Anal. Chem. 1997, 69, 2994-3001
Voltammetric Detection for Capillary Electrophoresis Sangryoul Park,† Michael J. McGrath,‡ Malcolm R. Smyth,‡ Dermot Diamond,‡ and Craig E. Lunte*,†
Department of Chemistry and Center for Bioanalytical Research, University of Kansas, Lawrence, Kansas 66045, and School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
Several approaches to implementing amperometric detection for capillary electrophoresis have been reported. This report describes the development of a voltammetric detector for CE. The detector is designed to minimize distortion of the voltammetry due to ohmic potential drop. This was accomplished by using a cast Nafion detection cell at the end of the separation capillary. The cast Nafion detection cell provided a low-dead-volume, low-resistance cell that minimized ohmic potential drop and peak band broadening. The ability to detect the current due to oxidation of analytes superimposed on a large background current was also improved. A dynamic background subtraction scheme was used in which a second working electrode, positioned in the electrochemical cell but outside of the detection cell, was used to compensate for the background current in real time. The output of the compensating working electrode was subtracted from the output of the detecting working electrode prior to analogto-digital conversion. Postexperimental digital background subtraction was also implemented. This approach provided optimal elimination of the background current with maximal detection of the analytical signal. The voltammetric detector developed produced high-quality voltammetric response of analytes with injected concentrations as low as 0.20 µM. The system was evaluated by obtaining CE voltammograms of a mixture of eight test phenolic acids. Electrochemical detection has proven to be well suited for capillary electrophoresis (CE).1-6 In particular, the ready miniaturization of electrochemical cells results in compatibility with the small dimensions of CE. Electrochemical detection can be performed either in the amperometric mode, in which the current is measured at a fixed potential, or in the voltammetric mode, in which the current is measured at several potentials. Amperometric detection is used most often because the instrumentation is simpler and the detection limits are much lower than for voltammetric detection. However, amperometric detection provides only the quantitative information of current versus time, †
University of Kansas. Dublin City University. (1) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (2) Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M. Anal. Chem. 1989, 61, 292A-303A. (3) O’Shea, T. J.; Telting-Diaz, M. W.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R. Electroanalysis 1992, 4, 463-468. (4) Ye, J. N.; Baldwin, R. P. Anal. Chem. 1993, 65, 3525-3527. (5) Lu, W. Z.; Cassidy, R. M. Anal. Chem. 1993, 65, 2878-2881. (6) Lin, B. L.; Colon, L. A.; Zare, R. N. J. Chromatogr. A 1994, 680, 263-270. ‡
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while voltammetry provides, in addition, electrochemical information of current versus potential.7,8 This voltammetric information can be used to aid in identification of individual compounds in a complex mixture, provide electrochemical resolution of poorly separated compounds, and evaluate the peak purity of separated compounds. To date, most reports on capillary electrophoresis with electrochemical detection (CEEC) have described amperometric detectors, while only two descriptions of voltammetric detectors have been reported.7,8 Several voltammetric detection approaches have been reported for liquid chromatography.9-14 The main disadvantage of voltammetric detection is the significantly poorer detection limits which can be achieved relative to those possible with amperometric detection. The poorer detection limits are a result of greater noise arising from the background currents associated with scanning the potential. The background current at a carbon fiber electrode has both faradaic and capacitive components. Unlike the relatively constant background currents of amperometric detection, the background currents with voltammetric detection vary dramatically with time (applied potential). The current arising from oxidation of the analyte is superimposed on this background current. Unfortunately, the background current is often several orders of magnitude larger than the analytical current. The higher efficiency separations of CE relative to those of LC necessitate use of faster potential scan rates, which results in larger background currents, thereby worsening the problem for CE detection. A variety of approaches have been described for improving the detection limits of voltammetric detection for liquid chromatography by decreasing the background current; however, these approaches have not been pursued for use with CE.15 In addition, exclusion of environmental and instrumental noise is more problematic for voltammetric detection than for amperometric detection. This is because the use of low-pass filters, while effective for amperometric detection, distort the higher frequency voltammetric data. (7) Ferris, S. S.; Lou, G.; Ewing, A. G. J. Microcolumn Sep. 1994, 6, 263-268. (8) Swanek, F. D.; Chen, G.; Ewing, A. G. Anal. Chem. 1996, 63, 3912-3916. (9) Caudill, W. L.; Ewing, A. G.; Jones, A. G.; Wightman, R. M. Anal. Chem. 1983, 55, 1877-1881. (10) Thomas, M. B.; Msimanga, H.; Sturrock, P. E. Anal. Chim. Acta 1985, 174, 287. (11) White, J. G.; St. Claire, R. L.; Jorgenson, J. W. Anal. Chem. 1986, 58, 293298. (12) Lunte, C. E.;Ridgway, T. H.; Heineman, W. R. Anal. Chem. 1987, 59, 761766. (13) Lunte, C. E.; Wheeler, J. F.; Heineman, W. R. Anal. Chim. Acta 1987, 200, 101-114. (14) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 436-441. (15) Lunte, C. E. LC-GC 1989, 7, 492-498. S0003-2700(97)00156-X CCC: $14.00
© 1997 American Chemical Society
Figure 1. Schematic diagram of the on-column electrical decoupler and electrochemical cell for voltammetric detection.
A second problem associated with voltammetric detection which is particularly severe for use with CE is ohmic potential drop (iR drop). The high resistance associated with typical detection cells for CE results in significant potential drop, even with small currents (V ) iR). Because of this ohmic potential drop, the potential of the working electrode relative to that of the reference electrode can be dramatically different from the applied potential. Ferris et al. have reported nonlinearity of CE voltammetry calibration curves due to iR drop.7 For chromatographic detection, iR drop can be minimized by using highly conductive mobile phases and positioning the working electrode close to the reference electrode. These solutions are often not feasible with CE. The use of highly conductive run buffers in CE results in Joule heating and a subsequent loss in separation efficiency and an increase in noise. Because the detection cell must be part of the separation capillary in order to minimize dead volumes, close placement of the working and reference electrodes is not possible. The development of a voltammetric detector for CE which addresses both the problems of background current and iR drop is described in this report. Dynamic background subtraction was used to better resolve the faradaic current from the background current. A microdetection cell cast with Nafion was designed to minimize the resistance between the working and reference electrodes and thereby reduce the ohmic potential drop. The CE voltammetry system was evaluated through the separation and detection of eight model phenolic compounds. EXPERIMENTAL SECTION Reagents. Catechol, chlorogenic acid, caffeic acid, protocatechuic acid, gentisic acid, sinapic acid, ferulic acid, vanillic acid, and p-coumaric acid were obtained from Sigma (St. Louis, MO). Stock solutions of these compounds were prepared in 0.1 M perchloric acid and were then diluted with buffer solution immediately before use. All chemicals were reagent grade or better and used as received. All solutions were prepared in NANOpure water (Sybron-Barnsted, Boston, MA). Capillary Electrophoresis System. The capillary electrophoresis system was the same as that described previously.16 A 65 cm long fused silica capillary with 50 µm i.d. was used for separation. A 1.0 mm long cast Nafion on-column electrical
decoupler was situated 2 cm from the detection end of the capillary, as described previously.16 A 1.0 mm long cast Nafion detection cell, prepared in the same manner as an end-column cast Nafion electrical decoupler,17 was fabricated on the detection end of the capillary.16 The electrophoresis run buffer was 10 mM sodium phosphate buffer, pH 7.5, unless specified otherwise in the text. For optimal electrochemical detection of the phenolic acids, postseparation pH modification was performed as described previously by immersing the Nafion on-column decoupler in 0.1 N HCl. This resulted in a pH of 2.3 at the outlet of the detection capillary. The separation voltage was set at 30 kV unless specified otherwise in the text. Sample introduction was carried out electrokinetically by applying 30 kV for 2 s. Voltammetric Detector. The voltammetric detector was constructed as described previously for an amperometric detector, except that two working electrodes with a bipotentiostat were used.16 Carbon fiber electrodes (33 µm diameter) were prepared as described previously.16,17 The length of the exposed portion of the carbon fiber was between 0.9 and 1.0 mm unless otherwise stated. The two electrodes showing the closest match in shape and magnitude of background current were selected from several carbon fiber working electrodes. Typically, from a batch of six electrodes, at least one well-matched pair was found. One working electrode was inserted into the cast Nafion detection cell, and the other working electrode was inserted into the electrochemical cell outside of the detection cell, as shown in Figure 1. A Ag/AgCl reference electrode and a Pt auxiliary electrode were also situated in the electrochemical cell. All potentials are referred to the Ag/ AgCl reference. Fresh carbon fiber electrodes were sonicated in 33% (v/v) Microcleaning solution (International Products, Trenton, NJ) before use. The electrodes were electrochemically activated before each experiment using a (1.8 V, 10 kHz square-wave applied for 10-30 s, depending on the degree of passivation of the electrode. The electrochemical cell was filled with 0.1 M sodium phosphate buffer, pH 2.3. Digital data acquisition and system control were carried out with a CIO-DAS1600 data acquisition/control board (Computer Boards, Mansfield, MA) which was connected to a 486/50 Gateway 2000 computer (Gateway 2000, N. Sioux City, SD). The
(16) Park, S.; Lunte, S. M.; Lunte, C. E. Anal. Chem. 1995, 67, 911-918.
(17) Park, S.; Lunte, C. E. Anal. Chem. 1995, 67, 4366-4370.
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interface board was controlled by software written locally using Turbo-C. The data acquisition board consisted of a 16-bit successive approximation type analog-to-digital converter (ADC) with a maximum sampling rate of 100 kHz, a programmable gain amplifier, an analog multiplexer (MUX) with eight differential input channels, and two 12-bit digital-to-analog converters (DAC). The MUX was used to monitor the individual output signals from the bipotentiostat as well as the difference in the two signals. The difference between the outputs of the two potentiostats was used for dynamic background subtraction. To compensate for possible differences in the surface areas of the two working electrodes, the signal from the second potentiostat (the output of the compensation electrode) was passed through an adjustable gain circuit prior to the subtraction circuit. The gain was balanced until the output of the subtraction circuit approached zero monitoring at 700 mV during continuous scanning. The programmable gain amplifier was set to a range of (1.25 V full-scale, which provided resolution of 0.0382 mV/bit. The conversion rate was fixed at 100 kHz. One DAC was used for external voltage programming of the high-voltage power supply for electrophoresis. In addition, the power supply was interlocked under software control, which provided accuracy in electrophoresis time and sample introduction. Several electrically actuated valves of the pressure system were also software controlled for automatic flushing and sample introduction. Data Acquisition and Waveform Generation. For performing voltammetry, a staircase waveform was generated using one of the 12-bit DAC converters of the interface board. To enhance the resolution of the DAC converter, external reference voltage sources (1.5 and 3.0 V) were built using an LM399 voltage reference and were used instead of the built-in 5.0 V reference voltage source. With a reference voltage source of 1.5 V, the bipolar resolution of the waveform generator for voltammetry was 0.73 mV/bit, compared to 2.44 mV/bit with the built-in 5.0 V source. Five staircase waveforms were used for the potential scan of voltammetry. In each case, a scan consisted of 50 potential points independent of the scan range. The time required for each scan was 400 ms for single-scan mode, 100 ms for two-scan mode, 70 ms for three-scan mode, 55 ms for four-scan mode, and 45 ms for five-scan mode. The scan rates of each waveform for a scan range of 1000 mV were 2.5, 10.0, 14.3, 18.2, and 22.2 V/s, respectively. Timing of the waveform generator was adjusted with software setting of the real-time clock/counter (8254C) on the interface board. The potential was held at its initial value for a delay time equivalent to that for the scan prior to each scan to minimize the interference from the large background currents caused by the rapid potential drop at the end of the previous scan. At the end of every cycle, extra time required for calculation and display was added to the regular delay period. The output of the waveform generator was degliched and smoothed with a low-pass filter with a time constant of 1 ms. The accuracy of the waveforms was confirmed using a digital oscilloscope (Model 2522A, BK Precision, Chicago, IL). Analog-to-digital conversion of the current signals started at the half-period of each step of the staircase. The numbers of A/D conversions for each data point were 150 for single-scan mode, 30 for two-scan mode, 20 for three-scan mode, and 10 for fourand five-scan modes. The converted current values were averaged to a representative value. Each voltammogram was acquired at 2996 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
Figure 2. Cyclic voltammetry with a carbon fiber microelectrode of 100 µM caffeic acid in 100 mM sodium phosphate buffer, pH 2.5. The scan rate was 10 mV/s. The solid line is for an open cell, while the dashed line was obtained in a 50 µm fused silica capillary.
a 0.5 s interval. If multiple scans were performed during this interval, the voltammograms were averaged to a representative voltammogram. Because of the large size of the data file (˜500 kB for 10 min run), a 1 MB RAM disk was used for real-time data storage. A collection of 240 voltammograms (2 min run) was saved as a block into the RAM disk. The data stored in the RAM disk were automatically transferred to the hard disk immediately after each electrophoresis run. For activation of the electrode, the voltage reference was switched to 3.0 V to allow the amplitude of the activation pulse to be up to 3.0 V. In addition, the time constant of the low-pass filter at the output of the waveform generator was changed from 1 to 0.01 ms to avoid smoothing the activation pulses. At the same time, the feedback resistor of the I/V converter of the potentiostat was switched to 2 kΩ to avoid saturation of the I/V converter output. These switching actions were automatically carried out with electromechanical switches controlled with digital outputs on the interface board. Software. The software for the data acquisition and system control was written in Turbo-C. The software provides real-time display of the 3-D voltammogram as well as a variety of system controls including generation of waveforms. Data were acquired in floating point format and saved in ASCII form. Data were ultimately transferred into Origin (Version 4.0, Microcal Software, Northampton, MA) files and then processed for a variety of displays and calculations. RESULTS AND DISCUSSION Distortion Due to Ohmic Potential Drop. Typically with electrochemical detection, the working electrode is inserted some distance into the end of the fused silica capillary. The reference electrode must be placed outside of the capillary, resulting in a high solution resistance between the working and reference electrodes. Voltammetric curves obtained in an open cell exhibited no distortion due to ohmic potential drop (iR drop) with buffers as dilute as 10 mM and at scan rates as high as 26 V/s (Figure 2). However, using a 50 µm i.d. fused silica capillary as the cell, serious distortion was observed with buffers up to 500 mM. A 1.0 M buffer was required in order to obtain a voltammogram comparable to those obtained in the open cell. Such high
Figure 3. Effect of CE run buffer concentration on the voltammetry of 100 µM protocatechuic acid. A 65 cm long fused silica column of 50 µm i.d. was used with a sodium phosphate buffer of pH 2.3. The voltammetric scan rate was 26 V/s. Buffer concentration: 9, 100; O, 50; 2, 25; and 4, 12.5 mM.
buffer concentrations are not practical for use with CE. White et al. have previously described the dependence of voltammetric performance on the mobile phase concentration in microbore liquid chromatography.11 Using 100 mM phosphate buffer, severe distortion of voltammograms obtained during a capillary electrophoretic run was observed (Figure 3). The cast Nafion on-column electrical decoupler used to shunt the electrophoretic current before the electrochemical cell can be used to modify the CE run buffer after separation but before detection. This has previously been used to change the run buffer pH to allow use of a higher pH run buffer for optimal separation and then to lower the pH for optimal electrochemical detection. In a similar manner, the ionic strength of the run buffer can be increased at the Nafion decoupler to decrease the solution resistance in the detection cell while using a lower ionic strength run buffer. Figure 4A shows voltammograms obtained after modification of a 10 mM phosphate run buffer with 1 M HCl at the Nafion decoupler. While this approach improves the voltammetric performance, a further decrease in the resistance was necessary for satisfactory performance. Initially, positive feedback iR compensation was applied in order to overcome the problem of iR drop distortion of the voltammograms. A positive feedback of 10 mV/nA (∆E/∆I) significantly improved the shape of the resulting voltammograms (Figure 4B) compared to those obtained without positive feedback (Figure 4A). However, the voltammograms still exhibit distortion due to iR drop. Increasing the positive feedback did not improve the voltammetric shape and led to instability in the detection circuitry. To minimize the resistance between the working and reference electrodes while maintaining the small volume of the detection cell, a modified detection cell was fabricated. The end-column electrical decoupler previously described to shunt the electrophoretic current was adapted as the detection cell. Using a cast Nafion end-column cell, the current path between the working and reference electrodes is through the Nafion and radial to the working electrode, rather than down the length of the cell and
Figure 4. Approaches to minimize iR drop: (A) postrun buffer modification; (B) positive feedback compensation; (C) use of a cast Nafion end-column detection cell. The CE run buffer was 10 mM sodium phosphate buffer, pH 7.5. For postrun buffer modification, the 1 mm on-column decoupler was placed in 1 M HCl, which produced a pH of 2.3 at the detector. Symbols: 0, chlorogenic acid; O, caffeic acid; 2, protochatecuic acid; and ], gentisic acid.
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Figure 5. Subtraction of the background current from the voltammetric response. (A) Raw data using a single working electrode; (B) digital background-subtracted data using a single working electrode; (C) raw data using two working electrodes with dynamic background subtraction; (D) digitial background-subtracted data with dynamic background subtraction.
along the working electrode as with a fused silica capillary cell. This minimizes the resistance between the working and reference electrodes. The cast Nafion tubing also serves as a diffusion barrier to maintain a very low dead-volume cell. The use of the cast Nafion cell significantly improved the shape of voltammograms relative to use of the fused silica capillary cells (Figure 4C). While this type of cell significantly improved voltammetric performance, optimal performance was achieved in combination with postcolumn buffer modification. Compensation for Background Current. The background currents associated with scanning the applied potential have been a major factor in limiting the utility of voltammetric detection. The background voltammetric currents in hydrodynamic systems are relatively constant, making digital background subtraction possible. This approach is typically used for voltammetric detectors for HPLC and previously described voltammetric detectors for CE. Digital background subtraction is effective at extracting the analytical signal from the large background currents (Figure 5A,B). However, because the background current is much larger than the analytical current, most of the range of the ADC is used for the background current. Therefore, a large digitization error arises in the analytical currents after background subtraction. This digitization error appears as noise in the data and defines the detection limits. For example, in this work, background currents were typically on the order of 200 nA, and sensitivity for analytes was approximately 5 nA/µM (peak current to concentration). Using a 12-bit ADC with a (1.25 V input range and gain of 500 2998
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nA/V in order to not saturate the current-to-voltage converter, each bit following analog-to-digital conversion corresponds to 0.31 nA. Therefore, the limit of detection considering only digitization error would be 61 nM. The detection limit can be decreased by using a higher resolution ADC. For example, changing to a 16bit ADC with all other parameters being the same, the detection limit would drop to 3.8 nM. This approach is, however, limited by the availability of ADCs with higher resolution. Dynamic Background Subtraction. The resolution of the analog-to-digital conversion could be improved if the background current was removed before the ADC. This was accomplished by using a second working electrode in a difference mode, as has been used in polarography.18 The second working electrode (compensating electrode) was positioned in the electrochemical cell but outside of the cast Nafion detection cell (Figure 1). Identical potential waveforms were applied simultaneously to both working electrodes. The output of the compensating electrode was subtracted from the output of the detecting working electrode before analog-to-digital conversion. This results in a dynamic background subtraction in real time prior to digitization of the signal. A similar approach has recently been described by Sturrock and O’Brien in which dynamic compensation was accomplished by feedback of a stored background scan.19 In this manner, the entire range of the ADC was used to digitize the (18) Bond, A. M. Modern Polarographic Methods in Analytical Chemistry; M. Dekker: New York, 1980; pp 157-160. (19) Sturrock, P. E.; O’Brien, G. E. Anal. Chim. Acta 1996, 324, 135-145.
Figure 6. Effect of scan rate on the background current and voltammetry of protocatechuic acid. CE conditions as in Figure 4. (A) voltammetry, (B) background current. Scan rates: 0, 2.5; b, 8.0; 4, 12.0; 1, 16.0; and ], 19.0 V/s.
analytical signal and not the background. While the background currents were similar at the two working electrodes, they were not identical. An adjustable gain was added to the compensating electrode circuit to adjust the magnitude of the compensating output to correct for differences in electrode surface area and activity. Even without perfect compensation, the two-electrode system reduced the background current at the ADC from over 200 nA to less than 10 nA (Figure 5C). The combination of dynamic background subtraction with postexperimental digital background subtraction provided the best signal-to-noise, as shown in Figure 5D. Effect of Scan Rate. The scan rate is a critical parameter to optimize in voltammetric detection for capillary electrophoresis. Both the background current and the sensitivity are affected by the scan rate. However, the scan rate is not directly selectable; rather, the potential range scanned and the interval between scans are the controlling experimental parameters. The potential range scanned is determined by the electrochemical properties of the analytes of interest, while the interval between scans is determined by the efficiency of the electrophoretic separation. A sampling frequency of 2 Hz in the electrophoretic domain was found to provide sufficient data density to fully describe the CE separation. A maximum voltammetric scan time of 400 ms was possible while providing overhead time for data processing. Using a potential range of 1.0 V, the slowest scan rate was, therefore, 2.5 V/s. In this work, the electrophoretic sampling frequency was held constant when the voltammetric scan rate was changed by making multiple scans within the set interval. The effect of scan rate on the background current and the signal current from 100 µM protocatechuic acid are shown in Figure 6. The background current was linearly dependent on the scan rate using the current in the plateau region of 700 mV.
Table 1. Effect of Separation Voltage on Electrochemical Response peak height (nA) CE voltage (kV)
electroosmotic flow (cm/s)
protocatechuic acid
gentisic acid
30 25 20
0.38 0.30 0.24
22.6 19.6 17.5
10.7 8.7 7.2
Effect of Electrophoretic Conditions on the Voltammetry. Unlike the case for other voltammetric detectors,7,8 the use of the on-column electrical decoupler with a high ionic strength solution in the cathodic buffer reservoir rendered the voltammetry independent of the run buffer ionic strength over a wide range of concentrations. As in the case of postseparation modification of pH, postseparation modification of ionic strength allows individual optimization of the separation and detection conditions. Run buffers as dilute as 5 mM can be used without deterioration of the voltammetric response. The shape of the voltammetric response was also not affected by the separation voltage. As the electroosmotic flow increases with increasing separation voltage, the peak current also increases with increasing separation voltage (Table 1). However, the normalized voltammetric response is insensitive to the electrophoresis conditions, providing considerable freedom in optimizing the separation conditions. CE Voltammetry of Phenolic Acids. To evaluate this voltammetric detection scheme for capillary electrophoresis, eight phenolic acids were selected as test compounds. These compounds were chosen because they exhibit a wide range of electrochemical behavior while having similar chemical structures. Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
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Figure 7. Typical CE voltammogram of a mixture of phenolic acids. Separation conditions: 65 cm long, 50 µm i.d. fused silica capillary with a 1 mm cast Nafion on-column electric decoupler immersed in 0.1 N HCl. The run buffer was 10 mM sodium phosphate, pH 7.5, with a separation voltage of 25 kV. Injection was made electrokinetically for 2 s at 25 kV. Detection conditions: a 1 mm long, 33 µm diameter carbon fiber electrode was inserted 700 µm into a 1 mm long cast Nafion end-column detection cell. The electrochemical cell was filled with 0.1 M sodium phosphate buffer, pH 2.5. Dynamic background subtraction using a second working electrode and postacquisition digital background subtraction were applied to the raw data. Peak identities: 1, chlorogenic acid; 2, sinapic acid; 3, ferulic acid; 4, caffeic acid; 5, p-coumaric acid; 6, vanillic acid; 7, protocatechuic acid; and 8, gentisic acid.
They have also been used previously in the evaluation of voltammetric detectors for liquid chromatography. The phenolic acids could be electrophoretically separated using a run buffer of 10 mM sodium phosphate, pH 7.5. The electrochemistry of the phenolic acids at carbon electrodes is kinetically unfavorable at high pH, likely due to repulsion of the anionic phenolic acids by anionic functionalities on the carbon electrode surface. This problem was overcome by using postseparation buffer modification, as previously described for amperometric detection of the phenolic acids with CE separation. This was accomplished by filling the cathodic buffer reservoir with 0.1 N HCl to produce a pH of 2.3 at the detector. Under these conditions, the phenolic acids exhibited well-behaved electrochemistry. A typical CE voltammogram is shown in Figure 7. This threedimensional presentation of the data is not generally useful; rather, two-dimensional presentations of either single-potential electropherograms or single-time-point voltammograms extracted from the entire data set are more useful. Electropherograms extracted from the entire data set of Figure 7 are shown in Figure 8. Each electropherogram of Figure 8 was collected at a different detection potential and corresponds to the electropherogram that would have resulted from amperometric detection at that potential. Likewise, Figure 9 shows the voltammograms extracted from the complete data set corresponding to the elution times of the phenolic acids. From these subsets of the complete data, both the normal electrophoretic and voltammetric parameters can be determined (Table 2). Detection Limits. Detection limits were determined using electropherograms corresponding to an applied potential of +1.13 V and are listed in Table 2. This potential is on the current plateau 3000 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
Figure 8. Single potential electropherograms extracted from the data set of Figure 7. Potentials: (-+-) +0, (- -) +400, (- - -) +600, (-‚-) +800, (-‚‚-) +1000, and (s) +1200 mV.
Figure 9. Voltammograms extracted from the data set of Figure 7. Symbols: O, caffeic acid; ], chlorogenic acid; b, ferulic acid; 0, gentisic acid; [, p-coumaric acid; 4, protocatechuic acid; 9, sinapic acid; and 2, vanillic acid. Table 2. Information Extracted from CE Voltammograms
compound
migration time (min)
half-wave potential (mV)
peak current (nA)
detection limit (µM)
chlorogenic acid sinapic acid ferulic acid caffeic acid p-coumaric acid vanillic acid protocatechuic acid gentisic acid
4.63 5.02 5.18 5.26 5.43 5.50 5.58 6.17
551 709 851 528 1072 990 656 624
6.8 15.1 3.9 16.4 6.4 12.9 21.1 7.9
0.59 0.32 1.3 0.42 0.87 0.44 0.20 0.62
for all of the phenolic acids (Figure 9). Somewhat lower detection limits could be achieved for the more easily oxidized phenolic acids by using an electropherogram from a less positive applied potential. However, unlike for amperometric detection, this is not because the noise is lower at lower applied potential. For voltammetric detection, most of the noise is associated with the scanning of the potential. Rather, the improvement in detection limits at less positive potential for easily oxidized analytes is a
result of greater selectivity at these potentials and, therefore, fewer interferences. CONCLUSIONS Voltammetric detection for capillary electrophoresis requires subtraction of large background currents associated with scanning the potential and severe ohmic potential drop associated with the high resistance of the small detection cell. The ohmic potential drop was minimized by fabricating a detection cell using cast Nafion as a conducting material. The cast Nafion detection cell provided a small volume, as required by CE, while achieving a low resistance to minimize iR drop. The background current was dynamically subtracted in real time using a second working electrode in a difference mode. These two innovations resulted in a voltammetric detector compatible with CE which provide well-
defined voltammetric curves. While the detection limits achieved were somewhat better than those previously reported for voltammetric detection, they are still significantly higher than those that can be achieved with amperometric detection. ACKNOWLEDGMENT This work was supported by Grant GM44900 from the National Institutes of Health.
Received for review February 7, 1997. Accepted May 9, 1997.X AC970156Q X
Abstract published in Advance ACS Abstracts, July 1, 1997.
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