Anal. Chem. lQ82, 5 4 , 2225-2228
2225
Consecutive Collision- Induced Dissociations in Fourier Transform Mass Spectrometry R. B. Cody, R. C. Burnler, C. J. Cassady, and B. S. Frelser” Department of Chernistty, Purdue University, West Lafayette, Indiana 47907
Consecutlve collision-induced dissociatlonci may be observed in a Fourler transform mass spectrometer (FTMS). No hardware modlficationr are necessary. The same pulse sequence used for regular C I D experlmenls can be employed to observe consecutlve C I D reactions in an MS/MS/MS experiment If one of the double resonance pulses normally used for ion ejection Is Instead employed for C I D acceleration. Examples of MS/MS/MS spectra obtained for both primary Ions and the products of ion-molecule reactions are given. “Uncontrolled” CXD may occur If the bandlwldth of the swept double resonance pulse used for daughter ion ejection overlaps the resonant frequency of the parent ion. Since the bandwidth for an RF pulse Is llmiled at short tlmes by the uncertainty prlnclple, thls Is most likely tal present problems when a double resonance pulse Is swept riapldiy over a range of frequencies. Am example of such uncontrolled C I D Is seen in the background spectrum for one aif the MS/MS/MS spectra.
Interest in Fourier transform mass spectrometry (FTMS) has been growing rapidly since the technique was first introduced by Connisarow in 1974 ( I ) . The capabilities of Fourier transform mas8 spectrometers Enclude ultra high resolution, exact mass determination, signal averaging, and simultaneous detection of all ions present m a mass spectrum ( 2 , 3 ) . These factors, along with the introduction of a commercially available spectrometer by Nicolet and the development of techniques such as low-pressure chemical ionization (4-6)and laser desorption of involatile samples (7,8), have combined to increase the attractiveness of FTMS to analytical chemists. Recently, we reported the observation of collision-induced dissociation (CID) in a Fourier transform mass spectrometer (9, JO),and have applied the CID technique to studies of proton-bound and metal ion bound alcohol dimers (11). A comparition study indicates that FTMS compares favorably with the triple quadrupole technique for obtaining low-energy CID spectra (12). In addition, we have shown that high-resolution detection of CID fragment ions is possible by FTMS and can ble used to separate isobaric ions and obtain elemental compositions (13). FTMS differs from all other methods for observing collision-induced dissociations in that a tandem instrument is not required. Ionization, mass selection, ion acceleration, collisiori with the target gm, and fragment ion detection all occur within the trapping cell. Software modifications, rather than hardware modifications (i.e., additional sectors), are employed to obtain CID spectra; the only change made in the experimental pulse sequence is the addition of an ion acceleration pulse (irradiation at the appropriate cyclotron resonance frequency) and a suitable delay time to allow collisions with the target gas. This fact suggests that the FTMS technique might be easily extended to observe consecutive collision-induced dissociations by further accelerating CID product ions and observing their fragmentation after collision with the target gas (14). This scheme is shown in reaction 1.
ml+
- N
m2+
N
mg+
Such consecutive reactions can be observed in two analyzer mass spectrometers (15-1 71, but the approach is subject to artifacts. Recently, the use of a triple analyzer mass spectrometer to observe consecutive CID reactions was reported (18,19). This approach was shown to be free of artifacts and was demonstrated to be useful for certain cases of complex mixture analysis. In particular, structural information for ions from a complex mixture was obtained in the presence of isobaric and isomeric ionic contaminants (18). Structural information for large molecules which give one dominant loss process can be increased by submitting the dominant collision fragment to a second CID stage (18). In this paper we demonstrate that spectra due to consecutive collision-induced dissociations may be easily observed in a Fourier transform mass spectrometer. These spectra, which may be termed “MS/MS/MS” spectra, can be obtained for both primary ions and products of ion-molecule reactions by making use of the pulse sequence available in the Nicolet FTMS software package without modification. In addition, a significant portion of the discussion is devoted to a problem with uncontrolled CID resulting from the broad bandwidth of the ejection pulse used to clear the cell of unwanted ions prior to CID acceleration of ions of the selected mass. This phenomenon is illustrated in the background spectrum for one of the “MS/MS/MS” experiments.
EXPERIMENTAL SECTION The methods and experimental conditions for obtaining CID spectra in the FTMS have previously been described in detail (9, 10). The pulse sequence employed is reproduced in Figure 1. For these experiments, the DR1 double resonance pulse was employed to eject unwanted ions before CID and the DR2 and DR3pulses were used to accelerate ions for the two CID stages. Since the current FTMS operating software limits the DR2pu;lse amplitude to the same RF level as DR1, ejection of ions during DR2was avoided either by keeping the pulse duration very short ( 1/(2?r A t )
MASS (amul
(2)
in the output frequency is greatest; hence the narrowest bandwidths will be attained with long pulses. From eq 2, we see that an uncertainty of 159 kHz will exist for a 1~s pulse, while a 1 ms pulse will only have an uncertainty of 159 Hz. In the FTMS, a broad bandwidth may create problems if the frequency “tail” of a double resonance pulse intended to excite or eject a given ion overlaps the resonance frequency of another ion, which can then absorb energy and be translationally accelerated (this is the reason ions can be irradiated “offresonance”) (9, IO). Although this limits “fronbend” resolution (resolution available for selecting the parent ion for which the CID spectrum is to be obtained), our experience to date suggests that unit “front-end” resolution is possible with the careful use of low RF levels and long excitation times (a few milliseconds). A much more difficult situation is evident in Figure 2a. Here we see the mass spectrum taken for a mixture of acetophenone a t 2.4 X lo-’ torr and argon a t 1.1 X torr. A swept double resonance pulse was employed to eject all of the daughter ions produced during the formation pulse. Despite ejection, a substantial peak at m / z 105 (loss of CW3.) appears along with the expected peak for the molecular ion at m / z 120. This peak is not the result of the inefficient or incomplete ejection. Instead, the broad bandwidth of the ejection pulse overlaps the resonance frequency of the molecular ion, which absorbs energy and is accelerated into the collision gas. This causes the molecular ion to undergo “uncontrolled” CID prematurely, which regenerates the fragment ions being ejected. Evidence for this phenomenon is found in the fact that these peaks are missing when an identical ejection pulse is applied in the absence of collision gas. Furthermore, increasing the CID interaction time increases the relative abundance of these fragments by allowing more time for collisions to occur in the same fashion as observed for “controlled” CID. Lastly, increasing the ejection R F level increases the fragment abundance rather than decreasing it. The nature of the swept ejection pulse, with necessarily high R F level and a short time for irradiating a t each individual resonance frequency, virtually ensures that a broad bandwidth be associated with the pulse. From eq 2, we can calculate that the uncertainty for a typical pulse (40 Hz/ps) is 6.4 MHz! Such uncontrolled CID is clearly undesirable and can often be limited by using a slow DR, pulse to eject the majority of fragment ions over the mass range where CID fragments are expected to occur. A low level DR2 pulse is then employed
111
M A S S lomu1
Figure 2. (a)Background spectrum for the acetophenone MS/MS/MS experiment. The DR, pulse is swept from 129.5 kHz to 216 kHz in 2.9 ms to eject fragment ions produced during the 10.6-eV formation pulse. The peak at m l z 105 arises from “uncontrolled“CID of the molecular ion (see text). (b) Same conditions as Figure 2a, with the exception that the molecular ion is irradiated with the DR, pulse and undergoes CID to increase the relative abundance of m l r 105. (c) Same conditions as 2b, but now the ions of m l z 105 produced by the first CID stage are accelerated by the DR, pulse and undergo CID to produce a peak at m / z 77. to eject the ions resulting from “uncontrolled” CID. In order to observe consecutive CID reactions without modifying the operating system, it was necessary to use the DR2 pulse as a CID pulse, eliminating the above (two pulse) method. Although it was not employed in this study, background subtraction is another approach to obtaining CID spectra in the FTMS that would avoid this problem entirely. The remarkable efficiency of the CID process for acetophenone is shown in Figure 2b, where the DR, pulse was added to accelerate the molecular ion to 11.6 eV. After a 10 ms delay, virtually all (-93%) of the C6H5COCH,+. ( m / z 120) undergoes CID and loses CH3-to form C&$O+ ( m / z 105). Now, the DR3 pulse is added to accelerate the C6H5CO+produced by the first CID reaction to 13.0 eV. After another 10 ms delay, the mass spectrum shown in Figure 2c was taken. The peak at m / z 77 and the decrease in the peak at m/z 105 clearly show the final step in the reaction sequence: CeHSCOCHs’. C&,CO+
N
C&bCO+ 4- CH,.
5C & j + 4- co
(3)
(4) The lowered efficiency (-17%) for this second CID reaction is due to ion ejection. Unlike primary ions, which are formed in the center of the cell by the electron beam, ion-molecule
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
C H
80
IW
00
Ir
NOi-
7 7
j
,eo
l
4Wm.J
50
I40
Flgure 3. MSIMSJMS spectrum for p -nitrotoluene. The ionizing electron energy is 10 eV. No ion ejection pulse was necessary, Since no fragment Ions are formeid at this energy.
products are formed throughout the cell. Some, being closer to the cell plates, are ejected rather than undergoing CID when the acceleration pulse causes an increase in the radius of their orbit. It is of interest to note that the isobaric ion CSHg+, produced by CII) of the cumene molecular ion (C&&H(CH3)2+*,m/z 120) will not undergo a second stage CID reaction within the kinetic energy range available in our instrument. Figure 3 shows a second stage CID spectrum (analogous to Figure 2c) taken for p-nitrotoluene. At the electron energy used for ionization (10 eV), the molecular ion (mlz 137) is the only ion appearing in the background mass spectrum (without CID); hence, no double resonance ejection pulse was necessay. The first stage CID reaction (75% efficient),shown in reaction 5, resulted from acceleration of the molecular ion to 6.0 eV, followed by a 20 ms delay. Acceleration of the resulting
(;7H7oC
N
C7H70’
+ NO.
-% C,jH7’ + co
(5) (6)
C&@+ -% C&5+ H&O (7) C7H70+to 20.1 eY, followed by a 40 ms delay, produced the peaks at m / z 79 (reaction 6) and m / z 77 (reaction 7). The efficiency of this last stage is somewhat greater than in the previous example, but still shows a decrease from the first stage efficiency. This decrease is again attributed to ion ejection. The products of ion-molecule reactions may also be examined by MS/MS/MS. One example is &own in the secondstage CID spectrum shown in Figure 4. Here the acetylation product ( m / z 14!$ resulking from a reaction of protonated acetic anhydride with its parent neutral (reaction 8) undergoes C4H703’
-
+ C4H603
C6Hlo04+4- CH3COOH (8)
CID and loses ketene (CH2CO)to produce C4H703’ (mle 103). This is shown in ireactioin 9. The resulting C4H703’ is then C6HIoO4+
IV
C4H703’
+ CHzCO
(9) accelerated and undergoles CID to produce a peak at m / z 43, corresponding to CH,CO+ (reaction 10). Again, a decrease +
70
90
I10
190
150
170
180
M A S S (ornu)
MASS (amul
C7H7N02’.
2227
C4H703+-% CH3CO+ + CHaCOOH (10) in efficiency from 43% (first stage) to 20% (second stage) is seen due to ion ejection. The smaller first stage efficiency (compared to the previous examples) exists for the same reason. One final example of an MS/MS/MS spectrum for an ion-molecule reaction product was obtained with isopropenyl
Figure 4. MS/MS/MS spectrum for the product of reaction 8. The ions at m / r 103, produced by first stage CID, undergo second stage CID to produce the ions at m / z 43. The unlabeled Ions are ionmolecule reaction products that were not ejected due to the limited number of pulses available wkh current software. The relative intensity of these ions did not change with CID of C6H,,O4” and C4H7O3+.
acetate. The product ion at m / z 143 corresponding to acetylated isopropenyl acetate was formed by a sequence of reactions analogous to that in the previous example. The first CID stage gives loss of ketene with 45% efficiency to produce the protonated ester at m / z 101. The second CID stage produces CH3CO+with 22% efficiency by loss of acetone. This fragmentation scheme is almost identical with that obtained with acetic anhydride and suggests that it will hold for esters in general. The efficiency in all of these examples could be improved considerably by making use of a larger cell and/or a larger magnetic field. A larger cell would allow larger ionic orbits to be achieved before ejection could occur. This increases the maximum attainable energy for each CID stage (9,lO). We are currently using a l-in.3 cell. A 3-in.3cell presently under construction should allow us to attain a 9-fold increase in kinetic energy for a single stage CID experiment. The efficiency of the second CID stage in MS/MS/MS experiments should also be greatly improved. In addition, the possibility of adding further CID stages to MS/MS/MS arises, making possible an “(MS)””experiment, with the maximum number of CID stages, n, limited by the competition between dissociation and ejection at the last stage. The higher magnetic fields attained by superconducting magnets should also improve efficiency by constraining the ions to move in tighter orbits. High-resolution detection of the CID products at each stage is possible at low enough pressures (13). It should be emphasized that this is high “back-end” or “MS-11”resolution, i.e., the resolution available for daughter ion analysis, and not “front-end” or “MS-I” resolution, which is defined as the resolution available for parent ion selection. The limitations on “front-end’’ resolution in the FTMS have not been fully evaluated yet. It is obvious, however, that the use of a very short acceleration pulse can limit “front-end’’resolution. This is evident from the appearance of a peak at m/z 108 in Figure 2b, due to CID of m / z 121. From eq 2, we can see that the 1OO-jm pulse used to irradiate mlz 120 has a bandwidth >1591 Hz. The resonance frequencies of m / z 120 and m / z 121 are only 977 Hz apart. Our experience has shown that with longer irradiation times, unit “front-end” resolution is possible (10). Despite these limitations, the utility of the MS/MS/MS technique in increasing the amount of information available for compounds such as acetophenone and p-nitrotoluene, which exhibit limited CID fragmentation, should be apparent. Information about fragmentation pathways and CID fragment ion structure is available from MS/MS/MS spectra, provided
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Anal. Chem.
1982,5 4 , 2228-2233
the fragments do not rearrange during the CID interaction time* The to examine the MS/MS/MS behavior Of ion-molecule reaction products could be useful in chemical ionization studies.
ACKNOWLEDGMENT The authors wish to thank the members of the Nicolet Instrument Co. Mass Spec. Group, and in particular R. B. Spencer, for their technical assistance. LITERATURE CITED (1) Comlsarow, M. 6.; Marshall, A. G. Chem. Phys. Lett. 1974, 25,282. (2) Parisod, G.; Comisarow, M. B. Adv. Mass Spectrom. 1980, 8 , 218. (3) Ledford, E. B., Jr.; Ghaderi, S.;White, R. L.; Spencer, R. B.; Kulkarni, P. S.;Wilkins, C. L.; Gross, M. L. Anal. Chem. 1980, 52, 463-468. (4) Hunter, R. L.; McIver, R. T. Anal. Chem. 1979, 5 1 , 699-704. (5) Ghaderi, S.; Kulkarni, P. S.; Ledford, E. B., Jr.; Wllkins, C. L.; Gross, M. L. Anal. Chem. 1981, 53,428-437. (6) Burnier, R. C.; Byrd, G. D.; Freiser, B. S. Anal. Chem. 1980, 52, 1841-1650. (7) Cody, R. 6.; Burnier, R. C.; Reents, W. D., Jr.; Carlin, T. J. McCrery, D. A.; Lengel, R. K.; Freiser, 8 . S. Int. J . Mass Spectrom. Ion Phys. 1980, 33,37-43. (8) McCrery, D. A.; Ledford, E. B., Jr.; Gross, M. L. Anal. Chem. 1982, 54. 1435-1437. (9) Coby, R. 6.; Freiser, B. S.Int. J . Mass Spectrom. Ion Phys. 1982, 4 7, 199-204.
(10) Cody, R. B.; Burnier, R. C.; Freiser, B. S. Anal. Chem. 1982, 54, 96-101. (11) Burnier, R. C.; Cody, R. B.; Freiser, B. S. J . Am. Chem. SOC.,in press. (12) Cody, R. 6.; Burnier, R. C.; Sallans, L.; McLuckey, S.; Verma, S.; Freiser, 8. S.;Cooks, R. G. Int. J . Mass Spectrom. Ion Phys., in press. (13) Cody, R. B.;Freiser, B. S. Anal. Chem. 1982, 5 4 , 1431-1433. (14) McIver, R. T. Workshop on Newer Aspects of Ion Cyclotron Resonance (Fourier Transform Mass Spectrometry), 29th Annual Conference of Mass Spectrometry and Allied Topics, Minneapolis, MN, 1981; D 798. (15) Proctor, C. J.; Brenton, A. G.; Beynon, J. H.; Kralj, B.; Marsel, J. Int. J . Mass Spectrom. Ion Phys. 1980, 35,393-403. (16) Proctor, C. J.; Kralj, B.; Brenton, A. G.; Beynon, J. H. Org. Mass Spectrom. 1980, 75, 619-631. (17) Boyd, R. K.; Shushan, B. Int. J . Mass Spectrom. Ion Phys. 1981, 3 7 , 355-368. (18) Burinsky, D. J.; Cooks, R. G.; Chess, E. K.; Gross, M. L. Anal. Chem. 1982, 5 4 , 295-299. (19) Maquestiau, A.; Meyran, P.; Flammang, R. Org. Mass Spectrom. 1982, 77, 96-101.
RECEIVEDfor review March 23,1982. Accepted July 30, 1982. Acknowledgment is made to the Department of Energy (DE-AC02-80ER10689) for supporting this research and the National Science Foundation (CHE-8002685) for providing funds to purchase the FTMS.
Linear Programmed Thermal Degradation Mass Spectrometry of Polystyrene and Poly(viny1 chloride) T. H. Risby" and J. A. Yergey Division of Environmental Chemistry, Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, 6 15 North Wolfe Street, Baltimore, Maryland 2 1205
J. J. Scocca Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 6 15 North Wolfe Street, Baltimore, Maryland 2 1205
The use of a computer-controlled temperature-programmable platinum filament as a probe for linear programmed thermal degradation mass spectrometry Is reported. A study of time-resolved pyrolysis of various polystyrene samples and 01 poly(vlny1 Chloride) was made: results show a stepwlse thermal degradation process. Energies of activation and preexponentlal factors for these thermal degradations were determined.
Linear programmed thermal degradation mass spectrometry (LPTD-MS) (1,2) as a monitor of thermal decomposition of high molecular weight samples has been used to distinguish between bacterial species and serotypes (3) and between various normal and abnormal human lymphocytes ( 4 ) . However, the fragments that were used to distinguish between specimens could not be chemically characterized; differentiation of samples was based on empirical data. Thermal decomposition as a means of characterizing high molecular weight compounds has been used in pyrolysis gas chromatography (PyGC) and pyrolysis mass spectrometry (PyMS) for a wide range of samples. Whereas PyMS and PyGC involve a single mass spectrum or gas chromatogram obtained after rapid heating of the samples to a fixed tem0003-2700/82/0354-2228$0 1.25/0
perature, LPTD-MS is based on a collection of sequential mass spectra during the programmed heating of the sample. The LPTD-MS data, with temporal dependence, allow detection of subtle differences in structure which would not be apparent with data from PyGC or PyMS. In addition, activation energies and preexponential factors for the decomposition processes can be obtained from LPTD-MS by changing the rate of sample heating; such results cannot be obtained by PyGC or PyMS. The major advance made in this study is the redesign of the temperature programmable probe and controller to allow accurate measurements of decomposition temperature. A similar probe is available commercially (Pyroprobe, Chemical Data Systems) but lacks the capability for computer-controlled variations in heating rates. Our improved design has been evaluated by studying the temperature-resolved pyrolysis of two well-characterized polymers, polystyrene and poly(viny1 chloride). The design of the probe and controller, data on the mechanisms of thermal decomposition, activation energies, and preexponential factors of these polymers are reported in this paper.
EXPERIMENTAL SECTION Apparatus. These studies were performed with a chemical ionization mass spectrometer (Scientific Research Instruments 0 1982 American Chemical Society
