News
Toxins ring loud and clear Detecting toxic gases in the environment remains a major concern. Laser-induced fluorescence (LIF) and similar techniques are popular but do not apply to all classes of molecules. In particular, LIF cannot be used for chlorinated aromatic molecules because of fast intersystem crossing that reduces the fluorescence quantum yield. R. Vasudev and colleagues at Mississippi State University propose the use of cavity ringdown spectroscopy (CRDS) and demonstrate its application to the detection of nitrogen dioxide and four chlorinated aromatic compounds. In CRDS, a laser pulse is trapped between two mirrors in a low-loss optical cavity. The long effective absorption pathlength allows small concentrations of toxins to be detected. Long pathlengths also can be achieved with conventional multipass absorption spectroscopy, but the researchers say it is easier with CRDS. Using a preliminary setup, Vasudev and colleagues have obtained a detection limit of several parts per million for nitrogen dioxide and the four chlorinated aromatics. The detection limits of the aromatics were independent of the extent and the sites of chlorination. With improvements, they say, the detection limits for nitrogen dioxide and chlorinated aromatics could be reduced significantly. The researchers add that CRDS provides absolute absorption cross sections making the technique self-calibrating and they conclude that CRDS has the potential to be a universal detector for gaseous toxins including dioxins or dioxin surrogates {Environ Sci Technol 1999 33 1936-39)
Cavity ringdown results (1/x versus pressure) for chlorobenzene at A. = 266 nm, d = 23.8 cm, and R = 0.9803.
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Combinatorial libraries by NMR There is nothing like an NMR spectrum to characterize an organic system in solution. But can this powerful method make the leap to the high-throughput demands of combinatorial chemistry? Bernd Meyer and co-workers at the University of Hamburg (Germany) demonstrate a promising new NMR method that can identify a ligand that binds to a target protein on a solid support within a mixture of potential ligands. The authors demonstrated their new method, saturation transfer difference (STD) NMR, by screening a mixture of seven oligosaccharides for their affinity to wheat germ agglutinin (WGA), a 36-kDa protein. WGA W3.S coupled to several solid phases of which controlled-pore glass turned out to work the best. Because this is a heterogeneous system STD NMR was combined with susceptibility-matched high-resolution magic angle spinning NMR
STD selectively saturates resonances of the receptor protein, leading to intramolecular transfer of the saturation throughout the protein and to bound ligands. Eventually all the ligand molecules are saturated because of the fast exchange of ligands. With all the bound ligands and protein saturated, the only peaks left are those of unbound ligands. The STD spectrum is then subtracted from an NMR spectrum of the unsaturated spectrum. The result is that only the bound ligand spectrum is left. In the example with WGA, N A^'-diacetylchitobiose was identified as the remaining spectrum and the bound ligand. A one-dimensional STD spectrum takes less than 5 min to record. However, the method so far does not work well with proteins on solid supports other than succinamidopropyl-derivatized controlled-pore glass, and even then problems occurred with molecules smaller than 250 Da. Current work is under way to remove this obstacle. (J. Am. Chem. Socc.999,121, 5336-37)
Pump-probe goes local
fa) Normal NMR spectrum of seven oligosaccharides in the presence of WGA on controlled-pore glass, (b) the STD spectrum of the same mixture, (c) a spectrum of N, N'-diacetylchitobiose for comparison, and (d) the STD spectrum of the seven oligosaccharides in the presence of just controlled-pore glass, which shows no spectrum and, hence, no binding.
Analytical Chemistry News & Features, August 1, 1999
Far-field pump-probe experiments can provide information about the dynamics of a system, but like all far-field methods, their spatial resolution is diffractionlimited. B. A. Nechay and co-workers at the Swiss Federal Institute of Technology Zurich propose to get around the diffraction limit by combining femtosecond pump-probe techniques with nearfield scanning optical microscopy (NSOM). Defining far-field as "global" and near-field as "local", Nechay and coworkers chose to combine pump-probe techniques and NSOM as global pump/ local probe because greater excitation intensity can be applied in the far field than in the near field. Their detection scheme allows degenerate (the pump and the probe pulses are the same wavelength) or two-color excitation. For the NSOM, S/N considerations led the researchers to choose illumination mode rather than collection mode and a transmission geometry rather than a reflection geometry The input pump and probe pulse trains were modulated at 1 and 1.05 MHz, re-
Diffusion NMR—be careful It seems like a straightforward idea: The diffusion coefficient of a molecule, such as a protein, changes when another molecule binds to it. As a result, diffusion NMR, which measures the diffusion coefficient noninvasively, has emerged as a powerful technique for identifying interacting systems and providing data to calculate binding constants. However, as Michael Shapiro and Aidi Chen of Novartis Pharmaceuticals report, an unintended nuclear Overhauser effect (NOE) can compromise the diffusion coefficient measurement. The problem arises during the diffusion time window (A) part of the NMR experiment (a stimulated echo-type of experiment)—when the diffusion is monitored. The magnetization is stored in the z direction during this window, and intermolecular NOE can take place, affecting the NMR signal decay and the subsequent results. Under conditions of slow-motion limit, strong NOE can build up rapidly during the A.
spectively. The probe transmission signal was measured with lock-in detection at the 50-kHz difference frequency. Unfortunately, the unaffected probe and back-reflected pump beams can affect the measurement by increasing the noise. The back-reflected pump beam caused greater problems, and two measures were taken to reduce its effects.
The Novartis scientists demonstrated the problem by running a series of diffusion experiments with human serum albumin and benzoic acid, a ligand known to bind to the protein. When the A is short, NOE is nol a problem, and an appropriate diffusion coefficient is determined. But, as A increases, the NOE affects the measurement, yielding diffusion coefficients that are less than 50% of the value determined at the shorter time. To further confirm the effect of the NOE the researchthe diffusion NMR Diffusion measurements with benzoic acid (H26), periment using the carboxyl showing the effect to NOE oo the proton signals as a carbon of benzoic acid which increases. The plotted data ara expected to be linear experiences little or no NOE with a slope yielding the diffusion coefficient; curved interactions In this case the lines indicatt the eresence of NOE. 13 C signals showed little variation with changing As nonbinding ligands by looking at how the Although it is a problem, this effect could decay rate is affected by varying A. .J. Am. be used to discriminate binding ligands from Chem. Soc. 1999,121, 5338-39)
Al0 3Gao 7As samples. The only diiffeence between the samples was the ion implantation pattern—200-nm implanted stripes with 400-nm spaces for the first sample and 100-nm stripes with 2-um spaces for the second. A two-dimensional image of the amplitude of the pump-probe signal contains a pattern that matches the periodicity of the implantation pattern. A similar pattern did not appear in the topographic images, indicating that it not a topographic artifact. Measurements indicated that the carrier dynamics in the unimplanted regions of the sample were affected by diffusion, which has previously been shown to affect the decay of carriers in other semiconductor nanostructures. Because of the small scale of the effects, they could not have been seen in far-field measurements. Global pump/local probe configuration (left). Experimental setup (right). AOM is an acousto-optic (Rev. Sci. Instrum. modulator and GVD is group velocity dispersion. (Adapted with permission. Copyright 1999 American 1999 70 2758-64) Institute of Physics.) The pump and probe beams were orthogonally polarized so that the pump could be filtered out while most of the probe was transmitted. In addition, a confocal arrangement consisting of a 50-iim pinhole filtered the llght totside oo f 1.25-um sample area. The instrument was used to measure undoped 80 A single quantum well Ga/As/
Analytical Chemistry News & Features, August 1, 1999
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