Anal. Chem. 1999, 71, 3070-3076
Nanoliter-Volume 1H NMR Detection Using Periodic Stopped-Flow Capillary Electrophoresis Dean L. Olson,†,‡ Michael E. Lacey,†,| Andrew G. Webb,§,| and Jonathan V. Sweedler*,†,|
Department of Chemistry, Electrical and Computer Engineering, and the Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Recent advances in the analysis of nanoliter volumes using 1H NMR microcoils have led to the application of microcoils as detectors for capillary electrophoresis (CE). Custom NMR probes consisting of 1-mm-long solenoidal microcoils are fabricated from 50-µm diameter wire wrapped around capillaries to create nanoliter-volume detection cells. For geometries in which the capillary and static magnetic field are not parallel, the electrophoretic current induces a magnetic field gradient which degrades the spectroscopic information obtainable from CE/NMR. To reduce this effect and allow longer analyte observation times, the electrophoretic voltage is periodically interrupted so that 1-min high-resolution NMR spectra are obtained for every 15 s of applied voltage. The limits of detection (LODs; based on S/N ) 3) for CE/NMR for arginine are 57 ng (330 pmol; 31 mM) and for triethylamine (TEA) are 9 ng (88 pmol; 11 mM). Field-amplified stacking is used for sample preconcentration. As one example, a 290-nL injection of a mixture of arginine and TEA both at 50 mM (15 nmol of each injected) is stacked severalfold for improved concentration LODs while achieving a separation efficiency greater than 50 000. Dissolving a sample in a mixture of 10% H2O/90% D2O allows H2O to serve as the nearly ideal neutral tracer and allows direct observation of the parabolic and flat flow profiles associated with gravimetric and electrokinetic injection, respectively. The unique capabilities of CE and the rich spectral information provided by NMR spectroscopy combine to yield a valuable analytical tool, especially in the study of mass-limited samples. A current trend in separation science is toward smaller-scale separations and more information-rich detection modes. Capillary separations can exhibit increased efficiency and are well suited to mass-limited situations. The importance of small-volume separations is growing, so that microbore liquid chromatography (LC) is now common, and capillary LC (cLC) and capillary electrophoresis (CE) have become accepted research tools. As analytes are separated from increasingly complex matrixes, detectors which can provide high information content have become more * Corresponding author. Tel.: 217-244-7359. Fax: 217-244-8068. E-mail:
[email protected]. † Department of Chemistry. ‡ Current address: Magnetic Resonance Microsensors Corp., Savoy, IL 61874. § Electrical and Computer Engineering. | Beckman Institute.
3070 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999
essential. Nuclear magnetic resonance (NMR) spectroscopy possesses a powerful ability to elucidate molecular structure. The use of NMR as a detector for LC continues to increase in importance and is the subject of recent reviews.1-3 Although electrophoretic NMR was used many years ago to measure physical properties such as molecular mobilities,4 no analytical separations per se were presented in that work. In fact, the combination of CE and NMR has been demonstrated only recently.5-8 The principal challenges posed by linking capillary separations to NMR detection arise from the relatively poor sensitivity of NMR and the short observation times available to measure flowing analyte bands. Thus, methods to enhance NMR sensitivity are required to make practical the coupling of these separation methods to NMR. Although the use of increased static magnetic field strengths (B0) represents the most common approach to augmenting NMR sensitivity, significant design challenges and extremely high costs associated with superconducting magnet development accompany these gains. A complementary method of sensitivity enhancement utilizes miniaturized (10 Hz. High-resolution NMR was subsequently demonstrated using improved microcoil probe designs11-13 which yielded line widths of
