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CONCLUSIONS High precision, accurate metal isotope ratios can be obtained using volatile metal chelates. Alkaline earth metals are best studied using electron ionization, since under chemical ionization polymeric ion species that are unsuitable for isotope ratio studies predominate in t h e spectrum. Chemical ionization mass spectrometry is most useful for transition metal studies; i t provides simpler spectra with intense pseudomolecular ions. The ability to measure metal isotopic abundances rapidly, accurately, and with high precision using a conventional mass spectrometer will provide biomedical scientists with a powerful tool for exploring trace metal metabolism.
LITERATURE CITED Prasad, A. S.; Oberleas, D. "Trace Elements in Human Health and Disease"; Academic Press: New York. 1976; Volume 2 , Chapters 26-46. Moore, L. J.; Rosman, K. J. R. Abstracts of the 1978 Conference of the Federation of Analytical Chemistry and Spectroscopy Societies, Boston, Mass., Oct. 30-Nov. 3, 1978. No. 219. Rabinowitz, M. B.; Wetherill, G. W.: Kopple, J. D. Science 1973, 782, 725-727. Schwartz, R.; Giesecke, C. C. Clin. Chim. Acta 1979, 97, 1-8. Donohue, D. L.; Carter, J. A,; Franklin, J. C. Anal. Lett 1977, 70, 371-379. Schulten, H. R . ; Lehmann, W. D. I n "Quantitative Mass Spectrometry in Life Sciences 11", de Leenheer, A. P.,Roncucci, R. R . Von Peteghem, C., Eds.; Elsevier Scientific Publishing Company: Amsterdam, 1978; pp 63-82. Tsuge. S.; Leary, J. J.; Isenhour, T. L. Anal. Chem. 1974, 46, 106-110. Frew, N. M.; Leary, J. J.; Isenhour, T. L. Anal. Chem. 1972, 44, 665-67 1,
Hui, K. S.; Davis, B. A,: Boulton, A. A . Neurochem Res. 1977, 2 , 495-506. Miller, D. D.: Van Campen. D. Fed. Am. SOC.f x p . Biol. Proc., 1978, 3 7 , 488. Hileman, F. D.; Athin, C. L.; Lee, G. R.; Smith, D. L.; Hughes, 6 .M. I n "Proceedings of the 26th Annual Conference of Mass Spectrometry and Allied Topics", 1978, 336-338. Veillon, C.; Wolf, W. R.; Guthrie, 6 . li. Anal. Chem. 1979, 51, 1022-1024. Uden, P. C.; Henderson, D. E. Analyst(London), 1977. 102, 889-916. Klein, P. D.; Haumann, J. R.; Hachey, D. L. Clln. Chem. 1975, 21, 1253-1 257. Martell. A. E.; Belford, R . L.; Calvin, M. J . Inorg. Nucl. Chem. 1958, 5 , 170-181. Brauman, J. I.Anal. Chem. 1966, 38, 607-610. Beynon, J. H.; Williams, A. E. "Mass and Abundance lables for Use in Mass Spectrometry"; Eisevier Publishing Company: Amsterdam, 1963. Belcher, R.; Cranley, C. R.; Majer, J. R.; Stephen, W. I.; Uden, P. D. Anal. Chim. Acta 1972, 6 0 , 109-116. Joshi. K. C.; Pothak, V. N. Coord. Chem. Rev. 1977, 2 2 , 37-122. Prescott, S. R.; Campana, J. E.; Jurs, P. C.; Risby, T. t i , ; Yergey, A. L. Anal. Chem. 1976, 48, 829-832. Prescott, S. R.; Campana, J. E.; Risby, T. H. Anal. Chem. 1977, 49, 1501- 1504. Rozett, R . W. Anal. Chem. 1974, 4 6 , 2085-2089. Matthews, D. E . ; Hayes, J. M. Anal. Chem. 1976, 48, 1375-1382. McLaughiin, E.; Rozett, R. W. J . Organomet. Chem. 1973, 62, 261-268. Schoeller, D. A. Biomed. Mass Spectrom. 1978, 3 , 265-271
RECEIVED for review December 13, 1979. Accepted March 18, 1980. This work was supported by the U. S. Department of Energy under contract No. W-31-109-ENG-38. T h e authors wish to acknowledge financial support of the Institut d u Radium, Paris, France, given to J-C. Blais during his sabbatical visit to Argonne National Laboratory.
Direct Coupling of a Liquid Chromatograph to a Continuous Flow Hydrogen Nuclear Magnetic Resonance Detector for Analysis of Petroleum and Synthetic Fuels James F. H a w , T. E. Glass, D. W. Hausler, Edwin Motell,' and H. C. Dorn" Department of Chemistry, Virginia Polytechnic Institute, a n d State University, Blacksburg, Virginia 2406 1
Initial results obtained for a flow 'H nuclear magnetic resonance (NMR) detector directly coupled to a liquid chromatography unit are described. Results achieved for a model mixture and several jet fuel samples are discussed. Chromatographic separation of alkanes, alkylbenzenes, and substituted naphthalenes present in the jet fuel samples are easily identified with the 'H NMR detector. Results with our present flow 'H NMR insert indicate that 5-Hz linewidths are readily obtainable for typical chromatographic flow rates. The limitations and advantages of this liquid chromatography detector are compared with more commonly employed detectors (e.g., refractive index detectors).
T h e widespread applicability of high performance liquid chromatography (HPLC) for separation of complex mixtures is well recognized. Although a number of different detectors (e.g., refractive index, UV, etc.) are commonly employed in high performance liquid chromatography, most of these are nonselective for identification of discrete compounds. A possible exception is the Fourier Transform Infrared detector Visiting Professor, Department of Chemistry, San Francisco San Francisco, Calif.
State University.
0003-2700/80/0352-1135$01 .OO/O
for liquid chromatography ( I ) . Fourier transform nuclear magnetic resonance (FT-NMR) is a proven technique for spectral analysis of pure compounds a n d simple mixtures. In addition, the relatively high sensitivity of the 'H nuclide to detection by NMR is attractive for applications requiring limited sample and/or ''apailable time window" for spectroscopic examination. The latter requirements characterize the initial requirements for a continuous flow detector in high performance liquid chroinatography utilizing 'H NMR as the detector (LC-'HNMR). Nonchromatographic applications of flow NMR have previously been reported. Rapid irreversible chemical reactions as well as transient phenomena such as Chemically Induced Dynamic Nuclear Polarization (CIDNP) have been studied by stopped-flow NMR ( 2 ) . The theoretical formalism for the study of fast transient chemical reactions by FT-NMR has also been reported ( 3 ) . An apparatus for continuous-flow F'T-NMR has previously been described by Fyfe et al. (4). The effect of line broadening due to decreasing residence time with increasing flow rates was discussed. A value of T,(ossD, (observed spin-spin relaxation time) for varying flow rates is defined in t h e equation below (1/T2)(0BSD)
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
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T h e spin-spin relaxation time (T21STAT)) is the value for static samples and T is the residence time in the receiver coil (related to flow rate). An analogous expression can be written for t h e observed value for spin-lattice relaxation times. Another obvious consideration in LC-NMR is solvent selection, but this is also an important consideration with virtually every LC detector. Freon 113 (1,1,2-trifluorotrichloroethane), chloroform-d,, and carbon disulfide are possible solvent candidates which span a fairly wide range of polarity. Although perhaps too expensive to use neat (even with recycle), methanol-& could serve as a polar modifier a t a level of several percent. All separations involving deuterated solvents can be optimized with the cheaper protonated analogues prior to L,C-'HNMR analysis. Chemical shift references such as tetramethylsilane (TMS) may be added directly t o t h e mobile phase with negligible effect upon elution characteristics so long as the reference does not strongly interact with the stationary phase. Similarly, quantitative references may also be added. T h e extension of this technique to analysis of fluorinated derivatives by I9FF T - N M R is not restricted by solvent considerations. In general, the solvents that are suitable for LC-'HNMR have reasonable spectral windows for LC-FT-IR. T h e two techniques could easily provide complementary information. Obviously, one of the major advantages of the LC-'HNMR approach is the savings in sample manipulation and analysis time. This is particularly valid when compared with the tedious process of individual chromatographic fraction collection, solvent evaporation, and preparation for static NMR or other spectroscopic examination. Recently, a liquid chromatography effluent stream coupled to a 'H NMR spectrometer has been described for static examination of chromatographic fractions ( 5 ) . We believe that our work is the first description of a continuous flow LC-'HNMR system. In this paper, we report the design of our LC-'HNMR insert and present the results for a typical model mixture to demonstrate the advantages of this technique. A simple mixture of four different hydrocarbons was first examined. In addition, four experimental military jet fuels were studied, since they
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model mixture represent relatively complex mixtures amenable t o t h e LC' H N M R approach.
EXPERIMENTAL The jet fuel samples were supplied by the Air Force Aero Propulsion Laboratory (Wright-Patterson Air Force Base, Ohio) and the Naval Research Laboratory (Washington, D.C.). The four jet fuels used in this study were: routine JP-4, modified JP-4 (sample modified by adding xylenes and naphthalenes), Shale Paraho JP-5, and Coal Coed process JP-5. The last two samples are experimental fuels derived from shale and coal, respectively. The model mixture sample was prepared by mixing: 1.00 g isooctane, 1.00 g 2-hexene, 1.25 g benzene, and 1.00 g naphthalene. Trichlorotrifluoroethane (Miller-Stephenson Chemical Co.) was degassed prior to use as the chromatographic solvent. Enough tetramethylsilane (TMS)was added to the solvent after degassing to make it - 0 . 3 7 ~ by volume. A Merck Silica Gel 60 Size B (310 mm x 25 mm i.d.1 column was used after solvent equilibration. The pump was a Waters M-45 solvent delivery system. A Valco injection valve with a 500-rL sample loop was used throughout. All samples were injected neat. A Laboratory Data Control Model 1107 refractive index (RI) detector was used t o obtain classical chromatograms. The NMR flow cell was connected directly to the outlet of the
RI detector via a length of Teflon tubing (id. 1 mm). The time delay between the RI detector and the NMR receiver coil was determined to be 40 s at a flow rate of 2.5 mL/min. Effluent was removed from the NMR flow cell through a Teflon tube and into a cold trap by a vacuum pump. A Jeolco PS-100 nuclear magnetic resonance spectrometer was used to obtain 'H spectra at 100.0 MHz for the flon studies. The spectrometer was used in the Fourier Transform (FT) mode with a Digilab Data System. A fixed head 128K Alpha Data Disc allowed sufficient data storage for 42 (2048 point) spectral files and required a minimum of 110 ms for each spectral file transfer from the computer (Nova 1200). Figure 1 is a schematic diagram of the 'H NMR insert used in these LC-'H NMR studies. The receiver coil is wound about a 5-mm NMR tube which is connected at the bottom to the 1.5-mm Pyrex tubing which introduces the effluent stream. The spectrometer was operated with an external proton lock system. The comparison 'H NMR spectra of the jet fuels were obtained at 90.0 MHz in the CW mode utilizing a Varian EM-390 spectrometer. Hexamethyldisiloxane was used as the reference and lock signal.
RESULTS AND DISCUSSION T h e four components of the model mixture have a n elution order of isooctane, 2-hexene, benzene, and naphthalene, respectively, utilizing the chromatographic conditions described in t h e Experimental section. T h e normal Refractive Index (RI) trace obtained for this mixture is presented in Figure 2. Good resolution is indicated in spite of relatively high sample loading. This is also indicated in Figure 3 which is the LC-'H N M R profile obtained for this chromatographic run. Spectra with no peaks other than t h e reference TMS have been omitted. T h e growth and decay of compounds as one examines progressively later spectra is readily apparent. Figure 4 shows spectra selected from near the maxima of t h e four chromatographic peaks. Resolution arid sensitivity may be more readily evaluated in this figure t h a n in the stacked plot. Although line widths of 6-7 Hz were typically obtained, line widths of 5 Hz were the best t h a t could be obtained with our present insert. Since Fyfe ( 4 ) was able to d o considerably better with similar conditions, a considerable improvement in resolution is certainly feasible with improved receiver insert design. Given our 5-Hz limitation on magnetic inhomogeneity, a n acquisition time of 0.4 s was selected as a compromise between sensitivity and further degradation of line widths. Doubling the flow rate (2.5 t o 5 mL/min) did not substantially degrade resolution so the line width con-
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
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