Anal. Chem. 1997, 69, 951-957
Integrated Capillary Electrophoresis/ Electrochemical Detection with Metal Film Electrodes Directly Deposited onto the Capillary Tip Phillip D. Voegel, Weihong Zhou, and Richard P. Baldwin*
Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
The practical application of electrochemical detection in capillary electrophoresis has been hampered by irreproducibility and inconvenience related to capillary/electrode alignment. In order to eliminate these problems, a simple, flexible method by which the capillary and the working electrode were integrated into a single operational unit was devised and evaluated. The electrodes were formed by sputtering a thin conductive layer of Au or Pt onto the exit tip of the capillary. Depending on the size of the capillary used (i.e., both inner and outer diameters), Au on-capillary electrodes (OCEs) gave detection limits at the micromolar level and slightly below for the test analytes dopamine and catechol. More important, operation of the OCEs required no alignment procedures beyond immersion in the CE buffer reservoir/detector cell. OCEs used in this manner exhibited relative standard deviations of 2-4% for repeated injections even if removed from solution between runs. Finally, the Au and Pt OCEs could themselves be modified further by conventional electrochemical procedures. Here, Cu OCEs, formed by electrodeposition onto Au, were used to detect carbohydrate compounds; and an enzyme OCE, formed by adsorption of glucose oxidase onto Pt, was used to detect glucose. The use of amperometric electrochemical (EC) detection techniques in capillary electrophoresis (CE) was first reported by Ewing1 in 1987. Since that time, EC methodologies have been shown to represent not just a viable but, in many cases, a highly attractive detection approach for CE.2 In particular, EC detection offers a sensitivity for easily oxidized or reduced analytes that often rivals that of laser-induced fluorescence, which is currently the method of choice for most CE applications. Furthermore, the small scale of CE presents no inherent difficulty for EC as it has become relatively easy to obtain microelectrodes of roughly the same size, 5-75 µm, as most capillaries. Finally, the instrumentation involved in EC detection is simple and relatively inexpensive, especially compared to laser-based approaches. This is not to say, however, that CEEC does not possess some characteristics that severely limit its applicability and utility. In particular, there are two problems that must be solved before CEEC techniques are likely to gain wide acceptance among (1) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (2) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A537A. S0003-2700(96)00748-2 CCC: $14.00
© 1997 American Chemical Society
nonelectrochemists. First, the number of analytically important species that are electroactive at modest potentials at the carbon electrodes conventionally used in CEEC is relatively small. Second, the use of EC detection schemes often requires a high degree of manipulative skill on the part of the analyst in order to align the microelectrode and capillary opening initially and then to maintain that alignment reproducibly over the course of hours of CEEC operation. The first of these difficulties has been addressed, at least in part, by the formation of electrochemically active derivatives of the compounds of interest or, better, by the use of more active electrodes that respond to a wider range of analytes. For example, the development of pulsed amperometric detection at Au electrodes3,4 and the use of Cu electrodes at constant potential5-8 have made possible the determination of a wide range of underivatized carbohydrate and amino acid compounds by CEEC. Also, the use of chemically modified electrodes has made it feasible to employ electrodes that more specifically target individual analyte species.9,10 The second CEEC problem is directly related to the small size of the capillaries employed in CE and the consequent need to employ microelectrodes of a similar dimension for the EC detection operation. For example, in Ewing’s original design, which has been the model for most subsequent CEEC work, the sensing electrode was a 10 µm diameter carbon fiber which was positioned at the outlet end of a 75 µm i.d. capillary.1 In this configuration, precise placement of the fiber is essential in order to ensure maximum exposure to analyte plugs emerging from the capillary. However, in practice, achieving optimum alignment of elements this size is not only a painstaking operation initially but, once established, ideal electrode positioning can be difficult to maintain reproducibly over an extended operating period. Over the years, several attempts at decreasing the difficulty of this step have been made. For example, Sloss and Ewing suggested the now commonly used practice of etching the exit end of the capillary by exposure to HF to create an enlarged opening into which the carbon fiber could be directly inserted.11 Our group (3) O’Shea, T. J.; Lunte, S. M.; LaCourse, W. R. Anal. Chem. 1993, 65, 948951. (4) Lu, W.; Cassidy, R. M. Anal. Chem. 1993, 65, 2878-2881. (5) Colon, L. A.; Dadoo, R.; Zare, R. N. Anal. Chem. 1993, 65, 476-481. (6) Ye, J.; Baldwin, R. P. Anal. Chem. 1993, 65, 3525-3527. (7) Ye, J.; Baldwin, R. P. Anal. Chem. 1994, 66, 2669-2674. (8) Ye, J.; Baldwin, R. P. J. Chromatogr., A 1994, 687, 141-148. (9) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307-311. (10) Zhou, J.; O’Shea, T. J.; Lunte, S. M. J. Chromatogr., A 1994, 680, 271-277. (11) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-81.
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investigated a wall-jet detector configuration in which the working electrode is the flat tip of a 100 µm (or larger) wire that is simply pushed up against the much smaller capillary outlet.6-8 With such an arrangement, the electrodes are large enough to be seen and worked with relatively easily, and electrode placement with respect to the capillary opening is less critical and easier to reproduce over time. Very recently, two relatively simple designs for capillary electrode holders have been described.12,13 The purpose of these devices was to allow the two to be brought together more stably and with less dependence on microscopes and micropositioners. Despite these developments, problems related to electrode/ capillary alignment and poor quantitative reproducibility continue to limit the attractiveness of EC detection for most CE practitioners. An obvious way to remove these problems completely would be to incorporate the capillary and the electrode into a single integrated unit. Very recently, Zhong and Lunte described the fabrication of such an “on-capillary electrode” (OCE) system constructed by mounting a Au wire across the capillary outlet.14 In this work, a Au wire, 25 µm in diameter, was glued onto one side of the capillary tip, bent across the capillary opening, and then glued to the other side of the capillary. This arrangement, as expected, succeeded in stabilizing electrode alignment while maintaining analytical performance roughly comparable to that of alternative detector designs. However, initial construction of the OCE system by this approach still requires a nontrivial degree of manipulative skill on the part of the experimenter (undoubtedly including continued reliance on microscopes and micropositioners during the fabrication step). In this work, we describe a simpler, more flexible, but equally effective scheme for OCE construction. Our approach makes use of ordinary metal-sputtering techniques to deposit a thin conductive metal coating on the exit end of the capillary. After making the necessary electrical contacts to the sputtered film and then insulating the film completely except for that on the capillary tip, EC detection could be performed without concern for electrode placement or drift. We describe below the analytical performance of CEEC systems fabricated in this fashion. Initially, OCEs were formed by sputtering either Pt or Au films onto the capillary tip. The separation and determination of several catechol species were then used as a test system to evaluate the performance of the new detector configuration. Finally, the sputtered Pt and Au films could themselves be subjected to additional modification in order to broaden detection capabilities further. Examples of such modifications carried out here include the formation of an OCE for carbohydrates by electrodeposition of Cu onto Au and formation of an enzyme OCE for glucose by incorporation of glucose oxidase onto Pt. In all cases, the OCEs were employed in what is generally referred to as an end column configuration in which there was no decoupling mechanism used to isolate the detection electrode from the CE voltage and current. This approach generally works well for CE systems employing capillaries with diameter of 25 µm or less.15 Although it is possible that improved performance (e.g., lower detection limits) might be obtained if some form of decoupling were included, our primary intent was the development of a CEEC device that is easy and reliable to construct and use (12) Chen, M.-C.; Huang, H.-J. Anal. Chem. 1995, 67, 4010-4014. (13) Fermier, A. M.; Gostkowski, M. L.; Colon, L. A. Anal. Chem. 1996, 68, 1661-1664. (14) Zhong, M.; Lunte, S. M. Anal. Chem. 1996, 68, 2488-2493. (15) Lu, W.; Cassidy, R. M.; Baranski, A. S. Anal. Chem. 1994, 66, 200-204.
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Figure 1. On-capillary electrode design (not drawn to scale): (1) metal film, (2) fused-silica capillary, (3) cyanoacrylate insulating layer, (4) nickel print, (5) connecting wire.
by a nonspecialist. Therefore, no effort was made here to include or evaluate decoupling strategies of any kind. EXPERIMENTAL SECTION Reagents. All reagents were purchased commercially and were used as received without further purification. A 5% Nafion solution was obtained from Aldrich Chemical Co. (Milwaukee, WI), and glucose oxidase from Aspergillus niger (EC 1.1.3.4, 150 units/mg) was obtained from Fluka (Ronkonkoma, NY). Apparatus. The CE instrument used has been described in detail previously.6 In all cases, the high-voltage electrode placed in the electrophoresis buffer reservoir served as the CE anode while the grounded electrode in the detection cell acted as the CE cathode. Thus, no decoupling of the CE and EC systems was employed. Separations were performed on fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with inner diameters of either 15 or 25 µm. EC detection was carried out with a threeelectrode cell configuration with an on-capillary working electrode constructed as described below, a Ag/AgCl (3 M NaCl) reference electrode, and a platinum wire counter electrode. Control of the applied potential and measurement of the resulting current were performed with a Bioanalytical Systems (West Lafayette, IN) Model LC-4B potentiostat. For operator safety, the buffer reservoir, capillary, and detection cell were all enclosed in a Plexiglas box equipped with a safety switch wired to shut off the power supply whenever the box was opened. To prevent clogging, the capillaries were always blown dry by passing nitrogen gas through them and storing in air until use in CE. Cyclic voltammetry (CV) was performed with a Bioanalytical Systems Model CV-1B potentiostat. Electrode Construction. The design of the OCE devices used in this work is illustrated in Figure 1. In general, the fabrication procedure involved the following four stages. Capillary Preparation. The desired length of the fused-silica separation capillary was obtained by scoring a long section of capillary (as obtained from the manufacturer) with a capillary cutter (Newport Inc., Irvine, CA) and breaking it off at the crack to form as smooth an end as possible. The resulting capillary tips were cleaned by dipping them into piranha solution (4:1 concentrated H2SO4/30% H2O2) for 30-60 s. They were then rinsed thoroughly with deionized water and allowed to air-dry. For “flat” OCEs, no further pretreatment of the capillary was necessary. For “cone” OCEs, a chemical etching of the capillary tip was carried out by following the procedure reported by Sloss and Ewing.11 This entailed immersing the end of the capillary into an aqueous 48% HF solution while maintaining a flow of helium gas through the system. After 15 min, the capillary was
removed from the HF, and the etching process was quenched by dipping the capillary into a saturated Na2CO3 solution to neutralize any residual acid and then rinsing it with deionized water. Sputter Coating. Following the above pretreatment, capillaries were wrapped in aluminum foil so as to leave a roughly 3 cm length exposed near the clean tip. They were then placed in the sputtering chamber (Model PS-2, International Scientific Instruments), either singly or in groups of four or eight, at an angle of ∼30° off the vertical defined by the sputter target and the counter electrode. The chamber was then sealed, pumped down to 0.1 Torr, and triply purged with argon. When the chamber pressure again reached 0.1 Torr after the final purging, the sputtering voltage was then set to the appropriate value (1.4 kV for Au and 1.2 kV for Pt). Sputtering was initiated by increasing the Ar pressure to 0.15 Torr at which point a bright blue-purple plasma became visible. This was continued for 4.5 min while the sputtering current was held at 25 mA for Au and 20 mA for Pt. The sputtering voltage was then reset to zero, and the vacuum was released. Examination of the resulting Au and Pt deposits by optical microscopy revealed that a thin metal film now covered the tip of the capillary and a 3 cm distance up the sides. Although intrusion of the sputtered film inside the capillary could not be observed visually because of the coating on its exterior surface, it would seem inevitable that some metal deposition down into the capillary must occur during the process. However, the capillary outlet was always clearly visible and was never at all obstructed by the sputtered material. Profilometry (Alfa Step 100 Contact Profilometer) measurements indicated that the resulting Au and Pt films were approximately 0.3 and 0.06 µm, respectively, in thickness. Electrode Connections. Electrical connection to the OCEs was made by wrapping a suitable length of 22-gauge stranded Cu wire around the side of the capillary ∼2 cm from the tip and in contact with the sputtered metal film. A drop of nickel print (GC Electronics, Rockford, IL) was then placed over the wire to ensure good conduction between it and the film. Subsequently, the wire lead, the nickel print, and the sputtered film on the sides of the capillary were carefully covered with a coating of cyanoacrylate adhesive (Loctite Corp., Hartford, CT) so that only the flat tip of the capillary actually remained uninsulated. The cyanoacrylate was allowed to dry overnight. At this point, the OCEs were ready for use in the CE system. Alternatively, the Au or Pt films could be modified further as described below. Electrode Modification. Two different electrode modification schemes were carried out on the Pt and Au OCEs. In the first, a Cu coating was electrodeposited onto a sputtered Au OCE prepared as above. This was done by immersing the Au OCE into a solution containing 21.0 g/L cupric acetate, 25.5 g/L sodium carbonate, 21.0 g/L sodium sulfite, and 25.5 g/L potassium cyanide and applying a potential of -3.0 V vs Ag/AgCl for 5 min.16 The second electrode modification carried out involved the formation of an enzyme OCE for glucose by incorporating glucose oxidase onto a Pt OCE according to the procedure of Jonsson and Gorton.17 In this case, a finished Pt OCE was immersed for 5 min in a pH 3.8 phosphate buffer containing 2 g/L glucose oxidase. The electrode was then rinsed with deionized water and coated with Nafion by dipping into a 0.5% Nafion solution in (16) Machinery’s Shop Receipts; Lindsay Publications, Inc.: Bradley, IL, 1990; pp 198-199. (17) Jonsson, G.; Gorton, L. Anal. Lett. 1987, 20, 839-855.
Figure 2. Cyclic voltammograms for a 15 µm i.d., 150 µm o.d. Au OCE in pH 8.0 phosphate buffer: (A) blank, (B) 1.4 mM catechol, (C) 1.0 mM dopamine, and (D) 1.1 mM 3,4-dihydroxybenzylamine. Scan rate: 50 mV/s.
methanol. Once formed, the glucose oxidase electrode assembly was stored in a refrigerator at 5 °C until used in CE. RESULTS AND DISCUSSION Electrode Characterization. When examined by CV, OCEs exhibited completely conventional electrochemical behavior. For example, as shown in Figure 2, the CVs obtained with a Au film sputtered onto a 15 µm i.d., 150 µm o.d. capillary gave both the background current expected for a blank buffer solution and the faradaic current expected for the oxidation of catechol and catecholamines. In all cases, the CVs were identical to those obtained under the same conditions at an ordinary Au disk electrode. The current for the catechol oxidation at the Au OCE was proportional to the catechol concentration and to the square root of the potential scan rate over the ranges studied. It is likely that Au OCEs would find some useful applications in CEEC. However, a dominant theme in current electroanalytical research is the availability of a wide range of electrode materials and modification schemes that permit electrode systems to be designed and optimized for the specific application at hand. Thus, if OCEs are to have broad usage in CEEC, then they must be able to be fabricated by relatively simple and effective procedures from a variety of substrate materials in addition to Au. One approach to accomplish this would be to identify sputtering methods for other electrode materials. For example, by altering the sputtering conditions slightly (see the Experimental Section), Pt OCEs can be constructed in a fashion analogous to that described above for Au. In principle, this approach should enable the formation of OCEs for many metals (although some metals require substantially higher sputtering voltages and currents and, perhaps, a more sophisticated sputtering apparatus). In addition, simple electrodeposition procedures and chemical modification schemes should be suitable for conversion of a directly sputtered Au or Pt OCE into one with a more interesting surface composition or structure. An example of the former possibility, involving the fabrication of a Cu OCE, is illustrated in Figure 3. In this instance, traditional procedures for the electrodeposition of Cu16 were employed to alter a Au OCE that had been formed by direct sputtering. As shown in curve A of the figure, the CV of the starting Au OCE exhibited the characteristic redox waves expected for oxide formation and removal (at +0.44 and -0.06 V vs Ag/AgCl, respectively) in the strongly alkaline Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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Figure 3. Cyclic voltammograms for a 25 µm i.d., 360 µm o.d. OCE in 100 mM NaOH: (A) blank at an unmodified Au OCE, (B) blank (- - -) and 10 mM glucose (s) at an electrodeposited Cu OCE. Scan rate, 50 mV/s.
solution used.18 The CV shown in curve B following Cu electrodeposition clearly represents that of a Cu electrodeswith oxidation waves at -0.49 and -0.21 V for Cu(I) and Cu(II) formation on the anodic scan and the corresponding reductions back to Cu(I) and Cu(0) on the reverse scan.19 Finally, addition of glucose produced a new oxidation at +0.56 V. This wave, which did not appear for glucose on the bare Au surface, is identical to that occurring for carbohydrate oxidation at metallic Cu electrodes in alkaline solution20 and will be employed below to illustrate the use of OCEs for carbohydrate detection in CE applications. Before use in CE, OCEs were routinely examined by CV as a precaution to make certain that the sputtering and assembly processes had been carried out successfully. Such examination of dozens of Au and Pt OCEs fabricated over the course of this work revealed the sputtering approach to be an extremely reliable means of electrode construction. Only rarely (much less than 10% of the time) was an OCE, prepared by sputter coating either Au or Pt, found to be defective. In these cases, although the sputtered electrode itself was invariably still fully active, the capillary had become blocked during the fabrication processs usually by improper application of the cyanoacrylate coating used to insulate the outside of capillary and the associated electrical connections. (18) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A-597A. (19) Miller, B. J. Electrochem. Soc. 1969, 116, 1675-1680. (20) Luo, P.; Zhang, F.; Baldwin, R. P. Anal. Chim. Acta 1991, 244, 169-178.
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Capillary Electrophoresis. The analytes that have been by far the most frequently studied in previous CEEC investigations are catechol compounds. The reason for this is that, in addition to their importance in the neurotransmission process, their oxidation occurs at relatively low potentials at most common electrode surfaces. Thus, the catechols were selected as our initial test system for characterization of OCE operation and performance. The electropherograms shown in Figure 4 were obtained for repeated injections of a mixture of dopamine and catechol with EC detection at a 15 µm i.d., 360 µm o.d. Au OCE. Because the capillary used was only 15 cm in length, the separation took place rather quickly, with the positively charged dopamine emerging from the capillary before the neutral catechol. These results were qualitatively the same as those obtained for the same test mixture with EC detection with a 50 µm diameter Au microwire which was operated in a conventional end column wall-jet configuration. However, two major differences resulting from the different detector designs were apparent. First, unlike the OCE, which was simply dipped into the CE cathode chamber at the start of the experiment, the wire electrode had to be painstakingly aligned, both visually and by repetitive injection of the sample mixture and movement of the microelectrode, until the maximum signal was obtained. Second, the OCE showed no run-to-run variation due to gradual movement of the electrode and capillary away from their initial placement. For example, the percent standard deviations for the 15 injections made in Figure 4 over nearly 1 h were only 3.8% for dopamine and 2.0% for catechol. Even these modest variations were more likely due to slight differences in injection volume than to any processes involving performance of the OCE. A more rigorous demonstration of the freedom of the OCE from alignment problems and associated irreproducibility is offered in Figure 5, where the Au OCE was actually taken out of solution (after removing the applied potential) and then reimmersed before each set of catechol injections. For both OCE systems shown, the run-to-run reproducibilityspercent standard deviations of 3.4% for dopamine and 3.8% for catechol over 15 different injectionsswas once again well within the uncertainty level introduced by sample injection despite the fact that the capillary/electrode element was simply dropped into the buffer reservoir/detector cell and then used immediately. The response of the OCEs over a longer termssuch as that required for routine laboratory usesalso proved to be quite attractive. During the course of this work, Au OCEs were used in CE, with the CE current and detection potential continuously applied, for periods of at least two days with a performance comparable to that described above. In normal operation, individual capillary/OCE units were used for three weeks or longer, as long as the capillaries were removed from the CE system when it was not in use and blown dry with N2 to prevent clogging. The Au and Pt OCE films were not thick enough or mechanically strong enough for polishing, but electrochemical cleaning procedures could be utilized without problem. In practice, however, this was not necessary in view of the ease of OCE preparation and their relative longevity compared to that of the capillaries themselves. Sputter coating could, of course, be applied with equal ease to capillaries of various size, allowing some flexibility in the choice of electrode area and in the relative capillary-to-electrode dimen-
Figure 4. Electropherograms for repeated injections of (1) 1.1 mM dopamine and (2) 2.3 mM catechol at a Au OCE. Electrophoresis conditions: 25 µm i.d., 360 µm o.d., 15 cm long capillary, 10 kV separation and injection voltages, 5 s injections, and pH 6.0 phosphate buffer. Table 1. Effect of OCE Dimensions on Analytical Performance.a Limits of Detectionb and Coulometric Efficienciesc for Different Capillary Dimensions 15 µm i.d./ 150 µm o.d.
dopamine catechol
15 µm i.d./ 360 µm o.d.
25 µm i.d./ 360 µm o.d.
LOD,d µM
Coulometric effic, %
LOD,d µM
Coulometric effic, %
LOD,d µM
1.2 (1.4) 0.9 (0.6)
46%
0.5 (0.6) 0.8 (0.6)
87%
0.6 (2.6)
81%
1.5 (3.2)
64%
a CE conditions: capillary length, 15 cm; electrophoresis medium, pH 6.0 phosphate buffer; separation voltage, 5 kV; detection potential, +0.60 V vs Ag/AgCl; 5 s injection at 5 kV. b For signal/noise ) 3. Values were estimated from linear calibration curves for dopamine and catechol concentrations from 5 to 800 µM. c Calculated by integrating CE peaks obtained for dopamine and catechol concentrations from 5 to 800 µM. d Values in parentheses are in femtomoles.
Figure 5. Electropherograms for repeated injections at Au OCEs with electrode removal between runs. Electrophoresis conditions: 5 kV separation and injection voltages, 5 s injections, pH 6.0 phosphate buffer, and 15 cm long capillary. (A) 15 µm i.d., 150 µm OCE for (1) 116 µM dopamine, and (2) 100 µM catechol. (B) 25 µm i.d., 360 µm o.d. OCE for (1) 116 µM dopamine and (2) 127 µM catechol.
sions. Accordingly, results were provided in Figure 5 for both a 25 µm i.d., 360 µm o.d. capillary and also a 15 µm i.d., 150 µm o.d. capillary. The principal difference in the results obtained for the two OCE systems was that considerably larger current levels were obtained for the larger capillary. This difference can be explained in large part by the fact that, for the same injection conditions, much more sample was actually injected into the larger capillary. Of course, the greater electrode area provided by the larger capillary may also contribute to its larger currents. The results of a systematic investigation of this size effect on OCE performance are shown in Table 1, where two cases of comparison were examined. In the first, the capillary inner diameter was kept constant at 15 µm while the outer dimension of the capillary was increased from 150 to 360 µm. Thus, the amount of sample injected was the same in both instances, and the only difference was the surface area of the Au OCE. This difference had relatively little effect on the analytical performance as only very slightly
improved detection limits were obtained for the larger OCE. In the second case, the capillary outer diameter was kept constant at 360 µm while the inner diameter was increased from 15 to 25 µm. Thus, the OCE area was kept nearly constant, and the injection amount was increased nearly 3-fold. Here, the concentration detection limit again remained nearly the same for both capillaries while the mass detection limit was somewhat poorer for the 25 µm system. In general, limits of detection remained remarkably the same for all conditions examined. An obvious point of comparison for different CEEC cell configurations is, of course, the limit of detection (LOD) attainable with each. As seen in Table 1, for the dopamine and catechol systems, these limits (signal/noise ) 3) were typically at or slightly below the micromolar (or femtomole) levels at the sputtercoated Au OCEs of interest here. Possible points of comparison to this are relatively numerous in the CEEC literature since catechols are far and away the most commonly encountered analytes in CEEC. However, nearly all of this work has been carried out with carbon electrodes, which can be expected to give improved catechol response compared to Au regardless of the detector configuration. Consequently, the most comparable results to ours are those reported recently by Zhong and Lunte, who examined both in-capillary detection at a 25 µm Au wire Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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inserted into a 50 µm capillary and on-capillary detection at a 25 µm Au wire mounted onto the tip of a similar capillary.14 The LODs reported (signal/noise ) 2) for dopamine for these systems were 0.50 and 0.12 µM, respectively. Although these values are marginally better than the 0.5-1.2 µM figures cited in Table 1, obvious differences in experimental conditions (pH 6.0 here vs pH 2.5; 15-25 µm i.d. capillaries here vs 50 µm i.d. capillary) and LOD definition (signal/noise ) 3 here vs signal/noise ) 2) and uncertainty as to the injection volume used in the earlier work make the detailed interpretation of these numbers meaningless. It is notable that the LODs reported by Zhong and Lunte were obtained for “off-column” detection schemes that utilized a decoupling junction to reduce the noise carried over from the CE system. As no such special measures were employed here, significantly better LODs might be expected for the sputter-coated OCEs from the use of decoupling techniques. Or, on the other hand, comparable performance was obtained here without the need for this additional experimental complication. Although the detection limits attainable with the on-capillary scheme were certainly acceptable with the simple configuration depicted in Figure 1, still lower detectability would, of course, be desirable. Accordingly, in an attempt to understand the masstransfer processes occurring with the on-capillary scheme and to try to improve the performance over that seen in Table 1, two slightly altered on-capillary geometries were examined. In the first such alteration, the tip of a 360 µm o.d. capillary was etched with HF prior to sputter coating of the metallic Au coating. The intent of this experiment was that the etching process might create an open, cone-shaped capillary tip with a larger opening than the 15 µm available in the capillary as received from the supplier. (This has, in fact, already been used by Ewing to provide a way to insert a microelectrode into the capillary exit for improved electrode placement and performance.11) It was postulated that a larger opening might allow the sputtering procedure to form the desired Au coating inside the actual end of the capillary. Such a coneshaped electrode might be able to carry out the detection before the analyte leaves the capillary and diffuses out into the bulk solution present in the detector cell. In the second, a plain glass microscope slide was carefully positioned near the OCE, again for a 360 µm capillary, to block the free diffusion or convection of the sample. This was done in an attempt to confine the sample emerging from the capillary into a thin layer around the OCE and thereby increase the proportion of sample seen by the detecting electrode. Unfortunately, neither of these changes resulted in any improvement in analyte signals or detection limits attained. Rather, detection limits were generally poorer with these modifications compared to the simple OCE systems initially characterized. The likely explanation for the failure of these two altered detection arrangements to improve performance is that the coulometric efficiency of the simple OCE configuration is already relatively high. For example, integration of the current obtained for dopamine and catechol peaks (as in Figure 4) indicated that as much as 80-90% of the injected amounts might actually be collected and oxidized in the course of these detection processes at the simple flat OCE. As shown in Table 1, this was the case at least for the 360 µm OCEs. Thus, for these electrodes, changes in the OCE shape or environment could produce little or no improvement in signal although they usually did cause a modest increase in detector noise. Because the collection efficiency of the 150 µm OCEs was somewhat lower, it is possible that such 956 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
Figure 6. Electropherogram of carbohydrate compounds at a Cu OCE: (1) glucaric acid, (2) glucuronic acid, (3) gluconic acid, (4) glucose, and (5) glucitol. Electrophoresis conditions: -3 kV separation and injection voltage, 2 s injection, 50 mM NaOH/0.25 mM CTAB separation buffer, and 25 µm i.d., 360 µM o.d., 20 cm long capillary. All analytes at 100 µM.
changes might have an effect on the response seen at OCEs constructed from small outer diameter capillaries. However, in all instances, the OCEs appeared to have higher efficiencies than most other approaches. For example, both in-column and oncolumn detection with a 25 µm Au wire have been reported to give only 10-25% conversions.14 OCE Applications. In practice, the determination of catechols would normally be performed at a carbon electrode where the background currents would be lower and the detection somewhat more sensitive than at Au or Pt. Thus, the detection of catecholamines by CE at Au OCEs comprised a useful test system for the OCE concept but is not likely to be widely used on its own. Therefore, in order to complete this study, we chose to examine briefly two other potential OCE applications of somewhat greater practical interest: the determination of carbohydrate compounds at a Cu OCE and the determination of glucose at an enzymecontaining OCE. An important strength of this approach is the variety of different OCEs that might be prepared with relative ease either directly, by sputtering different metals onto the capillary tip, or indirectly, by further modifying sputtered Pt or Au films. Cu OCEs. Several studies reporting the use of Cu electrodes for the detection of carbohydrates and related compounds in CE have appeared in recent years.5,6,8 In all earlier cases, the electrodes used consisted of small Cu wires operated in the endcolumn configuration. Here, a Cu OCE, prepared by electrodeposition onto a sputtered Au film (as in Figure 3), was employed for the detection of similar compounds. Figure 6 shows the electropherogram obtained at such a Cu OCE for a sample containing
members of the glucose family from glucitol to glucaric acid. With a capillary of only 20 cm length, nearly complete resolution of the mixture was achieved in less than 5 min with an electrophoresis medium of 50 mM NaOH/0.25 mM cetyltrimethylammonium bromide (CTAB). The latter is a cationic surfactant which was added in order to reverse the direction of the electroosmotic flow. As a result, both electroosmosis and analyte migration were in the same direction. Therefore, glucaric acid, the most negatively charged of the various sample species, appeared first, and glucitol, the least charged of the analytes, emerged from the capillary last. Quantitative reproducibility was excellent. For example, the relative standard deviation for 10 injections of glucose made over the course of 1 h was only 1.6%. Glucose Oxidase OCEs. Over the past two decades, a wide range of electrochemical biosensors, constructed by incorporating different biological entities onto an electrode surface, has been developed. In such biosensors, the biological species most commonly employed is an enzyme, and the most commonly employed enzyme has been glucose oxidase. In order to illustrate the potential of these devices for CE detection and the ease with which OCEs can be adapted for this purpose, a glucose oxidase OCE was constructed and tested. Of the many different methods available for immobilization of glucose oxidase onto an electrode surface, the one selected for use here involved simply adsorbing the enzyme onto a sputtered Pt OCE and then coating the surface with a Nafion film.17 This method was chosen not because it is the best or most effective of these possibilities but rather because it is extremely simple to perform. In addition, the enzyme layer that results should be relatively thin so as to provide rapid response and not compromise CE resolution. As with typical amperometric glucose oxidase sensors, the species actually giving rise to the oxidation current measured was hydrogen peroxide produced by action of the enzyme upon exposure to glucose. In practice, this glucose oxidase OCE gave the expected response, which consisted of an oxidation peak for glucose itself but not for other simple sugars. (Note that the slow rise in baseline consistently observed on sample injection is apparently due to slow re-equilibration of the Pt surface, a phenomenon that has been noted previously in LCEC studies.21 ) Typical electropherograms so obtained are shown in Figure 7. The unique aspect of these injections was that they were carried out, not one immediately after the other as is usually the case when electrode reproducibility is being evaluated, but over the course of three different days. Furthermore, in order to preserve the glucose oxidase activity between these injections, the OCE was removed from the CE instrument and placed in a laboratory refrigerator maintained at 5 °C. Nevertheless, upon removal of the OCE from the refrigerator, reconnection into the instrument, and reimmersion into the detector cell, the same glucose response (relative standard deviation 4.6%) was obtained each day. As always when employing an OCE in this work, no alignment operations wereÅperformed prior to sample injection.
Figure 7. Electropherograms of glucose (5 mM) at a glucose oxidase OCE: (A) day 1, (B) day 2, and (C) day 3. Electrophoresis conditions: 3 kV separation and injection voltage, 2 s injections, pH 7.0 phosphate buffer, and 25 µm i.d., 360 µm o.d., 20 cm long capillary.
CONCLUSIONS The OCE approach completely eliminates problems in CEEC associated with initially aligning the detecting electrode with the capillary outlet and later maintaining this placement stably and reproducibly throughout an extended series of CE experiments. Once constructed, OCEs require no specialized manipulations on the part of the CE operator and yet provide highly reproducible results. At the same time, other analytical performance characteristics are roughly the same as reported for other CEEC approaches. The only specialized apparatus required is a relatively simple sputter coating device which can be found in many electronics or microscopy laboratories. Most important, a wide variety of different OCEs can be fabricated relatively easilyseither by sputtering coatings of different metals or by altering Au and Pt coatings by conventional modification schemes. Because of these properties, the OCE approach should make CEEC much more attractive, especially to CE practitioners who do not possess an extensive background in electrochemistry. ACKNOWLEDGMENT The authors thank Kevin M. Walsh for use of the profilometer and sputter coating equipment and for numerous helpful discussions. This work was supported by the National Science Foundation through Grant EHR-9108764 of the Kentucky Advanced EPSCoR Program. Received for review July 25, 1996. Accepted December 20, 1996.X AC960748T
(21) Liu, Y.; Janle, E.; Huang, T.; Gitzen, J.; Kissinger, P. T.; Vreeke, M.; Heller, A. Anal. Chem. 1995, 67, 1326-1331.
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Abstract published in Advance ACS Abstracts, February 1, 1997.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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