Dean L. Olson (first row, right) completed his Ph.D. degree in Analytical Chemistry from the University of Illinois in 1994. He then joined the academic research group of Professor Sweedler performing the first high-resolution and sensitivity studies with nanoliter-volume NMR microcoils. He also conducted the first experiments using high-resolution NMR as a detector for capillary electrophoresis. He is now employed at MRM Corp. (Savoy, IL) conducting research toward the commercial development and application of capillarybased NMR probes. Andrew G. Webb (second row, right) received his Ph.D. degree in Medicinal Chemistry from the University of Cambridge in 1990 under Professor Laurie Hall. He was a postdoctoral researcher in the Department of Radiology at the University of Florida before joining the faculty in the Department of Electrical and Computer Engineering at the University of Illinois at Urbana−Champaign.He is currently an Associate Professor, with a full appointment in The Beckman Institute for Advanced Science and Technology. His research areas include the design of microcoils for NMR of mass-limited samples, the use of MRI temperature mapping for hyperthermia, and human brain mapping using functional MRI. Jonathan V. Sweedler (second row, left) received his Ph.D. degree in Analytical Chemistry from the University of Arizona in 1989 under Professor M. Bonner Denton and then spent 3 years at Stanford with Professors Richard Zare and Richard Scheller developing new methods to study neurotransmitters in individual neurons. He is currently a Professor of Chemistry, Neuroscience, and of the Beckman Institute for Advanced Science and Technology at the University of Illinois. His current research interests are twofold: first, he is developing information-rich methods with improved mass sensitivity for nanoliter-volume samples, including microcoil NMR, mass spectrometric imaging, and capillary-scale separations. In addition, he applies these techniques to understanding the role of neurotransmitter and neuropeptide co-transmission and the regulation of behavior in well-defined neuronal networks of opisthobranch molluscs.

An improved nanoliter-volume NMR probe design places the microcoil and capillary at the magic angle (57.7 degrees) with respect to the external magnetic field. Using an NMR probe which requires a total sample volume of just 200 nL, high-resolution 300-MHz 1H-NMR spectra (line width, 0.6 Hz) are presented of 10 mM alpha-bag cell peptide for an observe quantity of 45 ng (50 pmol in 5 nL). For the volume of sample inside the microcoil (the observe volume, Vobs), the 3 sigma limit of detection (LOD) is 9 ng (10 pmol, 2mM) for data obtained in 15 h. To reduce the data acquisition time, a probe with a greater Vobs is developed. As an example of a rapid, mass-limited analysis, a concentration corresponding to 400 ng of menthol dissolved in Vobs = 31 nL (82.6 mM) yields a spectrum in 9 min (LOD = 6.9 ng, 44 pmol, 1.4 mM). To illustrate improvements in concentration sensitivity, a spectrum is acquired in 45 min for 400 ng of menthol dissolved in a total sample volume of 200 nL (12.8 mM). Compared to a commercial nanoprobe for the same mass of menthol, these two examples reduce data acquisition time by at least 95%. Both model compounds demonstrate substantially improved concentration LODs compared to those obtained in previous high-resolution, microcoil NMR work. These advances illustrate the utility of enhanced sensitivity provided by NMR microcoils applied to nanoliter volumes of mass-limited samples.

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-micron 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.

Nanoliter-volume proton nuclear magnetic resonance (NMR) spectroscopy is used to monitor the electrolyte temperature during capillary electrophoresis (CE). By measuring the shift in the proton resonance frequency of the water signal, the intracapillary temperature can be recorded noninvasively with subsecond temporal resolution and spatial resolution on the order of 1 mm. Thermal changes of more than 65 degrees C are observed under both equilibrium and nonequilibrium conditions for typical CE separation conditions. Several capillary and buffer combinations are examined with external cooling by both liquid and air convection. Additionally, NMR thermometry allows nonequilibrium temperatures in analyte bands to be monitored during a separation. As one example, a plug of 1 mM NaCl is injected into a capillary filled with 50 mM borate buffer. Upon reaching the NMR detector, the temperature in the NaCl band is more than 20 degrees C higher than the temperature in the surrounding buffer. Such observations have direct applicability to a variety of studies, including experiments which utilize sample stacking and isotachophoresis.

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