Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase

Reversed-phase liquid chromatographic (RPLC) separation of isomers and homologues of similar polarity is challenging. Tocopherol isomers and homologues are one such example. alpha, beta, gamma, and delta-tocopherols have been successfully separated by RPLC on triacontyl (C-30) stationary phase. System suitability was tested by using four mobile phases, and observed chromatographic separations of beta and gamma-tocopherols were compared. Comparison indicated that methanol-tert-butyl methyl ether (TBME) 95:5 (v/v) at a flow rate of 0.75 mL min(-1) was the best mobile phase. Detection systems were also evaluated on the basis of limit of quantification; it was concluded that fluorescence detection was best. The method was validated by analysis of two homologues and two isomers of tocopherol in sesame, maize, and soybean samples. MS coupled with an ESI interface in negative-ion mode [M - H](-) was used for identification of individual components. It was concluded that addition of TBME to methanol was required to enhance the separation of beta and gamma-tocopherols, although methanol alone provided similar results. The applicability of the method to cereal, pulse, and oilseed samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the range 1.7-3.9 %. Recovery of tocopherols added to sesame samples ranged from 91 to 99 %.