Anal. Chem. 2001, 73, 3181-3186
Identification of Volatile Selenium Compounds Produced in the Hydride Generation System from Organoselenium Compounds Amit Chatterjee,* Yasuyuki Shibata, Minoru Yoneda, Rupendranath Banerjee,† Masao Uchida, Hiroyuki Kon, and Masatoshi Morita
Environmental Chemistry Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki-3050053, Japan, and Department of Inorganic Chemistry, Jadavpur University, Calcutta 700032, West Bengal, India.
We report a novel aqueous derivatization of selenomethionine (Semet), selenoethionine (Seet) and trimethylselenonium ion (TmSe) by NaBH4 and HCl to volatile selenium species, namely, diethyldiselenide (DeDSe), dimethyldiselenide (DMDSe), dimethylselenide (DmSe) and ethylhydrogenselenide (ESeH), in the hydride generation (HG) system. The volatile selenium compounds produced in the HG system were on-line trapped and concentrated in a U-tube that was immersed in the liquid nitrogen trap. The trapped volatile Se compounds were volatilized at 80 °C in a water bath, and 50-500 µL of volatile gas was injected into the GC/AED and GC/MS, respectively. It has been established that DmSe, DmDSe, and DeDSe are the predominant Se compounds that are produced in the HG system from TmSe, Semet, and Seet, respectively, followed by ESeH from Seet. Analytical methods previously employed have stated that these compounds are inactive in the HG system. Prior decomposition of Semet, Seet, and TmSe to selenous acid is essential before HG. To the best of our knowledge, current findings for the production and identification of volatile selenium compounds in the HG system are new and different from existing reports; hence, direct estimation of Semet, Seet, and TmSe is possible when coupling with a HG system using a suitable Se-specific detector. Selenium is one of the minor but biologically essential elements. It is widely distributed in the environment, especially in metal sulfide deposits. Organoselenium compounds are used as herbicides, fungicides, and bactericides in agriculture. Selenium having different oxidation states is present in the aquatic systems: selenide (both in organic and inorganic compounds), selenite, and selenate1. Selenium compounds are toxic, but organoselenium compounds have a different toxicity from that of inorganic ones. Dimethylselenide is considered to be 500 times less toxic than selenite2. Methylation is an effective detoxification * To whom correspondence should be addressed. Present address: Department of Chemistry and Biochemistry, 335 Mellon Hall, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282. Phone: 412-396-4106. Fax: 412-3965359. † Jadavpur University. (1) Gomez-Ariza, J. L.; Pozas, J. A.; Giraldez, I.; Moreles, E. J. Chromatogr. A, 1998, 823, 259-277. (2) Ganther, H. E.; Levander, O. A.; Baumann, C. A. J. Nutr. 1966, 88, 55-60. 10.1021/ac001356w CCC: $20.00 Published on Web 05/18/2001
© 2001 American Chemical Society
mechanism. There is evidence for the production of volatile organic selenium species, mainly dimethylselenide (DmSe) and dimethyldiselenide (DmDSe), from inorganic selenide salts as well as from selenocystine (Secys) and selenomethionine (Semet) by fungi, plants, and animals in the environment, and it has been well-documented in the literature.3-8 DmSe9 has been found in human breath at levels from 0.08 to 0.98 µg m-3. In recent years, bioanalytical interest of selenium has grown dramatically.10 Miscellaneous inorganic and organic selenium compounds were identified in biological samples over the past decades.3-8,10-19 Among these compounds are selenous acid, selenic acid, dimethylselenide, dimethyldiselenide, dimethyldiselenide sulfide, trimethylselenonium salts, selenoamino acids, selenium-containing carbohydrates, proteins, and nucleoside. Se compounds are widely used for dietary supplements in animals and humans.11 The large difference in its behavior depends on its chemical form and amount; thus, it is essential to develop fast and selective methods for the determination of selenium compounds. A variety of analytical methods are applied for Se estimation. Fluorimetry, hydride generation atomic absorption spectrometry (HGAAS) and HG inductively coupled plasma atomic emission spectrometry (ICPAES) are usually employed for the routine analysis of Se. Speciaton of Se has been reviewed by several (3) Challenger, F. Adv. Enzymol. 1951, 12, 429-491. (4) Baker, L.; Fleming, R. W. Bull. Environ. Contam. Toxicol. 1974, 12, 308311. (5) Lewis, B. G.; Johnson, C. M.; Delwiche, C. C. J. Agric. Food Chem. 1966, 14, 638-640. (6) Valsakova, V.; Benes, J.; Parizek, J. Radiochem. Radioanal. Lett. 1972, 10, 251-258. (7) Chau, Y. K.; Wong, P. T. S.; Silverberg, G. A.; Luxon, P. L.; Bengert, G. A. Science, 1976, 192, 1130-1131. (8) Jiang, S.; Robberecht, H.; Adams, F. Atmos. Environ. 1983, 17, 111-114. (9) Feldmann, J.; Riechmann, T.; Hirner, A. V. Fresenius J. Anal. Chem. 1996, 354, 620-623. (10) Gonzalez-LaFuente, J. M.; Dlaska, M.; Fernandez-Sanchez, M. L.; SanzMedel, A. J. Anal. At. Spectrom. 1998, 13, 423-429. (11) Pyrzynska, K. Analyst, 1996, 121, 77R-83R and references therein. (12) Jiang, S.; Chakraborty, D.; Adams, F. Anal. Chim. Acta, 1987, 196, 271275. (13) Elaseer, A.; Nickless, G. J. Chromatogr. A, 1994, 664, 77-87. (14) Olivas, R. M.; Donard, O. F. X.; Camara, C. Anal. Chim. Acta, 1994, 286, 357-370. (15) Challenger, F. Chem. Rev. 1945, 36, 315-361. (16) Cutter, G. A. Anal. Chim. Acta, 1978, 98, 59-66. (17) Dransfield, P. B.; Challenger, F. J. Chem. Soc. 1955, 1153-1160. (18) Reamer, D. C.; Zooler, W. H. Science, 1980, 208, 500-502. (19) Shibata, Y.; Morita, M.; Fuwa, K. Adv. Biophys. 1992, 28, 31-80.
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authors11,14 and performed using capillary electrophoresis20 and HPLC.21 Volatile organic selenides with Se (II) (R2Se, R2Se2, where RdCH3, C2H5) have been analyzed by gas chromatography (GC)., especially those in air samples.13 To increase the detection capabilities of these compounds, a cold trapping device is widely used.13,14 The most common one is the cold trap that is based on the liquid nitrogen cooling.22 A number of coupled GC/AAS systems with a previous purge and trap isolation technique have also been proposed for DMSe, diethylselenide (DESe), and DMDSe8,12 determination. However, most of the published methods for the determination of selenium using HGAAS demand previous conversion of selenium compounds to selenous acid that is so far very common, because only selenous acid [Se(IV)] is recorded to produce the volatile H2Se with NaBH4.23-28 Organoselenium compounds are inert and unable to form volatile Se compounds with NaBH411,23-29 so for selenium compounds except selenous acid, pretreatment is normally required. Thus, for the selenium speciation analysis, chemical decomposition steps are mandatory to convert Se compounds into selenic acid and for the succeeding reduction of selenic acid to selenous acid before HG.26-30 However, in our previous publications, we observed that the organic selenium compounds (selenocystine, selenohomocystine, selenomethionine, selenoethionine, trimethylselenonium iodide, selenocystathionine, selenocystamine, selenourea, selenocholine, and dimethyl(3-amino-3-carboxy-1-propyl)selenonium iodide) are HG active21,31,32. We have optimized the HG system and determined the selenium compounds by FI-HGAAS/HPLCHG-nitrogen-microwave induced plasma mass spectrometry (N2-MIPMS)21,31,32. But the volatile Se compounds, that originate in the HG system when the organoselenium compounds are reacted with NaBH4 and HCl have yet to be identified. The aim of this paper is to identify and to confirm the structure of volatile Se derivatives (compounds), formed in the optimized HG system from Semet, Seet, and TmSe when these compounds are reacted with the aqueous solution of NaBH4 and HCl, respectively, using GC/AED and GC/MS. EXPERIMENTAL SECTION A Hewlett-Packard (HP) model 5890 series II gas chromatograph (N2/CO2 built-in cooling system) was interfaced to a JEOL model JMS-700 mass detector. The GC/MS controlled by a 9836C HP data system was used for the identification of volatile Se compounds. The chromatograms and mass spectra thus obtained were printed as hard copies by a HP 7470A plotter and a HP (20) Gilon, N.; Potin-Gautier, M. J. Chromatogr. A, 1996, 732, 369-376. (21) Chatterjee, A.; Shibata, Y.; Morita, M. J. Anal. At. Spectrom. 2000, 15, 913919. (22) Pinillos, S. C.; Asensio, J. S.; Bernal, J. G. Anal. Chim. Acta, 1995, 300, 321-327. (23) Gonzalez-LaFuente, J. M.; Fernandez-Sanchez, M. L.; Sanz-Medel, A. J. Anal. At. Spectrom. 1996, 11, 1163-1169. (24) Henn, E. L. Anal. Chem. 1975, 47, 428-432. (25) Nygaard, D. D.; Lowry, J. M. Anal. Chem. 1982, 54, 803-807. (26) Itoh, K.; Chikuma, M.; Nishimura, M.; Tanaka, T.; Tanaka, M.; Nakayama, M.; Tanaka, H. Fresenius J. Anal. Chem. 1989, 333, 102-107. (27) Goulden, P. D.; Brooksbank, K. Anal. Chem. 1974, 46, 1431-1436. (28) Blocky, A.; Ebrahim, A.; Rack, E. P. Anal. Chem. 1988, 60, 2734-2737. (29) Gayon, J. M. M.; Gonzalez, J. M.; Fernandez, M. L.; Blanco, E.; Sanz-Medel, A. Fresenius J. Anal. Chem. 1996, 355, 615-622. (30) Pitts, L.; Worsfold, P.; Hill, S. J. Analyst, 1994, 119, 2785-2788. (31) Chatterjee, A.; Irgolic, K. J. Anal. Commun. 1998, 35, 337-340. (32) Chatterjee, A.; Shibata, Y. Anal. Chim. Acta, 1999, 398, 273-278.
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Table 1. Operating Conditions for GC/MS and GC/AED Gas Chromatography (HP built-in CO2/N2 cooling system) GC HP 5890 II column HP-1MS capillary column (100% dimethylposiloxane) split ratio 30:1 column size 30 m × 0.25 mm × 0.25 µm carrier gas; He; 1.0 mL min-1 inject temp 280 °C column temp -30 °C, 1 min isothermal; then 5 °C min-1 to 280 °C, 20 min isothermal separator temp 280 °C Mass Spectrum model ionization current ionization energy accelerating voltage ion mult magnetic field ion source ion source vacuum anal. tube vac chamber temp
JEOL JMS-700 300 µA 70 eV 8.0 KV 1.0 KV HS electron impact 5 × 10-6 Torr 1 × 10-6 Torr 200 °C
Gas Chromatography/Atomic Emission Detector model, GC 5890 II model/AED HP 5921 column HP-1 capillary column (100% dimethylposiloxane) column size 30 m × 0.25 mm × 0.25 µm inject vol, mode 0.05-0.5 mL, splitless inject temp 300 °C column temp 30 °C, 1 min isothermal; then 5 °C min-1 to 300 °C, 20 min isothermal oven temp (max) 325 °C element/wavelength Se/196.09 nm; C/193.0905 nm transfer line temp 300 °C cavity temp 300 °C He pressure 5.0 kgf cm-2 He supplied pressure 30 psi H2 pressure 4.2 kgf cm-2 cavity pressure 1.5 psi O2 pressure 5.2 kgf cm-2 N2 flow 2.0 L min-2 makeup gas flow 260 mL min-1 window purge 30 mL min-1 (cavity vent) reagent gas H2 reproducibility 5.2%
printer. Selenium compounds were separated on a capillary column, 30 m × 0.25 mm i.d. × 0.25 µm (HP-1MS, 100% dimethylpolysiloxane). A Hewlett-Packard (HP) model 5890 series II gas chromatograph was interfaced to a HP 5921 atomic emission detector. Control and operation of the system was achieved using a HP 35920 Pascal Chemstation with GC/AED software. Instrumental operating parameters are summarized in Table 1. For the GC/AED, the capillary chromatographic column (30 m × 0.25 mm i.d. × 0.25 µm) having specification identical to that used in GC/MS was used. Sample aliquots of 50-500 µL were manually injected into the capillary column. Helium was used as the carrier gas at a flow rate of 1.0 mL min-1. The details of the operating conditions for GC/AED and GC/MS are given in Table 1. Chemicals and Reagents. All reagents were of analytical grade and were used without further purification. Selenium
Table 2. Operating Conditions for FI-HG sodium borohydride, mL min-1 carrier flow (3 M HCl), mL min-1 sample loop fill time inject time He gas flow, mL min-1
Figure 1. Schematic diagram for the FI-HG with off-line GC/MS/ GC/AES.
compounds seleno-DL-ethionine (no. S-3750) and seleno-DL-methionine (no. S-3875) were purchased from the Sigma. Trimethylselenonium iodide was purchased from TRI Chemical Laboratory, Japan. Stock solutions were prepared with deionized Milli-Q water (18.0 MΩcm) from trimethylselenonium iodide (63.6 mg to 20 mL, 1000 µg Se mL-1), from selenoethionine (26.6 mg to 20 mL, 500 µg Se mL-1), from selenomethionine (124 mg to 100 mL, 500 µg Se mL-1). The stock solutions were stored in a refrigerator at -20 °C before use. Solutions of the selenium compounds with concentrations in the range of 2.00-50.0 ng Se mL-1 were prepared by an appropriate dilution of the stock solutions with Milli-Q water. The purity of the Semet, Seet, and TmSe were checked by a HPLC-ICPMS/ HPLC-HGAAS system using GS-220 gel-permeating, LC-SCX, and PRP X-100 columns21,31-33 in which a single peak for each Se standard was obtained. No peak for selenous acid was detected from any of the above Se standards used. Sodium tetrahydroborate (NaBH4) solutions (0.3 and 1.0% m/v) were prepared by dissolving NaBH4 powder (Merck, 1063710) of 96% purity in deionized Milli-Q-water and stabilizing the solutions with sodium hydroxide (0.1 and 0.2% m/v; Merck, 71690). The solutions were filtered before use to eliminate turbidity and then were stored in a refrigerator. Hydrochloric acid (3.0 M) was prepared by dilution of concentrated HCl (Merck P. A., No. 100319, further purified in a quartz sub-boiling distillation unit). System Description and Procedure. Figure 1 shows a schematic representation of HG with the trapping system used. The selenium compounds were reacted selectively with alkaline sodium tetrahydroborate (Semet and Seet with 0.3% and TmSe with 1.0% NaBH4) in a Perkin-Elmer FIAS-100 system equipped with a gas-liquid separator. The gaseous products were separated from the liquid in the gas-liquid separator, flashed into the U-tubes, and immersed in the dry ice-methanol bath and liquid nitrogen bath successively, by a stream of carrier gas (He). The operating conditions for the HG system are summarized in Table 2. After volatile hydride formation for 3 min, the dry ice-methanol bath was removed from the first U-tube. It was kept for 3 min at ambient temperature and then it was placed in the ∼80 °C water (33) Chatterjee, A.; Tao, H.; Shibata, Y.; Morita, M. J. Anal. At. Spectrom. Communicated, 2000.
4.5 5.6 500 µL 11 s 15 s 118 ( 15
bath so that any deposited volatile selenium compounds were transferred to the second U-tube where it was re-deposited in the liquid N2 bath. About 97% of the water vapor was removed from the gaseous products in the first U-tube. The remaining 3% of the water, which condensed in the second U-tube, could easily be removed by heating that U-tube for 20 s between determinations with a heat gun . The second U-tube was closed at both ends by stoppers containing a rubber septum, and removed from the liquid nitrogen bath. It was placed at ambient temperature for 3 min, then it was introduced to the hot water bath (80 °C) for one min. About 50-500 µL of the volatile Se hydrides was taken from the second U-tube by a gastight syringe through the septum, and was injected into the GC/MS and GC/AES, respectively. RESULTS AND DISCUSSION Optimization of Instrumental and Noninstrumental Parameters. Carrier Gas. To obtain the maximum sensitivity and good reproducibility for the determination of the investigated Se compounds, the influence of several instrumental and noninstrumental parameters on the analytical performance of the HG system was evaluated and optimized in detail, which were published previously21,31,32. We used the optimized HG system for the efficient production of volatile Se compounds. Volatile Se compounds thus produced were trapped in the U-tube by using cooling devices. Several cooling mixtures were used to trap the produced volatile Se compounds. Liquid nitrogen trapping is the most appropriate one. Three carrier gases (He, Ar, and N2) were used for the experiments; however, He was found to be the most suitable one, because it was not liquefied in the U-Tube at the liquid nitrogen temperature. Hence, we used He as a carrier gas for the experiment. Water Tap. It has already been established that water vapor produced from the HG system clogged the U-tube and may even decompose volatile Se compounds produced from the HG system. Calcium chloride is efficient as a desiccant,16 but it has to be replaced many times and also traps a portion of the produced volatile hydride.22 A U-tube immersed in the dry ice-methanol bath solves this retention problem. The dry ice trap could be used all day without regenerating or repacking. A liquid nitrogen trap shows that silanized glass wool is the best general packing material, and results remain quantitative and reproducible. A blank was run during measurement after each Se standard. We flushed the U-tubes with He every time for 3 min at room temperature between running of the Se standards so that no memory effect was observed. Hence, we used the liquid nitrogen trap for analysis. Separation and Identification of Organoselenium Compounds. The volatile Se gasses were trapped from Seet, Semet, and TmSe after HG. Portions (50-500 µL) of Se gass samples were injected directly into the GC/MS and GC/AED by a gastight Hamilton 1750 pressure-lock gas syringe. Se-containing fragments Analytical Chemistry, Vol. 73, No. 13, July 1, 2001
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Figure 2. GC/MS of volatile Se compounds produced in the HG system from Seet (0.3% NaBH4 and 3 M HCl were used for the HG; conditions are in Table 1).
are often easily recognized in mass spectra from the very characteristic groups of peaks resulting from the typical distribution of the six natural selenium isotopes. However, peaks resulting from the most abundant isotope, 80Se, are in general chosen to represent the Se-containing fragments. In Figure 2, a chromatogram and a mass spectrum are shown, corresponding to Se compounds produced from Seet using the HG system. MS clearly indicates the presence of DeDSe (m/z 218) at 24.63 min. The cluster of peaks from m/z 212-220 matches a molecule34 with empirical formula (C2H5)2Se2. This gives a clear picture of the prominent 80Se isotope that is implied by the presence of the appropriate metastable peaks as a result of the elimination of ethyl radical from the molecular ion as a dominant process (Figure 3, reaction A). This is the base peak. Fragmentation occurred, and we got two fragmentation products at m/z 190 and 160 corresponding to [Et-Se-Se-H]+ and [Se-Se]+ (Figure 3, reaction A). These mass spectra are similar to one obtained previously by using pure standard DeDSe1. We also got a peak at the retention time (RT) 6.25 min when m/z 110 was monitored on the MS (Figure 2c,d). The peak at the m/z 110 is the base peak of [Et-Se-H]+; the fragment peak at m/z 80 (Figure 3, reaction B) is due to [Se].+ (34) Heller, S. R.; Milne, G. W. A. EPA/NIH Mass Spectral Data Base; U.S. Department of Commerce: Washington, D.C., 1975; Vol. 2.
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Figure 3. Mass fragmentation patterns (reactions A-D) and probable chemical decomposition mechanism (parts 1-3) of Semet, Seet, and TmSe.
This peak agrees well with the corresponding peak in the GC/ AED (Figure 4a) chromatogram where two peaks are present, indicating two volatile Se compounds. Those are produced from Seet when it is reacted with sodium borohydride and HCl in the HG system. The probable decomposition pattern of Seet to DeDSe is given in Figure 3 (part 1). Although characteristic fragments at m/z 110 may also indicate DmSe, the sister peak at m/z 95 is not found in the fragmentation pattern13 of the mass spectrum (Figure 2d). Hence, the peak originating at m/z 110 is due to C2H5SeH. In Figure 4b, a chromatogram with GC/AED is shown corresponding to Se compounds produced from TmSe using the HG system. The GC/AED chromatogram confirmed Se compounds peaks at RT 6.695 and 12.112 min, so TmSe is borohydrideactive and it produced volatile Se compounds. To confirm the identity of volatile Se compounds produced in the HG system from TmSe, we injected these gaseous compounds into the GC/MS, as described previously. The chromatogram and the MS of volatile Se compounds are shown in Figure 5a,b. Mass fragment patterns are shown in Figure 3, reaction C. The base peak is obtained at m/z 110, thus indicating DmSe [CH3-Se-CH3]+. The peak produced at m/z 95 is due to the fragmentation of DmSe to [CH3-Se]+, and m/z 80 is due to [Se]+. This mass fragmentation agreed well with
Figure 5. GC/MS of volatile Se compound produced in the HG system from TmSe, (1.0% NaBH4 and 3 M HCl were used for the HG; conditions are in Table 1).
Figure 4. GC/AED chromatograms of the organoselenium compounds, produced from (a) Seet (9b) TmSe, and (c) Semet in the HG system (conditions are in Table 1).
previously published mass spectra of pure standard DmSe.13 Therefore, TmSe produces DmSe in HG system. The probable decomposition pattern of TmSe to DmSe with NaBH4 and HCl is shown in Figure 3, part 2. In Figure 6a,b, a chromatogram and a mass spectrum are shown corresponding to the Se compounds produced from Semet using the HG system. The compound identified is dimethyldiselenide (CH3)2Se2, which indicates the presence of the prominent 80Se isotope. The metastable peaks seen at m/z 175 and m/z 160 are for the elimination of a methyl radical from the molecular ion by the dominant process (Figure 3, reaction D). This mass spectrum agrees well with the previously reported mass fragmentation of DmDSe1. The base peak is found at m/z 190 (18.47 min), thus confirming DmDSe. Furthermore, in GC/AED, a peak (Figure 4c) at RT 12.122 min is identified. Therefore, Semet produces DmDSe in the HG system. However, in the GC/AED (Figure 4b) chromatogram, we detected another peak having the same RT as that of DmDSe (Figure 4a,b). The second peak is probably DmDSe. Because of a very low intensity of DmDSe, as compared to DmSe, we were unable to detect and identify the compound with our present GC/ MS; hence, some additional work is required to confirm this peak. Nevertheless, appreciable retention time differences between the GC/AED and GC/MS spectra of volatile Se compounds (Figures
2, 4-6) are observed. In fact, in GC/AED, the volatile Se compounds are eluted earlier than in the GC/MS method. This may be because of the dissimilar column temperatures that were applied for the GC/AED and the GC/MS (Table 1) or because two different analytical columns with same specifications were used. However, the reproducibility for GC runs between -30 °C and room temperature are reliable and within 5.2%. It is well-documented that Seet, Semet, and TmSe are inactive in the HG system.11,35 Most of the published HG methods are based on the decomposition of Semet, Seet, and TmSe to selenic acid before the HG10,35,36. But in our HG method, TmSe is decomposed with 1.0% borohydride and 3 M HCl and forms DmSe as the major volatile Se compound (Figures 4b, 5a,b). Amino acids such as Semet and Seet are decomposed directly by NaBH4 and HCl in the HG system, and DmDSe, DeDSe, and ethylhydrogenselenide are formed (Figures 2, 4, 6). This anomaly may be due to a dissimilar HG system and the different analytical conditions used for the HG of Semet, Seet, and TmSe. Methylation of selenium has been known for a long time.15 Volatile alkylselenium compounds are produced from inorganic selenium salts through a biomethylation process by various organisms.37 A variety of organisms, such as fungi, molds, and (35) Martinez, L.; Baucells, M.; Pelfort, E.; Roura, M. Fresenius J. Anal. Chem. 1996, 354, 126-127. (36) He, Y.; Moreda-Pineiro, J.; Luisa-Cervera, M.; Guardia, M. D. L. J. Anal. At. Spectrom. 1998, 13, 289-293. (37) Dauchy, X.; Potin-Gautier, M.; Astruc, A.; Astruc, M. Fresenius J. Anal. Chem. 1994, 348, 792-805.
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elenide, and ethylhydrogenselenide). It was stated that the Se-C bond in the organoselenium compounds is very rigid11,41 (As-C bond energy is 153 kcal/mol, higher than the Se-C bond energy 141.1 kcal/mol42,43), but the origination of volatile selenium species from organoselenium compounds with NaBH4 and HCl has clearly confirmed the instability of the Se-C bond. Probably, NaBH4 and HCl supplied sufficient agitation (dissociation energies) to the investigated Se compounds so that the Se-C bonds of organoselenium compounds were decomposed and produced the volatile Se compounds. However, the As-C bond in organoarsenic compounds (arsenocholine, arsenobetaine, and tetramethylarsonium ion) is more rigid than the Se-C bond and is unable to generate the borohydride-active volatile As compounds in the HG system. Nevertheless, the reaction of NaBH4 and HCl with each Se compound is specific. Hence, the conditions should be properly controlled to generate the volatile Se compounds from the investigated organoselenium compounds. Our proposed chemical decomposition mechanism for Semet, Seet, and TmSe are given in Figure 3, parts 1-3. It is already reported that biomethylation of Se to (CH3)2Se2 produces an intermediate compound, CH3SeH18,44,45. In our proposed chemical decomposition mechanism, Semet and Seet also produce the intermediate compounds CH3SeH and C2H5SeH. These compounds are rapidly combined and produce DmDSe and DeDSe (Figure 3, parts 1, 3).
Figure 6. GC/MS of volatile Se compound produced in the HG system from Semet (0.3% NaBH4 and 3 M HCl were used for the HG; conditions are in Table 1).
bacteria, are capable of producing volatile selenium compounds (DmSe, DmDSe, and (CH3)2SeO2) from inorganic or organic selenium compounds17,38,39. Until now, available literature has stated that the IV+ state for selenium is HG active,40 but current observation has implied that in addition to the IV+ oxidation state, the II- (TmSe, Semet, and Seet) oxidation state of selenium is also HG active and produces borohydride-active volatile Se compounds (dimethylselenide, dimethyldiselenide, diethyldis(38) Doran, J. A.; Alexander, M. Soil Sci. Soc. Am. J., 1977, 41, 70-73. (39) Chasteen, T. G.; Silver, G. M.; Birks, J. W.; Fall, R. Chromatographia, 1990, 30, 181-185. (40) Jackson, K. W.; Lu, S. Anal. Chem. 1998, 70, 363R-83R and references therein. (41) Cooke, T. D.; Bruland, K. W. Environ. Sci. Technol. 1987, 21, 1214-1219. (42) Smoes, S.; Drowart, J. J. Chem. Soc., Faraday Trans. 1977, 2, 73, 17461749. (43) Handbook of Chemistry and Physics, 1st student ed.; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1987, F121-F130. (44) Kolbl, G.; Kalcher, K.; Irgolic, K. J.; Magee, R. J. Appl. Organomet. Chem. 1993, 7, 443-466. (45) Oremland, R. S. In Selenium in the Environment; Frankenberger, W. T., Jr., Benson, S., Eds.; Marcel Dekker: New York, 1994, pp 389-419.
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CONCLUSIONS It has been established that TmSe, Seet, and Semet are NaBH4active and produce volatile Se compounds. DmSe, DmDSe, and DeDSe are the predominant Se compounds produced in HG system from TmSe, Semet, and Seet, respectively, followed by ESeH from Seet. To the best of our knowledge, current findings for the generation and identification of volatile selenium compounds are new and different from all other reported information. The accurate determination of different organoselenium species is still the major challenge for the analyst. This HG method is promising, can be coupled with selenium-specific detectors after HPLC separation for the determination of selenium compounds in environmental samples, and was successfully coupled with HPLC/MIPMS for the on-line separation and determination of Se compounds in human urine.21 ACKNOWLEDGMENT The authors gratefully acknowledge Dr. Sukti Hazra for helpful comments during the preparation of the manuscript, STA and JISTEC, Japan for financial support, and the Environmental Division for GC-MS measurements. Received for review November 20, 2000. Accepted March 7, 2001. AC001356W
