Use of isopropanol as a modifier in a hexane-acetonitrile based mobile phase for the silver ion HPLC separation of positional isomers of triacylglycerols containing long chain polyunsaturated fatty acids

A high resolution approach to silver ion HPLC was studied for the separatio n of positional isomers of triacylglycerols (TAGs) containing long chain po lyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docos ahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically syn thesized structured TAGs. Isopropanol was used as a novel modifier in a hex ane-acetonitrile based mobile phase for silver ion HPLC. Peak identificatio n was based on HPLC-mass Spectroscopy and selectivities of lipases. Positio nal isomers of TAGs containing one molecule of EPA, DHA, or DPA with satura ted fatty acids (FAs) such as caprylic acid and palmitic acid were separate d within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobi le phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved . The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of doub le bonds and their isomeric configuration. One isomer with an unsaturated F A in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have sho rter chain FAs, For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/ C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).