Anal. Chem. 1983, 55,2309-2312 (20) Done, J. N.; Kennedy, G. J.; Knox, J. H. “Gas Chromatography 1972”; Perry, S. G., Ed.; Applied Science Publishers Ltd: London, 1973; p 145. (21) Guiochon, G. J. Chromatogr. 1979, 785, 3. (22) Kennedy, G. J.; Knox, J. H. J. Chromatogr. Scl. 1972, 70, 549. (23) Martin, M.; Guiochon, G. Chromatographla 1977, 70, 194. (24) Glddlngs. J. C. “Dynamics of Chromatography. Part I . Principles and Theory”; Marcel Dekker: New York, 1965; Chapter 5. (25) Asshauer, J.; Halasz, I.J. Chromatogr. Scl. 1974, 72, 139. (26) Martin, M.; Eon. C.; Guiochon, G. J. Chromatogr. 1974, 99, 357. (27) Martin, M. Th6se de Doctorat d’Etat, UniversLB Paris VI, 1975, Chapter 2. (28) Lln. H.J.; Horvath, C. Chem. Eng. Sci. 1981, 36, 47.
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(29) Poppe, H.; Kraak, J. C.; Huber, J. F. K.; Van den Berg, J. H. M. Chromatographla 1981, 74, 515. (30) Abbott, S.; Achener, P.; Slmpson, R.; Klink, F. J. Chromatogr. 1981, 278, 123. (31) Katz, E.; Ogan, K.; Scott, R. P. W. J . Chromatogr. 1983, 260, 277. (32) Poppe, H.; Kraak, J. C. Lecture presented at the VIIth International Symposium on Column LC, Baden-Baden, May 1983. (33) Huber, J. F. K. Ber. Bunsenges. Phys. Chem. 1973, 77, 179. (34) Horvath, C.; Lin, H.-Y. J. Chromatogr. 1976, 726, 401.
RECEIVED for review May 23,1983. Accepted August 18,1983.
Coulostatic Pulse Amperometry for Liquid Chromatography/Electrochemistry Detection Anthony C. Barnes’ and Timothy A. Nieman*
School of Chemical Sciences, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
Small charge pulses are repetltively injected Into the cell to maintain the the worklng electrode at the deslred potential. Because the method Is not interfered with by charging current, the potential can be changed rapidly; 10-point voltammograms are run in 1-2 e. Caffeic acid and o-chlorophenol are quantitated at fixed potential In a flow Injection system over the range of 1-100 pM with 4 % preclslon. Hydrodynamic voltammograms were run for caffeic acid solutions as dilute as 0.5 pM. For a llquid chromatographic separation of 0 - , m-, and p-amlnophenol, the detection llmlt Is 40 pmol Injected; voltammograms can be run rapidly enough to obtain E,,2 values for all eluting substances from a single Injectlon.
In recent years liquid chromatography with electrochemical detection (LCEC) has become an established technique for the quantitation of electroactive species. General articles have covered the basics of the technique (1-4) and the large volume of recent literature is well covered in recent reviews (5,6) and a bibliography (7).LCEC has proven exceptional sensitivity and ease of use but has as a limitation, the general ineffectiveness of conventional LCEC techniques in applying potential scanning methods for qualitative characterization of eluting species within the time constraint of a chromatographic peak. This limitation arises from the large background and charging currents at solid electrodes and problems due to large and/or variable uncompensated resistances in flow cell configurations. Thus, LCEC detectors usually involve dc amperometry or coulometry at f i e d potential. To achieve greater selectivity, investigators have also used normal pulse (8, 9), differential pulse (8-11),ac (11,12),and square wave (13) techniques all at fixed potential. Some work has been done with square wave voltammetry as a scanning LCEC detector at a static mercury drop, and scan rates of 500 mV in 2 s were obtained (15). Because coulostatic methods are not interfered with by iR drop or charging current (16,17),a coulostatic LCEC detector should offer advantages over existing LCEC detectors. The coulostatic technique may be used to mimic controlled potential amperometry while maintaining the above mentioned ‘Present address: U p j o h n Co., 7000 S. Portage Rd., Kalamazoo,
MI 49001.
coulostatic characteristics. For this method we propose the name coulostatic pulse amperometry (CPA). The CPA method should provide the ability to perform potential scanning on a time scale short enough to be compatible with the time restrictions of eluting LC peaks and thus obtain qualitative information (hydrodynamic Ellz values) concerning eluting species. Furthermore, CPA should allow electrochemical detection for LC systems in which the mobile phase is of low conductivity. In earlier reports we have demonstrated use of coulostatics as a rapid scanning voltammetric technique (18) and preliminaryresults for application of coulostatic detection in flowing streams (19)and liquid chromatography (20). This paper details the development and application of an LCEC detector using coulostatic pulse amperometry. THEORY The development of computer-controlled electrochemical instrumentation has prompted a rebirth of interest in coulostatic analysis (18,21,22). In coulostatics, a charge pulse of known coulombic content is injected into a cell on a time scale much more rapid than the time scale for electron transfer between the electrode and electroactive species in solution. Therefore, essentially all of the injected charge goes toward charging the double layer capacitance with a resulting change in the potential of the working electrode. After pulse application, external circuit connections to the working electrode are broken. The only mechanism for charge to leave the double layer capacitance is then via faradaic reactions at the electrode-solution interface. The higher the concentration of electroactive species, the faster charge leaks off the double layer capacitance and the faster the electrode potential decays back toward its equilibrium value. Coulostatictechniques are unique in that the stimulation of the cell (via the charge pulse) and the measurement of electrode potential are separated in time. Because there is no current passing through the cell between successive charge injections, there is no iR drop in solution. Also, because double layer charging occurs only during the time of the pulse application, there is no interference due ‘to charging current during the time between successive pulses. In single-potential coulostatic pulse amperometry (CPA), small charge pulses are repetitively applied to the cell to maintain the working electrode at the desired potential within an acceptably narrow tolerance. Every time the electrode potential decays outside this tolerance range, a small current
0003-2700/83/0355-2309$01.50/00 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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t
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n I
I
t
1 i
1
Oa8
integrate lime hold time
outlet
inlet
outlet
J-spocer
pause time
eiectrode Ieod
1
Flgure 2. Wall jet electrode flow cell. Time
(SI
Flgure 1. Potential wave form for scannlng coulostatic pulse am-
perometry.
pulse is applied to bring the electrode potential back within the tolerance range. The sum of the charge injected divided by the time interval involved yields the current necessary to maintain the electrode at the desired potential. The current value obtained in this manner is used just as the current value obtained via conventional amperometry. A plot of current vs. concentration yields a working curve, a plot of current vs. potential yields a voltammogram, and a plot of current vs. time yields a chromatogram. In scanning CPA, the potential of the electrode is varied in a staircase manner. Potential control and current measuring for each potential step are accomplished as described for the single-potential method. The potential program is shown in Figure 1. There are three time intervals that can be adjusted to obtain optimum performance with regard to noise suppression and electrode potential control. These times are indicated in Figure 1as the hold time, the integrate time, and the pause time. Upon stepping from one potential to another, a period of time is required for the electrode double layer to charge and for the electrode surface to stabilize at the new potential. The hold time is a delay period prior to current measurement (charge integration) to allow for electrode stabilization. Hold times used are typically 70-100 ms for carbon paste electrodes and 7-30 ms for mercury electrodes. The integrate time is the period over which the quantity of injected charge is integrated. This interval can be adjusted as necessary such that the signal variance component due to counting discrete charge pulses is reduced to an acceptable level. In practice we find 100 ms to be a good compromise between scan time and signal to noise ratio considerations. After the integrate time period for the final potential step of a scan, the working electrode must be returned to the original potential so that a new scan can be initiated. This large potential step causes electrode stabilization to take longer, therefore requiring a longer period than the normal hold time prior to integration. This pause time is usually 1 to 2 s long and depends upon the characteristics of the specific electrode and solution being used. EXPERIMENTAL SECTION Instrumentation. The microprocessor-controlledcoulostat system is the same as that previously described (18). New software was written to accomplish the control and data acquisition for the CPA technique. Two different types of electrochemical flow cells were used. One was a TL-4A thin layer detector cell from Bioanalytical Systems. The other was a wall jet electrode cell constructed in-house and shown in Figure 2. Each cell had a spacer 0.13 mm thick and a 3 mm diameter carbon paste working electrode. The
Table I. Working Curves with Single Potential CPA with Flow Injection concn, pM
peak height, nA caffeic acid o-chlorophenol
100
1303
75 50 25 10 5
1008
coir coeff slope, mA M - I intercept, nA
689 351 185 102
600 453 322 167
91 42
0.9997
0.9993
12.7 .i. 0.2 47 2 9
5.8 ?: 0.1 24t 6
distance from the inlet of the wall jet cell to the face of the working electrode was 0.23 mm. The flow injection system consisted of a Sage Instruments Model 355 syringe pump connected by 0.8 mm i.d. Teflon tubing through an Altex Model 210 injection port (20 pL sample loop) to the flow cell. The HPLC system consisted of an Altex Model llOA pump, a silica column to saturate the mobile phase, an Altex Model 210 injection port (20 pL sample loop), a Whatman precolumn, a Whatman ODs-2 reverse-phase analytical column with 10-km particles, and a Bioanalytical Systems Model TL-4A flow cell. Procedures. All scanning CPA experiments involved measurements at 10 potentials separated by 50-70 mV. The hold, integrate, and pause times were 100 ms, 100 ms, and 1 s, respectively. For all experiments, the reference electrode was Ag/AgCl and all reported potentials are with respect to that reference. The HPLC work used a 0.1 M aqueous solution of HCIOl as the mobile phase. The flow rate was 2.0 mL/min. Reagents. All solutions were prepared with deionized water using reagent grade chemicals with no further purification. Caffeic acid (3,4-dihydroxycinnamicacid), o-chlorophenol,and the aminophenols were obtained from Aldrich. RESULTS AND DISCUSSION Quantitation with CPA. Flow injection with caffeic acid ( E l j 2= 0.32 V) and o-chlorophenol (Ell2= 0.76 V) was used to establish detection limits and linearity for CPA at a fixed potential. For caffeic acid the electrode was held at 0.6 V and for o-chlorophenol the electrode was held a t 1.0 V. Table I presents the results obtained along with least-squares statistics for the working curves. Eight replicate injections of a 10 pM caffeic acid solution yielded peak heights with a 4.4% relative standard deviation. The detection limits (for a signal to noise ratio of 2) are 1.3 p M (4.6 ng injected) for caffeic acid and 2.0 pM (5.1 ng injected) for o-chlorophenol. These detection limits result from the level of digital and 60-Hz electronic noise radiated from the various lines in the instrument. With improved instrument design, even more favorable detection limits could result. The working curves are highly linear over the
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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Time ( S I Chromatogram of a mixture of aminophenols (each at 100 pM) by use of single potential CPA. The elution order is p -, m-, and o -aminophenoi.
Flgure 3.
Potential ( V )
Flgure 4, Voltammograms for caffeic acid by use of potential scanning CPA. The number to the right of each curve is the concentration in
PM.
Table 11. Working Curves for Aminophenols by Use of Single Potential CPA with Liquid Chromatography para
concn, pM 100 75 50 25 10 7.5 5
corr coeff slope, mA M - ' intercept, nA
peak height, nA meta
ortho
1308 1020 682 338 128 78 58
1932 1422 936 414 150 114 70
648 460 370 164 52 40 34
0.9994 13.4 k 0.2
0.9997 19.7 r 0.2
0.9957 6.5 i 0.3
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-5
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14
entire ll/zdecade concentration range covered. The upper limit on linearity was not probed. Single Potential LCEC. To compare the CPA technique with normal controlled potential amperometry, a separation of a mixture of aminophenols was accomplished by using a reverse-phase column. The mobile phase was the same as used in a previous report of aminophenol separations using LCEC (23). The coulostat controlled the electrode at 1.2 V, and 900 ms integration times were used. A typical chromatogram is shown in Figure 3. The p-aminophenol elutes first, followed by m-aminophenol,and then o-aminophenol. Base line noise is 2 nA peak to peak. Working curve data for the three isomers are given in Table 11. Detection limits are 2-4 pM, which corresponds to 40 pmol or 5 ng injected. Potential Scanning CPA. T o test the effectiveness of potential scanning with CPA, the method was first applied to solutions of constant concentration flowing through the flow cell. Figure 4 shows the voltammograms obtained for solutions of caffeic acid ranging from 0.5 to 50 pM in a 0.1 M acetate buffer. The scans resemble linear scan voltammograms as would be obtained by conventional means. There is a slight negative slope to the diffusion limited plateau caused by the depletion effect of the electrode in solution. This effect was removed from subsequent experiments by changing the inlet of the wall jet cell from 3.1 mm i.d. to 1.6 mm i.d. and thus increasing the linear flow velocity. The observed EIl2values are very close to the expected value of 0.32 V, and the voltammograms show that this qualitative (Ell2)information can be obtained for concentrations as low as 0.5 pM. To determine the effect of modest changes in solution resistance, voltammograms were obtained for 50 pM caffeic acid
Figure 5.
Configuration of pulsing op amp and cell.
in 0.1, 0.01, and 0,001 M acetate buffer with no other electrolyte present. For this series of solutions, the resistance between the working and reference electrodes went from 8.6 kO to 6.1 MO; the resistance between the working electrode and counterelectrodewent from 63 O to 2.8 kO. Interelectrode resistances were determined by using a bipolar pulse conductance instrument (24). The voltammograms for these solutions had the same shape, same El12values, and same magnitude of currents. An important factor concerning the resistance between the working electrode and counterelectrode is that the cell is in the feedback loop of an operational amplifier as shown in Figure 5. As the solution resistance increases, the amplifier must reach a higher output voltage in order to pass the desired current through the cell. Our instrument uses a high voltage (f150 V) amplifier just for this reason (18). A current of 1 p A (very large by LCEC standards) could be passed through a cell with a resistance of 150 M Q (orders of magnitude larger than typical) between the working electrode and counterelectrode. Potential Scanning LCEC. To demonstrate the utility of potential scanning CPA for obtaining Ellz values of eluting peaks, the aminophenol separation was repeated. The coulostat scanned continuously between 0.55 and 1.18 V as the chromatogram was run. Figure 6 shows the result. In this figure, the current values obtained a t a given potential are connected and the data appear as a set of ten chromatograms, each one obtained at a different potential. It can be seen that p-aminophenol is oxidized even at the lowest potential, oaminophenol appears only above 0.6 V, and m-aminophenol does not appear until 0.9 V. If instead of plotting current at a given potential vs. time (as in the chromatograms of Figure 6) one plots current at a given time vs. potential, one can obtain the voltammograms shown in Figure 7. From plots like these one can readily
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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500
Time ( 8 1
Flgure 6. Chromatogram of a mixture of aminophenols (each at 333 pM) using potential scanning CPA. The number by each trace indicates
the electrode potential (V).
Figure 6 represents 130 voltammetricscans in a time period when only three species eluted, and for which only one scan for each peak would be necessary to obtain the desired Ellz data. Optimum use of the CPA technique results if the coulostat operates at a single potential to obtain maximum precision for quantitation. Once per peak a scan would be run; a convenient time is shortly after the peak maximum has passed. Such a strategy is easily accomplished with computer-controlled instrumentation. The half wave potentials obtained can serve as valuable information for identification of eluting species or to verify the presence or absence of a component in a mixture. If complete voltammograms are not required, the ability of the coulostat to change potential rapidly still can be a valuable asset. Species that are not chromatographically separated often can be separately quantitated by monitoring at more than one potential. Multiple potential monitoring, using multiple working electrodes and multiple amperometric detectors, has been used in LCEC for such purposes (25,26). By using CPA one could cycle between two or three selected potentials at a single electrode. Registry No. Caffeic acid, 331-39-5;o-chlorophenol,95-57-8; o-aminophenol,95-55-6;m-aminophenol,591-27-5;p-aminophenol, 123-30-8.
LITERATURE CITED
~ 4 0
0.60
0.80
1.00
1.20
Potential ( V )
Hydrodynamic voltammograms for (0)p amlnophenol, (A) rn-aminophenol, and (0) o-aminophenol from the chromatogram of Figure 6. Figure 7.
determine Ellz values which could then serve to identify eluting species. This experiment has been repeated with concentrations as low as 25 pM with comparable results. It should be emphasized that the data presented in Figures 6 and 7 were obtained from a single chromatographic run. If a similar set of 10-point voltammograms were to be obtained with a conventional amperometric LCEC detector (or using single potential CPA), one would need to do ten separate chromatograms, each one run at a different potential. We have done such an experiment (using a Bioanalytical Systems LC-4B detector) and obtained voltammograms essentially identical in shape with those presented in Figure 7. Voltammograms of micromolar levels of eluting species from a single chromatographicrun have also been reported by use of square wave voltammetry (15). Single-potential CPA yields chromatograms with a higher signal to noise ratio and a higher density of data points/unit time on a single chromatographic trace than does potential scanning CPA. For potential scanning CPA, the number of potential steps in the scan (10 in this example), and the hold, integrate, and pause times (100 ms, 100 ms, and 1 s in this example) determine the number of data points per potential acquired across the peak. With these experimental parameters, five to eight points per potential (i.e., five to eight complete voltammograms) were acquired across the width (at half height) of the chromatographic peaks shown in Figures 3 and 6. Depending upon the particular electrode, cell configuration, potential range, analyte, and concentration, scans can be run two to five times faster.
(1) Kissinger, P. T. Anal. Chem. 1977, 49, 447A-456A. (2) Rucki, R. J. Talanta 1980, 27, 147-156. (3) Toth, K.; Nagy, G.; Feher, 2.;Horvai, G.; Pungor, E. Anal. Chim. Acta 1980, 114, 45-48. (4) Stulik, K.; Pacakova, V. J . Necfroanal. Chem. 1981, 129, 1-24. (5) Ryan, M. D.; Wllson, G. S. Anal. Chem. 1982, 54, 20R-27R. (6) Majors, R. E.; Barth, H. G.; Lochmuller, C. H. Anal. Chem. 1982, 54, 323R-363R. (7) Shoup, R. E., Ed. "Recent Reports on Liquid Chromatography/ Electrochemistry"; Bloanalytical Systems, Inc.: West Lafayette, IN, 1982. (8) Dieker, J. W.; van der Linden, W. E.; Poppe, H. Talanta 1979, 26, 511-518. (9) Samuelsson, R.; Osteryoung. J. Anal. Chim. Acta 1981, 123, 97-105. (10) Swartzfager, D. G. Anal. Chem. 1978, 48, 2189-2192. (11) MacCrehan, W. A. Anal. Chem. 1981, 53, 74-77. (12) Kemula, W.; Kutner, W. J . Chromatogr. 1981, 204, 131-134. (13) Hanekamp, H. B.; Voogt, W. H.; Frei, R. W.; Bos, P. Anal. Chem. 1981, 53, 1362-1365. (14) Wang, J.; Ouzlel, E.; Yarnltzky, D. H.; Ariel, M. Anal. Chim. Acta 1978, 102, 99-112. (15) Samuelsson, R., ODea, J, J.; Osteryoung, J. Anal. Chem. 1980, 52, 2215-2216. (16) van Leeuwen, H. P. I n "Electroanalytical Chemistry"; Bard, A. J., Ed.; Marcel Dekker: New York, 1982; Voi. 12, pp 159-238. (17) Bard, A. J.; Faulkner, L. R. "Electrochemical Methods"; Wiley: New York, 1980; pp 270-276. (18) Reiss, J. J.; Nieman, T. A. Anal. Chem. 1983, 55, 1236-1240. (19) Reiss, J. J.; Nieman, T. A. Plttsburgh Conference on Analytical Chemlstry and Applled Spectroscopy, March 10-14, 1980; paper no. 105. (20) Barnes, A. C.; Niernan, T. A. Great Lakes Regional ACS Meeting, June 7-9, 1982; paper no. 84. (21) Schreiber, M. A.; Last, T. Anal. Chem. 1981, 53, 2095-2100. (22) Last, T. Anal. Chem. 1982, 54, 2327-2332. (23) Goto, M.; Koyanagl, Y.; Ishii, D. J . Chromatogr. 1981, 208, 261-268. (24) Powley, C. R.; Geiger, R. F., Jr.; Nieman, T. A. Anal. Chem. 1980, 52, 705-709. (25) Klssinger, P. T.: Roston, D. Curr. SeparaNons 1981, 3 (l), 7-8. (26) Mayer, 0 . S.; Shoup, R. E. Curr. Separations 1982, 4 (3). 40-42.
RECEIVED for review March 28, 1983. Resubmitted and accepted August 22,1983. This work was supported in part by the National Science Foundation (CHE-81-08816),a NIH Biomedical Research Support Grant (RR07030), and the Environmental Protection Agency. Although the research in this article has been funded in part by the United States Environmental Protection Agency through Cooperative Agreement CR 806819 to the Advanced Environmental Control Technology Research Center, University of Illinois, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.
