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z 1000 in an ion trap mass spectrometer
International Journal of Mass Spectrometry and Ion Processes, 124 (1993) l-10
Elsevier Science Publishers B.V., Amsterdam
Evaluation of storage of ions over m/z 1000 in an ion trap mass spectrometer Robert J. Strife and John R. Simms Miami
Valley Laboratories,
(First received 10 February
The Procter and Gamble Co., Cincinnati, OH 45239-8707
(USA)
1992; in final form 11 September 1992)
Abstract Precise quantities of volatile, perfluoroalkyl triazines with molecular masses over 1000Da were introduced to an ion trap via a capillary gas chromatograph. “High mass” ions were formed within the trapping field, by an electron ionization pulse, at various r.f.-voltage levels. The high mass ions were then scanned out of the trap by application of a supplementary a.c. voltage across the endcaps, during an r.f.-voltage ramp. Evaluation of these combined capillary GC-MS results showed that the chromatographic peak areas associated with ion current from triazine high mass ions fell rapidly when initial qz values of the ions were below 0.02. Implications for peptide sequencing are discussed. Keywords: ion trap; high-mass; storage efficiency.
Introduction
The ion trap mass spectrometer (ITMS, FinmganMAT) is a tandem-in-time device with which sequential product ion analyses, (MS)“, may be done. The observed collisionally-induced-dissociation (CID) efficiency approaches 100% in some cases. Therefore, there is interest in developing “high mass” applications of ion traps for sensitive protein and oligonucleotide sequencing [l]. Here, high mass will be used to refer to ions with masses beyond the “normal” range to m/z 650, of a commercial instrument (i.e., when operated in its normal mass selective instability mode, with ejection of ions at qZ > 0.908, a, = 0, by ramping the 1.1 MHz r.f.-voltage). It has been appreciated for some time that ion traps can store high mass species. The early work of Wuerker showed the storage of 20pm diameter Correspondence to: R.J. Strife, Miami Valley Laboratories, I’he Procter and Gamble Co., Cincinatti, OH 45239-8707, USA.
aluminum particles in a trap [2], to give an extreme example. In terms of organic analyses, Todd first reported the axial ejection of m/z 1466, generated by electron ionization of a probe sample of trisperfluorononyl triazine, through use of combined r.f. and dc. voltages in a “reverse scan”,technique along the /?, = 0 operating line [3]. Other extended-massrange methods along this boundary were presented. However, new developments in the analysis of high mass ions have utilized a technique referred to as axial modulation, using an additional a.c. voltage (104-10’ Hz range typically) on the ion trap endcaps and an operating line on the qZ axis of the stability diagram. In these appliations [4,5], compounds have generally been ionized in external sources. The ions are then injected into the trapping field. Injection and analysis of singly charged CsI cluster ions of over 20 000 Da have been reported. The Dehmelt pseudopotential-well model may be used to determine an appropriate r.f.-level and injection energy to ensure trapping of these externally formed ions [6].
R.J. Strife, J.R. Simms/Int. J. Mass Spectrom. Ion Processes 124 (1993) I-10
2
We are not aware of any studies which have attempted to address the inherent storage efficiency of the ion trap for high mass ions (that is, independent of injection parameters affecting storage). The storage efficiency of high mass ions should directly affect the observed sensitivity of practical analyses, and may become a limiting factor in ion-injection techniques. Within the trapping field, ions are stored according to the Mathieu a and q parameters, which, on ion traps, are of two types: those defining the axial (z) component of ion trajectories and those defining the radial (r) component. The following relationships apply, when the trap dimensions are r$ = 2.4 : 4Z = -2q, -4eV
qz= -mr$i2
a,=
-2u I -8eV
where V is the zero-to-peak amplitude of the applied r.f. voltage, m is the mass of an ion, r. is the radius of the ring electrode and Sz is the radial frequency of the applied r.f. voltage. The expression for qZ may be reduced, as discussed by March and Hughes [7], to qZ = 0.0808 Vm-’ where the frequency fl = Q/274 is 1.1 MHz on our commercial system. Furthermore, it was recently revealed that commercial systems are “stretched” in the z-direction so that z = 1.1 lz, [8]. Taking this factor into account, it can easily be derived that qZ on the stretched trap is closely approximated by O.O725Vm-‘. At a modest ring potential of 500 Vo-p9 an ion of m/z 1000 would have a low qZ value of only 0.036. The implications of low qZvalues are best understood by examining the stability diagram (Fig. l), a graph of the limits of a, and qZ values that give bounded solutions, describing stable trajectories of ions, for the Mathieu equation of motion. For our studies, a, = 0 (i.e., there’ is no d.c. voltage used) and ions of low qZ value (c 0.05) are very close to the stability limit. The objective of this study was to determine how efficiently such ions are stored. We have investigated the storage efficiency of high mass ions independent of external sources and
0.30 FOR
- RESONANT HIGH MASS
w
EJECTION OPERATION
.91 NORMAL EJECTION POINT
%
Fig. 1. Partial stability diagram for the quadrupole ion trap showing ejection points at qZ= 0.3 (by z-axial modulation) or at q, = 0.91 (by crossing the boundary into a region of z-axial instability in trajectory).
ion injection. Well-defined quantities of volatile, fluorinated, high mass triazine neutrals were introduced via a capillary gas chromatograph. Ions were formed within the trapping field by an electron ionization pulse applied at various r.f.-voltage levels, corresponding to various qZ values of the ions. Upon driving the ions out to the detector, the ion currents produced were measured accurately as the areas under the gaussian capillary GC peaks. The results of these combined capillary GC-MS studies are described herein. Experimental All solvents used were HPLC grade. Trisperfluoroheptyl triazine (TPFHT) and trisperfluorononyl triazine (TPFNT) were purchased from PCR Chemicals, Gainesville, FL. Linear alkyl benzenes were obtained from Aldrich Chemical Co., Madison, WI. An equimolar stock solution was made up at lOOpmol~L_’ per component in hexane and a dilution was made in hexane to 5 pm01 &' . The capillary GC (Hewlett-Packard 5890 Series II) and the ion trap mass spectrometer system (Finnigan-MAT, San Jose, CA) have been described previously [9]. On-column injection of the dilute solution at 70°C was used with a program using a 1 min hold followed by a temperature ramp of 5”min-’ to 80°C then 10”Cmin’ to 15O’C. The helium flow was 1.5 mlmin-’ (linear velocity of 5Ocm SK’), which was kept constant throughout the run.
3
R.J. Stri/e, J.R. Simms/Int. J. Mass Spectrom. Ion Processes I24 (1993) I-IO
The ion trap temperature was 15O’C and the r.f.-voltage ramp was calibrated with perfluorotributylamine (PFTBA). The fundamental frequency of this ITMS system is crystal-controlled at 1.l MHz. Zero-to-peak r.f.-voltages were measured with an oscilloscope, using a 100 : 1 probe, at several “cut-off masses” (explained within). A plot of VGp at ionization (v) versus the cut-off mass at ionization (x) was made. The linear relationship between the two variables, y = 12.41~ - 1 allowed V at any cut-off mass within the range to be calculated. The qZ value of an ion at any cut-off mass at ionization could then be calculated using the reduced equation shown. The ion trap was controlled by version 1.41 Scan Editor Software (Finnigan-MAT). The basic GC-MS operation of the ion trap was tested in a first set of analyses, carried out by injecting an equimolar mixture of alkyl benzenes (toluene, ethyl benzene, n-propyl benzene, n-pentyl benzene and n-decyl benzene), using a 0.1 ms ionization pulse at a given r.f.-voltage level, changing the r.f.-voltage to 495 V,, (a cut-off mass of 40), and then ramping the r.f.-v - age to drive the ions out to the detector at a qZ value of 0.908. This is the mass-selective instability method and is referred to below as the normal method of mass analysis. Two scans per second were adquired. In a second set of experiments, an equimolar mixture of the two triazines and n-propyl and in-decyl benzenes was injected, using a 0.1 ms lionization pulse at a given r.f.-voltage level. Immedi,ately after ionization, the r.f.-level was adjusted to 1495v,, (cut-off mass of 40) and a 2ms period lfor collisional cooling was used. Then, during the r.f.-voltage ramp (mass scan), an auxiliary voltage ‘was applied across the endcap electrodes at a frequency of 114 000 Hz to cause “early” ejection of all Cons of m/z values > 120 during the ramp, at a qZ value of 0.3 (viz. axial modulation [4,5l). This triples the mass range of the trap to beyond m/z 1900. It t&o triples the scan rate beyond m/z 120. The scan range was set to 500 Da in the software, or effectively 1500 Da with the auxiliary voltage in use. Mass ussignments were made by tripling the observed
W2M
(CH2W
Fig. 2. General structures for alkyl benzmes and pefiuorotriazincs; n = 7 and 9 for trisperlluoroheptyl and trispertborononyl triazines, designated TPFHT and TPFNT, respectively.
masses output by the data system. Averaging every two mass scans (called “micro-scans”) gave up to 15 scans across a capillary GC peak 7 s wide at its base. A final set of experiments was conducted, comparing cut-off masses of 15 and 40, over a range of 5-200ms post-ionization cooling times, with the cooling conducted at a cut-off mass of 40. In all cases, three replicate GC-MS runs were made in the full-scan mode. Particular chromatograms were reconstructed for a 5 Da window around the mass of interest and peak areas were calculated using the manual integration software in the cited release. Results and discus600
The compounds TPFHT and TPFNT (Fig. 2), with mol. wt. 1185 and 1485, respectively, present a good opportunity to study the storage of ions of m/z values > 1000, independent of ion-injection parameters. This is true because the compounds can be purchased with good purity and are sufficiently volatile to be introduced to the trap as well-defined quantities of neutrals, via capillary GC. Electron ionization can then be used to form high mass ions within the trapping field. By scanning the ions out and recording the mass spectra in the GC-MS mode, chromatographic peak areas proportional to the ion current are produced. Capillary triazines
GC properties
of alkyl
benzenes
and
The triazines were readily chromatographed, appearing at retention times between those of n-propyl benzene and n-decyl benzene. The
4
R.J. Strife, J.R. Simms/Int.
TABLE 1 Capacity ratios of selected compounds Compound
k
n-Propyl benzene Trispertluoroheptyl triazine Trispertluorononyl triazine n-Decyl Benzene
1.59 3.21 5.60 8.80
capacity ratios of the compounds, k, defined as 0R - Ulth4, where t, is the retention time of the analyte and tM is the gas hold-up of the column (estimated by the time of elution of an unretained component), are listed in Table 1. Each triazine gave only one chromatographic peak during analysis, which was symmetrical and had a peak width at half height of about 3s. A sample gas chromatogram, based on the total ion currents, is shown in Fig. 3. Reference mass spectra of triazines The mass spectra of TPFHT and TPFNT were first examined using a solids probe on a sector mass spectrometer of magnetic-electric (BE) configuration, using electron ionization, and were found to be in agreement with reference data [lo]. The spectra show intense ions at m/z 1166 and 1466, respectively (about 50% relative abundance), representing loss of F’ from the parent molecular ion. The base peaks appear at m/z 866 and 1066, respectively, and result from cleavage between the
L, E
3
*
w
4
5 w z 2 ii ,
1 :oo
,
I
I,
kl
8 I,,
TIME (minsec)
I,,
5:51
Fig. 3. Capillary gas chromatogram showing: 1, n-propyl benzene; 2, trisperfluoroheptyl triazine; 3, trispertluorononyl triazine, and 4, n-decyl benzene. Conditions: 15 m x 0.25 mm i.d. DB-1, injection at 250°C, oven program from 70°C at 30°C min-’ (later modified), constant flow He 50 cm s-’ .
J. Mass Spectrom.
Ion Processes
124 (1993)
I-10
a and /I carbons of one side chain. The only other ions listed in the reference data, above 10% in relative abundance, occur at m/z 119, 131 and 169. When the spectra are scanned to include m/z 69 from CF: , it is found to be the base peak, with the fragments from loss of F’ about 20% relative abundance. The reference spectrum for TPFNT is listed only to m/z 119. Normal ion trap mass spectromefry In the normal operation of the commercial ITMS system, ionization and trapping of ions are followed by simply ramping the r.f.-voltage applied to the ring electrode of the ion trap. This increases the qZvalues of ions beyond 0.908 (Fig. l), driving them axially out to the detector along the z-axis of the trap. Ions of increasing m/z value leave the trap sequentially in time, only up to m/z values of 650Da per unit charge. Ions of this mass require about 8OOOV(zero to peak) to be ejected in the stretched trap. The mixture of alkyl benzenes was analyzed in this mode, using triplicate injections. As a function of r.f.-voltage applied to the ring at the ionization event, the averaged capillary GC peak area for each alkyl benzene, derived from the total ion current produced, was greatest at about 185 V,, . This voltage corresponds to a cut-off mass of 15. Cut-off mass means the m/z ratio above which ions are stored and will be referred to in this paper assuming units of Da per unit charge for convenience. The r.f.-voltage levels are described by cut-off mass in the software running the instrument, since this allows scan functions to be set up more easily. So, in the example given, ions of m/z 15 have a qZvalue of 0.908. Thus, the cut-off mass is directly proportional to the ring voltage; e.g. a cut-off mass of 30 corresponds to about 370 V,, on our instrument. In Fig. 4, it can be seen that when the ring voltage is too low at ionization, ions are not stored efficiently even for the alkyl benzenes. That is, even m/z 91, the predominant ion in the spectra, is not stored well, though the qZvalue of this ion is greater than 0.10 at ionization.
R.J. Strife. J.R. Simms/Int. J. Mass Spectrom. Ion Processes 124 (1993) I-10 “PLATEAU” REGION FOR STUDY OF ION STORAGE
VARIABLE
g
RAMP TIME - SO ms
80
B ECYL $
BENZENE
-
60
II VOLTAGE
RELATIVE TIME
LOW MASS CUT-OFF (rf VOLTAGE)
36)
Fig. 4. Percent of maximum GC peak area observed (based on total ion current) vs. cut-off mass at the ionization event for toluene and n-decyl benzene, the most volatile and least volatile compound, respectively, in this study. Data points are from the average areas of three replicate runs.
However if the ring voltage becomes too high (> 500 V,,) at ionization, capillary GC peak areas for alkyl benzenes are seen to decline. We hypothesize that this observation is in accord with the decrease in ionization efficiency that is observed for EI at higher electron energies, as well as being due to less penetration of the electrons into the field. That is, the filament bias relative to ground is constant, so that higher ring voltages result in more direct hyperbolic trajectories of electrons toward the ring when the r.f.-phase is favorable, giving less penetration of the field. The area of “stable operation” during normal electron ionization was taken to be from cut-off masses in the range 10-40, though points outside of this range were explored. High mass experiments triazines
(> m/z 650 ejected)
using
In order to eject high mass, triazine-derived ions from the trap, the mass range of the instrument was increased by application of an a.c. voltage across the endcap electrodes [4,5] at a frequency identical to the z-axial component of motion of ions, at qZ = 0.3 (Fig. l), or about 114 kHz. That is, while
Fig. 5. ITMS scan function for high mass work plotted as relative voltage vs. relative time. A variable collisional cooling period (2-250 ms in these studies) follows ionization. The magnitudes of the voltages are not scaled to each other. Typical values are - 100 to + 100V for the electron gate and O-6 V for the supplementary a.c. voltage. The r.f.-voltage ramp occurs at 18 ms per 100 Da and is effectively triple this rate for ions ejected at q, = 0.3.
an ion’s trajectory is normally stable at qZ = 0.3 and a, = 0.0, it can be forced to leave the trap as it resonantly absorbs the energy from the field applied across the endcap electrodes. The high mass scan function, described in the Experimental section, is summarized in Fig. 5. As an example of how this affects the appearance of a mass spectrum obtained on the ITMS, consider scanning from an r.f.-voltage level corresponding to a cut-off mass of 40. Ions of m/z 120 and greater are stored at qZ values between 0.3 and zero. During the r.f-voltage ramp (mass scan) at the end of the experiment, these ions will be ejected as they attain a qZ value of 0.3. Their data-systemassigned m/z values will appear at one third of their true values, as the instrument data system is calibrated for the normal mass range mode only (ejection at about three times qZ = 0.3). The other ions, with initial qZ values above 0.3 (i.e. m/z 69), will never encounter the ejection frequency at !I== 0.3 during the mass scan and will be ejected as usual, at qZ = 0.908, as the r.f.-voltage is ramped. In the example chosen, CF: will be assigned as m/z 69 by the data system whereas m/z 866 would be assigned as near m/z 289.
6
R.J, Strije, J.R. Simms/Int.
J. Mass Spectrom. Ion Processes
124 (1993) I-10
TABLE 2 Values of ions from the test compounds at various ring voltages Cut-off mass”
lo(123) 20(247) 30(370) 4q495)
300
660
900 m/z
1200
1600
Fig. 6. Mass spectrum for trispertluorononyl triaxine on the ion trap mass spectrometer. The mass axis has been tripled and mass assignments shown are three times the data-system-assigned values. Values of m/z shown generally fell within l-2Da of reference spectrum values. Inaccuracies arise from a digitization rate effectively at only one third of the usual value and from mass shifts that are observed when axial modulation is used.
Ion trap mass spectra of standarch
The spectra for the alkyl benzenes are consistent under high mass conditions with the spectra obtained in the normal mode of operation. The only significant ions below m/z 120 for alkyl benzenes are those of m/z 91. The triazine mass spectra, obtained using an ionization cut-off mass of 30, were similar to those obtained at 70 eV on the sector instrument, with very intense ions for loss of F’ (m/z 1166 and 1466 for TPFHT and TPFNT respectively) and cleavage at the a and fl positions of the side chain being observed (m/z 866 and 1066 respectively, Fig. 6). Storage eficiency
curves
With these results in place, we were ready to investigate the storage efficiency of high mass ions. The r.f.-voltage level at ionization was varied such that the corresponding cut-off mass was between 10 and 40, the stable area of trap operation for the alkyl benzenes. The two triazines were mixed equimolar with n-propyl and n-decyl benzenes. The ql values of several ions representative of these com-
m/z 91
120
218
866
1166
1466
0.098 0.197 0.295 0.394
0.074 0.149 0.224 0.299
0.041 0.082 0.123 0.164
0.010 0.021 0.031 0.041
0.008 0.015 0.023 0.030
0.006 0.012 0.018 0.024
"The valuein parentheses is that of the corresponding voltage (V,,).
r.f.-
ponents, at the cut-off mass (and corresponding r.f.-voltage) present at the ionization event, are listed in Table 2. In the GC-full-scan MS mode, capillary GC peak areas reflect the total ion current at the detector. The current from individual ions can be evaluated post-run in reconstructed ion chromatograms. It is instructive to inspect the capillary GC peak areas for the triazines relative to the alkyl benzenes, when triazine high mass ions are efficiently trapped. One might expect the ionization crosssections to be slightly higher for the triazines, due to the presence of not only a A cloud of six electrons, but three lone pairs on each of the nitrogen atoms as well. Indeed, at an ionization level corresponding to a cut-off mass of 30, the capillary peak areas for the triazines are greater than for the equimolar alkyl benzenes (Fig. 7a). It is important to note here that this result established a benchmark for maximum storage efficiency. Since the obtained triazine mass spectra are similar to those on a beam instrument (BE configuration) and the GC peak areas are even greater than those for the low mass alkyl benzenes (efficient ion storage presumed) we conclude that the storage efficiencies for high mass ions can be on the order of those for the lower mass ions in the spectrum. At a cut-off mass of 10 at ionization, there is a large decline in the total ion current (and therefore the GC peak area) for the triazines while the alkyl benzene peak areas are almost unchanged (Fig. 7b).
1
R.J. Strife, J.R. Simms/Int. J. Mass Spectrom. Ion Processes 124 (1993) I-10 ION STORAGE ON STRETCHED TRAP
1 :oo
TIME (minsec)
I jooJ,, , ,
5:51
Fig. 7. Comparison of combined capillary GC-MS analysis of the compounds listed in Fig. 3, using cut-off masses at ionization of 30 (upper trace, (a)) versus 10 (lower trace, (b)). Only the triazine GC peak areas decrease because of lowered contribution to the total ion current by their high mass ion (see Fig. 3 for key to identities of peaks).
In Fig. 8, it can be se I that the ion current for m/z 1466 is falling rapidly while that for m/z 91 is maintained, at r.f.-voltages of lOO-2OOV,,. The decrease in the observed GC peak area is due to decreasing storage of the high mass ions, which :arry a substantial portion of the total ion current. The results become even clearer, when the GC peak areas associated with m/z 866, 1166, and 1466 ure plotted as a function of their qz values at ionization, over a range of several r.f.-voltages. As DECYL BENZENE
[
I
2
Be
‘!i:
~~-p’“’
40
&TPFNT
20 I
/
(l%,
&l-OFF MA&i (rf VOLTAGE)
;0 (465)
Fig. 8. Plot of percent of maximum GC peak area (based on ion current for a given mass) for m/z 91 of decyl benzene and m/z 1466 for TPFNT as a function of the cut-off mass at ionization and the corresponding r.f.-voltage. Data points represent the arverageof three replicate runs.
0.01
0.02
0.03
0.04
0.05
1 0.06
42 Fig. 9. Plots of percent of maximum GC peak area observed vs. qZat ionization for three principal high mass ions from the triazines. Three replicate analyses were carried out at each ionization level.
qz approaches a value around 0.02-0.03, the peak areas begin to decline rapidly for the triazine high mass ions (Fig. 9). Cooling time During review of this work, the question of cooling time after ionization was raised, since we had originally investigated only out to 5ms, without seeing significant change in our results from 2 ms. Triplicate GC-MS runs were performed with cooling periods of 5,50, 100, 150 and 200 ms post-ionization. As a control, ionization at a cut-off mass of 40 (relatively efficient storage of all ions) was first examined, with a varied post-ionization cooling period. Out to 2OOms, the ion populations fell steadily for m/z 91, 866, 1066 and 1466, to between 30 and 50% of their initial values. It was surprising to find the population of m/z 1166 rather stable over this period. Next, the cut-off mass at ionization was lowered to 15, and the same experiment was carried out (Fig. 10). The results for m/z 91,866 and 1066 were similar to those obtained at a cut-off mass of 40, falling to between 35 and 45% of their initial values, at the 2OOms time point. However, the
8
R.J. Strife, J.R. Simmsjlnt. J, Mass Spectrom. Ion Processes 124 (1993) I-IO
POST-IONIZATION COOLING EFFECT ON VARIOUS IONS
a”
0
100 POST-IONIZATION
200
COOLING (ms)
Fig. 10. Normalized relative intensities (based on GC peak area) for various ions as a function of post ionization cooling time (at a cut-off mass of 40), when a cut-off mass of 15 is used at ionization. The m/z value for eachcurve is shown and Cl0 refers to the total ion current (due mainly to m/z 91) from n-decyl benzene.
results for m/z 1166 and 1466 were directionally opposite. The ion populations were able to recover to 56% and 41% of their maximum values (i.e., observed at cut-off mass 40), using 200ms of cooling. However, the graph shows that this recovery reaches a plateau at this point. In considering an explanation for the results, the following sequence of events must be considered: (i) the neutral compounds must be uniformly delivered by the GC; (ii) ions must be formed by electron ionization with a certain efficiency; (iii) ions must be trapped and collisionally focussed down to the center area of the electrodes; (iv) ions must be ejected along the z-axis during the voltage ramp; (v) ions must pass through the perforated endcap and strike the conversion dynode, producing a response. Factors (i), (ii) and (iv) are controlled by the experimental design. The alkyl benzenes verify the chromatographic performance and delivery of the triazines during the experiment, because their retention times bracket those of the triazines. Also,
the areas of the alkyl benzenes remain the same while those for the triazines are rapidly falling. The ionization efficiencies have not changed greatly at various r.f.-voltage levels because alkyl benzene peak areas remain relatively constant in control experiments at different r.f.-voltage levels. Also, the triazine ions are always ejected at qz = 0.3, regardless of their initial qz value, thus striking the conversion dynode with the same momentum, kinetic energy and response factor. Thus factors (iii) and (v) need to be focussed on. Ions of low qz values are very close to the axial and radial stability limits for ion trajectories (Fig. 1). Moreover, the diagram in Fig. 1 reflects conditions for a perfect electrode. Therefore it is possible that low qz value ions are lost in unstable trajectories before the detection step of the experiment. Also, it has been suggested to us that ions of these low qz values may be approaching the physical boundaries of the trap. That is, the ion trajectories may be stable but exceeding the physical bounds of the electrode structure. We are investigating this possibility further. A third factor seems to be that ion losses work against the recovery of ions by collisional cooling and a practical limit is reached at 200 ms (He gas). Also, a decrease in the capillary GC peak area of a triazine may be due to the fact that the ion trajectory has not been damped enough. That is, when the condition for axial ejection is reached, the ions may be trapped, but they are not yet at the center of the trap. The ions do not exit along the z-axis through the perforated endcap and miss striking the conversion dynode. Implications These results show the importance of longcollisional cooling periods for high mass ions using He as a buffer gas. They also show that a minimum q2 value of 0.02 should be used to obtain efficient storage of ions between m/z 1000 and 1500. In a recent study [l 11, efficient trapping of injected CsI cluster ions was reported. However, this term was used only in a relative sense, in comparing trapping
R.J. Strife, J.R. Simms~Int. J. Mars Spectrom. ion Processes 124 (1993) I-10
of high mass ions using various bath gases (He compared to He with up to 5% of heavier noble gases). The actual trapping efficiency of the ions is unknown and it is interesting that a minimum qz of 0.032 was chosen for injection [12]. Previous reports _urion injection have revealed long integration times (minutes) to obtain extendedmass-range spectra. Here such spectra are obtained on 1 pm01 quantities using only 0.1 ms of ionization, in less than 1 s. Thus it is possible that there are substantial losses in ion injection. Comparisons of ion current initially generated by the two methods versus those finally detected could be useful. Nevertheless, the mass ranges and practical operating sensitivity demonstrated in peptide analyses by ion injection have been impressive, in our opinion. In this study, the results suggest that the trapping efficiency of high mass ions formed in the field approaches that of relatively low mass alkyl benzene ions formed in the field under some conditions. This hypothesis is based on the spectral comparison between the trap and beam instruments as well as the nearly equal capillary GC peak areas for triazines versus equimolar alkyl benzenes. The number of lions lost in the initial stages of any electron ionlization experiment is unknown to us, so that our rabsolute trapping efficiencies are also unknown. Another important result is found in the triazine spectra recorded at cooling times on the order of 100 ms. Ion/molecule reactions become significant and produce peaks at M + 69, no doubt from reaction of the CF: cation with neutral triazine molecules. If long cooling times are required, then spectral artifacts needing interpretation may occur. Also, the long cooling times may begin to broach the time domain needed for definition of very narrow chromatographic peak shapes as in capillary zone electrophoresis of proteins, if one assumes 8-10 scans are necessary for peak definition. Finally, in regard to injecting high mass ions formed externally, a calculation of the Dehmelt pseudopotential well-depth for an ion of m/z 4000, at a qz value of 0.02, shows that the corresponding cut-off mass is 88 (1103 V,,). The pseudopotential well depth is rather shallow, only 2.8eV on our
9
system under these conditions. If our hypothesis is corrrect, it is easy to see that the observed storage efficiency of the trap could be a complicating factor during injection of high mass ions from external sources. Conclusions The method described here allows for the first time a precise determination of apparent storage efficiencies of high mass ions relative to low mass ions. The low mass ions are presumed to be efficiently trapped and damped to the center of the trap but their absolute storage efficiency is unknown. Storage of high mass ions does not appear to be very efficient on the ion trap mass spectrometer when qz values fall below 0.02, using helium as a buffer gas. Given the recent results which noted deleterious scattering effects of heavier buffer gases, the prospect of extended-mass-range work on an ion trap mass spectrometer needs to be approached with guarded optimism. It is our opinion that further developments in the computerized modelling of ion trajectories, in the presence of various bath gases, will be valuable in further understanding ion trap behavior for ions of low qz value, near the stability limits of the storage diagram. Acknowledgment We thank Professor Ray March, Trent University, for helpful discussions and suggestions during the course of this investigation. References K.A. Cox, R.E. Kaiser Jr. and R.G. Cooks, Use of a Quadrupole Ion Trap Mass Spectrometer for Structural Determination of Biomolecules, Proc. 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June, 1990. R.F. Wuerker, H. Shelton and R.V. Langmuir, J. Appl. Phys., 30 (1959) 342. J.F.J. Todd, A. Penman and R.D. Smith, Int. J. Mass Spectrom. Ion Processes, 106 (1991) 117-135. R.D. Nourse and R.G. Cooks, Anal. Chim. Acta, 228 (1990) I-21.
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8
R.J. Strijie, J.R. Shuns/hi.
R.E. Kaiser, J.N. Louris, J.W. Amy and R.G. Cooks, Rapid Commun. Mass Spectrom, 3 (1989) 225-230. J.E. Fulford and R.E. March, Int. J. Mass Spectrom. Ion Phys., 30 (1979) 39-55. R.E. March and R.J. Hughes, Quadrupole Storage Mass Spectrometry, Chemical Analysis Series, Vol. 102, Wiley, New York, 1989, pp. 196-201. J. Louris, G. Stafford, J. Syka and D. Taylor, The Paul Ion Trap Mass Selective Instability Scan: Trap Geometry and Resolution, Proc. 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington D.C., May 31-
9 10 11 12
J. Mass Spectrom.
Ion Processes 124 (1993) I-10
June 5, 1992. R.J. Strife and J.R. Simms, J. Am. Sot. Mass. Spcctrom., l(l990) 265-271. Fisons Instruments, 32 Commerce Center, Danvers, MA 01923. K.L. Morand, K. Cox and R.G. Cooks, Rapid Commun. Mass Spectrom., 6 (1992) 520-523. J.R. Simms and J.R. Strife, Relative Ion Trap Storage Efficiencies of Ions Up to m/z 1500 by Capillary GC/MS; Proc. 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 19-24, 1991.
Elsevier Science Publishers B.V., Amsterdam
Evaluation of storage of ions over m/z 1000 in an ion trap mass spectrometer Robert J. Strife and John R. Simms Miami
Valley Laboratories,
(First received 10 February
The Procter and Gamble Co., Cincinnati, OH 45239-8707
(USA)
1992; in final form 11 September 1992)
Abstract Precise quantities of volatile, perfluoroalkyl triazines with molecular masses over 1000Da were introduced to an ion trap via a capillary gas chromatograph. “High mass” ions were formed within the trapping field, by an electron ionization pulse, at various r.f.-voltage levels. The high mass ions were then scanned out of the trap by application of a supplementary a.c. voltage across the endcaps, during an r.f.-voltage ramp. Evaluation of these combined capillary GC-MS results showed that the chromatographic peak areas associated with ion current from triazine high mass ions fell rapidly when initial qz values of the ions were below 0.02. Implications for peptide sequencing are discussed. Keywords: ion trap; high-mass; storage efficiency.
Introduction
The ion trap mass spectrometer (ITMS, FinmganMAT) is a tandem-in-time device with which sequential product ion analyses, (MS)“, may be done. The observed collisionally-induced-dissociation (CID) efficiency approaches 100% in some cases. Therefore, there is interest in developing “high mass” applications of ion traps for sensitive protein and oligonucleotide sequencing [l]. Here, high mass will be used to refer to ions with masses beyond the “normal” range to m/z 650, of a commercial instrument (i.e., when operated in its normal mass selective instability mode, with ejection of ions at qZ > 0.908, a, = 0, by ramping the 1.1 MHz r.f.-voltage). It has been appreciated for some time that ion traps can store high mass species. The early work of Wuerker showed the storage of 20pm diameter Correspondence to: R.J. Strife, Miami Valley Laboratories, I’he Procter and Gamble Co., Cincinatti, OH 45239-8707, USA.
aluminum particles in a trap [2], to give an extreme example. In terms of organic analyses, Todd first reported the axial ejection of m/z 1466, generated by electron ionization of a probe sample of trisperfluorononyl triazine, through use of combined r.f. and dc. voltages in a “reverse scan”,technique along the /?, = 0 operating line [3]. Other extended-massrange methods along this boundary were presented. However, new developments in the analysis of high mass ions have utilized a technique referred to as axial modulation, using an additional a.c. voltage (104-10’ Hz range typically) on the ion trap endcaps and an operating line on the qZ axis of the stability diagram. In these appliations [4,5], compounds have generally been ionized in external sources. The ions are then injected into the trapping field. Injection and analysis of singly charged CsI cluster ions of over 20 000 Da have been reported. The Dehmelt pseudopotential-well model may be used to determine an appropriate r.f.-level and injection energy to ensure trapping of these externally formed ions [6].
R.J. Strife, J.R. Simms/Int. J. Mass Spectrom. Ion Processes 124 (1993) I-10
2
We are not aware of any studies which have attempted to address the inherent storage efficiency of the ion trap for high mass ions (that is, independent of injection parameters affecting storage). The storage efficiency of high mass ions should directly affect the observed sensitivity of practical analyses, and may become a limiting factor in ion-injection techniques. Within the trapping field, ions are stored according to the Mathieu a and q parameters, which, on ion traps, are of two types: those defining the axial (z) component of ion trajectories and those defining the radial (r) component. The following relationships apply, when the trap dimensions are r$ = 2.4 : 4Z = -2q, -4eV
qz= -mr$i2
a,=
-2u I -8eV
where V is the zero-to-peak amplitude of the applied r.f. voltage, m is the mass of an ion, r. is the radius of the ring electrode and Sz is the radial frequency of the applied r.f. voltage. The expression for qZ may be reduced, as discussed by March and Hughes [7], to qZ = 0.0808 Vm-’ where the frequency fl = Q/274 is 1.1 MHz on our commercial system. Furthermore, it was recently revealed that commercial systems are “stretched” in the z-direction so that z = 1.1 lz, [8]. Taking this factor into account, it can easily be derived that qZ on the stretched trap is closely approximated by O.O725Vm-‘. At a modest ring potential of 500 Vo-p9 an ion of m/z 1000 would have a low qZ value of only 0.036. The implications of low qZvalues are best understood by examining the stability diagram (Fig. l), a graph of the limits of a, and qZ values that give bounded solutions, describing stable trajectories of ions, for the Mathieu equation of motion. For our studies, a, = 0 (i.e., there’ is no d.c. voltage used) and ions of low qZ value (c 0.05) are very close to the stability limit. The objective of this study was to determine how efficiently such ions are stored. We have investigated the storage efficiency of high mass ions independent of external sources and
0.30 FOR
- RESONANT HIGH MASS
w
EJECTION OPERATION
.91 NORMAL EJECTION POINT
%
Fig. 1. Partial stability diagram for the quadrupole ion trap showing ejection points at qZ= 0.3 (by z-axial modulation) or at q, = 0.91 (by crossing the boundary into a region of z-axial instability in trajectory).
ion injection. Well-defined quantities of volatile, fluorinated, high mass triazine neutrals were introduced via a capillary gas chromatograph. Ions were formed within the trapping field by an electron ionization pulse applied at various r.f.-voltage levels, corresponding to various qZ values of the ions. Upon driving the ions out to the detector, the ion currents produced were measured accurately as the areas under the gaussian capillary GC peaks. The results of these combined capillary GC-MS studies are described herein. Experimental All solvents used were HPLC grade. Trisperfluoroheptyl triazine (TPFHT) and trisperfluorononyl triazine (TPFNT) were purchased from PCR Chemicals, Gainesville, FL. Linear alkyl benzenes were obtained from Aldrich Chemical Co., Madison, WI. An equimolar stock solution was made up at lOOpmol~L_’ per component in hexane and a dilution was made in hexane to 5 pm01 &' . The capillary GC (Hewlett-Packard 5890 Series II) and the ion trap mass spectrometer system (Finnigan-MAT, San Jose, CA) have been described previously [9]. On-column injection of the dilute solution at 70°C was used with a program using a 1 min hold followed by a temperature ramp of 5”min-’ to 80°C then 10”Cmin’ to 15O’C. The helium flow was 1.5 mlmin-’ (linear velocity of 5Ocm SK’), which was kept constant throughout the run.
3
R.J. Stri/e, J.R. Simms/Int. J. Mass Spectrom. Ion Processes I24 (1993) I-IO
The ion trap temperature was 15O’C and the r.f.-voltage ramp was calibrated with perfluorotributylamine (PFTBA). The fundamental frequency of this ITMS system is crystal-controlled at 1.l MHz. Zero-to-peak r.f.-voltages were measured with an oscilloscope, using a 100 : 1 probe, at several “cut-off masses” (explained within). A plot of VGp at ionization (v) versus the cut-off mass at ionization (x) was made. The linear relationship between the two variables, y = 12.41~ - 1 allowed V at any cut-off mass within the range to be calculated. The qZ value of an ion at any cut-off mass at ionization could then be calculated using the reduced equation shown. The ion trap was controlled by version 1.41 Scan Editor Software (Finnigan-MAT). The basic GC-MS operation of the ion trap was tested in a first set of analyses, carried out by injecting an equimolar mixture of alkyl benzenes (toluene, ethyl benzene, n-propyl benzene, n-pentyl benzene and n-decyl benzene), using a 0.1 ms ionization pulse at a given r.f.-voltage level, changing the r.f.-voltage to 495 V,, (a cut-off mass of 40), and then ramping the r.f.-v - age to drive the ions out to the detector at a qZ value of 0.908. This is the mass-selective instability method and is referred to below as the normal method of mass analysis. Two scans per second were adquired. In a second set of experiments, an equimolar mixture of the two triazines and n-propyl and in-decyl benzenes was injected, using a 0.1 ms lionization pulse at a given r.f.-voltage level. Immedi,ately after ionization, the r.f.-level was adjusted to 1495v,, (cut-off mass of 40) and a 2ms period lfor collisional cooling was used. Then, during the r.f.-voltage ramp (mass scan), an auxiliary voltage ‘was applied across the endcap electrodes at a frequency of 114 000 Hz to cause “early” ejection of all Cons of m/z values > 120 during the ramp, at a qZ value of 0.3 (viz. axial modulation [4,5l). This triples the mass range of the trap to beyond m/z 1900. It t&o triples the scan rate beyond m/z 120. The scan range was set to 500 Da in the software, or effectively 1500 Da with the auxiliary voltage in use. Mass ussignments were made by tripling the observed
W2M
(CH2W
Fig. 2. General structures for alkyl benzmes and pefiuorotriazincs; n = 7 and 9 for trisperlluoroheptyl and trispertborononyl triazines, designated TPFHT and TPFNT, respectively.
masses output by the data system. Averaging every two mass scans (called “micro-scans”) gave up to 15 scans across a capillary GC peak 7 s wide at its base. A final set of experiments was conducted, comparing cut-off masses of 15 and 40, over a range of 5-200ms post-ionization cooling times, with the cooling conducted at a cut-off mass of 40. In all cases, three replicate GC-MS runs were made in the full-scan mode. Particular chromatograms were reconstructed for a 5 Da window around the mass of interest and peak areas were calculated using the manual integration software in the cited release. Results and discus600
The compounds TPFHT and TPFNT (Fig. 2), with mol. wt. 1185 and 1485, respectively, present a good opportunity to study the storage of ions of m/z values > 1000, independent of ion-injection parameters. This is true because the compounds can be purchased with good purity and are sufficiently volatile to be introduced to the trap as well-defined quantities of neutrals, via capillary GC. Electron ionization can then be used to form high mass ions within the trapping field. By scanning the ions out and recording the mass spectra in the GC-MS mode, chromatographic peak areas proportional to the ion current are produced. Capillary triazines
GC properties
of alkyl
benzenes
and
The triazines were readily chromatographed, appearing at retention times between those of n-propyl benzene and n-decyl benzene. The
4
R.J. Strife, J.R. Simms/Int.
TABLE 1 Capacity ratios of selected compounds Compound
k
n-Propyl benzene Trispertluoroheptyl triazine Trispertluorononyl triazine n-Decyl Benzene
1.59 3.21 5.60 8.80
capacity ratios of the compounds, k, defined as 0R - Ulth4, where t, is the retention time of the analyte and tM is the gas hold-up of the column (estimated by the time of elution of an unretained component), are listed in Table 1. Each triazine gave only one chromatographic peak during analysis, which was symmetrical and had a peak width at half height of about 3s. A sample gas chromatogram, based on the total ion currents, is shown in Fig. 3. Reference mass spectra of triazines The mass spectra of TPFHT and TPFNT were first examined using a solids probe on a sector mass spectrometer of magnetic-electric (BE) configuration, using electron ionization, and were found to be in agreement with reference data [lo]. The spectra show intense ions at m/z 1166 and 1466, respectively (about 50% relative abundance), representing loss of F’ from the parent molecular ion. The base peaks appear at m/z 866 and 1066, respectively, and result from cleavage between the
L, E
3
*
w
4
5 w z 2 ii ,
1 :oo
,
I
I,
kl
8 I,,
TIME (minsec)
I,,
5:51
Fig. 3. Capillary gas chromatogram showing: 1, n-propyl benzene; 2, trisperfluoroheptyl triazine; 3, trispertluorononyl triazine, and 4, n-decyl benzene. Conditions: 15 m x 0.25 mm i.d. DB-1, injection at 250°C, oven program from 70°C at 30°C min-’ (later modified), constant flow He 50 cm s-’ .
J. Mass Spectrom.
Ion Processes
124 (1993)
I-10
a and /I carbons of one side chain. The only other ions listed in the reference data, above 10% in relative abundance, occur at m/z 119, 131 and 169. When the spectra are scanned to include m/z 69 from CF: , it is found to be the base peak, with the fragments from loss of F’ about 20% relative abundance. The reference spectrum for TPFNT is listed only to m/z 119. Normal ion trap mass spectromefry In the normal operation of the commercial ITMS system, ionization and trapping of ions are followed by simply ramping the r.f.-voltage applied to the ring electrode of the ion trap. This increases the qZvalues of ions beyond 0.908 (Fig. l), driving them axially out to the detector along the z-axis of the trap. Ions of increasing m/z value leave the trap sequentially in time, only up to m/z values of 650Da per unit charge. Ions of this mass require about 8OOOV(zero to peak) to be ejected in the stretched trap. The mixture of alkyl benzenes was analyzed in this mode, using triplicate injections. As a function of r.f.-voltage applied to the ring at the ionization event, the averaged capillary GC peak area for each alkyl benzene, derived from the total ion current produced, was greatest at about 185 V,, . This voltage corresponds to a cut-off mass of 15. Cut-off mass means the m/z ratio above which ions are stored and will be referred to in this paper assuming units of Da per unit charge for convenience. The r.f.-voltage levels are described by cut-off mass in the software running the instrument, since this allows scan functions to be set up more easily. So, in the example given, ions of m/z 15 have a qZvalue of 0.908. Thus, the cut-off mass is directly proportional to the ring voltage; e.g. a cut-off mass of 30 corresponds to about 370 V,, on our instrument. In Fig. 4, it can be seen that when the ring voltage is too low at ionization, ions are not stored efficiently even for the alkyl benzenes. That is, even m/z 91, the predominant ion in the spectra, is not stored well, though the qZvalue of this ion is greater than 0.10 at ionization.
R.J. Strife. J.R. Simms/Int. J. Mass Spectrom. Ion Processes 124 (1993) I-10 “PLATEAU” REGION FOR STUDY OF ION STORAGE
VARIABLE
g
RAMP TIME - SO ms
80
B ECYL $
BENZENE
-
60
II VOLTAGE
RELATIVE TIME
LOW MASS CUT-OFF (rf VOLTAGE)
36)
Fig. 4. Percent of maximum GC peak area observed (based on total ion current) vs. cut-off mass at the ionization event for toluene and n-decyl benzene, the most volatile and least volatile compound, respectively, in this study. Data points are from the average areas of three replicate runs.
However if the ring voltage becomes too high (> 500 V,,) at ionization, capillary GC peak areas for alkyl benzenes are seen to decline. We hypothesize that this observation is in accord with the decrease in ionization efficiency that is observed for EI at higher electron energies, as well as being due to less penetration of the electrons into the field. That is, the filament bias relative to ground is constant, so that higher ring voltages result in more direct hyperbolic trajectories of electrons toward the ring when the r.f.-phase is favorable, giving less penetration of the field. The area of “stable operation” during normal electron ionization was taken to be from cut-off masses in the range 10-40, though points outside of this range were explored. High mass experiments triazines
(> m/z 650 ejected)
using
In order to eject high mass, triazine-derived ions from the trap, the mass range of the instrument was increased by application of an a.c. voltage across the endcap electrodes [4,5] at a frequency identical to the z-axial component of motion of ions, at qZ = 0.3 (Fig. l), or about 114 kHz. That is, while
Fig. 5. ITMS scan function for high mass work plotted as relative voltage vs. relative time. A variable collisional cooling period (2-250 ms in these studies) follows ionization. The magnitudes of the voltages are not scaled to each other. Typical values are - 100 to + 100V for the electron gate and O-6 V for the supplementary a.c. voltage. The r.f.-voltage ramp occurs at 18 ms per 100 Da and is effectively triple this rate for ions ejected at q, = 0.3.
an ion’s trajectory is normally stable at qZ = 0.3 and a, = 0.0, it can be forced to leave the trap as it resonantly absorbs the energy from the field applied across the endcap electrodes. The high mass scan function, described in the Experimental section, is summarized in Fig. 5. As an example of how this affects the appearance of a mass spectrum obtained on the ITMS, consider scanning from an r.f.-voltage level corresponding to a cut-off mass of 40. Ions of m/z 120 and greater are stored at qZ values between 0.3 and zero. During the r.f-voltage ramp (mass scan) at the end of the experiment, these ions will be ejected as they attain a qZ value of 0.3. Their data-systemassigned m/z values will appear at one third of their true values, as the instrument data system is calibrated for the normal mass range mode only (ejection at about three times qZ = 0.3). The other ions, with initial qZ values above 0.3 (i.e. m/z 69), will never encounter the ejection frequency at !I== 0.3 during the mass scan and will be ejected as usual, at qZ = 0.908, as the r.f.-voltage is ramped. In the example chosen, CF: will be assigned as m/z 69 by the data system whereas m/z 866 would be assigned as near m/z 289.
6
R.J, Strije, J.R. Simms/Int.
J. Mass Spectrom. Ion Processes
124 (1993) I-10
TABLE 2 Values of ions from the test compounds at various ring voltages Cut-off mass”
lo(123) 20(247) 30(370) 4q495)
300
660
900 m/z
1200
1600
Fig. 6. Mass spectrum for trispertluorononyl triaxine on the ion trap mass spectrometer. The mass axis has been tripled and mass assignments shown are three times the data-system-assigned values. Values of m/z shown generally fell within l-2Da of reference spectrum values. Inaccuracies arise from a digitization rate effectively at only one third of the usual value and from mass shifts that are observed when axial modulation is used.
Ion trap mass spectra of standarch
The spectra for the alkyl benzenes are consistent under high mass conditions with the spectra obtained in the normal mode of operation. The only significant ions below m/z 120 for alkyl benzenes are those of m/z 91. The triazine mass spectra, obtained using an ionization cut-off mass of 30, were similar to those obtained at 70 eV on the sector instrument, with very intense ions for loss of F’ (m/z 1166 and 1466 for TPFHT and TPFNT respectively) and cleavage at the a and fl positions of the side chain being observed (m/z 866 and 1066 respectively, Fig. 6). Storage eficiency
curves
With these results in place, we were ready to investigate the storage efficiency of high mass ions. The r.f.-voltage level at ionization was varied such that the corresponding cut-off mass was between 10 and 40, the stable area of trap operation for the alkyl benzenes. The two triazines were mixed equimolar with n-propyl and n-decyl benzenes. The ql values of several ions representative of these com-
m/z 91
120
218
866
1166
1466
0.098 0.197 0.295 0.394
0.074 0.149 0.224 0.299
0.041 0.082 0.123 0.164
0.010 0.021 0.031 0.041
0.008 0.015 0.023 0.030
0.006 0.012 0.018 0.024
"The valuein parentheses is that of the corresponding voltage (V,,).
r.f.-
ponents, at the cut-off mass (and corresponding r.f.-voltage) present at the ionization event, are listed in Table 2. In the GC-full-scan MS mode, capillary GC peak areas reflect the total ion current at the detector. The current from individual ions can be evaluated post-run in reconstructed ion chromatograms. It is instructive to inspect the capillary GC peak areas for the triazines relative to the alkyl benzenes, when triazine high mass ions are efficiently trapped. One might expect the ionization crosssections to be slightly higher for the triazines, due to the presence of not only a A cloud of six electrons, but three lone pairs on each of the nitrogen atoms as well. Indeed, at an ionization level corresponding to a cut-off mass of 30, the capillary peak areas for the triazines are greater than for the equimolar alkyl benzenes (Fig. 7a). It is important to note here that this result established a benchmark for maximum storage efficiency. Since the obtained triazine mass spectra are similar to those on a beam instrument (BE configuration) and the GC peak areas are even greater than those for the low mass alkyl benzenes (efficient ion storage presumed) we conclude that the storage efficiencies for high mass ions can be on the order of those for the lower mass ions in the spectrum. At a cut-off mass of 10 at ionization, there is a large decline in the total ion current (and therefore the GC peak area) for the triazines while the alkyl benzene peak areas are almost unchanged (Fig. 7b).
1
R.J. Strife, J.R. Simms/Int. J. Mass Spectrom. Ion Processes 124 (1993) I-10 ION STORAGE ON STRETCHED TRAP
1 :oo
TIME (minsec)
I jooJ,, , ,
5:51
Fig. 7. Comparison of combined capillary GC-MS analysis of the compounds listed in Fig. 3, using cut-off masses at ionization of 30 (upper trace, (a)) versus 10 (lower trace, (b)). Only the triazine GC peak areas decrease because of lowered contribution to the total ion current by their high mass ion (see Fig. 3 for key to identities of peaks).
In Fig. 8, it can be se I that the ion current for m/z 1466 is falling rapidly while that for m/z 91 is maintained, at r.f.-voltages of lOO-2OOV,,. The decrease in the observed GC peak area is due to decreasing storage of the high mass ions, which :arry a substantial portion of the total ion current. The results become even clearer, when the GC peak areas associated with m/z 866, 1166, and 1466 ure plotted as a function of their qz values at ionization, over a range of several r.f.-voltages. As DECYL BENZENE
[
I
2
Be
‘!i:
~~-p’“’
40
&TPFNT
20 I
/
(l%,
&l-OFF MA&i (rf VOLTAGE)
;0 (465)
Fig. 8. Plot of percent of maximum GC peak area (based on ion current for a given mass) for m/z 91 of decyl benzene and m/z 1466 for TPFNT as a function of the cut-off mass at ionization and the corresponding r.f.-voltage. Data points represent the arverageof three replicate runs.
0.01
0.02
0.03
0.04
0.05
1 0.06
42 Fig. 9. Plots of percent of maximum GC peak area observed vs. qZat ionization for three principal high mass ions from the triazines. Three replicate analyses were carried out at each ionization level.
qz approaches a value around 0.02-0.03, the peak areas begin to decline rapidly for the triazine high mass ions (Fig. 9). Cooling time During review of this work, the question of cooling time after ionization was raised, since we had originally investigated only out to 5ms, without seeing significant change in our results from 2 ms. Triplicate GC-MS runs were performed with cooling periods of 5,50, 100, 150 and 200 ms post-ionization. As a control, ionization at a cut-off mass of 40 (relatively efficient storage of all ions) was first examined, with a varied post-ionization cooling period. Out to 2OOms, the ion populations fell steadily for m/z 91, 866, 1066 and 1466, to between 30 and 50% of their initial values. It was surprising to find the population of m/z 1166 rather stable over this period. Next, the cut-off mass at ionization was lowered to 15, and the same experiment was carried out (Fig. 10). The results for m/z 91,866 and 1066 were similar to those obtained at a cut-off mass of 40, falling to between 35 and 45% of their initial values, at the 2OOms time point. However, the
8
R.J. Strife, J.R. Simmsjlnt. J, Mass Spectrom. Ion Processes 124 (1993) I-IO
POST-IONIZATION COOLING EFFECT ON VARIOUS IONS
a”
0
100 POST-IONIZATION
200
COOLING (ms)
Fig. 10. Normalized relative intensities (based on GC peak area) for various ions as a function of post ionization cooling time (at a cut-off mass of 40), when a cut-off mass of 15 is used at ionization. The m/z value for eachcurve is shown and Cl0 refers to the total ion current (due mainly to m/z 91) from n-decyl benzene.
results for m/z 1166 and 1466 were directionally opposite. The ion populations were able to recover to 56% and 41% of their maximum values (i.e., observed at cut-off mass 40), using 200ms of cooling. However, the graph shows that this recovery reaches a plateau at this point. In considering an explanation for the results, the following sequence of events must be considered: (i) the neutral compounds must be uniformly delivered by the GC; (ii) ions must be formed by electron ionization with a certain efficiency; (iii) ions must be trapped and collisionally focussed down to the center area of the electrodes; (iv) ions must be ejected along the z-axis during the voltage ramp; (v) ions must pass through the perforated endcap and strike the conversion dynode, producing a response. Factors (i), (ii) and (iv) are controlled by the experimental design. The alkyl benzenes verify the chromatographic performance and delivery of the triazines during the experiment, because their retention times bracket those of the triazines. Also,
the areas of the alkyl benzenes remain the same while those for the triazines are rapidly falling. The ionization efficiencies have not changed greatly at various r.f.-voltage levels because alkyl benzene peak areas remain relatively constant in control experiments at different r.f.-voltage levels. Also, the triazine ions are always ejected at qz = 0.3, regardless of their initial qz value, thus striking the conversion dynode with the same momentum, kinetic energy and response factor. Thus factors (iii) and (v) need to be focussed on. Ions of low qz values are very close to the axial and radial stability limits for ion trajectories (Fig. 1). Moreover, the diagram in Fig. 1 reflects conditions for a perfect electrode. Therefore it is possible that low qz value ions are lost in unstable trajectories before the detection step of the experiment. Also, it has been suggested to us that ions of these low qz values may be approaching the physical boundaries of the trap. That is, the ion trajectories may be stable but exceeding the physical bounds of the electrode structure. We are investigating this possibility further. A third factor seems to be that ion losses work against the recovery of ions by collisional cooling and a practical limit is reached at 200 ms (He gas). Also, a decrease in the capillary GC peak area of a triazine may be due to the fact that the ion trajectory has not been damped enough. That is, when the condition for axial ejection is reached, the ions may be trapped, but they are not yet at the center of the trap. The ions do not exit along the z-axis through the perforated endcap and miss striking the conversion dynode. Implications These results show the importance of longcollisional cooling periods for high mass ions using He as a buffer gas. They also show that a minimum q2 value of 0.02 should be used to obtain efficient storage of ions between m/z 1000 and 1500. In a recent study [l 11, efficient trapping of injected CsI cluster ions was reported. However, this term was used only in a relative sense, in comparing trapping
R.J. Strife, J.R. Simms~Int. J. Mars Spectrom. ion Processes 124 (1993) I-10
of high mass ions using various bath gases (He compared to He with up to 5% of heavier noble gases). The actual trapping efficiency of the ions is unknown and it is interesting that a minimum qz of 0.032 was chosen for injection [12]. Previous reports _urion injection have revealed long integration times (minutes) to obtain extendedmass-range spectra. Here such spectra are obtained on 1 pm01 quantities using only 0.1 ms of ionization, in less than 1 s. Thus it is possible that there are substantial losses in ion injection. Comparisons of ion current initially generated by the two methods versus those finally detected could be useful. Nevertheless, the mass ranges and practical operating sensitivity demonstrated in peptide analyses by ion injection have been impressive, in our opinion. In this study, the results suggest that the trapping efficiency of high mass ions formed in the field approaches that of relatively low mass alkyl benzene ions formed in the field under some conditions. This hypothesis is based on the spectral comparison between the trap and beam instruments as well as the nearly equal capillary GC peak areas for triazines versus equimolar alkyl benzenes. The number of lions lost in the initial stages of any electron ionlization experiment is unknown to us, so that our rabsolute trapping efficiencies are also unknown. Another important result is found in the triazine spectra recorded at cooling times on the order of 100 ms. Ion/molecule reactions become significant and produce peaks at M + 69, no doubt from reaction of the CF: cation with neutral triazine molecules. If long cooling times are required, then spectral artifacts needing interpretation may occur. Also, the long cooling times may begin to broach the time domain needed for definition of very narrow chromatographic peak shapes as in capillary zone electrophoresis of proteins, if one assumes 8-10 scans are necessary for peak definition. Finally, in regard to injecting high mass ions formed externally, a calculation of the Dehmelt pseudopotential well-depth for an ion of m/z 4000, at a qz value of 0.02, shows that the corresponding cut-off mass is 88 (1103 V,,). The pseudopotential well depth is rather shallow, only 2.8eV on our
9
system under these conditions. If our hypothesis is corrrect, it is easy to see that the observed storage efficiency of the trap could be a complicating factor during injection of high mass ions from external sources. Conclusions The method described here allows for the first time a precise determination of apparent storage efficiencies of high mass ions relative to low mass ions. The low mass ions are presumed to be efficiently trapped and damped to the center of the trap but their absolute storage efficiency is unknown. Storage of high mass ions does not appear to be very efficient on the ion trap mass spectrometer when qz values fall below 0.02, using helium as a buffer gas. Given the recent results which noted deleterious scattering effects of heavier buffer gases, the prospect of extended-mass-range work on an ion trap mass spectrometer needs to be approached with guarded optimism. It is our opinion that further developments in the computerized modelling of ion trajectories, in the presence of various bath gases, will be valuable in further understanding ion trap behavior for ions of low qz value, near the stability limits of the storage diagram. Acknowledgment We thank Professor Ray March, Trent University, for helpful discussions and suggestions during the course of this investigation. References K.A. Cox, R.E. Kaiser Jr. and R.G. Cooks, Use of a Quadrupole Ion Trap Mass Spectrometer for Structural Determination of Biomolecules, Proc. 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June, 1990. R.F. Wuerker, H. Shelton and R.V. Langmuir, J. Appl. Phys., 30 (1959) 342. J.F.J. Todd, A. Penman and R.D. Smith, Int. J. Mass Spectrom. Ion Processes, 106 (1991) 117-135. R.D. Nourse and R.G. Cooks, Anal. Chim. Acta, 228 (1990) I-21.
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R.J. Strijie, J.R. Shuns/hi.
R.E. Kaiser, J.N. Louris, J.W. Amy and R.G. Cooks, Rapid Commun. Mass Spectrom, 3 (1989) 225-230. J.E. Fulford and R.E. March, Int. J. Mass Spectrom. Ion Phys., 30 (1979) 39-55. R.E. March and R.J. Hughes, Quadrupole Storage Mass Spectrometry, Chemical Analysis Series, Vol. 102, Wiley, New York, 1989, pp. 196-201. J. Louris, G. Stafford, J. Syka and D. Taylor, The Paul Ion Trap Mass Selective Instability Scan: Trap Geometry and Resolution, Proc. 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington D.C., May 31-
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J. Mass Spectrom.
Ion Processes 124 (1993) I-10
June 5, 1992. R.J. Strife and J.R. Simms, J. Am. Sot. Mass. Spcctrom., l(l990) 265-271. Fisons Instruments, 32 Commerce Center, Danvers, MA 01923. K.L. Morand, K. Cox and R.G. Cooks, Rapid Commun. Mass Spectrom., 6 (1992) 520-523. J.R. Simms and J.R. Strife, Relative Ion Trap Storage Efficiencies of Ions Up to m/z 1500 by Capillary GC/MS; Proc. 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 19-24, 1991.