Anal. Chem. 1999, 71, 2578-2581
Technical Notes
High-Speed TOFMS Detection for Capillary Electrophoresis I. M. Lazar,† A. L. Rockwood,† E. D. Lee,† J. C. H. Sin,† and M. L. Lee*,‡
Sensar Corporation, 1662 West 820 North, Provo, Utah 84601, and Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602
A new, high-speed data acquisition system was tested for high storage rate time-of-flight mass spectrometry (TOFMS) detection in capillary electrophoresis (CE). For high spectral acquisition rates of 4 kHz, a spectral storage rate of 80 spectra s-1 was achieved. The resulting detection limit was in the low amol range (10-25 amol) for continuous infusion investigations. Present trends in analytical instrumentation are high speed and miniaturization. Fast separations which occur within seconds to a few minutes or high-speed separations which occur within milliseconds to seconds are being reported with increased frequency.1,2 Detection systems which can adequately monitor and provide quantitative results for these separations must be developed simultaneously. The mass spectrometer (MS) has become the preferred detector for chromatographic and electrophoretic separations because of the quality of identification information that it can provide; however, the intrinsic scanning characteristics of most mass spectrometers (quadrupole, ion trap, and sector) limit their applicability as detectors for high-speed separations. While only one mass spectrum acquired across an eluting peak can be sufficient for component-identification purposes, accurate peak-shape determination and precise quantitation require a minimum of 10 or, even better, 20 spectra collected across the peak. Assuming that 10 data points (which in MS language translates to 10 full mass spectra) are necessary to define a peak, the sampling frequency by the MS detector must increase for progressively narrower peak widths (see Table 1). For a 10-ms peak width, spectral generation must occur with a frequency of 1000 Hz, which corresponds to a 1-ms time window to produce a full mass spectrum. It is well-known that the signal/noise (S/N) ratio is proportional to the square root of n (where n is the number of times data are collected and averaged). Consequently, for a 1000 Hz sampling frequency, and for averaging only 10 spectra, an operating frequency of 10 000 Hz is needed, i.e., 10 000 full mass spectra must be generated per second. * To whom correspondence should be addressed: (tel.) 801-378-2135; (fax) 801-378-9357; (e-mail)
[email protected]. † Sensar Corp. ‡ Brigham Young University. (1) Monnig, C. A.; Jorgenson, J. W. Anal. Chem. 1991, 63, 802. (2) Jacobson, S. C.; Culbertson, C. T.; Dealer, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476.
2578 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
Table 1. Required MS Sampling Frequency and Resultant Spectrum Acquisition Time for Peaks of Different Widths peak width (s)
peak sampling frequencya (Hz)
spectrum acquisition time (ms)
5 1 0.1 0.01
2 10 100 1000
500 100 10 1
a
Assuming 10 data points (spectra) to define a peak.
This far exceeds the capabilities of all scanning MS instruments, and the only remaining alternative is the TOFMS,3,4 which is a nonscanning instrument capable of generating mass spectra with high frequency because of its relatively simple principle of operation: an ion packet which is accelerated with the same kinetic energy and is projected in a field-free drift tube will separate according to the individual velocities characteristic of each ion of a given mass/charge ratio. Since flight times in the drift tube (from the accelerating zone to the ion detector) are extremely short, 10-200 µs, complete mass spectra can be generated in the same short time window. However, even though such a high spectral production rate is typical of all TOFMS systems, an efficient solution to data manipulation and storage has not yet been reported until now. The enormous flow of information (gigabytes s-1) is extremely hard to handle with presently available data acquisition systems. In this paper we demonstrate that for high spectral acquisition rates (4 kHz) and MS data collected with 1-ns resolution (ion sampling rates of 109 s-1), a spectral storage rate of 80 spectra s-1 and attomole sensitivity have been achieved. EXPERIMENTAL SECTION Reagents. HPLC grade methanol and water were purchased from Mallinckrodt (Chesterfield, MO). Glacial acetic acid was obtained from EM Science (Gibbstown, NJ). Peptides were purchased from Sigma (St. Louis, MO). Capillary Electrophoresis. The research was performed using an in-house built CE system which allowed the use of short (3) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955, 26, 1150. (4) Price, D.; Milnes, G. J. Int. J. Mass Spectrom. Ion Processes 1990, 99, 41. 10.1021/ac981249q CCC: $18.00
© 1999 American Chemical Society Published on Web 05/19/1999
Figure 1. Schematic diagram of the TOFMS system.
CE capillaries. Uncoated fused silica columns from Polymicro Technologies (Phoenix, AZ) were conditioned prior to analysis by rinsing for 10-15 min with sodium hydroxide solution (1 M), followed by HPLC water (10-15 min). Continuous infusion of reserpine was performed using a Harvard 22 syringe pump (South Natick, MA). Time-of-Flight Mass Spectrometry. A Jaguar electrospray ionization (ESI)-TOFMS (Sensar/Larson-Davis, Provo, UT) system with orthogonal extraction was used as a detector. The Jaguar TOFMS incorporates a patented ion optics configuration based on circuit-board technology, which efficiently focuses and transports the ion beam from the atmospheric-pressure ion source to the launching region, and finally into the flight tube (Figure 1).5-7 A micro ESI source was used to bring the ions from the CE eluent into the gas phase. A sharp ESI tip (10-20 µm i.d., 40-60 µm o.d.) was connected to the CE capillary through a 27-gauge stainless steel needle tube, and the ESI voltage was applied to this metal union.8 After penetrating through a heated counter current of nitrogen gas, the ions were sampled from the source using a nozzle/skimmer arrangement. The ion beam was then efficiently collimated and transmitted by a radio frequency (RF)only quadrupole into the pulsing region of the mass spectrometer. The flight-tube design, based also on circuit-board technology, produced excellent resolution per effective unit length, such that the use of a reflectron was not necessary. Fine-tuning of the mass resolution was made possible by using a multianode detector design. Resolutions of approximately 2000-2200 could be routinely achieved using the whole detector area, while values as high as 5000 could be reached when the signal was collected on only 1 or 2 anodes. The monitored mass range can vary from 0-6000 to 400-10 000. A large dynamic range of 40 000 allows for the determination of trace components in the presence of major ones. The data acquisition system is capable of handling (collecting, compressing, and storing) gigabytes of data in extremely short times. There are two main types of data acquisition systems in (5) Rockwood, A. L.; Fabbi, J. C.; Harris, L.; Davis, L.; Lee, E. D.; Ogden, C.; Tolley, H.; Gunsay, M.; Sin, J. C. H.; Lee, H. G. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, USA, June 1-5, 1997; p 779. (6) Lee, E. D.; Rockwood, A. L.; Fabbi, J. C.; Sin, J. C. H.; Jones, J.; Davis, L. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, June 1-5, 1997; p 44. (7) Fabbi, J. C.; Rockwood, A. L.; Sin, J. C. H.; Woolley, C.; Lee, E. D.; Jones, J. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, CA, June 1-5, 1997; p 51. (8) Lazar, I. M.; Lee, E. D.; Rockwood, A. L.; Lee, M. L. J. Chromatogr., A 1997, 791, 269.
Figure 2. TOF mass spectra of reserpine acquired at high-speed data storage rates. Conditions: sample (0.5 µM) in CH3OH/H2O/ CH3COOH (50:50:1, v/v); continuous infusion (0.25 µL min-1); TOFMS data acquisition at 4000 Hz pulsing rate.
TOFMS: those based on a transient digitizer and those based on a time-to-digital converter. The transient digitizer has greater dynamic range, typically eight bit vs one bit in a time-to-digital converter. However, in other respects, the time-to-digital converter is superior, i.e., better time resolution, lower noise, lower cost, and better signal-averaging properties. The Simulpulse Recorder7 used by the Jaguar combines the advantages of both systems. The principle of operation of the Simulpulse Recorder is conceptually similar to a massively parallel time-to-digital converter, although it differs somewhat in specific technical details. In the Simulpulse Recorder the detector area is divided into smaller regions, each connected to a separate electronics module. The resultant digital signals from the separate modules are combined into a multibit digital word. Thus, it has high dynamic range, like a transient digitizer, but because it is based on threshold detection Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
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Figure 3. Signal stability plots at various acquisition rates. Conditions: same as in Figure 2. Data were collected for the protonated molecular ion of reserpine (609.28 m/z).
like a time-to-digital converter, it has the other desirable properties of a time-to-digital converter. A data system for ESI-TOFMS must address a mismatch between the repetition rate at which the TOFMS is fired (>103 s-1) and the spectral storage rate required for the separation to which the MS is interfaced (
