Anal. Chem. 1996, 68, 203-207
Dual-Electrode Detection for Capillary Electrophoresis/Electrochemistry Min Zhong, Jianxun Zhou, and Susan M. Lunte*
Center for Bioanalytical Research and Departments of Chemistry and Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047 Gang Zhao, Dean M. Giolando, and Jon R. Kirchhoff
Department of Chemistry, University of Toledo, Toledo, Ohio 43606
The extremely low sample volumes required for capillary electrophoresis and the high sensitivity and selectivity of electrochemical detection make capillary electrophoresis/ electrochemistry (CEEC) a very useful method for bioanalysis. In this paper, two types of dual-electrode detectors for CEEC are described. The first employs a ring-disk microelectrode placed in a wall-jet configuration and is used for the selective detection of substances undergoing chemically reversible oxidations. Collection efficiencies obtained for catecholamines with this configuration were between 25 and 35%. The second electrode design consists of two adjacent carbon fibers embedded in an epoxy matrix and is analogous to the parallel dualelectrode configuration used in liquid chromatography/ electrochemistry. This configuration can be used to confirm peak identity and purity by operating the electrodes at two different potentials. Alternatively, it is possible to perform simultaneous oxidative and reductive electrochemical detection. The small sample volume requirements and high separation efficiencies of capillary electrophoresis (CE) make it ideal for the analysis of volume-limited samples such as microdialysates, single cells, and solutions of recombinant proteins. Electrochemical (EC) detection provides many advantages for CE, including a high degree of selectivity and sensitivity and the ability to miniaturize the detector without a loss of sensitivity. Several different EC detectors for CE have been described in the literature, and this area has recently been reviewed.1-4 Dual-electrode electrochemical detection has been used in the past to enhance the selectivity of liquid chromatography/ electrochemistry (LCEC) for electroactive analytes in complex biological samples.5-12 The most common configurations used with LCEC are the parallel and series modes. In the parallel dual(1) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A536A. (2) Curry, P. D.; Engstrom-Silverman, C. E.; Ewing, A. E. Electroanalysis 1991, 3, 587-596. (3) Yik, Y. F.; Li, S. F. Y. Trends Anal. Chem. 1992, 11, 325-333. (4) Lunte, S. M.; O’Shea, T. J. Electrophoresis 1994, 15, 79-86. (5) Roston, D. A.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1982, 54, 1417A1434A. (6) Lunte, S. M.; Kissinger, P. T. In Laboratory Techniques in Electroanalytical Chemistry; Heineman, W. R., Kissinger, P. T., Eds.; Dekker: New York, in press. (7) Roston, D. A.; Kissinger, P. T. Anal. Chem. 1981, 53, 1695-1699. 0003-2700/96/0368-0203$12.00/0
© 1995 American Chemical Society
electrode mode, compounds eluting from the column pass over both electrodes simultaneously. If the two electrodes are set at different potentials, the ratios of current response obtained at each electrode can be used to verify peak identity and purity5-9 in a manner analogous to dual-wavelength monitoring in LC with UV detection (LCUV). Alternatively, oxidized and reduced species can be monitored simultaneously with this technique.8,9 The parallel dual-electrode mode has been used for the detection and identification of a variety of compounds in complex samples, including phenolic acids in beer7 and pterins9 and catecholamines10 in brain tissue. The second method of dual-electrode detection is the series mode, where analytes pass over each electrode sequentially.12 Using this configuration, it is possible to discriminate chemically reversible compounds from those undergoing chemically irreversible redox chemistry. The series dual-electrode mode has been used for the selective detection of catecholamines in brain tissue10 and for the determination of thiols and disulfides11 in a variety of matrices. A dual-electrode detector for the analysis of thiols and disulfides by capillary electrophoresis/electrochemistry (CEEC), in which two electrodes were set in series, has recently been reported.13 In this case, one electrode was placed inside the capillary through a laser-drilled hole and bent so that it was parallel to the flow. The second electrode was placed at the end of the capillary in the usual configuration. This arrangement made it possible to detect thiols and disulfides in a single CE run. However, the collection efficiencies using this configuration were fairly low (8%) due to the large distance between the electrodes, and there was a loss of separation efficiency due to the length of tubing (after the decoupler) needed to contain the two electrodes. In addition, the detection limits reported are significantly higher than those generally reported for other analytes with CEEC (millimolar level for disulfides). This method also requires access to a high-power laser to drill holes in the fused silica capillaries. In the present work, two kinds of micrometer-sized dualelectrode assemblies are described. In both cases, the electrodes (8) Radzik, D. M.; Brodbelt, J. S.; Kissinger, P. T. Anal. Chem. 1984, 56, 29272931. (9) Lunte, C. E.; Kissinger, P. T. Anal. Chem. 1983, 55, 1458-1462. (10) Mayer, G. S.; Shoup, R. E. J. Chromatogr. 1983, 255, 533-544. (11) Allison, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8-12. (12) Roston, D. A.; Kissinger, P. T. Anal. Chem. 1982, 54, 429-434. (13) Lin, B. L.; Co´lon, L. A.; Zare, R. N. J. Chromatogr. 1994, 680, 263-270.
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are simply aligned at the end of the capillary. A ring-disk microelectrode is used for the series configuration, and a dual carbon fiber microelectrode is employed for parallel detection. Advantages of this approach over previously reported methods include ease of construction and replacement of the electrodes as well as the capability to use these electrodes in both on-column and end-column EC detection. EXPERIMENTAL SECTION Apparatus. The construction of the CEEC system has been described previously.14 However, for these experiments, a fracture joint15 was used rather than a Nafion joint to isolate the electrochemical detector from the separation voltage. The parallel dual electrode was placed outside of the capillary, while the ring-disk microelectrode was inserted into the detection end of the capillary. A laboratory-built pressure injection system was used for sample injection, and a 90-cm-long fused silica capillary of 50 or 75 µm i.d., 360 µm o.d. was used in all separations. Dual-electrode experiments were carried out with two modified BAS LC-4B potentiostats (Bioanalytical Systems, West Lafayette, IN). Electropherograms were recorded with a Model BD-41 dual-pen strip chart recorder (Kipp & Zonen). For the parallel dual-electrode experiments, the system was deoxygenated as described previously.16 Construction of Dual Electrodes. Ring-Disk Carbon Microelectrode. A ring-disk microelectrode was prepared by chemical vapor deposition (CVD) of alternating concentric layers of silica and carbon onto 10-µm carbon fibers according to previously published procedures.17,18 In brief, the deposition of the silica layer is achieved by utilizing a precursor system of SiCl4, O2, and H2.17 When the desired thickness of silica had been deposited, the CVD reactor was purged with Ar, and a layer of pyrolytic carbon was deposited from acetone. The silica coating process was then repeated to insulate the carbon ring layer. The ring-disk microelectrode was prepared by cutting the conical end of the coated fiber with a diamond fiber-optics cleaver at the point along the cone that would produce the desired outside diameter for the analytical tip. Electrical connections to the two electrodes were made by etching away the outer and inner silica layers with an aqueous solution of 30% HF at the opposite end of the capillary. The exposed portions of the fiber and the ring were then independently connected to a 70-mm length of 0.2-mm-diameter copper wire with silver epoxy. For added mechanical strength and ease of handling, the electrode was sealed into a 1.5-mm glass capillary pulled to a tip diameter slightly greater than the coated fiber. The electrode extended 1-3 mm beyond the capillary sleeve. A SEM of the tip of a ring-disk microelectrode is shown in Figure 1a. The outer diameter of the resulting electrode tip is typically 45-65 µm; that of the electrode used in this work was 65 µm. Parallel Dual Carbon Fiber Electrode. Two pieces of 33-µmdiameter cylindrical carbon fiber (Avco Specialty Products, Lowell, MA) were cut to 4.5- and 6.0-cm lengths, respectively. These were (14) O’Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992, 593, 305-312. (15) Linhares, M. C.; Kissinger, P. T. Anal. Chem. 1991, 63, 2076-2078. (16) Malone, M. A.; Weber, P. L.; Lunte, S. M.; Smyth, M. R. Anal. Chem. 1995, 66, 3782-3787. (17) Zhao, G.; Giolando, D. M.; Kirchhoff, J. R. J. Electroanal. Chem. 1994, 379, 505-508. (18) Zhao, G.; Giolando, D. M.; Kirchhoff, J. R. Anal. Chem. 1995, 67, 14911495.
204 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996
Figure 1. Scanning electron micrographs of (a, top) ring-disk microelectrode and (b, bottom) parallel dual microelectrode.
coated with 3 and 5 cm of Nafion, respectively, by dipping them into liquid Nafion for several seconds and air-drying. The fibers were then threaded into 3-cm lengths of capillaries of 180 µm i.d. and 360 µm o.d. so that they protruded about 0.2 cm from one end of the capillary. This end of the capillary was packed with epoxy cement, allowed to cure, and polished until a smooth cross section of the carbon fibers was exposed. Electrical contact was made at the other end of the capillary with two copper wires using silver epoxy. The electrode was reinforced by sealing it into a glass capillary as described above. Figure 1b shows a typical SEM of the tip of a parallel dual microelectrode. Optimization of Electrochemical Detector. Alignment of the sensing electrode with the end of the capillary was quite important in this work. To accomplish this, 1 mM hydroquinone was pumped continuously through the capillary; based on the current response, the position of the electrode was adjusted with a micromanipulator. For the parallel dual electrode, electrodes were considered to be aligned when the current responses for hydroquinone at both electrodes achieved roughly the maximal signal, and the ratio of responses approached 1.0 (1.0 ( 0.1) when both electrodes were set at +800 mV vs Ag/AgCl. In the case of
Figure 2. Dual-electrode detection in the series configuration using a ring-disk working electrode. Detection of 100 µM dopamine (DA), epinephrine (Epi), norepinephrine (NE), catechol (CAT), and ascorbic acid (AA). CE separation conditions: 100 mM CAPS, pH 9.98; applied voltage, 30 kV; capillary, 77 µm i.d. × 90 cm. E1 ) +800 mV; E2 ) -400 mV.
the ring-disk configuration, the collection efficiency (Ne), i.e., the ratio of the current response at the ring electrode (at -400 mV) to that at the disk electrode (at +800 mV), was monitored until it reached the maximum level. All electrodes were electrochemically activated before use through application of a square wave of -2.0 to 2.0 V at 50 Hz for 30 s. Reagents. Phenolic compounds, catcholamines, L-ascorbic acid, and zwitterionic buffers CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) and TES (N-[tris(hydroxymethyl)methyl]-2aminoethanesulfonic acid) were purchased from Sigma (St. Louis, MO). All standards were made fresh daily and filtered prior to injection. Phenolic acid standards were prepared in NANOpure water (Sybron-Barnstead, Boston, MA), and catecholamine standards were prepared in 75 mM TES, pH 7.6. All stock solutions were prepared at 1 mM concentration, and standards for calibration were prepared by successive dilution of these stock solutions. CE Separation Conditions. A 77 µm × 90 cm capillary was used for separations employing the ring-disk microelectrode. Running buffers of 100 mM CAPS at pH 9.98 and 100 mM TES at pH 6.92 and applied voltages of 30 and 28 kV were employed for the separations of catecholamines and phenolic acids, respectively. A shorter capillary (50 µm × 82 cm) was used for the separation of vitamin B2 (VB2), NADH, and NAD+ with the parallel dual microelectrode. In this case, the running buffer was 100 mM CAPS at pH 9.8, and the applied voltage was 25 kV. In all experiments, sample introduction was accomplished by pressure injection for 3 s at 10 psi. This corresponded to a 15-nL injection. Sample Preparation. A Lipton tea bag (black tea) was dipped into 10 mL of boiling water for 30 min. The resulting solution
Figure 3. Series dual electrode detection of phenolic acids in (a) a standard solution (0.66 mM, equimolar) and (b) a tea sample. ChA, chlorogenic acid; CA, caffeic acid; p-CoA, p-coumaric acid. CE separation conditions: 100 mM TES, pH 6.92; applied voltage, 28 kV; capillary, 77 µm i.d. × 90 cm. E1 ) +850 mV; E2 ) -250 mV.
was allowed to cool to room temperature and was filtered through a 0.2 µm-pore-size membrane filter before injection. RESULTS AND DISCUSSION Detection of Catecholamines. To evaluate the dual-electrode CEEC in the series configuration using a ring-disk microelectrode, its use for the detection of catecholamines was investigated. A separation of dopamine (DA), epinephrine (Epi), norepinephrine (NE), catechol (CAT), and ascorbic acid (AA) at E1 (disk) is shown in Figure 2. For these experiments, E1 was set at +800 mV and E2 (ring) at -400 mV vs Ag/AgCl. These potentials were selected on the basis of the hydrodynamic voltammogram obtained for each compound by CEEC. All five of the compounds were detected at E1; however, only the four ortho phenols were detected at E2. AA was not detected at the collection (ring) electrode because its oxidation is chemically irreversible. The effective Ne values (calculated as the ratio of current responses of the two electrodes) were 28.4, 30.9, 33.7 and 34.4% for DA, Epi, NE, and CAT, respectively. The limit of detection (LOD) for the catecholamines at E2 using this system was determined to be 5 µM at S/N ) 2. The response was linear between 5 and 500 µM with correlation coefficients of 0.9957, 0.9974, and 0.9984 for NE, DA, and Epi, respectively. The LODs for these same compounds using dual-electrode LCEC are in the low nanomolar range.10 In the past, we have found the LODs for CEEC comparable to those for LCEC. However, in this case they are considerably higher. This could be due to several factors. For example, the ring of the ringdisk electrode is composed of vapor-deposited pyrolytic graphite, which may be a less active form of carbon than is commonly employed for LCEC. In addition, the surface area of Analytical Chemistry, Vol. 68, No. 1, January 1, 1996
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Table 1. Collection Efficiencies and Current Ratios Obtained for Phenolic Acids in Tea Ne (%)a
chlorogenic acid caffeic acid p-coumaric acid c
i2b/i1c
standard
tea
standard
tea
20.9 25.5 0.0
18.8 26.8 0.0
0.59 0.62 0.73
0.58 0.80 d
a Effective collection efficiency. b Current response at E ) +650 mV. Current response at E ) +800 mV. d Barely detectable.
Figure 5. Parallel dual-electrode detection of 0.01 mM VB2 and NADH and 0.1 mM NAD+. CE separation conditions: 100 mM CAPS, pH 9.98; applied voltage, 25 kV; capillary, 50 µm i.d. × 82 cm. E1 ) +850 mV; E2 ) -750 mV.
Figure 4. Parallel dual-electrode detection of phenolic acids in (a) a standard solution (0.66 mM, equimolar) and (b) a tea sample. CE separation conditions are the same as in Figure 3. E1 ) +650 mV; E2 ) +800 mV.
the two working electrodes is very small, leading to very small current responses. This is a particular problem in the CEEC system, since the noise generated by the separation voltage is more significant when measuring low currents. The peak efficiency for catecholamines obtained in Figure 2 is relatively poor compared to those reported for CEEC systems using single carbon fiber microelectrodes. However, in cases where we employed electrodes of smaller (45 µm) outer diameter, we did observe separations with column efficiencies much higher than that shown in Figure 2. It is, therefore, quite possible that this loss of peak efficiency results from the measurable system backpressure created when a 65-µm electrode is placed inside a 75-µm capillary. Determination of Phenolic Acids in Black Tea. Chlorogenic acid (ChA), caffeic acid (CA), and p-coumaric acid (pCoA) 206 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996
are phenolic acids commonly found in teas, beers, fruit juices, and many other food products.19 Dual-electrode systems have been used extensively in conjunction with LC for the detection of phenolic compounds in a variety of foods and beverages.20-22 Figure 3 compares an electropherogram of a standard mixture of phenolic acids obtained by CEEC with a ring-disk microelectrode with that of a tea sample. It can be seen that ChA and CA give a response at both electrodes, while there is no peak for CoA at E2, as expected. The effective collection efficiencies for ChA and CA in the tea sample were quite close to those obtained for the standards, ∼10-30%, as can be seen in Table 1. In this case, the collection efficiency for pCoA is zero because the oxidation is chemically irreversible. The peak identities of the compounds in the tea sample were further confirmed using the parallel dual-electrode detector, and these results are given in Figure 4 and Table 1. Peaks were purposely not well resolved in order to illustrate this feature of dual-electrode detection. The current ratios obtained for the peak with the same migration time as ChA present in the tea sample and for authentic ChA were very close, indicating that this peak is, indeed, ChA. However, the current ratios obtained for the peak migrating at the same time as CA in the tea sample were much different from those obtained for the authentic CA. This indicated (19) Khurang, A. L. ACS Symp. Ser. 1992, 506, 77-84. (20) Lunte, S. M.; Blankenship, K. D.; Read, S. A. Analyst 1988, 113, 99-102. (21) Lunte, S. M. J. Chromatogr. 1987, 384, 371-382. (22) Madigan, O.; McMurrough, I.; Smyth, M. R. Analyst 1994, 119, 863-868.
that there was an impurity comigrating with the CA peak in the tea sample, and a shoulder can actually be seen on the peak shown in Figure 4. Simultaneous Oxidative and Reductive Detection. NAD+ is one of the most important molecules in biological redox systems.23-28 The two-electron couple NAD+/NADH is a coenzyme for many dehydrogenases, including quinone reductases and cytochrome P450. It has also been used quite extensively in immunoassays and for monitoring enzyme activity.26-28 Because of its biological significance, extensive studies have been performed on the electrochemical behavior of the NAD+/NADH redox couple.23-25 The ability to perform simultaneous oxidative and reductive detection with the parallel dual microelectrode makes it possible to monitor NAD+ and NADH in the same run. Vitamin B6 isomers have been detected previously using CEEC with oxidative electrochemical detection.29 To demonstrate the use of the parallel dual electrode for monitoring both the oxidized and reduced forms simultaneously, a CE separation of VB2, NAD+, and NADH was developed. Figure 5 shows electropherograms of the sample obtained at E1 and E2 using the parallel dualelectrode configuration. It can be seen that both the oxidized and reduced forms of NADH were detected by this method in a single run. The VB2 and NADH were detected only at E1 (850 mV), and the NAD+ produced a response only at E2 (-750 mV). (23) Czochralska, B.; Bojarska, E.; Valenta, P.; Nurnberg, H. W. Bioelectrochem. Bioenerg. 1985, 14, 503-517. (24) Studnickova, M.; Paulova-Klukanova, H.; Turanek, J.; Kovar, J. J. Electroanal. Chem. 1988, 252, 383-394. (25) Moiroux, J.; Elving, P. J. J. Electroanal. Chem., 1979, 102, 93-108. (26) Avila, L. Z.; Whitsides, G. M. J. Org. Chem. 1993, 58, 5508-5512. (27) Tang, H. T.; Hajizadeh, K.; Halsall, H. B.; Heineman, W. R. Anal. Biochem. 1991, 192, 243-250. (28) Bao, J.; Regnier, F. E. J. Chromatogr. 1992, 608, 217-224. (29) Yik, Y. F.; Lee, H. K.; Li, S. F. Y.; Khoo, S. B. J. Chromatogr. 1991, 585, 139-144. (30) Bresnahan, W. T.; Moiroux, J.; Samee, Z.; Elving, P. J. Bioelectrochem. Bioenerg. 1980, 7, 125-155.
Under the reported separation conditions, the detection limits for VB2 and NADH at E1 (S/N ) 3) were 0.2 and 1 µM, respectively. The detection limits for NAD+ were ∼50 µM. The higher detection limits obtained for NAD+ are believed to be due primarily to the adsorption-induced deterioration of the electrochemical response and the higher background currents characteristic of the reductive mode.16,25,30 CONCLUSIONS Two electrochemical detection schemes for CE have been described. The designs were evaluated and found to be quite versatile. The ring-disk electrode mode was shown to be highly selective for chemically reversible compounds. The parallel dualelectrode mode was useful for verification of peak identity and purity as well as simultaneous oxidative and reductive detection. Future work will be focused on improving the detection sensitivity and the application of dual microelectrodes for the analysis of catecholamines in microdialysis samples. ACKNOWLEDGMENT The authors thank Bioanalytical Systems for the use of LC-4B potentiostats. The financial support of the Kansas Technology Enterprise Corp. and the Center for Bioanalytical Research is gratefully acknowledged. Support from the University of Toledo, Biomedical Research Small Grants Program, sponsored by the National Institutes of Health, and the College of Arts and Sciences at UT for funding and operation of the Arts and Sciences Instrumentation Center is also acknowledged. The authors thank Nancy Harmony for her assistance in the preparation on the manuscript. Received for review June 5, 1995. Accepted October 3, 1995.X AC950545P X
Abstract published in Advance ACS Abstracts, November 15, 1995.
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