Technical Notes Anal. Chem. 1996, 68, 1661-1664
Rudimentary Capillary-Electrode Alignment for Capillary Electrophoresis with Electrochemical Detection Adam M. Fermier, Michael L. Gostkowski, and Luis A. Colo´n*
Department of Chemistry, Natural Science and Mathematics Complex, State University of New York at Buffalo, Buffalo, New York 14260-3000
A capillary-electrode holder was constructed for electrochemical detection in capillary electrophoresis (CE). The device allows for positioning of the working electrode at the end of the capillary column without the aid of micropositioners or microscopes. The design facilitates the exchange of electrodes and capillaries without the need of refabricating the entire capillary-electrode setup. The system can be assembled in a very short period of time. Alignment with the self-guided system proved to be reproducible for the electrodes used (carbon, nickel, copper). The advantages of reduced downtime and low cost, make the device very attractive for the routine analysis of electroactive species by CE with electrochemical detection. Electrochemical detection schemes have proven to be very useful in monitoring the extremely small zone volumes of analytes separated by capillary electrophoresis (CE)1,2 and other capillary separation techniques.3 The selectivity and low detection limits of electrochemical detection schemes are desirable properties for routine analysis, particularly when combined with a powerful separation technique such as CE. Despite the high potential of electrochemical detection with CE schemes, it is not employed widely for routine analysis; CE with electrochemical detection is mainly used in research laboratories. One of the major difficulties with the implementation of CE with electrochemical detection is the crucial alignment between the separation capillary and the working electrode of the electrochemical setup.4 End-column detection5 has facilitated the coupling of CE with electrochemical detection; however, in most cases the working electrode still has to be positioned by means of micropositioners and with the aid of a microscope or simply by following a trial and error procedure.6,7 These approaches are time consuming (1) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (2) Gaitonde, C. D.; Pathak, P. J. Chromatogr. 1990, 514, 389-393. (3) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 479482. (4) Lu, W.; Cassidy, R. M.; Baranski, A. S. J. Chromatogr. 1993, 640, 433440. (5) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (6) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581. 0003-2700/96/0368-1661$12.00/0
© 1996 American Chemical Society
and require skilled personnel to operate the system. Such conventional setups are useful for one event only; they do not provide any means for effective electrode and/or capillary exchange. If the electrode needs to be polished or replaced by another material, the entire system needs to be refabricated. Furthermore, once the electrode and the capillary are aligned in the electrochemical cell, they cannot be moved without disrupting alignment or breaking the microelectrode. It has been demonstrated that it is possible to have a system without microscopes and micropositioners.8 However, the system used in such a demonstration, as with other conventional approaches, requires the column and the working electrode be fixed by means of epoxy, which again limits the use of the working electrode with only one capillary. In this technical note, we describe a simple electrochemical cell design that overcomes the above mentioned problems. A capillary-electrode holder secures the separation column and the working electrode without the aid of either microscopes or micropositioners. Furthermore, the electrode, as well as the capillary column, can be exchanged very easily. The system setup is very simple and suitable for routine analysis. We describe the use of the new approach with CE; however, the system can also be used with any other capillary separation technique. EXPERIMENTAL SECTION Reagents. Carbohydrates and epinephrines were purchased from Sigma Chemical Co. (St. Louis, MO) and were used as received without further purification. Stock solutions of these compounds were prepared using purified water from a Milli-Q UV plus water system (Millipore, Bedford, MA). Samples were made daily by diluting stock solutions with the appropriate amount of running electrolyte. The running electrolyte used in these experiments consisted of sodium hydroxide for the separation of sugars and morpholinoethanesulfonic acid (MES) for the separation of the epinephrines (Fisher, Pittsburgh, PA). Apparatus. The CE system was constructed in-house and is similar to that described previously.8 A high-voltage power supply from Glassman High Voltage Inc. (Whitehouse Station, NJ) was (7) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307-311. (8) Guo, Y.; Colo´n, L. A.; Dadoo, R.; Zare, R. N. Electrophoresis 1995, 16, 493497.
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Figure 2. Schematic of electrochemical cell.
Figure 1. Schematic of the capillary-electrode holder fabricated with two plexiglass plates.
employed for the separations. Fused-silica capillaries, 360 µm o.d. × 13 µm i.d., unless otherwise noted, were purchased from Polymicro Technologies, Inc. (Phoenix, AZ). Sample injection was performed by electromigration. Amperometric detection was performed using the end-column approach.5 A three-electrode potentiostat (CV-37) from Bioanalytical Systems, Inc. (West Lafayette, IN) was used to provide a constant potential to the working electrode. The potentiostat is equipped with a current amplifier that provided an output voltage signal proportional to the faradaic current. The voltage signal was fed into an A/D converter board (DT2804, Data Translation, Marlboro, MA) mounted on an IBM PC. Data acquisition was controlled by means of the GRAMS 386 for Chromatography software (Galactic Industries, Salem, NH). Disk-shaped copper and nickel working electrodes were constructed by threading 90-µm-diameter wires into a 100 µm i.d. × 360 µm o.d. × 2 cm length fused-silica capillary (California Fine Wire Co., Grover Beach, CA). With the wire protruding from both ends of the capillary piece, Epoxy 907 (Miller-Stephenson Chemical Co., Inc., Danbury, CT) was applied to seal and secure the wire at both ends. Once the epoxy was dried, the wire protruding from one end of the capillary tube was cut off with a stainless steel surgical blade. This leaves a shiny disk surface with a diameter of ∼90 µm. The piece of wire at the other end of the capillary was used for electrical contact. When required, the disk electrode was polished using a BioAnalytical Systems polishing 1662 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
kit. Carbon electrodes were constructed from 300-µm graphite rods (pencil lead) which were carefully sanded to a diameter near 200 µm and inserted into a 250 µm i.d. × 350 µm o.d. × 2 cm length fused-silica capillary such that the rod was exposed at both ends of the capillary. Epoxy 907 was again used to seal and secure the carbon rod firmly in place in the capillary. To one end of the carbon rod, electrical contact was made by winding a wire around the rod and applying nickel print (GC Electronics, Rockford, IL) to bind the wire to the exposed carbon. Once the nickel print had dried, Epoxy 907 was added to this connection to increase the stability of the linkage. At the other end, the carbon rod was then cut with a stainless steel blade to expose an active surface. A sheet of weighing paper was used to polish the electrode surface when necessary. Cell Design. End-column amperometric detection was conducted using the configurations shown in Figures 1 and 2. The capillary-electrode holder was fabricated from two small pieces of plexiglass with dimensions of 1-cm width, 2-cm length, and 0.3cm thickness. A small groove (350 µm width × 355 µm depth) was machined lengthwise at the center of one of the pieces. A hole with a diameter of 3.0 mm was also machined through the center of both plexiglass pieces. The capillary and the working electrode are accommodated in the groove, and the two small plexiglass plates are brought together and secured in place by means of four 1/4-in. (2-56) screws (see Figure 1). This configuration eliminates the need for plastic tubings around the capillary and the electrode,9 simplifying the overall design. Once the electrode and the capillary are secured in place, the assembly is introduced in the electrolyte solution contained in a 4-mL polyethylene vial (55 mm × 15 mm), as shown in Figure 2. The bottom piece of a 1000-µL plastic pipet tip containing a rubber (9) Chen, M.-C.; Huang, H.-J. Anal. Chem. 1995, 67, 4010-4014.
septum was accommodated at the bottom of the vial to guide the separation capillary. The septum prevented the electrolyte solution from escaping from the polyethylene vial. RESULTS AND DISCUSSION The most important feature of the capillary-electrode assembly, shown in Figure 1, is the groove on one of the plexiglass plates. The capillary column and the electrode both fit neatly in the holder groove because of their similar outer diameter (∼360 µm). Once in place, the second plate holds the capillary and the electrode in place. The use of a disk-shaped electrode having diameters as large as 10 times the inner diameter of the separation capillary in the wall-jet configuration does not have a major effect on separation efficiency.10 Therefore, electrodes with large diameters can be utilized without compromising separation performance, instead of small wires and fibers. We incorporated this configuration with the end-column detection5 approach in our design. However, the use of other configurations is also possible. Alignment is achieved without the aid of a microscope. Simply, the separation capillary is placed in the groove until it reaches the center of the holder, where it is secured in place with the two screws; the electrode is placed in the groove at the opposite end of the holder and slid in until it reaches the end of the secured separation capillary. Then, it is fastened in place with the two additional screws of the assembly. At this point, the disk-shaped electrode and the column are aligned. It should be noted that the capillary and the glass sheath of the electrode were in contact without any excessive force to push the capillary and electrode together. The capillary and the electrode are secured to the same support, preventing possible displacements caused by external movements, which could alter the electrode-capillary alignment. Moreover, the capillary-electrode holder can be moved around freely, without compromising the alignment. The use of microscopes, micropositioners, or any fixing glue is not required, only a small screwdriver is required to fasten the two plates of the assembly. Visual inspection of the capillary-electrode holder under the microscope after its assembly confirmed that the separation and electrode capillaries were in contact every time inspected (n > 50). The capillary-electrode holder is immersed in the electrolyte solution as shown in Figure 2. The hole at the center of the assembly allows for the diffusion of solution away from the detection point. Care must be taken to assure that bubbles have not become lodged inside the center of the holder, which may lead to an unstable signal. In our system, this is avoided by agitating the solution around the holder; however, a capillary-electrode holder with a larger hole diameter at the center should also reduce such effects. The complete assembly procedure is performed in a timely fashion. The capillary and the electrode can be mounted on the holder in ∼2 min. This is an extremely valuable asset for the combination of CE with an electrochemical detection scheme. Furthermore, if the electrode needs polishing or replacement, it can be removed from the holder, polished, and repositioned in place within a few minutes. This in turn reduces the downtime of the setup as refabrication of the entire system is not required. The performance of the capillary-electrode holder was assessed with three different types of electrodes; other materials can easily be used (e.g., carbon paste, platinum, etc.). Nickel and copper microelectrodes were used to monitor the separation of (10) Ye, J.; Baldwin, R. P. Anal. Chem. 1993, 65, 3525-3527.
Figure 3. (A) Separation of a sugar mixture detected on the nickel electrode (200 µM each): (1) sucrose, (2) lactose, (3) galactose, (4) glucose, (5) mannose, and (6) fructose. Separation conditions: fusedsilica capillary, 63 cm long (13 µm i.d. × 360 µm o.d.); electrolyte, 100 mM NaOH; field strength, 200 V/cm; 5-s electrokinetic injection at 12.6 kV; detection at 0.600 V vs Ag/AgCl. (B) Separation of a sugar mixture (200 µM each) detected on the copper electrode; (1) neutral marker (1% methanol), (2) sucrose, and 3) glucose. Separation conditions: fused-silica capillary, 80.9 cm long (25 µm i.d. × 360 µm o.d.); electrolyte, 100 mM NaOH; field strength, 309 V/cm, 5-s electrokinetic injection at 10.0 kV; detection at 0.650 V vs Ag/AgCl. (C) Separation of (1) norepinephrine and (2) epinephrine (50 µM each). Separation conditions: fused-silica capillary 53.5 cm long (50 µm i.d. × 360 µm o.d.); electrolyte, 25 mM MES at pH 5.70; field strength, 168 V/cm; 6-s electrokinetic injection at 9.0 kV; detection at 0.725V vs Ag/AgCl.
carbohydrates, while a carbon microelectrode was used to monitor the separation of epinephrine and norepinephrine. Representative electropherograms are illustrated in Figure 3. The separation conditions were similar to those previously reported for sugars and catecholamines.11,12 Capillary-electrode alignment can be evaluated by monitoring changes in peak heights.10 We evaluated the reproducibility of the alignment by measuring the peak height of glucose using a copper electrode under the conditions specified in Figure 3B (efficiencies were also monitored). The capillary and the electrode were mounted on the holder and set up as in Figure 2. At least three injections were performed and the electropherograms collected. The electrode was then removed from the holder and re-installed back to collect a new set of electropherograms. The procedure was repeated at least five times. The peak height reproducibility for glucose was 8% RSD. This reproducibilty represents an improvement over the 16% RSD reported by Ye and Baldwin for a 127-µm disk electrode operated (11) Colo´n, L. A.; Dadoo, R.; Zare, R. N. Anal. Chem. 1993, 65, 476-481. (12) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988, 60, 1975-1977.
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in the wall-jet configuration.10 It should also be noted that the run-to-run reproducibility obtained from at least six consecutive runs on a particular assembly was 5% RSD, without an automated injection system. The theoretical plates obtained for glucose were ∼150 000 (6% RSD, n ) 7); the plate count compares favorably with previous reports using a conventional configuration.8 Linearity was tested between 5 µM and 1 mM (e.g., r ) 0.997 for glucose). The limit of detection (LOD, S/N ) 3) for glucose was 1 fmol (2 µM). These results indicate that removal and repositioning of the working electrode does not compromise the performance of the CE-EC system. The figures of merit corresponding to the carbon and nickel electrodes using the present configuration were similar to those of the copper electrode. The theoretical plates for all sugars using the nickel electrode were above 100 000; linearity was observed for the range tested (12 µM to 1 mM, r ) 0.996) with an LOD of 0.5 fmol (1 µM) for glucose. Using the carbon electrode, the theoretical plates for norepinephrine and epinephrine were 115 000 and 70 000, respectively, and the LOD was 3 fmol (4 µM). Linearity with the carbon electrode was tested between the LOD and 150 µM (r ) 0.996). In summary, we have presented a simple and convenient device to perform electrochemical detection with CE; it can also be used
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with other liquid separation techniques using capillary tubes. The capillary-electrode holder has significant practical benefits over other conventional procedures, including the elimination of complicated arrangements (i.e., micropositioners, microscopes), ease of alignment, exchange of electrode or capillary, reproducibility from one cell setup to another, and convenient dimensions to work with capillaries. All of these make the present setup very advantageous to be used in routine analyses. One possible limitation of this system is the need to construct the electrode having a diameter similar to that of the separation capillary. Nonetheless, once one electrode has been fabricated, it can be used many times since the configuration allows for easy removal and polishing of the microelectrode to expose a new surface. ACKNOWLEDGMENT We gratefully acknowledge the financial support provided by The Whitaker Foundation. Received for review October 16, 1995. Accepted February 16, 1996.X AC9510346 X
Abstract published in Advance ACS Abstracts, March 15, 1996.
