Ultramicro fatty acid analysis of polar lipids: Gas-liquid chromatography after column and thin-layer chromatographic separation

A procedure is described for the analysis of the fatty acid composition of polar lipid classes in the nanogram range. The lipids are first fractionated by column chromatography followed by further separation into pure lipid classes by thin-layer chromatography. Lipid spots scraped from the thin-layer plates are esterified directly (i.e., without prior elution) with 6% sulfuric acid in methanol. The methyl esters are then analyzed by gasliquid chromatography with a hydrogen flame ionization detector. Samples of 200 nanograms or less give accurate results with helium as carrier gas, oxygen rather than air to support combustion, careful adjustment of the recorder and general attention to optimum electrical connections, dissociation of the column oven from the recorder and electrometer, and careful preconditioning of columns. Under proper conditions the base line is stable and a 10% of full scale deflection of the recorder can be obtained from 1 nanogram of a methyl ester, allowing highly precise analyses of fatty acid composition from the amount of lipid obtainable from one spot on a thin-layer chromatogram. Control studies demonstrated that extraneous peaks did not arise from the procedure or from the sphingosine and dihydrosphingosine of sphingolipids. The thin-layer chromatographic procedure did not influence the fatty acid composition of a pure sample of glucocerebroside isolated by column chromatography and the method was applied to lecithin and sphingomyelin or normal and pathological human brain specimens.