Anal. Chem. 1996, 68, 960-965
Detection of Ascorbic Acid Using a Carbon Fiber Microelectrode Coated with Cobalt Tetramethylpyridoporphyrazine Pavel Janda,*,† Jan Weber,† Lothar Dunsch,‡ and A. B. P. Lever§
J. Heyrovsky´ Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejsˇ kova 3, 182 23 Prague 8, Czech Republic, Institut fu¨ r Festko¨ rper- und Werkstofforschung, Dresden e. V., D-01069 Dresden, Helmholtzstrasse 20, Germany, and Department of Chemistry, York University, 4700 Keele Street, North York, Ontario M3J 1P3, Canada
A carbon microfiber electrode coated with an electrodeposited cobalt chelate of a tetra-N-methylpyridoporphyrazinocobalt derivative has been used for the potentiometric detection of ascorbic acid. It is effective over a fairly wide range of ascorbic acid concentration and may have value for in vivo analysis. L-Ascorbic acid, vitamin C, is an extremely important biological molecule with many roles including enzyme cofactor, reducing agent, involvement in neurotransmitter-related enzymes and, of course, essential nutritional factor.1-4 Various techniques have been employed for its measurement, 5-9 with a special need to develop in vivo methods. Electrochemical methods have long been used to determine ascorbic acid either directly10-15 or combined with other techniques.16-18 The direct application of cyclic voltammetry (CV) on solid electrodes or more sensitive differential pulse voltammetry (DPV) also allows in vivo measurements.1 Voltammetric techniques however, require a three-electrode potentiostatic system and periodic regeneration of the working electrode to obtain reproducible responses. Thus, CV or DPV does not permit con†
J. Heyrovsky´ Institute of Physical Chemistry. Institut fu ¨ r Festko¨rper- und Werkstofforschung. § York University. (1) Crespi, F.; Mo ¨bius, C. Kane, P. Pharm. Res. 1992, 26, 55. (2) Dryhurst G.; Kadish K.; Scherre F.; Rennberg R. Biological Electrochemistry; Academic Press: New York, 1982; Vol. 1, p 256. (3) Tolbert B. M.; Ward J. B. In Ascorbic Acid, Chemistry, Metabolism and Uses; Seib P. A., Tolbert, B. M., Eds.; Advances in Chemistry 200; American Chemical Society: Washington, DC, 1982. (4) Clemetson C. A. B. Vitamin C; CRC Press: Boca Raton, FL, 1989. (5) Kumar, V; Courie, P.; Haley, S. J. Chem. Educ. 1992, 69, A213. (6) Alwarthan, A. A. Analyst 1993, 118, 639. (7) Leon, L. E.; Captano, J. Anal. Lett. 1993, 26, 1741. (8) Backheet, E. Y.; Emara, K. M.; Askal, H. F.; Saleh, G. A. Analyst 1991, 116, 861. (9) Deutsch, J. C.; Kolhouse, J. F. Anal. Chem. 1993, 65, 321. (10) Brˇezina, M.; Zuman, P. Polarography in Medicine, Biochemistry and Pharmacy; Wiley: New York, 1958. (11) Jiang, Z. L.; Liang, A. H. Anal. Chim. Acta 1993, 278, 53. (12) Gao, Z. Q.; Ivaska, A.; Zha, T. X.; Wang, G. Q.; Li, P. B.; Zhao, Z. F. Talanta 1993, 40, 399. (13) Wring, S. A.; Hart, J. P.; Birch, B. J. Anal. Chim. Acta 1990, 229, 63. (14) Uchiyama, S.; Kobayashi, Y.; Suzuki, S.; Hamamoto, O. Anal. Chem. 1991, 63, 2259. (15) Kulys, J.; Dcosta, E. J. Anal. Chim. Acta 1991, 243, 173. (16) Leubolt, R.; Klein, H. J. Chromatogr. 1993, 640, 271. (17) Matsumoto, K.; Balza, J. J. B.; Mottola, H. A. Anal. Chem. 1993, 65, 1658. (18) Kobayashi, H.; Akamine, H.; Ohno, T.; Mizusawa, S. Electrochim. Acta 1991, 36, 649. ‡
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tinuous measurement but sampling with a frequency depending on the speed of the scan and the length of the regeneration period. The application of a less demanding potentiometric method yielding a continuous potential response to ascorbic acid concentration appears to be advantageous especially where monitoring over a long period of time is required. The use of a micro- or ultramicroelectrode would also provide a very mildly invasive procedure for in vivo monitoring and would permit the use of a two-electrode method. The potentiometric method19 with an immobilized redox mediator represents a convenient way to measure the equilibrium potential in systems with low mobility of direct electron transfer between the electrode and reacting species (electrochemical irreversibility).20 L-ascorbic acid (AA) can be oxidized to L-dehydroascorbic acid (DHAA) according to the scheme
C6H8O6 f C6H6O6 + 2e- + 2H+
(1)
with semidehydroascorbate as an intermediate product. The electrooxidative reaction is irreversible due to the electrochemical inactivity of L-dehydroascorbate, which is its final product. The exact mechanism, however, is subject to discussion.22,23 The direct oxidation of ascorbic acid by oxygen without any catalyst present, is relatively slow,20 especially at low pH. In this paper we describe a potentiometric method using a microelectrode employing the oxidation of ascorbic acid by oxygen, mediated by tetra-N-methylpyridoporphyrazinocobalt, deposited on the surface of a carbon fiber microelectrode. It is known that some metal complexes of the water-soluble tetrasubstituted pyridinoporphyrazines form conductive precipitates, possibly with sandwichlike structures24 after reduction of the central ion. Tetra-N-methyl-3,4-pyridoporphyrazinecobalt (CoTmtppa) can be reduced electrochemically from its green aqueous acidic or (19) Tse, Y. H.; Janda, P.; Lever, A. B. P. Anal. Chem. 1994, 66, 384. (20) Tur’yan Y. I.; Kohen R. J. Electroanal. Chem. 1995, 380, 273. (21) Lever, A. B. P.; Tse, Y. H.; Janda, P. U.S. Patent 5,342,490, 1994. (22) Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1986, 58, 1474. (23) Hu, I. F.; Kuwana, T. Anal. Chem. 1986, 58, 3235. (24) Kasuga, K.; Morisada, M.; Handa, M.; Sogabe, K. Inorg. Chim. Acta 1990, 174, 161. 0003-2700/96/0368-0960$12.00/0
© 1996 American Chemical Society
neutral solution, where it is present in its original Co(II) form. At potentials more negative than about +400 mV/SCE (pH 7), the cobalt center is reduced in a one-electron process19 according to the equation
[CoIITmtppa]4+ + e- T [CoITmtppa]3+
(2)
The Co(I) species is insoluble and forms a purple electrically conducting film19,21 on the electrode. The reduction of the Co(II) center can also be performed chemically using suitable electron donors. Ascorbic acid is such a donor and will convert the Co(II) to the Co(I) species. The Co(I) oxidation state is confirmed by spectrophotometric data and by the loss of the ESR signal of the Co(II) species.25,26 [CoITmtppa]3+ in common with other cobalt(I) phthalocyanine derivatives, can be reoxidized by dioxygen back to Co(II).25 EXPERIMENTAL SECTION Electrochemical measurements employed a three-electrode electrochemical cell with a carbon fiber microelectrode or highly oriented pyrolytic graphite electrode (HOPG) as a working electrode, a Pt counter electrode, and a SCE reference electrode, connected to a PAR 273A three-electrode digital potentiostatgalvanostat (EG&G Princeton). Potentiometric measurements were performed in a cell consisting of the phthalocyanine-coated microelectrode and a SCE reference electrode with stirrer, at 20 ( 2 °C, using a digital data acquisition unit. Electrochemical experiments were carried in a 0.1 M phosphate buffer, deoxygenated by argon (Linde). Solutions for the determination of ascorbic acid were equilibrated with oxygen (Linde) at room temperature. The phosphate buffer was prepared by mixing 0.1 M solutions of Na2HPO4 and KH2PO4 in distilled water, and the pH was adjusted by addition of KOH and H3PO4 solutions, respectively. Control samples of L-ascorbic acid (Merck) and Na2S (Lachema) were prepared as 10-2 M solutions in distilled water and standardized by iodometric titration. Tetra-N-methyl-3,4-pyridoporphyrazinecobalt(II) (CoIITmtppa) was prepared according to the literature.19,25,26 All other chemicals were reagent grade. Carbon fiber cylindrical microelectrodes were prepared from carbon fibers of 30 µm diameter (Avco), sealed into a glass capillary with epoxy resin (Devcon), leaving a 2 mm long carbon cylinder (area A, 1.89 × 10-3 cm2), which was connected to a Cu collector by conductive glue (Doduco). The microelectrodes were not pretreated in any fashion prior to use. The disk electrode was prepared from HOPG (Union Carbide). A HOPG disk of 8 mm diameter (area A, 0.503 cm2) was sealed into a Teflon cylinder with epoxy resin (Devcon). The HOPG surface was cleaned using adhesive tape by stripping off the surface layer. Electrodes were covered, stepwise, by a deposit of [CoITmtppa]3+ by polarizing the electrode at -400 mV/SCE for 2 min while immersed in a 10-4 M aqueous solution of [CoIITmtppa]4+ in 0.1 M phosphate buffer, pH 7, deoxygenated with argon. After each deposition step, the electrode was washed with distilled water and tested in a clean phosphate buffer using CV (25) Smith, T. D.; Livorness, J.; Taylor, H. J. Chem. Soc., Dalton Trans. 1983, 1391. (26) Woehrle, D.; Gitzel, J.; Okura, I.; Aono, S. J. Chem. Soc., Perkin Trans. 2 1985, 1171.
Figure 1. Cyclic voltammetry of a CoTmtppa/C electrode with coatings of varying thickness generated according to the procedure described in the Experimental Section: 0.1 M phosphate buffer, pH 7, deoxygenated by argon, scan rate 100 mV/s; 1-5, number of successive depositions. The charge corresponding to anodic peak 5 is 130 µC/cm2.
Figure 2. Diagram showing how the carbon fiber microelectrode (CFME) is protected during measurement and storage by being surrounded by a pipet. For measurement the pipet is immersed into the sample, while the microelectrode piston is pulled up, taking the sample into the pipet tip.
performed in the range of potentials -400 to -850 mV/SCE (pH 7), yielding a well-developed peak couple referred to as couple II in ref 19 and apparently corresponding to the first anion reduction localized on the porphyrazine ring. The area under the anodic peak of couple II was used to calculate the charge indicating the amount of [CoITmtppa]3+ deposited on the electrode surface. CV curves used for the calculation of charge for an increasing amount of the deposited [CoITmtppa]3+ are shown in Figure 1. The [CoIITmtppa]4+/[CoITmtppa]3+ redox couple was found to be less suitable for the determination of the charge because of dissolution of the deposited film during polarization of the electrode to the Co(I)/Co(II) redox potential. This procedure was repeated until a charge of about 200 µC/ 2 cm was attained. In such a case, the peak to peak separation for Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
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Figure 3. Cyclic voltammogram of ascorbic acid oxidation at a HOPG electrode, with various coverages of CoTmtppa as expressed by charge Q (see details in text): (a) Q ) 60 µC/cm2, peak A corresponds to ascorbic acid oxidation and peak B corresponds to oxidation of [CoITmtppa]3+ deposited on the electrode. (b) Curve 1, Q ) 10 µC/cm2; curve 2, bare surface. Ascorbic acid 10-4 M in 0.1 M phosphate buffer, pH 4.5, deoxygenated by argon; scan rate v ) 100 mV/s. Scan from 0 V/SCE, first sweep.
the anodic and cathodic redox reaction was less than 10 mV at a scan rate of 100 mV/s. The procedure described above, however, was followed only for the purpose of standardizing the preparation of the CoTmtppa (CoTmtppa/C) electrode for potentiometric measurements and the thickness of the CoTmtppa film was not determined. Once the electrode was completely covered, the amount of deposited [CoITmtppa]3+ was not important for correct establishment of its equilibrium potential and had no significant effect on the potential response of the electrode. After the deposition procedure, the electrode was washed with distilled water and stored dry in air. Some CoTmtppa/C microelectrodes were covered with an ionomer protective film, Nafion (Aldrich), using the procedure described in ref 28. They were subsequently dried under reduced pressure. Nafion films of different thickness were prepared by evaporation of different amounts of a 1 wt % Nafion solution. The fragile tip of the fiber microelectrode was protected during the measurement and storage in the way shown in Figure 2. This method still allows free access of the substrate from solution to the electrode.
observation of both L-ascorbic acid (A) and [CoITmtppa]3+ (B) oxidation reactions. The asymmetry of peak B corresponding to oxidation of [CoITmtppa]3+ can be explained by the fast consumption of [CoIITmtppa]4+ through chemical reduction by ascorbic acid. Figure 3b shows oxidation of L-ascorbic acid mediated by [CoIITmtppa]4+ shifted by some 80 mV more negatively (curve 1) relative to the oxidation at the bare electrode (curve 2), indicating the decrease of its overvoltage at the CoTmtppa/C electrode. This is in contrast with experiment described in Figure 3a, where the amount of deposited [CoTmtppa]3+ corresponded to a charge lower than 10 µC/cm2 and thus the surface redox couple was not identifiable in the presence of the relatively larger diffusion current of L-ascorbic acid. Apparently, the peak potential for ascorbic acid oxidation on the CoTmtppa/C electrode is shifted to values negative from both the potential of [CoITmtppa]3+ oxidation (eq 2) and the potential of ascorbic acid oxidation on the bare HOPG. This behavior can be explained by a catalytic EC mechanism of the ascorbic acid oxidation, where the [CoIITmtppa]4+ is chemically reduced by ascorbic acid according to the equation
RESULTS AND DISCUSSION
2[CoIITmtppa]4+ + C6H8O6 f
Voltammetry. (a) Oxidation of L-Ascorbic Acid. The processes associated with the L-ascorbic acid/L-dehydroascorbic acid redox couple and the surface [CoIITmtppa]4+/[CoITmtppa]3+ redox couple are illustrated in CV curves on the HOPG disk electrode in Figure 3. The amount of deposited [CoITmtppa]3+ and the concentration of L-ascorbic acid used in Figure 3a were set to allow simultaneous (27) Zagal, J.; Paez, M.; Tanaka, A. A.; dos Santos, J. R., Jr.; Linkous, C. A. J. Electroanal. Chem. 1992, 339, 13. (28) Weber, J.; Dunsch, L.; Neudeck, A. Electroanalysis 1995, 7, 255.
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2[CoITmtppa]3+ + C6H6O6 + 2H+ (3)
and the resulting [CoITmtppa]3+ is oxidized back by means of the electrode reaction described in eq 2. A plot of the experimental peak current values (Ip) vs the square root of the scan rate (v1/2) obtained from the CV of the ascorbic acid oxidation at a CoTmtppa/C electrode showed linear dependence indicating a diffusion-driven reaction. Comparison of the experimental data with theoretical values of Ip calculated
from eq 4 for an irreversible electron transfer:29
Ip ) 0.4958FnAC(Rna(F/RT)Dv)1/2
(4)
resulted in n ) 2 and Rna ) 0.3, indicating two-electron irreversible oxidation with the first oxidation step as the rate-determining step.23 Values for the parameters used in eq 3 are as follows: A ) 0.503 cm2, C ) 10-7 mol cm-3, D ) 10-5 cm2 s-1, where n is number of electrons in the overall process, A is the area of the disk electrode, R is the charge transfer coefficient, na the number of electrons in the rate-determining step, and C and D are the concentration and diffusion coefficient30 of L-ascorbic acid, respectively. Other symbols have their usual meanings. (b) Oxygen Reduction. Oxygen is reduced at the RDE coated with [CoITmtppa]3+ in a single wave at E1/2 ) -350 mV (pH 4.5) with a limiting current corresponding to a two-electron reduction. The CV peak current (Ip) for the reduction of oxygen at the CoTmtppa/C electrode, plotted vs v1/2, obeys, to a good approximation, eq 4 for n ) 2, Rna ) 0.5, A ) 0.503 cm2, C ) 10-6 mol cm-3, and D ) 1.9 × 10-5 cm2 s-1, where all parameters have meanings identical to those above, except for C and D, which are the concentration and diffusion coefficient of oxygen, respectively. The electrochemical reduction of oxygen on the CoTmtppa/C electrode can be described by means of an EC mechanism. The species [CoITmtppa]3+ is reoxidized by chemical reaction with O2 according to the equation
Figure 4. Scheme of reactions taking place on the CoTmtppa/C electrode and establishing the open circuit electrode potential.
2[CoITmtppa]3+ + O2 + 2H+ f 2[CoIITmtppa]4+ + H2O2 (5) and the resulting [CoIITmtppa]4+ is reduced back in an electrochemical reaction according to eq 2. These results are in good agreement with observations at many other cobalt phthalocyanines which in Co(I) form can reduce oxygen to hydrogen peroxide and where a one-electron ratedetermining step is assumed.27,31-35 Potentiometry. [CoIITmtppa]4+ can be reduced by ascorbic acid forming [CoITmtppa]3+ (eq 3) which can be re-oxidized back in chemical reaction with oxygen (eq 5), yielding hydrogen peroxide. These reactions can proceed both homogeneously with reactants in solution and heterogeneously by reactants immobilized on the electrode. Considering the two-electron oxidation of ascorbic acid and the two-electron reduction of oxygen, the redox processes associated with the [CoIITmtppa]4+/[CoITmtppa]3+ redox couple immobilized on the electrode surface can be described by the overall scheme shown in Figure 4. (29) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, Fundamentals and Applications; John Wiley: New York, 1980; p 222. (30) Heyrovsky´, J.; Ku˚ta, J. Principles of Polarography; Publ. House of Czech. Acad. Sci.: Prague, 1965; p 106. (31) Janda, P.; Kobayashi, N.; Auburn, P. R.; Lam, H.; Leznoff, C. C.; Lever, A. B. P. Can. J. Chem. 1989, 67, 1109. (32) Zecevic, S.; Simic-Glavaski, B.; Yeager, E.; Lever, A. B. P.; Minor, A. C. J. Electroanal. Chem. 1977, 83, 207. (33) Elzing, A.; Van der Putten, A.; Visscher, W.; Barendrecht, E. J. Electrochem. Chem. 1986, 200, 313. (34) Yeager, E. J. Mol. Catal. 1986, 38, 5. (35) Van der Putten, A.; Elzing, W.; Visscher, W.; Barendrecht, E. J. Electroanal. Chem. 1986, 214, 523.
Figure 5. Potential-time response of the CoTmtppa/C microelectrode to ascorbic acid in the range of concentrations of 10-4-10-3 M in 0.1 M phosphate buffer, pH 7, saturated by oxygen at 20 °C. Potential vs SCE.
Here [CoIITmtppa]4+/[CoITmtppa]3+ represents the reversible redox couple, while ascorbic acid and oxygen are the electron donor and acceptor, respectively. Equations 3 and 5 thus describe a net oxidation of ascorbic acid by oxygen mediated by CoTmtppa. Apparently, the [CoIITmtppa4+/[CoITmtppa]3+ concentration ratio on the electrode surface is determined by the ratio of the rates of chemical reactions: oxidation and reduction of cobalt porphyrazine according to eqs 3 and 5, respectively. In the case that the concentration of oxygen and other parameters such as temperature and pH are kept constant, the concentration of ascorbic acid in the solution is the only variable. Thus, the surface concentration ratio of the oxidized and reduced form [CoIITmtppa4+/[CoITmtppa]3+, which determines the open circuit potential of the electrode becomes a function of the ascorbic acid concentration. As concerns the actual chemistry occurring at the electrode, there are other possible scenarios which would require further experiments to substantiate. Alternatively, oxygen can be caAnalytical Chemistry, Vol. 68, No. 6, March 15, 1996
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Figure 6. Logarithmic plot of the potential of the CoTmtppa/C microelectrode vs logarithm of concentration of ascorbic acid CAA in 0.1 M phosphate buffer, saturated with oxygen at 20 °C. Statistics on 10 measurements in period of 2 months: (a) pH 4.5, slope -61 mV per decade of ascorbic acid concentration (correlation coefficient 0.9994). (b) pH 7, slope -62 mV per decade of ascorbic acid concentration (correlation coefficient 0.9993).
thodically reduced at the CoTmtppa/C electrode. If anodic oxidation of ascorbic acid is mediated at the same time, a mixed potential36,37 could be established at the electrode generated by these two opposing electrode reactions. Under such a circumstance, the CoTmppa surface need not vary its oxidation state but might mediate, for example, via formation of an oxygen adduct which is then reduced by the ascorbic acid. The potential-time response of the CoTmtppa/C microelectrode to increasing concentration of ascorbic acid under a constant concentration of oxygen is shown in Figure 5. The response time of the electrode depends only on the efficiency of stirring during injection of the sample. Calibration log plots of the potential dependence of the CoTmtppa/C microelectrode on the logarithm of the ascorbic acid concentration are presented in Figure 6 as a function of pH. Two CoTmtppa/C carbon fiber microelectrodes were used to collect data over a period of 2 months. Both electrodes showed linear dependence of potential on the logarithm of AA concentrations with a slope of -62 mV at both pH 4.5 (R ) 0.9994) and pH 7 (R ) 0.9993), through the entire range of concentrations 10-6-10-2 M L-ascorbic acid, with the relative standard deviation of 1.3% (pH 4.5) and 1.6% (pH 7). In the solution saturated with oxygen (1.1 × 10-3 M O2), the equilibrium potential (E) of the electrode was found to obey
E ) E′ + 60 log(CAA) (mV) (t ) 20 ( 2 °C)
(6)
where E′ (mV) is potential shift dependent on the pH and CAA (M) is the concentration of L-ascorbic acid in solution. As shown in Figure 6, the potential shift for pH change from 4.5 to 7 represents about -120 mV. Hence measurements should (36) Oldham, K. B.; Myland, J. C. Fundamentals of Electrochemical Science; Academic Press: New York, 1994; p 197. (37) Koryta, J.; Dvorˇa´k, J.; Kavan, L. Principles of Electrochemistry, 2nd ed.; John Wiley: New York, 1993; p 381.
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be carried out under pH-stabilized conditions if pH is expected to vary during the measurement. The electrode shows linear dependence of potential on the logarithm of concentration of oxygen in the solution with a slope of about 60 mV. Similarly, as in ref 19 it was found that in the solution saturated with air, potentials are 40 mV more negative vs solution saturated with oxygen. The slope of the AA calibration curve remains, however, unchanged. The potential shift can be avoided if measurements are performed at the equilibrium concentration of oxygen formed due to the exposure of sample to ambient atmosphere and temperature. The mean decay of the amount of CoTmtppa deposited on microelectrodes was measured as a change of the charge corresponding to the CoTmtppa ligand redox reaction as described in Experimental Section. Less than 10% decrease of charge was observed over the period of 2 months. During this time, data of 10 independent calibration plots were collected and summarized in Figure 6. No influence of the CoTmtppa decay on the potential response of the electrode to ascorbic acid was found during this period of time. The relatively good stability of deposited CoTmtppa film even in the presence of oxygen can be further improved by the Nafion overlay. It is of interest that [CoITmtppa]3+ deposits on the HOPG surface, from a solution of [CoIITmtppa]4+ even under open circuit conditions, i.e., no applied external potential, and in the presence of oxygen. An explanation of this phenomenon may be in the disproportionation reaction of [CoIITmtppa]4+ after its adsorption on the electrode surface. The exact mechanism of [CoITmtppa]3+ deposition however, is the subject of further work. The physiological level of ascorbic acid in the living body is relatively high compared to other vitamins; in plasma it is known to be about 15 µg/mL, i.e., about 10-4 M. The CoTmtppa/C microelectrode can clearly be used to monitor a broad range of ascorbic acid concentration beginning well below the physiological
Figure 7. Influence of a Nafion coating on the response of a CoTmtppa/C microelectrode to L-ascorbic acid and SH-, respectively. Potential response to L-ascorbic acid for concentrations ranging from 10-5 to 10-4 M in 0.1 M phosphate buffer, pH 7, saturated with oxygen at 20 °C. Potential vs SCE. (A-C) Increasing thickness of the protective Nafion film obtained by evaporation of a Nafion solution according to the procedure described in text: (A) 2, (B) 4, and (C) 8 µL of 2 wt % Nafion solution; (D) potential response to SH- in the concentration range 10-5-10-4 M in presence of 10-3 M L-ascorbic acid, using the same electrode as in (C). Arrows indicate injections of the ascorbic acid sample.
level with the detection limit of 10-7 M. This detection limit is comparable to other applications of direct potentiometry19 and lower than found for common voltammetric methods (10-6 M40,41). The lower accuracy of the potentiometric determination of ascorbic acid compared to amperometric methods with relative standard deviation typically of about 0.1%40,41 is compensated by a broad concentration range of linearity of calibration plot (over 4 orders of magnitude), simple instrumentation, and the absence of deactivation processes often accompanying voltammetry on solid electrodes.42 Potentiometric measurements are carried at ambient atmosphere, which further contributes to the simplification of the analytical procedure. Interference. Other compounds with reducing properties and redox potential close to ascorbic acid such as other aromatic diols (e.g., pyrogallol) may interfere if they are present. The ion-exchange property as well as the molecular sieve effect of an ionomeric film such as Nafion can be used to suppress the influence of some interfering compounds. This method was applied, for example, to the in vivo detection of neurotransmitters in the presence of ascorbic acid by DPV using electrodes covered by Nafion films,38 where the ion-exchange property as well as the molecular sieve effect of the Nafion film have been used. (38) Kristensen, E. W.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1987, 59, 1752. (39) Erdey, L.; Svehla, G. Ascorbino-metric Titrations; Akademiai Kiado: Budapest, 1973; p 10. (40) Doherty, A. P.; Stanley, M. A.; Vos, J. G. Analyst 1995, 120, 2371. (41) Korell, U.; Lennox, R. B. Anal. Chem. 1992, 64, 147. (42) Farrington, A. M.; Jagota, N.; Slater, J. M. Analyst 1994, 119, 233.
Further studies will seek a coating that will not inhibit ascorbic acid passage but will block other interferents. The CoTmtppa/C electrode has previously been used to determine the SH- ion19 while ascorbic acid is also accompanied by sulfhydryls in biological material; thus a combination of L-ascorbic acid and sulfhydryl ion was chosen as a model system for the interference study. Ascorbic acid gives a similar potential response at the CoTmtppa/C electrode as the sulfhydryl ion, and therefore, it is not possible to distinguish between them if they are present in a mixture. However, the CoTmtppa/C electrode with a protective Nafion film still responds to SH- while the film inhibits the ascorbic acid response to a degree dependent upon the thickness of the Nafion film. Figure 7 shows the potential response of the CoTmtppa/C microelectrode protected by a Nafion film of increasing thickness to ascorbic acid (Figure 7A-C) and to SH- (Figure 7D). Compared with the CoTmtppa/C electrode without Nafion (Figure 5), the potential response of the CoTmtppa/C electrode protected with Nafion is slower and its sensitivity toward ascorbic acid is diminished (Figure 7C). The response to SH- however, is affected substantially less (Figure 7D). The explanation may lie with the molecular sieve effect of Nafion film discriminating between the large ascorbate and small sulfhydryl ion. Both ascorbic acid, after dissociation into the first step at pH 7 (pK1 ) 4.2 39) and SH- carry negative charge and are therefore electrostatically repulsed by the anionic SO3- groups of the ionomer. The repulsion, however, appeared to be less significant for SH- even for relatively thick Nafion layers. Thus a Nafion-coated CoTmtppa/C electrode can be used to analyze for SH- in the presence of ascorbic acid. CONCLUSIONS The possible potentiometric detection of ascorbic acid using a CoTmtppa/C electrode has been presented. The microelectrode yielded fast and stable potential response to ascorbic acid in a broad range of concentrations. Such an electrode is suitable for the direct measurement of ascorbic acid concentration and for use as an end-point detector for ascorbic acid redox titrations.39 Application for measurements in vivo using an extremely small, mildly invasive, ultramicroelectrode seems feasible. ACKNOWLEDGMENT Our thanks are expressed to Yu-Hong Tse, for supply of CoTmtppa, and to Union Carbide, for the supply of HOPG. Partial support for this work from the Grant Agency of the Czech Republic, Grant 203/93/0244, is gratefully acknowledged. Received for review April 3, 1995. Accepted December 28, 1995.X AC950323R X
Abstract published in Advance ACS Abstracts, February 1, 1996.
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