Use of Disposable GRC Electrodes for the Detection of Phenol and Chlorophenols in Liquid Chromatography

ANALYTICAL SCIENCES MAY 2002, VOL. 18 549 2002 © The Japan Society for Analytical Chemistry

Jiye JIN,* Toshiya HIROI,** Kiyohito SATO,** Tomoo MIWA,** and Toyohide TAKEUCHI**

*Instrumental Analysis Center, Gifu University, 1-1 Yanagido, Gifu 501Ð1193, Japan **Department of Chemistry, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501Ð1193, Japan

In this study, a wall-jet flow cell with a GRC (graphite reinforced by carbon) electrode was designed for the amperometric detection of phenol and chlorophenols in liquid chromatography. The voltammetric responses of these analytes at the GRC electrodes are very similar to those at conventional glassy carbon electrodes. As the GRC electrodes were made of the same materials as commercially available mechanical pencil leads, they exhibit the advantages of low cost, simple surface renewability, lower residual current, and good electrode-to-electrode reproducibility, and thus can be used as disposable-type electrodes. Chromatographic separations of phenol, o-chlorophenol (o-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) were achieved with an ODS column using a

mobile phase containing a mixture of CH3CN and H2O (40:60) containing 25 mM L-(+)tartaric acid (pH = 4.5). Amperometric detections were based on the electrochemical oxidation of these compounds around +0.9 V vs. Ag/AgCl. Under the optimized conditions, linear calibrations were obtained in a range up to 100 µM for phenol, o-CP, 2,4-DCP, 2,4,6-TCP, and 200 µM for PCP, with the correlation coefficients r2 of 0.9992, 0.9997, 0.9986, 0.9992, and 0.9968, respectively. The chromatographic detection limits for the tested analytes were obtained at pmol levels.

(Received November 26, 2001; Accepted March 14, 2002)

renewed by polishing or washing with an organic solvent. So, Introduction such fouling becomes a major obstruction of this approach. In order to detect at much lower anodic potential, or to improve the Phenolic compounds are important industrial chemicals which stability of the electrodes, a number of researches have been cause environmental concern. Many phenols, especially reported by using chemically modified electrodes or biosensors chlorophenols (CPs), are known to be highly toxic to man and for the application in HPLC analysis.13Ð15 However, in most to aquatic organisms. In addition, certain thermal or photolytic applications, the electrode selection is still being focused on condensation of chlorophenols will result in the production of carbon-based materials because of their low background dioxins in the environment. Consequently, the development of currents and easy management in flow analysis systems. a sensitive and selective method for the detection of these The mechanical pencil leads have been used as electrode compounds is of particular importance. material in recent years. Aoki et al.16 first reported the use of Trace levels of phenol and CPs from environmental samples GRC (graphite reinforced by carbon) electrodes in are usually quantified using gas chromatography (GC),1,2 high- electroanalytical applications. These electrodes are made of performance liquid chromatography (HPLC),3Ð7 and capillary materials similar to commercially available mechanical pencil electrophoresis.8 In general, HPLC can offer the milder leads, which are usually made from carbon/carbon composites conditions for sample preparation, and consequently, the of neutral crystalline graphite powder and amorphous carbon. volatile phenolic compounds can be better prevented from being Therefore, the GRC electrodes provide good electrochemical lost as compared to GC approaches. On the other hand, as the properties arising from the edge of natural graphite powder, and conventional UV or fluorescence detectors in HPLC are not have been successfully applied in many voltammetric sufficiently sensitive to these compounds, post- or precolumn studies.17Ð21 So far, however, few reports have explained the derivation reactions are required to enhance UV or fluorescence application of such material as an electrochemical detector in sensitivity.5,6 HPLC. In this research, a wall-jet amperometric detector with Electrochemical detection coupled with HPLC can be an ideal the GRC electrode was designed for the detection of phenol and approach for such tasks because phenol and CPs are CDs in HPLC. The advantages of using GRC electrode were electroactive and can be detected with high sensitivity.9,10 their low cost, simple surface renewability, and good electrode- Moreover, the high selectivity of electrochemical detection to-electrode reproducibility. Therefore, they can be used as often requires less sample-pretreatment, in turn allowing rapid disposable-type electrodes which would facilitate phenolic and inexpensive detection. On the other hand, however, compound studies. Factors influencing both the performance of because high electrode potentials were required to observe the amperometric detection and the HPLC separation were useful oxidation signals, the electrodes became rapidly fouled investigated. due to the adsorption of dimeric or polymeric oxidation products.11,12 The fouled electrodes are usually not easily 550 ANALYTICAL SCIENCES MAY 2002, VOL. 18

Fig. 1 Construction of the flow cell. 1, PCTFE block (30 × 35 × 40 mm); 2, pencil lead (GRC working electrode); 3, Ag/AgCl reference electrode; 4, stainless-steel tube auxiliary electrode (0.25 mm i.d., 1.58 mm o.d.; 50 mm in length); 5 and 6, O-rings.

Experimental Fig. 2 Cyclic voltammograms of phenol and chlorophenols at a GRC electrode in CH3CNÐH2O (40:60, v/v) containing 25 mM of L- Reagents (+)tartaric acid. (A) blank, (B) phenol, (C), o-CP, (D) 2,4-DCP, (E) 2,4,6-TCP and (F) PCP. Analyte concentration, 1 mM; scan rate, 50 Phenol, o-chlorophenol (o-CP), 2,4-dichlorophenol (2,4- mV/s. DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) were purchased from Nacalai Tesque (Kyoto, Japan). The stock solutions of the analyte were prepared in methanol. The other chemicals used were of The HPLC system consisted of a CCMP pump system (Tosoh, analytical regent grade. Aqueous solutions were prepared with Tokyo, Japan), a 7000 injector with a 10-µl sample loop purified water (Milli-Q, Millipore System). (Rheodyne, Cotati, CA, USA) and a µBondapack C18 column (3.9 mm i.d. × 15 cm, Waters). The column was placed inside Preparation of the GRC electrode and construction of the flow into a column heater that was controlled by a TCM temperature cell control module (Waters). A solution of CH3CNÐH2O (40:60, The commercially available mechanical pencil leads (0.5-mm v/v) mixture containing 25 mM L-(+)tartaric acid was employed in diameter, 2 H grade, Mitsubishi Pencil, Japan) were used as as the mobile phase for HPLC separation. The pH of the mobile GRC electrodes. The electrode was prepared by inserting the phase was adjusted with 0.1 M NaOH or 0.1 M HCl solutions. pencil lead into a Teflon tube (0.5 mm i.d, 1.2 mm o.d; 15 mm The flow rate was set at 0.5 ml/min for flow injection in length) when it was being softened over a micro-flame experiments and at 1.0 ml/min for HPLC separations. heater. The GRC electrode can be tightly sealed in a Teflon Amperometric detections were performed with an HECS-972 tube after cooling. A glassy carbon (GC) working electrode potentiostat (Huso, Kanagawa, Japan). The chromatograms was prepared by sealing a GC rod (1-mm in diameter, Tokai were recorded on a Macintosh computer interfaced with a Carbon) in a glass tube with Epoxy resin. The electrodes were PowerLab data acquisition module, which was controlled by then polished with pieces of emery paper and soft paper to Power Chrom software (AD Instruments, NSW, Australia). expose a disk-shaped surface, and finally rinsed and sonicated in water. An electrochemical flow cell with a GRC electrode was Results and Discussion constructed, as shown in Fig. 1. The cell body was made of polychlorotrifluoroethylene (PCTEF), and a pencil lead Cyclic voltammetry of phenol and CPs at the pencil electrodes electrode can be easily set into the cell. The cell is based on Phenol and CPs are electroactive due to their electron- “wall jet” principle, in which the flow stream comes from the withdrawing substitution structures. In order to establish the chromatographic column perpendicular to the working electrode detection conditions for these compounds, their voltammetric surface. The reference electrode (Ag/AgCl, Hokuto Denko behavior at the pencil electrode was investigated. The Corp., Japan) was specially designed to house in the cell body. electrochemical responses of these compounds have been The tip of the reference electrode was 4 mm in diameter. A suggested to depend on the solvent-electrolyte and on the stainless-steel exit tube was used as an auxiliary electrode. electrode materials.15 In this study, the measurements were

carried out in a CH3CNÐH2O mixed solvent (40:60, v/v) Apparatus containing 25 mM of L-(+)tartaric acid as supporting Voltammetric measurements were performed with a CHI electrolyte.13 The mixed solution was chosen because it was Model 660 Electrochemical Analyzer (CH Instruments, Austin, used as mobile phase in HPLC experiments afterwards. Figure TX, USA). A three-electrode electrochemical cell was 2 shows the cyclic voltammograms of these compounds at pH comprised of an RE-1 reference electrode (Ag/AgCl; BAS, of 4.5. There was no redox peak in the background response Tokyo, Japan), a platinum wire auxiliary electrode and a (A), indicating that the impurities in the electrode material do working electrode. not interfere with the measurement in the applied potential ANALYTICAL SCIENCES MAY 2002, VOL. 18 551

Table 1 Voltammetric peak potentials (Epox, V vs. Ag/AgCl) and peak currents (Ip, µA) for the oxidation of phenol and chlorophenols at various pH values pH = 2.2 pH = 3.0 pH = 4.5 pH = 5.9 Compound Epox/V Ip/µA Epox/V Ip/µA Epox/V Ip/µA Epox/V Ip/µA Phenol +1.05 2.4 +0.95 2.3 +0.84 2.1 +0.82 2.0 o-CP +0.94 2.0 +0.90 2.0 +0.78 1.8 +0.76 1.7 2,4-DCP +0.92 1.4 +0.89 1.5 +0.81 1.5 +0.75 1.4 2,4,6-TCP +0.85 1.8 +0.82 1.4 +0.69 1.3 +0.68 1.2 PCP +0.95 2.1 +0.90 2.1 +0.82 2.0 +0.82 2.0

Fig. 3 Cyclic voltammograms of o-CP recorded at first and third scans in 1 M KCl. (A) GRC electrode; (B) GC electrode.

range. Voltammograms (B) Ð (F) are those for phenol, o-CP, easily renewed with a simple polishing procedure, or by 2,4-DCP, 2,4,6-TCP and PCP, respectively. The responses at washing with organic solvents. This thus limited the the GRC electrodes were almost identical to those obtained at a voltammetric studies on the phenolic compounds. This GC electrode. 2,4,6-TCP shows relative reversible behavior problem, however, can be easily overcome by using GRC with the redox potentials located at +0.68 and +0.6 V vs. electrodes because they are readily renewed by cutting of the Ag/AgCl, respectively. The irreversible electrochemical fouled surface, or simply by polishing on a piece of clean soft reactions were observed for the others (phenol, o-CP, 2,4-DCP paper. and PCP) with the oxidation potentials around +0.8 Ð +0.9 V vs. Ag/AgCl, while the reduction peaks of these compounds were Amperometric detection in flow system ill-defined. The voltammetric response of these compounds In the application of the pencil electrodes in a flow system, was also influenced by pH, as is summarized in Table 1. The the choice of the detecting potential is important. Figure 4 peak potentials shifted cathodically as the pH value was shows hydrodynamic voltammograms (HDVs) obtained by flow increased, indicating that the proton was involved in the injection of 100 µM each of phenol and CPs. The mobile phase electrochemical oxidation process. The peak currents were was a solution of CH3CNÐH2O (40:60, v/v) containing 25 mM almost unchanged. of L-(+)tartaric acid at a flow rate of 0.5 ml/min. The It is worth noting that the voltammograms in Fig. 2 were background current at each potential is also displayed in the recorded at the first CV scan. The peak currents were found to figure. The phenol and different CPs exhibited almost the same become smaller and the oxidation peaks were slightly shifted to trend in response as a function of applied potential; such effects positive potentials in the subsequent CV scans. Obviously, the which were in agreement with the cyclic voltammograms in Fig. adsorbed products are somewhat inert under the present 2. The maximum responses of these compounds were found experimental conditions, and thus inhibit the oxidation reactions around +0.9 V vs. Ag/AgCl. When one applied a potential more in the subsequent CV scans. In order to compare the positive than 1.0 V vs. Ag/AgCl, the responses decreased due to passivation behavior with that of a conventional GC electrode, a the oxidation of the electrolyte. Accordingly, a potential value set of voltammetric experiments on CPs in 1 M KCl supporting of +0.9 V vs. Ag/AgCl gave the best compromise between electrolyte was performed. Figure 3 shows cyclic signal and background amplitudes and was chosen to carry out voltammograms of 1 mM o-CP recorded at first and third scans the amperometric detection in the flow system. on GRC (A) and GC (B) electrodes, respectively. Clearly, a The features of the GRC electrodes are similar to those of more rapid and significant decrease in the peak current was graphite electrodes, which exhibit the large potential window observed at the GC electrode. This tendency was also observed and favorable signal-to-background characteristics. We found for the oxidation of other CPs. Although the reason is still that the GRC electrodes provided good stability in CH3CNÐH2O uncertain now, it seems that the pencil electrodes show better mixed solvent in comparison with carbon paste or some organic resistance to fouling than do the GC electrodes. According to polymer based graphite electrodes. So their analytical our experience, the poisoned GC electrodes are usually not applicability in flowing systems is expected. We screened 552 ANALYTICAL SCIENCES MAY 2002, VOL. 18

Fig. 4 Hydrodynamic voltammograms of phenol and CPs. The Fig. 6 Effect of pH on the retention behavior for ( ) phenol, ( ) analytes were 100 µM each of ( ) phenol, ( ) o-CP, ( ) 2,4-DCP, o-CP, ( ) 2,4-DCP, ( ) TCP, and ( ) PCP. Column, Waters ( ) 2,4,6-TCP, and ( ) PCP. The background current ( ) is also µBondapack C18; flow rate, 1 ml/min; detection potential, +0.9 V vs. plotted. Sample injection volume, 10 µl; mobile phase, CH3CNÐH2O Ag/AgCl; analyte concentration, 100 µM; sample injection volume, (40:60, v/v) containing 25 mM of L-(+)tartaric acid (pH = 4.5); flow 10 µl. rate, 0.5 ml/min.

lipophilic properties of the analytes. CH3CN was found to be preferable to methanol in terms of reducing the background current and improving the detector performance. 25 mM of L-(+)tartaric acid was added in the mixture to provide supporting electrolyte for electrochemical detection. Because the acidity of the phenolic compound increases with increasing

chloro-substitution, from a pKa value of 9.9 for phenol to 4.7 for pentachlorophenol,22 the retention factors depended on the pH of the mobile phase. Figure 6 shows plots of the retention factor (k) in logarithm scale versus the mobile phase pH. The retention factors for phenol, o-CP, 2,4-DCP and 2,4,6-TCP varied very little within the tested pH range. However, the retention factor for PCP was changed greatly by varying the pH from 2.2 to 5.8. At pH of 5.8, PCP was ionized to

pentachlorophenolate ion due to its lower pKa value. Fig. 5 The repeated FIA peaks for 100 µM phenol detected at a Consequently, it eluted before TCP. By performing the GRC electrode. Detection potential: +0.9 V vs. Ag/AgCl. The other separation at pH 4.5 with a flow rate of 1.0 ml/min, the conditions were the same as in Fig. 4. excessively long retention of PCP was reduced and the tested analytes were completely separated within 12 min. The influence of the temperature on both retention and detection behavior was also investigated. A decrease of pencil leads with different grades (2B, HB, 2H). The 2H pencil approximate 8% in retention time for PCP was observed when lead was found to be the most excellent in terms of its the temperature was increased from 20 to 50ûC. The retention mechanical strength and stability in amperometric response. of the other analytes remained almost unchanged with The repeated flow injection peaks for 100 µM phenol are shown increasing the temperature. On the other hand, an increase of in Fig. 5. The relative standard deviation (RSD) was estimated approximately 15% in peak area for each analyte was obtained as 1.2% according to the peak current measurements. The when the temperature increased from 20 to 30ûC, a consequence electrode fouling might not be a major problem when the of the background noise remaining almost constant. However, sample concentration was very low. In this study, one electrode when the temperature was set above 35ûC, the background noise could be used continuously for 3 days without deterioration of increased nearly 3 times as compared with that at 20ûC. Though the signals. The ease of the electrode surface regeneration was the peak area also increased with increasing the temperature, the a very important practical aspect. When the electrode is fouled, signal to noise ratio (S/N) decreased at temperatures over 35ûC. it can be replaced by a new one, or its surface can be renewed For these reasons, 30ûC was regarded as the optimum operating easily as described in the section above. temperature.

HPLC separation conditions Detection limit and reproducibility The HPLC separation experiments were carried out with a Figure 7 displays the chromatograms of the mixture standards

Waters µBondapack C18 column. The temperature was with increasing concentration (12.5 to 100 µM for phenol, o- controlled at 30ûC. As a mobile phase, the mixtures of CP, 2,4-DCP and 2,4,6-TCP; 25 to 200 µM for PCP). Defined

CH3CNÐH2O (40:60) were used in order to enhance the peaks and low noise level were observed. The retention times, ANALYTICAL SCIENCES MAY 2002, VOL. 18 553

Table 2 Determination of phenol and chlorophenols at the GRC electrode in HPLCa

Retention time/ Detection limitb Linear range y (nA) = a + bx (µM)c Compound RSDd, % min µM pmol abr2 Phenol 3.5 0.1 1.0 0.0869 0.6595 0.9992 1.7 o-CP 3.9 0.3 1.2 0.1958 0.2438 0.9997 2.0 2,4-DCP 5.1 0.5 2.2 0.3804 0.1852 0.9986 2.0 2,4,6-TCP 6.7 0.7 4.0 0.1500 0.1184 0.9992 2.6 PCP 10.2 2.5 12.0 0.0108 0.0362 0.9968 4.5

a. Detection potential, +0.9 V vs. Ag/AgCl; sample injection volume, 10 µl; mobile phase, CH3CNÐH2O (40:60, v/v) containing 25 mM of L-(+)tartaric acid (pH = 4.5); flow rate, 1.0 ml/min. b. Detection limits were estimated at S/N = 3. c. In the regression equation, y is the peak current (nA) and x is the concentration of the tested analytes. d. Relative standard deviation obtained from five repetitive injections of the sample containing 50 µM each of phenol, o-CP, 2,4-DCP, 2,4,6-TCP and 100 µM PCP.

with respect to the applied potential, mobile phase and operational temperature. Under the optimized conditions, linear calibrations were obtained in a range up to 100 µM for phenol, o-CP, 2,4-DCP, 2,4,6-TCP and 200 µM for PCP, with the correlation coefficients r2 of 0.9992, 0.9997, 0.9986, 0.9992, and 0.9968, respectively. The detector exhibited detection limits at the pmol levels and provided sufficient stability and reproducibility. Because the GRC electrodes have the advantage of easy surface renewability, and they are available to be used as a disposable-type electrode, a high practical utility is expected in analysis of phenol and its chlorinated derivatives.

Acknowledgements

The authors express their thanks to Dr. H. Hosono in Research & Development Department of Hokuto Denko Corporation for Fig. 7 Chromatograms of the mixture standards. a) 12.5 µM each the help with design and construction of the flow cell. of phenol, o-CP, 2,4-DCP and 2,4,6-TCP and 25 µM PCP; b) 25 µM each of phenol, o-CP, 2,4-DCP and 2,4,6-TCP and 50 µM PCP; c) 50 µM each of phenol, o-CP, 2,4-DCP and 2,4,6-TCP and 100 µM PCP; References d) 100 µM each of phenol, o-CP, 2,4-DCP and 2,4,6-TCP and 200 µ M PCP. Detection potential, +0.9 V vs. Ag/AgCl; sample injection 1. Tesarova and A. V. Pacakova, Chromatographia, 1983, 17, volume, 10 µl; mobile phase, CH CNÐH O (40:60, v/v) containing 25 3 2 269. mM of L-(+)tartaric acid (pH = 4.5); flow rate, 1 ml/min; temperature, 30ûC. 2. H. K. Lee, S. F. I. Li, and Y. H. Tay, J. Chromatogr., 1988, 438, 429. 3. S. Angelino and M. C. Gennaro, Anal. Chim. Acta, 1997, 346, 61. chromatographic detection limits, and dynamic ranges for the 4. X. Jin, M. Zhu, and E. D. Corte, Anal. Chem., 1999, 71, tested compounds are summarized in Table 2. Excellent 517. correlation coefficients were obtained over the range of 5. M. Wada, S. Kinoshita, Y. Itayama, N. Kuroda, and K. concentrations determined. Based on S/N ≥3, the detection limit Nakashima, J. Chromatogr. B, 1999, 721, 179. for phenol was estimated to be approximately 0.1 µM (1 pmol). 6. O. Fiehn and M. Jekel, J. Chromatogr. A, 1997, 769, 189. Under the same experimental conditions, the sensitivity was 7. S. Angelino and M. C. Gennaro, Anal. Chim. Acta, 1997, comparable to that obtained by the commercial electrochemical 346, 61. detector with a glassy carbon electrode. The electrode-to- 8. J. H. T. Luong, A. Hilmi, and A.-L. Nguyen, J. electrode reproducibility was evaluated by running the Chromatogr. A, 1999, 864, 323. chromatographic experiments with 5 individual pencil 9. R. E. Shoup and G. S. Mayer, Anal. Chem., 1982, 54, 1164. electrodes. When the base line got stable, the RSDs were 5.6% 10. M.-H. Chao and H.-J. Huang, Anal. Chem., 1997, 69, 463. for phenol (50 µM), 4.8% for o-CP (50 µM), 6.5 % for 2,4-DCP 11. C. D. Garcia and P. I. Ortiz, Anal. Sci., 1999, 15, 461. (50 µM), 6.2% for 2,4,6-TCP (50 µM), and 7.1% for PCP (100 12. M. Gattrell and B. MacDougall, J. Electrochem. Soc., 1999, µM), respectively. The accuracy of the method proposed here is 146, 3335. thus satisfactory. 13. K. B. Male, C. Saby, and J. H. T. Luong, Anal. Chem., 1998, 70, 4134. 14. O. Adeyoju, E. I. Iwuoha, M. R. Smyth, and D. Leech, Conclusions Analyst, 1996, 121, 1885. 15. T. Mafatle and T. Nyokong, Anal. Chim. Acta, 1997, 354, In conclusion, the properties of the pencil leads have been 307. exploited for electrochemical detection of phenol and 16. K. Aoki, T. Okamoto, H. Kaneko, K. Nozaki, and A. chlorophenols in HPLC. The performance of an Negishi, J. Electroanal. Chem., 1989, 263, 323. electrochemical detector with a GRC electrode was evaluated 17. J. Wang, A.-N. Kawdw, and E. Sahlin, Analyst, 2000, 125, 554 ANALYTICAL SCIENCES MAY 2002, VOL. 18

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