_-
Alan Campion Department of Chemistry Universlty of Texas at Austin Austin, rex. 78712
W. H. Woodrutr
INCI. MIS C346 LOS Alamos National Laborator! Los Alamos, N.M. 87545
fraction grating technology severely limited the sensitivity and applicahily of this type of instrumentation. The next quantum leap in Raman instrumentation was engendered hy the invention of the laser in 1960 (3). The extremely intense and monochromatic light produced by lasers made them a natural illumination source for Raman snectroscnnv. laser ~ . ~ ~ The ~ ~first ~ ~~ ~ ~~ Raman spectra were recorded by Porto and Wood in 1962 (4). They used the Landsherg-Mandelstam apparatus with a ruby laser suhstituted for the mercury arc illumination source. The extreme brightness of the laser source was, however, a mixed blessing. When used with a single-stage spectrograph, the laser produced very high ~~~~~~~~~~~
The history of Raman spectroscopy has been marked by several dramatic advances in instrumentation. At essentially the same time as the first qualitative ohservation of the effect hy C. V. Raman in 1928 (I), the first Raman spectrum was recorded hy Landsherg and Mandelstam (2). The apparatus used hy Landsherg and Mandelstam
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consisted of a mercury arc lamp as a stray light levels and accompanying arsource of “monochromatic” illuminatifacts such as grating ghosts, all of tion, a single-stage spectrograph to diswhich were serious interferences in the perse the spectrum of the scattered detection of Raman scattering. To cirlight, and a photographic plate to decumvent these problems, Landon and tect the dispersed spectrum. This type Porto in 1964 employed a two-stage of apparatus remained the standard in(double) monochromator (5). The strumentation for Raman spectroscopy function of the first-stage monochrofor more than 30 years. mator was to reduce the stray light enIt is worth noting that, by virtue of tering the second stage to a tolerable simultaneous photographic detection level. The second monochromator had of the entire Raman spectrum, the origthe detector at its exit slit. To record a inal Landsherg-Mandelstam apparaspectrum, the two monochromators tus was a multichannel device. By modwere scanned together, and the light ern standards, however, the feeble inintensity transmitted hy the second tensity of the mercury arc source and stage was recorded as a function of the the illumination geometry that it rewavelength setting of the monochroquired, the lack of speed and linearity mators. of the photographic film, and the dif-, ~~~“ Because the first-stage monochro0003-2700/87/A359-1299/501.50/0 @ 1987 American Chemical Sociely
1299 A
mator rejects stray light by rejecting all wavelengths except the one determined by its grating position, the double Raman spectrometer is by definition a single-channel spectrometric device. Thus the double monochromator solved the problem of high stray light and attendant artifacts by sacrificing the spectral multiplex advantage of the spectrograph. This loss was more than compensated for by the brightness of the laser source and its superior optical characteristics and by substitution of photomultiplier detection (having nearly quantum-limited sensitivity, linear response to light, and superior speed) for the photographic plate. The double Raman spectrometer is the standard instrument for “conventional” (i.e., scanning) Raman spectroscopy to the present day. Nevertheless, it is obvious that a laser Raman instrument that combined the spectral multiplex advantage of the spectrograph with the stray light rejection of the double monochromator and the sensitivity, speed, and linearity of the photomultiplier tube would be entirely superior. The first requirement for such an instrument is a detector that is capable of recording an image (as in photographic film) rather than simply an average light intensity (as in a photomultiplier tube), but that retains the aforementioned advantages of photoelectric detection. The answer to this requirement was suggested by television technology. Although standard television image tubes (and, later, solid-state array detectors) are insufficiently sensitive for Raman spectroscopy, photoelectric image intensification combined with such detectors can produce image detection with near quantumlimited sensitivity. This approach was first adopted by Delhaye, Bridoux, and co-workers in 1968, when they demonstrated the applicability of photoelectric image detection to Raman spectroscopy (6). Subsequent improvements in image-intensified detectors for Raman applications include the substitution of proximity-focused microchannel plate image intensifiers for the original electrostatically focused image intensifiers and improved spectral response (particularly ultraviolet sensitivity). Another improvement that has taken place is the substitution of solidstate array detectors for television image tubes. Currently the solid-state multichannel detectors are generally based on one-dimensional photodiode arrays. For most Raman applications, a one-dimensional display of light intensity versus position on the detector (i.e., position in the dispersed spectrum) is sufficient, and sacrificing the two-dimensional imaging capability of the television tube in favor of the supe1300A
rior spatial accuracy of the diode array is justified. The second requirement for successful multichannel Raman spectroscopy is a spectrograph that has good stray light rejection and a negligible tendency to produce grating artifacts. Modern diffraction grating technology has virtually eliminated the latter problem in that holographic techniques are used to produce diffraction gratings that are free from the systematic ruling errors of conventionally ruled gratings. Stray light rejection required reducing by several orders of magnitude the light intensity at the laser wavelength entering the spectrograph. This was accomplished either by using filters that absorb the laser wavelength but transmit the longer wavelength Stokes Raman scattering or (with more versatility) by employing a broad-band-pass foremonochromator that transmits the Raman frequencies of interest, but not the laser frequency, to the spectrographs. Based on the principles outlined above, the multichannel Raman spectrograph allows one to record a substantial segment of the Raman spectrum (ideally, the entire Raman spectrum) simultaneously without scanning the spectrometer’s wavelength setting. This results in two unique advantages over the conventional scanning double Raman spectrometer, one in spectrometric sensitivity and the other in time-resolved spectroscopy. For recent surveys of the impact of multichannel detection of Raman spectra, excellent conference proceedings are available ( 7-9) *
Concerning sensitivity (given equal conditions with regard to continuous wave [cw]laser illumination level, resolution, instrumental luminosity, and detector quantum efficiency), the multichannel spectrograph will outperform the scanning spectrometer in the time required to obtain a spectrum with a given signal-to-noise ratio by a factor equal to the number of spectral resolution elements that can be recorded simultaneously by the multichannel detector. This factor depends on detailed specifications of the detector and the spectrograph, but a typical improvement in spectral acquisition time is a factor of 1000. This result can be a crucial advantage when extreme sensitivity or sample durability is of concern, and it can be an enormous convenience for routine spectroscopy. Another way of stating the multiplex advantage is that the signal-to-noise ratio for a given spectral acquisition time will be better in the multichannel case by a factor equal to the square root of the number of resolution elements recorded simultaneously. From the above this is typically a factor of 30, which can be the difference between the observation of a good spectrum and
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
worthless noise. The time-resolved capability of multichannel Raman spectrometry emerges when the multichannel spectrograph is combined with pulsed-laser excitation. Because the multichannel spectrograph records an entire Raman spectrum a t once, Raman scattering a t all frequencies is recorded during a single laser pulse. If the laser pulse is sufficiently energetic and nonlinear effects do not intervene, a satisfactory spectrum can be obtained with single-pulse excitation on time scales of 25 ps or longer (IO). Even when multiple-pulse signal averaging is necessary the temporal resolution of the experiment is the laser-pulse duration, allowing vibrational study of a wide variety of dynamical phenomena (IO, 11) (see below). Scanning techiques can be used with pulsed-laser excitation for timeresolved Raman studies, but this requires that the sample be exposed to potentially damaging pulsed-laser irradiation for approximately 100-1000 times longer than with multichannel techniques. In the following sections we delineate certain instrumental aspects of the multichannel Raman technique and provide several case studies of the applications of these techniques.
Instrumentation All of the instrumentation required for multichannel Raman spectroscopy is commercially available; a user need only assemble off-the-shelf components for a particular application. Three items are required: a laser, a spectrograph, and a multichannel detector. A variety of laser sources are suitable for Raman scattering. Principal criteria for source selection include the frequency (to take advantage of resonance enhancement, if desired) and the pulse width (for time-resolved studies). Two choices for the spectrograph will provide the required dispersion and rejection of the Rayleigh scattered light. A single monochromator and a longwavelength pass filter are adequate for frequency shifts greater than about 800 cm-l. This system, which has been the work horse in our laboratories, is inexpensive and the stray light rejection is surprisingly adequate, even for the demanding surface Raman experiments. A triple spectrograph must be used when lower frequency shifts are of interest. The first two stages of this instrument act as a variable band-pass filter, and the third stage provides the dispersion. With either instrument, a trade-off must be selected between resolution and spectral coverage. Significant flexibility is provided by rapidly interchangeable gratings in the triple spectrograph; with our intensified diode array detector, for example, resolution and coverage range from 1.4 cm-l
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per channel with a spectral coverage of 980 cm-l to 5.6 cm-l per channel with a 3902 cm-1 spectrum. Given condensedphase Raman line widths that are typically 2-5 cm-l, the triple spectrograph configuration is appropriate to the task. A particularly exciting new development is the discovery of a very narrow notch filter with extremely high rejection of the laser line (12). This filter comprises a crystalline array of polystyrene spheres with lattice parameters that are comparable to the wavelength of light. The laser frequency is Braggdiffracted so that its transmission through the filter is less than 10-l" while Raman-shifted bands are transmitted with greater than 50%efficiency. Half-band-pass at 10% transmission is -40 cm-l, 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 aperture makes it easy to insert into the collection optical train. The great increase in throughput should more than offset the loss in flexibility that a triple spectrograph offers in changing exciting 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 manufacturers are currently available. The most common are the first-generation intensified vidicon (SIT) and the secondgeneration intensified diode array. Third-generation detectors based on charge-coupled device technology or resistive anode readout of microchanne1 plate image intensifiers are on the horizon. The SIT tube is essentially a television camera offering two-dimensional imaging. An integral image intensifier 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 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 levels. Unfortunately, however, the gating capability is lost at the lower temperature. The SIT tube is sensitive to wavelengths longer than the glass cutoff at 350 nm, the red response being typically that of an S-20 photocathode. A scintillator extends the ultraviolet response to shorter wavelengths but with a greatly diminished (2%) quantum efficiency. The second-generation detector, based on solid-state technology, was specifically designed for spectroscopic 1302A
applications. The self-scanned diode array is a one-dimensional detector that also uses photodiodes for the conversion of photons to separated electron-hole pairs, but the signal is read with on-chip circuitry rather than a scanning electron beam. A microchanne1 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 gated to better than 5 ns. To reduce the dark current, the photocathode is cooled to 0 O C and the diode array to -20 "C. The gating ability is unaffected by cooling. The current generation of multichannel optical detectors provides up to 1024 channels with a quantum 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 determined 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 divided 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 isolates the random thermal fluctuations, which are the real dark current, from the fixed pattern noise and AID bias, which both subtract out perfectly, and from the readout noise, which is negligible when using extended integration directly on the diode array. These characteristics are such that high-quality spectra can be produced with signal levels as low as 0.1 countsls in the absence of background. Although the intensified diode array is currently the most popular detector, two other devices are emerging as potentially competitive alternatives. Proximity-focused microchannel plate image intensifiers have been coupled to resistive anode arrays to produce a twodimensional true photon-counting detector (13).The advantages of this approach include its imaging capabilities and nearly complete absence of dark current. Unintensified charge-coupled devices (CCDs) look especially promising for very low-level detection when gating is not required. CCDs offer much higher quantum yields than detectors requiring image intensifiers (as high as 0.7), two-dimensional imaging, and exceptionally low dark currents when cooled to 77 K (14).
High-sensitivity Raman spectroscopy The dramatic increase in efficiency af-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
forded by multichannel detection makes it possible to obtain Raman spectra in situations in which conventional techniques require times that are much longer than those permitted by other physical constraints. We discuss 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 materials to be detected on low-area single crystal surfaces. Asher and co-workers were pioneers in the use of ultraviolet resonance Raman spectroscopy for detection and speciation of trace polycyclic aromatic hydrocarbons (15). This method has many advantages compared with mass spectrometry, which requires significant sample preparation, or fluorescence, which suffers from matrix effects such as quenching and interferences. Raman spectroscopy is chemically specific, and the resonance-enhanced cross sections produced by ultraviolet 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 spectrally well-separated from the Raman scattering. The experiments are conducted using a tunable pulsed ultraviolet laser source that comprises a frequency-doubled Nd:YAG laser pumping a dye laser. The dye laser pulses are then either doubled or mixed with the Nd:YAG fundamental to produce radiation tunable from 217 to 450 nm. Typical average powers are a few milliwatts. The detection system comprised a triple spectrograph and a microchanne1 plate image intensified photodiode array. Multichannel detection is essential in this application because the entire spectrum must be examined, and the low average powers available would make the time required in a scanning experiment prohibitively long. Figure 1shows the UV resonance Raman spectra of naphthalene, anthracene, phenanthrene, and pyrene as 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 signal-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
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Figure 1. UV resonance Raman spectra of polycycllc aromatic hydrocarbons. M Wiutions in aoelonitrib. Average I w r paww was a few miiliwaII6 al Um excilalion Shown are naphhiene, anmracene, phenanthrene.and pyrene 89 lrequencies listed. Specta wwe each acquked In about I O min. Excltalbn waveia@hs are indicatedon me figwe.
demonstrated to he 20 pph. By increasing the laser power a t the sample and multipassing the beam, it is estimated that this limit could he reduced hy 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 molecules is the detection of Raman spectra from molecules adsorbed on the surfaces of single crystal metals when, even under ultrahigh vacuum, experimental time scales are limited to an hour or so. Raman spectroscopy has been shown to be an especially powerful probe of surface and interfacial chemical reactions hecause of ita high chemical specificity and ita immunity to the presence of an ambient gas or liquid phase, in contrast to the powerful electron spectroscopies of surface science. If the method is to have an impact on the field, however, it must he sufficiently sensitive to detect less than a monolayer of molecules adsorbed on a well-characterized single crystal surface under ultrahigh vacuum. At that level, the method will he comparably sensitive to other surface science techniques with the additional advantages 1304A
ANALYTICAL CHEMISTRY, VOL. 59,
mentioned above. Suhmonolayer 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 surface can he estimated from I, = nF(ada/an)n where aalail is the differential Raman scattering cross section, n is the numher of scatterers in the laser beam, F is the laser flux,and il is the solid angle over which scattered photons are collected. Typical numbers for this system are a differential scattering cross section of 10-29 sr, a laser flux of 3 X 1021 photons $-I, 1Olo molecules in the focal region, and a collection solid angle of 181, resulting in a total scattered intensity of 300 photons 8-I. Assuming a total detection efficiency of 1%(a reasonable estimate based on a monochromator throughput of 10%and a photomultiplier quantum efficiency of lW), typical count rates are expected to he 3 countsls. Thus, a signal-to-noise ratio of 101, in the absence of any other noise sources, requires a counting time of -30 8 per wave number interval. For a complete spectral scan (3000 cm-') a t modest (5 em-') resolution, more than 5 h is required using a conventional scanning instrument. Even in ultrahigh
NO. 22, NOVEMBER 15. 1987
vacuum, a surface would become completely contaminated during this time, rendering the measurement meaningless. Using a multichannel detector of a t 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 faster, hut 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 (27). This system was chosen because the Raman scattering cross section for nitrobenzene is well-known and hecause nickel is a typical catalytic metal. Two experimental details should he mentioned. Maxwell's equations with appropriate boundary conditions decree that the electromagnetic field a t a conducting surface vanishes for most incident angles and polarizations. By solving Maxwell's equations, one fmds that substantial fields only along the surface normal can be supported when light incidence a t 60° to the surface normal and polarized in the plane of incidence is used. Similarly, the maximum intensity of the scattered radiation is found to peak at 60' to the surface normal. Thus we have designed our apparatus so that these conditions are satisfied, to maximize detectability.
The experiments were conducted in a standard ultrahigh vacuum chamber (hase pressure < torr) equipped with surface and gas diagnostics, which included low-energy electron diffraction, Auger electron spectroscopy, and quadrupole mass spectrometry. Raman scattering was excited hy 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 em-'. Figure 2a shows the Raman spectrum of a thin (50 A) film of nitrobenzene condensed onto a Ni (111)surface cooled to 100 K. All of the vihrational
hands of liquid nitrobenzene are ohserved with frequencies that are unshifted from the liquid hut are of quite different relative intensities. The unshifted frequencies are expected for this case of weak physical adsorption, and the intensity differences can he 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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.jure 2. Unenhanced Raman spectra of nitrobenzeneadsorbed on a Ni (111) surface under ultrahigh vacuum at 100 K.
(a) Multilayer (-50 A) nitrobenzene Condensed ~1 me surface. (b) halt a mnolayer h t has reacted lo form nitrosobenzene, as indicated. Laser power was -200 mW a1 514.5 nm. and me specba were acquired in -15 min.
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tained from about half a monolayer of nitrobenzene adsorbed on the Ni (111) surface a t 100 K. This spectrum is dramatically different from the first; detailed analysis shows that the spectrum results from chemically adsorbed nitrosobenzene. Apparently, even a t 100 K nickel is a sufficiently good catalyst that it can reduce aromatic nitro groups to nitroso groups. This spectrum is dramatic proof of the power of multichannel Raman spectroscopy in deducing the course of surface cbemical reactions. Multichannel detectors have made it possible to obtain Raman spectra from molecules adsorbed a t suhmonolayer coverages on low-area single crystal surfaces. Our method is perfectly general, requiring no special properties of either the adsorbate (as for resonance Raman scattering) or the surface (as for surface-enhanced Raman scattering). We currently operate over a wide frequency range (ZOO-SO00 cm-') a t moderate resolution (1cm-1) with relatively high sensitivity (5% of a monolayer). We expect improvements in sensitivity to better than 1%of a monolayer, and we have recently added highpressure (1atm) capabilities to launch studies of reacting systems.
T i m e - d v e d experiments Multichannel detectors have made possible time-resolved Raman spectroscopy, which has become an incredibly powerful probe of the time evolution of molecular structure in chemically reacting systems. In these studies, chemical reactions are initiated in a variety 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 resolution is attainable, or by pulse separation between the exciting laser and Raman laser pulses, in which case picosecond resolution is achievable. We present two examples that represent topics of great current interest: the spectra of excited states, which are important in photophysics and photochemistry, and the spectra of intermediates 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 understanding the isomerization mechanisms. 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 vibrational structure of the excited states, which may be used to investigate its 1306A
potential energy surface. Time-resolved resonance Raman spectroscopy is the ideal method with which to generate excited-state vibrational spectra because of its high spectral and temporal resolution. The SIlifetime of transstilbene is on the order of 100 ps, which establishes the time scale of the experiment. The optimum experiment should therefore comprise two independently 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 constructed a versatile pulse-probe picosecond ultraviolet Raman spectrometer that fulfills these requirements (18).The system consists of an amplified, synchronously pumped cavitydumped dye laser as the excitation source and a single spectrograph with an intensified photodiode array detector. The laser produces 25-ps pulses a t a repetition rate of 760 kHz with an energy of 140 nJ per pulse. Power available a t the sample is 6 nJ of frequencydoubled UV and 30 n J of visible. Figure 3 shows the Raman spectrum of the ground and first excited states of trans-stilbene. The ground-state spectrum was taken from a crystalline sampling using a conventional scanning instrument, whereas the excited-state spectrum was taken from a solution in hexane with the multichannel spectrometer. The photolysis pulse was at 296.3 nm and the probe pulse was at 592.7 nm; these wavelengths correspond 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 trans-stilbene. Very large frequency shifts are observed in the spectra. Mode mixing and normal coordinate rotation in the excited state make it difficult to make precise assignments based on the ground-state normal modes, but an approximate analysis follows. Some of these assignments have been made by comparison with spectra of the molecule in which the olefinic carbons have been substituted with 13C. The modes near 1144 and 1177 cm-I are so little shifted from their frequencies in the ground state that they are easily assigned as C-C-H bonds. Large frequency shifts and pronounced asymmetries are observed for bands near 1238 and 1565 cm-1, which correspond most closely to ground-state modes at 1194 and 1639 cm-I, 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 cm-I to higher energy (1238 em-' mode) and 60 cm-' (1565 cm-I mode) to lower energy, suggesting a distribution of conformers with varying degrees of phenyl ring rotation about the ethylenic bond. Part of the breadth of the 1565 cm-I band has recently been shown to arise from two overlapping bands, the ethylenic
\Navenumber shin Flgure 3. Ground (lower) and excited-state spectra of
I
eound-stale spectrumwas taken of lhe crysml using a convention Excited-stale Spectrumwas from a hexane SOlUtion taken wllh picosecondexcitation (h,,, = 296.3 nm. bobs = 592.7 nm) and multichanneldetection. The Spema Were acquiredin abart 10 min.
* 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 trans-stilbene radical anion. This similarity is appealing because both molecules have an electron in an antibonding *-**orbital. This interpretation immediately explains the observed frequency shifts The C=C olefinic bond is weakened, and the ethylenic carbon phenyl carbon single hond is strengthened. This pioneering study illustrates the power of picosecond resonance Raman spectroscopy in elucidating the structure and dynamics of electronically excited states. As our final example we present a study of the reaction of cytochrome oxidase with oxygen, in which a comhination of rapid mixing and pulse-probe time-resolved resonance Raman spectroscopy is used to detect intermediates in a chemical reaction of biological interest. Cytochrome oxidase catalyzes the reduction of oxygen to water. It is a mechanistically complex four-electronlfour-proton reaction involving oxygen binding a t one Fez+-Cu+ site followed by electron transfer from this site and a distant Fe2+-Cu+ site. Important questions include the nature of the initial oxygen adduct and partially reduced intermediates. To address these questions, Bahcock and co-workers rapidly mixed a solution of cytochrome oxidase, in which the heme binding site was blocked with carbon monoxide, with oxygenated buffer (19). The 532-nm second harmonic from a 7-11s pulsed NdYAG laser 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 Hz of the 355-nm third harmonic, was used to excite the resonance Raman scattering, which was detected by a 0.3-m single spectrograph and an intensified photodiode array. Figure 4 shows the time evolution of the spectrum. The 10-ns spectrum shows both an oxidation state marker (1355 cm-9 and a formyl stretching frequency (1666 cm-I) 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 proceeds indicate that the protein is being oxidized. The surprising feature of the data is that little change is observed over the first 50 FS of the reaction, during which optical experiments had clearly established that oxygen addition and partial oxidation of the metal centers had occurred. This mystery was
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1) Murrav. C. A,; Dierker, S. B. J. Opt. Sac. Am.,-in press. (15) Asher, S. A. Anal. Chem. 1984,56,120. (16) Johnson, C. R.; Asher, S . A. Anal. Chem. 1984.56,2261. (17) Campion, A.; 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, 1 FCO *""". (19) Bsbcock, G. T.; Jean, J. M.; Johnston.
C. N.;Palmer,G.; Woodruff, W. H. J.Am. Chem. SOC.1984,106,8305.
Figure 4. Time evolution of the resonance Raman spectrum of cytochrome oxidase as it reacts with molecular oxygen.
Each Spectrum was the average 01
115 pump-probe experiments. & -,
= 532 nm and &*,
= 416 nm,
wim -2 MJ pulse energy in each.
cleared up in a study of the power dependence of the spectra, in which it was clearly shown that the initial adduct was photolabile-the probe pulse simply photolyzed it. (This observation underscores the importance of varying the probe power in time-resolved Riman studies to ensure that it does not perturb the system.) The low-power spectrum taken at 40-fis delay has an oxidation state marker a t 1378 cm-' and a spin state marker a t 1588 cn-l, which are very similar to those in oxymyoglobin and oxyhemoglobin, providing the best data to date that the reoxidation of cytochrome oxidase involves an oxycytochrome moiety a t the socalled a3 site. This study illustrates very clearly the power of time-resolved resonance Raman spectroscopy in elucidating the mechanisms of chemical reactions a t the heart of biological processes.
generation of multichannel detectors promises to make possible an ever wider variety of interesting experiments in physics, chemistry, and biology.
C@nclwlOns Multichannel detectors have opened up many new areas for investigation using Fiaman spectroscopy. The multiplex advantage manifests itself in a greater effective sensitivity for the technique of 2 to 3 orders of magnitude and allows experiments to be conducted that were simply impossible before. We have presented examples that illustrate 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 molecules is very small (e.g., in trace analysis or in surface chemistry). The next
(7) Time-Resolved Vibrational Spectros-
1308A
It is a pleasure to acknowledge EG&G Princeton Applied Research Cop. for providing instrumentation for this research. The research conducted at The University of Texas at Austin WBS supported by the Nationd Science Foundation. the National Institutes of Health (DK362631, and The Robert A. Welch Foundation. We are very grateful to Sandy Aaher 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 a t Los Angeles, where he studied energy transfer in condensed-phase organic, inorganic, and biological systems using laser spectroscopy. He then did a postdoctoral fellowship a t the University of California a t Berkeley, studying energy transfer to surfaces. Currently he is associate professor of chemistry a t the University of Texas a t Austin. His research interests include surface physics, surface chemistry, and laser spectroscopy.
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. (1)
Soe. Am. 1962.52.251. ( 5 ) Landon, D.;Porto, S.P.S. Appl. Opt.
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(6) Delhaye, M. In Molecular Spectrosco-
py; The Institute of Petroleum: London, *oca lil"".
copy; Atkinson, G. H., Ed.; Academic: New York, 1983. (8) . . Time-Resolued Vibrational SoectrosCopy; Laybereau, A,; Stockburger, M., Eds.; Spnnger-Verlag: Berlin, 1985. (9) Tenth International Conference on Raman Soectroscoov: Peticolas. W. L.: Hudson. 8.. Eds.; Universitv of Oreeon: Eugene,'1986. (10) Woodyff, W. H. et al. In Time-Resolued VIbrotwnal Spectroscopy; G. H. Atkinson.. Ed.;. Academic: New York. 1984. (11) Woodruff, W. H. ACS Symp. Ser. 1983,211,473. (12) Asher, S. A.; Flaugb, P.; Washinger, G . Spectroscopy 1986,1,26. (13) Tsang, J. C. In Dynamics on Surfaces;
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
W. H. Woodruff received a B.A. in chemistry in 1962 from Vanderbilt University and a n 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 a t Princeton University. Currently he is on the staff of Los Alamos National Laboratory and is a n adjunct professor a t the University of New Mexico. His research interests are time-resolved laser spectroscopies applied to condensed-phased dynamics and chemical applications of lasers ingeneral, inorganic photochemistry, and biological electron transfer systems.
