STRUCTURAL CHARACTERIZATION OF SYNTHETIC POLYMERS AND COPOLYMERS USING MULTIDIMENSIONAL MASS SPECTROMETRY

Transcription

1 STRUCTURAL CHARACTERIZATION OF SYNTHETIC POLYMERS AND COPOLYMERS USING MULTIDIMENSIONAL MASS SPECTROMETRY INTERFACED WITH THERMAL DEGRADATION, LIQUID CHROMATOGRAPHY AND/OR ION MOBILITY SEPARATION A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Nadrah Alawani December, 2013

2 STRUCTURAL CHARACTERIZATION OF SYNTHETIC POLYMERS AND COPOLYMERS USING MULTIDIMENSIONAL MASS SPECTROMETRY INTERFACED WITH THERMAL DEGRADATION, LIQUID CHROMATOGRAPHY AND/OR ION MOBILITY SEPARATION Nadrah Alawani Dissertation Approved: Advisor Dr. Chrys Wesdemiotis Committee Member Dr. Michael J. Taschner Committee Member Dr. Peter L. Rinaldi Committee Member Dr. Matthew Espe Accepted: Department Chair Dr. Michael J. Taschner Dean of the College Dr. Chand K. Midha Dean of the Graduate School Dr. George R. Newkome Date Committee Member Dr. Yu Zhu ii

3 ABSTRACT This dissertation focuses on coupling mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to thermal degradation, liquid chromatography (LC) and/or ion mobility (IM) spectrometry for the characterization of complex mixtures. In chapter II, an introduction of the history and the principles of mass spectrometry (MS) and liquid chromatography (LC) are discussed. Chapter III illustrates the materials and instrumentation used to complete this dissertation. Polyethers have been characterized utilizing tandem mass spectrometry (MS/MS), as presented in Chapter IV and Chapter VI. Diblock copolymers of polyethylene oxide and polycaprolactone, PEO-b-PCL, have been characterized by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry (MALDI-Q/ToF) and LC-MS/MS (Chapter V). Thermoplastic elastomers have been characterized by thermal degradation using an atmospheric solids analysis probe (ASAP) and ion mobility mass spectrometry (IM-MS), as discussed in Chapter VII. Interfacing separation techniques with mass spectrometry permitted the detection of species present with low concentration in complex materials and improved the sensitivity of MS. In chapter IV, the fragmentation mechanisms in MS/MS experiments of cyclic and linear poly(ethylene oxide) macroinitiators are discussed. This study aimed at determining the influence of end groups on the fragmentation pathways. In the study iii

4 reported in Chapter V, ultra high performance liquid chromatography (UHPLC) was interfaced with MS and MS/MS to achieve the separation and in-depth characterization and separation of amphiphilic diblock copolymers (PEO-b-PCL) in which the architecture of the PEO block is linear or cyclic. Applying UPLC-MS and UPLC-MS/MS provides fast accurate information about the number and type of the blocks in the copolymers. Chapter VI reports MS/MS and IM-MS analyses which were performed to elucidate the influence of molecular size and collision energy on the fragmentation pathways of polyethers subjected to collisionally activated dissociation (CAD). Survival yields and collision cross-sections were derived for several oligomers of polybutylene oxide (PBO) and polytetrahydrofuran (PTHF) polymers by MS/MS and IM- MS, in order to understand their fragmentation energetics and fully characterize their structures. In Chapter VII, application of atmospheric solids analysis probe (ASAP) and ion mobility (IM) separation were coupled with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize commercially available thermoplastic elastomers. These compounds are mainly composed of thermoplastic copolymers, but also contain additional chemicals to enhance their properties or to protect them from degradation. Using ASAP-IM-MS enables fast analysis, involving mild degradation at atmospheric pressure (ASAP) and subsequent characterization of the desorbates and pyrolyzates by ion mobility mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS). Such multidimensional dispersion considerably simplifies the resulting spectra, permitting the conclusive separation and characterization of the multicomponent materials examined. iv

5 Chapter VIII summarizes the findings of this dissertation and is followed by appendices with supplemental data and the copyright permissions obtained for this dissertation. v

6 ACKNOWLEDGEMENTS First, thanks to my God (Allah) for my life through all tests in the past years. You have made my life more bountiful. May your name be exalted, honored, and glorified. I would like to express my gratitude to all those who gave me the possibility to complete this dissertation. I want to thank the Department of Chemistry at The University of Akron for giving me permission to commence this dissertation and to do the necessary research work. I especially want to thank my supervisor Prof. Chrys Wesdemiotis for his assistance, support and guidance during my research and study at The University of Akron. His perpetual energy and enthusiasm in research has motivated all his advisees, including me. In addition, he was always accessible and willing to help his students with their research. As a result, research life became smooth and rewarding for me. Dr. Michael J. Taschner, Dr. Peter Rinaldi, Dr. Matthew Espe and Dr. Yu Zhu deserve special thanks as my dissertation committee members. I would like to thank them for their time, precious assistance and invaluable comments. I would like also to acknowledge the faculty and staff of the Department of Chemistry for their assistance in my coursework. I am grateful to The Ministry of Higher Education in Saudi Arabia for granting me a scholarship to pursue my graduate degree in The University of Akron. vi

7 All my lab co-workers made it a pleasant place to work. In particular, I would like to thank some of the former group members; Dr. Michael Polce, Dr. Jody Modarelli, Dr. Sara Whitson, Dr. Nilüfer Solak, Dr. Aleer Yol, Dr. Bryan Katzenmayer, Dr. Vincenzo Scionti, Dr. Danijela Smiljanic, Dr. Madalis Casiano-Maldonado, Dr. Chunxiao Shi, Nhu Quynh Nguyen and the current group members; Xiumin Liu, Ahlam Alalwiat, Kai Guo, Michelle Kushnir and Lydia Cool. All these folks have inspired me in research and in life through our interactions during the long hours in the lab. Thanks. I want to thank Dr. Coleen Pugh, Dr. Li Jia, Dr. Gladys Montenegro, and Nishant Kumar for all the input, advice and for providing many samples which are part of this dissertation. I would like to give my special thanks to my husband, Mr. Saleh Alawami, whose patient love enabled me to complete this work. My deepest gratitude goes to my family for their unflagging love and support throughout my life; this dissertation is simply impossible without them. vii

8 TABLE OF CONTENTS Page LIST OF TABLES....xii LIST OF FIGURES......xii LIST OF SCHEMES....xix ACRONYMS AND ABBREVIATIONS...xxi CHAPTER I. INTRODUCTION...1 II. INSTRUMENTAL METHODES BACKGROUND Mass Spectrometry Ionization Methods Electrospray Ionization (ESI) Matrix Assisted Laser Desorption/Ionization (MALDI) Atmospheric Solids Analysis Probe (ASAP) Mass Analyzers Quadrupole Mass Analyzer Time-of-Flight Mass Analyzer Quadrupole Time-of-Flight (Q/ToF) Mass Analyzer viii

9 2.3.4 Quadrupole Ion Trap Mass Analyzer Detectors Ion Mobility Mass Spectrometry (IM-MS) Liquid Chromatography Mass Spectrometry (LC-MS) Columns and Stationary Phases Tandem Mass Spectrometry (MS/MS) III. MATERIALS AND INSTRUMENTATION Materials Instrumentation HCT Ultra II ESI-QIT Mass Spectrometer Ultraflex III ToF/ToF Mass Spectrometer MALDI-Q/ToF Mass Spectrometer Synapt HDMS Ion Mobility Mass Spectrometer Acquity UPLC...51 IV. STRUCTURAL CHARACTERIZATION OF CYCLIC AND LINEAR POLY(ETHYLENE OXIDE) MACROINITIATORS USING MULTIDIMENSIONAL MASS SPECTROMETRY Background Sample Preparation and Instrumentation Used Characterization of PEO Macroinitiators Characterization of 3,4-(42-Crown-14) Benzaldehyde PEO by ESI- QIT MS Characterization of 3,4-(42-Crown-14) Benzyl Alcohol PEO by ESI- QIT MS Characterization of PEO Methyl Ether, (4-hydroxymethyl)Phenyl Ether by ESI-QIT MS ix

10 4.4 Conclusions V. INTERFACING MASS SPECTROMETRY WITH LIQUID CHROMATOGRAPHY SEPARATION FOR THE CHARACTERIZATION OF POLY ETHYLENE OXIDE- POLYCAPROLACTONE (PEO-b-PCL) DIBLOCK COPOLYMERS Background Sample Preparation and Instrumentation Used Characterization of PEO-b-PCL Diblock Copolymers Using MALDI-ToF MS and UPLC-ESI-Q/ToF MS/MS Characterization of CH 3 -PEO-b-PCL Diblock Copolymer Characterization of ω-benzyl Alcohol PEO-b-PCL Diblock Copolymer Characterization of 3,4-(42-Crown-14) Benzyl Alcohol PEO-b-PCL Diblock Copolymer Conclusions VI. TANDEM MASS SPECTROMETRY OF POLYETHERS - SIZE AND COLLISION ENERGY EFFECTS Background Sample Preparation and Instrumentation Used Mass Spectrometry and Tandem Mass Spectrometry of Polyethers MS and MS n of Lithiated Polytetrahydrofuran ([PTHF+Li] + ) MS and MS n of Lithiated Poly(1,2-Butylene Oxide) ([PBO+Li] + ) MS and MS n of Lithiated Poly(1,2-Propylene Oxide) ([PPO+Li] + ) Evaluation of Dissociation Energetics of PTHF, PPO, and PBO by Energy- Resolved Tandem Mass Spectrometry x

11 6.5 Evaluation of the Collision Cross-Sections of PTHF, PBO, and PPO Using IM-MS Conclusions VII. ANALYSIS OF THERMOPLASTIC COPOLYMERS BY MILD THERMAL DEGRADATION COUPLED TO ION MOBILITY MASS SPECTROMETRY Background Sample Preparation and Instruments Used Thermoplastic Polyurethanes ASAP-IM-MS of Elastollan (PU-1) ASAP-IM-MS of Pellethane (PU-2) ASAP-IM-MS of Estane (PU-3) Thermoplastic Styrenic Copolymer Elastomers ASAP-IM-MS of SB-1 Elastomer Conclusions VIII. SUMMARY REFERENCES APPENDICES APPENDIX A. ADDITIONAL DATA APPENDIX B. COPYRIGHT PERMISSIONS xi

12 LIST OF TABLES Table Page 5.1 PEO-b-PCL components identified in the LC-MS fractions by the corresponding mass spectra and select MS/MS spectra PEO-b-PCL components identified in the LC-MS fractions of ω-bnpeo-b-pcl by the corresponding mass spectra and select MS/MS spectra PEO-b-PCL components identified in the LC-MS fractions of McPEO-b-PCL by the corresponding mass spectra and MS/MS spectra Mass (in Da) and center-of-mass collision energy for 50% SY (E 50 in ev) of the singly lithiated PTHF, PBO, and PPO oligomers investigated by energy-resolved MS/MS Collision cross-sections of sodiated PTHF, PBO, and PPO oligomers Collision cross-sections of lithiated PTHF, PBO, and PPO oligomers 148 xii

13 LIST OF FIGURES Figure Page 2.1 Generic instrument schematic of a mass spectrometer (A) Droplet formation at the needle tip of the ESI capillary; (B) decomposition of the droplet in the electrospray source according to Rayleigh s equation Schematic of the Coulombic explosion of a charged droplet Illustration of the MALDI process Illustration of atmospheric solids analysis probe, ASAP Schematic illustration the ionization mechanisms of ASAP Schematic representation of the quadrupole mass analyzer Schematic of a linear ToF mass analyzer Schematic of a reflectron ToF mass analyzer Schematic of the delayed extraction principle for ToF mass analyzer Schematic of the quadrupole ion trap mass analyzer Stability diagram for a QIT, in which four singly charged ions with the masses m 4 < m 3 < m 2 < m 1 were injected. If no dc is used (U=0 and a z =0), ion trajectories are determined only by the rf field (q z ). Ions with q z <0.908 remain trapped (m 1, m 2, m 3 ) and ions with q z >0.908 are ejected (m 4 ). The low-mass cut off the QIT is calculated from the equation q z = Schematic of a microchannel plate detector and the electron multiplication within the channels Schematic of a Daly detector Schematic of The Waters Synapt HDMS Q/ToF mass spectrometer 2006 Waters Corporation Schematic of the triwave section of the Synapt HDMS system Schematic of the operation of a traveling wave ion guide containing ring electrodes for transferring the ions through the buffer gas.34 xiii

14 2.18 Schematic of the basic components of an HPLC-MS system Van Deemter plot (dashed line) and individual plots of the terms of the van Deemter equation Schematic view of the Bruker HCT ultra II ESI-QIT mass spectrometer Schematic view of the Micromass Ultima MALDI-Q/ToF mass spectrometer Schematic view of the Acquity UPLC system ESI mass spectrum of lithium, sodium and potassium cationized 3,4-(42-crown-14) benzaldehyde PEO (M n 750) acquired with the QIT mass spectrometer CAD mass spectrum of the lithium cationized 13-mer of 3,4-(42-crown-14) benzaldehyde PEO obtained using ESI-QIT tandem mass spectrometry ESI-QIT MS 3 mass spectrum of the fragment ion at m/z 671 generated by CAD of m/z 699, the lithiated 3,4-(42-crown-14) benzaldehyde PEO 13-mer ESI-QIT MS 3 mass spectrum of the J 11 HH fragment ion at m/z 509 Da generated by CAD of m/z 699, the lithiated 3,4-(42-crown-14) benzaldehyde PEO 13-mer ESI mass spectrum of 3,4-(42-crown-14) benzyl alcohol PEO (M n 750 Da) acquired with the QIT mass spectrometer CAD tandem mass spectrum of the lithiated 13-mer of 3,4-(42-crown-14) benzyl alcohol, m/z 701, acquired with the ESI-QIT mass spectrometer ESI-QIT MS 3 mass spectrum of the fragment ion at m/z 683 generated by CAD of the lithiated 13-mer of 3,4-(42-crown-14) benzyl alcohol, m/z 701. A possible structure for the C 11 H 10 O 2 ion, which is 12 Da heavier bis(vinyloxy)benzene, is included as inset ESI mass spectrum of PEO methyl ether, (4-ydroxymethyl) phenyl ether, (M n 500), acquired with the QIT mass spectrometer CAD tandem mass spectrum of the lithiated 8-mer of PEO methyl ether, (4- hydroxymethyl) phenyl ether, m/z 497, acquired with the ESI-QIT mass spectrometer MALDI mass spectrum of sodium cationized PEO-b-PCL acquired on the MALDI- ToF/ToF mass spectrometer Expended view of molecular weight regions (I-III) in the MALDI mass spectrum of Figure 5.1. Roughly these regions correspond to PEO homopolymer (I) and PEO-b-PCL copolymer with shorter (II) or longer (III) PCL chains MALDI-MS/MS spectrum of the oligomer at m/z with the putative CH 3 - PEO 10 PCL 6 -OH structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 80 ev xiv

15 5.4 MALDI-MS/MS spectrum of the oligomer at m/z with the putative CH 3 - PEO 13 PCL 5 -OH structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 80 ev LC-MS total ion chromatogram of the CH 3 -PEO-b-PCL-OH diblock copolymer LC-MS mass spectra of LC fractions #5, #7, and #9 from the CH 3 -PEO-b-PCL-OH copolymer UPLC-MS/MS spectra of the ions at m/z in LC fraction #7 and in LC fraction #9. The collision energy was 60 ev LC-MS mass spectra of LC fractions #4, #6, and #8 from CH 3 -PEO-b-PCL-OH UPLC-MS/MS spectrum of the ion at m/z in LC fraction # 6. The collision energy was 60 ev MALDI mass spectrum of sodium cationized ω-bnpeo-b-pcl, acquired on the MALDI-ToF/ToF mass spectrometer Expended views of molecular weight regions I and II in the MALDI mass spectrum of Figure MALDI-MS/MS spectrum of the sodiated oligomer at m/z with the putative PEO 11 PCL 6 diblock composition, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 70 ev MALDI-MS/MS spectrum of the sodiated oligomer at m/z with the putative H-PCL 10 -OH structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 65 ev MALDI-MS/MS spectrum of the oligomer at m/z with the putative NaO- PCL 9 -H structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 65 ev LC-MS total ion chromatogram of the ω-bnpeo-b-pcl diblock copolymer LC-MS mass spectra of LC fractions #8, #10, and #12 from the ω-bnpeo-b-pcl diblock copolymer UPLC-MS/MS spectrum of the ion at m/z in LC fraction #10 and of the ion at m/z in LC fraction #12. The collision energy was 60 ev MALDI mass spectrum of sodium cationized McBnPEO-b-PCL, acquired on the MALDI-ToF/ToF mass spectrometer Expanded view of the MALDI mass spectrum shown in Figure Each copolymeric oligomer contains a C 7 H 5 O linking group and a OH end group at the PCL xv

16 chain end. The combined composition and mass of these substituents are C 7 H 6 O 2 and 112 Da, respectively MALDI-MS/MS spectrum of the sodiated oligomer at m/z with the putative diblock composition PEO 11 PCL 4, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 65 ev LC-MS total ion chromatogram of the McBnPEO-b-PCL diblock copolymer LC-MS mass spectra of LC fractions # 8, #10 and #11 from the McBnPEO-b-PCL diblock copolymer UPLC-MS/MS spectra of the ions at m/z in LC fraction #8 and the ion at m/z in LC fraction #10. The collision energy was 60 ev ESI mass spectrum of PTHF (M n 650) acquired with the QIT mass spectrometer. Lithium (A), sodium (B), and potassium (C) cationized oligomers are observed even though only LiTFA was added to the sample CAD mass spectrum of lithiated PTHF 8, m/z 601.7, obtained using the ESI-QIT mass spectrometer ESI-QIT MS 3 mass spectrum of the d 7 fragment ion (m/z 385.5) generated by CAD of the lithiated PTHF 8-mer ESI mass spectrum of PBO (M n 800) acquired with the QIT mass spectrometer, showing lithium (A), sodium (B) and potassium (C) cationized oligomers CAD mass spectrum of lithiated PBO 8, m/z 615.5, obtained using the ESI-QIT tandem mass spectrometer ESI-QIT MS 3 mass spectrum of the z 4 fragment ion (m/z 313.2) generated by CAD of the lithiated PBO 8-mer ESI mass spectrum of PPO (M n 1000) acquired with the QIT mass spectrometer, showing lithium, and sodium cationized oligomers in charge states 1+ and CAD mass spectrum of lithiated PPO 14, m/z 837.6, obtained using the ESI-QIT mass spectrometer ESI-QIT MS 3 mass spectrum of the b 13 fragment ion (m/z 761.5) generated by CAD of lithiated PPO 14-mer Fragmentation efficiency curves of the lithiated PTHF n (n = 7-10) oligomers, acquired using the Synapt ESI-Q/ToF instrument Fragmentation efficiency curves of the lithiated PBO n (n = 7-10) oligomers, acquired using the Synapt ESI-Q/ToF instrument..137 xvi

17 6.12 Fragmentation efficiency curves of the lithiated PPO n (n = 14-16) oligomers, acquired using the Synapt ESI-Q/ToF instrument Center-of-mass collision energy for 50% SY (E 50 ) vs. precursor ion mass for singly lithiated PPO oligomers a Two-dimensional IM-MS plot (m/z vs. drift time) of sodium cationized PTHF (M n 650) acquired with the Synapt Q/ToF mass spectrometer b Two-dimensional IM-MS plot (m/z vs. drift time) of sodium cationized PBO (M n 800) acquired with the Synapt Q/ToF mass spectrometer c Two-dimensional IM-MS plot (m/z vs. drift time) of sodium cationized PPO (M n 1000) acquired with the Synapt Q/ToF mass spectrometer a Mass spectrum extracted from the circled region in the ion mobility map of sodium cationized PTHF b Mass spectrum extracted from the circled region in the ion mobility map of sodium cationized PBO c Mass spectrum extracted from the circled regions in the ion mobility map of sodium cationized PPO. The minor distributions in the singly charged region correspond to [HO-(CH 2 CH(CH 3 )O) n -CH=CH-CH 3 +Na] + and [KO-(CH 2 CH(CH 3 )O) n -CH=CH- CH 3 +Na] + ions Ion mobility chromatogram extracted from the sodiated 8-mer and 9-mer regions in the 2D IM-MS map of the 8 and 9 oligomers of PTHF and PBO and showing the drift time distributions of these ions Figure 6.17 Collision cross-sections of sodiated and lithiated PTHF, PBO, and PPO oligomers D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of elastollan (PU- 1) at 250 C; the mass spectra extracted from mobility regions a and b (in ovals) are depicted in Figure Mass spectra extracted from regions a and b in the 2D IM-MS diagram of Figure D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of elastollan (PU- 1) at 450 C without and with MeOH in the ASAP source; the mass spectra extracted from mobility regions a-d (in ovals) are depicted in Figure (a) Mass spectrum extracted from regions a in the 2D IM-MS diagram of Figure 7.3. (b) ASAP-IM-MS/MS spectrum of the oligomer at m/z ([M] + ) which agrees with a species composed of the hard segment (340 Da) plus two soft segment repeat units (2x72 Da). Both radical losses via simple bond cleavages (for example, m/z 413.2, 341.2, xvii

18 323.2) and losses of closed-shell species via H-rearrangements (m/z 340.2, 322.2) are observed Mass spectrum extracted from regions b in the 2D IM-MS diagram of Figure7.3. The ions observed are molecular radical ions (*), sodiated ions (%), or protonated ion (#, $) Mass spectrum extracted from regions c in the 2D IM-MS diagram of Figure 7.3. See Figure A20 for the corresponding MS/MS spectrum Mass spectra extracted from regions d in the 2D IM-MS diagram of Figure 7.3 under dry (top) and wet (bottom) conditions D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of pellethane (PU- 2) at 450 C Mass spectrum extracted from IM region a in the 2D ASAP-IM-MS plot depicted in Figure Mass spectrum extracted from IM region b in the 2D ASAP-IM-MS plot depicted in Figure Mass spectrum extracted from region c in the 2D IM-MS diagram of Figure D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of estane (PU-3) at 450 C; the mass spectra extracted from mobility regions a and b (in ovals) are depicted in Figures 7.13 and 7.14, respectively Mass spectra extracted from IM regions a of the 2D ASAP-IM-MS diagram of estane (PU-3) acquired at 450 C (Figure 7.12) Mass spectra extracted from IM regions b of the 2D ASAP-IM-MS diagram of estane (PU-3) acquired at 450 C (Figure 7.12)..174 Figure 7.15 ASAP-IM-MS/MS spectra of (a) the oligomer at m/z ([M+H] + ), which agrees well with the structure of a poly (butylene adipate) macrocycles and (b) the oligomer at m/z ([M] +), which agrees well with a poly (butylene adipate) chain capped with one hard segment unit D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of styroflex (SB- 1) at 250 C (top) and the mass spectrum extracted from mobility region c (bottom). See Figure A29-A30 for the mass spectra extracted from IM regions a-b D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of styroflex (SB- 1) at 450 C (top) and the mass spectrum extracted from mobility region e (bottom). The labels S n BD m give the number of complete styrene and butadiene units, respectively. A double prime indicates saturated end groups at both chain ends (as in Scheme 7.3). Otherwise, the end groups contain one additional unsaturation. All labeled species are [M+H] + ions xviii

19 LIST OF SCHEMES Scheme Page 4.1. a Synthetic route to 3,4-(42-crown-14) benzyl alcohol, a microcyclic PEO b Synthetic route to a linear PEO with methyl ether and (4-hydroxymethyl) phenyl ether chain ends macroinitiator Proposed fragmentation pathways of lithiated 3,4-(42-crown-14) benzaldehyde PEO Charge-induced fragmentation pathway of J n HH ions (dihydroxy end groups) Charge-remote fragmentation pathway of series J n HV (hydroxy /vinyl end groups) Proposed fragmentation pathways of lithiated 3,4-(42-crown-14) benzene alcohol PEO Charge-remote fragmentation of the lithiated ethyl radical arising after phenoxy radical loss via free radical intermediates Synthetic scheme to methoxy PEO-b-PCL (CH 3 PEO-b-PCL), ω-benzyl alcohol PEOb-PCL (ω-bnpeo-b-pcl), and 3,4-(42-crown-14) benzyl alcohol PEO-b-PCL (McBnPEO-b-PCL) Charge-remote 1,5-H rearrangement (left) or intramolecular transesterification (right) in the caprolactone ester block of CH 3 -PEO m PCL n -OH Synthetic routes to widely used polyethers Charge-induced fragmentation pathway of lithiated PTHF to truncated fragments with hydroxyl/hydrogen (e n ), vinyl/hydrogen (d n ) and vinyl/vinyl (j n ) end groups Charge-remote hemolytic C-O bond cleavages in lithiated PTHF, leading to fragments with hydroxyl/carbonyl (c n ) and alkene/hydroxyl end groups. The b n series generated by this mechanism differs in the double bond position from bn series shown in scheme 6.2, but both series appear at the same m/z values (isomers) and hence, for simplicity, were given the same acronym Dissocation of d n fragments from lithiated PTHF via (a) the charge-induced pathway in Scheme 6.2 or (b) charge-remote unzipping through 1,5-H rearrangements..124 xix

20 6.5 Charge-induced fragmentations of lithiated PPO according to the mechanism detailed in Scheme 6.2. Each ether bond joins a secondary and tertiary C-atom. Cleavage of the CH-O bond leads to b n or z n fragments, whereas cleavage of the O-CH 2 bond leads to c n and x n fragments. Because both chain ends carry the same substituent (HO), c n and z n are identical, and b n and x n are MS-indistinguishable isomers General composition of polyurethanes The general composition of Styrene-butadiene elastomers Families of species formed during the thermal degradation of styrene-butadiene copolymers. In the species shown both end groups are saturated. Species containing an additional unsaturation in either or both end groups are also possible; their end groups masses are 2 Da or 4 Da lower, respectively xx

21 ACRONYMS AND ABBREVIATIONS APCI ASAP CAD CCS CE CE CHCA CI CID DCTB DHB DIOS DIT ECD EI EM EO EOID Atmospheric Pressure Chemical Ionization Atmospheric Solids Analysis Probe Collisionally Activated Dissociation Collision cross section Capillary Electrophoresis Collision Energy α cyano 4 hydroxycinnamic acid Chemical Ionization Collision Induced Dissociation trans 2 (3 (4 tert butylphenyl) 2 methyl 2 propenyliedene)malononitrile 2,5 dihydroxybenzoic acid Desorption/Ionization on Silicon Dithranol Electron Capture Dissociation Electron Ionization Electron Multiplier Ethylene Oxide Electro Optical Ion Detectors xxi

22 ESI ETD FAB FI FT ICR FWHM GC GPC HETP HPLC ICR IM IM MS IMS LAC LACCC LC LC MS LIT m/z MALDI MCP Electrospray Ionization Electron Transfer Dissociation Fast Atom Bombardment Field Ionization Fourier Transform Ion Cyclotron Resonance Full Width at Half Maximum Gas Chromatography Gel Permeation Chromatography Height Equivalent to a Theoretical Plate High Performance Liquid Chromatography Ion Cyclotron Resonance Ion Mobility Ion Mobility Mass Spectrometry Ion Mobility Spectrometry Liquid Adsorption Chromatography Liquid Adsorption Chromatography under Critical Conditions Liquid Chromatography Liquid Chromatography Mass Spectrometry Linear Ion Trap Mass to Charge Ratio Matrix Assisted Laser Desorption Ionization Multichannel Plate xxii

23 MS MS/MS MW MWD NMR oa PBO PDMS PEG PEO PMMA ppm PPO PSD PTHF PU Q Q/ToF QIT QqQ ROP RP LC Mass Spectrometry Tandem Mass Spectrometry Molecular Weight Molecular Weight Distribution Nuclear Magnetic Resonance Orthogonal Acceleration Poly(1,2-butylene oxide) Poly(Dimethyl Siloxane) Poly(Ethylene Glycol) Poly(Ethylene Oxide) Poly(Methyl Methacrylate) Parts Per Million Poly(1,2-propylene oxide) Post Source Decay Polytetrahydrofuran Polyurethane Quadrupole Quadrupole Time of Flight Quadrupole Ion Trap Triple Quadrupole Ring Opening Polymerization Reverse Phase Liquid Chromatography xxiii

24 SB SEC SFC SID SY TBE TFA TIC ToF ToF/ToF TPU TWIMS UHPLC Polystyrene and polybutadiene Size Exclusion Chromatography Supercritical Fluid Chromatography Surface Induced Dissociation Survival Yield Thermoplastic elastomer trifluroacetic acid Total Ion Chromatogram Time of Flight Tandem Time of Flight Thermoplastic polyurethane Traveling Wave Ion Mobility Spectrometry Ultra High Performance Liquid Chromatography xxiv

25 CHAPTER I INTRODUCTION Mass spectrometry (MS) is a powerful analytical technique not only for biological samples but also for synthetic materials due to its high sensitivity, low sample consumption, and speed of analysis. In order to determine the composition and the identity of an analyte, this analytical technique measures the mass-to-charge ratio (m/z) of the analyte or its fragments. In MS, gas-phase ions must be generated from the analyte in order to obtain a spectrum. 1 Electrospray ionization (ESI) 2, 3 and matrix assisted laser desorption/ionization (MALDI) 4-6 are considered as soft ionization techniques that enable the classification and characterization of synthetic polymers, because they can form intact gas-phase ions from such molecules. As a result, these two techniques have been used widely in structural and compositional studies of synthetic polymers. 7, 8 Although mass spectrometry is a powerful characterization technique, it suffers from limitations, for example, in the ionization of complex mixtures, such as copolymers, or of high molecular weight polymers, which may be impossible to analyze using MS only. Mass spectrometry (MS) separates ions based on their mass-to-charge ratio. Therefore, MS is unable to distinguish isomeric or even isobaric species. These problems are common with synthetic materials, which may consist of polymers, copolymers and sometimes additives to protect them from degradation. To overcome this limitation, MS 1

26 can be extended to tandem MS, which includes two stages of mass analysis (MS/MS) or coupled to pyrolysis or a separation technique such as size exclusion chromatography (SEC), gas chromatography (GC), capillary electrophoresis (CE), liquid chromatography (LC), or ion mobility spectrometry (IM). 1, 9 The resulting multidimensional technique can provide synthetic researchers with in-depth information about the composition, purity, the presence of additives, and degradation products of the sample analyzed. 7 Interfaced separation/mass spectrometry techniques have been applied to the characterization of polymeric materials. After the invention of soft ionization methods (ESI and MALDI), applications of mass spectrometry to polymers and copolymers have increased. In MALDI, a laser is used to irradiate a mixture of analyte and matrix which is a small organic molecule that absorbs the laser light and prevents the aggregation of the analyte molecules. This process transfers energy from the laser to the matrix leading to ionization of the analyte. 4-6 In ESI, a solution of the analyte in a volatile solvent is prepared and then introduced by a syringe into the mass spectrometer. 2, 3 Application of these techniques provides information about the molecular masses of the components in the polymer. Mass analysis of the ions formed by MALDI or ESI is called single stage mass spectrometry; the measured masses can be used to determine the mass of the monomer unit, the number of monomers present in the detected oligomers, and the end groups. By performing two or more mass analysis measurements using tandem mass spectrometry methods (MS/MS), more information about the molecular structure under examination can be obtained. In these experiments, collisionally activated dissociation (CAD) or collision-induced dissociation (CID) is commonly employed to cause fragmentation of a selected molecular 2

27 ion, and the resulting fragments are mass-analyzed in a second mass analysis step. Fragmentation of the selected molecular ion may take place in space or in time. Tandem mass spectrometry in space is preformed when two or more mass analyzers are coupled, usually in ion beam instruments; in contrast, tandem mass spectrometry in time is preformed in the same mass analyzer but over time using trap instruments. 1 The ion of interest, often called the precursor ion, is selected and its kinetic energy is increased to induce fragmentation by collision with neutral gas molecules. The resulting fragments are then analyzed to give clues about the structure of the examined molecule. 13 The fragments formed from an ionized molecule give information about the composition and structure of that molecule. As a result, tandem mass spectrometry has been a very useful tool to determine end groups and architectures of polymers. 21 In CAD, a fraction of the kinetic energy gained by ion acceleration (E lab ) is converted to internal energy causing bond breakage. The maximum internal energy that can be gained at a given E lab is equal to the corresponding center-of-mass collision energy (E CM ) during this process and causes bond breakage. It is important to elucidate how the precursor ion size and the internal energy gained in the collisions influence fragmentation pathways. Liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful analytical technique that combines the strength of LC and MS techniques. LC is used to separate nonvolatile mixtures into their components based on their physical or chemical interactions with the mobile phase and the stationary phase. 24 However, with common UV/Vis, refractive index, or light scattering detectors, LC lacks the specificity needed to identify chemical compound structures. This limitation can be overcome using MS which is suitable for molecular identification. Interfacing of LC and MS provides a powerful 3

28 22, 24 analytical tool with high sensitivity and specificity to analyze complex compounds. The most common LC method utilized to characterize synthetic polymers is SEC in which the polymers are separated based on their size (hydronamic volume). A different LC method is high pressure (performance) liquid chromatography (HPLC) which separates mixture components based on their polarity and their interaction and distribution between the stationary and mobile phases. Recently, LC has been coupled with ESI which allows the eluted fractions to be directly introduced to the ESI source where the mobile phase acts as the solvent The components of the mixture elute from the LC column based on their polarity and then are separated in the mass spectrometer based on their m/z ratio. Reverse phase (RP) liquid chromatography, where a polar mobile phase is used, is the method most easily coupled to ESI-MS because ESI requires protic or polar solvents. 3 In order for a sample to be analyzed by mass spectrometry, it needs to be ionized. There are two ionization techniques that are widely used for biological and synthetic materials. These two techniques are matrix assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI), which have the ability to generate intact, high-mass molecular ions with no or little fragmentation. Nevertheless, numerous synthetic polymers are still hard to analyze by ESI or MALDI mass spectrometry because they have very high molecular weight and/or very low polarity to gain charge by attachment of an atomic or small molecular ion. Such polymers can, however, be classified and characterized after thermal degradation, i.e. pyrolysis, or another type of degradation like hydrolysis. Pyrolysis is a technique using thermal energy to break down chemical bonds. 29, 30 Structural information about polymers not amenable to MALDI or ESI can be 4

29 obtained from fragments observed after pyrolysis. Most useful is information obtained using mild thermal degradation. Under these conditions, the initial pyrolyzates do not undergo extensive further decomposition. Mild pyrolysis followed by MS characterization of the resulting products allows one to determine the components of the original polymers. Such mild pyrolysis can be performed with an atmospheric solids analysis probe (ASAP), attached to a mass spectrometer. ASAP, first described by McEwen et al. in , is a sensitive technique that provides rapid and useful information about the composition of low-polarity and/or high molecular weight polymers. In this technique, hot nitrogen is used to thermally degrade the sample; the degradation products are then ionized by atmospheric pressure chemical ionization 31, 32 (APCI). Ion mobility spectrometry (IMS) is an analytical technique that separates ions based on their mobility against a buffer gas flow. 33 Separation of ions in an IMS tube takes place according to the ions size, charge, and interaction with the buffer gas. 34 A hyphenated technique that couples mass spectrometry with the separation capability of ion mobility (IM-MS) is available on the Waters Synapt HDMS mass spectrometer which is a commercially available mass spectrometer equipped with the traveling wave (Twave) variant of IMS. This instrument has a quadrupole/time-of-flight (Q/ToF) configuration and T-wave chamber between the Q and ToF analyzers. Ions created in the ionization source are separated by their mobilities in the T-wave chamber, where they drift against the flow of a buffer gas (N 2 ) under the influence of a pulsed electric field, and the mobility-separated ion are then sent to the mass analyzer. 35, 36 IM-MS has been applied to separate isomers, isobars, and conformers from a wide range of molecules 5

30 including biomaterials and synthetic materials Interfacing ASAP with IM-MS provides a means to rapidly identify multicomponent industrial materials. In this dissertation, the described multidimensional techniques have been optimized for applications to synthetic materials and tested with the characterization of materials that are challenging to analyze by other methods. In chapter II, an introduction of the history and the principles of mass spectrometry and liquid chromatography will be presented. Chapter III describes the materials and instrumentations used to complete this dissertation. The following four chapters are research project chapters. Chapter 8 summarizes the conclusions drawn from this dissertation. In chapter IV, fragmentation mechanisms of cationized cyclic and linear poly(ethylene oxide) macroinitiators are discussed. This study aimed at deciphering the influence of end groups on the fragmentation pathways of such polymers, cationized by a lithium salt which improves the fragmentation efficiency compared to the commonly used cationization by sodium salt. In Chapter V, ultra performance liquid chromatography (UPLC) was interfaced with MS and MS/MS to achieve the separation and in-depth characterization of amphiphilic diblock copolymers (PEO-b-PCL) in which the architecture of the PEO block was linear or cyclic. These copolymers were synthesized to form micelles in aqueous solvent. During synthesis, the starting materials often do not react completely and may be retrained as byproducts which can affect the micelles properties. Van 6

31 Leeuwen et al. reported that HPLC coupled with ESI-MS can be used to separate linoleic acid functionalized surfactants prepared from block copolymers of methyl polyethylene oxide (PEO) and poly(ε-caprolactone) PCL. 26 In other studies, Ahmed et al. reported the use of RP-HPLC and MALDI-ToF-MS for the analysis of PEO-PCL block copolymers. 37, 38 In these studies, oligomer separation was achieved by RP-HPLC according to the number of CL units. The fractions were collected and subjected to MALDI-ToF-MS to confirm the EO composition and molecular weight distribution of each fraction. The run time of their HPLC experiments was more than 40 min. Applying UPLC-MS and UPLC- MS/MS should provide faster, superior information to identify the types and sizes of the blocks in such copolymers. In Chapter VI, MS/MS and IM-MS analyses were preformed to elucidate the influence of ion size and collision energy variables on the CAD fragmentation pathways of poly(tetrahydrofuran) (PTHF), poly(1,2-proplene oxide) (PPO) and poly(1,2-butylene oxide) (PBO). Survival yields were calculated for several oligomers of PBO and PTHF cationized by lithium to compare the fragmentation energetic of the two polymers. IM- MS analyses were also utilized to determine the collisional cross sections of PBO and PTHF oligomers. The results from the IM-MS and fragmentation studies were combined to characterize the chemical properties of these polyethers. In Chapter VII, mild thermal degradation with the atmospheric solids analysis probe (ASAP) and ion mobility (IM) separation were coupled with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize commercially available thermoplastic elastomers. These elastomers are used in the manufacture of a wide variety 7

32 of packaging materials. Such compounds are mainly composed of thermoplastic copolymers, but also contain additional chemicals ( additives ), like antioxidants and UV stabilizers, for enhancement of their properties or protection from degradation. The traditional method for analyzing such complex mixtures has been by vacuum pyrolysis followed by electron or chemical ionization mass spectrometry (Py-EI/CI-MS), often after gas chromatography (GC) separation The technique used in this dissertation is an alternative, faster approach, involving mild degradation at atmospheric pressure (ASAP) and subsequent characterization of the desorbates and pyrolyzates by ion mobility mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS). As will be shown, such multidimensional dispersion considerably simplifies the resulting spectra, permitting the conclusive separation and characterization of the multicomponent materials examined. 8

33 CHAPTER II INSTRUMENTAL METHODS BACKGROUND 2.1 Mass spectrometry Mass spectrometry (MS) is an analytical method that is utilized widely in a variety of fields including polymers, drug discovery, natural products, proteomics, and metabolomics, pharmaceutical, environmental, and forensic science. This method is applied to determine the structure of individual substances or complex mixtures. To obtain a spectrum, the analyte molecules are converted to gas-phase ions which are separated according to their mass-to-charge ratio (m/z). Every mass spectrometer consists of five main components: inlet system, ion source, mass analyzer, ion detector and data system (Figure 2.1). 1 Mass analyzers and detectors are held under vacuum. The ion source can be held at ambient pressure or under vacuum ( torr). The vacuum is used to prevent collisions of the ions with other gaseous molecules. 1 Inlet system Ion source Mass analyzer Detector Vacuum system Data system Figure 2.1 Generic instrument schematic of a mass spectrometer. 9

34 The sample inlet system is used to introduce the sample to the mass spectrometer by direct injection or chromatography. After insertion, the sample is converted into gasphase ions in the ion source. The ions then are separated according to their mass-tocharge ratio (m/z) in the mass analyzer. The separated ions are detected by measuring their current and abundance with the detector, and converting them into electric signals. 1, 39 The data system is used to record these signals and produce a mass spectrum. Different ionization sources and mass analyzers are used within the field of mass spectrometry. The ionization methods and mass analyzers used to complete this dissertation will be discussed in-depth in the following sections. 2.2 Ionization Methods Ion creation is the most important step in mass spectrometric analysis. Success in the creation of ions from the analyte studied leads to successful analysis. This step precedes the separation of the ions in the mass analyzer based on their m/z. There are several methods to create gas-phase ions. They include charge transfer from charged species in the gas phase, electron capture, electron ejection, proton capture, proton ejection, as well as cationization of neutral species in the ion source. 40 The appropriate ionization method is selected based on the analyte of interest. Ionization methods can be classified to two types, hard and soft. In hard ionization, the analyte is extensively fragmented; as a result, the intact analyte peak is hardly observed. Hard ionization methods were initially used in mass spectrometry and these include electron ionization 10

35 (EI), chemical ionization (CI), fast atom bombardment (FAB), and field desorption (FD). Fragmentation of the analyte can help to determine the structural composition of the analyte but it can also complicate the spectrum and make it hard to interpret. This limitation has been overcome by the evolution of soft ionization methods. In soft ionization, the intact molecular ion species is detected with minimal or no fragmentation. 1, 7 Such methods include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). These two methods are widely used for the characterization of large biomolecules and synthetic polymers. In this chapter, only MALDI, ESI, and the atmospheric solids analysis probe (ASAP) will be discussed, which were the ionization methods used to complete the studies described in this dissertation Electrospray ionization (ESI) Electrospray ionization (ESI) is a soft ionization method that was first interfaced with MS in the late 1960s by Dole et al. 41 The modern ESI procedure used currently was developed by John Fenn in the late 1980s. He used ESI to characterize large protein molecules that produced multiply charged ions. 2, 3 In ESI, gas-phase ions are generated from a solution at ambient pressure under the influence of a high electric field. There are four steps in the ESI process: ion formation, nebulization, desolvation and ion desorption. 1, A solution of the sample is introduced in the ion source using a metal needle by means of either a syringe pump or an exit of an LC column. A strong electric field (2-6 kv) and nebulizer gas, usually nitrogen, are applied at the needle tip which induce charge accumulation on the surface of the analyte solution sprayed. This step 11

36 produces highly charged droplets from the solution. The droplets then take the shape of a Taylor cone and release smaller droplets because the repulsive forces between the accumulated charges on the droplet are usually higher than the surface tension (Figure 2.2). Figure 2.2 (A) Droplet formation at the needle tip of the ESI capillary (reproduced with permission from reference 42); (B) decomposition of a droplet in the electrospray source according to Rayleigh s equation (reproduced with permission from reference 1). When the repulsive forces between charges become equal to the droplet surface tension, i.e. at the Rayleigh limit, the charged droplets decompose. This decomposition is described by the Rayleigh equation (Equation 2.1) where q is the charge, ε 0 the permittivity environment, γ the surface tension, and D the diameter of a spherical droplet. 12

37 The decomposition of the droplets can occur before the Rayleigh limit because the 1, electric field mechanically distorts these droplets. q 2 =8π 2 ε 0 γd 3 (Equation 2.1) Solvent evaporation from the droplets is promoted by the coaxial flow of a heated dry gas, usually nitrogen. The droplets undergo shrinkage as the solvent evaporates. Charge remains the same while the droplets become smaller which increases their charge per volume unit and the electrostatic tension at the surface of the droplets. At the Rayleigh limit, where the charge repulsion becomes equal to the surface tension that holds the droplet together, the droplet forms a Taylor cone which then undergoes Coulombic explosion to form smaller droplets (Figure 2.3). The process of solvent evaporation and Coulombic explosion is repeated until a single ion with one or more charges remains. Alternatively, when the droplets become very small, the electric field on their surface is sufficiently high to allow the desorption of single ions with one or more charges. The ions formed by these processes are sent to the mass analyzer to determine their m/z values. Figure 2.3 Schematic of the Coulombic explosion of a charged droplet (adapted with permission from reference 40). 13

38 2.2.2 Matrix-Assisted Laser Desorption/Ionization (MALDI) MALDI is a soft ionization method which was first introduced in 1988 by Karas and Hillenkamp; 4, 5 soft laser desorption, which is similar to MALDI, was invented by Tanaka in the same year. 6 In MALDI, analyte ions remain intact in the gas phase, which facilitate the analysis of a wide range of large molecules such as proteins and polymers. The analyte is mixed with a large molar excess of a small organic compound, called the matrix. The matrix is used for many purposes. First, it absorbs laser energy at the laser wavelength used, which helps the analyte to desorb from the target which is a stainless steel plate. Second, it prevents the analyte from forming clusters. Third, it transfers protons to the analyte (if the analyte is basic enough to accept protons). Finally, it protects the analyte molecules from photo-induced decomposition. In some cases, a cationizing agent (e.g., Na +, Li +, Ag + ) is added to the mixture of analyte and matrix in order to promote ionization. This mixture is then deposited on the target and dried before insertion into MALDI source. In the MALDI process, the analyte is bombarded with a laser, usually a UV laser (e.g., N 2 or Nd:YAG laser), which leads to evaporation and ionization of the matrix. The matrix desorbs carrying with it the analyte form the surface of the target and creating a gas-phase plume that includes analyte molecules. The analyte molecules are subsequently ionized by gas phase proton transfer from the ionized matrix or cation transfer from the cationizing agent. 1 The MALDI process can be seen in Figure

39 Figure 2.4 Illustration of the MALDI process (adapted with permission from reference 1) Atmospheric Solids Analysis Probe (ASAP) Sample ionization using an atmospheric solids analysis probe (ASAP) was first reported by McEwen et al. in 2005; ASAP is a sensitive technique that provides rapid, direct, useful, and effective information about the composition of low-polarity and/or high molecular weight polymers. 31 ASAP requires only slight modification of commercial ESI or APCI ion sources. Volatile or semi-volatile liquid or solid materials analyzed by ASAP are ionized under atmospheric pressure chemical ionization conditions. 31, 32 Hot nitrogen is used to desorb volatile components from the sample and to thermally degrade the sample; the desorbates and degradation products are formed within an APCI source, where these are ionized under ambient pressure. Glass capillary tubes are used to insert the sample into the ASAP tip. 31, 32, 45 An illustration of the ASAP device can be seen in Figure

40 Typically, the ASAP option is operated in a similar manner as an APCI source under standard APCI conditions and in some cases without any solvent flow. Horning described in 1973 the two primary mechanisms of APCI; proton transfer and charge exchange. 46 In a proton transfer reaction, the proton is transferred from a reagent ion, such as a protonated water or methanol cluster, to the analyte via an ion-molecule reaction. Nitrogen cations (N + 2 ) are first formed by electron impact with electrons created by corona discharge. These cations then react with nitrogen molecules to form N + 4 which can ionize, via charge transfer, water vapor present as moisture, to form H 2 O + ( or a different solvent fed into APCI source) to form ionized solvent molecules. Subsequent reactions of H 2 O + with water vapor create H + (H 2 O) n which will react with the analyte molecules to form protonated ions. In the absence of moisture or solvent, the charge transfer mechanism predominates. In this case, the nitrogen radical cations formed by corona discharge ionization undergo charge transfer with analyte molecules to generate radical cations of the analyte. 31, 46, 47 This form of ionization is particularly useful for the analysis of non-polar compounds. The cations formed by either mechanism are then moved to the mass analyzer to be separated based on their m/z values. Figure 2.6 illustrates the proton transfer and charge transfer mechanisms. 16

41 Figure 2.5 Illustration of atmospheric solids analysis probe, ASAP 2009 Waters Corporation. (adapted with permission from reference 45). Charge transfer N 2 2N 2 M Corona Discharge N 2 + N 4 + M + M + M Proton transfer 2N 2 H 2 O 2H 2 O M Corona Discharge N 4 + H 2 O + H 3 O + + OH [M+H] + Figure 2.6 Schematic illustration of the ionization mechanisms of ASAP. 2.3 Mass Analyzers The most important component of each mass spectrometer is the mass analyzer in which the gas-phase ions are separated according to their mass-to-charge ratio (m/z). Mass analyzers are usually held under vacuum to prevent the neutralization of the gas- 17

42 phase ions before they reach the detector. Neutralization may occur by collision of the ions with other gaseous molecules. There are two main categories of mass analyzers. The first category is the scanning analyzer which allows a selected mass range at a given time to be transmitted and reach the detector. The other category can disperse all the ions in space inside the mass analyzer and allow them to travel simultaneously to the detector. Each mass analyzer has its strengths and limitations based on its characteristic features. The main features are the mass range, transmission, and resolution. The mass range determines the lowest and highest m/z values that the mass analyzer is able to measure. Transmission can be described as the ratio of the number of ions reaching the detector and the number of ions entering the mass analyzer. Resolution can be described as the ability of the mass analyzer to separate two adjacent ions having small difference in m/z value. 1, 23 These features determine the type of experiments that can be performed and the analytes that can be studied. The mass analyzers used to complete this dissertation will be discussed in the following section. These analyzers include the quadrupole, quadrupole ion trap (QIT), time-of-flight (ToF), and quadrupole/time-of-flight (Q/ToF) mass analyzers Quadrupole Mass Analyzer A quadrupole mass analyzer consists of four rods or electrodes arranged parallel to each other; ions can be separated with this device by using an electrostatic field 18

43 between the rods (Figure 2.7). This electric field is created by applying different directcurrent (dc), also referred as U, and radio-frequency potentials (rf), also referred as Vcosωt, to each pair of opposite rods. One set of opposite rods is subjected to positive dc and rf potentials (U- Vcosωt) while the other set is subjected to negative dc and rf potentials - (U- Vcosωt), where ω is the angular frequency of the rf potential. The ions which were formed in the ion source pass through a series of focusing or accelerating lenses before they enter the mass analyzer. These lenses provide the appropriate electric potentials to accelerate the ions in the direction of the z-axis. The ions start moving with a zigzag motion in the z-direction inside the quadrupole due to the potentials applied to the rods. The ions are attracted to the rods that have the opposite charge and vice versa. These ions change their motion when the sign of the potential changes. This process creates the zigzag motion in the z-direction. The rf potential is applied to the rods to focus the ions in the center of the quadrupole to prevent neutralization of the ions by collisions with the rods. The combination of dc and rf potentials creates a stability window that allows only ions with a specific m/z to pass 1, 7, 23 through the quadrupole. 19

44 Figure 2.7 Schematic representation of the quadrupole mass analyzer (adapted with permission from reference 1). The motion of the ions inside the quadrupole is defined by their x/y coordinates. The latter can be determined by solving the Mathieu equation, a mathematical equation that describes the ion motion inside the quadrupole (equations ). 48 d 2 u d( ωt 2 )2 + a u q u cosωt u = 0 Equation 2.2 a x = a y = 8zeU mω 2 r 0 2 Equation 2.3 q x = q y = 4zeV mω 2 r 0 2 Equation 2.4 In the Mathieu equation (equation 2.2), u represents either the x or the y coordinate which specify the transverse displacement of the ions moving inside the quadrupole in the x and y directions. The x and y coordinates depend on the a u and q u dimensionless parameters which are proportional to the dc potential (U) and rf amplitude (V), respectively, as well as to r 0 which is one half of the distance between opposite rods. 20

45 For certain values of a u and q u, these coordinates stay smaller than r 0 leading to a stable trajectory of the ions through the quadrupole. To transmit ions with successive m/z values to the detector, they are sequentially brought into stable trajectories by scanning U and V at a constant ratio Time-of-Flight Mass Analyzer The time-of-flight (ToF) mass analyzer is composed of a field-free flight tube where accelerated ions travel a distance L for a time t until they reach the detector. The ions created in the ionization source are separated according to their velocities and the time they spend to travel in the field-free flight tube (Figure 2.8). Figure 2.8 Schematic of a linear ToF mass analyzer (adapted with permission from reference 1). 21

46 Ions generated in a usually pulsed ion source (e.g., MALDI) are introduced to the ToF analyzer as packets and accelerated towards the field-free region. An ion with mass m and total charge q (= ze) is given a potential energy E el in the acceleration region which is converted to a kinetic energy E k as seen in Equation 2.5, where V is the potential used to accelerate the ions so that they gain the potential energy E el. Since all ions are accelerated by the same potential (V) to the same kinetic energy (E k ), their velocities (v) vary depending on the corresponding m/z. The m/z values of the ions can be derived from the time (t) needed to travel through the field-free flight tube (Equations ). E k = zev= ½ mv 2 Equation 2.5 t = L/v Equation 2.6 v = (2zeV/m) 1/2 Equation 2.7 t = (m/z) 1/2 L/(2eV) 1/2 Equation 2.8 Based on these equations and as mentioned above, all the ions have the same initial kinetic energy but different masses (m/z) which determine their velocities. The lighter ions move faster and spend less time in the flight tube than the heavier ions. A limitation exists in linear ToF instruments; ions with the same m/z value may be accelerated differently because they were formed at different sample locations or with different initial kinetic energies, which will cause their flight time in the analyzer to vary, leading to poor resolution. This limitation was resolved by the development of the reflectron (Figure 2.9) and delayed extraction techniques (Figure 2.10). The reflectron is an electrostatic mirror that is used to deflect the ions and increase their path in the flight 22

47 tube. For ions with the same m/z ratio, those with higher kinetic energy will penetrate deeper and spend more time in the reflectron while those with lower kinetic energy will penetrate less and spend less time in the reflectron. As a result, all the ions with the same m/z value reach the detector at the same time. 1 Figure 2.9 Schematic of a reflectron ToF mass analyzer (adapted with permission from reference 1). Delayed extraction is another method utilized to reduce the kinetic energy spread between ions with the same m/z value. Here, a time-delay is introduced between the ionization (MALDI) and acceleration steps to allow the ions formed to drift for nanoseconds to microseconds before being accelerated (Figure 2.10). This process separates the ions according to their initial kinetic energies before the extraction (i.e., acceleration) grid pulsed. The ions with higher kinetic energies drift closer to the grid than the ions with lower kinetic energies. The less energetic ions will therefore receive more kinetic energy and move faster than the initially more energetic ions, which enables them to catch up with the latter ions after the extraction grid is pulsed. As a result, all ions 23

48 with the same m/z value will reach the detector at the same time, leading to narrow peaks 1, 39 in the mass spectrum. Figure 2.10 Schematic of the delayed extraction principle for ToF mass analyzer (adapted with permission from reference 1) Quadrupole/Time-of-flight (Q/ToF) Mass Analyzer A hybrid mass analyzer combines various types of mass analyzers in order to enhance the performance of a mass spectrometer. The quadrupole/time-of-flight (Q/ToF) combination is one example of such hybrid instrument in which a quadrupole and a time- 24

49 of-flight analyzer are placed orthogonal to each other. A collision cell is located between the two mass analyzers. In MS mode, the quadrupole mass analyzer acts as an ion guide that transfers all ions coming from the source to the time-of-flight tube; for this, only an rf potential is applied to the quadrupole rods. In MS/MS mode, only a specific precursor ion is allowed to pass through by applying rf and dc potentials to the quadrupole. After passing the quadrupole, this precursor ion arrives at the collision cell where collisions with a neutral gas, usually argon (Ar), take place causing the precursor ion to form fragments. The precursor ion and its fragments then move towards the time-of-flight tube where they are separated according to their m/z value. Combining these two mass analyzers enhances the sensitivity to attomole levels, the resolution to > 10000, and the mass accuracy to 5-10 ppm Quadrupole Ion Trap Mass Analyzer The quadrupole ion trap (QIT) mass analyzer is a storage device to which an oscillating quadrupolar electric field is applied to trap and store ions in space by controlling their motions. It was developed by Wolfang Paul in the late 1950s. 49 This mass analyzer consists of one ring electrode located between two end cap electrodes. The ring electrode is exposed to an rf potential while the two end cap electrodes are grounded. A three-dimensional hyperbolic field is generated inside the trap due to the shape of the three electrodes (Figure 2.11). The end cap electrodes have small holes for the ions to enter and exit. Ions created in the ionization source are injected into the trap through the 25

50 small hole in the entrance end cap. The ions can be ejected through multiple holes on the exit end cap to reach the detector. Figure 2.11 Schematic of the quadrupole ion trap mass analyzer (adapted with permission from reference 42). The 3D hyperbolic field generated inside the trap forces the ions to move in stable trajectories towards the trap center where they are stored and trapped. Inside the trap, ions carrying the same charge polarity are repelled from each other which destabilizes their trajectories and may eject them from the trap. A buffer gas such as helium is introduced in the trap to avoid such ion loss. The buffer gas collides with the ions to remove excess translational energy which prevents the ions from ejecting and pushes them back toward 1, 42 the trap center. 26

51 The ion trajectories inside the quadrupole ion trap can be described by the Mathieu equation and the dimensionless parameters q z and q r (Equation 2.9). In the quadrupole mass analyzer, the ions travel in the z direction (vide supra); their motion in the x-y plane was controlled by the dc and rf potentials applied to the four rods. In the quadrupole ion trap mass analyzer, the motion of the stored ions is controlled by the potentials applied in three dimensions, x, y, and z. In equation 2.9, the x and y dimensions are expressed as r since x 2 + y 2 = r 2 due to the cylindrical QIT symmetry; V is the amplitude of the rf potential, ze the charge of the ions, m is the ion mass, r 0 is the inner radius of the ring electrode, and z 0 is the axial distance from the center of the trap (usually r 0 = 2 z 2 0 ). 1 The ions should never reach or exceed the r 0 and z 0 coordinates in order to have stable trajectories. For ions to remain inside the trap, the q z for the ions should be less than (Figure 2.12). q z = 2q r = 8zeV m( r z 2 0 )Ω2 Equation

52 Figure 2.12 Stability diagram for a QIT, in which four singly charged ions with the masses m 4 < m 3 < m 2 < m 1 were injected. If no dc is used (U=0 and a z =0), ion trajectories are determined only by the rf field (q z ). Ions with q z <0.908 remain trapped (m 1, m 2, m 3 ) and ions with q z >0.908 are ejected (m 4 ). The low-mass cut off the QIT is calculated from the equation q z = (reproduced with permission from reference 49). Application of an rf potential with the frequency Ω to the ring electrode is required to trap the ions inside the QIT from where they are later ejected according to m/z to acquire a mass spectrum. Sometimes, an auxiliary rf potential is applied to the end caps to isolate a selected precursor ion and subject it to an MS/MS experiment. Modern QIT mass analyzers are operated without dc potentials. The stability diagram (Figure 2.12) displays the range of rf and dc potentials that lead to stable ion trajectories for a range of m/z values. As mentioned above, no dc potentials are applied to modern QITs ( a z = a r = 0), i.e. only q z and q r determine trapping and motion in the z and r directions, respectively. For each trap, the frequency Ω is fixed and r 0, z 0, and e are constant values while q z depends on V and m/z. In Figure 2.12, ions 28

53 m 1, m 2, and m 3 have q z values < which keep them trapped in the QIT while m 4 has a q z value > which causes ejection from the trap in the axial z direction. In order to generate a mass spectrum of the trapped ions, V is scanned to eject these ions successively by increasing their q z values to After these ions exit the QIT, they 1, 49 reach the detector to produce an electrical signal proportional to their abundances. 2.4 Detectors Ions, that were generated in the ion source and separated in the mass analyzer, reach the detector and strike it creating an electric current that is proportional to their abundance. In general, the initial signal generated when the ions reach the detector is low because only a small portion of these ions reaches the detector at a specific time; thus, this signal is amplified to generate a measurable signal. The detectors of the mass spectrometers used in this dissertation were microchannel plate (MCP) and Daly (photon multiplier) detectors. The MCP detector is a device consisting of a plate in which parallel cylindrical channels have been drilled. Each channel is a few millimeters long and few micrometers in diameter. The function of these channels is to amplify the signal intensity which is achieved by covering each channel with a semiconductor material that emits electrons when struck with ions, thereby magnifying their number by around 10 5 times. The ion beam strikes the detector wall at an angle so that it can be reflected and magnified. The 29

54 resulting electron cascade is collected using a metal anode and the current is measured (Figure 2.13). 1 Figure 2.13 Schematic of a microchannel plate detector and the electron multiplication within the channels (adapted with a permission from reference 1). The Daly detector is an electro-optic device that converts ions to electrons and photons. This detector consists of a conversion dynode, a scintillator, and a photomultiplier tube (Figure 2.14). The ions coming from the analyzer hit the dynode which carries opposite charge generating secondary electrons. The electrons ejected from the dynode move towards a phosphorescent screen and strike it to generate photons. The photons are detected and amplified by a photomultiplier to produce a gain of 10 4 to The photomultiplier can be placed outside the vacuum system of the mass spectrometers, which extends its life time. 30

55 Figure 2.14 Schematic of a Daly detector (adapted with permission from reference 1). 2.5 Ion Mobility Mass Spectrometry (IM-MS) Convential mass spectrometry cannot separate ions with the same m/z values but different sizes, architectures, or conformations. Ion mobility spectrometry coupled with MS can overcome this limitation. Ion mobility (IM) describes the drift behavior of an ion traveling under the influence of an electric field in the opposite direction of the flow of a buffer. Ion mobilities depend on the size/charge ratios of the ions. The IM-MS technique provides two dimensional separation, based on the m/z in the mass analyzer and size in the ion mobility chamber. This technique has been used in biological analyses to reduce chemical noise and to separate ionic isomers and conformers. 50, 51 The IM-MS work in this dissertation was performed on the Synapt HDMS Q/ToF mass spectrometer, which 31

56 contains an ion mobility chamber between the quadrupole and time-of-flight mass analyzers (Figure 2.15). Figure2.15 Schematic of the Waters Synapt HDMS Q/ToF mass spectrometer 2006 Waters Corporation (adapted with permission from reference 52). The Synapt HDMS mass spectrometer consists of a traveling wave ion guide (TWIG), a quadrupole, the triwave region, and a time-of-flight tube. The ions generated in the ion source pass through the ion guide from where they are pulsed and move as a packet to the quadrupole. The quadrupole acts as an ion guide to move all ions to the triwave region in the MS mode. The triwave section consists of three components: trap, ion mobility separation, and transfer cell (Figure 2.16). In the trap cell, the ions are trapped as packets before they are sent to the ion mobility separation (IMS) cell. In the IMS cell, the ions travel against a stream of a buffer gas, usually nitrogen, by low voltage 32

57 pulses called traveling waves and, in this process, they are separated according to their mobilities. From the transfer cell, the packets of separated ions are transferred to the ToF mass analyzer. 36 MS/MS experiments on mobility-separated ions can be conducted in the transfer cell. Conversely, the trap cell can be used for MS/MS, if separation of the fragments by their mobilities is desired. Trap and transfer cells are filled with Ar at 10-2 mbar, whereas N 2 flows through the IMS call at ~ 1mbar. Opposite phases of an rf voltage are applied to adjacent ring electrodes throughout the triwave region to axially focus the ions traveling through this region. 36 Figure 2.16 Schematic of the triwave section of the Synapt HDMS system (adapted with permission from reference 36). A transient dc voltage is applied successively to the adjacent ring electrodes stacked in the IMS cell to drive the ions through the gas buffer. This voltage is applied in a repeating sequence at regular time intervals (Figure 2.17). The traveling waves that are created this way propel the ions through the IMS cell where they are separated. The 33

58 larger ions will have lower mobility against the buffer gas while the smaller ions will move faster in the IMS cell. 36, 53 In the Synapt HDMS instrument, traveling waves with heights (amplitudes) up to 25 V and velocities in the range between m/s are employed. Weak traveling waves (1-2 V and 300 m/s) are applied to the transfer cell to maintain the ion mobility separation of the ions until they reach the ToF mass analyzer, while the trap cell is not exposed to any traveling waves. Figure 2.17 Schematic of the operation of a traveling wave ion guide containing ring electrodes for transferring the ions through the buffer gas (adapted with permission from reference 53). The collision cross section of an ion can be deduced directly from the ion s drift time through the IMS cell only if a static low field is applied to this cell. Under these conditions, the ion velocity is given by Equation 2.10 and is proportional to the electric field. The proportionality constant, K, is called the mobility constant (Equation 2.11). 34

59 Equation 2.10 K = [ ] 1/2 Equation 2.11 In these equations, ν is the ion velocity, t d is the ion drift time through the IMS cell, L is the length of the IMS cell, E is the electric field applied to the IMS cell, N is the number density of the buffer gas, T is the absolute temperature, k is the Boltzmann constant, M is the mass of the ion, m is the mass of the buffer gas atom or molecule, and Ω is the collision cross section of the ion. The cross section of the ion reveals information about its structure and size. In traditional ion mobility spectrometry, which uses static, low fields in the IMS region, the measured drift time is directly proportional to the cross section of the ion (Equation 2.10 and 2.11). In contrast, with traveling wave (T-wave) spectrometers, there is no straightforward relationship between the electric field generated by the pulsed waves and the ion drift time through the IMS cell. Here, the drift time scale must be calibrated using the drift times of ions with known collision cross section before determining the collision cross section of the analyte ions. The collision cross section of an ion can provide useful information about its size and geometry. Ions with identical m/z ratios but different structure will have different velocities, based on their size and shape, when they travel through the IMS cell. Ions with larger cross sections will move slower because they collide with the buffer gas that moves against the ions and slow their motion leading to a longer drift time. Ions with higher molecular weight that carry multiple charges can have short drift times because of their higher charge state. On the other hand, if the molecular structure allows the ion to 35

60 fold around the charges in order to stabilize its structure through intramolecular solvation 36, 53 and minimize charge repulsion, a small size and shorter drift time would result. 2.6 Liquid Chromatography Mass Spectrometry (LC-MS) Liquid chromatography (LC) is one of the most efficient separation methods for the analysis of complex mixtures. High-performance liquid chromatography (HPLC) can be coupled to a mass spectrometer in order to combine the strength of the LC and MS techniques and facilitate the identification of the components in complex mixtures. LC was introduced in 1970s as a powerful analytical tool 54 for the separation of nonvolatile mixtures into their components based on their physical or chemical interactions with a mobile phase and a stationary phase. In order to analyze a complex mixture by HPLC, it must be dissolved in a proper solvent before it is introduced to the instrument. The analyte is carried by the mobile phase which contains one or more liquid solvents and passes through the column which is packed with a stationary phase. The components of the mixture are distributed in the 22, 24 mobile and stationary phases based on their interactions with both phases. In most cases, the HPLC system is interfaced with the ionization source, usually an ESI or APCI source, of the mass spectrometer. A basic LC-MS system includes six components; the mobile phase supply system, pump system, sample injector, column, detector, and data processing system (Figure 2.18). 54 A modern mobile phase supply system includes more than one mobile phase reservoir, usually between two to four 36

61 reservoirs. The pump is used to help the analytes to be carried through the column with a reasonable flow rate. The most important part in the HPLC system is the column, where the actual separation takes place. The column is packed with the stationary phase. After separation, the components of the analyte exit the column and reach the detector, which can be refractive index, fluorescence, conductivity, UV/Vis, or MS detector. In this dissertation, MS was interfaced with the LC system to provide a highly sensitive and specific analytical method for determining the components present in a mixture. 56 As any analytical method, traditional HPLC has strengths and limitations in its performance. The limitations include time and solvent consumption as well as low resolution and sensitivity. To overcome these limitations and improve HPLC, ultraperformance liquid chromatography (UPLC) has been developed. This enhances the 57, 58 sensitivity and efficiency of separation and identification of mixture components. Figure 2.18 Schematic of the basic components of an HPLC-MS system (adapted with permission from reference 55). 37

62 2.6.1 Columns and Stationary Phases The column is the heart of the LC system. It is usually a stainless steel tube packed with particles that can serves as the stationary phase or are coated with stationary phase. The most common packing material is silica gel. Packing particles of various sizes exist. There several HPLC variants depending on the on the stationary phase used: normal phase, reverse phase, ion exchange, size exclusion, affinity, and chiral chromatography. The most widely used variants have been normal-phase (NP) and reverse-phase (RP) HPLC, in which the stationary phase is an organic material that is attached covalently onto the packing particles. In the normal phase HPLC, the stationary phase is polar, containing nitro or amino groups, while the mobile phase is non-polar. In reverse phase HPLC, the stationary phase is non-polar, containing hydrophobic substituents (alkyl chains), while the mobile phase is polar. In the ion exchange HPLC, the stationary phase is a high-mass polymer containing acidic or basic groups that ionize easily; in size-exclusion chromatography, the stationary phase is silica or polymer particles with a network of uniform pores; in affinity HPLC, an affinity lignad, such as an antibody, is covalently bonded to the packing particles. Finally, in chiral chromatography, the stationary phase contains chiral molecules immobilized on the 59, 60 packing particles. Most current LC applications employ reverse phase liquid chromatography (RPLC). The most widely utilized stationary phases for these applications are organic coating carrying alkyl or aryl groups (R), such as octyl (C8), octadecyl (C18), or phenyl, which are bonded to silica through Si-O-Si-R linkages. Separation of complex mixtures is 38

63 achieved by the interactions developing between the analyte and the stationary phase. In RPLC, non-polar hydrophobic components are adsorbed on the hydrophobic alkyl or aryl groups of the stationary phase much more strongly than polar components, which travel faster along with the polar mobile phase (usually an aqueous mixture of organic solvents) and elute first. Commonly used mobile phase solvents in RPLC are acetonitrile, 24, 39, 55 methanol, ethanol, isopropanol, and tetrahydrofuran. The chromatographic separation is affected by the size of the packing particles. In HPLC, the columns are typically packed with spherical particles with a 3-5 µm diameter. In UPLC analysis, the particle diameter is less than 2 µm. The smaller particle size increases the efficiency of the column. The stationary phase particles are porous to increase the interactions of the sample with the stationary phase. 55 The distribution (partition) of the analyte between the stationary and the mobile phase controls the separation in chromatography. Components that share similar properties with the mobile phase move through the column faster and elute earlier than components that interact more strongly with the stationary phase. This distribution coefficient K, which is the ratio between the analyte concentrations in the stationary and mobile phases, describes the analyte distribution between the two phases. The retention time t R and the width of the peak at the half height W 1/2 can be used to identify the chromatographic peak. The column efficiency, which describes how well the sample is separated as it moves through the column, is measured by the number of theoretical plates (N); this parameter can be obtained from the chromatogram via equation 2.12 as: N = 5.54 (t R /W 1/2 ) 2 Equation

64 High column efficiency is achieved with a higher number of the theoretical plates; it gives rise to narrower and better resolved peaks in the chromatogram. The size of the pores of the packing material of the column also has an important impact on the separation of the analyte components. A bigger pore size can cause the analyte molecules to be trapped inside them, leading to broad bands and slower elution. There are three factors that control the distribution of the analyte components through the column. These factors, which need to be carefully considered to minimize the band broadening, are eddy diffusion, longitudinal diffusion, and mass transfer resistance. Eddy diffusion (A Term), also known as multipath effect, usually because the analyte molecules travel randomly via different paths, through the stationary phase. These differences in the path result from imperfections in the column packing or the unequal shape of packing particles. Some analyte molecules will travel fast (along a straight path) and elute early, while others will follow different and longer paths until they elute. This effect causes band broadening and poor resolution. This factor can be minimized using a column packed with particles of the same shape and by decreasing particle size which minimizes particle imperfections. Longitudinal diffusion (B Term) is also known as flow distribution and is caused by the diffusion of the analyte lengthwise along the column. This diffusion effect increases when the analyte components spend more time in the column. Hence, it can be minimized by increasing the flow rate of the mobile phase. 40

65 Resistance to mass transfer (C Term) refers to the relationship between the time it takes the analyte components to equilibrate between the two phases and the flow rate of the mobile phase. The time spent in the stationary phase is longer or shorter compared with the time required for the partition equilibration to take place which leads to band broadening. To decrease this effect, small stationary phase particles should be used. These have naturally small pores, facilitating analyte transfer from one particle to another. For analytes having a molecular weight of 3000 Da or less, a 100 Å pore size is used to decrease slow diffusion in and out the pore and, thus, reduce band broadening Only terms B and C depend on the flow rate of the mobile phase. Therefore, the flow rate should be optimized to minimize the effect of these terms. The actual effect of all three factors affecting column efficiency is described by the van Deemter equation (Equation 2.13), which reveals the dependence of the column efficiency on the mobile phase flow rate (Figure 2.19). The x axis of the plot is the mobile phase linear velocity (u in cm/s) and the y axis is the theoretical plate height, H = L/N, where L is the length of the column and N is the number of theoretical plates (vide supra). From this plot, the optimum flow rate that will reduce the dispersion (band broadening) and increase the 59, 61 separation efficacy can be predicted. H= A+ B/u + Cu Equation

66 Figure 2.19 Van Deemter plot (dashed line) and individual plots of the terms of the van Deemter equation. 2.7 Tandem Mass Spectrometry (MS/MS) Single stage mass spectrometry with soft ionization methods such as ESI and MALDI provides information about the mass of the intact molecular ion, which can be used to calculate the mass and number of monomer units and the mass of the total end groups of a polymer. With the help of two- or multi-dimensional mass spectrometry methods, also known as tandem mass spectrometry or multistage mass spectrometry methods (MS/MS or MS n, respectively), more information about the molecular structure under examination can be obtained. This method can be applied either in-space where two mass analyzers are coupled together and used for corresponding experiment or intime where the experiment is performed sequentially in an ion storage device

67 For in-space tandem mass spectrometry, the first mass analyzer is used to select and isolate the precursor ion. This ion collides with neutral gas molecules in a collision cell which leads to the production of fragment ions (product ions). The collision cell is located between the two analyzers. The fragment ions are separated in the second mass analyzer based on their mass-to-charge ratio (m/z). For in-time tandem mass spectrometry, the storage device is used to store the ion of interest and eject the other ions. The precursor ion is then fragmented during a certain time period. These two steps of ejection and fragmentation can be repeated to form fragments of the fragments (MS n ). 17 In this dissertation, a QIT instrument which has tandem in-time capability and a Q/ToF instrument which has a tandem in-space capability have been utilized. To perform fragmentation in the QIT, the ion of interest is isolated and subjected to collisions with the helium buffer gas inside the trap. These collisions convert translational (kinetic) energy to internal energy which induces the ions to fragment. The strength of a QIT instrument is the capability to perform multiple stages of tandem mass spectrometry (MS/MS). With a Q/ToF instrument, the quadrupole analyzer is used to select the ion of interest and allow it to enter a collision cell that is filled with argon gas. In the collision cell, collisions with the gas lead to the formation of the fragments. These fragments are then accelerated orthogonally to the ToF mass analyzer for separation according to their m/z ratio. Tandem mass spectrometry is used for structural identification based on the observed fragmentation pathways. Collisionally activated dissociation (CAD) is the most 43

68 common method to cause fragmentation; it consists of two steps, activation of the molecules by collisions and dissociation. The precursor ion is accelerated and collides with a neutral gas such as helium or argon. Some of the kinetic energy of the ion is converted to internal energy leading to bond breakage and fragment production during the collision process. The maximum internal energy (E int ) that an ion can gain is given by equation 2.14, where E kin represents the ion kinetic energy, M i represents the mass of the precursor ion and M g the mass of the atom of the neutral gas used in the collision. 62 This dissertation elucidated how the precursor ion size and the internal energy gained in the collisions influence the fragmentation pathways of polymer ions. E int = E kin M g / (M i + M g ) Equation

69 CHAPTER III MATERIALS AND INSTRUMENTATION 3.1 Materials Methanol, THF, water, acetonitrile, acetone, and hexane, all of HPLC grade, were purchased from Sigma-Aldrich (St. Louis, MO). Sodium trifloroacetate and lithium trifloroacetate, used to improve ionization of the samples, were obtained from Fluka (Buchs, Switzerland). Polytetrahydrofuran, PTHF, with a number average molecular weight of 650 g/mol and polypropylene oxide (PPO), with a number average molecular weight of 1000 g/mol were purchased from Sigma-Aldrich (St. Louis, MO). Poly(1,2- butylene oxide), PBO, with a number average molecular weight of 800 g/mol was received from Dr. Li Jia s group (University of Akron). Poly ethylene oxide macroinitiators and their copolymers were received from Dr. Coleen Pugh s group (University of Akron). Elastollan (PU-1) was obtained from BASF (Wyandotte, MI); pellethane (PU-2) was obtained from DOW Chemical (Midland, MI); and estane (PU-3) was obtained from BFGoodrich (Brecksville, OH). One styrenic copolymer sample, styrloflex (SB-1), was obtained from BASF (Wyandotte, MI). All materials were used in the condition received from their supplier without further purification. 45

70 3.2 Instrumentation The following sections describe the running conditions and instrument settings to obtain the data in this dissertation HCT Ultra II ESI-QIT Mass Spectrometer HCT Ultra II (Bruker Daltonics, Billerica, MA) is the electrospray ionization quadrupole ion trap mass spectrometer used to characterize the low molecular weight molecules/polymers discussed in Chapters IV and VI. A scheme of this instrument is shown in Figure 3.1. The samples are introduced to the electrospray chamber either by direct infusion or chromatography. Direct infusion mode was the method used to introduce samples in this instrumentation. In this mode, liquid solutions of samples are introduced through a fine metal needle at a flow rate of 180 μl/h. A syringe pump allows for control of the flow rate of the sample solutions. A nitrogen gas flows through the nebulizer which surrounds the grounded needle and joins the sample solution at the needle tip. The pressure of the nebulizer gas can be adjusted from 0 psi through 80 psi. To promote solvent evaporation in the spray chamber, a drying gas, heated nitrogen, is used. The drying gas temperature can be set between 35 ºC and 365 ºC at a flow rate of 0 L/min to 12 L/min. In this dissertation, the optimized settings were 8 L/min at 300 ºC and all experiments were recorded in positive mode. 46

71 Glass End plate Figure 3.1 Schematic view of the Bruker HCT ultra II ESI-QIT mass spectrometer (adapted with permission from reference 63). The analyte is introduced as a liquid solution into the spray chamber which is operated at atmospheric pressure. In the spray chamber, droplets are produced and subjected to the heated drying gas to help evaporate the solvent from the droplets, and to a high voltage to create an electrostatic gradient inside the spray chamber which helps in the ionization process. For positive mode, the entrance of the glass capillary is held between -4.5 kv and -1.5 kv relative to the spraying needle, and at -0.5 kv relative to the end plate. Migration of the charged droplets formed by ESI towards the glass capillary entrance is assisted by the electrostatic gradient created by the voltage differences. The glass capillary serves as transmission region which transfers the ions from atmospheric pressure to vacuum. The ions are pushed and focused through a sequence of skimmers and octapole lenses after they exit the glass capillary toward the ion trap. To allow transport of the ions produced by ESI to the ion trap and decrease interference of 47

72 background noise, the voltages of the skimmers and octapole lenses are set to certain values. The operation principle of a quadrupole ion trap was discussed in Chapter Ions ejected from the ion trap move to a Daly Detector to produce an electrical current that is proportional to the abundances of the ions. 63 All ESI spectra in Chapters IV and VI were measured in positive mode Ultraflex III ToF/ToF Mass Spectrometer The Ultraflex III ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA) consists of two time-of-flight analyzers. The first one is a short linear (ToF-1) which is interfaced with the second one, a reflectron analyzer (ToF-2). In single stage mass spectrometry mode, the two ToF analyzers operate as one combined linear or reflectron ToF analyzer while in tandem mass mode, the two ToF analyzers operate separately (LIFT mode). In LIFT mode, ToF-1 is employed to select the precursor ion and ToF-2 is used to separate the fragment ions produced from the selected precursor by increasing the laser power and by collisions in the LIFT cell. LIFT cell is a region between ToF-1and ToF-2. The laser employed in Ultraflex III is called smart beam 200 Hz laser; it is an Nd: YAG laser that emits light at the wavelength of 355 nm. All MALDI spectra in Chapters V and VI were measured in positive reflectron mode. Ions formed by MALDI were accelerated by 25 kv (IS1). In MS mode, the laser energy was adjusted as needed, to maximize the ion signals without causing any fragmentation. The IS2 voltage was set at 21.65, the lens voltage was set at 9.65 kv, and 48

73 the delay time 150 ns. The reflectron lenses 1 and 2 were set at and kv, respectively, in reflectron mode MS experiments. For MS/MS experiments, the LIFT parameters were set up as follows: IS1 at 8 kv, IS2 at 7.15 kv, lens potential at 3.6 kv, reflectron 1 and 2 lenses at and kv, respectively, and LIFT 1 and 2 settings at and 2.90 kv, respectively MALDI-Q/ToF Mass Spectrometer The Micromass Ultima (Waters, Milford, MA) MALDI tandem mass spectrometer (Figure 3.2), was used for the characterization of PEO-b-PCL diblock copolymers. This Q/ToF instrument was mainly used for the tandem mass experiments as described in Chapter V. The Micromass Ultima consists of quadrupole and time-of-flight analyzers. The quadrupole analyzer acts as either ion guide or mass selector. In MS/MS experiments, the quadrupole is operated in mass-selective mode. In this mode, rf and DC voltages are applied to the quadrupole to select the ion of interest and allow it to travel through collision cell which is pressurized with argon gas to induce fragmentation via collisionally activated dissociation (CAD). In the collision cell, the collision energy (CE) was adjusted to promote energetic collisions between this ion and the argon particles and to observe fragment ions. When the quadrupole is set in rf-only mode, it operates as an ion guide, transmitting all ions to the ToF analyzer. The two mass analyzers are placed orthogonally to each other. The reflectron ToF mass analyzer is used to separate the ions based on their mass-to-charge ratio, m/z, for both MS and MS/MS experiments. The 49

74 Micromass Ultima is equipped with a N 2 laser that emits light at a wavelength of 337 nm. The detector of this instrument is a microchannel plate detector. 64 For the MS/MS experiments in discussed in Chapter V, the CE was set between ev to obtain the desired fragmentation. All spectra were recorded in positive mode. Figure 3.2 Schematic view of the Micromass Ultima MALDI-Q/ToF mass spectrometer 2003 Waters Corporation. (adapted with permission from reference 64) Synapt HDMS Ion Mobility Mass Spectrometer The Synapt HDMS system (Waters, Milford, MA) is equipped with an electrospray ionization source and a quadrupole orthogonal acceleration time-of-flight 50

75 mass spectrometer as well as a tri-wave ion mobility region (Figure 2.15). The operation principle of this instrument and the triwave ion mobility function were explained in Chapter 2.5. The Synapt versatility is documented in Chapters VI, V, and VII Acquity UPLC The Acquity UPLC TM system (Figure 3.3) commercialized by Waters (Milford, MA) is equipped with a binary pump, auto sampler, and degasser. Acquity UPLC pumps can handle higher pressures (up to 15,000 psi) than HPLC pumps (up to 6000 psi). 65 The high pressure facilitates the usage of columns with smaller particle size to obtain narrow peaks, high resolution, and shorter elution times. A typical HPLC column has a particle size of 3-5 μm, whereas particle sizes in an analytical UPLC column are typically 1.7 μm. The Acquity UPLC system was coupled with the Synapt HDMS Q/ToF mass spectrometer to obtain the data explained in Chapter V. 51

76 Figure 3.3 Schematic view of the Acquity UPLC system 2004 Waters Corporation (adapted with permission from reference 65). 52

77 CHAPTER IV STRUCTURAL CHARACTERIZATION OF CYCLIC AND LINEAR POLY(ETHYLENE OXIDE) MACROINITIATORS USING MULTIDIMENSIONAL MASS SPECTROMETRY 4.1 Background Poly(ethylene oxide), (PEO), is a biocompatible and non-toxic polymer that is used widely in biological and medical applications. It has the ability to minimize cell and protein interactions. PEO can serve as initiator in polymerizations process aiming at the synthesis of diblock copolymers. An amphiphilic diblock copolymer is produced when a hydrophobic block is attached to PEO. The resultant copolymer is able to form self- 66, 67 assembled micelles in the solution, which may be used for drug delivery. Multidimensional mass spectrometry (MS n ), which includes more than one tandem mass spectrometry step, provides detailed information about the molecular structure under examination. This information can be used to characterize a polymer s end groups and sequence. In this technique, a specific oligomer ion is isolated and induced to dissociate into fragments by collisions with a static gas. Collisionally activated dissociation (CAD) is the most common means to cause fragmentation in MS n studies. Most of the ions generated by soft ionization techniques are closed-shell species that do not carry unpaired electrons. Dissociation of these ions proceeds via rearrangements in 53

78 which one bond is formed while another breaks down, or via radical intermediates formed by homolytic bond cleavage. Such reactions may have chargeinduced or charge-remote character. Bond cleavage and bond formation take place at or near the charge site in the charge-induced mechanism, while in charge-remote bond cleavages these processes take place away from the charge site and without its involvement. In charge-remote reactions via radical intermediates, radical rearrangements are possible. 17 This chapter describes the MS and MS n characterization of PEO macroinitiators synthesized with different architectures. The main goal was to elucidate the gas-phase fragmentation pathways of these macroinitiators and evaluate the structural information they provide. PEO polymers are readily ionized by alkali metal addition due to the presence oxygen in their backbones. 7 Lithium ion was used for the MS n studies because it is bound strongly by PEO, leading to cleavages in the polymer chain, and not to metal detachment, upon CAD In addition to lithium adducts, the mass spectra of PEO show sodium, potassium, and ammonium adducts, even if these were not added. The latter cations originate from impurities in the solvents, glassware, or sample. 4.2 Sample Preparation and Instruments Used Three PEO macroinitiators with different architectures were received from Dr. Pugh s group (courtesy of Dr. Gladys Montenegro). They were characterized using ESI- QIT MS (Bruker HCT Ultra II ion trap mass spectrometer). Sample solutions were prepared at a concentration of 1 μg/μl in THF: MeOH (70:30, v/v) and mixed with a 54

79 lithium trifluoroacetate solution (1μg/μL) in the ratio 100:1 (sample: salt) (v/v). All spectra were collected in positive mode. 4.3 Characterization of PEO Macroinitiators This chapter reports the procedure developed for the analysis of PEO macroinitiators with different architectures. A summary of the structures analyzed in this chapter is given in Scheme All polymers were synthesized by Dr. Gladys Montenegro in the Pugh laboratory. The synthesis of the macrocyclic PEO started with polyethylene oxide reacting with methanesulfonyl chloride to form the difunctional bis- (methanesulfuonate ester) of PEO which reacted with 3,4-dihydroxybenzaldehyde to form 3,4-(42-crown-14) benzaldehyde PEO that was reduced to form 3,4-(42-crown-14) benzyl alcohol PEO (Scheme 4.1.a). Note that a distribution of crown ether sizes is formed, with 42-crown-14 being the average size expected based on the average degree of polymerization of the PEO starting material (13-mer). On the other hand, the synthesis of the linear PEO macroinitiator started with monofunctional, PEO, in this case PEO monomethyl ether, which reacted with methanesulfonyl chloride to form the methanesulfuonate ester of PEO monomethyl ether, which reacted with 4-hydroxybenzyl alcohol to form a linear PEO with methyl ether and (4-hydroxy methyl)phenyl ether as end groups (Scheme 4.1.b). 55

80 Polyethylene oxide Methanesulfonyl chloride EG= Da bis-(methanesulfuonate ester) 3,4-dihydroxybenzaldehyde EG= Da 3,4-(42-crown-14) benzaldehyde EG= Da 3,4-(42-crown-14)benzyl alcohol Scheme 4.1.a Synthetic route to 3,4-(42-crown-14) benzyl alcohol, a microcyclic PEO. Polyethylene oxide Methanesulfonyl chloride EG= Da 4-hydroxybenzyl alcohol EG= Da Scheme 4.1.b Synthetic route to a linear PEO macroinitiator with methyl ether and (4- hydroxy methyl)phenyl ether. 56

81 Characterization of 3,4-(42-Crown-14) Benzaldehyde PEO by ESI-QIT MS Figure 4.1 shows the molecular weight distribution detected in the mass spectrum of 3,4-(42-crown-14) benzaldehyde PEO (inset). Four major series with the ethylene oxide repeat unit (C 2 H 4 O, 44 Da) are clearly visible in the expanded view. These series arise from lithiated, ammoniated, sodiated, and potassiated oligomers. The m/z values of all these ions agree well with the composition (C 2 H 4 O) n + C 7 H 4 O 2 (120 Da), confirming that the dihydroxy-benzaldehyde moiety, which would add a mass of 120 Da, has been incorporated in a macrocyclic PEO chain. [11-mer + Li] + [11-mer + NH 4 ] + [11-mer + Na] + [11-mer + K] + [12-mer + Li] + [12-mer + NH 4 ] + [12-mer + Na] + [12-mer + K] m/z m/z Figure 4.1 ESI mass spectrum of lithium, sodium and potassium cationized 3,4-(42- crown-14) benzaldehyde PEO (M n 750) acquired with the QIT mass spectrometer. Figure 4.2 shows the tandem mass spectrum of the lithium cationized 13-mer at m/z 699, obtained by ESI-QIT MS/MS. The base peak at m/z 671, marked by *, is formed 57

82 by the loss of CO (28 Da) from the precursor ion; this is a typical fragmentation of aldehydes. 68 The second most intense peak at m/z 509, marked by J HH 11, results from the loss of 1,2-bis (vinyloxy) benzene (C 14 H 18 O 4 ) from the base peak at m/z 671; such elimination produces a linear PEO chain with dihydroxy end groups. Two fragment series, labeled by J HH n and J HV n, are observed below J HH 11, i.e. after ring opening. The HH HV J n series corresponds to PEO with end groups of 18 Da (dihydroxy) and the series J n corresponds to PEO oligomers with the composition (C 2 H 4 O) n which is consistent with vinyl/hydroxy end groups. Series J HH HV n and J n are the major fragments from linear PEO chain with dihydroxy end groups. 17 Hence, these fragments are attributed to consecutive dissociation from J HH 11. Two further, minor series arise from the loss of one or more repeat unit from the precursor ion and from the ion at m/z 671. The fragmentation pathways observed are summarized in Scheme 4.2. * # J 11 HH O -CO O J HV J HV 6 5 J5 HH J HH 6 J HV 7 J HV J HV J HV J HH 9 J 7 HH J 8 HH J HV 11 J HH 10 #523.0 # 162 Da * # * # m/z Figure 4.2 CAD mass spectrum of the lithium cationized 13-mer of 3,4-(42-crown-14) benzaldehyde PEO obtained using ESI-QIT tandem mass spectrometry. 58

83 Scheme 4.2 Proposed fragmentation pathways of lithiated 3,4-(42-crown-14) benzaldehyde PEO. Triple-stage mass spectrometry (MS 3 ) experiments were performed on the ions at m/z 671 and 509 using the ESI-QIT mass spectrometer. The spectrum in Figure 4.3 confirms that the decomposition of the ion at m/z 671 mainly proceeds via the loss of 1,2- bis(vinyloxy)benzene (C 14 H 18 O 4 ) from the precursor ion followed by nominal losses of HH either 44n Da to form series J n with dihydroxy end groups, or of 44n + 18 Da to form HV series J n with vinyl/hydroxy end groups. The spectrum shown in Figure 4.4 confirms HH the ion generated after bis(vinyloxy)benzene loss, viz. J 11 (PEO 11-mer with dihydroxy HH chain ends), decomposes further to smaller J n and J HV n fragments. Both latter series can 59

84 be rationalized via competitive charge-induced C-O bond cleavages along the PEO chain of J 11 HH, as shown in Scheme 4.3. J 11 HH O * O J 5 HV J 5 HH J HH 6 J HH 7 J HV 6 J HV 7 J HV 8 J HH 8 J 10 HH J HV J HH 9 11 J HV 9 J HV Da * * * m/z Figure 4.3 ESI-QIT MS 3 mass spectrum of the fragment ion at m/z 671 generated by CAD of m/z 699, the lithiated 3,4-(42-crown-14) benzaldehyde PEO 13-mer. J 10 HH J 9 HH J 4 HH J 5 HH J 6 HH J 7 HH J 8 HH J 11 HH J 3 HH J 4 HV J 5 HV HV HV HV HV J 6 J 7 J 8 J 9 J 10 HV m/z Figure 4.4 ESI-QIT MS 3 mass spectrum of the J 11 HH fragment ion at m/z 509 Da generated by CAD of m/z 699, the lithiated 3,4-(42-crown-14)benzaldehyde PEO 13-mer. 60

85 In the charge-induced mechanism, lithium ion (Lewis acid) attached to an ether oxygen weakens the adjacent C-O bond, promoting heterolytic bond cleavage. This cleavage and concomitant the hydride shift between C-1 to C-2 lead to an ion-dipole complex between a lithium alkoxylate and an alkoxylethyl cation. Proton shift to the basic alkoxylate accompanied by rearrangement of the Li ligands gives rise to a Li-bond complex between two shorter PEO chains. This complex can dissociate to form series HH HV J n with dihydroxy end groups or series J n with hydroxyl/vinyl end groups (Scheme 4.3). 17 HV Once formed, series J n can unzip through charge-remote mechanism, involving the loss of acetaldehyde molecules (44 Da) via 1,5-H rearrangement over a six-membered ring, as shown in Scheme Scheme 4.3 Charge-induced fragmentation pathway of J n HH ions (dihydroxy end groups). 61

86 Scheme 4.4 Charge-remote fragmentation pathway of series J n HV (hydroxy/vinyl end groups) Characterization of 3,4-(42-crown-14)Benzyl Alcohol PEO by ESI-QIT MS The mass spectrum of 3,4-(42-crown-14)benzyl alcohol PEO shows four oligomer distributions. These distributions correspond to lithium, ammonium, sodium, and potassium adducts of 3,4-(42-crown-14)benzyl alcohol PEO, as marked in Figure 4.5. The measured m/z values of these adducts are consistent with a cyclic PEO architecture containing (C 2 H 4 O) n repeat units (44n Da) plus an in-cycle C 7 H 6 O 2 moiety (122 Da), cf. structure in Figure

87 701.2 [13-mer + Li] + [13-mer +Na] + [14-mer + Na] + [13-mer + NH 4 ] + [13-mer +K] + [14-mer + Li] + [14-mer + NH 4 ] [14-mer + K] m/z Figure 4.5 ESI mass spectrum of 3,4-(42-crown-14)benzyl alcohol PEO (M n 750 Da) acquired with the QIT mass spectrometer. The tandem mass spectrum of the lithiated 13-mer, m/z 701, shows very similar features to those observed for corresponding aldehyde, cf. Figure 4.6 and Figure 4.2. The same J HH n and J HV n product ions are present in both spectra. These two series are formed after the aromatic linking substituent has been lost (i.e. from J HH 11 ), justifying why both HH HV the benzaldehyde as well as the benzyl alcohol precursor yield identical J n and J n fragment series. The abundant fragment at m/z 671 further points out that the linking substituent is cleared stepwise in the form of formaldehyde (CH 2 O, 30 Da) and 1,2- bis(vinyloxy)benzene (C 14 H 18 O 4, 162 Da). The signals observed for loss of water (H 2 O) 63

88 H 2 O CH 2 O and formaldehyde (CH 2 O) from the precursor ion, which have significant abundances, fully agree with the benzyl alcohol # J 11 HH O O * 162 Da J 5 HV J 6 HV HH J J HH 6 J HH HV 7 5 J 7 J 8 HV J 9 HV J 8 HH J HH 10 J HV 11 J HH 9 HV J * #@ * m/z Figure 4.6 CAD tandem mass spectrum of the lithiated 13-mer of 3,4-(42-crown- 14)benzyl alcohol, m/z 701, acquired with the ESI-QIT mass spectrometer. Triple-stage (MS 3 ) experiments were performed on the fragment ions at m/z 671 and m/z 683. The MS 3 spectrum of the fragment ion at m/z 671 was identical within experimental error with that obtained for m/z 671 from the benzaldehyde functionalized precursor ion (Figure 4.3). The MS 3 spectrum of the fragment ion at m/z 683 (Figure 4.7) demonstrates that this ion mainly loses the aromatic linking unit, as a C 11 H 10 O 2 moiety (174 Da) to form an intense ion at m/z 509 which corresponds to J HH 11 fragment ion. The HH HV series J n with dihydroxy end groups and J n with hydroxyl/vinyl end groups (n < 11) 64

89 observed below J 11 HH are attributed to consecutive decompositions of this ion via C-O bond cleavages according to the mechanism in Scheme 4.3. The major fragmentation pathways of the benzyl alcohol substituted macrocycle are summarized in Scheme 4.5. J 11 HH J 10 HH -C 11 H 10 O Da J 5 HV J 5 HH J 6 HH J 7 HH J 6 HV J 7 HV J HH 9 J HH 8 J HV 8 J HV 9 J 10 HV J 11 HV m/z Figure 4.7 ESI-QIT MS 3 mass spectrum of the fragment ion at m/z 683 generated by CAD of the lithiated 11-mer of 3,4-(42-crown-14)benzyl alcohol, m/z 701. A possible structure for the C 11 H 10 O 2 ion, which is 12 Da heavier than bis(vinyloxy)benzene, is included as inset. 65

90 Scheme 4.5 Proposed fragmentation pathways of lithiated 3,4-(42-crown-14) benzene alcohol PEO Characterization of PEO Methyl Ether, (4-Hydroxymethyl)Phenyl Ether by ESI- QIT MS Figure 4.8 shows the mass spectrum of the linear PEO with methyl and (hydroxymethyl)phenoxy end groups. The main distribution in the spectrum results from the lithiated adducts of this polymer whose methyl and (hydroxymethyl)phenoxy end groups contribute a mass of 138 Da. The ammonium, sodium and potassium adducts of the polymer are also clearly observed as minor distributions. 66

91 [7-mer + Li] + [8-mer + Li] [7-mer + Na] + [7-mer + K] + [8-mer + Na] + [8-mer + NH 4 ] + [8-mer + K] + [9-mer + Li] + [9-mer + Na] + [9-mer + K] + [10-mer + Li] + [10-mer + Na] [10-mer + K] m/z m/z Figure 4.8 ESI mass spectrum of PEO methyl ether, (4-hydroxymethyl)phenyl ether, (M n 500), acquired with the QIT mass spectrometer. A tandem mass spectrometry experiment was performed on the lithiated 8-mer, m/z 497( Figure 4.9), using the ESI-QIT mass spectrometer. The product ion at m/z 467 corresponds to the loss of formaldehyde from the benzyl alcohol moiety in the (hydroxymethyl)phenyl end group, confirming the presence of the latter functionality. Further evidence for the aromatic end group is provided by the fragments labeled as b 8 (m/z 373) and c 8 (m/z 391), which are generated by elimination of this group in the form of C 7 H 8 O 2 (124 Da, 4-hydroxymethyl phenol) and C 7 H 6 O (106 Da, presumably benzaldehyde), respectively. Fragments lower in mass than b 8 and c 8 do not contain the aromatic end group and, hence, are most likely formed by consecutive dissociation of these abundant fragment ions (see Figure 4.9 for their structures). It should be noted at 67

92 H2O CH2O this point that the nomenclature b n and c n indicates fragments from RO- 1 CH 2-2 CH 2-3 O-R chains that are formed by cleavage of the 2 CH 2-3 O or the 3 O-R bond, respectively; the double prime denotes saturated chain ends, whereas the simple letters denote unsaturated chain ends in the end groups formed after fragmentation. 17 The c n series corresponds to PEO oligomers with methoxy/hydroxyl end groups of (32 Da), and the b n series to oligomers with methoxy/vinyl end groups (58 Da). C 7 C 8 b 7 b 8 e J HV 3 b 3 b J EV 5 5 b J HV J EV J 4 HV J HH 3 J HH 4 J HH 5 J 5 EV b 6 J HV 6 J 6 HH C 6 J 7 HV e m/z Figure 4.9 CAD tandem mass spectrum of the lithiated 8-mer of PEO methyl ether, (4- hydroxymethyl)phenyl ether, m/z 497, acquired with the ESI-QIT mass spectrometer. Since c 8 has an asymmetric structure (methoxy/hydroxyl end groups), its further dissociation via the charge-induced mechanism of Scheme 4.3 creates four sets of fragment series: viz. truncated b n and c n fragments (n < 8) that still carry the original methoxy end group (see structures in Figure 4.9), and analogous fragments that carry the 68

93 hydroxyl end group, viz. J n HV and J n HH (n < 8). The latter fragments are labeled by J n to indicate that they are internal and do not contain any of the original end groups of the precursor ion. 17 The consecutive fragmentation of b 8, on the other hand, should mainly follow the charge-remote mechanism of Scheme 4.4, which was found to be the lowest energy dissociation of PEO ions with double bonds in their end groups. 17 This reaction unzips b 8, giving rise to smaller ions in this series (vide supra). In addition to these series, there are two more fragments series in the lower mass region of the MS/MS spectrum, viz. c n (2 m/z units below c n ) and J EV n (ethoxy/vinyl end groups). These are ascribed to further fragmentation of the ethyl radical arising by hemolytic C-O bond cleavage at the aromatic chain end, which liberates a resonancestabilized 4-hydroxymethyl phenoxy radical. Radical migration from the terminal position to an internal C-1 position followed by β C-O cleavage results in the aforementioned fragments, cf. Scheme 4.6. The same reaction sequence after radical migration to an internal C-2 position would lead to truncated b n fragments (observed) and J CE HV n fragments with O=CHCH 2 O- and CH 2 CH 3 end groups which are isomers with J n (observed). The small fragment at m/z 409 originates from dioxane loss (C 4 H 8 O 2 ) from internal repeat units of the (C 2 H 4 O) n chain, which was established in an earlier study. 81 Finally, the fragment ions marked by e (m/z 461 and 417) are attributed to dissociation pathways promoted by the aromatic end group; their exact mechanism is still under investigation. 69

94 Scheme 4.6 Charge-remote fragmentation of the lithiated ethyl radical arising after phenoxy radical loss via free radical intermediates. 4.4 Conclusions The tandem mass spectra of PEO homopolymers with different end groups described in this chapter demonstrate the usefulness of this technique in the determination of individual end groups and substituents. For unequivocal structural assignments, knowledge of the fragmentation mechanisms is essential. This study builds upon the mechanisms elucidated in earlier studies, 17, 69, 70 corroborating that CAD of PEOs ionized by Li + attachment cause charge-induced and charge-remote fragmentations. Close inspection of the MS/MS spectra of the linear and cyclic precursor ions reveals two significant differences in their fragmentation patterns: the fragmentation 70

95 extent (fragment ion intensities divided by total ion intensities) is substantially higher for the linear architecture; less fragment ion series are produced from the macrocycles due to their higher symmetry. These features and the actual m/z values of the observed fragments permit conclusive differentiation and identification of the correct architecture. 71

96 CHAPTER V INTERFACING MASS SPECTROMETRY WITH LIQUID CHROMATOGRAPHY SEPARATION FOR THE CHARACTRIZATION OF POLYETHYLENE OXIDE POLYCAPROLACTONE (PEO-b-PCL) DIBLOCK COPOLYMERS 5.1 Background Block copolymer micelles are widely used as drug delivery devices. The polymer should be biodegradable and biocompatible to be used for this purpose. Poly(εcaprolactone) (PCL) is an aliphatic polyester which has been approved to be used for such application by the Food and Drug Administration (FDA) because of its chemical and physical properties. PCL is a hydrophobic polymer which limits its use on drug delivery devices. To overcome this limitation, amphiphilic diblock copolymers of PCL with hydrophilic polymers such as PEO have been synthesized to enhance the solubility of such copolymers in water or biological fluids. 67, 72 In aqueous solutions, PEO-b-PCL diblock copolymers form self-assembled micelles in which the core is hydrophilic and the tail hydrophilic. These micelles can be used as drug delivery devices to release the drug in the targeted organism. The synthesis of PEO-b-PCL diblock copolymers can be accomplished via ring opening polymerization, (ROP), of ε-caprolactone using various PEO macroinitiators with different architectures in the presence of a catalyst. 75, 76 This synthetic route can give 72

97 rise to a mixture of products having different physical and chemical properties. Such a mixture can contain unreacted starting materials, PEO homopolymer, or polymerized PCL (linear and cyclic homopolymers), and various side products. The complexity of the analysis of this mixture requires a powerful technique that can separate and identify each component. Liquid chromatography (LC) coupled with mass spectrometry (MS) has been used to solve such challenging analytical problems. Reverse-phase high-pressure liquid chromatography (RP-HPLC) has been used widely to characterize polymers in which the polymer units can be separated based on their distribution equilibria between the stationary and mobile phases. The more polar components elute first while the less polar ones elute last. Ahmed et al. reported the use of RP-HPLC and offline MALDI-ToF MS for the analysis of PEO-b-PCL. 37, 38 In these studies, oligomer separation was achieved by RP-HPLC according to the number of CL units (retention time increased with increasing CL content), with each fraction containing a distribution of EO units. The fractions were collected after the separation and subjected to MALDI-ToF MS to confirm the composition and molecular weight distribution of each fraction. The run time of the HPLC experiment was 40 min. 37, 38 Van Leeuwen et al. used HPLC coupled with online ESI to separate linoleic acid functionalized surfactants prepared by block copolymerization of methyl polyethylene oxide (PEO) and poly(ε-caprolactone) (PCL). 26 Mikhail and Allen reported the use of HPLC coupled with a UV detector to characterize a PEO-b-PCL diblock copolymer and a novel micelle-forming copolymer-drug conjugate containing docetaxel (DTX), (PEO-b-PCL-DTX). 78 UV chromatograms were used to compare the copolymer and the drug conjugated copolymer to prove the DTX attachment. 73

98 Traditional HPLC (UV detection) has limitations in its performance which include a long analysis time, significant solvent consumption, as well as low resolution and sensitivity. To overcome these limitations, an improved HPLC technique has been developed, called ultra performance liquid chromatography (UPLC). This technique improves the sensitivity and efficiency of separation and identification of mixture 57, 58 components and also shortens the analysis time. This chapter reports the first UPLC-Q/ToF MS and tandem MS on PEO-b-PCL. These techniques, as well as MALDI-ToF MS, MALDI-Q/ToF MS/MS, were employed to identify the structure, molecular weight, and impurities or unreacted materials in PEOb-PCL block copolymers with different architectures in the PEO block. 5.2 Sample Preparation and Instrumentation Used The PEO-b-PCL diblock copolymers were analyzed using MALDI-ToF/ToF MS (Bruker Ultraflex-III ToF/ToF mass spectrometer) and MALDI-Q/ToF MS (Waters Micromass Q/ToF Ultima mass spectrometer) instrumentation. The UPLC-MS study was performed using an Acquity UPLC TM system coupled with the Synapt HDMS TM hybrid quadrupole/time-of-flight (Q/oa-ToF) mass spectrometer (Waters, Beverly, MA). For MALDI analyses, the samples were dissolved in tetrahydrofuran (THF). Ionization was achieved using sodium trifluoroactate (NaTFA). Trans-2-[3-(4-tert-butylphenyl)-2- methyl-2-propenylidene] malononitrile (DCTB) served as the matrix. Solutions of the matrix (20 μg/μl), the sample (10 μg/μl), and salt (10 μg/μl) were mixed in the ratio 10:2:1, respectively. Chromatographic separation of the PEO-b-PCL diblock copolymers was performed on a Waters, Acquity UPLC BEH C µm, 2.1 x 50 mm column 74

99 using a flow rate of 0.25 ml/min and gradient elution which took 16 min. The copolymer components were eluted with water (solvent line A) and acetonitrile (solvent line B) via a gradient program. The mobile phase composition was changed from 30% acetonitrile (v/v) to 80% acetonitrile in the first 10 minutes and from 80% to 100% acetonitrile in the next 2 minutes. After using 100% acetonitrile to flush the column for 2 minutes, the column was regenerated from 100% to 30% acetonitrile in the last 2 minutes. The sample for UPLC-MS analysis was prepared by dissolving the copolymer in 70% water: 30% acetonitrile solution at the concentration of 0.1 mg/ml. 1% (v/v) of a 0.1 mg/ml NaTFA solution was added to improve ionization. All spectra were collected in positive mode. 5.3 Characterization of PEO-b-PCL Diblock Copolymers using MALDI-ToF MS and UPLC-ESI-Q/ToF MS/MS This chapter reports the procedures developed for the analysis of PEO-b-PCL diblock copolymers with different architectures on the PEO moiety. A summary of the structures analyzed are shown in Scheme 5.1. These copolymers were synthesized starting with monofunctional or difunctional PEO macroinitiators which reacted with ε- caprolactone via ring opening polymerization using tin 2-ethylhexanoate (Sn(Oct) 2 ) as a catalyst. With methoxy PEO or (i.e. (4-hydroxymethyl)phenoxy PEO), macroinitiators a linear-b-linear diblock copolymer is generated, while the cyclic-b-linear diblock copolymer analog is formed when 3,4-(42-crown-14) benzyl alcohol is used as initiator. The syntheses were performed by Dr. Gladys Montengro in professor Coleen Pugh s research group

100 Block copolymers are complex mixtures and may contain many byproducts that are formed during the synthesis. Therefore, the analysis of these copolymers can be challenging. The copolymers were examined with MALDI-ToF/ToF MS and MALDI- Q/ToF MS/MS to determine their molecular mass distribution and their structural composition prior to the LC-MS analysis. Scheme 5.1 Synthetic scheme to methoxy PEO-b-PCL (CH 3 PEO-b-PCL), ω-benzyl alcohol PEO-b-PCL (ω-bnpeo-b-pcl), and 3,4-(42-crown-14)benzyl alcohol PEO-b- PCL (McBnPEO-b-PCL) Characterization of CH 3 PEO-b-PCL Diblock Copolymer Single-stage MALDI mass spectrometry analysis was performed on the MALDI- ToF/ToF instrument while tandem MS experiments were performed on the MALDI- 76

101 Q/ToF mass spectrometer. In both cases DCTB was used as the matrix and NaTFA as ionization agent. The MALDI-ToF/ToF spectrum of CH 3 PEO-b-PCL is shown in Figure 5.1; it displays several distributions with different lengths of the two blocks, all arising from sodium adducts. Expanded views of regions (I-III), which contain distinct distributions, are shown in Figure 5.2. This copolymer carries methyl and hydroxyl end groups (CH 3 - and OH) based on the synthetic route described in Scheme 5.1. This information was used to build a spreadsheet (Appendix A) listing the m/z values of different sized copolymer blocks with the quoted end groups and sodium as ionization agent. The m/z values measured by MALDI-ToF MS were searched in the spreadsheet to determine the composition of the corresponding peaks. Based on the ions observed in the MALDI spectrum, which cover the range Da, the PEO block contains 7-15 repeat units and the PCL block contains 0-12 blocks. In order to confirm these assignments, tandem mass spectrometry on the MALDI-Q/ToF instrument was utilized. MS/MS can unveil information about the block sizes, sequences, and architecture of this copolymer as well as about its intrinsic fragmentation pathways. 77

102 MePEO13 OH MePEO11OH MePEO 12 OH I II III m/z Figure 5.1 MALDI mass spectrum of sodium cationized PEO-b-PCL acquired on the MALDI-ToF/ToF mass spectrometer I PEO9 PCL1 PEO7PCL2 PEO10 PCL1 PEO8 PCL m/z 78

103 PEO11 PCL5 PEO14 PCL4 PEO9 PCL6 PEO12 PCL PEO15 PCL4 PEO10 PCL6 PEO8 PCL7 PEO13 PCL5 PEO11 PCL6 PEO14 PCL5 PEO9 PCL7 PEO7 PCL8 PEO15 PCL5 PEO12PCL6 PEO10 PCL7 PEO13 PCL6 PEO8 PCL8 PEO11 PCL7 PEO14 PCL6 PEO9 PCL II m/z III PEO13 PCL7 PEO8 PCL9 PEO11 PCL8 PEO14 PCL7 PEO9 PCL9 PEO12 PCL8 PEO10 PCL9 PEO13 PCL8 PEO8 PCL10 PEO11 PCL9 PEO14 PCL8 PEO9 PCL10 PEO12 PCL9 PEO15 PCL8 PEO10PCL10 PEO13 PCL9 PEO11 PCL m/z Figure 5.2 Expended view of molecular weight regions (I-III) in the MALDI mass spectrum of Figure 5.1. Roughly, these regions correspond to PEO homopolymer (I) and PEO-b-PCL copolymer with shorter (II) or longer (III) PCL chains. 79

104 MALDI-Q/ToF MS/MS experiments were performed on several ions in region II. According to the spreadsheet, the ion at m/z contains six caprolactone (CL) and ten ethylene oxide (EO) units. This ion was isolated and dissociated by CAD at a collision energy of 80 ev. The MS/MS spectrum (Figure 5.3) shows a fragmentation pattern that agrees well with the predicted CH 3 -PEO 10 PCL 6 -OH diblock composition. Two major series are observed, generated by losses of either CL n (114n Da) or HO-CL n - H (114n +18 Da). These reactions can be rationalized via intramolecular transesterification (*) or 1,5-H rearrangement over the ester group (#), respectively (vide infra). 17 Both fragmentations stop after the loss of six CL units; no significant further decomposition within the EO units take place. They are probably charge-remote decompositions, involving the loss of CL n macrocycles (*) or the loss of linear HO- (CL) n -H chains (#), respectively. The fragment ions arising after the loss of these six CL units can be assigned to CH 3 -PEO 10 -OH for the series marked by * and CH 3 -PEO 9 - OCH=CH 2 for the series marked by #. Hence, the tandem spectra provide corroborating information about both block lengths of the examined oligomer: its PCL block size is unveiled by the number of CL n or HO-(CL) n -H PCL losses, and the PEO block size is reflected by the m/z values of the smallest fragments in the two fragment series (which only contain EO repeat units). 80

105 # # # * * * # * # * # * 132 Da Da m/z Figure 5.3 MALDI-MS/MS spectrum of the oligomer at m/z with the putative CH 3 -PEO 10 PCL 6 -OH structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 80 ev. An MS/MS experiment was also carried out for m/z which (according to the spreadsheet) has 13 EO units and five CL units; the resulting spectrum is shown in Figure 5.4. The same two series are observed in the spectrum, involving the loss of five CL units. The fragment ions after the loss of five CL units can be assigned to CH 3 - PEO 13 -OH for the * series and CH 3 -PEO 12 -OCH=CH 2 for the # series. This result fully agrees with the assignment predicted from the spreadsheet. 81

106 # # # # # * m/z Figure 5.4 MALDI-MS/MS spectrum of the oligomer at m/z with the putative CH 3 -PEO 13 PCL 5 -OH structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 80 ev. * * * * Scheme 5.2 illustrates the charge-remote 1,5-H rearrangement and intermolecular transesterification that can account for the fragment series # and *, respectively. Both these dissociations have been observed with sodiated polyesters. 17 Scheme 5.2 shows how such reactions proceed at the terminal ester group. Analogous reactions that cleave larger linear or cyclic PCL oligomers can occur at all other ester groups. The largest neutral losses and smallest fragment ions (in series # and *) result if these dissociations occur at the ester group connecting the PCL and PEO blocks; therefore, the latter fragments can be utilized to identify the corresponding block lengths. 82

107 Scheme 5.2 Charge-remote 1,5-H rearrangement (left) or intramolecular transesterification (right) in the caprolactone ester block of CH 3 -PEO m PCL n -OH The single-stage MS and tandem mass spectrometry data indicated the presence of unreacted starting material (CH 3 -PEO-OH) and several block copolymer distributions. To confirm this assignment, UPLC coupled with MS and MS/MS experiments were employed to separate the copolymer components based on polarity and identify their composition and connectivity by MS and MS/MS. The LC-MS total ion chromatogram acquired from the CH 3 -PEO-b-PCL-OH diblock copolymer using UPLC-ESI-Q/ToF MS is shown in Figure

108 (1) (5) (2) (3) (4) (6) (7) (8) (9) (11) (13) (12) (10) (14) (15) (16) Time (min) Figure 5.5 LC-MS total ion chromatogram of the CH 3 -PEO-b-PCL-OH diblock copolymer. The arrow indicates an impurity which is also observed with a blank sample. In the total ion chromatogram, there are more than 16 well separated fractions present and each one of these fractions corresponds to a different structure. The extracted spectra of the fractions indicate that oligomers having the same architecture and a constant number of caprolactone units are eluted at the same time. The number of caprolactone units increases with the retention time, as affirmed by Figure 5.6, which shows the extracted spectra of LC fractions #5, #7 and #9 from the copolymer sample. In fraction number 5 (eluting from 3.25 to 3.60 min), the ions in the singly charged major distribution (such as m/z , , and ) are sodiated oligomers with the same number of caprolactone units (three). The minor distributions corresponded to protonated, ammoniated, and potassiated oligomers (an expanded view of fraction #7 is shown in Appendix A). The distance between two adjacent peaks in these distributions is 44 Da, corresponding to one ethylene oxide unit, which indicates that they originate from CH 3 -PEO n -b-pcl m -OH with constant m and a range of n. Comparison of the m/z values of the main distributions in LC fractions #5, #7, and #9 further indicates that they all 84

109 contain oligomers with ~6-14 EO units and either 3 or 4 or 5 CL units, respectively. Oligomers with fewer caprolactone units elute faster than oligomers with higher caprolactone content because of the higher hydrophobicity of PCL vs. PEO segments. These assignments were confirmed by tandem mass spectrometry experiments on select oligomers from fraction #7 and #9. PEO8PCL3 PEO10PCL3 PEO7PCL PEO12PCL PEO13PCL Fraction # 5 CH 3 -PEO n -PCL 3 -OH PEO11PCL PEO8PCL PEO12PCL4 PEO7PCL PEO13PCL Fraction # 7 CH 3 -PEO n -PCL 4 -OH PEO10PCL5 PEO8PCL Fraction # 9 PEO11PCL5 PEO7PCL PEO12PCL5 CH 3 -PEO n -PCL 5 -OH PEO13PCL m/z Figure 5.6 Comparison of LC-MS mass spectra of LC fractions #5, #7, and #9 from the CH 3 -PEO-b-PCL-OH copolymer (m/z region of singly charged [M+Na] + ions, vide intra). Figure 5.7 shows the spectra obtained after UPLC-MS/MS. Under ESI, which usually deposits less internal energy than MALDI, 1 only HO-CL n -H losses (114n + 18 Da) are observed, pointing out that 1,5-H rearrangements, in sodiated PEO-b-PCL oligomers have lower activation energies than intramolecular transesterifications. The largest neutral loss contains 4 CL units from the ion at m/z and 5 CL units from 85

110 the ion at m/z which agrees well with the presence of PCL 4 blocks in the former and PCL 5 blocks in the latter. # Fraction # 7 CH 3 -PEO 9 -PCL 4 -OH # # Da # Da * Fraction # 9 CH 3 -PEO 9 -PCL 5 -OH # # # # Da # Da * Figure 5.7 UPLC-MS/MS spectra of the ions at m/z in LC fraction #7 and in LC fraction #9 at a collision energy of 60 ev. Figure 5.8 shows the extracted spectra of fractions #4, #6, and #8 whose main constituents are single oligomers of homopolymeric polycaprolactone with 3, 4, and 5 repeat units, respectively. These are observed as protonated, sodiated, and/or potassiated species. Further, fractions #6 and #8 also contain traces of cyclic PCL, observed in protonated form. The ion at m/z was isolated and fragmented to confirm that it is protonated PCL 4. The resulting spectrum, Figure 5.9, shows the fragments characteristic for PCL chain, arising by losses of 114n + 18 Da (series #) and 114n Da (series *). Proton charges promote charge-induced dissociations. The fragments observed are accounted for by proton-catalyzed intramolecular transesterifications by the OH 86

111 terminal group. Such reactions at the different ester group would generate CL 4 macrocycles (#) and truncated PCL n chains (*), either of which can keep the charge to be detected. The lightest ion at m/z is the protonated CL unit, formed by the loss of HO-CL 3 -H in agreement with having 4 CL units in this oligomer. [PCL 3 +Na] [PCL 3 +H] [PCL 3 +K] [PCL 4 +Na] [PCL 4 +H] + [PCL 4 +K] + [cpcl 4 +H] [PCL 5 +Na] [PCL 5 +H] [cpcl [PCL 5 +K] + 5 +H] m/z Figure 5.8 LC-MS mass spectra of LC fractions #4, #6, and #8 from CH 3 -PEO-b-PCL- OH diblock copolymer. cpcl n and PCL n designate macrocycles (no end groups) and linear chains with the connectivity HO-CL n -H, respectively. 87

112 * [PCL 4 +H] # * * # [PCL 2 +H] [PCL 3 +H] # # [PCL 1 +H] * m/z Figure 5.9 UPLC-MS/MS spectrum of the ion at m/z in LC fraction # 6 at collision energy of 60 ev. The LC-MS data indicate that the number of CL units increases with the eluting time. Further, the mass spectra of several neighboring peaks, for example #4 and #5, or #6 and #7, or #8 and #9, show that the homopolymer (#4, #6, #8) elutes faster than copolymer with the same number of CL unit (#5, #7, #9, respectively). Hence, the free acid group in the PCL homopolymer must increase the hydrophilicity more than a CH 3 - PEO n block. The MALDI-ToF mass spectrometry results presented in section revealed that the sample contains PEO starting material. In the LC-MS experiment, the PEO homopolymer with CH 3 - and OH end groups is eluted in fraction #1, as the most polar (hydrophilic) mixture component. 88

113 Although MALDI-ToF MS did not detect any PCL homopolymer, perhaps due to overlap with the other more intense and/or more easily ionizable distributions, LC-MS conclusively confirmed its presence in fractions #4, #6, #8, #10, #12, #13, #14, and #15. This finding underscores the usefulness of LC-MS for identifying minor mixture components. The compositions of fractions #1-16 determined by LC-MS and MS/MS analysis are detailed in Table 5.1. Table 5.1 PEO-b-PCL components identified in the LC-MS fractions by the corresponding mass spectra and select MS/MS spectra Fraction number Eluting time (min) Composition 1 ~0.75 CH 3 -PEO n -OH 2 ~1.12 CH 3 -PEO n -b-pcl 1 -OH 3 ~2.19 CH 3 -PEO n -b-pcl 2 -OH 4 ~ CL a 5 ~3.59 CH 3 -PEO n -b-pcl 3 -OH 6 ~ CL a 7 ~4.19 CH 3 -PEO n -b-pcl 4 -OH 8 ~ CL a 9 ~6.14 CH 3 -PEO n -b-pcl 5 -OH 10 ~ CL a 11 ~7.28 CH 3 -PEO n -b-pcl 6 -OH 12 ~7.99 ~ ~8.99 ~ ~9.84, ~ CL a CH 3 -PEO n -b-pcl 7 -OH 8 CL a CH 3 -PEO n -b-pcl 8 -OH 9 CL a CH 3 -PEO n -b-pcl 9 -OH 15 ~ CL a 16 ~11.02 CH 3 -PEO n -b-pcl 10 -OH a Mixtures of cyclic and linear oligomers (see Figure 5.8). 89

114 5.3.2 Characterization of ω-benzyl Alcohol PEO-b-PCL Diblock Copolymer The MALDI-ToF mass spectrum of ω-bnpeo-b-pcl diblock copolymer (Figure 5.10) displays several distributions of different combinations of the two blocks, all ionized by sodium ion adduction. Expanded views of the two main regions I and II, shown in Figure 5.11, demonstrate that this product contains one major and several minor oligomer distributions. The copolymer carries methyl and hydroxyl end groups at the PEO and PCL chain ends, respectively, and an internal 4-hydroxyl substituent at the block junction based on the synthetic route described in Scheme 5.1. The combined internal substituent and end groups have the composition C 8 H 10 O 2 (138 Da). This information was used to build a spreadsheet (Appendix A) listing the m/z values of copolymeric oligomers with the quoted central/end groups and sodium as ionization agent. The m/z values measured by MALDI-ToF MS were search in the spreadsheet in order to determine the composition of the corresponding ions. The major distribution with a 114-Da results from linear polycaprolactone homopolymer (PCL) with H- and - OH end groups (18 Da), while the minor distributions, with 114-Da or 44-Da repeat units, are attributed to combinations of variously sized PEO and PCL blocks. In order to confirm these assignments, MALDI-Q/ToF tandem mass spectrometry was employed. 90

115 I II m/z Figure 5.10 MALDI mass spectrum of sodium cationized ω-bnpeo-b-pcl, acquired on the MALDI-ToF/ToF mass spectrometer. I PEO8-PCL5 PEO11-PCL4 PEO9-PCL5 PEO12-PCL4 PEO10-PCL5 PEO8-PCL6 PEO11-PCL5 PEO9-PCL6 PEO12-PCL5 PEO10-PCL H--PCL9 -ONa H PCL10 -ONa H-PCL9-OH H-PCL10-OH H-PCL11-OH m/z 91

116 H-PCL16-OH II PEO13-PCL10 PEO8-PCL12 PEO11-PCL11 PEO9-PCL12 PEO12-PCL11 PEO10-PCL12 PEO13-PCL11 PEO8-PCL13 PEO11-PCL12 PEO9-PCL13 PEO12-PCL12 PEO10-PCL13 H-PCL17-OH H-PCL18-OH m/z Figure 5.11 Expended views of molecular weight regions I and II in the MALDI mass spectrum of Figure 5.5. Fragmentation of the oligomer at m/z resulted in the loss of CL n (114n Da) and HO-CL n -H (114n + 18 Da) moieties with n = 1-6, in agreement with the presence of a PCL 6 block in this oligomer (Figure 5.12). Such losses can be rationalized by the charge-remote 1,5-H rearrangement and intramolecular transesterification pathways depicted in Scheme 5.2, which generate fragment ions with a terminal double bond(# series) or terminal hydroxyl group (* series), respectively. 17 Note that the smallest fragment in series # cannot be formed by 1,5-H rearrangement because there is no H atom available 5 bonds away from the carbonyl oxygen of the ester group attached to the 92

117 benzyl linker. An alternative mechanism involving H-transfer from PEO block is presented in Scheme A1 in Appendix A. # # # # # * # * * * * * # # * # * # * # * # * m/z Figure 5.12 MALDI-MS/MS spectrum of the sodiated oligomer at m/z with the putative PEO 11 PCL 6 diblock composition, acquired with the MALDI-Q/ToF tandem mass spectrometer at collision energy of 70 ev. On the other hand, the oligomer at m/z from the major distribution (Figure 5.13) shows a series of CL n and HO-CL n -H losses with up to nine CL units, which agrees with the linear PCL 10 structure (H- and OH end groups) assigned with the help of the spreadsheet data. The smallest fragment ion at m/z corresponds to the sodiated CL unit, [C 6 H 10 O 2 +Na] +. With a linear PCL homopolymer, either of the mechanisms 93

118 presented in Scheme 5.2 can account for the observed fragments series (# and *). 1,5-H rearrangement over the ester group generates two fragments, one with a newly formed carboxyl group (*) and one with a terminal double bond (#); each one of these fragments is able to keep the Na + charge. Conversely, intramolecular transesterification at the ester groups generates a cyclic PCL (#) and a truncated linear PCL (*); again, either of these fragment may keep the charge to be detected. 17 Note that with a PEO-b-PCL copolymer, only the fragment retraining the PEO block is observed, because PEO provides a better coordination environments for the Na + ion than PCL. # # * # * # * # * # * # * # * # * Figure 5.13 MALDI-MS/MS spectrum of the sodiated oligomer at m/z with the putative H-PCL 10 -OH structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 65 ev. A minor distribution appearing at m/z 1089, 1203, 1317, etc. with a 114-Da repeat unit could not be assigned using the spreadsheet built to identify the copolymer 94

119 composition. This distribution is observed 22 Da higher than the major, homopolymeric HO-PCL n -H distribution strongly suggesting that it originates from COOH COONa exchange during MALDI. The tandem mass spectrum of the ion at m/z shows three fragment series with the 114-Da repeating unit (Figure 5.14). The first distribution (marked with *) arises from the loss of 114n Da (CL n units). The second distribution (marked with #) arises from losses of 114n + 18 Da (H-CL n -OH units). The third distribution (marked with $) arises from losses of 114n + 40 Da, which are consistent with H-CL n -ONa units. All these fragments can be generated from a linear PCL in which the COOH chain end was converted to COONa. The [M+Na] + ions of this product, contain two sodium ions. After fragmentation according to the pathways in Scheme 5.2, Na + /H + exchange in the separating fragments is possible. 85 Both Na + ions remain largely with the ionic fragment based on the ion series present in the MS/MS spectrum. * # * # * * * * * # * $ # $ $ # # $ $ $ # $ # Figure 5.14 MALDI-MS/MS spectrum of the oligomer at m/z with the putative NaO-PCL 9 -H structure, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 65 ev. 95

120 More detailed insight on the compositional heterogeneity of ω-bnpeo-b-pcl was sought by LC-MS. Figure 5.15 show the LC-MS chromatogram of this block copolymer, obtained by UPLC separation and ESI-Q/ToF MS detection. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (12) (11) (13) (14) (15) (16) Time (min) Figure 5.15 LC-MS total ion chromatogram of the ω-bnpeo-b-pcl diblock copolymer. More than 16 well separated fractions are present in the total ion chromatogram and each fraction represents different structures. As mentioned in the previous section, integration of consecutively detected peaks reveals that the number of caprolactone (CL) units increases with increasing retention time. Within each LC peak, the number of CL units stays constant as attested in Figure 5.16 by the LC-MS mass spectra of fractions #8, #10, and #12 which contain copolymer chains with 3, 4, and 5 CL units, respectively. For example in fraction number 8 (eluting from 5.10 to 5.30 min), the major oligomer series has the same number of CL units (three) and a distribution of EO units. To confirm these assignments, tandem mass spectrometry experiments were performed on the ions at m/z in fraction #10 and in fraction #12. The most prominent fragment series in their MS/MS spectra (Figure 5.17) arises by H-CL n -OH (114n + 18 Da) loses (#), with the largest such oligomer eliminated being the 4-mer (n = 4) for the precursor ion from 96

121 fraction #10 and the 5-mer (n = 5) for the precursor ion from fraction #12; this result agrees well with the assignments deduced from the spreadsheet (see above). The MS/MS spectra of m/z and include fragments that cannot arise from ω-bnpeo 9 -b-pcl n (n = 4 and 5, respectively). The ions, which have been marked by $ in Figure 5.17, are attributed to an isobaric impurity containing less EO and more CL repeat units than the major component in the LC fractions as well as a modified end group at PCL chain end, as shown in Scheme A2 (see Appendix A). A mass resolution of > 12,500 is needed to separate the isobars; this is not available in MS/MS mode with Q/ToF instruments, where precursor ion selection (by Q) is performed at unit mass resolution. 1 As found for the block copolymer discussed in the previous section, the benzylconnected block copolymer ω-bnpeo-b-pcl also contains PCL homopolymer, are eluted separately in fractions #7, #9, #11, #13, #14, and #16. Homopolymer with higher CL content elutes later because of the lower hydrophilicity of PCL than PEO chains. The compositions of fractions 1-16 determined by LC-MS and MS/MS are summarized in Table

122 PEO 10PCL PEO 8 PCL 3 PEO 7 PCL PEO 6 PCL PEO 12 PCL PEO 13 PCL PEO 14 PCL Fraction # 8 CH 3 -PEO n -Bn-PCL 3 PEO 8 PCL 4 PEO 7 PCL PEO 11 PCL PEO 12 PCL 4 PEO 6 PCL PEO 13 PCL Fraction # 10 CH 3 -PEO n -Bn-PCL 4 PEO 7 PCL 5 PEO 9 PCL 5 PEO 11 PCL 5 Fraction # CH 3 -PEO n -Bn-PCL 5 PEO 6 PCL PEO 13 PCL Figure 5.16 LC-MS mass spectra of LC fractions #8, #10, and #12 from the ω-bnpeo-b- PCL diblock copolymer (m/z region of singly charged [M+Na] + ions) 98

123 * Fraction # 10 CH 3 -PEO n -Bn-PCL 4 # 114 Da # 132 Da # $ $ $ $ # $ $ Fraction # 12 CH 3 -PEO n -Bn-PCL 5 $ $ # $ # $ # $ $ # Da # Da * Figure 5.17 UPLC-MS/MS spectra of the ion at m/z in LC fraction #10 and of the ion at m/z in LC fraction #12. The collision energy was 60 ev. See Scheme A1 for the fragmentation pathway to m/z (and 433.5) and Scheme A2 for a rationalization of series $ which is due to an isobaric impurity. 99

124 Table 5.2 PEO-b-PCL components identified in the LC-MS fractions of ω-bnpeo-b- PCL by the corresponding mass spectra and select MS/MS spectra Fraction number Eluting time (min) Composition 1 ~0.75 CH 3 -PEO n -OH 2 ~1.20 CH 3 -PEO n -Bn-OH 3 ~2.45 CH 3 -PEO n -Bn-PCL 1 -OH 4 ~ CL a 5 ~3.26 CH 3 -PEO n -PCL 3 -OH 6 ~ 3.83 CH 3 -PEO n -Bn-PCL 2 -OH 7 ~ CL a 8 ~5.15 CH 3 -PEO n -Bn-PCL 3 -OH 9 ~ CL a 10 ~6.30 CH 3 -PEO n -Bn-PCL 4 -OH 11 ~ CL a 12 ~7.42 CH 3 -PEO n -Bn-PCL 5 -OH 13 ~ CL a 14 ~8.44, ~8.90 CH 3 -PEO n -Bn-PCL 6 -OH 8 CL a 15 ~9.32 CH 3 -PEO n -Bn-PCL 7 -OH 16 ~10.13 CH 3 -PEO n -Bn-PCL 8 OH 9 CL a a Mixtures of cyclic and linear oligomers Characterization of 3,4-(42-Crown-14) Benzyl Alcohol PEO-b-PCL (McBnPEO-b- PCL) Diblock Copolymer Figure 5.18 shows the MALDI-ToF mass spectrum of McBnPEO-b-PCL diblock copolymer which displays several distributions of copolymeric oligomers, all ionized by Na +. An expanded view of the region with the most abundant peaks (Figure 5.19) demonstrates the presence of one major and several minor distributions. This copolymer 100

125 carries the phenyl ring of a benzyl group within a crown ether macrocycle based on the synthetic route described in Scheme 5.1. The aromatic linking group between the two blocks plus two EO units (minimum EO content for a macrocycle) contribute C 11 H 13 O 3 to the copolymer composition. This central substituent and the OH chain end of the PCL block provide a mass of 210 Da to each oligomer. This information was used to build a spreadsheet (Appendix A) listing the m/z values of variously sized copolymer blocks with the quoted central/end groups and sodium as ionization agent. The m/z values measured by MALDI-ToF MS were searched in the spreadsheet in order to determine the composition of the observed peaks. The major distribution with a 114-Da repeat unit originates from linear homopolymer of polycaprolactone (PCL) with 18-Da end groups (H- and OH) while the minor distributions, having 114-Da or 44-Da repeat units, can be attributed to copolymer with variously sized PEO and PCL blocks. In order to confirm these assignments, further information was sought by tandem mass spectrometry using the MALDI-Q/ToF instrument. 101

126 m/z Figure 5.18 MALDI mass spectrum of sodium cationized McBnPEO-b-PCL, acquired on the MALDI- ToF/ToF mass spectrometer. The linking substituent contributes 105 Da (C 7 H 5 O) and the end group of the PCL block 17 Da (OH) to the polymer mass (combined central/end group mass of 122 Da). 102

127 PEO11-PCL4 PEO14-PCL3 PEO9-PCL5 PEO12-PCL4 PEO7-PCL6 PEO15-PCL3 PEO10-PCL5 PEO13-PCL4 PEO11-PCL5 PEO14-PCL4 PEO79PCL6 PEO12-PCL5 PEO7-PCL7 PEO15-PCL4 PEO10-PCL6 PEO13-PCL H-PCL9 -ONa H-PCL10 -ONa H-PCL9-OH H-PCL10-OH H-PCL11-OH m/z Figure 5.19 Expanded view of the MALDI mass spectrum shown in Figure Each copolymeric oligomer contains a C 7 H 5 O linking group and a OH end group at the PCL chain end. The combined composition and the mass of these substituents are C 7 H 6 O 2 and 122 Da, respectively. The fragmentation pattern of the major distribution confirms that its members are PCL homopolymer with H- and OH end groups. The copolymeric oligomer at m/z with the putative composition PEO 13 PCL 4 was also examined by MS/MS. The resulting spectrum (Figure 5.20) shows losses of 1, 2, 3, and 4 units of CL, either as (CL) n (series *) or as H-(CL) n -OH (series #) which agreed with the mentioned assignment (PEO 13 PCL 4 ). The smallest fragment ion in series # at m/z 699 is rationalized 103

128 in Scheme A3. It only contains the linking substituent less one H atom (transferred in the H-rearrangement) and, hence, can be used to calculate the PEO block size: m/z ( Na + ) (C 7 H 4 O) = / (EO mass) = 13 (i.e. PEO 13 ). It is evident by comparing Figure 5.12 and in Figure 5.20 that the smallest fragment in series # is much more intense when the PEO block is cyclic. That can be reconciled by the fragmentation mechanisms presented in Scheme A1 and A3; the macrocylic PEO block gives rise to a sodiated polyether with a methylene cyclohexadienone end group, whereas the linear PEO block produces a sodium ion bound complex between methylene cyclohexadienone and a polyether which is less stable and can easily decompose. A common characteristic for both types of block copolymers is that the fragmentation predominantly occurs on the ester block irrespective of the PEO architecture, because ether bonds are more difficult to break than ester bonds under MS/MS (CAD) conditions. This in turn makes it possible to identify copolymer block lengths by tandem mass spectrometry. 104

129 # * # # # * * * m/z Figure 5.20 MALDI-MS/MS spectrum of the sodiated oligomer at m/z with the putative diblock composition PEO 11 PCL 4, acquired with the MALDI-Q/ToF tandem mass spectrometer at a collision energy of 65 ev. The block copolymer of McBnPEO-b-PCL was also studied by LC-MS. Figure 5.21 displays the total ion chromatogram obtained by UPLC, interfaced with ESI-Q/ToF MS. 105

130 (1) (2) (3) (4) (5) (8) (9,10) (6,7) (11) (12) (13) (14) (15,16,17) (18) Time (min) Figure 5.21 LC-MS total ion chromatogram of the McBnPEO-b-PCL diblock copolymer. There are more than 18 well separated fractions present in the total ion chromatogram and each fraction represents different macromolecular stoichiometries. As found for other copolymers, integration of consecutive peaks reveals that the number of caprolactone (CL) units increases with increasing retention time. The number of CL units stays constant within each LC peak as shown in Figure 5.21 which depicts the singly charged regions of the LC-MS mass spectra of three LC fractions (fractions #8, #10, and #11) from the copolymer sample, corresponding to copolymers containing 3, 4, and 5 CL units, respectively. For example, in fraction number 8 (eluting from 4.50 to 4.65 min), the ions in the major distribution (such as m/z , , , and ) have the same number of CL units (three) and a distribution of EO units. This assignment was confirmed by tandem mass spectrometry experiments on oligomers from fraction #8 and #10 as shown in Figure The ions at m/z and were isolated and fragmented. The resulting spectra show the loss of 3 CL unit from the ion at m/z and 4 CL unit from the ion at which agrees well with the assignments given in Table

131 PEO 11PCL 3 PEO 12PCL 3 PEO 10PCL PEO 13PCL PEO 14PCL 3 Fraction # 8 McPEO n -Bn-PCL PEO15PCL PEO 8PCL 4 PEO 9PCL PEO 10PCL PEO 13PCL PEO 14PCL 4 PEO PCL Fraction # 10 McPEO n -Bn-PCL 4 PEO 8PCL PEO 12PCL PEO PCL 5 Fraction # 11 PEO 10PCL McPEO n -Bn-PCL 5 PEO14PCL5 PEO 9PCL m/z Figure 5.22 LC-MS mass spectra of LC fractions #8, #10, and #11 from the McBnPEOb-PCL diblock copolymer. * # Da Fraction # 8 # 114 Da # McPEO n -Bn-PCL * Fraction # 10 # McPEO n -Bn-PCL # Da 114 Da # # Figure 5.23 UPLC-MS/MS spectra of the ions at m/z in LC fraction #8 and the ion at m/z in LC fraction #10. The collision energy was 60 ev. 107

132 As mentioned in the previously, oligomers from the PCL homopolymers coproduced during the synthesis are eluted separately, in fractions #1, #4, #6, #9, #11, #12, #13, #14, #15, #17, and #18. Again, oligomers with lower CL content elute faster than the heavier ones because of the lower hydrophilicity of PCL than PEO. The compositions of fractions 1-18 are detailed in Table 5.3. Table 5.3 PEO-b-PCL components identified in the LC-MS fractions of McPEO-b-PCL by the corresponding mass spectra and select MS/MS spectra Fraction number Eluting time (min) Composition 1 ~ CL a 2 ~1.12 Mc-PEO n 3 ~1.92 Mc-PEO n -PCL 1 -OH 4 ~ CL a 5 ~3.30 Mc-PEO n -PCL 2 -OH 6 ~ CL a 7 ~4.32 Contamination 8 ~4.64 Mc-PEO n -PCL 3 -OH 9 ~ CL a 10 ~5.87 Mc-PEO n -PCL 4 -OH 11 ~6.87 ~ ~7.97 ~ ~8.95 ~ ~9.84 ~ CL a Mc-PEO n -PCL 5 -OH 7 CL a Mc-PEO n -PCL 6 -OH 8 CL a Mc-PEO n -PCL 7 -OH 9 CL a Mc-PEO n -PCL 8 -OH 15 ~ CL a 16 ~10.90 Mc-PEO n -PCL 9 -OH 17 ~ CL a 18 ~12.90 Mc-PEO n -PCL 11 -OH a Mixtures of cyclic and linear oligomers. 108

133 5.4 Conclusions Mass spectrometry is a powerful technique for molecular characterization but the analysis of complex mixtures is still challenging. Interfacing MS with separation techniques and/or multiple MS stages enhances the ability for separation and identification the complex analytes. Copolymers are considered to be complex mixtures and their full characterization is significantly facilitated by such multidimensional techniques. This has been illustrated for the analysis of diblock copolymers consisting of poly(ethylene oxide) and polycaprolactone using MALDI-MS, MALDI-MS/MS, and UPLC interfaced with MS and MS/MS. MALDI-ToF/ToF MS and MALDI-Q/ToF MS/MS were applied to assess the compositional heterogeneity of the block copolymers and determine the fragmentation pathways of their sodiated oligomers, respectively. Diblock copolymer with varying PEO and PCL content as well as homopolymers of either PEO or PCL were detected by MALDI-MS. The results from MALDI-MS/MS showed that fragmentation pathways can be used to gain the block length information. Two major series were observed upon MS/MS of copolymeric oligomers, by losses of (CL) n macrocycles via intramolecular transesterifications (*) and HO-(CL) n -H chain via H-rearrangement mechanism (#), The latter dissociations proceed via 1,5-H transfers over the ester groups, except for the loss of the entire PCL block from benzyl-coupled block copolymers which involves H- transfer from more remote positions. The two pathways mentioned also dominate the MS/MS spectra of homopolymeric PCL oligomers. 109

134 Reverse-phase UPLC-MS and UPLC-MS/MS were also used to separate and identify individual components of the copolymers based on their polarity. The three copolymers were well separated by the number of caprolactone units. In these experiments, ESI was the ionization method. Hence, the LC-MS spectra contained both singly and multiply charged ions. The singly charged regions were less congested and most informative about the composition of the fractions; only these region therefore discussed. Examples of full LC-MS mass spectra are given in the Appendix. In general, ESI-MS/MS and MALDI-MS/MS spectra are quite similar. The relative abundances of the fragments generated by (CL) n losses are consistently lower with ESI (which is softer ionization method), pointing out that this pathway requires more internal energy than the competitive losses of HO-(CL) n -H chains. UPLC-MS conclusively showed that the block copolymers contained block PEO as well as PCL homopolymers. In contrast, MALDI-MS only detected one of the homopolymers, presumably because the other was at very low concentration in the sample analyzed. Moreover, UPLC-MS/MS revealed the presence of minor byproducts in one of the copolymers that could not be identified without chromatographic separation or two MS stages. Overall, this study clearly documented that a precise and sensitive analysis of the compositions and structures of complex mixtures can be achieved by multidimensional separation/mass spectrometry methods. 110

135 CHAPTER VI TANDEM MASS SPECTROMETRY OF POLYETHERS - SIZE AND COLLISION ENERGY EFFECTS 6.1 Background Mass spectrometry is a sensitive tool for the analysis of polymers. Soft ionization techniques such as electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) have enabled the classification and characterization of synthetic polymers because these techniques can form intact gas-phase ions from macromolecules. 1, 23 Tandem mass spectrometry (MS/MS) can give more information about the molecular structure under examination. Tandem mass spectrometry in space is performed when two or more mass analyzers are coupled, usually in beam instruments, while tandem mass spectrometry in time is performed in the same mass analyzer but over time and requires trap instruments. 1 The ion of interest, often called the precursor ion, is selected and then undergoes fragmentation by colliding with neutral gas molecules. The resulting fragments are analyzed to give clues about the structure of the examined molecule. 13 As a result, tandem mass spectrometry has become a very useful tool to characterize polymer end groups and architectures 20, 21 and to differentiate isomeric and isobaric structures. 86 Collisionally activated dissociation (CAD) or collision-induced dissociation (CID) is the most common method to cause fragmentation in MS/MS. In this 111

136 process, a neutral gas, such as helium and argon, collides with the accelerated precursor ion to give fragments. Bond cleavages can be charge-induced, occurring near or at the charge site. When the bond cleavage takes place at a location that is distant from the charge site and the charge does not participate in the dissociations, the process is called charge remote fragmentation. During CAD, a fraction of the ion kinetic energy (E lab ) is converted to internal energy causing bond breakage. The maximum internal energy that can be gained by an ion accelerated to E lab is the corresponding center-of-mass collision energy (E CM ), which can be calculated using the following equation: E CM = E lab x M gas /[M gas +M ion ] Equation 6.1 where is E lab is the laboratory-frame collision energy, M gas is the mass of the collision gas, and M ion is the mass of the precursor ion. The internal energy deposited must be higher than the energy of the bond(s) to be broken, so that dissociation can take place within the mass spectrometric time scale (ns to ms). Initially, the internal energy is redistributed over all rovibrational degrees of freedom (DoF) of the precursor ion. If the energy deposited exceeds the activation energy of a dissociation, the ion will be able to fragment via this reaction when enough energy quanta have accumulated on the bond(s) to be cleaved (reaction coordinate). Larger precursor ions have more DoF and require higher internal energies in order to fragment within the time span of MS/MS experiments, which is in the µs range for Q/ToF and ms range for QIT instrumentation. 1, 13 Hence, ion size is an important determinant of the fragmentation extent and pathways observed at a given collision energy. 87 For ions of comparable size (and DoF), on the other hand, the collision energy needed for similar fragmentation extents depends on the activation 112

137 energies of the induced fragmentation pathways, which are determined by the corresponding molecular structures. 88 The biocompatibility of polyethers has made these polymers useful for biological applications in many research areas. 90, 91 Commonly used polyethers for this purpose are poly(tetrahydrofuran) (PTHF), poly(1,2-propylene oxide) (PPO) and poly(1,2-butylene oxide) (PBO). These polyethers are typically prepared by ring-opening polymerization from tetrahydrofuran (THF), 2-methyloxirane, and 2-ethyloxirane, respectively, in 22, 89 presence of acid or base as a catalyst (Scheme 6.1). Tetrahydrofuran Polytetrahydrofuran 2-methyloxirane Polypropylene oxide 2-ethyloxirane Poly (1,2- butylene oxide) Scheme 6.1 Synthetic routes to widely used polyethers. In this study, MS/MS analyses were preformed to elucidate the fragmentation pathways of PTHF, PPO and PBO, and to determine the influence of collision energy and 113

138 of precursor ion mass (i.e., size) and structure on CAD fragmentation. In this context, it is noteworthy that PTHF and PBO carry isomeric repeat unit (both C 4 H 8 O, 72 Da) and that the repeat unit of PPO (C 3 H 6 O, 58 Da) is 14 Da smaller. The fragmentation extent upon MS/MS depends on how easily the precursor ion can be collisionally energized and on the activation energies of its dissociations. Ions with large cross-sectional areas are more readily activated by collisions. Such areas can be derived by ion mobility mass spectrometry (IM-MS). IM-MS is a technique that disperses ions based on their charge and collision cross-section, which is a function of the respective molecular size and shape. Therefore, isomers may be separated using IM-MS. In the ion mobility chamber, the ions travel against a buffer gas under the influence of an electric field; this process can be considered 36, 53, 104, 105 as a gas-phase ion chromatography, with the IM chamber acting as the column. The mass, charge, and shape of the ions determine their travel time through the IM chamber. This time, called drift time, can be converted to a collisionally cross-section (CCS) that is characteristic of the ions molecular size and shape. With the traveling wave version of IM-MS used in this dissertation, unknown CCS values are deduced by calibrating the drift time scale with ions of known CCS. Protonated polyalanine (PA) oligomers were used to build a calibration curve which was utilized to derive the CCS of 51, 92 the ions of interest. Polyethers are generally ionized by alkali metal adduction. Lithium and sodium ions were used as cationizing agents. Li + was employed in the MS/MS studies because it is tightly bonded by polyethers, thereby promoting fragmentations within the 114

139 macromolecular frame upon collisional activation. Sodium or lithium ion was used for the collision cross-section studies which did not involve fragmentation; sodiation leads to simpler, less congested spectra with more intense peaks. Here, IM-MS and CCS data are combined with results from collision energy studies to elucidate the fragmentation pathways of PTHF, PPO, and PBO, and to assess the influence of precursor ion mass, shape, and collision energy on CAD fragmentation. 6.2 Sample Preparation and Instrumentation Used MS and MS n experiments to decipher polymer were performed on a Bruker HCT Ultra II (Bruker Daltonics, Billerica, MA) quadrupole ion trap (QIT) equipped with an electrospray ionization (ESI) source. The collision energy and IM-MS studies were performed on a Synapt Q/ToF mass spectrometer (Waters corporation, Milford, MA) equipped with an ESI source and the traveling wave variant of IM-MS (Waters Corporation, Milford, MA). The QIT was operated in positive mode under the following, optimized conditions: nebulizer gas pressure, 10 psi; drying gas flow rate, 8.0 L min 1 ; drying gas temperature, 300 C; and capillary voltage, 4 kv. The helium bath gas in the trap was used as collision gas for the acquisition of MS/MS spectra via collisionally activated dissociation (CAD). Samples were prepared by dissolving the polymer in tetrahydrofuran (THF): methanol (MeOH), 70:30 (v/v), at a final concentration of 0.01 mg/ml in THF:MeOH (70:30 v/v). One percent (v/v) of lithium trifluoroacetate, LiTFA (1.0 mg/ml in THF), was added to these samples to promote cationization. The collision gas used was helium. The precursor ions for MS/MS were selected with an isolation width between 0.6 and

140 For the collision energy study, MS/MS spectra were acquired on the a Synapt HDMS quadrupole/time-of-flight (Q/ToF) mass spectrometer using the following parameters: ESI capillary voltage, 3.5 kv; sample cone voltage, 35 V; extraction cone voltage, 3.2 V; source temperature, 80 C; desolvation temperature, 180 C; cone gas flow, 50 L/h; and desolvation gas flow, 550 L/h (N 2 ). Mass spectra were acquired with the ToF analyzer (Q in RF only mode). For MS/MS, the quadrupole acts as a mass filter which allows for the passage of the ion of interest to the trap collision cell, located after the quadrupole, where CAD with argon gas takes place. The CAD fragments are then sent to the ToF section for mass analysis. The flight times of the incoming ions are converted in the ToF analyzer to provide their m/z values. Samples were prepared by dissolving the polymers in THF: MeOH (70:30, v/v) at a concentration of 0.01 mg/ml. Lithium trifloroacetate (LiTFA) (1% v/v of a 1.0 mg/ml solution in THF) was added to the samples to promote cationization. Argon was used as the collision gas and the CE was varied between 4-84 ev (laboratory frame) in increments of 10 ev. Collision energies were converted from the laboratory frame to center of mass (E CM ) CE by equation 6.1. MS/MS spectra were acquired in triplicate for each set of ions. The resulting data were averaged from each triplicate set for each ion. These results were used to plot survival yield curves using the Origin Pro 8 software. IM-MS experiments were conducted in positive ion mode with a capillary voltage of 3.5 kv, cone voltage of 35 kv, sampling cone voltage of 3.2 kv, source temperature of 80 C and desolvation temperature of 150 C. The instrument employs a moving electric field (traveling wave field) for IM separation; the sample solution was introduced to the ESI source by direct infusion. In IM-MS experiments, the ions coming from the 116

141 ionization source are pulsed as packets to the quadrupole. Then, they enter the traveling wave section which contains three confined regions called the trap, ion mobility cell and transfer cell. The trap collects the incoming ion packets and releases them to the IM device. In the IM chamber, the ions are propelled by low voltage waves against a stream of nitrogen gas, which causes separation according to the corresponding ion mobilities on the traveling waves. Lastly, the separated ion packets traverse the transfer cell and are directed to the ToF analyzer for mass analysis. Ion mobility separation was achieved by tuning the traveling-wave height and traveling-wave velocity in the IM cell to 11 V and 350 m s 1, respectively. Samples were prepared as for the collision energy studies, but were cationized by adding 1% (v/v) sodium trifluoroacetate (NaTFA) (1.0 mg/ml in THF). 6.3 Mass Spectrometry and Tandem Mass Spectrometry of Polyethers MS and MS n of Lithiated Polytetrahydrofuran ([PTHF+Li] + ) Figure 6.1 shows the ESI mass spectrum of the PTHF sample investigated [HO- (CH 2 CH 2 CH 2 CH 2 O)-H]. The added LiTFA salt leads to the formation of an abundant [M+Li] + series (A), where m/z values indicate a combined mass of 18 Da, which corresponds to the expected HO- terminal at both chain ends. The other distributions (B and C) arise from [M+Na] + and [M+K] + ions due to the presence of adventitious Na + and K + in the glassware. These ions have the same end groups as the lithiated oligomers. In each series, the mass difference between adjacent oligomers is 72 Da, which is the molecular weight of one tetramethylene oxide repeat unit (-CH 2 CH 2 CH 2 CH 2 O-). 117

142 [3-mer] Li+ [3-mer] Na Da A B [4-mer] Li+ A [4-mer] Na+ B [5-mer] Li+ A [5-mer] Na+ B [5-mer] K+ C [6-mer] Li+ A B C [7-mer] Li+ A [6-mer] Na+ [7-mer] Na+ B C [8-mer] Li+ A [8-mer] Na+ B [6-mer] K+ [7-mer] K+ [8-mer] K+ C [9-mer] Li+ [10-mer] Li+ A [9-mer] Na+ B [9-mer] K+ C A [10-mer] Na+ B [10-mer] K+ C [11-mer] Li+ A [11-mer] Na+ [11-mer] K+ B C [12-mer] Li+ A [12-mer] Na+ B [13-mer] Li+ A [13-mer] Na+ B [14-mer] Li+ A [14-mer] Na+ B [15-mer] Li+ A m/z Figure 6.1 ESI mass spectrum of PTHF (M n 650) acquired with the QIT mass spectrometer. Lithium (A), sodium (B), and potassium (C) cationized oligomers are observed even though only LiTFA was added to the sample (see text). The MS/MS studies performed on lithiated PTHF because sodiated PTHF exhibited very weak product ion signals. The binding energy of Na + is substantially smaller than that of Li +, leading to facile Na + detachment and few polymer fragments. The CAD tandem mass spectrum of the lithium cationized 8-mer (m/z 601), obtained on ESI-QIT instrumentation, is shown in Figure 6.2. Two main series, e n and d n, and two minor series, e n and J n, are clearly observed. For each series, the repeating unit is 72 Da. Series e n corresponds to product ions formed by fragmentation along the polyether backbone to yield fragment ions with the same end groups as the precursor ions (HO and H, 18 Da); these fragments arise by losses of (C 4 H 8 O) n moieties (72 Da). Series d n, with 118

143 alkene and HO end groups (72 Da), are the complementary fragments formed by the loss of HO-(C 4 H 8 O) x -H moieties ( 72n + 18 Da) from the precursor ions. Series e n appears two m/z units below e n and is consistent with an aldehyde group at one chain end (total end group mass of 88 Da). Series J n has alkene groups in both end, thus, represents internal fragments. The MS/MS spectrum of lithiated PTHF contains abundant e n and d n fragments, while series e n and J n ions appear with very low relative abundances. Series en, d n, J n can be rationalized by charge-induce fragmentation at the ether bonds, as shown in scheme 6.2. w x y z v e 7 a b c d e e 6 e 5 d 3 e 3 d 4 e 4 d 5 d 6 d 7 d 8 e 2 j 5 j6 j 7 j 8 e 4 e 5 e 6 e7 e m/z Figure 6.2 CAD mass spectrum of lithiated PTHF 8, m/z 601.5, obtained using the ESI- QIT mass spectrometer. The inset explains the nomenclature for terminal fragments (i.e., those containing one of the original end groups); a single letter (e.g., e n ) is used if the new chain end contains a double bond, and a double prime (e.g., e n ) is used to indicate a saturated chain end (see also Scheme 6.2). 17 Because of the symmetry of this macromolecule, a n = y n ; b n = x n ; c n = w n ; d n = v n. The J n symbols indicate internal fragments (see text). 119

144 1 e n series: m/z 72n b 2a 2c d n series: m/z 72n J n series: m/z 72n Scheme 6.2 Charge-induced fragmentation pathway of lithiated PTHF to truncated fragments with hydroxyl/hydrogen (e n ), alkene/hydrogen (d n ) and vinyl/vinyl (J n ) end groups. As a Lewis acid, Li + coordinates with an oxygen atoms. This Lewis acid-base interaction weakens the bond between the carbon and the oxygen atoms facilitating its cleavage. Heterolytic cleavage of the C-O bond accompanied by energetically favorable hydride (H ) shifts ultimately creates an ion-dipole complex between a lithium alkoxylate and an alkoxylbutyl cation. Proton transfer from the positively charged butyl group to the basic alkoxylate within this complex (route 1) forms the e n series. Alternatively (route 2a), proton transfer and ligand rearrangement in the ion-dipole complex leads to an isomeric Li + -bound complex between two truncated PTHF chains. The latter intermediate can dissociate to yield the e n series (route 2b) or d n series (route 120

145 2c). Series J n can be formed by consecutive dissociation of dn through the same chargeinduced mechanism (route 3). Consecutive dissociation of e n presumably also occurs, further truncating the polymer chain. Since series e n has the same structure of the precursor ion, its consecutive fragmentation does not produce any new fragments. The second minor fragment series, e n, is attributed to charge-remote C-O bond homolyses (Scheme 6.3). This route coproduces d n fragment ions. Judging from the relative intensities of e n and J n, hemolytic bond cleavages and consecutive dissociations are very minor fragmentation pathways under CAD conditions in a QIT. Scheme 6.3 charge-remote hemolytic C-O bond cleavages in lithiated PTHF, leading to fragments with hydroxyl/carbonyl (e n ) and alkene/hydroxyl end groups. The d n series generated by this mechanism differs in the double bond position from the dn series shown in Scheme 6.2, but both series appear at the same m/z values (isomers) and hence, for simplicity, were given the same acronym. 121

146 A triple-stage mass spectrometry (MS 3 ) experiments were performed on the d 8 fragment from lithiated PTHF in order to assess the complete dissociation pattern of a PTHF chain with alkene/hydroxyl end groups. Because d n ions are asymmetric (i.e., they have two different end groups), their dissociation via the charge-induced pathway of Scheme 6.2 would produce three different fragment series, viz. e n, J n, and d n which are depicted on the top of the scheme 6.4. Fragments with the structure of d n (alkene/hydroxyl end groups) are generated twice as frequently, explaining their higher relative intensities in the MS 3 spectrum (Figure 6.3). The dn ions can also depolymerize (unzip) via sequential 1,5-H rearrangements, as depicted on the bottom of Scheme 6.4; such unzipping has been observed for alkene/hydroxyl terminated ions of PEO 17 and provides an additional rationale for the high yield of d n fragments upon MS

147 d 7 d 6 d 8 d 3 d 4 d 5 e 5 e 6 j 7 e 3 j 4 e 4 j 5 j3 e 7 e 5 j 6 e 6 j m/z Figure 6.3 ESI-QIT MS 3 mass spectrum of the d 8 fragment ion (m/z 583.5) generated by CAD of the lithiated PTHF 8-mer. 123

148 Scheme 6.4 Dissociation of d n fragments from lithiated PTHF via (a) the charge-induced pathway in Scheme 6.2 or (b) charge-remote unzipping through 1,5-H rearrangements MS and MS n of Lithiated Poly(1,2-Butylene Oxide) ([PBO +Li] + ) Figure 6.4 shows the ESI mass spectrum of PBO, which has a formula of [CH 3 O- (CH 2 CH(CH 2 CH 3 )O)-H]. The presence of LiTFA salt leads to the formation of abundant charged [M + Li] + ions (major series A), whose m/z values indicate a combined end group mass of 32 Da, in agreement with the expected CH 3 O- and H- substituents at the chains ends of this polymer. The other distributions, B and C, arise from sodiated and potassiated ions with the same end groups, respectively. The mass difference between adjacent peaks in the same series is 72 Da, which is the molecular weight of one C 4 H 8 O repeat unit. Tandem mass experiments were performed to characterize the fragmentation 124

149 characteristics of chains with 1,2-butylene oxide and compare them to those of chains with tetramethylene oxide units. Figure 6.4 ESI mass spectrum of PBO (M n 800) acquired with the QIT mass spectrometer, showing lithium (A), sodium (B), and potassium (C) cationized oligomers. The CAD tandem mass spectrum of the lithium cationized PBO 8-mer (m/z 615), obtained on ESI-QIT instrumentation, is shown in Figure 6.5. Three main series (c n, z n, and c n) and three minor series (b n, z n, and x n ), are clearly observed. For each series, the repeat unit is 72 Da. The PBO investigated has an asymmetric structure, from which four different fragment ions (instead of two) can be generated by the reaction mechanism outlined in Scheme 6.2: cleavage of the O-CH 2 bonds according to this mechanism gives rise to series c n and x n, whereas cleavage of the CH(C 2 H 5 )-O bonds gives rise to series z n and b n. The first step in these processes is heterolytic dissociation of C-O bond to produce an alkoxylate anion and a carbocation that rearranges to a more stable oxonium 125

150 ion via hydride shifts. The incipient carbocation emerging after heterolytic bond dissociation is primary when O-CH 2 bonds are cleaved, but secondary when CH(C 2 H 5 )-O bonds are cleaved, justifying the predominance of series z n over c n, and of series b n over x n. As with PTHF, the series appearing 2 Da below z n and c n (viz. z n and c n ) are accounted for by charge-remote hemolytic C-O bond scission and consecutive β-loss of a hydrogen radical (cf. Scheme 6.3). The predominance of c n over z n suggests that the β- hydrogen loss must be the rate-determining step; this would favor the formation of c n where a tertiary H-radical is lost to form a keto end group. In contrast, z n formation requires the loss of a secondary H-radical to yield an aldehyde end group, which is energetically more demanding. 126

151 y z x a b c z 3 z z z c 3 z 6 z x 3 b 3 c c z c c m/z c 6 c z 2 x c 4 c 5 c x b 4 b c x 5 b c3 6 b 7 5 x6 z m/z Figure 6.5 CAD mass spectrum of lithiated PBO 8, m/z 615.5, obtained using the ESI-QIT mass spectrometer. The inset explains the nomenclature for terminal fragments from the PBO chain. An expanded view of the m/z region is shown at the left side. 127

152 The z 4 ion was examined further by MS 3 (Figure 6.6). Unlike lithiated PBO 8, the z 4 fragment has hydroxyl termini at both chain ends. For this reason, cleavage of either its O-CH 2 or its CH(C 2 H 5 )-O bond according to Scheme 6.2 would lead to only one pair of fragments with unique (different) m/z values, viz. truncated z n and x n (n < 4). Indeed, these are the only charge-induced product ions observed. Additionally, z n fragments from hemolytic C-O bond cleavages according to Scheme 6.3 are observed; these cogenerate x n fragments that overlap with the x n fragments arising from the charge-induced dissociations. Fragmentations at the side chain did not occur under MS 3 or MS/MS conditions. z z x 3 x 2 z z 3 x m/z Figure 6.6 ESI-QIT MS 3 mass spectrum of the z 4 fragment ion (m/z 313.2) generated by CAD of the lithiated PBO 8-mer. 128

153 MS and MS n of Lithiated Poly(1,2-Propylene Oxide ([PPO +Li] + )) Figure 6.7 shows the ESI mass spectrum of PPO, HO-(CH 2 CH(CH 3 ) n O)-H. Because of the higher molecular weight of this polymer (compared to the PTHF and PBO studied), ESI with LiTFA as cationization salt leads to the formation of singly and doubly charged ions. The major series are [M + Li] + ions with end groups of 18 Da which corresponds to the expected HO- and H- moieties at the polymer s chain ends. A second minor distribution arises from sodium ion adducts of the same polymer. The mass difference between two adjacent oligomers in the major or minor series is 58 Da, which is the molecular weight of one propylene oxide unit. 1+ charges [14-mer] Li+ [15-mer] Li+ 2+ charges [21-mer +Li + Na] 2+ [21-mer +2Li] 2+ [22-mer +2Li] 2+ [11-mer] Li+ [23-mer +2Li] 2+ [24-mer +2Li] 2+ [12-mer] Na+ [12-mer] Li+ [26-mer +2Li] 2+ [13-mer] Na+ [14-mer] Na+ [15-mer] Na+ [16-mer] Na+ [17-mer] Li+ [17-mer] Na+ [18-mer] Li+ [18-mer] Na+ [13-mer] Li+ [16-mer] Li m/z m/z Figure 6.7 ESI mass spectrum of PPO (M n 1000) acquired with the QIT mass spectrometer, showing lithium, and sodium cationized oligomers in charge states 1+ and

154 Singly charged PPO was investigated further by MS/MS. CAD tandem mass spectrum of the lithiated 14-mer (m/z 837), were obtained on the ESI-QIT instrumentation, is shown in Figure 6.8. Three series, viz. c n, c n, and b n, are clearly discerned. All have the PPO repeating unit of 58 Da. The PPO structure is asymmetric, containing two different types of C-O bonds at each ether linkage. In contrast to the PBO sample investigated, however, both chain ends of the PPO sample carry hydroxyl substituents. As a result, the charged-induced fragmentation pathway detailed in Scheme 6.2 gives rise to only two distinguishable product ion series, c n and b n, as explained in Scheme 6.4. These two series are the dominant fragments generated upon CAD. The third fragment series, c n, is again attributed to charge-remote hemolytic bond cleavages via the mechanism shown in Scheme 6.3; this pathway, which also contributes to the b n series, is summarized in Scheme A4 (see Appendix). 130

155 y z x a b c c 4 c 5 c 6 c 7 c8 c 9 c 10 b 11 c 11 b 12 c 12 b 10 b 13 b 4 b 5 b 6 b7 b8 b 9 b m/z Figure 6.8 CAD mass spectrum of lithiated PPO 14, m/z 837.6, obtained using the ESI-QIT mass spectrometer. The inset explains the nomenclature for terminal fragments from the PPO chain. See Scheme 6.5 for the structures of fragments b n and c n. A significant increase in the relative intensity of Series c n is observed at low masses. The higher intensities of the smaller c n fragments can be reconciled by consecutive fragmentation of the larger c n fragments. The c n ions from the PPO studied have the same end groups as the precursor ions which, as discussed above, largely dissociate through the charged-induced dissociation pathways depicted in Scheme 6.5. Fragmentation of the larger c n ions by this mechanism coproduces shorter c n and bn fragments; however, the latter fragments easily lose a 42- Da moiety to yield c n, as attested by the MS 3 spectrum of b 13 (Figure 6.9); this reactivity explains why the c n series increases in abundance, but the b n series does not. 131

156 c 12 b 13 b 12 c 4 c 5 c 6 b 7 c 7 b 8 c 8 b 9 c 9 b 10 c 10 b 11 c m/z Figure 6.9 ESI-QIT MS 3 mass spectrum of the b 13 fragment ion (m/z 761.5) generated by CAD of the lithiated PPO 14-mer. 132

157 b n c n z n x n Scheme 6.5 Charge-induced fragmentations of the lithiated PPO according to the mechanism detailed in Scheme 6.2. Each ether bond joins a secondary and a tertiary C- atom. Cleavage of the CH-O bond leads to b n or z n fragments, whereas cleavage of the O-CH 2 bond leads to c n and x n fragments. Because both chain ends carry the same substituent (HO), c n and z n are identical, and b n and x n are MS-indistinguishable isomers. 6.4 Evaluation of the Dissociation Energetics of PTHF, PPO, and PBO by Energy- Resolved Tandem Mass Spectrometry Information about the fragmentation energetic of an ion can be obtained by monitoring its CAD spectrum as a function of collision energy. From these energyresolved tandem mass spectra, 93 fragmentation efficiency curves (FECs) 94 can be constructed by plotting the relative intensity of the precursor ion (also called survival yield ) versus the corresponding laboratory-frame (E lab ) or center-of-mass (E CM ) 133

158 collision energy. The collision energy at which the survival yield (SY) drops to 50%, E 50, has been used as a measure of the gas-phase (intrinsic) stability of the ion under investigation E 50 values also under an estimate of the internal energies needed to fragment different ions 103 and are particularly useful for a qualitative comparison of the dissociation energetic of similarly sized ions (vide infra). Survival yields, of a series of PTHF, PBO, and PPO oligomers have been derived from MS/MS experiments preformed on the Synapt Q/ToF mass spectrometer, in order to examine internal energy and size effects on polymer fragmentation. The survival yield (SY) is defined by the following equation: SY = I p /( I p + ƩI f ), where I p is the intensity of the precursor ion, and ƩI f is the sum of all fragment ion intensities. Singly lithiated PTHF and PBO oligomers with 7-10 repeat units and PPO oligomers with repeat units were selected for the MS/MS experiments. The different average molecular weights of the polymer samples studied, precluded the use of the same n-mer from all these polymers. The collision energy by using the laboratory frame, E lab, was varied within the range 4-84 ev for each ion; the spectra were acquired in triplicate and then averaged for the calculation of the SYs. Collision energies were converted from laboratory to center of mass (E CM ) frame by Equation 6.1. E CM is the maximum internal energy that can be transferred in one collision to the percursor ion; the actual amount is usually less. 1 Further, CAD in Q/ToF mass spectrometers is generally performed under multiple collision conditions. For these reasons, the E CM amount of calculated via Equation 6.1 can only be used as an estimate of the internal energy 134

159 avialable to the percursor ion for dissociation during its residence time in the collision cell. Within the collision energy range probed, essentially the same charge-induced and charge-remote fragmentations were observed given rise to series e n, e n, J n and d n from PTHF oligomers, series c n, z n, c n, and x n from PBO oligomers, and series c n, b n, and c n from PPO oligomers. The relative fragment intensities change with E CM but, in all cases, they increase relative to the intensity of the precursor ion (see Figures A5-A15 in the Appendix A). The FECs, i.e. the plots of SY versus E CM, have sigmoid shape (Figures 6.10,6.11, and 6.12). As the degree of polymerization increases, the curves are shifted to lower E CM. The E 50 values determined for each oligomer from Figure are summarized in Table 6.1 and plotted against the corresponding masses in Figure This Figure indicates that E 50 values decrease with increasing number of monomer units and that the E 50 values for PTHF oligomers are consistently higher than those for PBO oligomers of the same size. The E 50 data are best fitted into second-order polynomial curves that approach a constant (minimum) value past a certain precursor ion size (correction coefficient R 2 = for PTHF, for PBO, and for PPO. 135

160 Syrvival Yield (SY) mer (529) 8-mer (601) 9-mer (673) 10-mer (745) Center-of-mass energy, E CM (ev) Figure 6.10 Fragmentation efficiency curves of lithiated PTHF n (n = 7-10) oligomers acquired using the Synapt ESI-Q/ToF instrument. 136

161 Survival Yiels (SY) mer (543) 8-mer (615) 9-mer (687) 10-mer (759) Center-of-mass energy, E CM (ev) Figure 6.11 Fragmentation efficiency curves of lithiated PBO n (n = 7-10) oligomers acquired using the Synapt ESI-Q/ToF instrument. 137

162 Survival Yield (SY) mer (837) 15-mer (895) 16-mer(953) Energy of center mass(e CM ) Figure 6.12 Fragmentation efficiency curves of lithiated PPO n (n = 14-16) oligomers acquired using the Synapt ESI-Q/ToF instrument. Table 6.1 Mass (in Da) and center-of-mass energy for 50% SY (E 50 in ev) of the singly lithiated PTHF, PBO, and PPO oligomers investigated by energy-resolved MS/MS. PTHF PBO PPO Mass (n-mer) E 50 Mass (n-mer) E 50 Mass (n-mer) E (7-mer) (7-mer) (8-mer) mer) (14-mer) (9-mer) (9-mer) (15-mer) (10-mer) (10-mer) (16-mer) Many factors that affect the amount of internal energy avialable to a collisionally acctivated ion for bond dissociation. These include the mass of the ion and the collision 138

163 gas, the degrees of freedom (DoF) of the ion and the number of collisions. For the same precursor ion, increasing the energy increases the fraction of such energy converted to internal energy, leading to a decrease of the survival yield from one at low energy to zero at high energy. A percursor ion with a higher number of repeatunits can distribute its internal energy over more DoF, with increasees the internal energy required for dissociation within limited time spent by the ion in the collision cell (higher kinetic shift). 1 On the other hand, more repeat units augment the molecular size and the ion collision cross-section (CCS), enabling more collisions per time unit and lowering the energy per collision needed for dissociation. According to Figure 6.13, the E 50 values of the PTHF, PBO, and PPO oligomers investigated decrease with mass, indicating that the the DoF effect is outweighted by the increasing number of collisions experienced by the percursor ion in the collision chamber due to a larger CCS. An additional reason for the decline E 50 with mass might be a better stabilization of the dissociating transition states for the larger oligomers. The smaller E 50 values of PBO oligomers, compared to those of PTHF oligomers with the same mass (Figure 6.13) could caused by differences in the CCSs of these polymers, which contain isomeric repeat units, or by differenced in the activation energies for disssociations in the PTHF vs. the PBO chain. For more information on there E 50 trends, the CCSs of cationized PTHF and PBO oligomers, as well as of PPO oligomers, were probed by IM-MS (see following section). Unfortunately, PPO oligomers of the same degree of polymerization or the same mass as the PTHF and PBO oligomers could not be investigated. The E 50 curves in Figure 139

164 6.13 suggest that the energy requirements for dissociation of lithiated PPO are intermidiate to those of PTHF and PBO. Interestingly, the PBO and PPO curves appear to approach very similar minimum E 50 values at higher mass, consistent with more similar fragmentation mechanisms between these polymers than between PBO and PTHF R² = E50 (ev) R² = R² = Precursor ion mass (Da) PTHF PBO PPO Figure 6.13 Center-of-mass collision energy for 50% SY (E 50 ) vs. precursor ion mass for singly lithiated PTHF, PBO, and PPO oligomers. 6.5 Evaluation of the Collision Cross-Sections of PTHF, PBO, and PPO Using IM-MS Ion mobility mass spectrometry (IM-MS) can be viewed as gas-phase ion chromatography, in which ions are separated by their shape, mass, and charge while they travel in the ion mobility region 12, 3451, 104 under the influence of an electric field and against a buffer gas. The time needed by an ion to pass through this region is called drift time and can be converted to collision cross-sections (CCS) of this ion. From drift times measured using the traveling wave variant of the IM-MS, CCS values are derived by 140

165 calibrating the drift time scale with ions of known CCS; polyalanine was used as the celebrant in this study. Following the procedure described by Scrivens et al., 105 the corrected CCSs of protonated polyalanine oligomers were plotted against their corrected drift times measured at the same traveling wave velocity and traveling wave height used for the polyethers, viz. 350 m/s and 8.5 V, respectively. The calibration curve resulting this way ( Figure A 16 in Appendix A) was then used to obtain the experimental CCSs for PTHF, PBO, and PPO, (Tables 6.2 and 6.3). Figure 6.14 shows the two-dimensional IM MS plots obtained after IM separation and subsequent mass analysis of the ions formed by ESI of PTHF, PBO, and PPO. The ions in these plots can be divided into one or more IM regions, depending on their separation in the IM chamber. Integration of these regions gives rise to the mass spectra displayed in Figure Only singly charged ions are observed from PTHF and PBO, while PPO gives rise to singly as well as doubly charged ions due to its higher molecular weight and higher weight fraction of metal binding O atoms. For PTHF and PBO, the extracted mass spectra contain one distribution with the C 4 H 8 O repeat unit (72 Da) and peaks corresponding to singly sodiated oligomers with HO-and H end groups for PTHF (Figure 6.15.a) and CH 3 O- and H end groups for PBO (Figure 6.15.b). The mass spectra extracted from the IM-MS plot of PPO contain several distributions with the C 3 H 6 O repeat unit (58 Da); in both the singly and doubly charged regions, the major product is PPO with the expected HO- and H end groups (Figure 6.15.c). The IM-MS plots of the lithiated polymers show similar characteristics, but the extracted mass spectra are more complex and less intense due to the admixture of sodiated and potassiated ions. 141

166 It is evident from Figure 6.14 that drift times increase with the number of repeat units. Figure 6.16 further provides evidence that PTHF oligomers have consistently somewhat shorter drift times than PBO oligomers with the same number of repeat units. These trends are confirmed by inspection of the experimental CCSs in Tables 6.2 and

167 m/z 1500 NA_090313_PTHF IM_1.raw : Load 3D Viewer for - NA_090313_PTHF_NA_IM.raw:1 Drift time (ms) Figure 6.14.a Two-dimensional IM-MS plot (m/z vs. drift time ) of sodium cationized PTHF (M n 650) acquired with the Synapt Q/ToF mass spectrometer m/z Figure 6.15.a Mass spectrum extracted from the circled region in the ion mobility map of sodium cationized PTHF. 143

168 m/z 1500 NA_090313_PBO IM_1.raw : Drift time (ms) Load 3D Viewer for - NA_090313_PTHF_NA_IM.raw:1 Figure 6.14.b Two-dimensional IM-MS plot (m/z vs. drift time) of sodium cationized PBO (M n 800) acquired with the Synapt Q/ToF mass spectrometer m/z Figure 6.15.b Mass spectrum extracted from the circled region in the ion mobility map of sodium cationized PBO. 144

169 m/z Drift time (ms) Figure 6.14.c Two-dimensional IM-MS plot (m/z vs. drift time) of sodium cationized PPO (M n 1000) acquired with the Synapt Q/ToF mass spectrometer m/z Figure 6.15.c Mass spectrum extracted from the circled regions in the ion mobility map of sodium cationized PPO. The minor distributions in the singly charged region 145

170 correspond to [HO-(CH 2 CH(CH 3 )O) n -CH=CH-CH 3 +Na] + and [KO-(CH 2 CH(CH 3 )O) n - CH=CH-CH 3 +Na] + ions. Figure 6.16 Ion mobility chromatogram extracted from the sodiated 8-mer and 9-mer regions in the 2D IM-MS map of PTHF and PBO and showing the drift time distributions of these ions. 146

171 Table 6.2 Collision cross-sections of sodiated PTHF, PBO, and PPO oligomers. PTHF n PBO n PPO n No.of repeat units m/z DT a CCS (n) (Å 2 ) b m/z DT a CCS (Å 2 ) b m/z DT a CCS (Å 2 ) b a Drift time in ms ± b Collision cross-section in Å 2 ±

172 Table 6.3 Collision cross-sections of lithiated PTHF, PBO, and PPO oligomers. No. of repeat units (n) PTHFn PBOn PPOn m/z DT a CCS (Å 2 ) b m/z DT a CCS (Å 2 ) b m/z DT a CCS (Å 2 ) b a Drift time in ms ± b Collision cross-section in Å 2 ± 1.3 For a more quantitative evaluation of the IM-MS results, the collision crosssections determined from the drift times of sodiated and lithiated PTHF, PBO, and PPO oligomers were plotted against the corresponding masses (m/z) in Figure These plots permit the comparison of oligomers of the same masses, which accounts for the slightly different end groups of PTHF (HO- and H) and PBO (CH 3 O- and H) and the different repeat units of PTHF and PBO (72 Da) versus PPO (58 Da). 148

173 Surprisingly, the branched PBO oligomers have higher CCSs than the linear PTHF oligomers. Branched structures generally lead to smaller hydrodynamic volumes and cross-sections than linear isomers. The opposite observation made here is, nevertheless, reconciled by considering that cationized oligomers are probed in the IM- MS experiments. The linear PTHF chains are able to form more compact Li + or Na + complexes than the PBO chains with their isomers, branched repeat units where ethyl side group could obstruct the metal ion coordination. More compact architecture result in smaller CCSs and faster drift times through the IM region. The strict hindrance provided by the branches to the metal coordination environment should be lower with shorter side groups. Indeed, sodiated or lithiated PPO oligomers (CH 3 side groups) show considerably lower collision cross-sections than PPO oligomers (C 2 H 5 side groups) of the same mass. The higher compactness of cationized PPO (vs. PBO) could also arise from the larger number of coordinating O sites in this polymer (vs. PBO of comparable mass). The CCS increases with the number of repeat units (and, thus, with mass) for all three polymers. This result corroborates the explanation provided earlier for the steady decline in E 50 values with mass, viz. that the collision energy required to fragment 50% of the precursor ions (SY = 0.5) decreases with oligomer size due to an increase in the number of collisions per precursor ion in the same direction, caused by the higher CCS (cf. Figures 6.13 and 6.17). Comparison of the E 50 data of lithiated PTHF and PBO, which contain isomeric repeat units, further reveals that adding one repeat unit reduce E 50 less than replacing the 149

174 PTHF with a PBO chain of the same mass (Figure 6.13). Meanwhile, according to Figure 6.17, the rise in CCS by replacing all PTHF with PBO units is substantially smaller than the rise caused by adding a repeat unit to either polymer. Consequently, the lower E 50 values observed for cationized PBO oligomers vis á vis PTHF oligomers (of the same size) must be largely caused by more favorable dissociation energetic for the alkali metal cationized PBO chains. The CAD fragments of lithiated (or sodiated) 17 polyethers predominantly result from heterolytic or hemolytic C-O bond cleavage followed by hydride shifts or H-atom losses, respectively. Both these processes should require less energy with PBO chains, where secondary H-atoms are available for β H-atom loss. 150

175 Collision cross-section (Å 2 ) Collision cross-section (Å 2 ) 280 R² = R² = R² = PTHF 180 PBO 160 PPO m/z R² = R² = R² = m/z PTHF PBO PPO Figure 6.17 Collision cross-sections of sodiated and lithiated PTHF, PBO, and PPO oligomers. 151

176 6.5 Conclusions The ESI mass spectra showed a major polymer distribution for each polyether. Each distributions showed the expected repeat unit (72 Da for PTHF and PBO; 58 Da for PPO) and its members had m/z values that agreed with HO- and -H as end groups for PTHF and PPO and with CH 3 O- and -H end groups for PBO. MS/MS (CAD) studies were performed on the lithiated oligomers of the main distribution of each polyether. For each polymer, three main series were observed; one corresponding to product ions with the same end groups as the precursor ion, a second series corresponding to fragments with vinyl (alkene) and -OH end groups (whose combined mass is equal to the repeat unit mass), and a third series having hydroxyl and carboxyl end groups. These series can be rationalized by a combination of charge-induced heterolytic C-O bond cleavages and charge-remote homolytic C-O bond cleavages at the ether linkages. These assignments were confirmed by multistage CAD (MS n ) experiments in the ion trap. CAD of the lithium cationized n-mers was also performed on the Synapt Q/ToF mass spectrometer. In these experiments, the collision energy was increased gradually in order to measure fragmentation efficiency curves (FECs) and elucidate internal energy and size effects on the polymer fragmentation. As the number of repeat units increased, the FECs shifted toward higher laboratory-frame collision energies (E lab ) and the E lab value needed to cause 50% of the precursor ions to fragment (E 50 ) increased, as also reported in other studies. 87, 103 The corresponding center-of-mass E 50 values, however, were quite similar indicating that the FEC shifts observed are primarily due to an increase in the number of degrees of freedom. A careful inspection of the trends in the E 50 values 152

177 in the center-of-mass frame showed a slight but reproducible decrease in E 50 with oligomer size, which was ascribed to the higher collision cross-sections of the larger ions. Further, the E 50 values of PTHF oligomers were consistently higher than those of PBO oligomers, even though these polymers are composed of isomeric repeat units. An IM MS study was performed for ions formed by ESI in order to obtain experimental collision cross-sections (CCSs) for metal cationized PTHF, PBO, and PPO oligomers. For all three polymers, the CCS increased with the number of repeat units, reconciling the above mentioned E 50 trend in center-of-mass collision energies vs. oligomer size. Comparison of PTHF and PBO oligomers of the same mass further showed that the completely linear PTHF chain formed ions with slightly smaller CCS than the branched PBO chain. The difference in CCS was, however, too small to justify the significant gap in E 50 values between metal-cationized PTHF and PBO. The latter differences were therefore largely attributed to the lower activation energies required for CAD of PBO vs. PTHF. 153

178 CHAPTER VII ANALYSIS OF THERMOPLASTIC COPOLYMERS BY MILD THERMAL DEGRADATION COUPLED TO ION MOBILITY MASS SPECTROMETRY 7.1 Background Thermoplastic copolymers are copolymers derived from two or more types of monomeric species; they melt and are pliable and moldable at high temperature, but return to the solid state when they are cooled down. 106, 108 Thermoplastic elastomers (TPEs) are a class of thermoplastic copolymers with elastomeric properties as well as softness and flexibility. Polymers must be amorphous above their glass transition temperature to be classified as elastomers. These elastomers are used widely in different applications, ranging from the automotive sector to the manufacture of flexible packaging materials. Polyurethanes (TPUs), styrenic block copolymers (TPE-s), and polyolefin blends (TPE-o) are the most common commercially available classes of thermoplastic elastomers. 109 Thermoplastic polyurethanes (TPUs) are a unique class of polymers with a wide range of physical properties based on the choice of their building blocks. The building blocks are called hard and soft segments. Depending on the composition of the soft segments, TPUs can be classified as polyester-based TPUs or polyether-based TPUs. 119 Thermoplastic styrenic block copolymers (TPE-s) consist of two or more polymeric 154

179 blocks (usually polystyrene and polybutadiene, SB). These blocks can be composed of different weight ratios of styrene and butadiene to influence their physical properties. 110 Additives are generally blended into elastomers to enhance their properties and to protect them from degradation due to exposure to oxygen, humidity, high temperature, or UV light. Additives are classified as antioxidants, UV stabilizers, fire retardants, plasticizers, dispersants, or fillers. 111, 112 The complexity and the generally high molecular weight of TPEs make the direct characterization of such materials by mass spectrometry (MS) or other spectroscopic methods of analysis challenging. The traditional method for analyzing such complex mixtures has been by thermal decomposition (vacuum pyrolysis) followed by electron or chemical ionization mass spectrometry (Py-EI/CI-MS) analysis of the degradation products, often after gas chromatography (GC) separation. The thermal decomposition of polyurethanes has been studied using several pyrolysis techniques, interfaced with mass spectrometry Lattimer et al. used chemical ionization (CI)113 and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to analyze the desorbates and pyrolyzates after offline pyrolysis at low (less than 200 C) and high (above 200 C) temperatures. 116 These studies proposed two mechanisms to explain the pyrolysis products, one involving a breakage of the urethane linkage and the other involving formation of cyclic oligomers. 116 Overall, linear polydiol oligomers, cyclic polydiol oligomers, as well as pyrolysis products from the diisocyanate were observed Mehl et al. applied MALDI-MS after size exclusion chromatography (SEC) fractionation to analyze the soft segment of polyether-based and polyester-based polyurethane (PU) soft segments. 117 On the other hand, Yontz et al. have utilized MALDI-MS to analyze the hard segment length after hydrolysis

180 Several studies reported the use of thermal decomposition for the analysis of polystyrene polymers and copolymer, and of blends of polystyrene and polydimethylsiolxane (PDMS). 126 All these studies indicated that a radical chain mechanism is the typical mechanism for thermal degradation of polystyrene and its blends. 127 Additives in polymers and plastics have been analyzed by various methods, including thermal desorption and supercritical fluid extraction. 108, 129, 130 Electrospray ionization (ESI) and tandem mass (MS/MS) has also been employed to identify organic additives in polymers based on their fragmentation pathways. 132 The described strategies for the characterization of elastomers and their additives include time-consuming steps and specific sample preparation procedures. Here, an alternative, faster approach is introduced, involving mild degradation at atmospheric pressure using an atmospheric solids analysis probe (ASAP) ionization source. This technique, first described by McEwen et al. in 2005, is a sensitive tool for obtaining rapid, useful, and effective information about the composition of low-polarity and/or high molecular weight polymers. 31, 32 The desorbates and pyrolyzates released in the ASAP source are characterized by ion mobility mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS). This strategy recently employed by Barrere et al. to analyze polypropylene materials and their additives and degradation products. 133 In this work, ASAP-IM-MS is used to characterize multicomponent industrial elastomers by interfaced separation-fragmentation-mass analysis, all performed in the mass spectrometer ( top-down strategy). 156

181 7.2 Sample Preparation and Instrumentation Used Three polyurethanes were examined in this study. Elastollan (PU-1), obtained from BASF; pellethane (PU-2), obtained from DOW Chemical; and estane (PU-3), obtained from BFGoodrich. One styrenic copolymer sample was used in this study, styrloflex (SB-1), obtained from BASF. HPLC-grade methanol was bought from Sigma- Aldrich (St. Louis, MO). All materials were used in the condition as received from their supplier or manufacturer. All experiments were performed using a Synapt HDMS Q/ToF mass spectrometer equipped with an atmospheric pressure solids analysis probe (ASAP) and the traveling wave variant of IM-MS (Waters Corp., Milford, MA). The samples were heated to their melting point in a glass vial. The ASAP capillary tube was dipped in the melted polymer and cooled to room temperature before being introduced into the ion source. Nitrogen gas flowing at 1200 L/h and heated at a temperature in the C range was used for thermal desorption. The thermal desorption and degradation products generated in this process were ionized by atmospheric pressure chemical ionization (APCI). A vial filled with methanol was placed in the ASAP source to enhance protonation in the experiments under wet conditions. The corona discharge current was 5 µa and the sampling cone voltage was 30 V. Mass spectra were acquired in positive ion mode for 10 min over the m/z ; during this time the N 2 temperature was varied in steps within the mentioned temperature range. Ion mobility separation was achieved by tuning the traveling-wave height to 9 V and the traveling-wave velocity to 300 m s -1. APCI of the desorbates and thermal degradation products gave rise mainly to molecular radical 157

182 cations and/or protonated species, depending on their structure and the conditions in the source (dry or wet, vide supra). Occasionally sodiated or potassiated ions were also formed from ASAP products with carbonyl groups which bind alkali metal ions quite strongly. These ions were transmitted through Q (set in rf-only mode), separated by their mobilities in the IM cell, and mass-analyzed in the ToF device. Select ions were isolated using Q as a mass filter for the acquisition of their MS/MS spectra. MS/MS experiments were performed in the trap cell, using argon as collision gas at a collision energy of 20 ev (unless noted otherwise). All ASAP-IM-MS analysis were performed under dry as well as wet conditions. With methanol vapor in ASAP source (wet conditions), the intensities of protonated species ([M+H] + ) increased, which help to distinguish them from radical ions (M + ) and make the correct peak assignments (see Figure A17 in the Appendix). In this chapter, spectra acquired under wet conditions are only shown if they differed substantially from those acquired under dry conditions (i.e., if they exhibited differences that went beyond the relative [M+H] + peak intensities). 7.3 Thermoplastic Polyurethanes Thermoplastic polyurethanes are copolymers prepared from three main components: a diisocyanate, a diol, and a chain extender. The synthesis of polyurethanes starts by the condensation of the diisocynate with a short diol, referred to as a chain extender, to form the hard segment. The hard segments then are condensed with the soft segment, which is either a polyether or a polyester diol (Scheme 7.1). The choice of the hard and soft segments determines the properties of the polyurethane. In addition to the 158

183 chemically bonded soft and hard segments, additives are blended into PUs to enhance their stability and protect them from degradation. In the case of elastollan (PU-1) and pellethane (PU-2), the soft segment is a polyether, while estane (PU-3) contains a polyester soft segment. Scheme 7.1 General composition of polyurethanes. Interfacing ASAP-MS with IM-MS seperates the polymer distributions formed by thermal decomposition from the desorbed additives, which usually have aromatic and relatively compact structures, and hence higher ion mobilities and lower drift times than similarly sized copolymer pyrolyzates ASAP-IM-MS of Elastollan (PU-1) Figure 7.1 shows the two-dimensional ASAP-IM-MS plot obtained after IM separation and subsequent mass analysis of the ions formed from elastollan (PU-1) at an ASAP source temperature of 250 C. The ions present in the 2D plot can be divided into two regions (a and b). Integration of these regions gave rise to the mass spectra displayed in Figure 7.2. In IM region a, molecular ions (M + ) and or fragments characteristic the additives phthalate (m/z149), which is used as plasticizer, and butylated hydroxytoluene 159

184 (BHT, m/z 220 and 205), which used as antioxidant, are observed, as well as the molecular ion of methylene diphenyl disocyanate (MDI, m/z 250), which confirms that an aromatic isocyanate was incorporated in this PU. The spectrum extracted from IM region b shows a peak distribution separated by 74 Da arising from polydimethylsiolxane (PDMS), which is often used as a dispersant or filler. Figure 7.1 2D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of elastollan (PU-1) at 250 C; the mass spectra extracted from mobility regions a and b (in ovals) are depicted in Figure

185 Figure 7.2 Mass spectra extracted from regions a and b in the 2D IM-MS diagram of Figure 7.1. At the highest temperature (450 C), four regions (a-d) can be detected in the ASAP-IM-MS plot under dry and wet ion source conditions (Figure 7.3). Region a and b contain ion series with a 72-Da repeat unit, pointing out that the soft segment is a (C 4 H 8 O) n - polyether. Figure 7.4.a shows the extracted mass spectrum from IM region a, which shows pyrolysis products indicative of the hard and soft segment of this polyurethane. The intense peaks at m/z 250 and 340 correspond to the radical ions (M + ) of MDI and the adduct of MDI and a C 4 H 10 O diol (90 Da), respectively, corroborating the isocyanate component revealed at the lower temperature and identifying butanediol (BDO, 90 Da) as the chain extender of the hard segment; this diol is a common chain extender in TPUs. In addition, a distribution containing the hard segment (340 Da) and 161

186 having a 72-Da repeat unit is observed, which is consistent with poly(tetrahydrofuran), PTHF, as the soft segment. PTHF has a C 4 H 8 O (72 Da) repeat unit and is widely used polyether soft segment in TPUs. These assignments are verified by the MS/MS spectra of ions at m/z 250 (Figure A18), 340 (Figure A19), and 484 (Figure 7.4.b), which agree well with the structures of the M + ions of MDI, MDI + BDO (hard segment), and MDI + BDO + (C 4 H 8 O) 2 (hard segment + 2 units of soft segment), respectively. The ion series at m/z 72n (hard segment + n soft segment units) extends into IM region b, in which oligomers with up to 7 (C 4 H 8 O) repeat units are observed (labeled by * in Figure 7.5). These longer MDI-BDO-(C 4 H 8 O) n chains are also observed as sodiated ions (labeled by % in Figure 7.5). IM region b also contains several distributions of the protonated soft segment oligomers with either macrocyclic architecture (#, m/z 72n) or linear architecture and water ($, m/z 72n + 18) end groups. In IM region c, an intense peak with unique ion mobility is observed at m/z (Figure 7.6). This mass-to-charge ratio is characteristic of the molecular ion (M + ) of the additive Irganox The corresponding ASAP-IM-MS/MS spectrum (Figure A20) shows abundant fragments from losses of C 4 H 8 (56 Da) and C 15 H 22 O (2,6-di-t-butyl-4- methylene cyclohexadiene, 218 Da), validating the proposed structure. IM region d is the only region for which substantially different mass spectra were observed under dry and wet ASAP ion source conditions. Under dry conditions, (Figure 7.7, top), a series of peaks 56 Da apart from each other appears with high relative intensity. Their m/z values correspond to the molecular ion (M + ) of the antioxidant Irganox 1010 (m/z ) and its in-source fragments, generated by successive isobutene (C 4 H 8, 56 Da) losses from the tert-butyl groups. A partial hydrolysis product of this 162

187 antioxidant, missing one ester substituent, is also observed at m/z Conversely, under wet conditions (Figure 7.7, bottom), the [M+H] + ions of the cyclic and linear soft segment oligomers dominate IM region d, suppressing the radical ions originating from Irganox It is evident that all the additives and pyrolyzates are not fully separated in the IM-MS plot due to comparable sizes and collision cross-sections. Nevertheless, IM-MS spreads the ions formed in the ASAP source into a 2D space from which select, small regions can be integrated to extract mass spectra that are easier to interpret than the convoluted mass spectrum without IM separation (Figure A21). Moreover, IM separation permits the detection of less abundant ions and, hence, of mass components than simple ASAP-MS, thus rendering a more comprehensive characterization of the thermoplastic copolymers analyzed. The ASAP-MS spectra of all samples investigated in this dissertation are given in the Appendix (Figure A21-A24). 163

188 Figure 7.3 2D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of elastollan (PU-1) at 450 C without and with MeOH in the ASAP source; the mass spectra extracted from mobility regions a-d (in ovals) are depicted in Figures If MeOH did not affect the spectral appearance, only the spectrum acquired under dry conditions (without MeOH) is shown. 164

189 (a) (b) Figure 7.4 (a) Mass spectrum extracted from region a in the 2D IM-MS diagram of Figure 7.3. (b) ASAP-IM-MS/MS spectrum of the oligomer at m/z ([M] + ) which agrees with a species composed of the hard segment (340 Da) plus two soft segment repeat units (2x72 Da). Both radical losses via simple bond cleavages (for example, m/z 413.2, 341.2, 323.2) and losses of closed-shell species via H-rearrangements (m/z 340.2, 322.2) are observed. 165

190 Figure 7.5 Mass spectrum extracted from regions b in the 2D IM-MS diagram of Figure 7.3. The ions observed are molecular radical ions (*), sodiated ions (%), or protonated ion (#, $). Figure 7.6 Mass spectrum extracted from regions c in the 2D IM-MS diagram of Figure 7.3. See Figure A20 for the corresponding MS/MS spectrum. 166

191 Without MeOH (dry) With MeOH (wet) Figure7.7 Mass spectra extracted from regions d in the 2D IM-MS diagram of Figure 7.3 under dry (top) and wet (bottom) conditions. 167

192 7.3.2 ASAP-IM-MS of Pellethane (PU-2) The 2D IM-MS plot of the ions generated by ASAP of pellethane (PU-2) at 250 C under dry conditions indicates the presence of small pyrolysis products that are very similar to those obtained from elastollan (PU-1), cf. Figure A25 in Appendix A. At 450 C, the ASAP-IM-MS plot includes more ions, which can be divided into three regions (a-c). IM region a (Figure 7.9) contains ions indicative of MDI (M + and [M+H] + at m/z 250 and 251, respectively) and the adduct of MDI + BDO (M + at m/z 340), affirming that pellethane and elastollan carry the same hard segment constituents. As mentioned above, the abundant ions at m/z 180, 208, and 221 in region a of both pellethane and elastollan are MDI fragments (cf. Figure A18). Further, both TPUs show ions at m/z 224 and 314 (cf. Figure 7.4 and 7.9), which are 26 Da lower in mass than the radical ions of MDI and MDI + BDO, respectively. These ions are attributed to the corresponding methylenediphenylamino isocyanates (MDAIs), which are also formed during the thermal degradation of TPUs. 117 In addition, m/z 224 would partly originate from fragmentation of m/z 340 (cf. Figure A19). IM region b (Figure 7.10) includes three prominent distributions with a 72-Da repeat unit, pointing out that the soft segment is a (C 4 H 8 O) n - polyether, seen in PU-1. All these distributions are comprised of protonated ions, [M+H] +, of soft segment oligomers with up to 10 soft segment units. Based on their m/z values, one distribution represents soft segment macrocycles (72n Da), the other soft segment chains with hydroxyl and hydrogen (i.e., H 2 O) end groups (72n + 18 Da), and the third hydroxylterminated soft segment chains capped with one hard segment at the opposite chain end 168

193 (72n Da). These distributions have been marked by #, $, and * labels in Figure The peak standing out in IM region c (Figure 7.11) corresponds to the molecular ion (M + ) of Irganox 1076 (m/z 530). Upon MS/MS, this M + dissociates mainly by methyl loss to yield m/z 515 and through benzylic C-C bond scission to yield m/z 219, which corresponds to C 15 H 23 O, in accordance with its structure (Figure A26). Although pellethane and elastollan have common hard and soft segment components, they can be distinguished by their unique additives. Further, longer soft segment distributions were detected in IM regionb for pellethane, suggesting that the soft segment chains are, an average, longer, in the latter TPU. Figure 7.8 2D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of pellethane (PU-2) at 450 C. 169

194 Figure 7.9 Mass spectrum extracted from IM region a in the 2D ASAP-IM-MS plot depicted in Figure

195 Figure 7.10 Mass spectrum extracted from IM region b in the 2D ASAP-IM-MS plot depicted in Figure 7.8. Figure 7.11Mass spectrum extracted from region c in the 2D IM-MS diagram of Figure

196 7.3.3 ASAP-IM-MS of Estane (PU-3) ASAP-IM-MS analysis of estane (PU-3) at 250 C indicates the presence of phathalate plasticizer in this TPU and identifies MDI as its diisocyanate component (Figure A27-A28). Repeating the analysis at 450 C generates ions that can be grouped into two regions, a and b, in the resulting IM-MS plot (Figure 7.12). IM region a displays ions characteristic of MDI (M + at m/z 250; [M+H] + at m/z 251) and their fragments (m/z 221, 209, 208, and 180) and also contains the ion at m/z 340 which is diagnostic for MDI + BDO (Figure 7.13). Hence, the hard segment of this TPU consists of the same components as the hard segments of pellethane and elastollan. It is further noteworthy that thermal degradation coproduces some MDAI and MDAI + BDO (M + at m/z 224 and 314, respectively) also in this case. Region b (Figure 7.14) shows partly the same ions and, more importantly, two additional peak distributions separated by 200 Da, a mass increment indicative of a polyester soft segment composed of butylene adipate (C 10 H 16 O 4 ) repeating units. The ions detected at m/z 201, 401, 601, and 801represent protonated poly(butylene adipate) macrocycles, which are a common thermal degradation product of such polyesters. The second distribution (m/z 340, 540, and 740) represents M + ions of soft segment chains capped with one hard segment unit. Note that in this case no Irganox additives were detected. Tandem mass spectrometry experiments were conducted on selected oligomers from these two distributions to confirm their composition (Figure 7.15). From the soft segment distribution, m/z 601 was subjected to CAD. The fragments observed are 172

197 reconciled by ring opening of the macrocyclic [M+H] + ion via a 1,5-H rearrangement, to form a linear isomer with alkene and carboxyl end groups, followed by typical dissociation pathways of polyester chains, 17, 69 which involve 1,5-H rearrangments over the ester groups and intramolecular dehydrations and alcohol eliminations. The former pathway can generate the ions at m/z 201, and 401, as well as m/z 255, and 455 (see structure in Figure 7.15.a). Proton-assisted alcohol elimination, on other hand, accounts for the fragments at m/z 129, 329, and 529, which can undergo consecutive dehydration to form m/z 111, 311, and 511, respectively. Similarly, dehydration of the series m/z 201, 401, and 601 provides a plausible route to m/z 183, 383, and 583, respectively. Upon MS/MS of m/z 740 from the distribution capped with one hard segment unit, a series of fragments arises by 1,5-H rearrangements over the ester groups (m/z 201, 340, 401, and 540) and by alcohol eliminations at the urethane groups (m/z 322 and 668), all of which support the proposed structure (cf. Figure 7.15.b). The intense peak at m/z 622 appears to origenate from a more complex stepwise process, involving the loss of 72 Da (C 4 H 8 O) from the hard segment moiety, to form m/z 668, followed by attack of the newly formed isocyanate group by the acid chain end, with concomitant loss of CO + H 2 O (46 Da), to form a cyclic product with m/z 662, cf. Scheme A5. The fragment at m/z 422 in Figure 7.15 is attributed to a similar fragmentation, commencing from m/z 540; this proposition is substantiated by MS/MS spectrum of m/z 540 formed upon ASAP, which shows m/z 442 as the dominant fragment (Figure A28). 173

198 drift time (ms) 450 o C b 5 a no Irganox additives m/z Figure D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of estane (PU-3) at 450 C; the mass spectra extracted from mobility regions a and b (in ovals) are depicted in Figures 7.13 and 7.14, respectively. MDI IM region a BDO hard segment MDI Figure 7.13 Mass spectrum extracted from IM region a of the 2D ASAP-IM-MS diagram of estane (PU-3) acquired at 450 C ( Figure 7.12). 174

199 # IM region b * # Da repeat unit indicative of a poly(butylene adipate) soft segment Da * # m/z * # soft segment macrocycles 1 hard + n (1-2) polyester soft segment units Figure 7.14 Mass spectrum extracted from IM region b of the 2D ASAP-IM-MS diagram of estane (PU-3) acquired at 450 C ( Figure 7.12). 175

200 (a) m/z 600 (b) hard + 2 soft segment units m/z Figure 7.15 ASAP-IM-MS/MS spectra of (a) the oligomer at m/z ([M+H] + ), which agrees well with the structure of a poly(butylene adipate) macrocycle and (b) the oligomer at m/z ([M] + ), which agrees well with a poly(butylene adipate) chain capped with one hard segment unit. 176

201 7. 4 Thermoplastic Styrenic Copolymer Elastomers Polystyrenes have a wide range of uses, including food packaging applications. Styrenic copolymers are composed of styrene and a different monomeric repeat unit connected through covalent bonds to form a block copolymer. The choice and the ratio of the monomeric unit, which builds the soft segment determines the properties of the copolymer. Introducing butadiene as comonomer leads to thermoplastic copolymer elastomers This type of elastomers contains blocks of styrene and butadiene units, as shown in Scheme 7.2. Additives are blended into such elastomers to optimize their properties. ASAP-IM-MS shows clearly separated copolymer and additive ions in the case of polyurethanes. Therefore, the same method was utilized to analyze a styrenebutadiene elastomer, viz. styroflex (SB-1). Scheme 7.2 The general composition of styrene-butadiene elastomers ASAP-IM-MS of SB-1 Elastomer At low temprature, the ASAP-IM-MS plot shows ions that can be divided into three mobility regions (a-c), region a contains ions from small degradation products with the styrene and/or butadiene repeat units, for example, m/z 105 and 209, m/z 284, 177

202 388, or m/z 207, 261, and 315 (see Figure A29). Region b contains ions characteristic of the additive Irganox 1076, including its M + ion at m/z 530 and its [M+H 3 O] + ion at m/z 549 (see Figure A30). Region c (Figure 7.16) shows the molecular ion of another antioxidant, Irgafos 168, along with the molecular ion of the oxidized form of this antioxidant (m/z 646 and 662, respectively). The MS/MS spectum of m/z includes an intense peak at m/z 441.3, origeniating from the loss of one phosphate arm (C 14 H 21 O), in agreement with the Irgafos 168 structure (Figure A31). Figure D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of styroflex (SB-1) at 250 C (top) and the mass spectrum extracted from mobility region c (bottom). See Figure A29-A30 for the mass spectra extracted from IM regions a-b. 178

203 At the higher temperatures (Figure 7.17), hydrocarbon fragments including ions characteristic of styrene and butadiene are observed at masses < m/z 450 Da, (region a). The corresponding mass spectrum is shown in Figure A32. The additive Irganox 1010 and its fragments are detected in IM region d (cf. Figure A33). On other hand region e shows larger degradation products (> m/z 500) composed of styrene and butadiene comonomers. The entire mass spectrum extracted from region e is provided in Figure A34, while Figure 7.17 shows an expanded range, covering the m/z region in which the most abundant ions are observed. The thermal decomposition of polystyrenes and polybutadienes has been investigated extensively The main degradation pathways involve hemolytic C-C bond scissions, followed by backbiting rearrangements and/or β bond scissions. In addition, intermolecular reactions are possible, especially when pyrolysis occurs in a confined environment. Based on the reported thermal degradation pathways of polystyrene and polybutadiene, the larger ASAP products from styroflex can be divided into three copolymeric families, having different end groups which may also vary in their degree of (un)saturation. 127 The three types with both chain ends saturated are shown in Scheme 7.3. The corresponding end group masses are 106 Da for type I, 120 Da for type II, and 92 Da for type III; these decrease by 2 Da if one of the chain ends is unsaturated and by 4 Da if both chain ends contain an additional double bond. All degradation products observed in IM region e can be assigned to one of the families mentioned above, as indicated in Figure This result unequivocally proves that the analyzed thermoplastic copolymer is SB rubber material. 179

204 Scheme 7.3 Families of species formed during the thermal degradation of styrenebutadiene copolymers. In the species shown both end groups are saturated. Species containg an additional unsaturated in either or both end groups are also possible; their end group masses are 2 Da or 4 Da lower, respectively. 180

205 IM region e Larger degradation products (> m/z 500), typical to those observed by pyrolysis of SB copolymers type III S 4 BD 2 " type III BD 10 type I BD 10 type II BD 10 type III S 3 BD 5 type I S 3 BD 5 type II S 3 BD 5 type III S 6 type I S 6 type II S m/z Figure D IM-MS plot (m/z vs. drift time) of the ions generated by ASAP of styroflex (SB-1) at 450 C (top) and the mass spectrum extracted from mobility region e (bottom). The labels S n BD m give the number of complete styrene and butadiene units, respectively. A double prime indicates saturated end groups at both chain ends (as in Scheme 7.3). Otherwise, the end groups contain one additional unsaturation. All labeled species are [M+H] + ions Conclusions The results presented in this chapter show that coupling mild thermal degradation (as with ASAP) with IM separation provides a fast and sensitive method for the 181

206 identification of multicomponent industrial elastomers. This method is ideally suitable for the determination of additives, which are detected usually in intact form and desorb already at relatively low temperatures. As the ASAP temperature is increased, oligomers characteristic of the material s (co)polymeric constituents are observed. The mixture of desorbates and degradants is often complex. Therefore, interfacing ASAP with IM separation considerably simplifies their identification. Further structural insight can be provided by MS/MS to confirm assignments made on the basis of m/z data. The 2D IM- MS separation allows for the detection of small differences that may provide hints as to the analyzed sample s origin. Finally, the complete analysis is fast and with minimal solvent use, as it is completely performed with the mass spectrometer without prior chemical modification or chromatography ( top-down approach). 182

207 CHAPTER VIII SUMMARY The widespread application of mass spectrometry to the structural analysis of small molecules and biomolecules is an indication of the power and usefulness of this analytical technique for chemical analyses. The development of soft ionization methods such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) has extended the use of this technique to large molecules such as biopolymers and synthetic polymers and copolymers. Although single-stage mass spectrometry is considered as a powerful analytical technique, it is unable to identify isomeric or isobaric species. Moreover, it faces challenges when used to identify complex mixtures as often encountered with industrial polymers. To overcome these limitations, mass spectrometry can be coupled with tandem mass spectrometry and/or separation techniques to facilitate the structural characterization of these compounds. High molecular weight polymers or copolymers are often hard or impossible to ionize, which precludes their analysis by mass spectrometry. In such cases, pyrolysis can be applied to achieve compositional characterization based on the pyrolyzates detected by mass spectrometry. The work described in this dissertation illustrated the capabilities resulting from interfacing mass spectrometry with thermal degradation, separation techniques, and/or tandem mass spectrometry for the analysis of complex polymers and copolymers. 183

208 Chapter IV reported the complete characterization of three polyethylene oxide macroinitiators with different architectures. The functionalized PEOs were synthesized by Dr. Pugh s group via substitution reactions utilizing mono- or dihydroxy benzylaldehyde and methanesulfonyl chloride as a catalyst. Tandem mass spectrometry was applied to obtain information on the connectivities of these compounds and confirm their architectures. In these studies, the ion of interest was isolated and subjected to energetic collisions with neutral gas atoms. The fragments unveiled insight about the end groups and linking substituents of the macroinitiators. In chapter V, the full characterization of diblock copolymers consisting of polyethylene oxide and polycaprolactone was reported, using MALDI-MS, MALDI- MS/MS, and UPLC interfaced with MS and MS/MS. These copolymers were synthesized by Dr. Pugh s et al. via ring opening polymerization of ε-caprolactone with PEO macroinitiators using stannous octoate as a catalyst. The results showed that tandem mass spectrometry can be used to determine the block lengths of the copolymers and that reverse-phase UPLC can separate the copolymers based on number of their CL units. In chapter VI, tandem mass spectrometry and ion mobility mass spectrometry were applied to polyethers. Polytetrahydrofuran (THF) and poly(1,2-propylene oxide) (PPO) were acquired from Sigma-Aldrich, while poly(1,2-butylene oxide) (PBO) was synthesized in Dr. Li Jia et al. This study was performed to elucidate the influence of size and collision energy variables on the fragmentation pathways of polyethers. The results from MS/MS and IM-MS were combined to allow a full explanation of dissociation mechanisms and energetic. 184

209 Lastly, Chapter VII reported the application of an atmospheric solids analysis probe (ASAP) ionization and ion mobility separation, coupled with tandem mass spectrometry, to characterize commercially available thermoplastic elastomers. Compared to ASAP-MS, the ASAP-IM-MS (MS/MS) technique simplified the analysis of such complex mixtures, which usually consist of copolymers and additives. Complete characterization of thermoplastic polyurethanes and styrenic copolymers was possible by separating pyrolyzates by their ion mobilities prior to MS and MS/MS analysis. In summary, this dissertation discussed the advantages of combining mass spectrometry techniques with thermal degradation and/or separation methods for the structural analysis of a variety of polymeric materials. This approach provided detailed structural information about the composition of such materials, including their repeat units, end groups, architectures, and additives and also permitted the fast separation as well as sensitive and conclusive characterization of the multicomponent materials examined. 185

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221 APPENDICES 197

222 APPENDIX A ADDITONAL DATA Table A1.Spreadsheet used to determine the PEO-b-PCL composition. All ions are sodiated with CH 3 - and -OH end groups. All m/z values are monoisotopic 200 Table A2. Spreadsheet used to determine the PEO-b-PCL composition. All ions are sodiated with CH 3 - and O 2 C 7 H 7 end groups. All m/z values are monoisotopic 200 Table A3. Spreadsheet used to determine the PEO-b-PCL composition. All ions are sodiated with an OH end group at the PCL chain end and with a C 11 H 13 O 3 substituent within the PEO macrocycle (See structure in Figure 5.18). The C 11 H 13 O 3 substituent includes two EO repeat units. All m/z values are monoisotopic.201 Table A4. Corrected drift times and collision cross-sections of protonated polyalanine oligomers.202 Figure A1. Expanded view of the m/z region of the LC-MS spectrum acquired from LC fraction #7 of the CH 3 -PEO-b-PCL-OH copolymer 203 Figure A2. Entire LC-MS mass spectrum of fraction # 7 from CH 3 -PEO-b-PCL..203 Figure A3. Entire LC-MS mass spectrum of fraction # 10 from ω-bnpeo-b-pcl Figure A4. Entire LC-MS mass spectrum of fraction # 10 from McBnPEO-b-PCL..204 Figure A5. CAD mass spectrum of lithiated PTHF, (m/z 529.4), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev 205 Figure A6. CAD mass spectrum of lithiated PTHF, (m/z 601.5), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev.205 Figure A7. CAD mass spectrum of lithiated PTHF, (m/z 673.5), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev.206 Figure A8. CAD mass spectrum of lithiated PTHF, (m/z 745.5), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev.206 Figure A9. CAD mass spectrum of lithiated PBO, (m/z ), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev.207 Figure A10. CAD mass spectrum of lithiated PBO, (m/z 615.6), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev

223 Figure A11. CAD mass spectrum of lithiated PBO, (m/z 687.6), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev 208 Figure A12. CAD mass spectrum of lithiated PBO, (m/z 759.7), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev Figure A13. CAD mass spectrum of lithiated PPO, (m/z 837.6), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev.209 Figure A14. CAD mass spectrum of lithiated PPO, (m/z 895.7), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev 209 Figure A15. CAD mass spectrum of lithiated PPO, (m/z 953.7), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev 210 Figure A16. Calibration curve for the derivation of collision cross-sections (CCSs), obtained by plotting the corrected CCSs of protonated polyalanine oligomers versus the corresponding corrected drift times.210 Figure A17. Mass spectra extracted from regions a and b in the 2D IM-MS spectrum of ellastolan (PU-1), acquired under wet ASAP source conditions. See Figure 7.2 for the spectra acquired under dry source conditions Figure A18. ASAP-IM-MS/MS spectrum of m/z 250, the radical ion of MDI (cf. Figure 7.4.a). Under dry conditions, MDI ionized mainly by electron removal (M +, m/z 250) and also by protonation ([M+H] +, m/z 251).212 Figure A19. ASAP-IM-MS/MS spectrum of m/z 340 (cf. Figure 7.4.a). Upon CAD, this radical ion undergoes simple bond scissions that expel radicals (m/z 269, 251) as well as rearrangements that expel closed-shell molecules (m/z 268, 250) A 20. ASAP-IM-MS/MS spectrum of m/z in region c of the ASAP-IM-MS/MS plot of elastollan at 450 ºC (cf. Figure 7.3).213 Figure A21. ASAP-MS spectrum (without IM separation) of elastollan at 250 and 450 ºC..213 Figure A22. ASAP-MS spectrum (without IM separation) of pellethane at 250 and 450 ºC Figure A23. ASAP-MS spectrum (without IM separation) of estane at 250 and 450 ºC..214 Figure A24. ASAP-MS spectrum (without IM separation) of styroflex at 250 and 450 ºC..215 Figure A25. 2D ASAP-IM map of pellethane at 250 ºC and the spectrum extracted from the entire IM space

224 Figure A26. ASAP-IM-MS/MS spectrum of m/z in the IM region c of the ASAP- IM-MS plot from pellethane (PU-2) at 450 ºC (cf. Figure 7.8)..217 Figure 27. 2D ASAP-IM-MS map of estane (PU-3) at 250 ºC and the spectrum extracted from the entire IM space..218 Figure A28. ASAP-IM-MS/MS spectrum of the oligomer at m/z ( M + ) from estane (PU-3), cf. Figure Figure A29. Mass spectrum extracted from IM region a in the 2D ASAP-IM-MS plot of styroflex at 250 ºC (Figure 7.16). 219 Figure A30. Mass spectrum extracted from IM region b in the 2D ASAP-IM-MS plot of styroflex at 250 ºC (Figure 7.16).220 Figure A31. ASAP-IM-MS/MS spectrum of Irgafos 168, desorbed from styroflex (SB-1) at 450 ºC Figure A32. Mass spectrum extracted from IM region a in the ASAP-IM-MS plot of styroflex at 450 ºC (see Figure 7.17) Figure A33. Mass spectrum extracted from IM region d in the ASAP-IM-MS plot of styroflex at 450 ºC (see Figure 7.17) Figure A34. Mass spectrum extracted from IM region e in the ASAP-IM-MS plot of styroflex at 450 ºC (see Figure 7.17) Scheme A1. H-rearrangement in sodiated ω-bnpeo-b-pcl leading to the smallest fragment in series # (Figure 5.17), i.e. the one formed by ion of the entire PCL block (in the form of H-CL n -OH) Scheme A2. Isobaric impurity (lower structure) in the main oligomer series (upper structure) in the LC-MS mass spectra of ω-bnpeo-b-pcl. This impurity gives rise to the ion marked by $ in Figure Scheme A3. H-rearrangement in sodiated McBnPEO-b-PCL leading to the smallest fragment in series # (Figure 5.20), formed by loss of the entire PCL block in the form of H-CL n -OH (474 Da for n = 4) 226 Scheme A4. Charge-remote hemolytic C-O bond cleavages in the lithiated PPO according to the mechanism shown in Scheme Scheme A5. Fragmentation pathway of m/z 740, formed by ASAP of estane (PU-3), cf. Figure 7.14, via sequential eliminations of butenol (72 Da), and CO + H 2 O (46 Da) to yield the fragments at m/z 668 and 622 (Figure 7.15)

225 Table A1.Spreadsheet used to determine the PEO-b-PCL composition. All ions are sodiated with CH 3 - and -OH end groups. All m/z values are monoisotopic. Table A2. Spreadsheet used to determine the PEO-b-PCL composition. All ions are sodiated with CH 3 - and O 2 C 7 H 7 end groups. All m/z values are monoisotopic. 201

226 Table A3. Spreadsheet used to determine the PEO-b-PCL composition. All ions are sodiated with an OH end group at the PCL chain end and with a C 11 H 13 O 3 substituent within the PEO macrocycle (See structure in Figure 5.18). The C 11 H 13 O 3 substituent includes two EO repeat units. All m/z values are monoisotopic. 202

227 Table A4. Corrected drift times and collision cross-sections of protonated polyalanine oligomers. n Ala n Measured drift time a t d (ms) Corrected drift time b t' d (ms) Reborted CCS c Ω (Å 2 ) Corrected CCS d Ω (Å 2 ) m/z a Traveling wave velocity, 350 m/s; traveling wave height, 11 V. b Obtained via t d = t d c (m/z) 0.5,where c is the enhanced duty cycle (EDC) dely coefficient (1.41). c From: 1. Henderson, S. C.; Li, J.; Counterman, A. E.; Clemmer, D. E. Intrinsic Size Parameters for Val, Ile, Leu, Gln, Thr, Phe, and Trp Residues from Ion Mobility Measurements of Polyamino Acid Ions, Journal of Physical Chemistry B, 1999, 103, Clemmer Group, Cross Section Database; base.php d Ω c = Ω / [z (1/m + 1/M g )], where z and m are the charge and the mass of the polyalanine oligomer and M g the molecular weight of the drift gas (N 2 ). 203

228 Figure A1. Expanded view of the m/z region of the LC-MS spectrum acquired from LC fraction #7 of the CH 3 -PEO-b-PCL-OH copolymer Doubly charged : [M+2Na] 2+ [M+2NH 4 ] 2+ [M+Na+NH 4 ] 2+ [M+Na+K] 2+ [M+2H] 2+ [M+2K] Singly charged : [M+Na] + [M+NH 4 ] + [M+H] + [M+K] m/z Figure A2. Entire LC-MS mass spectrum of fraction # 7 from CH 3 -PEO-b-PCL. See Figure 5.6 for the m/z singly charged ions. 204

229 PCL 4 +Na Doubly charged : PCL 4 +H [M+2Na] 2+ [M+2NH 4 ] 2+ [M+Na+NH 4 ] 2+ [M+Na+K] 2+ [M+2H ] 2 + [M+2K] 2 + Singly charged : [M+Na] + [M+NH 4 ] + [M+H] + [M+K] m/z Figure A3. Entire LC-MS mass spectrum of fraction # 10 from ω-bnpeo-b-pcl. See Figure 5.16 for the m/z singly charged ions Doubly charged : [M+2Na] 2+ [M+2NH 4 ] 2+ [M+Na+NH 4 ] 2+ [M+Na+K] 2+ [M+2H] 2 + [M+2K] Singly charged : m/z Figure A4. Entire LC-MS mass spectrum of fraction # 10 from McBnPEO-b-PCL. See Figure 5.22 for the m/z singly charged ions 205

230 529.4 (a) CE 14 ev e e 2 e 2 e e d (b) CE 64 ev d 4 d e 4 e d e d e j e d 7 j 3 e m/z Figure A5. CAD mass spectrum of lithiated PTHF, (m/z 529.4), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev (a) CE 14 ev e e e e 4 2 (b) CE 64 ev d e d e5 e e e 6 d e d 7 d d e 7 j 6 4 j j j m/z Figure A6. CAD mass spectrum of lithiated PTHF, (m/z 601.5), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. 206

231 (a) CE 14 ev (b) CE 64 ev e e 4 e 5 e d e 6 e 7 8 e 3 2 d d d d d d e e e 4 2 e j 4 j j m/z Figure A7. CAD mass spectrum of lithiated PTHF, (m/z 673.5), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. (a) CE 14 ev (b) CE 64 ev e 3 e e 2 e 4 e 5 e 6 e 7 8 e 9 d d d d d d d d m/z Figure A8. CAD mass spectrum of lithiated PTHF, (m/z 745.5), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. 207

232 543.5 (a) CE 14 ev z z (b) CE 64 ev z 2 z x 2 z c 2 x 3 c 3 x z c 4 z x 5 c z c m/z Figure A9. CAD mass spectrum of lithiated PBO, (m/z ), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev (a) CE 14 ev z z (b) CE 64 ev z z z 2 z 3 z c c c z 6 x x 3 x 4 x c 5 z c 7 x6 6 c 7 z m/z Figure A10. CAD mass spectrum of lithiated PBO, (m/z 615.6), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. 208

233 687.6 (a) CE 14 ev z z 4 z z z 6 x x z 7 z x x 5 z c 4 c c c 5 x6 c 6 c c (b) CE 64 ev m/z Figure A11. CAD mass spectrum of lithiated PBO, (m/z 687.6), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. (a) CE 14 ev z z (b) CE 64 ev z 5 z z z 7 z c c c 8 x z z 1 x 9 x x c x 6 c 6 c 7 c 8 c m/z Figure A12. CAD mass spectrum of lithiated PBO, (m/z 759.7), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. 209

234 837.6 (a) CE 14 ev (b) CE 64 ev c 2 c 3 c 4 c c c c c c C b 10 C b b 10 b b m/z Figure A13. CAD mass spectrum of lithiated PPO, (m/z 837.6), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev (a) CE 14 ev (b) CE 64 ev c 4 c 5 b c 3 c 6 c 7 c 8 c 9 c b 12 b 13 b 14 c m/z Figure A14. CAD mass spectrum of lithiated PPO, (m/z 895.7), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev. 210

235 Collision Cross Section (Å 2 ) (a) CE 14 ev (b) CE 64 ev c 3 c 4 c c 6 c 8 c 9 c 10 b 11 c 15 5 c 7 b 12 b 13 b m/z Figure A15. CAD mass spectrum of lithiated PPO, (m/z 953.7), acquired with the Synapt Q/ToF mass spectrometer at a E lab collision energy of (a) 14 ev and (b) 64 ev y = x R² = Corrected drift time (ms) Figure A16. Calibration curve for the derivation of collision cross-sections (CCSs), obtained by plotting the corrected CCSs of protonated polyalanine oligomers versus the corresponding corrected drift times. 104 See Table A4 for a list of the data used. 211

236 % % T=450 Flow=1200 hw 9 vw 300 NA_070213_elastollan_T450 with MeOH 19 (1.152) Cm (13:52) 100 IM region a 2: TOF MS 4.76e $ * * $ $ * $ m/z m/z T=450 Flow=1200 hw 9 vw 300 NA_070213_elastollan_T450 with MeOH 87 (5.504) Cm (87:127) 100 IM region b : TOF MS 1.03e3 $ # # m/z m/z * 1 hard + n soft segment units # soft segment macrocycles $ soft segment chains Figure A17. Mass spectra extracted from regions a and b in the 2D IM-MS spectrum of ellastolan (PU-1), acquired under wet ASAP source conditions. See Figure 7.2 for the spectra acquired under dry source conditions. 212

237 M NCO CO m/z Figure A18. ASAP-IM-MS/MS spectrum of m/z 250, the radical ion of MDI (cf. Figure 7.4.a). Under dry conditions, MDI ionized mainly by electron removal (M +, m/z 250) and also by protonation ([M+H] +, m/z 251) C 4 H 10 O 2 -NCO C 4 H 8 O M m/z Figure A19. ASAP-IM-MS/MS spectrum of m/z 340 (cf. Figure 7.4.a). Upon CAD, this radical ion undergoes simple bond scissions that expel radicals (m/z 269, 251) as well as rearrangements that expel closed-shell molecules (m/z 268, 250). 213

238 One t-bu group split off Da Da m/z A 20. ASAP-IM-MS/MS spectrum of m/z in region c of the ASAP-IM-MS/MS plot of elastollan at 450 ºC (cf. Figure 7.3). Upon CAD (at a collision energy of 30 ev), this radical ion (M + ) undergoes two consecctive C 4 H 8 (56 Da) losses from the t-bu groups to form intense fragments at at m/z and Benzylic cleavage (with accompanging H-atom rearrangement) leads to m/z (C 25 H 42 N 2 O 3 + ) which can lose C 2 H 2 O (ketene, 42 Da) to form m/z 376.4, ketyl radical (41 Da) to form m/z 377.4, or C 4 H 8 (56 Da) to form m/z A 56 Da loss is also observed from m/z ( to 321.3). This fragmentaion patterns is fully consistent with the structure of Irganox

239 530 MDI + BDO phthalate 149 MDI BHT T= 250 ºC 220 PDMS MDI 250 T= 450 ºC hard + n soft segment units 484 Irganox Figure A21. ASAP-MS spectrum (without IM separation) of elastollan at 250 and 450 ºC MDI 250 T= 250 ºC PDMS MDI T= 450 ºC 132 Irganox hard + n soft segment units Figure A22. ASAP-MS spectrum (without IM separation) of pellethane at 250 and 450 ºC. 215

240 Irganox 1076 Figure A23. ASAP-MS spectrum (without IM separation) of estane at 250 and 450 ºC T=250 C Irgafos T=450 C Irgafos m/z Figure A24. ASAP-MS spectrum (without IM separation) of styroflex at 250 and 450 ºC. 216

241 MDI PDMS m/z Figure A25. 2D ASAP-IM map of pellethane at 250 ºC and the spectrum extracted from the entire IM space. 217

242 m/z Figure A26. ASAP-IM-MS/MS spectrum of m/z in the IM region c of the ASAP- IM-MS plot from pellethane (PU-2) at 450 ºC (cf. Figure 7.8). The intense peak at m/z 515 corresponds to the loss of a methyl group and the peak at m/z 219 corresponds to C 15 H 23 O +. This fragmentation pattern agrees well with the structure of antioxidant Irganox

243 233.1 Drift time (ms) NA_020613_BFGOODRICH_IM250.raw : NA_020613_BFGOODRICH_IM250.raw : 1 m/z MDI PDMS m/z Figure 27. 2D ASAP-IM-MS map of estane (PU-3) at 250 ºC and the spectrum extracted from the entire IM space. 219