Elution time

Adsorption Chromatography Separates Lipids of Different Polarity

Complex mixtures of tissue lipids can be fractionated by chromatographic procedures based on the different polarities of each class of lipid. In adsorption chro-matography (Fig. 10-23b), an insoluble, polar material such as silica gel (a form of silicic acid, Si(OH)4) is packed into a glass column, and the lipid mixture (in chloroform solution) is applied to the top of the column. (In high-performance liquid chromatography, the column is of smaller diameter and solvents are forced through the column under high pressure.) The polar lipids bind tightly to the polar silicic acid, but the neutral lipids pass directly through the column and emerge in the first chloroform wash. The polar lipids are then eluted, in order of increasing polarity, by washing the column with solvents of progressively higher polarity. Uncharged but polar lipids (cerebrosides, for example) are eluted with acetone, and very polar or charged lipids (such as glycerophospholipids) are eluted with methanol.

Thin-layer chromatography on silicic acid employs the same principle (Fig. 10-23c). A thin layer of silica gel is spread onto a glass plate, to which it adheres. A small sample of lipids dissolved in chloroform is applied near one edge of the plate, which is dipped in a shallow container of an organic solvent or solvent mixture—all of which is enclosed within a chamber saturated with the solvent vapor. As the solvent rises on the plate by capillary action, it carries lipids with it. The less polar lipids move farthest, as they have less tendency to bind to the silicic acid. The separated lipids can be detected by spraying the plate with a dye (rhodamine) that flu-oresces when associated with lipids or by exposing the plate to iodine fumes. Iodine reacts reversibly with the double bonds in fatty acids, such that lipids containing unsaturated fatty acids develop a yellow or brown color. A number of other spray reagents are also useful in detecting specific lipids. For subsequent analysis, regions containing separated lipids can be scraped from the plate and the lipids recovered by extraction with an organic solvent.

Gas-Liquid Chromatography Resolves Mixtures of Volatile Lipid Derivatives

Gas-liquid chromatography separates volatile components of a mixture according to their relative tendencies to dissolve in the inert material packed in the chro-matography 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).

Specific Hydrolysis Aids in Determination of Lipid Structure

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidyl-choline) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure.

Mass Spectrometry Reveals Complete Lipid Structure

To establish unambiguously the length of a hydrocarbon chain or the position of double bonds, mass spectral analysis of lipids or their volatile derivatives is invaluable. The chemical properties of similar lipids (for example, two fatty acids of similar length unsaturated at different positions, or two isoprenoids with different numbers of isoprene units) are very much alike, and their positions of elution from the various chromato-graphic procedures often do not distinguish between them. When the effluent from a chromatography column is sampled by mass spectrometry, however, the components of a lipid mixture can be simultaneously separated and identified by their unique pattern of fragmentation (Fig. 10-24).

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