Separation of tocopherol and tocotrienol isomers using normal- and reverse-phase liquid chromatography.

This optimization study for tocopherols and tocotrienols involved both normal- and reverse-phase liquid chromatography using various columns and mobile phases. Normal-phase systems showed elution of the homologs in order of increasing polarity with separation based on methyl substituents on the chromanol moiety. Reverse-phase systems showed class separation based on the saturation of the phytyl side chain; the more saturated tocopherols were retained on the column longer. When the Zorbax ODS was used with an isocratic ternary acetonitrile:methanol:methylene chloride (60:35:5) mixture, the optimized resolution was greater than 2.0 and separation was achieved in less than 13 min, but there was no separation of beta- and gamma-tocopherols. The normal-phase silica and amino columns provided separation of all available isomers with resolution greater than 1.1 and separation times of less than 5.5 and less than 10 min, respectively. Optimized isocratic binary solvent mixtures of hexane:2-propanol were used for silica (99:1) and amino (98:2) columns. Derivative spectra showed differences depending on substituents in the chromanol moiety but not the phytyl side chain. Second- and fourth-derivative spectra gave the best differentiation of the vitamin E isomers.