Direct separation of regioisomers and enantiomers of monoacylglycerols by tandem column high-performance liquid chromatography

An HPLC-based method for direct separation of the regioisomers and enantiomers of monoacylglycerols (MAGs), i.e. sn-1-MAG, sn-2-MAG and sn-3-MAG, has been established. The method employs a tandem column system, in which two different columns (a conventional silica gel column and an enantioselective column) are connected in series. Three isomers of monooleoylglycerols (MOGs) and monolinoleoylglycerols (MLGs) were resolved on the system with resolution factor (Rs) of more than 1.1 between adjacent peaks. In addition, all types of oleoylglycerols, i.e. trioleoylglycerol (TOG), sn-1,2-dioleoylglycerol (DOG), sn-2,3-DOG, sn-1,3-DOG, sn-1-MOG, sn-3-MOG and sn-2-MOG, were successfully separated on the tandem column system, although baseline separation of the enantiomers was not achieved. By means of the established analytical method, the reaction course of Candida antarctica lipase B (CALB)-mediated esterification of glycerol with oleic acid was monitored. It was found that sn-1-MOG and sn-2,3-DOG were preferably generated over sn-3-MOG and sn-1,2-DOG, respectively, in the early stage of the reaction, and the maximal enantiomer excess (%ee) of sn-1-MOG and sn-2,3-DOG were 32 and 53%, respectively, at 2 h. The enantiomeric purities of these chiral acylglycerols decreased after prolonged reaction. The mechanisms for the formation of these chiral acylglycerols are discussed.