Triacylglycerols reversed-phase chromatography

Reverse-phase liquid chromatography is now virtually the only method used in the analysis of the TG mixtures. The first paper on TG-HPLC analysis was published in 1975 by Pei et al. (81). Triglycerides were separated on a VYDAC reverse-phase (35 - 44 /xm) column and eluted with methanol-water (9 1). Since Pei et al. first applied RP-HPLC to the separation of triacyl-glycerols, a number of reverse-phase systems have been developed as rapid and efficient resolution of complex triacylglycerol mixtures can be achieved. 

More recently, butterfat composition has been analyzed by capillary GC on polarizable liquid phases (122,123), reversed-phase high-performance liquid chromatography (124,125), and GC and HPLC with mass spectrometry (MS) (126), as well as by MS-MS (127). These studies have led to an extensive resolution of butterfat triacylglycerols, but the possible presence of small amounts of positional and reverse isomers as well as of species containing two or three short-chain acids per molecule has not been addressed. 

Maniongui, C., Gresti, J., Bugaut, M., Gauthier, S. and Bezard, J. (1991) Determination of bovine butterfat triacylglycerols by reversed-phase liquid chromatography and gas chromatography. J. Chrom., 543, 81-103. 

Lin, J. T., Woodruff, C. L., and McKeon, T. A. 1997. Non-aqueous reversed-phase high-performance liquid chromatography of synthetic triacylglycerols and diacylg-lycerols. J. Chromatogr. A, 782,41-48. 

Lin, J.T., Snyder, L.R., and McKeon, T.A. (1998) Prediction of Relative Retention Times of Triacylglycerols in Non-Aqueous Reversed-Phase High-Performance Liquid Chromatography, J. Chromatogr. A 808,43-49. 

Figure 5 Reversed phase HPLC (linear gradient of 10-90% isopropanol in acetonitrile at 25°C) of natural (top) and rearranged (bottom) butterfat triacylglycerols as obtained with light-scattering detector. Peak identification by carbon and double bond number. (Reproduced with permission from Marai L, Kuksis A, and Myher JJ (1994) Reversed-phase liquid chromatography-mass spectrometry of the uncommon triacylglycerol structures generated by randomization of butteroil. Journal of Chromatography A 672 87-99.)...

For samples with a high lipid content (e.g., milk), additional cleanup of the extracts may be necessary to eliminate other lipids such as triacylglycerols. In the past, open-column chromatography on magnesia, alumina, or silica gel has been used. More recently, solid-phase extraction (SPE) on disposable prepacked cartridges filled with silica or Cig reversed-phase packing material has been applied to vitamin A analysis. SPE is a refinement of the earlier open-column chromatographic systems. 

Kuksis, A., Marai, L. and Myher, J. J. (1991a) Reversed phase liquid chromatography-mass spectrometry of complex mixtures of natural triacylglycerols with chloride attachment negative chemical ionisation. J. Chromatogr., 588, 73-87. 

Marai, L., Myher, J. J. and Kuksis, A. (1983) Analysis of triacylglycerols by reversed phase high performance liquid chromatography with direct liquid inlet mass spectrometry. Can. J. Biochem. Cell Biol, 61, 840-59. 

Neff, W. E. and Byrdwell, W. C. (1995) Soybean triacylglycerol analysis by reverse-phase high performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry. J. Am. Oil Chem. Soc., 72, 1185-91. 

Leveque NL, Heron S, Tchapla A. Regioisomer characterization of triacylglycerols by non-aqueous reversed-phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent. J Mass Spec 2010 45 284-96. 

Nikolova-Damyanova, B. (1997). Reversed-phase high-performance liquid chromatography general principles and applications to the analysis of fatty acids and triacylglycerols. In Advances in Lipid Methodology - Four, W.W. Christie (ed.). Oily Press. 

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