Anal. Chem. 2004, 76, 3846-3850
Capillary Electrophoresis Coupled with Electrochemiluminescence Detection Using Porous Etched Joint Xue-Bo Yin, Haibo Qiu, Xiuhua Sun, Jilin Yan, Jifeng Liu, and Erkang Wang*
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, 130022, China
A new setup to couple capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection is described in which the electrical connection of CE is achieved through a porous section at a distance of 7 mm from the CE capillary outlet. Because the porous capillary wall allowed the CE current to pass through and there was no electric field gradient beyond that section, the influence of CE high-voltage field on the ECL procedure was eliminated. The porous section formed by etching the capillary with hydrofluoric acid after only one side of the circumference of 2-3 mm of polyimide coating of the capillary was removed, while keeping the polyimide coating on the other part to protect the capillary from HF etching makes the capillary joint much more robust since only a part of the circumference of it is etched. A standard three-electrode configuration was used in experiments with Pt wire as a counter electrode, Ag/AgCl as a reference electrode, and a 300-µm diameter Pt disk as a working electrode. Compared with CE-ECL conventional decoupler designs, the present setup with a porous joint has no added dead volume created. Moreover, the dead volume can be increasingly decreased by shortening the distance (∼100 µm) between the working electrode and the end of the separation capillary. The versatility in choice of capillaries and separation buffers within this design is the main advantage over the use of small i.d. capillary and low conductivity buffer in some CE-ECL systems. The performance of this setup is illustrated by the analyses of tripropylamine and proline. Electrochemiluminescence (ECL) allows the detection of analytes at low concentrations over a wide linear range partly due to the electrochemical excitation during ECL reaction1 and no external excitation light source, which gives an extremely low background signal.2-6 As a highly sensitive and selective detection * Corresponding author. Fax: (86)431 5689711. E-mail: ekwang@ ns.ciac.jl.cn. (1) Fa¨hnrich, K. A.; Pravda, M.; Guibault, G. G. Talanta 2001, 54, 531-559. (2) Knight, A. W.; Greenway G. M. Analyst 1995, 120, 2543-2747. (3) Lee, W. Y. Mikrochim. Acta 1997, 127, 19-39. (4) Egashira, N.; Kumasako, H.; Kurauchi, Y.; Ohga, K. Anal. Sci. 1994, 10, 405-408. (5) Martin, A. F.; Nieman, T. A. Anal. Chim. Acta 1993, 281, 475-481. (6) O’Connell, C. D.; Juhasz, A.; Kuo, C.; Reeder, D. J.; Hoon, D. S. B. Clin. Chem. 1998, 44, 1161-1169.
3846 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004
method, electrochemluminescent detection has been used in flow injection (FI) and high-performance liquid chromatography (HPLC) to detect pyryvate,2 amino acids,3 oxalate,4 NADH,5 and nucleic acids.6 Capillary electrophoresis (CE) has been developed as an efficient separation technique since its birth.7 The characters of high efficiency, powerful resolutions, short analysis time, and low instrumental cost of CE make it an alternative to HPLC.8,9 However, the dimensions of CE capillary have a considerable influence on its achievable sensitivity, and thus, more sensitive detection techniques are needed in CE than that used in HPLC.9 Although laser induced fluorescence (LIF) and mass spectrometry (MS) can offer suitable sensitivity for CE, the high instrument price limits their extensive applications. Moreover, because most of the analytes have no natural fluorescence, the derivation procedures before LIF detection make the analysis complex undoubtedly. ECL with a high sensitivity and a small consumption of reagents is a suitable detector for CE,10 and some technique for coupling CE with ECL has been investigated recently.10-21 Unlike the HPLC coupling with ECL detection, the electric currents in electrophoresis capillary under the high electric field can greatly affect the ECL procedure on the microelectrodes.10-18 Therefore, most of the CE-ECL papers reported are focused on eliminating that influence. No significant effect from the high electric field on ECL detection was observed if a capillary with a (7) Ewing, A. G.; Wallingford, R. A.; Olefurowicz, T. M. Anal. Chem. 1989, 61, 292A-303A. (8) Jorgensen, J. W.; Lukacs, K. D. Science 1983, 222, 266-272. (9) Swinney, K.; Bornhop, D. J. Electrophoresis 2000, 21, 1239-1250. (10) Knight, A. W. Trend Anal. Chem. 1999, 18, 47-62. (11) Cao, W.; Liu, J.; Yang, X.; Wang, E. Electrophoresis 2002, 23, 3683-3691. (12) Dickson, J. A.; Ferris, M. M.; Milofsky, R. E. J. High Resolut. Chromatogr. 1997, 20, 643-646. (13) Wang, X.; Bobbit, D. R. Anal. Chim. Acta 1999, 383, 213-219. (14) Hendrickson, H. P.; Anderson, P.; Wang, X.; Pittman, Z.; Bobbitt, D. R. Microchem. J. 2000, 65, 189-195. (15) Forbes, G. A.; Nieman, T. A.; Sweedler, J. V. Anal. Chim. Acta 1997, 347, 289-293 (16) Bobbit, D. R.; Jackson, W. A.; Hendrickson, H. P. Talanta 1998, 46, 565572. (17) Wang, X.; Bobbit, D. R. Talanta 2000, 53, 337-345. (18) Huang, X.; Zare, R. N.; Sloss, S. Ewing, A. G. Anal. Chem. 1991, 63, 189192. (19) Chiang, M.-T.; Lu, M.-C.; Whang, C.-W. Electrophoresis 2003, 24, 30333039. (20) Chiang, M.-T.; Whang, C.-W. J. Chromatogr. A 2001, 934, 59-66. (21) Cao, W.; Liu, J.; Qiu, H.; Yang, X.; Wang, E. Electroanalysis 2002, 14, 15711576. 10.1021/ac049743j CCC: $27.50
© 2004 American Chemical Society Published on Web 05/04/2004
small inner diameter (e25 µm) or low conductivity buffer was used for CE.18-21 Chiang et al.19,20 reported some CE-ECL systems with small ID capillaries and a low conductivity solution as a CE buffer without a decoupler. A CE system using a 25 µm i.d. capillary as a separation column to couple with an ECL detector without a decoupler for detecting tramadol and lidocaine in urine was achieved in our group.21 In previous work,11 we have investigated the influences of different separation factors without a decoupler on the CEL procedure. Although the influence of the CE high-voltage field on the ECL detection was decreased effectively by using a low-conductivity buffer and a small inner diameter capillary, it will limit the application of the CE-ECL system.11,18-21 The electric field decoupler to isolate ECL detections from the CE high voltage field has been used;12-17 however, the procedure of electrode alignment and decoupler fabrication is often tedious.22 Wang et al.13 used a Nafion tube which covered the fracture of the capillary as a decoupler. But the larger inner diameter of the Nafion tube may result in the diffusion of analytes and thus zone broadening. Etching the outside wall of the separation capillary was also used to separate the electrochemical or ECL current from the electrophoretic current.12,23 These works 12,23 need two cells to perform the experiments, one to support the fragile capillary and to isolate the CE high voltage, another to be used as the detection cell, which makes the fabrication process tedious and complex. Because of the disadvantages of such designs, a 2 cm23 or 5 cm12 detection capillary was used, hence resulting in a broadening peak and decreased separation efficiency. To protect the etched section of the capillary, the capillary must be fixed on a plate. While fixing the capillary made the fabrication of the joint system and the replacement of the capillary and the working electrode difficult, it was also troublesome to align the capillary and the working electrode.12,23 In this work, a simple and effective method to eliminate the influence of CE high-voltage field on the ECL detection is described. Similar to the previous work12,23,24 the joint was fabricated by etching the capillary wall with hydrofluoric acid after the polyimide coating was removed. But to enhance the robustness of the etched capillary, half of the circumference of polyimide in a 2-3 mm section was removed. Thus, only part of the circumference of the capillary wall was etched, and thus, it makes the capillary robust enough to fabricate the whole system. The etched joint has been used successfully to couple CE with ECL detection. The investigation of the influences of CE separation voltage, buffer concentration, and capillary inner diameter on ECL procedure indicated that the porous joint might eliminate the influence of CE electric field on the ECL detection significantly, and no added dead volume was created. EXPERIMENTAL SECTION Reagents. All the reagents employed were at least of analytical grade, and double distilled water (DDW) was used throughout. Tripropylamine (TPA) and tris(2,2′-bipyridine)ruthenium(II) chloride pentahydrate (Ru(bpy)3Cl2‚5H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Ru(bpy)32+ (10 mM) was dissolved (22) Park, S.; Lunte, S. M.; Lunte, C. E. Anal. Chem. 1995, 67, 911-918. (23) Hu, S.; Wang, Z. L.; Li, P. B.; Cheng, J. K. Anal. Chem. 1997, 69, 264267. (24) Wei, W.; Yeung, E. S. Anal. Chem. 2002, 74, 3899-3905.
Figure 1. The schematic diagram of the ECL detection cell (not to scale): (a) front view and (b) side view from Ru(bpy)32+ solution reservoir. (1) Working electrode; (2) PMT; (3) Ru(bpy)32+ solution reservoir; (4) CE ground electrode; (5) porous section of capillary; (6) separation capillary; (7) for reference electrode; (8) for counter electrode; (9) working electrode alignment screws; (10) sealon film.
in DDW as a stock solution and stored in a refrigerator. The working solution was prepared daily by diluting the stock solutions with 0.1 M phosphate salt solution (pH 7.5) just before use. TPA and L-proline (Pro, Shanghai Biochemical Co., Shanghai, China) were dissolved in DDW directly at a concentration of 1 and 100 mM, respectively, as stock solution. Sodium dihydrogen phosphate (Beijing Chemicals Co., Beijing, China) was used to prepare the electrolyte buffer solution. The pH of the buffer solution was adjusted with 0.1 mol L-1 NaOH (Beijing Chemicals Co.). Different concentrations of phosphate buffer solution for pH 8.0 were used to investigate the effect of the electrophoretic current on ECL. A 10 mM phosphate salt solution with pH 8.0 was used as the running buffer to separate TPA and proline. Instrumentation. A homemade CE setup with an ECL detector was used in this work. The high-voltage power supply was from the Shanghai Nucleus Institute (Shanghai, China). The power supply was operated in a voltage-controlled mode. Uncoated fused-silica capillaries (Yongnian Optical Fiber Co. Ltd., Hebei, China) with different inner diameters were cut to 40 cm in length and used to examine the effect of capillaries on ECL. Samples were injected in an electrokinetic mode at 10 kV for 5 s. The electrochemical measurement for ECL experiments was carried out with Model CH800 Voltammetric Analyzer (CH Instruments, Austin, TX). A three-electrode system was employed with Pt wire as a counter electrode, Ag/AgCl as a reference electrode, and 300-µm diameter Pt disk as a working electrode. The ECL emission was detected with a Model MCDR-A Chemiluminescence Analyzer Systems (Xi’An Remax Science & Technology Co. Ltd., Xi’An, China). The voltage of photomultiplier tube (PMT) used in Model MCDR-A Chemiluminescence Analyzer was set at 850 V in the process of detection. The CE-ECL setup used in this work is shown in Figure 1. The reference electrode and the counter electrode were inserted into the Ru(bpy)32+ solution reservoir 3 from both sides of the reservoir and were above both the capillary 6 and the working electrode 1 to avoid affecting the detection of ECL. The working Analytical Chemistry, Vol. 76, No. 13, July 1, 2004
3847
electrode 1 was adjusted and fixed by three screws 9 from three different directions to align with the separation capillary 6. The capillary polyimide coating in half of the circumference of some section (2-3 mm) was scraped 7 mm from the detection end of the capillary by a scalpel. The 7 mm of distance between the etched section and the end of the capillary is enough for separation of the ECL detection from the CE high-voltage field. Moreover, it does not reduce the effect length of the separation capillary significantly. The exposed section was immersed in 42% hydrofluoric acid for etching. (CAUTION: HF should only be handled with gloves under a ventilated hood.) Hydrofluoric acid etched only the polyimide-free region of the capillary. The etching procedure was monitored by measuring the current of the capillary full with 10 mM phosphate buffer (pH 8.0) periodically. As shown in Figure 1, after etching, the capillary was encircled with sealon film 1 mm in breadth (Fuji Photo Film Co. Ltd., Japan) between the etched section and the end of the capillary to isolate the Ru(bpy)32+ solution reservoir and CE ground cell and to align the separation capillary. The capillary was inserted through and held in place with ferrule fittings. The three screws around the working electrode accomplish alignment between the working electrode and the end of the capillary. The whole ECL detection system was held in a light-tight chamber, not given in Figure 1. Unlike the previous work,11 the distance between the end of the capillary and the working electrode can be about 100 µm in this work. The Ru(bpy)32+ solution reservoir and the CE ground cell were filled with phosphate buffer containing Ru(bpy)32+ and CE separation buffer, respectively. The volume of the Ru(bpy)32+ solution reservoir is about 300 µL. CE ground was achieved through a porous etched joint, a buffer in the CE ground cell, and a Pt electrode, which was inserted into the CE ground cell directly. RESULT AND DISCUSSION Construction of the Ground Porous Joint. The HF etching porous joint has been used in CE with electrochemical detection (CE-EC)23 and CE-ECL12 for isolating the electrochemical detector from the CE electric field, on-line concentration of proteins and peptides in CE,24 and the interface of CE-MS.25,26 Because the HF etching method is simple and effective to CE ground, and no added dead volume is introduced, we use it in the present system. We also remove all of the polyimide coating of the section of the capillary for etching as in the literature,12,23,24 but it is easy to break the capillary from the etched section. So, we only remove the polyimide coating in half of the circumference of some sections. With this approach, while the robustness of the capillary was significantly enhanced, the CE electric connection was realized. Hu et al.23 fixed the etched section on a Plexiglas slide and immersed it in the buffer to prevent breaking the etched thinwalled capillary. With the part of some section of the capillary etched in this work, the capillary is robust enough to align between the capillary and the working electrode and can last at least 2 months. Investigation of the etching time indicated that for a 75-µm i.d. capillary, an etching time lower than 120 min was insufficient (no current) and 140 min was too long (leakage from the etched section). The optimal etched time for a 75-µm i.d. capillary was (25) Janini, G. M.; Zhou, M.; Yu, L. R.; Blonder, J.; Gignac, M.; Conrads, T. P.; Issaq, H. J.; Veenstra, T. D. Anal. Chem. 2003, 75, 5984-5993. (26) Whitt, J. T.; Moini, M. Anal. Chem. 2003, 75, 2188-2191.
3848 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004
Figure 2. Cyclic voltammograms of Ru(bpy)32+ with different CE separation voltage. Capillary: 75 µm × 40 cm; separation buffer: 10 mM phosphate salt, pH 8.0; 100 µm of distance between the working electrode and the end of separation capillary; 5 mM Ru(bpy)32+. (a) Without the influence of HV field; (b) electrophoretic voltage, 8 kV; (c) 12 kV; (d) 15 kV; (e) 20 kV. Other conditions as shown in Figure 1.
120-130 min. Because the 7 mm of distance between the etched joint and the end of the capillary is short enough, no substantial difference in the current measurement was observed between the joint ground and the end ground when the same applied voltage and buffer were used. Hu et al.23 observed that when the wall of an exposed section is thinner than 20 µm, there is current in the separation capillary. Wei and Yeung24 presumed that the porous size of the porous section is so small that only certain small buffer ions can pass through it to carry the current. Janini et al.25 gave direct evidence by the surface scanning and the cross-sectional scanning electron images of 75-µm i.d. capillary. It reveals micrometer-sized pores on the outer surface of the etched segment but no observable changes across the wall thickness, indicating that the pores across the wall would have diameters in the low-nanometer range.25 Effect of High Voltage on ECL. The effect of high voltage on ECL was examined with a 40-cm long × 75-µm i.d. capillary, 10 mM phosphate buffer (pH 8.0), and 100 µm of distance between the end of the capillary and the working electrode. A convenient method to measure the extent of this effect is to record the cyclic voltammograms (CVs) in different high voltages. To observe the effect of different high voltages on a detector, the capillary was filled with 10 mM phosphate buffer (pH 8.0) to obtain different electrophoretic currents. Figure 2 shows the cyclic voltammograms (CVs) between 0.5 and 1.3 V (vs Ag/AgCl) at a scan rate of 50 mV s-1 in different high voltages. As illustrated in Figure 2, clearly, the presence of different high voltages did not result in the shift of the redox potential of Ru(bpy)32+. We also can find that the oxidizing current of Ru(bpy)32+ in CVs was decreased along with the increase in the separation voltage. Compared to the effect of the CE current leakage on the ECL procedure in the previous work,11 the dilution to the Ru(bpy)32+ solution near the working electrode surface by electrophoretic capillary effluent may be the main reason for the decrease of current in CV curves with an increasing high voltage due to the small distance (∼100 µm) between the working electrode and the end of the capillary. Influence of Buffer Concentration on ECL. The influence of buffer concentration on ECL was investigated by recording the CVs and the electropherograms of TPA and Pro with a 40-cm long
Figure 3. Electropherograms of 0.01 µM TPA and 0.1 mM Pro with different buffer concentrations (pH 8.0): (a) 5 mM; (b) 10 mM; (c) 15 mM; (d) 20 mM. Other conditions as shown in Figures 1 and 2.
× 75-µm i.d. capillary and 12 kV of separation voltage. Phosphate buffers (5, 10, 15, 20 mM, pH 8.0), corresponding to ∼25, 55, 90, 120 µA of separation current, were used to investigate the effect of different buffer concentrations on ECL. First, cyclic voltammograms (CVs) between 0.5 and 1.3 V (vs Ag/AgCl) at a scan rate of 50 mV s-1 in different buffer concentrations were recorded to examine the influence on ECL. Similar to the results of investigating the effect of high voltage, no shift of redox potential of Ru(bpy)32+ was found. Only small changes of the oxidizing current of Ru(bpy)32+ with a different buffer concentration were observed maybe due to the dilution of Ru(bpy)32+ by capillary effluent. The influence of different buffer concentrations on separation of TPA and Pro is shown in Figure 3. The use of a more concentrated phosphate buffer results in an increase in the migration time and the resolution between the two analytes as shown in Figure 3. Altria and Simpson27 observed that mobility is inversely proportional to the concentration of the buffer solution. High ionic strength decreases the electroosmotic flow (EOF), thereby increasing the migration time of analytes in the capillary.28 The results observed are consistent with some other work.27-29 Effect of the Distance between the Capillary and the Working Electrode on ECL. An accurate location of the working electrodes relative to the end of the separation capillary is important to obtain sensitive and reproducible results in CE with electrochemical detection, so the distance from the end of capillary to the working electrode plays an important role in amperometric detection.23,30 Similarly, the close distance would increase the influence of high voltage on the detection without a decoupler in the CE-ECL system.11 Therefore, the distance is also an important factor for achieving high sensitivity and efficiency of ECL detection. (27) Altria, K. D.; Simpson, C. F. Chromatographia 1987, 24, 527-532. (28) Tu, Q.; Qvarnstro ¨m, J.; Frech, W. Analyst 2000, 125, 705-710. (29) Tian, X. D.; Zhuang, Z. X.; Chen, B.; Wang X. R. Analyst 1998, 123, 899903. (30) Matysik, F. M. Anal. Chem. 2000, 72, 2581-2586.
Figure 4. Electropherograms of 0.01 µM TPA and 0.1 mM Pro with the distances between capillary end and working electrode: (a) 240 µm and (b) 100 µm. Inset, baseline before the peak. Other conditions as shown in Figures 1 and 2.
A 10 mM phosphate buffer (pH 8.0), a 40-cm long × 75-µm i.d. capillary, and 12 kV of separation voltage were used to investigate the influence of distance between the end of the capillary and the working electrode. From 80 to 250 µm, the change of capillary to electrode distance did not bring a significant shift in the redox potential of Ru(bpy)32+ on CV. Contrary to the previous work,11 the disappearance of the oxidizing current of Ru(bpy)32+ was not observed with the present porous joint design when the distance was smaller than 180 µm. The influence on the peak intensity and the peak sharp was observed with a different distance between the capillary and the working electrode. For distances smaller than 100 µm, the reproducibility of TPA and Pro signals is poor. It can be explained that too small a distance results in a great decrease of concentration and a disturbance to the Ru(bpy)32+ solution by the CE capillary effluent. With a longer distance (g150 µm), as shown in Figure 4, both peak height and resolution between TPA and Pro decrease with the increasing distance between the end of the capillary and the working electrode. The dispersion of analyte would be dominant leading to the decrease of signal intensity. The distance between the end of the capillary and the working electrode ranging from 100 to 150 µm was found to maintain the maximum signal with good precision for the 75-µm i.d. capillary. Influence of Capillaries with a Different Inner Diameter on ECL. The capillary inner diameter plays an important role in CE separation. While the narrow capillaries are efficient in dissipating Joule’s heating generated inside the capillary by the CE process, the broad capillaries can increase the sample loading and diminish the adsorption of solute molecules by the capillary surface because of the low surface-to-volume ratio of the column.8 In the CE-ECL technique,11 the high separation current due to broad capillaries may affect the ECL procedure and most of the CE-ECL reported focused on the elimination of this kind of Analytical Chemistry, Vol. 76, No. 13, July 1, 2004
3849
Figure 5. Electropherograms of 0.01 µM TPA and 0.1 mM Pro with different capillary inner diameters: (a) 50 µm; (b) 75 µm; (c) 100 µm. Other conditions as shown in Figures 1 and 2.
influence by using a small i.d. capillary.18-22 The effect of capillary inner diameters on ECL was examined using capillaries with 50, 75, and 100 µm i.d. and 40 cm length, 10 mM phosphate buffer (pH 8.0), and 100 µm of distance between the end of the capillary and the working electrode in this work. As shown in Figure 5, with the same injection time, the CE-ECL sensitivity increases with an increasing capillary i.d. since the broader i.d. capillaries can be injected more with a sample volume. The disadvantage of employing broad-diameter capillaries in our experience is that there is more turbulance on the Ru(bpy)32+ concentration near the detection region due to high EOF, that depresses the improvement of sensitivity partly. Analytical Performance of CE-ECL and Its Application. With the present design, the capillaries with different inner diameters and the buffers with different ion strength can be used, so an ideal CE-ECL detector was assembled, with which not only the intrinsic sensitivity of ECL was achieved but also the high performance of CE was maintained. The elimination of CE high voltage on the ECL procedure facilitated the optimization of the CE separation process independent of the detection. The inset of Figure 4b shows the noise after the elimination of the effect of high voltage on the ECL procedure. Different than the previous work,12,23,24 where the capillary must be rigidly fixed to a plate which serves to reinforce the capillary at the position because the removal of the polyimide coating in all of the circumference and the etching to the capillary makes the capillary fragile, the capillary in the present system is not fixed
3850
Analytical Chemistry, Vol. 76, No. 13, July 1, 2004
completely because it is strong enough to carry out all operations to fabricate the whole system. Retaining the coating on one side of the circumference of the capillary provided better support for the etched capillary wall compared to the procedure of completely stripping the polyimide coating.12,23-26 As a whole, the integration of CE ground and the ECL cell as well as the robustness of the etched capillary makes the fabrication of the present system much simpler in comparison with the previous systems.12,23 Moreover, because no glue is used in this work to fix, the replacement of the capillary and the electrodes is easier. The capillary with the porous joint in the present work has been used for a period of at least 2 months in the laboratory. Compared with porous joints containing an on-column fracture,13,15 where the analytes may diffuse through the fracture, and thus zone broadening and sample leakage are induced, the present etched joint does not create any dead volume and therefore provides better efficiency and minimum sample loss. The analytical characteristic data of the present CE-ECL system for the determination of TAP and Pro with 10 mM phosphate buffer (pH 8.0), a 75-µm i.d. capillary, and 12 kV of separation voltage are summarized as follows: the precisions (RSD) of the migration time for five replicate injections of 0.01 µM TPA and 0.1 mM Pro were 2.5 and 3.2%, and those of the peak height were 2.3 and 6.1%; the detection limits (signal-to-noise ratio, S/N ) 3) based on the peak height measurement of the two analytes were 2 × 10-10 and 1 × 10-7 mol L-1, respectively. CONCLUSIONS Coupling CE with ECL using porous joints was produced by HF etching in this work. The investigations of high voltage, buffer ion strength, and capillary inner diameter have demonstrated the feasibility of elimination of the effect of the CE current on the ECL procedure with porous joints. Compared with CE-ECL using a decoupler, the present system did not induce any dead volume and shorten the effective length of separation capillary. The more choice in capillary inner diameters and buffer ion strengths is the main advantage over the employing small inner diameter capillary or low ion strength buffer to control the influence of the CE current on ECL. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China with Grant Nos. 20299030, 39990570, and 20335040 and the National Key Basic Research Program 2002CB513110. X.B.Y. acknowledges the support of the China Postdoctoral Science Foundation for this project. Received for review February 14, 2004. Accepted February 25, 2004 AC049743J
