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Anal. Chem. 1986, 58, 1308-1311
Ion Source for Liquid Matrix Secondary Ionization Mass Spectrometry A. M. Falick,* G. H. Wang,l and F. C. Walls
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, S a n Francisco, California 94143
A new iiquld secondary Ionization mass spectrometry (LSIMS) ion source employing a Cs+ primary beam has been constructed, and its petformance has been compared against a standard Kratos MS-50 fast atom bombardment (FAB) source using Xe atoms. The LSIMS source has improved high-voltage feedthroughs (20 kV), a mechanical Cs' gun positlon adluster, and more eff iclent secondary ion collection optlcs. The LSIMS source was found to give approximately a factor of 2 increase In maximum quasi-molecular ion (MH+ or M Na') intensity when a 0.1 pA Cs+ primary beam was used, and a factor of 5 with a 1.0 pA primary beam. The sample spectra were also found to last slgniflcantiy longer with the LSIMS source. A measurement of integrated intensity over the total sample lifetime showed the LSIMS source to be a factor of 10-20 more sensitive than the FAB source. Both the improved optics and the focused primary beam contribute to the enhanced performance. The mass spectra obtained were observed to be essentially the same regardless of which prlmary beam was used.
+
Liquid matrix secondary ionization mass spectrometry (LSIMS) using a Cs+ ion primary beam has been used successfully in this laboratory for several years. The methods used and the spectra obtained are very similar to Xe fast atom bombardment (FAB) mass spectrometry (1). However, in operation, the use of a Cs+ primary beam has certain important advantages over a neutral Xe atom beam. First, an ion beam is much easier to control in intensity, energy, and focusing than a neutral beam. In the Cs+ gun used in all of these experiments (2) an oven containing a cesium alumina silicate is heated to produce Cs+. Control of total beam flux is easily achieved by varying the heater power. The primary particle energy is determined by the applied potential difference between the gun and the ion source block, and the gun is constructed with an Einzel lens so that the primary beam can be focused onto the sample. A second major advantage of the Cs+ gun over commonly used neutral atom guns is the reduced level of contamination of the ion source region, particularly the source slit. Cleaning of the source and source slit has been required much less frequently since the new Cs+ gun was placed in routine operation. In addition, the overall source pressure is significantly lower in operation than when a conventional neutral atom gun is used. Neutral atom guns use a charge exchange cell, which constantly bleeds neutral gas through the atom beam exit hole into the ion source housing. Finally, the Cs+ gun is compact enough to be mounted inside the source housing and requires no external gas or vacuum connections. In view of these advantages, it was appropriate to undertake to design a new ion source specifically tailored to make optimum use of a Cs+ primary beam, rather than simply using Present address: Institute of Chemistry, Academia Sinica, Beijing, China.
the Cs+ gun attached to a conventional FAB source (2). The new LSIMS ion source described here incorporates improved mass spectrometer collection efficiency for secondary ions, novel high-voltage feedthroughs, a mechanical Cs+ gun position adjuster, and easily modified optics.
EXPERIMENTAL SECTION Mass Analyzer. All experiments were performed on a Kratos (Manchester, UK) MS-50s mass spectrometer equipped with a 23-kG magnet and a postacceleration detector. The latter was operated at -10 kV for all of the experiments described. Spectra were recorded on a Gould ES-1000 electrostatic recorder (Gould, Inc., Cleveland, OH). Typical operating conditions were as follows: scan rate 100 s/decade, dynamic resolution M/AM = 3000. FAB Source. For the FAB experiments, a standard geometry (as of 1985) Kratos FAB source and an Ion Tech saddle field gun using Xe gas were used. The total flux of 8-keV Xe atoms irradiating the probe tip was not measured accurately; however, the indicated current on the saddle field gun power supply (40 MAin these experiments when Xe flow was adjusted for maximum sample signal) represents the measured current of Xe+ generated in the saddle field discharge region. The Xe+ beam is subsequently accelerated and passes through a charge exchange cell containing neutral Xe gas, resulting in a beam composed primarily (3) of fast Xe atoms. The most intense central portion of this beam should have a diameter of about 4 mm, based on the reported beam while the less intense portion would spread for similar sources (4), be 2-3 times larger. This is consistent with our observations of ion burns on the ion source, which indicated a beam diameter of 8-10 mm. The 3-mm-diameter copper probe tip used was cut so that a line normal to its surface was at a 20' angle with respect to the ion optical axis of the mass spectrometer. The primary Xeobeam formed an angle of 70' with respect to the target normal, resulting in a primary to secondary beam angle of 90'. LSIMS Source. The newly constructed LSIMS source is mounted on a standard Kratos MS-50 source flange so as to be interchangeable with other sources. Figure 1 shows the source flange on which are mounted the high-voltage feedthroughs, the gun tilt adjuster, and the source itself. The Cs' gun, which is mounted on the side of the ion source, is of a compact design developed in this laboratory ( 2 , 5 ) . The high-voltage feedthroughs are designed to withstand a 20-kV voltage difference between the conductors and the flange in which they are mounted. The increased voltage rating compared with the standard Kovar-glass feedthroughs normally used on Kratos source flanges was required for two reasons. First, there is good evidence that increasing the kinetic energy of the primary beam particles produces increased sensitivity (6, 7), so it was desirable to have as great a Cs+ accelerating voltage as feasible. Second, the Cs+ beam accelerating voltage floats on top of the normal instrument accelerating voltage, thus to obtain a 10-keV Cs+ beam in an instrument operating with 8-keV secondary ion energy requires feedthroughs capable of standing off 18 kV with respect to ground. (Of course, when negative secondary (sample) ions are being detected, the Cs+ source need only operate at 2 kV above ground in this example.) Eight feedthrough holes are cut in the source flange, one of which is used for the mechanical motion feedthrough. Each high-voltage feedthrough assembly has three conductors, so that a maximum of 21 contacts are available. In fact, only five feedthroughs are installed, the other holes being blanked off. The feedthrough bodies were machined from 19-mm-diameter polyimide resin rod (Vespel SP-1,Du Pont, Wilmington, DE). The
0003-2700/86/0358-1308$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58. NO. 7 . JUNE 1986
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focus ploter
h verpe1 POlIS
high voltoge feedthrough
probe tip
Flgure 2. LSIMS ion source. This view is rotated 90' from the view
in Figure 1.
L verpe1-
Table I. Comparison of Conditions Used in LSIMS and FAB Source Tests
10 m e r t , o n probe V O C Y Y ~loch
9"" tllt adprtmenl
condition primary beam gun
Flgure 1. LSIMS source, Cs' gun. and highvoltage and mechanical motion feedthroughs mounted on MS-50 source flange.
conductors are 2-56 threaded stainless-steel rod held in place hy means of slotted nuts at each end. The nut on the vacuum side is not bored through and is rounded to fit into a tapered hole in the polyimide rod. Careful tightening of the nuts produced a leak-free metal to polyimide seal. The feedthroughs are held in place in the source flange with a nut and Viton O-ring seal. No electrical leakage was observed when the feedthroughs were operated at up to 18 kV. The Cs+gun position can be mechanically adjusted from outside the vacuum system while the source is in operation. This feature is essential in order to operate the ion source in hoth positive and negative (secondary ion) modes. The electric fields used to focus the secondary ion beam in the source also affect the primary beam. When a change from positive to negative mode is made, the change in direction of the electric fields in the source causes the primary beam to shift. The micrometer adjustment of the gun tilt restores the beam to the optimum position. A change of 2-3O is required when changing from positive to negative operation. The gun tilt adjuster was constructed from the actuator of a Nupro SS-4BMG bellows valve (Nupro Co., Willoughhy, OH), mounted in the source flange in the same fashion as the electrical feedthroughs. Mechanical motion is transmitted through the bellows, so no sliding seals are used. One end of a polyimide rod is attached to the valve stem; on the other end of the rod is mounted a stainless-steel angle piece that supports the lower corner of the Cs' gun assembly. The gun rotates about an axis connecting two pivot points on the source block (see Figure 1) and is held in place against the adjuster hy a return spring. The source itself is supported on two 6.35-mm-thick X 50.8mm-long polyimide resin side plates identical in design with the original quartz blocks supplied by Kratos. The polyimide was found to he more robust and had no evident drawbacks in this application. The new ion source was designed to have improved mass spectrometer collection efficiency for secondary ions. In most FAB ion sources,including the standard MSM) FAB source, there is no electric field between the prohe tip and the extractor plate. The secondary ion collection system is essentially passive: ions whose initial trajectories are too divergent are lost. A previous study (8)dexrihed an immersion lens arrangement that impases a uniform electric field between the prohe tip and an extractor electrode. The present source uses an extraction field that is shaped (see below) to further increase the mass spectrometer collection efficiency. The central portion of the ion source is illustrated in Figure 2. The Cs+ beam lies in a plane perpendicular to both the plane of the page and the plane of the focus plates. The 3-mm-diameter probe tip sample surface is parallel to the extractor and focus plates. The Cs+ beam strikes the probe tip at an angle of about 65O with respect to target normal; thus the angle between the primary and secondary beams is about 65". The Cs+ beam di-
LSIMS
FAB
Ion Tech saddle field Cs+ gun (21, mounted on gun, Xe gas, Kratos mounting source 0.1 or 1.0 pA, 8 kV 40 PA," 8 kV
primary beam current, voltage 5 x 10" torr source pressureb primary to secondary 90° beam angle extraction field none
z x 10-7 torr 65'
present, see text
'Indicated current on the saddle field gun power supply. bIndieated pressure with primary beam operating but no sample on the prohe tip. As the ion gauge is in the pumping arm, this is not the true pressure in the ionization region. ameter a t the prohe tip is approximately 1 mm (2). An electric field of 1%-175 V/mm is established between the probe tip (which is electrically connected to the source block) and the extractor. The extraction field is shaped hy the addition of two stainless-steel tabs attached to the source base plate at a 45O angle and spanning its width (see Figure 2). The tabs and the bottom plate thus form a trough parallel to the extractor slit, which creates a focusing field for the secondary ions. A somewhat similar arrangement was employed by Todd, Glish, and Christie (9). The extractor plate is supported on four 6.35-mm-diameter polyimide resin posts at a distance of 6.35 mm above the source bottom plate and contains a 1.25-mm-wide X 12.7-mm-longslit. The focus plates are of similar design to the original Kratos parts and are also supported on polyimide posts at 6.35 mm above the extractar plate. The design of these lenses and posts permits easy adjustment of lens spacings by replacing the polyimide support posts. The Cs+ primary gun current was measured by temporarily installing a Faraday cage arranged so as to collect the entire Cs' beam. The Cs' current was then measured as a function of the voltage applied to the Cs' source heater so that when the Faraday cage was removed, any desired Cs+beam current could he obtained by adjusting the heater voltage accordingly. Cs+ primary beam currents of 0.1 and 1.0MAwere used in the experiments described below. Comparison Measurements. The two ion sources were compared by installing each in the mass spectrometer and ohtaining spectra of a series of samples with each source. Samples and matrix materials were taken directly from the same solutions in every m e . During the course of this series of experiments each source was removed, cleaned, aligned, and reinstalled. Data obtained before and after this procedure did not show any significant differences. Furthermore, data obtained with the LSIMS source were repeated after a number of FAB experiments; again no significant variations in the results were observed. The conditions used in the comparison measurements are summarized in Table I. Three test compounds were used to measure the ratio of maximum signal intensities obtainable with the two sources: gramicidin S, MetLys-bradykinin, and 6-0methylglucose polysaccharide (IO) (MGP). One microliter of solution containing approximately 0.9 nmol of sample was used in each case. One microliter of a 2 1 g1ycerol:thioglycerol matrix
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table 11. Relative Intensities of Quasi-Molecular Ion Peaks in the Spectra of Three Test Compoundsa
test compd gramicidin S MetLys-bradykinin MGP
relative intensity LSIMS, Cs+ mass of peak beam = beam = observed FAB 0.1 WA 1 . 0 ~ A 1141.7 1320.7 3539.3
1.0 1.0 1.0
3.3 2.6 1.6
9.4 5.5 4.7
was then added to the probe tip for all three compounds, and 1 pL of 1M HCl was added to the two peptides but not to the MGP. Excess water was pumped off in the probe lock. The same multiplier gain settings were used for each set of comparisons where practical. When the multiplier gain had to be changed, a separate measurement of the gain ratio was made. For measurements of sample signal as a function of time, a repetitive 3-s scan over the MH' region was set up using the peak-matching voltage scan. The output was then recorded continuously on the Gould recorder. The same sample quantity (approximately 0.9 nmol) and matrix were used as for the other experiments. The probe tips used in both sources were very similar in shape, material (copper),and weight (5 g). Only the angle of the tip was different. The same probe body (insulator, shaft, and handle) was used with both sources. Reagents. The MetLys-bradykinin and gramicidin S samples were obtained from Sigma (St. Louis, MO). The MGP (IO)was generously provided by Donald J. Walton (Queen's University, Kingston, Ontario), and the recombinant human insulin (Humulin) was kindly supplied by Ronald E. Chance (Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, IN). Samples were prepared by dissolving weighed amounts in distilled water or methanol. Calibration was achieved by using Ultramark 1621 (PCR, Gainsville, FL).
RESULTS AND DISCUSSION The goal of this work was to design and construct an ion source that would exploit the advantages of the Cs+ primary beam gun to the greatest extent possible. In order to ascertain the degree of success of this undertaking, the relative sensitivity of the LSIMS source was compared with a current commercially available FAB source. Because there were several major differences between the FAB and LSIMS sources, it was not possible to evaluate separately the contribution of each factor; only the overall relative performance of the LSIMS source was determined with respect to the FAB source. No attempt was made to determine detection limits or absolute sensitivities. (Work in progress in this laboratory will address these questions.) There are two different measures of sensitivity that can be used in this case: maximum signal intensity (e.g., maximum MH+ ion current attainable for a given quantity of sample) or integrated intensity (the total number of MH+ ions produced for a given sample quantity). The two sources were compared in both ways. The results of the two comparisons are different because the duration of the sample signal was significantly longer with the LSIMS source than with the FAB source (see below). Table I1 gives the results of the tests of maximum signal intensity. Only the heights of the peaks at the masses indicated in the table were measured. These peaks correspond to MH+ for the two peptides and to [M + Na]+ for the carbohydrate. For each compound the peak heights were normalized to the FAB data so that, for example, the measured peak height of the MH+ ion of gramicidin S at 0.1 MACs+ beam current was 3.3 times the height of the same peak in the spectrum obtained with the FAB source. As the table shows, the LSIMS source gave an improvement over the FAB source in terms of maximum signal intensity
250
LSIMS ( Q ' = l . O r A )
\ I
0
5
10
25
30
TIME (min) Flgure 3. Intensity of MH+ ion of MetLys-bradykinin ( m / z 1320) as a function of time for the FAB source and for the LSIMS source with two different Cs+ beam currents.
of roughly a factor of 2 at 0.1 MACs+ beam current, and about a factor of 5 at the higher Cs+ beam current. I t was observed during the experiments described above that the sample spectra lasted significantly longer in every case in the LSIMS source than in the FAB source. The extent. of the increase in the LSIMS signal duration was a function of the matrix used and a very strong function of the Cs+ primary beam current. Figure 3 shows an example of the effect. In it are plotted the height of the MH+ ion of MetLys-bradykinin as a function of time in the FAB source and in the LSIMS source at 0.1 and 1.0 pA Cs+ beam current. The integrated intensities for the three curves, which are proportional to the sensitivites expressed as coulombs per microgram of sample show that the LSIMS source has an advantage by a factor of 10-20 in this case. Not every sample showed this great an improvement, but on the average, at least a factor of 10 increase in integrated intensity could be achieved. Furthermore, since the Cs+ ion beam current is easily and continuously adjustable, one has the option of exchanging greater intensity for greater persistence as desired. As an example of the effectiveness of the new source, Figure 4 shows a single scan of the molecular ion region of a 1.7-nmol sample of human insulin. For this experiment, the CS' ion energy was increased to 10 kV. The scan rate (300 ppm/s) was such that the portion of the spectrum shown was scanned in approximately 30 s. This scan was acquired after the sample had been in the primary beam for more than 10 min. The long sample spectrum duration is especially useful when multiscan averaging is utilized. Previous workers using a similar Cs+ gun have established that there was little qualitative difference between spectra
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
MH'
Flgure 4. Single scan of molecular ion cluster of human insulin: Cs+ primary beam at 1 pA, 10 kV; scan speed 300 ppmls; accelerating voltage 3575 V; sample quantlty 1.7 nmol; matrix 1:l glycerol thio-
glycerol. obtained using Cs+ and Xeo primary beams (I),and in fact spectra obtained by plasma desorption as well as FAB and LSIMS have all been reported to be remarkably similar (11, 12). Our results agree with previous work (1):no differences between the FAB and LSIMS spectra were observed, apart from very minor variations in relative intensities that were comparable to scan-to-scan variations. There are two major factors that contribute to the enhanced performance of the LSIMS source: the ion optics and the focused primary beam. The use of uniform (8) and shaped (9) extraction fields was reported to improve sensitivity in previous studies. About a factor of 2 increase in sensitivity was observed in the LSIMS source described here when the two tabs forming the trough attached to the source bottom plate (see Figure 2) were added. It seems likely that adding similar focusing optics to the FAB source would produce an improvement in sensitivity, but a quantitative comparison was not possible in this study because of the differences in probe tip angle and primary to secondary beam angle. The indicated total beam current in the FAB source was 40 MA,vs. 1 pA maximum in the LSIMS source. The Cs' beam diameter is 1 mm, and it impinges on the target at an angle of 65O to target normal; therefore, an area of roughly 1 x 2.4 mm is irradiated (2.6 x (ion/s)/cm2) all of which is included in the 3-mm-diameter tip surface. Although some fraction of the XeO beam does not strike the sample, the more intense central portion ( 4 ) does, so the total primary beam current irradiating the sample is expected to be significantly less than 40 pA but still somewhat greater than for the 1.0 pA Cs+ beam. In addition to producing secondary ions, the primary beam can erode the sample surface and cause heating of the probe tip. For example, a 40-pA beam at 8 keV energy represents 0.32 W of power, enough to heat up an isolated 5-g Cu tip at a rate of about 7.5 OC/min. Of course, the entire Xe" primary beam does not strike the tip, and some heat is conducted away by the stainless-steel source block and probe shaft. Nevertheless, the XeO beam used in the FAB experiments has a greater ability to heat the probe tip than the Cs+ beam because it strikes the entire probe tip, not just the sample area. Experimentally, this was confirmed by the observation that the probe tip felt warm to the touch after several to tens of minutes of use in the FAB source, whereas warming was not observed in the LSIMS source with 0.1 pA Cs+ current. Some slight warming was observed with 1.0 p A Cs+ current after longer use h), but this is attributable to conduction and/or radiation from the hot Csf oven mounted nearby (Figure 2). The sample lifetime did not, however, appear to be a simple result of evaporation of the matrix from
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the probe tip. In the experiments illustrated in Figure 3, there was a visible amount of clear viscous liquid material remaining on the probe tip surface after the probe was removed from the instrument. The other effect of the primary beam that may be responsible for the increased sample lifetime with LSIMS is sputtering with preferential loss of surface layers containing increased sample concentrations (13, 14). Experiments described by Stoll et al. (15) using a Xe+ primary beam showed longer sample lifetimes (similar to Figure 3) as well as lower detection limits when a 1-pA focused (0.4 X 1.0 mm at the probe surface) beam was compared with a 9-pA unfocused beam. These authors ascribed both the prolonged desorption time and somewhat higher sensitivity to the fact that the primary beam was only sputtering sample from that portion of the sample surface from which secondary ions are accepted efficiently into the mass analyzer, thereby both minimizing sample loss and permitting sample molecules to diffuse in from nonirradiated areas to replenish the ionization region. Their conclusion was confirmed by noting that there was no difference in signal observed between the focused and unfocused primary beams when a dry NaI sample was used (15).
CONCLUSION A considerable improvement in sensitivity has been achieved over that obtainable with a conventional FAB (XeO) source: up to a factor of 5 in maximum intensity and greater than a factor of 10 in integrated intensity. Both the improved optics and the focused primary beam probably contribute to the enhanced performance. We anticipate that additional refinements of the LSIMS source design will improve its performance still further.
ACKNOWLEDGMENT We thank A. L. Burlingame for his support and encouragement and W. Aberth for useful discussions. Registry No. Cs+, 18459-37-5.
LITERATURE CITED (1) Aberth, W.; Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 5 4 , 2029. (2) Aberth, W.; Burlingame, A. L. I n Ion Formation In Organic Solids: Benninghoven, A., Ed.; Springer-Verlag: Berlin, 1983; Vol. 25, pp 167-171. (3) Ligon, W. V. Int. J. Mass Spectrom. Ion Phys. 1982, 4 1 , 205. (4) Mitchum, R. K.; Freeman, J. P.; Duhart, E. T.; Hunziker, J. Presented at the 32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonlo, TX, May 1984. (5) Commercially available from Antek, Palo Alto, CA. (6) Morita, Y.; Aberth, W.; Burlingame, A. L. Presented at the 32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, May 1984. (7) Falick, A. M.; Walls, F. C., unpublished data, (8) Aberth, W.; Burlingame, A. L. Anal. Chem. 1984, 5 6 , 2915. (9) Todd, P. J.; Glish, G. L.; Christie, W. H. I n t . J. Mass Spectrom. Ion Processes 1984. 8 1 . 215. (10) Forsbeig,-L. S.;'Dell; A.;-Walton, D. J.; Ballou, C. E. J. 6iol. Chem. 1982. 257. 3555. (11) Busch,-K. L.; Cooks, R. G. Science (Washington, D . C . ) 1982, 218, 247. (12) Fohlman, J.; Peterson, P. A.; Roepstorff, P.; Hojrup, P.; Kamensky, I.; Save, G.; Hakansson. P.: Sundauist, B. 6iomed. Mass Soectrom. 1985, 12, 380. (13) Llgon, W. V.; Dorn, S. 9. Int. J. Mass Spectrom. Ion Processes 1984, 57, 75. (14) Ligon, W. V.; Dorn, S. B. Int. J. Mass Spectrom. Ion Processes 1984. 6 1 . 113.
(15) Stoll,'R. G . ; Harvan, D. J.: Hass, J. R. Int. J . Mass Spectrom. Ion Processes 1984, 61, 71.
RECEIVED for review October 28, 1985. Accepted February 3, 1986. This work was supported by a grant from the Committee on Research of the UCSF Academic Senate (to A.M.F.) and by the NIH Division of Research Resources Grant RR01614 (A. L. Burlingame, principal investigator).
