Reversed-phase HPLC separation of 1,2-diacylglycerol regioisomers

We investigated the separation of the regioisomers (reverse isomers) of 1,2-diacyl-rac-glycerols by reversed-phase HPLC on columns containing endcapped and non-endcapped ODS phases. The reverse isomers of 1,2-diacylglycerols, having various pairs of acyl groups including saturated and unsaturated chains, were chromatographed as 3,5-dinitrophenylurethanes (DNPU) using acetonitorile as the mobile phase. Baseline separations were achieved for the reverse isomers of very different pairs of acyl groups, such as caprylate/palmitate and stearate/arachidonate, on both endcapped and non-endcapped ODS columns, which retained longer less-polar isomers with a lower degree of unsaturation or shorter chain at the sn-1(3) position. The latter columns, however, markedly shortened the elution times of the derivatives compared to the former columns. Most commercially available ODS columns are fully endcapped so as to decrease any undesirable influence of residual silanol. The present study, however, demonstrates that non-endcapped ODS phases are more suitable for reverse isomer separation than fully endcapped ones.