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

Abstract

Highly selective reversed phases (C(30) phases) are self-packed in 250 microm 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 microg of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow (1)H 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 (1)H,(1)H COSY NMR spectrum of alpha-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.

Building-Up a Comprehensive Database of Flavonoids Based on Nuclear Magnetic Resonance Data

“…Assignments of protons and carbons within each measured flavonoid (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) were made by data analysis of the 1D and 2D NMR spectra.…”

“…It combines the separation over a wide range of polarities (LC) with photo-spectrometric information (PDA), molecular mass value (MS) and full structural elucidation capabilities (NMR). Specifically, LC-solid phase extraction-NMR (LC-SPE-NMR) [5,6] and capillary LC-NMR (capLC-NMR) [7] methods, which have been developed recently, improve the isolation and efficiency of identification of compounds present in mixtures.…”

Magnetic Resonance Microimaging and Numerical Simulations of Velocity Fields Inside Enlarged Flow Cells Used for Coupled NMR Microseparations

“…The total data acquisition time was 6 h. In this study, a flow rate of 5 µL/min was used, since this is a typical value used in cLC separations. 11 For the flow cells used in the experiment, this flow rate corresponds to a residence time of ~6 s for an rf coil 2 mm in length, such as might typically be used for microseparations. Assuming a static line width of 1 Hz, the additional linebroadening introduced by flow, as described by eq 3, is negligible being only ~0.05 Hz.…”

Analysis of mass-limited mixtures using supercritical-fluid chromatography and microcoil NMR

“…3). 18,20 Fraction (i), with three methyl groups, is unambiguously α-tocopherol, and fraction (iv) must contain δ-tocopherol -it is noted that the apparent lack of splitting in the two aryl proton resonances is confirmation that these nuclei are not adjacently located on the ring. Fractions (ii) and (iii) each contain one of the aromatic hydrogens in (iv) and therefore must be β-tocopherol and γ-tocopherol.…”

“…This is significantly faster than normal LC, where complete separation of the fractions requires some tens of minutes. 18 Fig . 2 shows how the UPC 2 instrument was modified for collection of the separated fractions.…”