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 >2.0 and separation was achieved in >13 min, but there was no separation of β- and γ-tocopherols. The normal-phase silica and amino columns provided separation of all available isomers with resolution > 1.1 and separation times of <5.5 and < 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.