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Determination of methylmercury by electrothermal atomic absorption spectrometry using headspace single-drop microextraction with in situ hydride generation
Spectrochimica Acta Part B 60 (2005) 145 – 150 www.elsevier.com/locate/sab
Analytical note
Determination of methylmercury by electrothermal atomic absorption spectrometry using headspace single-drop microextraction with in situ hydride generation Sandra Gil, Sandra Fragueiro, Isela Lavilla, Carlos Bendicho* Departamento de Quı´mica Analı´tica y Alimentaria, Area de Quı´mica Analı´tica, Universidad de Vigo, Facultad de Ciencias (Quı´mica), As Lagoas-Marcosende s/n, 36200 Vigo, Spain Received 27 July 2004; accepted 26 October 2004 Available online 24 November 2004
Abstract A new method is proposed for preconcentration and matrix separation of methylmercury prior to its determination by electrothermal atomic absorption spectrometry (ETAAS). Generation of methylmercury hydride (MeHgH) from a 5-ml solution is carried out in a closed vial and trapped onto an aqueous single drop (3-Al volume) containing Pd(II) or Pt(IV) (50 and 10 mg/l, respectively). The hydrogen evolved in the headspace (HS) after decomposition of sodium tetrahydroborate (III) injected for hydride generation caused the formation of finely dispersed Pd(0) or Pt(0) in the drop, which in turn, were responsible for the sequestration of MeHgH. A preconcentration factor of ca. 40 is achieved with both noble metals used as trapping agents. The limit of detection of methylmercury was 5 and 4 ng/ml (as Hg) with Pd(II) or Pt(IV) as trapping agents, and the precision expressed as relative standard deviation was about 7%. The preconcentration system was fully characterised through optimisation of the following variables: Pd(II) or Pt(IV) concentration in the drop, extraction time, pH of the medium, temperatures of both sample solution and drop, concentration of salt in the sample solution, sodium tetrahydroborate (III) concentration in the drop and stirring rate. The method has been successfully validated against two fish certified reference materials (CRM 464 tuna fish and CRM DORM-2 dogfish muscle) following selective extraction of methylmercury in 2 mol/l HCl medium. D 2004 Elsevier B.V. All rights reserved. Keywords: Methylmercury hydride; Headspace single-drop microextraction; Pd(II); Pt(IV) sequestrating ions; ETAAS
1. Introduction Solid-phase microextraction (SPME) [1] and single-drop microextraction (SDME) [2] have emerged in last years as powerful tools for preconcentration and matrix separation prior to detection. Although originally developed for organic analytes, their potential for preconcentration of trace metals and organometals has been recognised [3]. While two sampling modes are available for performing microextraction techniques (i.e. direct and headspace), the use of headspace, although requiring volatile or semivolatile
* Corresponding author. Tel.: +34 986 812281; fax: +34 986 812382. E-mail address: [email protected] (C. Bendicho). 0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2004.10.008
analytes, avoids extraction of potentially interferent nonvolatile compounds [4]. Determination of methylmercury is of paramount importance owing to the toxicological effects associated with this Hg species [5]. Methylmercury derivatives are very volatile, which benefits their separation from the matrix. Methylmercury is commonly determined by several techniques such as atomic absorption spectrometry (AAS) [6], atomic emission spectrometry (AES) [7], atomic fluorescence spectrometry (AFS) [8] and inductively coupled plasmamass spectrometry (ICP-MS) [9] after gas chromatography separation. Methods involving preconcentration of methylmercury by SPME prior to its chromatographic separation have been published. After headspace sampling, gas chroma-
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tography (GC) coupled to AAS [10], MS [11], ICP-MS [12], AFS [13] and MIP-AES [14] have been used for detection. Recently, direct couplings between SPME and an atomic detector without chromatographic interface, as an efficient way to improve the detection limits of MeHg+ and avoid potential decomposition risk (artefact formation) existing in chromatographic separations have been described [15,16]. In a previous work [17], the authors reported for the first time, a headspace (HS)-SDME technique for preconcentration of hydride-forming elements such as As, Sb and Se onto a Pd(II)-containing aqueous drop prior to detection by electrothermal atomic absorption spectrometry (ETAAS). The sequestration mechanism proposed lied in the catalytic decomposition of the hydrides in the Pd(0) formed on the drop surface. Pd(0) arises as a result of the reducing action caused by the hydrogen gas that evolves in the headspace after the sodium tetrahydroborate (III) decomposition. In this case, Pd fulfils two functions, i.e. it behaves as both a trapping agent and a matrix modifier in the furnace. MeHg+ can be derivatised to form MeHgH upon reduction with sodium tetrahydroborate (III) [18,19]. According to the mechanism above indicated, this compound should also be efficiently trapped onto a drop containing a Pt-group element. In this work, the preconcentration and matrix separation of MeHg+ by HS-SDME following hydride generation is proposed. Pd(II) or Pt(IV) are employed as trapping agents in the aqueous drop. The enriched drop with mercury is subsequently injected in a graphite tube for Hg determination by electrothermal atomic absorption spectrometry (ETAAS). The preconcentration system is fully characterised through optimisation of the relevant variables influencing the generation and sequestration of methylmercury.
2. Experimental 2.1. Apparatus A Unicam (Cambridge, UK) Model Solaar 939 Spectrometer equipped with a GF-90 graphite furnace atomiser and an FS 90 autosampler was used. An Hg hollowcathode lamp was employed as the radiation source. Integrated absorbance was chosen as the analytical signal. Atomic absorption measurements were performed at 253.7 nm. The spectral band-pass was 0.5 nm. A deuterium background corrector was used when necessary. The thermal program for Hg is shown in Table 1. Pyrolytic graphite-coated graphite tubes with platform were employed. A high precision microsyringe (10 Al) with a plunger made of polytetrafluoroethylene (PTFE) (Hamilton) was employed for single-drop microextraction.
Table 1 Thermal program for determination of methylmercury by ETAAS following headspace single-drop microextraction Stage
Temperature (8C)
Hold time (s)
Ramp (8C/s)
Gas flow rate (ml/min)
Drying Ashing
120 200 400 1400 1500 2300
20 10 10 5 5 3
10 10 10 (off) (off) 500
300 300 300 0 0 300
Atomisation Cleaning
(Pd) (Pt) (Pd) (Pt)
Ultrasonic extraction of MeHg+ from fish tissue was carried out by a Sonics and Materials (Dambury, CT, USA) Model VC 100 probe ultrasonic processor. 2.2. Reagents All chemicals were of analytical reagent grade. A stock solution of MeHg+ (500 mg/l as Hg) was prepared by dissolving the appropriate amount of MeHgCl (Riedel-de H7en, Pestanal, Germany) in ultrapure water. Firstly, the MeHgCl was dissolved in a small amount of propan-2-ol (Merck, Darmstadt, Germany). The solution was stored at 4 8C prior to use. Diluted working standards were prepared fresh daily from the stock solution. Sodium tetrahydroborate (Merck), acetic acid and sodium acetate were used. CRM BCR 464 tuna fish and CRM NRCC DORM-2 dogfish muscle were used for validation. The trapping agent solutions were prepared from Pd(NO3)2d 2H2O (Merck) and H2PtCl6 (Fluka, Steinheim, Switzerland). L(+) ascorbic acid (Merck) was used to obtain a reduced Pd matrix modifier for direct determination of MeHg+ by ETAAS. 2.3. Procedure for headspace single-drop microextraction of methylmercury A 5-ml solution in 0.1 mol/l NaOAc/HOAc buffer is placed into a 40-ml vial closed with a silicone rubber septum. The septum was pierced by the microsyringe so that needle tip was located above the surface of the sample solution. Sampling was carried out by exposing to the headspace a 3-Al aqueous drop (50 mg/l of Pd(II) or Pt(IV) in 3% volume/volume HNO3) that is suspended at the needle tip. Then, 1 ml of sodium tetrahydroborate (III) (3.5% mass/volume) was injected into the vial while the solution was being stirred. After allowing trapping of the MeHgH onto the drop for 2 min, the drop is retracted back into the microsyringe and subsequently injected in the graphite furnace. The SDME device is depicted in Fig. 1. Optimal conditions for HS-SDME of MeHgH were the following: a 50 mg/l Pd(II) or Pt(IV) concentration in the drop; a 3.5% mass/volume sodium tetrahydroborate (III) (1 ml injection volume); a 3-min extraction time; medium composition: a 5-ml sample solution containing 0.1 mol/l HOAc/NaOAc buffer (pH 5) m1 g NaCl; a 300-rpm stirring
S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150
A B
G
C
D
E
F
Fig. 1. Scheme showing the headspace single-drop microextraction device. (A) Microsyringe (1–10 Al) for suspending the drop; (B) syringe for injecting the sodium tetrahydroborate (III) solution; (C) 3-Al aqueous drop containing Pd(II) or Pt(IV); (D) 40-ml volume vial; (E) sample solution containing MeHg+; (F) magnetic stirrer; (G) septum.
rate of the sample solution; a 3-Al drop volume; drop and sample temperature: 20 8C. A 100 ng/ml MeHg+ concentration (as MeHgCl) was used for optimisation.
3. Results and discussion 3.1. Optimisation of the HS-SDME method Sequestration of methylmercury hydride onto the drop containing the reduced noble metal on its surface could be explained through the catalytic decomposition mechanism, as proposed in a previous paper [17]. Unlike recent work dealing with SDME of organometals [20,21], an aqueous drop containing Pd(II) or Pt(IV) ions is employed here instead of an organic solvent drop. Other trapping agents tried in this work, which are based on the affinity of mercury for binding thiol groups, such as l-cysteine or diethyldithiocarbamate, did not provide efficient trapping of methylmercury hydride. These compounds act as strong complexing agents for mercury ions in solution, but are unable to sequestrate MeHgH from the headspace.
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Fig. 2 shows the effect of the noble metal concentration in the drop. Trapping is equally effective with Pd(II) and Pt(IV). As can be observed, maximum preconcentration was obtained with a concentration in the drop about 50 mg/l of Pd or 10 mg/l of Pt. These concentrations were similar to that found as optimal for sequestrating volatile covalent hydrides such as AsH3, SbH3 and SeH2 [17]. Despite HS-SDME being an equilibrium-based technique, optimisation of the extraction time is necessary to achieve an efficient sequestration. Fig. 3 shows the effect of the extraction time in the range 15– 300 s. For both Pt and Pd, increasing extraction occurs up to a ca. 180 s extraction time. This time is much shorter than that found for the use of HS-SPME [16] using the same derivatisation procedure. As in both cases, the mass transfer in the headspace is assumed to be identical, a faster mass transfer in the drop must occur in comparison with the SPME fiber coating. This could be an important advantage of SDME in comparison with SPME approaches for sample preparation prior to determination of methylmercury. Similar performance is observed for Pt and Pd. Pd was chosen for optimisation of the remaining variables. The use of an HOAc/NaOAc buffer has been recommended for generation of MeHgH [10]. In this study, both the buffer concentration and the pH achieved were optimised. The pH was studied in the range 2–9. While poor performance is observed at acid pH, a pH between 5 and 9 yielded similar results. The buffer concentration was studied in the range 0.1–1 mol/l. The higher the buffer concentration, the less stable the drop becomes at the tip of the needle. This effect was thought to be caused by the increased pressure reached inside the vial as the buffer concentration increased. A 0.1 mol/l buffer at pH 5 was considered as adequate for efficient MeHgH generation. The salting-out effect was studied by addition of NaCl. Additions of NaCl masses in the range 0–5 g to a 5-ml sample volume were performed. The extraction increased slightly up to 1 g NaCl and remained constant from that NaCl mass. A 1 g mass of NaCl was added to the sample solution for further experiments. The effect of both the sample solution and drop temperature was also tested. The optimisation curve for the sample solution temperature showed that extraction increased from 5 to 20 8C, and levelled off from 20 8C. On the contrary, when the drop temperature was studied, a steady extraction is observed in the range 10–30 8C, but extraction diminished from a 30 8C drop temperature. A 20 8C temperature was chosen as optimal for both sample solution and drop. Other variables optimised did not display any influence in the intervals studied. Thus, a constant trapping efficiency was observed when the stirring rate of the sample solution was varied in the range 100–900 rpm. Likewise, the sodium tetrahydroborate (III) concentration did not show any influence in the range 0.5–6% mass/volume. Sequestration experiments performed with Hg(II) salts under the optimal generation and trapping conditions established for MeHg+ showed that the trapping efficiency
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Integrated absorbance (s)
0,350 0,300 Pd Pt
0,250 0,200 0,150 0,100 0,050 0,000
0
50
100
150
200
250
300
350
Trapping agent concentration (mg/l) Fig. 2. Effect of the trapping agent concentration on Hg absorbance. Uncertainty intervals for N=3 replicate measurements are shown.
was at least five times less for Hg(II) in comparison with MeHg+. Some trapping observed for Hg(0) generated upon reaction between Hg(II) and sodium tetrahydroborate (III) could be due to the ability of Hg(0) to form amalgams with noble metals. Finally, optimisation of the method was accomplished by studying the effect of the drop volume on extraction. As expected, a larger drop surface exposed to the headspace brought about an improvement in the extraction efficiency. The maximum allowable drop volume was 3 Al. Larger drop volumes caused the detachment of the drop from the microsyringe tip during sampling. 3.2. Analytical characteristics Analytical characteristics for the sequestration of MeHg+ onto a Pd(II) or Pt(IV)-containing drop were established. The equation of the linear range of the calibration curves were the following: Pd(II): Y = 0.0025 [ MeHg+ ]– 0.0236; r 2 = 0.995 Pt(IV): Y = 0.0029 [MeHg+ ]–0.0693; r 2 =0.996 where Y is integrated absorbance, and [MeHg+] is the concentration of methylmercury (ng/ml).
The calibration curves were linear up to 300 ng/ml. Detection limits (LODs) (3r criterion) were 5 and 4 ng/ml for trapping with Pd(II) and Pt(IV), respectively. Quantification limits (10r criterion) were 18 and 14 ng/ml with both trapping agents, respectively. RSDs, calculated from 10 replicates, were about 7% with both sequestrating agents. The LOD of MeHg+, using the same instrument under optimal furnace conditions and without preconcentration, was 190 ng/ml for a 3-Al injection volume, which means that a preconcentration factor of ca. 40 is achieved. It is important to emphasize that direct determination (i.e. without preconcentration) of MeHg+ by ETAAS required the use of a reduced Pd modifier (0.25 Ag PdF0.5 Ag ascorbic acid) so that this species was thermally stabilised. Without the reduced Pd modifier, the LOD of MeHg+ was about 20 times worse. In the HS-SDME method proposed here, a reduced Pd is already achieved during sampling of the headspace as a result of the hydrogen evolved. The LOD obtained by HS-SDME–ETAAS is comparable to those obtained with HS-SPME–GC–ICPMS and HSSPME–GC–AAS (Table 2). Nevertheless, an improved LOD is obtained by direct couplings between microextraction and a detector such as SPME–ICPMS and SPME–QF–AAS. A disadvantage of SPME is the limited fiber lifetime and impaired precision with prolonged usage.
Integrated absorbance (s)
0,600 0,500 0,400 Pd Pt
0,300 0,200 0,100 0,000
0
50
100
150
200
250
300
350
Extraction time (s) Fig. 3. Effect of the extraction time on Hg absorbance using Pd(II) or Pt(IV) as trapping agents. Uncertainty intervals for N=3 replicate measurements are shown.
S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150 Table 2 Comparison of LODs found in the literature for determination of methylmercury after microextraction using headspace sampling Analytical technique
LOD (ng/ml)
SPME–GC–AAS SPME–HG–QF–AAS SPME–ICP-MS SPME–GC–ICP-MS SPME–GC–MS SPME–GC–AFS SPME–GC–MIP–AES SDME–ETAAS
2.6 0.4 0.2 3.7 1.3 3 0.1 4
RSD (%) 9 7 2.4 17 6 9 – 7
Ref. [10] [16] [15] [12] [23] [22] [14] This work
The SDME–ETAAS technique is fast, simple and costeffective as compared with more sophisticated couplings for determination of methylmercury. Precision (expressed as RSD) for the headspace sampling approaches reported in Table 2 is typically in the range 6–9%. 3.3. Method validation The method has been validated against CRM BCR 464 Tuna fish (certified MeHg+: 5.5F0.17 Ag/g) and CRM NRCC DORM-2 (certified MeHg+: 4.47F0.32 Ag/g). Marine biological tissues can contain both inorganic mercury and methylmercury. A separation of both species is needed so that the developed method can be applied to determination of methylmercury. The approach used here lies in the different sulphur binding strengths of both species (MeHg+bHg2+), which allows their separation in acidic media with variable HCl concentration. Selective extraction of MeHg+ is carried out according to the method established by Rio-Segade and Bendicho [24]. The standard addition method was used for calibration. The found values were 5.45F0.43 and 4.39F0.35 Ag/g (N=4) for CRM 464 and CRM DORM-2, respectively. These values was in excellent agreement with the certified ones, no significant differences being observed (t-test, P=0.05). A recovery study at the 50 ng/ml analyte level was also performed with CRM 464. For this purpose, the solid sample was spiked prior to extraction with an MeHg+ amount in order to reach that final concentration in the extract. The average recovery was 94F8% (N=3).
4. Conclusions An effective sequestration of methylmercury hydride onto a Pd(II) or Pt(IV)-containing aqueous drop (3 Al) is demonstrated. This sampling technique combined with ETAAS constitutes an attractive alternative to sophisticated couplings employed for methylmercury determination, being fast, simple and cost-effective. In contrast to other SDME methods, no toxic organic solvents are required, since the sequestration mechanism lies in the catalytic decomposition of the methylmercury hydride onto an aqueous drop containing Pd or Pt. The method is well
149
suited to determination of this Hg species in fish tissue by ETAAS provided that a selective extraction is applied as first sample pretreatment.
Acknowledgments This work has been financially supported by the Galician government (Xunta de Galicia) in the framework of Project PGIDT01PX13101PR.
References [1] C.L. Arthur, J. Pawliszyn, Solid phase microextraction with thermal desorption using fused silica optical fibers, Anal. Chem. 62 (1990) 2145 – 2148. [2] H. Liu, P.K. Dasgupta, Analytical chemistry in a drop. Solvent extraction in a microdrop, Anal. Chem. 68 (1996) 1817 – 1821. [3] Z. Mester, R. Sturgeon, J. Pawliszyn, Solid phase microextraction as a tool for trace element speciation, Spectrochim. Acta, Part B 56 (2001) 233 – 260. [4] A.L. Theis, A.J. Waldack, S.M. Hansen, M.A. Jeannot, Headspace solvent microextraction, Anal. Chem. 73 (2001) 5651 – 5654. [5] M. Reppeto, Toxicologı´a Avanzada, Diaz de Santos, Madrid, 1995, pp. 359 – 391. [6] C.M. Tseng, A. de Diego, F.M. Martin, D. Amouroux, O.F.X. Donard, Rapid determination of inorganic mercury and methylmercury in biological reference materials by hydride generation, cryofocusing, atomic absorption spectrometry after open focused microwaveassisted alkaline digestion, J. Anal. At. Spectrom. 12 (1997) 743 – 750. [7] M.S. Jimenez, R.E. Sturgeon, Speciation of methyl- and inorganic mercury in biological tissues using ethylation and gas chromatography with furnace atomization plasma emission spectrometric detection, J. Anal. At. Spectrom. 12 (1997) 597 – 601. [8] D.W. Bryce, A. Izquierdo, M.D. Luque de Castro, Pervaporation as an alternative to headspace, Anal. Chem. 69 (1997) 844 – 847. [9] R.C. Rodriguez Martin-Doimeadios, E. Krupp, D. Amouroux, O.F.X. Donard, Application of isotopically labelled methylmercury for isotope dilution analysis of biological samples using gas chromatography/ICPMS, Anal. Chem. 74 (2002) 2505 – 2512. [10] H. Bin, J. Gui-bin, N. Zhe-ming, Determination of methylmercury in biological samples and sediments by capillary gas chromatography coupled with atomic absorption spectrometry after hydride derivatization and solid phase microextraction, J. Anal. At. Spectrom. 13 (1998) 1141 – 1144. [11] L. Dunemann, H. Hajimiragna, J. Begerow, Simultaneous determination of Hg(II) and alkylated Hg, Pb and Sn species in human body fluids using SPME–GC/MS–MS, Fresenius’ J. Anal. Chem. 363 (1999) 466 – 468. [12] T. de Smaele, L. Moens, P. Sandra, R. Dams, Determination of organometallic compounds in surface water and sediment samples with SPME–CGC–ICPMS, Mikrochim. Acta 130 (1999) 241 – 251. [13] S. Diez, J.M. Bayona, Determination of methylmercury in human hair by ethylation followed by headspace solid-phase microextraction–gas chromatography–cold vapour-atomic fluorescence spectrometry, J. Chromatogr., A 963 (2002) 345 – 351. [14] S. Tutschku, M.M. Schantz, S.A. Wise, Determination of methylmercury and butyltin compounds in marine biota and sediments using microwave-assisted acid extraction, solid-phase microextraction and gas chromatography with microwave-induced plasma atomic emission spectrometric detection, Anal. Chem. 74 (2002) 4694 – 4701. [15] Z. Mester, J. Lam, R. Sturgeon, J. Pawliszyn, Determination of methylmercury by solid-phase microextraction inductively coupled
150
[16]
[17]
[18]
[19]
S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150 plasma mass spectrometry: a new introduction method for volatile metal species, J. Anal. At. Spectrom. 15 (2000) 837 – 842. S. Fragueiro, I. Lavilla, C. Bendicho, Direct coupling of solid phase microextraction and quartz tube-atomic absorption spectrometry for selective and sensitive determination of methylmercury in seafood: an assessment of chloride and hydride generation, J. Anal. At. Spectrom. 19 (2004) 250 – 254. S. Fragueiro, I. Lavilla, C. Bendicho, Headspace sequestration of arsine onto a Pd(II)-containing aqueous drop as a preconcentration method for electrothermal atomic absorption spectrometry, Spectrochim. Acta, Part B 59 (2004) 851 – 855. J.P. Craig, D. Mennie, N. Ostah, O.F.X. Donard, F. Martin, Novel methods for derivatization of mercury(II) and methylmercury(II) compounds for analysis, Analyst 117 (1992) 823 – 824. M. Fillipelly, F. Baldi, E.F. Brinckman, J.G. Olson, Methylmercury determination as volatile methylmercury hydride by purge and trap gas chromatography in line with Fourier transform infrared spectroscopy, Environ. Sci. Technol. 26 (1992) 1457 – 1460.
[20] H. Shioji, S. Tsunoi, H. Harino, M. Tanaka, Liquid-phase microextraction of tributyltin and triphenyltin coupled with gas chromatography tandem mass spectrometry: comparison between 4-fluorophenyl and ethyl derivatizations, J. Chromatogr., A 1048 (2004) 81 – 88. [21] V. Colombini, C. Bancon-Montigny, L. Yang, P. Maxwell, R.E. Sturgeon, Z. Mester, Headspace single-drop microextraction for the detection of organotin compounds, Talanta 63 (2004) 555 – 560. [22] Y. Cai, S. Monsalud, K.G. Furton, R. Jaffe, R.D. Jones, Determination of methylmercury in fish and aqueous samples using solid-phase microextraction followed by gas chromatography–atomic fluorescence spectrometry, Appl. Organomet. Chem. 12 (1998) 565 – 569. [23] A. Beichert, S. Padberg, B.W. Wenclawiak, Selective determination of alkylmercury compounds in solid matrices after subcritical water extraction, followed by solid-phase microextraction and GC–MS, Appl. Organomet. Chem. 14 (2000) 493 – 498. [24] S. Rio-Segade, C. Bendicho, Ultrasound-assisted extraction for mercury speciation by the flow injection-cold vapor technique, J. Anal. At. Spectrom. 14 (1999) 263 – 268.
Analytical note
Determination of methylmercury by electrothermal atomic absorption spectrometry using headspace single-drop microextraction with in situ hydride generation Sandra Gil, Sandra Fragueiro, Isela Lavilla, Carlos Bendicho* Departamento de Quı´mica Analı´tica y Alimentaria, Area de Quı´mica Analı´tica, Universidad de Vigo, Facultad de Ciencias (Quı´mica), As Lagoas-Marcosende s/n, 36200 Vigo, Spain Received 27 July 2004; accepted 26 October 2004 Available online 24 November 2004
Abstract A new method is proposed for preconcentration and matrix separation of methylmercury prior to its determination by electrothermal atomic absorption spectrometry (ETAAS). Generation of methylmercury hydride (MeHgH) from a 5-ml solution is carried out in a closed vial and trapped onto an aqueous single drop (3-Al volume) containing Pd(II) or Pt(IV) (50 and 10 mg/l, respectively). The hydrogen evolved in the headspace (HS) after decomposition of sodium tetrahydroborate (III) injected for hydride generation caused the formation of finely dispersed Pd(0) or Pt(0) in the drop, which in turn, were responsible for the sequestration of MeHgH. A preconcentration factor of ca. 40 is achieved with both noble metals used as trapping agents. The limit of detection of methylmercury was 5 and 4 ng/ml (as Hg) with Pd(II) or Pt(IV) as trapping agents, and the precision expressed as relative standard deviation was about 7%. The preconcentration system was fully characterised through optimisation of the following variables: Pd(II) or Pt(IV) concentration in the drop, extraction time, pH of the medium, temperatures of both sample solution and drop, concentration of salt in the sample solution, sodium tetrahydroborate (III) concentration in the drop and stirring rate. The method has been successfully validated against two fish certified reference materials (CRM 464 tuna fish and CRM DORM-2 dogfish muscle) following selective extraction of methylmercury in 2 mol/l HCl medium. D 2004 Elsevier B.V. All rights reserved. Keywords: Methylmercury hydride; Headspace single-drop microextraction; Pd(II); Pt(IV) sequestrating ions; ETAAS
1. Introduction Solid-phase microextraction (SPME) [1] and single-drop microextraction (SDME) [2] have emerged in last years as powerful tools for preconcentration and matrix separation prior to detection. Although originally developed for organic analytes, their potential for preconcentration of trace metals and organometals has been recognised [3]. While two sampling modes are available for performing microextraction techniques (i.e. direct and headspace), the use of headspace, although requiring volatile or semivolatile
* Corresponding author. Tel.: +34 986 812281; fax: +34 986 812382. E-mail address: [email protected] (C. Bendicho). 0584-8547/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2004.10.008
analytes, avoids extraction of potentially interferent nonvolatile compounds [4]. Determination of methylmercury is of paramount importance owing to the toxicological effects associated with this Hg species [5]. Methylmercury derivatives are very volatile, which benefits their separation from the matrix. Methylmercury is commonly determined by several techniques such as atomic absorption spectrometry (AAS) [6], atomic emission spectrometry (AES) [7], atomic fluorescence spectrometry (AFS) [8] and inductively coupled plasmamass spectrometry (ICP-MS) [9] after gas chromatography separation. Methods involving preconcentration of methylmercury by SPME prior to its chromatographic separation have been published. After headspace sampling, gas chroma-
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tography (GC) coupled to AAS [10], MS [11], ICP-MS [12], AFS [13] and MIP-AES [14] have been used for detection. Recently, direct couplings between SPME and an atomic detector without chromatographic interface, as an efficient way to improve the detection limits of MeHg+ and avoid potential decomposition risk (artefact formation) existing in chromatographic separations have been described [15,16]. In a previous work [17], the authors reported for the first time, a headspace (HS)-SDME technique for preconcentration of hydride-forming elements such as As, Sb and Se onto a Pd(II)-containing aqueous drop prior to detection by electrothermal atomic absorption spectrometry (ETAAS). The sequestration mechanism proposed lied in the catalytic decomposition of the hydrides in the Pd(0) formed on the drop surface. Pd(0) arises as a result of the reducing action caused by the hydrogen gas that evolves in the headspace after the sodium tetrahydroborate (III) decomposition. In this case, Pd fulfils two functions, i.e. it behaves as both a trapping agent and a matrix modifier in the furnace. MeHg+ can be derivatised to form MeHgH upon reduction with sodium tetrahydroborate (III) [18,19]. According to the mechanism above indicated, this compound should also be efficiently trapped onto a drop containing a Pt-group element. In this work, the preconcentration and matrix separation of MeHg+ by HS-SDME following hydride generation is proposed. Pd(II) or Pt(IV) are employed as trapping agents in the aqueous drop. The enriched drop with mercury is subsequently injected in a graphite tube for Hg determination by electrothermal atomic absorption spectrometry (ETAAS). The preconcentration system is fully characterised through optimisation of the relevant variables influencing the generation and sequestration of methylmercury.
2. Experimental 2.1. Apparatus A Unicam (Cambridge, UK) Model Solaar 939 Spectrometer equipped with a GF-90 graphite furnace atomiser and an FS 90 autosampler was used. An Hg hollowcathode lamp was employed as the radiation source. Integrated absorbance was chosen as the analytical signal. Atomic absorption measurements were performed at 253.7 nm. The spectral band-pass was 0.5 nm. A deuterium background corrector was used when necessary. The thermal program for Hg is shown in Table 1. Pyrolytic graphite-coated graphite tubes with platform were employed. A high precision microsyringe (10 Al) with a plunger made of polytetrafluoroethylene (PTFE) (Hamilton) was employed for single-drop microextraction.
Table 1 Thermal program for determination of methylmercury by ETAAS following headspace single-drop microextraction Stage
Temperature (8C)
Hold time (s)
Ramp (8C/s)
Gas flow rate (ml/min)
Drying Ashing
120 200 400 1400 1500 2300
20 10 10 5 5 3
10 10 10 (off) (off) 500
300 300 300 0 0 300
Atomisation Cleaning
(Pd) (Pt) (Pd) (Pt)
Ultrasonic extraction of MeHg+ from fish tissue was carried out by a Sonics and Materials (Dambury, CT, USA) Model VC 100 probe ultrasonic processor. 2.2. Reagents All chemicals were of analytical reagent grade. A stock solution of MeHg+ (500 mg/l as Hg) was prepared by dissolving the appropriate amount of MeHgCl (Riedel-de H7en, Pestanal, Germany) in ultrapure water. Firstly, the MeHgCl was dissolved in a small amount of propan-2-ol (Merck, Darmstadt, Germany). The solution was stored at 4 8C prior to use. Diluted working standards were prepared fresh daily from the stock solution. Sodium tetrahydroborate (Merck), acetic acid and sodium acetate were used. CRM BCR 464 tuna fish and CRM NRCC DORM-2 dogfish muscle were used for validation. The trapping agent solutions were prepared from Pd(NO3)2d 2H2O (Merck) and H2PtCl6 (Fluka, Steinheim, Switzerland). L(+) ascorbic acid (Merck) was used to obtain a reduced Pd matrix modifier for direct determination of MeHg+ by ETAAS. 2.3. Procedure for headspace single-drop microextraction of methylmercury A 5-ml solution in 0.1 mol/l NaOAc/HOAc buffer is placed into a 40-ml vial closed with a silicone rubber septum. The septum was pierced by the microsyringe so that needle tip was located above the surface of the sample solution. Sampling was carried out by exposing to the headspace a 3-Al aqueous drop (50 mg/l of Pd(II) or Pt(IV) in 3% volume/volume HNO3) that is suspended at the needle tip. Then, 1 ml of sodium tetrahydroborate (III) (3.5% mass/volume) was injected into the vial while the solution was being stirred. After allowing trapping of the MeHgH onto the drop for 2 min, the drop is retracted back into the microsyringe and subsequently injected in the graphite furnace. The SDME device is depicted in Fig. 1. Optimal conditions for HS-SDME of MeHgH were the following: a 50 mg/l Pd(II) or Pt(IV) concentration in the drop; a 3.5% mass/volume sodium tetrahydroborate (III) (1 ml injection volume); a 3-min extraction time; medium composition: a 5-ml sample solution containing 0.1 mol/l HOAc/NaOAc buffer (pH 5) m1 g NaCl; a 300-rpm stirring
S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150
A B
G
C
D
E
F
Fig. 1. Scheme showing the headspace single-drop microextraction device. (A) Microsyringe (1–10 Al) for suspending the drop; (B) syringe for injecting the sodium tetrahydroborate (III) solution; (C) 3-Al aqueous drop containing Pd(II) or Pt(IV); (D) 40-ml volume vial; (E) sample solution containing MeHg+; (F) magnetic stirrer; (G) septum.
rate of the sample solution; a 3-Al drop volume; drop and sample temperature: 20 8C. A 100 ng/ml MeHg+ concentration (as MeHgCl) was used for optimisation.
3. Results and discussion 3.1. Optimisation of the HS-SDME method Sequestration of methylmercury hydride onto the drop containing the reduced noble metal on its surface could be explained through the catalytic decomposition mechanism, as proposed in a previous paper [17]. Unlike recent work dealing with SDME of organometals [20,21], an aqueous drop containing Pd(II) or Pt(IV) ions is employed here instead of an organic solvent drop. Other trapping agents tried in this work, which are based on the affinity of mercury for binding thiol groups, such as l-cysteine or diethyldithiocarbamate, did not provide efficient trapping of methylmercury hydride. These compounds act as strong complexing agents for mercury ions in solution, but are unable to sequestrate MeHgH from the headspace.
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Fig. 2 shows the effect of the noble metal concentration in the drop. Trapping is equally effective with Pd(II) and Pt(IV). As can be observed, maximum preconcentration was obtained with a concentration in the drop about 50 mg/l of Pd or 10 mg/l of Pt. These concentrations were similar to that found as optimal for sequestrating volatile covalent hydrides such as AsH3, SbH3 and SeH2 [17]. Despite HS-SDME being an equilibrium-based technique, optimisation of the extraction time is necessary to achieve an efficient sequestration. Fig. 3 shows the effect of the extraction time in the range 15– 300 s. For both Pt and Pd, increasing extraction occurs up to a ca. 180 s extraction time. This time is much shorter than that found for the use of HS-SPME [16] using the same derivatisation procedure. As in both cases, the mass transfer in the headspace is assumed to be identical, a faster mass transfer in the drop must occur in comparison with the SPME fiber coating. This could be an important advantage of SDME in comparison with SPME approaches for sample preparation prior to determination of methylmercury. Similar performance is observed for Pt and Pd. Pd was chosen for optimisation of the remaining variables. The use of an HOAc/NaOAc buffer has been recommended for generation of MeHgH [10]. In this study, both the buffer concentration and the pH achieved were optimised. The pH was studied in the range 2–9. While poor performance is observed at acid pH, a pH between 5 and 9 yielded similar results. The buffer concentration was studied in the range 0.1–1 mol/l. The higher the buffer concentration, the less stable the drop becomes at the tip of the needle. This effect was thought to be caused by the increased pressure reached inside the vial as the buffer concentration increased. A 0.1 mol/l buffer at pH 5 was considered as adequate for efficient MeHgH generation. The salting-out effect was studied by addition of NaCl. Additions of NaCl masses in the range 0–5 g to a 5-ml sample volume were performed. The extraction increased slightly up to 1 g NaCl and remained constant from that NaCl mass. A 1 g mass of NaCl was added to the sample solution for further experiments. The effect of both the sample solution and drop temperature was also tested. The optimisation curve for the sample solution temperature showed that extraction increased from 5 to 20 8C, and levelled off from 20 8C. On the contrary, when the drop temperature was studied, a steady extraction is observed in the range 10–30 8C, but extraction diminished from a 30 8C drop temperature. A 20 8C temperature was chosen as optimal for both sample solution and drop. Other variables optimised did not display any influence in the intervals studied. Thus, a constant trapping efficiency was observed when the stirring rate of the sample solution was varied in the range 100–900 rpm. Likewise, the sodium tetrahydroborate (III) concentration did not show any influence in the range 0.5–6% mass/volume. Sequestration experiments performed with Hg(II) salts under the optimal generation and trapping conditions established for MeHg+ showed that the trapping efficiency
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Integrated absorbance (s)
0,350 0,300 Pd Pt
0,250 0,200 0,150 0,100 0,050 0,000
0
50
100
150
200
250
300
350
Trapping agent concentration (mg/l) Fig. 2. Effect of the trapping agent concentration on Hg absorbance. Uncertainty intervals for N=3 replicate measurements are shown.
was at least five times less for Hg(II) in comparison with MeHg+. Some trapping observed for Hg(0) generated upon reaction between Hg(II) and sodium tetrahydroborate (III) could be due to the ability of Hg(0) to form amalgams with noble metals. Finally, optimisation of the method was accomplished by studying the effect of the drop volume on extraction. As expected, a larger drop surface exposed to the headspace brought about an improvement in the extraction efficiency. The maximum allowable drop volume was 3 Al. Larger drop volumes caused the detachment of the drop from the microsyringe tip during sampling. 3.2. Analytical characteristics Analytical characteristics for the sequestration of MeHg+ onto a Pd(II) or Pt(IV)-containing drop were established. The equation of the linear range of the calibration curves were the following: Pd(II): Y = 0.0025 [ MeHg+ ]– 0.0236; r 2 = 0.995 Pt(IV): Y = 0.0029 [MeHg+ ]–0.0693; r 2 =0.996 where Y is integrated absorbance, and [MeHg+] is the concentration of methylmercury (ng/ml).
The calibration curves were linear up to 300 ng/ml. Detection limits (LODs) (3r criterion) were 5 and 4 ng/ml for trapping with Pd(II) and Pt(IV), respectively. Quantification limits (10r criterion) were 18 and 14 ng/ml with both trapping agents, respectively. RSDs, calculated from 10 replicates, were about 7% with both sequestrating agents. The LOD of MeHg+, using the same instrument under optimal furnace conditions and without preconcentration, was 190 ng/ml for a 3-Al injection volume, which means that a preconcentration factor of ca. 40 is achieved. It is important to emphasize that direct determination (i.e. without preconcentration) of MeHg+ by ETAAS required the use of a reduced Pd modifier (0.25 Ag PdF0.5 Ag ascorbic acid) so that this species was thermally stabilised. Without the reduced Pd modifier, the LOD of MeHg+ was about 20 times worse. In the HS-SDME method proposed here, a reduced Pd is already achieved during sampling of the headspace as a result of the hydrogen evolved. The LOD obtained by HS-SDME–ETAAS is comparable to those obtained with HS-SPME–GC–ICPMS and HSSPME–GC–AAS (Table 2). Nevertheless, an improved LOD is obtained by direct couplings between microextraction and a detector such as SPME–ICPMS and SPME–QF–AAS. A disadvantage of SPME is the limited fiber lifetime and impaired precision with prolonged usage.
Integrated absorbance (s)
0,600 0,500 0,400 Pd Pt
0,300 0,200 0,100 0,000
0
50
100
150
200
250
300
350
Extraction time (s) Fig. 3. Effect of the extraction time on Hg absorbance using Pd(II) or Pt(IV) as trapping agents. Uncertainty intervals for N=3 replicate measurements are shown.
S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150 Table 2 Comparison of LODs found in the literature for determination of methylmercury after microextraction using headspace sampling Analytical technique
LOD (ng/ml)
SPME–GC–AAS SPME–HG–QF–AAS SPME–ICP-MS SPME–GC–ICP-MS SPME–GC–MS SPME–GC–AFS SPME–GC–MIP–AES SDME–ETAAS
2.6 0.4 0.2 3.7 1.3 3 0.1 4
RSD (%) 9 7 2.4 17 6 9 – 7
Ref. [10] [16] [15] [12] [23] [22] [14] This work
The SDME–ETAAS technique is fast, simple and costeffective as compared with more sophisticated couplings for determination of methylmercury. Precision (expressed as RSD) for the headspace sampling approaches reported in Table 2 is typically in the range 6–9%. 3.3. Method validation The method has been validated against CRM BCR 464 Tuna fish (certified MeHg+: 5.5F0.17 Ag/g) and CRM NRCC DORM-2 (certified MeHg+: 4.47F0.32 Ag/g). Marine biological tissues can contain both inorganic mercury and methylmercury. A separation of both species is needed so that the developed method can be applied to determination of methylmercury. The approach used here lies in the different sulphur binding strengths of both species (MeHg+bHg2+), which allows their separation in acidic media with variable HCl concentration. Selective extraction of MeHg+ is carried out according to the method established by Rio-Segade and Bendicho [24]. The standard addition method was used for calibration. The found values were 5.45F0.43 and 4.39F0.35 Ag/g (N=4) for CRM 464 and CRM DORM-2, respectively. These values was in excellent agreement with the certified ones, no significant differences being observed (t-test, P=0.05). A recovery study at the 50 ng/ml analyte level was also performed with CRM 464. For this purpose, the solid sample was spiked prior to extraction with an MeHg+ amount in order to reach that final concentration in the extract. The average recovery was 94F8% (N=3).
4. Conclusions An effective sequestration of methylmercury hydride onto a Pd(II) or Pt(IV)-containing aqueous drop (3 Al) is demonstrated. This sampling technique combined with ETAAS constitutes an attractive alternative to sophisticated couplings employed for methylmercury determination, being fast, simple and cost-effective. In contrast to other SDME methods, no toxic organic solvents are required, since the sequestration mechanism lies in the catalytic decomposition of the methylmercury hydride onto an aqueous drop containing Pd or Pt. The method is well
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suited to determination of this Hg species in fish tissue by ETAAS provided that a selective extraction is applied as first sample pretreatment.
Acknowledgments This work has been financially supported by the Galician government (Xunta de Galicia) in the framework of Project PGIDT01PX13101PR.
References [1] C.L. Arthur, J. Pawliszyn, Solid phase microextraction with thermal desorption using fused silica optical fibers, Anal. Chem. 62 (1990) 2145 – 2148. [2] H. Liu, P.K. Dasgupta, Analytical chemistry in a drop. Solvent extraction in a microdrop, Anal. Chem. 68 (1996) 1817 – 1821. [3] Z. Mester, R. Sturgeon, J. Pawliszyn, Solid phase microextraction as a tool for trace element speciation, Spectrochim. Acta, Part B 56 (2001) 233 – 260. [4] A.L. Theis, A.J. Waldack, S.M. Hansen, M.A. Jeannot, Headspace solvent microextraction, Anal. Chem. 73 (2001) 5651 – 5654. [5] M. Reppeto, Toxicologı´a Avanzada, Diaz de Santos, Madrid, 1995, pp. 359 – 391. [6] C.M. Tseng, A. de Diego, F.M. Martin, D. Amouroux, O.F.X. Donard, Rapid determination of inorganic mercury and methylmercury in biological reference materials by hydride generation, cryofocusing, atomic absorption spectrometry after open focused microwaveassisted alkaline digestion, J. Anal. At. Spectrom. 12 (1997) 743 – 750. [7] M.S. Jimenez, R.E. Sturgeon, Speciation of methyl- and inorganic mercury in biological tissues using ethylation and gas chromatography with furnace atomization plasma emission spectrometric detection, J. Anal. At. Spectrom. 12 (1997) 597 – 601. [8] D.W. Bryce, A. Izquierdo, M.D. Luque de Castro, Pervaporation as an alternative to headspace, Anal. Chem. 69 (1997) 844 – 847. [9] R.C. Rodriguez Martin-Doimeadios, E. Krupp, D. Amouroux, O.F.X. Donard, Application of isotopically labelled methylmercury for isotope dilution analysis of biological samples using gas chromatography/ICPMS, Anal. Chem. 74 (2002) 2505 – 2512. [10] H. Bin, J. Gui-bin, N. Zhe-ming, Determination of methylmercury in biological samples and sediments by capillary gas chromatography coupled with atomic absorption spectrometry after hydride derivatization and solid phase microextraction, J. Anal. At. Spectrom. 13 (1998) 1141 – 1144. [11] L. Dunemann, H. Hajimiragna, J. Begerow, Simultaneous determination of Hg(II) and alkylated Hg, Pb and Sn species in human body fluids using SPME–GC/MS–MS, Fresenius’ J. Anal. Chem. 363 (1999) 466 – 468. [12] T. de Smaele, L. Moens, P. Sandra, R. Dams, Determination of organometallic compounds in surface water and sediment samples with SPME–CGC–ICPMS, Mikrochim. Acta 130 (1999) 241 – 251. [13] S. Diez, J.M. Bayona, Determination of methylmercury in human hair by ethylation followed by headspace solid-phase microextraction–gas chromatography–cold vapour-atomic fluorescence spectrometry, J. Chromatogr., A 963 (2002) 345 – 351. [14] S. Tutschku, M.M. Schantz, S.A. Wise, Determination of methylmercury and butyltin compounds in marine biota and sediments using microwave-assisted acid extraction, solid-phase microextraction and gas chromatography with microwave-induced plasma atomic emission spectrometric detection, Anal. Chem. 74 (2002) 4694 – 4701. [15] Z. Mester, J. Lam, R. Sturgeon, J. Pawliszyn, Determination of methylmercury by solid-phase microextraction inductively coupled
150
[16]
[17]
[18]
[19]
S. Gil et al. / Spectrochimica Acta Part B 60 (2005) 145–150 plasma mass spectrometry: a new introduction method for volatile metal species, J. Anal. At. Spectrom. 15 (2000) 837 – 842. S. Fragueiro, I. Lavilla, C. Bendicho, Direct coupling of solid phase microextraction and quartz tube-atomic absorption spectrometry for selective and sensitive determination of methylmercury in seafood: an assessment of chloride and hydride generation, J. Anal. At. Spectrom. 19 (2004) 250 – 254. S. Fragueiro, I. Lavilla, C. Bendicho, Headspace sequestration of arsine onto a Pd(II)-containing aqueous drop as a preconcentration method for electrothermal atomic absorption spectrometry, Spectrochim. Acta, Part B 59 (2004) 851 – 855. J.P. Craig, D. Mennie, N. Ostah, O.F.X. Donard, F. Martin, Novel methods for derivatization of mercury(II) and methylmercury(II) compounds for analysis, Analyst 117 (1992) 823 – 824. M. Fillipelly, F. Baldi, E.F. Brinckman, J.G. Olson, Methylmercury determination as volatile methylmercury hydride by purge and trap gas chromatography in line with Fourier transform infrared spectroscopy, Environ. Sci. Technol. 26 (1992) 1457 – 1460.
[20] H. Shioji, S. Tsunoi, H. Harino, M. Tanaka, Liquid-phase microextraction of tributyltin and triphenyltin coupled with gas chromatography tandem mass spectrometry: comparison between 4-fluorophenyl and ethyl derivatizations, J. Chromatogr., A 1048 (2004) 81 – 88. [21] V. Colombini, C. Bancon-Montigny, L. Yang, P. Maxwell, R.E. Sturgeon, Z. Mester, Headspace single-drop microextraction for the detection of organotin compounds, Talanta 63 (2004) 555 – 560. [22] Y. Cai, S. Monsalud, K.G. Furton, R. Jaffe, R.D. Jones, Determination of methylmercury in fish and aqueous samples using solid-phase microextraction followed by gas chromatography–atomic fluorescence spectrometry, Appl. Organomet. Chem. 12 (1998) 565 – 569. [23] A. Beichert, S. Padberg, B.W. Wenclawiak, Selective determination of alkylmercury compounds in solid matrices after subcritical water extraction, followed by solid-phase microextraction and GC–MS, Appl. Organomet. Chem. 14 (2000) 493 – 498. [24] S. Rio-Segade, C. Bendicho, Ultrasound-assisted extraction for mercury speciation by the flow injection-cold vapor technique, J. Anal. At. Spectrom. 14 (1999) 263 – 268.