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ANALYTICAL CURRENTS
RPLC/CZE/MS and the complex biological sample Because they are so complex, biological samples often overwhelm the separating capabilities of analytical techniques. Even powerful hyphenated methods such as LC/MS can fail to fully resolve difficult samples. James Jorgenson and his colleagues at the University of North Carolina (Chapel Hill) and Glaxo Wellcome have gone the extra step and combined three methods— reversed-phase liquid chromatography (RPLC), capillary zone electrophoresis (CZE), and mass spectrometry—to develop an on-line system that is fast and has significant resolving power. The reported system differs from "heart cut" techniques because the RPLC fraction is sampled repeatedly by CZE; in a 15-min RPLC run, the effluent is sampled 60 times by the CZE instrument. The RPLC column is coupled to the CZE instrument by a transverse flow-gating interface. The CZE, in turn, is connected to the mass spectrometer with a newly developed microelectrospray needle that allows the electrophoresis to be run at a high pH and still electrospray from a pH 3 buffer. Because the ideal way to separate peptide mixtures by CZE is on bare fused silica at pH > 10 whereas electrospray requires an acidic pH for protonation this new microelectrosprav needle combines the best of two worlds. The authors demonstrate the new system with peptide standards and tryptic digests of ribonuclease B. (J. Am. Soc. Mass Spectrom. 1997,8,495-500)
Schematic design of RPLC/CZE/MS. (Adapted with permission from Elsevier Science.)
Enhanced NMR of biomolecules Numerous signal-enhancement schemes for NMR spectroscopy have proven to be successful for specific systems. However, to date, there is no method that is generally applicable to macromolecular biological systems. Robert G. Griffin and colleagues at the Massachusetts Institute of Technology and the University of Oregon investigated one approach that might fit that description. Dynamic nuclear polarization (DNP) transfers the high-spin polarization of unpaired electrons to coupled nuclear spins through microwave irradiation at or near the electron paramagnetic resonance frequency. In this study, the analyte was placed in a frozen 60:40 glycerol-water solution doped with the nitroxide free radical 4-amino-TEMPO. Polarization is transferred from the nitroxide free radical and distributed throughout the solvent and solute. With this approach, arginine and the 18.7-kD protein T4-lysozyme were investigated using a DNP-cross-polarization NMR pulse sequence with magic-angle spinning. Samples were spun at speeds of up to 5 kHz and cooled to temperatures as
Using surfaceenhanced methods for chemical sensing
Top: DNP-cross-polarization spectra of 15 N-alanine-labeled T4 lysozyme after 64 acquisitions averaged with a 15-s recycle delay; bottom: identical conditions but no microwave irradiation. (Adapted with permission from the American Association for the Advancement of Science.)
low as 25 K. A custom-designed gyrotron produced 100 W of 140-GHz irradiation at 5 T. Signal enhancements of up to ~ 100 were observed in the solid-state NMR spectra. According to the authors, the technique could become a general approach for high-resolution solid-state NMR signal enhancement of biomolecules. (Science 1997,276, 930-32)
tection of the model compound p-nitrobenzoic acid and several aromatic and chlorinated pesticides. SEIRA was performed by placing the analyte on top of thin metal films of needle-like substrates that were deposited on single attenuated total-reflectance (ATR)
On-site, real-time monitoring has long been a goal of environmental analysis. Vibrational snectrosconic methods are attractive for such use, especially if their sensitivity can be improved. R. Kellner and coworkers at Vienna University of Technology (Austria) have demonstrated improved sensitivity with surface-enhanced IR absorption (SEIRA) and Principle of ATR spectroscopy using surface-enhancement surface-enhanced techniques. Metal clusters are deposiied on the ATR crystal, ,nd Raman spectroscopy a thin layer of analyte covers the surface. (Adapted with (SERS) for the depermission from the Society for Applied Spectroscopy.)
Analytical Chemistry News & Features, July 1, 1997 395 A
