Shape Selectivity of C30 Phases for RP-HPLC Separation of Tocopherol Isomers and Correlation with MAS NMR Data from Suspended Stationary Phases

Abstract

Vitamin E (tocopherol) acts in various organisms as the main free radical scavenger. This capacity, which is enhanced by the synergetic effect of vitamin C and carotenes, points to a possible application as anti-tumor agent in chemotherapy. There are several isomeric forms, namely α-, β-, γ-, and δ-tocopherol, having different antioxidative abilities, with α-tocopherol being the most biologically active. Using methanol as eluent and a C₃₀ stationary phase, we achieved complete separation of α-, β-, γ-, and δ-tocopherol and α-tocopherol acetate by RP-HPLC within 14 min. Detection was performed by UV and ¹H NMR spectroscopy. The advantage of NMR is the possibility of structural identification of chromatographic peaks. Also, coeluting peaks are easily recognized. The enhanced shape recognition of the C₃₀ phase has been attributed to the high order of the alkyl chains of the stationary phase. This has, so far, been proven by solid-state NMR spectroscopy. We now introduce the technique of ¹³C MAS NMR spectroscopy of suspended stationary phases. The resulting NMR spectra reveal that, in the presence of weak eluents, like methanol, the overall high order of the C₃₀ chains is slightly altered, whereas in stronger eluents, like MTBE, the alkyl chains possess a higher mobility.