Reversed-phase High-performance Liquid Chromatography of Triacylglycerols

The resolution of molecular species of triacylglycerols by reversed-phase high-performance liquid chromatography is discussed. Particular attention is given to the choice of stationary phase, mobile phase, injection solvent, detector and the effect of temperature. The influence of molecular structure on retention time is also explained. Triacylglycerols are major components of the human diet, so methods for their analysis are of great importance. Excellent resolution of triacylglycerols by reversed-phase high-performance liquid chromatography has been possible for many years, but many practical and theoretical aspects of the process are poorly understood, although excellent reviews are available on the topic [1,2]. With some honourable exceptions, little of interest is now being published in this area. Indeed, many chromatograms continue to be illustrated that are well below the standard that should be attainable. I suspect that this is because many analysts are concerned simply with obtaining a “recipe” to do the job. A little knowledge of the theory can help enormously to improve results. The nature of the separation is fairly well known, i.e. that the retention time is dependent on the total number of carbon atoms in the three fatty acyl moieties, with each double bond reducing the effective chain-length of the species by the equivalent of about two carbon atoms. This figure “two” is sometimes interpreted too literally, and I will say more below. Figure 1. Separation of triacylglycerols from a low-erucic acid rapeseed oil by reversedphase HPLC (Spherisorb ODS2; 250 x 4.6 mm column) with a gradient of acetone into acetonitrile (1 mL/min) and evaporative light-scattering detection (Abbreviations: P = palmitate, S = stearate, O = oleate, L = linoleate, T = triene (linolenate)). Reversed-phase HPLC of triacylglycerols W.W. Christie lipidlibrary.aocs.org 2 Figure 1 illustrates a separation of molecular species of triacylglycerols from a vegetable oil to show the nature of the separation and the quality of the resolution that should be attainable in all laboratories.