Human Biochemistry, Volume 1 - Murray R. 1993

Bioenergetics and Metabolism of Carbohydrates and Lipids
Physiologically Important Lipids
Methods for the Separation and Identification of Lipids Present in Biological Material

Traditional Methods of lipid Separation and identification, based on classical operations of crystallization, distillation, and extraction, have now been largely complemented by chromatographic techniques. Thin-Layer Chromatography is particularly useful for separating various lipid classes, whereas Gas-Liquid Chromatography (Fig. 15.32) is employed for the separation of individual Fatty acids. A preliminary stage in using these methods is the extraction of Lipids with a solvent system, most commonly a chloroform-methanol mixture (2:1).

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Fig. 15.32. Diagram of a gas-liquid chromatograph and the resulting separation of long-chain fatty acids (as methyl esters); a portion of the chromatogram is shown on the right.

Gas-liquid chromatography achieves the physical separation of a volatilized gas phase through the adsorption of its components on a stationary phase. This phase consists of an inert solid support, such as silica gel or inert granules of crushed firebrick, coated with a non-volatile liquid (e.g., lubricating grease or silicone oil). In practice, metal or Glass columns packed with the aforementioned sorbent are used; a mixture of fatty acid methyl esters in the gaseous state is passed through the Column, the entire length of which is maintained at a Temperature of 170—225° C (Fig. 15.32). A continuous stream of inert gas—argon or helium—carries the gaseous esters along. In accordance with general chromatographic principles, the separation of fatty acid esters is based on the differing affinities of the gas mixture components for the stationary phase material. Gases with a higher affinity for the stationary phase move more slowly through the column and emerge later than those with a relatively lower affinity. As individual fatty acid esters exit the column, they are automatically detected by physical or chemical methods as a series of peaks distributed in time according to their retention efficiency by the stationary phase (Fig. 15.32). The peak area is proportional to the concentration of each component in the mixture. The identification of each component is performed by comparison with the chromatogram of a standard gas mixture of known composition.

Fig. 15.33. Separation of major lipid classes by thin-layer chromatography. A hexane-diethyl ether-formic acid system (in an 80:20:2 volume ratio) is suitable as a solvent system.

Fig. 15.34. Formation of lipid membranes, micelles, emulsions, and Liposomes from Amphipathic Lipids, such as Phospholipids.

The advantages of gas-liquid chromatography include high sensitivity, which allows for the separation of very small amounts of a mixture, and the reusability of the column. Using this method, A large number of previously unidentified fatty acids have been discovered in natural fats.

Thin-layer chromatography is performed on glass plates coated with a thin layer of an adsorbent suspension, most commonly silica gel. After the suspension dries, the plates are baked in an oven for a specified time at a defined temperature. Next, the lipid mixture dissolved in a suitable solvent is applied to the cooled "activated" plate. Following the evaporation of the solvent, the edge of the plate closest to the applied spot is immersed in a mixture of appropriate Solvents; the plate is then "developed" in a closed chamber until the solvent reaches the opposite edge of the plate. Subsequently, the plate is dried to remove the solvent, and the positions of the spots are determined either by charring (Treatment with sulfuric acid followed by heating), by fluorescence (after treatment with dichlorofluorescein), or by exposure to iodine vapors (Fig. 15.33).