Anal. Chem. 1987, 59, 999-1002
(11) Holt, B. D.; Kumar, R. I n Fossli Fuels Utlllration Environmental Concerns; Markuszewski, R., Blaustein, B. D., Eds.; ACS Symposium Series 319; American Chemical Society: Washington, DC, 1986; pp 277-283. (12) Voihov, I. I.; Tsentsiper, A. B.; Chamova. V. N.; Latysheva, E. I.; Kuznetsova, 2 . I. Russ. J . Phys. Chem. (Engl. Trans/.) 1964, 38, 645-648. (13) Wok, B. D.; Kumar, R. Atmos. Environ. 1984, 18, 2089-2094. (14) Holt, B. D.; Kumar, R.; Cunningham, P. T. Science (Washington, D . C . ) 1982, 2 1 7 , 51-53. (15) Holt, 6. D.; Nielsen, E.; Kurnar, R. In Precipitation Scavenging, Dry Deposition, and Resuspension; Pruppacher, H. R., Semonin, R. G., Siinn, W. G. N., Eds.; Elsevier: New York, 1983; Vol. I , pp 357-368. (16) Zika, R.; Saltzman, E.; Chameides, W. L.; Davis, D. D. J . Geophys. Res. 1982, 87, 5015-5017.
999
(17) Yoshizumi, K.; Aoki, K.; Nouchi, I.; Okita, T.; Kobayashi, T.; Kamakura, S.; Tajima, M. Atmos. Envlron. 1984, 18, 395-401. (18) Schone, E. 2. Anal. Chem. 1984, 33, 127. (19) Lazrus, A. L.; Kok, 0. L.; Gltiln, S. N.; Lind, J. A.; McLiaren, S. E. Anal. Chem. 1985, 57, 917-922. (20) Klockow, D.;Jacob, P. Chemistry of Multiphase Atmospheric Systems; NATO AS1 Series, Vol G6; Springer-Veriag: Berlin, 1986. (21) Kok, G. L.; Thompson, K.; Lazrus, A. L. Anal. Chem. 1986, 58, 1192-1194. (22) Lazrus, A. L.; Kok, G. L.; Lind, J. A,; Gitlin, S. N.; Heikes, B. G.; Shetter, R. E. Anal. Chem. 1986, 58, 594-597.
RECEIVED for review May 30,1986. Accepted November 7, 1986.
Liquid Secondary Ion Time-of-Flight Mass Spectrometry James K. Olthoff, Jeffrey P. Honovich, and Robert J. Cotter* Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, Maryland 21205
A pulsed ion beam gun is interfaced to a pulsed drawout (Wiiey/McLaren) type thesf-flight analyzer. The field-free ion source is well-adapted to the use of a liquid matrix. Large primary ion pulses are used and the resulting secondary ions are recorded by us8 of fast analog to dlgltai techniques. Time delays between ion formation and extraction from the source region improve focusing and allow observation of fragment ions from metastable decompositions.
The use of primary ion beams, with energies in the kiloelectronvolt range, for the desorption of intact molecular ion species from nonvolatile organic compounds began with the introduction in 1970 of “molecular” or “static SIMS” (secondary ion mass spectrometry) by Benninghoven ( I , 2 ) . Primary ion current densities on the order of 1nA/cm2 used in this technique result in less sample damage and avoid the charging effects of “dynamic SIMS” which employs current densities of 1 pA/cm2 or more (3, 4 ) . Because of the low secondary ion currents which are a consequence of this approach, the use of time-of-flight analyzers, which have high ion transmission and the ability to record ions of all masses simultaneously, is particularly advantageous. In the SIMSTOF instrument first reported by Chait and Standing ( 5 ) ,a 1-nA beam was deflected onto the sample surface for approximately 10 ns, producing secondary ions which could be recorded by single ion counting techniques. The fast atom bombardment (FAB) technique employs primary neutral atom currents that are of the order of those used in dynamic SIMS (6),but the sample damage is reduced through the use of a liquid sample matrix (7,8). The use of neutral atoms is not essential (9),but convenient for highvoltage ion sources of double-focusing sector instruments on which the technique has been most successfully employed. Thus FAB has also been termed ”liquid SIMS” (IO). Recently we introduced the possibility of combining the FAB, or liquid SIMS, technique used on scanning instruments with the time-of-flight analyzer (11). Primary ion currents of 1pA were directed at a liquid sample in a field-free source for periods of 5 ps. This liquid SIMS-TOF instrument is also referred to as a “high flux TOF”, since the large primary ion pulse produces conditions which are similar to that of dynamic
SIMS, with secondary ion currents which are best recorded by analog (rather than ion counting) techniques. Spatial and energy focusing of secondary ions emitted from the less-defined surface are achieved by time-lag focusing as described by Wiley and McLaren (12). In this paper, the addition of fast (100 MHz), repetitive (50 spectra/s) transient recording techniques, postacceleration detection, optimization of primary ion pulse characteristics, and extension of the drawout pulse width, which leads to mass ranges above 13000 amu are reported. In addition, extension of the time-lag focusing technique provides a means for monitoring metastable fragmentation.
EXPERIMENTAL SECTION The time of flight mass spectrometer is a standard CVC (Rochester, NY) Model 2000 electron impact mass spectrometer with a 1-m fight tube, which has been modified as described below as to carry out high-flux SIMS measurements (Figure 1). A Kratos (Ramsey, NJ) Minibeam I ion gun has been fitted to the source chamber through a standard 2.75411. conflat port. The ion gun consists of an electron impact source and lens elements for accelerating, focusing, and rastering of the ion beam. The beam is focused onto a direct insertion copper probe tip located 2 in. from the front of the ion gun. A Keithley 410B electrometer connected to the probe is used to measure the average ion current reaching the probe tip. An additional 4-in. diffusion pumping system has been added to the source chamber for differential pumping of the ion gun. Argon or xenon gas pressures of up to 1 mtorr can be maintained in the ion source of the gun, while pressures of lo4 torr or less are maintained in both the source and analyzer regions of the mass spectrometer itself. In the Minibeam Control Unit, the emission regulator circuit has been replaced with a pulse amplifier which floats at the accelerating voltage. The 30-V electron grid pulse from the CVC timing circuitry is transmitted through a capacitor (680 pF, 6 kV) and amplified to 100 V. The pulse width can be varied from 1 to 10 ps and is used to control the grid to filament voltage of the ion gun source to turn the ion beam on and off. Following the primary ion pulse, the secondary ions are extracted by two negative voltage pulses (-150 and -300 V) applied, respectively, to the drawout and fiist acceleration grids. The rise time of the drawout pulse is 40 ns. The “time lag focusing” circuit on the CVC instrument has been modified to permit time delays of up to 20 ps between the primary ion beam pulse and the drawout pulse. In addition, the drawout and accelerating pulses have been lengthened to 8 ps in order that heavy ions leave the source with the full extraction field. The ions are accelerated t o their full
0003-2700/87/0359-0999$01.50/00 1987 American Chemical Society
1000
ANALYTICAL CMMISTRY. VOL. 59. NO. 7. APRIL 1. 1987
MASS
rl a
CYCLOSWRIN A FA0
Figure 3. TOF mass spectra of leu-enkephalh obtained (a) without and (b) wlth a iiqum mmx.
1203
CYCLOSWRM A La-SIMS-TOF
.
-
MASS F@n 2. Canprlson 01 lguid SIMS mass spectra ot c y c h p c h A 0btah-d (a)on a Kratos MS-50 doubb-fmushg mass spectrometer and (b) on me nqu!d SIMS-TOF instrument.
energy by two additional grids a t -1.5 and -3 kV, respectively. Horizontal and vertical deflectors are used to center the m n d a r y ion beam on the detector. The standard magnetic electron multiplier has been replaced with a Galileo (Sturbridge. MA) FTD 2003 dual channel plate detector. The front of the detector ia placed at -12 kV to provide postacceleration of the ions into the detector. Secondary electrons are colleded at ground potential on a collector anode terminated with a 504 connection to ground. While tuning the instrument. the signal generator is set a t 1 kHz and the spectrum is observed on an oscilloscope connected to the collector anode. The oaeilloseope is triggered by the drawout pulse. Horizontal, vertical deflection, ion focus (accelerating voltage pulse). %me lag focus" (delay time), and primary ion pulse length are adjusted for best resolution a t high mass using CsI clusters. In normal operation the repetition rate is set a t 50 or 100 Hz, and the collector anode signal is connected to a LeCroy (Spring Valley, NY) 6102 amplifier/attenuator, digitized by a LeCroy TR8818 100-MHz transient recorder and stored as 8K by 8-bit words on a LeCroy MM8103A memory module. The transient recorder is triggered by the drawout pulse. Successive spectra are added on a Lecroy 3500SA signal averaging system. Generally, loo0 spectra are obtained, requiring 10 to 20 9. Data are processed (smoothed, centroided. and mass calibrated) and plotted on the LeCroy signal averaging computer. Samples were dissolved in methanol, methanol/water, or methanol/HCI solutions, mixed with glycerol/thioglyceroI and
Flgu18 4. Oscilloscope @ace01 Csl clusters obtained at rate of 1 kH2 used fw tuning mass spectrometer.
a repetition
deposited on the probe tip. Delay times were adjusted for optimal focusing. or varied between 5 and 20 ps for metastable decomposition/fragmentation studies.
RESULTS AND DISCUSSION T h e cyclic peptide, cyclosporin A, produces a prominent protonated molecular ion peak and is used to provide a calibration peak for time-of-flight measurements. Figure 2 compares the spectrum obtained by the liquid SIMS-TOF instrument with that obtained by fast atom bombardment on a sector instrument. Similar fragment ions are observed in both spectra, and sensitivities are comparable. Under high flux conditions, small peptides produce good spectra dry or in a glycerol/thioglyceroI matrix, as is shown in the spectra of leu-enkephalin (mol w t 556) in Figure 3. With the liquid matrix, the protonated molecular ion signal is enhanced, and peaks due to the matrix are now observed. The liquid matrix also produces sample ion signals for much longer periods of time (several minutes), while dry sample signals last only seconds, presumably due to sample damage. The spectrum of dry CsI obtained in the tuning mode is shown in Figure 4 as a photographic recording of the oscilloscope trace when the ion gun is operated a t a repetition rate of 1 kHz. Peaks are observed out to 5894 amu. Signal averaged spectra. using the LeCroy, increase the dynamic range to allow for observation of cluster peaks beyond 13000 amu (Figure 5). Campana et al. (13),using a sector instrument, reported that spectra of CsI exhibited relatively high abun-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
1001
rmlnal Fragmanls n;26
CESUM IODIDE
a. MASS
Figure 5. Mass spectrum of dry CsI clusters obtained with the LeCroy signal averaging system. 10 000 spectra were add/averaged. The ion gun frequency was 100 Hz with a time lag of 20 ps.
EI
,120
1 -
>
c ;j io-' z W c
z
w
b. '
io+.
C. x tops 0 20ps
16'
'
h'd
285
b
' 6 ' 'ib'1L CLUSTER NUMBER
'Ib '16'118
Figure 6. Relative CsI cluster intensities as a function of cluster number, at three different time delays between the ionization and drawout pulse.
dances for certain cluster numbers (n = 6, 9,13, etc.), representing particularly stable ion structures. However, Standing and co-workers (14),using a SIMS-TOF instrument with prompt extraction, observed a smoother decrease in intensity with increasing cluster number. The difference in spectra observed on those two instruments arises from metastable fragmentation of some clusters into more stable ones. Since ions fragmenting in the flight tube will be recorded as parent ions (15),the prompt extraction time-of-flight analyzer records the ion distribution present only at the end of their residence time in the source, a few nanoseconds after ion formation. However, the liquid SIMS-TOF instrument can be used to directly observe metastable decay in the microsecond time scale by changing the time delay between the primary ionization pulse and the ion extraction pulse. The results presented in Figure 6 compare relative ion cluster intensities at different delay times. The cluster ion intensities at 5 ps resemble those obtained by Standing, while those at 20 pus resemble more closely those obtained on sector instruments. The introduction of a time delay between ion formation and extraction (mass analysis) has interesting effects on the mass spectra, particularly with respect to the appearance and relative intensities of fragment ion peaks. Figure 7a shows the mass spectrum of a pentapeptide containing all phenylalanine residues obtained 10 ps after the primary ion pulse. By use of the notation suggested by Roepstorff (16),all of the A and B (C terminal) and Y" (N terminal) sequence ions are observed, with those of lower mass having larger relative abundance. Increasing the time delay to 15 or 20 ys (Figure 7b,c) decreases the observed relative abundances of the low mass fragment ions compared with the protonated molecular ion intensity. In the field-free ion source, ion velocities are dependent only upon their mass (m)and initial kinetic energy (KE), so that the average time before the ion drifts toward
MASS Flgure 7. Liquid SIMS-TOF mass spectrum of the peptide penta-cphenylalanine at (a) 10 ps, (b) 15 ps, and (c) 20 ps time delay.
I
r5
OXYTOCIN
A
MASS
7241
Cys-Tyr-lle-Gln-Asn-Cys Pro-Leu-GlyNH,.H' C
285
Figure 8. Liquid SIMS-TOF mass spectrum of oxytocin.
the wall of the source at a distance, d, from the ionization region is
t = d(m1/2KE)~/~ This has been measured experimentally to be about 1 ps for m / z 28 at thermal velocities (17). One would expect average lifetimes of 3.2 and 10 ps for ions of masses 300 and 3000, respectively. The time delay thus discriminates against ions of low mass but also against ions with high initial kinetic energies. While increases in time delay generally improve focusing of ions of higher mass (12),the fact that the peak widths of the low mass ions in Figure 7 are narrower at long delay times suggests that many of these ions were formed with high kinetic energy and are the result of higher energy ("hard") desorption events. Unlike the above example, the peptide, oxytocin, shows a pronounced preference for fragmentation at the N-terminal
1002
1
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
MELITTIN MASS
Figure 9. Liquid SIMS-TOF mass 20-ps delay time.
spectrum of melliiin obtained at
end of the proline residue, resulting in either an N-terminal or C-terminal ion (Figure 8). This cleavage is adjacent to a cyclic structure (formed by the disulfide bond between two cysteines). Also, the amide bond on the N-terminal end of proline is generally longer and weaker than that of other residues, as the five-membered ring in the proline structure forces this residue to bend out of the normal a-helical or @-sheetconfiguration of the peptide (18). Spectra of high mass peptides, obtained on prompt extraction time-of-flight analyzers, are generally characterized by a large base line rising at lower mass, a molecular ion peak, and very few fragment ion peaks, leading to the suggestion that one can only measure molecular weights by such methods. The large base line can be attributed in part to metastable fragmentation which occurs during ion acceleration in the source leading to indeterminate flight times (19). Making use of the field free source and delayed ion acceleration, we attempted to hold the ions produced from the desorption of melittin in the source until much of the metastable fragmentation had occurred. At 10-ps time lag, peaks corresponding to fragment ions could be observed above the base line and were more pronounced a t 20 ps (Figure 9). While the peaks are not well-resolved, they are reproducible and represent real fragmentation processes which occur in a time frame observable on instruments which provide prompt acceleration only by the use of retarding grids (20)or ion mirrors (21, 22). It is interesting to note that the increase in delay time has the opposite effect (reported in Figure 7) of enhancing the fragment ions, since the time frame for fragmentation is now on the same order as the delay time. In addition, the major fragmentation o c c w at the amide bond between leucine and proline.
CONCLUSION In the fast atom bombardment technique, the composition of the liquid matrix has profound effects on the secondary
ion yield. Desorption of protonated molecular ion species is enhanced by the addition of acids to glycerol (23) or by the use of thioglycerol (24) or a mixture of dithioerythritol and dithiothreitol (25) as matrices. The relative intensities of singly and multiply charged molecular ions for insulin have been shown to follow directly the pH of the matrix (26)and suggest the importance of considering solution pK,'s and isoelectric points (PI) in promoting desorption of either positive or negative ions. Thus strong bases, diethanolamine and triethanolamine, are suitable matrices for the desorption of negative (M - H)-ions, and addition of ammonium or alkali metal salts to the matrix generates positive (M + NHJ+ and MNa+ ions from samples which are not sufficiently basic to be protonated. The instrumental combination described above, therefore, brings to time-of-flight mass spectrometry the ability to exploit these and other properties of the liquid matrix.
LITERATURE CITED Benninghoven, A. Z.Phys. 1970, 230, 403. Benninghoven, A.; Jaspers, D.; Sichtermann, W. Appl. Phys. 1978.
11,35. Colton. R. J. J. Vac. Sci. Techno/. 1981, 18,731. Turner, N. H.; Colton, R. J. Anal. Chem. 1982, 54,293R. Chait, B. T.; Standing, K. G. I n t . J. Mass Spectrom. I o n Phys. 1981, 40. 185. Ross, M. M.; Wyatt, J. R.; Colton, R. J.; Campana, J. E. I n t . J. Mass Spectrom . Ion Processes 1983, 54 237. Surman, D. J.; Vickerman, J. C. J. Chem. Res., Synop. 1981, 6, ~
170. Barber, M.; Bordoli, R. J.; Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54,645A. Aberth, W.; Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 54,
2029. Burlingame, A. L.; Baiiiie, T. A.; Derrick, P. J. Anal. Chem. 1988, 58, 165R. Cotter, R. J. Anal. Chem. 1984, 56, 2594. Wiley, W. C.; McLaren, I.H. Rev. Sci. Insfrumen. 1955, 26, 1150. Campana, J. E.; Bariak, T. M.; Colton, R. J.; DeCorpo, J. J.; Wyatt, J. R.; Dunlap, 8. I.Phys. Rev. Lett. 1981, 47, 1046. Ens, W.; Beavis, R.; Standing, K. G. Phys. Rev. Lett. 1983, 50, 27. Schueler, B.; Beavls. R.; Boibach, G.; Ens, W.; Main, 0.E.; Standing, K. G. I n Secondary I o n Mass Spectrometry SIMSV; Colton, R. J., Ed.; Sprlnger-Verlag: Berlin, 1986 pp 57-59. Roepstorff, P.; Folman, J. Biomed. Mass Spectrom. 1984, 11, 601. van Breemen, R. B.; Snow, M.; Cotter, R. J. I n t . J. Mass Spectrom. I o n Phys. 1983, 49, 35. CantW, C. R.; Schimmel, P. R. Biophysical Chemistry; W.H. Freeman: San Francisco, CA, 1980;Vol. I, pp 270-272. Chait, 8. T. I n t . J. Mass Spectrom. I o n Phys. 1983, 53, 227. Chait, B. T.; Field, F. H. I n t . J. Mass Spectrom. I o n Phys. 1981, 41,
17. Della-Negra, S.;LeBeyec, Y. Anal. Chem. 1985, 57, 2035. Standing, K. G.;Ens, W.; Beavis, R.; Bolbach, G.; Main, D.; Schueler, B.; Westmore, J. B. I n I o n Formation from Organic Solids IFOS I I I ; Benninghoven, A., Ed.; Springer-Verlag: Berlin, 1986; pp 37-41. Malorini, A.; Marino, G.; Milone, A. Biomed. Mass Spectrom. 1988, 73, 477. Fenselau, C. Anal. Chem. 1982, 54, 104A-IllA. Finke, J.; Oroege, M. W.; Cook, J. C.; Suslick. K. S. J. Am. Chem. SOC. 1984, 106,5750-5753. Schronk, L. R.; Cotter, R. J. Biomed. Environ. Mass Spectrom. 1988,
73.395-400.
RECEIVED for review July 21, 1986. Accepted December 8, 1986. Research was supported in part by a Grant, GM 33967, from the National Institutes of Health.
