Fatty Acid analysis by gas chromatography

• Principle
• Preparation
• Separation
• Quantitation

Principle

The discovery in the mid-1950's of gas-liquid chromatography (GLC or in short GC) has revolutionized the analysis of fatty acids and, undoubtedly, this technique is the most frequently used. It remains one of the most powerful analytical procedures for separating and analyzing the properties of any acylated lipids, especially when combined with techniques which can be used to identify the chemical structure of the peaks, e.g., mass spectrometry or NMR. A chromatographic analysis involves passing a mixture of the molecules to be separated through a column which contains a matrix capable of retarding the flow of the molecules. Molecules in the mixture are separated because of their differing affinities for the matrix in the column. The stronger the affinity between a specific molecule and the matrix, the more its movement is retarded, and the slower it passes through the column. Thus different molecules can be separated on the basis of the strength of their interaction with the matrix. After being separated by the column, the concentration of each of the molecules is determined as they pass by a suitable detector (e.g., UV-visible, fluorescence, or flame ionization). Chromatography can be used to determine the complete profile of molecules present in a lipid. This information can be used to: calculate the amounts of saturated, unsaturated, polyunsaturated fat and cholesterol; the degree of lipid oxidation; the extent of heat or radiation damage; detect adulteration; determine the presence of antioxidants. Various forms of chromatography are available to analyze the lipids in foods, e.g. gas chromatography (GC), high pressure liquid chromatography (HPLC), thin layer chromatography (TLC).

Preparation

Intact triacylglycerols and free fatty acids are not very volatile and are therefore difficult to analyze using GC (which requires that the lipids be capable of being volatized in the instrument). For this reason lipids are usually derivitized prior to analysis to increase their volatility. Triacylglycerols are first saponified which breaks them down to glycerol and free fatty acids, and are then methylated.

Separation by GC

Fatty acids are the group of lipids most commonly analyzed by GLC. This method is applicable to biological samples containing compounds with chain length in the range C14 to C24.
GLC analysis of fatty acids is performed following their conversion to apolar, methyl ester derivatives. Columns with polar phases are used, as polyethylene glycol stationary phase (Carbowax), to coat capillary column. The majority of commercially available columns are coated with phases bonded or immobilized on the sillica column wall. This technology leads to a great durability with a strong thermal stability.
Analytical conditions must be adapted from published values to obtain reliable and precise results. The temperature gradient program is the main parameter to be modified according to the nature of the column and the complexity of the fatty acid mixture.

The identification of a peak can be made from its retention time but must be confirmed by other investigations such as TLC mobility, column fractionation or GC-MS. In practice, it is convenient to work not with retention time which is temperature and gas flow rate dependent, but with retention time relative to that of a suitable standard (stearic acid present in all samples is the most convenient). The logarithm of the relative retention time and the number of carbon atoms is linearly related and may be of some help to identify unknown fatty acids. A plot of these parameters with homologous series of saturates, monoenes, dienes, trienes, etc. gives a series of parallel lines.

Cut-open illustration of a gas chromatograph

Scheme of a gas chromatograph

Only the principle of this determination is presented here (as an example we restrict the demonstration for peaks situated between palmitic and stearic acids): if t_16:0 is the retention time of palmitic acid and t_18:0 is the retention time of stearic acid and t_i the retention time of the unknown peak, the ECL value of this peak is calculated as : 100 x [[(18-16) x (log t_i - log t_16:0)/(log t_18:0 - log t_16:0)] + 16].

Chromatogram of the fatty acid profile from a green algae (Enteromorpha sp). Notice the presence of 17:0 as an internal standard, and the three unusual plant fatty acids: 16:2n-6, 16:3n-3 and 16:4n-3

Quantitation and expression of results

The lipidologist must be aware of the non exact linearity of the detector response to the fatty acid mass. The responses of the detector used should be checked with a calibrated standard mixture. This correction is more important in studies concerning highly unsaturated fatty acids that are often prevalent in marine products.
Peak areas are now measured with an electronic integrator, this is the most accurate and convenient procedure. Nevertheless, the lipidologist must be aware of the limitations of his integrator and, the use of an adapted chromatographic software is highly recommended to verify the integration process.
Results are frequently expressed as weight percent, this is currently used in nutrition works. In biochemical studies, as for membrane structure, results should be expressed on a molar percentage basis.