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surements. Though linear dynamic ranges (LDR) are poor, excellent sensitivity and detectability are achievable over a narrow interval of analyte concentrations. We are presently investigating antibody-sandwich and direct assays in order to extend FIS applications. Manipulation of these techniques, along with the preincubation competitive assay, will allow full exploitation of the in situ measurement possibilities offered by fiber-optic sensor instrumentation. ACKNOWLEDGMENT The authors thank C. J . Wust of the University of Tennessee for his valuable advice and R. N. Compton of Oak Ridge National Laboratory for his loan of laboratory equipment and space. Registry No. GOPS, 2530-83-8. LITERATURE C I T E D Munkholrn, C.; Walt, D. R.; Mllanovich, F. P.; Klainer, S. M. Anal. Chem. 1986, 58, 1427-1430. Seitz. W. R. Anal. Chem. 1984, 56, 16A-34A. Milanovich, F. P. Environ. Sci. Technol. 1986, 20,441-442. Arnold, M. A. Anal. Chem. 1985, 57, 565-566. Andrade, J. D.; Vanwagenen, R. A,; Gregonis, D. E.; Newby, K.; Lin, J. N. I€€€ Trans. Electron Devices 1985. ED-32, 1175-1 179. Peterson, J. I.; Fitzgerald. R. V.; Buckhold, D. K. Anal. Chem. 1984, 56,62-67. Zhujun, 2.; Seitz, W. R. Anal. Chim Acta 1984, 160, 47-55. Schultz, J. S.;Mansouri, S.: Goldstein, I. J. Diabetes Care 1982, 160, 47-55.
Ross, I. N.; Mbanu, A. Opt. Laser Technol. 1985, Feb., 31-35. Tromberg, B. J.; Eastham, J. F.: Sepaniak, M. J. Appl. Spectrosc. 1984. 38. 38-42. Schwab. S.D.; McCreery, R. L. Anal. Chem. 1984, 56, 2199-2204. Vo-Dinh, T.; Griffin, G. D.; Arnbose, K. R.: Sepaniak, M. J.: Trornberg. B. J. Proceedings of the Tenth International Symposium on Polycylic Aromatic Hydrocarbons: Battelle: Columbus, OH, in press. Dakubu, S.; Ekins, R.; Jackson, T.; Marshall, N. J. I n Practical Immunoassay: The State of the Art; Butt, W. R., Ed.; Marcel Dekker: New York, 1964; Chapter 4. Sutherland, R. M.; Dahne, C.; Place, J. F.: Ringrose, A. S. Clin. Chem. (Winston-Salem, N C ) 1984, 30, 1533-1536. Hirschfeld, T. E. U S . Patent 4 447 546, May 6, 1964. Sportsman, J. R.; Wilson, G. S.Anal. Chem. 1980, 52,2013-2018. Sanderson, C. J.; Wilson, D. V. Immunology 1971, 20, 1061-1065. Thorell, J. I.; Larson, S. M. Radioimmunoassay and Related Techniques; C. V. Mosby: St. Louis, MO, 1976; Chapter 5. Aloisi, R. M. Principles of Immuncdlagnostics: C. V. Mosby: St. Louis, MO, 1979; Chapter 9. P. C. Rodgers I n Practical Immunoassay: The Stare of the Art; Butt, W. R., Ed.; Marcel Dekker: New York, 1984: Chapter 10. Nygren, H.; Stenberg, M. J . Colloid Interface Sci. 1985, 107, 561-5166, Stenberg, M.; Elwing. H.; Nygren, H. J . Theor. Biol. 1982, 98, 307-320.
RECEIVED for review November 12, 1986. Accepted January 12, 1987. This work has been supported by the National Institutes of Health (GM 34730) and the Office of Health and Environmental Research, U.S. Department of Energy, under Contract Number DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.
CORRESPONDENCE On-Line Mass Spectrometric Detection for Capillary Zone Electrophoresis Sir: Mikkers, Jorgenson, and co-workers ( I , 2 ) have reported on the use of capillary zone electrophoresis (CZE) for high-resolution separations of amino acids, peptides, proteins, and complex salt mixtures. This technique has been shown to provide separation efficiencies of up to lo6 theoretical plates, often in less than 20 min ( 3 ) . Capillary zone electrophoresis is particularly useful in the separation of ionized and partially ionized species in aqueous solvents, although nonaqueous solvents have also been used ( 4 ) . In CZE separation occurs in a capillary tube filled with a buffer and immersed in buffer reservoirs a t each end (Figure 1). The sample is typically introduced as a sample plug by electromigration from a separate sample reservoir (2). Electroosmotic flow in the capillary is caused by the migration of ions from the diffusive layer of the electrical double layer at the capillary surface, under the influence of an electrical field imposed tangentially to the surface, causing the ions to migrate toward the oppositely charged electrode ( 5 ) . The resulting bulk electroosmotic flow can be sufficiently fast so that positively charged ions, neutral species, and negatively charged ions elute in short times, with the separation due to differences in the electrophoretic mobilities of the analytes. Detection of the eluting species in CZE is usually performed by on-line fluorescence or UV absorbance detection. Such detection techniques have been adequate for species that fluoresce, absorb, or are amenable to derivatization with fluorescing or absorbing chromophores ( I , 2). However these detectors impose difficult cell volume and sample size limitations if high separation efficiencies are to be realized. These
limitations constitute a major drawback in the use of CZE for the separation and identification of complex mixtures. The ideal detector for CZE would provide universal detection, selectivity, and sensitivity without degrading separation eff ici en cy . We have developed a viable alternative to CZE detection based on mass spectrometric interfacing. A capillary zone electrophoresis-mass spectrometry (CZE-MS) interface obviously requires a substantial departure from the conventional CZE arrangement; it is clear that the interface design and ionization method are crucial to success. The liquid flow rate in CZE (- 1 pL/min) is highly compatible with conventional mass spectrometers even if the total column effluent was introduced directly. The direct liquid introduction interfaces developed for LC-MS suffer from orifice plugging at low flow rates and the thermal degradation of high mass-low volatility components (6,7).Thermospray ionization, though attractive, has not been shown to be effective for liquid flow rates below a few tenths of a mL/min. Therefore, our evaluation of the requirements for a mass spectrometer interface suggested an approach that incorporates the electrospray ionization technique developed by Dole et al. (8)and the more recent work reported by Fenn and co-workers (9). In this communication, we report the successful development of CZE-MS instrumentation for the separation and analysis of ionic species in aqueous solutions. EXPERIMENTAL SECTION Apparatus. A schematic of the CZE-MS instrument is given
0003-2700/67/0359-1230$01.50/0 1967 American Chemical Society
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Fluorescence Fused Silica Capillary
\Buffer I
L
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Schematic illustration of the apparatus used for capillary zone electrophoresis. Flgure 1.
C
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Electrospray ionization mass spectrum of a mixture of five quaternary ammonium salts at M concentration introduced by continuous electromigration. The dominant peaks are due to the quaternary ammonium cations of tetramethylammonium bromide (mIz 74), tetraethylammonium perchlorate (m /z 130), trimethylphenylammonium iodide (m /z136), tetrapropylammonium hydroxide (mIz 186),and tetrabutylammonium hydroxide (m/ z 242) and a background peak due to Na-MeOH' (m l z 55). Flgure 3.
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i
Schematic illustration of the apparatus developed for capillary zone electrophoresis-mass spectrometry (CZE-MS): (A) electrically insulated sampling box; (B) anode and sample injection reservoir; (C) fused silica capillary: (D) cathode and electrospray needle; (E) electrospray; (F) focusing ring; (G) nozzle: (H) skimmer; (I) rf only quadrupole: (J) ion entrance aperture: (K) quadrupole mass spectrometer; (L) channeltron electron multiplier. Flgure 2.
in Figure 2. Capillary zone electrophoresis is carried out with a 0-60 kV dc power supply, Glassman High Voltage, Inc. (Whitehouse Station, NJ), Model LG60P2.5. The high-voltage electrode and capillary end (anode) and solution vials are contained in an insulating sampling box with a remote controlled sampling arm and injection timer to facilitate the interchange and injection of solutions. Fused silica capillaries, 100 pm i.d. and 100 cm long, from Polymicro Technologies, Inc. (Phoenix, AZ), were used in all experiments without further treatment. The cathode (low voltage end) of the fused silica capillary is terminated in a stainless steel capillary sheath, 300 pm i.d. and 450 pm 0.d. The sheath potential is controlled with a 0-5 kV dc power supply and functions as both the CZE cathode and electrospray needle. The stainless steel capillary ensures immediate electrical contact with the solution flowing out of the fused silica capillary, thus, terminating the CZE circuit and intializing the electrospray (see Figure 2). Electrospray ionization is carried out at atmospheric pressure in a 2.54 cm long by 2.29 cm i.d. stainless steel cylinder. The cylinder terminates in an electrically biased (190 V dc) focusing ring with a 0.475-cm aperture. The ion sampling nozzle has a 0.5 mm i.d. orifice, is made from copper, and is in contact with a copper cylinder at ground potential. This cylinder surrounds the electrospray assembly and is heated to 60 "C by a system of cartridge heaters. The electrospray needle, focusing ring, and ion sampling nozzle are concentric with the mass analyzer. These components can be positioned independently relative to the fixed skimmer (with the aid of linear motion drives), even while high voltage is on, in order to maximize ion formation and transmission. A curtain of N l , at a flow rate of 2.5 L/min, is fed between the focusing ring and the nozzle and allowed to flow counter to the electrospray to aid in the desolvation process. The vacuum system consists of a three-stage differentially pumped chamber. The first stage allows for a supersonic beam expansion through the ion sampling nozzle. This region is pumped to 0.85 torr by a 150 L/s roots blower. A portion of the supersonic beam is sampled by a 1.2 mm i.d. beam skimmer, Beam Dynamics, Inc. (Minneapolis, MN), Model 1. The second differentially pumped stage houses a 22 cm long, 0.95 cm diameter quadrupole filter. This quadrupole is operated in the rf only mode with a -1.8 V dc rod bias and facilitates ion transmission. The pressure in this region is maintained at 8 X torr with a 1500 L/s turbomolecular pump. An electrically isolated stainless steel plate (-28 V dc), with a 0.635 cm i.d. orifice, allows the mass spec-
trometer chamber to be maintained at 2 X lo4 torr, using a 550 L/s turbomolecular pump. The 2000 amu range quadrupole mass filter, Extrel Co. (Pittsburgh, PA), Model CQPSlHV, and a channeltron electron multiplier, Detector Technologies, Inc. (Brookfield,MA), Model 203, operated in the analog mode, reside in this chamber. Data acquisition and mass scanning are performed with a Teknivent Corp. (St. Louis, MO) Model 1050 interface IBM PC/XT based system. Procedures. Injection of samples was performed using the electromigration technique (2). In electromigration, the anode end of the column is introduced into the analyte solution, the injection voltage is turned on for a predetermined amount of time, the voltage is turned off, and the buffer replaced; the CZE applied voltage (V,,, = 40 000 V dc) and electrospray voltage (V,, = 3000 V dc) are then turned on and the separation is allowed to continue. The injected sample size is given by the equation Cu,tiVi/t,VczE where C is the concentration of the analyte, u, is the column volume, ti is the injection time, Vi is the injection voltage (with no electrospray voltage applied), t , is the analyte's retention or elution time, and VC, is the CZE voltage. This equation is valid if the retention times (or net mobilities) are proportional to the CZE voltage over the entire voltage range. The CZE voltage ( VCm) here refers to the voltage drop across the CZE column which has been modified from the traditional sense because the cathode is maintained at the electrospray voltage; thus VczE = V,,, - VEsI.
RESULTS AND DISCUSSION In our initial work with CZE-MS we have used (50/50) water-methanol with M KC1 as the separation and electrospray medium (9). We have observed that watermethanol provides a considerable electroosmotic mobility (3.6 X cm2/(V s)). Thus, positively ionized compounds should elute in less than 12.5 min from a 100 cm long column (with VcZE = 37000 V). As our initial test mixture we chose five ammonium salts: tetramethylammonium bromide, tetraethylammonium perchlorate, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, and trimethylphenylammonium iodide. These quaternary ammonium salts all gave good electrospray signals with the dominant peak in the mass spectrum being the quaternary ammonium cation. Figure 3 shows the electrospray mass spectrum for the five components injected continuously without CZE separation. The first CZE-MS separation of such a mixture, taken under multiple ion monitoring of the corresponding quaternary ammonium cation peaks, is shown in Figure 4. The amounts injected for the quaternary ammonium salts, 14-17 fmol, gave single ion electropherograms with good peak shapes and signal/noise ratios. Figure 5 shows the same separation obtained for a
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R
1
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Figure 4. Reconstructed total ion electropherogram of five quaternary ammonium salts, at lo-' M (14-17fmol injection) concentration, obtained by CZE-MS: (A) tetramethylammonium bromide; (B) trimethylphenylammonium iodide; (C) tetraethylammoniumperchlorate; (D) tetrapropylammoniwn hydroxide; (E) tetrabutylammonium hydroxide.
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limitation of the technique but only as the first medium used in CZE-MS. Of course, other buffers may be more desirable for particular CZE applications. Nonvolatile buffers, for example, should pose few problems based on the work of Fenn and co-workers (9). The ability to combine CZE with mass spectrometry is not as surprising as it might appear a t first glance. The cathode need not be in a buffer reservoir, but only biased negative with respect to the anode. Thus, a metalized segment of capillary tubing or other electrical contact with the buffer provides the essential control of the electric field. This approach (necessary for mass spectrometric interfacing) does not alter the electroosmotic flow, at least to an extent that is detectable with fluorescence detection just prior to the electrospray. The success of this approach is further supported by the high efficiency separations presented in this communication. On the basis of these initial results, electrospray ionization appears to provide an ideal interface for the marriage of a highly efficient separation technique, capillary zone electrophoresis, with the sensitive and highly specific detector provided in the mass spectrometer. Future work will aim at obtaining enhanced sensitivity and separation efficiencies and exploring the role of various instrumental parameters relevant to CZE separations and MS detection.
LITERATURE CITED Mikkers, D. W. P.; Everaerts, F. M.; Verhegge, Th. P. E. J . Chromatogr. 1979, 169, 11. Jorgenson, J. W.; Lukacs, K. D. Science 1984, 222, 266. Lauer, H. H.; McManigllL D. Anal. Chem. 1988, 58, 166. David, P. A.; Pellechia, P. J.; Manning, D. L.; Maskarlsha, M. P. Report ORNL/TM-9141, April 1984. Pretorius, V.; Hopkins, 9. J.; Schieke, J. D. J . Chromatogf. 1974, 9 9 , 23. Arpino, P. J.; Beamgrand, C. Int. J . Mass Spectrom. Ion Processes 1985, 6 4 , 275. Bruins, A. P. J . Chromafogr. 1985, 323, 99. Mack, L. L.; Kralik. P.; Rhonde, A,; Dole, M. J . Chem. Phys. 1970, 52, 4977. Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. 9. Anal. ChSm. 1985, 57, 675.
Figwe 5. Reconstructed total ion electropherogram of five quaternary ammonium salts, at lo-' M (0.7-0.9 fmol injection) concentration, obtained by CZE-MS: tetramethylammonium bromide (rn /I 74);trimethylphenylarnmonium iodide (rn/ z 136);tetraethylammonium perchlorate (rn/z 130);tetrapropylammoniumhydroxide (rnl z 186);tetrabutylammonium hydroxide (rn / z 242).
0.7-0.9 fmol injection, obtained by decreasing Vi to 20000 V and C to lo-' M. Though the separation efficiencies in Figure 4 vary from 26 000 and 100 000 theoretical plates, they are increased to between 35000 and 140000 theoretical plates in Figure 5. Such increases in efficiency with decrease in sample concentration and size suggest further improvement may be possible with higher buffer ionic strength (2). The water-methanol solution used in this preliminary work should not be construed as a
Jos6 A. Olivares Nhung T. Nguyen Clement R. Yonker Richard D. Smith* Chemical Methods and Separations Group Chemical Sciences Department Pacific Northwest Laboratory P.O. Box 999 Richland, Washington 99352 RECEIVED for review October 31, 1986. Accepted December 18,1986. We thank the U.S. Department of Energy through Contract DE-AC06-76RLO-1830and the U.S. Army Medical Research Institute of Infectious Diseases for support of this work. Pacific Northwest Laboratory is operated by Battelle Memorial Institute.
Measurement of Isotope Ratios by Doppler-Free Laser Spectroscopy Applying Semiconductor Diode Lasers and Thermionic Diode Detection Sir: In a recent correspondence (1)we pointed out why the application of tunable lasers in analytical spectroscopy is not common outside research laboratories. Although laser-based techniques like laser-induced flourescence (LIF), laser-enhanced ionization (LEI), and resonance ionization spectros-
copy (RIS) have already shown their potential and their extreme detection sensitivities (1-7), the complexity, the high cost, and the limited wavelength ranges of the most commonly used dye lasers are the reasons for the slow establishment of analytical laser spectroscopy. However, we are convinced that
0003-2700/87/0359-1232$01.50/0 0 1987 American Chemical Society
