New Directions in Electrophoretic Methods ^
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lectrophoresis is the most powerful process available for the separation and analysis of complex mixtures of charged biopolymers. This new book offers scientists the latest developments in elec trophoretic separations and state-of-the-art electrophoretic technology and applications. It covers both the basic theory and devel opment of electrophoretic methods and the separation and identification tools used by developers. The sixteen chapters in this book focus on several electrophoretic methods: • isotachophoresis
• zone electrophoresis • isoelectric focusing • two-dimensional electrophoresis • pulsed electrophoresis
Specific topics discussed with these meth ods include: • • • • •
polyacrylamide gels immobilized pH gradients silver-stain detection of proteins agarose gels synthetic ion-containing polymers
Electrophoretic methods play a major role in scientific advances in medicine, agricul ture, chemistry, and biotechnology. This book will prove a valuable reference for anyone working with electrophoretic methods. James W. Jorgenson, Editor, University of North Carolina Marshall Phillips, Editor. U.S. Department of Agriculture Developed from a symposium sponsored by the Divi sions of Agricultural and Food Chemistry and Analyti cal Chemistry of the American Chemical Society ACS Symposium Series No. 33S
284 pages (1987) Clothbound LC 87-1777 ISBN 0-8412-1021 -7 US & Canada $64.95 Export $77.95 Order from: American Chemical Society Distribution Office Dept. 52 1155 Sixteenth St. N.W. Washington. DC 20036 or CALL TOLL FREE
800-227-5558 and use your credit card!
Raman instruments is the difficulty in obtaining high-resolution data. To work at high spectral resolution, very narrow slits and high dispersion grat ings are required. Under these condi tions, the throughput of the instru ment falls drastically. A number of nonlinear techniques have been suc cessfully applied to the problem of high resolution, but the problems of exces sive background and photodecomposition still dominate the practice of Ra man spectroscopy. Recent work by Jennings et al. (2) has demonstrated that FT-Raman spectroscopy success fully addresses the problem of limited resolution in gas-phase studies. In such an experiment, however, the Rayleigh line is relatively weak and there is no fluorescent background. Many approaches have been taken in an attempt to minimize the fluores cence problem. Temporal-based tech niques, in which the difference in time scale between Raman scattering and fluorescence is exploited, have been promising, but they are not universally applicable. In addition, temporalbased techniques do hot effectively al leviate the photodecomposition prob lems. The traditional drench-quench method, in which the sample is irradi ated with the laser for an extended time in an effort to photobleach the system, is fine if the background arises from an impurity, but if the sample it self fluoresces, this approach is useless. The recent discovery of the lumines cence-quenching properties of a silver surface (3) can be exploited in many cases, but the film deposition proce dure is not always amenable to all sam ples. Nonlinear experiments such as coherent anti-Stokes Raman scattering provide a high degree of fluorescence discrimination, but again, they are not universally applicable—especially to solid samples. Because the fluorescence and the photodecomposition processes have certain minimum energies associated with them, the most logical approach would be to reduce the energy of the photons striking the sample to a value lower than the threshold for excitation. In this way, the first excited electronic state (of the sample or of an impurity) would never be populated. This ap proach has often been discussed in the past. The krypton laser was supposed to have been the answer to the fluores cence problem because it provided a strong line at 6471 A. Unfortunately, the Raman effect itself is wavelengthdependent, and the cross section for scattering falls off as l/λ 4 . In addition, it often appears that excitation in the red is still sufficient to produce fluores cence at a reduced level. The excitation probably occurs through hot band-as sisted processes. The overall gain in the
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Raman-to-fluorescence ratio is not suf ficient. To completely avoid the excitation process, Hirschfeld suggested that Ra man spectroscopy performed with a Nd-YAG laser might be the answer. This laser operates at 1.06 μιη or 9395 cm -1 , which should be well below the threshold for any fluorescence process. Unfortunately, the cross section for Raman scattering at 1.06 μια is down by a factor of 16 from that at 5145 À. An additional problem is the lack of good detectors (i.e., shot noise limited) comparable to a photomultiplier. F T - R a m a n instrumentation
To compensate for the loss in cross section and the poor detectors, a multiplexing instrument would be required. This is the basic argument for attempting FT-Raman spectroscopy. No drastic improvement in performance over a conventional system operating in the visible is expected unless there is a background present. Then the FT-Raman instrument operating at 1.06 /urn should allow the acquisition of spectra, whereas the instrument operating at 5145 À fails completely. The basic FT-Raman instrument is similar to a conventional grating instrument in that the scattered light must be collected and then passed through a spectrometer. The collection optics in an FT-Raman experiment serve the same purpose as in a conventional Raman experiment. We need to collect as much light as possible for analysis. There are, however, some constraints. The half-angle divergence of the collected beam must not exceed the resolution requirements of the interferometer, or the collected beam must be passed through the limiting aperture stop of the interferometer. This condition is easily met for low-resolution experiments (1-4 cm - 1 ). The second constraint involves the type of optical element employed. Normally, collection of scattered light is accomplished using lenses that have a wavelength-dependent chromatic aberration. In the visible region, the chromatic aberrations are usually acceptable because the entire Raman spectrum may cover only 0.23 μτα. In the near-IR the Raman spectrum would span close to 1 μια in wave length, lenses would not be able to col lect and refocus all wavelengths to the same point, and we would have a wave length-dependent distribution of in tensities across the detector. However, recent work by Rabolt, Zimba, and Hallmark (4) has shown that with the proper choice of optical materials the chromatic aberration problem is mini mal, and excellent results are obtained using lenses as collection elements. Our initial approach utilized a para bolic mirror with a hole at the apex
