Determination of Mercury Compounds in Water Samples by Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry With an In Situ Nebulizer/Vapor Generator CHIA-CHING WAN, CHIH-SHYUE CHEN AND SHIUH-JEN JIANG* Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, 80424, Taiwan A preliminary study of a cold vapor generation system as the reversed-phase LC with 0.5% m/v L-cysteine solution as the ion pairing reagent and the mobile phase. The various mercury sample introduction device for liquid chromatography– inductively coupled plasma mass spectrometry (LC–ICP-MS) species studied included inorganic mercury (HgII), methylmercury (methyl-Hg) and ethylmercury (ethyl-Hg).Euent from is described. Samples containing ionic mercury compounds were subjected to chromatographic separation before injection the LC column was delivered to the vapor generation system and ICP-MS system for mercury determination. The optimiz- into the cold vapor generator.The species studied include inorganic mercury (HgII ), methylmercury and ethylmercury. ation of the CV generation LC–ICP-MS technique and its analytical figures of merit, and also its application to the The sensitivity, detection limits and repeatability of the LC–ICP-MS system with a cold vapor generator were determination of mercury compounds in NASS-4 open ocean sea-water reference material and a tap water sample collected comparable to or better than those for an LC–ICP-MS system with conventional pneumatic nebulization or other sample from National Sun Yat-Sen University, are described.introduction techniques. The limits of detection for various mercury species were in the range 0.03–0.11 ng ml-1 Hg based EXPERIMENTAL on peak height. The concentrations of mercury compounds in open ocean sea-water reference material NASS-4 and a tap ICP-MS Device and Conditions water sample collected from National Sun Yat-Sen University An ELAN 5000 ICP-MS instrument (Perkin-Elmer SCIEX, were determined.Thornhill, ON, Canada) was used. Samples were introduced Keywords: Inductively coupled plasma mass spectrometry; with an in situ nebulizer/vapor generation sample introduction liquid chromatography; mercury speciation; in situ system. ICP conditions were selected that maximized the nebulizer/vapor generator; water mercury ion signal using a flow injection (FI) method. A simple FI system was used for all the FI work performed in this study.It was assembled from a six-port injection valve In recent years, it has become recognized that trace metal (Type 50, Rheodyne, Cotati, CA, USA) with a 200 ml sample analysis must involve true metal speciation, in addition to loop. A solution of 50 ng ml-1 HgII, methyl-Hg and ethyl-Hg total metal analysis. Biological, biomedical and toxicological in the mobile phase (to be used for subsequent chromatoproperties depend on the specific form in which the metal is graphic separations) was loaded in the injection loop and present, and combinations of metals have dierent eects on injected into the mobile phase, which worked as the carrier of the environment depending on the nature of the mixture.the FI system. The cold mercury vapor generated was then Information about the various species in a sample can be transported to the ICP-MS system for mercury determination. obtained by a newer form of chromatographic separation with The sensitivity of the instrument may vary slightly from day element-selective/specific final detection.to day. The ICP-MS operating conditions used are summarized Environmental pollution caused by mercury is almost in Table 1. entirely due to industrial application in the production of The data acquisition parameters used are given in Table 1. pesticides, electrical apparatus, paints and dental applications, and the toxicity of these species is well documented. A knowledge of speciation is important when assessing the mobility of Table 1 ICP-MS equipment and operating conditions mercury in the environment.1,2 Several methods of liquid chromatography (LC) and gas ICP-MS instrument Perkin-Elmer SCIEX ELAN 5000 chromatography (GC) coupled with element-specific detection Plasma conditions— for mercury speciation have appeared, including electrochemi- Rf power 1100W cal detection (EC),3 cold vapor atomic absorption spectrometry Plasma gas flow rate 15 l min-1 Intermediate gas flow rate 0.74 l min-1 (CVAAS),4–7 atomic emission spectrometry (AES)8,9 and induc- Aerosol gas flow rate 0.98 l min-1 tively coupled plasma mass spectrometry (ICP-MS).10–14 The Mass spectrometer settings— cold vapor (CV) generation sample introduction technique has Bessel box lens +15.34 V been applied in several LC–atomic spectroscopic applications Bessel box plate lens -79.10 V for mercury speciation.4–7,9,10 The use of the cold mercury Photon stop lens -10.05 V vapor generation technique increases the signal of mercury Einzel lenses 1 and 3 -0.04 V Resolution Normal significantly.In this work, a simple in situ nebulizer/ Dwell time 120 ms vapor generator system was employed as a sample intro- Sweeps per reading 5 duction device in LC–ICP-MS for mercury speciation Points per spectral peak 1 determination.15–17 Isotope monitored 202Hg Ionic compounds containing mercury were separated by Journal of Analytical Atomic Spectrometry, July 1997, Vol. 12 (683–687) 683The element-selected chromatograms were recorded in real HgII, methyl-Hg and ethyl-Hg in the LC mobile phase were prepared. These stock solutions were then loaded into the time and stored on the hard disk with Graphic software. The dwell time, sweeps per reading and points per spectral peak injection loop and injected into the CV generation system. Several operating parameters aected the eciency of CV parameters were set so that each data point could be obtained in less than 1 s.formation. The concentration of sodium tetrahydroborate (NaBH4), concentration of acid and the volume of the mixing coil were studied to obtain the optimum conditions. Chromatographic Apparatus and Conditions A Hitachi Model L-7100 LC pump, injector (Rheodyne Model Reagents 7225i) and reversed-phase column (Spherisorb ODS-2, 5 mm Analytical-reagent grade chemicals were used without further diameter particles, 150×4.6 mm id) comprised the LC system.purification. Sodium tetrahydroborate was obtained from Samples were loaded with a syringe into a 100 ml sample loop. Janssen Chemical (Geel, Belgium), L-Cysteine from TCI All separations were performed at room temperature under Chemical (Tokyo, Japan), mercury nitrate and methylmercury isocratic conditions. Separations were attempted with several chloride from Merck (Darmstadt, Germany) and ethylmercury combinations of column, organic modifier concentration, Lchloride from TCI Chemical.Standards containing 200 mg l-1 cysteine concentration and pH. The conditions given in Table 2 (as element) of each individual species in 2% v/v H2SO4 were are those which yielded the best chromatographic resolution prepared. These standards were combined and diluted with for the various sets tested. The column outlet was connected the LC mobile phase and analyzed by CV–ICP-MS. To prepare to the vapor generation device with Teflon tubing (Fig. 1).the solutions to be used as the mobile phase, a suitable amount of L-cysteine was dissolved in pure water to the desired Cold Vapor Generation System and Conditions concentration. A simple in situ nebulizer/vapor generation sample introduction system was coupled with LC–ICP-MS for mercury speci- Sample Preparation ation determination. A schematic diagram of the LC The applicability of the method to real samples was demon- nebulizer/vapor generator system is shown in Fig. 1. With this strated by the analysis of National Research Council of Canada sample introduction system, the entire injected sample was (NRCC) NASS-4 (open ocean sea-water reference material for nebulized.The nebulization process, in which the liquid is trace metals). The reference sample was diluted twofold with shattered into fine droplets in an argon stream, is a very the LC mobile phase, then a 100 ml portion of the sample eective way to purge Hg vapor from the liquid, probably solution was injected into the LC–CV–ICP-MS system.A tap more so than bubbling argon through a static reservoir of water sample collected from National Sun Yat-Sen Univer- bulk liquid, as in a conventional gas–liquid separator. Cold sity was treated with same procedure and analyzed by vapor generated from the vapor generation system was LC–ICP-MS. delivered to ICP-MS system for mercury determination. The operating conditions for cold mercury vapor generation were optimized using an FI method.The LC pump and column RESULTS AND DISCUSSION were removed from the system during these studies. Since HgII, Selection of LC Operating Conditions methyl-Hg and ethyl-Hg show dierent behaviors and dierent sensitivities in the CV generation process, dierent mercury The eect of an organic solvent on the plasma is generally to species were studied successively to obtain compromised reduce significantly its excitation properties. In this study, no operating conditions for the vapor generation system.Stock organic solvent was added to the LC mobile phase. The eects standard solutions of various mercury species at 50 ng ml-1 of the concentration of L-cysteine and flow rate of the mobile phase on the liquid chromatogram were studied to obtain the best LC separation. Table 2 Liquid chromatography and hydride generation conditions Fig. 2 shows the eect of the concentration of L-cysteine on the chromatogram. Each mercury species was present at L C conditions— Pump Hitachi Model L-7100 50 ng ml-1.As shown in Fig. 2(a), the concentration of Column Spherisorb ODS-2, 5 mm, L-cysteine did not aect the chromatogram significantly. On 150×4.6 mm id the other hand, as shown in Fig. 2(b), the concentration of Mobile phase 0.5% m/v L-cysteine (pH 5) L-cysteine did aect the signal-to-background value of the Mobile phase flow rate 1.6 ml min-1 mercury ion signal and an optimum concentration of Sample loop 100 ml 0.5% L-cysteine was obtained.Hydride generation conditions— Fig. 3 shows the eect of the LC mobile phase flow rate on NaBH4 solution 0.1% m/v in 0.02 mol l-1 NaOH the chromatogram. The retention times decreased with increase NaBH4 solution flow rate 1.0 ml min-1 in the mobile phase flow rate. In another experiment, we found that the pH of the mobile phase did not aect the retention times of the mercury species studied significantly. For the best LC resolution, a solution containing 0.5% m/v L-cysteine (pH 5) at a flow rate of 1.6 ml min-1 was adopted in subsequent experiments. Selection of Hydride Generation Conditions Since HgII and organomercury show dierent behaviors and dierent sensitivities in the CV generation process,4,6,9 dierent mercury species were studied successively to obtain compromise operating conditions of the CV generation system.The concentration of NaBH4 is critical in the determination of Fig. 1 Schematic diagram of LC–CV system. mercury by CV generation.We therefore investigated the eect 684 Journal of Analytical Atomic Spectrometry, July 1997, Vol. 12Fig. 4 Eect of NaBH4 concentration on Hg ion signal. All the data points were relative to the signal obtained with conventional pneumatic nebulization (concentration of NaBH4 0%). The concentration of HNO3 was 0.05 mol l-1 at a flow rate of 0.5 ml min-1. system. The enhancement factors for mercury were dierent for these three Hg species.This may be attributed to the variation of the vapor generation eciency of the various mercury species. Fig. 5 shows the peak height of the FI peaks as a function of the concentration of HNO3. Since the LC mobile phase already contained 0.5% m/v L-cysteine, the concentration of HNO3 did not aect the mercury signal significantly. In fact, the ion signals of all three mercury species decreased when extra HNO3 was added. In the following experiments, no extra Fig. 2 Eect of L-cysteine concentration on (a) retention time and HNO3 was used in the CV generation system.(b) signal-to-background ratio of various mercury species. The mobile Although not illustrated here, in another experiment we phase flow rate was 1.0 ml min-1. Each mercury species was present found that the volume of the mixing coils (0–2 ml) did not at 50 ng ml-1. aect the mercury signal significantly. In fact, the mercury ion signals decreased slightly when an extra mixing coil was used.In the following experiments, no extra mixing coil was used, except for the necessary connecting tubing (40 cm×0.5 mm id). A summary of the optimum operating conditions of the vapor generation system is given in Table 2. Mercury Speciation A typical chromatogram (ICP-MS detection) for a solution containing HgII, methyl-Hg and ethyl-Hg is shown in Fig. 6. Fig. 3 Eect of mobile phase flow rate on liquid chromatogram. Each mercury species was present at 50 ng ml-1.Other LC conditions are given in Table 2. of NaBH4 concentration on the generation of mercury vapor. The results are shown in Fig. 4. As the NaBH4 concentration increased, the peak heights of various mercury species increased rapidly and reached the maximum when the NaBH4 concentration was about 0.1%. In order to avoid any possible matrix interference, the concentration of NaBH4 used was as low as possible. In the following experiments, 0.1% m/v NaBH4 was used. Compared with the conventional nebulization (0% Fig. 5 Eect of HNO3 concentration on Hg ion signal. All the signals NaBH4), as shown in Fig. 4, the mercury ion signals increased were relative to the first point. For concentrations and flow rates of other reagents, see Fig. 1. 8–36-fold when 0.1% NaBH4 was used in the CV generation Journal of Analytical Atomic Spectrometry, July 1997, Vol. 12 685Table 4 Calibration parameters (0.1–20 ng ml-1) for the mercury species Sensitivity/ Correlation Detection Compound counts s-1 ng-1 ml coecient limit/ng ml-1 HgII 590 0.9989 0.11 Methyl-Hg 2140 0.9994 0.03 Ethyl-Hg 1440 0.9995 0.04 Table 5 Mercury detection limits (ng ml-1) Method HgII Methyl-Hg Ethyl-Hg LC–CV–ICP-MS* 0.11 0.03 0.04 LC–CV–ICP-MS† 1.2 0.6 1.2 LC–PN–ICP-MS‡ 7 16 16 LC–USN–ICP-MS§ 0.12 0.22 0.26 LC–DIN–ICP-MS¶ 4.0 4.0 4.0 PC–LC–ICP-MSd 0.017 0.016 — LC–CV–MIP-AES** 0.15 0.35 — * This work, 100 ml sample loop.† Ref. 10, 100 ml sample loop. ‡ Ref. 10, 100 ml sample loop.PN=pneumatic nebulizer. Fig. 6 Typical Hg-selective chromatogram for A, HgII, B, methyl-Hg § Ref. 11, 200 ml sample loop. USN=ultrasonic nebulizer. and C, ethyl-Hg. Each mercury species was present at 5 ng ml-1 (as ¶ Ref. 13, 2 ml sample loop. DIN=direct injection nebulizer. Hg). For LC and CV conditions, see Table 2. d Ref. 14, 100 ml sample loop and preconcentration of 1.0 l of sample solution. ** Ref. 9, 100 ml sample loop. As shown, all three species studied were fully resolved and the separation was complete in less than 6 min.The background was used as a standard reference. A 100 ml injection of the at m/z 202 increased when CV generation sample introduction sample solution was analyzed for mercury using the CV was used, which could be due to the trace mercury contamigeneration system. The chromatogram obtained for this deter- nation of the reagents used for LC separation and CV genermination is shown in Fig. 7. As can be seen, both inorganic ation and to the better analyte transport eciency with CV mercury and methylmercury were present in this sample.A generation sample introduction or the memory of mercury small dip in the background in front of the HgII peak was from the spray chamber. Peak area measurements indicated found which was due to the matrix of the sea-water sample. that the response for mercury was dierent for these three Apparently, the chromatographic separation has isolated the mercury species.This may be attributed to variations in the mercury from the matrix. As shown in Table 6, the recoveries CV generation eciency of the various mercury species menof various mercury species from the sea-water sample were in tioned earlier. Similar results were observed when the analyte the range 93–97%. The amount of mercury present in this was determined in the FI mode. water sample was quantified by the external calibration method Repeatability was determined using five injections of a test and the results are given in Table 6.The concentration of mixture containing 5 ng ml-1 HgII, methyl-Hg and ethyl-Hg. mercury determined by LC–CV–ICP-MS is higher than the As shown in Table 3, the RSD of the peak heights was less reference value given in Ref. 21. It should be mentioned that than 4% for all the species, which is similar to the precision the reference value for mercury in the sample is given for obtained in previous ICP-MS experiments with LC separations total mercury.using other types of sample introduction device.11,16,18–20 A tap water collected from National Sun Yat-Sen University Calibration curves based on peak heights were linear for each was also analyzed for mercury. As shown in Table 6, no mercury compound in the range tested. The detection limits mercury was detected in this sample, possibly because the were calculated from these calibration curves and based on the amount (or concentration) necessary to yield a net signal equal to three times the standard deviation of the background.The absolute detection limits were 3–11 pg, which corresponds to relative values of 0.03–0.11 ng ml-1 (see Table 4). The use of more purified reagent should lower the detection limit. As shown in Table 5, the detection limits obtained in this work are comparable to or better than previous results with similar techniques.9–11,13,14 Determination of Mercury inWater Samples In order to prove that the system works for practical analysis, an open ocean sea-water reference material (NRCC NASS-4) Table 3 Repeatability of retention time and peak height of the LC elution peaks (n=5) Retention time Repeatability of Compound ±s/s peak height (RSD) (%) HgII 84±1 1.6 Fig. 7 Typical Hg-selective chromatogram of open ocean sea-water Methyl-Hg 143±1 1.5 NASS-4. The concentrations of HgII and methyl-Hg in the injected Ethyl-Hg 326±2 3.3 solution are 0.75 and 0.09 ng ml-1, respectively. 686 Journal of Analytical Atomic Spectrometry, July 1997, Vol. 12Table 6 Recoveries and concentrations of mercury in water samples as measured by LC–CV–ICP–MS. Values are means±standard deviations for three determinations Concentration Reference Sample Compound Recovery (%) found/ng ml-1 value/ng ml-1 NASS-4 sea-water HgII 93±6 1.50±0.12 0.9† Methyl-Hg 97±4 0.18±0.03 Ethyl-Hg 93±4 ND* Tap water HgII 102±7 ND Methyl-Hg 100±1 ND Ethyl-Hg 101±3 ND * ND=not detectable.† Ref. 21. 6 Aizpun, B., Fernandez, M. L., Blanco, E., and Sanz-Medel, A., concentrations of the mercury species in this sample were J. Anal. At. Spectrom., 1994, 9, 1279. below the detection limits of the LC–CV–ICP-MS system. 7 Schickling, C., and Broekaert, J. A. C., Appl. Organomet. Chem., Recoveries of various Hg species in a spiked tap water sample 1995, 9, 29. were in the range 100–102%. 8 Bulska, E., Emteborg, H., Baxter, D. C., Frech, W., Ellingsen, D., and Thomassen, Y., Analyst, 1992, 117, 657. 9 Costa-Fernandez, J. M., Lunzer, F., Pereiro-Garcia, R., Sanz- CONCLUSION Medel, A., and Bordel-Garcia, N., J. Anal. At. Spectrom., 1995, 10, 1019. 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Received August 19, 1996 5 Lupsina, V., Horvat, M., Jeran, Z., and Stegnar, P., Analyst, 1992, 117, 673. Accepted February 24, 1997 Journal of Analytical Atomic Spectrometry, July 1997, Vol. 12 687