26 1
Anal. Chem. 1991, 63,201-268
phenol are much more reactive than toluene, whereas nitrobenzene does not react. The following combinations of substrate and reagent have been found unreactive in our apparatus. BF3/methanol does not esterify aliphatic carboxylic acids. Hydrazine does not form hydrazides with aldehydes and ketones. Phosphites are not converted to phosphates by oxygen, m-chloroperbenzoic acid, or hydrogen peroxide. Hydrogen peroxide does not oxidize amines. Acetyl chloride does not convert aliphatic alcohols to esters. Trifluoromethanesulfonic acid does not increase the fraction of protonated molecular ion observed for amines and ketones. Hydrogen sulfide and sulfuric acid independently and in combination do not attack isolated double bonds. Phosphorus oxychloride does not react with benzoic acid. A boraneltrimethylamine complex does not reduce nitrobenzene. Although this list of failures may seem impressive, it is a significant advantage of this apparatus that a very large number of potential reagents can be evaluated for a representative variety of substrates in a very short period of time. We feel that perhaps the most important lesson learned in this work is that postcolumn derivatization can be quite simple to perform. Accordingly, people should be encouraged to try the thousands of potential reactant/reagent combinations that this technique makes possible.
variety of organic reactions on GC peaks as they elute. The reagents are introduced and removed very efficiently, and it is possible to carry out reactions on a peak by peak basis. It is important that the reaction region is maintained at or near atmospheric pressure. The products of these postcolumn reactions can be monitored with a sequentially connected mass spectrometer. Reactions which enhance the information obtained from a mass spectrum have been identified. For example , we have demonstrated deuteration, molecular ion enhancement and remote charge site fragmentation.
LITERATURE CITED Markey, S. P.; Abramson, F. P. Anal. Cbem. 1982, 54, 2375-6. Chace, D. H.; Abramson, F. P. Anal. Cbem. 1889, 67, 2724-30. Zaikin, V. G.; Mikaya, A. T. J . Chrometog. 1984, 76, 77-91. Mikaya, A. I.; Zaikin, V. G.; Vdovin, V. M. J . Mol. Cats/. 1985, 32, 353-5. Giebov, L. S.; Mikaya, A. I.; Smetanin, V. I.; Zaikin, V. G.; Klinger, G. A,; Loktev, S. M. J . Catal. 1985, 93, 75-82. Mikaya, A. I.; Zakin, V. G.; Ushakov. N. V.; Vdovin, V. M. Mass Spectrom. Rev. 1990, 9 , 115-32. Teeter, R. M.; Spencer, C. F.; &em, J. W.; Smithson, L. H. J . Am. 0 1 Chem. Soc. 1868, 43, 82-6. Chaffee, A. L.; Lspa, L. Anal. C t " . 1965, 57, 2429-30. Ligon, W. V.; Grade, H. Proceedings of the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Diego, CA, 1985; Paper RE-9, p 645. Munson, 0. Ami. C t " . 1977, 49. 772A-3AB 775A-6A, 778A. Jensen, N. J.; Tomer, K. B.; Gross, M. L. J . Am. Cbem. SOC.1985, 107, 1863-1868. Aiiev. 2. E.; Guseinov, K. 2.;Aiiev, S. A. Arerb. Kbim. Zb. 1976, 7. 56-81.
CONCLUSION It has been found that the relatively simple addition of a conventional split-type GC injector directly after the GC column via a "tee" type connection provides an effective apparatus to introduce reagent and thereby carry out a wide
RECEIVED for review August 2, 1990. Accepted October 29, 1990.
Mass Discrimination Due to z Axis Ion Cloud Coherence in the Fourier Transform Mass Spectrometry Trapped-Ion Cell D. E. Riegner, S. A. Hofstadler, and D. A. Laude, Jr.* Department of Chemistry, The Uniuersity of Texas a t Austin, Austin, Texas 78712
A mass dlscrlminatlon effect common to Fourier transform mass spectrometry (FTMS) experiments involving the Introductlon of extemally generated Ions to the analyzer trappedIon cell Is described. The combination of Inhomogeneous excitation fields with the coherent z axls motion of focused ion packets Is shown to Introduce tlmedependent varlatlons in apparent relatlve ma88 abundance8 that are as large as 60 % . This effect Is evaluated for the dualcell Ion equlllbratlon experiment by modeling the z axls motlon of Isomass packets between adjacent trappedion cells durlng the pulse sequence. Predlcted oscillations In the FlMS signal profile with period equal to haif the expected cubic cell trapping period are then verifled experimentally. Mechanlsms for abrupt termlnatlon of the massdependent osdllatlons at a tlme 2-25 ms after lnitlatlon are also consldered; for example, Ion-neutral and Ion-Ion interactions are discounted, and correlation with the onset of large amplitude magnetron motion Is established. Finally, the merits of the dualcell Ion equllibratlon pulse sequence as a direct probe of z axis e v e lution of the Ion cloud durlng the FTMS experiment are demonstrated.
Adjacent Fourier transform mass spectrometry (FTMS) trapped-ion cells share a common trap plate that can serve 0003-2700/91/0363-0261$02.50/0
conductance limit to facilitate ion transfer between source and analyzer regions of the vacuum system while a large pressure differential is maintained (1-8).In this way highsensitivity FTMS spectra are generated at low pressures from ions formed in high gas load sources. An essential element of all dual-cell FTMS pulse sequences is an event during which the common trap plate is set to ground potential to allow ion transfer between the cells. Two classes of dual-cell pulse sequences can be distinguished on the basis of the position and duration of the ion-transfer event in the pulse sequence. For example, if the event time is on the order of a single trapping oscillation, then a coarse mass filter is achieved through appropriate selection of ion-transfer times because of mass-dependent differences in ion velocities. Giancaspro has studied this type of pulse sequence in detail to understand the mass discrimination effects (1,Z).More recently, we have used it to explicitly measure z axis trapping frequencies in elongated trapped-ion cells (3). A second class of dual-cell ion-transfer pulse sequences, termed equilibration sequences, allows an ion population to be detected in the analyzer region that is representative of the original ion population in the source. This is accomplished by grounding the common trap plate during a continuousionization event for a time that is long compared to the axial trapping period of the ions. The ibn equilibration sequence is commonly used in electron ionization experiments in which BS a
0 1991 American Chemical Society
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complete analyzer cell mass spectra are desired, as for example with FTMS detection of gas chromatrographic effluent (45). Despite its utility for many analytical measurements, a detailed evaluation of this type of pulse sequence has not been presented. Of particular concern is the validity of the assumption that no mass discrimination is associated with the equilibration event, since this is the basic premise of any quantitative FTMS measurment of relative mass intensities. We report here an examination of the dual-cell ion equilibration pulse sequence which indicates that considerable mass discrimination can occur. Fluctuations in apparent relative ion abundances are as high as 60%. As will be demonstrated, this variation in mass intensities is a consequence of the gated trapping event that follows ion transfer and can arise in any FTMS experiment in which trap plate potentials are altered to inject ions into the cell. In most single-cell experiments, the ions are formed in the cell and randomly distributed along the z axis in a symmetrical and continuously maintained trapping field. This random z axis distribution of the ions is not ensured if the ion cloud is tightly focused at the time of formation or if the ions are injected into the trapped ion cell. As will be shown, the dual-cell ion equilibration pulse sequence is particularly susceptible to the effect because the preponderance of ions are initially located near the conductance limit trap plate when trapping potentials are altered to retain ions in the individual cells. Each isomass group of ions then undergoes a coherent oscillatory motion along the z axis of the cubic cell at a characteristic trapping frequency. Coherent motion of the ion ensemble is not by itself responsible for the variation in ion abundance observed in FTMS spectra. An additional requirement is derived from the manner in which the cyclotron orbits of ions are excited prior to detection. It has been demonstrated that the radio frequency pulse used to increase the cyclotron radius during an FTMS excitation or ejection event is not uniform over the length of the cell (9). Specifically, there is a significant reduction in the applied excitation field near the trap plates and ions do not experience homogeneous excitation as they traverse the cell (10-12). Nonuniform excitation is transparent to most FTMS measurements because the ion cloud is randomly distributed with respect to z axis position. However, the effects of an inhomogeneous excitation field should be observed for a focused ion bundle with large z amplitude and will be manifested by changes in apparent relative mass intensities in FTMS spectra as a function of each isomass packet's position in the cell at the time of excitation. A profile of changing signal intensity as a function of offset time between the gated trapping event and excitation would then exhibit a mass-dependent periodicity corresponding to twice the trapping frequency of an ion packet in the cell. In this work, experimental evidence for this m a s discrimination effect is presented and supported by a model for the evolution of the ion cloud during the ion equilibration pulse sequence.
THEORY A mathematical model to predict the instantaneous spatial and kinetic energy distributions of the ion cloud along the z axis during the ionization and subsequent delay events of the dual-cell equilibration pulse sequence was developed. T o perform this simulation it was necessary to obtain equations of motion for both ion flight in the anharmonic well established between the dual cells during the beam event and ion flight in the cubic cell during subsequent events in the pulse sequence. Derivation of the z axis equation of motion in the anharmonic well follows from an approximation of the exact potential expression for a right paralleIepiped with a nonzero potential applied to a single face (13). The potential along the z axis is analogous to that derived by Sharp, Eyler, and Li (13) and is given by
v, = v, + k = {[(2m
+ l)(c/a)I2 + [(2n + l ) ( ~ / b ) ] ~ ] ' / ~
Here Vt is the trapping potential a t the source and analyzer trap plates which are separated by the grounded conductance limit and V , is the potential applied to the excitation and detection plates. Each side of the conductance limit consists of a cubic cell with dimensions a = b = c = 5 cm. Here, the conductance limit is at z = 0 and the trap plate to which a potential is applied is at z = c. As derived elsewhere (15)and shown in eq 2, a velocity expression with m, the mass of the
ion, and q, the charge, can be obtained. For greater accuracy in smaller wells, the factor d27r can be replaced by a variable exponential term, {, obtained from a numerical fit of the exact electric field in the cell. This numerical-fitting procedure is unnecessary for elongated cells and may be avoided in smaller cells in which the quadrupolar approximation of the electric field is often adequate. Integration of the expression for an ion with initial position, zo, yields an instantaneous ion position in the dual cell, z , at any time during the beam event. To obtain an equation for subsequent z axis motion in a cubic cell, the exact potential expression from Sharp, Eyler, and Li for a right parallelepiped with nonzero potentials applied to two parallel trap plates was used (14). In this case a quadrupolar approximation of the electric field is acceptable, due to the harmonic nature of the potential well (3,14-18), and an explicit expression for z axis displacement and for the trapping frequency, shown in eq 3, is obtained (16). Here p, equivalent to 2a in a cubic cell, is equal to 2.7737 (16). ut
-4
=
(3)
A simulation of the z axis motion of an ion population created by electron ionization during an ion equilibration pulse sequence was performed. T o begin, ions were formed with an initial Maxwell-Boltzmann distribution of thermal velocities at random positions on the source side of the dual cell over the course of the beam event. Each ion then oscillated over the length of the dual cell at a characteristic trapping frequency determined by ita mass and initial z amplitude, in accordance with eq 2. Instantaneous ion position and kinetic energy were obtained for each ion at termination of the beam event. Upon reinstating the conductance limit potential, new potential wells in the cubic cells were calculated and total ion energies were reassigned. Trajectories in the new 5-cm wells were calculated by using the quadrupolar equation for trapping frequency in eq 3. In this simulation, no consideration was given to ion-neutral or ion-ion interactions or to ion motion not along the x = y = 0 center line. These simplifications are reasonable for electron ionization that confines ions to the center of the cell and for the low analyzer pressures and small ion densities encountered in most FTMS measurements. Presented in Figure 1 are ion density plots generated from the modeling studies at various stages during the equilibration pulse sequence. The data are for 50000 ions of mass 128 formed in the source cell over the course of a 5-ms beam event.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
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dependence on ion kinetic energy.
EXPERIMENTAL SECTION
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Figure 1. Simulations of ion density distribution for the m l z 128 ion of naphthalene in a dual cell with 2.0 V wells at (a) the terminus of a 5-ms beam event and with a postbeam delay of (b) 27.2 ps and (c) 54.3 gs after the potential of the conductance limit is raised to 2.0 V. The solid lines represent the potential in the cell corresponding to the y axis. Ion density is shown by dots along the center line of the cell.
Figure l a depicts the equilibrium distribution of the ion population between the two cells at the clwe of the beam event just before the conductance limit trap potential is raised. As expected, the ion cloud exhibits an approximately symmetrical kinetic energy and spatial distribution with respect to the conductance limit. Ion density is concentrated in the bottom of the potential well with about 46% of the ions a t any time within 10 mm on either side of the conductance limit. When a 2.0-V potential is applied to the conductance limit a t the end of the ionization event, about 20% of all ions possess a new z amplitude in excess of the trapping potential and are ejected from the cell (15). Many that remain assume a significantly larger z amplitude and are more susceptible to z axis ejection during the excitation event (17-19). Of particular interest here is the observation that once the conductance limit is raised, the ion ensemble is not symmetrically distributed about the center of the cubic cell potential well. It is expected that the z axis motion of the ion bundle will become coherent as depicted in the ion density distributions of Figure I b and c. Ion density distributions are shown at offset times of 27.2 and 54.3 ps, respectively, prior to excitation. The oscillation period for this ion packet is in agreement with a 108.6-gs trapping period predicted by the harmonic oscillator model for the z axis trapping motion of a single ion. Because ion motion is harmonic in this cubic cell, the coherent z axis oscillation should not dephase until ion coulombic or other electric field effects defocus the ion cloud or until collisional damping drives the cloud to the bottom of the potential well. Note that this oscillating behavior is not expected in single-cell experiments for most ionization and ion dissociation processes because, although individual ions exhibit trapping motion, there is no coherence to the z axis motion of the ions as an ensemble. Neither should the oscillation be observed in anharmonic elongated wells, since the ion cloud rapidly dephases because of trapping frequency
Instrumentation. The Fourier transform mass spectrometer used for these experiments is described elsewhere (4). Briefly, the instrument includes a 3.0-T superconducting magnet and a differentially pumped dual trapped-ion cell assembly consisting of stainless steel cubic cells of 5-cm length. A 2-mm aperture at the common trap plate also serves as a conductance limit (CL) that separates source and analyzer regions of the spectrometer and allows pressure differentials of a factor of 102-103. System base pressures are in the low Torr range. During a gated ionization event, a low-microamp electron beam originating in the fringing magnetic field traverses the length of the vacuum chamber for several milliseconds. Ions are formed in proportion to the neutral analyte population within a narrow cylindrical volume along the center line of both cells. The FTMS experiment was performed with a standard equilibration pulse sequence during which, after a quench event, source (ST) and analyzer trap (AT) plates were maintained at trapping potentials of 0.5-2.0 V. During an electron beam event of several milliseconds duration, the CL was maintained at 0.0 V to allow ions to traverse both trapped-ion cells with a trapping period determined by ion mass, trap potential, and initial z axis amplitude. Because the beam event was usually much longer than a single trapping oscillation,the ions established an equilibrium distribution in the source and analyzer cells. Upon termination of the beam event, the potential applied to the CL was raised nonadiabatically to the trapping potential for the balance of the experiment. Most experiments were performed with broad-band swept excitation over 2.66 MHz at a 3200 Hz/ps sweep rate, followed by acquisition of 16K data points over the same bandwidth. Some single-frequency excitation experiments utilized a 100-ps duration pulse at the cyclotron resonance frequency of the ion of interest. Data processing of the transient included baseline correction, sinebell apodization, and addition of 16K zeros prior to a magnitude mode Fourier transform. The data to be presented are in the form of FTMS isomass intensity profiles that probe the z axis evolution of the ion cloud. The profile is a plot of relative ion intensity of a particular mass ion as a function of increasing delay time between the beam and excitation events, usually in 5-ps steps in order to adequately define periodic behavior. The software does not permit detection of the ion profile at times between 0 and 100 ps after restoration of the trap potentials. The actual oscillation period before excitation includes not only the variable delay but also settling times associated with electronic components and any double-resonance excitation or ejection events. The time during the swept excitation event prior to the onset of resonance must also be included; for example, for the excite conditions noted above, resonance excitation of the mass 128 ion with a cyclotron frequency of 362.2 kHz, occurs 113 ps into the 812.5-ps excitation event. Volatile samples were introduced to the source region of the spectrometer through a high-precision variable leak valve. Stable Torr were used source pressures ranging from 5 X lo4 to 2 x with analyzer pressures maintained below 2 X lo4 Torr. For most profiles, the mlz 128ion of naphthalene with a cyclotron frequency of 362.2 kHz was evaluated. A -15-eV electron beam was used to reduce fragmentation of the molecular ion. Control experiments were also performed with a -70-eV electron beam, and similar intensity profiles were obtained. Perfluorotributylamine (PFTBA) ions at mass 69 (673.4 kHz), mass 131 (353.9 kHz), and mass 219 (211.1 kHz) were formed by a -70-eV electron beam and were monitored in mass-dependence studies.
RESULTS AND DISCUSSION As mentioned earlier, observation of this coherent z axis motion is dependent upon the excitation process; specifically, if a homogeneous rf field is used to excite the cyclotron orbit of the ions or if the excitation event is long compared to the period of the ion oscillation, then it is not detected by FTMS. However, in the usual FTMS experiment the rf field falls off near the trap plates and the excitation event is short; thus the average ion orbit for the ensemble should be reduced near the trap plates if excitation conditions are optimized for an
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
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approaching 30% of the maximum intensity; trapping periods of 155 and 66 ps are measured for the m/z 128 ion in 1.0- and 5.0-V wells, respectively. These values are within 4 % of expected trapping periods. While ion cloud asymmetry may be the driving force behind the observed oscillations, it is most likely not the only mechanism contributing to the observed periodic fluctuations in signal intensity. For example, it has been well documented (18-21) that the amplitude of axial oscillation can be increased by excitation a t twice the trapping frequency, 2w,. It is conceivable then, that while the initial impetus for these coherent oscillations is supplied by the initial asymmetry of the ion cloud, interaction with the swept frequency excitation pulse at 2wt may alter the amplitude of the trapping oscillation. The change in amplitude is most likely small due to the brief time excitation a t 2wt is applied. For example, when the trapping amplitude is to be excited by irradiation a t 2wt, a single-frequency excitation pulse is typically applied for several milliseconds (18-21). In this case using a 2.66 MHz bandwidth and sweep rate of 3200 Hzlps, the time during which excitation is applied near 20, is on the order of a few nanoseconds. Similarly, excitation at the trapping sidebands of the cyclotron frequency has been shown to increase or decrease the amplitude of the axial oscillation (22). As will be demonstrated, it appears that these resonance effects make only slight contributions to the observed signal intensities. The mass dependence of the oscillating ion signal is shown in Figure 2d and e for the mass 69 and 131 ions of PFTBA. The profiles are for an ion population generated with a 5-ms, 5-pA beam event with a 2.0 X lO-'-Torr pressure of neutral analyte in the source region. Measured trapping periods of 76 ps for the m / z 69 ion and 125 ps for the mlz 131 ion with 2.0-V trapping potentials are each within 5% and 10% of the predicted values, respectively. Their ratio is also within 15% of that predicted for an inverse square root of mass relationship predicted from the quadrupolar approximation of trapping potential. This mass dependence is particularly troublesome for quantitative measurements that require re-
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ion cloud centered in the cell. It would be expected then that a profile of ion intensity generated by FTMS detection a t increasing delay times after the beam event would exhibit an oscillating signal that is a t a maximum when the ion cloud passes through the center of the cell and a t a minimum as it approaches the trap plates. A cubic cell trapping frequency extracted from the profile should conform to harmonic motion in accordance with eq 3. Figure 2 presents experimental results that verify a periodicity predicted from the simulated profiles. In Figure 2a-c the mass 128 ion of naphthalene formed by a 5-pA electron beam a t a source pressure of 5 x Torr is monitored. Shown first in Figure 2a is a control experiment in which ions are formed and detected in the source region between the ST and CL in a single-cell experiment with trapping potentials continuously maintained at 2.0 V. Because the ion population is symmetrically distributed between the trap plates, oscillations are not observed. Nonperiodic fluctuations in signal intensity are attributed to changes in neutral naphthalene populations. In contrast, the electric field dependent oscillations predicted for the dual-cell experiment are evident in Figure 2b and c, with changes in apparent relative abundances
ANALYTICAL CHEMISTRY, VOL. 63,NO. 3, FEBRUARY 1, 1991
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producibility between relative ion intensities. An idea of the magnitude that these fluctuations can assume from spectrum to spectrum can be gained from Figure 3, where two PFTBA spectra, acquired at postionization delays of 140 and 180 ps, are shown. (Actual delays including the nonresonance delay during the swept excitation event are 350 and 390 ps for the m f z 69 ion and 250 and 290 ps for the m / z 131 ion, respectively.) The relative abundance of the 131 and 219 ions to the base peak are 49% and 39% in Figure 3a and 71% and 53% in Figure 3b. Thus, apparent relative mass intensities are not necessarily reproducible from experiment to experiment in the dual cell as long as this mass discrimination effect is uncontrolled. As will be shown, any change in experiment pulse sequence parameters that alter the delay between reinstatement of trapping potentials and the excitation event will perturb the apparent relative mass intensities that are measured. The magnitude of the fluctuation in signal intensity is dependent on several factors associated with ion formation and detection. This is demonstrated with the collection of ion profiles in Figure 4 for the naphthalene molecular ion detected in a dual cell with the equilibration sequence. To
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begin, the profile in Figure 4a from a 100-ps duration single-frequency excite, does not exhibit periodic behavior. This is because the resonance excitation event is so long compared to the trapping period that each ion experiences an average of the excitation power over the length of the cell. It would be expected that any lengthy excitation process, for example the stored waveform inverse Fourier transform (SWIFT) method (231,would be less susceptible to periodic fluctuations in signal intensity and yield a better representation of relative ion abundances. Figure 4b-d presents profiles acquired with swept excitation for different combinations of beam time and current expected to generate approximately equal detectable ion populations below the space charge limit. The three are profiles of similar shape and intensity with a variation in the intensity of about 60%. Evidently, the possibility that at longer beam events the ion cloud would be increasingly damped due to source side collisions is not the case, since increased amplitude associated with a more tightly focused ion cloud are not observed. The only discernable difference between the three profiles is a phase shift from short to long beam times that may occur because the spatial and kinetic energy distributions in the 10-cm well during the beam event have not been fully ran-
266
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
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domized. Once an equilibrium distribution was established, it would be expected that ion motion in the cell would have a constant phase initiated by the change in conductance limit potential. Further evidence that collisional damping during the beam event does not significantly alter the shape of the ion cloud comes from profiles presented in Figure 4e and f. In these examples, analyzer side oscillations follow ion formation a t source pressures of 3 X lo4 and 2 X Torr, respectively. An invariance of oscillation amplitude to pressure again indicates that typical FTMS experiment conditions are incompatible with significant collisional damping of the ion cloud and argues against it as the mechanism for termination of the oscillation. Oscillation profiles were next examined as a function of the size of the ion population to determine the significance of ion coulombic repulsive effects on the periodic nature of the signal. Profiles in Figure 4g and h are for ion populations that are factors of 10 and 20 higher than those presented in Figure 4b-e. The broadening and distortions displayed in these profiles would indicate that ion-ion interactions are significant. For example, in Figure 4g the ion cloud formed with a 2-ms, 25-pA beam a t source pressures of 2 X lo-' Torr exhibits a reduction in the relative amplitude of the oscillation that is attributed to a smearing of the ion cloud along the z axis. The periodic motion is still observed but is higher in frequency than expected, which suggests the ion cloud makes a significant contribution to the effective electric field. Finally, the profile indicates that the ion population splits into two discrete ion packets with a phase offset of 20 ps, each exhibiting its
own local maxima. Presented in Figure 4h is the ion profile for an ion population now well above the space charge limit. At this point only a single period is distinguishable as the oscillation is quickly damped. The duration of the oscillating signal and mechanism by which it is damped or terminated are of interest for an understanding of macroscopic behavior of the ion cloud detected by FTMS. Several competing mechanisms can be postulated. One possibility is a dephasing of the coherent ion packet due to coulombic repulsive effects; in this case the signal damps as the ion cloud distribution smears along the z axis. As shown in Figure 4g and h, this mechanism is important for large ion populations. However, as the profiles in Figure 4b-e indicate, the magnitude of the repulsive effects in smaller ion populations is insufficient to promote a rapid damping of the signal and other processes must be responsible. An alternate mechanism for oscillation damping would involve thermalization of the ion ensemble due to ion-neutral collisions that would drive the ion cloud to the bottom of the well. However, a t low pressures commonly used for FTMS detection, this damping would occur at best on a several hundred millisecond times scale and is not observed. Note that whatever the mechanism, it is different from that responsible for damping in the Giancaspro experiment that also generates trapping oscillation profiles in dual cells. In that case an exponential decay process was observed, but was attributed to clipping of ions a t the orifice of the common trap plate. Profiles were acquired for much longer times than shown in Figures 2 and 4 in an effort to observe the termination of the oscillations. In some cases oscillations were observed a t
ANALYTICAL CHEMISTRY, VOL. 63,NO. 3,FEBRUARY 1, 1991
delay times of 10-25 ms, but more often they terminated within about 5 ms. Moreover, the profiles never exhibited an exponential decay indicative of collisional damping but rather ended abruptly. For example, Figure 5a and b depicts oscillation profiles for 1.0- and 2.0-V trapping wells, respectively, acquired over a several millisecond interval until the oscillations ceased. The duration of the oscillation was found to be dependent upon trapping potential and to vary significantly with cell and electron beam alignment in the magnetic field. An expanded region of the termination point for oscillations in the 2.0 V well is shown in Figure 5c. A relatively smooth and well-defined periodicity is replaced by an increasingly noisy signal during the period between 1.3 and 2.3 ms. Interestingly, the standard deviation for scan to scan reproducibility measured at various times along the profile was found to vary a great deal. For example, early in the oscillation period (less than 1ms) when well-shaped profiles are observed, it is less than 2% of the maximum signal oscillation. However, during the transient period when the oscillations break down a t around 1.8ms, the scan to scan reproducibility is 1 order of magnitude worse. Later, after oscillations have ceased, the standard deviation in scan to scan reproducibility returns to its original level. The source of this turbulence is evidently associated with the mechanism by which oscillations are terminated. We have observed some correlation between the cessation of trapping oscillation and the appearance of a strong periodic signal corresponding to magnetron motion, which Giancaspro has also observed ( 1 , 2 ) . For example, for some experiments in a 1.0-V well the magnetron motion produced an oscillation amplitude similar in intensity to that expected for the trapping motion, yet no trapping oscillations were observed even at very short delays. Under identical conditions in a 2.0-V well, the oscillation amplitude at the magnetron frequency was much smaller while the trapping oscillation amplitude was large. The magnetron motion was observed in both source and analyzer cells, but the amplitude was larger at higher pressures as would be expected (24). Figure 6 presents representative examples of the observed magnetron motion at 1.0- and 2.0-V trap potentials. Shown in Figure 6a and b are source and analyzer cell oscillations over a 40-ms time interval; the periodicity of the signal is 15.1 ms, in good agreement with calculated values for cells of these dimensions (16). Shown in Figure 6c and d are source and analyzer cell profiles acquired over the same time interval for a 2.0-V well; here the 8-ms period exhibited in the source cell is in agreement with theory, while a weak periodic signal a t twice the magnetron frequency is observed in the analyzer cell. We point out that Giancaspro and Dunbar have also observed the magnetron motion directly (2,24)but no good explanation for the source of such a large magnetron amplitude at short times and low pressures is provided. We speculate that a coupling of magnetron and trapping motion for the ion ensemble in appropriate electric fields is responsible for the large fluctuation in scan to scan reproducibility, and possibly for the cessation of trapping oscillation and onset of a pronounced magnetron amplitude. The disappearance of trapping oscillation within a few milliseconds in our system is fortunate because such massdependent fluctuations in amplitude at longer intervals would have important negative consequences for quantitative measurements in ion-molecule reactions. However, even with the rapid damping, it is necessary to be aware of effects on apparent relative ion abundances when selecting pulse sequence parameters. Within the period that these oscillations occur, any change in experiment parameters that alters the delay time between reapplication of the trapping potential and subsequent excitation of the ion population will alter the
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Figure 7. Ion intensity profiles as a function of swept excitation condtioins for a duaCcell equilibration experiment in a 1.0-V well. The mlz 128 ion of naphthalene is detected. (a) Excite conditions include a sweep of 3200 Hzlps from 2600 kHz to ( + ) 0 Hz, (0) 100 kHz, (+) 200 kHz, and (X) 300 kHz. (b) Excite conditions include a sweep 100 kHz, (+) 200 kHz, and (X) of 3200 Hzlps from ( + ) 0 Hz, (0) 300-2600 kHz.
relative and absolute signal intensity of each ion in the spectrum. In addition to reaction delay events, other pulse sequence event times including ejection and excitation events must be considered. As an example, presented in Figure 7 are absolute signal intensities for the m / z 128 ion of naphthalene obtained for different excitation sweep parameters. In all experiments a 3200 Hz/ps sweep rate is used while the bandwidth is reduced by increments of 100 kHz. In Figure 7a, the sweep originates at 2.66 MHz and a constant delay of 83 ps occurs prior to excitation of the 128 ion at 362 kHz; consequently, the profiles are similar in intensity. In contrast, in Figure 7b the experiment was performed by initiating the excite event at the variable lower frequency. In this case the delay until excitation of the 128 ion is reduced in 31-ps intervals, which results in phase shifts in the observation of the maximum ion signal. Additionally, Figure 7 demonstrates the difference in oscillation intensity when the swept frequency bandwidth does and does not include excitation at 2w,. In both Figure 7a and b the full sweep bandwidth (+) includes excitatioin a t 2w, = 12994 Hz, while the other excitation bandwidths (0,+, and X) do not excite at 2w,. The resulting ion intensity profiles are very similar in magnitude implying that the interaction of the oscillating ion bundle with the
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applied excitation a t 2w, is minimal.
CONCLUSIONS A new form of mass discrimination in FTMS has been observed and is attributed to interaction of a nonuniform excitation field with ion clouds that are initially asymmetrically distributed in the cell. Trapping frequencies in a single section cell may be observed directly in this way. Evaluation of the effect in a dual-cell instrument during an ion equilibration experiment indicates mass-dependent shifts as high as 60% occur, which can have significant impact on quantitative measurements. A mechanism for damping of the oscillation is unclear, but truncation of the periodic behavior within a few milliseconds is not consistent with ion-ion or ion-neutral collisions; alternately, there appears to be some correlation between the onset of a strong magnetron oscillation and the cessation of the trapping event. The data presented also indicate that the dual-cell ion equilibration pulse sequence will be a useful tool in probing the z axis evolution of the ion cloud during FTMS experiments. LITERATURE CITED Giancaspro. C.; Verdun, F. R . Anal. Chem. 1988, 5 8 , 2097-2099. Giancaspro, C.; Verdun. F. R.; Muller, J. F. Int. J . Mass Spectrom. Ion Processes 1988, 7 2 , 63-71. Hofstadier, S. A,; Laude, D. A., Jr. J. Am. SOC.Mass Spec. 1990, 7 , 35 1-360. Hogan, J. D.; Laude, D. A., Jr. Anal. Chem. 1990, 6 2 , 530-535. Hogan, J. D.: Laude, D. A.. Jr. Unpublished materials. Cody, R. B.; Kinsinger, J. A.; Ghaderi, S.; Amster. I.J.; McLafferty, F. W.; Brown, C. E. Anal. Chim. Acta 1985, 178, 43-66.
(7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
(21) (22) (23) (24)
Wise, M. B. Anal. Chem. 1987, 5 9 , 2289-2293. Kerley. E.; Russell. D. Anal. Chem. 1989. 61, 53-57. Dunbar, R. C. Int. J. Mass Spectrom. Ion Processes 1985. 56, 1-7. Wang, M.; Marshall, A. G. Anal. Chem. 1990, 62, 515-520. Hanson, C. D.; Castro, M. E.: Kerley. E. L.; Russell, D. H. Anal. Chem. 1990, 6 2 , 520-526. Wang, M.; Marshall, A. G. Anal. Chem. 1989, 61, 1288-1293. Morse, P. M.; Feschback, H. Methods of Theoretical Physlcs; McGraw-Hili: New York, 1953; pp 1252-1260. Sharp, T. E.; Eyler, J. R.; Li, E. Int. J. Mass Specfrom. Ion Phys. 1972, 9 , 421-439. Hofstadler, S. A.; Laude, D. A., Jr. Int. J . Mass Specfrom. Ion Processes, in press. Hunter, R. L.; Sherman, M. G.; McIver, R. T., Jr. Int. J. Mass Specfrom. Ion Phys 1903, 50, 47-54. Alleman, M.: Kofel, P.; Kellerhals, Hp.; Wanczek, K. P. Int. J . Mass. Spectrom. Ion Processes 1987, 75, 47-54. Van De Guchte, W. J.; Van Der Hart, W. J. Int. J. Mass Spectrom. Ion Processes 1990, 95, 317-326. Hwng, S. K.; Rempei, D. L.; Gross, M. L. Int. J . Mass Specfrom. Ion Processes 1986, 72, 15-31. Van De Guchte, W. J.; Van Der Hart, W. J. Int J. Mass Spectrom. Ion Processes 1908, 8 2 , 17-32. Delong, S. E.; Mitchell, D. W.; Cherniak, D. J.; Harrison, T. M. Int. J . Mass Spectrom. Ion Processes 1909, 9 1 , 273-282. Alleman, M.: Kellerhals, H. P.; Wanczek, K. P. Chem. Phys. Lett. 1981, 84. 547-551. Chen, L.; Marshall, A. G. Int J. Mass Spectrom. Ion Processes 1987, 79, 115-125. Dunbar, R. C.; Chen, J. H.; Hays, J. D. Int. J. Mass Specfrom. Ion Processes 1984, 5 7 , 39-56.
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RECEIVED for review August 27,1990. Accepted October 29, 1990. This work is supported by the Welch Foundation and by a grant from the Texas Advanced Technology and Research Program.
Immunoassay Method for the Determination of Immunoglobulin G Using Bacterial Magnetic Particles Noriyuki Nakamura, Kohji Hashimoto, and Tadashi Matsunaga*
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan
We have developed a novel immunoassay method using bacterlai magnetic partlcles for the determination of Immunoglobulin G (IgG). FluoresceinIsothbcyanate (FITC) conJugatedant1 IgG-bacterial magnetk partlcles were prepared. The fluorescence quenching caused by agglutination of FITC-ant1 IgG antibody-bacterial magnetic partlcie conjugates was measured by uslng a fluorescence spectrophotometer. The aggregates based on speclfk knmunoreactlon were separated by a gelatin solution. The aggregation of bacterial magnetic partlck conjugates was enhanced by appllcatlon of a magnetic field. The relative fluorescence intensity correlated itnearly with a concentration of IgG in the range 0.5-100 ng/mL.
INTRODUCTION Determination of serum immunoglobulin levels is of value in various clinical fields. A variety of techniques has previously been employed in solid-phase immunoassay, for example, the use of latex particles ( I ) and liposomes (2). Agglutination reactions which use carriers are often used due to their im-
* Corresponding author.
proved handling in the laboratory. In these methods, the solid phase on which the immunological reaction takes place must be separated from the reaction solution by centrifugation before the measurements are carried out. The use of magnetic particles allows separation of the bound and free antibody fractions by application of a magnetic field. This technique also overcomes the problem of mixing during the incubation period (3-7). Magnetotactic bacteria, which orient and swim along geomagnetic fields, are known to produce magnetic particles (8). Purified bacterial magnetic particles (BMPs) are small in size (500-1000 A) and disperse very well because they are covered with a stable lipid membrane (9-11). Large quantities of bioactive substances may be immobilized on BMPs due to the presence of this membrane. Moreover, the location of the immobilized bioactive substance can be controlled with a magnetic field. This paper describes the immobilization of anti-mouse IgG antibody onto BMPs. In addition, a novel solid-phase fluoroimmunoassay using antibody immobilized onto BMPs has been developed for the rapid determination of mouse IgG concentration. EXPERIMENTAL SECTION Materials, FITC-conjugated goat anti-mouse IgG antibody was obtained from Sigma Chemical Co. (St. Louis, MO). The
0003-2700/91/0363-0268$02.50/00 1991 American Chemical Society
