the injection port, and allowed to warm up for 5 seconds (Ulberth, 2003)
Separation by GC
The group of lipids most commonly analyzed by GLC are fatty acids. This method is applicable to biological samples containing compounds with chain length in the range C14 to C24.
After the conversion of Fatty acids into polar, methyl ester derevatives, GLC analysis is performed. For this, columns with polar phases are used, as polyethylene glycol stationary phase (Carbowax), to coat capillary column (Analytical Techniques in Aquaculture Research).
Large number of detectors operating on different principles have been developed for use in Gas Chromatography.
I. Flame ionization detectors:
The ions are generated by burning of the organic matter by the fame of hydrogen and air. The ion current is measure by establising the potential beween the collector and the jet tip, which is passed to an amplifier and then to recorder (Ulberth, 2003).
II. Mass spectroscopy
Mass spectrometer capture, ionize, accelerate, deflect, and detect the ionized molecules.It ionizes the compound and sorts the ions based on their mass-to-charge ratio.