AMI. Chem. 1883, 65, 3525-3527
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TECHNICAL NOTES
Amperometrlc Detection In Capillary Electrophoresis With Normal Size Electrodes Jiannong Ye and Richard P. Baldwin' Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
INTRODUCTION Since its introduction over a decade ago, capillary electrophoresis (CE) has become a firmly accepted, if not yet completely optimized, analytical technique suitable, often uniquely, for the separation and analysis of complex mixtures. C E s primary strength is its ability to provide extremely high separation efficiencies in relatively short times and to do so with sample volumes in the nanoliter to picoliter range. Its major limitation lies in the inability of common detection systems to respond to such small volumes with sufficient sensitivity.1 For example, the response of conventional absorbance-based methods of detection is limited by the short optical path length afforded by capillaries that are generally 100 pm or less in inside diameter. Among the more promising high-sensitivity detection approaches for CE are electrochemical (EC) techniques anaolgous to those developed earlier for high-performance liquid chromatography. Such amperometricmethods possess a high inherent sensitivity and, as they involve measurements that are independent of path length, are naturally compatible with detection in very narrow capillaries. TWO principal difficulties must be overcome in order for the significant potential of EC detection to be realized in practice. First, there is the need to isolate the high voltage employed in the CE separation from the orders of magnitude smaller electrochemical potential used to control analyte oxidation and reduction. Initially, this problem was solved by placing a small fracture near the downstream end of the separation capillary and covering the crack with a porous glass" or Ndion"' coating that permits this point of the capillary to be held at ground but maintains the electroosmotic flow out of the end of the capillary. More recently, it has been shown that the electrophoretic current and associated iR drop in the detector are small enough in most cases that the exit end of the capillary column can simply be placed into the electrochemical cell very near the working electrode surface.a1o Second, there is the need to employ microsize working electrodes, typically wires or fibers 5-50 mm in diameter and lOO-5OO pm in 1ength.S"J This requirement, invoked in order to match the electrode size to that of the separation capillary, greatly complicates electrode fabrication, makes electrode/
capillary alignment difficult to maintain both during an electrophoresis run and from run to run, and severly limits the experimenter's choide of possible electrode materials. In this work, we describe the design and characterization of a simple wall-jet electrochemical detector for CE which allows the use of normal size (i.e., >100-pm diameter) working electrodes without introducing significant postcapillary zone broadening and thereby compromising separation efficiency. In the wall-jet approach, a disk-shaped electrode consisting of a metal wire with only its tip cross section exposed was positioned immediately in front of the much smaller capillary opening; detection was performed on solution exiting the capillary and flowing radially across the face of the electrode. Principal advantages of being able to use larger electrodes ranged from easier and more rugged electrode construction to easier and more reproducible capillary/electrodealignment. These advantages were demonstrated here by comparing the performance of the wall-jet configuration to that of a conventional microelectrode system for the detection of mixtures of sugars a t copper electrodes.
EXPERIMENTAL SECTION Reagents. Carbohydrateswere purchased from Sigma Chemical Co. and were used as received without further purification. Stock solutions of carbohydrate samples were prepared in deionized water and then diluted to the desired concentration with the 0.10 M NaOH separation electrolyte just prior to use. Apparatus. All experimentswere performed with a CE system constructed in-house. The high-voltage power supply (Model R 301B, HipotronicsInc., Millerton, NY) was capable of delivering 0-30 kV. Protection of the operator from accidental exposure to high voltages was achieved by enclosing the entire capillary and all electrodes in a Plexiglas box equippedwith a safetyswitch to shut off the power supply whenever the box was open. In addition, the outlet end of the capillary was always maintained at ground. Separationswere performed on 80-cm lengths of fused silica capillaries (PolymicroTechnologies, Phoenix, AZ)with 25pm i.d. and 360-gm 0.d. The electrophoresis medium was always 0.10 M NaOH. Sample injection was carried out by electromigration by applying a potential of 10 kV to the sample solution for a duration of 5 s; the resulting injection volume was 1 nL. An overview of the EC detector employed is shown in Figure 1. The design was based on the end-column approach suggested by Huang et al.8 in which the sensing electrode is simply placed at the outlet of the separation capillary and detection is carried out in the same solution reservoir that contains the grounding (1) Albin, M.; Grossman,P. D.; Moring, 5.E. Anal. Chem. 1993,65, electrode for the CE instrument. The end of the capillary was 489A-497A. (2jWallingford, R. A.;Ewing, A. G.Anal. Chem. 1988,60,258-263. cut to the desired length and to as high a degree of flatness as (3)Wallingford, R. A,;Ewing, A. G.Anal. Chem. 1989,61,98-100. possible with a fiber cleaver (Newport Corp., Irvine, CA) and (4)Enptrom-Silverman, C. E.;Ewing,A.G.J.MicrocolumnSep. 1991, then inserted into a small slot cut into the side of a 1-in.-diameter 3,141-145. (5)O'Shea,T. J.;Greenhagen,R.D.;Lunte,S.M.;Lunte,C.E.;Smyth,plastic vial which served both as the cathode compartment for the electrophoresis and as the electrochemical cell for the EC M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr. 1992,593,305-312. (6)OShea, T. J.; Lunte,S. M. Anal. Chem. 1993,65,247-250. detection. Also placed into the cell were a copper working (7)O'Shea,T.J.;Lunte,S. M.; Lacourse, W. R. Anal. Chem. 1993,65, electrode (described below), a Ag/AgCl (3 M NaC1) reference 948-951. electrode, and a platinum wire counter electrode. Control of the (8)Huang, X.;&re, R. N.; Sloes, S.; Ewing, A. G.Anal. Chem. 1991, applied potential and measurement of the resulting current was 63,189-192. performed with a Bioanalytical Systems (West Lafayette, IN) (9)Colon,L.A.;Dadoo, R.;&re, A.G. Anal. Chem. 1993,65,476-481. Model LC-3 amperometric detector. (10)Sloss, S.;Ewing, A. G.Anal. Chem. 1993,66,577-581. 0003-2700/93/0365-3525$04.00/0
0 1993 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993
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Flgure 1. Top view of the wall-Jet end-column detection apparatus: A, separationcapillary;B, electrochemlcal cell; C, Cu worWng electrode; D, Pt counter electrode; E, cathode for electrophoresis (grounded);F, AglAgCl reference electrode; 0, pipet tip, H, hollow plastic rod; I,
micropositioner.
3
The workingelectrodeswere constructedby carefully inserting a copper wire of the desired dimensions into a polypropylene universal pipet tip (Cat. No. 21-278-51, Fisher Scientific Co.) and sealing the wire in place with epoxy glue (GC Electronics, Rockford, IL). The pipet tip was then fit into a hollow plastic rod held in place by an Oriel Corp. (Stratford, CT) Model 14901 micropositioner. Using the rod adjuster, the Cu electrode was inserted into a slot in the opposite side of the cathode compartment/electrochemicalcell and, with the aid of a smallmicroscope, was positioned up against the outlet of the capillary. Four different Cu wires were used in fabrication of the working electrodes: 127- and 320-rm-diameter magnet wires (Newark Electronics, Chicago, IL) whose side areas were covered with an insulating coating and 25- (CaliforniaFine Wire Co., Grover City, CA) and 127-pm (Newark Electronics) diameter bare wires containing no insulated surfaces. For the former,3-5-mmlengths were allowed to protrude from the pipet tip, while for the latter, only 300-400-pm lengths were exposed.
RESULTS AND DISCUSSION The size of the capillaries typically employed in CE generally falls in the 5-75-rm (i.d.1 range. Previous studies on the application of EC detection followingthe CE separation have generally employed working electrodes made from bare carbon fibers or metal wires of approximatelythe same size.”10 In order to optimize the current generated by any electroactive analytes emerging from the capillary, accurate alignment of the electrode and the bore of the capillary is therefore critical. Unfortunately, given the small dimensions of both of these elements, achieving the optimum alignment and maintaining this condition long enough to allow reproducible operation represents a difficult and irreproducible process, even with visual assistance from a low-power microscope. In an attempt to improve the stability of the resulting arrangement, the electrode is sometimes made smaller than the capillary bore and actually inserted inside the end of the capillary channe12,6,6JO In the wall-jet approach suggestedhere, the cross-sectional area of the disk-shaped working electrode was intentionally made much larger than the bore of the capillary. It was anticipated that, as a result, efficient alignment would be easier to achieve for a single set of CE experiments and easier to reproduce from day to day. Furthermore, it was hoped that small displacementsof either the capillary or the electrode following the initial setup would have reduced effect, simply shifting the point of contact of the capillary effluent to a slightly different location on the electrode surface. In order to determine whether the wall-jet configurationwould in fact produce these improvements, ita performance was evaluated by comparing the detection capabilities of different size Cu disk electrodes operated in a wall-jet arrangement with those of Cu wire microelectrodes operated in the conventional
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FIgw 2. Electropherogramsof sugar mixture wtth differentCu working electrodes at +0.60 V vs Ag/AgCI: A, 127-pm dlsk; B, 320-pm disk; C, 127-pm bare wire with 300-400-pm exposed length; and D, 25-pm bare wire wtth 300-40O-pm exposed length. The peaks correspond to (1) sucrose, (2) lactose, (3) glucose, (4) fructose, and (5) ribose, each at a 200 pM concentration. Separation voltage, 25 kV.
fashion after carefully aligning them with the capillary opening. The test sample consisted of a mixture of five sugars (sucrose, lactose, glucose, fructose, ribose) whose oxidation at a Cu electrode was recently reported by Colon, Dadoo, and ZareQto offer an attractive CE detection scheme. The results obtained for this test mixture with the four different electrode arrangements are illustrated by the electropherogramsin Figure2 and summarized quantitatively in Table I. For a 127-pm Cu disk electrode operated in the wall-jet configuration, the corresponding electropherogram in Figure 2A shows that all five sugars were readily separated and well-detected. The limit of detection (S/N = 3) measured for ribose under these conditions was only 1fmol (or 1p M for the 1-nL injection volume used). More important, each of the peaks appeared quite sharp and well-shaped,and the efficiency as indicated by a theoretical plate number N estimated to be at least 74 0o0 (for ribose) was respectable. Even for the larger 320-pm Cu disk electrode, a perfectly acceptable electropherogram (Figure 2B) was still observed, with only a modest decreasein Nbecause of slightly increased band-broadening due to the larger electrode surface area. For the 127-pm disk, the coulometric efficiency for carbohydrate oxidation, which is irreversible at Cu, was estimated to be 10% or less while the larger electrode appeared to increase this by a factor of 4-5. Thus, the larger electrode was able to electrolyze more of the sample and therefore give larger currents for the same concentrations. Nevertheless, no improvement in signal-to-noise ratio or detection limit occurred as a result. The third configuration examined employed a bare 127pm Cu wire electrode whose entire 300-400-rm length had been stripped of any insulating coating and was exposed to
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993
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Table I. Performance Characteristics of Wall-Jet and Conventional Electrode Configurations conventional config wall-jet-like config 127-pm disk 320-pm disk exposure surface area, m m 2 detection limit," fmol (pM)
linear range' no. of theor plates' 7% RSDb
0.0127 1(1) 1pM-1 mM 74 OOO 16
0.0804 1(1) 1pM-1 mM 64 OOO 11
127-pm Cu wire (300-400 pm long)
0.173 2 (2) 2 pM-1 mM 22 OOO 17
26-pm Cu wire
(300-400pm long) 0.0319 5 (5) 5 pM-1 mM 86OOO
65
a Calculated on the basis of the ribose peak,conditions as in Figure 1;the number of theoretical plates was calculated by the formula N = 6 . 6 4 ( t J ~ 1 / 2 ) ~ .Calculated for five to six cycles of capillary/electrodealignment,cell disassembly,and realignment; values include injection
uncertainties of about 8%.
the solution. The electropherogram obtained here (Figure 2C) closely matched that seen for the 127-pm disk electrode except that the peaks for all five sugars wer substantially broader and the efficiency was now greatly diminished. Apparently, analyte was not electrolyzed quickly as it was carried across the tip of the electrode continued to undergo oxidation more slowly along the exposed sides of the Cu wire and thereby produced noticeable peak tailing. Finally, a Cu wire microelectrode,only 25 pm in diameter and with a 300-400-pm exposed length, was used in order to simulate the conventional setup employed in previous EC detection schemes. AB expected, this approach yielded an electropherogram (Figure 2D) containing sharp and wellshaped analyte peaks (N = 86 OOO). Presumably, the effluent from the capillary formed an efficient sheathlike flow pattern around the length of the microelectrode,which, in this set of experiments, was actually the same size as the internal bore of the capillary. Thus, analyte oxidation occurred rapidly despite the length of the wire. Of the four electrodes, this one gave the smallest current levels (estimated coulometric efficiency of
