Hyphenation of Capillary HPLC to Microcoil 1H NMR Spectroscopy for the Determination of Tocopherol Homologues

Abstract

Highly selective reversed phases (C₃₀ phases) are self-packed in 250 μm inner diameter fused-silica capillaries and employed for capillary HPLC separation of shape-constrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC−ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLC- NMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 μg of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow ¹H NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D ¹H,¹H COSY NMR spectrum of α-tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample.