Anal. Chem. 2003, 75, 3637-3642
Technical Notes
Miniaturized Tris(2,2′-bipyridyl)ruthenium(II) Electrochemiluminescence Detection Cell for Capillary Electrophoresis and Flow Injection Analysis Jifeng Liu, Jilin Yan, Xiurong Yang,* and Erkang Wang*
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
The design and performance of a miniaturized chip-type tris(2,2′-bipyridyl)ruthenium(II) [Ru(bpy)32+] electrochemiluminescence (ECL) detection cell suitable for both capillary electrophoresis (CE) and flow injection (FI) analysis are described. The cell was fabricated from two pieces of glass (20 × 15 × 1.7 mm), and the 0.5-mmdiameter platinum disk was used as working electrode held at +1.15 V (vs silver wire quasi-reference), the stainless steel guide tubing as counter electrode, and the silver wire as quasi-reference electrode. The performance traits of the cell in both CE and FI modes were evaluated using tripropylamine, proline, and oxalate and compared favorably to those reported for CE and FI detection cells. The advantages of versatility, sensitivity, and accuracy make the device attractive for the routine analysis of amine-containing species or oxalate by CE and FI with Ru(bpy)32+ ECL detection. Since a series of mechanisms for the electrochemiluminesecnce (ECL) observed upon reaction of tris(2,2′-bipyridyl)ruthenium(III) [Ru(bpy)33+] with reductants [e.g., Ru(bpy)3+, amines, or oxalate] were reported,1-5 Ru(bpy)32+ ECL detection technique has gained enormous analytical utility. Most of the analytical applications of Ru(bpy)32+ ECL are found in flowing streams such as flow injection (FI) and high-performance liquid chromatography (HPLC) to detect alkylamine, amino acids, peptides, and rutheniumlabeled proteins and nucleic acid probe assays, and its analytical importance has been intensively reviewed recently.6-9 For FI * Corresponding author. Tel: +86 431 5262003. Fax: +86 431 5689711. E-mail:
[email protected]. (1) Tokel, N. E.; Bard, A. J. J. Am. Chem. Soc. 1972, 94, 2862-2863. (2) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512-516. (3) Rubinstein, I.; Martin, C. R.; Bard, A. J. Anal. Chem. 1983, 55, 15801582. (4) Noffsinger, J. B.; Danielson, N. D. Anal. Chem. 1987, 59, 865-868. (5) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 37, 3127-3131. (6) Lee, W.-Y. Mikrochim. Acta 1997, 127, 19-39. (7) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1-41. (8) Fa¨hnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-559. (9) Knight, A. W. Trends Anal. Chem. 1999, 18, 47-62. 10.1021/ac034021y CCC: $25.00 Published on Web 05/17/2003
© 2003 American Chemical Society
detection, the instrumentation usually is assembled from the conventional thin-layer amperometric detection cell with working electrode placed against a transparent Plexiglas window for detection of chemiluminescence (CL) emission.6,10-14 This conventional ECL flow cell is also avaliable for HPLC with ECL detection after being coupled directly to the HPLC column output in a Ru(bpy)32+ postcolumn addition or mobile-phase addition method.12,15-17 On the working electrode surface, in situ ECL reaction occurs between Ru(bpy)33+ and the analyte to emit CL emission. The volumes of the thin-layer cell are usually 100,11 25,10 15,12 12,17 9.2,18 and 1.5 µL.19 When transparent conductive glass electrodes are used, this flow cell configuration can be simplified, for the working electrode can be used as the observation window directly.20,21 Development of a miniaturized ECL flow cell has aroused the researcher’s interest, because a microcell can be mounted close to a photomultiplier tube (PMT), or bonded to a more elegant silicon photodiode, thus becoming an intrinsic component of the device itself.22-24 The electrodes used in such miniaturized flow cells are thin metal plates,22-24 metal coatings on inert substrates,25 or metal interdigitated arrays.26,27 Though the overall size of the (10) Shultz, L. L.; Stoyanoff, J. S.; Nieman, T. A. Anal. Chem. 1996, 68, 349354. (11) Lee, W.-Y.; Nieman, T. A. Anal. Chem. 1995, 67, 1789-1796. (12) Jackson, W. A.; Bobbitt, D. R. Anal. Chim. Acta 1994, 285, 309-320. (13) Hogan, C. F.; Forster, R. J. Anal. Chim. Acta 1999, 396, 13-21. (14) Forster, R. J.; Hogan, C. F. Anal. Chem. 2000, 72, 5576-5582. (15) Lee, W.-Y.; Nieman, T. A. J. Chromatogr., A 1994, 659, 111-118. (16) Skotty, D. R.; Nieman, T. A. J. Chromatogr., B 1995, 665, 27-36. (17) Collinson, M. M.; Wightman, R. M. Anal. Chem. 1993, 65, 2576-2582. (18) Skotty, D. R.; Lee, W.-Y.; Nieman, T. A. Anal. Chem. 1996, 68, 15301535. (19) Chen, X.; Sato, M. Anal. Sci. 1995, 11, 749-754. (20) Wilson, R.; Akhavan-Tafti, H.; DeSilva, R.; Schaap, A. P. Anal. Chem. 2001, 73, 763-767. (21) Wilson, R.; Akhavan-Tafti, H.; DeSilva, R.; Schaap, A. P. Electroananlysis 2001, 13, 1083-1092. (22) Arora, A.; de Mello, A. J.; Manz, A. Anal. Commun. 1997, 34, 393-395. (23) Greenway, G. M.; Knight, P. J. Anal. Proc. 1995, 32, 251-253. (24) Knight, A. W.; Greenway, G. M. Analyst 1995, 20, 2543-2547. (25) Hsueh, Y.-T.; Collins, S. D.; Smith, R. L. Sens. Actuators, B 1998, 49, 1-4. (26) Fiaccabrino, G. C.; de Rooij, N. F.; Koudelka-Hep, M. Anal. Chim. Acta 1998, 359, 263-267.
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cell is small, the volume of the cell remains as large as 3 22 and 150 µL.27 The Ru(bpy)32+ ECL technique has recently been coupled with CE (CE-ECL),28-38 but this technique is not employed widely for routine CE analysis. Unlike the HPLC technique, where the injected sample volume (microliter level) is compatible with that of the ECL flow cell, the use of CE should require a new design of detection cell to maintain the separation efficiency.9 In the previously reported CE-ECL systems, a microelectrode was inserted into the capillary tip28 or positioned at the detection capillary outlet, thus creating an interface between the capillary end and the working electrode for in situ ECL detection.29 To avoid adsorption of cationic Ru(bpy)32+ onto the silica wall of the separation capillary, addition of Ru(bpy)32+ postcolumn is preferred. This can be done in two approaches: Ru(bpy)32+ solution is added into a small reservoir at the separation capillary outlet;29-32,37,38 Ru(bpy)32+ is continuously delivered by using a syringe pump to the capillary-electrode interface located in an in situ ECL cell.33-35 The main problems associated with these two approaches are the negative effects of variability of Ru(bpy)32+ concentration caused by reservoir evaporation and dilution of CE effluent on the sensitivity and reproducibility of the CL signal and instrumental complications. A new type of chip CE-ECL technique has been reported;36 however, further work should be done to improve the detector sensitivity and performance for the purpose of analytical application. In our laboratory, we had constructed an end-column ECL cell used for CE37,38 and a flow cell used for FI.39,40 Now we are developing an instrument with ECL detection in flow systems for laboratory research purposes. The goal of this work is to construct a detection cell as an affiliated part of the instrument that could be used for both CE and FI to facilitate our ongoing work in developing an ECL assay for clinical and biological applications. In this technical note, we describe the design and characteristics of this new type of ECL detection cell. Results of analysis of model analytes such as tripropylamine (TPA), proline, and oxalate are presented and demonstrate the favorable analytical characteristic in both CE and FI modes. EXPERIMENTAL SECTION Reagents. All reagents and chemicals used were at least analytical reagent grade. Tris(2,2′-bipyridyl)ruthenium(II) chloride (27) Michel, P. E.; van der Wal, P. D.; Fiaccabrino, G. C.; de Rooij, N. F.; Koudelka-Hep, M. Electroanalysis 1999, 11, 1361-1367. (28) Forbes, G. A.; Nieman, T. A.; Sweedler, J. V. Anal. Chim. Acta 1997, 347, 289-293. (29) Dickson, J. A.; Ferris, M. M.; Milofsky, R. E. J. High Resolut. Chromatogr. 1997, 20, 643-646. (30) Chiang, M.-T.; Whang, C.-W. J. Chromatogr., A 2001, 934, 59-66. (31) Hendrickson, H. P.; Anderson, P.; Wang, X.; Pittman, Z.; Bobbitt, D. R. Microchem. J. 2000, 65, 189-195. (32) Bobbitt, D. R.; Jackson, W. A.; Hendrickson, H. P. Talanta 1998, 46, 565572. (33) Wang, X.; Bobbitt, D. R. Anal. Chim. Acta 1999, 383, 213-220. (34) Wang, X.; Bobbitt, D. R. Talanta 2000, 53, 337-345. (35) Huang, X.-J.; Wang, S.-L.; Fang, Z.-L. Anal. Chim. Acta 2002, 456, 167175. (36) Arora, A.; Eijkel, J. C. T.; Morf, W. E.; Manz, A. Anal. Chem. 2001, 73, 3282-3288. (37) Liu, J.; Cao, W.; Qiu, H.; Sun, X.; Yang, X.; Wang, E. Clin. Chem. 2002, 48, 1049-1058. (38) Cao, W.; Liu, J.; Yang, X.; Wang, E. Electrophoresis 2002, 21, 3683-3691. (39) Wang, H.; Xu, G.; Dong, S. Analyst 2001, 126, 1095-1099. (40) Xu, G.; Dong, S. Electronanlysis 2000, 12, 583-587.
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Figure 1. Working electrode assembly: 0.5-mm-diameter platinum wire (1), cable (2), glass tubing (3), solder joint (4), sealon film (5), stainless steel tubing (6), and heat-shrinkable tubing (7). Dimensions are not in scale.
hexahydrate (Aldrich Chemical Co.), TPA (Acros Chemical Co.), L-proline (Shanghai Biochemical Co.), and sodium oxalate (Beijing Chemical Factory) were dissolved in pure water at concentrations of 10, 1, 100, and 100 mmol/L as stock solutions, respectively. The buffers used in this experiment were sodium dihydrogen phosphate and disodium hydrogen phosphate. All solutions were prepared with water purified by a Milli-Q system (Millipore) and stored at 4 °C in a refrigerator. Before use, the stock solution was diluted to the desired concentrations and all samples and buffer solutions were filtered through 0.22-µm cellulose acetate filters (Shanghai Xinya Purification Material Factory). Platinum Disk Electrode (Figure 1). A 2.5-cm length of 0.5mm-diameter platinum wire was soldered at one end to the core wire of a cable and threaded into a 2-cm length of 0.55-mm-i.d. × 0.65-mm-o.d. glass tubing, which was filled with epoxy resin (6101, Xinchen Chemicals Co., China), allowing the platinum wire to extend out ∼1 mm from the end of the glass tubing. After the solder point and the exposed wire was sealed by a piece of sealon film (Fuji Photo Film Co.), a 3-cm length of 0.7-mm-i.d. × 0.8mm-o.d. stainless steel tubing filled with epoxy resin slid over the electrode assembly until the glass tubing just protruded out from the end of the stainless steel tubing, and the excess epoxy resin was wiped away. After the curing process was completed, a piece of heat-shrinkable tubing was used to cover about 5-mm length of the stainless steel tubing and 5-mm length of the cable. The protruding platinum wire was cut with a sharp scalpel blade to expose the 0.5-mm disk, then the electrode was polished with 1and 0.3-µm R-Al2O3, respectively, and rinsed with deionized water to give a smooth electrode surface. The stainless steel tubing provided adequate holding for the working electrode. After each CE or FI run, the electrode was cyclic voltammetrically treated in the potential range of -0.5 to 0 V at a scan rate of 100 mV/s for 2 min. It was found by this treatment that the long-term ECL response stability could be retained for the detection cell. ECL Detection Cell Construction. The chip-type cell was fabricated using wet chemical etching from two pieces of soda lime glass (20 × 15 × 1.7 mm; Figure 2), which was purchased from Shaoguang Microelectronic Co. (SMC, Changsha, China). The glass came with a chrome film (145-nm thickness) deposited on one surface. The two pieces of glass plates were used as cover and bottom plates, respectively, and were wrapped with adhesive tape, which was adhered to the surface of glass tightly by exerting a force on it. The pattern of flow channel and accommodating grooves of working electrode, reference electrode, and capillary were made on the glass plates by removing appropriate sections of the tape using a sharp scalpel blade and the chrome film was exposed (Figure 2A). After etching the chrome film [Ce(NH4)2(NO3)6/CH3COOH, SMC], the glass plates were placed in etching solution (HF/NH4F, SMC) at room temperature and agitated every 2 h during the etching process. The etching time for the plates
Figure 2. Constructing process of the chip-type ECL flow cell and schematic view of CE and FI measurement modes. Design pattern of flow channel and accommodating grooves (A), etched glass plates (B), accommodating pipet tips and guides (C), CE (D), and FI (E) measurement modes: cover plate (1), bottom plate (2), pattern designs of flow channel (3), working electrode (4), reference electrode (5) and capillary (6) accommodating grooves, flow channel (7), accommodating grooves of working electrode (8), capillary (9) and reference electrode (10), Ru(bpy)32+ solution reservoir (11), waste reservoir (12), working electrode guide (13), capillary guide (14), rubber septum [15; accommodated at the bottom of the (10)], connecting capillary (16), working electrode (17), separation capillary (18), silver wire quasi-reference electrode (19), inlet capillary (20), outlet tubing (21), and rubber septa seal (22).
in this work was 12 h, after which the adhesive tape and chrome film were removed and the plates were washed thoroughly with
water. The etched thickness was ∼0.6 mm using this method (Figure 2B). Flow channel access holes (2-mm diameter) were Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
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drilled, using a diamond driller, on the cover plate at the two ends of the channel, and two 200-µL PVC pipet tips, used as Ru(bpy)32+ solution and waste reservoirs, were glued around the access holes using epoxy resin (Figure 2C). A 1-cm length of 0.85-mm-i.d. × 0.95-mm-o.d. and a 2-cm length of 0.45-mm-i.d. × 0.7-mm-o.d. stainless steel tubing (acting as working electrode and capillary guide, respectively) were glued using epoxy resin in the electrode and capillary accommodating grooves on the bottom plates, respectively (Figure 2C). The cover and bottom glass plates were allowed to cure for 2 h, and then they were glued together at the edges around the flow channel using epoxy resin. A 2-cm length of 0.2-mm silver wire was glued in the reference electrode accommodating groove, with a 0.5-cm length of the wire inserted into the channel, and acted as quasi-reference electrode. This assembly was left overnight for proper curing, and the flow channel (15 × 3 × 1.2 mm) was formed. The bottom plate also acted as an observation window. The stainless steel working electrode guide was glued at the outer surface on the part protruding out of the cell, with a cable using silver electrically conductive glue (Loctite Co.), and acted as counter electrode. CE Measurement Mode. Electrophoresis in the capillary was driven by a high-voltage power supply (Shanghai Nucleus Institute). A piece of uncoated fused-silica capillary (25-µm i.d., 360µm o.d.; Hebei Yongnian Optical Conductive Fiber Plant) was cut to 50 cm in length and was used as a separation capillary. Before use, the capillary was filled with 0.1 mol/L NaOH solution for 30 min; then it was flushed with filtered water and the running buffer for 15 min by means of a syringe. The running buffer in the CE experiment was 10 mmol/L phosphate solution (pH 9.0). The Ru(bpy)32+ solution used for CE-ECL detection was 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate solution (pH 9.0). The separation capillary and the 0.5-mm platinum disk working electrode were inserted into the guides (Figure 2D) and were aligned under a microscope (×72 magnification). A distance of 70 ( 5 µm between working electrode and the end of separation capillary was found to be optimal. After the proper alignment was achieved, working electrode and separation capillary were glued in situ, using cyanoacrylate adhesive, to the ends of the guides, respectively. Ru(bpy)32+ solution was added into the flow channel, then a rubber septum was accommodated at the bottom of the Ru(bpy)32+ solution reservoir, and a 0.5-mm length of 75-µm-i.d. × 360-µm-o.d. fused-silica capillary was inserted through the rubber septum connecting the Ru(bpy)32+ solution reservoir to the flow channel (Figure 2D). The Ru(bpy)32+ solution reservoir was also filled with Ru(bpy)32+ solution, which could flow into the flow channel from the reservoir through the connecting capillary and finally arrive into the waste reservoir under hydrostatic pressure. After 1 h, the Ru(bpy)32+ solution reservoir was refilled with fresh Ru(bpy)32+ solution, and the waste reservoir was drained. During this process, the mean flow rate of Ru(bpy)32+ solution was ∼100 µL/h. A positive high voltage (15 kV; 6 µA) was applied at the injection end with the detection cell held at ground potential through the separation capillary guide. Electromigration was used for sample introduction (10 kV × 10 s, 4.5 nL). FI Measurement Mode. Acetone was dropped on the cyanoacrylate adhesive joint, which was used to glue the separation capillary with the guide, and then the adhesive joint was softened 3640
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and the separation capillary slid out the capillary guide. A 40-cm length of 320-µm-i.d. × 420-µm-o.d. fused-silica capillary (Hebei Yongnian Optical Conductive Fiber Plant) used as the inlet of the flow channel slid into the guide (Figure 2E). When the inlet capillary was aligned with the working electrode and the distance between them was set at 0.3 mm, the inlet capillary was glued in situ, using cyanoacrylate adhesive, to the ends of the capillary guide and connected with PTFE connecting tubing. Ru(bpy)32+ solution and waste reservoirs were sealed tightly using rubber septa, and then a 3-cm length of 0.5-mm-i.d. × 0.7-mm-o.d. stainless steel tubing was inserted through the septum accommodated in the waste reservoir and acted as outlet of flow channel (Figure 2E). The process of changing from CE to FI measurement mode and vice versa took ∼10 min. The FI apparatus was a model IFIS-C flow injection processor (Xian Remex Electronic and Technological Co.) consisting of dual variable-speed peristaltic pumps and an eight-channel injector valve fitted with a 100-µL sample loop. All PTFE connecting tubing was 0.5-mm i.d.. The carrier stream was 0.2 mmol/L Ru(bpy)32+ solution prepared in 50 mmol/L phosphate buffer, and sample solutions were prepared in 0.2 mmol/L Ru(bpy)32+ solution. For oxalate determination, the solution pH was adjusted to 6.8. For TPA and proline determination, the solution pH was set as 9.0. In this experiment, under this pH condition, higher signal-to-noise ratio could be obtained. Sample solution was injected and passed through the flow cell, on the working electrode surface, Ru(bpy)32+ was oxidized to Ru(bpy)33+, and ECL reaction occurred between Ru(bpy)33+ and analyte. Light generated in the cell was transmitted through the observation window to the PMT. ECL Detection System. For both CE and FI measurements, the working electrode was held at +1.15 V (vs silver wire quasireference electrode) controlled using a model 800 electrochemical analyzer (CH Instruments). CL emission was detected by a model MCDR-A multifunctional chemistry analytical processing system (Xian Remex Electronic and Technological Co.). The cell was placed directly in front of the PMT window and was enclosed in a light-tight box to prevent stray room light from contributing to background noise. The PMT was biased at 800 V. RESULTS AND DISCUSSION The most important features of this chip-type ECL detection cell are its suitability for CE and FI measurements and the continuous Ru(bpy)32+ solution flow formed by hydrostatic pressure flowing through the capillary-electrode interface without the use of a syringe pump, thus making our ECL instrument design simplified. Ru(bpy)32+ ECL detection has been a mature technique in FI and HPLC analysis but is relatively new in CE analysis. The capillary-electrode interface adopted in CE-ECL is different from the coaxial flow and Ru(bpy)33+ static reservoir mode adopted in those reported for other Ru(bpy)32+ CE-CL systems.41,42 With this chip-type ECL detection cell, the CE effluent comes directly in contact with the surface of the working electrode in CE measurement mode, so the dead volume of the cell is greatly decreased. Ru(bpy)33+ is generated in situ at the surface of the (41) Tsukagoshi, K.; Miyamoto, K.; Saiko, E.; Nakajima, R.; Hara, T.; Fujinaga, K. Anal. Sci. 1997, 13, 639-642. (42) Barnett, N. W.; Hindson, B. J.; Lewis S. W.; Purcell, S. D. Anal. Commun. 1998, 35, 321-324.
working electrode by electrochemical oxidation; however, the flow of effluent from the electrophoresis capillary over the electrode may reduce the concentration of Ru(bpy)33+, thereby reducing the efficiency of the light-producing reaction.43 So an optimum distance between the separation capillary outlet and the working electrode should be taken into consideration to obtain higher separation efficiency and ECL signals. Under the experimental conditions adopted here (15 kV separation voltage, 10 mmol/L phosphate running buffer, 10 kV × 10 s for sample injection), we found that this distance could be 70 µm ((5 µm) adjusted under a microscope (72× magnification). For the CE-ECL system, the alignment between separation capillary and working electrode is also necessary. To some extent, this procedure is similar to that adopted for CE with end-column amperometric detection system.44-46 However, with this detection cell, the outer diameter of separation capillary was comparable to the diameter of the platinum disk, so the alignment between them can be achieved easily even without the aid of a microscope if they are accommodated in the guides properly. Ru(bpy)32+ ECL is a sensitive and selective detection technique; nevertheless, in a CE-ECL system, when Ru(bpy)32+ was added into a reservoir postcolumn, the variability of Ru(bpy)32+ concentration caused by reservoir evaporation or dilution of CE effluents, the depletion of Ru(bpy)32+, and potential interferences from reaction products accumulated during analysis may have negative effects on sensitivity and reproducibility of ECL responses. So the relative standard deviations (RSDs) of run to run were as large as 7.8% for 10 µmol/L TPA29 and 8.4 and 6.4% for 10 µmol/L proline determination.29,30 Though Ru(bpy)32+ solution can be replenished at a given time,32 these effects will not be eliminated completely. As an alternative, Ru(bpy)32+ can be delivered continuously to the electrode-capillary interface by a syringe pump,33-35 the Ru(bpy)32+ solution will remain fresh during the whole analytical process, and the run-to-run RSDs were only 5 33 and 1.4%35 for 20 and 100 µmol/L proline determination, respectively. However, syringe pumps, reaction tubes, and mixing tee were often used, thus making the equipment complicated. The ECL detection cell designed here provides a continuous introduction of Ru(bpy)32+ into the detection zone (electrodecapillary interface). For CE-ECL analysis, Ru(bpy)32+ solution flows from its reservoir through the flow channel to the waste reservoir under hydrostatic pressure. During this process (within 1 h), the mean flow rate was 100 µL/h. While the flow rate of electrophoresis effluent was 2.7 µL/h, so ample supply of Ru(bpy)32+ in the reaction was assured. Though CE operation has to be interrupted to refill the Ru(bpy)32+ reservoir with fresh solution and drain the waste reservoir, at the electrode-capillary interface, Ru(bpy)32+ solution could keep fresh during the whole electrophoresis runs. Shown in Figure 3 are typical electropherograms of six consecutive runs of 1 µmol/L TPA and 100 µmol/L proline. The run-torun RSDs of ECL signals were 1.7-2.5% in the investigated concentration range of 0.01-5 µmol/L (γ ) 0.9982) for TPA and 1-1000 µmol/L (γ ) 0.9981) for proline. These RSD values were (43) Gilman, S. D.; Silverman, C. E.; Ewing, A. G. J. Microcolumn Sep. 1994, 6, 97-106. (44) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (45) Chen, M.-C.; Huang, H.-J. Anal. Chem. 1995, 67, 4010-4014. (46) Fermier, A. M.; Gostkowski, M. L.; Colo´n, L. A. Anal. Chem. 1996, 68, 1661-1664.
Figure 3. Precision of repetitive TPA and proline runs. Electropherograms for a mixture containing 1 µmol/L TPA and 100 µmol/L proline. Sample solution was injected for 10 s at 10 kV (∼4.5 nL). Separation was carried in 10 mmol/L phosphate buffer, pH 9.0; 15 kV of separation voltage was applied to the fused-silica capillary [50 cm × 25 µm (i.d.)]; 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate buffer was added into the reservoir pH 9.0. The potential of working electrode was +1.15 V (vs silver wire quasi-reference electrode).
Figure 4. Effect of flow rate on ECL intensity for 10 µmol/L oxalate (9), 1 µmol/L TPA (b), and 5 µmol/L proline (0) determined by FIECL. The relative standard deviation of each point was less than 3% (n ) 3). The carrier stream was 0.2 mmol/L Ru(bpy)32+ in 50 mmol/L phosphate buffer at pH 6.8 for oxalate determination and at pH 9.0 for TPA and proline determination. Sample solutions were prepared in carrier stream solutions.
lower than those reported for other CE-ECL systems.29,33 The detection limits of TPA (1.4 × 10-9 mol/L) and proline (1.6 × 10-7 mol/L) were also lower than those reported for other CEECL systems (37 µmol/L29 for TPA and 13,29 1,30 0.9,31 0.2,33 and 1.2 µmol/L for proline35). The calculated theoretical plate numbers for TPA and proline were 52 000 and 105 000/m, respectively. These plate counts also compare favorably with that previously reported (4225/m for proline38). This detection cell can be easily changed from CE to FI measurement mode after replacing the separation capillary with inlet capillary, inserting outlet tubing, and sealing the Ru(bpy)32+ solution reservoir. In FI measurement mode, Ru(bpy)33+ reacts with the analyte before it flows out of the detection cell. To obtain the greatest CL intensity, the flow velocity must be optimized relative to the reaction time course. It has been reported that the Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
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oxalate and 1.64 mL/min for TPA and proline determination. The FIA peaks of oxalate, TPA, and proline are shown in Figure 5. Sampling throughputs were estimated to be about 70 samples/h for oxalate and 50 samples/h for TPA and proline. The RSDs of ECL intensities of consecutive injections at the same concentration were 1.5-2.4% for three analytes. The linear ranges of the three analytes were over 3 orders of magnitude. The detection limits obtained for oxalate, TPA, and proline were 4.4 × 10-8, 1.5 × 10-8, and 3.1 × 10-8 mol/L, respectively, which were lower than those reported with a thin-layer ECL flow cell (0.5 µmol/L for oxalate and proline11). The concentrations 1 µmol/L TPA and 10 µmol/L proline solution were used to investigate reproducibility (four chip cells were tested), and we found the RSDs of cell to cell are less than 15 and 10% for CE and FI, respectively. As for cross-assembly, the RSDs are less than 10% in both CE and FI cases.
Figure 5. Repetitive injections of standard solutions of (A) oxalate, (B) TPA, and (C) proline. Concentrations on the peaks refer to 1 µmol/L analyte. The carrier stream was 0.2 mmol/L Ru(bpy)32+ in 50 mmol/L phosphate buffer at pH 6.8 for oxalate determination and pH 9.0 for TPA and proline determination. Sample solutions were prepared in carrier stream solutions. Carrier streamflow rate 2.05 mL/ min for oxalate and 1.64 mL/min for TPA and proline determination. Insets: Magnified profiles of ECL intensities of 0.1 µmol/L oxalate (A), TPA (B), and proline (C).
maximum CL emission is obtained at low linear velocities for slow reactions (e.g., proline), at intermediate linear velocities for reactions with intermediate kinetics (e.g., TPA), and at high linear velocities for fast reactions (e.g., oxalate).10 Detection limits may be enhanced if the effect of flow on the CL intensity is known for the analyte of interest. Figure 4 shows the effect of flow velocity on ECL intensities. With this chip-type ECL detection cell, we found the changes of ECL intensities with increasing flow velocity were similar to those obtained with a thin-layer ECL flow cell,10 and this phenomenon has been discussed in detail.10 As a compromise, to obtain high ECL signals and high sampling throughout, we selected flow rates of 2.05 mL/min for
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CONCLUSION We have presented a versatile, low-cost, and easily constructed chip-type cell to perform Ru(bpy)32+ ECL detection with CE and FI. In CE measurement mode, by using two pipet tips as Ru(bpy)32+ solution and waste reservoirs, Ru(bpy)32+ could be continuously delivered to the detection zone, thus eliminating the need of syringe pump and mixing tee. When the separation capillary was replaced with an inlet capillary and an outlet tubing was accommodated, the cell could be changed easily from CE to FI mode. With a 0.5-mm-diameter platinum disk electrode, high sensitivity (10-9-10-8 mol/L level detection limits) and wide linearity (>103 with γ >0.998) can be achieved for both CE and FI measurements. These results compare favorably to those reported previously for CE and FI flow cells with ECL detection. By examining the reproducibility of the detection cell, we found the run-to-run RSDs were less than 2.5% for both CE and FI measurements in the investigated linear concentration ranges. We think that this detection cell can also be used with HPLC after accommodating the column outlet into the capillary guide, so this cell has significant practical benefits for routine analysis in flow streams. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant 20299030, 39990570).
Received for review January 9, 2003. Accepted April 16, 2003. AC034021Y
