Nanoliter-Volume ¹H NMR Detection Using Periodic Stopped-Flow Capillary Electrophoresis

Abstract

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.

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection

“…As most of the native amino acids absorb only slightly or not at all ultraviolet light (UV), different detection approaches were used in combination with the CZE and MEKC separation techniques. UV and fluorescence or laser-induced fluorescence (LIF) detection after a suitable precolumn, postcolumn and on-column derivatization with a UV chromophore [8][9][10][11][12][13][14] and fluorophore [7,[15][16][17][18], respectively, direct UV detection at a very low wavelength [19,20], indirect UV detection [21], electrochemical detection [22,23], mass spectrometry [24,25], and some other detection techniques [26,27] were applied to the analyses of amino acids.…”

Recent advances in the study of biomolecular interactions by capillary electrophoresis

“…The major drawback is still the low capacity of standard CE systems, equipped with UV detectors to yield specific information about sample components. CE coupled with other detectors, such as CE-MS [132,151], CE-inductively coupled plasma (ICP)-MS [152,153], CE-NMR [154,155], CE-FTIR [156], CERaman [157,158], and CE-X-ray [159] is capable to provide more structural elucidation. At present, however, only the on-line MS technique has a sufficient detection sensitivity for structural identification.…”

Detection in capillary electrophoresis

“…For example, on-line NMR detection was first shown to work with CE by Wu et al [94,95] and has been used for the analysis of peptides [96]. More recently, Sweedler and co-workers [97] have shown that NMR detection can be used in stop-flow CE analysis and achieve nanogram sensitivity for arginine and triethylamine (TEA), 7 ng (330 pmole; 31 mM), and 9 ng (88 pmole; 11 mM), respectively. Although other methods are more sensitive (i.e., LIF), using NMR detection structural information can be obtained.…”