Instrumentation
spectroelectrochemistry Combination of Optical and Electrochemical Techniques for Studies of Redox Chemistry William R. Heineman Department of Chemistry University of Cincinnati Cincinnati, Ohio 45221
The combination of two quite different techniques, electrochemistry and spectroscopy, has proved to be an effective approach for studying the redox chemistry of inorganic, organic, and biological molecules. Oxidation states are changed electrochemically by addition or removal of electrons at an electrode. Spectral measurements on the solution adjacent to the electrode are made simultaneously with the electrogeneration process. Thus, spectroscopy is used as a probe to observe the consequences of electrochemical phenomena that occur in the solution undergoing electrolysis. Such "spectroelectrochemical" techniques are a convenient means for obtaining spectra and redox potentials and observing subsequent chemical reactions of electrogenerated species. This article describes several of the more commonly used spectroelectrochemical methods and presents illustrative examples of their application. A variety of optical methods that have been coupled with electrochemistry are summarized in Figure 1. The most frequently used technique is absorption spectroscopy in the ultraviolet-visible-infrared region. Absorption spectroscopy can be implemented by any of three methods. Transmission spectroscopy (Figure 1A, IB) involves passing the optical beam directly through a transparent electrode and
390 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
the adjacent solution (7-3). In specular reflectance spectroscopy (Figure 1C) the beam is passed through solution and reflected from the electrode surface back through the solution (4, 5). Internal reflectance spectroscopy (Figure ID) involves introducing the optical beam through the back side of a transparent electrode at an angle greater than the critical angle so that the beam is totally reflected (1-3, 6). Spectral changes near the electrode are observable due to the small penetration of the electric field vector into the solution. Sensitivity for both types of reflectance spectroscopies can be enhanced by multiple reflections. Luminescence and scattering spectroscopic techniques have been coupled with electrochemistry. In Raman and resonance Raman spectroelectrochemistry (Figure IE) the excitation is by a laser beam directed through solution at an electrode and the Raman back-scattering is observed (7). A particularly important aspect of Raman spectroelectrochemistry is the structural information about electrogenerated species which is contained in a Raman spectrum. A beam of excitation light can be passed through an electrochemical cell, and the resulting fluorescence of electrogenerated species observed (8). A variety of electrode reactions are accompanied by the emission of light. Such electrogen0003-2700/78/0350-390A$01.00/0 © 1978 American Chemical Society
Figure 1.
Spectroelectrochemicai techniques ABSORPTION SPECTROSCOPY
LUMINESCENCE AND SCATTERING SPECTROSCOPIES
ESR NMR
UV, VISIBLE, IR
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REFLECTANCE
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erated chemiluminescence results from decay of an excited state formed by the solution reaction of electrogenerated cations and anions (9). Electrochemical cells have been placed in the sample cavities of ESR and NMR spectrometers to record the absorption spectra of electrogenerated species (10,11). Recently, mass spec troscopy has been coupled to an elec trochemical cell (12). Two types of solution geometries are commonly used in conjunction with the above optical techniques. The usual cell (Figures IB, 1C, ID) is analogous to a conventional electro chemical cell in that the electrode is in contact with an electrolyte solution much thicker than the diffusion layer adjacent to the electrode (1-3). By contrast, the thin-layer cell (Figure 1A) confines a thin ( range. With remarkable dispersion (e.g., 3.0nm/mm with our I200g/mm grating). • Finest resolution in its field. Better than 0.6nm with 1200 g/mm grating). On our parabolic-mirror model Mark X, it's 0.3nm! • Minimal scattered light. At 500nm, it's less than 0.05%! • A beautiful selection of accessories to make your lab life easier. Like digital Omni-Drive (1-200nm/minute) . . . quantum photometer. . . filter wheels to expedite fluorescence, phosphorescence, luminescence, polariza tion and photometry studies . . . and much more. Use Mark X as a heavy-duty monochromator or as a spectrograph. In instruction or research. It's all in a handsome little package with a handsome little price. Not $2500, not $2000, but — thanks to volume production — a little over $1000. Incidentally, we call it a quarter-meter. Actually we've made it 275mm for greater dispersion, improved focal plane. The extra 25mm are on the house.
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396 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
The complete mechanism now con sists of generation of R at the elec trode (Equation 1) with reaction of Λ as it diffuses away from the electrode and encounters Ζ (Equation 3). When the potential-step experiment is re peated, the absorbance response caused by generation of R is less, due to its reaction with Z. Curves b-d show how the absorbance due to R is diminished as the rate constant in creases. The value for k can be calcu lated from the magnitude by which the absorbance is decreased by the re action. The appropriate diffusion equations must be solved or simulated to calculate rate constants (2, 3). Since the shape of the absorbance-time re sponse varies depending upon the mechanism of the reaction, mechanis tic information can also be obtained. Although this discussion has fo cused on optical observation of the reaction by a light beam passing through an OTE and the solution, specular reflectance (4), internal re flectance (1-3), and resonance Raman (7) spectroscopies have also been used. These techniques are capable of monitoring extremely fast reactions with rates up to the diffusion-con trolled limit. Consequently, instru mentation requirements are more stringent. Fast potentiostats and spec trometers interfaced with a computer for signal-averaging of repetitive puls es are necessary to realize the full capabilities of the technique. Rapid scanning spectrometers that can re cord several hundred complete spectra per second are utilized for obtaining spectra of short-lived intermediates in more complicated reaction mecha nisms (19). The thin-layer electrode is also use ful for studying reaction mechanisms (20, 21). In this case, a potential is ap plied to the cell for about 30 s to com pletely convert all of Ο in the thin so lution layer to R. The reaction of β is then monitored optically by light passing through electrode and solu tion. This approach is analogous to conventional kinetic methods such as stopped-flow. Consequently, the ab sorbance-time response can be mathe matically treated with conventional kinetic equations. Since about 30 s is required to completely convert Ο to R, this technique is used for slower re actions. As such, it complements the faster technique described above. A variety of chemical reactions have been studied by the various spectroelectrochemical techniques. A few rep resentative examples are listed in Table I. Indirect Coulometric Titration with Optical Monitoring. The titra tion of molecules by reductant or oxi dant quantitatively generated at an electrode is known as coulometry.
Table I.
Reactions Studied by Spectroelectrochemistry
When the generating electrode is opti cally transparent, the course of the ti tration can be monitored optically by light passing through the electrode and the stirred solution (Figure IB). A titration curve is obtained by plot ting the absorbance at the monitored wavelength as a function of charge passed through the cell, the charge being proportional to the amount of titrant generated. The shape of the ti tration curve is determined by the op tical properties, redox potentials, and η values of both the molecule being titrated and the electrogenerated ti trant. Redox potentials of complex bi ological molecules can be obtained by careful measurement of the shape of titration curves (1, 16, 28). An example of the capabilities of this technique is the titration of cyto chrome c oxidase in the presence of cytochrome c (29). As the terminal component in the oxidative phospho rylation chain, cytochrome c oxidase transfers electrons from cytochrome c to oxygen, reducing the latter to water. It is a complex molecule con taining two heme irons and two cop pers (for a total of four redox centers) imbedded in a lipoprotein matrix. By contrast, cytochrome c is a relatively simple molecule with only a single heme iron as the redox center.
Figure 5 shows spectra recorded during the reductive titration of a mixture of cytochrome c oxidase and cytochrome c. The biocomponents were initially in their fully oxidized state. Each spectrum was recorded after the coulometric addition of 5.0 nequiv of the reductant methyl violo gen radical cation, MV.+, which was generated at a tin oxide transparent electrode by the following reaction:
The absorbance increase at 605 nm corresponds to the reduction of the two heme components of cytochrome c oxidase; the increase at 550 nm cor responds to the reduction of the heme in cytochrome c. The inset figure shows the absorbance changes at 605 and 550 nm as a function of the amount of MV.+ generated in terms of charge passed through the cell. Evalu-
398 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
ation of the relative shapes of the two titration curves indicates the sequence of titration: one heme of cytochrome c oxidase is reduced first, followed by the heme in cytochrome c and then the second heme in cytochrome c oxi dase. Overlap occurs as a result of the close proximity of the redox poten tials. Comparison between such exper imental titration curves and com puter-simulated curves for various values of redox potentials enables the exact redox potentials of the hemes and coppers in cytochrome c oxidase to be evaluated. The total amount of charge required for complete reduc tion of the two cytochromes corre sponds to a five-electron stoichiometry: one electron for cytochrome c and four electrons for the two hemes and two coppers in cytochrome c oxidase. Cytochrome c, myoglobin, cyto chrome c oxidase, blue copper laccases, and spinach ferredoxin have been .studied by indirect coulometric titra tion. The technique exhibits several advantageous features. Nanoequivalent aliquots of oxidant or reductant can be accurately and precisely gener ated. Small-volume (one mL and less) cells require only small amounts of ex pensive biocomponents for measure ments. The biocomponent can be repetitively reduced and oxidized.
Redox potentials can be measured for optically nonabsorbing redox compo nents (30). No volume changes occur during titrations, eliminating dilution corrections for spectral data. Solutions are easily rendered and maintained anaerobic by vacuum degassing proce dures (28). S u r f a c e Studies. T h e importance of surface studies stems from the in fluence of the surface condition on the rate and mechanism of an electrode reaction. T h e n a t u r e of the electrode surface itself can be probed by reflec tance, internal reflection, transmis sion, R a m a n , and ellipsometric spec troscopies (1-3, 5, 6, 31, 32). T h e for mation of oxide layers on metal elec trodes and the absorption of ions, or ganic molecules, and proteins on the electrode surface have been observed optically. Techniques such as ESCA and Auger spectroscopy have yielded valuable information about the sur faces of electrodes after their removal from the electrochemical cell (33, 34). American Chemical Society members receive Chemical and Engineering News each week. C&E News brings you the up-to-date happenings in the chemical world plus official ACS news. AND THERE ARE MANY OTHER BENEFITS: Publications—Members enjoy sub stantial savings on world renowned ACS publications. Meetings—Two national meetings each year plus a host of regional and l o c a l m e e t i n g s are h e l d f o r y o u r benefit. Local Sections—provide you with activities of local interest and an op portunity to participate in Society affairs. Divisions—28 subject divisions help you keep up with yourspecial chemical interest. Educational Activities— short courses, audio courses and interaction courses help you expand as a professional. Employment Aids—give you a helping hand in today's tight job market. But most important, your membership helps support the scientific and edu cational society that represents you as a professional. 110,000 Chemists and Chemical Engi neers know the value of ACS member ship. Send coupon below today for an application. American Chemical Society Office of Member Services 1155 Sixteenth Street, N.W. Washington, D. C. 20036 Yes, I am interested in membership in the American Chemical Society. Please send information and application. Name Add ress City State Zip
400 A ·
Conclusion
T h e precision with which oxidation states can be controlled, coupled with the capability for obtaining spectral information, is stimulating the adop tion of spectroelectrochemical tech niques for the study of inorganic, or ganic, and biological redox species.
Figure 5. Indirect coulometric titration of cytochrome c (17.5 μΜ) and cytochrome c oxidase (6.3 μΜ) by reduction with MV. + e l e c t r o generated at S n 0 2 optically transparent electrode Each spectrum recorded after generation of 5 X 10~ 9 equiv(0.5 millicoulombs) of MV.+. Inset shows titration curves recorded at 550 and 605 nm (29)
A N A L Y T I C A L CHEMISTRY, V O L . 5 0 , NO. 3, M A R C H
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References (1) T. Kuwana and W. R. Heineman, Ace. Chem. Res., 9, 241 (1976). (2) N. Winograd and T. Kuwana, "Spectroelectrochemistry at Optically Trans parent Electrodes", in "Electroanalytical Chemistry", Vol 7, A. J. Bard, Ed., Mar cel Dekker, New York, N.Y., 1974. (3) T. Kuwana, Ber. Bunsenges. Phys. Chem., 77,858(1973). (4) A.W.B. Aylmer-Kelly, A. Bewick, P. R. Cantrill, and A. M. Tuxford, Faraday Discuss. Chem. Soc, No. 56, 96 (1973). (5) J.D.E. Mclntyre, "Specular Reflection Spectroscopy of the Electrode-Solution Interphase", in "Advances in Electro chemistry and Electrochemical Engi neering", Vol 9, R. H. Muller, Ed., Wiley-Interscience, New York, N.Y., 1973. (6) W. N. Hansen, "Internal Reflection Spectroscopy in Electrochemistry", ibid. (7) TD. L. Jeanmaire, M. R. Suchanski, and R. P. Van Duyne, J. Am. Chem. Soc, 97, 1699 (1975). (8) F. M. Hawkridge and B. Ke, Anal. Biochem., 78,76(1977). (9) L. F. Faulkner and A. J. Bard, in "Elec troanalytical Chemistry", Vol 10, A. J. Bard, Ed., Marcel Dekker, New York, N.Y., 1977. (10) T. M. McKinney, "Electron Spin Res onance in Electrochemistry", in "Elec troanalytical Chemistry", Vol 10, A. J. Bard, Ed., Marcel Dekker, New York, N.Y., 1977. (11) J. A. Richards and D. H. Evans, Anal. Chem., 47,964(1975). (12) M. Petek and S. Bruckenstein, J. Electroanal. Chem., 47, 329 (1973). (13) R. W. Murray, W. R. Heineman, and G. W. O'Dom, Anal. Chem., 39, 1666 (1967).
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