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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
CORRESPONDENCE Speciation of Organometallic Compounds by Zeeman Atomic Absorption Spectrometry with Liquid Chromatography Sir: Over the past several years we have devised and expanded the capabilities of Zeeman atomic absorption spectrometry (ZAA) (1-6). Using this technique, trace elements in a complex matrix can be directly analyzed with high accuracy even when there is only one atom of interest contained in several million atoms of the host material. Quantities in the nanogram, or in some cases picogram, range can be determined within 15 s for more than 30 elements. Because of its high selectivity and high sensitivity, ZAA can be used as a new technique for organometallic species determination by interfacing with a high pressure liquid chromatograph (HPLC). The HPLC separates various molecular species. Different kinds of eluents can be directly introduced in the ZAA detection system such as organic solvents or even high concentration salt solutions. Then organometallic species in the ppb range are separately detected according to their retention times. This technique has a much larger field of application than HPLC coupled with conventional AA (7-9). The advantages of the ZAA technique are described in recent publications ( 5 , I O ) . In the case of polarized Zeeman AA ( 4 ) , a steady magnetic field at 11 kgauss is applied to the sample vapor perpendicular to the incident light beam. The difference in absorption of the polarized constituents P , and PI,is proportional to the atomic density, but is not affected by the various kinds of spectral interferences caused by thermal decomposition of the eluents. The recently developed HPLC technique has many advantages over gas chromatography. Nonvolatile, polar, thermally unstable molecules or high molecular weight compounds can be separated ( I I). In the present system, the main requirement is that the sample of organometallic species be soluble in the mobile phase without alteration of its chemical form. A demonstration of the operation of this system is provided by the analysis of a mixture of vitamin BIZand C O ( N O ~ ) ~ . Vitamin BIZis a large molecule (molecular weight = 1340.9 in cyanocobalamine form) which has a Co in its functional center (12). Two samples were prepared. Sample 1 contained a mixture of Co in vitamin Biz, 0.813 pg/mL, (cyanocobalamine form, Glaxo Laboratories Ltd., Greenfield, England) and Co introduced as Co(NoJZ, 25.0 pg/mL. Sample 2 contained only vitamin BIZat the same concentration as that in Sample 1. In the present system a high pressure liquid chromatograph with a high pressure sampling valve (Hitachi Model 635 and 635-0575)was coupled with a polarized Zeeman AA spectrophotometer by an automatic injector (a modified Perkin-Elmer Model AS-l), and 0.5 mL of the sample was eluted with CH3COONH4(2 M) through an anion-exchange column (Hitachi Ion Exchange Resin No 2611) at a flow rate of 1.0 mL/min and pressure of 20 kg/cm2. The separation between vitamin BIZ and Co(NoJ2 was confirmed by using a UV absorption detector a t 254 nm and a coulometric detector (Hitachi Model 630-0130) (23) a t the same time (Figure 1). The upper and the middle traces in Figure 1 show the signals from the coulometric detector and the UV absorption detector, respectively. The first peak in the UV absorption signal shows the appearance of the vitamin R,, and the second peak in the coulometric signal shows that 0003-2700/78/0350-1700$01 .OO/O
Table I. Concentration of Co in Each Fraction Determined by ZAA, ppm collection periods 2- 5 5-8 8-11 min min min Sample 1 0.126 0.007 3.63 (vitamin B,, and Co(NO,),) Sample 2 0.120 0.007 0.127 (vitamin B,,only) of the Co(N0J2. The retention times for vitamin Blz and Co(N0JZwere 3 and 9 min, and the half widths were 35 and 50, respectively. After separation, 10 pL from the collected fractions of the eluent was intermittently introduced into the PZAA spectrophotometer by utilizing the autosampler to measure the concentration of Co with time at the wavelength at 240.7 nm. The lower trace in Figure 1 shows an example of the ZAA signal in the case where 10 pL from each 3 mL of the eluent was measured. (The first fraction was 2 mL.) For Sample 1 (vitamin B12+ Co(NOJ2) two separate peaks of Co concentration were observed a t the retention times of ~ . Sample 2 (vitamin B12 the vitamin B12and C O ( N O ~ ) For only), the large peak corresponding to C O ( N O ~disappeared. )~ Table I shows the concentration of Co in fractions with retention times between 2-5, 5-8, 8-11 rnin of both Sample 1 and Sample 2. About 0.12 ppm of Co was detected in the 2-5 min fraction of both Sample 1 and Sample 2. This result shows that about 90% of Co bound t o vitamin BIZwas recovered in this fraction. A comparatively high concentration of Co (3.63 ppm) was detected in the 8-11 min fraction of Sample 1. However, in the 8-11 rnin fraction of Sample 2, only 0.127 ppm of Co, which might be attributed t o contamination from the reagents and the flowing system of HPLC, was observed. From Table I we can safely conclude that the concentration of Co bound to the vitamin Blz molecule could be obtained without interference in the presence of a comparatively high concentration of coexisting inorganic Co. Also the concentration of the inorganic Co could be obtained separately from the Co in vitamin BIZ. In recent experiments 250-pL fractions have been used in this system for the speciation of various organometallic compounds. It was found that, if the resin and eluent are properly chosen, there is little dilution of the sample upon passage through the HPLC column. In this case, the sensitivity is determined by the ZAA. The detection limits of various elements and the linear range of the Zeeman AA spectrophotometer have already been reported ( 5 ) . Species determinations by the present technique have applications to the field of biochemistry because many enzymes and coenzymes have a metal atom in their functional center. This type of analysis is also important in the environmental field because the toxicity of a metal depends upon its chemical form. Further investigations of the present system including the development of a new type of furnace for speciation will be reported soon.
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
(3) H. Koizumi and K. Yasuda, Spectrochim. Acta, Part B , 31, 237 (1976). (4) H. Koizumi and K. Yasuda. Spectrochim. Acta, Parts. 31, 523 (1976). (5) H. Koizumi, K. Yasuda, and M. Katayama, Anal. Chem., 49, 1106 (1977). (6) H. Koizumi. Anal. Chem., 50, 1101 (1978). (7) F. E. Brickman, W. R. Blair, K. L. Jewett, and W. P. Iverson, 173rd National Meeting, American Chemical Society, New Orleans, La., 1977. (8) D. R. Jones and S. E. Manahan, Anal. Chern., 48, 502 (1976). (9) S.C. Van Loon, D. Radziuk, N. Kahn, J. Lichwa, F. J. Fernandez, and J. D. Kerber, At. Abs. News/., 16, 79 (1977). (10) S. D. Brown, Anal. Chem., 49, 1269A (1977). (1 1) P. R. Brown, "High Pressure Liquid Chromatography, Biochemical and Biomedical Applications", Academic Press,, New York and London, 1973. (12) Albert L. Latner, "Clinical Biochemistry", 7th ed., W. 8. Saunders Co., Philadelphia, Pa., 1975. (13) Y. Takata, J . Chfomatogr., 108, 255 (1975).
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Hideaki Koizumi* Naka Works, Hitachi Ltd. Katsuta, Ibaraki, 312, Japan Figure 1. Separation of vitamin B,, and Co(N0J2 for Sample 1 of Table I. Upper trace: coulometric detector; middle trace: UV absorption detector (254 nm); lower trace: ZAA spectrophotometer(240.7 nm)
ACKNOWLEDGMENT We are grateful to K. Tsukada and M. Iwabuchi of Naka Works of Hitachi Ltd. for their help in this experiment. LITERATURE CITED (1) T. Hadeishi and R. D. McLaughlin, Science, 174, 404 (1971). (2) T. Hadeishi, Appl. Phys. Lett., 21, 438 (1972).
Tetsuo Hadeishi Ralph McLaughlin Lawrence Berkeley Laboratory University of California Berkeley, California 94720
RECEIVED for review March 2, 1978. Accepted July 7 , 1978. Work supported by the U.S. Department of Energy.
Identification of Elemental Sulfur in Deep-sea Ferromanganese Nodules Sir: The fine structure in the X-ray emission spectra of sulfur-containing compounds has been used to distinguish the various oxidation states of sulfur. The high resolution sulfur LII,IIIemission spectra ( I ) as well as the sulfur KP spectra (2) have been reported recently and the results have been compared with the results obtained from photoelectron spectroscopic studies and interpreted on the basis of molecular orbital theory. Both the X-ray emission technique as well as the photoelectron spectroscopic method have the advantage of being nondestructive. If, however, sulfur is present in low concentrations in a complex matrix, both methods are relatively insensitive. For example, in an attempt to obtain the X-ray photoelectron spectrum of sulfur in a finely powdered homogeneous sample of deep-sea ferromanganese nodules which contained between 0.1 and 0.2% sulfur, the sulfur 2p,/2,3/2spectrum could not be detected above the background after scanning for 10 h in a McPherson ESCA 36 photoelectron spectrometer with A1 K a irradiation. Attempts t o use wavelength dispersive X-ray emission spectroscopy for the sulfur LnJn spectra in the 150-eV region were equally fruitless. Ferromanganese nodules are small concretions, 1-20 cm in diameter, that are found on the deep ocean floor. They are a potential source of copper, nickel, cobalt, and molybdenum and are considered to be an important mineral reserve. Each nodule is heterogeneous and consists mainly of amorphous hydrous oxides of iron and manganese in which a variety of microcrystalline structures are occluded. We have used proton-induced X-ray emission (PIXE) to determine the composition of the heterogeneous surface of several nodule fragments 0.25 to 0.50 cm in diameter. The surfaces of these nodule fragments had varying textures and a range of colors from dark grey to a very pale yellow. The nodule fragments were mounted on planchets in an evacuated sample chamber that was maintained a t a pressure of -lo+ Torr, and were 0003-2700/78/0350-1701$01 .OO/O
subjected to bombardment with a 1.0-MeV proton beam that was focused on a 5 mm2 area of the nodule surface. The energy spectrum of the emitted X-rays was obtained with the aid of an energy dispersive Si(Li) detector with a resolution of 150 eV a t 8.046 keV, together with a 1000-channel pulse height analyzer. A typical spectrum that was obtained in the low energy region, between 0 and 8 keV, for a nodule fragment is shown in Figure 1. An important feature of this spectrum is that the Bremsstrahlung has been eliminated almost completely by the use of a low energy (1.0 MeV) proton beam and a carbon foil beam diffuser. The ;sulfur line a t 2.307 keV and the silicon line a t 1.740 keV are of particular interest in this spectrum. The Si/S ratio calculated from the relative intensities of the Si and S lines is 20:l. This ratio varied between 1O:l and 30:l in finely powdered homogeneous samples of nodules and in the surfaces of the majority of the nodule fragments that were examined by PIXE. An unexpected result, shown in Figure 2, was obtained when the surfaces of the nodule fragments in which the pale yellow granules were embedded were bombarded with protons. The X-ray emission spectrum between 0 and 8 keV showed only the Si and S lines and the calculated ratio of Si/S was about 1:E; this is almost the reciprocal of the normal Si/S ratio that is found in manganese nodules. The lines in the X-ray emission spectrum (Figure 2) that correspond to any of the major cation components, such as Mn and Fe, were absent. The absence of any positively charged counterions is proof that the very large sulfur peak cannot originate from an anionic species of sulfur, such as sulfate or sulfide. The only reasonable conclusion is that the sulfur peak originated from elemental sulfur that was present in the nodule fragment. Confirmatory evidence for the presence of elemental sulfur in manganese nodules was obtained from the X-ray photoelectron spectrum of the same nodule fragment with the pale @ 1978 American Chemical Society
