Li-Hong Tseng

On-Line Coupling of Capillary Electrochromatography, Capillary Electrophoresis, and Capillary HPLC with Nuclear Magnetic Resonance Spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.

Capillary HPLC−NMR Coupling: High-Resolution 1H NMR Spectroscopy in the Nanoliter Scale

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.

Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance.

Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous‐flow CEC‐NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE‐NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two‐dimensional (2‐D) spectrum of the synthetic peptide aspartame.

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.

Metabolite Profiling of Human Amniotic Fluid by Hyphenated Nuclear Magnetic Resonance Spectroscopy

The metabolic profiling of human amniotic fluid (HAF) is of potential interest for the diagnosis of disorders in the mother or the fetus. In order to build a comprehensive metabolite database for HAF, hyphenated NMR has been used, for the first time, for systematic HAF profiling. Experiments were carried out using reverse-phase (RP) and ion-exchange liquid chromatography (LC), in order to detect less and more polar compounds, respectively. RP-LC conditions achieved good separation of amino acids, some sugars, and xanthines. Subsequent NMR and MS analysis enabled the rapid identification of 30 compounds, including 3-methyl-2-oxovalerate and 4-aminohippurate identified in HAF for the first time, to our knowledge. Under ion-exchange LC conditions, a different set of 30 compounds was detected, including sugars, organic acids, several derivatives of organic acids, and amino acids. In this experiment, five compounds were identified for the first time in HAF: D-xylitol, amino acid derivatives (N-acetylalanine, N-acetylglycine, 2-oxoleucine), and isovalerate. The nonendogenous nature of some metabolites (caffeine, paraxanthine, D-xylitol, sorbitol) is discussed. Hyphenated NMR has allowed the rapid detection of approximately 60 metabolites in HAF, some of which are not detectable by standard NMR due to low abundance (microM) and signal overlap thus enabling an extended metabolite database to be built for HAF.

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

Comparison of the separation of cis/trans isomers of tretinoin with different stationary phases by liquid chromatography-nuclear magnetic resonance coupling

Abstract The comparison of two different types of RP stationary phases in their ability to separate cis/trans isomers of retinoic acid (tretinoin) has been investigated by LC-NMR coupling. Only by recording of 1 H NMR spectra the structural identification of the separated compounds was possible, since their absorption coefficients are very similar and their mass is identical, and therefore identification by UV-Vis is not unambiguous and identification with LC-MS fails due to identical fragmentation patterns. A commonly used C 18 phase and a recently developed C 30 phase have been used for the separation of a mixture of thermal isomerized retinoic acids. Three isomers could be separated and identified with the separation on a C 18 column, whereas five cis/trans isomers could be identified by the use of a C 30 column. It could be shown that even under optimized chromatographic conditions the separation efficiency of the two phases varies and that these differences are probably due to differences in the alkyl chain organization of the stationary phases.

On-flow identification of metabolites of paracetamol from human urine using directly coupled CZE–NMR and CEC–NMR spectroscopy

Direct NMR spectroscopic detection on-flow to capillary electrophoresis (CE) or capillary electrochromatography (CEC) was applied to the separation of metabolites of paracetamol from an extract of human urine. The detection and characterisation of the major metabolites, the glucuronide and sulfate conjugates of the drug as well as identification of the endogenous material hippurate was achieved. This demonstrates that NMR detection and identification of drug metabolites is possible with nanolitre volumes of analyte.