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Anal. Chem. 1996, 68, 355-359
Carbon Film-Based Interdigitated Ring Array Electrodes as Detectors in Radial Flow Cells Osamu Niwa* and Masao Morita
NTT Basic Research Laboratories, 3-1 Morinosato, Wakamiya, Atsugi, Kanagawa, Japan
We have fabricated the first carbon film-based interdigitated ring array (IDRA) electrodes for application as detectors in thin-layer radial flow cells. The limiting current of dopamine (DA) at the electrode is proportional to the one-third power of the volume flow rate (v1/3) when two electrodes in the IDRA are held at 750 and 50 mV, respectively. In contrast, it is not proportional to v1/3 when only one electrode is held at 750 mV with the other electrode disconnected, due to the high conversion of DA in the flow cell at a low flow rate. The collection efficiency (CE) and redox cycles (Rc) increase with decreasing flow rate, since the redox cycling of the IDRA suffers less influence from the flow. The IDRA electrode was used in combination with microbore liquid chromatography (LC) for catecholamine detection. The CE and Rc are particularly high in the usual flow rate range for microbore LC, which is from 0.02 to 0.1 mL/min. Rc values of 4.3 and 3.2 were achieved for DA and epinephrine, respectively, at a flow rate of 0.02 mL/min, and the noise level of the electrode is low. This indicates that the IDRA electrode is very useful as a highly sensitive electrochemical detector in a radial flow cell. A dual electrochemical detector with two working electrodes is widely used in flow injection analysis and liquid chromatography (LC)/electrochemistry.1-5 This is because dual electrochemical detectors are superior to single detectors in terms of their selectivity and sensitivity. Traditionally, three types of electrochemical detector have been studied: two electrodes in series, two electrodes in parallel, and two electrodes facing each other.6 Of these geometries, the two electrodes facing each other have interesting properties because they enable the signals of electrochemically reversible analytes to be enhanced selectively. This is because redox cycling is established between these two electrodes. This current enhancement is more effective when there is only a small distance between the two electrodes. Therefore, the use of the interdigitated array (IDA) microelectrode is becoming more widespread than the use of two electrodes facing each other because the gap between two filar electrodes (1) Blank, C. L. J. Chromatogr. 1976, 117, 35. (2) Roston, D. A.; Kissinger, P. T. Anal. Chem. 1981, 53, 1965. (3) Roston, D. A.; Kissinger, P. T. Anal. Chem. 1982, 54, 429. (4) Schieffer, G. W. Anal. Chem. 1980, 52, 1944. (5) MacCrehan, W. A.; Durst, R. A. Anal. Chem. 1981, 53, 1700. (6) Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry; Marcel Dekker: New York, 1992; Chapter 22. (7) Aoki, K.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1988, 256, 269. (8) Aoki, A.; Matsue, T.; Uchida, I. Anal. Chem. 1990, 62, 2206. (9) Takahashi, M.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1992, 335, 253.
in an IDA can be reduced to a micrometer or less using lithographic technique.7-9 Radial flow cells, including wall-jet or thin-layer radial flow cells,10-19 are useful when designing dual electrodes. Ring-disk or carbon fiber multielectrodes have been used as series or parallel dual (or multi-) electrodes in radial flow cells.20-22 Thin-layer radial flow cells are advantageous in terms of achieving high sensitivity because they are small in volume and the distance between the column end and the electrochemical detector can be minimized by installing the former directly above the latter. Kissinger et al. reported a thin-layer radial flow cell which realizes a low detection limit using a glassy carbon (GC) electrode.19 Therefore, the combination of a small-volume radial flow cell and a dual electrode which effectively enhances the current in addition to an IDA in a cross flow cell may lead to the realization of a low detection limit. We have been developing electrochemical detectors from carbon film in order to apply them as LC detectors.23 Film electrodes are advantageous in terms of application to smallvolume chromatographic detectors. This is because carbon film can be fabricated into an electrode of any size and shape and many electrodes can be integrated close together in a small area. We reported a low detection limit of 5 fg (32 amol) for dopamine (DA) by using a carbon film-based IDA microelectrode as a microbore LC detector.24 Since the radial flow cell is capable of achieving a higher signal, the combination of a cell and a dual electrode as with the IDA should achieve high sensitivity and a low detection limit. This paper discusses the electrochemical properties of carbon film-based interdigitated ring array (IDRA) microelectrodes in a thin-layer radial flow cell. We also report the determination of catecholamines by combining the electrodes with microbore LC. EXPERIMENTAL SECTION Chemicals and Reagents. Dopamine hydrochloride (DA) and epinephrine (E) were obtained from Funakoshi (Tokyo, (10) Hanekamp, H. B.; De Jong, H. G. Anal. Chim. Acta 1982, 135, 351. (11) Fleet, B.; Little, C. J. J. Chromatogr. 1974, 12, 747. (12) Yamada, J.; Matsuda, H. J. Electroanal. Chem. 1973, 44, 189. (13) Gunasingham, H.; Fleet, B. Anal. Chem. 1983, 55, 217. (14) Gunasingham, H.; Fleet, B. Anal. Chem. 1983, 55, 1409. (15) Albery, W. J.; Bret, C. M. A. J. Electroanal. Chem. 1983, 148, 201. (16) Chin, D.-T.; Chandran, R. J. Electrochem. Soc. 1981, 128, 1904. (17) Do Duc, H. J. Appl. Electrochem. 1980, 10, 385. (18) Bohs, C. E.; Linhares, M. C.; Kissinger, P. T. Curr. Sep. 1994, 12 (4), 181. (19) Huang, T.; Kissinger, P. T. Curr. Sep. 1995, 13 (4), 114. (20) Hanekamp, H. B.; van Nieuwkerk, H. J. Anal. Chim. Acta 1980, 121, 13. (21) Hoogvliet, J. C.; Elferrink, F.; Van der Poel, C. J.; Van Dennekom, W. P. Anal. Chim. Acta 1983, 153, 149. (22) Ji, H.; Wang, E. Talanta 1991, 38, 73. (23) Tabei, H.; Takahashi, M.; Hoshino, S.; Niwa, O.; Horiuchi, T. Anal. Chem. 1994, 66, 3500. (24) Niwa, O.; Tabei, H.; Solomon, B. P.; Xie, F.; Kissinger, P. T. J. Chromatogr. B 1995, 670, 21-28.
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Figure 1. Schematic representation and photograph of carbon film-based IDRA electrode.
Japan). They were dissolved in 0.1 M perchloric acid (Wako, Osaka, Japan) to a concentration of 20 mM. Mobile phase buffer solutions were prepared using water purified with Milli-Q (Millipore, Bedford, MA), 1-octanesulfonic acid (OSA), sodium citrate dihydrate, phosphoric acid, methanol, diethylamine-HCl (Wako, Osaka, Japan), ethylenediaminetetraacetic acid disodium salt (EDTA), dimethylacetamide (DMA) (Aldlich), and sodium dihydrogen phosphate monohydrate (Merck, Darmstadt, FRG). Electrodes. The carbon film-based IDRA electrodes were fabricated by photolithography and dry etching from a carbon film of pyrolyzed 3,4,9,10-perylenetetracarboxylic dianhydride on thermally oxidized silicon wafers.25,26 Figure 1 shows a schematic representation and a photograph of the IDRA electrodes. One of the dual electrodes (W1) has a 1 mm disk at its center, and each of the other electrodes is separated with a 2 µm gap. The width of the ring increases from 42 to 207 µm with decreasing diameter in order to collect the analyte efficiently in the high flow at the center. On the other hand, the width of the outer ring is narrow because the flow rate is lower at the edge of the radial flow cell. Each ring electrode has almost the same area (about 0.0079 cm2). The total surface areas of W1 and W2 are 0.142 and 0.134 cm2, respectively. Apparatus and Experimental Conditions. All the continuous flow and chromatographic measurements were performed using a BAS 200B system (Bioanalytical Systems, West Lafayette, IN). The mobile phase was pH 3.2 buffer containing 50 mM sodium citrate dihydrate, 25 mM sodium dihydrogen phosphate monohydrate, 10 mM diethylamine hydrochloride, 27 µM EDTA, 2.2 mM OSA, 30 mL/L methanol, and 22 mL/L DMA. The pH was adjusted with 0.1 M phosphoric acid. The mobile phase was thoroughly filtered through regenerated cellulose with 0.2 µm pores. All samples were diluted in deionized water from 20 mM/ mL stock solutions prepared in 0.1 M perchloric acid. (25) Tabei, H.; Morita, M.; Niwa, O.; Horiuchi, T. J. Electroanal. Chem. 1992, 33, 25. (26) Niwa, O.; Tabei, H. Anal. Chem. 1994, 66, 285.
In the continuous flow measurement, a mobile phase containing 10 µM DA flowed into a thin-layer radial flow cell. The potentials of W1 and W2 were held at 750 and 50 mV (dual mode), or W1 was held at 750 mV and the other was disconnected from the potentiostat (single mode) in order to measure the collection efficiency (CE) and number of redox cycles (Rc). For the LC measurement of catecholamines, we used a Rheodyne 8125 injection valve (Berkley, CA) with a 5 µL sample loop for sample injection. We used a SepStik microbore C18 reversed-phase column (100 mm × 1 mm i.d., 5 µm particle size) (Bioanalytical Systems). The potential of each electrode was the same as that in the continuous flow measurement. The flow rate of the mobile phase was varied from 0.02 to 0.07 mL/min, the thickness of the gasket for the thin-layer flow cell was 12 µm, and the effective cell volume was 340 nL. The column was kept at 30 °C. The mobile phase was purged with argon for 5 min and then kept under a 27.6 kPa blanket of argon. RESULTS AND DISCUSSION Limiting Current. Since the Coulomb efficiency at the electrode in a thin-layer radial flow cell is very high, the analyte concentration may decrease at the edge of the electrode due to the consumption of the analyte by the electrochemical reaction. The Coulomb efficiency becomes greater when the flow rate is decreased because the analyte stays over the electrode for a longer time. Therefore, a higher signal should be obtained if the analyte can be regenerated by a reverse electrochemical reaction. Figure 2 shows the variation in DA oxidation and reduction currents at W1 and W2 of an IDRA electrode in a thin-layer radial flow cell as a function of the cube root of the volume flow rate (v1/3). The DA concentration is 10 µM, and the thickness of the flow cell is 12 µm. The limiting current at W1 in the dual mode is proportional to v1/3 from 0.02 to 1.0 mL/min. In contrast, the current in the single mode is proportional to v1/3 only when the flow rate is higher than 0.25 mL/min. The limiting current is lower than the theoretical line at flow rates lower than 0.25 mL/
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Figure 2. Flow rate dependence of the DA limiting current at an IDRA electrode in the dual and single modes. The DA concentration is 10 µM, and the flow cell is 12 µm thick. The W1 and W2 electrodes in the IDRA are held at 750 and 50 mV, respectively, vs Ag/AgCl in the dual mode, whereas W1 is at 750 mV and W2 is disconnected from the potentiostat in the single mode.
Figure 3. Coulomb efficiency of DA at the IDRA electrode in the dual and single modes. The experimental conditions are same as those in Figure 2.
min. The Coulomb efficiency was calculated from the limiting current and the amount of DA flowing through the cell. Figure 3 shows the Coulomb efficiency at an IDRA electrode in the dual and single modes. The Coulomb efficiency in the single mode is 0.18 when the flow rate is 1.0 mL/min, but it is about 1.0 at a flow rate of 0.02 mL/min. This clearly indicates that the DA consumption causes a DA shortage at the edge of the electrode, which makes the limiting current lower than the theoretical line in the single mode. In contrast, the oxidized DA is reduced immediately by W2 in the dual mode. Therefore, the limiting current is proportional to v1/3 in the thin-layer radial flow cell despite the very large apparent Coulomb efficiency (∼3.6 at 0.02 mL/min). The limiting current
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Figure 4. Variation in CE and Rc as a function of volume flow rate. CE was calculated by dividing the reduction currents at W2 by the oxidation currents at W1. Rc was calculated by dividing the oxidation currents at W1 in the single mode by those in the dual mode.
at W2 is lower than that at W1, but it approaches the W1 level, suggesting that the DA oxidized at W1 was reduced at W2 effectively at a low flow rate. Collection Efficiency and Redox Cycling. A higher CE in the flow cell is very important for electroanalytical purposes, such as the determination of analyte in the presence of an electroactive interferent or the determination of the analyte once it has been activated at an upstream electrode. Large redox cycling is also useful because the signal enhancement brought about by redox cycling improves the sensitivity and the detection limit. The flow rate dependence of CE was studied with an IDRA electrode in a thin-layer radial flow cell. Figure 4 shows the CE and Rc of DA as a function of the flow rate. The variation in CE is small at higher flow rates but increases rapidly at lower flow rates. A CE of 0.83 was obtained at a flow rate of 0.02 mL/min. This value is lower than that achieved with an IDA electrode in a thin-layer cross flow cell (more than 0.9 at a flow rate of 0.03 mL/min for DA).24 The IDA electrode consists of multiband electrodes, which are easier to miniaturize by lithography. We have applied IDAs with bandwidths of 2-5 µm as LC detectors. On the other hand, it is very difficult to fabricate a ring array with a width similar to that of the IDA because of its ring geometry. The CE at the IDRA electrode is lower than that at the IDA because of the difference in the electrode size. This is because the CE decreases as the bandwidth and gap are increased. A CE similar to that at the IDA will be achieved if an IDRA electrode with narrow ring arrays can be fabricated. Large redox cycling at a low flow rate is very important in thinlayer radial flow cells. This is because the signal in the single mode is much lower than the theoretical line shown in Figure 2 due to the consumption of the analyte in the cell. The Rc values are calculated by dividing the limiting current in the dual mode by that in the single mode. Rc is about 1.5 at higher flow rates but reaches 3.5 at a flow rate of 0.02 mL/min. This indicates that redox cycling is particularly important for obtaining a higher signal at a low flow rate. The Rc at higher flow rates (about 1.6) is much higher than the Coulomb efficiency (0.27) shown in Figure 3.
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Table 1. Comparison of the Anodic Peak Current Densities of 1 pmol of DA at IDRA and 6 mm Disk Electrodes current densities/nA cm-2 IDRA electrode
Figure 5. Chromatograms of E and DA at an IDRA electrode. The flow rate is 0.02 mL/min. (a) W1 in the dual mode, (b) W2 in the dual mode, and (c) W1 in the single mode. The injected amount of E and DA was 1 pmol in a 5 µL sample.
When the Coulomb efficiency is corrected using Rc, we find that only 18% of the molecules collect at the electrode. In contrast, it is very close to the Coulomb efficiency at the lower flow rate (Rc and Coulomb efficiency are 3.5 and 3.6), suggesting taht almost all the DA molecules are cycled at the IDRA electrode. Determination of Catecholamine in Combination with Microbore LC. Small-volume analysis is particularly necessary for determining biological substances such as neurotransmitters obtained from microdialysis sampling.27,28 Catecholamine has been determined by LC/electrochemistry because electrochemical detectors are very sensitive and no derivatization reaction is needed. An IDRA electrode in a thin-layer flow cell is promising for use in such analyses because of the small cell volume (about 340 nL for a 12 µm thick cell), low dead volume from the column end to the electrode, and high sensitivity due to redox cycling. Figure 5 shows typical chromatograms of DA and E at an IDRA electrode in the dual and single modes. The concentration of each analyte was 0.2 µM. The currents of both E and DA were enhanced with 5 µL sample injections, as shown in Figure 5a,c. In the LC measurement, the CEs of DA and E are 0.67 and 0.64, and the Rcs of DA and E are 2.1 and 1.6 at the flow rate of (27) Robinson, T. E., Justice, J. B., Jr., Eds. Microdialysis in the Neurosciences; Elsevier: Amsterdam, 1991; p 117. (28) Huang, T.; Shoup, R. E.; Kissinger, P. T. Curr. Sep. 1990, 9, 139.
flow rate/ mL min-1
dual mode
single mode
6 mm disk electrode
0.07 0.05 0.03 0.02
264 238 228 190
81.3 72.1 60.1 43.8
69.6 53.7 35.0 24.4
0.07 mL/min, respectively. However, both the CE and Rc values of E and DA increase with decreasing flow rate, similar to the results of the continuous flow measurement. DA exhibits a higher CE and Rc than E because of its better electrochemical reversibility. An Rc of 4.3 was obtained at a flow rate of 0.02 mL/min, suggesting that the IDRA electrode is advantageous as an LC detector, particularly at low flow rates. The current densities of DA at an IDRA electrode in the single and dual modes and at a conventional 6 mm GC disk electrode are compared in Table 1. The IDRA electrode in the single mode exhibits higher current densities than those at the 6 mm disk electrode, particularly at low flow rates. There are two possible reasons for the higher current density in the IDRA electrode in the single mode: radial diffusion around each ring electrode or depletion layer recharge.29,30 However, the radial diffusion may contribute only at the edge of each ring because the narrowest ring electrode is wider than 42 µm. In contrast, a replenishing of the diffusion layer between each ring electrode by convective diffusion may contribute more than radial diffusion because of the large distance from one ring element for oxidation to the next oxidation electrode. The current density at the IDRA electrode in the dual mode is much higher than that in the single mode due to redox cycling. At 0.02 mL/min, the current density in the dual mode is about 8 times higher than that at the disk electrode. The signal to noise ratio (S/N) is very important in terms of achieving a low detection limit. Therefore, the noise level of the IDRA electrode was measured in a higher sensitivity range. At a flow rate of 0.05 mL/ min, the noise level of an electrode at 750 mV in the dual mode is about 1.3 pA. No significant difference was observed in the noise in the dual and single mode measurements. By calculating the signal of E and DA at 1 pmol injection (34.0 and 33.7 nA), a detection limit of 0.12 fmol can be expected at an S/N of 3. These results indicate that a low detection limit can be achieved with a small-volume sample by combining an IDRA electrode and a small-volume column for LC measurement, because such columns are usually operated at very low flow rates. Further improvement in the detection limit may be achieved by optimizing the electrode area and the ratio of the ring width and gap and by miniaturizing each ring electrode. CONCLUSION Carbon film-based IDRA electrodes show excellent electrochemical properties as detectors in thin-layer radial flow cells. The (29) Cope, D. K.; Tallman, D. E. J. Electroanal. Chem. 1986, 205, 101. (30) Cope, D. K.; Tallman, D. E. J. Electroanal. Chem. 1986, 188, 21.
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limiting current is proportional to v1/3 in the dual mode in spite of a very high apparent Coulomb efficiency (about 3.6) because the redox cycling regenerates the species. Both CE and Rc increase with decreasing flow rate, suggesting that IDRA electrodes are particularly useful for application as small-volume LC detectors which are usually operated at low flow rates. The E and DA signals were also enhanced at the IDRA electrode with a 5 µL sample volume, similar to the case with continuous flow measurement. Rc values of 4.3 and 3.2 were
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achieved for dopamine and epinephrine, respectively, at a flow rate of 0.02 mL/min, and the DA current density was about 8 times higher than that at a bulk carbon disk electrode. Received for review August 16, 1995. Accepted November 1, 1995.X AC950832T X
Abstract published in Advance ACS Abstracts, December 1, 1995.
