High-performance liquid chromatography of sterols: yeast sterols

Abstract

It is evident that the high-pressure liquid chromatograph is an excellent tool for studying sterol metabolism. As noted in the text, the individual effects of unsaturations and alkyl groups on reverse phase elution volumes cannot be extrapolated to predict quantitative effects of multiple functional groups. The mechanism(s) of retention seems more complex than can be explained simply by polarity or hydrophobicity. Since the molecular location of these functional moieties seems critical, retention and separation of sterols may involve specific structural configurations and hence specific interactions with the stationary phase material. The association we have drawn between polarity and HPLC elution may indeed be a secondary effect of another phenomenon. Future studies may unveil the true mechanism(s) of HPLC retention and separation, and allow for the construction of HPLC systems which will separate all isomeric combinations of sterols at the analytical level. The simplicity, rapidity, and reproducibility of these methods make the coupled technique very useful for investigating sterol metabolism. Application of this technique to analyzing putative sterol mutants, purifying sterols for auxotrophic feeding, and analyzing the metabolism of supplemented sterols in auxotrophs provides for significant advances in membrane physiology.