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Anal. Chem. 1988, 58,848-852
preparing microelectrodes is greatly appreciated. Registry No. NAD', 865-05-4; NAD., 50958-71-9; Pt, 744006-4; K,Fe(CN)G,13943-58-3;C, 7440-44-0.
LITERATURE CITED
V Flgure 8. Cyclic voltammograms of 1 mM NAD' in 0.1 M sodium pyrophosphate, pH 8.0. Voltammograms taken (a) directly at a 3 mm diameter glassy carbon electrode and (b) at a 25 pm diameter platinum electrode set at 0.0 V positioned in diffusion layer of the glassy carbon electrode. Positive potential limit for scans was -0.50 V; negative limit was -1.35 V.
converted to the dimer, indicating a half-life substantially shorter than a few milliseconds. With our present capabilities concerning microelectrode construction and positioning, a reaction intermediate would need to have a lifetime in the millisecond domain to be detected. Admittedly, other electrochemical techniques are capable of detecting intermediates with shorter lifetimes than that. Efforts are under way to decrease the interelectrode distance further so that species with shorter lifetimes can be observed. Because of the second-order dependence of diffusional time on distance, decreasing the interelectrode distance by a modest amount could result in a significant payoff in the detection of short-lived species.
ACKNOWLEDGMENT The carbon fibers were a gift from Gary Mabbott of Colby College. The help of Mark Wightman and his students in
(1) Pruiksma, R.; McCreery, R. L. Anal. Chem. 1979, 57,2253. (2) Pruiksma, R.; McCreery, R. L. Anal. Chem. 1981, 53,202. (3) Rossi, P.; McCurdy, C. W.; McCreery, R. L. J. Am. Chem. SOC.1981, 703,2524. (4) Rossi, P.; McCreery, R . L. J. Nectroanal. Chem. 1983, 757,47. (5) Jan, C.; McCreery, R. L.; Gamble, F. T. Anal. Chem. 1985, 57,1764. (6) McLarnon, F. R.; Muller, R. H.; Tobias, C. W. J Electrochem. SOC. 1982, 729, 2201. (7) Awadura, Y.; Kondo, Y. J . Electrochem. Soc. 1976, 723, 1184. (8) Isaacs, H. S.;Kissei, G. J. Nectrochem. SOC.1972, 779, 1628. (9) Isaacs, H. S.;Kendig, M. W. Corroslon (Houston) 1980, 36,269. (10) Isaacs, H. S. Localized Corros.-Cause of Metal Failure 1972, 576, 158. (11) Isaacs, H. S.; Vyas, B. Nectrochem. Corros. Test. 727. (12) Engstrom, R. C. Anal. Chem. 1984, 56,890. (13) Engstrom, R. C.; Weber, M. W.; Werth, J. Anal. Chem. 1985. (14) Wightman, R. M. Anal. Chem. 1981, 53, 1125A. (15) Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1980, 8 2 , 846. (16) Ewing, A. G.; Dayton, M. A.; Wightman, R. M. Anal. Chem. 1981, 53, 1842. (17) Hepel, T.; Osteryoung, J. J. Phys. Chem. 1982, 8 6 , 1406. (16) Bard, A. J.; Faulkner, L. "Electrochemical Methods"; Wiiey: New York, 1980; p 566. (19) Sleszynski, N.; Osteryoung, J.; Carter, M. Anal. Chem. 1984, 56, 130. (20) Welsshaar, D. E.; Tallman, D. E. Anal. Chem. 1983, 55, 1146. (21) Johnson, D. C.; Allen, R. 6.Talanta 1973, 20,305. (22) Laser, D.; Ariel, M. J . Nectroanal. Chem. 1974, 4 9 , 123. (23) Schieffer, G. W.; Blaedel, W. J. Anal. Chem. 1977, 49, 49. (24) Bard, A. J.; Faulkner, L. "Electrochemical Methods": Wllev: New York, 1980; p 180. (25) Adams, R. N. "Electrochemistry at Solid Electrodes"; Marcel Dekker: New York, 1969; p 219. (26) Fleischmann, M.; Lasserri, F.; Roblnson, J.; Swan, D. J . Electroanal. Chem. 1984, 777,97. (27) Schmakel, C. 0.; Santhanam, K. S. V.; Eiving, P. J. J. Am. Chem. SOC. 1975, 75,5083. (28) Burnett, J. N.; Underwood, A. L. Blochemisfry 1965, 4 , 2060. (29) Wilson, A. M.; Epple, D. 0.Biochemistry 1988, 5,3170.
RECEIVED for review August 9,1985. Accepted November 22, 1985. This work was supported in part by the National Science Foundation, Grant CHE-8411000.
Electrocatalytic Response of Cobalt Phthalocyanine Chemically Modified Electrodes toward Oxalic Acid and a-Keto Acids Leone1 M. Santos and Richard P. Baldwin* Department of Chemistry, University of Louisville, Louisville, Kentucky 40292 Oxalic acid and several a-keto acids (pyruvic, phenylpyruvic, a-ketobutyric, a-ketoglutaric, and a-ketolsocaprolc) exhibited electrocataiytlc behavior at chemically modified carbon paste electrodes contalnlng cobalt phthaiocyanlne Incorporated Into the paste mixture. All underwent oxldation at unmodified carbon electrodes at potentlais more posltlve than +1.2 V vs. Ag/AgCI but gave substantlal anodlc peaks between +0.75 and 0.90 V at the cobalt phthalocyanine contalnlng surface. When used In LCEC, the modified electrodes permitted detection of the aclds at correspondingly lower potentlais than at conventlonal electrodes. As a result, quantltatlon in urine samples was possible with no sample treatment other than dllutlon and partlculate filtratlon. The detection llmlt for oxalic acid at +0.75 V was 0.3 pmol injected and less than 1 nmol for the a-keto aclds.
Over the past decade, electrochemical measurement tech-
niques have achieved wide acceptance as a sensitive and selective approach for the detection of numerous compounds following high-performance liquid chromatography. For example, liquid chromatography with electrochemical detection (LCEC) now represents a powerful method for the determination of phenols and catechols, aromatic amines, thiols, and nitro compounds in complex physiological and environmental samples (1,2). However, because the principal requirement of the technique is that the analyte of interest undergoes oxidation or reduction at a comparatively low potential, many important but difficult-to-electrolyzespecies are not accessible to LCEC. A particular problem arises for compounds whose electrode reactions involve slow electron transfer kinetics and therefore occur at an appreciable rate only at potentials drastically exceeding their thermodynamic redox potentials. For these compounds, LCEC detection usually cannot provide optimum sensitivity and selectivity and, in extreme cases, can provide no usable quantitation at all.
0003-2700/86/0358-0848$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
One approach that has shown promise for minimizing such overvoltage effects is the use of electrocatalytic chemically modified electrodes (CMEs) containing a surface-bound redox mediator selected for its ability to chemically oxidize or reduce solute species which are themselves electrolyzed only irreversibly at conventional electrode surfaces. Thus, the use of carbon paste CMEs modified by the incorporation of cobalt phthalocyanine (CoPC) has been shown to decrease the OVerpotential for the oxidation of thiols such as cysteine and glutathione by several hundred millivolts ( 3 , 4 ) . As a result, detection limits orders of magnitude below those obtained at unmodified carbon electrodes and comparable to those at gold amalgam electrodes were obtained. A similar effect with the same CoPC CMEs was found for hydrazine, which at conventional electrodes, is oxidized only at potentials in excess of +1.0 V vs. Ag/AgCl but can be quantitated at the picomole level in the 0 to +0.5 V range at the modified electrode (5). During the course of our initial work with CoPC CMEs, it became apparent that these electrodes can catalyze numerous electron transfer processes in addition to those of NzH4and SH compounds. This, of course, was not entirely surprising as the metallophthalocyanines have commonly been employed as catalysts for the redox reactions of a wide variety of organic and inorganic species (6). Of particular interest from the LCEC point of view was the CME behavior of oxalic acid and the a-keto acids-whose determination in physiological media such as urine and blood is an important clinical assay for the diagnosis of numerous human disease conditions (7).Previous analysis approaches for these compounds have usually favored either gas (7,8)or liquid (9,10) chromatography. However, because of the volatility constraints of the former and the lack of a strongly absorbing chromophore suitable for the optical monitoring methods most commonly employed in the latter, preliminary derivatization steps have usually been required for either approach to be effective. Recently, LCEC involving the oxidation of the acids at glassy carbon electrodes has been suggested to provide an alternate and potentially preferable analysis procedure (11-13). But, although some success in this area was reported, the extreme potentials required to carry out the oxidations severely limited both the sensitivity and the selectivity of the approach in real sample analysis. In this paper, we will describe the electrocatalysis of oxalic and a-keto acids at the CoPC CME and will examine the utility of the effect for LCEC quantitation of the compounds in urine. The CMEs chosen for this application consisted of carbon paste mixtures containing appropriate quantities of CoPC simply added to the graphite powder during paste preparation. Despite the fact that other potentially useful strategies were available for CoPC CME formation, the ease of carbon paste CME preparation, the flexibility with which the CoPC surface coverage could be adjusted, and the rapidity and reproducibility with which fresh CME surfaces could be regenerated made this approach especially attractive.
EXPERIMENTAL SECTION Reagents. Cobalt phthalocyanine was obtained from Eastman Kodak Co. and was used as received without further purification. Stock solutions of oxalic acid (Matheson, Coleman, and Bell), pyruvic acid (Sigma), a-ketoglutaricacid (K and K Laboratories), a-ketobutyric acid (Aldrich),and a-ketoisocaproic acid (Sigma) were made up fresh each day. The urine samples were obtained from a healthy volunteer, filtered by passing through a 10-15-pm glass filter, and diluted with the mobile-phase solution. Working Electrodes. Carbon paste for unmodified electrodes was made by thoroughly hand mixing 5 g of graphite powder (Fisher Scientific Co.) with 3 mL of Nujol oil (McCarthyScientific Co., Fullerton, CA) in a mortar and pestle. Modified carbon pastes were prepared similarly except that 0.10 g of CoPC was fint added to the graphite powder to give a mixture that was 2.0% CoPC
,
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___..I
1.2
I .O
0.8
0.4
O*L
0.2
0
V o l t s vs. A g l A g C l
Figure 1. Cyclic voltammograms of (A) 1.0 X loJ oxalic acid and (B) 8.0 X M pyruvic acid at unmodified and 2% CoPC (-) carbon paste eiectrodes. Electrolyte was 0.10 M phosphate buffer (pH 2.0),and scan rate was 64 mV/s. (.e.)
by weight. The Nujol oil was then added and ground with the graphite mixture for 30 min. Apparatus. Cyclic voltammetry wm performed with an IBM Model EC/225 voltammetric analyzer. A carbon paste working electrode (with or without CoPC), a saturated Ag/AgCl reference electrode, and a platinum wire counter electrode were used for all experiments. Flow injection analysis and liquid chromatography were performed with a Perkin-Elmer Series 10 pump, a Rheodyne (Berkeley, CA) Model 7125 injector with a 6-pL loop, and an IBM Model EC/230 amperometric detector. Chromatography was performed with a Regis 25pm, 5-pm octadecylsiiane column. The mobile phase was a 0.10 M phosphate buffer adjusted to pH 2. the flow rate was always 1.0 mL/min.
RESULTS AND DISCUSSION CME Electrocatalysis. A typical cyclic voltammogram (CV) obtained for oxalic acid at a conventional carbon paste surface is shown in Figure 1A. The CV is characterized by a single, nearly pH-independent oxidation wave with a peak potential, Ep,of roughly +1.2 V vs. Ag/AgC1;no corresponding reduction wave was seen on the reverse scan under the conditions employed. Nearly identical behavior has also been described for this compound at glassy carbon electrodes subjected to the usual regimen of alumina polishing followed by thorough rinsing in water (14,15).The need to apply such high potentials to effect the oxidation indicated that the compound is at best only marginally compatible with LCEC analysis, and although some success has been reported in this area (11-13), much less than optimum sensitivity and selectivity for the determination would be expected under these conditions. A roughly 100-mV decrease in the Ep value for oxalic acid has been obtained by dispersion of micrometer-size
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
alumina particles on glassy carbon (1416). However, a more substantial decrease in the overvoltage is still required for LCEC to approach its ideal analytical capabilities-especidy for determination of the analyte directly in the relevant physiological matrices. The CV obtained for oxalic acid under the identical solution conditions but a t a carbon paste electrode containing 2% COPCby weight is shown for comparison in Figure 1A. The electrocatalytic response of the CME was reflected by the appearance of a new oxidation wave with an E, of approximately +0.75 V. In addition, one or more smaller anodic processes were also seen a t higher potentials. The peak potential of the principal oxidation varied somewhat with the oxalic acid concentration employed, apparently shifting to higher potential as the analyte concentration was decreased. variations in solution pH over the investigated range of 2-7 produced practically no change in the voltammogram. Peak currents were proportional to the oxalic acid concentration and to the square root of the potential scan rate employed (up to 400 mV/s). It would appear that the enhanced current observed at the CME is best explained on the basis of electrocatalysis by the incorporated CoPC. Although cycling of the CoPC CME in a solution containing only phosphate buffer and no oxalic acid revealed only an extremely small oxidation in the +0.80-V region, the potential of the oxalic acid oxidation at the modified electrode closely coincides with that previously reported for the Co(II)/Co(III) redox process of the phthalocyanine compound. This has been confirmed for CoPC in solution as the tetrasulfonate by electrochemical (17,18) and spectroelectrochemical (19) means and, by analogy, for CoPC itself incorporated onto graphite and carbon paste surfaces (3,20). In fact, a nearly identical solution-phase electrocatalysis response toward oxalic acid was obtained a t an unmodified electrode when a small quantity of CoPC tetrasulfonate was simply added to the oxalic solution. In virtually all respects, the behavior of oxalic acid resembled that previously seen for thiol-containing compounds such as cysteine and glutathione at CoPC CMEs ( 3 , 4 ) . For both sets of compounds, maximum currents occurred near +0.8 V, and continued operation of the electrodes a t more positive potentials resulted in a decrease in electrocatalytic activity. This deactivation, which could be due to a number of factors such as the further oxidation and decomposition of the phthalocyanine or an irreversible complexation of the Co(II1) center, was evidenced by a noticeable sharpening of the catalytic wave over that normally expected for a simple diffusion-limited process and a marked decrease in the current level seen on subsequent CV scans. Similar changes in the current-voltage characteristics of several a-ketocarboxylic acids were also observed when these compounds were examined a t CoPC-containing electrodes. The compounds studied included pyruvic, phenylpyruvic, a-ketobutyric, a-ketoglutaric, and a-ketoisocaproic acids. All underwent oxidation at plain carbon paste electrodes only at potentials more positive than +1.3 V vs. Ag/AgCl but yielded substantial anodic currents some 300-400 mV lower a t the CoPC CMEs. In general, the catalytic activity of these electrodes toward the keto acids (astypified by the CV shown in Figure 1B for pyruvic acid) was not entirely identical with that seen for oxalic acid. Major differences included much smaller current levels for the same Concentration of the keto acids and the fact that the catalytic currents observed for them occurred primarily at somewhat higher potentials. Analogous carboxyl compounds not possessing keto substitution in the a position (e.g., malonic, maleic, and succinic acids) failed to exhibit a similar electrocatalysis at the modified electrode surface.
A
81 0
5
10
Retention
15
20
25
Time [rnln]
Figure 2. Chromatograms of (a) 3.8 X lo-' M oxalic acid, (b) 1.6 X M pyruvic acid, (c) 4 X M a-ketoglutaric acid, (d) 6 X M aketobutyric acid, (e)1.4 X M a-ketoisocaproic acid obtained at 2.0% CoPC electrode (A) and unmodified carbon paste (B); E = +0.75 V vs. Ag/AgCI.
LCEC. In view of the marked decrease in overpotential resulting from the use of the CoPC CMEs in voltammetry, it appeared likely that these electrodes might also provide significantly enhanced performance in LCEC applications. In particular, detection of the acids should be possible at lower potentials than were required when conventional electrodes were employed. The chromatograms shown in Figure 2 demonstrate that this was indeed the case. Although no oxidation currents at all were observed at plain carbon paste (or glassy carbon) for a mixture of oxalic and several of the a-keto acids following reverse-phase liquid chromatography and detection at +0.75 V vs. Ag/AgCl, well-formed peaks were obtained for the CMEs under the same chemical and electrochemical conditions. Hydrodynamic voltammograms (HDVs) obtained separately for each of the compounds served to confirm that the potential dependences of the electrocatalyses under flow conditions were virtually the same as those found above in the CVs. (See Figure 3 for the HDVs for oxalic, pyruvic, and a-ketobutyric acids.) At the CoPC electrodes, the oxidation wave for oxalic acid exhibited a peak a t +0.8 V, while those for the a-keto acids showed a small response at +0.8 V but gave a maximum current in the vicinity of +0.9 V. As in CV, the current level obtained for the keto acids was much smaller than that seen for oxalic acid at a comparable concentration level-the difference in response being approximately a factor of 100. None of the HDVs exhibited the simple plateau shape ordinarily observed for an uncomplicated diffusion-limited oxidation. Rather, decreases in chromatographic peak current occurred a t higher potentials for all of the analytes and resulted in peak-shaped HDVs. This was undoubtedly related to the potential-dependent electrode deactivation seen earlier in CV and severely hindered the high-potential operation of the CMEs in LCEC. The problem was most severe by far for oxalic acid, for which operation at potentials greater than +OS5 V for even a few chromatograms produced a permanent decrease in response that continued to manifest itself even after the CME was returned to low potentials. A similar phenomenon was observed previously for sulfhydryl oxidation at the CoPC electrodes ( 3 ) .
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
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1.0
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.-
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Flgure 4. Chromatograms of (A) 0.20 mL of urine diluted to 50 mL at plain carbon paste electrode, (B) 0.10 mL of urine diluted to 50 mL at 2.0% CoPC electrode, and (C) same sample as in B but spiked with M pyruvic acid, (3) 2.0 X M a-ketoglutaric acid, (2) 4.0 X M a-ketobutyric acld, and (7) 1.4 X M a-keto(5) 6.0 X isocaproic acid. Peaks 1, 4, and 6 correspond to oxalic acid, ascorbic acid, and uric acid, respectively; E = +0.75 V vs. AgIAgCI.
0.2
0.1
0 0.6
0.7
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1.0
1.1
Volts vs.Ag/AgCI
Figure 3. Normalized hydrodynamic voltammograms of (a) oxalic acid (---), (b) pyruvic acid (-), and (c) a-ketobutyric acid (-) at 2.0%
CoPC electrode; (d) oxalic acid at plain carbon paste.
On the basis of the HDVs, it was apparent that no single potential provided optimum detection and quantitation for all compounds examined. Rather, it would be expected that the selection of applied potential for any given sample should constitute an ad hoc compromise between maximum sensitivity (i.e., high potential) and stringent selectivity and electrode stability (i.e., low potential). As all of the analytes gave a ready response a t +0.75 V, this potential might represent a reasonable choice for most work. For all but oxalic acid, however, operation at more positive potentials would be expected to increase signal levels commensurately. At +0.75 V, the current response of the CME toward oxalic acid was linear from 1 X lo-' to 2 X loe6M (i = 4.64 nA/wM*C 0.053 nA, correlation coefficient = 0.999 for eight concentrations over the range). The limit of detection (SIN = 2) was 5 X low8M, or 0.3 pmol injected. At the same potential, the detection limits for the a-keto acids ranged from 150 pmol injected for a-ketoglutaric acid to 1nmol for a-ketoisocaproic acid (which, under the chromatographic conditions employed, produced a very broad, strongly retained peak). However, by simply increasing the detector potential to +OB0 V, the limits of detection for all except the latter compound could be decreased to less than 50 pmol. The stability of the CME response was evaluated by placing the CoPC-containing electrode in the flowing mobile-phase stream and following its current response toward the above analytes over an extended length of time. Typical results obtained for repeated injections of 2.0 X IO4 M oxalic acid showed that, as long as the electrode was maintained at +0.75 V vs. Ag/AgCl or lower, peak heights were practically unchanged-with the CMEs retaining more than 95% of their original activity after 3 h of continuous usage. But, as expeded from the HDV in Figure 3, operation at more positive potentials produced such a rapid decrease in electrocatalytic response that, over the same time period, the current was
+
reduced to only 65% of its initial level. The response of the CoPC CMEs toward the a-keto acids was even better as stable currents were obtained for these compounds at potentials as high as +0.85 V. In any case, the CoPC/carbon paste surface could be rapidly and reproducibly renewed if necessary. The practical utility of the CME detection approach was well-illustrated by chromatograms obtained for urine samples, which for healthy individuals, are expected to contain appreciable concentrations of oxalic acid. The a-keto acids, on the other hand, are generally present in comparable proportions only in abnormal subjects experiencing one of several characteristic diseases. Curves A and B of Figure 4 are +0.75-V chromatograms for a urine sample from a healthy volunteer obtained, respectively,at plain and 2% CoPC carbon paste electrodes. In the former case, only two peaks (retention times = 5.0 min and 12.3 min, respectively) were detected. By matching of the retention times with those of standard compounds, we determined the earlier eluting of the pair to be ascorbic acid while the second was uric acid. Both of these compounds are customary urine components and are known to be electroactive at this value of applied potential. The chromatogram obtained with the CoPC CME exhibited not only these two peaks but also a third more rapidly eluting peak whose retention time was identical with that of oxalic acid standards. The slight shoulder on the trailing edge of this peak indicates the presence of a minor, as yet unidentified urine component. In addition, the ascorbic acid peak was increased somewhat compared to that observed at the unmodified electrode; this latter aspect is accounted for by the fact that CoPC can also serve as an efficient electrocatalyst for ascorbic acid oxidation and therefore produces a larger response fo this analyte as well. Over the course of this work, which necessitated the examination of numerous different urine samples, peak heights observed for all three components (oxalic, ascorbic, and uric acids) varied considerably from instance to instance, as would be expected for specimens collected without strict control of diet, time of sample collection, etc. However, no serious complication was ever observed involving the coelution of an electroactive species at or near the oxalic acid peak-despite the fact that no sample treatment was employed for the urine samples prior to
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Anal. Chem. 1986, 58. 852-860
chromatographic analysis other than dilution and particulate filtration. Oxalic acid peak height for any single sample was quite reproducible (standard deviations of 1-2% for three replicate measurements) and quantitation via a calibration curve approach yielded values that fell within the range expected for healthy individuals (
