Normal Phase High-Performance Liquid Chromatography of Tocopherols on Polar Phases

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Abstract
Five plant tocopherols were separated by normal phase high performance liquid chromatography (HPLC) on aminopropylsilica or diol-bonded silica with fluorescence detection. HPLC characteristics of these compounds were studied under various mobile phase conditions. Mobile phases employed binary solvent systems comprising a hydrocarbon and an alcohol, an ether or an ester. Separation factors (α) of adjacent ring-methylated tocol components were determined. Employment of aprotic weakly polar modifiers in the mobile phases increased the α values for the pair of 5,8- and 7,8-dimethyltocol, but decreased those for the 5,7-and 5,8-dimethyltocol pair. Both component resolution and detection sensitivity were adversely affected by the use of a diol-bonded silica phase of large particle size. HPLC with cyclohexane-binary eluents invariably led to lower values of analyte capacity factors (k′) than with hexane counterparts. Further, regardless of the type of column evaluated, the tocol isomers eluted in an ethereal mobile phase in the order of increasing k′ values: k′(tetrahydrofuran) <k′(dioxane) < k′(t-butyl methyl ether < k′(diisopropyl ether). In HPLC with protic co-solvents, the observed differences in k′ values among the three dimetyltocols were interpreted in terms of methyl substitution effects.