ml concentration levels by capillary electrophoresis with amperometric detection

Analytica Chimica Acta 364 (1998) 63±74

Speciation of mercury at ng/ml concentration levels by capillary electrophoresis with amperometric detection Edward P.C. Laia,*, Weigang Zhanga, Xenia Triera, Anett Georgia, Sebastian Kowalskia, Scott Kennedya, Tini MdMuslima, Ewa Dabek-Zlotorzynska1,b a

Centre for Analytical and Environmental Chemistry, Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada b Analysis and Air Quality Division, Environmental Technology Centre, Environment Canada, Ottawa, Ontario K1A 0H3, Canada Received 9 September 1997; received in revised form 10 January 1998; accepted 22 January 1998

Abstract A technique based on capillary electrophoresis and amperometric detection (CE-AD) has been developed for the speciation of mercury. This technique has the unique capability to detect only cationic mercury species that are electrochemically active. Capillary electrophoresis with electrokinetic injection allows ef®cient separation of inorganic mercury and organomercury cations in 8 min. Selective detection of these electrochemically active species is attained by controlling the reduction potential applied on the micro-electrode. For Hg2‡, an optimal potential of ÿ0.2 V can be used to prevent interference by less electroactive toxic metals and other substances found in complex environmental samples. The amperometric signal is linearly proportional to the Hg2‡ concentration over three orders of magnitude, with a detection limit of 0.2 ng/ml. The mass detection limit corresponds to 8 fg of Hg2‡ in an injection volume of 40 nl. For CH3Hg‡, the detection limit is 3 ng/ml when a potential of ÿ0.5 V is used. These detection sensitivities are attractive for environmental monitoring of contaminated sediments in ecosystems. Steam distillation is evaluated for the extraction of CH3Hg‡ from the sediment matrix. It yields an unknown mercury species which is unsuitable for CE-AD determination under the speci®ed conditions. # 1998 Elsevier Science B.V.

1. Introduction Metal speciation is becoming more and more signi®cant since the environmental toxicity and biological availability of many metals depend on their different chemical forms and oxidation states. It is important to develop reliable speciation methods that allow a better understanding of the dose-effect role and the water±soil±plant±animal±human transfer *Corresponding author. Tel.: +1613 520 2600 (Ext.) 3835; e-mail: [email protected] 1 E-mail: [email protected] 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00147-0

mechanism. Of particular toxicological interest is mercury and its speciation. Some forms of mercury, especially methylmercury, are known to accumulate in the food chain and lead to extremely toxic effects on biota and humans [1,2]. Mercury contamination is a widespread environmental problem; even the apparently pristine Arctic region is nowadays full of toxic mercury in whale skins and seal livers, at levels many times health guidelines. Different methods have been reported in the last ®ve years for the separate determination of mercury species [3,4]. The most widely used separation technique is gas chromatography, followed by the determination

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with electron capture detection [5], atomic spectrometry [6±8], microwave-induced plasma emission spectrometry [9], mass spectrometry [10], or inductively coupled plasma mass spectrometry [11]. Highperformance liquid chromatography (HPLC) can be applied, coupled with UV detection [12] or atomic spectrometry [13±16]. Also, ion chromatography [17] or cryogenic trapping [18,19] can be performed using atomic spectrometry for detection. However, an interlaboratory study using different methods revealed a high dispersion of results and the presence of uncontrolled sources of error in measured methylmercury concentrations [19]. In comparison with other organometallic species, the method development related to mercury speciation is not completely satisfactory. Most of the instrumental methods are either indirect, cumbersome to apply, or expensive to set up. Efforts to improve mercury species determination in the environment are continuing and new, independent mercury speciation methods are important to develop. Recently, there has been an increasing interest in the separation and determination of ionic species using capillary electrophoresis (CE). Its high separation power for different ionic species in aqueous buffer solution covers a broad ®eld of applications. Application of CE for metal speciation studies has especially received much attention where it excels or supplements other separation methods like ion chromatography and HPLC [20±24]. To date, UV absorption is the simplest detection mode in CE. For inorganic mercury and organomercury species, however, chemical derivatization with cysteine or dithizone sulphonate is required and errors may occur due to incomplete derivatization in unknown sample matrices [25±27]. Another on-going problem faced by researchers is the relatively poor sensitivity of UV absorption, which restricts the use of CE-UV in ultratrace analysis. Therefore, the interfacing and application of more sensitive detection modes such as mass spectrometry, laser-induced ¯uorescence and amperometry is becoming increasingly important. Several research groups have succeeded in applying amperometric detection (AD) in CE, using cathodic reduction [28±31] and anodic oxidation [32±39] for the determination of inorganic and biochemical species. Although the development of CE-AD is still in its initial phase, detection limits in the order of 10ÿ9 and 10ÿ18 mol/l have already been reported.

The objective of this work was to develop an inexpensive, selective and sensitive CE-AD method for studying the chemical speciation of mercury. A laboratory-constructed system is presented, together with results on the direct separation and detection without chemical derivatization of inorganic mercury and methylmercury present in environmental waters at the ng/ml level. For sediments, CE-AD is unique in its ability to determine only those mercury species that are charged and electrochemically active after extraction by steam distillation. 2. Experimental 2.1. Capillary electrophoresis The experimental setup for CE-AD analysis is schematically shown in Fig. 1. Polyimide-coated fused silica capillaries (50 and 65 mm i.d., 360 mm o.d.) were obtained from Polymicro Technologies (Phoenix, AZ), and capillary lengths between 60 and 70 cm were used. The capillary was ®lled with a buffer solution. Electrophoresis in the capillary was driven by a high-voltage power supply (Spellman Electronics CZE 1000R, Plainview, NY). In the normal polarity mode, the positive high voltage (20± 25 kV) on the injection side was connected to a Pt anode that was immersed in the same buffer solution contained in a 2 ml polyethylene microtube. All components of the injection side were housed inside a plexiglas shield for electrical isolation. Sample introduction to the capillary was accomplished using electrokinetic injection at 25 kV on a sample solution for 5 s under the control of a computer interface program. The detection end of the capillary and three electrodes for AD were all immersed in a plastic cell containing the electrophoretic buffer solution. 2.2. Capillary conditioning New or deteriorating capillaries were treated by ¯ushing with 0.1 M HCl for 5 min, water for 5 min, and the electrophoretic buffer solution for 50 min under water pump suction and N2 gas pressurization. The buffer solution was composed of 0.1 M creatinine in distilled deionized water and adjusted to pH 4.8 with acetic acid. Each day after the experiment, it was

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Fig. 1. Experimental setup for CE-AD analysis.

found satisfactory to keep the buffer under hydrodynamic ¯ow until next time (anywhere from 1 to 48 days). The hydrodynamic ¯ow via a height differential of 5 cm prevented any deposition of creatinine on the inside walls of the capillary. Before each daily use, the capillary was preconditioned by performing a blank run for 15±30 min. 2.3. Amperometric detection The AD cell was a 101048 mm acrylic cuvette (Sarstedt, W. Germany) with small holes drilled through to pass a gold (Au) micro-disk working electrode and the capillary. A platinum coil counter electrode (which also served as the cathode for the high voltage drop through the capillary) was wrapped around the micro-electrode tip while a Ag/AgCl reference electrode (World Precision Instruments DRIREF-2, Sarasota, FL) was suspended separately. As no commercial sources were available to supply the right size of Au micro-electrode, the working electrode was prepared by sealing a 25 mm diameter Au wire (Goodfellow Metals, Cambridge, UK) in a glass pipette. The open end of the glass pipette was enclosed in a plastic syringe which was ®lled with silver epoxy for contact with a lead wire. The micro-electrode was normally stored in air. Before every experiment, the Au-disk end was polished by ®ne grades of alumina (Bioanalytical Systems, West Lafayette, IN). After rinsing with water, the electrode was activated by

successive cyclic sweeps in the buffer solution between ÿ0.5 and ‡1.0 V vs. Ag/AgCl until a reproducible voltammogram was obtained. Alternatively, electrochemical cleaning was effective when a potential of ‡1.0 V was applied to the micro-electrode for 10 s to ensure that all deposited mercury and impurities were oxidized and stripped from the Au-disk surface. In the present end-column detection scheme, alignment with the ®xed micro-electrode was accomplished by adjusting the capillary which was mounted on an XYZ translational stage. Two microscopes were employed to inspect the alignment from both the top and the side views. The three electrodes for AD were controlled by a potentiostat circuit which was designed and fabricated in-house. This potentiostat was designed to ensure that the output signal was directly proportional to the current ¯owing into the Au micro-electrode. The choice of ultra-low input bias op-amps (Analog Devices AD549LH, Norwood, MA) guaranteed that this current was due to the analyte electrochemistry only. Good sensitivity was attained by selected resistors (100 k and 1 M ) for high gains, which were made possible by the very low input voltage noise of the op-amps. Special attention was paid to the layout of the components on the printed circuit board and within the potentiostat enclosure. All components of the AD cell and the potentiostat were shielded from external electromagnetic noise by a Faraday cage. All connections between the potentiostat and other equip-

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ment were made using shielded cables of minimal lengths and in a manner to eliminate ground loops. Noise that might arise from mechanical vibrations to perturb the micro-electrode and capillary alignment was eliminated by setting up the AD cell assembly on a sturdy aluminum platform with thick rubber feet. A programmer (EG&G PARC 175, Princeton, NJ) was used to control the micro-electrode potential through the potentiostat. AD of mercury species was performed at a controlled potential between ÿ0.2 and ÿ0.5 V vs. Ag/AgCl, and all electropherograms were recorded on an integrator (Dionex 4270, Sunnyvale, CA). 2.4. Cyclic and hydrodynamic voltammetry All cyclic voltammetry experiments were carried out on the creatinine buffer solutions, with and without a mercury salt added, using the AD cell described above. The scan rate was typically 50 mV/s. Hydrodynamic voltammograms were obtained by running standard solutions of Hg2‡, CH3Hg‡ and C2H5Hg‡ separately, and measuring the CE-AD peak areas from successive injections of the same standard solution with 100 mV potential increments to cover the range from 0.0 to ÿ0.9 V for AD. 2.5. Reagents All chemicals were purchased in the highest purity available, and were used without further puri®cation. All standards and electrophoretic buffer solutions were prepared using 18 M cm distilled deionized water (DDI) (Millipore Milli-Q water system, Bedford, MD). A 1000 mg/ml Hg2‡ stock solution was prepared in DDI water from mercuric chloride (J.T. Baker, Phillipsburg, NY) for all CE-AD experiments and from mercuric acetate (Alfa, Ward Hill, MA) for cyclic voltammetry only. The methylmercury and ethylmercury stock solutions were prepared from methyl mercuric chloride (ICN Biomedicals, K‡K Labs, Plainview, NY) and from ethyl mercury chloride (Pfaltz & Bauer, Stamford, CT), respectively, by dissolving the appropriate quantity of each salt in the minimum volume of methanol and diluted to the ®nal volume with DDI water to give a concentration of 500 mg/ml. The organomercury solutions were stored away

from light to prevent decomposition. Diluted working standard solutions were prepared daily.2 Consult local authorities as to the proper disposal of mercury compounds. Creatinine buffer solutions were prepared by dissolving the solid (Sigma, St. Louis, MO) in DDI water and adjusted to pH 4.8 with acetic acid. The working buffer solution was dispensed through 0.45 mm syringe ®lters (Gelman Sciences, Ann Arbor, MI) before use in CE. 2.6. CH3Hg‡ distillation Steam distillation of CH3Hg‡ was performed in an apparatus taken from Horvat et al. [40]. An aliquot of 15 ml of 20 ng/ml CH3Hg‡, 0.2 ml of 20% KCl and 0.5 ml of 8 M H2SO4 were added into a 30 ml PTFE vial. Nitrogen gas was bubbled through the mixture, and the PTFE vial was electrically heated up to a temperature of 1458C. The distillation rate was controlled in order to collect 7 ml/h, and distillation continued for 1.5 h. The distillate was collected by 5 ml of DDI water contained in another PTFE vial which was kept cool in an ice bath. Approximately 12.7 ml of distillate solution was obtained for analysis by CE-AD, inductively coupled plasma-mass spectrometry, gas chromatography-electron capture detection and gas chromatography-mass spectrometry. A Dionex sample pretreatment cartridge in the hydroxide form was prepared by treating the anion exchanger cartridge in bicarbonate form with 2 ml of 0.1 M NaOH, followed by 10 ml of DDI water. This converted the counterions at the exchange sites from ÿ HCOÿ 3 to OH . A 3 ml aliquot of distillate solution was then passed through the cartridge, so that Clÿ and ÿ SO2ÿ 4 were taken up from the solution and OH was released to neutralize an equivalent amount of H‡. This sample pretreatment was tested with a 10 ng/ml CH3Hg‡ standard solution which was acidi®ed to pH 2 by adding 0.1 M HCl to simulate the acidic distillate. The trace metal grade HCl containing 6 ng/l mercury was purchased from Mallinckrodt (Paris, KY). 2

Caution: Mercury compounds, especially organomercury compounds, are highly toxic and appreciably volatile. Perform all solution preparations in a well ventilated fume hood.

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3. Results and discussion 3.1. Cyclic and hydrodynamic voltammetry The redox behavior of creatinine buffer solutions and dissolved mercury species was ®rst investigated by cyclic voltammetry. It is well known that the Au electrode has a higher overpotential (0.24 V) than Pt towards H‡ reduction, providing a useful potential range of 1 V vs. the Ag/AgCl reference electrode for

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the AD of mercury (and other toxic metal) ions. Fig. 2(a) shows the current-potential response during the cyclic voltammetric scan for 0.1 M creatinine, pH adjusted to 4.8 with acetic acid, on the Au microelectrode. When the potential is initially scanned from ‡0.5 towards ‡1.0 V, O2 evolution and/or gold anodization produces a strong down-going current signal. On reversing the scanning potential direction at ‡1.0 V, a cathodic peak is produced at ca. ‡0.3 V probably due to the reduction of surface oxide as

Fig. 2. Cyclic voltammograms for 0.1 M creatinine buffer solution, pH adjusted to 4.8 with acetic acid, containing (a) 0 mg/ml Hg2‡, (b) 100 mg/ml Hg2‡ from HgCl2, (c) 100 mg/ml Hg2‡ from Hg(CH3COO)2, (d) 100 mg/ml CH3Hg‡ from CH3HgCl, and (e) 100 mg/ml C2H5Hg‡ from C2H5HgCl. Scan rateˆ50 mV/s.

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previously observed in a different buffer system [33]. The next cathodic signal from ca. ÿ0.3 to ÿ0.7 V corresponds to the reduction of dissolved oxygen [41]. Hydrogen evolution is eventually observed for water cathodization as a strong current signal increasing beyond ÿ1.0 V. During the last part of potential scan from ÿ1.0 back to ‡0.5 V, no further current peaks arise. Evidently, 0.1 M creatinine is a useful buffer solution for AD with the Au electrode in the range of potential studied. Creatinine itself does not generate any reduction wave to interfere with cathodic analytical signals; on the anodic side, an oxidation wave will appear at ca. ‡0.7 V if the concentration of creatinine is increased to 0.3 M. Mercury(II), when dissolved in the 0.1 M creatinine buffer, produces a two-electron reduction peak in the potential region between ÿ0.1 and ÿ0.6 V (Fig. 2(b) and (c)). The signi®cant overlap of this peak with the background oxygen reduction wave limits the choice of cathodic AD potentials for background-free Hg2‡ determination. In the case of Hg2‡ prepared from HgCl2, the Clÿ in solution facilitates the anodic stripping of deposited Hg and a much sharper peak is observed at ‡0.1 V in Fig. 2(a) than that obtained from Hg(CH3COO)2 in Fig. 2(b). The second anodic response is exhibited at ‡0.4 V for both Hg2‡ solutions. The organic mercury compounds, on the contrary, show a one-electron reduction peak in the potential region between ÿ0.3 and ÿ0.4 V (Fig. 2(d) and (e)). Apparently, this is followed by further reduction of the organomercurial species on polarity reversal of the scanning potential at ÿ1.0 V, to produce a high cathodic wave from ÿ0.8 to ÿ0.2 V. A single anodic response is exhibited at ‡0.3 V for both CH3Hg‡ and C2H5Hg‡. The dif®culty with the AD of these two organomercury compounds is modest sensitivity, as can be seen from their relatively weak reduction peaks. While cyclic voltammetry provided information on the redox behavior of mercury species dissolved in the buffer solution, electrophoretic removal of the Clÿ anion (which migrated towards the anode) and neutral methanol solvent molecules from the injected CH3Hg‡ and C2H5Hg‡ analytes could change their AD responsivities. For con®rmatory reasons, the CEAD sensitivity toward organomercury species was next examined by hydrodynamic voltammetry (HDV) [30]. Measurements of the CE-AD peak areas

in successive sample injections were carried out, with 100 mV potential increments applied on the Au microelectrode over the range from ‡0.0 to ÿ0.9 V. Within this range of potentials, the reduction of dissolved oxygen would only generate a constant background signal but not a peak. As shown in Fig. 3 for methylmercury, the CE-AD peak area varied with the working electrode potential. Maximal signal-to-noise ratio was obtained at a potential of ÿ0.5 V for CH3Hg‡. Similarly, the optimum potentials are ÿ0.2 V for Hg2‡, and ÿ0.6 V for C2H5Hg‡. These values compare favorably with their cyclic voltammetry behaviors, as well as previous observations that Hg2‡ was preconcentrated cathodically at ÿ0.2 V on an Au-disk electrode for the determination by anodic stripping voltammetry [42] and that the reduction of CH3Hg‡ occurred in two steps at ÿ0.2 and ÿ0.5 V [43]. These results suggested that the selectivity and sensitivity of CE-AD analysis could be enhanced by using a characteristic reduction potential to determine each mercury species. For instance, amperometric detection at ÿ0.2 V would allow Hg2‡ to be selectively determined with maximal sensitivity despite the coexistence of organomercury species and other less electroactive toxic metals in complex environmental samples. Alternatively, a compromised potential of ÿ0.5 V might be selected for the simultaneous detection and quanti®cation of all mercury species. Cyclic voltammetry was also used regularly to check the operating conditions of the CE-AD system. A voltammogram of the detector buffer solution was examined, before a CE-AD run, to see if any undesirable changes in shape and peak potentials had occurred. Such changes might reveal a deteriorating micro-electrode, a contaminated buffer solution, a fouled reference electrode, and/or any accumulation of gas bubbles. Efforts could immediately be made to correct the problem. In addition, the electrode behavior and the CE-AD reproducibility were more satisfactory when cyclic voltammetry was used to activate the Au micro-electrode. Alternatively, the micro-electrode was conditioned by jumping the applied potential from ÿ0.20 or ÿ0.5 to ‡1.0 V for 10 s and back to the optimum value before each CE-AD analysis was started. The effect of electrochemical pretreatment on electrode response was judged by the observation of reproducible electropherograms in replicate analyses of a standard Hg2‡/CH3Hg‡ solution.

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Fig. 3. Hydrodynamic voltammogram for CH3Hg‡ in the working electrode potential range from 0.0 to ÿ0.9 V vs. Ag/AgCl reference electrode.

3.2. Effect of electrode alignment on amperometric detection A major technical dif®culty associated with CE-AD is to isolate the micro-electrode circuit from the high electric ®eld for CE separation (which gives rise to noise on the electropherogram) [29,30,35,37,38]. While the high electric ®eld is a problem for microelectrodes that are placed inside the capillary, such a problem existed to a smaller extent when the Au micro-disk electrode was positioned outside the capillary. The gap distance between the Au disk and the capillary end had a signi®cant effect on sensitivity, noise level and peak ef®ciency. As the micro-electrode body had a larger front end than the capillary outlet dimension, the CE ef¯uent impinged directly on (and perpendicular to) the Au disk for AD (see Fig. 1). A very large distance would therefore decrease the contact of the diverging ef¯uent stream with the Au disk, resulting in a lower detection sensitivity. On the other hand, the noise level decreased with increasing gap distance. An optimal electrode position was determined by a good signal-to-noise ratio while maintaining a low detection volume to achieve reasonably good separation ef®ciency. Typically 80 mm was found to be the best distance for the CE-AD setup. Experimental

data also showed that the migration time recorded for the Hg2‡ peak increased linearly by 1.2 min if the micro-electrode was moved away from the capillary by 60 mm (from a gap distance of 75 to 135 mm). The migration time increased substantially with gap distance within such a small range, probably because the electric ®eld strength decreased rapidly from the capillary outlet end. A velocity of 0.051 mm/min was determined for the migration of Hg2‡ ions through this small range, as compared to a migration velocity of 162 mm/min inside the capillary. This has the implication that the gap distance must be kept constant in order to obtain reproducible migration times from day to day. The counter electrode coil was wrapped around the micro-electrode front end to establish a symmetric electric ®eld which directed a spatially cohesive ef¯uent stream of analyte ions towards the Au disk for AD. A better peak shape and more reproducible results could be obtained. In principle, the smaller the microelectrode front end and hence the counter electrode coil, the more focused the ef¯uent stream and the greater the detection sensitivity would be. In this work, the best micro-electrode was fabricated by sealing a 25 mm Au wire in a glass pipette of 1 mm in ®nal diameter. This dimension of micro-electrode is

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fairly robust, preventing frequent breakage during polishing or assembly. 3.3. Optimization of CE-AD parameters Several parameters were optimized to achieve the highest AD sensitivity and the shortest CE time. For creatinine concentrations between 0.01 and 0.3 M, a low concentration resulted in a low electroosmosis current, a high electroosmotic ¯ow and a low buffer conductivity. This offered the advantage of low baseline noise, but the separation and electrokinetic injection ef®ciencies were poor. As the buffer concentration was increased, both the separation and electrokinetic injection ef®ciencies became better but more baseline noise appeared owing to the increased electrophoretic current and its leaking residue on the detecting electrode. It was decided that 0.1 M creatinine was the best overall buffer concentration to use. Next, increasing the injection voltage from 500 V to 30 kV enhanced the CE-AD peak area for a 0.1 mg/ml Hg2‡ standard solution by 2.5105 integrated units (or 1.28 nC) per kV. Since 30 kV occasionally produced poor peak area reproducibility due to voltage instability problems, 25 kV was used throughout this work. Last, increasing the injection time from 0.2 to 5 s resulted in a proportional increase in peak area at the rate of 2.5106 integrated units per second. Longer injection times (up to 30 s), however, began to cause peak broadening and delays in the migration time. A 5 s electrokinetic injection time was the best compromise. 3.4. Mercury speciation by CE-AD Using the optimized parameters, Hg2‡ could be separated from CH3Hg‡ and C2H5Hg‡ under an electric ®eld gradient of 20 kV/65 cm (Fig. 4). Experimentally, the migration time for Hg2‡ increased by 1.55 min with every kilovolt decrease in electrophoretic voltage, which would result in better separation from the organomercury species if the high voltage was decreased below 20 V. On the other hand, the wall-jet effect on amperometric detection in the endcolumn con®guration would be enhanced by increasing the electrophoretic voltage, as the migration rates of analytes out of the capillary became faster. Note that the separation of two organomercury species with

Fig. 4. Separation of 83 ng/ml Hg2‡ (tˆ6.51 min), 8 mg/ml CH3Hg‡ (tˆ6.82 min) and 6 mg/ml of C2H5Hg‡ (tˆ7.12 min) by CE-AD, using a selective WE potential of ÿ0.2 V. Capillary was 65 mm inner diameter, running buffer was 0.1 M creatinine adjusted to pH 4.8 with acetic acid, electrophoresis voltage was ‡25 kV, and injection was electrokinetic at ‡25 kV for 5 s.

identical charges in Fig. 4 was mainly due to a difference in size. The smaller of the two species, CH3Hg‡, migrated at a faster rate, followed by the larger C2H5Hg‡ species. The CE-AD system was calibrated with a series of Hg2‡ standards up to 500 mg/ml. A linear dynamic range of nearly three orders of magnitude was observed from 0.2 to 100 ng/ml after the stacking effect of electrokinetic injection is taken into account using the ratio of buffer to sample solution conductivities [44,45]. A least-squares regression analysis of the seven points in the linear portion of the standard calibration graph produced the following equation: Aˆ2.5104 [Hg2‡], where A is the analyte peak area (in integrated units) and [Hg2‡] the concentration of Hg2‡ (in ng/ml). The correlation coef®cient was better

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Fig. 5. Determination of 6.9 ng/ml CH3Hg‡ (tˆ7.75 min) by CE-AD, using an optimized working electrode potential of ÿ0.5 V. Capillary was 50 mm inner diameter, running buffer was 0.1 M creatinine adjusted to pH 4.8 with acetic acid, electrophoresis voltage was ‡20 kV, and injection was electrokinetic at ‡20 kV for 30 s.

than 0.95. The relative standard deviation (RSD) of the CE-AD peak area was 9% for 100 ng/ml Hg2‡ in nˆ7 measurements, and 21% for 0.5 ng/ml Hg2‡. There were no visible signs of system performance degradation after 6 h of CE-AD operation. Any deterioration of AD response due to off alignment of the microelectrode with respect to the capillary was corrected by regular inspection. The detection limit based on three standard deviations of the baseline (7103 integrated units) is 0.2 ng/ml, thanks to on-line preconcentration of ionic analytes at trace levels with electrokinetic injection. This great performance is also made possible by the use of an optimized working electrode potential of ÿ0.2 V, which is suf®ciently negative for the reduction of Hg2‡ but not negative enough to start the reduction of dissolved oxygen in the buffer solution. It is as good as the typical detection limit of 0.2 ng/ml for mercury in water using permanganate oxidation and cold vapor atomic absorption detection (U.S. EPA Method 245.2) [46] and better than the 0.8±1.9 ng/ml for HPLC with AD at ÿ0.8 V [47] or the 1 mg/ml for CE-pulsed AD in 0.035 M creatinine [31]. At present, the regulatory level is 0.2 mg/ml inorganic mercury for hazardous wastewater [48]. With an equivalent injection volume of 40 nl, the mass detection limit for CE-AD is estimated at 8 fg (ˆ40 atomoles) of Hg2‡ which is lower than the picogram capability of atomic ¯uores-

cence spectroscopy and the sub-nanogram quanti®cation for cold-vapor AAS. Similarly, the detection limits for CH3Hg‡ and C2H5Hg‡ were 23 and 150 ng/ml, respectively, when the AD was performed at a compromised potential of ÿ0.2 V which is not the best for the two organomercury species. An improved detection limit of 3 ng/ml can be obtained for CH3Hg‡ by using an optimized potential of ÿ0.5 V to achieve its two-electron reduction (Fig. 5). This detection capability is quite adequate for the interim environmental quality guidelines of 140 ng/g for methylmercury in marine and freshwater sediment [49]. For CH3Hg‡ detection at ÿ0.5 V, the system performance is compromised by O2 reduction interference in terms of background current, detector noise and consequently limit of detection. Further improvement in detection limit can be expected if special efforts are made to remove O2 from the whole CE-AD system. Separation of Hg2‡ and CH3Hg‡ from other toxic metals, such as Cd2‡, Cu2‡ and Zn2‡ was also studied. When 0.1 M creatinine and 20 kV were used, the CEAD peaks for Hg2‡, CH3Hg‡ and Cu2‡ were well resolved and detected with a working electrode potential of ÿ0.8 V (Fig. 6) although the Cd2‡ peak would overlap with Hg2‡ if added. This overlap is not a problem because the working potential may be set at ÿ0.2 V to selectively detect Hg2‡ but not Cd2‡. If both Hg2‡ and Cd2‡ must be determined, a lower

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nine, as the high buffer concentration generated a large electroosmotic ¯ow current inside the capillary under the 25 kV electric ®eld. If the electric ®eld was decreased to 15 kV, the 0.3 M creatinine buffer produced excellent separation of Hg2‡ at 17.06 min, Cu2‡ at 18.82 min, Cd2‡ at 20.14 min and Zn2‡ at 23.28 min. 3.5. Environmental mercury speciation

Fig. 6. Analysis of 10 mg/ml Cd2‡ (tˆ6.72 min), 11 mg/ml Hg2‡ (tˆ6.83 min), 10 mg/ml CH3Hg‡ (tˆ7.52 min) and 0.8 mg/ml Cu2‡ (tˆ7.78 min) by CE-AD, using a more negative working electrode potentioal of ÿ0.8 V. Capillary was 50 mm inner diameter, running buffer was 0.1 M creatinine adjusted to pH 4.8 with acetic acid, electrophoresis voltage was ‡20 kV, and injection was electrokinetic at ‡30 kV for 4 s.

electric ®eld can be used to obtain better separation. For instance, 15 kV gave the elution of Cd2‡ at 11.2 min and Hg2‡ at 11.5 min, whereas 9 kV produced the peaks at 16.2 min for Cd2‡, 16.9 min for Hg2‡, 18.8 min for CH3Hg‡ and 20.9 min for Zn2‡. Alternatively, a stronger buffer solution of 0.3 M creatinine at pH 4.8 and 25 kV could be employed to achieve good resolution in a shorter analysis time: 6.85 min for Hg2‡, 7.77 min for Cu2‡, 8.21 min for Cd2‡ and 10.20 min for Zn2‡. Note that this elution order is different from that obtained with 0.1 M creatinine, probably due to a change in the interactions between creatinine and the toxic metals. There was, however, excessive Joule heating with 0.3 M creati-

Methylmercury (chloride) is the most prevalent form of organomercury found in environmental waters, typically at the 10 fg/ml level which is too low for CE-AD analysis. Previous studies have shown that inorganic mercury can be methylated by microbes (bacteria, yeasts and fungi) living in the bottom sediments of lakes and rivers [4]. Assessment of mercury contamination in an ecosystem is naturally best done at the source of methylmercury production ± the sediments. Sediments containing methylmercury in the low ng/ml range is amenable to CE-AD analysis, yet this technique requires desorption of the organomercury species from sediment surfaces to provide a sample solution. Several extraction methods from the literature were considered, and the one selected for evaluation was a steam distillation technique that had been ®rst developed by Horvat et al. [40] and recently applied for the determination of methylmercury in sediments and ®sh tissue [50]. In this technique, H‡ ions from H2SO4 presumably separate CH3Hg‡ from any physical matrix that binds it without breaking the C±Hg bond, while Clÿ ions from KCl form an association product with CH3Hg‡ which can be carried out of the 1408C distillation mixture by nitrogen bubbling. This distillation procedure has advantages over solvent extractions with regards to percentage recovery and selective isolation of CH3Hg‡ from the majority of Hg2‡ interference. However, the distillate was found to be acidic with a pH in the range from 2.0 to 3.8. Ion-chromatography analysis also indicated the presence of chloride and sulfate in the distillate, at the 60 and 1.5 ppm levels, respectively. Consequently, the ef®ciency of electrokinetic injection suffered greatly from the high conductivity of the distillate sample which was measured to be 2 mS/cm. One solution was to pass the distillate through a sample pretreatment cartridge in the hydroxide form, were removed from the where the Clÿ and SO2ÿ 4

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distillate by anion exchange and an equivalent amount of OHÿ was released to neutralize the H‡. This sample pretreatment was tested with a 10 ng/ml CH3Hg‡ standard solution which was acidi®ed to pH 2 by adding 0.1 M HCl to simulate the acidic distillate. At the end of treatment, the standard solution had a pH of 5. Electrokinetic injection of the CH3Hg‡ ions could then be performed with good ef®ciency. Approximately 70% recovery of the original quantity of CH3Hg‡ was attained, as determined by CE-AD analysis. Next, 300 ng of CH3Hg‡ was processed by the steam distillation. After cartridge treatment of one portion of the distillate, however, no CH3Hg‡ or Hg2‡ peak could be observed in the CE-AD electropherogram. Analysis of another portion of the distillate by ICP-MS showed a 80% recovery of the CH3Hg. A third portion of the distillate was extracted by chloroform for both GC-ECD and GC-MS analyses, and the chromatograms showed no peaks for CH3HgCl and CH3HgCH3. These results are very intriguing. One question is whether CH3Hg‡ is converted to some unknown species during the steam distillation. Horvat et al. [40] originally developed this method to extract CH3Hg‡ from sediments for GC-AFS analysis after the distillate was chemically derivatized by aqueousphase ethylation to form CH3HgC2H5. Can it be that the distillated species is not CH3HgCl, which usually dissociates in water to yield CH3Hg‡? This hypothesis contradicts the previous work of Meuleman et al. [51] which separated CH3Hg from biological tissue by treatment with concentrated H2SO4 in a closed vial and conversion of CH3Hg into volatile CH3HgI using iodoacetic acid. Till further investigation identi®es the CH3Hg species, the steam distillation technique is not suitable for the extraction of CH3Hg‡ from sediments for CE-AD analysis. The above evaluation of the steam distillation technique, however, has indirectly demonstrated the unique capability of CE-AD to detect only cationic mercury species that are electrochemically active. Finally, a recent report has described a new alkaline tetrabutylammonium bromide (0.25 M Bu4NBr in 4 M KOH) digestion medium for sample treatment and mercury speciation in seagrass, followed by hydride generation-AFS [52]. The large alkyl groups serve as phase transfer catalysts by transporting OHÿ into hydrophobic sites to increase alkaline hydrolysis of the plant material. It

73

is the combination of Bu4NBr and KOH that promotes the alkaline hydrolysis necessary for quantitative release of CH3Hg‡ from eelgrass. Recovery of CH3Hg‡ from the alkaline speciation agent is in the 94.46.5% range, as compared to 85% for vacuum distillation range. It will be interesting to conduct an experiment using this new development for mercury speciation in sediments. 4. Conclusions The resolution provided by capillary electrophoretic separation, in conjunction with selective amperometric detection, is useful for the speciation and simultaneous determination of inorganic mercury and methylmercury. Constant-voltage amperometric detection is simple to implement, does not cost very much, and provides good sensitivity. The detection limits of 0.1 ng/ml for Hg2‡ and 3 ng/ml for CH3Hg‡ are potentially attractive for the monitoring of mercury contamination in sediments. A suitable CH3Hg‡ extraction method, however, must be developed before it is feasible for environmental scientists to adopt this CE-AD technique for mercury speciation in sediment analysis. Acknowledgements This work was funded by the Tri-Council of Canada. The authors thank Michel Grenier and Jim Logan for fabricating the potentiostat and creating the computer interface program. References [1] Ph. Quevauviller, Appl. Organomet. Chem. 8 (1994) 715. [2] M. Hempel, Y.K. Chau, B.J. Dutka, R. McInnis, K.K. Kwan, D. Liu, Analyst 120 (1995) 721. [3] R. Puk, J.H. Weber, Appl. Organomet. Chem. 8 (1994) 293. [4] Ph. Quevauviller, J. Chromatogr. A 750 (1996) 25. [5] R.A. Lorenzo, A. Carro, E. Rubi, C. Casais, R. Cela, J. AOAC International 76 (1993) 608. [6] R. Fischer, S. Rapsomanikis, M.O. Andreae, Anal. Chem. 65 (1993) 763. [7] M. Ceulemans, F.C. Adams, J. Anal. At. Spectrom. 11 (1996) 201. [8] Y. Cai, R. Jaffe, A. Alli, R.D. Jones, Anal. Chim. Acta 334 (1996) 251.

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