2577
Anal. Chem. 1991, 63, 2577-2580
Continuous-Flow Fast Atom Bombardment and Field Desorption Mass Spectrometry of Oligomers of 3,3,3=Trifluoro- I-phenylpropyne Richard B. van Breemen,*Chien-Hua Huang, and Carl L. Bumgardner Department of Chemistry, Box 8204, North Carolina State University, Raleigh, North Carolina 27695-8204
Fleld derorptlon mass spectrometry was used to determlne the chdn length, mokculsr wdghl range, and klenrmeS of the end-capphg groups of synthetk ollgomen, of 3,3,3-trluorol-phenylpropyne. I n order to obtain structurally slgnlflcant fragment Ions of selected molecular Ion precursors, the MS/MS technique of BE-llnked scannlng wlih collisional actlvatlon (CAD) was used. Because of the short duratlon and low Intendy of the slgnal, no useful MS/MS data could be obtalned using FD as the Ionization method. However, B/€-IWed scans folkwing CAD could be obtained using fast atom b 0 m b a r M (FAB) matis spectrometry. To overcome kn "ruppressbn"of components In the unpurtfled digomerlc mlxture and reduce the chemlcal nolse during FAB, continuous-Mw FAB llquld chromatography/mass spectrometry (LC/MS) was used to obtaln molecular weight Information comparable to fleld desorptlon ma88 spectrometry. Finally, B/€-llnked scannlng wlth CAD was canled out durlng LC/MS In order to obtain fragment Ions confirmlng the presence of phenyl and trmuoromethyl substituents on the polyacetylene chaln.
INTRODUCTION Polyacetylenes and related synthetic polymers such as polyphenylacetylenes have been demonstrated to conduct electric current because of their extended series of conjugated double bonds (I). Potential applications of these polymers include use as organic semiconductors or use as components of electrolytic capacitors or rechargeable batteries (2). In order to determine the factors that influence the conductivity of these polymers, accurate methods are needed to measure their chain length, molecular weight, and structure, including defects in the polymeric chain. In studies of polyanilines (31, gel permeation chromatography was used to show a bimodal distribution of molecular weights that consisted of a light fraction weighing approximately 4800 and a heavy fraction weighing between 200 000 and 350000. However, this method provided no information regarding the structure of the polymer such as the mass of the monomeric unit, sites of defects in the chain, or the nature of the end-cappinggroups. For the determination of molecular weight range, structure of the repeating unit, and identity of the capping groups that terminate the polymeric chains, field desorption (FD) mass spectrometry has been used to analyze oligomers and low molecular weight polymers such as poly(ethylenimine) ( 4 ) and polybutadiene (5). A newer technique than FD, fast atom bombardment (FAB) mass spectrometry has been applied to the analysis of a variety of compounds including biological polymers such as peptides (6) and some synthetic organic oligomers and polymers (4). During FAB, sample ions are desorbed into the gas phase from a liquid matrix of low volatility as a result of bombardment
* Corresponding author. 0003-2700/91/0363-2577$02.50/0
by a beam of energetic atoms (7).Although FD can generate molecular ions with relatively low background noise, the signal is transient and typically lasts only seconds to a few minutes at most. Compared to FD, FAB ionization has the advantage of generating ions over a longer period of time, which can extend from a few minutes to 1 h or more. Prolonged ionization facilitates collisional activation of selected-ion precursors and then structural analysis of the product ions by using MS/MS. Recently, FAB mass spectrometry has been combined online with reversed-phase HPLC in a system called continuous-flow FAB mass spectrometry. Continuous-flow FAB mass spectrometry has been applied to the analysis of a variety of compounds including peptides (8),oligonucleotides (9),and chlorophylls (IO). However, no continuous-flow FAB mass spectrometric analyses of polyacetylenes or other conductive polymers have been reported. In this study, a frit-FAB version of continuous-flow FAB mass spectrometry will be used. During continuous-flow FAB using a frit, the HPLC mobile phase is pumped through a fused-silica capillary and then through a stainless steel frit located inside the ion source of the mass spectrometer (11). The fast atom beam is focused onto the opposite side of the frit from the capillary, so that sample ions are desorbed into the gas phase as they flow through the frit. The HPLC solvent rapidly evaporates and is pumped away by the vacuum pumps of the mass spectrometer. As part of our studies on the free radical reactions of fluorinated alkynes and related compounds, we have been investigating the free radical polymerization of 3,3,3-trifluoro-1-phenylpropyneto form a fluorinated polyacetylene. Although fluorinated polyacetylenes lack heteroatoms that might serve as sites of protonation or deprotonation, which are common ionization pathways in FAB mass spectrometry, molecular ions, Me+,can be formed during FAB, especially if the analyte contains delocalized T electrons. We report here the analysis of oligomers of 3,3,3-trifluoro-l-phenylpropyne (1) using continuous-flowFAB liquid chromatography-mass spectrometry (LC/MS) and, for comparison, FD mass spectrometry.
Q-c = c- c F3 1
EXPERIMENTAL SECTION The monomer, 3,3,3-trifluoro-l-phenylpropyne(l),was synthesized according to the method of Bumgardner and Bunch (12) and purified by distillation under reduced pressure. Oligomerization was carried out without solvent in a sealed vial at 90 'C for 72 h using benzoyl peroxide as the free radical initiator. The molar ratio of monomer to benzoyl peroxide was 10:l. The products of the reaction were dissolved in methylene chloride and diluted to a final concentration of approximately 1pg/pL. All analyses were carried out using aliquots of this stock solution. Further details of the chemistry of the polymerization of 1 and related acetylene derivatives are presented elsewhere (13). 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63,NO. 22, NOVEMBER 15, 1991
HPLC sepmtion of oligomers was carried out using an Applied Biosystems (Foster City, CA) model 140A dual-syringe solvent delivery system, which had been modified so that the dynamic mixer was replaced with a 'T" union to minimize dead volume. The HPLC system was equipped with a Rheodyne (Cotati, CA) Model 8125 injector and Vydac (Hesperia, CA) CISnarrow-bore column (15 cm X 2.1 mm) packed with 300 A pore size, 5 pm diameter silica particles. Oligomers were eluted from the column using a 20-min gradient from 50:50:0:0.25 (v/v/v/w) to 103060:0.25 water/methenol/ethyl acetate/3-nitrobenzyl alcohol. The solvent flow rate was 70 pL/min, and 20 pL was injected onto the reversed-phase column per analysis. Positive-ion FAB mass spectra were obtained using a JEOL (Tokyo,Japan) JMS-HX11OHF double-focusingmass spectrometer equipped with a JMA-DA5000 data system and continuous-flow FAB interface. Xenon fast atoms at 6 kV were used for FAB ionization. For standard probe FAB mass spectrometry, 2 fig of the oligomericmixture in methylene chloride was loaded onto 1 fiL of the FAB matrix, 3-nitrobenzyl alcohol. For continuous-flow FAB mass spectrometry, 3-nitrobenzyl alcohol in the mobile phase functioned as the matrix. The accelerating voltage was 10 keV, and the resolving power was lo00 for all FD and low-resolution FAB measurements. Exact mass measurements were carried out using FAB mass spectrometry at a resolving power of 1Oo00. The ion source was maintained at a temperature of 46 O C . The range m/z 10-1500 was scanned over approximately 10 s. For compatibility with the vacuum system of the mass spectrometer, the HPLC column eluate was split so that approximately 5 pllmin entered the continuous-flowFAB interface. At a column flow rate of 70 fiL/min, this resulted in a split ratio of 1:14. For analysis using LC/MS, 20 fiL of a 1pg/fiL solution of the oligomeric mixture in methylene chloride was injected onto the HPLC column. Because the column eluant was split, approximately 1.4 pg of the oligomers was analyzed by continuous-flow FAB mass spectrometry. Under controlled-pressureconditions, acetone was introduced into the FD ion source using the reservoir inlet. The FD ion source, equipped with a silicon emitter, was tuned while monitoring the acetone signal at m / z 58. For each analysis using FD mass spectrometry, approximately 2 fig of the oligomeric mixture was loaded onto the silicon emitter from a microsyringe. Silicon emitters were used because they are more durable and less expensive than carbon emitters, although carbon emitters can produce a more abundant ion current for some compounds. However, Schulten and Lattimer reported that there is no particular advantage to using silicon emitters (14). Using a cathode potential of -1.2 kV and emitter and acceleratingvoltage of +10 kV, FD mass spectra were recorded continuously over the range of emitter currents from 10 to 35 mA. The higher molecular weight oligomers desorbed at higher emitter currents. These spectra were summed to produce a single mass spectrum representingthe entire mass range of oligomers. The sensitivities of FD and static FAB maas spectrometrywere compared for the analysis of the oligomer detected at m / z 834 in 2 pg of the unpurified oligomeric mixture and 2.7 X A/pg, respectively. and were 1.4 X MS/MS analyses were carried out using B/E-linked scanning and collisional activation (CAD). Fragmentation of the precursor ion was enhanced by CAD using helium gas in the first field-free region of the double-focusingmass spectrometer. The helium gas pressure was adjusted so that the abundance of the selected-ion precursor was attenuated 70%.
RESULTS AND DISCUSSION After polymerization, a 2-pg aliquot of the unpurified reaction product mixture in methylene chloride was analyzed using FD mass spectrometry to determine the extent of polymerization and the molecular weights of the products. This FD mass spectrum is shown in Figure 1. Molecular ions, M + , of the different oligomers were detected over a mass range extending from the cyclic dimer a t m / z 416 to the linear octamer at m/z 1514. Three series of oligomers were detected, one containing the capping groups -H and -C6H5 (series A), another containing two phenyl capping groups (series B), and a less abundant third series consisting of two hydrogen capping groups (series C) (Figure 2). Ions within each series differed
m/Z
Figure 1. Positive-ion field desorption (FD) mass spectrum of the unpurified oligomeric products following polymerization of 3,3,3-trC flwro-lphenylpropyne (1). Approxknately 2 pg of the reactkn product mixture was used In the analysis. The structues of the kns designated A, 8, and C are shown In Figure 2.
Series A R=H , R'=Ph Series B R=R'=Ph Series C R=R'=H Figure 2. Structuresof the three series of oligomers formed by free radical polymerization of 3,3,3-trlfluoro-l-phenylpropyne (1). in mass by multiples of 170 mass units, which corresponded to the molecular weight of the expected monomeric unit, -(CF3)C=C(C6H5)-. The ion a t m J t 416 was probably an A-series dimer that had undergone cyclization with loss of a hydrogen atom. The structure of the ion at m / z 416 was investigated more rigorously using FAB mass spectrometry as described below. The corresponding linear dimer was detected a t m / z 418, although at lower relative abundance. Because the FD mass spectrum contained primarily molecular ions, B/E-linked scanning of a selected precursor ion was carried out following CAD in order to obtain structurally significant fragment ions. The most abundant ion in the FD mass spectrum, m / z 416, was selected as the initial molecular ion precursor. Because the number of ions formed during FD mass spectrometry of each oligomer in the mixture was low and the duration of ionization for each oligomer was less than approximately 30 s, the signal-to-noise ratio of fragment ions in the BJE-linked scan was not adequate for the recording of an MS/MS spectrum. For the same reason, exact mass measurements at high resolution are difficult to obtain using field desorption. In order to generate more abundant molecular ions over a longer period of time for MSJMS analysis, FAB ionization was investigated as an alternative to FD. The unpurified mixture of polyacetylene oligomers was analyzed by positive-ion FAB mass spectrometry using either glycerol, thioglycerol, or 3-nitrobenzyl alcohol as the matrix. No molecular ion species were observed using glycerol or thioglycerol. However, using a matrix of 3-nitrobenzyl alcohol, M'+ ions were detected at m / z 664,834, and 1004, which corresponded to trimers, tetramers, and pentamers in the B series (Figure 3). Oligomers in the B series were less polar than those in the A series because both end-capping groups were phenyl
ANALYTICAL CHEMISTRY, VOL. 63,NO. 22, NOVEMBER 15, 1991
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groups instead of one phenyl and one hydrogen. In the analysis of peptide mixtures by FAB mass spectrometry, hydrophobic peptides in the mixture are usually detected in much greater abundance relative to the more polar peptides (15,16). Similar surface activity properties probably facilitated the selective ionization of the more hydrophobic polyacetylenes. Collisional activation and B/E-linked scanning were used to obtain structurally significant fragment ions of the molecular ion of the tetramer at m/z 834 in the positive-ion FAB mass spectrum (Figure 4). An abundant fragment ion was detected at m / z 765, indicating loss of a trifluoromethyl radical. Although less abundant, fragment ions were observed corresponding to [M - F]+ at m/z 815, [M - Ph]+ a t m/z 757, [M - CF3 - Ph]+ a t m/z 688, and [M - 2Ph]+ a t m / z 680. These fragment ions confirmed the identities of the substituents on the polyacetylene chain as phenyl and trifluoromethyl groups. Finally, the fragment ions detected a t m / z 417 and 587 were formed by cleavage of carbon-carbon bonds of the oligomer, as indicated in Figure 4. These ions were unusual because they were formed by fragmentation of the backbone of the oligomer instead of elimination of substituents from the chain. Although a partial set of B-series ions from the oligomeric mixture was detected using FAB mass spectrometry, ions correspondingto several less abundant B-series oligomers and the entire A series were not observed. Therefore, reversedphase HPLC separation followed on-line by continuous-flow FAB mass spectrometry was investigated as a method to obtain a more complete profile of oligomers in the mixture. The reconstructed total-ion and selected-ion chromatograms for the continuous-flow FAB LC/MS analysis of the polyacetylene mixture is shown in Figure 5. Because abundant FAB matrix ions were continuously detected in the low mo-
lecular weight region of the LC/MS mass spectra, the total-ion chromatogram shows relatively little variation during the analysis. Necessary for FAB ionization, the 3-nitrobenzyl alcohol matrix was included in the mobile phase for simplicity of analysis instead of being added postcolumn ( I 7)or by using coaxial flow (18). Compared to reversed-phaseHPLC without the presence of the FAB matrix (data not shown), addition of 3-nitrobenzyl alcohol to the mobile phase reduced chromatographic resolution so that some overlapping of A- and B-series oligomers was observed. However, chromatographic resolution was sufficient to at least partially resolve each oligomer belonging to each series. During the LC/MS analysis, both series of oligomers were detected beginning with A2 and B2and extending through A, and Be An example of the FAB mass spectrum of B, at m/z 664 obtained using LC/MS is shown in Figure 6. Background subtraction was used to eliminate matrix ions and ions from other oligomers that partially coeluted with B3. Because the oligomer A4 virtually coeluted with B3, the A4 molecular ion was detected at m / z 758 in Figure 6. The only ions detected using FD mass spectrometry that were not observed during LC/MS were the heptamers and octamers, probably because these oligomers were present in trace amounts and were lost in the chemical noise during FAB ionization. Like FD, continuous-flow FAB provided a molecular ion profile for a mixture of synthetic polyacetylenes with little fragmentation. However, MS/MS with CAD could be carried out successfully during continuous-flowFAB in order to obtain structurally significant fragment ions of molecular ion precursors. For example, the B/E-linked scan with CAD of m/z 416 obtained during LC/MS is shown in Figure 7. Fragment ions were observed that confirmed the presence of -F, -CF, and phenyl groups on the precursor ion. For example, [M F]+ was detected at m / z 397, [M - CF3]+at m / z 347, [M -
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 22, NOVEMBER 15, 1991
of sample ionization, FAB mass spectrometry can be used to obtain MS/MS spectra and exact mass measurements,as well as molecular weights of synthetic polymers. However, because of chemical noise and ion ‘suppression”, not all components of oligomeric mixtures can be detected using FAB ionization. In addition to FD mass spectrometry, continuous-flow FAB LC/MS of a substituted polyacetylene has been demonstrated to be a useful technique for the determination of the chain lengths of the various oligomers present and the identities of their end capping groups. Finally, LC/MS/MS was used to obtain fragment ions that confirmed the identities of the substituent groups along the polyacetylene chain. 100
150
200
250
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7. posltlvekn BIEmced scan wlth cobbnal advation of mlz 416 obtalned durlng continuous-flow FAB LC/MS analysis of oligomers of 1.
2CF3]+ at m / z 278, [M - F - HF]+ at m/z 377, [M - CF, HF]+ at mlz 327, [M - CF3 - 2HF]+ at m / z 307, and loss of a phenyl group was detected at m / z 339. The same limitations that precluded MS/MS analysis of the molecular ions formed during FD prevented exact mass measurements of these ions from being carried out using FD maas Spectrometry. Therefore,the exact mass of the molecular ion at mlz 416 was determined by high-resolution FAB mass spectrometry to be 416.0100, which corresponded exactly to an elemental composition of Cz4HI4Fs.Prior to analysis, this compound had been purified by using HPLC. The structure of this molecule was probably a dimer with one phenyl endcapping group that eliminated a hydrogen atom to form a cyclic, aromatic molecule. The absence of fragment ions of the polymeric chain supports the assignment of this structure as a resonance stabilized, cyclic molecule.
CONCLUSIONS Although FD mass spectrometry continues to be a useful technique for the determination of the relative chain length of low molecular weight polymers, MS/MS analysis of selected molecular ions of the oligomeric mixture can be hampered by low abundance of individual molecular ions and the short duration of their formation. Because of the longer duration
LITERATURE CITED (1) Tokura, Y.; Koda, T.; Itsubo, A.; Mlyabayashi, M.; Okuhara, K.; Ueda, T. J . Chem. phvs. 1988, 85. 99-104. (2) Baughman. R. H.; Bredas, J. L.; Chance, R. R.; Elsenbaumer, R. L.; Shacklette, L. W. Cbem. Rev. 1982, 82, 209-222. (3) MacDiarmid, A. G.; Epstein, A. J. Faraday Dlscuss. chem.Soc. 1089, 88, 317-332. (4) LatHnw, R. P.; Schulten, H.R. Int. J . Mass Spectfom. Ion Processes 1085, 67, 277-284. (5) Craig, A. 0.; Cullis, P. 0.; Derrick, P. J. Int. J . Mass Spectrom. Ion p t r V ~ .1981, 38, 297-304. (6) Blemann, K.; Scoble, H. A. Science 1087, 237, 992-998. (7) Fenselau, C.; Cotter, R. J. Chem. Rev. 1087, 87. 501-512. (8) Caprioll, R. M.; Moore, W. T.; Dague, B.; Martin, M. J . Chromatogr. iaaa. 443. 355-362. ... ..(9) vanireemen. R. B.; Martin, L. B.; Le, J. C. J . Am. Soc. Mass Spectrm. 1901. 2 . 157-163. (IO) van Breemen, k. B.; Canjura, F. L.; Schwartz, S. J. J . Chromatogr. 1991, 542, 373-383. (11) Ito, Y.; Takeuchi, T.; Ishii, D.;Goto, M. J . Chromatogr. 1985, 346, 161-166. (12) Bumgardner, C.; Bunch. J. J . Fluorine Chem. 1987, 3 6 , 313-317. (13) Bumpardner, C. L.; Huang, C.-H.; van Breemen, R. 8. J . Fluorhe Chem., in press. (14) Schulten, H.R.; Lattimer, R. P. Mess Spectrom. Rev. 1984, 3. 231-315. (15) Caprioli, R. M.; Moore, W. T.; Fan, T. Rapid Commun. Mass Spectram. 1987, 1 , 15-18. (16) Caprioli, R. M.; Moore, W. T.; Petrle, 0.;Wilson, K. Int. J . Mass Spectrom. Ion Processes 1088, 8 6 , 187-199. (17) Games, D. E.; Pleasance, S.; Ramsey, E. D.;McDowail, M. A. Bbmed. Envfron. 1988. 15. 179-182. . Mass _ _ _ Saectrom. -r-- (18) Deterdlng, L. J.; Moseley, M. A.; Tomer, K. BYJorgenson, J. W. Ami. Chem. 1989, 6 1 , 2504-2511.
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RECEIVED for review June 10,1991. Accepted August 22,1991. This research was supported by the North Carolina Biotechnology Center (R.v.B.) and the Ethyl Corp. (C.L.B.).
