Anal. Chem. 1984. 56. 575-578
was calibrated after the in vivo experiment suggested that this height corresponded to a DA concentration between 15 and 25 nM. As regards in vitro experiments, DNPV, as compared with DPV, represents a moderate improvement; but in vivo, in an attempt to monitor DA release, it represents a decisive one (Figure 3). It is likely that, in vivo, the diffusion of DA in the extracellular space is hindered. This limits the DA amount which can reach the electrode surface. In these conditions it is likely that a DPV scan led to a DA depletion in the vicinity of the electrode. On the other hand, since with DNPV the electrode is at a resting potential during four-fifths of the scan duration, this minimizes the number of DA molecules getting oxidized during one scan and, thus, the use of DNPV instead of DPV results in a large increase in the in vivo DA signal. Moreover, as expected from studies employing NPV (3),DNPV signals were more stable than DPV ones. In fact the DPV signal disappeared after a few seans while DNPV ones were stable for more than 1 h (Figure 4). When recorded from the striatum of a pargyline-treated rat, DNPV allowed us to record a peak at +85 mV. This peak was attributed to DA rather than to a residual DOPAC contribution since it appears at the same potential as DA and since it is greatly increased by amphetamine injections (Figure 4). In fact, numerous studies demonstrated that DA release is strongly stimulated by amphetamine while the DOPAC level is simultaneously decreased (4-6). Our conclusion has been reinforced by additional arguments (10): This presumed DA peak was completely suppressed following a specific degeneration of striatal dopaminergic terminals, it was decreased by injections of dopaminergic agonists and increased by antagonists; these drug effects were much more rapid than the effect of the same drugs on the striatal DOPAC level. As estimated here from in vitro calibrations, striatal DA concentration in the extracellular space was between 15 and 25 nM. This concentration has been recently estimated to be 54 nM by means of perfusion with dialysis tubing implanted in the striatum of a normal rat (6). Although these two techniques are very different these estimations are in agreement; this suggests that pargyline treatment did not induce a dramatic change in the extracellular DA concentration. It has been also observed that striatal DA level, as measured post-mortem, is moderately increased (+25 % ) by pargyline (11). However, these estimations question that recently reported by Ewing et al. (12):In their electrochemical study the extracellular DA concentration was measured following electrical stimulation of the nigrostriatal pathway and estimated to be 35.5 pM. This seems in contrast with a previous report by the same authors who showed that their technique
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was not sensitive enough for monitoring the amphetamine stimulated DA release (3). In conclusion, this study demonstrates that in vivo electrochemical monitoring of spontaneous DA release is feasible providing that the DOPAC contribution to the catechol peak is pharmacologically suppressed by inhibition of the DOPAC synthesis. In order to reach this goal, DNPV appears to be superior to other already used techniques. Moreover, it points out that DNPV used in combination with treated carbon fiber electrodes makes the detection of some molecules such as catecholamines possible within nanomolar concentrations. ACKNOWLEDGMENT We thank the Solea Tacussel Company which designed the new apparatus used in this study accordingly to our specifications. Registry No. Dopamine, 51-61-6. LITERATURE CITED Adams, R. N.; Marsden, C. A. In “Handbook in Psychopharmacology”; Iversen, L. L., Iversen, S. D., Snyder, S.H., Eds.; Plenum Press: New York, 1963; Vol. 15, pp 1-74. a n o n F.; Cespugllo R.; Buda M.; Pujol J. F. In “Methods in Blogenic Amine Research”; Parvez, S.,Nagatsu, T., Nagatsu, I., Parvez, H., Eds.; Elsevier: Amsterdam, 1983. Ewing, A. G.; Dayton, M. A.; Wightman, R. M. Anal. Chem. 1981, 53, 1842-1847. Gonon, F.; Buda, M.; Cespugllo, R.; Jouvet, M.; Pujol. J. F. Nature (London) 1980, 286, 902-904. Gonon, F.; Buda, M.; Cespugllo, R.; Jouvet, M.; Pujol, J. F. Brain Res. 1981, 223, 69-80. ZetterstrBm, T.; Sharp, T.; Marsden, C. A.; Ungerstedt, U. J . Neurochem., In press. Gonon, F.; Fombarlet, C. M.; Buda, M. J.; PuJol, J. F. Anal. Chem. 1061, 53, 1386-1389. Borman, S. A. Anal. Chem. 1982, 5 4 , 698A-705A. Gonon, F.; Buda, M.; Pujol, J. F. In “Measurement of Neurotransmitter Release”; Mardsen, C. A., Ed.; Wiley: London, In press. Gonon, F.; Buda, M.; Pujol, J. F. 5th International CatecholamineSymposium, Gijteborg, 1983; Abstract No. 177 (Supplement to Progress in Neuro-Psychopharmacology and Blologlcal Psychiatry). Westerink. B. H. C. Eur. J . Pharmacal. 1979. 56, 313-322. Ewing, A. G.; Blgelow, J. C.; Wightman, R. M. Science 1983, 227, 169-1 71.
’
Present address: Laboratolre de Chimie Organlque et CIn(tlque, ICPI, rue du Plat, 65002 Lyon, France.
F. G. Gonon* Florence Navarre’ M. J. Buda INSERM U 171 H8pital Ste. Eug6nie Pavillon 4 H 69230 St. Genis Laval, France
RECEIVED for review August 4,1983. Accepted November 1, 1983.
Variations in Electron-Transfer Rate at Polished Glassy Carbon Electrodes Exposed to Air Sir: Glassy carbon has found applications as an anode and as a cathode in numerous electroanalytical methods (1).This material is not inert, however, and redox reactions of functional groups on glassy carbon surfaces have been observed (1-9). In order to obtain reproducible electroanalyticalresults, a wide variety of pretreatment procedures have been recommended. Specific techniques range from polishing the electrode surface with alumina or other abrasives of submicron 0003-2700/84/0356-0575$01.50/0
particle size (1-3, 7-12) to polishing followed by electrochemical ( I , 6,12)or chemical (4)pretreatments. Ultrasonic cleaning (5,12,13) has been used to remove traces of polishing materials from the surface. That such a variety of pretreatments have proliferated is related to a relatively poor understanding of the nature and reactivity of the electrode surface before and after pretreatment. In this report, we provide evidence that polishing of glassy carbon produces a @ 1984 American Chemlcal Soclely
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surface which reacts in air. This unstable condition is manifested by a slowly changing rate of electron transfer when the electrode is exposed to air after polishing. EXPERIMENTAL SECTION Chemicals. Ferrocene was obtained and purified as previously described (14).1,2,3,4-Tetrahydrocarbazole(99%) was used as received from Aldrich Chemical Co. Water was prepared with a Sybron/Barnstead NANOpure water-purification system and had a specific resistivity greater than 10 MS1 cm. All other chemicals were analytical reagent grade. Apparatus and Procedures. A Princeton Applied Research Corp. (PARC) Model 174A polarographic analyzer and a PARC Model 170 electrochemistry system were used for rotating-disk voltammetry (RDV) and cyclic voltammetry (CV). For RDV, a Sargent synchronous rotator (Type KYC-22) operated at 1800 rpm was used. Construction of the glassy carbon (Normar Industries,A = 0.08 cm2) electrodes has been described previously (14,15). All electrodes were surrounded by a cylindrical Teflon collar of thickness 3.0 mm. Three different glassy carbon (GC) electrodes were employed. GC1 had been used extensively in previous electrochemical work; GC2 and GC3 were newly constructed for this study. GC surfaces were prepared by polishing on a Buehler metallographic polishing wheel, successively, with 400 and 600 grit silicon carbide paper, using water to reduce frictionalheating. These steps were followed by polishing with Metadi 0.25-pm diamond paste on Buehler microcloth and ultrasonication and washing in spectrograde methanol for 2-3 min. The final pretreatment, employed prior to each scan, consisted of polishing on felt cloth on the metallographic wheel with either 0.3-pm alumina or Raybright gem polish (PARC) for 2-3 min, followed by ultrasonication for 2-3 min in water. GC electrodes prepared in this way were either used immediately or allowed to stand in air or various solutions before use, as noted below. Before the anodic scans, electrodes were held at a potential of 0.0 V vs. SCE for 2 min. Cathodic CV scans were initiated immediately after application of the initial potential. Three-electrode cells of the Metrohm type and Pt-wire counterelectrodes were employed for all voltammetric experiments. For aqueous solutions, a PARC Model KO065 SCE was the reference electrode; a Ag/Ag+ (0.001 M) reference was used with acetonitile solutions. Assembled cells had resistances of 400-900 Q. Prior to initiation of anodic scans, solutions were purged with purified nitrogen (15)for 10-15 min. To ensure complete removal of oxygen from solutions analyzed by cathodic scans, purging with nitrogen was extended to 1h. All experiments took place at the ambient temperature of the laboratory (25 A 2 "C). A Leybold-Heraeus LHS 10 electron spectrometer housed at the Leybold-Heraeus facility in Export, PA, was used for X-ray photoelectron spectroscopy (XPS). Computations. A Radio Shack TRS-80 Model I microcomputer and the level I1 BASIC language were employed for all computations. Current-potential curves obtained by RDV were analyzed by a computer program designed to provide automatic mechanistic classification (16)based on nonlinear regression analysis and deviation-pattern recognition (17). Current-potential curves were digitized and corrected for residual current as previously described (14).In all regression analyses, The absolute errors in the currents were assumed to be randomly distributed. RESULTS AND DISCUSSION The procedure for polishing GC electrodes yielded reproducible voltammograms for oxidations known to involve fast, reversible electron transfer. For example, CV of ferrocene in acetonitrile containing 0.2 M LiC104(18)produced anodic and cathodic peaks of equal height with peak separations of 59 f 2 mV, as expected for a reversible electron transfer (19). Similarly, 1,2,3,4-tetrahydrocarbazole,which oxidizes via reversible one-electron transfer followed by dimerization, gave reproducible anodic peaks similar to those obtained on carbon-paste and platinum anodes (15). Results for these two compounds were not influenced by small variations in the polishing procedure. Because the electron-transfer reactions involved are fast and reversible, the voltammograms appear
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Flgure 1. Influence of time of storage In air of pollshed GC electrodes on E3,, - E,,, for oxidation of 0.5 rnM ferrocyanlde in 0.5 M KNO:, by RDV: (0)GC1, (0)GC2,(0)GC3.
to be relatively insensitive to changes in the nature of the electrode surface. Ferrocyanide is oxidized a t platinum and glassy carbon electrodes in a quasi-reversible reaction (4,6,20-22).The rate of electron transfer for this reaction is very sensitive to the surface state of the electrode; therefore, voltammetric oxidation of ferrocyanide was used as a probe to investigate electron-transfer properties of the electrodes. The shape of the current potential curve for quasi-reversible oxidation in RDV is given by (20)
i = ij(1 + 8 + 8'T7ZR/ksho)-'
(1)
where 6 = exp[(nF/RT)(El/2 - E)], 6' = exp[(l - a)(nF/ RT)(Eo' - E ) ] , kb" is the standard heterogeneous rate constant for electron transfer, mR is the mass-transfer coefficient for the electroactive species, and il is the limiting current. Provided a remains constant, eq 1shows that an increase in ksho causes the voltammetric wave to increase in steepness. Thus, changes in kaho can be monitored by observing the difference between the three-quarter-wave potential (E3I4)and the quarter-wave potential (E1/4); i.e., decreases in E314- El/1 reflect increases in ksho. When ferrocyanide in 0.5 M KNo3 was oxidized by RDV using freshly polished GC electrodes, broad waves with E3/4- E,,? = 193 f 26 mV and Ellzof 430 f 80 mV vs. SCE were obtained. When GC electrodes were allowed to stand in air for about 1-2.5 h after polishing and then used for RDV, a dramatic decrease in E3/4- Ell4 was observed (Figure 1). This reflects an increase in ksho with time of exposure to air. As the time of exposure to air increased beyond 3 h, ksho again became smaller, indicated by a relaxation in E3/4- Elj4to larger values. Half-wave potentials (El,2)of the voltammetric waves followed a similar trend, first decreasing with time of air exposure of the GC electrodes and then increasing. Minima in ESJ4- E114 (Figure 1)and Ellz (0.240 vs. SCE) were observed after GC electrodes stood in air for about 1.5 h. Observation of the increase in ksh" was not influenced by small changes in the polishing procedure. For example, indistinguishable results were obtained whether the final polishing step employed 0.3-pm alumina or Raybright polish, Likewise, doubling the final polishing time did not influence the observable variations in ksh".
Data obtained by RDV after different times of exposure of the GC electrodes to air were classified as quasi-reversible or showed only small deviations from a quasi-reversible reaction when analyzed by the mechanistic classification program (16).Voltammograms calculated by nonlinear regression
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984 E , V vs. SCE v
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Figure 2. Rotating-disk vottammograms of 0.5 mM ferrocyanide in 0.5 M KNOB. Solid lines are experimental curves; circles were calculated by using parameters, given below, found by nonlinear regression onto
eq 1 (76): (a) GC1, immediately after polishing, Eo’ = 0.260 V, CY = 0.72, mR/kao = 3.85; (b) GC2, after polishing and 1.75 h In air, Eo’ = 0.208 V, LY = 0.64, m,/k,O = 1.83, (c) GC2,after polishing and 5 h in air, Eo‘ = 0.183 V, CY = 0.68, m,/k,’ = 25.3.
onto eq 1 showed good agreement with experiment in all cases (Figure 2), and the value of a! was reasonably constant at about 0.68. Thus no drastic changes in the electrode process take place when electrodes are stored in air for different periods, and changes in wave shape indeed reflect changes in k s h o . Three sets of voltammetric data obtained with electrodes stored in air for 1to 2.5 h gave the following average parameter values from regression onto eq 1: Eo’ = 458 f 11 mV VS. NHE (cf. Eo’ (m KCl) = 474 mV (20) vs. NHE), CY = 0.635 f 0.006, and m R / k s h o = 1.69 f 0.16. Determination of mR from the limiting current (20) enabled estimation of ksho = 0.0022 cm s-l for these conditions. This value is close to the 0.0054 cm s-l measured for ferrocyanide at electrochemically pretreated GC electrodes (21). Thus, both electrochemical pretreatment and air exposure gives increases in rates of reaction of similar magnitude. Comparatively larger rate constants (0.002-0.1 cm s -l) were observered for oxidation of ferrocyanide on platinum in alkali chlorides and nitrates (22). Recent evidence suggests that adsorption of electroinactive species is involved in the redox process at Pt (22),but the role of adsorption has not yet been investigated at glassy carbon. Anodization (+1.5 V vs. SCE) followed by cathodization (-1.5 V vs. SCE) of GC electrodes has been shown to produce an enhanced reversibility for the anodic oxidation of ferrocyanide (6). When this electrochemical pretreatment (5 min per cycle) was used after polishing our GC electrodes, an average E3I4- E l l 4 of 92 f 6 mV and an Ellz of 246 f 5 mV was obtained for ferrocyanide by using freshly activated electrodes. When electrochemicallypretreated electrodes were allowed to stand in air for 1-1.5 h, a small decrease in activity was observed. A pronounced memory effect following electrochemical pretreatment was also noted. In order to observe increases in k h o from air treatment of a GC electrode which had been previously activated electrochemically, extensive refinishing of the electrode surface was necessary. No increase in ksho was observed when freshly polished electrodes were stored in nitrogen-saturated or air-saturated solutions of 0.5 M KNO,. These results appear to rule out any contribution to observed phenomena of mechanical relaxation of the surface after polishing. X-ray photoelectron spectroscopy (XPS) was used to examine GC surfaces which had been completely refinished and exposed to air for several days. GC3 was analyzed before it had ever been used for electrochemistry. Spectra for this electrode were qualitatively similar to those obtained for GC1 and GC2, which had extensive electrochemical histories at the time of XPS analysis. The spectra revealed no traces of alumina on the GC surface, but the presence of traces of silicon (
