Anal. Chem. 1985, 57, 675-679
detector but not by the mass spectrometer. Those compounds account for the four unidentified peaks appearing between 15 and 30 min in the absorbance chromatogram (Figure 3). The utility of reconstructed mass chromatogramsis evident in Figure 4. BHT and Irganox 1076 are easily detected in the reconstructed mass chromatogram, but in the total ion current chromatogram, Irganox 1076 is obscured by the peak at scan 557.
675
(3) Rodriguez, F. "Principles of Polymer Systems", 2nd ed.; McGraw-Hill: New York, 1982; pp 277-302. (4) "Cyasorb UV 531 Light Absorber"; Technical Bulletin D-42, American Cyanimld Co.: Wayne, NJ, 1982. Llchtenthaler, R. G.; Ranfelt, F. J. Chromafogr. 1978, 149, 553. Schabron, J. F.; Fenska, L. E. Anal. Chem. 1980, 52, 1411. Pasteur, G. A. Anal. Chem. 1977, 49, 363. , Perlstein, P. Anal. Chim. Acta. 1983, 149, 21. (9) Hanley, M. A.; Dark, W. A. J . Chromafogr. Sci. 1980, 18, 655. (10) Hayes, M. J.; Lankmayer, E. P.;Vouros, P.; Karger, B. L.; McGulre, J. M. Anal. Chem. 1983, 55, 1745. ~
LITERATURE CITED (1) Wheeler, D. A. Talanta 1968, 75, 1315. (2) Wlms, A. M.; Swarin, S . J. J . Appl. Polym. Sci. 1975, 79, 1243.
RECEIVED for review October 18,1984. Accepted December 7, 1984.
Electrospray Interface for Liquid Chromatographs and Mass Spectrometers Craig M. Whitehouse, Robert N. Dreyer,' Masamichi Yamashita? and John B. Fenn* Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520-2159
Electrospraying LC effluent into a dry bath gas creates a dlsperslon of charged droplets which rapidly evaporate. As the droplets grow smaller the Increase In surface charge density and the decrease in radtus of curvature result In electric fleids strong enough to desorb solute Ions. Part of the resuttlng dispersion of ions In bath gas passes through a small orlflce or channel Into an evacuated region to form a supersonic free jet. The core of this jet passes through a conical skimmer orlflce and transports the Ions to the Inlet of a mass analyzer. The reported results suggest that from the standpolnts of flexibillty, convenience, sensitlvlty, cleanliness, and ease of maintenance, this ESPI source may comprise an effective and practical LC-MS Interface.
The successful union of gas chromatography with mass spectrometry spawned a revolution in chemical analysis. An important impediment to the marriage was a basic incompatibility between vital features of each partner's mode of operation. The lifeblood of a gas chromatograph is the flow of carrier gas in which the species of analytical concern are usually present in only trace amounts. To the mass spectrometer, which is happy only under high vacuum, the prospective flood of carrier gas diluent was anathema. Clearly needed to overcome this impediment was a means of removing and discarding a large fraction of the carrier gas before the stream to be analyzed entered the mass spectrometer. One of the early and most successful devices for accomplishing this removal was the so-called jet separator first described by Ryhage and still widely used in one form or another (I). This device derived from the research of E. W. Becker and his colleagues who were exploring the use of supersonic free jets expanding into vacuum for molecular beam sources as had been proposed by Kantrowitz and Grey (2). Surprisinglylarge species separation effects were observed when the free jet gas comprised a mixture of heavy and light molecules (3, 4). 'Present address: Department of Pharmacology, Yale University. Present address: Institute of Space and Astronautical Science, Tokyo 153, Japan. 0003-2700/85/0357-0675$01 ._. S O / O -
Originally attributed to pressure diffusion during the expansion, this separation was subsequently shown to stem from preferential inertial penetration of heavier species into the stagnation zone behind the bow shock wave on the sampling probe immersed in the supersonic flow (5, 6). More recently the rapid and highly successful development of high-performance liquid chromatography (HPLC) has reached the stage where its practitioners have for some time also been contemplatingthe advantages of mass spectrometric detection. As was the case in GC-MS there is an impediment to the union of LC with MS that is similar but even more formidable. To be successful an LC-MS interface must not only divert a large fraction of the mobile phase from the mass spectrometer inlet but it must also make possible the transformation of nonvolatile and fragile species from solutes in a liquid to ions in a vacuum ready for mass analysis. To accomplish the latter step has been an exceedingly refractory problem to which until recently there have been no really satisfactory solutions even when the sample is static and there are no harsh constraints on the time available for preparation. In the last few years there has emerged a new breed of ion sources that to a much greater extent than their predecessors seem at once to be effective, easy to use, and compatible with both partners of the LC-MS union. Similar in some sense to the field ionization (FI) and field desorption (FD) sources introduced by Beckey and his collaborators, the newcomers apparently share a common mechanism: field ion desorption from liquids (FIDL) (7). Their membership includes the electrohydrodynamic(EHD) source of Evans and co-workers, the atmospheric pressure ion evaporation (APIE) source of Iribarne and Thomson, and the Thermospray (TS) source of Vestal and his colleagues (8-18). Along with other techniques for the ionization of nonvolatile molecules, these FIDL sources have recently been reviewed by Vestal (19). In this paper we report some experience and results with another variation on the FIDL theme, an electrospray ion (ESPI) source that has its roots in some experiments performed 15 years ago by Malcolm Dole and his collaborators. They attempted to produce beams of charged macromolecules by electrospraying a solution of polystyrene molecules into a bath gas to form a dispersion of macroions that was ex0 1985 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
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panded through an orifice as a supersonic free jet into vacuum (20-22). Those authors believed that as the droplets evaporated, the increasing coulomb repulsive forces came to exceed the surface tension so that the droplet underwent fission. Successive fissions ultimately gave rise to droplets containing a single solute molecule that retained the droplet charge as the remaining solvent evaporated. In the case of smaller solute ions with which we have been working, it appears that the high field formed at the surface of an evaporating charged droplet desorbs solute ions into the bath gas in much the same way as in APIE and T S (23). ESPI is operationally different from TS and APIE in the way it produces charged droplets. The latter two techniques disperse liquid into droplets by hydrodynamic forces. Statistical fluctuations in the distribution of solute anions and cations result in a net positive or negative charge on each droplet. In TS the sample liquid is passed through a small bore tube whose walls are hot enough to vaporize most of the solvent. The consequent expansion accelerates the flow and atomizes the remaining liquid so that a dispersion of approximately equal numbers of positively and negatively charged droplets in solvent vapor issues from the tube as a supersonic jet into the first stage of the vacuum system. In APIE the liquid sample is nebulized by a jet of air in the vicinity of a polarizing electrode at high voltage. The resulting field not only determines the sign of the droplet charge but also greatly enhances its magnitude. Whereas TS and APIE produce charging by atomization, ESPI produces atomization by charging. The sample liquid is injected into the bath gas through a metal hypodermic tube a t a potential of several kilovolts relative to the surrounding chamber walls. Charge is thus deposited on the surface of the emerging liquid and produces coulomb repulsion forces sufficient to overcome surface tension so that the liquid is dispersed in a fine spray. In all three techniques evaporation of solvent from the droplets increases the surface charge density and decreases the radius of surface curvature. The resulting increase in electric field strength finally reaches levels high enough to desorb ions into the ambient gas. Also common to APIE, TS, and ESPI is the use of a supersonic free jet to transport the ions from relatively high pressure in the desorption region into the vacuum of the mass spectrometer chamber. In sum, it would appear that history has once again succumbed to repetition, if not bigamy. The same supersonic free jet that aided the marriage of MS and GC now promises to abet the union of MS with LC! EXPERIMENTAL SECTION Figure 1shows the essential features of our most recent version of an ESPI source. Sample solution at flow rates typically between 5 and 20 pL/min enter the electrospray chamber through a stainless steel hypodermic needle. Representative values of applied voltage are in parentheses after each of the following components: needle (ground),cylindrical electrode (-35001, metalized
inlet and exit ends of the glass capillary that passes ion-bearing gas into the first stage of the vacuum system (-4500 and +40, respectively),the skimmer aperture between the first and second vacuum stages (-20), the ion lens in front of the quadrupole (-100). To produce negative ions, voltages of similar magnitude but opposite sign are applied. We have not yet operated this new apparatus in the negative ion mode but in an earlier apparatus we obtained equivalent results with both polarities (24). A first glance the indicated potential differences of 4540 V between the inlet and exit ends of the glass capillary may seem startling. We have found that with carrier gas at a pressure of about 1 atm, the ion mobility is so low that the flow can lift the ions out of the potential well at the capillary inlet. Consequently, only a few elements inside the apparatus need be at high voltage. All the external parts are at ground potential and pose no hazard to an operator. Indeed, we have been able to “pump”ions through a potential difference of at least 15 kV. Thus we can readily provide the ion energies necessary for injection into a magnetic sector analyzer. This possibility is not so readily realized with some of the other sources. The capillary, with a bore of 0.2 X 60 mm, for given inlet conditions passes just about the same flux of both gas and ions as the thin plate orifice, 0.1 mm in diameter, that it replaced. The high field at the needle tip charges the surface of the emerging liquid which, as mentioned above, becomes dispersed by coulomb forces into a fine spray of charged droplets. Driven by the electric field the droplets migrate to the inlet of the glass capillary through a countercurrent stream of bath gas (nitrogen) at a pressure that is slightly above 1 atm, typically 1000 torr, a temperature that we have varied from 50 “C to 80 “C or more, and a flow rate typically in the range of 150 cm3/s. The performance of the source is not at all sensitive to these variables. The droplets rapidly evaporate and the solvent vapor along with any other uncharged material is swept away by the flow of bath gas. Desorbed ions arriving in the vicinity of the capillary inlet are entrained in dry bath gas and transported into the first vacuum chamber where they emerge in the supersonic jet of carrier gas leaving the capillary. Pressure in this first vacuum chamber is or less by an oil diffusion pump with an maintained at 5 X effecti.ve speed of about 1000 L/s. A portion of the free jet flow passes through the 2-mm aperture of the skimmer into a second vacuum chamber containinga quadrupole mass spectrometer (VG Micromass 1212) whose output is monitored by a UV chart recorder. Pressure in the quadrupole chamber is maintained at about lo4 torr. When the field at the needle tip exceeds a critical value determined by temperature, pressure, and composition of bath gas, configuration of electrodes, and solution composition,electrical breakdown occurs and results in a sustained corona discharge. Typically, this discharge starts at voltages between 4 and 6 kV. Under these “high voltage” conditions the spectra are markedly different and are characterized by substantial attenuation of the parent solute peaks along with the appearance of other peaks corresponding to species found in discharges as a result of ion molecule reactions. All of the spectra shown in this report were obtained at voltages below the breakdown value. Thus, essentially all of the peaks correspond to solute ions present in the original solution, sometimes in aggregation with one or more un-ionized solvent or solute molecules. We have not yet studied them in detail, but it seems quite likely that the differences between spectra obtained at high and low voltage will contain information useful in identifying the structure of the unfragmented parent ions. RESULTS AND DISCUSSION The ESPI results in our previous papers (23,24) were obtained with an apparatus in which the upper limit for the mass range of the quadrupole analyzer was between 300 and 400 daltons. They showed that for a wide variety of species, the primary peaks in both positive and negative modes comprised solute ions in aggregation with one or more nonionic solvent or solute molecules. In the new apparatus described in the previous section the quadrupole analyzer can resolve masses up to 1500 daltons. Thus, one principal objective of the present study was to determine whether ESPI would work
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985 CYCLOSPORIN A
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as effectively in the higher mass range that our new apparatus can probe. However, for a first test of the new system we tried some of the same species we had used in the earlier studies.
Figure 2a shows the spectrum obtained with a solution of adenosine in a 50-50 methanol-water solvent at a concentration 0.1 pg/mL. The liquid flow rate was 6 pL/min during the 5 min it took to obtain this spectrum. The principal peak is at a mass of 268 daltons and corresponds to a protonated adenosine molecule. Also labeled is the peak at 300 daltons which we think corresponds to the same species solvated with one molecule of methanol. The fairly pronounced peak labeled 136 could be due to a doubly charged parent ion peak but is more likely due to a combination of the mass 133 and 134 fragments into which adenosine can readily split. We emphasize that all of the peak assignments in this report must be regarded as tentative. The quadrupole is still not working well, so that with these relatively dilute sample solutions the actual masses may be up to three units in error in the low mass range and ten or more at the high masses. We believe that all of the other peaks correspond to small cations, e.g., Na+ or H+,with one or more molecules of solvation, as was the case in the very similar spectrum obtained in the old system. Figure 2b,c shows spectra obtained respectively for the monoand diphopsphates of adenosine. The peak corresponding to the protonated parent molecule is identified in each case. It is interesting that a strong peak corresponding to the parent together with one methanol molecule of solvation appears in the AD case but not the AMP case. We have not tried to make definite assignments for the other peaks because of the uncertainty in mass mentioned above. However, the similarity between these results and those obtained in the original system, where we were able to make assignments with more confidence, persuade us that no appreciable fragmentation of the parent species occurs. Figure 3 shows the spectrum obtained with a solution comprising 0.1 pg/pL of the peptide cyclosporin A, an antilymphocytic agent, in an 85-15 acetonitrile-water solution. The primary peak is the singly protonated parent molecule. The width of the peak at the base clearly reveals the inadequate resolution of our quadrupole, so we make no attempt to interpret the small shoulders on either side of the peak base. The smallest peak of the triplet near mass 600 is probably due to a doubly protonated parent molecule. Our confidence in this assignment stems from experiments with high concentrations at which the improved resolution make it quite clear that the apparent mass difference in isotopic peaks is half that to be expected for singly charged ions. The two slightly larger peaks seem likely to stem from the presence respectively of one and two water molecules of solvation. Relative to the singly charged ion the higher polarity of the doubly charged ion may enhance its affinity for water enough
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985 2+
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to compensate for the relatively hydrophobic exterior of the parent molecule. Figure 4 shows the spectrum obtained with the antibiotic gramicidin S at a concentration of 0.01 pg/pL in 50-50 methanol-water. It is interesting that the predominant peak is due to the doubly charged ion, identified from isotopic mass intervals when experiments were carried out with higher concentrations of analyte as mentioned above. In this case, the presence of double charge does not give rise to the solvation that seemed to occur with cyclosporin A. It is appropriate to remark on sensitivity. The spectrum shown in Figure 4 required 5 min for the complete mass scan because at higher scan rates the mass markers do not appear in the chart output of the recorder that is our only present means of collecting data. The sample liquid flow rate was 6 pL/min so that 0.3 pg of analyte passed through the system during the scan. We estimate that the fraction of the total scan devoted to the primary peak was roughly 15/1500 or 0.01. Thus, the amount of analyte in the peak is about 3 ng. when we use an oscilloscope to monitor the output signal, we can carry out a complet scan in 1 s and obtain relative peak heights exactly the same as those for the slow scan. We conclude that we would thus need only 1/300 of 3 ng or 10 pg of analyte to obtain a peak equivalent to that in Figure 4. The apparent signal/noise in the recorder spectrum is at least 200 and probably more. If we assume that S I N or 2 represents detectability, we conclude that we can detect as little as 100 fg of this material. This argument assumes that S I N is not significantly greater in the fast scan, a not unreasonable assumption for the dc mode in which we operate. Just because the peak that we discern contains only 100 fg of analyte does not mean that we can detect this amount of material in any arbitrary sample introduced at random into the ion source. Detectability in any unconstrained sense will always have a marked dependence on scan rate, resolution, concentration, and injection time. These factors will all be different from laboratory to laboratory and experiment to experiment. A perhaps more meaningful basis upon which to compare instrument senstivities, especially in the case when sample is introduced continuously, albeit for short periods, rather than in batches, is the minimum steady state flow rate of analyte
that will give rise to a discernible mass peak. On this basis the experiments that led to Figure 5 provide a useful perspective. For the lowest concentration of gramicidin S, 5 x pg/mL, we could obtain a discernible peak with S I N of order 2 at a flow rate of 1.0 pL/min with a single mass scan at a rate of 20 amu/s. In other words we were detecting an analyte flow rate of 8 pg, or roughly 7 fmol, per second. If enough sample is available to permit multiple scans and signal averaging, the minimum detectable flow rate could be substantially less. These numbers are not ultimate in any sense. We are still on the steep part of the learning curve for this technique and have every reason to egpect substantial improvements in sensitivity. We have obtained entirely similar results with the peptides bleomycin and "Substance P" that have molecular weights respectively of about 1375 and 1348. The actual predominant peak with the sample of commercial bleomycin that we used (Blenoxane) was the complex with copper having a mass 1440. For both of these substances, as well as for gramicidin S, the doubly charged ion peaks predominate. This propensity toward multiple charging is provocative. If we can determine when, why, and how it occurs, we may be able to promote it and thus to extend substantially the effective mass range, not only of the ESPI source but of available mass analyzers as well. It is important for quantitative analysis that the mass spectrometer signal have a near linear dependence upon the concentrationof analyte. In Figure 5 is an example that shows such a near-linear dependence over 4 orders of magnitude in the concentration of gramicidin S in methanol-water. Moreover, the data for gramicidin S in Figure 6 demonstrate that the signal does not depend strongly on the liquid flow rate. These features of ESPI behavior are reassuring because they mean that peak heights (areas) obtained with LC effluents will not be distorted by an ESPI interface. Of course, the key concern in this forum is how well that the ESPI source will stabilize the LC-MS union. Before definitive conclusions can be reached, we must actually couple a chromatograph with a mass spectrometer by means of an ESPI source. Thanks to Whatman, Ltd., we have in fact obtained a microbore column with a 1mm i.d. and a length of 250 mm and, except for appropriate injection valves, are ready to make the acid test. Indeed, in some preliminary experiments, we have injected a sample of cyclosporin A into the column and passed the effluent into the source. We obtained a spectrum essentially identical with the one shown in Figure 3. Unfortunately, the injected sample was 20 ALL,
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so large that we overloaded the column with the result that the "elution" time or peak width was 52 min for 2 pg of analyte! With an oscillating mass scan of 8 daltons centered a t mass 1203, we were able to determine the shape of the leading and trailing edges of the peak. They were very abrupt and sharp, indicating minimal tailing. Thus, the ESPI source has virtually no dead volume. It is clear that until we have acquired more experience with an actual LC-ESPI-MS combinationwe will be in no position to make any meaningful judgement on the extent to which ESPI can be considered a solution to the interface problem. However, our experience to date seems to provide grounds for optimism. We close with some observations on practical operational features of ESPI that seem pertinent. 1. The system is not prone to fouling. The countercurrent flow of bath gas sweeps away all the solvent vapor and other uncharged material. In effect, only ions are carried into the vacuum system by the free jet of bath gas. We have run pretty steadily for periods as long as a month without any decrease in performance. Even then, there was no appreciable fouling in the vacuum system. The major problem was a slight accumulation of salt deposit a t the end of the hypodermic injection tube and on the side walls of the electrospraychamber. Both of these elements are in the high pressure section of the system and thus readily accessible for cleaning. 2. Thus far we have found no evidence to suggest that sensitivity depends upon the molecular weight of the analyte beyond what one would expect for decreases in quadrupole transmission and multiplier response for ions of increasing mass. In other words, the ionization efficiency seems to be relatively constant over the mass range. Of course, we have aa yet tried relatively few materials, but our confidence in this conclusion is increasing. 3. The system seems inherently stable. Small fluctuations in liquid flow rate do not cause ripples in the signal output. Modest changes in temperature and flow rate of bath gas do not markedly affect the signal. Consequently,these variables do not need to be controlled all that carefully. The variable of most immediate and direct influence on performance is the voltage applied to the liquid injection needle so that a reasonably well stabilized power supply is needed. Optimal voltage, bath gas flow rate, and temperature settings are
879
identical for all compounds run thus far. Consequently, adjustments need not be made to optimize sensitivity from one compound to the next. 4. The ability to use viscous drag forces during bath gas flow through the capillary to "pump" ions to any voltage that would be required for injection into a mass magnetic sector mass analyzer is very important. It means that all the advantages of such instruments in the analysis of very heavy ions will be accessible to LC/MS analysis. In sum, though our experience is thus far limited, results with ESPI along with those obtained by Vestal with TS and by Iribarne and Thomson with APIE give more reasons for hope than despair in the quest for a practical LC-MS interface. ACKNOWLEDGMENT We gratefully acknowledge the interest, support, and encouragement of VG Analytics who provided the quadrupole mass spectrometer and other reenforcement. We also record our gratitude for the cooperation and support that our colleagues at Yale have so generously provided. In particular, the yeoman efforts of Chin-Kai Meng and Ted Grabowski have been invaluable. LITERATURE CITED Ryhage, R. Anal. Chem. 1964,5 1 , 359. Kantrowltz, A.; Grey, J. Rev. Sci. Instrum. 1951,2 2 , 328. Becker, E. W.; Bier, K.; Burghoff, H. Z . Natufforsch., TellA 1955, IO, 565. Becker, E. W.; Bler, K.; Ehrfeld, W.; Schubert, K.; Schutte, R.; Seldel, D. In "Nuclear Energy Maturlty"; Zaieskl, P., Ed.; Pergamon: Oxford, 1975; p. 172. Reis, V. H.;Fenn, J. B. J . Chem. Phys. 1963,39, 3240. Sherman, F. S. Phys. Fluids 1965,8 , 773. Beckey, H. D. "Principles of Field Ionization and Field Desorption Mass Spectrometry"; Pergamon: New York, 1977. Stimpson, B. P.; Simons, D. S.; Evans, C. A,, Jr. J . Phys. Chem. 1978,8 2 , 660. Evans, C. A. Jr.; Hendrlcks, C. D. Rev. Sci. Instrum. 1972,43, 1527. Simons, D. S.; Colby, B. N.; Evans, C. A., Jr. Int. J . Mass Spectrom. Ion. Phys. 1974, 15, 291. Stlmpson, B. P.; Evans, C. A., Jr. Biomed. Mass Spectrom. 1978,5 , 52. Iribarne, J. B.; Thomson, B. A. J . Chem. Phys. 1976,6 4 , 2287. Thomson, B. A.; Irlbarne, J. B. J . Chem. Phys. 1979, 7 1 , 4451. Iribarne, J. 8.; Dziedzic, P. J.; Thomson, B. A. Int. J . Mass SpectfO/??. Ion. P h p . 1983,5 0 , 331. Blakley, C. R.; Carmody, J. J.; Vestal, M. L. J . Am. Chem. SOC. l980,-102, 5931. Blakiey, C. R.; Carmody, J. J.; Vestal, M. L. Clln. Chem. (Winston&/em, N . C . ) 1980,2 6 , 1467. Blakley, C. R.; Carmody, J. J.; Vestal, M. L. Anal. Chem. 1980,5 2 , 1636. Blikiey, C. R.; Vestal, M. L. Anal. Chem. 1983,55, 750. Vestal, M. L. k s s Spectrom. Rev. 1983,2 , 447. Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J . Chem. Phys. 1968,49, 2240. Mack, L. L.; Kralik, P.; Rheude, A.; Dole, M. J . Chem. Phys. 1970, 5 2 , 4977. Dole, M.;Cox, H. L., Jr.; Gieniec, J. Adv. Chem. Ser. 1973,No. 125, 73. Yamashita, M.;Fenn, J. B. J . Phys. Chem. 1964, 8 8 , 4451. Yamashita, M.; Fenn, J. B. J . Phys. Chem. 1984,8 8 , 4671.
RECEIVED for review October 8,1984. Accepted December 6, 1984. This research has been sponsored in part by the National Science Foundation (Grant ENG-7910843),the U.S. Department of Energy (Grant ET-78-G-01-3246),and the National Cancer Institute (Grant C288-52/04). C.M.W. acknowledges with appreciation a fellowship award from the Exxon Corp. M.Y. acknowledges support from the Institute of Space and Aeronautical Sciences that made his participation possible.
