Anal. Chem. 1997, 69, 1070-1076
Imaging of Oxygen Evolution and Oxide Formation Using Quinine Fluorescence Joseph E. Vitt and Royce C. Engstrom*
Department of Chemistry, University of South Dakota, Vermillion, South Dakota 57069
Quinine was used as a fluorescent acid-base indicator to image electrode reactions that consume hydroxide ions. The fluorescence intensity was used as a measure of the microscopically local current at the electrode surface during O2 evolution and oxide formation. Highest resolution images were obtained 300-600 ms after application of the potential in an unbuffered (pH 10) 0.5 mM quinine solution. The increase in fluorescence intensity observed at later times was offset by a decrease in spatial resolution. Imaging anodic reactions using quinine fluorescence is complementary to the previously established method for imaging cathodic processes using fluorescein fluorescence. Similarly, this new method should be able to provide information about microscopically local reaction rates and concentration profiles at electrode surfaces for a variety of anodic reactions. Evaluation of electrochemical data frequently involves the assumption that the observed current is representative of the electrochemical reaction at all points on the electrode surface. This assumption is valid as long as the electrode surface is both uniformly active and uniformly accessible to the analyte. However, many frequently encountered conditions exist where the total current is not indicative of the local current density at specific sites on the electrode surface. Some experimental conditions that lead to a nonuniform distribution of current density include convergent diffusion at the edges of inlaid disk electrodes,1-5 inhomogeneities due to the presence of adsorbed species6-11 or oxide films,12 differential reactivities at various crystal faces of an electrode,13 depletion of analyte at the downstream portion of an electrode in a flow-through cell,14-16 and the use of composite electrodes.17-19 Several techniques have been developed to detect chemical species involved in electrochemical processes in a spatially resolved manner. One such technique, commonly referred to as
scanning electrochemical microscopy (SECM),20-22 uses an ultramicroelectrode to make electrochemical measurements within the diffusion layer of a substrate electrode.23 SECM has been used to map spatial variations in electrochemical activity,24-26 which could be useful in identifying the characteristics that lead to increased activity. Also, concentration profiles within the diffusion layer can be measured directly,27 which is useful for characterizing mass-transport processes to and from a surface and for determining the fate of transient products of electrochemical reactions. SECM has recently been extended to imaging of nonelectrochemical events as well, such as iontophoretic transport through microscopic pores in membranes28 and reactions at immobilized enzymes.29,30 One condition of SECM is that, when operated in the feedback mode,31 a chemically reversible couple is required, which limits the number of oxidation-reduction reactions that can be studied. Another method that has been used to image electrochemical activity at electrode surfaces is electrogenerated chemiluminescence (ECL) combined with optical microscopy.5,18,32-35 In ECL imaging, electrochemistry is used to initiate a chemiluminescent reaction, and the emitted light is collected with an optical microscope equipped with an imaging detector such as a video camera. The ECL reaction of luminol has been used most frequently because of its large intensity, and images with resolutions approaching the limits of optical microscopy have been
(1) Bard, A. J. Anal. Chem. 1961, 33, 11-15. (2) Lingane, P. J. Anal. Chem. 1964, 36, 1723-1726. (3) Oldham, K. B. J. Electroanal. Chem. 1981, 122, 1-17. (4) Aoki, K.; Osteryoung, J. J. Electroanal. Chem. 1981, 122, 19-35. (5) Engstrom, R. C.; Pharr, C. M.; Koppang, M. D. J. Electroanal. Chem. 1987, 221, 251-255. (6) Landsberg, R.; Thiele, R. Electrochim. Acta 1966, 11, 1243-1259. (7) Scheller, F.; Muller, S.; Landsberg, R.; Spitzer, H.-J. J. Electroanal. Chem. 1968, 19, 187-198. (8) Trukhan, A. M.; Povarov, Yu. M.; Lukovtsev, P. D. Elektrokhimiya 1970, 6, 425-429. (9) Povarov, Yu. M.; Trukhan, A. M.; Lukovtsev, P. D. Elektrokhimiya 1970, 6, 602-611. (10) Povarov, Yu. M.; Lukovtsev, P. D. Electrochim. Acta 1973, 18, 13-18. (11) Engstrom, R. C.; Nohr, P. L.; Vitt, J. E. Colloids Surf. A 1994, 93, 221-227. (12) Casillas, N.; Charlebois, S. J.; Smyrl, W. H.; White, H. S. J. Electrochem. Soc. 1993, 140, L142-L145. (13) Ross, P. N. J. Electrochem. Soc. 1979, 126, 67-77.
(14) Weber, S. G.; Purdy, W. C. Anal. Chim. Acta 1978, 100, 531-544. (15) Cope, D. K.; Tallman, D. E. J. Electroanal. Chem. 1986, 205, 101-123. (16) Magee, L. J., Jr.; Osteryoung, J. Anal. Chem. 1990, 62, 2625-2631. (17) Anderson, J. E.; Tallman, D. E.; Chesney, D. J.; Anderson, J. L. Anal. Chem. 1978, 50, 1051-1056. (18) Petersen, S. L.; Weisshaar, D. E.; Tallman, D. E.; Schulze, R. K.; Evans, J. F.; DesJarlais, S. E.; Engstrom, R. C. Anal. Chem. 1988, 60, 2385-2392. (19) Tallman, D. E.; Petersen, S. L. Electroanalysis 1990, 2, 499-510. (20) Liu, H.-Y.; Fan, F.-R. F.; Lin, C. W.; Bard, A. J. J. Am. Chem. Soc. 1986, 108, 3838-3839. (21) Bard, A. J.; Fan, F.-R. F.; Kwak, J.; Lev, O. Anal. Chem. 1989, 61, 132138. (22) Engstrom, R. C.; Pharr, C. M. Anal. Chem. 1989, 61, 1099A-1104A. (23) Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Winquist, S. Anal. Chem. 1986, 58, 844-848. (24) Wipf, D. O.; Bard, A. J. J. Electrochem. Soc. 1991, 138, 469-474. (25) Wipf, D. O.; Bard, A. J. J. Electrochem. Soc. 1991, 138, L4-L6. (26) Engstrom, R. C.; Small, B.; Kattan, L. Anal. Chem. 1992, 64, 241-244. (27) Engstrom, R. C.; Meaney, T.; Tople, R.; Wightman, R. M. Anal. Chem. 1987, 59, 2005-2010. (28) Scott, E. R.; White, H. S.; Phipps, J. B. Anal. Chem. 1993, 65, 1537-1545. (29) Horrocks, B. R.; Mirkin, M. V.; Pierce, D. T.; Bard, A. J.; Nagy, G.; Toth, K. Anal. Chem. 1993, 65, 1213-1224. (30) Wittstock, G.; Yu, K.; Halsall, H. B.; Ridgway, T. H.; Heineman, W. R. Anal. Chem. 1995, 67, 3578-3582. (31) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227. (32) Engstrom, R. C.; Johnson, K. W.; DesJarlais, S. Anal. Chem. 1987, 59, 670673. (33) Bowling, R. J.; McCreery, R. L.; Pharr, C. M.; Engstrom, R. C. Anal. Chem. 1989, 61, 2763-2766. (34) Pantano, P.; Kuhr, W. G. Anal. Chem. 1993, 65, 2452-2458. (35) Hopper, P.; Kuhr, W. G. Anal. Chem. 1994, 66, 1996-2004.
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© 1997 American Chemical Society
obtained. However, a map of ECL intensity specifically measures the ability of each site on the electrode surface to oxidize luminol and/or hydrogen peroxide. (Oxidation of hydrogen peroxide is necessary to produce the superoxide radical required by the chemiluminescent reaction36,37). Therefore, there is no guarantee that the map of electrode activity is generally applicable to a variety of reactions since a site that is active toward luminol oxidation may be inactive toward some other analyte of interest. In addition, a study of the effect of electrode material on the luminol ECL reaction found that the ECL intensity was lower at electrode materials that produced more current for the oxidation of luminol,38 complicating the assignment of relative activity. Another approach to luminescence imaging of electrode surfaces is based on the electrochemical initiation of fluorescence of the common fluorescent acid-base indicator, fluorescein.39 Since fluorescein is highly fluorescent only when deprotonated,40 the fluorescence intensity increases during the electrochemical reduction of water, which produces hydroxide ions. The resulting fluorescence is collected by a video microscope as described above for ECL imaging, resulting in an image of the electrochemically altered pH of the diffusion layer. Recently, the electrochemically generated pH gradients over arrays of ultramicroelectrodes were imaged using fluorescein as the fluorescent probe in order to evaluate a method of electrotitration.41 Imaging of cathodic reactions using fluorescein has also been extended to include the reduction of oxygen in solution.42 Fluorescence imaging is likely to be more versatile than ECL imaging since any electrochemical reaction that produces hydroxide ions or hydrogen ions should alter the fluorescence intensity. However, the spatial resolution obtained with fluorescence imaging is expected to be significantly worse than that obtained with ECL imaging because the OH- can diffuse away from the electrode, whereas the chemiluminescent product of luminol oxidation emits light before diffusion occurs to a significant distance. Nevertheless, fluorescence imaging using fluorescein is an excellent method for spatially resolved monitoring of cathodic processes. In fact, there may be more information available from fluorescence imaging than from ECL imaging since concentration profiles can be obtained in addition to locating sites of electrochemical activity at a surface. In this study, quinine was evaluated as a fluorescent acidbase indicator for anodic reactions that consume hydroxide ions, since quinine is highly fluorescent only at pH values less than about 9.5.43 The anodic reaction studied in this work was the oxidation of H2O to O2 which consumes OH- according to the reaction
4OH- h O2 + 2H2O + 4e-
(1)
Images of oxygen evolution were obtained at a variety of electrode (36) Misra, H. P.; Squatrito, P. M. Arch. Biochem. Biophys. 1982, 215, 59-65. (37) Lind, J.; Merenyi, G.; Eriksen, T. E. J. Am. Chem. Soc. 1983, 105, 76557661. (38) Vitt, J. E.; Johnson, D. C.; Engstrom, R. C. J. Electrochem. Soc. 1991, 138, 1637-1643. (39) Engstrom, R. C.; Ghaffari, S.; Qu, H. Anal. Chem. 1992, 64, 2525-2529. (40) White, C. E.; Argauer, R. J. Fluorescence Analysis: A Practical Approach; Marcel Dekker: New York, 1967; p 104. (41) Fiedler, S.; Hagedorn, R.; Schnelle, T.; Richter, E.; Wagner, B.; Fuhr, G. Anal. Chem. 1995, 67, 820-828. (42) Bowyer, W. J.; Xie, J.; Engstrom, R. C. Anal. Chem. 1996, 68, 2005-2009. (43) Schulman, S. G.; Threatte, R. M.; Capomacchia, A. C.; Paul, W. L. J. Pharm. Sci. 1974, 63, 876-880.
materials, and the fluorescence intensity and spatial resolution were studied as functions of current density, quinine concentration, initial pH, and time in order to determine the optimum experimental conditions. This technique was used to successfully map O2 evolution at Ru sites separated by about 25 µm in a Kel-F composite electrode with both graphite and Ru electroactive sites. EXPERIMENTAL SECTION Apparatus. The video imaging system and fluorescence microscope used in this study have been described.39 The excitation source was a 50 W Hg arc lamp, and the microscope was fitted with a 365 nm low-pass excitation filter, a 400 nm highpass emission filter, and a 395 nm dichroic beamsplitter. Electrochemical experiments were performed using a Pine Instrument Co. potentiostat/galvanostat (Model AFRDE5). The gold disk electrode (area 0.007 85 cm2) was made by sealing a 1 mm gold wire in Kel-F (poly(chlorotrifluoroethylene), Kel-F-81, 3M Co.). The Kel-F composite electrodes were fabricated according to published procedures17,44 and consisted of Ru, graphite, and Kel-F. The compositions of the various electrodes are given here as volume percent; for example, 5% Ru/10% Kelgraf indicates 5% Ru and 10% graphite in Kel-F. The reference electrode was a Ag/AgCl electrode with 3 M NaCl gel filling solution from Bioanalytical Systems and had a potential of 0.197 V vs the SHE. A platinum wire was used as the auxiliary electrode. Reagents. All solutions were prepared by dissolving reagent grade chemicals in deionized water (NanoPure, Barnstead/ Thermolyne, Dubuque, IA). Quinine sulfate was obtained from Mallinckrodt (St. Louis, MO). Procedure. All images were obtained with the sample electrode inserted through the bottom of the cell so that the electrode surface was facing up and was covered by a minimal amount of electrolyte (about 2-3 mm). The compositions of the various solutions used in imaging are given in the text or the figure captions. All experiments were performed in unstirred solutions. The fluorescence intensities are shown in arbitrary units and cannot be directly compared because the sensitivity of the camera was adjusted between experiments. RESULTS AND DISCUSSION Voltammetry. A series of fluorescence images were obtained during a potential sweep from -0.20 to 1.60 V at a 1.0 mm Au disk electrode. No anodic or cathodic current was observed for quinine over the potential range used. The resulting images are shown in Figure 1, beginning with 0 V in the top left frame and increasing by 50 mV in each successive frame until the potential sweep was reversed at 1.60 V. The fluorescence intensity first increased above background levels at a potential of 1.10 V (frame 23). The fluorescence intensity continued to increase during the positive potential sweep, and the resulting images of the electrode surface were consistent with the geometry of the electrode. The fluorescence intensity was relatively uniform across the surface of the electrode, which is consistent with the expected uniform current density for O2 evolution where the solvent is the reactant. However, the fluorescence intensity was not uniform across the electrode surface during the negative potential sweep. Instead, the fluorescence was limited to a smaller and smaller portion at the center of the electrode as the potential was decreased. This (44) Vitt, J. E.; Johnson, D. C.; Tallman, D. E. Anal. Chem. 1993, 65, 231-237.
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Figure 1. Fluorescence images of a 1.0 mm Au electrode obtained during a voltammetric sweep from -0.20 to 1.60 V at a scan rate of 100 mV s-1. The solution contained 0.50 mM quinine and 50 mM K2SO4 adjusted to pH 10. The first frame was obtained at 0.0 V during the positive potential sweep and each successive frame is 50 mV greater until the reversal of the potential sweep, at which point the potential of each successive image is 50 mV more negative. Each image represents an area 1600 µm on a side. Figure 3. Current and total fluorescence intensity as a function of potential obtained at a 1.0 mm Au electrode during a voltammetric sweep from -0.20 to 1.60 V at a scan rate of 100 mV s-1. The solution contained 0.50 mM quinine and 50 mM K2SO4 adjusted to pH 10.
Figure 2. Three-dimensional image of fluorescence intensity as a function of position obtained at the 1.0 mm Au electrode at 1.50 V, corresponding to frame 31 of Figure 1.
resulted from the diffusional relaxation of the pH gradient once O2 evolution ceased. The electrode edges returned to bulk solution pH before the electrode center. This relaxation occurred slowly and caused significant hysteresis in the fluorescence vs potential curves, which are discussed later. A three-dimensional plot of fluorescence intensity vs position is shown in Figure 2 for the image obtained at 1.50 V during the positive potential sweep. (This corresponds to frame 31 in Figure 1.) The image shows clearly the boundary of fluorescence intensity that marks the edge of the electrode. Using the three-dimensional plot in Figure 2, the diameter of the electrode was calculated to be 1050 µm, as compared to the nominal value of 1000 µm and the value of 1030 µm measured by ordinary light microscopy. Therefore, a small amount of diffusional broadening is apparent in that particular image. The total fluorescence intensity at each potential was measured by integrating the fluorescence intensity for each frame shown in Figure 1. The resulting fluorescence vs potential curve is shown in Figure 3, along with the current vs potential curve. During separate voltammetric experiments, we observed that the presence of quinine decreased the value of the current obtained as compared to the residual response for O2 evolution and oxide formation and stripping, although the voltammetric curves were 1072 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
very similar in shape and peak potentials whether or not quinine was present. Also, the voltammetric curve obtained when the positive potential limit was 1.60 V (Figure 3, lower curve) differs from the residual curve expected at a Au electrode in that two peaks were observed for oxide reduction instead of one. The peak at about 0.25 V during the negative potential scan corresponds to oxide reduction at the expected potential for a Au electrode in pH 10 electrolyte. The anomalous peak observed at about 0.65 V was probably due to the shift in the pH to more acidic values, which caused oxide reduction to shift to more positive potentials. Once oxide reduction proceeded, the pH returned to about pH 10 due to the production of OH- during cathodic reduction of the oxide layer. This behavior is similar to that reported by Larew and Johnson,45 where oxide stripping was used to generate an alkaline pH in the diffusion layer to enhance the detection of glucose at Au electrodes. For the voltammetric data shown in Figure 3, oxide formation proceeded on the Au electrode at potentials from about 1.00 V to the positive potential limit of 1.60 V. The characteristic exponential increase in current due to anodic evolution of O2 occurred for potentials greater than about 1.40 V during the positive potential scan, although the potential corresponding to the onset of O2 evolution is difficult to measure from these results since oxide formation was contributing to the current at these potentials. The fluorescence intensity (Figure 3, upper curve) increased sharply for potentials greater than 1.10 V, and, during the positive potential scan, the fluorescence intensity appears to reflect the processes of both Au oxide formation and O2 evolution. However, during the negative potential scan, the current for O2 evolution ceased for potentials negative of about 1.40 V, while the fluorescence intensity was still above background levels when the imaging was stopped at 0 V. At slower scan rates, the hysteresis between the current for O2 evolution and the fluorescence intensity was not (45) Larew, L. A.; Johnson, D. C. J. Electroanal. Chem. 1989, 264, 131-147.
Figure 4. Current and total fluorescence intensity as functions of potential obtained at a 1.0 mm Au electrode during a voltammetric sweep from -0.20 to 1.40 V at a scan rate of 100 mV s-1. The solution contained 0.50 mM quinine and 50 mM K2SO4 adjusted to pH 10.
as noticeable; that is, the fluorescence returned to baseline levels at more positive potentials during the negative scan. Conversely, at faster scan rates, the delay between the cessation of current for O2 evolution and the return of the fluorescence intensity to background levels was more severe. This behavior is consistent with a finite time needed for restoration of interfacial pH to bulk solution values. The voltammetric and fluorescence vs potential curves obtained when the positive potential limit was only 1.40 V are shown in Figure 4. The voltammetric results (lower curve) are similar to those shown in Figure 3, except only one peak is evident for oxide stripping (Epeak ≈ 0.25 V) and the current corresponding to O2 evolution was greatly decreased due to the lower positive potential limit. The fluorescence intensity (upper curve) increased sharply at potentials greater than about 1.00 V during the positive potential scan. In fact, the fluorescence intensity appears to be as sensitive a measure of Au oxide formation and O2 evolution as is the current. The largest contribution to the increase in fluorescence intensity is probably due to the pH shift from OH- consumption by Au oxide formation, since the current due to O2 evolution is much smaller than that due to oxide formation. The hysteresis between the current for O2 evolution and the fluorescence intensity was also greatly decreased during the negative potential scan as compared to the results when the positive potential limit was 1.60 V. This results from the smaller shift in interfacial pH, as evidenced by the lower fluorescence intensity, due to the decrease in the current for O2 evolution. From the results shown in Figure 4, it is evident that the total fluorescence intensity is a good indication of the total current for O2 evolution and Au oxide formation at a uniformly active disk electrode. Therefore, microscopic measurements of the fluorescence intensity are also likely to be a good measure of the microscopic current. From these voltammetric results, we concluded that quinine would be an effective fluorescent probe of anodic processes that consume hydroxide ions. However, optimization of the conditions
Figure 5. Fluorescence images of a 1.0 mm Au disk electrode as a function of current density: (b) 2.0 µA, 1.56 V; (c) 1.0 µA, 1.48 V; (d) 0.50 µA, 1.38 V; (e) 0.30 µA, 1.37 V; (f) 0.20 µA, 1.26 V; and (g) 0.10 µA, 1.18 V. The solution consisted of 0.50 mM quinine and 50 mM K2SO4 adjusted to pH 10. Image a is the Au electrode under ordinary visible illumination. Each image represents 1600 µm × 1600 µm and was recorded by integrating for 2 s after adjusting the camera sensitivity to maximize the fluorescence intensity.
using voltammetry was complicated because both potential and time are varied. The imaging conditions were optimized under steady-state, or constant current, conditions as described in the following sections. The effects of various experimental parameters on the fluorescence intensity and spatial resolution were used to thoroughly characterize the application of quinine fluorescence imaging to anodic reactions. Effect of Current Density. The effect of the rate of O2 evolution on fluorescence intensity and image resolution was determined by applying a constant anodic current to the electrode. After initiation of the current, the potential gradually increased until reaching a constant value. The fluorescence intensity increased at the electrode surface when the potential was sufficiently positive that the current being passed corresponded to anodic evolution of O2, after which the fluorescence image gradually achieved a constant appearance. To ensure that the image corresponded to the steady-state value, the current was applied for a minimum of 5 min before the image was recorded. The images obtained at a 1 mm Au disk electrode as a function of current density are shown in Figure 5. For comparison, the optical micrograph of the Au surface under ordinary visible illumination is shown in image a of Figure 5. The steady-state potential at the electrode for each current density is given in the figure caption. At 2.0 µA (image b), the fluorescence extends at least a few hundred micrometers away from the Au electrode, indicating that the region of decreased pH extends to dimensions much larger than the electrode dimensions. From the image, it is apparent that the pH gradient does not extend symmetrically away from the electrode surface. This effect may be due to convection from either incomplete vibration isolation or thermal effects from the microscope light source. It was noted that, for repeat experiments, similar images were observed, but the fluorescence did not necessarily extend in the same direction away from the electrode for each experiment. At lower current values, the fluorescence images indicated that the decrease in pH is Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
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Figure 6. Fluorescence images of a 1.0 mm Au disk electrode held at 0.50 µA as a function of quinine concentration: (a) 0.50 mM, 1.38 V; (b) 0.10 mM, 1.38 V; (c) 0.050 mM, 1.39 V; (d) 0.010 mM, 1.43 V. Each solution also contained 50 mM K2SO4 supporting electrolyte and was adjusted to pH 10. Each image represents 1600 µm × 1600 µm and was recorded by integrating for 2 s after adjusting the camera sensitivity to maximize the fluorescence intensity.
restricted to near the electrode surface, with 0.50 µA (image d) being the value that best reproduced the geometry of the electrode while maintaining the maximum fluorescence intensity as compared to the background intensity. As the current was decreased (images e, f, and g), the maximum fluorescence intensity over the electrode systematically decreased compared to the background intensity. At 0.10 µA (image g), the image of the electrode has nearly disappeared into the background. Effect of Quinine Concentration. Constant current experiments as described above were performed at various quinine concentrations to determine the optimum concentration of the fluorescent probe. The results of these experiments are shown in Figure 6. It is important to note that the absolute intensities from frame to frame should not be compared because the camera sensitivity was increased prior to obtaining each image to provide adequate exposure. However, the signal-to-background ratio is clearly discernible from the figures. Image a is identical to that shown in Figure 5, image d, for 0.50 mM quinine at 0.50 µA. Good resolution of the Au electrode was also achieved using quinine concentrations of 0.10 mM (image b) and 0.050 mM (image c), although the fluorescence intensity was decreased significantly as compared to the background. The fluorescence intensity over the electrode surface was barely above the background level when the quinine concentration was reduced to 0.010 mM, and the image was no longer representative of the geometry of the electrode, as shown in image d. The change in buffer strength of the solution due to the change in quinine concentration may have had a slight effect on the pH gradient at the electrode surface. However, the degradation in image quality shown in Figure 6 primarily reflects the detection limit of fluorescence over background. Knowing the detection limit is important because a minimal concentration of quinine is desirable in order to minimize any interference with the electrochemical reaction. A horizontal profile was taken from each of the images in Figure 6, and the results are plotted in Figure 7. Because of the sensitivity normalization, the background intensity appears to 1074 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
Figure 7. Fluorescence intensity as a function of horizontal position across the center of the fluorescence images in Figure 6: (a) 0.50 mM; (b) 0.10 mM; (c) 0.050 mM; (d) 0.010 mM quinine. The edge of the electrode as determined under ordinary visible illumination is depicted by the vertical dashed lines.
increase, even though the absolute intensity actually decreased with decreasing quinine concentration. The largest signal-tobackground fluorescence intensity was obtained using 0.50 mM quinine (curve a). Also, Figure 7 shows that, for 0.50 mM quinine, the fluorescence intensity increased above the background level at a position very near to the edge of the electrode (indicated by the dashed lines in Figure 7), thus providing good spatial resolution. However, the fluorescence intensity was not uniform over the electrode surface, but instead increased gradually until reaching a maximum near the center of the electrode. This is the result of diffusion from bulk solution to neutralize the protonated quinine and to restore the OH- concentration to its bulk solution value. Because of the steady-state nature of this experiment, diffusion has a large effect on the pH gradient. Compared to the transient images of Figure 1, it is obvious that steady-state conditions do not provide the best spatial resolution. Chronoamperometry. The effect of time on fluorescence imaging using quinine was determined by stepping the electrode potential to a series of values corresponding to anodic evolution of O2. The fluorescence intensity at various times after a potential step from 0 to 1.60 V is plotted versus position across the Au electrode in Figure 8. The background fluorescence (curve a) present at 0 V was essentially equal for the Au electrode and the Kel-F shroud. After the potential was stepped to 1.60 V, a sharp increase in fluorescence occurred at the edge of the electrode, and a roughly constant value of fluorescence intensity was present over the electrode surface. This behavior is in contrast to the intensity profiles discussed above for steady-state experiments where the fluorescence intensity was not constant over the electrode surface. The intensity profiles shown in Figure 8 are more consistent with the uniform distribution of current density expected at a disk electrode undergoing a surface-controlled reaction, that is, in the absence of a concentration gradient for the electroactive species. The profiles shown in Figure 8 are somewhat noisier than those in Figure 7, because the images were integrated for only 1/30 of a second instead of 2 s. Figure 8 shows that the fluorescence intensity increased as a function of time after the potential step. However, the advantage
Figure 8. Fluorescence intensity as a function of position across the center of the 1.0 mm Au disk electrode after a potential step from 0 to 1.60 V: (a) before the potential step; (b) 0.30 ( 0.30 s; (c) 0.60 ( 0.30 s; (d) 1.80 ( 0.30 s; and (e) 3.60 ( 0.30 s. The solution consisted of 0.50 mM quinine and 50 mM K2SO4 adjusted to pH 10. The edge of the electrode as determined under ordinary visible illumination is depicted by the vertical dashed lines.
gained by higher signal-to-background ratios was accompanied by a deterioration in spatial resolution at longer times due to diffusion. As time increased, significant diffusional broadening of the fluorescence profile occurred. The problem is most apparent for the profile obtained after 3.60 s (curve e), where the fluorescence intensity was above background levels at distances greater than 100 µm from the edge of the electrode. These results support the conclusion that the time after initiation of the electrochemical reaction must be minimized if the fluorescence intensity is to correspond to local current density. For potential steps to lower potentials than shown in Figure 8, and thus lower current densities, the increase in diffusional broadening was slower, while it occurred more quickly at higher potentials. In summary, both time and current density affect the growth of the pH gradient, and both should be minimized for the best spatial resolution. The application of less positive potentials resulted in a slower deterioration of spatial resolution. As shown in Figure 9, a potential step to 1.20 V resulted in an increase in fluorescence intensity that achieved a constant value in less than about 1 s. From the voltammetric data, the potential step to 1.20 V corresponded to Au oxide formation rather than O2 evolution. Although the current from oxide formation diminished quickly after the application of the potential, the fluorescence intensity lasted substantially longer because the pH gradient must be neutralized by diffusion of hydroxide from bulk solution. The fluorescence appears to be related to the integral of the current, with the resulting intensity lasting for a few seconds after the current has decreased to almost zero. Effect of Initial pH. Steady-state images of the gold electrode were obtained at a constant current of 0.5 µA as a function of the initial pH in bulk solution. Images comparable to those shown previously at pH 10 were obtained for pH values from 7 to 11. Images obtained at pH values less than 7 showed a significant decrease in fluorescence intensity as compared to the background, but even at pH 5 an image of the electrode was evident. Imaging of Ru/Kelgraf Composite Electrodes. Images of oxygen evolution obtained at a 5% Ru/10% Kelgraf composite
Figure 9. Fluorescence intensity as a function of position across the center of the 1.0 mm Au disk electrode after a potential step from 0 to 1.20 V: (a) before the potential step; (b) 0.30 ( 0.30 s; (c) 0.60 ( 0.30 s; and (d) 1.80 ( 0.30 s. The solution consisted of 0.50 mM quinine and 50 mM K2SO4 adjusted to pH 10. The edge of the electrode as determined under ordinary visible illumination is depicted by the vertical dashed lines.
Figure 10. Fluorescence images of a 5% Ru/10% Kelgraf composite electrode after the potential was stepped from -0.6 to 1.1 V. Image a is the 5% Ru/10% Kelgraf electrode under ordinary visible illumination. Image b was integrated for 0.5 s and image c for 2 s. The solution contained 0.10 mM quinine and 50 mM KNO3 adjusted to pH 10. Each image represents an area 160 µm on a side.
electrode are shown in Figure 10. The electrode under normal illumination is shown in image a. Kel-F is translucent, so there are regions that appear to be graphite or Ru when observed under normal illumination. However, whether or not these regions are electrochemically active cannot be determined by examining the normally illuminated image. When subjected to fluorescence imaging, as shown in Figure 10b,c, regions that support electron transfer become visible. Since Ru evolves oxygen at much less positive potentials than graphite, the fluorescence images in Figure 10 reveal only the Ru sites that are on the surface and electrically connected. As shown in Figure 10, this imaging technique easily resolved active Ru sites that were separated by only 25 µm. CONCLUSIONS This work has demonstrated that, in short-duration electrochemical experiments, fluorescence imaging using quinine can provide spatial resolution of microscopic electrode activity for O2 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
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evolution. At longer times, fluorescence imaging may provide some indication of the shape of concentration profiles for anodic reactions that consume OH-. A current density of about 64 µA/ cm2 was the optimum value for imaging O2 evolution under steadystate or constant current conditions, and about 13-26 µA/cm2 was the minimum detectable current. Contrary to our expectation, the highest quinine concentration used (0.5 mM) provided the best fluorescence intensity and spatial resolution. In fluorescence imaging, the presence of the probe species, in this case quinine, does alter the pH gradient in the diffusion layer since hydroxide must be consumed to generate fluorescence. However, that does not interfere with the utility of the technique for imaging electrode processes. Quinine fluorescence imaging of anodic reactions is complementary to the established method for cathodic processes using
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fluorescein fluorescence.39 Similarly, this new method should be able to provide information about microscopically local reaction rates and concentration profiles at electrode surfaces for a variety of anodic reactions. ACKNOWLEDGMENT This work was supported by the National Science Foundation under Grant No. OSR-9108773 and by the South Dakota Future Fund. Received for review August 12, 1996. Accepted December 30, 1996.X AC960816B X
Abstract published in Advance ACS Abstracts, February 1, 1997.
