Technical Notes Anal. Chem. 1997, 69, 264-267
Amperometric Detection in Capillary Electrophoresis with an Etched Joint Shen Hu, Zong-Li Wang, Pei-Biao Li, and Jie-Ke Cheng*
Department of Chemistry, Wuhan University, Wuhan 430072, China
A system for coupling amperometric detection with capillary electrophoresis (CE) is demonstrated. In CE amperometric detection, the separation and detection capillaries are usually coupled with a porous joint to isolate the detector from the high applied voltage when the capillary inner diameter is larger than 25 µm. In this report a simple method is suggested. The joint was prepared by etching the capillary wall with HF after the polyimide coating of the capillary had been removed. This etched joint can efficiently isolate the detector from the high applied voltage. The performance of the CE-amperometric detection with an etched joint was evaluated with hydroquinone. The successful separation and amperometric detection of catecholic compounds, diphenols, and phenol have been carried out by this system. Capillary electrophoresis (CE) has attracted much attention in recent years as an efficient separation technique.1 It has been used widely for its advantages of small injection volume, good efficiency, and high speed. One of the major areas of research in CE is the development of sensitive detectors. Amperometric detection is an important method of detection for CE because it has attractive features including high sensitivity, good selectivity, and low cost. CE with amperometric detection has been established as a powerful analytical technique, especially for the analysis of biological microenvironments such as single cells.2,3 No significant effects from the high electric field on amperometric detection will be observed if capillaries with very small inside diameters (e25 µm) are used for CE.4-6 The microelectrode can be positioned at the end of the capillary directly without using the porous joint to reduce the background noise resulting from the high electric field employed for electrophoresis. This technique is termed “end-column detection”, but this detection mode will not be available if capillaries with large inside diameters (>25 µm) are used for CE. The electric currents in these capillaries under the high electric field can greatly affect the (1) Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M. Anal. Chem. 1989, 61, 292A-303A. (2) Olefirowicz, T. M.; Ewing, A. G. Chimia 1991, 45, 106-108. (3) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62, 1872-1876. (4) Lu, W.; Cassidy, R. M.; Baranski, A. S. J. Chromatogr. 1993, 640, 433440. (5) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (6) Lu W.; Cassidy, R. M. Anal. Chem. 1994, 66, 200-204.
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measurement of the faraday currents on the microelectrodes. Therefore, it is necessary to isolate the amperometric detector from the high electric field. The first off-column amperometric detector in CE was introduced by Wallingford and Ewing.7 An electrically conductive porous glass joint was used to couple amperometric detection with CE to reduce the effects of the high electric field on amperometric detection. Afterward, similar designs including the use of porous graphite tubing,8 Nafion tubing9 or cellulose acetate film10 were used to circumvent the same problem. The common feature of these joints is to make a suitable fracture on the capillary and then couple two pieces of column with porous tubing or polymer film over the fracture. In this work, a simple method for fabricating the conductive joint without fracturing the capillary is described. The joint was fabricated by etching the outside wall of the capillary with hydrofluoric acid after the polymer coating had been removed. When the etched wall is thin enough, it becomes a porous glass membrane which allows only small buffer ions to pass through it. The fabrication of this conductive joint is simple and inexpensive without using porous tubing or polymer film. The whole system coupled by the etched joint has been used successfully to perform amperometric detection for CE. EXPERIMENTAL SECTION Construction of the Joint Assembly. Fused-silica capillaries (Yongnian Optical Fiber Factory, Hebei, China) of 50-µm i.d., 375µm o.d., and 60-cm length were used in this study. Before use the capillaries were washed with 0.1 M NaOH, double-distilled, deionized water and operating electrolyte. A short section (∼3 mm) of polyimide coating was scraped 2 cm from the detection end of the capillary by a scalpel. The exposed section was fixed on a 2.5 cm × 1 cm Plexiglas slide with epoxy glue and then was immersed in 40% hydrofluoric acid for etching. An optical microscope (Shanghai Optical Instrument Corp., Shanghai, China) was used to observe the etched capillary wall and measure the wall thickness. After the exposed section was etched for ∼3 h, its wall was thinner than 20 µm. Then the slide together with the etched section of capillary was placed on the bottom of a 10(7) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (8) Yik, Y. F.; Lee, H. K.; Li, S. F. Y.; Khoo, S. B. J. Chromatogr. 1991, 585, 139-144. (9) 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. (10) Chen, I. C.; Whang, C. W. J. Chromatogr. 1993, 644, 208-212. S0003-2700(96)00331-9 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Schematic diagram of etched joint assembly.
mL Plexiglas vial with two opposite 1-mm holes drilled on the walls, which allowed the separation capillary (58 cm long) and detection capillary (2 cm long) to stretch out of the vial. With the capillaries in position, the holes were sealed with epoxy glue. The vial was then filled with running buffer and a platinum wire electrode was dipped in the buffer as the cathode of the high applied voltage. Since the etched segment was fragile, it was fixed on a Plexiglas slide and kept immersed in buffer to prevent breaking the etched thin-walled capillary. The etched joint was durable; no apparent deterioration of it was observed after daily use over a 4-month period. Otherwise, the etched joints are easily commercial; we completed the construction with a 100% success rate. A schematic diagram of the etched joint assembly is illustrated in Figure 1. Apparatus. Electrophoresis in the capillary was driven by a high-voltage dc power supply (30 kV, Huazhong University of Technology, Wuhan, China). The high-voltage end was housed in a Plexiglas box equipped with an interlock system to protect the operator. The coupled capillary and the container holding the etched joint were filled with buffer. Platinum wires were used as the electrodes. Sample introduction was performed by electromigration. The carbon fiber microelectrode was prepared by inserting a single carbon fiber (8-µm diameter, Goodfellow Co., London, England) through a 1-mm-i.d. capillary tube until the fiber protruded ∼0.5 cm from the tip of the capillary tube; then the tip was sealed with a little drop of epoxy glue. After the epoxy glue was dried at room temperature for 2 days, the carbon fiber was connected to a copper lead via graphite powder. The exposed fiber was then cut to the designed length (0.3-0.5 mm) with a scalpel. Amperometric detection was performed using a two-electrode configuration. The microelectrode was inserted into the detection capillary using an XYZ micromanipulator (Wuhan Instrument Corp., Wuhan, China) with the aid of an optical microscope. A saturated calomel electrode (SCE) was employed as reference electrode. Potential control was provided by an HDV-7 potentiostat (Sanming Electric Factory, Fujian, China). The electrochemical currents produced at the detector were measured by a picoammeter (Dept. of Chemistry, Wuhan University, Wuhan, China) and were recorded on a strip-chart recorder (Sichuan Instru. Corp., Sichuan, China). The detector and the electrochemical cell were housed in a faraday cage to reduce noise. Reagents. Unless stated otherwise, all the chemicals were of analytical-reagent grade (Shanghai Reagent Corp., Shanghai, China). Norepinephrine (NE), isoproterenol (IP), and sodium dodecyl sulfate (SDS) were obtained from Sigma and used without further purification. All analytes were prepared from 0.01 M stock
Figure 2. Currents in the separation capillary vs different applied potentials: column, 50 µm i.d., 60 cm total length; buffer, 10 mM NaH2PO4-10 mM Na2HPO4 at pH 6.8.
solutions in 0.1 M perchloric acid. All solutions were prepared with double-distilled, deionized water and filtered through a 0.22µm cellulose acetate filter (Shanghai Institute of Medicine and Industry). RESULTS AND DISCUSSION When the thickness of the exposed capillary wall was etched to 30-40 µm, current (0.2-0.3 µA) was observed in the separation capillary filled with 10 mM NaH2PO4-10 mM Na2HPO4 buffer (pH 6.8) if high voltage (20 kV) was applied. The current increased as the etching time extended. However, no obvious change in the current was observed when the thickness of the exposed section was less than 20 µm. If the same applied voltage and buffer were used, the difference in the current measurement was less than 1.6% for 8 h. A linear relationship (R ) 0.9995) was obtained between the current in the separation capillary and the applied voltage (Figure 2), indicating that the electric conductivity in the separation capillary was constant. It coincided with the results obtained in the capillary without the joint. The resistance of the joint was calculated to be 1.5 × 109 Ω. No substantial difference in the current measurement was observed between capillaries with the etched joint and those without the etched joint when the same applied voltage and buffer were used. After the etched joint is constructed, the etched segment of the capillary becomes a porous glass membrane that allows permeation of small buffer ions and therefore allows the application of the high electric field across the separation capillary. Electroosmotic flow is a fundamental constituent of CE operation and affects the electrophoretic separation dramatically. The generation of the electroosmotic flow in the separation capillary was easily observed since a drop of buffer formed at the end of the detection capillary while the high potential was applied. The electroosmotic flow drove the solvents with the solute zones to pass through the etched segment to the end of the detection capillary. Hence, the detector could be isolated from the high electric field to reduce the background noise. Hydroquinone (HQ) was chosen to manifest the feasibility of the CE-amperometric detection system described above. An electropherogram of 1.0 × 10-7 M HQ in phosphate buffer (pH 6.8) is shown in Figure 3. The corresponding number of theoretical plates was determined to be ∼190 000, indicative of the high separation efficiency achievable with this system. A linear relationship between peak current and sample concentration was obtained. Analytical Chemistry, Vol. 69, No. 2, January 15, 1997
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Table 2. Effect of the Electrode Length on the Peak Current of Hydroquinone (0.1 µM) and Peak-to-Peak Noise (60 s)a
Figure 3. Electropherogram of hydroquinone (1.0 × 10-7 M): separation voltage, 20 kV; injection, 20 kV for 1 s; column, 50 µm i.d., 60 cm total length; detection potential, 0.8 V vs SCE; buffer, 10 mM NaH2PO4-10 mM Na2HPO4 at pH 6.8.
electrode length (µm)
peak current (pA)
noise (pA)
300 350 400 450 500
5.1 6.1 7.0 7.8 8.7
0.35 0.40 0.45 0.55 0.65
a Column, 50 µm i.d., 60 cm total length; buffer,10 mM NaH PO 2 4 10 mM Na2HPO4 at pH 6.8; separation voltage, 20 kV; electrode potential, 0.8 V vs SCE; electrode insertion depth, 200 µm.
Table 1. Effect of the Electrode Insertion Depth on the Peak Current of Hydroquinone (1.0 µM) and Peak-to-Peak Noise (60 s)a insertion depth (µm)
peak current (pA)
noise (pA)
50 100 150 200 250 300
58 60 73 81 92 105
0.50 0.60 0.60 0.65 1.10 1.75
a Column, 50 µm i.d., 60 cm total length; buffer, 10 mM NaH PO 2 4 10 mM Na2HPO4 at pH 6.8; separation voltage, 20 kV; electrode potential, 0.8 V vs SCE; electrode length, 500 µm.
The linear range of the calibration curve was from 1.0 × 10-7 to 1.0 × 10-4 M with a correlation coefficient of 0.996 (n ) 8). The detection limit (S/N ) 3, peak-to-peak noise) was 2.0 × 10-8 M and corresponds to 72.0 amol with an injection volume of 3.6 nL. After the working electrode was carefully aligned with the capillary outlet and fixed firmly in position, reproducible results were obtained. Relative standard deviations for the migration time and the peak current of HQ were 1.0% and 2.1% (n ) 8) respectively. Accurate location of the working electrode relative to the detection end of the capillary is important to obtain sensitive and reproducible results in CE with amperometric detection.4,6 In our study, electrode position also affects detection dramatically. Table 1 lists the peak current of HQ (1.0 µM) and the peak-to-peak noise vs different insertion depths of the microelectrode. As can be seen, the sensitivity increases as the insertion depth into the detection capillary increases. However, the noise also increases as the insertion depth increases. This is much more obvious when the depth is over 200 µm. To get the maximum S/N, an insertion depth of 200 µm was chosen for all detections. Furthermore, the sensitivity decreases as the microelectrode is moved from the center point. The analyte in the zone is not uniformly distributed because the friction of the zone against flow at the capillary surface. The analyte concentration drops off rapidly at the capillary wall, and the relative response at the capillary wall is 5% of that at the center point. So the working electrode is placed at the center of the detection capillary with micromanipulator and this increases the sensitivity. Electrode length also affects the sensitivity and noise of the detector. An electrode length of g300 µm should be necessary for obtaining a long enough electrode insertion depth. Both the sensitivity and noise increase as the 266 Analytical Chemistry, Vol. 69, No. 2, January 15, 1997
Figure 4. Electropherogram of (1) norepinephrine (10 µM), (2) isoproterenol (10 µM), and (3) catechol (10 µM): injection, 1 s at 10 kV; other conditions as in Figure 3.
Figure 5. Electropherogram of (1) p-diphenol (5 µM), (2) m-diphenol (10 µM), (3) o-diphenol (10 µM), and (4) phenol (20 µM): buffer, 15 mM NaH2PO4-5 mM Na2B4O7 with 10 mM SDS (pH 6.8); other conditions as in Figure 4.
electrode length increases. Table 2 lists the peak current of HQ (0.1 µM) and the peak-to-peak noise vs different electrode lengths. It can be seen that the optimal electrode length is 400 µm. Otherwise, when optimal electrode position and length were chosen, the noise of the detector was steady if the same buffer and applied voltage were used. The relative standard deviation of the noise for six capillaries with joints was 6.8% (all conditions as in Figure 3), indicating that the construction of the joints was
reproducible. Figure 4 shows an electropherogram of equimolar (10 µM) NE, IP, and catechol (CAT) in phosphate buffer (pH 6.8). Detection limits (S/N ) 3, peak-to-peak noise) of 2.2 × 10-8 (45 amol), 8.5 × 10-8 (180 amol), and 2.3 × 10-8 M (32 amol) were obtained, respectively. The corresponding plate numbers were about 140 000, 170 000, and 220 000, respectively, demonstrating the high performance of this system. This system was also applied for separation and detection of isomeric diphenol and phenol (Figure 5). Complete separation was obtained in a phosphate-borate buffer containing 10 mM SDS (pH 6.8). Compared with the results in phosphate buffer containing SDS,11 the tailing of CAT is reduced significantly. Pronounced tailing results from the interactions between the analytes and the capillary wall. Nonionic CAT is transformed into anion due to the complexation of boric acid with CAT,12 and then the interactions between CAT and the capillary wall are limited by charge repulsion. Hence, tailing is inhibited. Furthermore, much better separation efficiencies of 390 000, 430 000, 580 000, and 470 000 (11) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988, 60, 258-263. (12) Wallingford, R. A.; Ewing, A. G. J. Chromatogr. 1988, 441, 299-309.
theoretical plates for p-diphenol, o-diphenol, m-diphenol, and phenol were obtained, respectively. The band broadening for offcolumn amperometric detection results from the laminar flow and the back pressure generated in the detection capillary.7 If the porous joints that contain an on-column fracture are used, the back pressure may force the analyte ions to diffuse through the fracture. It will cause zone broadening and sample leakage. In comparison with them, the etched joint does not create a dead volume and therefore provides better efficiency and minimum sample loss. ACKNOWLEDGMENT This work was supported by the Fund of the National Natural Science Foundation of China.
Received for review April 3, 1996. Accepted September 30, 1996.X AC960331N X
Abstract published in Advance ACS Abstracts, November 15, 1996.
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