Separation and quantitation of free fatty acids and fatty acid methyl esters by reverse phase high pressure liquid chromatography (original) (raw)

Reverse phase high pressure liquid chromatography (HPLC) on octadecylsilyl columns separates mixtures of either free fatty acids or fatty acid methyl esters prepared from mammalian tissue phospholipids. Acetonitrile-water mixtures are used for the elution of esters. Aqueous phosphoric acid is substituted for water for the separation of the free acids. Unsaturated compounds are detected and quantitated by their absorption at 192 nm. Saturates are detected better at 205 nm. T h e order of elution of fatty acids in complex mixtures varies as a function of acetonitrile concentration. At any given concentration, some compounds overlap. However, by varying the solvent strength, any fatty acid of interest can be resolved including many geometrical and positional isomers. Methyl esters prefractionated according to unsaturation by argentation thin-layer chromatography (TLC) are rapidly and completely separated by elution with CH'CN alone. Argentation TLC-reverse phase HPLC can be used as an analytical as well as a preparative procedure. Octylsilyl columns are used for rapid resolution and improved detection of minor or low ultraviolet-absorbing components in the fractions. For example, monoenoic fatty acids with up to 32 carbons have been detected in bovine brain glycerophospholipids. Specific radioactivities of 'H-and ''C-labeled fatty acids and the distribution of radioactivity among acyl groups from complex lipids are measured. T h e method is not recommended for complete compositional analysis, but is useful for determinations of specific radioactivities during studies on turnover and metabolic Abbreviations: GLC, gas-liquid chromatography; HPLC, high pressure liquid chromatography: TLC, thin-layer chromatography; FFA, free fatty acids; FAME, fatty acid methyl esters. Fatty acids are abbreviated by the convention, number of carbon atomsmumber of double bonds.