Nominations Sought for Ninth Annual Analytical Chemistry Starter

NEWS

Nominations Sought for Ninth Annual Analytical Chemistry Starter Grant Awards Nominations are currently being solicited for two Analytical Chemistry Starter Grant Awards sponsored by the Society for Analytical Chemists of Pittsburgh. Each award consists of a grant of $10,000 and is given to an assistant professor who is currently working in the field of analytical chemistry. The grants are designed to encourage high-quality, innovative research by new analytical chemistry professors and to promote the training and development of graduate students in this field. Assistant professors who have accepted a college or university appointment since Dec. 31, 1984 are eligible to apply. Application forms can be obtained from Robin Garrell, Department of Chemistry, University of Pittsburgh, Pittsburgh, Pa. 15260 (412-6248580). Completed applications must be received by Feb. 15, 1988. Award winners will be announced on April 1,1988.

Hewlett-Packard Purchases License to Commercialize MAGIC Hewlett-Packard Corp. recently agreed to give Georgia Institute of Technology a percentage of the gross profits on sales in return for the exclusive right to commercialize the Monodispersive Aerosol Generation Interface Combining Liquid Chromatography with Mass Spectrometry (MAGIC). In a research project that started in 1978, Georgia Tech professor of chemistry Richard Browner and coworkers developed a device that interfaces a liquid chromatograph with a mass spectrometer. The work was first presented in ANALYTICAL CHEMISTRY in 1984 (see Anal.

Chem. 1984,56, 2626). The fluid that carries a compound for analysis leaves the chromatograph and enters the interface, where it is directed into a tube in the form of an aerosol jet spray. The droplets in the aerosol are sized uniformly so that the liquid readily evaporates. This process is accomplished by blowing helium gas across the aerosol jet. According to Browner, since Hewlett-Packard began working with MAGIC, company researchers have successfully run about 100 compounds through the interface. Browner and Ross Willoughby, co-inventors of the technology, will receive half of the royalty payments made to Georgia Tech.

Applications Sought for 1988 Pittsburgh Conference Memorial National College Grants Award Program The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Inc., and its cosponsoring technical societies, the Spectroscopy Society of Pittsburgh and the Society for Analytical Chemists of Pittsburgh, are current-

ly soliciting applications for the 15th Pittsburgh Conference Memorial National College Grants Award Program. Based on submitted proposals, at least eight colleges will be selected to receive awards for the purchase of scientific equipment, audio-visual and other teaching aids, and library materials for teaching science at the undergraduate level. The awards consist of a maximum of $2500. To be eligible for an award, a school must meet these criteria: • Enrollment must not exceed more than 2500 students. • No more than 25% of its operating budget can be provided by the state or federal government. (Two-year community colleges sponsored by political subdivisions of a state are not bound by these requirements.) • Funding will not be provided for materials to be used solely for research purposes. • Previous awardee schools are not eligible for an award for a three-year period following receipt of their award. (Thus the 1985,1986, and 1987 awardee schools cannot apply for the 1988 program.) In addition, this award can be used as part of a "matching grant" program for undergraduate studies. Interested faculty members should submit completed applications and proposals (original and three copies of each) to Richard Danchik, The Pittsburgh Conference, Inc., 12 Federal Drive, Suite 322, Pittsburgh, Pa. 15235. The deadline for submission is April 1,1988. Award winners will be announced by May 1, 1988.

For Your Information Scientists at Lawrence Berkeley Laboratory (LBL) have designed and constructed a Raman spectroscopy system sensitive enough to detect monolayer coverages on surfaces. Researchers can study molecules and interfaces under conditions not readily amenable to standard vacuum-based surface analytical techniques and can also obtain one-dimensional compositional images of a material along the path of a narrow illuminating laser beam. Use of an imaging photomultiplier tube as a detector increases the sensitivity of the system by at least one order of magnitude over that of conventional systems while background and associated noise is reduced by several orders of magnitude, according to Kirk Veirs, who developed the system together with Gerd Rosenblatt and Victor Chia. For more information, contact the LBL Public Information Department, 1 Cyclotron Road, Berkeley, Calif. 94720. Molecular Devices Corp. and E.I. du Pont de Nemours and Co. have signed an agreement that will accelerate the development of Molecular Devices' patented biosensor technology. In exchange for licensing fees and R&D funding, Du Pont receives exclusive worldwide marketing rights for the physician's office and hospital bedside diagnostic testing markets. The biosensors involved are silicon-based devices that can detect a wide range of substances, including hormones, drugs, proteins, antibodies, microorganisms, and enzymes from samples as small as a drop of blood. Additional information can be obtained from Gregory Sessler, 3180 Porter Drive, Palo Alto, Calif. 94304.

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 ·• 1297 A

per channel with a spectral coverage of 980 c m - 1 to 5.6 c m - 1 per channel with a 3902 c m - 1 spectrum. Given condensedphase Raman line widths that are typi­ cally 2-5 cm - 1 , the triple spectrograph configuration is appropriate to the task. A particularly exciting new develop­ ment is the discovery of a very narrow notch filter with extremely high rejec­ tion of the laser line (12). This filter comprises a crystalline array of poly­ styrene spheres with lattice parameters that are comparable to the wavelength of light. The laser frequency is Bragg diffracted so t h a t its transmission through the filter is less than 10 - 1 0 while Raman-shifted bands are trans­ mitted with greater than 50% efficien­ cy. Half-band-pass at 10% transmis­ sion is ~40 cm - 1 , so that low-frequency bands are easily accessible. The great advantages of this filter are that the multiple stages of a triple spectrograph with attendant alignment difficulties and low overall transmission can be eliminated, and the large, clear aper­ ture makes it easy to insert into the collection optical train. The great in­ crease in throughput should more than offset the loss in flexibility that a triple spectrograph offers in changing excit­ ing laser lines. Prototypes of this filter have been evaluated, and we hope that a commercial version will be available soon. Several types of detectors supplied by a number of different manufactur­ ers are currently available. The most common are the first-generation inten­ sified vidicon (SIT) and the secondgeneration intensified diode array. Third-generation detectors based on charge-coupled device technology or resistive anode readout of microchannel plate image intensifiers are on the horizon. The SIT tube is essentially a television camera offering two-dimen­ sional imaging. An integral image in­ tensifier boosts the gain of the device so that it can detect approximately two photoelectrons ejected from the photocathode, resulting in a net quantum yield that approaches 10%. The SIT tube may be gated to ~10~ 8 s for timeresolved studies. For very low-lightlevel work it is necessary to cool the detector to —40 °C or so to reduce the target dark current to manageable lev­ els. Unfortunately, however, the gating capability is lost at the lower tempera­ ture. The SIT tube is sensitive to wave­ lengths longer than the glass cutoff at 350 nm, the red response being typical­ ly that of an S-20 photocathode. A scin­ tillator extends the ultraviolet re­ sponse to shorter wavelengths but with a greatly diminished (2%) quantum ef­ ficiency. The second-generation detector, based on solid-state technology, was specifically designed for spectroscopic

applications. The self-scanned diode array is a one-dimensional detector that also uses photodiodes for the con­ version of photons to separated elec­ tron-hole pairs, but the signal is read with on-chip circuitry rather than a scanning electron beam. A microchannel plate image intensifier is used to boost the gain to provide single photoelectron sensitivity. The output of the image intensifier is phosphor-coupled into the diode array. The wavelength response is generally much broader than that of the SIT tube, primarily because there is no glass cutoff in the ultraviolet. Quantum efficiencies as high as 20% at 300 nm are available. The intensified diode array can be gat­ ed to better than 5 ns. To reduce the dark current, the photocathode is cooled to 0 °C and the diode array to - 2 0 °C. The gating ability is unaffect­ ed by cooling. The current generation of multichannel optical detectors pro­ vides up to 1024 channels with a quan­ tum efficiency of 0.1 and an effective dark count rate in the range of 0.1 to 1 counts/s for a detector cooled to —20 °C. The effective dark count rate for a multichannel detector is deter­ mined by taking a pair of scans with a dark detector and subtracting one from the other. The root-mean-square noise is then calculated, squared, and divid­ ed by the total integration time of the two scans to arrive at a dark count rate that is the equivalent of a photomultiplier dark current. This procedure iso­ lates the random thermal fluctuations, which are the real dark current, from the fixed pattern noise and A/D bias, which both subtract out perfectly, and from the readout noise, which is negli­ gible when using extended integration directly on the diode array. These char­ acteristics are such that high-quality spectra can be produced with signal levels as low as 0.1 counts/s in the ab­ sence of background. Although the intensified diode array is currently the most popular detector, two other devices are emerging as po­ tentially competitive alternatives. Proximity-focused microchannel plate image intensifiers have been coupled to resistive anode arrays to produce a twodimensional true photon-counting de­ tector (73). The advantages of this ap­ proach include its imaging capabilities and nearly complete absence of dark current. Unintensified charge-coupled devices (CCDs) look especially promis­ ing for very low-level detection when gating is not required. CCDs offer much higher quantum yields than de­ tectors requiring image intensifiers (as high as 0.7), two-dimensional imaging, and exceptionally low dark currents when cooled to 77 Κ (14). High-sensitivity Raman spectroscopy The dramatic increase in efficiency af­

1302 A • ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

forded by multichannel detection makes it possible to obtain Raman spectra in situations in which conven­ tional techniques require times that are much longer than those permitted by other physical constraints. We dis­ cuss two examples: chemical analysis, which allows very low concentrations of aromatic hydrocarbons to be detected in a mixture, and surface science, which allows submonolayer quantities of ma­ terials to be detected on low-area single crystal surfaces. Asher and co-workers were pioneers in the use of ultraviolet resonance Ra­ man spectroscopy for detection and speciation of trace polycyclic aromatic hydrocarbons (15). This method has many advantages compared with mass spectrometry, which requires signifi­ cant sample preparation, or fluores­ cence, which suffers from matrix ef­ fects such as quenching and interfer­ ences. Raman spectroscopy is chemi­ cally specific, and the resonance-en­ hanced cross sections produced by ul­ traviolet excitation are large enough to allow trace analysis at the 20-ppb level. The strong wavelength dependence of the resonance enhancement can be used for selective emphasis of certain components in a mixture. As a bonus, exciting higher electronic states in this region of the spectrum also eliminates fluorescence, which originates from the lowest excited state and thus is spec­ trally well-separated from the Raman scattering. The experiments are con­ ducted using a tunable pulsed ultravio­ let laser source that comprises a fre­ quency-doubled Nd:YAG laser pump­ ing a dye laser. The dye laser pulses are then either doubled or mixed with the Nd:YAG fundamental to produce radi­ ation tunable from 217 to 450 nm. Typical average powers are a few milli­ watts. The detection system comprised a triple spectrograph and a microchan­ nel plate image intensified photodiode array. Multichannel detection is essen­ tial in this application because the en­ tire spectrum must be examined, and the low average powers available would make the time required in a scanning experiment prohibitively long. Figure 1 shows the U V resonance Ra­ man spectra of naphthalene, anthra­ cene, phenanthrene, and pyrene as 10~6 M solutions in acetonitrile. They were each excited near the maximum in their electronic absorption spectra in this region; exciting wavelengths are given next to each spectrum. These spectra were taken with ~3-10 mW of average power and accumulated for about 10 min. Note the excellent sig­ nal-to-noise ratios and the abundance of distinct spectral features available for speciation. The Raman intensity was shown to be linear over at least 3 orders of magnitude in concentration, and the current detection limit was

Figure 1. UV resonance Raman spectra of polycyclic aromatic hydrocarbons. Shown are naphthalene, anthracene, phenanthrene, and pyrene as 10~5 M solutions in acetonitrile. Average laser power was a few milliwatts at the excitation frequencies listed. Specta were each acquired in about 10 min. Excitation wavelengths are indicated on the figure.

demonstrated to be 20 ppb. By increas­ ing the laser power at the sample and multipassing the beam, it is estimated that this limit could be reduced by at least 2 orders of magnitude, to 20 parts per trillion. These methods have been applied to study the composition of complicated mixtures of hydrocarbons such as coal liquids and demonstrate the power of multichannel Raman spectroscopy in chemical analysis (16). A second example of the importance of multichannel detection in problems involving very small numbers of mole­ cules is the detection of Raman spectra from molecules adsorbed on the sur­ faces of single crystal metals when, even under ultrahigh vacuum, experi­ mental time scales are limited to an hour or so. Raman spectroscopy has been shown to be an especially power­ ful probe of surface and interfacial chemical reactions because of its high chemical specificity and its immunity to the presence of an ambient gas or liquid phase, in contrast to the power­ ful electron spectroscopies of surface science. If the method is to have an impact on the field, however, it must be sufficiently sensitive to detect less than a monolayer of molecules adsorbed on a well-characterized single crystal sur­ face under ultrahigh vacuum. At that level, the method will be comparably sensitive to other surface science tech­ niques with the additional advantages

mentioned above. Submonolayer sensitivity for unenhanced (surface or resonance) Raman spectroscopy is essentially impossible without multichannel detection. For example, the signal expected from a monolayer of benzene adsorbed lying flat on a typical transition metal sur­ face can be estimated from IR = nF(da/dQ)U where θσ/θΩ is the differential Raman scattering cross section, η is the num­ ber of scatterers in the laser beam, F is the laser flux, and Ω is the solid angle over which scattered photons are col­ lected. Typical numbers for this system are a differential scattering cross sec­ tion of ΙΟ - 2 9 sr, a laser flux of 3 X 1021 photons s - 1 ,10 1 0 molecules in the focal region, and a collection solid angle of 1 sr, resulting in a total scattered inten­ sity of 300 photons s _1 . Assuming a total detection efficiency of 1% (a rea­ sonable estimate based on a monochromator throughput of 10% and a photomultiplier quantum efficiency of 10%), typical count rates are expected to be 3 counts/s. Thus, a signal-to-noise ratio of 10:1, in the absence of any other noise sources, requires a counting time of ~30 s per wave number interval. For a complete spectral scan (3000 cm - 1 ) at modest (5 cm - 1 ) resolution, more than 5 h is required using a conventional scanning instrument. Even in ultrahigh

1304 A • ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

vacuum, a surface would become com­ pletely contaminated during this time, rendering the measurement meaning­ less. Using a multichannel detector of at least 600 channels, however, the same measurement could be completed in the 30 s it took to record one data point by the conventional method. Clearly, multichannel detection has not only made this measurement fast­ er, but it has given it meaning. As an example of our technique for surface Raman spectroscopy we offer the adsorption of nitrobenzene on a Ni (111) surface under ultrahigh vacuum (17). This system was chosen because the Raman scattering cross section for nitrobenzene is well-known and be­ cause nickel is a typical catalytic metal. Two experimental details should be mentioned. Maxwell's equations with appropriate boundary conditions de­ cree that the electromagnetic field at a conducting surface vanishes for most incident angles and polarizations. By solving Maxwell's equations, one finds that substantial fields only along the surface normal can be supported when light incidence at 60° to the surface normal and polarized in the plane of incidence is used. Similarly, the maxi­ mum intensity of the scattered radia­ tion is found to peak at 60° to the sur­ face normal. Thus we have designed our apparatus so that these conditions are satisfied, to maximize detectability.

bands of liquid nitrobenzene are observed with frequencies that are unshifted from the liquid but are of quite different relative intensities. The unshifted frequencies are expected for this case of weak physical adsorption, and the intensity differences can be used to determine the structure and orientation of the molecules in the film. This spectrum, requiring only 8 min to acquire, clearly demonstrates the utility of multichannel Raman spectroscopy in the study of ultrathin molecular films. Potential applications include the study of photoresists on semiconductors or polymer-modified electrodes. Figure 2b is the Raman spectrum ob-

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The experiments were conducted in a standard ultrahigh vacuum chamber (base pressure < 10~10 torr) equipped with surface and gas diagnostics, which included low-energy electron diffraction, Auger electron spectroscopy, and quadrupole mass spectrometry. Raman scattering was excited by an argon ion laser and detected using a 0.3-m single spectrograph fitted with an intensified, cooled vidicon. A colored glass filter rejects the scattered Rayleigh light but restricts the accessible frequency range to > 900 cm - 1 . Figure 2a shows the Raman spectrum of a thin (50 À) film of nitrobenzene condensed onto a Ni (111) surface cooled to 100 K. All of the vibrational

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Figure 2. Unenhanced Raman spectra of nitrobenzene adsorbed on a Ni (111) surface under ultrahigh vacuum at 100 K. (a) Multilayer (~50 A) nitrobenzene condensed on the surface, (b) half a monolayer that has reacted to form nitrosobenzene, as indicated. Laser power was ~200 mW at 514.5 nm, and the spectra were acquired in ~15 min.

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ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 • 1305 A

tained from about half a monolayer of nitrobenzene adsorbed on the Ni (111) surface at 100 K. This spectrum is dra­ matically different from the first; de­ tailed analysis shows that the spectrum results from chemically adsorbed nitrosobenzene. Apparently, even at 100 Κ nickel is a sufficiently good cata­ lyst that it can reduce aromatic nitro groups to nitroso groups. This spec­ trum is dramatic proof of the power of multichannel Raman spectroscopy in deducing the course of surface chemi­ cal reactions. Multichannel detectors have made it possible to obtain Raman spectra from molecules adsorbed at submonolayer coverages on low-area single crystal surfaces. Our method is perfectly gen­ eral, requiring no special properties of either the adsorbate (as for resonance Raman scattering) or the surface (as for surface-enhanced Raman scatter­ ing). We currently operate over a wide frequency range (200-5000 cm - 1 ) at moderate resolution (1 cm - 1 ) with rela­ tively high sensitivity (5% of a mono­ layer). We expect improvements in sensitivity to better than 1% of a mono­ layer, and we have recently added highpressure (1 atm) capabilities to launch studies of reacting systems. Time-resolved experiments Multichannel detectors have made possible time-resolved Raman spec­ troscopy, which has become an incredi­ bly powerful probe of the time evolu­ tion of molecular structure in chemi­ cally reacting systems. In these studies, chemical reactions are initiated in a va­ riety of ways (e.g., flash photolysis, pulse radiolysis, rapid mixing) after which the Raman spectra are taken as a function of time. The time resolution is usually provided either by gating the detector, in which case nanosecond res­ olution is attainable, or by pulse sepa­ ration between the exciting laser and Raman laser pulses, in which case pico­ second resolution is achievable. We present two examples that represent topics of great current interest: the spectra of excited states, which are im­ portant in photophysics and photo­ chemistry, and the spectra of interme­ diates in biological and photobiological reactions. The diphenylpolyenes are important models for isomerization reactions in general and for visual transduction and photosynthesis in particular. The structures of excited states of these molecules are clearly important in un­ derstanding the isomerization mecha­ nisms. Cis-trans isomerization in stilbene has been extensively studied by time-resolved absorption and emission spectroscopy, but these methods do not provide information about the vibra­ tional structure of the excited states, which may be used to investigate its

potential energy surface. Time-re­ solved resonance Raman spectroscopy is the ideal method with which to gen­ erate excited-state vibrational spectra because of its high spectral and tempo­ ral resolution. The Si lifetime of transstilbene is on the order of 100 ps, which establishes the time scale of the experi­ ment. The optimum experiment should therefore comprise two inde­ pendently tunable pulsed lasers with pulse widths of a few picoseconds and high average powers, because Raman scattering is a linear spectroscopy. Gustafson and co-workers have con­ structed a versatile pulse-probe pico­ second ultraviolet Raman spectrome­ ter that fulfills these requirements (18). The system consists of an ampli­ fied, synchronously pumped cavitydumped dye laser as the excitation source and a single spectrograph with an intensified photodiode array detec­ tor. The laser produces 25-ps pulses at a repetition rate of 760 kHz with an energy of 140 nJ per pulse. Power avail­ able at the sample is 6 nJ of frequencydoubled UV and 30 nJ of visible. Figure 3 shows the Raman spectrum of the ground and first excited states of irarcs-stilbene. The ground-state spec­ trum was taken from a crystalline sam­ pling using a conventional scanning in­ strument, whereas the excited-state spectrum was taken from a solution in hexane with the multichannel spec­ trometer. The photolysis pulse was at 296.3 nm and the probe pulse was at 592.7 nm; these wavelengths corre­ spond to the maximum of the groundand excited-state absorptions, respec-

tively. Thus resonance enhancement of the excited-state spectrum is achieved through the participation of higher electronic states. The excited-state spectrum is fully developed within 25 ps and all features decay with a time constant that is characteristic of the Si state of irarcs-stilbene. Very large frequency shifts are ob­ served in the spectra. Mode mixing and normal coordinate rotation in the ex­ cited state make it difficult to make precise assignments based on the ground-state normal modes, but an ap­ proximate analysis follows. Some of these assignments have been made by comparison with spectra of the mole­ cule in which the olefinic carbons have been substituted with 13C. The modes near 1144 and 1177 c m - 1 are so little shifted from their frequencies in the ground state that they are easily as­ signed as C—C—H bonds. Large fre­ quency shifts and pronounced asym­ metries are observed for bands near 1238 and 1565 cm" 1 , which correspond most closely to ground-state modes at 1194 and 1639 c m - 1 , respectively. These modes are approximately the C—C stretch of the ethylenic carbon phenyl carbon single bond and the C = C ethylenic stretch, respectively. These bands tail 30 c m - 1 to higher en­ ergy (1238 c m - 1 mode) and 60 c m - 1 (1565 c m - 1 mode) to lower energy, sug­ gesting a distribution of conformers with varying degrees of phenyl ring ro­ tation about the ethylenic bond. Part of the breadth of the 1565 c m - 1 band has recently been shown to arise from two overlapping bands, the ethylenic

Figure 3. Ground (lower) and excited-state spectra of frans-stilbene. Ground-state spectrum was taken of the crystal using a conventional scanning double monochromator. Excited-state spectrum was from a hexane solution taken with picosecond excitation (Xpuise = 296.3 nm, \jrobe = 592.7 nm) and multichannel detection. The spectra were acquired in about 10 min.

1306 A • ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

C = C stretch as well as a phenyl C = C stretch, but it is still thought that much of the remaining line width is inhomogeneous because of the presence of conformers. The excited-state spectrum is very similar to the ground-state spectrum of the irans-stilbene radical anion. This similarity is appealing because both molecules have an electron in an antibonding 7Γ-7Γ* orbital. This interpreta­ tion immediately explains the observed frequency shifts: The C = C olefinic bond is weakened, and the ethylenic carbon phenyl carbon single bond is strengthened. This pioneering study il­ lustrates the power of picosecond reso­ nance Raman spectroscopy in elucidat­ ing the structure and dynamics of elec­ tronically excited states. As our final example we present a study of the reaction of cytochrome ox­ idase with oxygen, in which a combina­ tion of rapid mixing and pulse-probe time-resolved resonance Raman spec­ troscopy is used to detect intermedi­ ates in a chemical reaction of biological interest. Cytochrome oxidase catalyzes the reduction of oxygen to water. It is a mechanistically complex four-elec­ tron/four-proton reaction involving oxygen binding at one Fe 2 + —Cu + site followed by electron transfer from this site and a distant Fe 2 + —Cu + site. Im­ portant questions include the nature of the initial oxygen adduct and partially reduced intermediates. To address these questions, Babcock and co-workers rapidly mixed a solu­ tion of cytochrome oxidase, in which the heme binding site was blocked with carbon monoxide, with oxygenated buffer (19). The 532-nm second har­ monic from a 7-ns pulsed Nd:YAG la­ ser was used to photodissociate the carbon monoxy-heme complex, making the binding site available for oxygen. At variable delay times, a 416-nm probe pulse, generated by Stokes-shifting in H 2 of the 355-nm third harmonic, was used to excite the resonance Ra­ man scattering, which was detected by a 0.3-m single spectrograph and an in­ tensified photodiode array. Figure 4 shows the time evolution of the spectrum. The 10-ns spectrum shows both an oxidation state marker (1355 cm - 1 ) and a formyl stretching frequency (1666 cm - 1 ) that prove that the reduced deoxy enzyme has been generated. The shift of the oxidation state marker to higher frequency and the decrease in intensity of the formyl stretching mode as the reaction pro­ ceeds indicate that the protein is being oxidized. The surprising feature of the data is that little change is observed over the first 50 μ& of the reaction, dur­ ing which optical experiments had clearly established that oxygen addi­ tion and partial oxidation of the metal centers had occurred. This mystery was

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(14) Murray, C. Α.; Dierker, S. B. J. Opt. Soc. Am., in press. (15) Asher, S. A. Anal. Chem. 1984,56, 720. (16) Johnson, C. R.; Asher, S.A. Anal. Chem. 1984, 56, 2261. (17) Campion, Α.; Brown, J. K.; Grizzle, V. M. Surf. Sci. 1982,115, L153. (18) Gustafson, T. L.; Roberts, D. M.; Chernuff, D. A. J. Chem. Phys. 1983, 79, 1559 (19) Babcock, G. T.; Jean, J. M.; Johnston, C. N.; Palmer, G.; Woodruff, W. H. J. Am. Chem. Soc. 1984,106, 8305.

Raman frequency, cm"1 Figure 4. Time evolution of the resonance Raman spectrum of cytochrome oxidase as it reacts with molecular oxygen. Each spectrum was the average of 115 pump-probe experiments. Xpump = 532 nm and Xprobo = 416 nm, with ~2 m j pulse energy in each.

cleared up in a study of the power de­ pendence of the spectra, in which it was clearly shown that the initial adduct was photolabile—the probe pulse sim­ ply photolyzed it. (This observation underscores the importance of varying the probe power in time-resolved Ra­ man studies to ensure that it does not perturb the system.) The low-power spectrum taken at 40-/xs delay has an oxidation state marker at 1378 c m - 1 and a spin state marker at 1588 cm - 1 , which are very similar to those in oxymyoglobin and oxyhemoglobin, provid­ ing the best data to date that the reoxidation of cytochrome oxidase involves an oxycytochrome moiety at the socalled as site. This study illustrates very clearly the power of time-resolved resonance Raman spectroscopy in elu­ cidating the mechanisms of chemical reactions at the heart of biological pro­ cesses. Conclusions

Multichannel detectors have opened up many new areas for investigation using Raman spectroscopy. The multi­ plex advantage manifests itself in a greater effective sensitivity for the technique of 2 to 3 orders of magnitude and allows experiments to be conduct­ ed that were simply impossible before. We have presented examples that illus­ trate how this improved sensitivity can be used in time-resolved experiments in which low duty cycles effectively limit the applicable laser power and in studies in which the number of mole­ cules is very small (e.g., in trace analy­ sis or in surface chemistry). The next

generation of multichannel detectors promises to make possible an ever wid­ er variety of interesting experiments in physics, chemistry, and biology. It is a pleasure to acknowledge EG&G Princeton Applied Research Corp. for providing instrumen­ tation for this research. The research conducted at The University of Texas at Austin was supported by the National Science Foundation, the National Institutes of Health (DK 36263), and The Robert A. Welch Foundation. We are very grateful to Sandy Asher and Terry Gustafson for permitting us to discuss their work in this article.

Alan Campion received a B.A. in chemistry in 1972 from New College (Sarasota, Fla.) and a Ph.D. from the University of California at Los Ange­ les, where he studied energy transfer in condensed-phase organic, inorgan­ ic, and biological systems using laser spectroscopy. He then did a postdoc­ toral fellowship at the University of California at Berkeley, studying ener­ gy transfer to surfaces. Currently he is associate professor of chemistry at the University of Texas at Austin. His re­ search interests include surface phys­ ics, surface chemistry, and laser spec­ troscopy.

References

(1) Raman, C. V.; Krishnan, K. S. Naturwissenschaften 1928,121, 501. (2) Landsberg, G.; Mandelstam, L. Naturwiss. 1928,16, 557. (3) Maiman, T. H. Nature 1960,187, 493. (4) Porto, S. P. S.; Wood, D. L. J. Opt. Soc.Am. 1962,52,251. (5) Landon, D.; Porto, S.P.S. Appl. Opt. 1965,4,762. (6) Delhaye, M. In Molecular Spectrosco­ py; The Institute of Petroleum: London, 1968. (7) Time-Resolved Vibrational Spectros­ copy; Atkinson, G. H., Ed.; Academic: New York, 1983. (8) Time-Resolved Vibrational Spectros­ copy; Laubereau, Α.; Stockburger, M., Eds.; Springer-Verlag: Berlin, 1985. (9) Tenth International Conference on Raman Spectroscopy; Peticolas, W. L.; Hudson, B., Eds.; University of Oregon: Eugene, 1986. (10) Woodruff, W. H. et al. In Time-Re­ solved Vibrational Spectroscopy; G. H. Atkinson, Ed.; Academic: New York, 1984. (11) Woodruff, W. H. ACS Symp. Ser. 1983, 211, 473. (12) Asher, S. Α.; Flaugh, P.; Washinger, G. Spectroscopy 1986,1, 26. (13) Tsang, J. C. In Dynamics on Surfaces; Pulman, B.; Tortner, J.; Nitzan, Α.; Gerber, B., Eds.; D. Reidel: Dordrecht, Holland, 1984, p. 379.

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W. H. Woodruff received a B.A. in chemistry in 1962 from Vanderbilt University and an M.S. (1969) and a Ph.D. (1972) from Purdue University. He then studied the resonance Raman spectroscopy of metalloproteins as a National Institutes of Health Fellow at Princeton University. Currently he is on the staff of Los Alamos National Laboratory and is an adjunct profes­ sor at the University of New Mexico. His research interests are time-re­ solved laser spectroscopies applied to condensed-phased dynamics and chemical applications of lasers in gen­ eral, inorganic photochemistry, and biological electron transfer systems.