Distinction of synthetic dl-α-tocopherol from natural vitamin E (d-α-tocopherol) by reversed-phase liquid chromatography. Enhanced selectivity of a polymeric C18 stationary phase at low temperature and/or at high pressure

• Distinction of synthetic dl-α-tocopherol from natural vitamin E.
• Separation of dl-α-tocopherol diastereomers by reversed-phase LC.
• Enhanced selectivity at low temperature and/or at high pressure for this system.

Separation of diastereomers of dl-α-tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3 mmID, 20 cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic dl-α-tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0 °C, 536 bar, while three peaks were observed when the separation was carried out either at 12.0 °C at low pressure or at 20 °C at 488 bar. Higher temperature (30.0 °C) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0 °C and 441 bar, although the stationary phase afforded greater resolution for β- and γ-tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0 °C and 463 bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of dl-α-tocopherol.