Jens Schewitz

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.

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-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.

Direct coupling of capillary electrophoresis and nuclear magnetic resonance spectroscopy for the identification of a dinucleotide

SummaryCoupling of capillary electrophoresis (CE) with nuclear magnetic resonance (NMR) spectroscopy has been achieved by use of a recently developed capillary NMR interface. The technique has been used for the analysis of synthetic nucleic acids. A crude adenosine dinucleotide synthesized by solid-phase chemistry has been investigated by continuous-flow CE-NMR. The combination of the separation technique and NMR spectroscopy proved well suited to identification of the main components, and to detection of by-products of the synthesis of the nucleotide. Two-dimensional CE-NMR spectra were also acquired in the stopped-flow mode and the TOCSY NMR spectrum thus acquired afforded confirmatory evidence for the structure of the adenosine dinucleotide. All experiments were performed in fully deuterated solvents and using nanoliter amounts of the dinucleotide sample.

Peer Reviewed: On-Line Coupling of Capillary Separation Techniques with 1H NMR.

Improved sensitivity, deuterated solvents, and less sample make this approach feasible for many applications.

OPTIMIZED LARGE SCALE SYNTHESIS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES ON PS-PEG SUPPORTS

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 μmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG