I
I
much greater and, in fact be equal to the total absorbance change seen a t 550 nm. Analysis of the data for CZ/CA = 1.0 and 2.6 gave kf values of 1.0 X 106M-1 sec-l for both cases (see Table 111). This is consistent within experimental error with the rate constant calculated a t 602 nm, suggesting that the MV.+ is reacting with cyt clI1 via a catalytic mecanism (12). These results are in good agreement with the recent findings of Kuwana (13,14).
ACKNOWLEDGMENT We thank D. L. Langhus for his assistance with the programming and computer interfacing. I50
300
''ME
4 50
LITERATURE CITED
(HILLISEC)
Figure 8. Chronoabsorptometry of cytochrome c at 550 nm. CcytC M ~= + 2.6 Upper curve, total absorbance change (experimental); solid line, absorbance due to cytochrome species only (experimental): open boxes, theoretical points for kf = 1.0 X 1 0 6 W ' sec-', 60 steps averaged
chrome oxidation states a t 550 nm, Equation 16 must be combined with Equation 37 to give Equation 38.
(1) C. Li and G. S . Wilson, Anal. Chem., 45, 2370 (1973). (2) M. Ito and T. Kuwana, J. Nectroanal. Chem., 32, 415 (1971). (3) N. Winograd, H. N. Blount, and T. Kuwana, J. Phys. Chem., 73, 3456 (1969). (4) H. N. Blount. N. Winograd, and T. Kuwana, J. Phys. Chem., 74, 3231 (1970). (5) G. C. Grant and T. Kuwana, J. Electronal. Chem., 24, 11 (1970). (6) L. Ramaley and G. S.Wilson, Anal. Chem., 42, 606 (1970). (7) F. M. Hawkridge and T. Kuwana, Anal. Chem., 45, 1021 (1973). ( 8 ) E. F. Caldin, "Fast Reactions in Solution", Blackwell. Oxford, 1964. (9) H. R. Mahler and E. H. Cordes, "Biological Chemistry", Harper and Row, New York. NY. 1966. (10) J W Hayes, D E Glover, D E Smith, and M W Overton, Anal Chem, 45. 277 11973) (11) A.Ehrenberg and S.Paleus, Acta Chem. Scand., 9, 538 (1955). (12) E. Steckhan and T. Kuwana, Ber. Bunsenges. Phys. Chem., 78, 253 (1974). (13) T. Kuwana, Second international Symposium on Bioelectrochemistry, Pont a Mousson, France, October 1973. (14) T. Kuwana, private communication, 1974.
AAcll,clll is calculated by subtracting the absorbance due to MV.+ as measured at 602 nm from the measured absorb0 and Equation 28). ance a t 550 nm (see Equation 19, In Figure 8, the absorbance a t 550 nm as a function of , time is shown. The absorbance due to cyt c, A A c ~ ~ ,isc ~ ~ ~RECEIVED for review September 3, 1974. Accepted January shown on the curve and is calculated by subtracting out the 20, 1975. Presented in part a t the 168th National Meeting, absorbance due to MV.+ as measured a t 602 nm. In this figAmerican Chemical Society, Atlantic City, NJ, September ure is also shown the theoretical line for hf = 1 X 106M-l 1974. This investigation was supported in part by National sec-l and D c y t c / D ~ ~=. +0.2 (11). If the diffusion coeffiScience Foundation Grants GP-38416X and GP-32909 and cient ratio were 1, the absorbance due to cyt clI would be the Office of Naval Research.
=
Simultaneous Electrochemical-Electron Spin Resonance Measurements with a Coaxial Microwave Cavity R.
D. Allendoerfer, G. A.
Martinchek, and Stanley Bruckenstein
Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14214
A new design for a cell to make simultaneous electrochemical-electron spin resonance measurements (SEESR) is described. The 22 cm2 helical working electrode of the electrolysis cell forms the center conductor of a coaxlal microwave cavity providing optimum ESR sensitivity while maintaining good electrochemical parameters. The ESR signals resulting from potential steps, constant current pulses, cyclic and square wave voltammetry are discussed. It is estimated that electrochemically generated radicals with lifetimes as short as sec can be observed by ESR using this cell.
The electrochemical generation of organic free radical ions for study by electron spin resonance has become a standard technique since it was first introduced by Maki and Geske ( I , 2) in 1959. The technique has yet to realize 890
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, M A Y 1975
its full potential as a means of identifying intermediates in electrochemical processes, however, largely because the conventional thin sample cells used for ESR studies produce large ohmic drops when adapted for electrochemistry. Effective potential control during in situ electrolysis in conventional ESR cells is virtually impossible. This difficulty is well illustrated by the distorted cyclic voltammogram of nitrobenzene presented by Piette, Ludwig, and Adams ( 3 ) whose voltammetry was done in a flat cell required for ESR measurements in a TE102 rectangular cavity. Their particular cell design was the forerunner of the widely used Varian E-256 electrolytic sample cell, so the above comments apply to it also. A variety of other designs for controlled potential electrolysis in the ESR cavity have been proposed and reviews of this subject are available (4-6). In 1971, Goldberg and Bard (7) described a thin flat cell designed to make simultaneous electrochemical-electron spin resonance measurements (SEESR) with better
i i
U Figure 1. Schematic diagram of the coaxial microwave cavity
standard TEo1l X-band microwave cavity. (8)is a cylindrical metallic conductor. (C)is a quartz tube
( A ) is a
electrode potential control than the original Piette et al. ( 3 ) cell, and which thus provides a standard against which new cells should be compared. We describe below a radically new design for SEESR cells based on a coaxial microwave cavity. This coaxial design allows use of electrodes with ten times greater surface area than was previously possible ( 7 ) and thus provides greater ESR sensitivity. I t also effectively places the electrode diffusion layer in the region of maximum ESR sensitivity while the vast majority of the solution lies outside of this region. Hence, electrode potential (or current) changes which alter the concentration of reactants in the diffusion layer produce an immediate ESR response, and this response is not superimposed on that from species originating a t the counter electrode as can be a problem in the Goldberg-Bard design ( 7 ) .The larger electrode area increases the electrolysis current which superficially might be thought to make the ohmic potential control requirements even more stringent than for a flat cell, but the coaxial electrode design provides a nearly ideal geometry for potential control so these problems are easily overcome.
EXPERIMENTAL The ESR experiments were performed on a Varian V-4502 EPR spectrometer and the electrolysis experiments were performed using a Princeton Applied Research Corp. 173D/176 potentiostat/ galvanostat. Synchronous detection of the ESR signal during square-wave voltammetry was accomplished by using the V-4270B second derivative accessory to the ESR spectrometer. The squarewave used for potential modulation and to trigger the synchronous detector was derived from an HP-209A audio oscillator. Cell resistance measurements were made with an ac Wheatstone bridge a t 1000 Hz. Hexamethylphosphoramide (HMPA) (Aldrich) was dried with sodium metal and vacuum distilled twice before use. Tetrabutylammonium perchlorate (TBAP) (Eastman) was recrystallized, dried over P205, and stored under vacuum until used. Nitrobenzene, perylene, and sulfuric acid were reagent grade and used as received. The gold helix was constructed from No. 24 gauge 24K gold wire. The mercury amalgamated gold helix was prepared by dipping the gold electrode into a pool of mercury, then shaking off the excess mercury. 9
CELL DESIGN The Microwave Cavity. Coaxial microwave cavities have been used relatively little as detectors in ESR experi-
ments (8) though, as we will show below, they can be made a t least as sensitive for solvents with high microwave loss tangents as the standard rectangular TEloz cavity. A block diagram of such a cavity is shown in Figure 1. It is constructed by placing a quartz test tube (C) containing a metallic conductor (B) in the center of a standard TEoll cylindrical cavity (A) and putting the liquid sample in the unfilled volume of the tube. The resonant modes of such a cavity are only slightly perturbed from the corresponding ones in the cylindrical cavity and the Bessel functions describing them are labeled according to the same convention ( 8 ) , Le., we have constructed a TEoll coaxial cavity. The node in the electric field along the principal axis of the cylindrical TEoll cavity is expanded to lie along the entire surface of the central conductor in the coaxial cavity, and thus the cylinder immediately adjacent to the cylindrical center conductor becomes the optimum place to put a lossy liquid sample for an ESR experiment. For our experiments, the cylindrical cavity we used was the Varian V-4533 Rotating X-band Cavity with the microwaves coupled to it in the normal manner. This cavity has a diameter and cylinder length of 41 mm. The diameter of the central conductor was 6 mm and its length was approximately 50 mm. The resonant frequency of this coaxial cavity is higher than the 9.0-9.8 GHz range of our X-band klystron, thus quartz or Teflon cylindrical inserts had to be placed around the central conductor in order to lower the resonant frequency. When the central conductor is solid and is made of a good conducting metal, the cavity Q of this system is not measurably different from that of the unperturbed cylindrical cavity. However, this arrangement is not practical for ESR measurements since the 100-kHz magnetic field modulation tends to heat a solid central conductor, and the ac magnetic field is distorted by its presence. These problems can be circumvented, as they are in the walls of the Varian V-4533 cavity, by constructing the central conductor as a finely wound shallow pitched helix. Various plastic supports for the helix were tried but none proved to be universally inert to the variety of free radicals and solvents met in electrochemistry, so we have chosen to use a free standing helix. The helix was wound to fit snugly against the inner wall of a 6-mm i.d. quartz test tube. In this configuration, the microwaves penetrate the solution adjacent to the helix to varying degrees depending on the wire diameter and spacing before being reflected. Thus, one can vary the effective sample volume by stretching or compressing the helix. By using a concentrated sulfuric acid solution of the perylene radical cation as a test solution and a helix wound from No. 24 gauge gold wire we found that a helix with 16 turns/cm gave the maximum ESR signal. A larger number of turns/cm gave a higher Q but the smaller sample volume resulted in less signal. Fewer turns/cm provided a larger sample volume but a lower Q, also resulting in less signal. Other lossy, high dielectric constant solvents gave similar results, i.e., coils with 20-30% open space gave the optimum ESR sensitivity. An estimate of the effective sample volume can be made by measuring the cavity resonance frequency of a given helix-solvent combination and comparing this frequency to the resonance frequency of the same cavity when the same quartz tube is filled with the same solvent and various solid conductors of known diameter. The diameter of the solid conductor having the same resonance frequency as the helix is then taken as the effective diameter of the helix. Using the helix described above and water as the solvent, we find that the microwaves penetrate one wire radius f lo%, as is shown in Figure 2. Since the effective ESR sample volume is poorly defined, it is difficult to measure the ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
891
Ful
C
A
G
t
I MY
.I
Flgure 2. Diagram of the quartz-metal helix-solution interface (A) is the wall of the quartz tube. ( B ) is a cross section of a helix wire. (C)is the liquid sample. The shaded portion of area (C)represents the portion of the sample seen by the microwaves
I
Flgure 4. Schematic diagram of the SEESR cell mounted in the microwave cavity.
(a
(A) is the auxiliary electrode lead. (8) is the Pt auxiliary electrode. is the Luggin capillary for the reference electrode. (D) is the lead for the working electrode. ( E ) is the Au helical working electrode. (F)is a quartz tube. (0) is a cylindrical TEo11 microwave cavity
Flgure 3. ESR spectrum of the nitrobenzene radical anion. Solvent, hexamethylphosphoramide; supporting electrolyte, tetrabutyi ammonium perchlorate
absolute sensitivity of this cavity directly but relative measurements are readily made. For example, we selected the central line of the ESR spectrum of the perylene cation radical dissolved in concentrated sulfuric acid as a standard, then we compared the signal intensity obtained from the coaxial cavity containing the gold helix to that obtained from the same solution placed in a Varian E-248 aqueous solution sample cell mounted in the V-4531, TEloz rectangular cavity. In both cases, the field modulation amplitude and microwave power were adjusted to give the maximum ESR signal so these parameters were identical, although the instrument settings were different. The result was that within 10% the signal heights were the same for both cavities. Varian Associates (9) report that this signal height ratio, obtained by comparing the TEoll cylindrical cavity to the TE102 rectangular one when using the same radical, is 0.25. Thus the coaxial cavity is a fourfold improvement over the cylindrical one for lossy, saturable samples. Figure 3 shows the ESR spectrum of a 10-3M solution of the nitrobenzene anion radical produced electrolytically in HMPA with TBAP as the supporting electrolyte. The signal-to-noise ratio is over 1OOO:l which illustrates graphically the sensitivity of the coaxial cavity. More importantly, the spectrum illustrates that normal undistorted absorption mode ESR spectra can be obtained from this cavity. The variation in linewidths observed here and for all other radicals recorded using the gold helix are readily rationalized on chemical grounds. However, when platinum wire is used for the helix, this is not the case. Then, the signals are distorted. For a first derivative line with a 100 mG peak to peak width, the low field peak is about twice as intense as the high field peak. This distorted lineshape is probably due to an inhomogeneity in the magnetic field induced by 892
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
the platinum helix which has an appreciable positive magnetic susceptibility. When gold or silver wire is used for the helix (where the magnetic susceptibility is tenfold less and negative) no such distortion is observed. Since the overall sensitivity of the platinum helix is not noticeably different from that found for the gold helix, unless lineshape analysis is the object of the experiment, platinum is also a satisfactory material for the helix. The Electrolysis Cell. The coaxial electrolysis cell is shown mounted in the ESR cavity in Figure 4. The helical conductor used in the coaxial microwave cavity described above forms the working electrode ( E ) of the electrolysis cell. Since the microwaves do not penetrate beyond the helix, virtually any material may be placed inside the helix without affecting the ESR experiment. Thus, this space is available for the bulk of the solution, auxiliary and reference electrodes, etc. The auxiliary electrode ( B ) and its lead ( A ) are platinum. This electrode is a 50 mm long, 1 mm in diameter cylinder formed from 1-mil foil. It is positioned along the axis of the helical working electrode so the current flow is radial. This geometry would give a uniform current density over the entire surface of a cylindrical working electrode. Since the current density of the working electrode would be uniform, potential control of the whole electrode could be obtained by monitoring a single point as is done with Luggin capillary (C). The helical working electrode is a good electrochemical approximation to the ideal cylindrical electrode and this conclusion is substantiated below. The Luggin capillary goes down the hollow center of electrode ( B ) and out a small hole, ending as close to electrode ( E ) as possible. The capillary is drawn from Pyrex tubing and is open at both ends. Reference electrodes of standard design are then placed in the wide end of the capillary above the actual cell. For the experiments described herein, we used the SCE supplied with the Varian E-256 Electrochemistry Accessory. The active surface area of the working electrode has not been measured but should be only slightly less then the geometric area of the wire used to construct it, which is 22 cm2, since only a small fraction of the electrode will be shielded from the electrolyte solution by its contact with the walls of quartz tube ( F ) . The cell has a resistance of 13 Q when filled with a 0.1M TBAP dimethylformamide solution which compares “favorably:’ with the 5-10 kR resistance estimated by Goldberg and Bard (7) for the Piette et al. (3) type flat cell containing this solution. We have not observed any effects attributable to uncompensated I R drop for the coaxial cell even
,/
OFF
/
ON
\
~~
~
o
io
20 30 40 50 TIME ( S E C I
60
Flgure 5. ESR signal during and following a constant current pulse of 1 mA
-4
-6
-8
-10
-12
-14
-16
-18
-20
V o l t s v s SCE
when poorly conducting electrolyte solutions such as TBAP in HMPA or T H F are used. As is shown elsewhere (IO),cyclic voltammograms recorded using this cell are indistinguishable from those run in a normal polarographic cell at a HMDE except for the 1000-fold increase in current due to the increased electrode area. A 10-mV error in a cyclic voltammetric peak potential could easily have been detected and, since the typical peak current for a millimolar solution in the coaxial cell is 10 mA, the uncompensated resistance must be less than 1 R .
SEESR RESULTS The ESR and electrochemical results given above show that the coaxial cell approaches ideal behavior both as a microwave cavity and as an electrolysis cell but provide no information about its usefulness as an SEESR cell. In an ideal SEESR cell, the ESR signal intensity responds instantaneously and is proportional to the charge associated with the diffusion controlled electrochemical process proceeding in the cell. In what follows, we discuss the range of experimental conditions over which this ideal behavior is approached by our cell. The halfwave potential for the one electron reduction of nitrobenzene in HMPA containing 0.1M TBAP as supporting electrolyte is -1.1 V vs. SCE. If the controlled potential is stepped rapidly from -0.60 to -1.60 V, the ESR signal begins to increase immediately (
