Anal. Chem. 1996, 68, 4409-4413
Effects of Capacitive Coupling on Ion Remeasurement Using Quadrupolar Excitation in High-Resolution FTICR Spectrometry Cynthia C. Pitsenberger, Michael L. Easterling, and I. Jonathan Amster*
Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556
Axial ejection is found to be the principal cause of the loss of ions during Fourier transform mass spectrometry remeasurement experiments. Ion remeasurement improves to near unit efficiency when an open-ended cylindrical analyzer cell is used with capacitive coupling of the excitation signal to the adjacent trapping plates. With capacitive coupling enabled for the entire experiment, 100% remeasurement efficiency is observed over a narrow mass range (10 Da) for 200 scans of ions of poly(ethylene glycol) formed by internal matrix-assisted laser desorption/ionization. Greater than 99.5% efficiency per remeasurement cycle has been achieved for wide mass range (1000 Da) remeasurement with capacitive coupling enabled. Further evidence for axial ejection during remeasurement comes from a comparison of remeasurement efficiencies obtained at different trapping potentials. In recent years, dramatic improvements have been achieved in the analytical performance of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry as researchers continue to push the limits of high resolution, sensitivity, mass accuracy, and high mass. One technique that has contributed to these advances is quadrupolar excitation (QE). This method, originally introduced by Savard et al.,1 can be used to direct mass-selected ions to the center of an ICR analyzer cell by converting magnetron motion to collisionally damped cyclotron motion, a process called axialization. In the absence of quadrupolar excitation, collisional damping of the magnetron motion causes an outwardly directed radial drift of ions from the ICR analyzer cell. With QE, ions can undergo multiple collisions without radial ion loss and, in fact, are directed to the center of the analyzer cell. Therefore, highmass ions that are formed and trapped in the cell with high kinetic energies can be collisionally thermalized to improve their detection.2-4 This is useful for capturing and collisionally relaxing ions that have been transferred from an external ion source.5,6 High-resolution analysis has been possible by using QE with (1) Savard, G.; Becker, S.; Bollen, G.; Kluge, H.-J.; Moore. R. B.; Schweikhard, L.; Stolzenberg, H.; Wiess, U. Phys. Lett. A 1991, 158, 247-252. (2) Easterling, M. L.; Pitsenberger, C. C.; Kulkarni, S. S.; Taylor, K. T.; Amster, I. J. Int. J. Mass Spectrom. Ion Processes, in press. (3) Pitsenberger, C. C.; Easterling, M. L.; Amster, I. J. Anal. Chem. 1996, 68, 3732-3739. (4) Salvador, J. P.; Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 379384. (5) Marto, J. A.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1994, 8, 615-620. (6) Hendrickson, C. L.; Laude, D. A. Anal. Chem. 1995, 67, 1717-1721. S0003-2700(96)00659-2 CCC: $12.00
© 1996 American Chemical Society
pulsed gas collisional damping2 or with a dual-analyzer cell,7 either of which permits detection at low pressures. QE permits massselective ion isolation for MS/MS experiments.8,9 Another important capability of analytical ICR enabled by QE is ion remeasurement. The capability to remeasure ions is a significant advantage of ICR’s nondestructive image current detection, which stands in contrast to other mass spectrometry methods. Efficient ion remeasurement improves signal-to-noise and is useful for ionization conditions that yield low ion abundances or for samples of limited quantity.10 Remeasurement has been demonstrated without QE, but radial ion loss and low resolution make this a less attractive alternative. As the original pioneer in FTICR ion remeasurement, McLafferty demonstrated 92-98% efficiency for remeasuring high-mass ions using a lowmass collision gas to relax ions to the center of the analyzer cell.11 Experimental conditions (high magnetic field) were such that cyclotron damping occurred much faster than radial ion loss through magnetron expansion. Guan et al. found unit remeasurement efficiency in a closed elongated cell for 200 scans of multiply charged protein ions formed by electrospray ionization.12 Campbell et al. demonstrated that, under specific conditions, unit remeasurement efficiency for an electrosprayed protein can be achieved for 100 co-added scans in an open-ended, elongated cell.13 However, each of these remeasurement experiments was conducted without QE, and experimental conditions were very specific for each case, thus appearing impractical for routine use. Implementation of QE has made remeasurement highly efficient and reproducible for both single-frequency and broad-band quadrupolar excitation.2,3 This laboratory was the first to demonstrate remeasurement using QE.14 Remeasurement efficiencies greater than 99.5% for a single m/z in a cubic cell at 1 T were reported. For good remeasurement efficiency, ion loss for each detection cycle must be minimized. Remeasurement efficiency was maximized in these early demonstrations by using high trapping potentials and low dipolar excitation amplitudes for ion (7) Pasˇa-Tolic´, L.; Huang, L.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825-833. (8) Guan, S.; Marshall, A. G.; Wahl, M. C. Anal. Chem. 1994, 66, 1363-1367. (9) Guan, S.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Chem. Rev. 1994, 94, 2161-2182. (10) Solouki, T.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Anal. Chem. 1995, 67, 4139-4144. (11) Williams, E. R.; Henry, K. D.; McLafferty, F. W. J. Am. Chem. Soc. 1990, 112, 6157-6162. (12) Guan, Z.; Hofstadler, S. A.; Laude, D. A. Anal. Chem. 1993, 65, 15881593. (13) Campbell, V. L.; Guan, Z.; Vartanian, V. H.; Laude, D. A. Anal. Chem. 1995, 67, 420-425. (14) Speir, J. P.; Gorman, G. S.; Pitsenberger, C. C.; Turner, C. A.; Wang, P. P.; Amster, I. J. Anal. Chem. 1993, 65, 1746-1752.
Analytical Chemistry, Vol. 68, No. 24, December 15, 1996 4409
detection. However, the application of a trapping potential creates an electric field gradient which is radially repulsive. This radial electric field reduces the effective magnetic field strength, shifts the ion’s cyclotron frequency, and reduces the upper mass limit of the analyzer cell. The use of high trapping voltages and low dipolar excitation amplitudes led to low resolution. Thus, using lower trapping potentials is desirable. Isotopic resolution was not observed with this first example of QE remeasurement, because of the coupling of ion motions that occurs when the ratio of the magnetic field to the trapping voltage is low.15-17 Several approaches have been taken to create a more spatially uniform radio frequency (rf) electric field to prevent axial ejection.18-22 Beu and Laude have shown that capacitive coupling of the trap plates to their adjacent excite plates can lead to a dramatic reduction of the axial component of the excitation field, reducing the axial ejection of ions.21 Since energy deposition along the trapping axis is minimized during excitation with capacitive coupling, lower trapping potentials may be used during detection. Although a number of other designs have sought to reduce both axial ejection and the radial electric field,23,24 Beu and Laude’s capacitive coupling is, by far, the simplest to implement. In this report, we show that axial ejection during ion excitation is the major mechanism for ion loss during single-frequency and broad-band QE remeasurement. The results furthermore show that, by capacitively coupling excitation signals to the trapping plates of an open-ended cylindrical analyzer cell, high remeasurement efficiency is achieved. Others have studied ion loss during remeasurement as a function of electric field and cell geometry.12,13,25 Campbell et al.13 and Guan et al.12 have both concluded that radial diffusion was the primary mechanism for ion loss during remeasurement without QE. These authors did not discuss the possibility of axial ion loss, as there was no way to differentiate between the two ion loss mechanisms. By using QE, we eliminate radial ejection as an ion loss mechanism and examine axial ejection independently. These findings allow remeasurement with high efficiency and high resolution. EXPERIMENTAL SECTION A 4.7 T FTICR spectrometer that was designed and fabricated at the University of Georgia was used for all experiments. The instrument consists of a custom-designed vacuum chamber, a shielded 4.7 T superconducting magnet, and a commercial data system (IonSpec, Irvine, CA). The main vacuum chamber is pumped to a base pressure of 1 × 10-9 Torr with a 330 L/s turbomolecular pump backed by a 150 L/s diffusion pump. Housed in the main vacuum chamber, in the homogeneous field of the magnet, is an open-ended, capacitively coupled analyzer (15) Huang, J.; Tiedemann, P. W.; Land, D. P.; McIver, R. T.; Hemminger, J. C. Int. J. Mass Spectrom. Ion Processes 1994, 134, 11-21. (16) Wang, T. L.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1986, 68, 287-301. (17) Chen, S.; Comisarow, M. B. Rapid Commun. Mass Spectrom. 1991, 5, 450455. (18) Wang, M.; Marshall, A. G. Anal. Chem. 1990, 62, 515-520. (19) Hanson, C. D.; Castoro, M. E.; Kerley, E. L.; Russell, D. H. Anal. Chem. 1990, 62, 520-526. (20) Caravatti, P.; Allemann, M. Org. Mass Spectrom. 1991, 26, 514-518. (21) Beu, S. C.; Laude, D. A. Anal. Chem. 1992, 64, 177-180. (22) Grosshans, P. B.; Chen, R.; Limbach, P. A.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1994, 139, 169-189. (23) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1995, 146/ 147, 261-296. (24) Caravatti, P.; Allemann, M. Org. Mass Spectrom. 1991, 26, 514-518. (25) Vartanian, V. H.; Laude, D. A. Anal. Chem. 1996, 68, 1321-1327.
4410
Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
cell with a diameter of 2.25 in. and an aspect ratio (length of detection or excitation electrodes divided by the diameter of the analyzer cell) of 1. An ancillary chamber that is evacuated by a 1100 L/s cryogenic pump is used to pump sample targets to a base pressure of 1 × 10-7 Torr before they are introduced to the main vacuum chamber. This two-stage sample introduction permits laser desorption analysis under ultrahigh vacuum. Nitrogen (>99.999% purity) for collisionally induced axialization is introduced through a pulsed valve inlet. The pulsed valve is opened for less than 20 ms, and the analyzer pressure rises to approximately 1 × 10-4 Torr. The pressure returns to approximately 5 × 10-9 Torr within 20-25 s. Two-plate quadrupolar excitation was used for all experiments. Data accumulation and processing are controlled by the data system (IonSpec OMG/ 586). The instrument is equipped with an internal preamplifier (IonSpec) mounted directly on the analyzer cell plate to reduce the adverse effects of distributed capacitance on the image current. Samples are desorbed using the 355 nm output of a Nd:YAG laser (New Wave Research, ACL-MS-355, Sunnyvale, CA), which is focused with a telescopic arrangement of two plano-convex lenses. The sample target, a 1 in. diameter stainless steel cylinder, can be rotated under remote control to examine many different samples without moving the focal point of the laser beam. Poly(ethylene glycol) with an average molecular mass of 2000 Da was used for these experiments. The poly(ethylene glycol) samples are prepared as 1 nmol/µL solutions using a 70:30 solution of water (0.1% TFA) and acetonitrile. A 2 µL sample of the poly(ethylene glycol) solution is mixed on the probe with 2 µL of a saturated solution of sinapinic acid in a 70:30 solution of water (0.1% TFA) and acetonitrile. Both single-frequency and broad-band QE remeasurement experiments follow a similar sequence of events, depicted in Figure 1. Ions are formed and detected at trapping potentials of 0.5-2 V. After ions are formed, an experimental sequence for remeasurement is loaded by the data system and used for the remeasurement cycles. At the beginning of this sequence, the trapping potential is raised to 3 V, and a relay is activated to switch the electronics from normal dipolar excitation to quadrupolar excitation. A buffer gas is introduced, and the ions undergo quadrupolar excitation for 3.9 s. The trapping potential is reduced, and the ions remaining in the analyzer cell are detected approximately 25 s after the start of the sequence. The quadrupolar excitation signal originates from either the internal frequency synthesizer of the data system or an external function generator. The external generator provides several types of wave forms, including fixed frequency, linear and logarithmic frequency sweeps, and white noise. A SPDT relay that is activated by a TTL pulse from the data system switches between excitation by the external function generator and the internal frequency synthesizer. The signal is then amplified and applied to the excitation plates of the analyzer cell. Linear frequency sweeps of sinusoidal wave forms were used for repetitive chirp quadrupolar excitation. For these experiments, the frequency range of 47 924-28 767 Hz, corresponding to a mass-to-charge range of 1500-2500, was repeated at a rate of 1000 Hz, i.e., with a frequency sweep rate of 20 kHz/ms. Capacitive coupling was used to extend the rf excitation to the ends of the analyzer cell to linearize the excitation field and reduce the axial ejection of ions.21 The circuit that is (26) Dahl, D. A. SIMION 3D, Version 6.0; Idaho National Engineering Laboratory: Idaho Falls, ID, 1995.
Figure 1. Experimental sequence for single-frequency and broad-band QE remeasurement studies using capacitive coupling. The last event is a trigger pulse for turning capacitive coupling on and off.
used to achieve capacitive coupling with this instrument is described in a separate paper.2 Plots of the rf electric field lines for this cell were generated using SIMION 6.0.26 RESULTS AND DISCUSSION As with many analytical methods, signal-to-noise in FTICR is improved by summing the signals produced during multiple measurement cycles. With FTICR, ions are detected in a nondestructive manner, and so they can be formed once and measured several times. This process, called remeasurement, produces significant improvements in signal-to-noise, provided that the remeasurement efficiency is high. Remeasurement efficiency is defined as the percentage of ions present in the analyzer cell after a measurement cycle that are returned to the center of the analyzer cell and are available for a subsequent cycle of measurement. Remeasurement efficiency is determined experimentally by observing the decrease in the intensity of a mass spectral peak during several cycles of remeasurement. For the experiments reported here, the best remeasurement efficiencies are found when capacitive coupling is enabled during the entire experimental sequence. Figure 2 shows a matrix-assisted laser desorption/ ionization (MALDI) mass spectrum of an oligomer of poly(ethylene glycol) containing 41 repeating units. These ions, formed by one laser desorption event, underwent 200 cycles of remeasurement using a low-amplitude, single-frequency QE3,27,28 with capacitive coupling enabled during the entire experiment. The isotope peaks for the 200th scan are clearly resolved, and their absolute abundances remain constant for the 200 remeasurement cycles, demonstrating 100% remeasurement efficiency. Ion loss by axial ejection is known to decrease with the implementation of capacitive coupling. This is demonstrated by examining remeasurement efficiencies as a function of trapping potential, shown in Figures 3 and 4. Figure 3 shows a plot of (27) Pastor, S. J.; Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 379-384. (28) Wood, T. D.; Ross, C. W.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1994, 5, 900-907.
Figure 2. MALDI spectra of the 1st and 200th remeasurement scans of a 41-mer of poly(ethylene glycol) (m/z 1846), showing 100% remeasurement efficiency. A low-amplitude (1 V), single-frequency QE signal was set to m/z 1835, and the trapping voltage at detection was 2 V. Capacitive coupling was enabled for the entire remeasurement experiment. Absolute intensities were used so that each spectrum could be compared directly.
signal intensity for the m/z 1846 ion of poly(ethylene glycol) as a function of remeasurement cycle for three trapping potentials, all performed with capacitive coupling. At trapping voltages greater than 1 V, the remeasurement efficiency is 100%. Remeasurement efficiency decreases as trapping voltage is reduced further; for example, it is approximately 99.5% per measurement cycle at 0.5 V trapping potential. This decrease in remeasurement efficiency is consistent with axial ejection. With detection excitation energies remaining constant, a reduction in the trapping potential well allows ions to sample regions of the cell where the rf electric field gradient can impart axial excitation to the ions. Furthermore, ions require less z-axis energy to escape the cell as the trapping potential is reduced. This phenomenon is demonstrated more clearly when capacitive coupling is disabled during remeasurement, as shown in Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
4411
Figure 3. Relative intensity of the signal produced by singlefrequency QE remeasurement of a 41-mer of poly(ethylene glycol), plotted against the scan number. The solid lines are for calculations of signal intensity at different remeasurement efficiencies. At 2.0 and 1.0 V trapping (Vt), 100% efficiency is noted for 50 scans, and capacitive coupling is enabled for the duration of the remeasurement. A remeasurement efficiency of 99.5% is observed for a trapping potential of 0.5 V. Figure 5. (Top) Isopotential contours of a 30 V rf excitation applied to an uncoupled, cylindrical analyzer cell of aspect ratio 1, calculated using SIMION. Trapping potentials are set at 1 V. (Bottom) Isopotential contours for a 30 V rf excitation signal applied to a capacitively coupled, open-ended cylindrical cell with a 1 V trapping potential.
Figure 4. Single-frequency QE remeasurement curves of a 41-mer of poly(ethylene glycol). The solid lines show the signal intensity predicted for remeasurement efficiencies between 90 and 100%, as labeled. For each experiment, capacitive coupling is disabled for the entire remeasurement cycle. At a trapping potential of 2.0 V, 100% remeasurement efficiency is observed. At trapping potentials of 1.0 and 0.5 V, the efficiency is observed to decrease to 96-98%.
Figure 4. At 2.0 V trapping potential, unit remeasurement efficiency was obtained for the 41-mer of poly(ethylene glycol). Aside from disabling capacitive coupling, all other experimental conditions were the same as for the data in Figure 3. At such high trapping potentials, the ions are confined to the center of the analyzer cell and thus do not experience axial ejection. However, by dropping the trap potential to 1.0 and 0.5 V, the remeasurement efficiency is decreased significantly. With capacitive coupling disabled, ions acquire more z-axis energy and can escape the cell. Therefore, at the m/z range examined here, axial ejection can contribute to ion loss. The data in Figures 3 and 4 are consistent with axial ejection and can be described in terms of the theoretically generated rf electric field in the analyzer cell. Shown in Figure 5 are plots of the isopotential electric field contours resulting from dipolar excitation in a cylindrical open-ended analyzer cell, contrasting the electric fields that are present both with and without capacitive coupling. As shown in the top of Figure 5, the finite dimensions of the excitation electrodes of the cell create a curvature in the 4412 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
electric field resulting from the rf signal applied to the transmitter plates. This curvature leads to the axial excitation of ions when the z-axis energy gained by the ions is greater than the trapping potential. Capacitively coupling the trapping plates to the adjacent excite plates decreases the curvature of the rf electric field, as can be seen in the plot displayed in the bottom panel of Figure 5. The rf excitation field is uniform throughout the cell volume, and its component along the z-axis is zero, so ions experience a force only in the xy-plane. With capacitive coupling, ions can occupy a larger volume of the cell without experiencing axial ejection. In the recent study by Campbell et al., radial ion loss was attributed as the dominant ion loss mechanism for remeasurement without QE in an open-ended cylindrical cell.13 They argued that axial ejection would be unlikely to contribute to ion loss at high m/z. Axial ejection is usually considered to affect only the detection of ions of low mass-to-charge.29 However, this assumption is based on the efficiency of detecting ions in a single measurement cycle. Axial ejection occurs to a smaller degree for ions of high mass-to-charge but has a measurable effect on the efficiency of their remeasurement. It should be noted that, in their prior study, Campbell et al. used an elongated cylindrical cell which produced less curvature of the excitation field than does the analyzer cell used in our study, which therefore reduced the effects of axial ejection. Capacitive coupling will reduce the curvature of the excitation field for a cylindrical cell of any aspect ratio, and our results suggest that the efficiency of remeasurement of high mass-to-charge ions will improve when the electric field is linearized by these or any other means. Axial ejection is also found to be the primary mechanism for ion loss during broad-band QE remeasurement, that is, remeasurement over a broad mass range. Figure 6 shows mass spectra collected for 50 remeasurement cycles of poly(ethylene glycol) with an average molecular mass of 2000 Da. A sweep of the (29) Huang, S. K.; Rempel, D. L.; Gross, M. L. Int. J. Mass Spectrom. Ion Processes 1986, 72, 15-31.
Figure 6. First and 50th remeasurement scans for broad-band QE axialization of poly(ethylene glycol) with an average molecular mass of 2000 Da. Continuous, repeated frequency chirps over the mass range m/z 1500-2500 were used for axialization. Capacitive coupling is enabled for the experiment, and the trapping potential is set to 1 V. An x-axis expansion for each scan is shown to illustrate isotopic resolution. Absolute intensities were used for each spectrum.
Figure 8. Comparison of the 1st and 50th remeasurement scans for broad-band QE axialization of poly(ethylene glycol), at 1.0 V trapping potential, illustrating the reduction in remeasurement efficiency when capacitive coupling is not used during remeasurement experiments. All experimental details are the same as for the data of Figure 6, except that capacitive coupling was not used.
intensities. After close to only 20 remeasurement cycles, all ions are lost through axial ejection. If capacitive coupling is enabled only during the detection excite event, or if capacitive coupling is enabled only during QE, the remeasurement efficiency is reduced compared to that obtained using QE for the entire experimental sequence, as shown in Figure 7. This suggests that, if capacitive coupling is disabled, axial loss can occur both during QE and during the dipolar excitation used for ion detection. Optimum remeasurement conditions are obtained by using capacitive coupling for the entire remeasurement sequence.
Figure 7. Plot of the relative intensities of ion signals for broadband QE of poly(ethylene glycol) with an average molecular mass of 2000 Da, with axialization over the mass range of m/z 1500-2500, as in Figure 6. The data in Figure 6 are plotted here, showing >99.5% remeasurement efficiency. The same experiment, but without capacitive coupling, exhibits99.5%, as shown in Figure 7. We have illustrated the capabilities of broad-band quadrupolar excitation remeasurement in another report,3 but here we emphasize the effectiveness of capacitive coupling for efficient remeasurement. For example, at a trapping potential of 1.0 V, the broad-band QE efficiency is less that 80% with capacitive coupling disabled. Shown in Figure 8 are the mass spectra for this remeasurement experiment at 1 V, both plotted as absolute
CONCLUSION We have shown that the major ion loss mechanism during QE remeasurement is axial ejection. Capacitively coupling the excitation plates to the trapping plates significantly reduces axial ejection and permits efficient, high-resolution ion remeasurement with either single-frequency or broad-band quadrupolar excitation. To obtain the highest remeasurement efficiency for a given set of conditions, we have found that capacitive coupling must be enabled during the entire experiment, not exclusively during QE or during detection. A 100% remeasurement efficiency with isotopic resolution is observed for 200 remeasurement cycles of the 41-mer of poly(ethylene glycol). Greater than 99.5% remeasurement efficiency has been achieved for broad-band QE over a 600 m/z range as well. Ion remeasurement and capacitive coupling are currently being used in conjunction with our broadband quadrupolar excitation techniques to study multiple-stage mass spectrometry (MSn) in ICR. ACKNOWLEDGMENT We gratefully acknowledge the financial support of the National Science Foundation, Grant CHE-9412334. Received for review July 8, 1996. Accepted October 3, 1996.X AC960659G X
Abstract published in Advance ACS Abstracts, November 1, 1996.
Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
4413
