Search

A method is described for fractionation and determination of the lipid and steroid components in very small amounts of substrates. Samples as small as 20 suprarenal glands of either normal or pantothenic acid deficient rats were extracted with chloroform-methanol (2:1). The total lipid extracts were separated by thin-layer chromatograms, the solvent system being benzene-ether-acetic acid (70:30:1). Spots corresponding to cholesterol esters, triglycerides, fatty acids, cholesterol, and phospholipids were obtained; corticosteroids lag behind as the least mobile fraction, together with the phospholipids. The areas were eluted with appropriate solvents and the main components were determined. Corticosteroids were estimated by a colorimetric method with tetrazole blue and by U.V. spectrophotometry. A second chromatogram was then performed on the corticosteroid eluate; the solvent system was chloroform-methanol-water (90:10:0.25). A clear-cut separation of the principal corticosteroids present in the suprarenal glands of rats was obtained.

A method is described for the fractionation of neutral lipids into classes by means of silicic acid column chromatography. Elution is performed with a concave gradient of ethyl ether in petroleum ether; the gradient is achieved by means of two metering pumps. This gradient is simple to obtain and can be easily modified for different requirements following our scheme of calculation. The effluent is monitored continuously by means of a hydrogen flame ionisation detector, a part of the effluent being continuously drawn off. Our method can be used for analytical purposes as well as for isolation of pure lipidic fractions. The quantitative analysis of the components of a lipid mixture is performed following a weighing microtechnique, using a Cahn electrobalance. Examples are reported of the separation of reference lipid mixtures of oils and tissue lipids and of oil solutions of steroids for pharmaceutical use. Our method is also useful for detecting lipidic contaminants in solvents and materials for chromatography. The necessity of very carefully controlled solvents and materials to avoid contamination is underlined.