Signal Enhancement in HPLC/Microcoil NMR Using Automated Column Trapping

Abstract

A new HPLC-NMR system is described that performs analytical separation, pre-concentration, and NMR spectroscopy in rapid succession. The central component of our method is the online preconcentration sequence that improves the match between post-column analyte peak volume and the micro-coil NMR detection volume. Separated samples are collected on to a C18 guard column with a mobile phase composed of 90% D 2 O/10% acetonitrile-D3, and back-flashed to the NMR microcoil probe with 90% acetonitrile-D3/10% D 2 O. In order to assess the performance of our unit, we separated a standard mixture of 1 mM ibuprofen, naproxen, and phenylbutazone using a commercially available C18 analytical column. The S/N measurements from the NMR acquisitions indicated that we achieved signal enhancement factors up to 10.4 (±1.2)-fold. Furthermore, we observed that pre-concentration factors increased as the injected amount of analyte decreased. The highest concentration enrichment of 14.7 (±2.2)-fold was attained injecting 100 μL solution of 0.2 mM (~4 μg) ibuprofen.

Zero-field remote detection of NMR with a microfabricated atomic magnetometer

“…We realize pulsed NMR linewidths of Ϸ26 Hz, limited, we believe, by residence time and flow dispersion in the encoding region. Estimates of the fundamental sensitivity limit for an optimized system, assuming a modest 10-kG prepolarizing field, indicate detection limits competitive with those demonstrated by microcoils in superconducting magnets (9)(10)(11)(12)(13)(14). Hence, the technique described here offers a promising solution to NMR of mass-limited samples-for example, in the screening of new drugs-without requiring superconducting magnets.…”

Operating nanoliter scale NMR microcoils in a 1tesla field

“…When samples are mass-limited, reducing the detection volume to match the sample size offers enhanced signal-to-noise ratio (SNR) performance, and significant efforts have been undertaken to perfect high-resolution spectroscopy in very small coils [1][2][3][4][5][6][7][8][9]. The integration of NMR with separation techniques such as liquid chromatography (e.g., [10]) or capillary electrophoresis [11,12] proceeds more naturally when the NMR detection volume can be made compatible with the very small sample volumes and fluid handling tubing typical of the separation step. Researchers have also sought the integration of NMR with microfluidic lab-on-a-chip devices, in which case the NMR detector coil is often formed in a lithographic-type process [13,14].…”

Design and construction of a microcoil NMR probe for the routine analysis of 20‐μL samples

“…Probe SNR was measured as 9.2 for the anomeric proton doublet in a single scan experiment on a sample of 100 mM sucrose. This is approximately a factor of 3 lower in SNR compared with our previous homebuilt microcoil probes (16). Typical spectra are shown in Fig.…”

“…The current SNR of this probe is diminished compared with the commercially available small volume microcoil probes (3,25,38) or those previously constructed in our laboratory (10,13,16). We believe that a significant loss of signal results from the use of the transmission line element.…”

“…Hyphenated microcoil NMR techniques include popular separation methods such as HPLC (15)(16)(17)(18)(19)(20)(21)(22), MS (23), SPE (16,24,25), CE (26,27), cITP (28)(29)(30)(31)(32)(33)(34), and most recently, GC (35). Although the combination of these techniques has been straightforward, there is often a potential loss of SNR and thus information, as only a small percentage of the available sample is located and detected within the probe coil (7).…”