Anal. Chem. 1990, 62, 1319-1324
equilibrium constants and thermodynamic properties are in good agreement. As the hydrophobicity of a solute becomes very large, the results obtained in this study support the proposal that the retention mechanism is shifting toward a direct transfer of the solute from micellar pseudophase to the surfactant-modified stationary phase. Future studies involving the effect of organic modifier and its role on efficiency can be evaluated in terms of equilibrium and thermodynamics. ACKNOWLEDGMENT The authors are grateful to Paul Ander and Joseph Fett for many helpful discussions. LITERATURE CITED (1) Khaledl, M. 0.; Breyer, E. D. Anal. Chem. 1989. 67, 1040-1047. (2) Okada, T. Anal. Chem. 1988, 60, 2116-2119. (3) Palmisano, F.; Guenieri, A.; Zambonln. P. G.; Cataldl, T. R. I . Anal. Chem. 1989. 61, 946-950. (4) Posluszny, J. V.; Weinberger, R. Anal. Chem. 1988, 60, 1953-1958. (5) DeLuccia, F. J.; Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1985, 57, 1564-1568. (6) Herries, D. G.; Bishop, W.; Richards, F. M. J . phys. Chem. 1964, 68, 1842- 1852.
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(7) Armstrong, D. W.; Nome, F. Anal. Chem. 1981. 53, 1662-1666. (8) Arunyanart, M.; Cline love, L. J. Anal. Chem. 1964, 56, 1557-1561. (9) Berthd, A.; Girard. I.; Gonnet, C. Anal. Chem. 1986, 58, 1359-1362. (10) Khaledl. M. G.; Peuler, E.; Ngeh-Ngwainbi, J. Anal. Chem. 1987, 59. 2738-2747. (11) Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1985. 57, 2837-2843. ( 12) Borwding, M. F.; Quina, F. H.; Hinze, W. L.; Bowermaster, J.; McNair, H. M. Anal. Chem. 1988, 60,2520-2527. (13) Dorsey, J. G.; DeEchegaray, M. T.; Landy, J. S. Anal. Chem. 1983, 55. 924-928. (14) Wells, M. J.; Clark, C. R. Anal. Chem. 1981, 53. 1341-1345. (15) Melander, W.; Campbell, D. E.; Horvath, C. J . Chromatogr. 1978, 158, 215-225. (16) Knox, J. H.;Vasvari, G. J . Chromtogr. 1973, 83, 181-194. (17) Melander, W. R.; Horvath, C. High Performance Liquhi Chromatogrephy: Advances and Perspectives; Horvath, C., Ed.; Academic Press: New York, 1980; Voi. 2, p 198. (18) Colin. H.;Diez-Masa. J. C.; Guiochon, G.; Czajkowska, T.; Miedziak, I. J . Chrometogr. 1978. 167, 41-65. (19) Berthod. A.; Girard. I.; Gonnet, C. Anal. Chem. 1986, 5 3 , 1362-1367. (20) Mukerjee, P. J . Phys. Chem. 1982, 66, 1733-1735.
RECEIVED for review November 27, 1989. Accepted March 2, 1990.
Liquid Chromatography/Time-of-flight Mass Spectrometry with High-speed Integrated Transient Recording W. Bart Emary, Ihor Lys, and Robert J. Cotter*
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, Baltimore, Maryland 21205 Richard Simpson
Department of Chemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228 Andrew Hoffman
Kratos Analytical, Manchester M31 2LD, England
High-performance iiquld chromatography (HPLC) has been interfaced to a tkneof-flight mass spectrometer. The interface Is a continuous flow probe and ions are desorbed from the iiquld matrix by energetlc ion bombardment. Pulsed and delayed ion extraction from the source permits the use of broad ionization times, results in the production of analog signals in each tkne-of-fllght cycle, and provides both energy and spatial focusing. A high-speed Integrated transient recording system has been developed and isabm reported. TMS instrument Is the prototype for devebpment of a high-speed, highinass range LC detector with high duty cycle. Its performance is demonstratedfor the separatlon of several mixtures of small peptides.
INTRODUCTION A number of techniques have been developed for interfacing bonded phase high-performanceliquid chromatography (LC) to mass spectrometers. The most common (and commercially available) instrumental configurations employ moving belt interfaces ( 1-3), thermospray ionization (4),and continuous flow fast atom bombardment (5-8)with quadrupole and sector (double focusing) mass analyzers. Because the ionization sources in sector instruments are generally at a very high 0003-2700/90/0362-1319$02.50/0
electrical potential (4-8 kV),the high (>1mL/min) flow rates used with the thermospray technique have made this method unquestionably easier to accomplish on quadrupole-based mass analyzers. Quadrupole instruments also have the advantage that they can be scanned more rapidly than sector instruments but are limited in mass range to (typically) around 3000 amu. Alternatively, lower flow rates (1-10 pL/min) and microbore columns may be used with the continuous flow fast atom bombardment (FAB) technique and can therefore more easily take advantage of the high resolution and high mass range (10000 amu) of high-performance sector instruments. Recently electrospray ionization has been introduced as a means for interfacing liquid chromatography and mass spectrometry (9). This technique has been used primarily with quadrupole analyzers and, since it produces ions with very high charge states, has extended the mass range of such instruments so as to permit the recording of molecular ions of proteins ( 1 0 , l l ) . In addition, both continuous flow FAB and electrospray have been used as interfaces for capillary zone electrophoresis (CZE), while thermospray has been used for high-performance anion exchange (HPAE) chromatography (12). T h e Time-of-Flight Mass Spectrometer as a n LC Detector. Time-of-flight (TOF) mass analyzers have not been employed as detectors for high-performance liquid chromatography. They have, however, the ability to record mass 0 1990 American Chemical Society
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ranges as high or higher than those that can be recorded by sector instruments at scanning speeds that are faster than those of the quadrupole:a combination of qualities that would seem to have great potential for the rapid analysis of tryptic peptides during fractionation. Time-of-flight mass spectrometers have been used to record molecular ions of proteins with masses in excess of 200000 daltons, using matrix assisted UV laser desorption (13),suggesting that they may ultimately be the analyzer of choice for monitoring chromatographicand electrophoretic separations of proteins as well. Since the high mass range of the TOF permits the recording of these ions as singly charged species, rather than a series of multiply charged ions, this would appear to be an advantage in the case where several coeluting species would result in a rather complex mass spectrum. Time-of-flight analyzers are generally regarded as low resolution devices; however, a number of laboratories have demonstrated mass resolutions comparable to those of magnetic instruments using picosecond lasers (14), supersonic cooling (14), and reflectrons (14-16). Integrating Transient Recording. A critical factor for the development of the time-of-flight mass spectrometer as a chromatographic detector is the need for continued development of recording devices that are compatible with both the mass spectral and chromatographic time frames. In 1955, when the first practical time-of-flight instrument designed by Wiley and McLaren (17) was commercialized by the Bendix Corp. the existing electronics technology did not permit the recording of the complete 100 ps transient that comprised the mass spectrum from each pulsed ionization event, or timeof-flightcycle. Instead, a gated detector enabled the recording of a single 10-ns slice of the signal from each transient. In successive cycles that gate was advanced to permit recording of the entire spectrum over many cycles, in a time frame that was compatible with direct recording by an oscillographicor strip-chart recorder. While that instrument continues to be manufactured (CVC Products, Rochester, NY) and used for combined gas chromatography/mass spectrometery, the low duty cycle of the time slice detection (18)scheme results in considerable sacrifice in ultimate sensitivity. Over the past several years, recording devices have been developed and manufactured that are compatible with the mass spectral time frame but not with the chromatographic time frame. High-speed time-to-digital conoerters (TDCs) are currently capable of recording up 15 ions in each timeof-flight cycle and are compatible with plasma desorption (19) and static SIMS (secondary ion mass spectrometry) (20) time-of-flight instruments which produce only a few secondary ions from each ionization event. While TDCs perform what Holland et al. (18) have termed time array detection, such instruments are not well suited as chromatographic detectors, since many cycles are required to assemble a completed mass spectrum with reasonable dynamic range. Transient recorders are high-speed analog-to-digital converters that have been used for some time to record and digitize the single shot analog signals (spectra) produced in laser microprobes (21). The Biomation 8100 waveform recorder manufactured by Gould (Cupertino, CA) was an 8-bit 100-MHz recorder that could digitize and store up to 2048 10-ns points from a 20-ps transient. That recorder was incorporated into early commercial microprobes and was used as well in our initial infrared laser desorption time-of-flight mass spectrometer (22). In our instrument, digitized transients were downloaded to an Apple computer and the memory slices added to produce mass spectra with dynamic ranges in excess of the 8-bits provided from a single shot. Holland et al. (18) have noted that such integrating transient recording (ITR) techniques are the basis for producing time-of-flight recording that is compatible with both the mass spectral and chroma-
tographic time frames. In their scheme, every 10-ns time bin (for a 100-MHzrecorder) through the course of the experiment would be digitized, in order to produce the maximum duty cycle. In addition, the number of transients that would be integrated to form each spectrum could be varied to maximize the dynamic range or the number of spectra obtained across each chromatographic peak. Integrating transient recorders which achieve the ideal proposed by Holland (18) have not been commercia!ly available. While the 100-MHz recording rate does not generally result in limitations on the mass resolution, the commercially available instruments are generally limited in repetition rate and downloading time. The 3500 SA signal averaging recorder and the 9400 digital oscilloscope manufactured by LeCroy (Spring Valley, NY) are 100-MHz 8-bit transient recorders that can digitize 8k 10-nspoints in a single 80-ps transient and have both been used with our more recent instruments (23-26). They are also integrating transient recorders that are capable of integrating up to 50 transients/s. It is, however, possible to generate 12000 80-ps transients per second, so that the repetition rate is a severe limitation on the duty cycle. In addition, considerable time is lost while downloading the accumulated spectra into a host computer and clearing the transient recorder memory for the next group of transients. Thus, the Michigan State group has devoted considerable time and made significant progress in developing a recording system that would operate at higher duty cycle (27,28). Their efforts are aimed primarily at the development of an integrating transient recording system that would provide high chromatographic integrity as well as dynamic range for capillary GC/MS. In this paper we present our frst efforts in the development of time-of-flight mass spectrometers as detectors for highperformance liquid chromatography. Our aims are 2-fold: (1) to show that HPLC and time-of-flight mass spectrometers can be made compatible by using an instrument which incorporates pulsed ionization and pulsed secondary ion extraction and (2) to introduce a general design for a high-speed integrating transient recorder with high duty cycle.
DESIGN CONSIDERATIONS In both sector and time-of-flight analyzers, mass analysis depends upon the acceleration of ions to high kinetic energies. Liquids vaporized in the high voltage ionization sources can easily lead to electrical breakdown. In addition, collisions between sample ions and solvent vapor in the accelerating region result in degradation of their kinetic energies leading to poor mass resolution and low ion transmission. In sector instruments, this can be minimized by differential pumping of the ion source and the accelerating region. In the TOF analyzer, ion formation and acceleration are generally accomplished in the same region, and high ion transmission is achieved because the source and analyzer regions are not separated by beam-defining slits. Thus, differential pumping is not a practical option. In the time-of-flight analyzer mass resolution depends upon minimization and/or correction of the initial time, kinetic energy, and spatial distributions of the ions formed in the ion source (17, 23, 29). Generally these are addressed by employing short (pico- to nanosecond) ionization pulses, high accelerating voltages, and thin samples coated on an equipotential surface, respectively. In this case, however, it was necessary to employ a TOF analyzer capable of handling the thick, nonconducting glycerol droplets formed by the LC effluent. In addition, broad (microsecond) ionization pulses were to be employed, in order to produce high secondary ion yields for each time-of-flight cycle. These were to be detected as analog signals and recorded by high speed analog/digital conversion, so that signal averaged mass spectra could be
ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
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Figwe 1. Block diagram of the IqM s8coI)c19ry ion time-of-ft&ht mass spectrometer (LSIMS-TOF) and data acquisition system used for combined LC-TOF.
obtained in time frames compatible with those of the chromatographic peaks. These problems are addressed by using a continuous flow probe, low (1pL/min) flow rates, and ionization by primary ion bombardment in a grounded ion source. The ionization pulse is broad (1-10 ps), and time, energy, and spatial focusing are acheived by a time-delayed ion extraction pulse as proposed initially by Wiley and McLaren (17). Analog signals (transients) from each time-of-flight cycle are digitized, accumulated, and downloaded to a PC using a high repetition rate 100-MHz transient recorder developed for this instrument. EXPERIMENTAL SECTION The Liquid SIMS Time-of-Flight Mass Spectrometer. A schematic diagram of the time-of-flight mass spectrometer is shown in Figure 1. This instrument, known as a liquid secondary ion time-of-flight mass spectrometer (LSIMS-TOF), has been described previously (24,25);however, in this case the sample probe has been replaced by a continuous flow probe, and spectra are recorded by a 100-MHzhigh repetition rate transient recorder developed specifically for LC-TOFMS. In this instrument the output from the PULSE GENERATOR is fed to the TOF TIMING CIRCUIT and determines the number of mass spectra/second (100 Hz to 10 kHz) to be acquired. The TOF TIMING CIRCUIT is used to set the pulse width for the primary ion gun (1-10 ps) ,the delay time and pulse heights for the ion drawout, backing plate, and acceleration pulses, the duration of the low mass blanking pulse, and the delay time for triggering the transient recorder. The primary ion gun is a Kratos (Manchester, UK) MINIBEAM I, whose control unit has been modified by replacing the emission regulator circuit with a constant current source (to control the filament current) and a pulse amplifier (25). The incoming pulse is amplified to 100 V and applied to the electron energy grid, which switches the bkeV Xe+ beam on for 1-10 ps for desorption of ions from the sample probe. The secondary ions are extracted by pulses applied simultaneously to the ion drawout and accelerating lenses (0 to -250 V) and the backing plate (0 to +250 V). The fast rise time of these pulses (25 ns) provides time focusing, while their voltages and delay times provide spatial and energy focusing, respectively (17). The secondary ions are then further accelerated to 3 keV as they enter the 65-cm drift region. A t the end of the drift region, the ions are postaccelerated to 12 keV into a Galileo (Sturbridge,MA) dual channelplate detector. Low mass blanking plates (1250 V) are used to deflect ions below a predetermined mass to reduce saturation of the channelplate detector. The detector signal is amplified by a Stanford Research (Palo Alto, CA) Model SR440 wideband (dc to 300 MHz) amplifier and fed to the analog input of the Kratos (Manchester, UK) MARK I 100-MHz data acquisition system (described below). In this system transients are recorded and digitized as 8 bit X 8K words;
ready
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Figure 2. Block dagram of the Kratos MARK I high-speed 100-MHz
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and the accumulated 16 bit X 8K spectrum from 256 added transients are downloaded to an Compaq (Houston,TX) 16-MHz 386-based PC with a Newbury Data Drive Model 32805 (Cheghire, England) 220-Mbyte hard drive. The TOF MARK I High-speed Data Acquisition System. The MARK I data acquisition system was designed to provide continuousrecording of analog signals with minimum "dead time". A block diagram of the system is shown in Figure 2. The amplified detector signal is fed directly to the analog input of an Analog Devices (Norwood, MA) AD9002 150 MHz 8-bit flash analog-to-digitalconverter. Conversion is enabled when a trigger pulse from the TOF TIMING CIRCUIT is received at the start input; the conversion rate is controlled by a 100-MHzclock. The system clock is fed to a LATCH CONTROL which outputs five phased clock pulses to the LATCH and ECL/TTL CONVERTERS and directs the ECL data from the flash ADC sequentially to each of the five latches, which transfer data to their respective ACCUMULATORS at the rate of 20 MHz. The ACCUMULATORS each consist of two 4K x 16-bit RAM banks and an arithmetic/logic unit (ALU), which adds the data point from the current transient to the corresponding summed point in RAM controlled by the ADDRESS GENERATOR. A t the conclusion of each transient recording, 20 ns is required to reset the ADDRESS GENERATOR, prior to accepting the next start signal. When 256 transients have been recorded, the contents of the ACCUMULATORS are downloaded to a Compaq 386 PC and stored on the SCSI 220-Mbyte drive. When this operation is completed, a ready signal indicates that the system is ready to accept the next group of transients. The ACCUMULATORS are capable of storing transients up to 20K in length, corresponding to 204.8 ps. In practise, shorter transients are recorded, with the maximum length loaded into the CONTROL REGISTER from the PC. Thus, for example, one can record and accumulate 256 8K transients (corresponding to 81.92 ps and a mass range of 0-7400 daltons) in less than 21 ms, which corresponds to a repetition rate of 12.2 kHz. In the results reported below, a somewhat lower repetition rate (1800 Hz) was used, which enabled acquisition, storage and display of up to five summed (256 transient) 16 bit X 8K bytes spectra per second. The LC/TOF System. Although the LC/TOF instrument has been designed for use with a microbore LC system, our initial experimentswere carried out with a reversed-phaseHPLC system with the effluent split to reduce the flow rate into the mass spectrometer. The reversed-phase HPLC column (83 mm X 4.6 mm) contained 3 pm base deactivated C18 packing material obtained from Perkin-Elmer (Norwalk, CT). The mobile phase consisted of 5% by volume glycerol and 0.1% trifluoroaceticacid in degassed H20/CH,CN (3:l). The flow rate was maintained at 1 mL/min by a Beckman (Berkeley, CA),Model 114 binary solvent delivery system;all separations were achieved ismatically. Ultraviolet (UV) detection was performed at 280 nm using a Kratos (Ramsey,NJ) SpectraflowModel 757 variable wavelength
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detector. At the outlet of the UV detector, a 1:lOOO split of the chromatographiceffluent resulted in delivery of mobile phase to the continuous flow probe of approximately 1pL/min. The low flow rate was achieved with a 1-m length of SGE (Austin, TX) 60 pm i.d. capillary between the splitter tee and the continuous flow probe tip. The remaining effluent was collected at the tee as waste. The continuous flow probe is a modified version of that previously reported (26),and uses a metal seal (solder) around the silica capillary to provide better electrical and heat conductivity, thereby reducing charge buildup and providing more stable pressures. Addition of heat is essential for stable operation of the flow probe and, since the open source design of the TOF does not permit heating of the probe tip by contact with a heated source block, heating was provided in the source tip itself. A thermocouple was added to the probe tip, adjacent to the silica capillary, for temperature measurement. The temperature of the probe tip was maintained at 50 OC although no dramatic differences in performance was noted over the range from 40 to 60 OC monitoring the molecular ion peak of a peptide continuously flowing into the source. At this flow rate and temperature, a base pressure of 5 X Torr was maintained in the mass spectrometer. Peptides used in this study were obtained from Sigma (St. Louis, MO)).Peptide mixtures were prepared as solutiom in water and mixed immediately before injection onto the HPLC column. Data were acquired and processed on the Compaq 386 PC system. Mass spectra were base-line subtracted, and the data were plotted as total ion (reconstructed) chromatograms and single ion chromatograms.
RESULTS AND DISCUSSION Two-Component Mixture. Figure 3a shows the UV chromatogram of a mixture of 50 nmol each of methionineenkephalin (MW 574) and Tyr-Met-Gly-Phe-ProNH2(MW 613) separated by HPLC. The 1OOO:l split results in a total injected sample into the flow probe of 50 pmol of each peptide. Figure 3b shows the reconstructed ion chromatogram (RIC) obtained simultaneously. The UV and RIC chromatograms have similar peak widths and separation of maxima, indicating minimal broadening of the peak profile during transfer to the mass spectrometer. Figure 4 shows the two single ion chromatograms corresponding to the MH+ ions of each peptide. In these and subsequent experiments, the trigger pulse from the TOF TIMING CIRCUIT was fed to a divide-by-ten circuit so that only every tenth cycle (transient) was recorded by the transient recorder. Even at the lowered repetition rate of 1800 Hz, a 20-min chromatogram would result in storage of 93 Mbytes of data. While this can be accommodated by the
Time (minutes) Flgure 4. Single ion chromatograms for the MH+ ions of the peptides
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220-Mbyte drive, it increases the time needed for off-line processing (e.g. base-line subtraction of each spectrum, addition of spectra for display, etc.). However, the quality of the mass spectra can be maintained at the higher acquisition rates, and efforts are underway to develop real-time processing to reduce the amount of stored data. In addition, it can be expected that higher sensitivities (lower detection limits) can be achieved when the entire ion signal can be recorded. Three-Component Mixture. Figure 5 shows the UV and reconstructed ion chromatograms of a mixture of methionine-enkephalin (MW 574), Tyr-Met-Gly-Phe-ProNH2(MW 613), and Leu-Trp-Met (MW 448). Figure 6 shows the single ion chromatograms of the MH+ ions for each of the three peptides. Figure 7 shows the mass spectrum of Tyr-Met-Gly-PheProNH2 obtained in the same experiment. Mass resolution is reduced from that which could be obtained by using a normal FAB probe and is due primarily to collisons in the source and accelerating region due to the higher base pressure in the mass spectrometer with the introduction of volatile solvents carried by the mobile phase. Such collisions are also
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responsible for the increase in base-line signal and undoubtably, in reduction of the sensitivity. At the same time, we note that (unlike scanned instruments) complete masa spectra and selected ion monitoring are achieved with the same sensitivity on a time-of-flight instrument using time array detection (18). Components Eluting with Similar Retention Times. Figure 8a shows the UV chromatogram of a four-component mixture containing methionine enkephalin (MW 574)) TyrMet-Gly-Phe-ProNH2 (MW 6131, a-casein fragment ArgTyr-Leu-Gly-Tyr-Leu (MW 784)) and Leu-Trp-Met (MW 448). Parts b and c of Figure 8 show the single ion chromatograms for the two components (a-casein and Leu-Trp-Met) which coelute.
CONCLUSIONS Holland et al. (18) have noted that the ability to acquire mass spectra rapidly is an advantage for capillary GC/MS for which the GC peaks themselves are very narrow in the time frame. The integrated transient recording system permits the recording of several spectra over the course of the GC peak and thereby reduces the distortions in relative abundances that would occur when mass spectral scan times are of the order of the GC peak widths. In addition, each of the spectra acquired represent the accumulation of many transients, thereby improving the dynamic range. In the most optimal
Figure 8. (a) UV chromatogram of a mixture of four peptides: TyrGlyOly-Phe-Met (peak I), Tyr-Met-Gly-Phe-ProNH,(peak 2), a-casein fragment (peak 3),and Leu-Trp-Met (peak 4). Single ion chromatograms of the MH+ regbn of (b) a-casein fragment and (c)Leu-Trp-Met.
case, transients are continuously recorded without dead time between the transients to produce an instrument with the duty cycle limited only by the percentage of time that ionization is occurring. We have developed such a system for LC time-of-flight mass spectrometry. While the broader peaks obtained from reversed phase HPLC are not so nearly demanding of chromatographic integrity as those from capillary GC, the integrating transient recording system developed in this work would be equally applicable to that technique as well. When the time-of-flight mass spectrometer is used as an LC detector, the time lag focusing scheme (17) not only permits focusing of ions desorbed from an irregular and nonconducting liquid surface but also allows the use of broad ionization pulses. In this case, ionization occurs for 10 ps of the 80-ps cycle, so that the duty cycle can be expected to be as high as 12% assuming that the ions formed during that time remain in the ion source until application of the drawout pulse and that acquisition can itself be accomplished with zero dead time. Based upon previous lifetime calibrations on a similar system (30))the half-life of ions formed at thermal energies remaining in the focusible region of the source is approximately 6 ps for m / z 1000,8.5I.CS for m l z 2000,13 ps for m l z 5000, etc., so that high ionization duty cycle can be expected to be achieved for high mass ions. In this system, acquisition and integration of transients can be achieved with nearly zero dead time (except for a 20-ns resetting time) for the first 256 transients, while some time is lost in downloading accumulated data to the host computer. Since the downloading time is comparable to or less than the accumulation time, acquisition, integration of transients, and storage of averaged spectra with zero dead time can be achieved by using parallel accumulators, a feature that will be incorporated into the next version of this acquisition system. As we have noted, however, the data obtained for this initial report utilized considerably lower duty cycles, in order to reduce the amount of stored information and subsequent processing. Both total ion and single ion chromatograms obtained on this instrument reveal chromatographic quality comparable to that obtained by UV detection. At the same time, we have noted that mass resolution is reduced. In general, mass resolution is effected by collisions with volatile solvent vapor and improved pumping speed, lower flow rates, etc. will be
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
incorporated into this developing instrument. we have noted (23) that the best mass resolution which we have obtained on a similar linear time-of-flight instrument is about 1/800. Ion reflectrow (15) can be used to improve the mass resolution, and such devices have been used by Wollnik (16) in conjunction with pulsed ion extraction. One objective of this work has been to demonstrate that the time-of-flight mass analyzer can be used in conjunction with high-performance liquid chromatography (HPLC) or microbore LC and that the key to such methods is a combination of broad ionization times to produce analog transients and to raise the duty cycle, and pulsed ion extraction for spatial focusing of the ions desorbed from the chromatographic effluent. Our second objective has been to develop a suitable high-speed, high-duty cycle integrating transient recording system that is capable of handling the high rates from such an instrument. In its present state we envision that this instrumental configuration will be useful for the simultaneous mapping and sequencing of peptide fragments, as they are fractionated by reversed-phase or microbore HPLC. In the longer term, we expect to develop configurations that are compatible with capillary zone electrophoresis (CZE), using both pulsed particle beam bombardment and electrospray. For the latter, a pulsed ion extraction, reflectron instrument is being developed to provide spatial and energy focusing under continuous ionization conditions.
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ACKNOWLEDGMENT The authors wish to acknowledge the helpful support of Kratos Analytical (Manchester, England), and in particular the support of David Finbow, for their interest in time-of-flight mass spectrometry and development of the MARK I data acquisition system.
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RECEIVEDfor review January 12,1990.Accepted March 19,
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1990. R.J.C., W.B.E., and I.A.L. were supported by an NIH grant (GM 33967) from the National Institutes of Health. R.S. was supported by a grant (GM 21248)to Dr. Catherine Fenselau from the NIH. This work was carried out at the Middle Atlantic Mass Spectrometry Laboratory, an NSF supported Regional Instrumentation Facility.
