Cyclic Chronopotentiometry as a Detection Tool for Flowing Solution

The analytical signal is obtained by monitoring the change in the average electrode potential (calculated for either a cathodic or an anodic half-cycl...
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Anal. Chem. 2006, 78, 6747-6755

Cyclic Chronopotentiometry as a Detection Tool for Flowing Solution Systems Anna Basa,† Jolanta Magnuszewska,† Tadeusz Krogulec,*,† and Andrzej S. Baranski*,‡

Institute of Chemistry, University of Białystok, Hurtowa St. 1, 15-399 Białystok, Poland, and Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada, S7N 5C9

Cyclic chronopotentiometry provides a very simple detection method, which may be particularly useful in capillary electrophoresis (CE) and microseparation systems. It has been shown that for disk microelectrodes it is possible to define safe reduction and oxidation currents that would never lead to the formation of H2 or O2 gas bubbles, even if they are applied for an indefinitely long time period. During end-column CE detection, currents passing through the working microelectrode can be completely controlled by the external electronic circuit and they are not affected by the separation current. Consequently, problems created by the offset potential in CE can be completely eliminated. The detection can be accomplished through a variety of different mechanisms; however, generation of the electrode response as a result of analyte adsorption seems to be most common. The method is applicable to many analytes, which do not have to be electroactive. The analytical signal is obtained by monitoring the change in the average electrode potential (calculated for either a cathodic or an anodic half-cycle) caused by an analyte interacting with the electrode. The analytical signal is proportional to the analyte concentration, within a concentration range extending over ∼2 orders of magnitude. Chronopotentiometry is one of the oldest electroanalytical techniques, but until recently, it was relatively rarely used in electrochemical analysis. Classic chronopotentiometry involves the measurement of the electrode potential during an electrode process occurring at constant current. At some moment (called the transition time), the analyte concentration at the electrode surface drops to zero causing an abrupt change of the electrode potential; consequently, the transition time can be used to obtain information about the analyte concentration.1,2 More recently, a new version of chronopotentiometry, called cyclic reciprocal derivative chronopotentiometry,3-6 was devel* Corresponding authors. E-mail: [email protected]. Tel. (+48) 857457803. Fax (+48) 857470113. E-mail: [email protected]. Tel. (306) 9664701. Fax (306) 9664730. † University of Białystok. ‡ University of Saskatchewan. (1) Bergveld, R.; Eijkel, J. C. T.; Olthuis, W. Biosens. Bioelectron. 1997, 12, 905-916. (2) El-Hallag, I. S. Monatsh. Chem. 1998, 129, 625-632. (3) Bi, S.; Yu, J. J. Electroanal. Chem. 1996, 405, 51-58. (4) Molina, A.; Gonza´lez, J.; Saavedra, F.; Abrantes, L. M. Electrochim. Acta 1999, 45, 761-773. (5) Wang, J.; Tian, B. Anal. Chem. 2000, 72, 3241-3244. 10.1021/ac060331f CCC: $33.50 Published on Web 09/01/2006

© 2006 American Chemical Society

oped. Alternative names for this technique are derivative,7 adsorptive,8 depletive,9 or galvanostatic10 stripping chronopotentiometry. In this version of chronopotentiometry, a dependence of dt/dE versus E is being measured and analytic signals are recorded in the form of peaks (which resemble voltammetric peaks, particularly in the case of surface electrode processes or stripping processes); recording the response in that form simplifies the interpretation of the results. The main advantage of the application of chronopotentiometry in analysis arises from the fact that determination can be carried out in highly resistive media with dynamic range and reproducibility similar to that observed under voltammetric conditions in well-conductive solutions.5 Typically, without a preconcentration of analytes, the analytical measurements can be performed from about 2 to 50 µM;5 however, when the preconcentration step is included, the limit of determination can be decreased well below 1 ppb.7-10 The relative standard deviations of chronopotentiometric measurements are typically in the range of 1-5%. Another technique somewhat related to chronopotentiometry is potentiometric stripping analysis (sometimes incorrectly called stripping chronopotentiometry). This technique is used for trace determination of metal ions, which are first preconcentrated in a mercury electrode for several minutes by an electrochemical reduction carried out at constant potential. The experimental setup used in this case does not include a circuit for controlling the current; instead, preconcentrated analytes are stripped from the mercury electrode by an oxidizing agent (typically Hg2+) added to the analyzed solutions. Occasionally, potentiometric stripping analysis has been used in flow-through systems.11,12 However, to our best knowledge, cyclic chronopotentiometry, in any form, has never been used for the detection of analytes in flowing systems such as flow injection analysis (FIA), high-performance liquid chromatography (HPLC), or capillary electrophoresis (CE). (6) Gonzales, J.; Molina, A. Langmuir 2001, 17, 5520-5526. (7) La Pera, L.; Lo Curto, R.; Di Bella, G.; Dugo, G. J. Agric. Food Chem. 2005, 53, 5084-5088. (8) Dugo, G.; La Pera, L.; Lo Turco, V.; Di Bella, G.; Salvo, F. J. Agric. Food Chem. 2004, 52, 1829-1834. (9) Van Leeuwen, H. P.; Town, R. M. Environ. Sci. Technol. 2003, 37, 39453952. (10) Szłyk, E.; Szydłowska-Czerniak, A. J. Agric. Food Chem. 2004, 52, 40644071. (11) Schulze, G.; Han, E.; Frenzel, W. Fresenius Z. Anal. Chem. 1989, 332, 844848. (12) Beinrohr, E.; Dzurov, J.; Annus, J.; Broekaert, J. A. C. Fresenius Z. Anal. Chem. 1998, 362, 201-204.

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Most experiments described in this work were carried out under flow injection conditions; a more extensive study of chronopotentiometric detection in CE will be presented in separate publications. Nevertheless, in this work, we will show that chronopotentiometric detection is particularly well suited for capillary electrophoresis. Electrochemical detection in CE is complicated by interference of the high separation potential (typically >10 kV) with the detection circuit. This interference is particularly severe when a cylindrical microelectrode (acting as a detector) is inserted into the capillary. The problem can be resolved by employing the offcolumn detection method13 in which the detector is preceded by a fracture decoupler. The decoupler is prepared by fracturing the capillary near the detection end and joining the two parts with an ion-conductive material, such as porous glass13 or Nafion;14 consequently, the separation current can pass through the joint and reach ground without polarizing the solution around the working electrode. An alternative solution to the problem was proposed by Ewing et al.,15 who placed a microdisk working electrode at the outlet of the capillary. This arrangement, called end-column detection, was originally proposed for very narrow capillaries (i.d. e5 µm); however, later it was shown that this detection method is also very useful in the case of wider capillaries.16,17 End-column detection reduces the interference arising from the separation potential, but it does not eliminate that interference completely because, traditionally, end-column electrochemical detection in CE is carried out under so-called controlled-potential conditions (pulsed amperometric18,19 or voltammetric20). A microelectrode that serves as a detector is placed at a distance of several micrometers from the capillary outlet, and it is polarized by the electrical potential, usually changing periodically with time in a predefined fashion. A small electrical current passing through the electrode changes upon the arrival of analytes separated by electrophoresis, and it can be recorded in the form of peaks, whose height and surface area depend on concentration. Unfortunately, under CE conditions, it is impossible to control the electrode potential completely. Potential at the electrode/solution interface depends not only on the setting of a potentiostat but also on the offset potential created at the end of the capillary by the separation current. That offset potential may exceed 1 V, and it depends on many experimental conditions. The inability to fully control the potential of the detector creates a variety of problems, including the following: decrease of signal-to-noise ratio, systematic errors, loss of signal, and in some cases formation of H2 or O2 bubbles at the electrode. Gas bubbles dramatically increase the noise level in the measurements, and in some cases, they may (13) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (14) O′Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N. J. Chromatogr.. A 1992, 593, 305-312. (15) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (16) Lu, W.; Cassidy, R. M.; Baranski, A. S. J. Chromatogr., A 1993, 640, 433440. (17) Matysik, F.-M.; Backofen, U. Fresenius’ J. Anal. Chem. 1996, 356, 169172. (18) Kappes, T.; Hauser, P. C. Electroanalysis 2000, 12, 165-170. (19) LaCourse, W. R. In Carbohydrate Analysis by Modern Chromatography and Electrophoresis; El Rassi, Z., Ed., Elsevier Science B. V.: Amsterdam, 2002; pp 905-945. (20) Wen, J.; Baranski, A.; Cassidy, R. Anal. Chem. 1998, 70, 2504-2509.

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cause a break in the high-voltage circuit (used in electrophoresis) that leads to electrical sparks damaging the microelectrode. Chronopotentiometry is a controlled-current technique, and the current needed to stimulate the electrode can be fully controlled by an external electronic circuit; consequently, problems created by the offset potential can be completely eliminated. EXPERIMENTAL SECTION Reagents. All studied analytes, sodium metaarsenite NaAsO2 (International Enzymes Ltd.), zinc(II) sulfate (Fluka), sodium thiocyanate (Sigma), and quercetin (Sigma) were analytical grade (Warning: NaAsO2 is very toxic). Mobile phases used in the determination of As(III), Zn(II), SCN-, and quercetin were prepared, respectively, from NaOH (Aldrich), acetic acid and sodium acetate (POCh), phosphoric acid (POCh), and Borax (Sigma). All solutions were prepared in deionized water (KB-5522 DW from Cobrabid-AQUA, Warsaw, Poland). Solutions of analytes were brought to the desired concentration by dilution with a mobile phase prior to use. Electrochemical measurements were carried out without the removal of dissolved oxygen, at a room temperature of ∼22 °C. Electrochemical Cells. Working electrodes were made by sealing 12.5-µm (in radius) Au wires (Mennica Panstwowa, Warsaw, Poland) into Corning Kovar Sealing glass tubing 7052 (World Precision Instruments). A lead was made by inserting the ends of the microwire and a thicker copper wire into a stainless steel tubing ∼1 cm long (cut off from a syringe needle, gauge 27) and squeezing the tubing with pliers. The capillary tubes were then cut perpendicular to their length, to expose the wires. Next, the electrodes were polished with 1000- and 2000-grade carborundum paper, and finally mirrorlike polishing was accomplished using 0.3-µm aluminum oxide finishing films (TrueView Products Inc.). The electrode surface was cleaned before each experiment, by performing ∼10 voltammetric scans between potentials of hydrogen and oxygen evolution in the mobile phase alone. The reproducibility of the electrode surface was checked by measuring the magnitude of oxide formation/reduction peaks under cyclic voltammetry conditions. The auxiliary electrode was made of a platinum wire, 0.5 mm in diameter. The reference electrode was Ag/AgCl/1 M KCl. The cell used in FIA experiments was similar to one previously described,21 and it is shown in Figure 1a. The flow was induced by a difference in height between reservoirs containing the studied solutions and the cell outlet, and the flow rate of mobile phases and analytes was equal to 1.4 mL/min. Setup Used in Capillary Electrophoresis Experiments. Electrophoresis in a fused-silica capillary was performed in a plexiglass safety box (Warning: danger of high voltage). Separation voltage was provided by a high-voltage (0-25 kV) power supply model 25A12-P4-F-M (UltraVolt). The capillary electrophoresis cell was similar to one used previously21 and is shown in Figure 1b. The working electrode and the capillary were aligned under a microscope within 10 ( 2 µm using a three-way micropositioner. The electrochemical detection cell was placed at the cathodic end of a capillary housed in a Faraday cage. The operation of all parts of the experimental setup was controlled by a computer. (21) Magnuszewska, J.; Krogulec, T.; Baranski, A. S. Chem. Anal. (Warsaw) 2000, 45, 189-203.

and plotting it versus the starting time of each cycle. The second depiction of the response does not carry any information about the mechanism of the electrode process, but as will be shown later, it provides sufficient information to determine the concentration of analytes interacting with the electrode process. The software for the microcontrollers was written in assembly language (with an assembler provided by Microchip), and the PC software (for data acquisition and processing) was written using Microsoft Visual C++, version 6.0, and Visual Basic, version 6.0. The experimental data were further processed in Microsoft Excel; three-dimensional graphs were prepared using Harvard Chart 3.0 graphing software.

Figure 1. Diagrams of electrochemical cells used in flow injection experiments (a) and in capillary electrophoresis experiments (b).

Electronic Circuit. All cyclic voltammetric and chronopotentiometric measurements were carried out with a custom-built electronic system that performed all necessary data acquisition functions. The system was based on a microcontroller (Microchip PIC18F452), which was interfaced with a computer via a serial link. The electrode response (current or potential) was sampled with a 16-bit analog-to-digital converter (Texas Instruments TLC4545), after passing through an antialiasing filter made of a clock-tunable fifth-order Bessel low-pass filter (Linear LTC1065). The system was also equipped with several 16- and 12-bit digitalto-analog converters, which were used to control a potentiostat and a galvanostat. The design of the galvanostat was typical, similar to the one described in the literature,22 with the working electrode connected to the inverting input of an operational amplifier (Texas Instruments TL072). In chronopotentiometric experiments, the current was applied to the electrode in the form of a square wave (i.e., alternating between positive and negative values). The magnitude of current could be set to any value between 0 and 1 mA, but in this paper, only magnitudes between 5 and 200 nA were used (in this current range, the accuracy of the current was ∼0.5%). The potential of the working electrode was measured using a voltage follower made of a low-noise JFET-input operational amplifier (Texas Instruments TL072). That voltage was continuously sampled at a frequency selected by the user (up to 50 kHz), and typically, 128 data points were collected during each (cathodic and anodic) halfcycle. Different sampling rates in cathodic and anodic half-cycles were allowed by the data acquisition system, but that option was not used in this work. The response of the electrode could be presented in various forms. For example, in three-dimensional plots, the electrode potential is shown as a function of the time (measured in milliseconds) from the beginning of each squarewave cycle on one axis and the starting time of each cycle (measured in seconds) shown on the other axis. The other, simpler representation of the response is obtained by averaging the electrode potential within each (anodic or cathodic) half-cycle (22) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York; 2001; p 644.

RESULTS AND DISCUSSION Background Electrode Processes. Let us first consider very common processes that often occur during chronopotentiometric detection, i.e., reduction of water (or hydronium ion) or oxidation of water (or hydroxide ion)

2H2O + 2e- f H2 + 2OH-

(1)

2H2O f O2 + 4H+ + 4e-

(2)

and

These two reactions could potentially produce gas bubbles that could block the microelectrode, but the gas bubbles would never be formed if the concentrations of O2 and H2 near the electrode never exceeded solubility of these gases in aqueous solutions. For an electrode reaction occurring at a spherical electrode, the concentration of the product near the electrode surface, C sk, is described by the following equation:23

C sk(t) ) C bk +

[ ( ) (x )]

ir0 Dkt 1 - exp 2 erfc nFADk r0

Dk t

(3)

r02

where C bk is the bulk concentration of species k, A is the surface of the electrode, n is the number of electrons, Dk is the diffusion coefficient, F is Faraday’s constant, and i is the current passing through the electrode. Diffusional mass transport at a disk microelectrode of radius a resembles the mass transport observed in the case of a hemispherical electrode of radius r0 ) 2a/π.24 Using that analogy, we can obtain an approximate description of the concentration of a soluble product generated at a disk microelectrode:

C sk(t) ) C bk +

[ ( ) (x )]

π2Dkt |i| 1 - exp erfc 4nFDka 4a2

π2Dkt 4a2

(4)

This equation shows that the concentration increases with time but it never exceeds the limiting value given by (23) Based on appendix written by: Mamantov, G.; Delahay, P. In Delahay, P.; Mattax, C. C.; Berzins, T. J. Am. Chem. Soc. 1954, 76, 5319. (24) Oldham, K. B. J. Electroanal. Chem. 1981, 122, 1-17.

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C max ) C bk + k

|i| 4nFDka

(5)

Using this equation, one can easily derive an expression for the maximum value of a safe current (i.e., a current that will not produce gas bubbles on a microelectrode): b |imax | ) 4nFDka(C sol k - C k)

(6)

where C sol k is the solubility of a given gas in the solution. Using the following data (applicable to dilute aqueous solutions sol at 25 °C),25 DH2 ) 5.11 × 10-5 cm2 s-1, C H ) 7.83 × 10-7 mol 2 b sol -3 -5 2 -1 cm , C H2 ) 0, DO2 ) 2.42 × 10 cm s , C O2 ) 1.27 × 10-6 mol b cm-3, and C O ) 2.67 × 10-7 mol cm-3 (for an air-saturated 2 solution), we can calculate that for a microelectrode (12.5 µm in radius) the safe oxidation current should not be larger than 47 nA, and the safe reduction current should not be more negative than -38.6 nA. However, practice shows that these limits can be exceeded a few times without causing the formation of gas bubbles because probability of nucleation in a slightly supersaturated solution, in a very small volume around the microelectrode, is rather low. Also, convection associated with a hydrodynamic flow causes an increase of these limits and, conversely, placing the microelectrode very close to a solid surface reduces them (in this latter case a more accurate description can be obtained by using mass transport equations derived for scanning electrochemical microscopy26). Let us now consider electrode processes that occur at a gold microelectrode in an air-saturated 0.1 M NaOH solution. A cyclic voltammogram obtained in such a solution is shown in Figure 2. The characteristic feature of a cyclic voltammogram at a gold electrode is a set of peaks associated with the formation and dissolution of a surface oxide layer at about 0.4 and 0.03 V, respectively. In addition, two waves (at -0.145 and -0.95 V) are visible, which are attributed to the reduction of oxygen dissolved in the solution (these waves disappear when the oxygen is removed by bubbling argon through the solution). Figure 3 shows chronopotentiometric curves recorded in the same solution. Dashed lines in Figure 3 present potentials attained by a gold microelectrode polarized in 0.1 M NaOH with a constant current for more than 100 s. When i ) 0, the electrode potential is close to an equilibrium value determined by the O2(air)/H2O redox couple (this potential is close to zero with respect to the reference electrode used in these experiments). When a constant current of -45 nA is applied, the electrode potential reaches a value characteristic of the hydrogen evolution process (curve f), but when a constant current of 45 nA is applied, the electrode potential attains a value characteristic of the oxygen evolution process (curve g). When small negative currents are applied (e.g., -10 nA), the electrode potential attains a value characteristic of the oxygen reduction process (curve e). When an alternating square-wave current is applied, the electrode potential oscillates in a very reproducible fashion (in (25) CRC Handbook of Chemistry and Physics, 86th ed.; Lide, D. R., Ed.; CRC Press: Boca Raton, FL., 2005; pp 6-193, 8-80. (26) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York; 2001; p 671.

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Figure 2. Cyclic voltammograms recorded for a Au microelectrode in air-saturated (solid line) and argon-saturated (dashed line) 0.1 M NaOH at 0.1 V s-1.

Figure 3. Cyclic chronopotentiograms recorded for a Au microelectrode in 0.1 M NaOH at different currents: (a) ia ) -ic ) 10 nA; (b) ia ) 10 nA, ic ) -45 nA; (c) ia ) 45 nA, ic ) -10 nA and limiting potentials reached at constant current equal to 0 (d), -10 (e), -45, (f) and 45 nA (g).

Figure 3, only single cycles of such oscillations are shown as solid lines). Depending on the current setting, these oscillations occur in different potential ranges. For example, ia ) -ic ) 10 nA (curve a) causes the oscillations between the onset potential of gold oxide formation and the potential of oxygen reduction; ia ) 10 nA and ic ) -45 nA drives the oscillations to a region between the potential of gold oxide formation and the potential of hydrogen evolution (curve b); and finally, ia ) 45 nA and ic ) -10 nA (curve c) drives the oscillations to the region between the potential of oxygen evolution and the potential of gold oxide reduction. As shown in Figure 4, the magnitude of the oscillations depends on the magnitude of the current (here only curves for ia ) -ic are shown). When the magnitude of the current is sufficiently large (larger than 70 nA), the oscillations extend over the whole potential window (i.e., from the potential of oxygen evolution to the potential of hydrogen evolution). In these cases, transient steps associated with the electrode processes of gold oxide formation and gold oxide reduction are clearly visible. It should be stressed however, that the shape of the chronopotentiometric curves also depends on the time period of the square-wave cycle; transient steps associated with gold oxide electrode processes can be observed only if the electrical charge passing during the cathodic and anodic half-cycles exceeds the electrical charge associated with the formation of the gold oxide layer.

and Pt include the following: the charging of the double layer, the formation (or reduction) of the oxide layer, the surface reconstruction, the reduction of dissolved oxygen, and the evolution of dissolved hydrogen and oxygen. In addition, on Pt electrodes the adsorption/desorption of hydrogen is taking place. It should be stressed that the formation/reduction of the oxide layer on noble metal electrodes is a complex, multistep process, which is not completely understood at the present time. The average electrode potential can be expressed as a sum of two fully independent contributions, the ohmic polarization of the solution and the interfacial polarization of the electrode, Figure 4. Cyclic chronopotentiometric curves for a Au microelectrode in 0.1 M NaOH recorded at different currents. Measuring conditions: ia ) - ic ) 5, 20, 45, 60, 80, and 100 nA from lowest to highest curve, respectively.

Oscillations of the electrode potential are important for cleaning the electrode surface from contaminations;27 however, practice shows that oscillations over too large a potential range also reduce the sensitivity of detection. General Mechanism of the Response Generation. The chemistry involved in the proposed detection method is essentially the same as in voltammetric detection schemes that we devised in the past27-29 for surface-active analytes. The new elements involve a different method of driving the electrode reaction and a different method of acquiring the electrode response; in this new method, both the magnitude of the alternating (square-wave) current passing through the electrode-solution interface and its time period are constant and the average electrode potential, ∆Eav, (within the oxidation or the reduction half-cycle) is measured. The average electrode potential is related to the electrical work (WCT) needed to transfer the detection charge from a source (at the reference electrode potential) to the working electrode. For example, for the first half-cycle we can write

WCT )

∫

τ/2

0

iD∆E(t) dt ) iDτ 2 2 τ

∫

τ/2

0

∆E(t) dt )

i Dτ ∆Eav (7) 2

where ∆E(t) is the working electrode potential, iD is the detection current, and τ/2 the duration of the half-cycle. However, the case considered here is very different from one encountered during potentiometric measurements. Here, WCT is not directly related to the chemical potential of the analyte, because there is no equilibrium between the electrode and solution, and in addition, analyte molecules do not interact exclusively (or selectively) with the electrode. Under normal circumstances, many different processes occur simultaneously on the electrode, and the major fraction of the electrical charge is consumed by the background electrode processes (BEP) involving the solvent and the supporting electrolyte. These processes at Au (27) Baranski, A. S.; Krogulec, T.; Nelson, L. J.; Norouzi, P. Anal. Chem. 1998, 70, 2895-2901. (28) Baranski, A. S.; Norouzi, P. Can. J. Chem. 1997, 75, 1736-1749. (29) Gerhardt, G. C.; Cassidy, R. M.; Baranski, A. S. Anal. Chem. 2000, 72, 908-915.

WICT iDτ

∆Eav ) RSitot + 2

(8)

where WICT is the work of transferring the detection charge (iDτ/2) through the electrode-solution interface, RS is the solution resistance, and itot is the total current (i.e., the detection current and, in the case of CE, also the separation current) passing through the solution zone between the working electrode and the reference electrode. This relationship demonstrates the fundamental advantage of controlled-current methods over controlled-potential methods (particularly under CE conditions) because is shows how to remove the effect of the offset potential without employing traditional decoupling methods (this issue will be explored later in this paper); it also shows that the detection method is practically universal. When an analyte is introduced into the solution it affects the ∆Eav in three major ways: (i) All ionic analytes affect to some extent the solution resistance around the working electrode, so in the case of nonelectroactive and non-surface-active analytes (such as alkali or alkaline earth metal ions), the detector can operate as a conductivity detector, although the sensitivity of such detection is relatively low. (ii) Analytes that undergo an electron-transfer process (AEP) at the electrode consume a part of the electrical charge passed through the interface. As a result, less charge is consumed by the BEP. Since the energy needed to run BEP is, in general, different from the energy needed to run AEP, the WICT changes. (iii) Analytes that undergo adsorption on the metal/solution interface alter the kinetics of one or more background electrode processes; as a result, the WICT changes. Option iii allows for quite sensitive detection of analytes that are surface active but do not possess redox properties. However, is should be stressed that many (perhaps most) redox species also adsorb on the electrode, and in such cases, all three mechanisms are involved in the response generation. Of course, the proposed detection method is nonselective and can only be used in combination with some separation method. In general, ∆Eav depends on many variables (including, temperature, separation current, iD, τ, and solution composition), but if all variables are constant except for the concentration of the analyte, the relationship between ∆Eav and the concentration can be represented as the Maclaurin series, which is linear at low concentrations. Experiments show that in many cases the linear range extends over more than 2 orders of magnitude. Analytical Chemistry, Vol. 78, No. 19, October 1, 2006

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Response Generation for Selected Analytes. Changes of the electrode potential caused by a flow injection of 10-4 M AsO2in 0.1 M NaOH into 0.1 M NaOH are shown in Figure 5 (this is a three-dimensional plot, which shows potential oscillations within individual square-wave cycles as a function of the starting time of each cycle). These changes of electrode potential were caused by a square-wave current with ia ) - ic )10 nA and the time period of 300 ms. It can be noted that, on the arrival of the analyte, the electrode potential drops substantially. Electrode processes of As(III) are quite complex; in basic solutions, As(III) present as the arsenite ion can be reduced at a gold microelectrode to elemental As(0).30 However, in 0.1 M NaOH, this process takes place only at potentials more negative than -0.9 V. In an acidic environment, one may also observe oxidation of As(III) to As(V) at a potential less positive than the potential of gold oxide formation; however, this oxidation process is not observed at all in alkaline solutions (presumably because of sluggish electrontransfer kinetics). Consequently, within the potential range of oscillations shown in Figure 5, i.e., between about -0.8 and 0.4 V, As(III) is not involved in any reduction or oxidation processes, yet its presence even at very small concentrations affects the electrode potential. The explanation of this behavior is provided by Figure 6, which shows the effect of As(III) on the potential of oxygen reduction. An addition of As(III) into the solution inhibits the oxygen reduction (presumably because As(III) is adsorbed on gold) and shifts the wave of the oxygen reduction toward more negative

potentials. Now, since for ia ) -ic )10 nA oscillations of the electrode potential occur between the potential of gold oxide formation and the potential of oxygen reduction, the inhibition of the oxygen reduction process by As(III) shifts the whole E versus t curve toward more negative potentials (later it will be shown that in a certain concentration range that shift is directly proportional to the concentration of As(III) in the solution). A similar effect to one described for As(III) is also observed for many different analytes (both organic and inorganic) because most species adsorb on gold to some extentsthat provides an almost universal but not selective method of detection. Figure 7 shows how chronopotentiometric curves change during the injection of four different analytes in four different electrolytes. Quercetin (one of most common flavonoids) was dissolved in 0.04 M Na2B4O7 and injected into 0.04 M Na2B4O7. The electrolyte for SCN- determination was 0.1 M H3PO4, for the As(III) ion-0.1 M NaOH, and for the Zn(II) ion-acetate buffer (pH 4.8). These figures show superimposed E versus t curves recorded in subsequent current cycles during injections of the analytes. The solid lines represent the electrode response to the supporting electrolyte alone, and the dotted lines represent the electrode response in the presence of the analytes. The insets show the change of the average electrode potential (within an anodic or a cathodic half-cycle) plotted versus the starting time of each cycle. In the case of quercetin, SCN-, and As(III), the response was obtained using ia ) - ic )10 nA, and in all three cases, the changes in chronopotentiometric curves are very similar. This could suggest that the mechanism of the response generation for quercetin and SCN- is similar to the one previously described for As(III), although in the case of quercetin, a kinetically controlled oxidation at the potential gold oxide formation may also take place. In the case of Zn(II), a relatively large cathodic current (ic ) -45 nA) was used, and that current forced the electrode potential to approach the hydrogen evolution potential in the cathodic halfcycle, causing the deposition of Zn(0) on a Au microelectrode (actually this must have been an underpotential deposition process,31 because the standard potential of Zn2+/Zn(s) couple was not been reached under these conditions). Subsequently, Zn(0) was stripped off the electrode in the anodic half-cycle producing a transient step at ∼ -0.45 V. Clearly, the mechanism of the response generation in this case was different from that in other cases; in the anodic half-cycle, the average potential of the electrode decreased in the presence of the analyte, but in the cathodic half-cycle, the change of the average potential was the opposite (for other analytes the change of the average electrode potential had the same sign in both half-cycles). The responses shown in Figure 7 were generated by relatively large concentrations of analytes, but actually this detection method can react to very small changes in the average electrode potential. At this stage, with our instrumentation, the noise in the average potential measurement is between 20 and 50 µV, and further reduction in the noise level is possible. From an analytical point of view, the relation between analyte concentration and the electrode response is the most important. Although with numerical data processing one can easily handle

(30) Billing, C.; Groot, D. R.; van Staden, J. F. Anal. Chim. Acta 2002, 453, 201-208.

(31) Quaiyyum, Md. A.; Aramata, A.; Moniwa, S.; Taguchi, S.; Enyo, M. J. Electroanal. Chem. 1994, 373, 61-66.

Figure 5. Three-dimensional potential-time curves obtained at Au microelectrode during a three consecutive injection (3 s each) of 10-4 M AsO2- in 0.1 M NaOH to 0.1 M NaOH. ia ) -ic ) 10 nA.

Figure 6. Cyclic voltammograms recorded at 0.05 V s-1 for a Au microelectrode in air-saturated 0.1 M NaOH (dashed line) and after addition of 10-3 M AsO2- (solid line).

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Figure 7. Superimposed cyclic potential-time curves obtained for a Au microelectrode for flow injection (3 s each) of (a) 1 × 10-3 M quercetin in 0.04 M Na2B4O7 (ia ) -ic ) 10 nA), (b) 1 × 10-3 M NaSCN in 0.1 M H3PO4 (ia ) -ic ) 10 nA), (c) 1 × 10-4 M NaAsO2 in 0.1 M NaOH (ia ) -ic ) 10 nA), and (d) 1 × 10-3 M ZnSO4 in 0.1 M acetate buffer (ia ) 10 nA, ic ) -45 nA). Solid lines represent overlapping cyclic chronopotentiograms recorded in the absence of the analyte before and after the injection. The dotted lines illustrate changes caused by the injection of the analyte.

Figure 8. Calibration curves obtained for AsO2- in 0.1 M NaOH at a 12.5-µm (in radius) Au electrode in 0.1 M NaOH. The peak height corresponds to the maximum change of the average electrode potential during the cathodic half-cycles (the average of six replicate measurements was used to calculate each data point). The oxidation and reduction currents were equal to 10 and -10 nA, respectively.

nonlinear calibration curves, a linear relationship between the response and the concentration is still preferred. We did not expect to obtain linear calibration curves because of the complex mechanisms of the response generation in our detection method. However, to our surprise, for all studied compounds the calibration curves were linear within a concentration range extending for ∼2 orders of magnitude. The calibration curves obtained for As(III) in 0.1 M NaOH, using the change in the average electrode potential in the cathodic half-cycle of chronopotentiometric curves are shown in Figure 8. The calibration curve was obtained for 21 concentrations ranging from 0.1 to 1000 µM; for each concentration, at least 7 replicate measurements were made. The relative standard deviation (RSD) of the response obtained at 0.1 µM (13 replicates) was

9%, and for concentrations larger than 2 µM the average RSD was 2.5%. The calibration curve is linear in the concentration range from about 0.5 to 50 µM, with the slope of 1.56 ( 0.01 mV/µM, the intercept of 0.5 ( 0.3 mV, and R2 ) 0.9994. The small intercept (clearly visible on the logarithmic plot) was probably caused by a residual electronic coupling (possibly through ground) between the injection circuit and the detection circuit. At higher concentrations, the response becomes a nonlinear function of the concentration and finally reaches a plateau because, most likely, the electrode coverage with the adsorbed As(III) ion reaches saturation. Preliminary results indicate that similar calibration curves can be obtained for different analytes (Zn2+, Pb2+, quercetin, and glucose were studied). The detection limits obtained for the studied compounds under chronopotentiometric conditions are similar to those obtained under voltammetric conditions providing that the detection time is comparable. For the 300-ms detection period, the detection limits are ∼0.1 µM (except ∼1 µM for glucose). If necessary, the detection period can be reduced even to 3 ms, but the preliminary results indicate that this causes some increase in the detection limits. The detection limits obtained with PAD are typically between 0.01 and 1 µM.18 In the case of the As(III) determination under FIA conditions with PAD, a detection limit of ∼5 µM has been reported.32 Application of Cyclic Chronopotentiometry in Capillary Electrophoresis. As was emphasized in the introduction, this detection method was designed for capillary electrophoresis. Figure 9a illustrates problems with end-column electrochemical detection under voltammetric conditions. The solid line represents a cyclic voltammogram recorded for a gold microelectrode positioned at the end of the capillary, but with the separation (32) Williams, D. G.; Johnson, D. C. Anal. Chem. 1992, 64, 1785-1789.

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Figure 9. Cyclic voltammetric (a) and chronopotentiometric (b) curves recorded during capillary electrophoresis experiments before (solid lines) and after (dotted lines) switching on the separation high voltage. The analyte was 10-3 M NaAsO2, and the running buffer was 0.1 M NaOH. Measured “offset” was 320 mV. CE conditions: fused-silica capillary 50 cm long, 25 µm i.d., separation voltage 10 kV. Detection condition for a Au microelectrode 12.5 µm in radius: (a) scan rate 2 V/s and (b) ia ) -ic ) 25 nA.

voltage switched off. The dotted line represents a cyclic voltammogram recorded in the same system after switching the separation voltage (10 kV) on. The separation current passing through the capillary creates, at the detection end, an ohmic polarization of the electrolyte around the capillary outletsthat ohmic polarization is called the offset potential. In this particular experiment, the electrode was positioned in a spot where the offset potential is ∼0.32 V. Since voltammetry is a controlled-potential technique, the potential offset alters the quantity that is supposed to be controlled, and as a result, the response of the electrode changes in a very complicated way: peaks move horizontally and change their height. In addition, a new electrode process (hydrogen evolution) starts at negative potentials. In extreme cases, that process may lead to the formation of gas bubbles, which cause a dramatic increase in noise and, in some cases (e.g., reversed electroosmotic flow), that may also lead to a break in the highvoltage circuit causing electrical sparks that could damage the microelectrode and possibly even the electronic measuring circuit. Chronopotentiometry is a controlled-current technique; therefore, in this case, the offset potential does not alter the controlled quantity. Also, the separation current does not affect the current passing through the electrode. In electrochemical instruments, current and potential are controlled differently. Potential is always controlled in a relative sense (versus the reference electrode potential); this is why it is affected by the offset potential, which 6754 Analytical Chemistry, Vol. 78, No. 19, October 1, 2006

Figure 10. Electropherograms obtained with the cyclic chronopotentiometric detection and a fused-silica capillary 68 cm in length, 25-µm i.d. (a) Pt electrode (10 µm in radius), ia ) -ic ) 70 nA, averaging in the reduction half-cycle; separation potential 18 kV; 0.05 M H3PO4 running buffer; hydrodynamic injection of 0.45 nL solution containing 8 × 10-4 M histidine (1), 5 × 10-3 M asparagine (2), 6 × 10-4 M methionine (3), 1 × 10-4 M phenylalanine (4), and 4 × 10-4 M cysteine (5). (b) Au electrode (12.5 µm in radius), ia ) 16 nA, ic ) -60 nA, averaging in the oxidation half-cycle; separation potential 15 kV; 1 M CH3COOH running buffer; hydrodynamic injection of 0.3nL solution containing Na+ (1) and Li+ (6) at 1 × 10-3 M each, and Zn2+ (2), Cd2+ (3), Pb2+ (4), and Cu2+ (5) at 1 × 10-4 M each.

appears between the working and the reference electrode. However, the current in a galvanostat is controlled in the absolute sense; therefore, it is not affected by the separation current. In chronopotentiometric experiments, the offset potential changes only the measured quantity. As a result, the electrode response shifts vertically by a constant value equal to the offset potential (as shown in Figure 9b). Since the analytical response is expressed as a difference in the average electrode potential (the average potential in the presence of an analyte minus the average potential in the running buffer), it is not affected at all by a constant offset potential. On the other hand, in chronopotentiometric measurements, the offset potential can be easily monitored by comparing the average electrode potential in the presence of the separation voltage with one obtained for the separation voltage switched off. Monitoring of the offset potential is great help in the precise manual positioning of the microelectrode at the capillary outlet, but it can also be used in automatic positioning of a microelectrode by computer-controlled motorized micropositioners. In the latter case, a computer program could use the change in the offset potential as feedback information while driving the microelectrode into an optimum position. Preliminary electropherograms obtained with chronopotentiometric detection for amino acids at a Pt microelectrode and alkali metal ions and some transition metal

ions at a Au microelectrode are shown in Figure 10. It should be noted that alkali metal ions are neither reduced nor adsorbed on the gold electrode; in this case, the detection was possible because of changes in the solution resistance as shown by eq 8; however, the sensitivity of this detection was poor because the conductivity of the background electrolyte was rather high. CONCLUSIONS It has been shown that cyclic chronopotentiometry provides a very simple and very useful detection method, particularly for capillary electrophoresis and microseparation systems. For chronopotentiometry that employs disk microelectrodes, safe reduction and oxidation currents can be defined. Such currents never lead to the formation of H2 or O2 gas bubbles, even if they are applied to the microelectrode for an indefinitely long time. Important benefits of using chronopotentiometric detection in CE arise from the fact that current passing through the working microelectrode can be controlled in an absolute sense and is not affected by the separation current; consequently, problems created by the offset potential can be completely eliminated. Another potential application of this detection method can be in HPLC, particularly in the case of poorly conductive eluents. The detection response in this method can be generated through many different mechanisms. Some analytes may undergo reduction or oxidation processes; others may adsorb on the electrode and influence the kinetics of one (or more) of the electrode processes that occur on the electrode (i.e., hydrogen evolution, gold oxide formation, oxygen evolution, gold oxide reduction, or oxygen reduction). The adsorbed analytes may also affect the double layer capacitance, and in some systems, analytes may affect the solution conductivity. However, practice shows that

the electrode response generation via the adsorption mechanism is the most common. Therefore, the method is applicable to analytes, which do not have to be electroactive providing that they adsorb on the gold surface, and fortunately, most organic compounds (and many inorganic) behave in that way. In this work, quercetin, SCN-, and AsO2- were used to illustrate that mechanism of the response generation, but we observed similar behavior for many other analytes. The method can also be used for the detection of metal ions (including Cu2+, Zn2+, Cd2+, Pb2+), which produce a response through a different mechanism that involves the underpotential deposition of metal ions on gold. Even ions that are neither reduced nor adsorbed at the electrode (such as alkali metal ions) can be detected, because they affect the solution resistance. Consequently, cyclic chronopotentiometry provides an almost universal but (in most cases) a nonselective method of detection. The analytical signal in this method is represented by a change in the average electrode potential caused by an analyte interacting with the electrode (calculated for either a cathodic or an anodic half-cycle). The analytical signal is proportional to the concentration of analytes within a concentration range extending over ∼2 orders of magnitude. Preliminary results indicate that, for many analytes, detection limits of ∼0.1 µM can be obtained. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the State Committee for Scientific Research (KBN), Poland (Grant 4 T09A 028 24) Received for review February 22, 2006. Accepted July 17, 2006. AC060331F

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