Anal. Chem. 1997, 69, 463-470
Application of a 32-Microband Electrode Array Detection System for Liquid Chromatography Analysis Ming-Huei Chao and Hsuan-Jung Huang*
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan, ROC
An electrode array consisting of 32 microband electrodes and a generator electrode was constructed. With this electrode array, both the oxidation and reduction threedimensional hydrodynamic chromatovoltammograms of a reversible reaction can be obtained in a single voltammetric run. The versatility of this electrode array was demonstrated by running solutions containing various phenolic compounds with an FIA and an HPLC system. From the oxidation and reduction chromatovoltammograms obtained, individual components in the sample solution can be determined quantitatively, even when overlapping of the chromatography peaks occurred. With this microband electrode array, detection limits down to 2.0 × 10-8 M for the studied phenolic compounds were obtained. Electrochemical detection is one of the most versatile and sensitive detection methods used in liquid chromatography.1-6 Among the various electrochemical techniques applied, amperometric determination, due to the inherent high selectivity and low detection limit, is the most frequently used method.1-4 Besides the conventional single- and dual-electrode detectors used,2-7 versatility of electrochemical determination has been improved by the development of multielectrode detectors. Electrode arrays consisting of 4,8-10 8,11,12 11,13 and 1614-16 electrodes having various flowing patterns have been proposed. To accommodate the control of these multielectrodes, multichannel potentiostats, which are capable of controlling up to 16 electrodes, have also been developed.14-16 Mastue et al.14,15 reported a multichannel elec(1) Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry, 2nd ed.; Marcel Dekker: New York, 1996; Chapter 27. (2) Roston, D. A.; Kissinger, T. P. Anal. Chem. 1982, 54, 429-433. (3) Allison, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8-12. (4) Hamada, M. A.; Rabenstein, D. L. Anal. Chem. 1988, 60, 2283-2287. (5) Martinez, R. C.; Gonzalo, E. R.; Garcia, F. G.; Mendez, J. H. J. Chromatogr. 1993, 664, 49-58. (6) Yuebe, L.; Zbang, R. Electroanalysis 1994, 6, 1126-1131. (7) Shoup, R. E.; Mayer, G. S. Anal. Chem. 1982, 54, 1164-1168. (8) Wang, J.; Rayson, G. D.; Lu, Z.; Wu, H. Anal. Chem. 1990, 62, 1924-1928. (9) Wang, J.; Chen, Q.; Rayson, G.; Tina, B.; Lin, Y. Anal. Chem. 1993, 65, 251-254. (10) Fang, T.; McGrath, M.; Diamond, D.; Smyth, M. R. Anal. Chim. Acta 1995, 305, 347-358. (11) Fielden, P. R.; McCreedy, T. Anal. Chim. Acta 1993, 273, 111-121. (12) Fielden, P. R.; McCreedy, T.; Ruck, N.; Vaireanu, D. I. Analyst 1994, 119, 953-958. (13) Harrington, M. S.; Anderson, L. B.; Robbins, J. A.; Karweik, D. K.; Rev. Sci. Instrum. 1989, 60, 3323-3328. (14) Mastue, T.; Aoki, A.; Ando, E.; Uchida, I. Anal. Chem. 1990, 62, 407-409. (15) Mastue, T.; Aoki, A.; Uchida, I. Anal. Chem. 1992, 64, 44-49. (16) Hoogvliet, J. C.; Reijn, J. M.; van Bennekom, W. P. Anal. Chem. 1991, 63, 2418-2423. S0003-2700(96)00546-X CCC: $14.00
© 1997 American Chemical Society
trochemical detection system that consisted of a 16-microelectrode array detector and a homemade computer-controlled 16-channel potentiostat. The multichannel detector developed by Hoogvliet et al.16 was a circular array of 16 working electrodes which performed in a parallel configuration. Although the three-dimensional (3-D) hydrodynamic voltammograms can be obtained, the multichannel electrodes can only be operated in either a parallel or series mode according to their original designs. The hydrodynamic voltammograms obtained usually represent only either the oxidation or the reduction reaction, regardless of whether the studied electrochemical reaction is reversible or not. To improve the function of a multichannel electrochemical detector, a renewable electrode array consisting of 32 microband electrodes and a generator electrode was designed. With this electrode array, both the oxidation and reduction 3-D hydrodynamic voltammograms of a reversible reaction can be obtained in a single voltammetric run. Electrochemical characteristics of analytes can thus be easily obtained from the oxidation and reduction 3-D hydrodynamic voltammograms. To demonstrate the versatility of the 32-microband electrode array (32-MEA) detector, solutions containing various phenolic compounds were run through an FIA and an HPLC system and monitored with the 32-MEA detector. The feasibility for analyzing parathion, a high reduction potential species, was also investigated. EXPERIMENTAL SECTION Fabrication of Microband Electrode Array. The microelectrodes in the array were made by a technique similar to that of the screen printing process.17-19 A paste of a specially prepared printing material was squeezed through a mask onto the surface of a glass substrate beneath. This substrate was then cured to leave a thin pattern of microelectrodes on its surface. As only the print on the edge of the glass substrate was exposed to solution and used as the microelectrode, an array of microband electrodes was formed by stacking the printed substrates together. The printing material used in this experiment was prepared by thoroughly mixing an appropriate amount of carbon powder (1.0 g of Sigradur-G powder, from HTW, Germany) and Epofix resin (0.5 mL, from Struers A/S, Copenhagen, Denmark). The substrate used was the conventional cover glass with dimensions of 0.12 mm × 22 mm × 22 mm. The printing pattern of the (17) Craston, D. H.; Jones, C. P.; William, D. E.; Murr, N. E. Talanta 1991, 38, 17-26. (18) Wring, S. A.; Hart, J. P.; Bracey, L.; Brich, B. J. Anal. Chim. Acta 1990, 231, 203-212. (19) Wang, J.; Tian, B. Anal. Chem. 1992, 64, 1706-1709.
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Figure 1. Scheme of the microelectrodes-printed substrate (a), top view of the spacer covered 32-MEA resin block (b), and the crosssectional view of the flow cell (c).
microelectrode was made from a piece of polyester film (120 µm thick), whereby two parallel rectangular strips (each with dimensions of 2 mm × 16 mm and 5 mm apart from each other) were cut and removed from the polyester film. The paste of the printing material was applied onto the substrate, excess paste was removed, and the substrate was left to cure at room temperature for 24 h. A piece of thin copper wire was then connected to the end of the cured microelectrode for use as electrical contact. Figure 1a shows the scheme of a microelectrodes-printed substrate. As there were two microelectrodes on each piece of cover glass, 16 pieces of the microelectrode-printed glass were stacked and glued together to form the microband electrode assembly. The assembly was then soaked with the electrode face side down in a pool of liquid Epofix resin to form a microelectrode array resin block (with dimensions of 25 mm × 30 mm × 30 mm) and left to cure for 24 h at room temperature to form the 32 microband electrodes. After successively polishing the electrode side, two sets of microband electrodes were exposed. The dimensions of the microband electrode in the assembly are approximately 2 mm length and 120 µm width, and the distance between two neighboring microband electrodes is about 130 µm. A cavity with an approximate dimension of 2 mm × 10 mm × 8 mm was made right at the center of the two sets of microband electrodes to make the generator electrode. A piece of glassy carbon with dimensions of 2 mm × 10 mm × 5 mm was embedded in the cavity and secured with the same epoxy resin. For the electrical contact of the generator electrode, a piece of thin copper wire was connected to the generator electrode from the other end of the block. The generator electrode formed had dimensions of 2 mm × 10 mm. The finished MEA consisted of two sets of 16 microband electrodes located respectively at the upstream and the downstream of the flow and a generator electrode located in between and was about 1.5 mm apart from the two sets of electrode arrays. Figure 1b shows the top view of the spacer-covered 32-MEA block. The dimensions of the generator and microband electrodes are also indicated. All of the electrodes in the array can be renewed by appropriate polishing. 464
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The thin-layer flow cell used in this experiment was similar to that of the BAS (LC-17A). Besides the block of multichannel electrodes, a block of stainless steel was used as the counter electrode, in which an Ag/AgCl reference electrode (filled with 3 M NaCl solution) was included. Figure 1c shows a cross-sectional view of the thin-layer flow cell. The flow of solution in the thinlayer flow cell was regulated by a piece of PTFE spacer (50 µm thickness) on which a channel in a longitudinal oval shape was made. The dead volume of this thin-layer flow cell was estimated to be 4.2 µL. Though the Epofix resin was designed for cold mounting, the cured resin also showed very good resistance to chemicals. A 32-MEA Epofix resin block was deliberately soaked in solutions of HCl (0.1 M), NaOH (0.1 M), and acetonitrile (100%) respectively for 24 h with no appreciable damage. Multichannel Potentiostat. To facilitate the control of the 32-MEA detector, a multichannel potentiostat which is capable of applying programmable potentials on each of the 32 microelectrodes was built. The design of the multichannel potentiostat was modified from that of Mastue et al.14 and Hoogvliet et al.16 Besides doubling the number of electrode control to 32, software control for the offset of background current for each microelectrode was made. Figure 2 shows the circuitry of the software-controlled 32channel potentiostat. For a proper control of this 32-channel potentiostat, two PCLD-889 amplifier/multiplexer boards (from BQC Microsystem, Sunnyvale, CA) and one PCL-818 highperformance data acquisition card with programmable gain (from BQC Microsystem) were used with a 486 DX4-100 personal computer. As the multichannel potentiostat can accommodate only 32 electrodes, control of one of the upstream microband electrodes was replaced with the control of a generator electrode. The potential of the generator electrode (designated as w1) was controlled by one of the two DACs (DAC1) on the PCL-818 card. The potentials of the microband electrodes in the array (w2-w32) were controlled by passing an appropriate potential generated from DAC2 of the PCL-818 card through a resistor ladder. The generated potential was divided by the resistor ladder evenly into 31 steps. Each of the evenly spaced potentials was then used as the potential source for the microband electrode. The current responses of the 32 electrodes (I1, I2, ...) were converted to voltage (V1, V2, ...) by the voltage follower and then connected respectively to the 32 inputs on the two 16-channel amplifier/multiplexer boards (PCLD-889). The signals selected from the two 16-to-1 multiplexers were properly amplified and fed separately into the two 12-bit ADCs on the PCL-818 high-performance data acquisition card. Currents in a range of 1 nA/V to 2 µA/V could be amplified to an appropriate voltage level for the analog-to-digital conversion. The conversion time of the ADC used in the PCL-818 card is 10 µs, and the current resolution of this conversion system can be lowered down to 0.5 pA. To eliminate the high-frequency noise, low-pass filters with a time constant of 50 ms were also included in the circuitry. The function of background current offset was achieved by connecting a differential amplifier (OP8) to the inverting input of an operational amplifier (OP5, 1/4 LF444A), which played the role of maintaining a constant potential for the microband working electrode (w2).16 The two inputs of the differential amplifier were connected respectively to the ground and the offset voltage supplied for background current correction. Different from the design of Hoogvliet et al., two DACs with two sets of 1-to-16 analog
Figure 2. Circuitry of the software-controlled 32-channel potentiostat.
multiplexers (AD7506) instead of 31 DACs were used for the generation of appropriate offset voltages for the 31 microelectrodes. As only two DACs were used, the offset voltages could only be generated and fed to each of the differential amplifier and activate the offset function successively. The generated offset voltage needs to be held constant for a certain period of time while the DAC is used for generating offset voltages for other microelectrodes. This requirement was fulfilled by incorporating a 2 µF capacitor to the input of the voltage follower. It was found that 187 ms was needed for the capacitor to reach the required offset voltage of approximately 30-100 mV, and the generated voltage could be held for at least 10 s without appreciable change (less than 5%). A charging period of 200 ms was thus used for the initial charging of each capacitor. After that, each capacitor was recharged periodically every second. A total of four DACs were used in this software-controlled 32channel potentiostat. Among them, two DACs were used for the control of the applied potentials to the 32 working electrodes, and the other two DACs were used for the offset control of 31 background currents. Computer Software. Programs for functional control, data acquisition, storage, display, and processing were written in Turbo-C (V3.0, Borland). Voltammograms shown were obtained from the average of 100 repetitively sampled results. This developed data acquisition and display program allows voltammogram from the 31 microelectrodes to be displayed on the screen twice a second. Several formats for data representation were also developed. The 3-D plots of the current vs time and vs potential (or number of channel), 2-D plots of the current vs time at any specified electrode potential (chromatogram or FIA response), and plots of the current vs potential at any specified time (hydrodynamic voltammogram) can be readily obtained. FIA and HPLC Systems. The FIA measurements were conducted with a Gilson Miniplus 3 peristaltic pump, a Rheodyne 7125 injector with a 100-µL sample loop, and the 32-MEA detector. In HPLC measurement, the flow of solutions was controlled by a Spectra-Physics isocratic pump (SP8810), and a 4.6-mm-i.d. × 250-
mm stainless steel column packed with ODS2 silica (Fisons 5-µm particle size) was used as the separation column. The mobile phase used for phenolic acid separation was a solution of 0.1 M phosphate with the addition of 10% methanol at pH 4.6. The mobile phase used for parathion separation was a solution of 0.025 M phosphate with 50% methanol. The solution was filtered through a 1.0-µm filter prior to use. The flow rate was controlled at 1.2 mL/min throughout the experiment. Reagents. All chemicals and solvents used were of analytical grade and were used as received. The deionized RO water prepared from a Milli-Q system (Millipore) with R no less than 16 MΩ-cm was used for the solution preparation. Hydroquinone, gentisic acid, caffeic acid, o-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, and sulfanilic acid were obtained from Tokyo Kasei Organic Chemicals Co., and catechol was obtained from Jassen Chemicals Co. Solutions of the studied phenolic compounds were prepared from stock solutions before use. Procedures. The newly constructed MEA was polished successively with different grades of polishing paper (120-1000 grade) before use. After polishing, the 32-MEA should be activated by applying a potential of +1.2 V in a 0.1 M NaOH solution for 30 min.20,21 For daily pretreatment of the 32-MEA, potentials of +1.2 V for 5 min and -0.6 V for another 5 min in a phosphate solution were applied. For properly conditioned electrodes, variation of peak potential among different electrodes should be less than 30 mV when monitoring the same electrochemical reaction. The effective working period of the MEA was about 4-5 days. By going through the polishing and activation procedures, activity of the 32-MEA can be reinstated. Normalization of the responses of the microband electrodes and the generator electrode was achieved by applying the same potential (0.8 V vs Ag/AgCl) on each of these electrodes and comparing their responses for the same oxidation reaction of 0.1 (20) Anjo, D. M.; Kahr, M.; Khodabakhsh, M. M.; Nowinski, S.; Wanger, M. Anal. Chem. 1989, 61, 2603-2608. (21) Motta, N.; Guadalupe, A. R. Anal. Chem. 1994, 66, 566-571.
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mM hydroquinone. The normalization procedures were performed daily prior to the FIA or HPLC analyses. The normalization factors changed insignificantly during the same working day. With the 32-MEA detector, the flow at the center microband electrodes will be faster than those at the edges, and the flow stream will not be perfectly parallel with the band electrodes (especially for those at the edges), which might induce the possibility for the product from one band electrode at the edge to interact with its inner neighboring band electrode. But as the width of the electrode array is small (∼4 mm) and the potential difference across the neighboring electrodes is only about 50 mV, the crossover interaction for those band electrodes at or near the edges should be insignificant. Besides the normalization procedures, no further efforts were made to correct the possible errors inherent with the flow effect. RESULTS AND DISCUSSION Characteristics of the 32-MEA Detector. The characteristics of the 32-MEA detector were studied by running solutions of 1.0 mM hydroquinone with an FIA system. The 3-D chronovoltammograms obtained without and with the application of the generator electrode in the 32-MEA are shown in Figure 3a and 3b, respectively. The flow rate of carrier was controlled at 0.5 mL/min. For the 32-MEA detector, the applied potential for the generator electrode was set at 900 mV, those for the 15 upstream electrodes were varied in the range of 865.5-367.5 mV, and those for the 16 downstream electrodes were in the range of 332.5 to -200 mV. The step potential for the adjacent microband electrode was -35.5 mV. From Figure 3, both the oxidation and reduction chronovoltammograms were found, but the reduction chronovoltammograms shown in Figure 3a were very small in magnitude. Without the application of a generator electrode, the quinone could only be produced from the upstream electrodes, and only a small fraction of the produced quinone passed over the downstream electrodes would be reduced. The reduction currents obtained from the downstream electrodes were thus small compared with the oxidation currents produced at the upstream electrodes. The collection efficiency of this MEA detector for hydroquinone (based on the ratio of the limiting reduction current to the limiting oxidation current) was estimated to be about 0.05 (Figure 3a). The reduction chronovoltammograms shown in Figure 3b were much larger than those shown in Figure 3a and were comparable in magnitude with the oxidation chronovoltammograms. With the application of a generator electrode, the collection efficiency of the 32-MEA detector was found to be about 0.45. The collection efficiency for hydroquinone determined with a standard dual electrode was reported to be 0.375.2 The rather small collection efficiency obtained from Figure 3a was due to the very thin microband electrode used and the very large gap between the upstream and downstream electrodes. The relatively larger collection efficiency obtained from Figure 3b should be attributed to the participation of the generator electrode in the electrolysis. As the area of the generator electrode was much larger than that of the upstream electrodes and a high enough potential was applied on the generator electrode, the amount of quinone produced by the generator electrode should be much larger than that produced solely by the upstream electrodes. The small distance between the generator electrode and the downstream electrodes should also increase the reduction efficiency of quinone on the downstream electrodes. 466 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Figure 3. 3-D chronovoltammograms obtained by running solutions of 1.0 mM hydroquinone with an FIA system without (a) and with (b) the application of generator electrode in the 32-MEA detector. Applied potential for generator electrode, 900 mV; upstream electrodes, 865.5-384 mV; downstream electrodes, 332.5 to -200 mV; step potential, 35.5 mV. Carrier for FIA, 0.1 M KH2PO4 with 5% methanol; flow rate, 0.5 mL/min; injection volume, 100 µL.
Figure 4 shows the oxidation and reduction hydrodynamic voltammograms of eight different phenolic acids (each with concentration of 10 ppm) obtained with the 32-MEA detector in an FIA system. The potentials applied for the upstream electrodes were in a range of 1203-550 mV, while those for the downstream electrodes ranged from 503 to -200 mV. The potential step of the adjacent electrode was -47 mV, with the potential of the generator electrode set at 1250 mV. All of the hydrodynamic voltammograms obtained showed sigmoidal oxidation and reduction waves. The limiting oxidation currents in plots of Figure 4 were normalized to unity. Three types of redox behavior were found in Figure 4. A welldefined oxidation-reduction wave and a collection efficiency higher than 0.40, characterized a reversible reaction, were found for the reactions of hydroquinone (Figure 4f), gentisic acid (Figure 4e), caffeic acid (Figure 4g), and catechol (Figure 4h). In contrast to the reactions discussed above, a relatively poorly defined reduction wave and a rather small collection efficiency of 0.15,
Figure 4. Oxidation-reduction hydrodynamic voltammograms of eight phenolic acids (each with concentration of 10 ppm) determined with the 32-MEA detector: 2,6-dihydroxybenzoic acid (a), 2,4-dihydroxybenzoic acid (b), o-hydroxybenzoic acid (c), sulfanilic acid (d), gentisic acid (e), hydroquinone (f), caffeic acid (g), and catechol (h). The limiting oxidation currents were normalized to unity. Applied potentials for the 32-MEA detector were as follow: generator electrode, 1250 mV; upstream electrodes, 1203-549.5 mV; downstream electrodes, 502.8-200 mV; step potential, -46.7 mV.
characterized by the poor reversibility, were found for the reactions of 2,4-dihydroxybenzoic acid (Figure 4b), 2,6-dihydroxybenzoic acid (Figure 4a), and o-hydroxybenzoic acid (Figure 4c). The reduction wave of sulfanilic acid (Figure 4d) showed characteristics in between those of the two groups of compounds discussed above. Its reversibility should also be in between those of the two groups. With this 32-MEA detector, the hydrodynamic voltammograms together with the collection efficiency could be readily obtained and used as an index of reversibility for the studied electrochemical reactions. Table 1 summarizes several parameters, such as the half-wave potential of the oxidation and reduction reactions, the collection efficiency, and the differences in the oxidation and reduction half-wave potentials determined from Figure 4. From
Table 1, it was found that the larger the collection efficiency obtained, the smaller the difference of half-wave potential and the more reversible the reaction. Although the magnitude of the collection efficiency depended on the dimensions of the generator electrode and the design of the electrode assembly, the collection efficiency obtained with the 32-MEA detector should be useful for diagnosing the reversibility of an electrochemical reaction. The collection efficiency was found to vary with the change of flow rate in the studied system, but it became constant as the flow rate was kept lower than 1.5 mL/min. The 32-MEA Detector as an Electrochemical Detector for HPLC. To demonstrate the versatility of the 32-MEA detector, synthetic samples containing 2 ppm each of sulfanilic acid, gentisic acid, 2,4-dihydroxybenzoic acid, catechol, o-hydroxybenzoic acid, Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
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Table 1. Summary of Oxidation and Reduction Half-Wave Potentials, Difference of Half-Wave Potentials, Retention Time, and Collection Efficiencies from Hydrodynamic Voltammograms in Figure 4
2,6-dihydroxybenzoic acid 2,4-dihydroxybenzoic acid o-hydroxybenzoic acid sulfanilic acid gentisic acid hydroquinone catechol caffeic acid
EOx 1/2a (mV vs Ag/AgCl)
ERed 1/2b (mV vs Ag/AgCl)
∆E1/2c (mV vs Ag/AgCl)
retention time (s)
collection efficiencyd (%)
937 ( 23.4 923 ( 23.4 998 ( 23.4 965 ( 23.5 409 ( 23.4 340 ( 23.4 456 ( 23.4 380 ( 23.4
234 ( 23.4 undefinable 346 ( 23.4 314 ( 23.4 225 ( 23.4 197 ( 23.4 290 ( 23.4 380 ( 23.4
703
489 230 367 168 224 220 338 582
15.3 ( 0.4 14.6 ( 0.2 13.5 ( 0.2 23.2 ( 0.4 44.8 ( 0.6 45.2 ( 0.5 39.2 ( 0.7 37.8 ( 0.4
652 651 184 143 166
a Oxidation half-wave potential. b Reduction half-wave potential. c Difference of oxidation and reduction half-wave potentials. d Average of three chromatographic runs.
Figure 6. Hydrodynamic voltammograms at peak times of 224 (a) and 334 s (b) obtained from Figure 5. Figure 5. 3-D chromatovoltammograms obtained by HPLC analysis of a synthetic sample containing (2 ppm each) sulfanilic acid (1), gentisic acid (2), 2,4-dihydroxybenzoic acid (3), catechol (4), ohydroxybenzoic acid (5), 2,6-dihydroxybenzoic acid (6), and caffeic acid (7). For the 32-MEA detector: applied potential for generator electrode, 1250 mV; upstream electrodes, 1203-549.5 mV; downstream electrodes, 502.8 to -200 mV; step potential, 46.7 mV. Mobile phase for HPLC, 0.1 M KH2PO4 with 5% methanol; flow rate, 1.2 mL/min; injection volume, 100 µL.
2,6-dihydroxybenzoic acid, and caffeic acid were analyzed by the 32-MEA detector-incorporated HPLC. The potential scheme used was the same as that used for obtaining the hydrodynamic voltammograms shown in Figure 4. Figure 5 shows the 3-D chromatovoltammograms obtained. From Figure 5, only five voltammetric peaks were found at a glance. But after careful examination, two sets of overlapped waves were found in the voltammetric peaks located at peak times 224 and 334 s, respectively. To have a closer observation, the hydrodynamic voltammograms at the peak times of 224 and 334 s were displayed and shown as curves a and b in Figure 6. The waves that responded for two sets of overlapped peaks were shown clearly. The halfwave potentials for these two sets of reactions were 409, 923 and 456, 998 mV, respectively. By referring to the half-wave potential and retention time data listed in Table 1, oxidation of gentisic acid (component 2), 2,4-dihydroxybenzoic acid (component 3), catechol (component 4), and o-hydroxybenzoic acid (component 5) should be the response for the two sets of overlapped peaks shown. 468 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Besides the components determined above, components 1, 6, and 7 could be identified easily by referring to the half-wave potential and the retention time data listed in Table 1. They were sulfanilic acid, 2,6-dihydroxybenzoic acid, and caffeic acid, respectively. For quantitative determination, a different strategy has to be used for solving the peak-overlapping problem on the chromatograms. The oxidation half-wave potentials for the overlapped components were 409, 923 mV for components 2 and 3, and 456, 1000 mV for components 4 and 5, respectively. With an applied potential in a range of 600-850 mV, only components 2 and 4 in the solution could be oxidized. The limiting currents obtained would thus respond to the oxidation of components 2 and 4, respectively. With an applied potential larger than 1000 mV, the limiting current corresponding to the oxidation of components 2, 3 and 4, 5 in solutions should be obtained. A chromatogram having the response of component 3 or 5 could thus be obtained from the difference chromatogram which resulted upon the subtraction of a chromatogram obtained at lower oxidation potential (600-850 mV) from that at higher potential (e.g., 1200 mV). Figure 7a,b shows the chromatograms obtained from electrodes with applied potential at 1203 (electrode 2) and 690 mV (electrode 13), respectively, and Figure 7c shows the difference chromatogram obtained. The originally overlapped chromatography peaks in Figure 7a were thus resolved into the interference-free peaks, as shown in Figure 7b,c. The resolved chromatogram (Figure 7c) should be good enough for both the
Figure 7. Chromatograms at 1203 mV (electrode 2, a) and 690 mV (electrode 13, b) from Figure 5, and difference chromatogram obtained by numerical subtraction of b from a (c). Also shown are chromatograms obtained at 1203 mV (electrode 2) with solutions containing 4.0 ppb each of phenolic compounds (d).
qualitative and quantitative determinations of the originally overlapped components. The applicability of this numerical subtraction procedure depended on the extent of overlapping and the half-wave potential difference of the overlapped species. Erroneous results could be obtained if the half-wave potential difference was too small (smaller than 250 mV for completely overlapped peaks and smaller than 150-200 mV for partially overlapped peaks).22 For further application, the versatile 32-MEA detector was used for the analysis of parathion, an analyte of high reduction potential. With the same HPLC system, a solution containing 10.0 ppm of parathion was analyzed. Figure 8a shows the 3-D chromatovoltammogram obtained. The applied potentials for the upstream electrodes were in the range of -1148.4 to -426 mV, while those for the downstream electrodes were in the range of -337.4 to 400 mV. The potential step for the adjacent microelectrode was 51.6 mV, with the potential of the generator electrode set at -1200 mV. The mobile phase used was a 0.025 M phosphate solution (22) Last, T. A. Anal. Chem. 1983, 55, 1509-1512.
Figure 8. 3-D chromatovoltammograms obtained by HPLC analysis of 10.0 ppm parathion (a), and the hydrodynamic voltammogram at peak time of 410 s (b). For the 32-MEA detector: applied potential for generator electrode, -1200 mV; upstream electrodes, -1150 to -400 mV; downstream electrodes, -350 to 400 mV; step potential, 51.6 mV. Mobile phase for HPLC, 0.025 M KH2PO4 with 50% methanol; flow rate, 1.2 mL/min; injection volume, 100 µL.
(with 50% methanol), and the flow rate was controlled at 1.2 mL/ min. Deaeration of the mobile phase and standard solutions was achieved by bubbling the solutions with nitrogen for 30 min prior to analysis and continued throughout the experiment. The chromatographic peaks found at retention times of 300 and 410 s were peaks of solvent and parathion, respectively. The hydrodynamic voltammogram at peak time 410 s was displayed and shown as Figure 8b, from which the reduction and oxidation half-wave potentials were found at approximately -810 and 100 mV, respectively. Though the limiting current of reduction was much larger than that obtained for oxidation, the rather high reduction potential applied (e.g., -1100 mV) might induce various interfering reactions, e.g., the reduction of oxygen and other reducible components in solution and even the electrolysis of Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
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solvent in the mobile phase. Adoption of the interference-free oxidation current obtained at lower oxidation potential should thus be a better choice for the quantitative analysis purpose. With the 32-MEA detector, both the reduction and oxidation chromatovoltammograms of analyte (parathion) can be readily obtained, which would provide ample information for the selection of optimal conditions for quantitative analysis. In addition, due to the much larger generator electrode used in the MEA, larger amounts of parathion could be reduced, thus resulting in a larger oxidation current at the downstream electrodes. Better sensitivity can be obtained with this 32-MEA detector than that obtained with the conventional dual-electrode or multielectrode detector. Linearity and Detection LImits. The linearity and detection limits of the 32-MEA detector were evaluated by running the standard solutions of phenolic compounds with the HPLC system. From the calibration graphs plotted, good linearity was obtained for most of the phenolic compounds studied (except caffeic acid) in a concentration range of 10-4-10-8 M. The detection limits (based on S/N ) 3) for the studied compounds were found to be at a level of 2.0 pmol (with an injection volumn of 100 µL), or 2.0 × 10-8 M. Compared with the detection limits evaluated to be (23) Mattusch, J. M.; Baran, H.; Schwett, G. Fresenius J. Anal. Chem. 1991, 426430. (24) Wang, J.; Naser, N.; Angnes, L.; Wu, H.; Chen, L. Anal. Chem. 1992, 64, 1285-1288. (25) Youqin, X.; Huber, C. O. Anal. Chem. 1991, 63, 1714-1719. (26) Wang, J.; Goldrn, T.; Ruiliang, L. Anal. Chem. 1988, 60, 1642-1645. (27) Wring, S. A.; Hart, J. P.; Brich, B. J. Analyst 1989, 114, 1571-1573. (28) Niwa, O.; Morita, M. Anal. Chem. 1996, 68, 355-359. (29) Takahashi, M.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1992, 335, 253-263. (30) Aoki, A.; Matsue, T.; Uchida, I. Anal. Chem. 1990, 62, 2206-2210.
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about 10-700 pmol for various phenolic compounds by Roston and Kissinger2 and about 150 fmol by Mattusch et al.,23 the performance of this 32-MEA detector should be satisfactory. Chromotograms obtained from the injection of sample solutions with a concentration of 4.0 ppb (about 2.0 × 10-8 M) for each phenolic components are shown in Figure 7d. From Figure 7d, due to the inherent difference in sensitivity, peaks for components 1, 2, 3, and 5 could be identified clearly, while peaks for components 6 and 7 were hard to define. The relative standard deviation (n ) 3) for the determination of sulfanilic acid at a concentration of 4.0 ppb was found to be 12.3%. Beyond the analytical applications demonstrated above, the detection capability of the 32-MEA can be enhanced by the incorporation of various electrocatalysts, such as Pd, Pt, Ru,24 copper oxide,25 cobalt phthalocyanine,26,27 etc., into the carbon paste for the construction of microelectrodes. With the application of the lithographic technique,28-30 the number of microband electrodes in the MEA can be increased substantially. That should promote the function of the MEA detector and make its function comparable with that of a spectrophotometric diode array detector. ACKNOWLEDGMENT The authors thank the National Science Council of ROC for financial support of this work (Contract No. NSC 82-0208-M-110047). Received for review June 4, 1996. Accepted November 4, 1996.X AC960546X X
Abstract published in Advance ACS Abstracts, December 15, 1996.
