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Anal. Chem. 1999, 71, 3637-3641

Determination of End Groups of Synthetic by Matrix-Assisted Laser Desorption/ : High-Energy Collision-Induced Dissociation

Andrew R. Bottrill, Anastassios E. Giannakopulos, Carl Waterson, David M. Haddleton, Ken S. Lee, and Peter J. Derrick*

Institute of Spectrometry and Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom

Matrix-assisted laser desorption/ionization has been com- MALDI-time-of-flight (TOF) to obtain values bined with high-energy collision-induced dissociation for for molecular mass averages, Mn and Mw, especially in combination the analysis of poly(ethylene glycols) with butanoyl, with size exclusion chromatography.17-20 benzoyl and acetyl end groups, using novel technology Techniques of synthesis are becoming increasingly comprising a magnetic-sector mass and sophisticated with a move toward the attempt to design macro- buncher with an in-line quadratic-field ion mirror. High- molecules for specific application. This has usually been centered energy (>8 keV) collision-induced dissociation facilitated around living, or pseudoliving, polymerization which allows the unambiguous end-group determination of these polymers, incorporation of different functionalities at precise positions by providing masses of end groups and structural informa- judicious choice of reaction conditions.28-34 For example, R and tion. The high-energy collision-induced dissociation also â terminally functional polymers may have very different proper- provided information regarding repeat units. (12) Williams, J. B.; Gusev, A. I.; Hercules, D. M. Macromolecules 1997, 30, 3781-3787. (13) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F.; Sepulchre M. Mass spectrometry of synthetic polymers is gaining in popular- Macromol. Chem. Phys. 1996, 197, 2615-2625. (14) Zammit, M. D.; Davis, T. P.; Haddleton, D. M.; Suddaby, K. G. Macromol- ity due to the unprecedented high-quality information accessible ecules 1997, 30, 1915-1920. to the synthetic polymer chemist. Matrix-assisted laser desorp- (15) Guttman, C. M.; Blair, W. R.; Danis, P. O. J. Polym. Sci., Part B: Polym. tion/ionization (MALDI) has been shown to be an especially Phys. 1997, 35, 2409-2419. (16) Duncalf, D. J.; Wade, H. J.; Waterson, C.; Derrick, P. J.; Haddleton, D. M.; 1-8 useful tool for the direct analysis of synthetic polymers. The McCamley, A. Macromolecules 1996, 29, 6399-6403. results obtained for polymers from MALDI mass spectrometry (17) Nielen, M. W. F. Anal. Chem. 1998, 70, 1563-1568. experiments typically give qualitative molecular weight information (18) Montaudo, M. S.; Puglisi, C.; Samperi, F.; Montaudo, G. Rapid Commun. Mass Spectrom. 1998, 12, 519-528. but normally not structural information. Nevertheless, MALDI has (19) Jackson, C.; Larsen, B.; McEwen, C. Anal. Chem. 1996, 68, 1303-1308. been utilized to gain information on monomer reactivity ratios,9 (20) Kassis, C. E.; DeSimone, J. M.; Linton, R. W.; Lange, G. W.; Friedman, R. end groups,10-13 and mechanisms of polymerization.14-16 Many M. Rapid Commun. Mass Spectrom. 1997, 11, 1134-1138. (21) Barber, M.; Bordoli, R. S.; Sedgewick, R. D.; Tyler, A. N. Nature 1981, attempts, with a variety of success, have also been made to use 293, 270-275. (22) Arberth, W.; Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 54, 2029- (1) Bahr, U.; Depe A.; Karas, M.; Hillenkamp, F.; Giessmann, U. Anal. Chem. 2034. 1992, 64, 2866-2869. (23) Craig, A. G.; Derrick, P. J. J. Chem. Soc., Chem. Commun. 1985, 891- (2) Belu, A. M.; DeSimone, J. M.; Linton, R. W.; Lange, G. W.; Friedman, R. 892. M. J. Am. Soc. Mass Spectrom. 1996, 7,11-24. (24) Craig, A. G.; Derrick, P. J. Aust. J. Chem. 1986, 39, 1421-1434. (3) Lloyd, P. M.; Suddaby, K. G.; Varney, J. V.; Scrivener, E.; Derrick, P. J.; (25) Jackson, A. T.; Jennings, K. R.; Scrivens, J. H. J. Am. Soc. Mass Spectrom. Haddleton, D. M. Eur. Mass Spectrom. 1995, 1, 293-300. 1997, 8,76-85. (4) Hoberg, A.-M.; Haddleton, D. M.; Derrick, P. J. Eur. Mass Spectrom. 1997, (26) Jackson, A. T.; Yates, H. T.; Scrivens, J. H.; Critchley, G.; Brown, J.; Green, 3, 471-473 M. R.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1996, 10, 1668- (5) Creel, H. S. Trends Pol. Sci. 1993, 1, 336. 1674. (6) Jackson, C. A.; Simonsick, W. J. Curr. Opin. Solid. State Mater. Sci. 1997, (27) Andersen, U. N.; Colburn, A. W.; Makarov, A. A.; Raptakis, E. N.; Reynolds, 2, 661-667. D. J.; Derrick, P. J.; Davis, S. C.; Hoffman, A. D.; Thomson, S. Rev. Sci. (7) Danis, P. O.; Karr, D. E.; Simonsick, W. J.; Wu, D. T. Macromolecules 1995, Instrum. 1998, 69, 1650-1660. 28, 1229-1232. (28) Webster, O. W. Science 1991, 251, 887-893. (8) Danis, P. O.; Karr, D. E.; Xiong, Y.; Owens, K. G. Rapid Commun. Mass (29) Davis, T. P.; Haddleton, D. M.; Richards, S. N. J. Macromol. Sc., Rev. Spectrom. 1996, 10, 862-868. Macromol. Chem. Phys. 1994, C34, 243-324. (9) Suddaby, K. G.; Hunt, K. H.; Haddleton, D. M. Macromolecules 1996, 29, (30) Haddleton, D. M.; Waterson, C.; Derrick, P. J.; Jasieczek, C.; Shooter, A. J. 8642-8649. Chem. Commun. 1997, 683-684. (10) Maloney, D. R.; Hunt, K. H.; Lloyd, P. M.; Muir, A. V. G.; Richards, S. N.; (31) Matyjaszewski, K. J. Phys. Org. Chem. 1995, 8, 197-207. Derrick, P. J.; Haddleton, D. M. J. Chem. Soc., Chem. Commun. 1995, 561- (32) Yeates, S. G.; Richards, S. N. Surf. Coat. Int. 1996, 10, 437-442. 562. (33) Matyjaszewski, K.; Wang, J.-L.; Grimaud, T. J.; Shipp, D. A. Macromolecules (11) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. J. Pol. Sci. A Pol. 1998, 31, 1527-1534. Chem. 1996, 34, 439-447. (34) Perec, V.; Kim, H. J.; Barboiu, B. Macromolecules 1997, 30, 8526-8528.

10.1021/ac990523t CCC: $18.00 © 1999 American Chemical Society , Vol. 71, No. 17, September 1, 1999 3637 Published on Web 07/24/1999 ties in certain applications. Thus it is important for techniques to be developed that can confirm, or refute, synthetically targeted macromolecular structures. In addition to empirical molecular mass data, it is important to be able to determine information regarding the end groups of and the sequence of monomers/ groups within a synthetic macromolecule. Tandem mass spec- trometry can in principle provide such structural information for a synthetic polymer. has been widely used in the biological area, combined with ionization techniques such as liquid secondary ion mass spectrometry (LSIMS)21,22 and . Early investigations with showed how tandem mass spectrometry could be used to investigate the structure of synthetic polymers.23,24 End-group information for poly(methyl methacrylate) (PMMA) has been derived from LSIMS using collision-induced dissociation (CID) at 4-keV collision energy in magnetic-sector instruments.25 The envelopes of peaks due to cationized molecules in these LSIMS experiments were centered at lower mass-to-charge ratios than the molecule-ion envelopes with MALDI for the same Figure 1. Structures of the differing end-terminated poly(ethylene polymers, presumably as a result of reduced ionization efficiency glycol) oligomers chosen for MALDI-CID. in the high-mass region in the LSIMS case. MALDI-CID results for poly(ethylene glycol)s (PEGs), MALDI-CID results for PMMA, possible full control over the laboratory-frame collision energy. and LSIMS-CID results for PMMAs have been reported by The expectation was that the high laboratory-frame collision (103- 4 Jackson et al.,26 utilizing laboratory collision energy of 800 eV on 10 eV) accessible in MAG-TOF would be advantageous as regards a magnetic-sector/orthogonal TOF instrument. For the PMMAs, fragmentation of large . The high laboratory-frame collision better signal-to-noise ratios were reported for the MALDI-CID energies access interaction times shorter than those accessible spectra compared to the LSIMS tandem mass spectra, due at least at lower energies. For example, center-of-mass collision energy in part to the higher sensitivities of MALDI. With orthogonal TOF, is approximately the same for a large ion at a laboratory-frame ion energies prior to orthogonal acceleration, and hence collision collision energy of 800 eV with Ar collision gas as at a laboratory- energies, need to be low (ideally <100 eV), and the 800-eV frame collision energy of 8000 eV with He collision gas. The collision energy employed in these experiments would be very physical processes differ, however, as is evidenced by the results much an upper limit. The restriction to low collision energies is presented in this paper, with one significant difference being the the major shortcoming of orthogonal TOF in the context of tandem interaction times. The results presented here are consistent with mass spectrometry. The novel instrument27 used in this study has the fragmentation induced by collision being more localized within E been designed to overcome any limitation or restriction of collision the macromolecule at the higher LAB’s and shorter interaction times, when energy transfer must occur within a smaller time energy to low values (see below). window. In this paper, we report CID experiments with poly(ethylene glycol)s with different R, terminal functionalities performed using â EXPERIMENTAL SECTION novel hybrid magnetic-sector/time-of-flight instrumentation (MAG- Cationized molecules were produced using a MALDI ion 27 TOF). The instrumentation consisted of a Kratos Concept H source and accelerated to 8 keV at the . Particular ions double-focusing magnetic sector and associated ion buncher as were selected for fragmentation by adjusting the the first mass spectrometer (MS-1) and a planar hyperbolic (so- strength and exit slit in MS-1. It was necessary to time focus and called quadratic field) ion mirror as the second mass spectrometer space focus the ions of the selected ion packet at the focal point (MS-2). The ion mirror was in-line with the optical axis of the of the ion mirror (MS-2) to achieve optimum resolution of the magnetic-sector instrument and the buncher. The collision ener- fragments. The spread in arrival times of the ions arising from gies employed spanned the range 8-12 keV. Full details of the the ion formation and from the passage through the electric and design have been published.27 The method of ionization employed magnetic sector of the instrument was reduced to a minimum was MALDI. MALDI-TOF instrumentation1-8 can in principle be (1-2 ns) by ion bunching. The ion buncher was positioned after used to perform high-energy CID, but the usefulness of such the exit slit of MS-1 and a short distance (230 mm) in front of the experiments is limited by the relatively low resolution achievable grounded collision cell. An additional translational energy spread in the selection of the parent ion and by the inherent difficulty in of up to 4 keV was imposed upon the ion packet before collision measuring the fragment ions. by the ion buncher. The pressure of collision gas, usually helium, The MAG-TOF mass spectrometer27 was designed and built argon, or xenon, was set to give 40% attenuation of the selected in order to be able to perform collision-induced dissociation on ion. large ions at high laboratory-frame collision energy ELAB and with Equal sensitivity to all fragment ion masses was achieved by high sensitivity for the tandem mass spectra. High sensitivity has combining an inclination of the ion mirror with a slight electro- been achieved through the use of TOF to measure the tandem static vertical deflection in the region after the collision cell and mass spectra. The MAG-TOF instrument used in this study made before the ion mirror.27,36 The sample probe tip in the MALDI

3638 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999 Figure 2. MALDI-CID spectrum of acetyl end-terminated poly(ethylene glycol) using He (a) or Xe (b) as the collision gas. source could be rastered in the x and y directions, providing a chloride, benzoyl chloride, and butyryl chloride were obtained fresh area for the laser to desorb ions at every laser shot. from Aldrich and used without further purification. PEG600 was R,ω-Dihydroxyl terminal poly(ethylene glycol), (number-aver- esterified by condensation with acetyl, 1, benzoyl, 2, and butyryl, age molar mass, n, of 600) (PEG600), triethylamine, acetyl 3, chloride (Figure 1). PEG 600 (10.2 g, ∼17 mmol) and 40 mL of tetrahydrofuran (THF) (freshly distilled from sodium/benzo- (35) Matyjaszewski, K.; Coca, S.; Nakagawa, Y.; Xia, J. Polym. Mater. Sci. Eng. phenone) were added to an oven-dried Schlenck tube under 1997, 76, 147-148. (36) Makarov, A. A.; Raptakis, E. N.; Derrick, P. J. Int. J. Mass Spectrom. Ion nitrogen. Triethylamine (5.21 mL, 37.4 mmol) was added to the Processes 1995, 146, 165-182. solution prior to the acid chloride (acetyl chloride, 2.6 mL (37.4

Analytical Chemistry, Vol. 71, No. 17, September 1, 1999 3639 Figure 4. Low-m/z region of the MALDI-CID spectrum of benzoyl Figure 3. Low-m/z region of the MALDI-CID spectrum of butanoyl end-terminated poly(ethylene glycol) for Xe collision gas. end-terminated poly(ethylene glycol) for Xe collision gas. The spectra for 1 using He and Xe are similar to each other, mmol), benzoyl chloride, 5.15 mL (37.4 mmol), or butyryl chloride, although not identical. The center-of-mass collision energy was 3.9 mL (37.4 mmol)) being slowly added. The reactions were left approximately 30 times higher with Xe (Figure 2b) as compared at room temperature for 14 h. Triethylammonium chloride formed to He (Figure 2a); the interaction times were approximately the as a white precipitate, which was removed by filtration prior to same for both. The center-of-mass collision energy with He (Figure the filtrate being passed down a column of basic alumina, to 2a) would be no more than half that for collision of the same ion remove excess unreacted acid chloride. The volatiles were with Xe at a laboratory collision energy of 800 eV (as in the removed by rotary evaporation and in a vacuum oven at 60 °C for experiments using orthogonal TOF26). Both the He spectrum 2 days. (Figure 2a) and the Xe spectrum (Figure 2b) at higher laboratory R-Cyano-4-hydroxycinamic acid (Sigma-Aldrich, Poole, Dorset, collision energy (but lower center-of-mass collision energy in the U.K.) was the matrix used for all of the MALDI experiments. A He case) differ from the Xe spectrum26 at the lower laboratory matrix solution 0.1 M in 30:70 water/methanol (analytical grade, collision energy in the low-m/z region. The end-terminated PEGs Fisher Scientific, Loughborough, U.K.) was used throughout the studied were symmetrical (i.e., same group at each end), and the experiments. A 0.5-µL aliquot of the matrix solution was deposited same fragment ions would arise from cleavages from either end on the probe tip and was dried using a stream of air. The sodium of the chain. In each spectrum (Figures 2-4) there are several cation in the form of a salt solution (100 µL, 0.1 M) was added to clear low-m/z peaks that can be attributed to the end-groups and 1 mL of the analyte, 3 × 10-3 M in 30:70 water/acetone (analytical fragments of the end groups. grade, Fisons Scientific, Loughborough, U.K.). A 0.5-µLofthe Acetyl end-terminated poly(ethylene glycol), 1, showed a very + cation/analyte solution was deposited on top of the matrix layer intense peak at m/z 43, attributed to the [CH3CO] fragment and + and dried using a stream of air. a second peak at m/z 87 due, it is proposed, to [CH3CO + C2H4O] . RESULTS AND DISCUSSION Butanoyl end-terminated poly(ethylene glycol), 2, showed + + [M + Na] ions of 1, 2, and 3 were selected in turn by strong peaks at m/z 71, attributed to [CH3CH2CH2CO] , and m/z + adjusting the magnetic-field strength in MS-1. Collision-induced 115, attributed to [CH3CH2CH2CO + C2H4O] . An intense peak dissociation was performed upon the selected ion by an inert gas was observed at m/z 43 that is proposed to arise from the [CH3- + (He and Xe were used) into the collision chamber. The fragments CH2CH2] fragment. Benzoyl end-terminated poly(ethylene gly- + from these collisions were observed at a planar array detector col), 3, showed intense peaks at m/z 105, attributed to [C6H5CO] + situated after MS-2. The resulting spectra are shown in Figures and m/z 149, attributed to [C6H5CO + C2H4O] . A peak at m/z + 2-4. 77 is attributed to the aryl ion [C6H5] .

3640 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999 the lower energy orthogonal TOF CID.26 The internal fragment m/z 53 corresponds to a sodiated fraction of the repeat unit, the formation of which would again be expected to be a high-energy process. The fragment m/z 53 (and other members of the C series) is not significant in lower energy CID. A second relevant comparison is between the CID spectra (Figures 2-4) of MALDI- Figure 5. Diagram showing sites of bond dissociation to produce generated ions at high energy and the CID spectra at high energy A and B fragment ions. of sodiated PEGs formed by field desorption.38,39 These field desorption spectra38,39 resemble those at low energy when the In addition to fragments arising from decomposition of the sodium-containing ions are formed by MALDI. The conclusion polymer end groups, three repeating series of fragment ions were to be tentatively drawn is that the energy driving the fragmentation observed. These are shown in Figure 2 and are labeled A, B, and following MALDI and CID at 8 keV derives to a significant extent C. from both the ionization process and the collision process. It would The repeating series of fragment ions labeled A and B in Figure then be the combination of global energization during ionization 2 were consistent with the fragmentation pathways of adducts of and local energization by collision that leads to the formation of PEG with ions proposed on the basis of LSIMS tandem the especially structurally informative fragment ions, elucidating 40 mass spectrometry,37 utilizing a marginally lower energy regime both end groups and repeat units. Preliminary experiments with - (8 keV). Fragments A and B are proposed to arise from dissocia- the PEGs 1 3 using Fourier transform ion cyclotron resonance tion of a -oxygen bond in the poly(ethylene glycol) (FTICR) and sustained off-resonance irradiation (SORI) have backbone. The m/z’s of these fragments were observed to change, confirmed that the low-mass fragments are absent from these low- as expected, with end group (Figure 5). energy tandem mass spectra of the sodiated molecules. A series of [M + Na]+ fragments, labeled C, was observed in Matrix-assisted laser desorption/ionization and time-of-flight all spectra at m/z (53 + 44n). Formally, this results from cleavage mass spectrometry have led to the development of several of the polymer backbone without retention of the end groups. This commercial instruments used in the identification of synthetic series was very intense at low m/z with the acetyl end-terminated polymers. Structural information so far has been provided by PEG, but became weaker at higher m/z and was not observable means of metastable decay and collision-induced dissociation in < above m/z 400. The m/z 53 peak was intense in all spectra. the low-energy region ( 1000 eV) because of design and funda- The distinguishing feature of the high-energy (short interaction mental limitations. time) CID spectra (Figures 2-4) compared to lower energy The selected results shown here demonstrate that high-energy spectra from magnetic-sector/orthogonal TOF instruments is the collision-induced dissociation performed on MALDI-generated ions high intensity of low-mass fragments. These intense low-mass can provide unambiguous structural and end-group information fragment ions fall into two categories of fragments: end groups on synthetic polymers. and internal fragments. The end-group fragments do not contain ACKNOWLEDGMENT sodium, indicative of their formation being due to high-energy The authors thank Unilever for their generous financial processes. These sodium-free ions are much less significant in support. The support of the Engineering and Physical Sciences Research Council is gratefully acknowledged (GR/K38892). (37) Selby, T. L.; Wesdemiotis, C.; Lattimer, R. P. J. Am. Soc. Mass Spectrom. 1994, 5, 1081-1092. (38) Agma, M. Ph.D. Thesis, University of New South Wales, 1989. Received for review May 18, 1999. Accepted June 8, 1999. (39) Craig, A. G. Ph.D. Thesis, University of New South Wales, 1984. (40) Bottrill, A. R.; Feng, X.; Wallace, J.; Derrick, P. J., unpublished results. AC990523T

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