Ulrich Braumann

Characterization of the aromatic composition of some liquid foods by nuclear magnetic resonance spectrometry and liquid chromatography with nuclear magnetic resonance and mass spectrometric detection

Abstract This paper describes the first application of NMR spectroscopy and LC-NMR/MS to the direct analysis of the aromatic composition of beer, grape juice and a wine phenolic extract. 1 H NMR spectroscopy provides non-invasive information on the overall aromatic profile and enables the identification of some compounds. However, a more comprehensive assignment is hindered by the low peak intensity and strong signal overlap in the low-field spectral region, as well as by the inherent lack of scalar coupling information for many aromatic compounds present. LC-NMR/MS can overcome these problems and is shown to aid significantly in the identification of aromatic compounds composing all samples analyzed. Some examples are the identification of several cinnamic acids (e.g. p-coumaric, trans-coutaric and trans-caftaric) in grape juice, the identification of 2-phenylethanol, tyrosol and tryptophol in beer and the detection of phenolics such as catechin, epicatechin, trans-resveratrol, tyrosol and caffeic acid in the wine extract.

On-line coupling of separation techniques to NMR

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.

Direct On-Line Coupling of Capillary HPLC with 1H NMR Spectroscopy for the Structural Determination of Retinyl Acetate Dimers: 2D NMR Spectroscopy in the Nanoliter Scale

This paper presents the application of directly coupled capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance spectroscopy (NMR) for structural elucidation of a so-far unknown kitol isomer. One- and two-dimensional continuous- and stopped-flow NMR spectra were recorded in a 180 μm i.d. capillary, corresponding to a detection volume of only 200 nL. Unequivocal structural assignment on the basis of 1D and 2D stopped-flow capillary HPLC-NMR experiments was performed. The kitol isomer mixture was present in a sample of thermally isomerized retinyl acetate and separated on a capillary column.

Application of on-line capillary high-performance liquid chromatography-nuclear magnetic resonance spectrometry coupling for the analysis of vitamin A derivatives

Abstract The direct on-line coupling between capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance (NMR) spectrometry has been used to derive structural information about constituents of a mixture of vitamin A derivatives. 1 H NMR spectra were recorded in the stopped-flow and continuous-flow mode within a 180 μm I.D. capillary column mounted in a micro probe on a 600 MHz NMR spectrometer. The resolution of the 1 H NMR spectra obtained in capillary HPLC-NMR coupling experiments is sufficient to determine coupling constants in the order of 1.5 Hz. The detection limit is in the lower nanogram range. A stopped-flow 2D-TOCSY experiment of a 1% solution of vitamin A acetate acquired within 4 h reveals that the acquisition of 2D NMR spectra is possible in the nanoliter detection scale without any loss of structural information.

Separation and identification of vitamin A acetate isomers by supercritical fluid chromatography—1H NMR coupling

Abstract Unlike HPLC-NMR coupling, the combination of supercritical fluid chromatography (SFC) with NMR spectroscopy is not yet a routine method, but the feasibility of this coupling technique has already been demonstrated with synthetic mixtures. In this paper the first real life application is presented and the potential of this new method is shown. A mixture of five cis/trans isomers of vitamin A acetate is separated by SFC using supercritical CO 2 as eluent. Therefore no solvent signal suppression is necessary and the unrestricted observation of the whole spectral range is possible, contrary to the HPLC separation in n -heptane, where in the aliphatic region almost 2 ppm of the 1 H NMR spectrum are affected by the suppression technique.

Application of Directly Coupled High-performance Liquid Chromatography–Nuclear Magnetic Resonance–Mass Spectrometry to the Detection and Characterisation of the Metabolites of 2-Bromo-4-trifluoromethylaniline in Rat Urine

Directly coupled HPLC–NMR–MS was used to identify the major urinary metabolite of 2-bromo-4-trifluoromethylaniline in the rat as the sulfate conjugate of 2-bromo-4-trifluoromethyl-6-hydroxyaniline. This metabolite could not have been fully characterised using HPLC–NMR or HPLC–MS alone as the sulfate group is ‘NMR silent’ and MS would not have enabled the position of substitution on the aromatic ring to have been assigned. The need to carefully select HPLC solvents for directly coupled HPLC–NMR–MS so that NMR and MS data can both be obtained is also shown.

Nuclear magnetic resonance monitoring of centrifugal partition chromatography in pH-zone-refining mode

The coupling of centrifugal partition chromatography to NMR spectroscopy as detection and analysis method is investigated. Separation of a test mixture of three N-2,4-dinitrophenyl amino acids was performed in pH-zone-refining elution mode. The NMR and chromatography instrumentation was very similar to the one of the LC-NMR technique.

Application of direct on-line coupling of HPLC and SFC with 1HNMR spectroscopy for the investigation of monomeric acrylates

The separation of monomeric acrylates was performed with High Performance Liquid Chromatography (HPLC) and Supercritical Fluid Chromatography (SFC). A direct on-line structural assignment of all compounds by continuous-flow 1HNMR spectroscopy is possible with both separation techniques. The direct SFC-NMR coupling offers the advantage that the recorded continuous-flow 1HNMR spectrum is not obscured by solvent signals.

Chapter 5:Application of LC-NMR to the Study of Natural Products

NMR spectroscopy has been applied for many years to perform structure elucidation on pure compounds. Therefore, prior to NMR analysis, it was necessary to separate mixtures by means of extraction and preparative chromatography. Such procedures required large amounts of material and a chromatographic separation good enough to produce a more or less pure compound, a situation that often needed multiple chromatographic steps. In addition, NMR sensitivity required milligram amounts in order to be able to run 2D heteronuclear experiments, the cornerstone of structure elucidation. Over the years, NMR sensitivity has been enhanced by improving probehead technology and increasing magnetic field strength. With the introduction of cryogenic probes, a major enhancement in signal-to-noise ratio was achieved, commonly a factor of 4 in most solvents used. It is now possible to run the relevant experiments for structure elucidation by NMR in the low microgram range. This chapter shows how LC-NMR can be integrated into the structure verification and identification of natural product mixtures. It describes various tools that allow the increase of NMR sensitivity in such a way that <1 μg components in the active volume can be accessed by NMR. Additionally, this chapter provides examples where these tools can be applied to natural product-related samples.