Dimuthu A. Jayawickrama

Hyphenation of capillary separations with nuclear magnetic resonance spectroscopy

The hyphenation of small-volume separations to information-rich detection offers the promise of unmatched analytical information on the components of complex mixtures. Nuclear magnetic resonance (NMR) spectroscopy provides information about molecular structure, although sensitivity remains an issue for on-line NMR detection. This is especially true when hyphenating NMR to capillary separations as the observation time and analyte mass are decreased to the point where reduced information is obtained from the eluting analytes. Because of these limitations, advances in instrumental performance have a large impact on the overall performance of a separation-NMR system. Instrumental aspects and the capabilities of cLC-NMR, CEC-NMR and CE-NMR are reviewed, and applications that have used this technology highlighted. Recent trends towards small volume capillary scale separations are emphasized, as is the recent success of capillary-isotachophoresis (cITP)-NMR.

Mobile phase compensation to improve NMR spectral properties during solvent gradients

A solvent compensation method based on flow injection analysis is used to obtain high quality nuclear magnetic resonance (NMR) spectra during solvent gradients. Using a binary solvent system containing D2O and CD3OD, NMR line broadening and chemical shift changes are observed with a 10% methanol per min solvent composition gradient. However, by creating a second equal but reverse gradient and combining the two solvent gradients before the NMR detector, the composition of solvent reaching the NMR flow cell is kept constant. We demonstrate a system using flow injection analysis of combining solvent gradients and show constant NMR spectral performance as a function of time as the combined flow has a constant solvent composition irrespective of the initial solvent gradient. Using this approach, methods can be developed to measure high quality NMR spectra during on-flow gradient LC-NMR experiments. The ultimate ability of this approach depends on the ability to compensate for the disturbance of the solvent gradient and reverse gradient by a pair of LC columns (the analytical and reverse gradient columns).

Protein Dynamics Study Using a Continuous-Flow Microfluidic Mixer

A major goal ofprotein dynamics studies is to obtain structural and kinetic information on intermediate states. Under the continuous flow, the conversion of a reaction time coordinate (time after mixing) to a stable (in time) distance coordinate well suited to spectroscopy that requires the accumulation of spectra, such as NMR and FT-IR. Although stopped-flow method was usually used for protein dynamics studies, this continuous flow method could be an alternative. The feasibility of this technique is demonstrated by measuring the native to A-state transition kinetics of ubiquitin (Ub), a well-characterized protein with 76 amino acid residues by both NMR and FT-IR.