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Anal. Chem. 1981, 53, 1700-1704
Dual-Electrode, Liquid Chromatographic Detector for the Determination of Analytes with High Redox Potentials Wllllam A. MacCrehan” and Richard A. Durst Organic Analytical Research Division, Center for Analytlcal Chemistry, Natlonal Bureau of Standards, Washlngton, D.C. 20234
An electrochemical detection approach for ilquld chromatography Is described utilizing two sequential, generator/detector electrodes. Anaiytes are first electrolyzed, and the reaction products are then detected electrochemically at a second electrode. In some cases, this allows the detectlon of analytes with high redox potentlals, wlth good sensltlvlty and selectivity. The technique Is tested on several organic analytes: dichlone, nltrobenzene, and three organotln catlons.
Electrochemical detection in liquid chromatography has been shown to be a powerful approach for the trace measurement of many important organic analytes ( I , 2). However, most applications of this technique have focused on the determination of analytes that have low oxidation of reduction potentials. Detection at potentials more positive than +1 V or more negative than -1 V (vs. a Ag/AgCl reference), using either carbon-based or mercury-based electrodes, results in a severe loss in sensitivity and selectivity in the aqueous solvents employed in liquid chromatography (3). Ideally, the analyst would like to convert analytes that have high reduction or oxidation potentials into more easily detectable forms. In some situations, precolumn or postcolumn chemical derivatization reactions can be used very effectively (4), but these approaches are often cumbersome. A second alternative has recently appeared (5),in which an “on-line” coulometric generator is used in a precolumn electrolysis to convert the analyte of interest. However, from an analytical viewpoint, the fact that many electrode reactions produce multiple products may render the precolumn reaction approach unsuitable for many determinations. A second approach overcomes this limitation, where the electrochemical conversion occurs after the chromatographic separation. One application by Eggli and Asper (6) employs a porous silver/amalgam electrode for the coulometric reduction of disulfide bonds. A second thin-layer detector cell with a mercury pool electrode is attached “downstream” for the detection of the electrogenerated sulfhydryl compounds, by measuring the anodic reaction current with mercury. In this paper, a single-cell, dual-electrode detector with an amperometric generator electrode is described. Several aspects of this mode of detection are considered, including the analytical advantages and liabilities, cell configurations, electrode materials and residual current, detection limits, and the use of a modulated potential at the generator electrode. EXPERIMENTAL SECTION Apparatus. The liquid chromatographemployed was modified specifically for use in conjuction with reductive electrochemical detection. A borosilicate-glass solvent reservoir was purged continuously with high-purity argon. Stainless steel tubing was used for all inlet and outlet tubes. A commercially available solvent metering pump, incorporating a two-piston, pressurefeedback design, was modified to reduce oxygen leakage into the system by replacing the Teflon tube inlet with stainless steel tubing. A fixed volume (20 pL) injector was used for the argon-purged samples. The dual-electrode voltammetric cell design is illustrated in Figure 1. This thin-layer flow cell consists of two individual,
three-electrode halves. The potentials of the generator and detector electrodes are measured with respect to separate reference electrodes (Ag/AgCl, 3 mol/L C1-). The generator potential was controlled either by a potentiostat or manually with a battery, resistor, and high-impedance input digital voltmeter. In all cases, only the detector electrode was grounded. A commercially available potentiostat was used for the amperometric measurementa at the detector electrode. A simple, commercially available analog lock-in-amplifier,with single phase-angle capability, was used for detection in the generator-modulation experiments. Reagents. HPLC-grade water, methanol (MeOH),and acetonitrile (MeCN)were obtained from a commercial supplier. The ammonium acetate (NH,OAc), used as supporting electrolyte, was prepared by neutralizing acetic acid with ammonium hydroxide to a pH of 5.0. Analytical standard dichlone (2,3-dichloro-1,4naphthoquinone) was obtained from the EPA Quality Assurance Laboratory, Research Triangle Park, NC.
RESULTS AND DISCUSSION Dual-Electrode Cells. The dual-electrode approach consists of two electrodes used in a manner conceptually similar to the rotating ring-disk pair but adapted to a thinlayer flow-cell arrangement. The principle is illustrated in Figure 2, with relative dimensions of one of our cells shown to scale. The first electrode encountered by the flowing solvent, designated the generator electrode, is held a t a potential sufficient to electrolyze the analytes of interest. The downstream electrode, the detector electrode, is poised at a different potential, but one that is sufficient to detect the electrochemically generated products. Thus, in order for the approach to be applicable, the analytes must be electroactive and form reaction products that are detectable at low electrode potentials. By conversion of the difficult-to-detect analytes to much more easily detectable products, detection can be at potentials where selectivity and sensitivity are high. The principle of the dual-electrode, electrochemical flow cell was first developed by Gerischer et al. (7). Their voltammetric cell consisted of two closely spaced gold-foil electrodes similar in principle to Figure 2. The hydrodynamic response of this cell was evaluated assuming laminar flow (8, and a detailed mathematical model was developed by Braun (8).
Another design (9) using dual-electrodes but in radially symmetric arrangement was developed by Schieffer and Blaedel for “anodic stripping with collection” using mercury-coated glassy carbon. These cells with equal-sized closely spaced electrodes provide the advantage of a well-defined, short-transport time for the electrogenerated products. This is particularly useful in kinetics studies and, as we shall demonstrate, for modulated experiments. However, the product generating efficiency of the small upstream electrode provides relatively low sensitivity. T o increase the efficiency of electrochemical product formation, some workers have used a coulometric electrode as generator. Porous silver (IO),glassy carbon ( I I , I 2 ) , and silver amalgam (6) have been used. One disadvantage of the use of the coulometric generator electrode is the necessity of applying a substantially larger potential than the voltammetric plateau requires (to ensure 100% current efficiency and to
Thls artlcle not subject to U S . Copyright. Published 1981 by the Amerlcan Chemical Society
ANA1.YllCAL CHEMISTRY. VOL. 53. NO. 11. SEPTEMBER 1981
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lncorpora~a large (5.0 mm) generator and small (1.0 mm) detector elecbode; (E) “sandwicW cell. wing equaCslzed. parallel elecbodes. detector, the relative signal, expressed as a percentage (%RS), was evaluated by using a reversible test compound. Dichlone was selected by virtue of ita reversible behavior a t silveramalgam electmdea in reverse-pulse voltammetry (vide infra). The direct, reductive signal (chromatographic peak height) was measured for the two-electron reduction (to form the hydroquinone) a t the detector when poised at 4 . 4 V, but with no potential applied to the generator. A second experiment was then run under the same conditions, except a potential of 4 . 4 V was applied to the generator and a potential of +0.2 V (sufficient for the two-electron oxidation of the hydroquinone) was applied to the detector. The %RS was then calculated
%RS = Ox signal (for electrogenerated hydroquinone) x loo Red signal (for quinone) mm+
Flgun 2. Duaklecbode concept.
overcome the substantial iR drop often encountered at hiah current). This requirement lowers the obtainable selectivity and restricts the effective redox potential that can be applied without solvent-discharge bubble formation. Secondly, coulometric electrodes have an inherently large hold-up volume; for example, the generator in ref 6 required a volume of over 200 rL. Such large dead volumes are unsuitable for highefficiency liquid chromatography. For the work presented in this paper, amperometric generator electrodes were chosen. Figure 3 shows two electrode configurations, viewed perpendicularly to the flowing solvent, with the cell spacer gasket in place. The upper cell employs a large diameter (5 mm) circular electrode as generator, in conjunction with a closely spaced 1 m m diameter detector electrode. This “horseshoe cell” gives a large dual-electrode signal, by virtue of the large surface area of the generator; however, the transport time for the reaction products is poorly defined. The second design in the lower part of Figure 3 overcomes this problem at the sacrifice of the generator area. This demountable “sandwich cell” consists of two parallel 1.0 X 3.5 mm plates, separated by a replaceable polyethylene spacer gasket (0.1 mm). For comparison of the results obtained with the two dual-electrode cell designs to that of a direct, single-electrode
(1) which expresses the relative amount of analytical signal in the dual-electrode mode compared to the direct reduction. The %RSfor the horseshoe cell was 60% as compared to 37% for the sandwich design, when identical electrode materials were used a t a flow rate of 1.0 mL/min. The %RS measured in this manner reapresents a relatively ideal case, the detection of a reversibly behaved compound. However, many analytea that are irreversibly behaved can also be detected by this dual-electrode approach, as long as an electroactive product is formed. I t should be noted that the dual-electrode detection approach may be matrix sensitive, since the detector response depends critically on the chemical reactions that follow the electron transfer at the generator electrode. Sample matrix constituents may influence the course of the reaction product formation, thereby affecting the sensitivity. This is particularly likely for highly reactive free-radical products. In many cases, however, the initial electron transfer products react rapidly with the solvent (in aqueous systems) to produce stable products (on the time scale of the dual-electrode experiment), and the response of such analytes should have low matrix dependence. Analyte Detectability. In order to evaluate the applicability of the dual-electrode detection approach to a specific analyte, a study of the electrode reaction must.be made. For this work, reversepulse voltammetry was used to provide the
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
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Potential in V 4. Normal and reverse pulse voltammetry of nitrobenzene: upper trace, normal-pulse mode; lower trace, reverse-pulse mode. Conditions: electrode Hg/Ag, 2.0 mm button; scan rate, 5 mVls; semple rate, 1.0 Hz; solvent, 50% MeOHlH,O, 0.1 mol/L NH,OAc (pH 5.0); concentrations, 2.0 X lo-‘ mol/L. ~~~~~~
information necessary for the detection of electrogenerated products. The important principles of this method were described in detail by Osteryoung et al. (13). Briefly, in reverse-pulse voltammetry, a potential is applied to a static elwtrode that is sufficient to cause electrolysis of the analyte, and periodically, potential pulses of linearly increasing magnitude and short duration are applied. The resulting current, measured at the end of the pulse, is a characteristic of the reaction products formed at the base potential. The time scale for the formation of products is similar to that for the dual-electrode experiment. Figure 4 shows both the normal-pulse and the reverse-pulse behavior of nitrobenzene on a mercury-coated silver (Hg/Ag) button electrode. The negative-scanningmode reveals the reduction of the compound with Ell2 = -0.58 V. The reverse-pulse scan, based a t a potential of -0.8 V, shows a very small amount of the reverse reaction (oxidation of the nitrobenzene radical) but also shows a more substantial amount of the oxidation of phenylhydroxylamine, one major product of the reduction (14) with Ell2 = $0.03 V. Thus, for dual-electrode detection, a generator potentid of about -0.8 V and detector potential of +0.1 V will allow the detection of nitrobenzene. Residual Current at Different Electrodes. In order to apply the dual-electrode technique to reducible analytes at high sensitivity, it was necessary to evaluate detector electrode materials for their residual current (since the magnitude of this current frequently determines the detection limit (15)). In this work, two dual-electrode material pairs were chosen: generator-Hg/Ag, detector-Hg/Ag; generator-Hg/Ag, detector-GC (glassy carbon). The Hg/Ag electrode is prepared by adhesion of a thin layer of mercury to a polished silver substrate. The resulting amalgam electrode has a hydrogen overvoltage nearly the same as pure mercury, but in a convenient “solid” electrode form. The detector residual current was measured over a potential range of +0.3 V to -0.2 V, as a function of negative potentials applied to the generator. When a Hg/Ag detector was used in unbuffered media, an anodic wave with Ell2 = +0.05 V appeared as the generator potential was advanced more negative than -0.2 V. The anodic wave was found to arise from the product (OH-) of the reduction of traces of oxygen in the chromatographic solvent O2 2e- 2H20 H 2 0 2 20H(2)
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(4) The effect of reactions 2 and 3 on the anodic current of reaction 4 could be largely suppressed by the use of an acidic
nitrobenzene
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buffer as supporting electrolyte. In this manner, the effect of traces of dissolved oxygen on the base line current can be largely eliminated. Figure 5 compared the results obtained by direct reduction of nitrobenzene and dichlone in the presence of trace oxygen to those obtained in the dual-electrode mode. The current in the direct mode is measured at the Hg/Ag generator of the horseshoe cell. The relative current is much larger than that obtained at the detector because of the difference in electrode areas. However, although the magnitude of the detector current is smaller, the increase in signal-to-noise ratio, resulting from the discrimination against residual current [and hence flow noise (15)l is quite evident. Glassy carbon was also used as the detector in conjunction with a Hg/Ag generator in the horseshoe cell. The residual current of the detector was virtually independent of the generator potential up to -1.7 V. As expected, no anodic current from the electrogenerated OH- from oxygen reduction was found. However, when dichlone was used as a test analyte the %RS was found to be only 14%,resulting from the irreversible behavior of dichlone on glassy carbon. Figure 6 shows the detection of dichlone in an unpurged sample. The dual-electrode mode provides a response to 0 2 that is about 3 orders of magnitude smaller than an equivalent amount of dichlone. Usable Potential Range. The greatest advantage in the application of dual-electrode detection is not for the measurement of easily-reduced analytes such as dichlone and nitrobenzene. Such species can be measured with high sensitivity by direct reduction. The most important application will be for analytes that have very high reduction or oxidation potentials such as the organotin cations (16, 17). Usually in the reductive detection mode, the residual current and associated noise increase sharply as the potential of mercury- and carbon-based electrodes is increased more negative than -1.2 V at pH 5 (18);the useful potential for
ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
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dichlone
Flgure 8. Relationship of (EGE),and hydrodynamic
offset
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8 10 12 Minutes Flgure 7. Trace organotin cation detection in dual-electrode mode. Conditions: cell, sandwich with two Hg/Ag electrodes; potentials, generator -1.2 V, detector +0.2 V; sample, 2.5 X lo-' moVL of each; separation, as in ref 16.
highest sensitivity is limited by the reduction current of the solvent. However, generator potentials up to -1.7 V, at pH 5, can be used under similar circumstances without the attendant increase in detector noise. Potentials greater than -1.7 V give rise to microscopic hydrogen bubbles that generate intolerable detector noise. Since the residual current at the detector is nearly independent of the generator potential, the limit of detection (LOD) for any given analyte will be determined primarily by the %RS and the residual current at the low applied potential of the detector. Figure 7 shows the low-level detection of three organotin cations at a generator potential of -1.2 V and detector at +0.2 V. Detection was performed with two Hg/Ag electrodes in the sandwich cell. The LOD obtained is about a factor of 5 better than can be achieved with the direct reductive mode (16). When a calibration curve was made in the to mol/L range, a linear resplonse was obtained at the low end, but significant negative curvature occurred at concentrations above mol/L. This effect can be explained by the formation of a nonelectroactive dimer (16, 1 7 ) that fouls the generator at the higher concentrations and resulting in a lower
modulation interval (A€), base GE potential response for a hypothetical analyte.
concentration of the detectable free-radical product reaching the detector. Selectivity, The selectivity of the dual-electrode mode should be equal to, or better than, direct amperometry, provided that the %RS for the analytes of interest is high. The different sensitivities of the direct and dual-electrode modes can be seen in Figure 5. In the direct mode (upper chromatogram), a larger signal is obtained for nitrobenzene than for dichlone by virtue of the four electrode reduction process vs. two electrode. However, in the dual-electrode mode (lower chromatogram), the sensitivity for nitrobenzene has been decreased (relative to that for dichlone). The nitrobenzene has a less sensitive product-forming and product-detecting reaction, w'hich gives a %RS of only 17% (under conditions where dichllone has a %RS of 60%). In some analytical situations, the interference of coeluting, electroactive matrix constituents could be eliminated by the use of the dualelectrode approach, provided the interferences do not generate detectable products with high %RS. Generator Modulation. The selectivity of the dualelectrode approach may be increased (if necessary) by the modulation of the generator electrode potential. The principle is illustrated in Figure 8. In the upper quadrant of the current/po tential plot, the hydrodynamic response for the direct reduction of a hypothetical analyte is shown. A base potential of' the generator electrode (&E) at the E1/Pof the reactant is chosen for the center of the sine wave modulation interval. The lower quadrant of the current/potential curve shows the hydrodynamic response of the oxidation of the electrochemical product. Thus, if the generator potential is modulated as shown, the concentration of reaction product will also be modulated. When a potential on the limiting current platr?au is chosen for the detector (E,&, the magnitude of the product current will vary in a sinusoidal manner. A lock-in-amplifier is used to selectively detect the modulated component of the resulting current, while ignoring other dc components. This approach enhances the selectivity by limiting the response to only those analytes which have their current/potential wave in the modulation interval chosen and forming a detectable product at the chosen detector potential. Figure 9 shows a preliminary example of how this approach of increasing the selectivity might be applied. The upper chromatogram shows the selective detection of nitrobenzene, using the sandwich cell, a low modulation frequency, an arbitrary lock-in phaaie angle, and an E G E equal to the Ellz of nitrobenzene. The response is limited almost exclusively to nitrobenzene. Conversely, when the E G E is chosen to equal the Ellz of dich!lone, only this compound is detected. It is evident that the signal-to-noise ratio is very poor in this preliminary experiment. This is probably a result of a poor choice of detection phase angle. Unfortunately, the optimum phlase angle for the situation cannot be easily determined empirically with this simple lock-in-amplifier nor can it be readily predicted theoretically as is the case with ac voltammetry at a single electrode. Rather, the optimum phase
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Anal. Chem. 1981, 53, 1704- 1706
compounds. This technique can be used to extend the useful available potential range for reductive or oxidative detection, without the attendant loss in sensitivity and selectivity that accompanies the application of large detector electrode potentials in direct amperometry. There is a special advantage in the use of the dual-electrode mode in the detection of reducible analytes, since the noisy residual current resulting from traces of oxygen can be largely eliminated. Future work will evaluate the applicability of this new approach to samples of environmental importance.
LITERATURE CITED Heineman, W.; Kissinger, P. Anal. Chem. 1980, 52, 138R-151R. Kissinger, P. Anal. Chem. 1977, 49, 447A-456A. Kissinger, P.;Bratin, K. "Recent Developments in the Reductive LCEC of Organic Compounds Using Mercury Film and Glassy Carbon Electrodes" Dresented at the Eastern Analvtical Symooslum. New . . York, Sept 2'1, 1980. Klsslnger, P.; Bratin, K.; Davld, 6.; Pachla, L. J. Chromatogr. Scl. 1070. 17. 137-146.
Inject Flgure 9. Selective detection of nitrobenzene and dichlone using modulated dual-electrode detection: upper chromatogram, E, = -0.60 V; lower chromatogram, E, = -0.25 V. Conditions: chromatography as in Figure 5; A € = 100 mV; modulation frequency 15 Hz; phase angle 12'; sample, 1 X mol/L of each.
angle for product current detection will be a function of several variables: the flow rate, hydrodynamic mass transfer between electrodes, the electrode follow-up chemical reaction kinetics, and the modulation frequency. These relationships must be investigated in the future to optimize this approach for a specific analytical problem. CONCLUSION The dual-electrode approach provides the trace analyst with another tool for the selective detection of certain electroactive
Schieffer,' G- Anal.- Chem. 198 1, 53, 126- 127. Eggii, R.; Asper, R. Anal. Chlm. Acta 1978, 101, 253-259. Gerischer, H.; Mattes, I.; Braun, R. J. Electroanal. Chem. 1965, 70, 553-567. Braun, R. J. Electroanal. Chem. 1968, 79, 23-35. Schieffer, G.; Blaedel, W. Anal. Chem. 1977, 49, 49-53. Kenkei, J.; Bard, A. J. Electroanal. Chem. 1974, 54, 47-54. Klhara, S.; Yamamoto, T.; Motojima, K.; Fujlnaga, T. Tahnta 1972, 19, 657-668. Fujlnaga, T.; Kihara, S. CRC Crit. Rev. Anal. Chem. 1977, 22, 223-254. Osteryoung, J.; Klrowa-Eisner, E. Anal. Chem. 1980, 52, 62-86. Kemula, W.; Krygowskl, T. I n "Encyclopedia of the Elements"; Bard, A., Lund, H., Eds.; Marcel Dekker: New York, 1979. Swartzfager, D. Anal. Chem. 1978, 48, 2189-2192. MacCrehan, W. Anal. Chem. 1981, 53, 74-77. Mairanovskii, S. Russ. Chem. Rev. 1978, 45, 298-317. MacCrehan, W.; Durst, R. "Optimizing Reductive Electrochemical Detectlon in Liquid Chromatography": ACSlCSJ Chemlcal Congress, Honolulu, HA, April 5, 1979, Abstract No. 207.
RECEJYED for review March 3,1981. Accepted June 15,1981. The authors wish to thank the Environment,al Protection Agency for partial support of this work through the Interagency Energy/Environment Program (EPA/IGA/05-E684).
CORRESPONDENCE Sequence Analysis of Qligopeptides by Secondary IonKollision Activated Dissociation Mass Spectrometry Sir: Sequence analysis of proteins and oligopeptides by mass spectrometry presently involves enzymatic and/or chemical degradation of the sample to a complex mixture of small peptides containing two to eight residues, chemical derivatization of the mixture components to enhance their volatility, and the combination of chromatography and mass spectrometry for characterizing the primary structure of each peptide in the mixture (1). Recently, we have described the use of collision activated dissocation mass spectrometry on a triple quadrupole instrument for the direct sequence analysis of' 26 oligopeptides in a mixture without prior separation of the components (2). In this approach the peptides are converted to N-acetyl and N-acetyl-d3N,O-permethylated derivatives (3)which are then volatilized directly into the ion source of the mass spectrometer and converted to M iH" ions under chemical ionization conditions. Quadrupole 1 is set to pass ions with a particular
m1.z value or a group of ions with mlz values within a 1-6 amu window. When the selected ions enter quadrupole 2, they suffer collisions with either argon atoms or nitrogen molecules, become vibrationally excited, and dissociate to produce fragment ions characteristic of the amino acid sequence in the peptide. These fragment ions are then transmitted to quadrupole 3 where they undergo mass analysis. A mass spectrum of the fragments derived from each ion entering quadrupole 2 results. Eliminat,ion of all or most of the derivatization steps in the above scheme would further simplify polypeptide sequence analysis by mass spectrometry. Recently, it has been shown that secondary ion mass spectrometry, with either energetic argon ions ( 4 ) or argon neutrals (5-7) as projectiles, can be employed to facilitate volatilization and ionization of nonvolatile, thermally labile, polar organic molecules. With 3-5 kV argon atoms as projectiles, oligopeptides are sputtered into
0003-2700/81/0353-1704$01.25/00 1981 American Chemical Society
