450
Anal. Chem. 1983, 55, 450-453
0 2
4
6
8
10
0 2 4 Time,
6
8
0
2
4
6
8
101214
minutes
Figure 9. Separation of naphthylamine (a), N,Ndimethylnaphthylamine (b), and nitronaphthalene (c) isomers. Chromatographic conditions are
the same as those given in Figure 8, except for mobile phase for (b), methanoVwater (40:60). density of the cyano group as well as of the inside of the cyclodextrin cavity (1). In a reversed-phase system, however, no pairs of isomers such as shown in Figure 7 could be separated by a column of LiChrosorb RP-8 and methanol/water (1:l). Chromatographic Separation. The performance of the P-CD-bonded column was tested by attempting a chromatographic separation of the isomers of some typical compounds, and satisfactory results were obtained. Though it is generally difficult to separate nitroaniline isomers by a conventional RP-HPLC system if the pH of the eluent is not adjusted, they could be separated completely on a diamino-0-CD column by using a mixture of methanol/water (20:80) as eluent without adjusting the p H of the eluent, as shown in Figure 8a. Cresol isomers could also be separated easily by the present system as shown in Figure 8b, though the RP-HPLC system failed to separate the ortho and the meta isomers. The order of elution of monosubstituted benzene isomers on the CD-bonded column is generally ortho < meta < para
and is the reverse of the one observed when alkyl-bonded silica and aqueous organic solvent containing cyclodextrin are used as stationary and mobile phase, respectively. It reflects that the sorption is based on the inclusion complex formation. An exception is the order of elution of nitroaniline isomers, which is meta < ortho < para on a CD-bonded column and different from the one (para < meta < ortho) observed when cyclodextrin is used as eluent. The reason for this apparent anomaly is not clear at present. For naphthalene derivatives, each pair of isomers could be separated successfully except for naphthol and naphthonitrile isomers. A diamine column or a conventional alkyl-bonded silica column could not achieve the separation of these isomers. As Figure 9 shows, 2-substituted isomers are, without exception, eluted after 1-substituted isomers, demonstrating the easier formation of inclusion complexes of the former with P-cyclodextrin. In conclusion, an attempt was made successfully to bond cyclodextrin molecules chemically on the surface of porous silica gels for HPLC. The silica gels thus obtained showed a characteristic property of cyclodextrin of forming inclusion complexes with various organic compounds, just as cyclodextrin itself does in aqueous solution. Registry No. Cyclodextrin, 12619-70-4; 0-cyclodextrin, 7585-39-9; 1-naphthylamine, 134-32-7;a-cyclodextrin, 10016-20-3.
LITERATURE CITED (1) Bender, M. L.; Komiyama, M. "Cyclodextrin Chemistry"; Springer-Verlag: New York, 1978. Cramer, F . ; Hettler, H. Naturwissenshaften 1967, 5 4 , 625-632. Bergeron, R. J. J . Chem. Educ. 1977, 5 4 , 204-207. Saenger W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344-362. Smolkov6-Keulemansov& E.; Krqsl, S. J . Chromatogr. 1980, 184, 347-361. (6) Hinze, W. L. Sep. f u r i f . Methods 1981, IO, 195-237. (7) SmolkovB-Keulemansov6, E. J . Chromatogr. 1962, 251, 17-34. (8) Gelb, R. I.; Schwartz, L. M.; Cardelino, B.; Fuhrman, H. S.;Johnson, R. F.; Laufer, D. A. J . Am. Chem. SOC. 1961, 703, 1750-1757.
(2) (3) (4) (5)
RECEIVED for review August 2, 1982. Accepted November 4, 1982.
Determination of Alkylmercury in Seawater at the Nanogram per Liter Level by Gas Chromatography/Atmospheric Pressure Helium Microwave-Induced Plasma Emission Spectrometry Koichi Chlba, Kazuo Yoshida, Klyoshl Tanabe,' Hlroki Haraguchl, * and Keiichlro Fuwa Department of Chemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 713, Japan
The determination of alkylmercury compounds has been Investigated by atmospheric pressure hellurn mlcrowave-lnduced plasma emisslon spectrometry combined with gas chromatography. The detection llmits for CH,HgCI, C,H,HgCI, and (CH,),Hg were 0.09, 0.12, and 0.40 pg/L, respectively, In the case of peak height analysls, and the dynamic ranges for these compounds were more than 4 orders of magnltude. Furthermore, they could be detected separately by the above GC/MIP system. I n addition, the present method has been applied to the determination of alkylmercury compounds In seawater, where the preconcentratlon of the seawater samples was performed with a benzene cysteine extractlon.
Present address: National Institute for Public Health, Minato-
ku, Tokyo 108, Japan.
In recent years, a sensitive and accurate determination of organomercury compounds has been required in biological and environmental fields. These mercury compounds are more toxic than metallic mercury, and they may cause serious illness in extremely polluted areas. I t has been reported that organomercury compounds are significantly concentrated in fish ( 1 4 3 , predominantly as methylmercury compounds ( 2 , 7). The syntheses of methylmercury compounds by microorganisms in freshwater sediments have been investigated by some workers (8, 9). There are, however, only a few reports on the determination of methylmercury compounds in seawater (10, 11). This may be because the concentrations of organomercury 'compounds in seawater are generally much lower than those of inorganic mercury compounds. The most widely accepted method for the determination of methylmercury compounds is based on the Westoo's pro-
0 1983 American Chemical Society 0003-2700/83/0355-0450$01.50/0
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1963
451
Table I. Operating Conditions for the Gas Chromatograph exptl conditions for determination of column column packing column temp, "C injector temp, "C detector oven temp, "IC transfer tube temp, "IC carrier gas carrier gas flow rate, rnL/min a
CH,HgCl and C,H,HgCl Pyrex, 1 m X 3 m m i.d. 16% DEGSa on 80/100mesh Chromosorb W 160 180
(CH, ),Hg Pyrex, 3 m x 3 mm i.d. 3% OV-17 on 80/100mesh Uniport HP 70 130 130 140 helium 80
180 190 helium 80
DEGS = diethylene glycol succinate.
cedure (I-3), which involvei3 the formation of a water-soluble adduct of methylmercury and cysteine, its extraction into water, acidification, arid finally the extraction of the liberated CH3HgX with an aromatic solvent. The CHgHgX is then determined by gas chromatography with electron capture detection (GC/ECD) The IECD provides high sensitivity but is not highly selective for the mercury determination. Alternate gas chromatographic techniques were employed with the ECD replaced by a reduced pressure helium microwave plasma (4) and argon microwave plasma emission detectors (11-13). A colorimetric method ( 5 ) and cold-vapor atomic absorption spectrometry (6) also have been used for the determination of methylmercury. T h e atmospheric pressure helium microwave-induced plasma (He-MIP) with a Beenakker-type cavity (14) has been investigated as a n excitation source for spectrochemical analysis of metallic and nonmetallic elements. We previously reported the determination of inorganic mercury compounds by He-MIP emission spectrometry with the cold-vapor generation technique (15). Furthermore, the He-MIP has been shown t o be useful as an element-selective detector for gas chromatography (16, 17). In this paper, a n atmospheric pressure He-MIP for GC detection has been adapted t o determine organomercury compounds, after 500-fold preconcentration has been applied to the determination of methylmercury chloride in seawater.
EXPERIMENTAL SECTION Chemicals. All chemicals used are of analytical reagent grade. The stock solutions of methylmercury chloride, ethylmercury chloride, and dimethylmercury are prepared by dissolving them in benzene. All the analytical standard solutions are obtained by diluting the stock solution with benzene to the proper concentration. The extractant solution for alkylmercury compounds is prepared by dissolving 10 g of L-cysteine and 8 g of sodium acetate in 1 L of distilled water. Instrumentation. The GC/MIP system, described in detail elsewhere (16, 19, corcsists of a Shimadzu GC-6A gas chromatograph (Shimadzu Seisakusho, Ltd.), a chemically deactivated four-way valve for solvent ventilation (GC-604-A, Nippon Kuromato Kogyo Co., Ltd.), a heated transfer tube interface, a Beenakker-type TMolo microwave resonance cavity, and an Eberbtype monochromator (0.5m focal length; Nippon Jarrell-Ash Co., Ltd.). A Shimadzu GC-6A dual column gas chromatograph equipped with a thermal conductivity detector (TCD) is employed. The interface between the gas chromatograph and the discharge tube of the MIP is constructed from a chemically deactivated four-way valve and a heated transfer tube (16, 17). The gas chromatographic columns and olptimum operating conditions are summarized in Table I. As is seen in Table I, the DEGS column was used for the measurement of methylmercury chloride and ethylmercury chloride. and the OV-17 column for that of dimethylmercury. The former column was treated with dimethylsilane and KBr in order to deactivate the surface. The TMolomicrowave cavity is constructed in the laboratory from pure copper metal after Beenakker's description (14) and some modifications (18-20). The microwave generator, which
HC1 40 m L B e n z e n e 50 m L
1
S h a i e V i g o r o u s l y f o r 1 0 rnin Colfect t h e Organic Phase
7
S o l u t i o n Containing 1 % L-Lystein a n d 0 . 6 Z, CH3COOKa, 5 m L
F
C o l l e c t A ueous Phase tic1 0.6 m!
Benzene 1 mL S h a k e Yig-sly
f o r 1 0 min
7 trans^ Figure 1. Extraction procedure for alkylmercury compounds in seawater sample.
provides 20-200 W of microwave power at 2.45 GHz, is run at 7 5 W forward power. The width and the height of both monochromator entrance and exit slits are 10 pm and 1 mm, respectively. A photomultiplier tube (R955; Hamamatsu TV Co., Ltd.) with low dark current and high gain over a wide wavelength region is used as a detector. The measurement of mercury is carried out at the 253.7-nm mercury line. Procedure. The flow rate of carrier helium gas is adjusted at 80 mL/min for both columns, and then the plasma is ignited with a Tesla coil. About 30 min later, the plasma and the temperature of the gas chromatograph stabilize. The adjustments of wavelength and observation position in the plasma are performed as follows. A proper concentration of methylmercury chloride standard solution (1-2 wL) is injected very slowly into the OV-17 column, and a broad mercury peak appears. During the appearance of the peak, wavelength and observation position (both vertical and horizontal) are adjusted quickly. This procedure is repeated two or three times for exact wavelength and observation position adjustment. This adjustment has to be performed daily before the measurement. The extraction procedure for the ultratrace levels of alkylmercury in seawater is presented in Figure 1. This technique was devised for 500-fold preconcentration of alkylmercury with reference to the Westoo extraction procedure ( 2 , 3 ) . All reagents used in this extraction must be monitored for alkylmercury contamination before the extraction is performed. The chromatograms were detected with the TCD and the MIP detectors in series. When the MIP was used as a detector, the emission signals were monitored at 253.7 nm, and the solvent was vented through a four-way valve before reaching the MIP.
RESULTS AND DISCUSSION Optimization. T h e optimum conditions for the plasma and the optics in the GC/MIP system were discussed previously (16, 17).
452
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
Table 11. Analytical Figures of Merit in the Determination of Alkylmercury Compounds by the GC/MIP System
compound methylmercury chloride ethylmercury chloride dimethylmercury
detection limit, M/L
re1 std dynamic dev,a range, % decades
0.09 (0.02 pg/s)
2.0
5
0.12 (0.02 pg/s)
2.0
5
0.40 (0.03 pg/s)
3.0
4.5
Measured with 1 yg/L of mercury for each compound. T h e optimal gas chromatographic conditions were determined by the following experiments. In the case of the DEGS column, the mercury signal increased gradually when the carrier gas flow rate changed from 50 to 80 mL/min. Methylmercury chloride and ethylmercury chloride were not adequately separated from each other, when the carrier gas flow rate was increased to more than 80 mL/min. The mercury signal (peak height) increased markedly as the column temperature was raised. The increase of mercury emission intensity was linear in the temperature region from 130 "C to 160 "C, but the emission signal rose suddenly up over 160 "C. Tailing was observed a t 1170 "C. This may be due to the partial decomposition of methylmercury chloride. Therefore the column temperature was optimized at 160 "C. The same optimum conditions were obtained for ethylmercury chloride. The optimized conditions are also shown in Table I. A typical chromatogram obtained under optimum conditions is shown in Figure 2. As can be seen from Figure 2 (lower curve), only the peaks of methylmercury and ethylmercury chlorides corresponding to 10 yg/L are observed with the M I P detector (retention time of each compound was 260 s and 470 s, respectively). These peaks are adequately separated from each other. On the other hand, in the chromatogram obtained with the TCD, only a peak corresponding to the benzene solvent was observed, while the peaks for the mercury compounds could not be detected. The experiment took about 10 min for one sample with the present system. The column packed with OV-17 used for the measurement of dimethylmercury was optimized in a similar manner in terms of carrier gas flow rate and column temperature. Detection Limit and L i n e a r D y n a m i c Range. The detection limit, the linear dynamic ranges, and the reproducibility for the methylmercury chloride, ethylmercury chloride, and dimethylmercury are listed in Table 11, where all the values were obtained without preconcentration. The detection limit was defined as the signal level corresponding to twice the standard deviation of background emission a t the analytical wavelength. The detection limits shown on the first column in Table 11were obtained from the peak height, using 4 yL of the standard solution, and those in parentheses were obtained in the unit of area intensity, pg/s. The latter indicates the absolute detection limit. As can be seen from Table 11, the absolute detection limits of these compounds are virtually the same. This result suggests that the mercury compounds were decomposed almost completely in the plasma. Dynamic ranges of each compound were linear over 4.5 to
0
2
4
6
6
1
0
Time (min)
Flgure 2. Gas chromatograms detected by TCD (upper) and MIP (lower):(A) benzene, (B) CH,HgCI (1 yg of Hg/L), (C) C,H,HgCI (1 yg
of Hg/L).
5 orders of magnitude and are almost the same as that obtained for inorganic mercury compounds (15). P r e c o n c e n t r a t i o n T r e a t m e n t . The present GC/MIP system was applied to the determination of alkylmercury compounds in seawater with the preconcentration-extraction technique. Before the application, the extraction efficiency of each extraction step (shown in Figure 1) and the reproducibility of the entire procedure were investigated by use of methylmercury chloride. Generally, the contents of alkylmercury compounds in seawater are extremely low, and only the analytical values for methylmercury have been reported so far (10,11,21). Therefore, only the preconcentration of methylmercury chloride was investigated in the following experiment. The extraction efficiency of the first step (extraction of methylmercury chloride from sample solution into benzene) was 49%. This procedure was repeated three times, resulting in a total extraction efficiency of 87%. The reverse extraction efficiency of the second step (extraction from benzene to 1% L-cysteine aqueous solution) was 100%. The extraction efficiency of the final step (extraction from aqueous phase to benzene) was 48%. As the result, the total extraction efficiency of the complete procedure was 42%. As can be seen from Figure 1, the sample volume was finally reduced by 1/500. The detection limit with the above extraction treatment was 0.4 ng/L in the case of 4 FL sampling for GC/MIP. The relative standard deviation of the present procedure was 6 % , which was obtained with a 20 ng/L sample solution and 10 measurements. These analytical characteristics in determining methylmercury are summarized in Table 111, along with the data for the direct determination (see Table 11). The effects of interferences of some substances in the present extraction procedure were investigated. The results are summarized in Table IV. NaCl was examined since it is the major salt constituent in seawater. L-Cysteine and EDTA are well-known to produce strong complexes with mercury. As can be seen from Table IV, they do not detract from the extraction. The interference of inorganic mercury compounds was not investigated because it has been already reported that inorganic mercury is virtually unextracted into organic solvents (11, 12).
Table 111. Analytical Characteristics of the Solvent Extraction Technique for Methylmercury Determination sample volume injected into detection extraction re1 std sample treatment GC, uL limit, ng/L efficiency, % dev, 70 without extraction with extraction
4
4
2 .o
90
0.4
42
6.0
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
be detected in seawater. The analytical results are presented in Table V, where inorganic mercury compounds were also measured by cold-vapor atomic absorption spectrometry (22, 23). Since this seawater sample was collected off-shore near a big city, the concentrations of inorganic mercury compounds were rather high (23). The concentration of methylmercury chloride was about 2 ng/L, which was about 2.3% of total mercury compounds.
Table IV. Interference Effects on the Recovery in Methylmercury Determination
a
amt of interferant
% recovery of
interferant Na C1 L-cysteine EDTA
5% 10-3 M 10-3 M
99.8 99.2 103
CH,HgCl (1.0
CH3HgCl
of Hg/L).
kg
-
Table V. Analytical Results of Mercury in Seawater amt of
amt of CH3HgC1, ng/L
av
inorganic
recovery, %
1.58 1.63 1.95 2.45 2.52 2.03 i 0.44.
mercury, ng/L
89.5
85
4
I
1'
4-
a,
I Y Q
TCD
a,
n
B
+
0
ACKNOWLEDGMENT The authors thank N. Fujioka and J. Takahashi for their experimental help and valuable discussion in the determination of inorganic mercury in seawater. Registry No. CH3HgC1, 115-09-3; C2H6HgC1,107-27-7; (CH&Hg, 593-74-8; water, 7732-18-5; cysteine, 52-90-4; benzene, 71-43-2. LITERATURE CITED
I_-
c .-w
453
A
I
2
4
MIP 6
8
Time (min) Figure 3. Gas chromatogram of methylmercury chloride extracted from seawater: (A) benzene, (8)CH,HgCI.
Determination of M e t h y l m e r c u r y i n Seawater. The seawater sample was collected in Aburatsubo Bay near Tokyo, Japan, and preconcent,rated as shown in Figure 1. The gas chromatogram of the seawater sample (500-fold preconcentrated with the extraction technique) is shown in Figure 3. As may be seen from Figure 3, methylmercury chloride could
(1) (2) (3) (4) (5)
WestoB, G. Acta Chem. Scand. 1966, 20, 2131-2137. Westoo, G. Acta Chem. Scand. 1967, 21, 1790-1800. Westoo, G. Acta Chern. Scand. 1968, 22, 2277-2280. Bache, C. A.; Lisk, D. J. Anal. Chem. 1971, 43,950-952. Jones, P.; Nickless, G. Analyst (London) 1978, 103, 1121-1126. (6) Coliett, D. L.; Fleming, D. E.; Taylor, G. A. Analyst (London) 1980, 105, 897-901. (7) Vostal, J. "Mercury in the Environment"; CRC Press: Cleveland, OH, 1972. (8)Jensen, S.; Jernelov, A. Nature (London) 1969, 223, 173-174. (9) Wood, J. M.; Rosen, C. G.; Kennedy, S. F. Nature (London) 1968, 220, 173-174. (10) Fltzgerald, W. F.; Lyons, W. 9. Nature (London) 1973, 242,452-453. (11) Talmi, Y.; Norwell, V. E. Anal. Chim. Acta 1976, 85, 203-209. (12) Grossman, W. E. L.; Eng, J.; Tong, Y. C. Anal. Chim. Acta 1972, 6 0 , 447-449. (13) Talmi, Y. Anal. Chim. Acta 1975, 7 4 , 107-117. (14) Beenakker, C. I. M. Spectrochim. Acta, Part8 1976, 318,483-486. (15) Tanabe, K.; Chlba, K.: Haraauchl. H.; Fuwa, K. Anal. Chem. 1981. 53, 1450-1453. (16) Tanabe, K.; Haraguchl, H.; Fuwa, K. Spectrochim, Acta, Parts 1981, 368,633-639. (17) Chiba, K.: YoshMa, K.; Tanabe, K.; Ozaki, M.; Haraguchi, H.; Winefordner, J. D.; Fuwa, K. Anal. Chem. 1982, 5 4 , 761-764. (18) Tanabe, K.; Matsumoto, K.; Haraguchi, H.; Fuwa, K. Anal. Chem. 1980, 52, 2361-2365. (19) Tanabe, K.; Haraguchi, H.; Fuwa. K. Spectrochim. Acta, Part 6 1981, 368, 119-127. (20) Tanabe, K.: Haraauchi, H.; Fuwa. K. SDectrochim. Acta, Part 8.in press. (21) Fujita, M.; Iwashima, K.; Fukuoka, I.; Takabatake, E.; Yamagata, N. Suishitsu Odaku Kenkyu 1978 1 , 133-139. (22) Haraguchi, H.; Takahashi, J.; Tanabe, K.; Fuwa, K. Spectrochim. Acta, Part 8 1961, 366, 719-728. (23) Takahashi, J.; Haraguchi, H.; Fuwa, K. Chem. Lett. 1981, 7-10.
RECEIVED for review May 24,1982. Accepted November 22, 1982. The present research has been supported by the Grant-in-Aid for Environmental Science (No. 56030019) from the Ministry of Education, Science and Culture, Japan (1981).
