Anal. Chem. 1999, 71, 873-878
Development of Electrokinetic Capillary Electrophoresis Equipped with Amperometric Detection for Analysis of Explosive Compounds Abdelkader Hilmi, John H. T. Luong,* and An-Lac Nguyen
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2
Cyclic voltammograms of trinitrotoluene (TNT) and other related explosive compounds obtained by using glassy carbon, platinum, nickel, gold, and silver electrodes revealed the applicability of gold and silver in capillary electrophoresis (CE) amperometric detection. The selected electrode, gold or silver, was inserted into a specially designed detection cell that was easily adapted to a commercial CE apparatus. The electrochemical reduction of TNT, octahydro-1,3,5,7-tetranitro-1,3,5,7tetrazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and 10 other explosives could be reliably monitored at -700 mV vs Ag/AgCl. CE analyses were performed with a borate buffer (15 mM, pH 8.7) containing 25 mM sodium dodecyl sulfate (SDS) to attain baseline resolution of the selected compounds. A bimetal electrode, prepared by depositing silver on gold, offered a superior performance by exploiting the sensitivity of gold while suppressing its response toward acetonitrile to achieve a 10-fold lower detection limit than UV measurement. The CE system equipped with amperometric detection was used to determine the explosive content of soil extracts and groundwater, yielding results in good agreement with those obtained by the liquid chromatographic method recommended by the U.S. Environmental Protection Agency. Decommissioning of military bases in several locations throughout the world requires thorough verification before the sites can be developed for other uses, including being allowed to reintegrate with nature. Trinitrotoluene (TNT) and other related explosive compounds must be monitored in soil, groundwater, and surrounding waterways since these mutagenic, toxic, and persistent pollutants can leach from the contaminated soil to accumulate in the food chain.1 The need for on-site TNT monitoring has been adequately satisfied with the development of immunochemical sensors2 and electrochemical probes3 that are virtually free of interference from contaminants such as benzidine, phenol, hydrazine, and metal ions. However, nitrophenol, nitrobenzene, and dinitroaniline, when present in the sample, will significantly affect (1) Walker, J. E.; Kaplan, D. L. Biodegradation 1992, 3, 369-385. (2) Shriver-Lake, L. C.; Breslin, K. A.; Charles, P. T.; Conrad, D. W.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1995, 67, 2431-2435. (3) Wang, J.; Bhada, R. K.; Lu, J.; MacDonald, D. Anal. Chim. Acta 1998, 361, 85-91. 10.1021/ac980945n CCC: $18.00 Published on Web 01/14/1999
© 1999 American Chemical Society
the response of these specific monitors. In addition, samples with high levels of endogenous humic materials require complicated treatment and/or instrumentation arrangement involving microdialysis sampling. In practice, TNT is often found in a matrix, soil or water, that also contains several related compounds, including TNT degradation intermediates and metabolites as well as nontoluene based explosives. Therefore, detection of these explosives in complex environmental matrixes by either immunochemical or electrochemical sensors is difficult and will require new analytical methods. The search for sensitive and rapid procedures to simultaneously quantitate explosives in complex samples has been earnestly conducted, and practically all separation techniques, including gas chromatography (GC),4 liquid chromatography (LC),5 and supercritical fluid chromatography (SFC),6 have been explored and developed for routine assay. The current U.S. Environmental Protection Agency (EPA)-recommended method (Method 8330) for explosive analysis requires two LC runs for complete identification. Effort has also focused on the development of highly efficient capillary electrophoretic (CE) procedures, particularly electrokinetic chromatography (EKC) to improve the resolution for several explosive compounds which cannot be resolved by LC. An EKC procedure using a sodium dodecyl sulfate (SDS)-phosphate, pH 7, electrolyte was first attempted by Oehrle7,8 to separate 14 common explosives including 2,4-DNT (dinitrotoluene), 2,6-DNT, 2-NT (nitrotoluene), 3-NT, and 4-NT. Recently, even better resolution, where 10 positional nitroaromatic isomers were baseline resolved, was achieved with a mixed-mode EKC technique that utilized SDS and negatively charged sulfobutyl ether β-cyclodextrin, as a pseudo-stationary phase with different selectivities toward the analytes.9 The resolution power of EKC is thus well established, but UV measurement is not sufficiently sensitive for detection of explosives in environmental samples. Furthermore, many extractable components in contaminated soils, particularly soils that have supported vegetation, often interfere with absorbency measurement. (4) Hable, M.; Stern, C.; Asowata, C.; Williams, K. J. Chromatogr. Sci. 1991, 29, 131-136. (5) Kleibohmer, W.; Camman, K.; Robert, J.; Musenbrock, E. J. Chromatogr. 1993, 638, 349-356. (6) Wallenborg, S. R.; Markides, K. E.; Nyholm, L. J. Chromatogr. A 1997, 785, 121-128. (7) Oehrle, S. A. J. Energ. Mater. 1996, 14, 47-56. (8) Oehrle, S. A. Electrophoresis 1997, 18, 300-302. (9) Luong, J. H. T.; Guo, Y. J. Chromatogr. A 1998, 811, 225-232.
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Table 1. Structures, Abbreviations, and Peak Assignments of the Nitroaromatics, Nitramine Explosives, RDX, and HMX
Electrochemical detectors, which promise high sensitivity, simplicity, and low cost, have been coupled with CE to detect carbohydrates, amino acids, neurotransmitters, and chlorophenols.10-13 Since nitroaromatic explosive compounds are known to exhibit well-defined redox behavior,14 amperometric detection appears to be promising for analysis of such analytes. The incentive is also augmented by the fact that most of the UVabsorbing compounds, including humic acids from soils, are not electroactive at the potential required for detection of nitroaromatics (-700 mV). The objective of this work is to develop a simple end-column amperometric detector in conjunction with EKC for analysis of TNT and related compounds. Gold, silver, and silver-modified gold electrodes are investigated for sensitive detection of the explosive compounds including RDX and HMX in soil extracts and groundwater, and their detection limit is compared with absorbency measurement. Cyclic voltammetry is applied to characterize electrochemical reduction and oxidation of TNT and other explosives to provide a basis for CE with amperometric detection of the explosives. EXPERIMENTAL SECTION Materials. All explosives were purchased from Chem Service (West Chester, PA), whereas other chemicals were obtained from Aldrich (Milwaukee, WI). Table 1 gives the structures, common abbreviations, and peak assignments for the explosives investigated in this study. The soil and groundwater samples were taken from contaminated sites. Metal wires were purchased from Alfa Aesar (Ward Hill, MA) and Aldrich. Sample Preparation. Stock solutions of explosives were prepared in acetonitrile (ACN), and an appropriate volume of each (10) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1765. (11) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62, 1872-1876. (12) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307-312. (13) Hilmi, A.; Luong, J. H. T.; Nguyen, A. L. J. Chromatogr. A 1997, 761, 259268. (14) Bratin, K.; Kissinger, P. T.; Briner, R.; Bruntlett, C. Anal. Chim. Acta 1981, 130, 295-311.
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component was added to ACN to obtained desired concentrations. Prior to CE or LC analysis, the groundwater sample was passed through a 0.45-µm filter. Extraction of the explosives in contaminated soils was carried out by agitating 1 g of soil and 10 mL of ACN in a capped container for 30 min, followed by centrifugation and filtration with a 0.45-µm disk filter. All samples were protected from light and kept at 4 °C. Instrumentation. Cyclic voltammetric (CV) experiments used a potentiostat/galvanostat (model 263A, EG&G, Princeton Applied Research, Princeton, NJ) to operate a three-electrode system consisting of a saturated Ag/AgCl reference electrode (RE-1, Bioanalytical Systems (BAS), West Lafayette, IN), a platinum wire counter electrode, and a working electrode. Glassy carbon electrodes and gold electrodes were also purchased from BAS, while the platinum, silver, or nickel wires (0.127 mm) served as the corresponding working electrodes. Analog signals from the voltammograph were digitized by an A/D board (DP 500-AD supplied with an interface box, Labtronics, Guelph, ON, Canada) that was installed on a PC 486 computer. The data were stored in ASCII files and converted to PRN files for treatment by a graphic program. All experiments were performed at room temperature (21-23 °C). Preparation of Detecting Electrodes. The amperometric detection cell required a working electrode prepared with metal wire. A 3-cm wire (0.127 mm diameter) was inserted into a glass capillary (1.2 mm i.d., commonly used for melting point determination, 1 cm long), with the wire recessing about 2 mm from one capillary end. This capillary end was heated with a Bunsen burner to seal the glass around the wire. The resulting capillary end was then polished on fine silicon carbide abrasive paper aided by an alumina slurry (CF-1050, BAS). Microscopic inspection was performed in order to retain only those having a circular cross section well sealed with the glass capillary. A piece of 0.5-mm silver wire was attached to the other end of the small wire so that the 0.5-mm wire almost touched the end of the glass capillary. Epoxy glue was used to secure a rigid connection between the 0.5-mm wire and the glass capillary (Figure 1). Preparation of Silver-on-Gold Electrodes. A gold electrode, prepared as described above, was installed as the working electrode in a three-electrode system, and 1 mM silver nitrate in 0.1 M nitric acid was placed in the voltammetric cell. A potential of -2 V was then applied for 5 min to deposit silver on the gold surface. Amperometric Detection and Electrophoretic Capillary Arrangement. Polyimide-coated fused silica capillaries of 20 µm i.d. (internal diameter) were purchased from Polymicro Technologies (Phoenix, AZ). Vacuum injection (10 s), 20 kV, and an 80cm capillary length were used in all cases. Electropherograms were obtained with the Applied Biosystems CE instrument (ABI270A, Perkin-Elmer, Foster City, CA). The factory-installed detector was used in experiments requiring UV measurement, set at 230 mm. For amperometric detection, one capillary end was top mounted into the anodic reservoir. However, the other capillary end was inserted into the cathodic reservoir through a septum opening, created at the bottom (Figure 1). Three other septa were installed on the side of the reservoir to allow insertion of the three electrodes. The reference electrode was a silver wire with AgCl formed at the tip by electroformation, while a platinum wire served
Figure 2. Cyclic voltammogram of 200 ppm TNT in 15 mM borate, pH 8.7, containing 25 mM SDS. Glassy carbon working electrode vs Ag/AgCl reference at 50 mV/s (scan rate).
Figure 1. Amperometric detection cell with details of the detecting electrode. Components are not drawn to scale. Electrodes: (1) detecting or working, (2) counter, (3) reference.
as the counter electrode. The metallic working electrode, as described above, was used as the detecting electrode and positioned directly at the capillary outlet. The separation capillary was positioned so that its outlet was as close to the tubing center as possible. Due to the large diameter of the detecting electrode (0.127 mm) compared to the internal diameter of the capillary (20 µm), good electrochemical efficiency was achieved without precise microalignment. During electrophoresis, a CV-1B voltammograph (BAS) was used to apply -700 mV to the detecting electrode. The time response data were digitized and treated by the same procedure used for cyclic voltammetry. Peak Identification and Concentration Calculation. The electropherogram peaks were identified by spiking with individual components. For each component, a series of runs at different concentrations was performed to obtain a peak height vs concentration plot, used for calculating concentration in unknown samples. Liquid Chromatography (LC). The system consisted of a pump (model 590, Waters, Milford, MA) a 20-µL injection loop (model 7725, Rheodyne, Cotati, CA), a C18 column (15 cm length packed with 5-µm spheres, Supercosil LC-PAH, Supelco, Mississauga, ON, Canada), and an UV detector at 230 nm (Waters LC spectrophotometer, model 481). Safety Considerations. TNT and other explosive compounds cause headache, weakness, anemia, and liver injury, and vapor of such chemicals is very dangerous. Stock solutions of the explosives (a few ppm or less) must be prepared and handled in a ventilated hood. Disposable latex gloves and then Ansell Edmont shoulder-length neoprene gloves (Aldrich) must be worn to avoid any contact or exposure while working with explosive compounds because they may be absorbed through the skin. The stock solutions must be stored in closed small glass containers (100 mL or less), remote from any reducing reagents. These explosives are also toxic and in part carcinogenic and mutagenic, and therefore special care must be taken to dispose of waste solutions.
RESULTS AND DISCUSSION Cyclic Voltammograms of TNT and Other Explosives. The nitro groups of TNT and other nitroaromatics were reported to undergo reduction to hydroxylamines, followed by conversion to amine groups.3 Schmelling et al.15 also studied the cyclic voltammetric behavior of TNT and concluded that sequential reduction of the three nitro groups resulted in three distinct reductive waves. The half-wave potentials were reported as -0.22, -0.39, and -0.57 V when the supporting electrolyte was nitrogen-sparged 0.1 M phosphate buffer, pH 5.1, with 8% methanol and the working electrode was glassy carbon measured against an Ag/AgCl reference electrode. Initial experiments conducted in this study confirmed that the reduction of TNT in a milieu suitable for capillary electrophoretic separation (15 mM borate buffer, pH 8.7, containing 25 mM SDS) also yielded three well-defined reduction waves (Figure 2). The CV experiment was initially conducted using a glassy carbon working electrode since carbon fibers were commonly used in CE amperometric detection.11 Unfortunately, difficulties were encountered when the glassy carbon or carbon fiber electrode was connected to the separation capillary due to the fragility of the former. Consequently, the search was directed toward the rigid metal electrodes prepared with platinum, nickel, silver, gold, and silver-plated gold. Platinum poised at -0.4 V vs normal hydrogen electrode (NHE) was shown to reduce TNT.15 However, in this study, the cyclic voltammogram obtained with either the platinum or nickel electrode displayed progressively reduced peaks with each cycle, an indication of electrode fouling. In contrast, the CV obtained for TNT using a gold electrode exhibited a well-defined reduction peak (-0.77 V) and an oxidation shoulder (-0.25 V) that did not diminish with repeated cycles (Figure 3A, dotted line). In this system, both 3-NT and 2,3-DNT (Figure 3B and C, dotted lines) displayed stable voltammograms. The CVs obtained with the silver electrode were also reproducible with repeated sweeps (Figure 3, solid lines), although they possessed different current magnitudes (compared with CVs monitored with the gold electrode) due to the difference in the electrode sensing areas. Based on such results, only gold and silver were used for subsequent CE amperometric detection. The cyclic voltammograms obtained with the gold and silver electrodes were notably different from those monitored with a glassy carbon electrode. With the carbon electrode, the CV displayed well-defined peaks, with the number of peaks corresponding to the number of nitro groups on the aromatic ring. Such (15) Schmelling, S. C.; Gray, K. A.; Kamat, P. V. Environ. Sci. Technol. 1996, 30, 2547-2555.
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Figure 3. Cyclic voltammograms at 50 mV/s scan rate obtained with gold electrode (dotted lines) and silver electrode (solid line). (A) TNT, (B) 3-NT, (C) 2,3-DNT in 15 mM borate, pH 8.7, containing 25 mM SDS.
characteristics were not obtained with either gold or silver electrodes. The underlying reason requires further investigation which is beyond the scope of this study. Nevertheless, the cyclic voltammograms obtained by the gold or silver electrodes exhibited good reductive electroactivity at -700 mV; therefore, this applied potential was used to pursue CE with amperometric detection. Amperometric Detection Cell. Fragility is a major drawback of early devices10-12 designed to perform amperometric detection in CE systems, and more sturdy designs have recently been reported.16,17 The fragility is a direct consequence of the need to decouple the detecting electrode from the high voltage applied across the separating capillary. This is a prerequisite since the electrophoretic current (up to 200 µA) produced in the capillary is about 50-60-fold higher than the electrochemical currents measured at the amperometric detector (
