Working with Lipids:- Gas-Liquid Chromatography Resolves Mixtures of Volatile Lipid Derivatives
Gas-liquid chromatography separates volatile compo nents of a mixture according to their relative tendencies to dissolve in the inert material packed in the chromatography column and to volatilize and move through the column, carried by a current of an inert gas such as helium. Some lipids are naturally volatile, but most must first be derivatized to increase their volatility (that is, lower their boiling point). For an analysis of the fatty acids in a sample of phospholipids, the lipids are first heated in a methanol/HCl or methanol/NaOH mixture, which converts fatty acids esterified to glycerol into their methyl esters (in a process of transesterification; Fig. 10–23d). These fatty acyl methyl esters are then loaded onto the gas-liquid chromatography column, and the column is heated to volatilize the compounds. Those fatty acyl esters most soluble in the column material partition into (dissolve in) that material; the less soluble lipids are carried by the stream of inert gas and emerge first from the column. The order of elution depends on the nature of the solid adsorbant in the column and on the boiling point of the components of the lipid mixture. Using these techniques, mixtures of fatty acids of various chain lengths and various degrees of unsaturation can be completely resolved (Fig. 10–23e).
FIGURE 10–23 Common procedures in the extraction, separation, and identification of cellular lipids. (a) Tissue is homogenized in a chloroform/methanol/water mixture, which on addition of water and removal of unextractable sediment by centrifugation yields two phases. Different types of extracted lipids in the chloroform phase may be separated by (b) adsorption chromatography on a column of silica gel, through which solvents of increasing polarity are passed, or (c) thin layer chromatography (TLC), in which lipids are carried up a silica gel coated plate by a rising solvent front, less polar lipids traveling farther than more polar or charged lipids. TLC with appropriate solvents can also be used to separate closely related lipid species; for example, the charged lipids phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol are easily separated by TLC. For the determination of fatty acid composition, a lipid fraction containing ester-linked fatty acids is trans esterified in a warm aqueous solution of NaOH and methanol (d), producing a mixture of fatty acyl methyl esters. These methyl esters are then separated on the ba sis of chain length and degree of saturation by (e) gas-liquid chromatography (GLC) or (f) high-performance liquid chromatography (HPLC). Precise determination of molecular mass by mass spectrometry allows unambiguous identification of individual lipids.
