Anal. Chem. 1993, 65, 1027-1033
1827
Broad-Band Excitation in the Quadrupole Ion Trap Mass Spectrometer Using Shaped Pulses Created with the Inverse Fourier Transform Randall K.Julian, Jr., and R. Graham Cooks. Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
This paper reports on broad-band excitation of ions in the quadrupole ion trap mass spectrometer (ITMS) using shaped pulses. In place of a singlefrequency excitation signal, applied to the end caps of the ITMS, a shaped pulse which excites a broad spectrum of frequencies is used. Shaped pulses are time domain signals created by taking the complex inverse Fourier transform of a frequency domain function whose magnitude represents the desired excitation spectrum. In mass spectrometry these signals are termed SWIFT (stored wave form inverse Fourier transform) pulses. By selection of a frequency spectrum which includes ion secular frequencies, SWIFT pulses can be constructed to excite a wide range of m l z values in the quadrupole ion trap. Using the phase modulation method described by Chen et al., the frequency domain spectrum is converted to a complex function prior to being transformed to the time domain. The time domain signal is then processed and loaded into an arbitrary wave form generator (ARB) connected to the end-cap electrodes and applied in a dipolar fashion. Three basic applications of SWIFT pulses are demonstrated in the quadrupole ion trap: (i)broad-band ejection of desorbed matrix ions by application of SWIFT pulses during ion injection from an external source, (ii) broad-band ejection of trapped ions for selective ion isolation, (iii) broad-band excitation which results in collision-induced dissociation (CID) of selected ions. Applying SWIFT pulses while ions are being injected from a Cs+ desorption source results in ejection of matrix ions, which reduces space charge and greatly improves parent ion intensity and overall sensitivity. SWIFT pulses are effective at ejecting ions which have been stored for ion isolation, and the method shows good mass resolution.
INTRODUCTION Manipulation of the frequency domain of excitation energy has been an important step toward improved performance in both FT-NMR1 and FT-ICR293experiments. Shaping pulses has become a very powerful method for specifying the frequency content of excitationenergy acrossa wide frequency range.‘ When the desired sample excitation profile is linear (l)Tomliion,B. L.; Hill, H. D. W. J. Chem. Phys. 1973,59, 1776. ( 2 ) Marshall, A. G.; Wnng, T.4. L.; Ricca, T. L. J. Am. Chem. SOC.
1985,107,7893. (3) Wang, T.-C. L.; Ricca, T. L.; Marshall, A. G. A n d . Chem. 1986,58, 2936.
(or nearly so), with the magnitude spectrum of the excitation energy, the inverse Fourier transform can be used to create the pulse. When the relationship between the frequency spectrum of a pulse and the sample excitation profile is not linear, there are other numerical approaches to creating pulses.4 In the case of the FT-ICR experiment, there is a direct relationship between the postexcitation ion cyclotron radius and the excitation spectral magnitude at the cyclotron frequency. Using this relationship, Marshall and co-workers synthesized excitationpulses for the FT-ICR using the inverse Fourier transform with excellent results.2.3 The use of Fourier synthetic methods to create shaped pulses for the FT-ICR has been termed SWIFT (stored wave form inverse Fourier transform).3 SWIFT pulses have a more uniform excitation distribution than is possible with either rectangular pulses or swept frequency chirps.”7 In FT-ICR instrurnenta,therefore, shaped pulses are used to create an excitation signal which has a very flat energy distribution as a function of ion frequency. SWIFT is also useful in selectively ejecting ions for MS” experiments or selectively exciting ions for CID experiments. In the magnetic (ICR) ion trap, stored ions move in trajectories with cyclotron frequencies which are directly related to the mass-to-chargeratio (mlz)of the ions. It is by coupling the pulse energy to this motion that excitation is accomplished in FT-ICR. In radiofrequency quadrupole ion traps, ion motion is complex with trajectories containing a large number of frequency components. The largest amplitude frequency component is termed the secular frequency of motion, which is similar to ion cyclotron motion in the ICR in that it is mass dependent.3 By applying an excitationsignal to the end caps of the quadrupole ion trap (viz. along the z axis) and matching ita frequency to the secular frequency of motion in the z direction for a given ion, it is possible to cause resonant excitation. This may lead either to collision-induced dissociation (CID) or, a t a sufficient amplitude, to ion ejection. Resonant excitation has been widely studied and is covered in a broad review of ion trap mass spectrometry by March and Hughes.9 Typically, the excitation energy used contains a single frequency component a t a selected amplitude, giving a very narrow band of excitation. McLuckey and co-workers have demonstrated the use of swept frequencies in the quadrupole ion trap to cause excitation10 and also filtered noise signals to cause broad-band activation.11 Vedel et al. also reported the use of broad-band excitation in a quadrupole (4) Warren,W. S.; Silver, M. 5.Adu. Mag. Reson. 1988,12, 247. (5) Comkarow, M. B.; Marshall, A. G. Chem.Phy8.Lett. 1974,26,282. (6) Comiearow,M.B.; Marshall, A. G. Chem. Phys. Lett. 1974,26,489. (7) Groshana, P. B.; Marshall, A. G. A d . Chem. 1991,63,2067. (8)Fischer, E. 2.Phys. 1969, 156, 26. (9) March, R. E.; Hughes, R. J. Quadrupole Storuge M w Spectrometry; Chemical Analysis Series 102; Wiley: New York, 1989. (10) McLuckey, S. A.; Goeringer, D. E.; Glieh, G. L. J. Am. SOC.M u s Spectrom. 1991,2, 11. (11) McLuckey, S. A.; Goeringer, D. E.; Glieh, G. L. 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington DC, 1992, p 1013.
0003-2700/93/036C1827$04.00l0 0 1993 American Cbmlcal Soclety
1828
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
ion trap12 using a swept frequency generator13 that includes a frequency randomizer to reduce memory effects created by a monotonic frequency sweep. This paper reports on broadband excitation signals created using the inverse Fourier transform method (SWIFT). With this method, more control over the frequency content of the excitation energy is possible than with the previously demonstrated broad-band techniques. The experimental apparatus required to use these pulses with the quadrupole ion trap is described and several applications are presented.
of the ion in (a, q) space and not on the initial ion conditions. To compute the ion frequency, p, can be computed using the following continuing fraction? p,2
= a,
+ " 2
+
VU
4 u2 - 2
THEORY
(P, - 2 T - cy,
Resonant excitation in the quadrupole ion trap is performed by applying an auxiliary signal containing a frequency component which couples to the secular motion of a stored ion. When this signal is applied in dipolar fashion to the end-cap electrodes of the device, the secular frequency of motion in the z direction is used as the excitation frequency. SWIFT pulses are generated by computing the secular frequency in the z direction for all the mlz values to be excited a t fixed rf and dc levels. A frequency domain spectrum,which includes all of these frequencies, is then created. Ions move within the quadrupole ion trap according to the Mathieu equation:14 (d2U/dt2)+ (a, - 2q,
COS
2F)u = 0
(1)
where, for the end-cap grounded operating mode, a, and q, are defined as15
+ 22,2)Q2 = -8eV/m(r,2 + 2 2 2 ) ~ '
a, = -2a, = -16eU/m(r,2 qr = -2q,
(2)
(3)
In these equations u corresponds to an axis of motion in cylindrical coordinates (r, z ) , U is the dc potential, V is the amplitude (0 to peak) of the rf potential applied to the ring electrode, and e is the electric charge of the electron. Here m is the mass-to-charge ratio (mlz),ro is the internal radius of the ring electrode, and zo is the inscribed radius of the end-cap electrodes. 5 is the generalized time term, 5 = Qti2, where 52 is the rf drive frequency. Equations 2 and 3 depend on the form of eq 1, which is derived from the quadrupole potential equation. Because various sign conventions can be used in eq 1 and because there are several operating modes which change the magnitude of the quadrupole potential, different expressions for a and q can be found in the literature and care must be taken when numerical values are being computed. Ion secular frequencies may be computed from the solution of eq 1, which has been given by McLachlan:15
(P, - 4)' - a , - qU2/((p,- 6)' - a, - ...etc.)
From eq 4, the secular frequency, urdescribed by n = 0, is given as u, =
(@,/an
(12)Vedel, F.;Vedel, M.; March, R. E. Int. J . Muss Spectrom. Ion Processes 1991, 108, R11. (13)Vedel, F.;Vedel, M.; March, R. E. Int. J . Muss Spectrorn. Ion Processes 1990, 99,125. (14)McLachlan, N. W. Theoryund ApplicutionofMuthieuFunctions:
Dover: New York, 1964. (15)Knight, R. D.Int. J . Mass Spectrom. I o n Phys. 1983, 51, 127.
(6)
Equation 5 can be easily implemented in a computer program16 to allow values for p, to be computed for any value of a, and qz with arbitrary accuracy. Once the ion frequency has been computed for all the ions in the range to be excited using eq 6, the frequency domain spectrum is constructed to include these frequencies. It should be noted that the magnitude of the ion excitation will be linear with ion frequency only within limits. As proposed by Demelt,17the motion of an ion at qz < 0.4 can be approximated by a harmonic oscillator which will respond linearly with frequency. For qz > 0.4, the ion response is nonlinear and can only be approximated as linear over narrow regions. The nonlinearity of ion excitation has implications in experimental design; however, it poses no difficulty for most practical experiments involving broadband excitation. Such a spectrum is shown as a magnitude-frequency plot in Figure la. The frequency spectrum is transformed to the time domain using the inverse Fourier transform (IFT),which is carried out using an adaptation of the Cooly-Tukey fast fourier transform algorithm.lS The transform algorithm used generates output which must be reflected about the N / 2 axis to generate the actual time domain signal.18 Figure l b gives the reflected IFT of the magnitude spectrum of Figure la. As shown in Figure l b , the narrow width of this time domain signal is poorly suited for excitation in the ion trap mass spectrometer. A large amplitude would be required to obtain the power required for excitation using this pulse. The solution to this problem, developed by Chen et al.,19 is to convert the magnitude spectrum in (la) to a complex, phasemodulated signal as shown in Figure IC. The real, &, and imaginary, Ii, components are created from the magnitude data, Magi, using the following relationship: R, = Mag, cos di
Zi = Magi sin 4i Here u(5) describes the path of the ion along an axis ( r ,z ) as a function of generalized time. A andE are coefficients which are determined from the initial position and velocity of the ion. The Cz, coefficients describe the amplitude of the individual frequency components of the motion, and 0, is a parameter which describes the actual frequencies of those components. Czn and p, depend only on the working point
(5)
Yu -
Where
$~i
(7)
varies quadratically with frequency: @i= 4o + Ji
+ (K/2)i2
(8)
Here &is the initial phase, i is the frequency index (frequency data array element number), and J and K are the quadratic terms. J a n d Kmust be chosen to satisfy the Nyquist criteria, such that the rate of phase change per frequency domain data point is less than ?r.l9 Chen et al. observed the best (16) Louris, J. N. QCALCS Program, unpublished work based on: Lawson, Todd, J. F. J.; Bonner, R.J. Dyn. Muss Spectrom. 1975,4,46. (17) Demelt, H. G. Ado. Mol. Phys. 1967, 3, 53. (18)Embree, P.M.; Kimbel, B. C. Language Algorithms for Digital Signal Processing; Prentice-Hall: Englewood Cliffs, NJ, 1991. (19) Chen,L.;Wang,T.-C.L.;Ricca,T. L.;Marshall,A.G.Anal. Chem. 1987, 59,449.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993 1829 Normal Pulse
Phase Modulated Pulse
Frequency Domain
F v q u n c y Ooman (real)
0
I
Inverse Fourier
zm Hn
IW
Inverse~ourier Inverse Fourier
Transform
Reflected at N/2
Reflected at Nf2 T-~-
400 500 600 100 Frequency (LHz)
Mo
9ca
1 m ,100
I
I
Normol Pulse
Phose Modulated Pulse
Time Domain
Time DDmom
100 75
.
h'
Y
"
'0.
M
3
E
25
0 -lM 1 ._
-25
0
Time (ma)
1
2
3
4
5
5
7
l m e (ms)
Flgurr 1. Comparison betwen normal and phase-modulated frequency domain data for creating SWIFT pulses. The frequency domain is first specifled for a SWIFT pulse (a). If the magnitude spectrum Is transformed via the inverse Fourier transform directly, the result Is a narrow sync function In the tlme domain (b). If the magnitude spectrum Is phase modulated before the transform (c), the transformed signal (d) is broader,
requiring less amplitude to achieve the same power.
results when the rate of change per data point was kept at or below half the Nyquist limit.1S All pulses generated for this study followed this guideline, selecting an initial phase ($0) of zero, J = 0 . 5 and ~ K = -l/N,, where N,is the number of nonzero data points in the frequency spectrum. The real component of the complex functionresulting from eq 7 is shown in Figure IC,and Figure I d gives the real component of the reflected complex IFT. The time domain signal transformed from the phase-modulated pulse has a much improved active signal width, allowing more control over the excitation power applied;however, as shown in Figure 2a, it does not terminate at zero and must be windowed to eliminatespectralleakage.lsJ9 A quarter-wave sine function,lg which is shown in Figure 2b, is used which consists of a sine function scanned from Oo to 90° during the first quarter of the signal time, a dc portion during the second and third quarters of the signal time with a value of 1, and a cosine function scanned from Oo to 90" during the fourth quarter of the signal time. As shown in Figure 2c, this window forces the time domain signal to start and end with a zero amplitude and gives no attenuation or phase shift during the central portion of the signal giving maximum signal power. (20) Kaiser, R. E., Jr.; Cooks, R. G.; Stafford, G. C., Jr.; Syka, J. E. P. Int. J. Mass Spectrom. Ion Processes 1991,106,79. (21) Louris,J. N.; Cooks,R. G.; Syka,J. E. P.; Kelley, P. E.; Stafford, G.C.; Todd, J. F. J. Anal. Chem. 1987,59,1677. (22) Julian, R. K.; Rieser, H.-P.;Cooks,R. G. Znt. J. Mass Spectrom. Ion Processes, in press. (23) Kaiser, R. E., Jr.; Louris, J. N.; A m y , J. W.; Cooks, R. G . Rapid Comm. Mass Spectrom. 1989,3, 225.
EXPERIMENTAL SECTION The time domain signal is downloaded into an arbitrary wave form generator (ARB) (Wavetek Model 75, Wavetek San Diego, San Diego, CA). The Model 75 ARB has a 12-bit DAC output with a maximum amplitude of 5 V,,, operating at a maximum sample rate of 2 MHz. The output of the ARB is connected to the ion trap mass spectrometer, (ITMS, Finnigan, San Jose, CA) as shown in Figure 3. Because it is also useful to apply a singlefrequencyITMS ac signal during portions of the rf scan,an analog switch circuit is used to choose the source of the signal applied to the end caps. The user controls the time at which the ARB output is applied by activating the switch with a TTL signal originating from the ITMS electronics. Due to signal loss within the analog switchand amplitude limitations of the Wavetek Model 75, a broad-band amplifier is used to amplify the signal prior to being split by the balun.20 The maximum amplitude available from this hardware is 30 V,,, well above threshold required for resonant ejection. The amplified signalis passed through a balun to apply the signal between the end caps in a dipolar fashion. The ITMS used in these experiments is a prototype instrument which is described elsewhere.21For computation of q. values the dimensions ro and zo were obtained from the ITMS electrodes.22 Measured dimensions used in all calculations are ro = 1.000 cm and zo = 0.7811 cm. Ions are created externally by a 7-kV Cs+ desorption source and injected via a lens system which has also been described elsewhere.23 For the experiments using the peptide substance P, a solid substance P sample was obtained commercially (Sigma Chemical Co., St. Louis, MO). The solid was dissolved in a 5050 water-methanol to form a 371 pmol/pL solution. This solution was diluted by mixing 85% methanol-
1830
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993 P h c s e h."od,la:ec
Dulse
T i m e Doma " ~
I
..
-. u
2-
ARE TRIGGER
-
80 60 -
5
-
I
I AREOUT
AC
PC
40 -
200 -
TRIGGER
I
Analog Switch
-20 -43 -
-60
-
-eo -100
Balun
1
Flgure 3. Hardware setup for applying SWIFT pulses to the end caps of the ion trap mass spectrometer In a dlpolar fashlon. TTL pulses produced by the prototype ITMS electronics are used to activate control circuitry and trigger an arbitrary wave form generator.
,
-60 -80
1
1
-
-inn.
I
"l
C
1
2
3
4 Time
5
6
7
8
(TPS'
Figure 2. Inverse Fourier transform of the phase-modulated pulse. The IFT does not terminate at zero amplltude (a). Therefore, the quarter-wavesine window (b)from Chen et al.leIs usedasan apodizatlon functlon. Apodizing with this function reduces spectral leakage in the final wave form (c)by reducing Its amplltude to zero at the start and finish of the signal without attenuating the active portion of the pulse.
water, 5% glacial acetic acid, and 10% sample to produce the 37.1 pmol/pL sample solution used in the experiments. The experiments using bradykinin (Sigma Chemical Co.) followed the same sample preparation method; however a 97.1 pmol/pL solution after dilution was used. On a gold-plated probe tip, 1 pL of a 5050 glycerol-thioglycerol matrix was mixed with 1 FL of the peptide sample. For the experiments using Cs,+lIn+ clusters, a solid CsI sample was obtained commercially Aldrich Chemical Co., Milwaukee, WI), dissolved in water, and applied to the gold-plated probe where the solvent was evaporated in vacuo.
RESULTS AND DISCUSSION Selective, broad-band excitation of ions in the ion trap is useful for performing several different experiments where a wide range of masslcharge ratios need to be excited. Using large-amplitude SWIFTpulses (>2 Vhp), broad-band ejection
of ions is accomplished. Such SWIFT pulses can be applied during ionization to selectively trap ions of interest while ejecting matrix ions, thus increasing the analyte signal without exceeding the overall space-charge limitations of the instrument. Ejection pulses may be applied at any time during the rf scan to perform broad-band isolation. At lower amplitudes, SWIFT pulses can be used to resonantly excite a broad range of mlz values so causing CID. Broad-Band Ejection during Ion Injection. Peptide sample ions which are formed during desorption ionization are co-injected with large numbers of matrix ions.22 Because of space-charge,the total number of ions which may be trapped in an ion trap is limited.I7 Sensitivity is reduced when spacecharge, created by unwanted matrix ions, limits the total number of analyte ions which may be trapped. Further, mass spectra recorded using the mass-selective instability scan suffer reduced sensitivity, resolution, and CID efficiency as the space-charge trapping limit is reached.24 One method for eliminating unwanted matrix ions is to raise the rf potential during injection. This procedure works well for internally created ions; however, there is an optimum rf voltage for trapping externally created ionszswhich is usually too low to remove a large portion of the matrix background. By applying a broad-band excitation signal during injection, matrix ions which would normally be trapped by the rf field are resonantly ejected. The rf voltage is optimized for trapping the analyte, and because matrix ions are ejected while the analyte is not, the analyte is actually concentrated over the course of the ionization period. This procedure increases the number of analyte ions which may be trapped before space-charge begins to negatively affect the spectrum. To perform broad-band ejection during ionization, a scan function is used which activates a train of SWIFT pulses during the ionization stage, as shown in figure 4. In one experiment, performed using substance P,the injection energy was optimized at 7.8eV, which results in an optimum rf voltage of 998 Vhp25 (low-mass exclusion limit m/z 80). A pulse was designed to eject low-mass ions from mlz 0 to 350. At the given rf level, ions of mlz 350 have a qr value of 0.207 55 which gives a p, value of 0.148 03 and a secular frequency (fL) of 81.415 kHz. Since the highest secular frequency possible for an ion trap with a 1.1-MHz drive frequency (Q)is 550 kHz, a pulse which ranges from 80 to 550 kHz will eject all low-mass ions from 0 to approximately m/z 356. A pulse was constructed to produce a time domain signal with 8192 data points which would be transmitted a t 1.1-MHz sample rate (909.1 nsldata point) producing a signal with a 7.45-ms (24) Johnson, J. V.; Yost, R. A.; Kelley, P. E.; Bradford, D. C. Anal. Chern. 1990. 62. 2162. (25) Kaiser, R. E., Jr. Ph.D. Thesis, Purdue University, 1990.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
Broad band excitation
=
AC Shaped
During Ion Injection
a
I
,
w
0 10
-
(M + H)' 1348 m/z
-
I
SURSTANCE P (37.1 pmol) NO SWIFT PULSE 80 ms Ionization
C
) I A I B I
-1
1891
I
210
I#) Tme (ms) NOTTO SCALE
2k
4k
Q b
675
13b
1125
mlz
Flgure 4. Scan functlun to control the rf voltaqe for applying the SWIFT pulse durlng 1n)eCtion. The switch Is activated and the wave form generator is triggered durlng the ion Injection p d o d A. The pulse Is allowed to continue Into the storage period B to ensure total ejectlon of matrlx ions. The switch is deacUvated prlor to the mass analysls ramp In period C, wMch allows the standard a0 signal to be applied for resonant ejection and hence mass range extension.
SUBSTANCE P (37.1 pmol) SWIFT PULSE 80 ms Ionization
-
+
(M H)' 1348 m h
h 225
0
1
m
I
m
'
m
I
'
a
I
x
'
O
I
a
'
I
m
'
m
I
'
a
I
m
Fnsumv Wit Flgwe 5. Spectrum analyzer results giving the frequency content of the Injection SWIFT pulse (80-60O-kHz pulse) as generated by the arbitrary wave form generator at Its maximum output amplitude (5 VGp). The plot shows that while the SWIFT pulse covers the desired frequency range with good frequency cutoff resolutlon, there Is a 15 % drop in signal amplitude at the hlgh-frequency end of the spectrum. Thls nonllnearltyIscaused by the imperfectrepresentatknof the SWIFT pulse in the ARB and the sampbrate used. A Wear frequency response Is not needed for thIs experiment since the threshold amplitude for ejectkncenbeachlevedfortheen~frequQncyrangewlthoutdlstortlng the frequency cutoffs. For experknents requklng llnear frequency response, the nonllnear output of the actual hardware should be considered.
duration. The pulse was designed with an end frequency of 600 kHz to ensure that power would be applied through the stability limit. The pulse was phase modulated with 40 = 0, J = 0.5*, and K = -258.13 X 1Vr. As shown in Figure 4, the SWIFT excitation must fill the ionization period A and is usually allowed to continue after ionization is complete (period B) to ensure complete ejection. Due to the short duration of the ejection pulse, the 80-ms ionization time and 5-ms electron multiplier settle time intervals are filled by applying multiple pulses; in this case 11pulses are used. At the beginning of period C, the analog switch is activated and the normal resonant ejection signal is applied at a frequency of 162.64 kHz and an amplitude of 6 VO-~. Given that real hardware cannot exactly duplicate the SWIFTwaveform stored in the computer memory, a spectrum analysis was performed on the SWIFT pulse generated by
450
675
900
1125
1350
mlz Figure 6. Resonant ejection mass-selective instability scan of a 37.1pmol sample of substanceP. Ionsare created vladesorptlon ionlzatlon from a glycerol-thlogiyceroi matrix. The sampk was bombarded by a 7 k V Cs+ beam for 80 ms durlng which tlme Ions were Injected into the lon trap: (a) no SWIFT pulse appiled; this spectrum shows the severeeffectsof spacecharge, with low parent ion Intensity;(b) SWIFT pulse applied during Ion Injection. By resonant ejectlon of low-mass matrix ions, the total spacecharge is reduced.
the ARB to determine ita actual frequency characteristics. The resulta are shown in Figure 5 for the injection pulse. This plot was collected by configuringthe ARB to send a continuous stream of pulses to a spectrum analyzer at the maximum ARB amplitude. Unlike typical spectrum analysis plota which give amplitude in decibels, the amplitude axis is linear to show the nonlinearity in the actual frequency content of the pulse. The SWIFT pulse generated by the ARB does cover the entire region from 80 to 600 kHz, although not nearly as linearly as the computer data. There is a 15% drop in amplitude as the frequency approaches the sample frequency of the ARB. The nonlinearity could be removed if a faster ARB were used. For a resonant ejection experiment, the linearity of the frequency domain is of much less consequence since a threshold amplitude for ejection can be reached for all ions within the frequency range of the pulse without distorting the frequency cutoffs of the pulse. Figure 6a shows the mass spectrum of 37.1 pmol of substance P recorded using 80-ms ionization and a resonance ejection mass range extension factor of 2.25. The spectrum is dominated by matrix ions and is apace-charged so badly that the protonated molecular ion (M + H)+ is not visible. Figure 6b shows the effect of applying 11SWIFT pulses during ionization. The SWIFTpulses all have a maximum amplitude of 9 VQ. The low-mass ions are ejected, eliminating the space-charged condition which prevented the molecular ion from being ejected efficientlyby the mass-selectiveinstability
1832
ANALYTICAL CHEMISTRY, VOL. 65, NO. 14, JULY 15, 1993
RF Isolation Microscan Broad band excitation
Table I. Cs.+Jn+Clusters, m / z , qn 8, and Secular Frequency in the z Direction (f.) for V = 3761 Vhp (Low-Mass Cutoff m / z 301.4). n
mlz
PI
8.
1
392.7 652.5 912.3 1172.1 1432.0 1691.6
0.696 93 0.419 44 0.300 00 0.233 50 0.191 12 0.161 79
0.559 70 0.307 92 0.216 06 0.166 92 0.136 13 0.115 00
2 3 4 5 6 a
fz
I
(kHz)
I IAl
8
307.837 169.354 118.833 91.808 74.869 63.248
8, values were computed using QCALC3.I6
a)
-
50
0 2 0 1 2 0
520530
Tim (
L
$ / I
L
988
+
No SWIFT Pulse i
i
3
D
!c/
o{2m--
k
Broad Band Ejection
cs (CSI)"
AC Shaped
590
650
~ NOT j TO SCALE
Flgure 8. Scan function to control the rf voltage for isolation and actlvation of a parent ion to cause CID. The forward-reverse isolation scan2sis used, requiring the ac signal to be active prior to the SWIFT excitation. Several SWIFT pulses can be applied during stage C to increase total excitation power.
Table 11. Isotopes of Protonated Bradykinin, m/z, qn fin and Secular Frequency in the z Direction (f.) for V = 2936.9 Vhp (Low-Mass Cutoff m / z 235.4). miz PI Bz fz (kHz) 1060.6 1061.6 1062.6 1063.6 1064.6
Mass/Charge
SWIFT Pulse 0
Mass/Charge Flgure 7. Broad-band ion isolation of Cs,,+, I,,+ ions: (a)shows ClUSterS n = 1 to n = 6 prior to application of the SWIFT pulse; (b) shows the ejection of clusters 3 and 4 when a SWIFT pulse is applied containing a range of frequencieswhich include the secular frequenciesof cs413+ and cS&+.
scan. This example confirms that even when experiments require long ionization times, sensitivity restrictions imposed by space-charge can be lifted using broad-band excitation during ionization. Broad-Band Ejection for Ion Isolation. The objective of this experiment is to use a single pulse to eject ions of specified mlz values. In the case chosen for illustration, CsI is used as the source of cluster ions and selected ions of a very wide range of masslcharge ratios are removed from the trap. A simple scan function was used which consisted of an ionization step, SWIFT ejection stage, and mass analysis. During the SWIFT ejection step the rf voltage was set to 3761 V,, placing the Cs&+ cluster (mlz 912.3) at a qz of 0.3000. During the mass analysis, resonant ejection was used with a frequency of 120.067 kHz, to extend the mass range by a factor of 3. Table I gives the theoretical mass, qz, &, and the secular frequency for the Csn+lIn+clusters from R = 1 to n = 6. A SWIFT pulse was synthesized to cause the ejection of the clusters at both m/z 912.3and 1172.1. Figure 7a shows the mass spectrum of the CsI cluster 1-6 before the SWIFT pulse was applied, and Figure 7b shows the same spectrum when the SWIFT pulse is applied with a 5 . 0 - V ~ amplitude. ~ The selected clusters are ejected with only a slight effect on
0.201 56 0.201 37 0.201 18 0.201 00 0.201 80
0.143 68 0.143 55 0.143 41 0.143 27 0.143 13
79.026 78.950 78.874 78.798 78.732
8, values were computed using QCALC3.ls
the abundance of neighboring ions. Broad-Band Excitation for CID. Experiments were done to compare broad-band excitation to single-frequency excitation. This comparison was made by fragmenting protonated bradykinin, to generate an MS/MS spectrum. In this experiment, a reverse-forward isolation scan was used to prepare the protonated moleculefor dissociation and mass analysis. The mass analysis step was performed using a mass range extension factor of 2.0. The scan function used is shown in Figure 8 where the forward-reverse rf scan is used for molecular ion isolation.2e This is labeled A and B and creates a window around the molecular ion region 35 Da wide. The isolation steps eject any dissociation product ions formed prior to the CID step. In preparation for dissociation, the rf voltage was set to 2937 V, bringingthe mlz 1060.6isotope of protonated bradykinin to a qz value of 0.201 562. Table I1 gives the mass/charge ratio, qr, pz, and the secular frequency cfi) of each of the protonated bradykinin isotopes. A frequency domain spectrum was synthesized which was 855 Hz wide centered at 78.983 kHz, ensuring that all the molecular ion isotopes would be resonantly excited. The time domain signal was generated by performing the complex inverse fast Fourier transform on the phase-modulated frequency spectrum and then reflecting the result about N/2. The resulting wave form had a significant start and stop amplitude; therefore it was windowed prior to downloading to the ARB. This resulted in trade-off between spectral leakage and frequency domain spectral distortion, caused by attenuating signal in the first and last quarter of the time domain wave form. Setting the wave form sample frequency to 500.8 kHz in the ARB results in a frequency spectrum with 61.13 Hz/data point and a total signal time of 16.357 ms. The analog switch is activated and the SWIFT pulse is applied during step C, where a burst of four pulses is output. ~
(26) Kaiser, R. E., Jr.; Cooks,R. G.; Syka, J. E. P.; Stafford, G. C., Jr. Rapid Commun. Mass Spectrom. 1990, 6,30.
ANALYTICAL CHEMISTRY, VOL. 65, 370
a)
'k
j &\\r-
Bradyklnln Slngk Frequency Excitation 71 mr
I
I. 1
600
700
800
800
1 m
1100
1200
1100
1200
MWChW 420
1 Bradyklnln m - b & l d Exdtalion 78.558 * 79.412 ~ H Z 71 mo 800 mVp (ma)
-
I 800
700
800
800
I
1 m
MWChw
Figure 0. MS/MS spectrum of the molecular ion region of bradykinin, exclted for 71 ms using a maxhnum peak vottage of 800 mVbP The upper spectrum show the result of using a singlafrequency excitation at 78.940 kHz. The Inset shows a hlgh-resolution scan of the molecular ion region obtalned by reducing the rf scan rate by a factor of 10. In the upper spectrum, not all of the molecular ion region Is exclted due the nanow wldth of thesingle-frequency excltatkm. The lower spectrum shows the result of using four 85542-wlde SWIFT pulses centered at 78.983 kHz. Each SWIFT pulse is 16.36 ms In duration. In the Inset, Is evklent that more of the molecular ion reglon has been exclted uslng broabbend pulses.
The total time of step C was set to 71 ma. The analog switch was deactivated at the end of step C and the rf level lowered in preparation for the mass analysis scan. A standard ITMS scan from m/z 100 to 650 was performed with an axial modulation frequencyof 184.638 khresulting in a mass range extension factor of 2.0. Figure 9 shows the MS/MS spectrum of a 97.4-pmol sample of bradykinin using the traditional single-frequencyexcitation method. Good fragmentation is achieved with nearly complete removal of the parent ion. Figure 9b shows the results of using the broad-band pulse in place of the single frequency. Slightly better fragmentation is achieved using the SWIFT pulse due to the excitation of the entire molecular ion isotope envelope. The insets in Figure 9 show the high-resolution
NO. 14, JULY
15, 1993
1888
spectra of the molecular ion region after excitation by each of the two methods. Comparing the insets of Figure 9a,b, it is evident that the broad-band pulse is exciting more of the isotope envelope than the single-frequencymethod. Since the product ion intensity is as good or better than the singlefrequency method, it is apparent that the broad-band signal is not simply ejecting ions at a greater rate. In conclusion,it has been demonstrated that inverse Fourier synthesized wave forms can be used in the ITMS for both selective and broad-band excitation. There are obvious limitations to the technique at low ion mass/charge ratios which correspondto very high ion secular frequencies. There has been a trade-off made between the quality of the frequency domain spectrum and the SWIFT pulse length. The pulse length is longest when the ARB output rate is set at the Nyquist limit for the highest frequency component present. The irradiation period is still very short compared to quadrupole ion trap time frames. Problems with short pulse duration and frequency domain quality can be overcome by applying multiple pulses with larger amplitudes. Large amplitudes distort the frequency domain cutoffs slightly causing the pulse to have an even wider frequency range, which can interfere with ions close in mass. All of these problems can be overcome through the use of a significantly better ARB than was used here; however, there are fundamental limits to resonant excitation which cannot be overcome. At very high mass, the ion frequencies become increasinglyclose, and because ions absorb energy in a range near their secular frequency, there is a fundamental limit to the resolution achievablewith any type of resonance excitation at very low secular frequencies. SWIFT excitation is complementary to traditional excitation-ejection methods such as rf/dc and rf-only isolation and single-frequencyresonance techniques which are used for selecting and exciting ions in MS/MS experiments. There are many possible benefits of broad-band signals which could not be explored with the ionization methods described in this paper. Ionization methods such as electrospray and laser desorptionproduce molecular ion envelopes which have a wide frequency range which cannot be activated by single-frequencyexcitation methods. In such cases SWIFT pulses can be constructed to cover a frequency range as broad as needed to ensure all the ions of interest can be resonantly excited. SWIFT pulses, unlike other broad-band signals such as filtered noise, allow total control over the excitation profile. They therefore allow utilization of multiple-frequencyranges each having different or continuously varying power levels.
ACKNOWLEDGMENT This work was supported by the National Science Foundation CHl387-21768. R.K.J. acknowledges Fellowship support from Eli Lilly and Co. RECEIVED for review November 6, 1992. Accepted March 24, 1993.
