Daryl A. Roston
lnstrumentation
Chemistry Department Northern Illinois University
Dekalb, 111.
Ronald E. Shoup Research Laboratories Bioanalytical Systems, Inc. West Lafayene, Ind.
Peter 1.Kissinger Chemistry Department Purdue University and Bioanalylical Systems Inc. West Lafayetie, Ind.
LiquidChromatography/Electroc hemistry: Thin-Layer Multiple ElectrodeDetection Since 1972,over 600 reports concerning the development or utilization of liquid chromatographylelectrochemistry (LCEC) have been published ( I ). This rapid increase in the use of LCEC during the past decade attests to the capabilities of the technique. It has been used effectively to address problems in areas as diverse as drug metabolism, forensic chemistry, food science, and neurochemistry. An approach to electrochemical detection that is currently the focus of growing interest is the simultaneous use of two or more working electrodes in LCEC, rather than only one. Dualelectrode detection can be used to improve selectivity, detection limits, and peak identification. It can also extend the accessible potential range of an electrochemical detector. Multipleelectrode detection in a variety of formats will help sustain the growth in LCEC applications during the next several years. The purpose of this article is to provide an introduction to dual-electrode detection for liquid chromatography. Dual-electrode instrumentation and detector cell design considerations are briefly described, with additional emphasis on the improvements that can be manifested by using two detectors instead of one. Due to space limitations, the thinlayer approach will be emphasized. There are, of course, many other possibilities, a few of which have been published ( I ) . 0003-2700/82/A351-1417$01.00.0
0 1982 American Chemical Society
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Figure 1. Circuitry for dual-electrode amperometric detection VI and V2 are Ue output voltages tor the two working ektrades. W, and WI, held at -El and -4 n. Ref. the reference electrw!+ OA = operational amplifier. R = resistor. i = current, Aux = auxiliary Slectrode
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
1417A
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Figure 2. Options for a thin-layer amp e r m t r i c detector: single, paralleladjacent, and series configuration The stainless steel auxiliary elecbode (Aux) is the entire top half 01 me cube. Ref = reference eleG bode,W = warking electrade
lnrtrumentatlon Electronics for Dual-Electrode Detection. Instrumentation required for implementing amperometric or voltammetric experiments simultaneously at different electrodes has been used by electrochemists for years. Dual-electrode“potentiostats” have been developed for use with rotating ring-disk systems ( 2 )and twinelectrode thin-layer cells (3).Two reports have described circuitry for a dual-electrode potentiostat specifically designed for liquid chromatographic dual-electrode detection ( 4 , 5 ) . The principles of dual-electrode potentiostat operation can be understood by considering the circuit shown in Figure 1. The difference in potential between the solution and the working electrode dictates the chemistry that occurs at the working-electrode surface. For single-electrode operation, solution potential control is achieved with a feedback system consisting of an operational amplifier voltage follower (OA-l), the reference electrode, and the auxiliary electrode. The reference electrode “monitors” the difference in potential between the input voltage of OA-l and the solution. The difference is adjusted to zero by OA-1 by changing the solution potential with the auxiliary electrode. Since W1 is held at virtual ground, the potential of W1 with respect to the reference electrode is -El. 1418 A
For dual-electrode detection, the xoblem is that of controlling the poentia1 at the solution-surface interface of a second working electrode (Wz). Nothing can be done to incorporate an independent second working electrode if this action would change the potential of the bulk solution. Otherwise the interfacial potential of the first working electrode would be changed. The recourse is to vary the interfacial potential difference of the iecond electrode by changing its po.entia1 external to the electrochemical cell. If the external potential of Wz is at the potential of the circuit common, then it would necessarily have the same interfacial potential as WI, since the bulk solution potential would also De the same for both electrodes. The iolution is to float the circuit common for the second current-to-voltage converter (OA-3) to a potential which is the difference between the desired electrode potentials for W1 and Wp. Wp is now held at E1 - E Z ,so its potential with respect to the reference electrode is -E*. Obviously, a great deal more is involved in producing a practical device than is depicted in Figure 1. Factors such as time constants, background currents, and offset circyits have to be
considered. Some details are available in previously published reports (2-5). Cell Design Considerations for Dual-Electrode Detection. Many of the considerations involved in designing a dual-electrode detector are the same as those involved in designing a single-electrodedetector. Fundamen.tal aspects of electrochemical cell design have been discussed in several recent reviews (S9). Minimizing cell impedance is important for singleelectrode detection; however, problems arising from high impedance can be particularly acute for dual-electrode operation. In dual-electrode detection, a current response at one electrode causes an iR drop, which results in a change of the solution potential and affects the potentials of both electrode-solution interfaces. This resulting “cross-talk” can render dualelectrode detection impossible to use if the uncompensated resistance ( R ) is too large. Cross-talk problems can he minimized by positioning the auxiliary electrode as close as possible to the working electrodes, minimizing cell impedance. For multiple electrodes in a thin-layer cell, this may be achieved by placing the auxiliary electrode directly across the thin layer from the working electrodes.
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Figure 3. Cyclic voltammogram for caffeic acid Condnlans medla. w a t ( l - ~ m n o ~ - p o p ~ n o l - a c e acd t s (40.4 1 1). 0 018 M ammonium acelate. scan-rale 100 mV16. glassy carbon e ectrde (OCEl Reprmled hom Reterence 13
ANALYTICAL CHEMISTRY, VOL. 54. NO. 13, NOVEMBER 1982
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The basis of the popularity of thinlayer amperometry lies in the adaptability of the thin-layer detector cell for many different types of analytical tasks. One dual-electrode thin-layer amperometric detector is shown in Figure 2. The parameters responsible for the excellent performance characteristics of a single-electrodethinlayer amperometric cell are not altered by introducing the second detector electrode in the thin layer. By sim ply changing or rotating the bottom half of the thin-layer cell, it is possible to use a single electrode or dual electrodes in the parallel-adjacent or series orientations (Fipure 2). A wide variety of electrode materials have heen utilized in this design, including glassy carbon, carbon paste, platinum, gold, nickel, and mercury-gold amalgam. By changing the working electrode size and shape an enormous number of possibilities exist. Detection Improvements with DualElectrode Detection Series Dual-Electrode Detection. The majority of dual-electrode detection publications have dealt with series detection (note Figure 2) (1&20). With series detection the eluant is modified by the upstream detector so that a more useful chromatogram can he recorded at the downstream detector. Several approaches to series detection have been reported. Schieffer utilized a series coulometric-amperometric detector to improve selectivity. Interfering compounds that were more easily oxidized than the compounds of interest were removed with the upstream coulometric cell (10.11).An early use of series dual-electrode detection was reported by King and Kissinger (12).By adding Br- to the aqueous mobile phase, the upstream detector could be used for the constant current generation of bromine. Use of a reaction coil between the upstream and downstream detector allowed the generated bromine to react with eluting analytes, such as unsaturated fatty acids and prostaglandins. The corresponding decrease in the bromine level resulted in a decrease in current at the downstream electrode where the back-reduction of unreacted bromine to bromide was amperometrically monitored. Most series dual-electrode detection work has involved the use of two amperometric detectors positioned in series in a conventional thin-layer cell (13-17,19,20). Series thin-layer amperometric detection can he more clearly understood by considering the cyclic voltammogram of caffeic acid shown in Figure 3. Well-defined oxidation and reduction currents are evident because the oxidized and reduced forms of caffeic acid are stable. Figure 1422A
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Figure 4. Series duaI-electrOde chromatograms for caffeic acid Condi1ions:glassy ~ d e l e c t o r p o l m l a l ~ . ( a ) W = +~l . O V , W e = t l . O V , ( b ) W 1 =+l.OV.WI= 0.0 Y: 25cm Biophaae C4*column: flow rate = 1 mL/mln: mobile phase, wamr-me~no1-popBnoIn s :circular OCEdetectars. acetic acid(404l:l). 0.018 M ammmium a&b. D ~ t B * ~ d i " ~ ! ~ i odual 3" diameter: elechda spacing 0.1 mn: mln-layer channel uoss-seclion dlmensims. 0.013 cm X 0.5 mm. Reprinted hom Reference 19
4a depicta the series chromatogram for caffeic acid when both electrodes are poised at +LO V. Oxidative responses are observed at the upstream (WJ and downstream (Wp) electrodes. The response of the downstream electrode is smaller because of the depletion of caffeic acid at the upstream electrode. Figure 4b shows the series chromatograms resulting when the downstream electrode potential was changed to 0.00 V. An oxidative response was observed at WI and a reductive current was observed at Wp. The reductive current at WZcorresponds to the reduction of the oxidized form of caffeic acid produced at WI. Improvements resulting from series amperometric detection are a function of the downstreamhpstream peak current ratio, the collection efficiency (13,141: Collection efficiency = 'dpsmatream lupstr-
Because the collection efficiency is a complex function of several parameters, a range of collection efficiencies is often observed for compounds in a given sample. Maximum collection achievable with a given detector is determined by critical cell dimensions, namely, the distance between the electrodes and the ratio of the lengths of the electrodes along the flow-axis (21-23). Less than maximum collection is usually the result of chemical
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
reactions (such as hydrolysis or oxidative coupling) of the upstream detector product that render the product electroinactive at the downstream detector potential; however, numerous additional factors can decrease collection (13).Maximum values for series detectors comprised of equal-surfacearea planar electrodes in a thin-layer cell range from 37% to 42% (13-16, 22).Also, Roston and Kissinger reported collection values ranging from 3%to 37% for a series of monocyclic phenolic acids separated by reversedphase chromatography (13). If the electrode surface areas are different, then the collection values will reflect this. In the limit, for large surface areas, the collection value will approach 100%for chemically reversible redox couples. Several aspects of electrochemical detection can be improved with series amperometric detection. Selectivity can be improved for compounds which have collection values that are significantly higher than coeluting compounds. Because of differences in collection, the relative responses of eluting compounds change, with the responses of "high" collection compounds becoming more dominant. Improvements in selectivity achieved with series amperometric detection are illustrated by Figure 5, which shows simultaneous series detector chromatograms of ethylacetate extracts of a commercial beer sample. In
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