Anal. Chem. 1085, 57, 2726-2728
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CORRESPONDENCE Pulsed Valve Reagent Addition for Chemical Ionization Gas Chromatography/Fourier Transform Mass Spectrometry: Mass Measurement Accuracy Sir: The mass measurement accuracy of gas chromatography/Fourier transform mass spectrometry (GC/FTMS) has been demonstrated to routinely provide low ppm errors over a wide mass range, even in the absence of calibrant (1). T o date, all accurate mass data has been acquired from electron impact (E11 spectra (1,2);application to chemical ionization (CI) GC/FTMS is more difficult because of the high reagent gas concentrations in the cell (3). Efficient chemical ionization of the GC effluent has been demonstrated by use of a pulsed valve for introduction of the CI reagent gas (4). This approach has the advantage that low base pressures are achieved prior to data acquisition, so that in principle resolution and mass measurement accuracy in the CI mode should be similar to that obtained for E1 spectra. Dramatic improvements in resolution have previously been achieved by the use of pulsed valves for reagent gas addition in collision-induced dissociation (CID) (5) and as an interface for GC/FTMS (2, 6). In this communication, the first low ppm error accurate mass measurement of chemical ionization GC/FTMS data is presented. From the alternate EI-CI GC/FTMS analysis of a six-component mixture, average errors of 5.3 ppm for the E1 spectra and 7.8 ppm for the quasi-molecular ions in the CI mode are obtained in the absence of calibrant. Such accurate mass measurement of ions provides a useful complement to the computerized search of mass spectral libraries; a post-search algorithm that implements this information has recently been reported (7). The molecular formula for each compound, now obtained from accurate mass measurement of the quasi-molecular ion in the CI spectrum, further increases the information available. Acquisition of both E1 and CI spectra from a single GC injection provides an additional demonstration of the potential of the technique. A significant time savings is achieved because, in addition to identification of unknowns by computerized mass spectral searches, both molecular weight and elemental composition data are obtained from a single GC run. Alternate EI-CI FTMS analysis, easily obtained through a minor software modification, has been previously demonstrated for GC/FTMS. However, in that simpler experiment the continuous presence of reagent gas during the collection of both E1 and CI data degraded the spectral resolution (3). The potential for crossover between the two types of spectra which exists is also disadvantageous when using electron impact information for computerized library searching. These limitations are overcome through the use of a pulsed valve which provides a pressure burst of reagent gas only during the CI sequence; crossover contamination is eliminated, while spectral resolution is significantly improved. EXPERIMENTAL SECTION A Nicolet Analytical Instruments FTMS-1000 Fourier transform mass spectrometer with a 3.0-T superconducting magnet and a 2.54 X 2.54 X 7.62 cm3 trapped-ion cell was interfaced to a Varian 3700 gas chromatograph. Deactivated fused silica (10 fim x 19 mm) was used to restrict the helium carrier gas flow into the FTMS. This restriction produced a FTMS background
pressure of (4-5) X lo4 torr with an approximate 200:l split. The FTMS was maintained at 95 OC to minimize pumpoil background. The restrictor and a 3-ft, glass-lined, 4-mm i.d. stainless-steel transfer line were heated to 250 "C. The vacuum system was pumped by an Alcatel Crystal 100 diffusion pump and had a conductance of 120 L/s helium at 150 "C. GC Conditions. A temperature program from 80 to 200 OC permitted separation of a six-component mixture (Table I) using a 60 m X 0.33 mm i.d. J+W DB-5 bonded-phase (1-fim film) capillary column. A 0.2-hL equivolume mixture of the six components was injected with a 10:1 split. Helium at 5 mL/min (measured at 100 OC) was used as the carrier gas. FTMS Conditions. Electron impact spectra were obtained at a 70-eV ionizing potential, by using a 5-ms, 310-nA electron beam. Trap plates were maintained at 0.5 V. Chemical ionization conditions differed from E1 conditions by pulsed addition of the reagent gas prior to ionization, followed by a 700-ms delay before detection. The experimental sequence (Figure 1)consisted of nine signal-averagedscans per E1 file followed by a single scan for the CI file, collected at the rate of one file/s. These conditions permitted adequate definition of the GC peak profile with 4-8 spectral files collected for each eluting component. Acquisition parameters of 64K data collected over a 1.0-MHz bandwidth yielded complete mass spectra with a low-mass cutoff of 45 amu. Each file was baseline corrected, sine bell apodized, augmented by 64K zeroes, and magnitude mode Fourier transformed. For CI measurements a General Valve Corporation (Fairfield, NJ) Series 9 solenoid valve was used to pulse methane reagent gas into the vacuum system. This event was triggered from the FTMS-10oO console immediatelyfollowing the 5-ms quench pulse. Reagent gas was introduced for a period of 1-3 ms providing cell pressures e 5 X lo-' torr (measured with an uncalibrated Granville Phillips ionization gauge). Experimental Protocol. Calibration was accomplished prior to the run by introduction of perfluorotributylamine (PFTBA) (PCR Research Chemicals, Gainesville,FL) at a pressure similar to that generated by the GC eluents. (This pressure was determined by injecting 0.05 rL of PFTBA into the GC and monitoring the ion gauge reading as the PFTBA eluted; pressures typically varied from 4 X lo4 to 7 X lo4 torr.) The effects of trap potential, emission current, reaction delay, and electron beam time on resolution were determined by continuously leaking PFTBA into the analyzer cell and introducing pulses of methane via the solenoid valve. The resolution for four ions from PFTBA at m/z 69, 131, 219, and 264 was measured and compared with that obtained under E1 conditions. In the pulsed-valve CI mode, it was found that a delay of 600-700 ms was necessary for neutral concentrations to decrease to a level permitting resolution equivalent to that for static E1 (ca. 100oO fwhh at m/z 69). Using such delays, it would be possible for an eluting compound to undergo self-CI. To ensure that methane CI was responsible for the majority of quasi-molecular ions, a series of static CI measurements was obtained both in the absence of methane and with to 1 X lo4 torr. These meamethane pressures from 4 X surements verified the efficiency of reaction with CHSf to produce quasi-molecular ions ( 4 ) . RESULTS AND DISCUSSION For static low-pressure CI-FTMS, reagent gas pressures of ca. lo4 torr are utilized. Obviously, under these conditions resolution is significantly degraded compared to that obtained
0003-2700/85/0357-2726$01.50/00 1985 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
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Table 11. Measured Masses of Benzaldehydes for Alternate EI-CI GCIFTMS
El 5rns
n
QUENCH
error, ppm
I ms
measured mass
DELAY 5ms
n
50.01504 51.02295 52.03071 74.01494 77.03841 105.03326 106.04083 107.04899
ELECTRON BEAM I rns
EXCITE 33ms
L --- -DETECT
E1
CI
1.2
0.5 0.8 2.2 2.1 2.2
4.6 1.4
1.9 av 5 ms
Jl 2
”
QUENCH ’
a) CI
I SCAN
TRIGGER V A L V E
r l O o 0 - ?
DELAY 5ms
ELECTRON BEAM 700ms
r----1 REACTION DELAY
Ims
EXCITE 33 rns
r - - - l - D E T E C T
Flgure 1. Experimental sequence for the alternate EI-CI GC/FTMS analysis. Typical pulse and delay times are included with dashed lines representing variable time periods.
Table I. Average Mass Measurement Errors for the Six-Component Mixture EI-CI GC/FTMS Experiment” mol wt peaks cyclohexane ethyl acetate benzaldehyde 3-heptanone p-fluoroacetophenone 2-chlorobromobenzene
84 88 107
114 138 190
4 7 5
E1 av, ppm 7.8
-
5
1.9 4.0 4.8
10
u
1.3
1.4
I
T b) E l
9 SCANS
CI mol ion, ppm 5.1 2.7 1.4
0.6 3.0
13.2
7.8
11.4
12.0
av5.3
6.3
7.8
12.2
OThese measurements were made for a large mass range (1.0 MHz, lower mass cutoff = 45.5), in the absence of calibrant, for a six-componentmixture. under normal E1 conditions. This problem is compounded when GC/FTMS is performed because of the additional background of carrier gas entering the analyzer cell. Pulsed-valve introduction of reagent gas circumvents this limitation, and resolution for similar mass peaks in the E1 and CI spectra should be similar. For example, E1 resolution of 14 000 (fwhh) and CI resolution of 12 000 were obtained for the two ions of 3-heptanone at mlz 57 ( 4 ) . These were direct-mode measurements over a wide mass range from m / z 45 to above m/z 500 (1.0 MHz at 3 T). Higher resolution could be obtained if heterodyne measurements (mixer mode) were used to monitor a narrower mass range. However, for an unknown mixture analysis, it is preferable to observe the entire mass spectrum rather than selected mass regions. A natural outgrowth of resolution improvement for CIFTMS is the ability to obtain useful, accurate mass measurements of quasi-molecular ions. In order to limit errors to the low ppm range, centroids of spectral peaks must be measured precisely. At low detection pressures, there is less collisional damping, permitting acquisition of more data points and improved resolution. The present studies establish that
Flgure 2. Comparison of C I and E1 spectra for p-fluoroacetophenone. The C I spectrum (a) Is from a single scan and the E1 spectrum (b) is the average of nine signal-averaged scans. The large (M 4- H)’ ion observed at m l z 139 in (a) results from methane C I with the analyte. Because no ejection sweeps were between electron beam and reaction delay events, E1 contaminant is observed in the C I spectrum.
it is possible to obtain low ppm error for all ions observed in the absence of calibrant during alternate EI-CI GC/FTMS analysis. Table I summarizes the results for a six-component test mixture. Table I1 lists the measured masses and errors for one component of the mixture, benzaldehyde. Under the conditions utilized, the accuracy of these measurements appears to be data point rather than pressure limited. When accurate mass measurements are obtained in the absence of calibrant for FTMS, it is necessary that the space charge effects for sample and calibrant be nearly identical. This requires similar conditions for sample and calibrant, e.g., identical trap potentials, electron beam times, and emission currents. Although these parameters can be controlled, a potential difficulty in the CI mode would be pressure fluctuation caused by poor valve-pulse reproducibility. However, this was not a problem, since the valve-open time was re-
,
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Anal. Chem. 1985,57,2728-2733
producible to about f10%, and a constant base pressure was reached prior to detection. Average mass measurement errors for E1 and CI measurements were approximately the same although the errors in the E1 mode were higher than expected (based on previous GC/FTMS results (4)). These higher errors likely resulted from the compromise between the conflicting requirements for accurate mass measurement and chemical ionization. Optimum mass measurement accuracies are obtained for E1 spectra as space-charge effects are minimized; best results are obtained when the emission current is low (e120 nA), the electron beam time is short, and low trap potentials (0.3-0.5 V) are used. For chemical ionization, however, a higher trap potential (1.0 V) is required to minimize the loss of signal observed due to inefficient trapping during the long delays necessary to achieve base pressures. In addition, to obtain an abundant yield of CH6+and CzH5+in the presence of the GC effluent, it is necessary to use high emission currents (>E100 nA). Compromise conditions included a trap potential of 0.5 V and an emission current in excess of 300 nA. Under the conditions used for this alternate EI-CI GC/ FTMS analysis, it was possible to obtain approximately 2-3 CI and 2-3 E1 spectral files per chromatographic peak. Excellent S/N was obtained for CI spectra even though only one scan per file was collected (see Figure 2), presumably because column effluent continuously entered the analyzer cell during the 700-ms delay, allowing CI to take place. Ideally, this delay should be minimized to permit either ensemble averaging for increased spectral S/N or better chromatographic time resolution. However, because of the low conductance in the current system, this was not possible. Improved conductance
or the use of a differentially pumped dual cell design would be expected to improve performance (8). Ejection of excess reagent ions, which would improve resolution and mass measurement accuracy a t shorter pump down times, was unnecessary with the the 700-ms delay used in this analysis. LITERATURE CITED Johiman, C. L.; Laude, D. A., Jr.; Wilkins, C. L. Anal. Chem. 1985, 57, 1040-1044. Sack, T. M.; McCrery, D. A.; Gross, M. L. Anal. Chem. 1985, 57, 1290-1 295. Laude, D. A., Jr.; Brissey, G. M.; Ijames, C. F.; Brown, R. S.; Wiikins, C. L. Anal. Chem. 1984, 56, 1163-1168. Laude, D. A., Jr.; Johiman, C. L.; Brown, R. S.; Ijames, C. F.: Wiikins, C. L. Anal. Chim. Acta, in press. Carlin, T. J.; Freiser, B. S. Anal. Chem. 1983, 55, 571-574. Sack, T. M.; Gross, M. L. Anal. Chem. 1983, 55, 2419-2421. Laude, D. A., Jr.; Johiman, C. L.; Cooper, J. R.; Wiikins, C. L. Anal. Chem. 1985, 57, 1044-1049. Settine, R. L.; Ghaderi, S.; Littlejohn, D, presented at 1985 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, LA, Paper 1266.
Carolyn L. Johlman David A. Laude, Jr. Robert S . Brown Charles L. Wilkins* Department of Chemistry University of California Riverside, California 92521 RECEIVED for review April 29, 1985. Accepted July 1, 1985. This research was supported by the National Science Foundation under Grants CHE-82-08073 and CHE-82-17610 (a department research instrument grant), which are gratefully acknowledged.
Tandem Quadrupole-Fourier Transform Mass Spectrometry of Oligopeptides Sir: Fourier transform mass spectrometers provide a potentially attractive alternative to magnetic sector, quadrupole, and time-of-flight instruments for the characterization of large biological molecules (1-3). Equipped with superconducting magnets, Fourier transform instruments are expected to operate with a mass range in excess of 10 000 and a resolution of at least 10000 a t this mass. Fourier transform instruments function as ion storage devices and can therefore accumulate ions produced in low abundance from small amounts of sample. In addition, all masses in a spectrum can be recorded simultaneously a t low resolution without sample ion destruction (4). Because of these two features, it should be possible to record complete mass spectra at the picomole sample level for molecules having molecular weights of several thousand. Direct analysis of biological molecules in mixtures should also be possible by using the double resonance technique to eject unwanted species prior to mass analysis of the ions stored in the cell. Consecutive collision-activated dissociation ( 5 , 6 )or laser photodissociation experiments (7-10) are expected to provide detailed structural information on the population of stored (M + H)’ ions. Release of kinetic energy during ion fragmentation under the above experimental conditions severely restricts the resolution that can be achieved in spectra recorded on sector instruments. Mass measurement on Fourier transform mass spectrometers is independent of ion translational energy. Consequently, main beam and daughter ion spectra can both be recorded at high resolution. One necessary requirement for the realization of the long ion storage times, picomole detection limits, and ultrahigh0003-2700/85/0357-2728$0 1.50/0
resolution mass analysis on large biomolecules is that the ion cyclotron resonance cell in the Fourier transform instrument must be maintained a t or below IO-* torr. Unfortunately glycerol and other liquid matrices employed in the highly successful particle bombardment ionization methods all have vapor pressures in excess of torr. Interfacing these techniques with a Fourier transform mass spectrometer has proved to be difficult. Recently, two approaches have been suggested for overcoming problems associated with high pressure accompanying sample introduction and ionization in Fourier transform instruments. The first involves use of a differentially pumped, two-compartment cell within a conventional Fourier transform mass spectrometer. Nicolet Instruments now markets an instrument embodying this concept. The second approach utilizes a tandem quadrupole-Fourier transform mass spectrometer. In this instrument, sample introduction and ionization are carried out in a differentially pumped quadrupole ion source and only the ions of interest are then transferred to the ion cyclotron resonance cell for mass analysis (11, 12). Results presented in an earlier paper demonstrated the feasability of using this type of instrument for mass analysis of ions produced by cesium bombardment of nonvolatile samples dissolved in a volatile liquid matrix (13). At that time the instrument was operating with a prototype pumping system of marginal capacity. Pressure in the ion cyclotron cell could only be maintained at 2 X torr, with or without sample in the ion source. As a result, much of the data obtained was of limited analytical utility. 0 1985 American Chemical Society
