1677
Anal. Chem. 1087. 59. 1677-1685
separated and analyzed after a simple sample cleanup and without requiring derivatization. Mass spectra of monosaccharides yield molecular weight information with little fragmentation. IDMS, using [U-13C]glucose as internal standard, shows linearity even at very low isotopic enrichment (0.1% to 1%). The accuracy of this method was determined on standard mixtures at low percentage of [U-13C]glucose rather than 50% (1:l ratio) since the accuracy is lower with the smaller percentage enrichment. When used on biological samples (e.g., glucose from human serum), this method shows the same degree of sensitivity and accuracy. The data given show that combined LC/MS can be successfully applied to quantitative analysis of glucose or sorbitol in small biological samples. Since the use of stable isotopes is a general accepted procedure for the measurement of daily glucose production and utilization, and it is considered superior to radioisotopes in accuracy, specificity, and patient safety (14),this analytical method, using ~ - [ ~ ~ C ~ ] g l uisc olikely s e to prove useful in kinetic studies where good precision, sensitivity, and specificity are required. We are currently applying this method in our laboratory to determine production rates in type I GSD patients with no glucose-6-phosphatase enzyme activity, as determined by liver biopsy, and to further elucidate mechanisms by which these patients can produce glucose. Future studies
will be performed in patients with other metabolic disorders. Registry No. D-Glucose, 50-99-7; D-sorbitol, 50-70-4.
LITERATURE CITED Matthews, D. E.; Bier, D. M. Annu. Rev. Nutr. 1983, 3 , 309. Bier, D. M.: Arnold, K. J.; Sherman, W. R.; Holland, W. H.; Holmes, W. F.; Klpnis, D. M. Dlabetes 1977, 26, 1005. Dan, P.; Clemons, P. M.; Sperllng, M. I.; Gelfand, M. J.; Chen, I.W.; Sperling, M. A.; Norman, E. J. Anal. Lett. 1983, f6(B9), 655. Tserng, K.; Kalhan, S. C. Am. J. Physlol. 1983, 245, E476. Whlte, E.; Welch, M. J.; Sun, T.; Snlegoski, L. T.; Schaffer. R.; Hertz, H. S.;Cohen, A. Biomed. Mass. Spectrom. 1982, 9 , 395. Bier, D. M.; Sherman, W. R.; Holland, W. H.; Klpnis, D. M. I n Proceedings of the First International Conference for Stable Isotopes in Chemistry, Blology and Medicine, May 1973, USAEC Conf-730525, p 397. Cadleux, S. Biomed. Mass Spectrom. 1983, 70, 130. Pelletler, 0.; Roboz, J.; Kappatos, D. C.; Greaves, J.; Holland. J. B. Clin. Chem. (Winston-Salem, N . C . ) 1984, 3 0 , 1611. Voyksner, R. D.; Bursey, J. T.; Pelllzzari, E. D Anal. Chem. 1984, 56, 1507. Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750. Games, D. E.; McDowal, M. A.; Levsen, K.; Schafer, K. H.; Dobberstein, P.; Gower, J. L. Biomed. Mass Spectrom. 1984, 7f(2), 87. Jakobs, C.; Warner, T. G.; Sweetman, L.; Nyhan, W.L. fedlatr. Res. 1984, 18, 714. Houk, R. S.;Fassel, V. A.; Svee, J. Org. Mass Spectrom. 1982, 77, 240. Bier, D. M. Nutr. Rev. 1982, 4 0 ( 5 ) , 129.
RECEIVED for review August 8,1986. Accepted March 13,1987.
Instrumentation, Applications, and Energy Deposition in Quadrupole Ion-Trap Tandem Mass Spectrometry John N. Louris and R. Graham Cooks*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 John E. P. Syka, Paul E. Kelley, and George C. Stafford, Jr. Finnigan MAT, 355 River Oaks Parkway, San Jose, California 95134 John F. J. Todd Chemical Laboratory, University of Kent, Canterbury, Kent, CT 27NH, U . K . The use of an Ion-trap mass spectrometer to generate daughter spectra of selected parent Ions Is shown. Ions are created wlthln the trap by electron impact, undeslred Ions are ejected from the trap, and the selected parent Ion undergoes cdlisbnally acthrated dlgsoclatbn (CAD). The CAD efflclency can be greatly Increased by applylng a small supplementary ac voltage of appropriate frequency. The energy transferred to the selected Ion, whlch depends upon the radlo frequency voltage applled to the trap as well as the supplementary ac voltage and frequency, Is explored with the molecular lorn of tetraethylsllane, n-butylbenrene, and nltrobenrene. Extraordlnarlly high CAD efflclency ( 100 % ) Is readlly achleved although the Internal energy deposlted Is small ( 5 2 eV) under these condltlons. Under condltlons that yield greater average Internal energy deposltlon, dlssoclatlon efflclencles are smaller and activated Ions with a wide range of Internal energles are generated. Scan modes, which allow daughter spectra to be recorded for the products of Ion/ molecule reactions and for fragment Ions generated In a previous collision (sequential daughter MS/MS/MS), are also Illustrated.
-
Mass spectrometers that allow two stages of mass analysis (MS/MS instruments) have found application in qualitative
analysis, quantitative analysis, and mechanistic studies ( I ) . They are used to study such gas-phase ion reactions as collisionally activated dissociation (CAD) and photodissociation (2), as well as electron-transfer reactions (3, 4 , and ion/ molecule reactions (5). Spectrometers capable of MS/MS experiments are of two types: those in which the stages of mass analysis are performed in different mass analyzers, and spectrometers in which the several stages of mass analysis are performed in a single mass analyzer. Multiple quadrupole, multiple sector, and hybrid sector/quadrupole instruments are of the first type, while ion cyclotron resonance (ICR) instruments were, until recently, the only MS/MS instruments that used a single mass analyzer for the sequential stages of mass analysis. The quadrupole ion trap has been used for the trapping and mass analysis of ions since the early work of Fischer (6, 7 ) . The ion trap is now widely used for applications ranging from mass spectrometry to optical spectroscopy of trapped atomic and molecular ions ( 8 , 9 ) . The device consists of a chamber formed between metal surfaces in the shape of a hyperboloid of one sheet (the ring electrode) and a hyperboloid of two sheets (the two end-cap electrodes, Figure 1). Ions are created within the chamber by electron impact from an electron beam admitted through a small aperture in one of the surfaces. Radio frequency (rf) voltages (and sometimes direct current voltage offsets) are applied between the ring electrode and
0003-2700/87/0359-1677$01.50/0 0 1987 American Chemical Society
1678
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table I. Typical Operating Parameters trap size rf frequency BC
= 1.0 cm 1.1 MHz 0.15-15 kV (pp) 50-550 kHz 10 mV to 15 V (pp)
enalyte pressure
1 ms 5 ms 1-50 ms 180 ps/Da 1 x 10-3 torr (2-4) x 10-7 torr
rf voltage BC frequency
voltage ionization gate time interval P, figure 4 interval D, figure 4 interval S,figure 4 He pressure
T~
If the ring voltage is then reduced so ions of lower mass-tocharge ratio may be trapped, any fragment ions produced after the drop in voltage will accumulate in the trap and may be observed by a second scan of the ring electrode voltage to higher values. Thus, if a molecular ion is selected to be trapped, a spectrum can be obtained of the fragment ions it produces over time. This paper discusses the use of a quadrupole ion trap for such MS/MS experiments. Special attention is given to the methodology for CAD within the ion trap and to the internal energy deposited in the ion selected for dissociation. Flgure 1. lon-trap electrodes. An r l field is established between the ring electrode and the two end-cap electrodes. The assembled structure is shown in cross section. the two end-cap electrodes establishing a quadrupole electric field. This field is uncoupled in the three directions so that ion motion can be considered independently in each direction; the force acting upon an ion increases with the displacement of the ion from the center of the field but the direction of the force depends on the instantaneous voltage applied to the ring electrode. A restoring force along one coordinate (such as the distance, r , from the ion trap's axis of radial symmetry) will exist concurrently with a repelling force along another coordinate (such as the distance, z, along the ion trap's axis), and if the field were static the ions would eventually strike an electrode. However, in an rf field the force along each coordinate alternates direction so that a stable trajectory may be possible in which the ions do not strike a surface. In practice, ions of appropriate mass-to-charge ratios may be trapped within the device for periods of many seconds. Various methods of mass analysis of the trapped ions have been available since the invention of the ion trap, but the usefulness of the device as a mass spectrometer has recently been greatly increased by a new method of operation, the mass-selective instability mode (8). In this method, a radio frequency voltage (without a direct current component) is applied to the ring electrode, while the two end-cap electrodes are held a t ground potential. All ions whose mass-to-charge ratio exceeds a minimum cut-off value (which is linearly proportional to the magnitude of the radio frequency voltage) are trapped. To record a mass spectrum, the radio frequency voltage is increased with time so that ions of successively greater mass-to-charge ratios develop unstable trajectories, are ejected through perforations in an end-cap, and are detected with an electron multiplier. The resolution attained with this mode of operation is greatly enhanced by the presence of about a millitorr of helium, presumably because this buffer gas damps the otherwise stable ion trajectories to the center of the trap. In the mass-selective instability mode, all ions of less than a given mass-to-charge ratio may be excluded from the trap by raising the radio frequency voltage to the appropriate level. For example, if a single compound is introduced into the trap, the molecular ion and its isotopic variants may be trapped.
INSTRUMENTATION AND EXPERIMENTAL SECTION The ion-trap mass spectrometer used in this study was constructed from components of a Finnigan MAT model 100 ion-trap detector (ITD). The trap was removed from the commercially supplied vacuum manifold and modified to permit installation on an optical rail in a large, rectangular vacuum manifold (Figure 2). Also, ceramic insulators were added to permit the electrical isolation of both end-caps. The upper surface of the manifold is formed by glass plates (sealed with Viton seals), which allow ready access to the system. The manifold is evacuated by using a turbomolecularpump (170 L/s, Bakers) backed by a mechanical torr. Sample is introduced via pump. Base pressure is 2 X a direct insertion probe or via a precision leak value. Helium buffer gas is introduced hy using a separate leak valve. Ionization is effected by electron impact, occurring within the trap, obtained hy using the gated electron heam technique employed in the commercial ITD. Ion detection is by a channel electron multiplier (Detech), which is external to the trap and placed near small perforations in an end-cap, A single-hoard microcomputer is used to control timing sequences in the instrument, including the scanning of the rfvoltage, the application of the supplementary ac voltage, and the gating of the electron heam and the electron multiplier voltage. The supplementary ac voltage is provided by a single-hoard frequency synthesizer (SCI-TEK),which is also interfaced to the single-hoard computer. The singlehoard computer is also used to acquire and transmit data to the main data system, which is based on an IBM XT. Figure 3 shows a block diagram of the ion trap electronics. Typical operating voltage sequences are shown in Figure 4. Typical voltage levels and frequencies, as well as other typical operating parameters, are gathered in Table I. A number of these parameters were varied and the results of such changes form part of the present paper. RESULTS AND DISCUSSIONS Comparison w i t h O t h e r MS/MS Spectrometers. The quadrupole ion trap confines ions by using an electric field while an ICR spectrometer confines ions with a combination of magnetic and electric fields. Both devices may be operated in a variety of ways, but in a typical mode of operation for both, a mass spectrum is acquired by pulsing an electron beam into the chamber and trapping all ions formed by electron impact; the contents of the trap are then subjected to mass analyses. For the ICR spectrometer, mass analysis is often performed by exciting the ions with a radio frequency pulse, observing the resulting current induced in plates of the cell
ANALYTICAL CHEMISTRY. VOL. 59. NO. 13. JULY 1, 1987
1679
End Cop Electrode
Flgure 2. Ion trap mounted on an ion optical rail in a vacuum manifold which allows easy access to wmponents.
Figure 3. Ion-trap control system including the supplementary ac supply needed fw MSlMS experiments.
by the motion of the ions, and recovering the spectrum of frequencies present (and thus the mass spectrum) via the Fourier transform (10-12). In the quadrupole ion trap, mass analysis is performed by sequentially ejecting ions from the trap and detecting them with an electron multiplier. In both types of spectrometers, the mass analysis step may be modified or replaced by a step that removes unwanted ions from the trap and allows only those ions of a narrow mass range to remain trapped. These ions may then be allowed to undergo some reaction such as CAD in which collision with a gas induces fragmentation, and the products of this reaction may be subjected to mass analysis to yield a daughter MS/MS spectrum. In comparison, MS/MS spectrometers in which the stages of mass analysis are performed in different mass analyzers
must convey the ions from one mass analyzer to the next. While both classes of MS/MS spectrometers are capable of producing daughter spectra, there are important differences in the other types of MS/MS scan modes that are possible. For example, FT-ICR and quadrupole ion-trap spectrometers can, in principle, perform any number of sequential steps of reaction and ion isolation (MS"). Spectrometers that use separate mass analyzers are limited by the number of stages of mass analysis that are present in the instrument. However, such spectrometers are capable of two important experiments, the parent scan and the neutral loss scan (131, that are not directly accessible in the ICR or quadrupole ion trap spectrometer. Ion Motion in the Quadrupole Ion Trap. The theory of ion motion within conventional quadrupoles and within quadrupole ion traps was presented by Paul and co-workers in their early papers describing the devices (6, 7,141. Later authors have also presented this theory (15,16). Essentially, it is possible to generate a parametrized stability diagram (Figure 5 ) that characterizes the solutions to the derived equations of motion. The coordinates of this stability diagram ~ ~ qr ~ are parameters a, and q. such that a, = - 8 ~ U / r n r ,and = -4zV/mrnZQz,where U is the direct current voltage, Vis the radio frequency peak voltage level, r, is the radius of the trap, Q is the angular frequency of the radio frequency voltage, m is the mass of the ion, and z is the charge of the ion. The most important characteristic of the trajectory is whether it is stable or unstable; if the a,, 9. pair falls within the region indicated (Figure 5 ) , the trajectory can be stable (although an unfavorable initial velocity and position may produce an amplitude so large that the ion strikes an electrode). Outside the region indicated, the trajectory is unstable regardless of the initial velocity and position. Another important characteristic of the trajectory is the component frequencies in the axial and radial directions. The parameters
1880
ANALYTICAL CHEMISTRY, VOL. 59,
NO. 13, JULY 1, 1987
1
STANDARD MS SCAN
A
Fundamental R F Voltage
ir' Electron Gate Ian Signal
n_
DAUGHTER SCAN ( M S / M S ) Fundamental RF Voltage Gate AC Voltage Signol I on
n
u (MS3)
SEQUENTIAL DAUGHTER SCAN Fundamental RF Voltage
--]I
Gate
AC Voltage \On
Sianal
JL
A
I
4
h
I
I
C I DAUGHTER SCAN ( M S I M S )
Jk&
Fundamental GoteVoltage Electron RF
AC voltage Ion Signal
I
--
Figwe 4. Ion-trap scan programs used to obtain the types of spectra indicated. The selection of the parent ion occurs in the interval P, daughters are generated during the period D, and the mass analysis scan is indicated as S.
0 2 0 4 06 OB
9,
10 1 2
14
0
5
10
15
20
TIME (microseconds)
Figwe 5. Stabilii diagram (left) and calculated trajectqries (right) of three ions trapped using no dc offset (Le. a, = 0) and the same ring electrode voltage. The displacement from the center along the axis of the trap (z-coordinate, Figure 1) is plotted vs. time: (a) m / z 502, qr = 0.123, 0, = 0.087;(b) m / z 119. q, = 0.519,p, = 0.390; (c) m l z 69, q, = 0.895, p, = 0.892.
p, and pr, which indicate these frequencies, are obtained from a, and qz by calculation (16) or from the stability diagram. The component frequencies for each direction are given by f, = (s + p/2)Q, where s = 0, i l , =t2, ... The relative contribution of each frequency component may also be calculated. Representative trajectories for trapped ions of different mass (under fixed conditions and with no dc voltage) are shown in Figure 5. In particular, the axial components of the
trajectories for ions of m / z 502,119, and 69 are shown for the case where the rf voltage is adjusted to bring m / z 68 to the boundary between stability and instability. The q, parameter is then easily calculated for any other m / z value by using the known qz value (0.908) for the boundary of the stability region where qJO.908 = (m/z)/68. Since p, may be determined once qz is known, the frequency components of the trajectory are also readily determined. Examination of the trajectories of Figure 5 shows that the fundamental frequency (s = 0) makes the most important contribution to the frequency spectrum; a t low values of 0, the contribution of the higher order frequencies (s > 0) is relatively slight but their importance increases with @., Most importantly, a t larger m / z values, q2 is lower, p, is lower, and the ions move more slowly. An important consequence for MS/MS experiments of this dependence of @, (and thus the fundamental frequency) on the qz value is the measure of control it permits over the average energy deposited in a collision of an ion and a neutral compound. At higher q, values collisions are more energetic, provided the amplitudes of the oscillations are the same. However, more energetic collisions are achieved at the expense of the mass range of the fragment ions that can be trapped for subsequent mass analysis. In the example of Figure 5, while m / z 502 has a low q, value, all fragment ions of m / z greater than 68 can be trapped. If the qz value of m / z 502 is increased (by increasing V) so that the ion trajectory resembles Figure 5b, more energetic collisions are possible but only ions of m / z greater than 287 can be trapped. Fragments of lower m / z will be lost from the trap. Another way of increasing the energy deposition is to increase the amplitude of the trajectories. This can be achieved by applying between the end-cap electrodes a small sinusoidal voltage that is of the same frequency as the axial component of motion of the parent ion. This supplementary voltage excites the parent ion to a larger trajectory. To avoid confusion with the radio frequency voltage on the ring electrode, this supplementary voltage will be referred to as the ac voltage. The frequency, amplitude, and duration of the ac voltage can all be shown to affect the nature of the MS/MS spectrum. Several conference proceedings have shown examples of these MS/MS capabilities (17-19). Fischer (6, 7) used a small ac voltage applied across the end-caps of an ion trap for the resonance detection of ions. Similarly, Paul et al. (14),applied a small ac voltage across opposing electrodes of a quadrupole mass filter to selectively remove ions by resonance ejection. In both cases, the perturbation produced in the field within the quadrupole device was treated theoretically by assuming a superimposed homogeneous field. Fischer pointed out that this approximation leads to some simple generalizations about the effect of the supplementary ac voltage on the ion trajectories (7): (a) Resonance occurs if the frequency of the ac voltage coincides with any of the frequencies ( s + p / 2 ) Q . (b) During resonance, the average amplitude of the trajectory increases linearly with time. (c) As the amplitude increases, all frequency components increase equally, not just the frequency component that coincides with the ac frequency. (d) The rate of increase in amplitude is proportional to the relative contribution of the frequency component that coincides with the ac frequency. In addition, the rate of increase in the amplitude is proportional to the ac voltage and inversely proportional to the mass-to-charge ratio of the ion. While the simple theory only treats the case of an isolated ion, the general, qualitative results are nevertheless useful in understanding the effect of the ac voltage on ion trajectories under the actual experimental conditions in which a high
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
":I
a)
m / z 93
m/2
1681 123
0.8
b) X
: , :
C)
0.I
0.0 0.0
25
0.1
0.2
0.3
0.5
0.4
0.6
0.7
0.8
0.9
qz Flguro 6. Comparison of theoretical (line) and experimental (circles) values for the stabilii parameter 0, as a function of 9,, which is proportional to the main rf voltage. Data are for the molecular ion of chrysene in the presence of a millitorr of helium.
pressure (1 mtorr) of helium is present. For example, the fundamental frequency may be determined experimentally at a particular rf voltage by determining the ac frequency (0,) a t which particular ions are ejected from the trap when using the lowest possible ac voltage. Figure 6 compares a typical set of such determinations (circles) with theoretical values (smooth curve); within the region of qz values which are most useful for MS/MS work (0.2-0.8), the experimentally determined fundamental frequencies agree (within experimental error) with the theoretical values. Significant deviations do appear at the lower qz values, however. Similarly, the nonfundamental frequencies may be determined and are found to agree well with the theoretical values. Larger voltages must be used to achieve the same ejection time with these higher frequencies, as expected. Effects of Experimental P a r a m e t e r s on MS/MS Spectra. The presence of helium does have some important consequences. Figure 7 shows daughter spectra of the molecular ion of nitrobenzene recorded in the presence and the absence of helium. The results are surprising. First, the addition of helium has a small but favorable effect in improving the resolution, but it actually causes a decrease in the abundance of the daughter ion of m / z 93 (compare scans a and b in Figure 7). This decrease may in part be due to collisional deexcitation of internally excited molecular ions which would otherwise undergo unimolecular dissociation. Alternatively, the helium may tend to damp the trajectory of the parent ion without inducing fragmentation, so that the meaa velocity of the parent ion decreases so much that all collisions (with helium and with nonionized nitrobenzene) are ineffective a t producing fragments. The molecular ions can be given increased kinetic energy by excitation from a supplementary ac source. In the absence of helium gas (Figure 7c) this broadens the peak associated with the parent ion without leading to significant fragmentation. However, in the presence of helium, collisionally activated dissociation occurs and the daughter spectrum (Figure 7d) shows characteristic fragments a t m / z 93 and 65. The parent ion is completely removed, the isotopic peak at m / z 124 forming the remaining high-mass signal. These results are typical of those for other systems, and one concludes that, in the presence of helium, the use of a supplementary ac voltage (in resonance with the motion of a mass-selected ion) is an effective means of obtaining MS/MS spectra. Note that two separate means are used to select the parent ion. First, the parent ion is selected by scanning the ring electrode (rf) voltage so as to empty the trap of lower mass ions and, second, the parent ion is brought into resonance by using the supplementary ac voltage.
'
d)
m / z 65
m / z 93
Flgure 7. MSlMS daughter spectra of ionized nitrobenzene ( m l z 123) (a) without helium and without application of the supplementary ac voltage, (b) with helium and without the ac voltage, (c) without helium and with the ac voltage, and (d) with helium and with the ac voltage.
The preceding observations, including the considerations regarding ion motion in the trap, lead to some simple guidelines about the manipulation of the daughter (MS/MS) spectrum through the choice of the scan parameters. Firstly, the qz value (at which the parent ion is trapped) influences the collision energy by determining the frequency of the ion motion. The voltage of the ac excitation signal also influences the collision energy by affecting the rate of increase in the amplitude of the parent ion trajectory which affects the mean velocity of the ions. The interaction between these two effects is complex. Secondly, as Fischer showed (7), excitation a t a resonant frequency other than the fundamental frequency will require a larger voltage to achieve the same rate of increase in the amplitude of the trajectory, but the relative contribution of each frequency component will be the same as if the fundamental frequency were excited. Lastly, before the ac voltage is applied, the parent ions are in trajectories of low amplitude; by selecting a low ac voltage the average velocity of ions can be made to increase so gradually that low-energy dissociations are promoted. The efficiency of CAD in the ion trap is frequently quite high as is illustrated in Figure 7 . Following previous practice (11) with MS/MS instruments with separate mass analyzers, the overall CAD efficiency is defined as the sum of the fragment ion abundances measured after mass analysis divided by the abundance of the mass-analyzed parent ion prior to dissociation. Since all spectra in Figure 7 are recorded by using the same detector sensitivities, it is clear that the total fragment ion abundance is approximately equal to the parent ion abundance and that the CAD efficiency approaches 100%. Energy Deposition. n-Butylbenzene was ionized by electron impact and its CAD in the ion trap was examined to obtain information on the internal energy deposited in the parent ion ( m / z 134). The ratio of the daughter ions of mlz 91 and 92 has been used repeatedly to gauge internal energy deposition in experiments ranging from photodissociation (20, 21) through collisional activation (22,23) and charge exchange (24). By the use of a qz value of 0.36 as shown in Table 11, ionized n-butylbenzene is dissociated, giving m / z 91 and 92 in the ratio 1:2. Since the ions examined in this study do not show significant ion dissociation in the absence of excitation, this ratio can be used directly to estimate that most molecular ions, after excitation, have internal energies between 1 and 2 eV (the activation energies for the two dissociations are 1.1and 1.7 eV, respectively (21)). A value for the average internal energy
1682
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
IO000
V
50
60
, 70
, , , , , 90 100 I10 120 130 140
I
80
, 150
m/z F W e 8. MS/MS daughter spectra of ionized tetraethylsilane. The scan program of Figure 4b was used with all parameters being constant except that each spectrum was acquired with a different excitation ac voltage.
Table 11. Ratios of [91+]/[92+] from the Dissociation of the Molecular Ion of Butylbenzene ac voltage, V
q 2
2.1 3.8 1.6 2.5 14.4
0.36 0.36 0.60 0.60 0.60
0.26 0.26 0.46 0.46 0.46
0.069 0.50 0.36 1.7 4.2
of the activated ions of about 2 eV also is consistent with ratios of these fragment ion abundances published for charge exchange (24). The [91+]/[92+]ratio may be varied over a wide range by varying the ac voltage and frequency 6, (by working at different q2values). The parent ion could be efficiently converted into 92' a t the lower qr value, with very little 91+ being produced. At the higher qz value, the production of 91+ could be promoted. As the ac voltage was increased, a ratio of [91+]/[92+]larger than 41 could be produced, but the absolute intensity of the two daughter ions decreased as an increasing fraction of the ions were rapidly ejected from the trap; under these conditions the parent may simply be ejected before daughters are produced, or the daughter ions may be created with initial conditions of velocity and position that prohibit trapping. This behavior of MS/MS in the trap appears to be general: a fragment ion associated with a low-energy process may be produced in excellent yield; fragment ions associated with higher energy processes may also be produced, but often a t the expense of the efficiency of the production of detectable fragments. Note that the ratio of [91+]/[92+] of 4.2:l approaches the highest value (7:l)recorded for CAD (22-24); based on charge exchange (24) and calculation (20) such ratios correspond to internal energies of the excited n-butylbenzene molecular ions in excess of 5 eV. However, since most of the ions are ejected from the trap when such high ratios are observed, it cannot be concluded that this is the average internal energy of the excited ions. A rough measure of the distribution of internal energies associated with a population of internally excited ions can be obtained when one knows the activation energy for each fragmentation reaction and records the fragment ion abundances. In the simple case where a linear sequence of reactions is available to the selected ion, the abundance of each fragment ion represents the probability that the excited parent ion has an internal energy sufficient to access its activation energy but insufficient to cause more extensive dissociation. By considering the series of fragment ions, the energy distribution of the initially excited ion population is approximated. Evidence has been given (25) that this is a useful method of characterizing distributions of internal energy and of following
the effects of experimental conditions on them. The fragmentation of the tetraethylsilane molecular ion was chosen to study the effect of the operating parameters on internal energy deposition. Its main fragmentation pathway consists of the following reaction sequence of known activation energies (to) (17, 18). CSi(C2Hs),I'+ rn/z 144
'o,
CSi(C2Hs),l+
-
m / r 115
CHSi(C2Hs)21+ m/z 07
,O
[H2Si(C2H5)I+ m / r 59
A study of the effect on the CAD process of varying the excitation voltage while all other parameters remain constant is shown in Figure 8. When no ac voltage is applied, only a small amount of fragmentation occurs, as already noted. At an ac voltage of 100 mV the parent tetraethylsilane ion is efficiently converted into m / z 115. At higher voltages, fragments corresponding to more energetic processes appear. Also, unit mass specificity for the excitation of the parent ion is achieved at the lower voltages, but not at the higher voltages. At the highest voltages used the number of ions detected is rather small, probably because of the rapid ejection of ions from the trap. These data indicate that the distribution of internal energies associated with the excited molecular ions, which lead to detectable fragments, broadens as the ac voltage increases. Low-energy processes still occur but those of higher energy accompany them. For example, the fragment ions of m/z 115, 87, and 59, which are significant products at 1000 mV of excitation, require internal energies of 0.5, 2.0, and 3.5 eV, respectively. Figure 9 shows the effect on the same daughter spectrum, of varying qz while keeping the other parameters constant. Changes in y2 are made by varying the rf voltage while maintaining a constant ac excitation voltage. At very low qz, the parent ion is ejected from the trap without producing fragments. At somewhat larger q2 values, only the m / z 115 fragment is produced. The fragments corresponding to more energetic processes begin to appear at a yr value of about 0.2. Note that the qr value of the parent ion determines the minimum mass of the daughter ion that can be trapped. As qz is increased, the energy deposition increases, but the mass range is diminished so that m f z 87 and 59 appear at intermediate yz values but are not recorded at high qz values. The extent of conversion of parent ions to fragment ions is also dependent upon the duration of the excitation. For example, at an ac voltage of 50 mV, the dissociation of the parent ion required an excitation time of 10 ms (Figure lo), but with an ac voltage of 160 mV, the reaction was complete in about 2.1 ms. In both experiments, the parent ion is
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1. 1987
,
1
50
,
60
,
,
I
80
70
,
,
I
110
100
90
120
130
I
I
140
150
1683
m/z Flgure 9. MSlMS daughter spectra of ionized tetraethyisilane. The scan program of Figure 4b was used with ail parameters being constant except that each spectrum was acquired at a different qz value. The ac voltage was 500 mV (pp) for all spectra.
/
50
/
60
/
/
70
80
/
90
/
100
/
/
/
110
120
130
/
140
/
150
m /z Flgure 10. Time course of the decomposition of the molecular ion of tetraethylsilane. The scan program of Figure 4b was used with all parameters being constant except that each spectrum was acquired with a different duration of the application of the ac excitation voltage. The ac voltage is 50 mV (pp).
/
50
/-
60
/
70
/
80
/
90
/
100
/
/
/
110
120
130
/
140
/
150
m /z Figure 11. Time course of the decomposition of the molecular ion of tetraethylsiiane. The conditions are the same as for Figure 10, except that an ac voltage of 5000 mV (pp) was used. The vertical scale for this plot is increased over that used for Figure 10 because of the diminished peak height due to the ejection of ions from the trap (cf. Figure 8).
converted quantitatively into fragments. At an ac voltage of 5000 mV, all parent ions disappeared within 1ms (Figure ll), but the CAD process was no longer 100% efficient. The daughter spectrum of tetraethylsilane molecular ion is dominated by the molecular ion and the dissociation
products m / z 115,87, and 59. However, even in this simple system ion/molecule reactions occur and their products appear in the daughter spectrum, almost always in very low abundance. Noteworthy among the products of these reactions is mlz 133, formally due to addition of water to triethyl-
1684
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1 , 1987
IO00
/
50
/
/
60
70
/
80
1
90
/
100
/
/
/
1
110
120
130
140
/-
150
m/z Figure 12. Reaction of the molecular ion of tetraethylsiiane with adventitous water. The scan program shown in Figure 4b was used, except that no excitation voltage was used. siliconium ion, m / z 115. This ion appears in the spectra presented above, in which the effects of experimental variables upon the dissociation of tetraethylsilane are examined. In the absence of a supplementary excitation voltage, little dissociation occurs and the ion/molecule reactions can be studied in detail (Figure 12). As the delay time between ion selection and product ion analysis is increased, products due to ion/ molecule reactions increase in abundance. After a 1-s delay, the entire initial ion current has been quantitatively converted into m / z 133 product ions. Obviously, the presence of appropriate neutral reagent gases allows ion/molecule reactions to be efficiently promoted and studied in the ion trap by using MS/MS scan techniques. Other Modes of Operation. While the emphasis of this study is on the energization and subsequent dissociation of ions trapped in a three-dimensional quadrupole field, additional experiments become possible with the microprocessor-controlled ac generator. The ion trap could be cleared of undesired ions by sweeping the frequency of the ac generator through the approximate range. Alternatively dc voltage offsets may be used for this purpose. In particular, the use of a dc offset during the ionization period has proven to be useful when the desired parent ion is present as a small proportion of the total number of ions. Successive ions may be dissociated (MS") by changing the frequency of the excitation voltage during the experiment or by changing the ring rf voltage so that first a parent ion and then its daughter ion are brought into resonance at the same frequency (Figure 4c). In the experiment shown the daughter ions are not isolated so ions may be present in the sequential daughter spectrum that are not due to two steps of CAD. To demonstrate the selective daughter scan capability, protonated dimethylmorpholinophosphoramidate ( m / z 196) was generated under conditions that promote ion/molecule reactions (vide supra). This ion was either dissociated under CAD conditions to yield m / z 152 as the major fragment or else the scan program of Figure 4c was used to further fragment mlz 152, yielding a sequential daughter spectrum. This simple procedure demonstrates the feasibility of successive steps of MS/MS, but a more complex experiment that could isolate each product would be more desirable. Applications. The use of chemical ionization in conjunction with the mass selective instability mode of operation has recently been described (26,27).Reagent gas is introduced at a low pressure. Then time is allowed for the formation of reagent gas ions, and for those ions to react with neutral
7% I
I
I1 /
60
l
I
I
80
l
I36
119
I, 137 137
I ~
l
100
1
l
I
l
I20
1
I
I
J
140
m/Z
Figure 13. CIMSIMS of amphetamine (a) C I scan without MS/MS, (b) the scan program of Figure 4, but without an excitation ac voltage, and (c)the scan program of Figure 4,but with an ac voltage to excite m l z 136.
analyte; the MS/MS part of the experiment is then performed as usual. Figure 13 compares an ion-trap CI mass spectrum of amphetamine with a CIMS/MS daughter spectrum of protonated amphetamine (mz 136) obtained by using the scan program of Figure 4d. Note that the degree of fragmentation of the protonated molecule can be controlled by the time of application and amplitude of the supplementary ac voltage. Photodissociation can also be performed in conjunction with the mass selective instability mode of ion-trap operation. The scan program of Figure 4b was used except that the ac voltage was not used; instead light from a pulsed Nd:YAG laser was admitted into the ion trap during the excitation period (28). In favorable cases complete conversion of the parent ion into fragment ions was achieved.
CONCLUSIONS The most important observation made in this study is that very efficient CAD may be achieved in an ion trap. The scanning procedure developed here provides a practical me-
Anal. Chem. 1987, 5 9 , 1685-1691
thod of producing MS/MS spectra. These spectra show a strong dependence on the scan parameters used (and by implication, the energy deposition) so that care must be exercised in choosing and maintaining particular instrumental parameters. This is a consequence of the complex ion motion in the trap and hence the distribution of velocities in the ion population. The possibility of studying ion/molecule reactions adds further information and complexity. However, ion trap MS/MS spectra can be made highly reproducible, and the three principal factors that determine the appearance of these spectra, qL,supplementary ac voltage, and ac voltage duration are readily controlled.
LITERATURE CITED (1) Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley: New York, 1983. (2) Harris, R. M.: Beynon. J. H. I n Gas Phase Ion Chemistry; Bowers. M. T., Ed.; Academic: New York, 1984; Vol. 3, Chapter 19, pp 100-127. (3) Kenttlmaa, H. I.; Wood, K.: Busch, K. L.; Cooks, R. G. Org. Mass Spectrom. 1983, 18, 561-567. (4) Beynon, J. H.; Caprioli, R. M.; Baitinger, W. E.; Amy, J. W. Org. Mass Spectrom. 1970, 3, 455. (5) Kinter, M. T.; Bursey, M. M. J . Am. Chem. SOC. 1988, 108, 1797. (6) Fischer, E. Z . Phys. 1959, 156, 1-26. (7) Fischer, E. Doctoral Dissertatlon, University of Bonn, 1958. (8) Stafford, G. C.. Jr.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J . Mass Spectrom. Ion Processes 1984, 6 0 , 85-98. (9) Wineiand, D. J.; Itano, W. M. Adv. At. Mol. Phys. 1983, 19, 135. (10) Comisarow. M. 6.: Marshall, A. G. Chem. Phvs. Lett. 1974. 25. 282-283. (I 1) Wanczek, K. P. Int . J . Mass Spectrom. Ion Processes 1984, 6 0 , 11-80. (12) Lande, D. A,. Jr.; Johlman, C. L.; Brown, R. S.;Weil, D. A,; Wilkins, C L. Mass Spectrom Rev. 1986, 5 , 107-166.
1685
(13) Yost, R. A.; Enke, C. G. Anal. Chem. 1979, 5 1 , 1251A-1264A. (14) Paul, W.; Reinhard, H. P.; von Zahn, V. Z . Phys. 1958, 152, 143-182. (15) Lawson, G.; Todd, J. F. J.; Bonner, R. F. Dyn. Mass Spectrom. 1976, 4 , 39-81. (16) Dawson, P. H. Quadrupole Mass Spectrometry; Elsevier: New York, 1976; Chapters 2-3. (17) Kelly, P. E.; Stafford, G. C.. Jr.; Syka, J. E. P.; Reynolds, W. E.; Louris, J. N.; Amy, J. W.! Todd, J. F. J. 33rd Annual Conference of Mass Spectrometry and Allied Topics, San Diego, CA, 1985; p 707. (18) Louris, J. N.; Brodbelt, J.; Cooks, R. G.; Stafford, G. C., Jr.; Syka, J. E. P. 34th Annual Conference of Mass Spectrometry and Allied Topics, Cincinnati, OH, 1986; p 685. (19) Kelley, P. E.; Syka, J. E. P.; Ceja, P. C.; Stafford, G. C.. Jr.; Louris, J. N.; Grutzmacher, H. F.; Kuck. D.; Todd, J. F. J. 34th Annual Conference of Mass Spectrometry and Allied Topics, Cincinnati, OH, 1986: p 963. (20) Griffiths, I.W.; Harris, F. M.; Mukhtar, E. S.;Beynon, J. H. I n t . J . Mass Spectrom. Ion Phys. 1981, 4 1 , 83. (21) Chen, J. H.; Hayes, J. D.; Dunbar, R. C. J . Phys. Chem. 1984, 8 8 , 4759-4764. (22) McLuckey, S. A.; Sallans, L.;Cody, R. 6.; Burnier, R. C.; Verma, S.; Freiser, B. S.;Cooks, R. G. Int. J . Mass Spectrom. Ion Phys. 1982, 4 4 , 215. (23) McLuckey, S.A.; Ouwerkerk, C. E. D.; Boerboom, A. J. H.; Kistemaker, P. G. Int. J . Mass Spectrom. Ion Phys. 1984, 5 9 , 85. (24) Nacson, S.; Harrison, A. G. Int. J . Mass Spectrom. Ion Processes 1985, 63, 325. (25) Wysocki, V. H.; Kenttamaa, H. I.; Cooks, R . G. Int. J . Mass Specfrom. Ion Processes in press. (26) Kelley, P. E.; Stafford, G. C.; Syka, J. E. P.; Reynolds, W. E.; Louris, J. N.;Todd, J. F. J. Adv. Mass Spectrom. 1985, 10, 869-870. (27) Brodbelt, J. S.;Louris, J. N.; Cooks, R . G. Anal. Chem., in press. (28) Louris, J. N.; Brodbelt, J. S.;Cooks, R. G. Int. J . Mass Spectrom. Ion Processes, in press.
RECEIVED for review December 23, 1986. Accepted March 19, lgg7* This work was supported by the National %ence Foundation CHE 84-08728.
Focused, Rasterable, High-Energy Neutral Molecular Beam Probe for Secondary Ion Mass Spectrometry Anthony D. Appelhans, James E. Delmore,* and David A. Dah1
Idaho National Engineering Laboratory, EG&G Idaho, P.O. Box 1625, Idaho Falls, Idaho 83415
Beams of focused (-0.5 mm dlameter at 2.5 m), rasterable, 3-17 keV neutral SF, molecules produced via autoneutrallzatlon from the correspondlng anlon have been demonstrated to be effective for produclng secondary Ions from electrically insulating speclmens (e.g., Mylar, Teflon) without sample charging problems. Secondary ion currents measured with A ( lo5 counts/s equlvalent) for the quadrupole were major peaks when uslng a picoampere (neutral equivalent) level prlmary beam which was more than adequate to characterize the speclmens. No contamlnatlng effects from S or F were seen In the spectra. The results demonstrate the feaslblllty of using the autoneutrallrlng SF, fast neutral source for a variety of static secondary Ion mass spectrometry applications that require electrically neutral primary particles focused into a near-parallel small diameter beam (over distances of 18 cm to several meters). Appllcatlons could include the analysis of polymers, ceramics, and biospeclmens or the generation of Ions lnslde the magnetically conflned cell of an Fourler transform Ion cyclotron resonance mass spectrometer.
-
Secondary ion mass spectrometry (SIMS) and fast atom bombardment mass spectrometry (FABMS) have found many distinct areas of application in recent years (1). A variety of
ion and neutral atom sources have been developed for these applications, ranging from micrometer-dimensioned ion beams capable of imaging to a variety of schemes for producing broad, unfocused beams of neutral atoms. However, one area that has seen little development is that of focused (as opposed to collimated), rasterable neutral atom/molecule beams. There are several areas of applicability for such a primary beam probe. The principal application of static SIMS/ FABMS has been the study of large, fragile molecules most of which are electrically nonconducting. A focused, rasterable, small diameter (millimeters and less) neutral primary beam capable of producing easily measurable secondary ions at low (nondamaging) fluence would simplify analysis of small areas (interfaces, boundary regions) on polymers, ceramics, and biological specimens. Extending this to spatial analysis/imaging requires a microfocused neutral beam that can be rastered over the sample. This type of imaging has recently been demonstrated by Eccles e t al. ( 2 ) ,although using an approach substantially different from the one taken in this study. In addition to the obvious applications of a focused neutral beam for SIMS there is another area where such a beam has significant potential application: sputtering of secondary ions from samples in the magnetically confined ion cell of Fourier transform ion cyclotron resonance (FTICR) mass spectrometers. The magnetic and electric fields of the FTICR cells make it difficult to inject a beam of charged particles into the
0003-2700/87/0359-1685$01.50/0 0 1987 American Chemical Society
