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amperometric detection for liquid chromatography
Analytica Chimica Acta, 200 (1987) 101-114 Elsevier Science Publishers B.V., Amsterdam -Printed
VOLTAMMETRIC/AMPEROMETRIC CHROMATOGRAPHY
in The Netherlands
DETECTION
FOR LIQUID
CRAIG E. LUNTEa, JOHN F. WHEELER and WILLIAM R. HEINEMAN* Department
of Chemistry,
University of Cincinnati, Cincinnati, OH 45221
(U.S.A.)
(Received 7th April 1987)
SUMMARY A voltammetric/amperometric detector based on a dual-electrode electrochemical detector is described for liquid chromatography. The detector combines the advantages of both voltammetric and amperometric detection. A threedimensional data array of current response as a function of both time (chromatographic domain) and potential (electrochemical domain) is obtained. From the chromatographic point of view, this allows postexperimental choice of the optimal detection potential. Different detection potentials can even be chosen for each chromatographic peak. Having the voltammetric data as well as the chromatographic data provides ready identification of chromatographitally unresolved compounds and the ability to resolve such co-eluting compounds voltammetrically, The voltammetric data also provide a second method of peak identification for greater certainty in peak assignments. Voltammetric detection limits of less than 10 pmol of material injected on the column were achieved with this detection method. From the electrochemical perspective, voltammetric/amperometric detection provides a technique for obtaining hydrodynamic voltammograms with small amounts or small volumes of sample. Voltammograms can also be obtained for the individual components of complex mixtures without the need for isolation steps.
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*William R. Hememan received his B.S. degree from Texas Tech University m 1964 and his Ph.D. from the University of North Carolina at Chapel Hill in 1968. He was a research chemist at the Hercules Research Center from 1968 to 1970 before becoming a research associate with Professor Ted Kuwana at Case Western Reserve Umversity and The Ohio State University. He joined the faculty at the University of Cincinnati in 1972 where he is Professor and Chairman of the Analytical Division. Heineman’s research interests include thin-layer spectroelectrochemistry, EKAFS spectroelectrochemistry, electrochemical immunoassay, polymer modified electrodes, stripping voltammetry and the analytical chemistry of technetium radiopharmaceuticals. aCurrent address: Department of Chemistry, The University of Kansas, Lawrence, KS 66045, U.S.A. 0003-2670/87/$03.50
o 1987 Elsevier Science Publishers B.V.
Development of acceptable voltammetric detectors has been hindered by the much higher detection limits that can be achieved with these detectors because of the large charging currents associated with scanning the applied potential. To overcome this charging-current problem, several techniques have been reported. Pulse techniques are the most commonly used methods to diminish charging-current contributions. With these techniques the charging current is allowed to decay before the faradaic current is sampled. Several pulse waveforms have been used, including staircase [2] , differential pulse [3] , normal pulse [ 41, and squarewave [ 5--91. In addition to pulse techniques, alternating current (AC) voltammetric techniques have been used to discriminate against the charging current [lo] . Recently, microelectrodes have been used with electrochemical detectors to alleviate charging-current problems [ll, 121 . Coulostatic detectors have also been designed to obtain voltammetric information [ 13, 141. Even with these advanced techniques, the detection limits achieved with voltammetric detection are several orders of magnitude higher than for amperometric detectors. In recent work from this laboratory, a voltammetric detector was described for flow-injection systems [15, 161 . A series-configuration dualelectrode thin-layer flow cell is used in which the potential at the upstream electrode is scanned while the potential of the downstream electrode is held constant. A similar approach has been described by Trubey and Nieman [17], using a coulostatic technique to achieve the potential sweep. The downstream electrode is used to monitor either the formation of product or depletion of reactant at the upstream electrode. If products are detected, the technique is termed collection mode, while if depletion of reactant is determined the technique is termed shielding mode. In this way, the current response at the downstream electrode reflects the voltammetric behavior of the analyte at the upstream electrode without the charging current associated with scanning the potential. These processes are illustrated in Fig. 1 along with the upstream excitation waveform and a typical downstream response. A detection limit of lo-’ M was achieved by flow injection analysis during the initial characterization of this detection technique. In this report, the detection technique, termed voltammetric/amperometric detection, is extended to application to liquid chromatography. Instrumental improvements are described and data analysis techniques are demonstrated. Sample analysis is illustrated by the detection of oxidizable compounds in a beer extract. EXPERIMENTAL
Reagents
and instrumentation
Caffeic acid, p-coumaric acid, gentisic acid, and vanillic acid were obtained from Sigma Chemical Co. Ferulic acid and sinapic acid were purchased from Aldrich Chemical Co. All chemicals were used as received. Standard solutions
103
Shleldtng
Fig. 1. Timing diagram for dual-electrode voltammetric/amperometric detection in the collection and shielding modes. Wl, upstream electrode; W2, downstream electrode; E, upstream (Wl) potential; I,, upstream (Wl) current; Id, downstream (W2) current; At, time delay between electrodes.
were prepared in 0.1 M ammonium phosphate buffer, pH 4. Stock solutions were prepared fresh weekly and standards were prepared daily from the stock solutions. The chromatographic system was a Bianalytical Systems (West Lafayette, IN) LC-400 liquid chromatograph. A 2007.~1 sample loop was used for all experiments. A Brownlee RP-18 ODS, 5 pm (4.6 mm X 10 cm) column was used. The mobile phase was 0.1 M ammonium phosphate buffer, pH 4 with 15% (v/v) methanol. A flow rate of 1 ml min-’ was used unless otherwise noted. A BAS dual-electrode thin-layer cell with glassy-carbon working electrodes was used. The reference electrode was Ag/AgCl for all experiments and potentials were reported versus this reference. Waveform generation and data acquisition and analysis were controlled by a Zenith 158 personal computer (Franklin Park, IL) with 640 kbyte of memory and 20-Mbyte hard disk. The computer was interfaced with an ADALAB-PC interface card (Interactive Microware, State College, PA) equipped with a 12-bit fast analog-to-digital converter (A/D), a 12-bit integrating A/D, two 12-bit digital-to-analog converters (D/A), and four 16-bit timers. The D/As had output ranges of -2.5 to + 2.5 V. The integrating A/D had a range of -1.0 to + 1.0 V while the fast A/D had a range of -10 to +lO V. The rate of data acquisition and waveform output during an
104
individual potential scan was controlled by one of the timers. The ADALABPC interface card was connected to a Pine Instrument RDE-3 bipotentiostat (Grove City, PA) through its external potential inputs and current jacks. The bipotentiostat provided the reference and current-to-voltage converter circuitry but was not used to generate the waveform. Software for data acquisition and instrument timing was written in assembly language while software for data analysis and processing was written in both BASIC and assembly language. Data was stored in memory during the chromatography and could be transferred for permanent storage to a disk (either hard or floppy) after data collection was complete. Real-time display of the current at a userdefined potential was used to monitor the chromatogram. The waveform consists of 100 equally spaced steps from the initial potential to the final potential. After the final step, the potential is returned to its initial value until the next scan is initiated. The initial potential, final potential, and scan rate are userdefined parameters which set the step height and step width. The scan frequency is user-defined with the limitation that there be sufficient delay between initiation of each scan for the scan to occur plus approximately 450 ms of overhead time for data to be transferred to high memory. In general, a scan rate of 2.0 V s-l over a 1.0-V range at a frequency of 1 scan s-l provides good voltammetric information while maintaining good density in the chromatographic data. Because of memory limitations of the personal computer system, a maximum of 1600 scans can be acquired for any chromatographic run. Sample preparation The beer sample (1 ml) was acidified to pH 2 with acetic acid. The acidified sample was then applied to a C-18 Sep-Pak cartridge (Waters Associates). The Sep-Pak was rinsed with 10 ml of water and the phenolic acids then eluted with 2 ml of 0.5 M ammonia solution. The basic extract was acidified with acetic acid and injected onto the analytical column. RESULTS
AND DISCUSSION
Background subtraction In the ideal case, voltammetric/amperometric detection would exhibit no charging-current response at the downstream amperometric electrode. However, the two working electrodes in the dual-electrode cell are not completely independent and a small current is observed at the downstream electrode when a large current is present at the upstream electrode. This nonfaradaic current at the amperometric electrode is termed cross-talk and arises from small changes in the interfacial potential at the amperometric electrode caused by the iR drop of the scanned potential electrode. The absolute cross-talk current is scan-rate dependent but the relative crosstalk is independent of scan rate and is approximately 0.1% of the upstream charging current for the electrochemical cell used in these experiments.
105
Even in the presence of the cross-talk background current, voltammetric/ amperometric detection provides lower detection limits than direct voltammetric detection because the background is 3-4 orders of magnitude smaller. In addition, because this background is now on the same order of magnitude as the signal (nA), subtraction of the background signal is possible to lower the detection limit further. Similar background subtraction techniques have previously been used with direct voltammetric detection [2, 111. In these experiments, background voltammograms are collected prior to sample injection until a steady background is achieved. This steady background voltammogram is stored in memory and can be subtracted from subsequent chromatographic voltammograms to lower the detection limit. Figure 2 shows the raw and background-subtracted voltammograms for a chromatographic injection of 85 pmol of caffeic acid, All subsequent voltammograms shown are background-subtracted. Flow characterization Under the hydrodynamic conditions of the series dual-electrode thin-layer flow cell, there is a finite time delay between when any given segment of solution flows over the upstream electrode and when it reaches the downstream electrode. This time delay must be known in order to correlate the downstream current response to the upstream potential. This time delay is dependent upon the flow rate and cell geometry. Because the cell geometry is constant for all experiments reported here, only the flow-rate dependence need be determined. The flow-rate dependence was found as previously described [16] . A plot of the inverse of delay time (s-l) versus flow rate (~1 s-l) gave a straight line of slope 1.03 ~1~’ with an intercept of -1.11 s-l and a correlation coefficient (rZ) of 0.9991. This relationship is incorporated into the software to correlate the current to the potential. The shape of the voltammetric wave is also dependent on flow rate. The voltammograms of chromatographic peaks exhibited maxima followed by a plateau region at higher potential. This behavior cannot be ascribed to a change in the concentration of the analyte during the course of the voltammetry because peaking is observed in voltammograms from both the leading and tailing edge of the chromatographic peak. The degree of “peaking” was found to increase as the flow rate increased (Fig. 3). This is in contrast to the results of White et al. [ 111 who observed an inverse relationship between “peaking” and flow rate for a fiber microelectrode thin-layer cell. For the present flow-injection system, this inverse relationship has also been observed, but only at low flow rates (<0.5 ml min-‘). At the flow rates used here, welldefined hydrodynamic voltammograms were observed when the usual flow-injection configuration was used to introduce the sample. While this result does not detract from the utility of voltammetric/amperometric detection with this cell, work is currently under way to attempt to explain these anomalous results.
106
Fig. 2. Background current subtraction. (A) Background current (----) and raw voltammogram for 85 pmol of caffelc acid injected (-). (B) Background-subtracted voltammogram. The scan rate was 2.0 V 6-I and the retention time for caffeic acid was 4.5 min.
Effect of scan rate Because the resistance of the thin-layer flow cell is high, iR drop considerations are important. With the flow cell used in these experiments, scan rates up to 2.0 V s-l could be used with little apparent iR drop. At scan rates of greater than 5.0 V s-l, the iR drop is apparent as a skewing of the voltammetric curve (Fig. 4). A scan rate of 2.0 V s-l is sufficient for chromatographic detection. This allows a large potential window to be scanned while maintaining good density in the chromatographic data. For example, a typical experiment would be to scan a 1.0-V potential window at a frequency of 1 scan s-l. The scan rate also affects the degree of “peaking” of the voltammograms as described previously. The slower the scan rate, the more peaked the voltammograms, while the plateau current is independent of scan rate. This behavior is also contrary to that observed in flow injection analysis and reported by White et al. [ll] for a carbon fiber microelectrode. Because of this scan rate dependence of the peak current, quantitation at the peak potential will be more sensitive but dependent on scan rate, However, quantitation at a potential on the current plateau will be slightly less sensitive but the current response will be independent of scan rate. While the results shown for the previous sections have been for collection-mode detection, the results are the same when the shielding mode is used. Chromatographic detection Collection mode. The collection mode is achieved by operating the downstream electrode at a potential to reverse the reaction occurring at the upstream electrode. In other words, this detection mode involves redox cycling of the analyte and is amenable only to chemically reversible compounds. The chemical reversibility of a compound determines its collection efficiency. The collection efficiency is defined as the ratio of downstream response to upstream response when both responses are on their respective mass transport-limited plateaus, In this application, the collection efficiency can be
107
t 04
Fig. 3. Effect of flow rate on collection mode voltammetry. Caffeic acid (5 nmol) injected with a scan rate of 2.0 V s-l ; 9 is the normalized current response. Flow rate: (...) 1 ml min-’ , (----) 2 ml min-’ ; (-) 3 ml inn?. Fig. 4. Effect of scan rate on collection mode voltammetry. Caffeic acid (5 nmol) injectedataflowrateof3mlmin~‘.Scanrate:(-)0.5Vs“,(----)1.0Vs~‘;(~~~)2.0Vs~‘; (-*-) 5.0 v s-l.
considered as a response factor for a given compound. The collection efficiencies of the phenolic acids used in this study are listed in Table 1. In addition to depending upon the compound, the collection efficiency is a function of the cell geometry. For the cell used in these studies, a collection efficiency of 0.3 corresponds to a chemically reversible compound. Voltammetric/amperometric detection results in a three-dimensional data array of current response versus both time and applied potential. A chromatovoltammogram for a mixture of phenolic acids is shown in Fig. 5. The optimal detection potential depends on the compound and experimental conditions. An advantage of voltammetric/amperometric detection is that all of the information needed to make the choice of the optimal detection potential is generated at the time of the experiment. Voltammograms of the eluting peaks can be obtained by looking at a potential scan at the chromatographic peak. Figure 6 shows the voltammograms of the peaks from the chromatovoltammogram of Fig. 5. From these voltammograms, the proper choice of potential for chromatographic detection can be made from the actual data instead of relying on predetermined values. For example, to determine all of the phenolic acids in the sample, a potential of 1.2 V is required as shown in Fig. 7A. If it is preferred not to detect the phenolic acids which are difficult to oxidize, a potential of 0.9 V can be chosen as shown in Fig. 7B. At this potential, gentisic acid, caffeic acid, and sinapic acid are still on their mass-transport limited plateau, whereas vanillic acid, p-coumaric acid, and ferulic acid give very little response at 0.9 V. If these hard-to-oxidize compounds are of interest and the other compounds are interferences, a difference mode approach can be used
108 TABLE 1 Collection and shielding efficiencies for phenolic acids Compound
Collection efficiency
Shielding efficiency
Compound
Collection efficiency
Shielding efficiency
Gentisic acid Vanillic acid Caffeic acid
0.30 0.24 0.30
0.68 0.68 0.69
p-Coumaric acid Ferulic acid Sinanic acid
0.07 0.23 0.13
0.68 0.69 0.70
as previously described [ll, 161. A difference chromatogram of i(1.2 V) i(0.9 V) is shown in Fig. 7C. All the chromatograms in Fig. 7 are from a single chromatographic run. Voltammetric/amperometric detection allows choice of optimal detection method with little pre-experimental planning. Because all of the voltammetric and chromatographic data are stored in memory, several presentation techniques can be evaluated for optimal information retrieval. By using a flow rate of 1.0 ml min-’ and a scan rate of 2.0 V s-l with a scan frequency of 1 scan s-l, current response was linear over at least four orders of magnitude (320 nmol-32 pmol gentisic acid injected, slope = 0.095 nA pmol-‘, intercept = -0.74 nA, r2 = 0.9998) using either the peak or the plateau currents. A voltammetric detection limit of 7.4 pmol of gentisic acid (retention time, 1.5 min) and 32 pmol of caffeic acid (retention
POTENTIAL
I’.‘)
\s
Fig. 5. Chromatovoltammog*of phenolic acid mixture obtained in the collection mode. Scan rate 2.0 V s-i , flow rate 1.0 ml min-’ , downstream (amperometric) detection at -0.2 V. Injection of 80 pmol of gentisic acid, 130 pmol of vanillic acid, 220 pmol of caffeic acid, 520 pmol of pcoumaric acid, 410 pmol of ferulic acid, and 580 pmol of sinapic acid. Fig. 6. Voltammograms of the chromatographic peaks extracted from the chromatovoltammogram of Fig. 5. Compounds: (a) caffeic acid; (b) gentisic acid; (c) sinapic acid; (d) ferulic acid; (e) vanillic acid; (f) pcoumaric acid. 0 is the normalized current response.
109
Fig. ‘7. Individual chromatograms extracted from the chromatovoltammogram of Fig. 5: (A) single-potential chromatogram at 1.2 V, (B) single-potential chromatogram at 0.9 V; (C) difference chromatogram of i(1.2 V) - i(0.9 V). Peak identities: (1) gentisic acid, (2) vanillic acid; (3) caffeic acid; (4) p-coumaric acid, (5) ferulic acid ; (6) sinapic acid.
time, 4.5 min) injected on column was achieved with acceptable voltammetric response (Fig. 8). The detection limit is obviously dependent on the retention time of the compound with later eluting compounds having higher detection limits because of dilution during the chromatographic separation. However, voltammetric detection of 10 to 100 pmol injected is readily achieved. As can be seen from Fig. 8, the detection limit is determined by the resolution of the analog-to-digital converter and not by the noise of the system. Because the background is subtracted after data collection, at low analyte concentrations the background signal limits the gain that can be used without saturating the output. This gain and the resolution of the A/D converter then determine the smallest current changes that can be differentiated. This digitization is readily apparent in Fig. 8. In order to achieve reasonable voltammetric curves at least a lo--20 bit change is necessary. With the system used for these experiments, the digitization limited the ability to obtain good voltammetric curves rather than the noise in the voltammetry. The use of an A/D converter with greater resolution should lower the detection limits. Shielding mode. The shielding mode is achieved by operating the downstream electrode at a potential on the mass transport-limited plateau for the reaction occurring at the upstream electrode. The upstream voltammetry will then cause a decrease in the response at the downstream electrode. Shielding-mode detection is characterized by the shielding efficiency which is analogous to the collection efficiency. The shielding efficiency, as shown
110
Fig. 8. Voltammograms of caffeic acid (B).
from the injection of 7.4 pmol of gentisic acid (A) and 32 pmol
in Table 1, is independent of the electrochemistry of a given compound and is dependent only on the cell geometry. Because the shielding mode makes use of mass-transport phenomena while the collection mode makes use of electrochemical phenomena, these are complementary techniques. The collection mode offers greater selectivity while the shielding mode is more universal. As the hardware and software requirements are identical, both methods can be applied to the same sample on alternative chromatographic runs to establish the most useful method for a given sample. Figure 9 shows the chromatovoltammogram obtained m the shielding mode for the same phenolic acid mixture used previously with the collection mode. As can be seen, the response is markedly different when the shielding mode is used. The maximum chromatographic response is obtained at the smallest upstream potential because all material passes to the downstream
Fig. 9. Chromatovoltammogram of phenolic acid mixture obtained in the shielding mode. Scan rate 2.0 V s-’ , flow rate 1.0 ml min-‘, downstream detection (amperometric) at +1.2 V. The mixture was the same as for Fig. 5.
111
electrode to be detected. As the potential of the upstream electrode reaches values sufficient to oxidize the eluting material, the downstream response decreases because the amount of material in the reduced form reaching the downstream electrode is decreased. While the three-dimensional presentation may appear confusing, the same information is obtained with this experimental arrangement as with the collection mode. Voltammograms can be obtained by taking the “spine” of a chromatographic peak and are identical to those obtained in the collection mode. The same types of chromatographic presentations can be done with the shielding-mode data as with collection-mode data. However, in the case of shielding mode, the most sensitive response is at the lowest upstream potential (Fig. 10A and B). Difference-mode presentation can also be used to detect hard-to-oxidize compounds selectively in the presence of easily oxidized compounds, as shown in Fig. 1OC. The shielding-mode response was linear over four orders of magnitude (320 nmol or 16 pmol of gentisic acid injected, slope = 0.093 nA pmol-‘, intercept = -0.19 nA, r * = 0 .9999). The detection limit for the shielding mode is slightly lower than for the collection mode with this detector configuration. While it would seem that the detection limit should be lower for the collection mode because the downstream potential is more optimal for amperometric detection, in practice the downstream noise is lower in the shielding mode. This is because the major source of noise in the voltammetric signal arises from the cross-talk interference which is somewhat lower in the shielding mode for the cell and potentiostat design of these
TIME (m(n)
Fig. 10 Individual chromatograms extracted from the chromatovoltammogram of Fig. 9: (A) single-potential chromatogram at 0.1 V, (B) single-potential chromatogram at 1.2 V; (C) difference chromatogram of i(1.2 V) - i(0.9 V). Peak identities as in Fig. 7.
112
experiments. A voltammetric detection limit of 3.7 pmol of gentisic acid and 16 pmol of caffeic acid injected on column was achieved. Test sample. To test the utility of this detector, a sample of extracted beer was injected onto the chromatographic column. The potential was scanned from 0.0 V to 1.2 V at 2.0 V s-l at a frequency of 1 scan s-l. The three-dimensional chromatogram is shown in Fig. 11. A chromatogram extracted from the three-dimensional data is shown in Fig. 12A. Chromatographic peaks B, C, E, F and G elute at the same time as standards of vanillic acid, caffeic acid, pcoumaric acid, ferulic acid and sinapic acid, respectively. The voltammetric responses of these peaks are also the same as for the standard compounds (Fig. 6). Combining both chromatographic and voltammetric comparisons provides a high degree of certainty for these assignments of peak identity. The voltammetry can also provide information about unidentified peaks. For example, examination of the voltammetry of chromatographic peak A (Fig. 13) indicates that there are actually two unresolved components. This can be seen by comparing voltammograms from the front, peak and tail and noting how the voltammetry changes across the peak. While chromatographitally unresolved, the two components can be resolved voltammetrically. This can be done either by using the extracted voltammogram or by using the difference-mode chromatographic presentation as shown in Fig. 12B. A more complete evaluation of the utility of voltammetric deconvolution of chromatographically unresolved peaks is currently underway.
Fig. 11. Chromatovoltammogram
of beer extract.
113
T
IOnA I
G
T
4nA 1
Fig. 12. Individual chromatograms (A) single-potential chromatogram
i(0.9 V).
extracted from the chromatovoltammogram ofFig. 11: at 1.2 V; (B) difference chromatogram of i(1.2 V) -
Conclusions Voltammetric/amperometric detection provides advantages relative to either amperometric or direct voltammetric detection individually. The voltammetric data generated during the chromatographic analysis can be used for confirmation of peak identity or classification. Having the entire chromatovoltammogram allows post-experimental choice of the optimal detection potential, including the use of different potentials for each chromatographic peak. Adding voltammetric resolution to the chromatographic resolution permits identification of coeluting compounds and the ability to resolve them voltammetrically. Voltammetric/amperometric detection realizes these advantages while achieving detection limits lower than direct voltammetric techniques and approaching amperometric detection. Improvements in the computer interPOTENTIAL
IVolts,
-. 02
-- 04
0 --06
-- 06
-
Fig. 13. Voltammograms
10
of the front (a..), peak (-)
and tail (---) of peak A.
114
face and the flow cell should lower the detection limits even further. From detection an electrochemrcal point of view, voltammetric/amperometric provides a method of obtaining voltammograms with small amounts or small volumes of sample. Voltammograms of the individual components of a complicated mixture can also be obtained without the need for isolation. This last capability should find great use in bioelectrochemical investigations. REFERENCES 1 R. E. Shoup, Ed., Bibliography of Recent Reports of Electrochemical Detection, BAS Press, West Lafayette, IN, 1982. 2 W. L. Caudill, A. G. Ewing, S. Jones and R. M. Wightman, Anal. Chem., 55 (1983) 1877. 3 W. A. MacCrehan, Anal. Chem., 53 (1981) 74. 4 M. Stastny, R. Volf, H. Benadikova and I. Vit, J. Chromatogr. Sci., 21 (1983) 18. 5 R. Samuelson. J. O’Dea and J. G. Osteryoung, Anal. Chem., 52 (1980) 2215. 6 J. Wang, E. Ouziel and C. H. Yarnitzky, Anal. Chim. Acta, 102 (1978) 99. 7 J. J. Scanlon, P. A. Flaquer, G. W. O’Brien and P. E. Sturrock, Anal. Chim. Acta, 158 (1984) 169. 8 P. A. Reardon, G. E. O’Brien and P. E. Sturrock, Anal. Chim. Acta, 162 (1984) 175. 9 M. B. Thomas, H. Msimanga and P. E. Sturrock, Anal. Chim. Acta, 174 (1985) 287. 10 R. E. Panzer and P. J. Elving, Electrochim. Acta, 20 (1975) 635. 11 J. G. White, R. L. St.Claire III and J. W. Jorgenson, Anal. Chem., 58 (1986) 293. 12 J. G. White and J. W. Jorgenson, Anal. Chem., 58 (1986) 2992. 13 T. A. Last, Anal. Chim. Acta, 155 (1983) 287. 14 T. A. Last, Anal. Chem., 55 (1983) 1509. 15 C. E. Lunte, S.-W. Wong, K. W. Chan, T. H. Ridgway and W. R. Heineman, Anal. Chim. Acta, 188 (1986) 263. 16 C. E. Lunte and W. R. Heineman, Anal. Chem., 59 (1987) 761. 17 R. K. Trubey and T. A. Nieman, Anal. Chem., 58 (1986) 2549.
VOLTAMMETRIC/AMPEROMETRIC CHROMATOGRAPHY
in The Netherlands
DETECTION
FOR LIQUID
CRAIG E. LUNTEa, JOHN F. WHEELER and WILLIAM R. HEINEMAN* Department
of Chemistry,
University of Cincinnati, Cincinnati, OH 45221
(U.S.A.)
(Received 7th April 1987)
SUMMARY A voltammetric/amperometric detector based on a dual-electrode electrochemical detector is described for liquid chromatography. The detector combines the advantages of both voltammetric and amperometric detection. A threedimensional data array of current response as a function of both time (chromatographic domain) and potential (electrochemical domain) is obtained. From the chromatographic point of view, this allows postexperimental choice of the optimal detection potential. Different detection potentials can even be chosen for each chromatographic peak. Having the voltammetric data as well as the chromatographic data provides ready identification of chromatographitally unresolved compounds and the ability to resolve such co-eluting compounds voltammetrically, The voltammetric data also provide a second method of peak identification for greater certainty in peak assignments. Voltammetric detection limits of less than 10 pmol of material injected on the column were achieved with this detection method. From the electrochemical perspective, voltammetric/amperometric detection provides a technique for obtaining hydrodynamic voltammograms with small amounts or small volumes of sample. Voltammograms can also be obtained for the individual components of complex mixtures without the need for isolation steps.
To date, raphy
have
most
reports
dealt
with
on electrochemical
amperometry
of operation
of such detectors
can
be
achieved.
because
they
readily
increasing in both
the potential
and the extremely
Interest provide
detection
in voltammetric
improved
(electrochemical)
for liquid
[ 11. This has been
resolving
because
low detection detectors, power
chromatogof the ease limits
by obtaining
and time (chromatographic)
which
however,
is data
domains,
*William R. Hememan received his B.S. degree from Texas Tech University m 1964 and his Ph.D. from the University of North Carolina at Chapel Hill in 1968. He was a research chemist at the Hercules Research Center from 1968 to 1970 before becoming a research associate with Professor Ted Kuwana at Case Western Reserve Umversity and The Ohio State University. He joined the faculty at the University of Cincinnati in 1972 where he is Professor and Chairman of the Analytical Division. Heineman’s research interests include thin-layer spectroelectrochemistry, EKAFS spectroelectrochemistry, electrochemical immunoassay, polymer modified electrodes, stripping voltammetry and the analytical chemistry of technetium radiopharmaceuticals. aCurrent address: Department of Chemistry, The University of Kansas, Lawrence, KS 66045, U.S.A. 0003-2670/87/$03.50
o 1987 Elsevier Science Publishers B.V.
Development of acceptable voltammetric detectors has been hindered by the much higher detection limits that can be achieved with these detectors because of the large charging currents associated with scanning the applied potential. To overcome this charging-current problem, several techniques have been reported. Pulse techniques are the most commonly used methods to diminish charging-current contributions. With these techniques the charging current is allowed to decay before the faradaic current is sampled. Several pulse waveforms have been used, including staircase [2] , differential pulse [3] , normal pulse [ 41, and squarewave [ 5--91. In addition to pulse techniques, alternating current (AC) voltammetric techniques have been used to discriminate against the charging current [lo] . Recently, microelectrodes have been used with electrochemical detectors to alleviate charging-current problems [ll, 121 . Coulostatic detectors have also been designed to obtain voltammetric information [ 13, 141. Even with these advanced techniques, the detection limits achieved with voltammetric detection are several orders of magnitude higher than for amperometric detectors. In recent work from this laboratory, a voltammetric detector was described for flow-injection systems [15, 161 . A series-configuration dualelectrode thin-layer flow cell is used in which the potential at the upstream electrode is scanned while the potential of the downstream electrode is held constant. A similar approach has been described by Trubey and Nieman [17], using a coulostatic technique to achieve the potential sweep. The downstream electrode is used to monitor either the formation of product or depletion of reactant at the upstream electrode. If products are detected, the technique is termed collection mode, while if depletion of reactant is determined the technique is termed shielding mode. In this way, the current response at the downstream electrode reflects the voltammetric behavior of the analyte at the upstream electrode without the charging current associated with scanning the potential. These processes are illustrated in Fig. 1 along with the upstream excitation waveform and a typical downstream response. A detection limit of lo-’ M was achieved by flow injection analysis during the initial characterization of this detection technique. In this report, the detection technique, termed voltammetric/amperometric detection, is extended to application to liquid chromatography. Instrumental improvements are described and data analysis techniques are demonstrated. Sample analysis is illustrated by the detection of oxidizable compounds in a beer extract. EXPERIMENTAL
Reagents
and instrumentation
Caffeic acid, p-coumaric acid, gentisic acid, and vanillic acid were obtained from Sigma Chemical Co. Ferulic acid and sinapic acid were purchased from Aldrich Chemical Co. All chemicals were used as received. Standard solutions
103
Shleldtng
Fig. 1. Timing diagram for dual-electrode voltammetric/amperometric detection in the collection and shielding modes. Wl, upstream electrode; W2, downstream electrode; E, upstream (Wl) potential; I,, upstream (Wl) current; Id, downstream (W2) current; At, time delay between electrodes.
were prepared in 0.1 M ammonium phosphate buffer, pH 4. Stock solutions were prepared fresh weekly and standards were prepared daily from the stock solutions. The chromatographic system was a Bianalytical Systems (West Lafayette, IN) LC-400 liquid chromatograph. A 2007.~1 sample loop was used for all experiments. A Brownlee RP-18 ODS, 5 pm (4.6 mm X 10 cm) column was used. The mobile phase was 0.1 M ammonium phosphate buffer, pH 4 with 15% (v/v) methanol. A flow rate of 1 ml min-’ was used unless otherwise noted. A BAS dual-electrode thin-layer cell with glassy-carbon working electrodes was used. The reference electrode was Ag/AgCl for all experiments and potentials were reported versus this reference. Waveform generation and data acquisition and analysis were controlled by a Zenith 158 personal computer (Franklin Park, IL) with 640 kbyte of memory and 20-Mbyte hard disk. The computer was interfaced with an ADALAB-PC interface card (Interactive Microware, State College, PA) equipped with a 12-bit fast analog-to-digital converter (A/D), a 12-bit integrating A/D, two 12-bit digital-to-analog converters (D/A), and four 16-bit timers. The D/As had output ranges of -2.5 to + 2.5 V. The integrating A/D had a range of -1.0 to + 1.0 V while the fast A/D had a range of -10 to +lO V. The rate of data acquisition and waveform output during an
104
individual potential scan was controlled by one of the timers. The ADALABPC interface card was connected to a Pine Instrument RDE-3 bipotentiostat (Grove City, PA) through its external potential inputs and current jacks. The bipotentiostat provided the reference and current-to-voltage converter circuitry but was not used to generate the waveform. Software for data acquisition and instrument timing was written in assembly language while software for data analysis and processing was written in both BASIC and assembly language. Data was stored in memory during the chromatography and could be transferred for permanent storage to a disk (either hard or floppy) after data collection was complete. Real-time display of the current at a userdefined potential was used to monitor the chromatogram. The waveform consists of 100 equally spaced steps from the initial potential to the final potential. After the final step, the potential is returned to its initial value until the next scan is initiated. The initial potential, final potential, and scan rate are userdefined parameters which set the step height and step width. The scan frequency is user-defined with the limitation that there be sufficient delay between initiation of each scan for the scan to occur plus approximately 450 ms of overhead time for data to be transferred to high memory. In general, a scan rate of 2.0 V s-l over a 1.0-V range at a frequency of 1 scan s-l provides good voltammetric information while maintaining good density in the chromatographic data. Because of memory limitations of the personal computer system, a maximum of 1600 scans can be acquired for any chromatographic run. Sample preparation The beer sample (1 ml) was acidified to pH 2 with acetic acid. The acidified sample was then applied to a C-18 Sep-Pak cartridge (Waters Associates). The Sep-Pak was rinsed with 10 ml of water and the phenolic acids then eluted with 2 ml of 0.5 M ammonia solution. The basic extract was acidified with acetic acid and injected onto the analytical column. RESULTS
AND DISCUSSION
Background subtraction In the ideal case, voltammetric/amperometric detection would exhibit no charging-current response at the downstream amperometric electrode. However, the two working electrodes in the dual-electrode cell are not completely independent and a small current is observed at the downstream electrode when a large current is present at the upstream electrode. This nonfaradaic current at the amperometric electrode is termed cross-talk and arises from small changes in the interfacial potential at the amperometric electrode caused by the iR drop of the scanned potential electrode. The absolute cross-talk current is scan-rate dependent but the relative crosstalk is independent of scan rate and is approximately 0.1% of the upstream charging current for the electrochemical cell used in these experiments.
105
Even in the presence of the cross-talk background current, voltammetric/ amperometric detection provides lower detection limits than direct voltammetric detection because the background is 3-4 orders of magnitude smaller. In addition, because this background is now on the same order of magnitude as the signal (nA), subtraction of the background signal is possible to lower the detection limit further. Similar background subtraction techniques have previously been used with direct voltammetric detection [2, 111. In these experiments, background voltammograms are collected prior to sample injection until a steady background is achieved. This steady background voltammogram is stored in memory and can be subtracted from subsequent chromatographic voltammograms to lower the detection limit. Figure 2 shows the raw and background-subtracted voltammograms for a chromatographic injection of 85 pmol of caffeic acid, All subsequent voltammograms shown are background-subtracted. Flow characterization Under the hydrodynamic conditions of the series dual-electrode thin-layer flow cell, there is a finite time delay between when any given segment of solution flows over the upstream electrode and when it reaches the downstream electrode. This time delay must be known in order to correlate the downstream current response to the upstream potential. This time delay is dependent upon the flow rate and cell geometry. Because the cell geometry is constant for all experiments reported here, only the flow-rate dependence need be determined. The flow-rate dependence was found as previously described [16] . A plot of the inverse of delay time (s-l) versus flow rate (~1 s-l) gave a straight line of slope 1.03 ~1~’ with an intercept of -1.11 s-l and a correlation coefficient (rZ) of 0.9991. This relationship is incorporated into the software to correlate the current to the potential. The shape of the voltammetric wave is also dependent on flow rate. The voltammograms of chromatographic peaks exhibited maxima followed by a plateau region at higher potential. This behavior cannot be ascribed to a change in the concentration of the analyte during the course of the voltammetry because peaking is observed in voltammograms from both the leading and tailing edge of the chromatographic peak. The degree of “peaking” was found to increase as the flow rate increased (Fig. 3). This is in contrast to the results of White et al. [ 111 who observed an inverse relationship between “peaking” and flow rate for a fiber microelectrode thin-layer cell. For the present flow-injection system, this inverse relationship has also been observed, but only at low flow rates (<0.5 ml min-‘). At the flow rates used here, welldefined hydrodynamic voltammograms were observed when the usual flow-injection configuration was used to introduce the sample. While this result does not detract from the utility of voltammetric/amperometric detection with this cell, work is currently under way to attempt to explain these anomalous results.
106
Fig. 2. Background current subtraction. (A) Background current (----) and raw voltammogram for 85 pmol of caffelc acid injected (-). (B) Background-subtracted voltammogram. The scan rate was 2.0 V 6-I and the retention time for caffeic acid was 4.5 min.
Effect of scan rate Because the resistance of the thin-layer flow cell is high, iR drop considerations are important. With the flow cell used in these experiments, scan rates up to 2.0 V s-l could be used with little apparent iR drop. At scan rates of greater than 5.0 V s-l, the iR drop is apparent as a skewing of the voltammetric curve (Fig. 4). A scan rate of 2.0 V s-l is sufficient for chromatographic detection. This allows a large potential window to be scanned while maintaining good density in the chromatographic data. For example, a typical experiment would be to scan a 1.0-V potential window at a frequency of 1 scan s-l. The scan rate also affects the degree of “peaking” of the voltammograms as described previously. The slower the scan rate, the more peaked the voltammograms, while the plateau current is independent of scan rate. This behavior is also contrary to that observed in flow injection analysis and reported by White et al. [ll] for a carbon fiber microelectrode. Because of this scan rate dependence of the peak current, quantitation at the peak potential will be more sensitive but dependent on scan rate, However, quantitation at a potential on the current plateau will be slightly less sensitive but the current response will be independent of scan rate. While the results shown for the previous sections have been for collection-mode detection, the results are the same when the shielding mode is used. Chromatographic detection Collection mode. The collection mode is achieved by operating the downstream electrode at a potential to reverse the reaction occurring at the upstream electrode. In other words, this detection mode involves redox cycling of the analyte and is amenable only to chemically reversible compounds. The chemical reversibility of a compound determines its collection efficiency. The collection efficiency is defined as the ratio of downstream response to upstream response when both responses are on their respective mass transport-limited plateaus, In this application, the collection efficiency can be
107
t 04
Fig. 3. Effect of flow rate on collection mode voltammetry. Caffeic acid (5 nmol) injected with a scan rate of 2.0 V s-l ; 9 is the normalized current response. Flow rate: (...) 1 ml min-’ , (----) 2 ml min-’ ; (-) 3 ml inn?. Fig. 4. Effect of scan rate on collection mode voltammetry. Caffeic acid (5 nmol) injectedataflowrateof3mlmin~‘.Scanrate:(-)0.5Vs“,(----)1.0Vs~‘;(~~~)2.0Vs~‘; (-*-) 5.0 v s-l.
considered as a response factor for a given compound. The collection efficiencies of the phenolic acids used in this study are listed in Table 1. In addition to depending upon the compound, the collection efficiency is a function of the cell geometry. For the cell used in these studies, a collection efficiency of 0.3 corresponds to a chemically reversible compound. Voltammetric/amperometric detection results in a three-dimensional data array of current response versus both time and applied potential. A chromatovoltammogram for a mixture of phenolic acids is shown in Fig. 5. The optimal detection potential depends on the compound and experimental conditions. An advantage of voltammetric/amperometric detection is that all of the information needed to make the choice of the optimal detection potential is generated at the time of the experiment. Voltammograms of the eluting peaks can be obtained by looking at a potential scan at the chromatographic peak. Figure 6 shows the voltammograms of the peaks from the chromatovoltammogram of Fig. 5. From these voltammograms, the proper choice of potential for chromatographic detection can be made from the actual data instead of relying on predetermined values. For example, to determine all of the phenolic acids in the sample, a potential of 1.2 V is required as shown in Fig. 7A. If it is preferred not to detect the phenolic acids which are difficult to oxidize, a potential of 0.9 V can be chosen as shown in Fig. 7B. At this potential, gentisic acid, caffeic acid, and sinapic acid are still on their mass-transport limited plateau, whereas vanillic acid, p-coumaric acid, and ferulic acid give very little response at 0.9 V. If these hard-to-oxidize compounds are of interest and the other compounds are interferences, a difference mode approach can be used
108 TABLE 1 Collection and shielding efficiencies for phenolic acids Compound
Collection efficiency
Shielding efficiency
Compound
Collection efficiency
Shielding efficiency
Gentisic acid Vanillic acid Caffeic acid
0.30 0.24 0.30
0.68 0.68 0.69
p-Coumaric acid Ferulic acid Sinanic acid
0.07 0.23 0.13
0.68 0.69 0.70
as previously described [ll, 161. A difference chromatogram of i(1.2 V) i(0.9 V) is shown in Fig. 7C. All the chromatograms in Fig. 7 are from a single chromatographic run. Voltammetric/amperometric detection allows choice of optimal detection method with little pre-experimental planning. Because all of the voltammetric and chromatographic data are stored in memory, several presentation techniques can be evaluated for optimal information retrieval. By using a flow rate of 1.0 ml min-’ and a scan rate of 2.0 V s-l with a scan frequency of 1 scan s-l, current response was linear over at least four orders of magnitude (320 nmol-32 pmol gentisic acid injected, slope = 0.095 nA pmol-‘, intercept = -0.74 nA, r2 = 0.9998) using either the peak or the plateau currents. A voltammetric detection limit of 7.4 pmol of gentisic acid (retention time, 1.5 min) and 32 pmol of caffeic acid (retention
POTENTIAL
I’.‘)
\s
Fig. 5. Chromatovoltammog*of phenolic acid mixture obtained in the collection mode. Scan rate 2.0 V s-i , flow rate 1.0 ml min-’ , downstream (amperometric) detection at -0.2 V. Injection of 80 pmol of gentisic acid, 130 pmol of vanillic acid, 220 pmol of caffeic acid, 520 pmol of pcoumaric acid, 410 pmol of ferulic acid, and 580 pmol of sinapic acid. Fig. 6. Voltammograms of the chromatographic peaks extracted from the chromatovoltammogram of Fig. 5. Compounds: (a) caffeic acid; (b) gentisic acid; (c) sinapic acid; (d) ferulic acid; (e) vanillic acid; (f) pcoumaric acid. 0 is the normalized current response.
109
Fig. ‘7. Individual chromatograms extracted from the chromatovoltammogram of Fig. 5: (A) single-potential chromatogram at 1.2 V, (B) single-potential chromatogram at 0.9 V; (C) difference chromatogram of i(1.2 V) - i(0.9 V). Peak identities: (1) gentisic acid, (2) vanillic acid; (3) caffeic acid; (4) p-coumaric acid, (5) ferulic acid ; (6) sinapic acid.
time, 4.5 min) injected on column was achieved with acceptable voltammetric response (Fig. 8). The detection limit is obviously dependent on the retention time of the compound with later eluting compounds having higher detection limits because of dilution during the chromatographic separation. However, voltammetric detection of 10 to 100 pmol injected is readily achieved. As can be seen from Fig. 8, the detection limit is determined by the resolution of the analog-to-digital converter and not by the noise of the system. Because the background is subtracted after data collection, at low analyte concentrations the background signal limits the gain that can be used without saturating the output. This gain and the resolution of the A/D converter then determine the smallest current changes that can be differentiated. This digitization is readily apparent in Fig. 8. In order to achieve reasonable voltammetric curves at least a lo--20 bit change is necessary. With the system used for these experiments, the digitization limited the ability to obtain good voltammetric curves rather than the noise in the voltammetry. The use of an A/D converter with greater resolution should lower the detection limits. Shielding mode. The shielding mode is achieved by operating the downstream electrode at a potential on the mass transport-limited plateau for the reaction occurring at the upstream electrode. The upstream voltammetry will then cause a decrease in the response at the downstream electrode. Shielding-mode detection is characterized by the shielding efficiency which is analogous to the collection efficiency. The shielding efficiency, as shown
110
Fig. 8. Voltammograms of caffeic acid (B).
from the injection of 7.4 pmol of gentisic acid (A) and 32 pmol
in Table 1, is independent of the electrochemistry of a given compound and is dependent only on the cell geometry. Because the shielding mode makes use of mass-transport phenomena while the collection mode makes use of electrochemical phenomena, these are complementary techniques. The collection mode offers greater selectivity while the shielding mode is more universal. As the hardware and software requirements are identical, both methods can be applied to the same sample on alternative chromatographic runs to establish the most useful method for a given sample. Figure 9 shows the chromatovoltammogram obtained m the shielding mode for the same phenolic acid mixture used previously with the collection mode. As can be seen, the response is markedly different when the shielding mode is used. The maximum chromatographic response is obtained at the smallest upstream potential because all material passes to the downstream
Fig. 9. Chromatovoltammogram of phenolic acid mixture obtained in the shielding mode. Scan rate 2.0 V s-’ , flow rate 1.0 ml min-‘, downstream detection (amperometric) at +1.2 V. The mixture was the same as for Fig. 5.
111
electrode to be detected. As the potential of the upstream electrode reaches values sufficient to oxidize the eluting material, the downstream response decreases because the amount of material in the reduced form reaching the downstream electrode is decreased. While the three-dimensional presentation may appear confusing, the same information is obtained with this experimental arrangement as with the collection mode. Voltammograms can be obtained by taking the “spine” of a chromatographic peak and are identical to those obtained in the collection mode. The same types of chromatographic presentations can be done with the shielding-mode data as with collection-mode data. However, in the case of shielding mode, the most sensitive response is at the lowest upstream potential (Fig. 10A and B). Difference-mode presentation can also be used to detect hard-to-oxidize compounds selectively in the presence of easily oxidized compounds, as shown in Fig. 1OC. The shielding-mode response was linear over four orders of magnitude (320 nmol or 16 pmol of gentisic acid injected, slope = 0.093 nA pmol-‘, intercept = -0.19 nA, r * = 0 .9999). The detection limit for the shielding mode is slightly lower than for the collection mode with this detector configuration. While it would seem that the detection limit should be lower for the collection mode because the downstream potential is more optimal for amperometric detection, in practice the downstream noise is lower in the shielding mode. This is because the major source of noise in the voltammetric signal arises from the cross-talk interference which is somewhat lower in the shielding mode for the cell and potentiostat design of these
TIME (m(n)
Fig. 10 Individual chromatograms extracted from the chromatovoltammogram of Fig. 9: (A) single-potential chromatogram at 0.1 V, (B) single-potential chromatogram at 1.2 V; (C) difference chromatogram of i(1.2 V) - i(0.9 V). Peak identities as in Fig. 7.
112
experiments. A voltammetric detection limit of 3.7 pmol of gentisic acid and 16 pmol of caffeic acid injected on column was achieved. Test sample. To test the utility of this detector, a sample of extracted beer was injected onto the chromatographic column. The potential was scanned from 0.0 V to 1.2 V at 2.0 V s-l at a frequency of 1 scan s-l. The three-dimensional chromatogram is shown in Fig. 11. A chromatogram extracted from the three-dimensional data is shown in Fig. 12A. Chromatographic peaks B, C, E, F and G elute at the same time as standards of vanillic acid, caffeic acid, pcoumaric acid, ferulic acid and sinapic acid, respectively. The voltammetric responses of these peaks are also the same as for the standard compounds (Fig. 6). Combining both chromatographic and voltammetric comparisons provides a high degree of certainty for these assignments of peak identity. The voltammetry can also provide information about unidentified peaks. For example, examination of the voltammetry of chromatographic peak A (Fig. 13) indicates that there are actually two unresolved components. This can be seen by comparing voltammograms from the front, peak and tail and noting how the voltammetry changes across the peak. While chromatographitally unresolved, the two components can be resolved voltammetrically. This can be done either by using the extracted voltammogram or by using the difference-mode chromatographic presentation as shown in Fig. 12B. A more complete evaluation of the utility of voltammetric deconvolution of chromatographically unresolved peaks is currently underway.
Fig. 11. Chromatovoltammogram
of beer extract.
113
T
IOnA I
G
T
4nA 1
Fig. 12. Individual chromatograms (A) single-potential chromatogram
i(0.9 V).
extracted from the chromatovoltammogram ofFig. 11: at 1.2 V; (B) difference chromatogram of i(1.2 V) -
Conclusions Voltammetric/amperometric detection provides advantages relative to either amperometric or direct voltammetric detection individually. The voltammetric data generated during the chromatographic analysis can be used for confirmation of peak identity or classification. Having the entire chromatovoltammogram allows post-experimental choice of the optimal detection potential, including the use of different potentials for each chromatographic peak. Adding voltammetric resolution to the chromatographic resolution permits identification of coeluting compounds and the ability to resolve them voltammetrically. Voltammetric/amperometric detection realizes these advantages while achieving detection limits lower than direct voltammetric techniques and approaching amperometric detection. Improvements in the computer interPOTENTIAL
IVolts,
-. 02
-- 04
0 --06
-- 06
-
Fig. 13. Voltammograms
10
of the front (a..), peak (-)
and tail (---) of peak A.
114
face and the flow cell should lower the detection limits even further. From detection an electrochemrcal point of view, voltammetric/amperometric provides a method of obtaining voltammograms with small amounts or small volumes of sample. Voltammograms of the individual components of a complicated mixture can also be obtained without the need for isolation. This last capability should find great use in bioelectrochemical investigations. REFERENCES 1 R. E. Shoup, Ed., Bibliography of Recent Reports of Electrochemical Detection, BAS Press, West Lafayette, IN, 1982. 2 W. L. Caudill, A. G. Ewing, S. Jones and R. M. Wightman, Anal. Chem., 55 (1983) 1877. 3 W. A. MacCrehan, Anal. Chem., 53 (1981) 74. 4 M. Stastny, R. Volf, H. Benadikova and I. Vit, J. Chromatogr. Sci., 21 (1983) 18. 5 R. Samuelson. J. O’Dea and J. G. Osteryoung, Anal. Chem., 52 (1980) 2215. 6 J. Wang, E. Ouziel and C. H. Yarnitzky, Anal. Chim. Acta, 102 (1978) 99. 7 J. J. Scanlon, P. A. Flaquer, G. W. O’Brien and P. E. Sturrock, Anal. Chim. Acta, 158 (1984) 169. 8 P. A. Reardon, G. E. O’Brien and P. E. Sturrock, Anal. Chim. Acta, 162 (1984) 175. 9 M. B. Thomas, H. Msimanga and P. E. Sturrock, Anal. Chim. Acta, 174 (1985) 287. 10 R. E. Panzer and P. J. Elving, Electrochim. Acta, 20 (1975) 635. 11 J. G. White, R. L. St.Claire III and J. W. Jorgenson, Anal. Chem., 58 (1986) 293. 12 J. G. White and J. W. Jorgenson, Anal. Chem., 58 (1986) 2992. 13 T. A. Last, Anal. Chim. Acta, 155 (1983) 287. 14 T. A. Last, Anal. Chem., 55 (1983) 1509. 15 C. E. Lunte, S.-W. Wong, K. W. Chan, T. H. Ridgway and W. R. Heineman, Anal. Chim. Acta, 188 (1986) 263. 16 C. E. Lunte and W. R. Heineman, Anal. Chem., 59 (1987) 761. 17 R. K. Trubey and T. A. Nieman, Anal. Chem., 58 (1986) 2549.