Anal. Chem. 2003, 75, 2251-2255
Selective Measurement of Ultratrace Methylmercury in Fish by Flow Injection On-Line Microcolumn Displacement Sorption Preconcentration and Separation Coupled with Electrothermal Atomic Absorption Spectrometry Xiu-Ping Yan,*,†,‡ Yan Li,‡ and Yan Jiang‡
State Key Laboratory of Functional Polymer Materials for Adsorption and Separation, and Central Laboratory, Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China
A novel nonchromatographic speciation technique for ultratrace methylmercury in biological materials was developed by flow injection microcolumn displacement sorption preconcentration and separation coupled on-line with electrothermal atomic absorption spectrometry (ETAAS). In the developed technique, Cu(II) was first online complexed with diethyldithiocarbamate (DDTC), and the resultant Cu-DDTC was presorbed onto a microcolumn packed with the sorbent from a cigarette filter. Selective preconcentration of methylmercury (MeHg) in the presence of Hg(II), ethylmercury (EtHg), and phenylmercury (PhHg) was achieved at pH 6.8 through loading the sample solution onto the microcolumn due to a displacement reaction between MeHg and the presorbed Cu-DDTC. The retained MeHg was subsequently eluted with 50 µL of ethanol and on-line determined by ETAAS. Interferences from coexisting heavy metal ions with lower stability of their DDTC complexes relative to Cu-DDTC were minimized without the need of any masking reagents. No interferences from 5.5 mg L-1 Cu(II), 4.5 mg L-1 Cd(II), 2.5 mg L-1 Cr(III), 3 mg L-1 Fe(III), 10 mg L-1 Ni(II), 10 mg L-1 Pb(II), and at least 25 mg L-1 Zn(II) were observed for the determination of MeHg at the 50 ng L-1 level (as Hg). With the consumption of only 3.4 mL of sample solution, an enhancement factor of 75, a detection limit of 6.8 ng L-1 (as Hg) in the digest (corresponding to 3.4 ng g-1 in original solid sample for a final 50 mL of digest of 0.1 g of solid material), and a precision (RSD, n ) 13) of 2.3% for the determination of methylmercury at the 50 ng L-1 (as Hg) level were achieved at a sample throughput of 30 samples h-1. The recoveries of methylmercury spike in real fish samples ranged from 97 to 108%. The developed technique was validated by determination of methylmercury in a certified reference material (DORM-2, dogfish muscle), and was * Corresponding author. Fax: (86)22 23503034. E-mail:
[email protected]. † State Key Laboratory of Functional Polymer Materials for Adsorption and Separation. ‡ Research Center for Analytical Sciences. 10.1021/ac026415f CCC: $25.00 Published on Web 04/19/2003
© 2003 American Chemical Society
shown to be useful for the determination of methylmercury in real fish samples. It is well known that methylmercury (MeHg) is the most toxic and most commonly occurring organomercury compound. The main concern lies in its ability to accumulate in fish tissues, which causes mercury poisoning, as was observed for marine life and humans in Japan1 and marine birds in Sweden.2 Because of these risks, MeHg is included in the black list of national and international regulations and is monitored in fish samples by a number of organizations to check the contamination levels. The results are often used to check whether the levels in fishes available from markets are below the threshold values and may be consumed safely by humans. Consequently, the development of rapid, simple, selective, and sensitive techniques for the determination of MeHg at ultratrace levels in environmental and biological materials is of particular significance. Methods available for the determination of organomercury compounds in environmental and biological samples include three main approaches, depending on the sample type and nature: (i) the cold vapor technique, by using different reductants, such as SnCl2 or NaBH4, to reduce mercury to the volatile elemental form;3-6 (ii) gas chromatography (GC) coupled to different spectrometry detectors;7-13 (iii) high-performance liquid chroma(1) Kiyoura, R. In Advances in Water Pollution Research; Pearson, E. A., Ed.; Pergamon Press: New Yourk, 1964; p 291. (2) Johnels, A. G.; Westermark, T. In Chemical Fallout; Miller, M. W.. Berg, G. G., Eds.; Charles C. Thomas: Springfield, IL, 1969. (3) Cai, Y.; Monsalud, S.; Furton, K. G.; Jaffe, R.; Jones, R. D. Appl. Organomet. Chem. 1998, 12, 565-569. (4) Oda, C. E.; Ingle, J. D. Anal. Chem. 1981, 53, 2305-2309. (5) Ubillu´s, F.; Alergrı´a, A.; Barbera´, R.; Farre´, R.; Lagarda, M. J. Food Chem. 2000, 71, 529-533. (6) Bagheri, H.; Gholami, A. Talanta 2001, 55, 1141-1150. (7) Rapsomanikis, S.; Craig, P. Anal. Chem. Acta 1991, 248, 563-567. (8) Fisher, R.; Rapsomanikis, S.; Andrease, M. O. Anal. Chem. 1993, 65, 763766. (9) Filipelli, M. Anal. Chem. 1987, 59, 116-118. (10) Ceulemans, M.; Adams, F. J. Anal. At. Spectrom. 1996, 11, 201-206. (11) Kato, T.; Uehiro, T.; Yasuhara, A.; Morita, M. J. Anal. At. Spectrom. 1992, 7, 15-18. (12) Hintelmann, H.; Evans, R. D.; Villeneuve, J.Y. J. Anal. At. Spectrom. 1995, 10, 619-624. (13) Prange, A.; Jantzen, E. J. Anal. At. Spectrom. 1995, 10, 105-109.
Analytical Chemistry, Vol. 75, No. 10, May 15, 2003 2251
tography (HPLC) coupled to various spectrometry detectors.14-19 The main disadvantage of most methods reported is the timeconsuming sample treatment and analysis required.20 Although the most widely used method for the mercury speciation is GC with capture electrode or with other element-specific detectors, the use of HPLC for mercury speciation has the advantage of simplified sample preparation.16 In GC analysis, it is essential to form volatile, thermally stable derivatives, whereas this is not necessary for HPLC. Compared with conventional chromatographic techniques for speciation analysis with which high dilution factors are always associated, a distinct advantage of the on-line hyphenated flow injection (FI) precocentration and separation technique with electrothermal atomic absorption spectrometry (ETAAS) is that the analyte species is at the same time preconcentrated.21-23 Chromatographic techniques also suffer potential risks in disturbance on existing equilibrium between different species due to long interaction between the analytes and the column packing material. Therefore, the phenomenon arising in uncontrolled species transformations during the chromatographic process has to be carefully checked.24 Because FI preconcentration and separation were completed in fractions of a second, shifts of the equilibrium between the analyte species after one part had been removed were almost impossible within the time frame of the experiment.21-23 FI on-line sorption preconcentration and separation coupled with high-sensitivity ETAAS has been proved to be a very powerful technique for the determination of total ultratrace element and individual oxidation state of an element in a variety of sample matrixes.21,25-29 With FI on-line techniques, the drawbacks of batchwise operation can be overcome to a great extent, while preconcentration efficiency can be further enhanced.21,25 Besides, FI on-line preconcentration offers the possibility for the differential determination of individual oxidation states of an element by selective preconcentration.21,22,30,31 However, no work on the application of FI on-line preconcentration and separation hyphenated with ETAAS for speciation of organometallic compounds has been reported before. (14) Sanz-Medel, A.; Aizpun, B.; Marchante, J. M.; Segovia, E.; Fernandez, M. L.; Blanco, E. J. Chromatogr., A 1994, 683, 233-243. (15) Costa, J. M.; Lunzer, F.; Pereiro, R.; Sanz-Medel, A.; Bordel, N. J. Anal. At. Spectrom. 1995, 10, 1019-1025. (16) Sa´nchez, D. M.; Martı´n, R.; Morante, R.; Marn, J.; Munuera, M. L. Talanta 2000, 52, 671-679. (17) Falter, R.; Ilgen, G. Fresenius’ J. Anal. Chem. 1997, 358, 401-406. (18) Carro, A. M.; Mejuto, M. C. J. Chromatogr., A 2000, 882, 283-307. (19) Harrington, C. F. Trends Anal. Chem. 2000, 19, 167-179. (20) Ebdon, L.; Foulkes, M. E.; Rouxa, S. L.; Mun ˜oz-Olivas, R. Analyst 2002, 127, 1108-1114. (21) Welz, B. Spectrochim. Acta, Part B 1998, 53, 169-175. (22) Yan, X.-P.; Kerrich, R.; Hendry, M. J. Anal. Chem. 1998, 70, 4736-4742. (23) Yan, X.-P.; Yin, X.-B.; He, X.-W.; Jiang, Y. Anal. Chem. 2002, 74, 21622166. (24) Emons, H. Trends Anal. Chem. 2002, 21, 401-411. (25) Fang, Z.-L. Spectrochim. Acta, Part B 1998, 53, 1371-1379. (26) Burguera, J. L.; Burguera, M. Spectrochim Acta, Part B 2001, 56, 18011829. (27) Yan, X.-P.; Jiang, Y. Trends Anal. Chem. 2001, 20, 552-562. (28) Fang, Z.-L. Flow Injection Atomic Absorption Spectrometry; Wiley: Chichester, U.K., 1993. (29) Alonso Vereda, E.; Garcı´a de Torres, A.; Cano Pavo´n, J. M. Talanta 2001, 55, 219-232. (30) Yan, X.-P.; Van Mol, W.; Adams, F. Analyst 1996, 121, 1061-1067. (31) Yan, X.-P.; Sperling, M.; Welz, B. Anal. Chem. 1999, 71, 4353-4360.
2252 Analytical Chemistry, Vol. 75, No. 10, May 15, 2003
Table 1. Graphite Furnace Temperature Program for Determination of Methylmercury step
temperature/°C
time/s
argon flow/ mL min-1
1 (drying) 2 (atomization) 3 (cleaning)
50-150 1000 2200
50 7 3
150 0 150
The purpose of this work is to develop a simple, rapid, selective, and sensitive method for the determination of ultratrace MeHg in fish samples by on-line coupling of FI microcolumn displacement sorption preconcentration and separation to ETAAS and to compare the proposed method with conventional chromatography-based methodologies for MeHg speciation. EXPERIMENTAL SECTION Instrumentation. An Hitachi model 180-80 polarized Zeeman atomic absorption spectrometer fitted with a Hitachi autosampler (Part No. 170-9608-1) and a Hitachi graphite cup atomizer (Part No. 180-7402) was used throughout. A high-intensity mercury hollow cathode lamp (Ningqiang Light Sources Co. Ltd., Hengshui, China) was operated at 2 mA with a spectral slit width of 1.3 nm to isolate the 253.7-nm analytical line. Integrated absorbance (peak area) was set as measurement mode for the sound repeatability. The detailed graphite furnace temperature program used for the determination of methylmercury was shown in Table 1. A model FIA-3100 flow injection system (Vital Instruments Co. Ltd., Beijing, China) was used for the displacement sorption separation and preconcentration. It consists of two peristaltic pumps and a standard rotary injection valve (eight ports on the rotor and eight ports on the stator). The rotation speed of the two peristaltic pumps, their stop-and-go intervals, and the actuation of the injection valve were programmed (see Table 2). Ismaprene pump tubes were used to deliver the samples and reagents. Smallbore (0.35-mm-i.d.) PTFE tubings were adapted for all connections, which were kept the shortest possible length to minimize the dead volume. The PTFE microcolumn (1.5-cm × 2-mm i.d.) used for the preconcentration and separation was packed with the sorbent from a cigarette filter. Reagents. All reagents were of the highest available purity and at least of analytical grade. Doubly deionized water (DDW) was used throughout. The chelating agent solution was prepared by dissolving diethyldithiocarbamate (DDTC) (Guangzhou Chemicals Co., Guangzhou, China) in DDW just prior to use. Copper solutions containing 2-20 µg L-1 Cu were prepared by stepwise dilution of the stock solution of 1000 mg L-1 Cu (National Research Center for Standard Materials, Beijing, China) immediately before use. Diluted nitric acid and hydrochloride acid used for the acidification of the standard or sample solutions were prepared from the concentrated nitric acid and hydrochloride acid (The Third Chemical Co., Tianjin, China). Inorganic mercury stock solution of 1000 mg L-1 was prepared by dissolving the chloride mercury (The Second Chemical Co., Beijing, China) in DDW. The stock solutions of 1000 mg L-1 (as Hg) of MeHg, ethylmercury (EtHg), and phenylmercury (PhHg)
Table 2. Operational Sequence of FI On-Line Displacement Sorption Preconcentration System Coupled with ETAAS for Determination of MeHg flow rate/mL min-1
valve position step 1 (Figure 1a) 2 (Figure 1b) 3 (Figure 1b) 4 (Figure 1b) 5 (Figure 1a) 6 (Figure 1b) 7 (Figure 1b) 8 (Figure 1a)
function
injector
electromagnatic
duration/s
medium pump L-1
pump 1
pump 2
10 µg Cu 0.005% m/m DDTC air
off
3.2
3.0
off
50
sample
4.0
off
off
15
air
3.0
off
inject
off
5
ethanol
3.6
off
capillary into the graphite tube
fill
off
4
off
off
elution and eluent introduction
fill
off
25
2.8
off
capillary into the waste
inject
off
4
off
off
presorption
inject
off
15
air segmentation
fill
off
5
displacement sorption
fill
on
remove residual solution
fill
fill the eluent loop
were prepared by dissolving methylmercury, ethylmercury, and phenylmercury chloride (Alfa) in acetone, respectively. Working solutions were prepared from the stock solutions by stepwise dilution just before use. Warning: these organomercury solutions should be prepared in a well-ventilated hood, and care should be taken to avoid direct contact of these reagents with skin because of their high toxicity. Samples. A certified reference material DORM-2 (dogfish muscle, NRCC) was analyzed to check the accuracy of the developed technique. Fresh sea fish and shellfish samples were collected from local markets. The tissue was homogenized in a blender. The homogenized sample was split into two parts. One was used directly for extraction. The other was dried in an oven at 60 °C for 24 h before extraction, as described by Ubillu´s et al..5 Extraction Procedure for MeHg Determination. An alkaline digestion procedure as reported by Fisher et al.8 was employed to liberate MeHg from biological samples. Briefly, 10 mL of 25% m/v KOH/methanolic solution was added to 10 mg of the certified reference material DORM-2 or 100 mg of the homogenized fresh tissue or the pulverized dried fish tissue in a 50-mL flask. The flask was then shaken in an ultrasonic bath until the tissue was completely dissolved. The digest was diluted to volume with DDW and adjusted to pH 6.8 with KH2PO4 buffer solution just before determination. Precocentration and Separation Procedure. The developed FI manifold for the two different valve positions is shown in Figure 1. Details of the duration and function of each step for on-line microcolumn displacement sorption preconcentration are given in Table 2. A complete cycle of the separation and preconcentration required 123 s with a sample loading time of 50 s. RESULTS AND DISCUSSION Consideration of an FI On-Line Microcolumn Displacement Sorption Preconcentration System. It is well known that the cigarette filter can efficiently adsorb many poisonous organic compounds and, hence, alleviate the poisonous effect on smokers. Our experiments demonstrated that the sorbent also favored the adsorption of neutral hydrophobic organometallic chelates other than free metal ions. Therefore, the cigarette filter material was used as the sorbent for the preconcentration and separation of
air
Figure 1. FI manifold for the on-line microcolumn displacement sorption preconcentration and separation ETAAS for determination of methylmercury: P1 and P2, peristaltic pumps; MC, microcolumn; DT, delivery tubing; EL, eluent loop; EC, eluent container; W, waste; EV, electromagnetic valve; “off” for air flow and “on” for sample solution; ETA, electrthermal atomizer. Valve position: (A) inject; (B) fill.
methylmercury in this work. Such sorbent exhibited substantial sorption power for the MeHg-DDTC chelate. To minimize the interferences from concomitant ions in complex matrixes, a newly proposed displacement sorption preconcentration and separation concept32 was applied to develop an FI on-line microcolumn displacement sorption preconcentration and separation system for ETAAS determination of ultratrace MeHg. To this end, Cu(II) and DDTC solution merged on-line (32) Yan, X.-P.; Li, Y.; Jiang, Y. J. Anal. At. Spectrom. 2002, 17, 610-615.
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Figure 2. Effect of pH in sample solution on the displacement sorption for (a) 100 ng L-1 MeHg, (b) 10 ng L-1 Hg(II), (c) 500 ng L-1 EtHg, and (d) 500 ng L-1 PhHg. All other conditions as in Tables 1 and 2.
and passed through the microcolumn; the formed Cu-DDTC chelate was then presorbed onto the microcolumn. Subsequently, the sample solution was loaded onto the column, and the methylmercury in the sample solution was preconcentrated by the column as a result of the displacement reaction between MeHg(I) and the presorbed Cu-DDTC because of the relatively higher stability of MeHg-DDTC than Cu-DDTC. Those coexisting heavy metal ions with poorer stability of their DDTC chelates than Cu-DDTC could not displace Cu(II) from the presorbed Cu-DDTC and, hence, would be left in the solution and removed to the waste. Factors Affecting the Presorption of Cu-DDTC. The displacement sorption for MeHg relates to the on-line chelating and presorption of Cu-DDTC and, hence, to such factors as the concentration of copper solution, the concentration of chelating reagent DDTC, and the pH for Cu-DDTC formation and presorption. Studies on the effect of Cu concentration showed that the integrated absorbance of 100 ng L-1 (as Hg) MeHg increased linearly with the Cu concentration until 6 µg L-1 Cu and then gradually leveled off with further increase in Cu concentration up to 20 µg L-1. For further experiments, 10 µg L-1 Cu was selected for presorption. The effect of DDTC concentration on the integrated absorbance was examined using 100 ng L-1 (as Hg) methylmercury. The maximum integrated absorbance was observed in a DDTC concentration range of 0.003-0.075% m/m. Higher (>0.075% m/m) or lower (
