cellulose acetate chemically modified

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Anal. Chem. 1988, 6 0 , 1642-1645

as about 10 min, we did not experience any effect due to the diffusion through the edge. Finally, it is interesting to observe spectroelectrochemically that the TLEC experiences a large solution resistance voltage drop. This is illustrated in Figure 5, where the potentiostatic current (a) and its corresponding dA/dt plot are shown. While the current follows the pattern characteristic of TLEC measurements, the dA/dt curve shows a maximum rate of MV" production at about 1s. This is because the optical fiber probe is smaller than the electrode; thus the effect of the iR drop is shown in the form of the time delay. In the case of the current, the total current is recorded, while the dA/dt curve records the response shown in the center of the electrode. This observation indicates that spatial mapping is possible, provided the optical probe is small enough. In conclusion, we have constructed a thin-layer electrochemical cell with which both electrochemical and spectroelectrochemical measurements can be made. It has been demonstrated that the CV currents are characteristic of those recorded from a typical thin-layer cell. The cell is simple to assemble reproducibly. Also, equations relating electrochemical and optical parameters in thin-layer cells have been derived. The DCVA signal is directly proportional to the cell thickness, which allows a straightforward determination of the thickness. Registry No. Pt, 7440-06-4; quartz, 14808-60-7; methyl viologen, 1910-42-5.

LITERATURE CITED Hubbard, A. T.; Anson, F. C. I n Electroanalytical Chemistty; Bard, A. J., Ed.; Marcel Dekker: New York, 1970; Vol. 4, Chapter 2. Hubbard, A. T. CRCCrit. Rev. Anal. Chem. 1973, 2 , 201. Winograd, N.; Kuwana, T. I n Nectroanalytical Chemistry; Bard, A. J., Ed.; Marcel1Dekker: New York, 1974; Vol. 7, Chapter 1. Heineman, W. R.; Norris, 8. J.; Goelz, J. F. Anal. Chem. 1975, 45, 79.

Kobayashi, T.; Yoneyama, H.; Tamura, H. J. Nectroanal. Chem. Interfacial Electrochem. 1984. 161, 419. Kobayashi, T.; Yoneyama, H.; Tarnurla. H. J. Nectroanal. Chem. I n terfacial Electrochem. 1984, 177, 293. Szentrlmay. R.; Yeh, P.; Kuwana, T. I n Nectrochemical Studies of Biologcal Systems; Sawyer, D. T., Ed.; ACS Symposium Series 38, American Chemical Society: Washlngton, DC, 1977; Chapter 9. Pyun, C.-H.; Park, S.-M. Anal. Chem. 1986, 58, 251. Bard, A. J.; Faulkner, L. R. Nectrochemical Methods; Wlley: New York, 1980; pp 406-413. Bancroft, E. E.; Sldwell, J. S.;Blount, H. N. Anal. Chem. 1981, 5 3 , 1390. Bancroft, E. E.; Blount, H. N.; Hawkridge, R. M. I n Nectrochemical and Spectrochemical Studies of Biological Redox Components ; Kadlsh, K. M., Ed.; Advances in Chemistry 201; American Chemical Society: Washlngton, DC, 1962. Bird, C. L.; Kuhn, A. T. Chem. Sac. Rev. 1981, 1 0 , 49. Schwarz, W. M., Jr. Ph.D. Dissertation. 1961, University of Wisconsin. Kosower, E. M.; Cotter, J. L. J. Am. Chem. Soc. 1964, 8 6 , 5524. Watanabe, 1.;Honda, K. J. Phys. Chem. 1982, 86, 2617.

RECEIVED for review November 4,1987. Accepted March 9, 1988.

Cobalt Phthalocyanine/Cellulose Acetate Chemically Modified Electrodes for Electrochemical Detection in Flowing Streams. Multifunctional Operation Based upon the Coupling of Electrocatalysis and Permselectivity Joseph Wang,* Teresa Golden, and Ruiliang Li Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003 Flow-through amperometric detectors have proven themselves as highly sensitive means for measuring electroactive species and are the instrumentation of choice in numerous laboratories around the world (I). Particularly successful has been the combination of the resolution of a chromatographic column with electrochemical detectors ( 2 ) . Electrochemical detectors offer excellent sensitivity, wide linear range, discrimination against nonelectroactive species, low dead volume, and low cost. However, improvements in the stability, selectivity, and scope of flow detectors are highly desirable to meet new challenges posed by clinical and environmental samples. The utility of solid-electrode-based detectors is often hampered by a gradual fouling of the surface due to adsorption of large organic surfactants or of reaction products. Amperometric detection lacks the ability to discriminate between solutes possessing similar redox characteristics. Finally, the detection of many important solutes is often hindered by their slow electron-transfer kinetics at the commonly used electrode materials. One field that offers great potential for alleviating the above problems, and hence for enhancing the power of electrochemical flow detectors, is that comprising chemically modified electrodes (CMEs) ( 3 , 4 ) . Although modified electrodes have frequently been used for voltammetric measurements in batch systems, the sophistication currently available in tailoring surfaces has not been widely utilized for flow analysis.

So far, the main approach for using CMEk in flowing streams has been electrocatalysis. This scheme exploits the ability of certain surface-bound redox mediators to enhance electron-transfer kinetics and thus lower the operating potential (5-8). In particular, modified carbon paste electrodes containing added cobalt phthalocyanine (CoPC) were shown to decrease by several hundreds millivolts the potential required for electrooxidation of several irreversibly oxidizable species (5, 7). Because of its electrocatalytic capability toward a wide variety of redox systems, CoPC is expected to play an important role in future detection schemes. Permselectivity is another promising avenue for utilizing CMEs in flowing streams. The size or charge exclusion characteristics of polymeric coatings such as cellulose acetate (9), Nafion (IO), or poly(viny1pyridine) (II) have offered substantial improvements in the selectivity and stability of amperometric measurements. For example, surface fouling problems are eliminated, as the cellulose acetate (CA) forms an effective barrier for the transport of organic surfactants. We have demonstrated that the permeability of cellulose acetate coatings can be controlled and manipulated by hydrolyzing them in alkaline media for different time periods (9). In the work reported here, we describe the incorporation of the catalyst cobalt phthalocyanine into the cellulose acetate domain. At this new microstructure, the cellulose acetate

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Potential (VI






Polrnllal (V)

Flgure 1. Cyclic vottammograms for 2 X lo-‘ M oxalic acid recorded at the nonhydrolyzed (a) and 40-mln-hydrolyzed(b) CoPC/CA-coated electrodes, as well as at plain Cow-coated (c) and bare (d) glassy carbon electrodes. The supporting electrblyte was a phosphate buffer (pH 4.7).

serves as a template that establishes the structure and transport characteristics, while CoPC serves to enhance the electron-transfer kinetics. Base hydrolysis is shown to control the permeability of the two-domain structure in a manner similar to that common at single-domain cellulosic films.The mixed CoPC/CA coating thus exhibits properties superior to those of the two components alone and offers great potential for electrochemical monitoring of flowing streams. In particular, we will demonstrate the improved stability, scope, and selectivity of amperometric measurements resulting from such multifunctional operation. In addition, the study suggests that cellulose acetate could be an ideal host material for the preparation of other multifunctional coatings.

EXPERIMENTAL SECTION Apparatus and Reagents. The flow injection system was described previously (9). A sample loop of 20 pL and a glassy carbon thin-layer detector (Model TL-5, Bioanalytical Systems) were used. The flow rate was 1.0 mL/min. An Ag/AgCI reference electrode (Model RE-1,Bioanalytical Systems) was used in all experiments. The auxiliary electrode was a platinum wire or a stainless steel tube (static and flow experiments, respectively). Cyclic voltammograms were recorded, at a rate of 50 mV/s, with an EG&G PAR Model 264A voltammetric analyzer and a Houston Instrument X-Y recorder. A 3-mm-diameter glassy carbon disk (Bioanalytical Systems Model MF2012) was used, together with a Bioanalytical Systems Model VC-2 electrochemical cell. All solutions were prepared with doubly distilled water. Cobalt phthalocyanine, oxalic acid (Kodak), cellulose acetate (Aldrich), hydrazine sulfate, ascorbic acid (Baker), L-cysteine, uric acid, and penicillamine (Sigma) were used without further purification. Hydrogen peroxide (H202,27.5%) was purchased from Hydrox Chemical Co. Solutions (2000 ppm) of organic surfactants were prepared daily by dissolving the reagent grade materials. A 0.05 M phosphate buffer (pH 4.7) was used as supporting electrolyte and carrier solution in most experiments. Measurements of hydrogen peroxide were performed by using a phosphate buffer (pH 7.4) solution. Electrode Coating Procedures. The “mixed” CoPC/CA solution was prepared by adding 1.2 mg of CoPC to 23.3 mL of a 1% cellulose acetate solution. The latter was prepared by dissolving 0.20 g of cellulose acetate in a solution containing 12.7 mL of acetone and 10.6 mL of cyclohexanone and stirring for 1 h. The “mixed“ CoPC/CA solution was stirred for 3 h. Prior to its coating, the electrode was polished with 0.05-pm alumina particles, sonicated for 10 min, and allowed to air-dry. The electrode was coated with 10 p L of the CoPC/CA solution, placed to cover the active disk and its surroundings. The solvents were allowed to evaporate during a 10-min period. Hydrolysis proceeded in a stirred 0.07 M KOH solution for the desired time.

Flgure 2. Hydrodynamic voltammograms of 1 X lo-‘ M oxalic acid (A) and hydrazine sulfate (B) at CoPCICA electrodes hydrolyzed for 20 (a) and 40 (b) mln.

Plain CoPC-coated electrodes were prepared by syringing 10 pL of a 1 X lo4 M CoPC solution (in sulfuric acid) on the electrode and allowing it to dry for 3 min.

RESULTS AND DISCUSSION The combined catalytic and size exclusion functions of CoPC/CA-coated electrodes are demonstrated by the cyclic voltammograms shown in Figure 1. The catalytic function is demonstrated by the substantial (ca. 250 mV) cathodic shift in the oxalic acid peak potential (compare curves b and d). The single-domain CoPC (c) and mixed CoPC/CA (b) coatings exhibit a similar lowering of the overvoltage, indicating that CoPC maintains its catalytic activity while being “host” in the cellulosic matrix. Base hydrolysis can be used to manipulate the permeability of the CoPC/CA film; for example, the nonhydrolyzed f i i excludes the oxalic acid, as indicated from the absence of voltammetric peaks (a). The ability to control the transport characteristics of CoPC/CA coatings was illustrated for two additional high-overvoltagesolutes, hydrazine and cysteine, known (5, 7) to undergo electrocatalysis at CoPC (not shown). In both cases, the nonhydrolyzed film excluded the solute from reaching the catalytic sites; in contrast, the catalytic 40-min-hydrolyzed coating yielded the expected (57) response. A similar behavior was observed for penicillamine, which yielded a well-defined oxidation peak (EP= +0.65 V) when the 40-min-hydrolyzed CoPC/CA film was used and no response with bare and mixed-CoPC/ CA (0-min hydrolysis) coated electrodes. The above discussion suggests that CoPC/CA coatings effectively couple the electrocatalytic power of CoPC with the desired structural features (and hence permselectivity) of cellulose acetate. Such multifunctional character forms the basis for the analytical utility and advantages of CoPC/CAcoated flow detectors. The hydrodynamic voltammograms (HDVs), shown in Figure 2, demonstrate the permselectivity and catalytic functions of CoPC/CA CMEs under flowing conditions. These voltammograms were constructed from a series of flow injection experiments performed a t different applied potentials. The unmodified electrode (dotted lines) requires extremely high operating potentials to detect the oxalic acid and hydrazine, and hence it is prone to large background currents and potential interferences. The CoPC/CA-based detector exhibits well-defined peak-shaped HDVs, with marked decreases in overpotentials. Such a peak-shaped response was observed previously at plain CoPC CMEs (7, 12). The potential dependence of the catalytic response under flow conditions is similar to that found in cyclic voltammetry. The use of different hydrolysis times increases the film permeability and hence the HDV peak current (compare a and b). For both 20- and 40-min hydrolysis, the HDVs are identical in shape. In the following sections we will illustrate two major improvements in amperometric detection



1, 1988





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Flgure 3. Detection peaks at electrodes coated with CoPC (a) and CoPC/CA (b): repetitive injections of 1 X lo4 M ascorbic acid (A) and 2X M Lcystelne (B) solutions, containing 200 mg/dL gelatin and 100 mg/dL albumin, respectively; hydrolysis time, 45 (A(b)) and 40 (B(b)) min; applied potential, +0.10 (A) and 4-0.75(B)V.

(based upon the coupling of electrocatalysis and permselectivity), including the elimination of the catalyst poisoning by organic surfactants and the differentiation between solutes undergoing catalysis a t the CoPC. Figure 3 demonstrates the utility of the CoPCJCA film as a protective layer in the presence of surface-active materials. With the plain CoPC-coated electrode (a), a rapid decrease of the ascorbic acid (A) and L-cysteine (B) catalytic peaks (up to 93 and 87%) is observed in the presence of gelatin and albumin, respectively. Such effects are attributed to the interaction of these surfactants with the catalytic layer. This passivation problem is eliminated by using the CoPC/CA microstructure (Figure 3b). No loss of catalytic activity is observed, indicating effective exclusion of the surfactant. Similar improvements were observed for the detection of hydrazine in the presence of gelatin (not shown). The data of Figure 3 indicate also that the CoPCJCA-coated electrode responds rapidly to dynamic concentration changes that characterize analytical flow systems. Stability problems often characterize the electrocatalytic response a t CoPC-based detectors, even in the absence of coexisting surfactants ( 5 , 1 3 ) . Often the initial exposure of fresh CoPC CMEs to the flowing solution results in a gradual decrease in the response for 10-20 min (5);more severe is the potential-dependent deactivation that produces a permanent decrease in the response (12). Surprisingly, the mixed CoPCJCA coating offers substantial improvements in the stability of the electrocatalytic response of certain solutes. For M example, a series of 25 repeated injections of 2 X hydrogen peroxide yielded a relative standard deviation of 570, with the last injection producing more than 90% of the current exhibited by the first one. In contrast, the plain CoPC-coated detector exhibited a severe (85%) current diminution and a relative standard deviation of 65% (operating potential, +0.70 V). Similar improvements were observed for successive injections of an ascorbic acid solution. No such improvements were observed for the high-potential deactivation in the presence of oxalic acid and L-cysteine, with both CoPC and CoPC/CA coatings yielding about 30% current diminutions within 30 successive injections. The fact that improved stability is observed at the CoPCJCA film for certain solutes appears to reflect the nature of deactivation of CoPC CMEs. Most likely, the incorporation of CoPC into the CA domain alleviates stability problems associated with leaching of the modifier from the surface (with the cellulosic matrix holding the CoPC more firmly than does the carbon surface). In contrast, the high-potential deactivation (attributed to further oxidation of the phthalocyanine or irreversible com-







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Figure 4. Flow injection peaks for ascorbic acid solutions containing increasing hydrogen peroxide concentrations at electrodes coated with CoPC/CA (A) and CoPC (B): (a) 1 X lo-' M ascorbic acid and (b-d) same as (a) but after successive additions of 2 X lo-' M hydrogen peroxide: applied potential, +0.7 V; hydrolysis time (A), 10 min.

plexation of the Co(II1) center (13))is not affected by the use of mixed coating. Improved selectivity is another important feature of CoPCJCA CMEs. CoPC possesses a powerful electrocatalytic activity toward a wide variety of redox systems. Because flow injection systems usually lack a separation power, it is not possible to differentiate between species exhibiting similar redox potentials at CoPC-modified electrodes. For example, both hydrogen peroxide and ascorbic acid undergo electrocatalysis at CoPC-coated electrodes to yield cyclic voltammetric peak potentials of 0.65 and 0.08 V, respectively (not shown). Obviously, the selective flow injection measurement of hydrogen peroxide in the presence of ascorbic acid (which is of great interest for many biosensing applications) is not feasible at plain CoPC CMEs. In contrast, the ability to manipulate the transport characteristics of the CoPCJCA coating permits differentiation between small solutes, e.g., hydrogen peroxide, and larger ones. Such selective flow injection amperometric measurements of hydrogen peroxide are illustrated in Figure 4. The 10-min-hydrolyzed mixed coating effectively excludes the ascorbic acid from the surface (A(a)), allowing convenient quantitation of hydrogen peroxide (A(b-d)). In contrast, the large ascorbic acid peak, observed when the single-domain CoPC coating (B(a)) is used, precludes the measurement of hydrogen peroxide (B(b-d)). A selective hydrogen peroxide flow injection response was obtained also in the presence of uric acid (not shown). In contrast, and as expected, an additive response was obtained a t the CoPCcoated detector. The mixed coating yielded also a stable hydrogen peroxide response, as compared with the rapid peak diminution that characterized the response at the single-domain CoPC film. Such an observation is consistent with the improved stability reported earlier in this paper. Many enzyme transducers can greatly benefit from the ability to selectively measure hydrogen peroxide in the presence of the endogenous compounds ascorbic and uric acids. The possibility of incorporating a biocatalyst into the electrocatalyticJpermselective CoPCJCA matrix is currently under investigation. The concentration dependence of the CoPCJCA-coated detector was evaluated for successive injections of hydrazine solutions of increasing concentration, 10-100 /IM (operating potential, +0.60 V; hydrolysis time, 40 min). The electrocatalytic peak current increased linearly with increasing concentrations of hydrazine; the slope of the resulting calibration plot corresponded to a sensitivity of 1.1 nA/WM (correlation coefficient, 0.999). Similar injections of a 5 /IM hydrazine solution were used to estimate the detection limit. The sig-

Anal. Chem. 1988, 60, 1645-1648

nal-to-noise characteristics ( S I N = 3) indicated a detection limit of 1pM, i.e., 0.64 ng, in the 20-pL sample. Hence, the excellent sensitivity of amperometric detection is not compromised by the electrode coating. In conclusion, amperometric detection for flowing streams is greatly improved by the multifunctional character of CoPC/CA coatings. The incorporation of CoPC into the CA domain has essentially no effect on the catalytic behavior. Such effective coupling of electrocatalysis and permselectivity provides a means for minimizing problems and extending the scope of electrochemical measurements in flowing streams. This study suggests also that cellulose acetate could be an ideal host material for the preparation of other powerful multifunctional coatings.

Registry No. CoPC, 3317-67-7; CA, 9004-35-7; carbon, 7440-44-0; hydrogen peroxide, 7722-84-1; hydrazine, 302-01-2; cysteine, 52-90-4; oxalic acid, 144-62-7.


LITERATURE CITED (1) Stulik, K.; Padkovi, V. €/echoanalytical Measurements h Flowing Liquids; Ellis Horwood: Chichester, England, 1987. (2) Kisslnger, P. T. Anal. Chem. 1977, 49, 447A. (3) Bard, A. J. J. Chem. Educ. 1983, 60,302. (4) Murray, R. C.; Ewing, A. G.; Durst, R. A. Anal. Chem. 1987, 59, 379A. (5) Korfhage, K. M.; Ravichandran, K.; Baldwin, R . P. Anal. Chem. 1984, 56, 1514. (6) Wang, J.; Freiha, B. Anal. Chem. 1984, 56, 2266. (7) Halbert, M. K.; Baldwin, R. P. Anal. Chem. 1985, 57, 591. (8) Marko-Varga, G.; Appelqvist, R.; Gorton, L. Anal. Chim. Acta 1988, 179, 370. (9) Wang, J.; Hutchins, Anal. Chem. 1985, 57, 1536. (10) Wang, J.; Tuzhi, P.; Golden, T. Anal. Chim. Acta 1987, 794, 129. (11) Wang, J.; Golden, T.; Tuzhi, P. Anal. Chem. 1987, 59, 740. (12) Santos, L. M.; Baldwin, R. P. Anal. Chem. 1986, 58, 848.

RECEIVED for review December 29,1987. Accepted March 28, 1988. This work was supported by the National Institutes of Health (Grant No. GM 30913-04) and Battelle Pacific Northwest Laboratory.

Thin-Layer Spectroelectrochemical Cuvette Cells with Long Optical Path Lengths Yupeng Gui,’ Steven A. Soper, and Theodore Kuwana* Center for Bioanalytical Research, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66046 Thin-layer transmission spectroelectrochemistry has been widely used to investigate various heterogeneous and homogeneous electrochemical processes in both aqueous and nonaqueous solutions (1-6). However, the conventional thin-layer spectroelectrochemical cell, in which the light is passed through an electrode, has two major disadvantages. One is its short optical path length, which is defined by the thickness of the thin solution layer, and provides poor optical sensitivity for observing solution species. Another is the requirement of optically transparent electrodes (OTE), such as metal-coated and metal oxide coated glass of Pt and Au minigrid electrodes. Recently, a new spectroelectrochemical cell, the long optical path length thin-layer cell (LOPTLC), has been developed in this and other laboratories (7-9) to overcome the above disadvantages. In this LOPTLC configuration, the light irradiates the thin solution layer along the axis of the electrode/solution interface, thus greatly improving its optical sensitivity (about 100 times more sensitive than a conventional thin-layer cell) while all other thin-layer spectoelectrochemical behaviors are maintained. Several studies (410-1 7)have demonstrated the excellent utility of this LOPTLC to investigate various heterogeneous and homogeneous phenomena, such as adsorption, desorption, catalytic oxidation, catalytic hydrogenation, and solution chemical reactions associated with a solid electrode. However, there are a few shortcomings with this LOPTLC: (1) Fragile electrodes, such as glass-coated and Si semiconductor electrodes, are difficult to use because pressure must be applied to the body of the electrode to seal for vacuum-tight conditions. (2) It is very inconvenient and time-consuming to remove the electrode from the cell in order to pretreat the electrode. (3) The current cell does not allow simultaneous transmission spectroelectrochemistry to be conducted with other spectroscopy methods, such as luminescence and surface reflectance. (4) It is difficult to perform flowing solution experiments. ( 5 ) It is difficult to rigorously exclude oxygen within the cell which is made from Teflon or Kel-F (10,16) and the LOPTLC made from a material that is not O2 Present address: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172.

permeable should help to circumvent this problem. T o overcome these shortcomings, two new thin-layer spectroelectrochemical cells with long optical path lengths were designed. These cells are called “cuvette” cells because they are simply made from commercially available quartz cuvettes. In this communication, the construction and characterization of two different types of cuvette cells are reported. The use of one of these cells in flow injection analysis (FIA) is also reported.

EXPERIMENTAL SECTION Cell Fabrication. A. A schematic diagram of the glassy carbon cuvette cell (cell A) is shown in Figure 1. Figure 1A is the front view of the cell, viewed along the optical path. Parts B and C of Figure 1are the left and right views, respectively, of the cross section S-S as marked in Figure 1A. The cell body is an ordinary quartz cuvette with a 10-mm optical path length. Its internal height and width are 23 mm and 2.0 mm, respectively. A small hole was made at the bottom of the cuvette by polishing with the tip of a fine file and finally drilling with a heated tungsten wire. At the top of the cuvette there are two small pieces of 125 wm thick Teflon film (type 500C, Du Pont, Wilmington, DE) on each of the two cuvette walls, as indicated in Figure 1B. The Teflon film was melted onto the cuvette wall at a temperature of ca. 300 “C. This film controlled the thickness of the solution layer. To eliminate any stray light caused by the light passing through the cuvette wall, both ends of the wall were coated with black water-insoluble ink. The coating was applied with an extra fine point marker while viewing the wall under a 4 5 x microscope (Fisher Scientific Co., Fair Lawn, NJ). The working electrode is a piece of glassy carbon (GC) plate (GC-20 grade, Tokai Carbon Co., Tokyo, Japan). It was first cut to the approximate internal size of the cuvette with a glass saw, polished subsequently with fine sandpaper (grit 0000, Buehler, Ltd., Lake Bluff, IL), and finally polished on a glass polishing plate (Barnes Analytical Division, Stamford, CT) successively with 1-,0.3-, and 0.05-wm alumina slurry (Buehler,Ltd.). Prior to each polishing and after the fiial polishing, the electrode was sonicated in water. The final electrode was about 1.7 mm wide, 9.8 mm long, and 28 mm high. The geometric area of the electrode, which is exposed to solution, was 5.46 cm2. The electrical contact to the electrode was provided by clamping with a three-finger stainless steel tricep (Universal Technical Products, Inc., New York). Rigid contact to the electrode by the tricep was achieved

0003-2700/88/0360-1645$01.50/00 1988 American Chemical Society