Characterization of Column Packings in Normal-Phase Liquid Chromatography. I. Retention Behavior of Fat-Soluble Vitamins in Silica Gel-Binary Solvent Systems

Abstract

To characterize packing columns in high performance liquid chromatography, the retention indices of ten fat-soluble vitamins were systematically measured using binary solvents each containing ethyl acetate, tetrahydrofuran and 2-propanol in n-hexane for silica gel chromatography. A linear relationship between the logarithm of the capacity ratio and that of the concentration of the polar solvents was confirmed. The retention sequence of the solutes was determined as follows: retinol > ergocalciferol = cholecalciferol > δ- > γ- > β- > α - tocopherol > menadione > phylloquinone. The retention behavior of retinal was similar to that of tocopherol derivatives, but varied depending on the polar solvent used. Such a retention sequence of fat-soluble vitamins may be explained on the basis of hydrogen bonding interactions between the active functional group on the solute molecules and silanol groups on the silica gel surface. Based on the adsorption selectivity given by the phase systems used, the resolution of each class of vitamins but not that of vitamin D homologues was successfully carried out.