Separation of carotenoids during reversed-phase HPLC: retention mechanism and methylene selectivity on a monomeric stationary phase
Abstract
Fractions of carotenoids of orange bell pepper (Capsicum annuum) obtained by means of normal phase thin layer chromatography were used to study certain features of chromatographic retention of carotenoids during reversed-phase HPLC on a traditional monomeric stationary phase. The difference in the retention of α- and β-carotenoids on stationary phases of the same make (Kromasil) with different lengths of implanted radicals (C18, C8, and C4) in the same composition of the mobile phase demonstrates the presence of sorption distribution of carotenoids on the monomeric C18 phase. We compared the eluting ability of acetone, ethyl acetate, and 2-propanol used as additives to acetonitrile towards carotenoids. The study determined that ethyl acetate has the greatest eluting ability. A similar ability demonstrated by 2-propanol is explained by the literature data regarding the self-association of the compound and its association with acetonitrile. To determine the methylene selectivity a fraction of zeaxanthin diesters was used. In the article we also explain why it is not advisable to use all the substances of the homologous series to determine the methylene selectivity based on the concept of atoms of the same type suggested by V.M. Tatevsky. The article describes a method of calculating the quantitative characteristics of retention of the elements of the homologous series using the “dead” time determined based on the retention of uracil and homologues. The obtained increments (contributions to the retention of a pair of methylene groups) varied significantly. The article presents the discussion of the obtained results. It compares the behaviour of homologous series based on zeaxanthin and capsanthin, the latter being more hydrophilic. The article demonstrates the advantages of determining the “dead” time based on the retention of homologous series. We also observed a predicted earlier dependence of the increments (or methylene selectivity) on the structure of the homologous series: while for all the transitions between diesters (from dilaurate to dipalmitate) the increments are the same, the increments of capsanthin diesters retained less strongly are by 0.002 logarithmic units lower than those of zeaxanthin diesters.
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