Anal. Chem. 1994,66, 200-204
Background Noise in Capillary Electrophoretic Amperometric Detection Wenrhe Lu and Rlchard M. Cassldy’ Chemistty Department, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0 WO, Canada
Six electrolytes were used to evaluate the performance (background noise) of amperometric detection with four electrode materials (C, Pt, Au, Ag). Both filament and disk electrodes (10-25-pm diameter) were placed either internal or external to the capillary. The large potential fields in the capillary were sufficient to cause oxidation and reduction of the electrolytes, often with bubble formation, along the length of the electrode placed inside the capillary,and electrodes could be dramatically altered when they were exposed to these potential fields. Measurement of the potential fields around the ends of capillaries with differentinternal diameters showed that the potential field had negligible effects on background noise for capillary internal diameters of 125 pm. Normal peak-to-peak noise observed with a simple potentiostat design was in the range of 0.05-0.1 pA for end-capillary detection (i.d. 1 25 p m ) . Potential fields were significant (-1 V) at the end of 75-pmcapillaries, and the use of grounded detection zones gave only moderate improvements in peak-to-peaknoise.
The attractive potential of electrochemical detection in capillary electrophoresis (CE) has been shown for a wide range of analytes, including inorganic ions, small organic molecules, and large biomolecules.1-12 Attractive features of amperometric detection with ultramicroelectrodes include small detection volume, high sensitivity, good selectivity, and low cost. Most initial detector designsa were based on the principle that to reduce detector noise from the potential field at the end of the capillary, the current in the capillary had to be connected to ground prior to the analyte reaching the working electrode. Indeed, the opinion has been expressed3 that some short-circuiting technique, such as cracking the capillary in front of the working electrode, is a requirement for effective electrochemical detection. These “grounded” detection cells can reduce the amount of the separation current passing through detection zone, but construction of such cells is difficult. It has also been n0ted~9~ that the background noise in these grounded systems is still affected by the ( I ) Curry, P. D., Jr.; Engstrom-Silverman, C. E.; Ewing, A. G. Electroandysis 1991, 3, 587-596. (2) Gordon, M. J.; Huang, X.;Pentoney, S.L.. Jr.; Zare. R. N. Science 1988,242, 224-228. (3) K0k.W.T. Prsentationin Fifth InternationalSymposiumon High Performance Capillary Electrophoresis, Orlando, FL, 1993. (4) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. ( 5 ) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1989, 61,98-100. (6) O’Shea, T. J.; Greenhagen, R. D.; Lunte, S.M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabc, N. J. Chromatogr. 1992, 593, 305-312. (7) Huang, X.;Zare, R. N.; Sloss, S.;Ewing, A. G. Ana/. Chem. 1991,63,189192. (8) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988, 60, 258-263. (9) Lu, W.; Cassidy, R. M.;Baranski, A. S.J . Chromatogr. 1993,640,433-440. (IO) Lu, W.; Cassidy, R. M. A n d . Chem. 1993, 65, 1649-1653. (11) Lu, W.; Cassidy, R. M. Anal. Chem. 1993, 65, 2878-2881. (12) Colon, L. A . ; Dadoo, R.; Zare, R. N . Anal. Chem. 1993, 6 5 , 476-481.
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Analytical Chemistry, Vol. 66, No. 2, January 75, 1994
separation voltage, but no quantitative data on this have been presented. An alternative to the grounded detection zone is ~ will reduce the to use small capillaries (55 p ~ m ) ,which separation current and cause the potential field at the end of thecapillary todecay more rapidly.1~~ While it has been shown that such small capillaries improve detector performance, systematic studies have not been made on the relationship between capillary internal diameter (Ld.), decay of the potential fields, and detector noise. Most reported applications of electrochemical detection have made use of cylindrical electrodes in a effort to maximize oxidation/reduction e f f i ~ i e n c y , ~ but - ~ J a~ disadvantage reported for small-bore capillaries is the difficulty in placement of the electrode inside the~apillary.~ It has been shown that enlargement of the end of the capillary can be useful in this regard;I3however, potential gradients should exist within such geometries, and it is not clear what effect this arrangement may have on separation efficiency. To further develop electrochemical detection in CE it is important to obtain quantitative information about the effect of the separation voltage on detector noise. Unfortunately, detailed studies have not been reported on the effects of the separation voltage on the performance of amperometric detection in CE. The information available is usually in the form of generalized statements or isolated data on equipment that varies in design from worker to worker. In some of our studies of electrochemical detection we have observed little or no effect from the separation voltage with ungrounded detection zones, even with relatively large capillaries (10 and 25 pm) and both “in-capillary”and “end-capillary”placement of the working e l e c t r ~ d e . ~ -Consequently, ]~ there is a need to examine the factors that affect detector noise. Some of the parameters examined in this study are as follows: grounded and ungrounded detection zones; electrolyte composition; potential field within and outside the capillary; the effect of electrode placement in-capillary or end-capillary; electrode size and shape. These evaluations were obtained with six commonly used background electrolytes, three different electrode materials, and four different capillary i.d. sizes (1075 pm). EXPER I MENTAL SECT1ON Apparatus. Polyimide-coated fused-silica capillaries, 1075-pm i.d., were obtained from Polymicro Technology Inc. (Phoenix, AZ). Before use the capillaries were washed with water, 0.1 mol/L HC1, water, and operating electrolyte. The high-voltage power supply, with reversible polarity (0 to f30 ( 1 3 ) S l o s s , S.;E w i n g , A . G . A n a l . C h e m . 1993, 6 5 , 5 7 7 - 5 8 1 .
0003-2700/94/03660200$04.50/0
0 1994 American Chernlcal Society
over the scratch, and a piece of platinum wire (3 cm X 0.1 mm diameter) was placed between the tubing and the capillary. The Teflon tubing was then gently heated to shrink it around the capillary and the Pt wire; both ends of the Pt wire were exposed outside theTeflon tube. Thecapillary was fixed with 5-min epoxy glue to the detection cell, with the joint assembly inside the cell (see Figure 1). After the glue hardened the capillary was gently bent to cause it to fracture at the scratch. This cell was then filled with background electrolyte to cover the joint assembly. The working electrode was adjusted into position at theend of thecapillary, and this separation reservoir 1 -... was filled with electrolyte. The Pt wire provided a space 1 5 CB between the capillary and the tubing for electrolyte and thus Flguro 1. Joint assembly for grounded detection zone: (a) three-way allowed the current in the capillary to flow to ground. The podtkm#;(b) plastk clamp; (c)heet9hrhrkableTeflontube; (d) ~8Pfila1~; switch in this ground circuit (fin Figure 1) was used to convert (e) platinurn wke; (f) pound swltch; (g) potentlostat; (h) eleotrolyte COWS;(I) capi~latyfracture plane; (co) counter electrode; (w) working from grounded to ungrounded detection. electrode ; (r) reference electrode. Cbemicals. All solutions were prepared from the doubly distilled deionized water (Corning, Mega-Pure system, MPkV), was obtained from Spellman (Model RHR30PN30, 6A & D2). The background electrolytes had the following Plainview, NY). The voltage input was housed in a plexiglass compositions: (A) 0.0050 mol/L N,N-dimethylbenzylamine box with a interlock on the access door to protect the operator. and 0.0065 mol/L a-hydroxyisobutyric acid (HIBA) (9876, The detection cell and detector were housed in a faradaic cage Aldrich, Milwaukee, WI) with the pH value adjusted with to minimize the interference from external sources of noise. acetic acid to 4.90;(B) 0.01 mol/L monosodium phosphate, Electrochemicaldetection was based on a chronoamperometric phosphoric acid (BDH, Toronto, Canada) and 0.010 mol/L mode with a three-electrode system. Electrode position was sodium dodecyl sulfate (SDS) (9996,Sigma, St. Louis, MO) adjusted with a x , y , z micropositioner (f1 pm, Model MR3, with phosphoric acid added to give a pH of 6.90;(C) 0.010 Klinger, Garden City, NY). The detection process was mol/L phosphate as in (B) without SDS; (D) 0.030 mol/L controlled via a 386 DX/40 MHz IBM personal computer creatinine (Sigma, St. Louis, MO) and 0.0080 mol/L equipped with PCL-8 18 or PCL-8 12 high-performance data a-hydroxyisobutyricacid (98% Aldrich, Milwaukee,WI) with acquisition card (B & C Microsystem Inc., Sunnyvale, CA); the pH adjusted with acetic acid to 4.80; (E) 0.020 mol/L each data point consisted of an average of 10 000conversions 2-(n-morpholino)ethanesulfonicacid (MES; Sigma) adjusted over a 200-ms period. The computer programs, which to pH 6.00 with NaOH; (F) 0.0050 mol/L sodium chromate controlled the application of potential to the electrode and the with OFM surfactant (Waters/Millipore, Milford, MA) at collection and display of the data, were written locally. The pH 8.00. The electrolyte in the separation reservoir was detector, which had low-pass filters with a time constant of replaced daily to avoid chemical and pH changes. All pH 500 ms, has been described previo~sly.~All voltages were values were measured with a combination glass electrode measured vs a saturated calomel reference electrode. calibrated at pH 4.00and 7.00 (Aldrich, hydrion dry buffers). The construction of the detection cell assembly and the All solutions, including electrolytesand samples, were filtered ultramicroelectrodes (5-25 pm in diameter) was described through a 0.2-pm Nylon-66 membrane syringe filter (Colepreviou~ly.~J~ The alignment between the capillary (mounted Parmer). on a micropositioner) and the electrode was measured with an optical scale under a microscope (to within -3 pm) for RESULTS AND DISCUSSION both top view and side view directions. For end-capillary Electrochemistry of Electrolytes. The current/potential placement, the electrode was positioned directly in front of response for Pt, Au, and carbon fiber ultramicroelectrodes in the capillary exit at a distance of -3-5 pm. The reference the background electrolytes listed in the ExperimentalSection electrode was saturated calomel electrode (SCE; Miniature were examined by cyclic voltammetry to provide information model, Fisher Co., Ottawa, Canada), and the auxiliary on how the current at the working electrode might be affected electrode was platinum foil with an exposed surface area of by the electric field from the separation voltage. These 0.5 cm2. The diameters of the platinum (Goodfellow, electrode materials and electrolyteswere selected because they Cambridge Science Park, Cambridge, England), gold (Goodare commonly used in CE with electrochemical detection. fellow), and silver (Goodfellow) filaments were 25 pm, and Typical results for one of the electrolytesstudied, creatinine/ the diameter for carbon fibers was 10pm (AmocoPerformance HIBA (D), are shown in Figure 2. The residual current was Products). small and relatively constant at E > --300 mV for carbon Grounded Detection Zone. Figure 1 shows a schematic of fiber and platinum electrodes, and at E > 0 mV for gold disk the arrangement used for studies with a grounded detection electrodes. Any current in these positive potential ranges was zone. A 62-cm capillary (75-pm i.d. and 354-pm 0.d.) was likely generated by the electrochemical reactions of trace scratched with a capillary cutter at 1.5 cm from the end of impurities. At more negative potentials, the current began the capillary. An 8-mm length of heat-shrinkable Teflon to increase at a rate that depended on the electrode material tubing (860-pm i.d. before shrinking and 380-pm i.d. after (different overpotentials for the electrochemical reactions). shrinking;Cole-Parmer Instrument, Chicago, IL) was placed
t
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Ana&tlcal Chemistty, Vol. 66. No. 2,January 15, 1994
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noU0 2. CycHc voltammograms for different electrodes in creatinine OIeCtrdytO: (a, top) carbon Rber (200 X 10 pm), (b, M b )goki dbk (25 pm), and (c, bottom) platinum disk (25 pm). Experlmental condltkns: electrolyte, 0.03 moVL creatinine and 0.008 mollL HIBA at pH 4.80; scan rate, 50 mVls, potential measured vs SCE.
The more important reactions in this negative potential range would include reduction of dissolved oxygen, slow hydrogen evolution, and/or slow decomposition of supporting electrolyte. Because the background current increases rapidly in the negative potential region, any small changes in electrode potential would be expected to induce relatively large changes in the background current in this region. Although the six electrolytes gave different voltammograms, the general trend of increasing currents at negative applied potentials was similar, and thus only three voltammograms are shown here. Oxygen was not removed in these studies, and this offered a high and gradually increasing current for the evaluation of noise under conditions of high background signal. Oxygen removal is also not as critical for measurement with ultramicroelectrodes as it for larger electrodes. With ultramicroelectrodes there is a rapid approach to steady-state conditions, and this feature can permit much more accurate background correcti0n.1~Thus, reasonably sensitive detection under reductive conditions is possible,'O and it may be possible (14) Baranski, A.
S.Anal. Chrm. 1987,59, 662-666.
202 Analytical Cht?misf?y,Vol. 66, No. 2, Januty 15, 1994
to further reduce the importanceof oxygen removal with more modern approaches.15 Effect of Separation Voltage on End-Capillary Detection. In ungrounded detection, the paths of the separation and detection currents are not independent. Electric fields from the separation current (IR drop) can change the interfacial potential at the working electrode and, consequently, alter the current observed. To assess the effect of these electric fields short-term (5 s) and long-term (60 s) noise was measured as peak to peak (maximum minus minimum current) for seven different electrodes, both with and without an applied separation voltage; five different electrolytes (A, B,and D-F in the Experimental Section) were studied with the seven electrodes adjusted to within -3 pm of the end of a 60-cm, 25-pm4.d. capillary. Two-working electrode potentials (+700 and -700 mV vs SCE) were selected to test detector performance. In the evaluation of these results it must be emphasized that results observed at low current values depend on the electrode arrangement and instrumentation used. The instrumentation used in these studies is relatively simple, and thus the noise levels observed should be representative of situations where medium-priced potentiostats are used. Although some significant differences between the noise levels were observed with the five electrolytes, there were a number of similar trends, and typical results for one of the electrolytes studied is shown in Table 1. For eachelectrolyte, -50% of the values of short-term noise were 10.1 PA and -78% of these values were observed when the potential of the working electrode was +700 mV. The low noise observed for +700 mV was consistent with cyclic voltammetric studies, (IS) Wojciechowski, M.;Go, W.;Osteryoung, J. Anal. Chem. 1985,J7,155-158.
which showed that background currents in this region were small (see Figure 2). At -700 mV, the background current was usually large. Under such conditions where the current at the working electrode is not limited by concentration polarization, a small change in the electrode/electrolyte potential can have a large effect on background current. For example, even a modest slope of 0.1 pA/ 100 mV corresponds to a peak-to-peak noise of 1 PA. In these studies, the average of the peak-to-peak noise values that were >0.1 pA was 0.6 (k0.8)PA. In general there was no direct relationship between the separation voltage and the short-term noise. For those situations where the noise was C0.1 PA, 40% of these values were observed when the separation voltage was on. The application of the separation voltage had the largest effect on noise when the reactions at the electrodewere not in a diffusionlimited region (Le,, -700 mV). To evaluate the effect of separation voltage on large currents that were diffusion limited, noise levels were studied at a 200 X 10 pm carbon fiber electrode for the oxidation (+700 mV) of catechol in the DMBA electrolyte. Detection current was linear with the mol/L, 1 nA concentration of the catechol: 10 nA for mol/L. The measured for 10-6 mol/L, and 0.1 nA for current changed by only - 5 pA on application of the separation voltage (20 kV), and the signal-to-noise ratio (- 500: 1) also remained essentially constant for these three concentrations. The above results suggest that the separation voltage has no significant effect on the detection noise for end-capillary detection at 25-pm capillaries. Normally one would expect that the observed noise should be related to electrode surface area. The results for the creatinine electrolyte (Table 1) suggest a general increase in noise with electrode surface area, but there was little evidence of this trend with the other electrolyte systems. Apparently, other parameters such as instrumental and environmental noise9 and the chemistry at the surface of the electrode are more important for the small currents observed here. The noise in these studies was normally in the range of 0.2-0.5 PA, which is essentially that expected from the electronic components used in this study.I6 Effect of SeparationVoltage on Detectioninside Capillaries. Several papers have reported detection systems where a cylindrical working electrode is placed inside the capillary3"J3 to provide greater oxidation/reduction efficiency. Although increased noise from the separation voltage is expected for ungrounded detection zones, we previously observed for a phosphate electrolyte? and in these studies for a chromate electrolyte, that the background noise did not increase with the application of the separation voltage (30 kV). With a 10-pm carbon fiber placed 180 pm inside the end of the capillary, peak-to-peak noise was in the 0.1-0.2-pA range. When short sections of filaments (Pt, C, Ag, or Au) were placed inside a capillary, and not connected electronically, vigorous bubble formation (most likely 0 2 and H2 evolution) was observed along the length of the filament when the separation voltage was applied. These reactions were caused by the large potential differences (in solution) along the length of the electrode. If the potential field within the capillary is 300 V/cm, then across a 200-pm section of the electrolyte the (16) Keithley, J. F.;Yeagcr,J.R.;Erdman,R. J.Low~ue/Meeruremcnrs;Kcithley Instruments,Inc.: Cleveland, OH,1984.
potential difference is expected to be 6 V. However, since the electrode has essentially zero resistance relative to the electrolyte, the potential difference must be -0 inside the electrode. Thus, at the positive end of the capillary the electrode must be -3 V relative to the electrolyte, and at the negative end +3 V relative to the electrolyte. Consequently, H+is consumed to give H2 at the positive end and 02 and H+ are produced at the negative end with the net result that part of the current in the capillary is transferred to the electrode via chemicalreactions. Visual examinationunder a microscope showed that gas began tobe evolved slowly when theseparation voltage was increased to 5 kV (the IR drop across the 200pm fiber was 1.7 V), and the rate of evolution increased with increasing separation voltage. When the electrode was connected to a potentiostat, a small part of the current from the oxidation or reduction process (depends on polarity of separationvoltage) passed into the detection circuit, and under conditions where gas evolution was observed, the residual current was -2OOO nA and peak-to-peak noise was -200 nA (20-kV separation voltage). Photographs obtained with an electron microscopeshowed the large potential differencesacross the electrode/electrolyte surface could produce significant changes in the chemical composition of the electrodes. In some instances, instead of bubble production, the electrode changed its color and shape in only a few minutes. Although these electrodes usually exhibited low noise and an electrochemical response to electroactive analytes, it is unlikely that they would exhibit long-term analytical stability. End-Capillary Detection. The above results show that it is difficult to obtain reproducible results when the working electrodeis placed in a potential gradient. Some of our studies have shown there are rapid changes in the appearance of the electrode in the vicinity of the end of the capillary, indicating that end-capillary detection may expose the electrode to significant potential fields. Although the effect of such a potential field has been estimated to be unimportant for very small capillaries ( - 5 pm1.7), it is of interest to determine more exactly its effect on the working electrode. Results for a study of background noise observed with a R (25 pm) disk electrode (phosphate + SDS electrolyte) adjusted to -3 pm of the end of lo-, 2 5 , 50- and 75-pm capillaries are shown in Figure 3. These results show that the background noise began to increase appreciably for capillaries with internal diameters greater than 25 pm. The results in Figure 3 indicate that there is a potential gradient at the end of the capillaries but do not provide any information on the shapeof the gradient as a function of distance from the end of the capillary. Consequently,a series of electrodes were used to measure the potential (vs ground) as a function of distance away from the end of the capillary, and the resutls are shown in Figure 4; positive values for distance refer to movement of the tip of the test electrode into the capillary. The curves are displaced to permit display on one graph; the absolute voltage of each initial reading, which varied due to changes in the interfacial potential, are given in the caption for Figure 4. The results obtained with 75-pm capillarieswere similar for both platinum wire (curve a) and platinum disk (curve b) electrodes. Both of these curves show that there was a significant rise in the potential field as the electrodewas brought into close proximity
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Ana&tilcel &emisby, Vd. 66, No. 2, January 15, 1994
209
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Figure 3. Effect of capillary i.d. on peak-to-peak noise for end-capillary detection. Experimental conditions: electrolyte, phosphate with SDS; electrode, 25-pm disk platinum electrode; separation voltage, 20 kV; capillary length, 60 cm.
-700
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27
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60 cm.
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Distance (Jm) Figure 4. Voltage between working electrode and ground as a function of the distance of electrode relative to the capillary. Experimental conditions: electrolyte, phosphate; separationvoltage, 20 kV; capillary length, 60 cm; electrode, adjusted to -3 pm of end of capillary; potential, (a) platinum wire with 75-pm capillary and the initial voltage at -80 pm (0.65 V), (b) platinum disk electrode with 75-pm capillary and initial voltage at -120 pm (1 V), (c) platinum disk electrode with 25-pm capillary and initial voltage at -60 pm (0.0015 V), and (d) carbon fiber with 25-pm capillary and initial voltage at -40 pm (0.01 V).
of the capillary and that this effect extended well out into the solution. With 25-pm capillaries, however, results with both platinum disk (curve c, Figure 4) and carbon fiber (curve d) electrodes show that the potential dropped off very rapidly at the end of the capillary. Consequently, on the basis of the results in Figures 3 and 4,it should be possible to reduce the effect of separation voltage to negligible levels if end-capillary detection is used with capillaries having internal diameters of 1 2 5 pm. This conclusion should be valid for peak-to-peak noise levels of 2-0.05 PA, which was the lower limit for the equipment used in these studies. The results in Figures 3 and 4 show that the significant potential fields at the ends of large capillaries ( 7 5 pm) can result in large background noise. Consequently, detection at the end of 75-pm capillaries with grounded detection zones was briefly studied to determine whether this approach could
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Analytical Chemistry, Vol. 66, No. 2, January 15, 1994
eliminate this problem. Grounded and ungrounded detection schemes were compared for end-capillary detection with the same 75-pm capillary by changing the location of ground electrode, as shown in Figure 1. The results in Table 2 show that in some situations grounded systems gave a significant reduction in background current relative to ungrounded detection. However, a comparison of the value for the grounded system with that obtained when noseparation voltage was applied showsthat in many instances the separation voltage was not completely isolated from the detection zone. Indeed, in some situations there was no appreciable difference between both detectisn modes. The reduction of background noise by use of grounded detection zones might be expected to improve with smaller capillaries, but maintaining peak resolution might be more difficult, and construction of these systems would be more complex. Thus the experimentally complex technique of grounded detection zones does not appear to be particularly effective for reduction of the residual potential fields at the ends of capillaries. The results reported here suggest that end column detection, and capillaries of
