Anal. Chem. 2008, 80, 355-362
Tandem Mass Spectrometry Characteristics of Silver-Cationized Polystyrenes: Internal Energy, Size, and Chain End versus Backbone Substituent Effects Michael J. Polce,†,‡ Manuela Ocampo,§ Roderic P. Quirk,§ Alyison M. Leigh,† and Chrys Wesdemiotis*,†
Departments of Chemistry and Polymer Science, The University of Akron, Akron, Ohio 44325 The Ag+ adducts of polystyrene (PS) oligomers with different sizes (6-19 repeat units) and initiating (r) or terminating (ω) end groups mainly decompose via free radical chemistry pathways upon collisionally activated dissociation. This reactivity is observed for ions formed by matrix-assisted laser desorption/ionization as well as electrospray ionization. With end groups lacking weak bonds (robust end groups), dissociation starts with random homolytic C-C bond cleavages along the PS chain, which lead to primary and benzylic radical ions containing either of the chain ends. The primary radical ions mainly depolymerize by successive β C-C bond scissions. For the benzylic radical ions, two major pathways are in competition, namely, depolymerization by successive β C-C bond scissions and backbiting via 1,5-H rearrangement followed by β C-C bond scissions. The extent of backbiting decreases with internal energy. With short PS chains, the primary radical ions also undergo backbiting involving 1,4- and 1,6-H rearrangements; however, this process becomes negligible with longer chains. If the polystyrene contains a labile substituent at a chain end, this substituent is eliminated easily and, thus, not contained in the majority of observed fragments. Changes in the PS backbone structure can have a dramatic effect on the resulting dissociation chemistry. This is demonstrated for poly(r-methylstyrene), in which backbiting is obstructed due to the lack of benzylic H atoms; instead, this backbone connectivity promotes 1,2-phenyl shifts in the primary radical ions formed after initial C-C bond homolyses as well as H atom transfers between the incipient primary and benzylic radicals emerging from these homolyses. Tandem mass spectrometry (MS/MS) is emerging as an important analytical method for the structural characterization of synthetic polymers and materials. This potential is indicated by recent applications, in which MS/MS was employed to conclu* To whom correspondence should be addressed. E-mail: wesdemiotis@ uakron.edu. Phone: 330-972-7699. Fax: 330-972-6085. † Department of Chemistry. ‡ Present address: Lubrizol Inc., 9911 Brecksville Rd., Brecksville, OH 44141. § Department of Polymer Science. 10.1021/ac701917x CCC: $40.75 Published on Web 12/18/2007
© 2008 American Chemical Society
sively identify end groups and central substituents of functional polymers,1-5 distinguish isomers with different architectures,3,6,7 and determine copolymer sequences.8-10 Mass spectrometry analyses begin with the formation of gasphase ions from the sample under examination. For an MS/MS experiment, a specific oligomer from the distribution of ions created upon ionization is mass-selected and induced to decompose, typically by collisionally activated dissociation (CAD). Reconstruction of the original structure from the fragments detected in CAD spectra requires knowledge of the corresponding fragmentation mechanisms, which provide a template on how to correctly reconnect the observed fragments.11 Such information has been gathered for several types of biopolymers, in particular peptides,12,13 oligosaccharides,14,15 and nucleic acid derivatives,16 leading to precise interpretation rules for their tandem mass spectra. (1) Jackson, A. T.; Bunn, A.; Hutchings, L. R.; Kiff, F. T.; Richards, R. W.; Williams, J.; Green, M. R.; Bateman, R. H. Polymer 2000, 41, 7437-7450. (2) Yalcin, T.; Gabryelski, W.; Li, L. Anal. Chem. 2000, 72, 3847-3852. (3) Wollyung, K. M.; Wesdemiotis, C.; Nagy, A.; Kennedy, J. P. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 946-958. (4) Jackson, A. T.; Bunn, A.; Priestnall, I. M.; Borman, C. D.; Irvine, D. J. Polymer 2006, 47, 1044-1054. (5) Jackson, A. T.; Green, M. R.; Bateman, R. H. Rapid Commun. Mass Spectrom. 2006, 20, 3542-3550. (6) Wesdemiotis, C.; Polce, M. J.; Harris, F. W.; Baek, J. - B. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics, Montreal, QC, Canada, June 8-12, 2003. (7) Jackson, A. T.; Scrivens, J. H.; Slade, S.; Simonsick, W. J. Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 23-27, 2004. (8) Urakami, K.; Akimoto, N.; Nishijima, K.; Kitanaka, Y.; Echigoya, M.; Hashimoto, K. Chem. Pharm. Bull. 1999, 47, 1068-1072. (9) Arnould, M. A.; Wesdemiotis, C.; Geiger, R. J.; Park, M. A.; Buehner, R. W.; Vandervorst, D. Prog. Org. Coat. 2002, 45, 305-312. (10) Wesdemiotis, C.; Pingitore, F.; Polce, M. J.; Russel, V. M.; Kim, Y.; Kausch, C. M.; Connors, T. H.; Medsker, R. E.; Thomas, R. R. Macromolecules 2006, 39, 8369-8378. (11) Polce, M. J.; Wesdemiotis, C. In Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Synthetic Polymer Characterization; Li, L., Ed.; Wiley: New York, In press. (12) Wang, P.; Kish, M. M.; Wesdemiotis, C. Encycl. Mass Spectrom. 2005, 2, 139-151. (13) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508-548. (14) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (15) Park, Y.; Lebrilla, C. B. Mass Spectrom. Rev. 2005, 24, 232-264. (16) Monn, S. T. M.; Tromp, J. M.; Schuerch, S. Chimia 2005, 59, 822-825.
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In contrast, the fragmentation mechanisms of synthetic polymer ions have remained largely unknown. To overcome this problem, our group undertook a comprehensive study of the fragmentation pathways of silverated polystyrenes carrying robust end groups.17 [M + Ag]+ ions from unlabeled, deuterated, and chain-end functionalized polystyrene (PS) oligomers were formed by matrix-assisted laser desorption ionization (MALDI)18,19 and activated to dissociate via CAD in a quadrupole/time-of-flight (Q/ TOF) tandem mass spectrometer. Based on the fragmentation patterns observed and estimates of select bond energies in the [M + Ag]+ complexes, it was concluded that fragmentation proceeds via free radical chemistry, beginning with charge-remote homolytic C-C bond cleavages in the PS chain.17 Such cleavages create incipient radical ions with benzylic or primary radical sites, which dissociate consecutively to the ultimately observed products by typical radical site reactions, viz. bond scissions in β position to the unpaired electron and hydrogen atom rearrangements (backbiting) followed by β scissions.17 The present study continues this work with an investigation of additional determinants of fragmentation behavior, including the internal energy deposited in the CAD step, the ionization method used for [M + Ag]+ formation, and the size of the fragmenting PS oligomer; the dissociation characteristics of polystyrenes with labile functional groups at the initiating (R) or terminating (ω) chain end, as well as a polymer with a modified backbone connectivity, i.e., poly(Rmethylstyrene), are also examined. EXPERIMENTAL SECTION MALDI Studies. MALDI-MS/MS experiments were performed on a Waters Q/ToF Ultima quadrupole/orthogonalacceleration time-of-flight mass spectrometer (Milford, MA), equipped with a pulsed nitrogen laser emitting at 337 nm. Solutions of dithranol matrix (20 mg/mL), polystyrene (10 mg/ mL), and silver trifluoroacetate cationizing agent (10 mg/mL) were mixed in the ratio 10:2:1, and ∼1.0 µL of the final mixture was deposited on the 96-well sample holder plate that is inserted into the MALDI source. This sample preparation led to the formation of abundant [M + Ag]+ ions from the polystyrenes studied. The ions exiting the MALDI source were directed toward the quadrupole mass filter, which was set to transmit one oligomer mass only (mass-selective mode). The precursor ion resolution can be adjusted to select one isotope or the complete isotopic cluster of an oligomer. The selected ion proceeded to an rf-only hexapole collision cell, pressurized with Ar at ∼0.9-1.0 bar, where CAD took place at laboratory frame kinetic energies that can be varied up to Elab ) 200 eV (multiple collision conditions). Laboratoryframe and center-of-mass collision energies are related through the equation Ecm ) ElabmAr/(mAr + mion), where mAr and mion are the masses of Ar (collision gas) and the mass-selected precursor ion, respectively; Ecm gives the upper limit of internal energy that can be transferred to the precursor ion per collision. The fragment and undissociated precursor ions exiting the collision cell were focused through an rf-only hexapole lens, and the focused ion packet was accelerated orthogonally by ∼10 kV into the TOF (17) Polce, M. J.; Ocampo, M.; Quirk, R. P.; Wesdemiotis, C. Anal. Chem. 2008, 80, 347-354. (18) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (19) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301.
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region for mass analysis. Control mass spectra were measured with the TOF mass analyzer by setting the quadrupole mass filter to rf-only mode, so that it transmitted all ions produced in the MALDI source. The ion abundances of several TOF MS or MS/ MS scans were summed to obtain spectra with good signal/noise ratio. The quoted m/z values are monoisotopic. ESI Studies. ESI-MS/MS experiments were performed on a Bruker Esquire-LC quadrupole ion trap mass spectrometer (Billerica, MA) equipped with electrospray ionization.20 Polystyrene [M + Ag]+ ions were formed using THF/methanol (9:1) solutions of the polymer (1 mg/mL) and silver trifluoroacetate (0.5-1 mg/ mL). Higher polymer concentrations than those typically used for polar polymers were necessary to obtain tandem mass spectra with adequate signal/noise ratio. The polymer and salt solutions were combined in the ratio 1:1 (v:v) and the mixed solution was sprayed into the ion source by a syringe pump at a rate of 350 µL/h. The spraying needle was grounded, and the entrance of the sampling capillary was set at -4 kV for the analysis of positive ions. Nitrogen was used as the nebulizing gas (10 psi) and drying gas (8 L/min, 300 °C), and He was the bath gas in the ion trap (10-8 bar). The desired polystyrene ions ([M + Ag]+) were isolated and induced to fragment by CAD inside the trap by resonance excitation with a rf field. The excitation time was set at 40 ms and the rf amplitude (Vp-p) was adjusted in the 1.202.50 V range to maximize fragment ion abundances. A total of 50-65 scans/spectrum were averaged. The quoted m/z values are monoisotopic. Materials. Polystyrene standards, C4H9-(C8H8)n-H, were obtained from Goodyear (Akron, OH) or Scientific Polymer Products (Ontario, NY). Poly(R-methylstyrene), Mn ) 1300, was purchased from Polymer Source (Dorval, QC, Canada). Chainend functionalized polystyrenes were synthesized via alkyllithiuminitiated living anionic polymerization by Quirk and co-workers, using procedures established in the Quirk group.21,22 Polystyrene functionalized at the initiating chain end with a tert-butylthio group, (CH3)CS-, was synthesized via chain-transfer radical polymerization23 by Harwood and co-workers (University of Akron). Dithranol, the MALDI matrix, was purchased from Alpha Aesar (Ward Hill, NY), and the solvents and silver trifluoroacetate were purchased from Aldrich (Milwaukee, WI). The polymers, solvents, and salt were used in the condition received without any further purification. RESULTS AND DISCUSSION Fragmentation of Silverated Polystyrenes with Robust End Groups. In a polystyrene with robust end groups, the intrinsically weakest bonds are located within the PS chain. Standards with the connectivity C4H9-(C8H8)n-H and functionalized samples carrying stable end groups, for example, the ω-hydroxylated polystyrene C4H9-(C8H8)n-CH2CH2OH, belong to this category. CAD of silverated oligomers from such polystyrenes proceeds via the free radical chemistry summarized in Scheme 1.17 Representa(20) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (21) Hsieh, H. L.; Quirk, R. P. Anionic Polymerization. Principles and Practical Applications; Marcel Dekker, Inc.: New York, 1996. (22) Quirk, R. P.; Mathers, R. T.; Wesdemiotis, C.; Arnould, M. A. Macromolecules 2002, 35, 2912-2918. (23) Lian, B.; Thomas, C. M.; Navarro, C.; Carpentier, J. - F. Organometallics 2007, 26, 187-195.
Scheme 1. (a) Nomenclature for Fragments from Polystyrene Ions.17 (b) Primary (an•/yn•) and Benzylic (bn•/zn•) Radical Ions Arising by Random Homolytic C-C Bond Cleavages in the Chain of a Polystyrene with the Connectivity C4H9-(C8H8)-H, and Closed-Shell Fragments Produced by Consecutive β Scissions from These Radical Ions. (c) Backbiting via 1,5-H Rearrangement (1,5-rH) and Subsequent β C-C Bond Scissions in the Benzylic Radical Ions bn•/zn•. R ) C4H9 for an, anb, bn, and bn•, or CH2Ph for yn, ynb, zn, and zn•
tive spectra, obtained from the Ag+ adduct of C4H9-(C8H8)19-H (m/z 2142.2) using Q/TOF tandem mass spectrometry and different collision energies, are depicted in Figure 1. Except for the losses of 1-4 styrene units, which are significant only at the lowest collision energies (vide infra), all CAD fragments are accounted for by random C-C bond homolyses in the PS chain, followed by typical radical site reactions. Only a concise description of these dissociations is presented here, as their detailed mechanisms and energetics, as well as the nomenclature used, have been discussed in a preceding article.17 The initial C-C bond cleavages generate primary (an•/yn•) and benzylic (bn•/zn•) radical ions containing either the R (an•/bn•) or the ω (yn•/zn•) chain end, cf. Scheme 1. The ultimately observed products arise by consecutive reactions, involving β scissions of H• or phenyl (Ph•), as shown in Scheme 1b, or backbiting in the benzylic radical ions, as shown in Scheme 1c. In addition to these reaction sequences, the initially formed radical ions also depolymerize by successive monomer evaporations. The energetically most favorable dissociations of the benzylic radical ions (bn•/zn•) are monomer evaporation and backbiting via 1,5-H rearrangement; both have comparable energy requirements.17,24 In contrast, β-H• loss from bn•/zn• is associated with a considerably higher energy barrier,17,24 in accord with the low yield (24) The activation energy for the β C-H scission bn•/zn• f bn/zn + H• (Scheme 1b) is ∼200 kJ/mol, while that for the β C-C bond scission bn•/zn• f bn-1•/ zn-1• + C8H8 (monomer loss) is ∼80 kJ/mol; dissociation of bn•/zn• via backbiting and subsequent β C-C bond scission to yield bn-3•/zn-3•, an-2/ yn-2, K3, or J2• (Scheme 1c) also requires ∼80 kJ/mol.17 The activation energies for the competitive β scissions an•/yn• f an-1•/yn-1• + C8H8 (monomer loss), an•/yn• f an/yn + H• (Scheme 1b), and an•/yn• f anb/ynb + Ph• (Scheme 1b) are ∼80, ∼140, and ∼160 kJ/mol, respectively.17
of bn/zn in the CAD spectra. Monomer evaporation contributes to the abundance of the internal fragment K1, i.e. C8H8-Ag+. Backbiting in the benzylic radical ions bn•/zn• transfers the unpaired electron from a terminal to an internal benzylic position, from where it can promote two different β scissions (indicated by dotted and dashed lines in Scheme 1c). The latter scissions lead to the methylene-terminated fragments an/yn, which dominate the medium- and high-mass ranges of the CAD spectra (Figure 1), or to shorter benzylic radical ions, which can undergo anew backbiting, if they have the size and internal energy necessary to do so. As a result, only small benzylic radicals are detected in the CAD spectra (up to b4• and z5• in Figure 1). Repeated backbiting would also cause the relative abundances of an/yn to increase with decreasing n, as indeed observed. The β scissions following backbiting coproduce the dimeric internal radical ion J2• (m/z 302) and the trimeric internal closed-shell ion K3 (m/z 419). The same J2• and K3 fragments are generated in all backbiting events, irrespective of the size of the original bn•/zn• radical, explaining the high abundances of these low-mass ions. For silverated C4H9(C8H8)n-H, the internal ions J2• and K3 are identical with the terminal ions z2• and y3, respectively. Isotopomers deuterated at the ω chain end or in the benzylic positions, and the ω-hydroxylated polystyrene C4H9-(C8H8)n-CH2CH2OH, with which the overlapping fragments are separated, show that J2• and z2• have comparable abundances ((50%), whereas K3 is g5 times more abundant than y3.17,25 The energetically preferred dissociation of the primary radical ions (an•/yn•) is monomer evaporation;17,24 hence, an•/yn• must be a major source of K1. The β scission of Ph• (Scheme 1b) requires roughly twice the energy needed for monomer loss,17,24 consistent Analytical Chemistry, Vol. 80, No. 2, January 15, 2008
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Figure 1. MALDI-CAD spectra of silverated C4H9-[CH2CH(Ph)]19-H (m/z 2142.2). The complete isotopic cluster was mass-selected for CAD with Ar at (a) Elab ) 120 eV (Ecm ) 2.2 eV), (b) Elab ) 140 eV (Ecm ) 2.6 eV), and (c) Elab ) 160 eV (Ecm ) 2.9 eV). The intensity ratio [m/z 302.0]/[m/z 2142.2] is (a) 0.045, (b) 2.1, and (c) 110. S abbreviates the styrene (C8H8) unit.
with the very low abundance of series anb/ynb (near noise level in Figure 1). The competitive β scission of a hydrogen radical (Scheme 1b) has an intermediate energy requirement and should (25) A silverated styrene dimer (K2, m/z 315) is also observed and attributed to β C-C scission after two consecutive backbiting events, involving sequential 1,7- and 7,3-H rearrangements.17
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be a contributor to the an/yn series; the major route to an/yn proceeds, however, via backbiting in bn•/zn• (Scheme 1c), which is associated with a ∼40% lower energy barrier.17,24 Internal Energy Effects. The dependence of the polystyrene fragmentation pathways on internal energy was investigated by acquiring MALDI-CAD spectra for the [M + Ag]+ ion of C4H9-
(C8H8)19-H (m/z 2142.2) as a function of collision energy. With laboratory frame collision energies (Elab) up to ∼40 eV, corresponding to center-of-mass collision energies (Ecm) of e0.7 eV, only one fragment is observed, 416 u below the [M + Ag]+ peak. This fragment arises by the nominal loss of four styrene units, possibly a tetramer. In the Elab window 40-100 eV (Ecm ) 0.71.8 eV), the losses of 1, 2, and 3 styrene units are also observed with smaller relative abundances. The low-mass radical ions and internal ions dominating the spectrum of Figure 1 start appearing at ∼100 eV. These ions, as well as all other fragments resulting from random homolytic cleavages of the PS backbone and subsequent backbiting/β scissions, become the major CAD products at Elab ) 120 eV (Ecm ) 2.2 eV), cf. Figure 1a, but the losses of 1, 2, and especially 4 styrene units are still competitive at this collision energy. Upon further increases of the collision energy, the losses of 1-4 styrene units essentially disappear, cf. Figure 1b,c. The latter losses had been observed upon the spontaneous dissociation (postsource decay) of silverated polystyrenes formed at high laser power in a reflectron TOF mass spectrometer.26 They presumably involve rearrangement dissociations with low critical energies but tight transition states, so that they proceed efficiently only at the lowest internal energies. At the lower internal energies favoring homolytic cleavages (Figure 1a), terminal radical ions are readily detected up to b4• (m/z 580) and z5• (m/z 614). Further, series an/yn, which originate largely from backbiting, have considerable relative intensities, and the internal trimer ion K3, a backbiting signature, is the second most abundant fragment. As the internal energy is increased (Figure 1b,c), the intensities of an/yn and K3 decrease significantly, while those of b1+, z1•, K1, b1•, and z2• rise substantially (b1+ does not contain Ag). These results suggest that the extent of depolymerization via monomer evaporation increases with internal energy at the expense of backbiting. Even though the energy requirements for backbiting and monomer loss are predicted to be similar (vide supra),24 backbiting involves a rearrangement and, thus, should be slower than monomer fission at increased internal energies, in keeping with the observed trend. Ionization Method and Oligomer Size Effects. The fragmentation behavior of silverated polystyrene was also examined with oligomer ions produced by ESI in a quadrupole ion trap (QIT). Singly charged [M + Ag]+ ions are most efficiently formed from small PS oligomers. The ESI-CAD spectrum of such an ion, viz. silverated C4H9-(C8H8)7-H, is shown in Figure 2a. The MALDI-CAD spectrum of a comparably small oligomer is included in Figure 2b. ESI- and MALDI-CAD spectra are fairly similar, both containing the fragment ions expected from the random homolytic cleavages of the PS chain discussed. It is noteworthy that the internal fragments K3 and J2• and series an/yn, which are produced by backbiting in the benzylic radical ions bn•/zn•, are more abundant in the ESI-CAD spectrum, consistent with the lower average internal energies deposited by ESI-CAD in an ion trap vis a` vis MALDI-CAD in the collision cell of a Q/TOF (beam) mass spectrometer;11 as mentioned, backbiting is more competitive at lower internal energies. Also, the longer fragmentation time available in QITs (tens of ms) versus Q/TOF instrumentation (
