High-performance liquid chromatographic resolution of reverse isomers of 1,2-diacyl-rac-glycerols as 3,5-dinitrophenylurethanes

The resolution of reverse isomers remains a major unsolved problem in glyce rolipid chromatography. We have investigated the separation of the reverse isomers of 1,2-diacyl-rac-glycerols under a variety of high-performance liq uid chromatography (HPLC) conditions. The reverse isomers of diacylglycerol s having various pairs of acyl groups including short and highly unsaturate d chains, which were prepared by partial Grignard degradation of the corres ponding triacylglycerols, were chromatographed as 3,5-dinitrophenylurethane s. Excellent resolution was achieved for me reverse isomers of very differe nt pairs of acyl groups, such as acetate-palmitate and docosahexaenoate-pal mitate, by chiral-phase HPLC on columns containing (R)- and (S)-1-(l-naphth yl)ethylamine polymeric phases, reversed-phase HPLC on a highly efficient C -18 column (4 mu m particle size) and silver ion HPLC on a silver loaded ca tion-exchange column. The chiral-phase HPLC also permitted complete enantio mer resolution for all the reverse isomers examined. No satisfactory resolu tion by any of the HPLC methods, however, was obtained for the reverse isom ers possessing minor differences in chain lengths and degree of unsaturatio n, such as laurate-palmitate and oleate-linoleate. The limitations of resol ution and characteristics of elution are described. (C) 2000 Elsevier Scien ce B.V. All rights reserved.