ANALYSIS AND FRACTIONATION OF NATURAL SOURCE DIACYLGLYCEROLS AS URETHANE DERIVATIVES BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Reversed-phase high-performance liquid chromatography on a thermostatted octadecylsilyl column was used to separate and fractionate mixtures of diacylglycerols after derivatization with 3,5-dinitrophenyl isocyanate (urethane derivatives). In addition to the separation of commercial diacylglycerol species, the separation of diacylglycerols obtained from peanut oil and cottonseed oil triacylglycerols by chemical hydrolysis is reported. Acetonitrile - acetone mixtures were used for elution of the diacylglycerol urethane derivatives. Unsaturated and saturated derivatives were detected by their refractive indices. They were then collected and their fatty acids analysed as methyl esters by capillary gas chromatography. The elution order of diacylglycerol derivatives in complex mixtures varies as a function of chain length, unsaturation and positional isomerism of the constituent fatty acids. The elution order and resolution vary as a function of temperature. The diacylglycerol composition of mixtures calculated from peak areas is similar to the composition reconstituted from the fatty acid composition of the collected diacylglycerol fractions. The method can be applied to complete compositional analysis but is especially useful for the collection of pure fractions of diacylglycerols during studies of the stereospecific distribution of fatty acids in triacylglycerols.