Reversed-phase liquid chromatography of astacene

Abstract

Carotenoids can be efficiently separated by non-aqueous reversed-phase liquid chromatography on Zorbax ODS. However, astancene, an acidic kwetocarotenoid was found to display a peculiar behaviour in that it could not be eluted from this support using mixtures of acetonitrile—methanol—dichloromethane. The importance of astacene as a degradation product of astaxanthin warranted the development of a new liquid chromatographic system. Organic acids promoted the elution of the compound, but failed to suppress the accompanying excessive peak tailing. Efficient chromatography resulted from the incorporation of bis(2-ethylhexyl) phosphate (BEHP), an acid with a high degree of lipophilicity, in the mobile phase. Optimum separation of astacene and astaxanthin was achieved using methanol-free, semi-aqueous eluents, i.e., acetonitrile—water, containing 0.01—0.05 M BEHP. Increasing amounts of BEHP significantly reduced retention while, unexpectedly, water had little effect over the concentration range studied (1–5%). To illustrate the practical usefulness of the new system, astacene was demonstrated in a saponified extract of flower petals of Adonis annua.