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(3) Martin, G. J.; Martin, M. L. Tefrabedron Lett. 1981,3525. (4) Martln, G. J.; Martin, M. L. C.R. Hebd. Seances Acad. Sci. 1981, 293, 31. 151 Martin. M. L.: Delouech. J. J.: Martin. G. J. “Practical NMR Spectroscopy”t Heyden: London, Philadelphia, 1980; Chapter 9. Mantsch, H. H.; Salto, H.; Smlth, I. C. P. I n “Progress in NMR Spectroscopy”; Emsley, J. W., Feeney, J., Sutcliffe, L. H., Eds.; Pergamon Press: London, 1977; Vol. 1I, p 237. Hatch M. D.;Slack C. R. B/ocbem. J . 1986, 101, 103. Calvin, M.; Bassham, J. A. “Photosynthesis of Carbon Compounds”; W. A. Benjamin: New York, 1962. Bricout, J. Rev. CyW. Biol. Veg. 1978, 1 , 133. Bricout, J.; Fontes, J. C.; Merlivat, L.; Pusset, M. Ind. Agric. Aliment. 1975,375.
(11) Rauschenbach, P.; Slmon, H.; Stichler, W.; Moser, H. Z.Naturforscb ., C: Biosci. 1979, 34C, 1.
Gerard J. Martin* Maryvonne L. Martin Franpoise Mabon Marie-Jo Michon Laboratoire de Chimie Organique Physique, ERA 315 Facult6 des Sciences (F), 44072 Nantes Cedex, France RECEIVED for review April 19, 1982. Accepted July 21, 1982.
AIDS FOR ANALYTICAL CHEMISTS Long Optical Path Electrochemical Cell for Absorption or Fluorescence Spectrometers Michael J. Slmone, Wililam R. Heineman, and George P. Kreishman* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 1
Since their introduction, optically transparent electrodes have been extensively exploited to make a variety of electrochemical measurements. Presently, their use in the determination of Eo’ (formal redox potential) and n (electron stoichiometry) is commonplace. Of particular interest to the bioanalytical chemist has been the use of spectroelectrochemistry in the study of biological redox components ( I ) . The heterogeneous electron transfer rate can be very slow for biological materials whose redox centers are shielded from interaction with the electrode surface. The use of mediator titrants, together with optically transparent electrodes, has provided effective coupling of the biomolecule to the electrode and has allowed for the subsequent characterization of these molecules with optical techniques (2). Yildiz et al., capitalizing on the inherently greater sensitivity of fluorescence over absorbance, used an optically transparent thin-layer electrode (OTTLE) in conjunction with a spectrofluorometer to study the highly fluorescent molecule perylene (3). In a similar manner, previous work in our laboratory attempted to make use of the inherent fluorescence of tryptophan-59 of horse heart cytochrome c to study possible conformational changes upon oxidation and reduction ( 4 ) . A difference in the fluorescence intensity between the oxidized and reduced forms of cytochrome c was detected and the observed change was quantitatively consistent with previously postulated conformational changes (5) and with that observed for tuna cytochrome c in the solid state (6). A number of technical difficulties associated with the use of the conventional OTTLE cell complicated the preliminary studies. In order to compensate for the relatively short path length (0.02 cm) of the OTTLE, high concentrations of cytochrome c coupled with high instrumental gain settings were used. These conditions resulted in unusually high Rayleigh light scattering relative to fluorescence intensity. In addition, because of the right angle geometry of the fluorometer, difficulties were encountered in reproducibly positioning and taking spectra while thermostating the OTTLE. The present paper is a report on the construction and evaluation of a new long optical path electrochemical cell which can be used for both absorbance and fluorescence studies. Several investigators have reported novel long optical path cell designs optimized for particular uses (7-12). These cells, although elegant in design, generally require large outlays of time and/or expense due to their complexity. The cell
described in this report has as its attributes, (1) ease and quickness of construction from inexpensive, commercially available materials, (2) the ability to be used with relatively inexpensive instrumentation, and (3) small sample volume (0.5 mL) requirement.
EXPERIMENTAL SECTION The gold resinate solution (GOLD #8300,28% Au content) was purchased from Englehard Corp., East Newark, NJ. K,Fe(CN), and o-tolidine were purchased from Fisher Scientific; NaC1, NaH2P04,and Na2HP04(reagent grade) were purchased from MC/B chemicals. All chemicals were used without further purification. Cyclic voltammograms were obtained with a Bioanalytical Systems (BAS) Model CV IB potentiostat coupled to a Keithly Model 178 digital multimeter and a Hewlett-Packard Model 7015A X-Y recorder. Potential step experiments were performed with a Princeton Applied Research (PAR) Model 173 potentiostatgalvanostat. All potentials are reported relative to the standard calomel electrode (SCE). Absorbance measurements were made with a Gilford Model 250 absorbance spectrophotometer and fluorescence spectra were obtained with a Perkin-Elmer Model 650-10s fluorescence spectrophotometer equipped with an Hewlett-Packard Model 7015A X-Y recorder. In a typical experiment, the absorbance or fluorescence spectrum was taken after equilibrium had become established at the applied potential. Applied potentials were selected randomly between the oxidizing and reducing potential limits. The equilibration time was from 30 to 40 min and was indicated by a cessation in spectral response change. This equilibration time is relatively long when compared to the few minutes necessary for complete electrolysis in the OTTLE cell and illustrates the pseudo-thin-layernature of the long optical path cell. The increase in equilibration time due to the leakage from the bulk solution is minimal due t o the fact that the working electrode surface extends from the bottom of the insert to the four sides separating the active zone solution and the bulk solution. This electrode area is similar to the reactant-getter electrode element utilized previously (3). Eo’and n values were obtained from the y intercepts and slopes of Nernst plots with lines drawn using linear least squares analysis of the data points. A diagram of the long optical path cell is shown in Figure 1. A Teflon block (Cincinnati Plastics) was cut and milled so that the base fit snugly into a standard 1 X 1cm quartz cuvette (Fisher Scientific). A set of four “feet” were milled into the bottom of the base providing a space of 0.5 mm in depth (active zone volume -25 wL. A small hole was drilled into the center of the base t o hold the auxiliary (Pt wire) and reference (SCE) electrodes. A
0003-2700/82/0354-2382$0 1.2510 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY. VOL. 54. NO, 13. NOVEMBER 1982 2383
A
B
Flgure 2. Cyclic voltammogram of 1.0 mM K,Fe(CNk in 0.5 M NaCI; E,, = +0.400 V vs. SCE. initial scan negative; scan rate 1 mV/s.
hex? C Fipue 1. (A) Goldcoated Teflon working electrode in&, areas 1 and 2 as in C. (6)carpleted atl magam showing (a) PI auxilii e!sdnxb, (b) SCE reference electrode. (c) radiation path, (d) Teflon bottom spacer,(e)quam cwelte. and (0gok!-coated TeROn working electrode. (C) Top view of cell showing radiation paths in absorption and fluorescence experiments, areas 1 and 2 as in A.
lo?
small pin hole was made in the bottom to expose the auxiliary and reference electrodes. Once completed, a thin mat of the gold resinate was brushed on certain areas of the Teflon as shown. These areas include the bottom, the four sides extending up 1 em from the bottom, and a narrow (0.3 em) strip extending up one side for electrical contact to the potentiostat. The coated Teflon was placed in a drying oven for approximately 10 min at 125 OC. Several coats of the resinate were applied in this manner. After drying, the coated Teflon was placed in an annealing oven for 10 min at 350 OC. This air firing drove off the organic resin and fixed the remaining film of gold onto the Teflon (13). This procedure was repeated until a conducting film of gold was deposited. The fluorescence spectrophotometer utilized in the present study is designed with horizontal slits for excitation and emission radiation. For this reason the cell described here is designed with a horizontal radiation path. For spectroelectrochemicalexperiments using a spectrometer of conventional vertical slit design, the secondary filter of the instrmnent, which masks the radiation beam and allows the use of smaller sample volumes, was found to work satisfactorily. Presumably a secondary slit system could be utilized to modify the radiation beam of conventional fluorescence spectrophotometers.
RESULTS AND DISCUSSION Initially, the electrochemical properties of the long optical path length cell were evaluated by using 1.0 mM K3Fe(CN)6 in 0.5 M NaCl. A representative cyclic voltammogram is shown in Figure 2. Eo‘ estimated using the equation E O ‘ = (E, + EA)/!&where E, and E , are the cathodic and anodic peak potentials, respectively, is 0.213 V vs. SCE and is in very good agreement with literature values (14,15). Even though the reference and auxiliary electrodes were positioned in order to minimize iR drop, AEp from this cyclic voltammogram indicates that a potential gradient across the working electrode does exist. Since the spectra were taken only after equilibrium had been estahlished a t the applied potential, this iR drop presented no difficulties. Spectroelectrochemical experiments were performed in order to evaluate the utility of the long optical path cell for the determination of EO’ and n values. A Nemst plot of absorbance values for a 1.0 mM K,Fe(CN), solution at various applied potentiah yielded values for a line with a slope of 60.9
\VI
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Figure 3. Cyclic witammogram of 1.0 mM 0-tolidine in 0.5 M H&COOH and 1.0 M tKl0,; E,, = 0.410 V vs. SCE. initial scan pOSRive; scan rate 2 m V h .
mV which correspondsto an n value of 0.97 and E O ’ , calculated f ” the y intercept, of ,207 V vs. SCE, which agrees well with values obtained through cyclic voltammetry. The long optical path cell was evaluated for spectrofluoroelectrochemical use with o-tolidine. The spectroelectrochemistry of o-tolidine has been studied extensively and involves a two-electron transfer upon oxidation and reduction in acidic aqueous media (16,17). Previously, o-tolidine has been used for the evaluation of spectroelectrochemical cells due to the intense absorhance of the oxidized form a t 438 nm and near transparency of the reduced form a t that wavelength. In addition, with the moderate fluorescenceintensity (excitation, 270 nm; emission, 415 nm) exhibited by reduced o-tolidine and the decreased fluorescence intensity of the oxidized form, the results of the spectroelectrochemical studies can he compared with the spectrofluoroelectrochemical studies. A typical cyclic voltammogram of 1.0 mM o-tolidine in 0.5 M acetic acid and 1.0 M perchloric acid is given in Figure 3. E O ’ from this cyclic voltammogram is calculated to be 0.619 V vs. SCE, in agreement with previous determinations (15). Nernst plots of spectroelectrochemical experiments with M o-tolidine yielded Eo’ values 3.0 x M and 1.0 X of 0.621 V and 0.616 V vs. SCE, respectively. Calculated n values from the slopes were 1.8 for both concentrations. The fluorescence intensity of o-tolidine at various Ewp can be used to determine Eo‘ and n values in a manner analogous to that using absorbance. For very dilute solutions the fluorescence intensity can be defined by an equation comparable to Beer’s law (181,F = &bc, where + i s the quantum
2384
ANALYTICAL CHEMISTRY, VOL.
54, NO. 13, NOVEMBER 1982
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yield (photons emitted/photons absorbed) and e, b, and c are as in Beer's law. Using this equation, therefore
-[OI- [Rl
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500
Figure 4. Fluorescence spectra of 5.0 pM o-tolidine in 0.5 M H,CC0 6 H and 1.0 M HC104at various applied potentials (E*): (a)+0400 V, (b) +0.560 V, (c)4-0.590 V, (d) +0.596 V, (e)+OB00 V, (f) 4-0.605 V, (9) +0.611 V, (h) +OB20 V, (i) 4-0.640V, (j) 4-0.659 V, (k) +0.600 V; A, 270 nm, A, 415 nm.
(F3 - J'J/& F3 - Fz =(F2 - F J / @ F2 - Fl
where F3 is the fluorescence intensity at Eapp set at the limiting reducing potential, F1 is the fluorescence intensity when Eapp is set at the limiting oxidizing potential and F, is the fluorescence intensity at an intermediate ICapp. The fluorescence spectra of 5.0 X lo4 M o-tolidine in 0.5 M acetic acid and 1.0 M perchloric acid at various kapp are shown in Figure 4. Spectrum a represents the limiting con= +0.400V vs. SCE) where [O]/[Rj < 0.001 while dition (Eapp spectrum k (Eapp= +0.800 V vs. SCE) represents [D]/[R] >lOOO. Intermediate spectra (kj) correspond to intermediate Espp's.A Nernst plot taken from these fluorescence spectra yielded a slope of 28.9 mV, which corresponds to an n value of 2.0 E"' calculated from the y intercept is 0.618 V vs. SCE, which agrees very well with those values obtained using cyclic voltammetry and spectroelectrochemistry. As with spectroelectrochemical experiments, interferences from other absorbing species may complicate the data obtained. Figure 5 represents, & spectrofluoroelectrochemical experiment using 3.0 X M o-tolidine in 0.5 M acetic acid and 1.0 M perchloric acid. Figure 5 illustrates the influence of high concentrations of oxidized a-tolidine on the fluorescence spectra due to the close proximity of the absorbance maximum (438 nm) and fluorescence emission maximum (415 nm). Spectrum a represents the limiting conditions when nearly all of the o-tolidine molecules are in the reduced form. As can be seen there is little influence by the oxidized form. As the potential is stepped to more positive values, however, the concentration of oxidized o-tolidine increases and a resulting absorbance of the emitted radiation (secondary absorbance) occurs. A Nernst plot of the data shown in Figure 5 yields a calculated n value of 1.85 which compares favorably to those obtained from spectroelectrochemical experiments using 3.0 X M and spectrofluoroelectrochemical experiments using 5.0 X lo4 M o-tolidine. The E"' value, however, is 0.597 V vs. SCE. This Eo' is 0.024 V less than previously obtained from spectroelectrochemical experiments with 3.0 x M o-tolidine. This difference illustrates that care must be taken when interpreting data from spectra where highly
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Flgure 5. Fluorescence spectra of 30 pM o-tolidine in 0.5 M H,CCOOH and 1.O M HCIO4 at varlous applied potentials (E,,,,,,): (a)+0.400 V, (b) +0.580 V, (c) +0.600 V, (d) +0.620 V, (e) +0.640 V, (f) 4-0.660 V, (9)
+0.800 V.
absorbing species may be present. In coficlusion, the long optical path cell described can be used to determine the electron stoichiometry, n, and the formal redox potential, E"', when coupled with an absorbance or fluorescence spectrophotometer. The cell, by virtue of its long optical path, can be used to analyze electroactive materials at concentrations lower than those usually associated with thin layer cells. In addition, compounds which are both electroactive and fluorescent can be analyzed at even lower concentrations.
ACKNOWLEDGMENT The authors wish to thank Ralph Magnotti, Jane Lewis, and Emory DeCastro for helpful discussion on the contents of this paper and Harry B. Mark for the use of his PAR potentiostat. The authors thank Stanley Bruckenstein for information about the properties of gold resinate.
LITERATURE CITED (1) Helneman, W. R.; Anderson, C. W.; Halsall, H. B.; Hurst, M. M.; Johnson, J . M.; Kreishman, G. P.; Norris, B. J.; Simone, M. J.; Su, C.-H. Adv. Chem. Ser. 1982, N o . 201, 1. (2) Szentrlmay, R.; Yeh, P.; Kuwana, T. ACS Symp. Ser. 1977, N o . 3 8 , 143. (3) Yildiz, A,; Klssinger, P. T.; Reilley, C. N. Anal. Ch'em. 1968, 40, 1018. 14) Simone, M. J.; Heineman, W. R.; Kreishman. G. P. J . Colloid Interface Sci. 1982, 8 6 , 295. (5) Salemme, F. A. Annu. Rev. Biochem. 1977, 46, 299, and references therein. (6) Takano, T.; Dickerson, R . E. Proc. Natl. Acad. Sci. USA 1980, 7 7 , 6371. (7) Kuwana, T.; Heineman, W. R. Acc. Chem. Res. 1978, 9, 241. (8) Stankovich, M. J. Anal. Biochem. 1980, 109, 295. (9) Hendler, R. W.; Songco, D.; Clem, J . R. Anal. Chem. 1977, 49, 1980. (10) Anderson, J. L. Anal. Chem. 1979, 5 1 , 2312. (11) Baumgartner, C. E.; Marko, G. T.; Aikens, D. A,; Richtol, H. H. Anal. Chem. 1980, 5 2 , 267. (12) Pruiksma, R.; McCreery, R. L. Anal. Chem. 1979, 5 1 , 2253. (13) Data Sheet EM-11660, Revised 5/81, Engelhard Industries Division, 1 West Central Av., East Newark, NJ 07029. (14) Kolthoff, I. M.; Tomsicek, W. J. J . fhys. Chem. 1935, 39, 945. (15) DeAngelis. T. P.; Heineman. W. H. J . Chem. Educ. 1976, 5 3 , 594. (16) Kuwana, T.; Strojek, W. Discuss. Faraday SOC. 1968, 45, 134. (17) Murray, R. W.; Heineman, W. R.; O'Dom, G. W. Anal. Chem. 1987. 39, 1666. (18) Pringsheim, P. "Fluorescence and Phosphorescence": Interscience: New York, 1949; p 347.
RECEIVED for review June 7,1982. Accepted August 4,1982. This work was supported in part by a grant from the University of Cincinnati Research Council (G.P.K.). M.J.S. was the recipient of a Lowenstein-Schubert-TwitchellFellowship and a University Research Council Summer Fellowship.
