Non-aqueous reversed-phase high-performance liquid chromatography of synthetic triacylglycerols and diacylglycerols

Reversed-phase high-performance liquid chromatography (HPLC) methods were developed for the separation of molecular species of 45 synthetic triacylglycerols and diacylglycerols. These methods used linear gradients of methanol-isopropanol and UV detection at 205 nm as well as radioactive how detection, which we add for metabolic studies. The elution orders of triacylglycerols and diacylglycerols depend on the polarity of their fatty acid constituents with elution time increasing as polarity decreases. The elution orders of triacylglycerols depending on their fatty acid constituents were as follows: ricinoleic acid<linolenic acid<palmitoleic acid<myristic acid<palmitelaidic acid<linoleic acid<linolelaidic acid<oleic acid<palmitic acid<elaidic acid<petroselinic acid<petroselaidic acid<stearic acid, while the elution orders of diacylglycerols were: palmitic acid<oleic acid<stearic acid. For both classes of glycerides, elution corresponded closely with chain length, degree of unsaturation and presence of polar groups and they were similar to the elution orders of fatty acids on an aqueous C-18 HPLC which we reported recently. Other structural features also affect elution order, as triacylglycerol containing a cis-fatty acid elutes slightly earlier than its isomer containing a trans-fatty acid. Additionally, higher polarity in the sn-2 position causes earlier elution; Diacylglycerol with a hydroxy group al sn-2 position of the glycerol backbone and triacylglycerol with a polar group on the fatty acid chain at sn-2 position elute slightly earlier than their respective sn-1(3) isomers.