Application of Radioisotopes to Polymer Chemistry: Investigation of Radiolabelled Atom Transfer Polymerization

Transcription

1 Application of Radioisotopes to Polymer Chemistry: Investigation of Radiolabelled Atom Transfer Polymerization A thesis submitted to The University of Manchester for the degree of PhD in the Faculty of Engineering and Physical Sciences 2015 Mark Long SCHOOL OF CHEMISTRY

2 TABLE OF CONTENTS Table of Contents 2 List of Tables 4 List of Schemes 6 List of Figures 8 Abstract 14 Declaration 15 Copyright Statement 16 Acknowledgements 17 The Author 18 CHAPTER 1 Introduction and Thesis Content 19 CHAPTER 2 Free Radical and Living Polymerization: Atom Transfer Radical 30 Polymerization CHAPTER 3 Application of Fluorescent Spectroscopy to Polymer Chemistry 48 CHAPTER 4 Application of 14 C and 3 H Radioisotopes to Polymer Chemistry 58 CHAPTER 5 Utilising 14 C-Radiolabelled Atom Transfer Radical Polymerization 97 Initiators CHAPTER 6 Monitoring Atom Transfer Radical Polymerization using C Radiolabelled Initiators 2

3 CHAPTER 7 Controlled Synthesis of Radiolabelled Amine Methacrylate 140 Water-soluble Polymers with Varying End-groups and Studies Of Adsorption Behaviour CHAPTER 8 Summary & Conclusions 161 Final Word Count

4 LIST OF TABLES CHAPTER 1 Introduction and Theses Content Table 1 Percentage fraction of the molecular weights of initiators, 24 relative to the molecular weight of poly N,N diethyl amino ethyl methacrylate (DEAEMA) CHAPTER 2 Free Radical and Living Polymerization: Atom Transfer Radical Radical Polymerization Table 1 Physical Properties of Tritium ( 3 H) and 14 C 60 CHAPTER 6 Monitoring Atom Transfer Radical Polymerization using 14 C Radiolabelled Initiators Table 1 Summary of ATRP of 2-hydroxypropyl methacrylate (2-HPMA) 113 with labelled and non-labelled initiators to target DPn =50 monomer units Table 2 Summary of the impact of silica flash chromatography on 122 unreacted/terminated initiator removal (shown as percentage of total activity of sample) from poly(2-hpma) with different target chain lengths. Calculated DPn ( 1 H NMR end group analysis) is also shown before and after flash chromatography Table 3 Impact of repeat precipitation on the removal of unreacted 123 /terminated initiator (shown as percentage of total activity of sample) from poly(2- HPMA) with different target chain lengths. Calculated DPn ( 1 H NMR end group analysis) is also shown before and after precipitation 4

5 Supporting Information: Utilising 14 C-Radiolabelled Atom Transfer Radical Polymerisation Initiators Table 1 Rf values 115 Table 2 NMR & GPC data of 2-HPMA ATRP polymerisation with a 117 target DPn= 50 monomer units. CHAPTER 7 Controlled synthesis of radiolabelled amine methacrylate watersoluble polymers with varying end-groups and sudies of adsorption behaviour Table 1 Summary of the ATRP polymerisation of N,N diethylamino 145 ethyl methacrylate with labeled and non-labelled initiators (Target: DPn=50 monomer units; Mn=9260+ initiator). GPC data calculated from poly(methyl methacrylate) standards using THF as eluent Supporting Information: Controlled synthesis of radiolabelled amine methacrylate water soluble polymers with varying end groups and studies of adsorption Table 1 Radioactivity levels of Cyclone Phosphore Imager standards 152 5

6 LIST OF SCHEMES CHAPTER 2 Free Radical and Living Polymerization: Atom Transfer Radical Radical Polymerization Scheme 1 Free Radical Polymerisation 31 Scheme 2 Atom Transfer Radical Addition 34 Scheme 3 Mechanism of Atom Transfer Free Radical Polymerisation 36 CHAPTER 3 Application of 14 C and Radioisotopes to Polymer Chemistry Scheme 1 Decomposition of 1, 1 azo-iso-butyronitrile 67 Scheme 2 Conversion of iso-butyronitrile with methacrylonitrile to 68 tetramethyl succinodinitrile in the presence of cyano-2-propyl radical Scheme 3 Decomposition of azo-iso-butyronitrile in the presence of 68 oxygen Scheme 4 Decomposition of benzyl peroxide and subsequent initiation 70 Scheme 5 Polymerization of iso-butene initiated by aluminium trichloride 72 Scheme 6 Polymerization of iso-butyl vinyl ether initiated using boron 73 trifluoride 14 C etherate Scheme 7 Polymerization of 14 C styrene with high molecular weight 76 poly(styrene) Scheme 8 Chain transfer using zinc 14 C diethyl 78 Scheme 9 Termination mechanism combination and disproportionation 80 Scheme 10 Pyrolysis of poly(styrene) poly(methyl styrene) copolymer 81 6

7 CHAPTER 5 Utilizing 14 C-Radiolabelled Atom Transfer Radical Polymerization Initiators Scheme 1 Strategies for synthesising 14 C labelled ATRP initiators, 100 esterification of 14 C labelled benzyl alcohol, synthesis of 14 C labelled 2-bromo-iso-butyric acid, reaction of C -labelled 2-bromo-iso-butyric acid with benzyl alcohol. CHAPTER 6 Monitoring Atom Transfer Radical Polymerization using 14 C Radiolabelled Initiators Scheme 1 Synthesis 14 C radiolabelled ATRP initiators, esterification of C labelled benzyl alcohol, synthesis of 14 C labelled 2-bromo-iso-butyric acid, reaction of 14 C labelled 2-bromo-iso-butyric acid with benzyl alcohol. CHAPTER 7 Controlled Synthesis of Radiolabelled Amine Methacrylated Water- Soluble Polymers with Varying End Groups and Studies of Adsorption Behaviour Scheme 1 Synthesis of 14 C radiolabelled ATRP initiators i) reaction 145 with 14 C labelled primary alcohols and ii) reaction of 14 C-labelled 2-bromo-iso-butyric acid with 9-hydroxyfluorene. 7

8 LIST OF FIGURES CHAPTER 2 Free Radical and Living Polymerization: Atom Transfer Radical Polymerization Figure 1 ATRP first order kinetics 38 CHAPTER 3 Application of Fluorescent Spectrroscopy to Polymers Figure 1 Jablonski diagram 49 CHAPTER 5 Utilizing 14 C-Radiolabelled Atom Transfer Radical Polymerization Initiators Figure 1 Radio TLC of 14 C labelled 2-bromo-iso-butyrate 14 C methylene 101 labelled initiator (A); 14 C methyl labelled initiator (B). Insets show background radioactivity. (Eluent: Et2O CH3CO2H 90/10 v/v.) Figure 2 Monomer and polymer structures formed during this study: hydroxypropyl methacrylate; unlabelled poly(hpma); 14 C methylene labelled poly(hpma); 14 C methyl labelled poly(hpma). Figure 3 Radio TLC of poly(hpma) with target DPn=50 using (A) 14 C 102 methylene labelled initiator; (B) 14 C methyl labelled initiator initiator; (C) DPn=25; 14 C methyl labelled initiator initiator; (D) DPn = 10; 14 C methyl labelled initiator. Insets show intermediate region radioactivity. (Eluent: Et2O CH3CO2H 90/10 v/v.) 8

9 CHAPTER 6 Monitoring Atom Transfer Radical Polymerization using 14 C Radiolabeled Initiators Figure 1 GPC analysis of benzyl 2-bromo-iso-butyrate studied using 124 refractive index detection (concentration detection) and scintillation counting of fractions from the GPC eluent (radioactivity detection). Figure 2 GPC analysis of ATRP of 2-HPMA using 14 C methylene labelled 125 initiator. (A) Sample taken after 30 minutes polymerisation; (B) sample after 24 hours polymerisation. Refractive index (concentration detection) and scintillation counting of fractions collected from the GPC eluent (radioactivity detection) are shown, overlaid with scintillation of fractions for 14 C methylene labelled initiator for comparison. Figure 3 Radio TLC of: (A) 14 C methyl labelled 2- bromo-iso-butyrate 125 initiator; (B) 14 C methylene labelled 2-bromo-iso-butyrate initiator over layed with poly(2-hpma) synthesised from 3 with a target DPn= 50 monomer units, after 24 hours polymerisation time (>97% conversion); (eluent: Et2O CH3CO2H 90/10 v/v.). Figure 4 Comparison of Radio TLC, GPC/liquid scintillation, 1 H NMR and 126 GPC/RI analysis of ambient ATRP of 2-HPMA (target DPn= 50) with benzyl 2-bromo-iso-butyrate in methanol. (A) 14 C methylenelabelled 2-bromo-iso-butyrate initiator and (B) 14 C methyl-labelled 2- bromo-iso-butyrate initiator. Figure 5 Radio TLC of: (A) poly(2-hpma) synthesised from 14 C methylene 127 labelled 2-bromo-iso-butyrate initiator with a target DPn= 50 monomer units: polymer sample at 24 hours polymerisation time (>97% monomer conversion), polymer sample after silica flash chromatography, polymer sample after three precipitations; (B) overlayed supernatant of repeated precipitations: precipitation supernatant 1, precipitation supernatant 2, precipitation supernatant 14 C methylene labelled 2-bromo-iso-butyrate initiator. Radio TLC data have been normalised to trace initiator peak to 9

10 show comparative peak height with removed polymer. (Eluent: Et2O CH3CO2H 90/10 v/v.) CHAPTER 7 Controlled Synthesis of Radiolabelled Amine Methacrylated Water- Soluble Polymers with Varying End Groups and Studies of Adsorption Behaviour Figure 1 Radiolabelled polymers with different end groups; A) methyl, 144 B) benzyl and C) 9-hydroxyfluorene terminated. D) labelled polymers in aqueous solution and E) adsorption onto surfaces Figure 2 Radio TLC of 14 C-labelled initiators: A) methyl 2-bromo-iso- 145 butyrate, 7 (Eluent: 60:40 petroleum ether 40-60/Et2O v/v), B) benzyl 2- bromo-iso-butyrate, 8 (Eluent: 90:10 petroleum ether 40-60/Et2O v/v), and C) 9-hydroxyfluorenyl 2-bromo-iso-butyrate, 9 (Eluent: 90:10 petroleum ether 40-60/Et2O v/v) Figure 3 Comparative kinetic evaluation of the ambient polymerisation 146 of N,N-diethylamino ethyl methacrylate with initiators 7 (red data), 8 (green data) and 9 (blue data). Conversion is shown as coloured circles and ln(m0)/(m) is given as triangles. Figure 4 Autoradiography (storage phosphor imaging) images of 146 radiolabelled poly(deaema) adsorbed onto A) hair, B) filter paper and C) photographic paper. Poly(DEAEMA) has varying initiatorderived end-groups i) methyl, ii) benzyl and iii) 9-hydroxyfluorenyl. Experiment relevant autoradiography standards are shown for comparison. Figure 5 Adsorption of radiolabelled poly(deaema) onto filter paper 147 (circles) and photographic paper (squares) measured by autoradiography. Initiator-derived end-group variation includes methyl (red), benzyl (green) and 9-hydroxyfluorenyl (blue) groups. 10

11 Figure 6 Adsorption of radiolabelled poly(deaema) onto A) filter paper 147 (circles), B) virgin hair (triangles) and C) photographic paper (squares) as measured by oxidation and liquid scintillation counting. Initiator-derived end-group variation includes methyl (red), benzyl (green) and 9-hydroxyfluorenyl (blue) groups. Figure 7 Surface tensiometry of poly(deaema)with different chain-end 148 modification; methyl (red triangles), benzyl (green squares) and 9- hydroxy fluorenyl (blue circles) at A) ph=2. 11

12 Supporting Information: Utilising 14 C-Radiolabelled Atom Transfer Radical Polymerisation Initiators Figure S1: 1 H NMR spectrum of benzyl 2-bromo iso-butyrate 104 Figure S2: 13 C NMR spectrum of benzyl 2-bromo iso-butyrate 105 Figure S3: Mass Spectrum of benzyl 2-bromo iso-butyrate 105 Figure S4: 1 H NMR spectrum of Figure S5: 13 C NMR spectrum of Figure S6: Radio TLC of 3 in various solvent mixtures 108 Figure S7: 1 H NMR spectrum of diethyl dimethyl malonate 109 Figure S8: 1 H NMR of 2-bromo iso-butyric acid 110 Figure S9: 13 C NMR of 2-bromo iso-butyric acid 110 Figure S10: 1 H NMR of diethyl ( 14 CH3) dimethyl malonate (5) 111 Figure S11: 1 H NMR of 2-bromo ( 14 CH3) iso-butyric acid (8) 111 Figure S12: 13 C NMR of 2-bromo ( 14 CH3) iso-butyric acid (8) 112 Figure S13: Radio TLC of bromo ( 14 CH3) iso-butyric acid (8) 112 Figure S14: 1 H NMR of benzyl 2-bromo ( 14 CH3) iso-butyrate (11) 113 Figure S15: 13 C NMR of benzyl 2-bromo ( 14 CH3) iso-butyrate (11) 114 Figure S16: Radio TLC Trace of benzyl 2-bromo ( 14 CH3) iso-butyrate (11) 115 Figure S17: Kinetic analysis of ATRP polymerisation of 2-HPMA using 116 radiolabelled and non-labelled initiators. Figure S18: Overlaid GPC chromatograms of samples taken during the ATRP 117 Figure S19: polymerisations of 2-HPMA. Radio TLC of poly(2-hpma) using THF as eluent

13 Monitoring Atom Transfer Radical Polymerisation using 14 C- Radiolabelled Initiators Figure S1: 1 H NMR spectrum of benzyl 2-bromo iso-butyrate 131 Figure S2: 13 C NMR spectrum of benzyl 2-bromo iso-butyrate 131 Figure S3: Mass Spectrum of benzyl 2-bromo iso-butyrate 132 Figure S4: 1 H NMR spectrum of Figure S5: 13 C NMR spectrum of Figure S6: 1 H NMR spectrum of diethyl dimethyl malonate 134 Figure S7: 1 H NMR of 2-bromo iso-butyric acid 135 Figure S8: 13 C NMR of 2-bromo iso-butyric acid 135 Figure S9: 1 H NMR of diethyl ( 14 CH3) dimethyl malonate (5) 136 Figure S10: 1 H NMR of 2-bromo ( 14 CH3) iso-butyric acid (8) 136 Figure S11: 13 C NMR of 2-bromo ( 14 CH3) isobutyric acid (8) 137 Figure S12: Radio TLC of bromo ( 14 CH3) iso-butyric acid (8) 137 Figure S13: 1 H NMR of benzyl 2-bromo ( 14 CH3) iso-butyrate (11) 138 Figure S14: 13 C NMR of benzyl 2-bromo ( 14 CH3) iso-butyrate (11) 139 Supporting Information: Controlled synthesis of radiolabelled amine methacrylate water soluble polymers with varying end groups and studies of adsorption behaviour Figure S1: 1 H NMR Spectra for Initiators 154 Figure S2: 13 C NMR Spectra of Initiators 155 Figure S3: GPC chromatogram of poly(deaema) with a target DPn monomer units using benzyl ( 14 CH2) 2-bromo-iso-butyrate (DRI detector) Figure S4: Adsorption of radiolabeled poly(deaema) onto different 157 substrates. Comparison within each chain end group Figure S5: Radio TLC of poly(deaema) polymers to determine unreacted 158 initiator content Figure S6: Regression of linear region of surface tension measurements

14 ABSTRACT University The University of Manchester Name Mark Long Degree Degree of PhD in the Faculty of Engineering and Physical Sciences Title Application of Radioisotopes to Polymer Chemistry: Investigation of Radiolabelled Atom Transfer Polymerization Year 2015 The use of the radioisotope 14 C in polymer chemistry has been reviewed, showing how it has been used to investigate the mechanistic aspects of free radical polymerizations, and the use of polymers in other scientific disciplines such as environmental, physical, chemical and medical sciences. An overview of the application of fluorescent spectroscopy to polymer chemistry is also reported. It covers the fundamentals of fluorescence chemistry, its application and the potential problems of the use of fluorescent labels in polymer chemistry. The application of radioisotopes to atom transfer radical polymerisation (ATRP) to investigate the fate of initiators used in the ATRP of 2-hydroxypropyl methacrylate (2- HPMA) is also reported. By using 14 C radiolabelled initiators, radio thin layer chromatography (Radio TLC) and the liquid scintillation counting of fractions, collected from gel permeation chromatography (GPC), the fate of the initiating species where monitored during the polymerization of samples of 14 C poly(2-hpma), with degrees of polymerization of 10, 25 and 50 was assessed. GPC and Radio TLC, data showed that there was an under-utilisation of the initiator, 16% clearly observable at high monomer conversion (>97%), which could result in the initiation of new chains at monomer conversions of >90% and as late as 300 minutes after the polymerisation had started. These results contradict ATRP theory which states all initiator is consumed immediately at the commencement of the polymerization. 14 C poly(2-hpma) was also used to determine the efficiencies of the polymer purification methods, flash chromatography and precipitation. Although repeated precipitation increased fractionation, it was shown to be superior to flash chromatography in removing residual unreacted or terminated initiator. Finally, the possible effects of fluorescent labels on adsorption of low molecular weight 14 C poly(deaema) onto real surfaces (filter paper, photo graphic paper and hair) from aqueous solutions at ph=2 were investigated. Three low molecular weight samples of 14 C poly(deaema) were prepared by ATRP using 14 C labelled initiators synthesized from alcohols of increasing hydrophobicity i.e. methyl, benzyl and 9-hydroxyfluorene (fluorescent label). The levels of adsorption were determined using phosphor imaging, oxidation of organic samples and liquid scintillation counting. Results indicated that differences in the chemistry of the polymer end groups can affect adsorption of the 14 C poly(deaema) and polymer assembly at the air/water interface. There was greater adsorption of polymers with a fluorescent end group. The increasing deposition was attributed to the increasing hydrophobicity of the polymer end group. Moreover, the controlled placement of one fluorescent label per polymer chain can influence the polymer s properties, prompting the question, is the use of fluorescent groups to assess polymer behaviour and properties viable? 14

15 DECLARATION No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. 15

16 COPYRIGHT STATEMENT i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the Copyright ) and he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, inaccordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the Intellectual Property ) and any reproductions of copyright works in the thesis, for example graphs and tables ( Reproductions ), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see in any relevant Thesis restriction declarations deposited in the University Library, The University Library s regulations (see and in The University s policy on Presentation of Theses 16

17 ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisors, Professor Steve P Rannard, Professor Francis R Livens and Professor David W Thornthwaite for all the guidance and support, and the advice and training given by Dr Suzanne H Rogers with respect to Atom Transfer Radical Polymerization. I would like to acknowledge funding from Unilever Research and Development Wirral for funding this program. Finally, I want to say a very big thank you to my wife and children for persevering with me during my studies. 17

18 THE AUTHOR The author commenced employment with Unilever Research and Development Port Sunlight Wirral in 1987 in the Organic Chemistry section to train as a synthetic Organic Chemist. In 1990, he subsequently moved to the Radiotracer Laboratory as Radiation Protection Supervisor and, initially, deputy Radiotracer Laboratory Manager then as Radiotracer Manager from 1995 to the present day. Work carried out in the Radiotracer Laboratory comprised small, micro scale synthesis of organic, polymeric and inorganic compounds, and their subsequent use in radiochemical physical and environmental studies. From 2013 the Radiotracer Laboratory is now based at the University of Liverpool University where the author continues to be employed by Unilever Research in his role of radiochemist and also as advisor to Professor Steve Rannard with respect to the mangment of the Radiotracer Laboratory, now owned/managed by the University, and Steve s research program into the use and preparation of radiolabelled polymers. During the twenty eight years of employment at Unilever Research and Development the author has studied part time for a number of academic and professional qualifications and is a member of a number of professional societies. Qualifications Bsc Applied Chemistry Humberside University attained BSc Hons 2: Graduate Royal Society Chemistry Part II Graduate Royal Society Chemistry Part I Higher National Certificate in Chemistry Society Memberships 1. Society for Radiological Protection 2. International Isotope Society 3. Association of University Radiation Protection Officers 18

19 CHAPTER 1 INTRODUCTION AND THESIS CONTENT 19

20 CHAPTER 1 INTRODUCTION AND THESIS CONTENT 1.0 Introduction A radiochemical study relies on three components, the availability of radioisotopes, their incorporation into a molecule without affecting the molecule s chemical, physical or biological properties, and the development of an in vivo or in vitro experiment, in which the radiolabelled molecule can be used to examine a hypothesis. When all three components are available, radiochemical experiments can be carried out, permitting questions to be answered, even to those, that may have not yet been posed. As a consequence, radioisotopes have frequently been used to assist with the development of many areas of scientific interest including polymer chemistry. The value of using radioisotopes lies in the high sensitivity and specificity that can be achieved in studies under virtually any experimental condition. By radiolabelling at specific positions within the polymer using radiolabelled initiators or monomers, the presence of the radioactivity allows a clear signal to be measured without interference from other chemical species or the system s physical properties. This allows, for example, aspects of the polymerization mechanisms to be explored, quantification of products and by-products, and the chemical, physical behaviour of the monomers, initiators or polymers to be determined. Application to polymer chemistry enables the dilution and quantification of molecules or groups present from the pico to the micro mole scale without the need for lengthy purification procedures that may alter the analyte. By ensuring that the radiochemical and chemical purities are >95% the accuracy of the radiochemical measurements can be as good as +/- 0.5%. However in recent years, with advances in new techniques, increasing use of fluorescent labels, increases in levels of legislation and spiralling costs, the use of radiotracers to investigate mechanistic polymer chemistry has reduced considerably, most notably when investigating the mechanisms of controlled living polymerizations, such as Atom Transfer Free Radical (ATRP) Polymerization. This PhD aims to rectify this shortfall by using 14 C initiators to investigate the mechanistic aspects of ATRP, and purification techniques to remove the copper catalyst. Additionally, by using radiolabelled and radio/fluorescent labelled polymers, the effects of the fluorescent label on the physical deposition of the polymers were also investigated. 20

21 1.1 Aims and Objectives The aims and objectives of research are four fold 1. Review the historical and current applications of the radioisotope 14 C in polymer chemistry. 2. Investigate the mechanism of Atom Transfer Free Radical Polymerization (ATRP) and determine the fate of its initiators during the ambient alcoholic ATRP polymerization of 2-hydroxypropyl methacrylate (2-HPMA) using 14 C labelled initiators. 3. Determine and compare the efficiencies of the purification of the final 14 C labelled poly(2-hpma) polymers using flash column chromatography or precipitation. 4. Using samples of 14 C labelled poly(diethyl amino ethyl methacrylate) (DEAEMA) with a DPn50 to investigate whether the presence of the fluorescent label 9-hydroxyfluorene affects the physical deposition of low molecular weight poly(deaema). 21

22 1.2 Thesis Layout Chapter one gives details of the structure of the thesis and the contents of the later chapters. Chapter two gives an overview of Aton Transfer Radical Polymerization (ATRP) and chapter 3 gives an overview of the application of fluorescent spectroscopy to polymer chemistry. Chapter four reviews the development and application of 14 C to polymer chemistry. These developments originate in the pioneering use of radioisotopes by John Cuthbert Bevington and George Ayrey, in the 1950s, to investigate the mechanisms of free radical polymerizations, using the 14 C labelled initiators, azo-iso-butyronitrile (AIBN) and dibenzoyl peroxide, to prepare homo polymers and copolymers using non-labelled monomers and their subsequent use to investigate the methods of purification, physical/chemical properties of polymers and the application of radiolabelled polymers over the last years. Chapters five, six and seven comprise papers published in Chemical Communications (1) and Polymer Chemistry. (2,3) These papers detail experimental work carried out using 14 C labelled initiators, to investigate the role of the initiators in the mechanism of ATRP, and subsequent polymer purification to remove residual initiator. ATRP (4, 5, 6) is a controlled polymerisation and its use allows the polymer chemist to control the polymer architecture and chemistry, including chain length, dispersity, and end group chemistry. For the end-user, accurate knowledge of the polymer architecture and chemistry is important for structure-property relationships and the final application of the polymer. Since the earliest reports by Matyjaszewski (7) and Sawamoto, (8) ATRP has grown to become a leading controlled radical polymerisation technique in many academic and industrial research groups, with reports of successful synthesis of branched, (9,10,11) block, (12,13) and star polymers, (14,15) polymerisation in emulsion conditions, (16,17) polymerisation using ionic liquids, (18,19) hydrophobic solvents, (20,21) and aqueous environments, (22,23) heterogeneous polymerisation from surfaces, (24,25,26) and modification of natural polymers, (27) using a range of monomers. Arme s successful development (28) of aqueous and alcoholic ATRP has significantly broadened the scope of the technique, allowing water soluble polymers to be controllably synthesised at ambient temperatures under protic solvent conditions. ATRP is a multi-component system consisting of a monomer, an initiator which is either an alkyl or aryl halide, a 22

23 transition metal (I) halide, usually Cu(I)Br/Cl, and a nitrogen-based, bidentate complexing ligand such as 2,2 bipyridyl. The stoichiometry of the initiator: transition metal halide: ligand is 1:1:2, based on the number of moles of monomer and degree of polymerization of the final polymer. ATRP has been used to prepare well defined polymers with predetermined molecular weights, narrow dispersities (<1.5) and precise architectures. This thesis describes the first radiolabelled ambient methanolic-atrp polymerization of 2-hydroxypropyl methacrylate, (2-HPMA) using three variants of the same initiator benzyl 2-bromo-iso-butyrate, two with a 14 C -label at a different position formed using 14 C - 2-bromo-iso-butyric acid and 14 C benzyl alcohol, and a nonlabelled version, used to establish there were no adverse effects due to the 14 C radiolabel s presence and position. Selectively labelling the end groups of poly(2- HPMA) using a 14 C initiator enables all chains to have a single 14 C radioactive site with equal levels of radioactivity, avoiding statistical label incorporation along the polymer chain. It also allows the fate of the ATRP initiator during the polymerisation to be determined. 14 C poly(2- HPMA) with targeted chain lengths of 10, 25 and 50 monomer units were synthesised. By monitoring the presence of radioactivity during the preparation and analysis of the final 14 C polymers, new observations relating to the fate of initiating groups during the polymerization where made. Additionally the efficiency of the purification of the polymers by either flash chromatography or precipitation has been determined by monitoring polymer and initiator residues during purification and analysis. In practice using 14 C labelled initiators for ATRP is the only method to determine directly the fate of the initiator under real conditions. Radio thin layer chromatography (Radio TLC) and liquid scintillation counting of fractions collected from conventional gel permeation chromatography (GPC) have been used to study the fate of the 14 C labelled initiators. Comparison of GPC and Radio TLC showed a significant under-utilisation of the initiator with approximately 16% clearly observable at high monomer conversion (>97%). New chains appeared to be initiated at monomer conversions >90% and as late as 300 minutes after polymerisation had commenced. Purification by repeated precipitation was far more efficient compared to flash chromatography, using alumina or silica, in removing residual unreacted or terminated initiator, although increased fractionation could be seen with each repeat. 23

24 Chapter six comprises a paper published in Polymer Chemistry, (3) detailing the investigation into the effects of the fluorescent tag 9-hydroxyfluorene on the deposition of poly(deaema) on to real substrates. Three 14 C radiolabelled ATRP initiators were synthesized having groups of increasing hydrophobicity i.e. methyl benzyl and fluorophore 9-hydroxyfluorene. They were used to investigate the possible effects of the presence of the fluorophore 9-hydroxyfluorene on the deposition of poly(deaema) from buffered (ph=2) aqueous solutions on to negatively charged hydrophilic cellulose-based filter, and poly(ethylene) coated hydrophobic photo papers and negatively charged, human, undamaged root hair (keratin) which are representative of real test surfaces. The three substrates were chosen to establish variability of adsorption under identical controlled conditions using commercially relevant and non-ideal surface chemistry. The polymers were prepared using aqueous ATRP with a degree of polymerization (DP) of units. The 14 C radiolabelled initiators used were: methyl ( 14 CH3) bromo iso-butyrate (A Log P = 1.494), to give a polymer with a relatively non hydrophobic end group, 14 C benzyl ( 14 CH2) bromo iso-butyrate, (A Log P = 3.077) and 14 C 9-hydroxyfluorene bromo ( 14 CH3) iso-butyrate (A Log P= 4.378) which gave polymers with increasing hydrophobic end groups. Using a 14 C will only increase the molecular weight by two atomic mass units relative to the unlabelled material, and is therefore very unlikely to affect the overall hydrophobicity of the polymer. Table 1 provides details of end group molecular weights and their percentage relative to the molecular weight of monomer DEAEMA. End Group Molecular Weight of End Molecular Weight of Diethyl % of DP50 Number Average Molecular Weight Group Amino Ethyl Methacrylate Methyl % Benzyl % 9-Hydroxyfluorene % Table 1 Percentage Fraction of the Molecular Weights of Initiators Relative to the Molecular Weight of Poly diethyl amino ethyl methacrylate (DEAEMA) 24

25 DEAEMA has a molecular weight of Deprotonated 9-hydroxyfluorene has a molecular weight of , approximately the weight of a single DEAEMA unit and 1.94% of the molecular weight of DPn50 polymer. To model a conventional polymerization, the three radiolabeled polymers were purified by flash column chromatography to remove catalyst and ligand residues, dried to remove all traces of reaction solvent, and subsequently used to prepare a 0.14% w/w solution at ph=2. Several samples of the three test substrates were suspended in solutions of the radiolabelled polymers for up to 48 hours. On removal, all samples were allowed to drain back into the solutions before air drying for 24 hours prior to analysis. Two radio analytical techniques were employed to analyse each sample. Non-destructive, storage phosphor imaging enabled the visualisation and quantification of the surface radioactivity associated with the sample. Destructive sample oxidation enabled the total radioactivity levels associated with the sample after exposure to be determined. Both radio analytical methods were used in combination with liquid scintillation counting. The resulted showed that the polymers deposition increased as the end group hydrophobicity increased. The deposition was highest for a 9-hydroxyfluorene end group followed by the benzyl and finally the methyl end groups. Indicating that the hydrophobicity of the end group affects the physical and chemical properties of the resulting polymer. Additionally for each polymer the deposition level differed for each test surface. The deposition of poly(deaema) with 9-hydroxyfluorene and benzyl end groups increased as the hydrophobicity of the surface increased from filter paper to hair to photographic paper. This suggested that poly(deaema) with hydrophobic end groups preferentially deposit on to hydrophobic surfaces, due to their lower solubility in ph 2 buffer and the surface which repelled water the most. Poly(DEAEMA) with the relatively non hydrophobic methyl end group preferentially deposits on to hydrophilic wet surfaces. 25

26 1. M Long, D.W.Thornthwaite, S.H. Rogers, G Bonzi, F.R. Livens and S.P. Rannard, Chem. Comm., 2009, M. Long, D. W. Thornthwaite, S. H. Rogers, F. R. Livens and S. P. Rannard, Polym. Chem., 2011, 2, M. Long, D. W. Thornthwaite, S. H. Rogers, F. R. Livens and S. P. Rannard, Polym. Chem., 2012, 3, T. Pintauer and K. Matyjaszewski, Chem. Soc. Rev., 2008, 37, S. Wang and K. Matyjaszewski, J. Am. Chem. Soc., 1995, 117, M. Kato, M. Kamigaito, M. Sawamoto and T. Higashimura, Macromolecules, 1995, 28, S. Wang and K. Matyjaszewski, J. Am. Chem. Soc., 1995, 117, M. Kato, M. Kamigaito, M. Sawamoto and T. Higashimura, Macromolecules, 1995, 28, T. He, D. J. Adams, M. F. Butler, C. T. Yeoh, A. I. Cooper and S. P. Rannard, Angew. Chem., Int. Ed., 2007, 46, T. He, D. J. Adams, M. F. Butler, A. I. Cooper and S. P. Rannard, J. Am.Chem. Soc., 2009, 131, I. Bannister, N. C. Billingham, S. P. Armes, S. P. Rannard and P. Findlay, Macromolecules, 2006, 39, J. V. M. Weaver, Y. Tang, S. Liu, P. D. Iddon, R. Grigg, N. C. Billingham, S. P. Armes, R. Hunter and S. P. Rannard, Angew. Chem,, Int. Ed., 2004, 43, E. S. Read, K. L. Thompson and S. P. Armes, Polym. Chem., 2010, 1, P. T. Hammond, Macromolecules, 2009, 42, H. Gao and K. Matyjaszewski, Macromolecules, 2006, 39, K. Min, H. Gao, J. A. Yoon, W. Wu, T. Kowalewski and K. Matyjaszewski, Macromolecules, 2009, 42, G. Chambard, P. De Man and B. Klumperman, Macromol. Symp, 2000, 150, M. Xie, Y. Kong, H. Han, J. Shi, L. Ding, C. Song and Y. Zhang, React. Funct. Polym, 2008, 68, 1601, 19. T. Biedron and P. Kubisa, J. Polym. Sci., Part A: Polym. Chem., 2002, 40,

27 20. X.-S. Wang, S. F. Lascelles, R. A. Jackson and S. P. Armes, Chem. Comm., 1999, S. McDonald and S. P. Rannard, Macromolecules, 2001, 34, C. Perruchot, M. A. Khan, A. Kamitsi, S. P. Armes, T. Von Werne and T. E. Patten, Langmuir, 2001, 17, C.-D. Vo, A. Schmid, S. P. Armes, K. Sakai and S. Biggs, Langmuir, 2007, 23, J. Lindqvist and E. Malmstrom, J. Appl. Polym. Sci., 2006, 100, D. Bontempo, G. Masci, P. De Leonardis, L. Mannina, D. Capitani and V. Crescenzi, Biomacromolecules, 2006, 7, S. P. Rannard, S.H. Rogers and R. Hunter, Chem. Comm., 2007, X.-S. Wang, S. F. Lascelles, R. A. Jackson and S. P. Armes, Chem. Comm., 1999, E.J. Ashford,V. Naldi, R. O Dell, N.C. Billingham, S.P. Armes, Polymer Prep., 1999, 40,

28 1.3 Status of Manuscripts CHAPTER 5 Utilising 14 C-radiolabelled Atom Transfer Radical Polymerization Initiators Status: Published Journal: Chemical Communications Reference Mark Long, David W. Thornthwaite, Suzanne H. Rogers, Gwénaëlle Bonzi, Francis R. Livens and Steve P. Rannard, Chem. Comm., 2009, CHAPTER 6 Monitoring Atom Transfer Radical Polymerization using 14 C Radiolabelled Initiators Status: Published Journal: Polymer Chemistry Reference Mark Long, Suzanne H. Rogers, David W. Thornthwaite, Francis R. Livens and Steve P. Rannard, Polym. Chem., 2011, 2, CHAPTER 7 Controlled synthesis of radiolabelled amine methacrylate water-soluble polymers with varying end-groups and studies of adsorption behaviour Status: Published Journal: Polymer Chemistry Reference Mark Long, Suzanne H. Rogers, David W. Thornthwaite, Francis R. Livens and Steve P. Rannard, Polym. Chem., 2012, 3,

29 Authors and Contributions: Dr Ben Carter Assistance with critical micelle concentration measurements using Kibron surface tensiometer Mr Brian Harrison Assistance with Gel Permeation Chromatography polymer molecular weight determinations Professor Francis Livens Supervision throughout and review of each manuscript Mr Ian Osbourne Assistance with Gel Permeation Chromatography polymer molecular weight determinations Professor Steve Rannard Supervision throughout and review of each manuscript Dr Matthew Reid Assistance with Nuclear Magnetic Resonance Spectroscopy Dr Suzanne Rogers Assistance Atom Transfer Radical Polymerizations Techniques Professor Dave Thornthwaite Supervision throughout and review of each manuscript 29

30 CHAPTER 2 FREE RADICAL AND LIVING POLYMERIZATION: ATOM TRANSFER RADICAL POLYMERIZATION 30

31 CHAPTER 2 FREE RADICAL AND LIVING POLYMERIZATION: ATOM TRANSFER RADICAL POLYMERIZATION 2.0 Free Radical and Living Polymerisation Free radical polymerisation (FRP) is one of the most common and practical techniques for producing polymers on an industrial scale. It exhibits a great tolerance to material and operational impurities, and a compatibility with a wide range of vinyl monomers with different functional groups, making it a robust and cost effective process that accounts for a significant portion of polymer production. Mechanistically, FRP can be described as four independent steps: initiation, propagation, chain transfer and termination (combination or disproportionation) as shown in Scheme 1; however, in practice these may occur simultaneously and continually during any polymerisation. Dissociation of Initiator (I) to give two Free Radicals (R. ) I 2 R. Initiation of Monomer Unit (M), to give Growing Polymer Chain (P1. ) R. + M P1. Propagation of Growing Polymer Chain (Pn. ) Pn. + M Pn+1. Chain Transfer to Monomer Unit (M) Pn. + M Pn + M. Termination: Combination of chains (Pn. and Pm. ) gives a terminated polymer chain Pn+m Pn. + Pm. Pn+m Termination: Disproportionation of chains (Pn. and Pm. ) gives two terminated polymer chains Pn and Pm Pn. + Pm. Pn + Pm Scheme 1 Free Radical Polymerisation 31

32 The major limitations of FRP are that the polymers produced usually have a broad molecular weight distribution and there is poor control of polymer architecture. These limitations are due in the main to the termination processes (combination, disproportionation and chain transfer) that can occur from the onset of the polymerisation. However, termination mechanisms do help to control the exotherm during the polymerisation by maintaining a lower concentration of radicals than would be present if termination were eliminated. Termination reflects the high reactivity of transient radical species, leading to polymer chains having short lifetimes during the propagation steps. This makes it difficult to prepare more complex and defined structures which are often required for advanced applications. Chain-growth polymerisations may also be performed in such a way that, on average, the propagating chains grow under identical conditions and are consequently quite monodisperse. These reactions are called living polymerisations, a term first used during by M Szwarc (1) during his work using anionic polymerizations of styrene in A polymerisation is termed living when side reactions such as termination or chain transfer are absent, at least for the timescale of the polymerisation, and all polymer chains are initiated simultaneously. This results in growing polymer chains whose ends are permanently active. A living polymerisation should fulfil the following criteria: First order kinetics with respect to the monomer. Fast initiation compared to propagation to ensure that all chains grow simultaneously and retain functionality. Propagation rates are independent of degree of polymerisation Single propagating species to prevent the inter-conversion of propagating radicals. On consumption of a first monomer the polymerisation can continue by the addition of more of the same monomer or the addition of a second different monomer. 32

33 The number of simultaneously growing chains will be equal to the final number of polymer molecules, giving a dispersity equal to one. The degree of polymerisation is directly proportional to the ratio of monomer concentration to initiator concentration. The number average molecular weight (Mn) of the polymer should have a linear relation with conversion. Living polymerisations are interesting from both a scientific and an industrial viewpoint. The discovery of these techniques has opened up a range of possibilities regarding design of novel polymeric architectures and compositions. Szwarc stated, no reaction can be truly living, however, some newly reported polymerisations systems, termed controlled free radical polymerisations, are not far from achieving this goal. 2.1 Controlled Free Radical Polymerisation A continuing problem in controlling FRP is that the active chain ends can terminate or produce unwanted side reactions. There is a commercial drive to develop methods for eliminating termination or side reactions and to ensure that only a fraction of the growing polymer chains are activated at any given time, to produce monodisperse polymers. This polymerization is called Controlled Free Radical Polymerisation (CRP). The International Union of Pure and Applied Chemistry (IUPAC) has stated that, although the term controlled free radical polymerisation is acceptable, due to the proliferation of different techniques a single term, reversible-deactivation radical polymerisation, is required which best describes the general polymerisation strategy. (2) Within this thesis, the term controlled radical polymerisation will be used. In the last twenty years, several methods of CRP have been developed that allow for much greater control over polymer architecture. One of the most studied methods of CRP is atom transfer radical polymerisation and this is the technique used in the research described within this thesis. 33

34 2.2 Atom Transfer Radical Polymerisation (ATRP) The pioneering work on ATRP was conducted independently by two groups in Kato, Kamigaito, Sawamoto, and Higashimura (3) demonstrated the polymerisation of methyl methacrylate using a ruthenium-based complex. Simultaneously, Wang and Matyjaszewski (4) showed the polymerisation of styrene using a copper based mediator. The term Atom Transfer Radical Polymerisation was coined by Matyjaszewski and coworkers, and since 1995 a vast amount of papers have been published on ATRP in bulk and solution Atom Transfer Radical Addition The concept of using transition metal complexes to mediate radical polymerisations was a modification of Atom Transfer Radical Addition (ATRA) (5) which itself was a modification of the Kharasch addition, (6,7,8) which originally used light to generate a radical. In ATRA, transition metal complexes were used to promote 1:1 halogen addition to alkenes through a redox process, as shown in Scheme 2. Scheme 2 Atom Transfer Radical Addition 34

35 In ATRA, a copper (I) complex undergoes a one-electron oxidation with abstraction of a halogen atom from the halogenated organic compound. This reaction generates an organic radical and a copper (II) complex. Substituents on the organic halide can facilitate the reaction by stabilizing the resulting radical. The organic radical can then add to an unsaturated group (e.g. alkene) via, an inter or intramolecular mechanism. The new radical re-abstracts the halogen from the copper (II) complex to form the original copper (I) complex and generate the product. For efficient ATRA, trapping of the product radical should be faster than the subsequent reaction step and adduct reactivation should be very slow, maximizing the product yield. Compounds resulting from the termination processes comprise very little of the product, because the copper (II) complex acts as a persistent radical (9) which controls the concentration of the intermediate radicals. Substrates for this reaction are chosen to ensure that once an addition occurs, the newly formed radical is far less stabilized than the initial radical and will essentially react irreversibly with the copper (II) complex to form the inactive alkyl halide. The rate of deactivation (kdact) >> rate of activation (kact). In ATRA, typically only one addition step occurs; however, if the starting and product alkyl halides, have similar reactivities toward atom transfer, it may be possible to repeat the catalytic cycle and add multiple unsaturated groups, as in a polymerisation. Because of the involvment of transition metals in the activation and deactivation steps, chemo-, regio-, and stereoselectivities in ATRA and Kharasch additions may be different Atom Transfer Radical Polymerisation (ATRP) The fundamental assumption in ATRP is that all initiator is consumed immediately, leading to the instantaneous growth of the maximum number of polymer chains, of equal length, at the start of the polymerization where bimolecular termination is supressed by maintaining a low radical concentration throughout the polymerisation. (10,11) ATRP achieves a low radical concentration by having a reversible dynamic equilibrium between active and dormant states, where the equilibrium must favour a dormant state (halogen capped R-X). The formation of dormant chains should be fast. The equilibrium, which is rapidly attained, is dependent on the atom transfer of a halogen 35

36 atom between the growing polymer chains and the transition metal catalyst. The transfer of the halogen atom is controlled by the reversible redox properties of a transition metal catalyst. ATRP uses a copper catalyst as shown in scheme 3. The radicals are generated via a reversible redox process of a halogenated transition metal complex Cu(I)X/L, comprising of Copper (I) cation, a halogen (X) plus a multidentate ligand (L). This undergoes a one-electron oxidation via an inner sphere electron transfer and an abstraction of a halogen X from the dormant species, (inititator or polymer chain) via a homolytic cleavage, forming the Cu(II)X2/L complex. The initiator or polymer chain is reduced giving a transient organic free radical which can then undergo propagation with a monomer M. Scheme 3 Mechanism of Atom Transfer Free Radical Polymerisation During the onset of the polymerization during the non stationary stage, when the equilibrium is being established, <5% of the transient organic radicals terminate. This termination results in the formation of a persistent radical effect (PRE) as first defined by Fischer. (9) The ATRP persistent radical X., which cannot terminate with itself, forms a irreversible deactivator Cu(II)X2/L complex, As the concentration of the deactivator increases, the equilibrium is pushed to the dormant state, reducing the concentration of the growing radicals, and rate of the initiation whilst minimizing further termination, resulting in a dynamic equilibrium between the propagating radicals and dormant species. Once attained the dynamic equilibrium ensures a steady state of growing radicals is established through the activation-deactivation process. 36

37 2.3 Kinetics of the ATRP Equilibrium ATRP has rate constants of activation, kact, and deactivation kdeact where kdeact>>kact kinetically favouring deactivation and a dormant state which results in a low concentration of active radical species during the attainment of the equilibrium. The equilibrium constant Keq is equal to: Keq = kact kdeact Eq1 Provided the dynamic equilibrium and quantitative initiation are fast and a good rate of polymerisation is maintained, targeted molecular weights and narrow dispersities can be obtained. In the absence of any side reactions other than termination, the magnitude of the equilibrium constant (Keq) determines the success of the ATRP. An equilibrium constant with a high value, there will be little or no deactivator present, resulting in the loss of control of the polymerization, for a low value, there will be a high concentration of deactivator resulting in a slow or no polymerisation. This shows that the concentration of propagating radicals and rate of deactivation can be adjusted to maintain control of the polymerisation. As ATRP is a catalytic process, the overall position of the equilibrium not only depends on the active and dormant species, but also on the reactivity of the added transition-metal catalyst. ATRP polymerization rate can be approximated to: Rp=kp(M)(P. ) Eq 2 Where kp is the propagation rate constant and (M), (P ), are the concentrations of monomer, and propagating chains respectively and it is assumed there is negligible bimolecular termination. In practice a low level of termination does occur with a rate constant kt. Increasing the temperature of ATRP increases the rate of propagation leading to a greater activation energy for termination. This leads to greater kp/kt ratios and equilibrium constant resulting in a greater control of the polymerisation. As chain transfer and other side reactions become more pronounced at higher temperatures, the optimal temperature is dependent on the monomer, catalyst used, and the targeted molecular weight. In the literature ambient temperature ATRP is often used, especially in the case of reactions containing water as a solvent or co-solvent. 37

38 For ATRP, conversion varies linearly with time while the concentration of the active species is constant. This leads to first order kinetics with respect to the monomer as shown in Fig1. Conversion ln ((M)o/(M)t) Time Fig 1 ATRP First Order Kinetics ATRP methodology has been developed to ensure the polymerisation follows first order kinetics, and the molecular weights increase linearly with conversion resulting in dispersities <1.5 and the number average degree of polymerisation (DPn) determined by the ratio of monomer to initiator. At high monomer conversions, the rate of propagation slows down, but the rates of side reactions including termination do not, resulting in an increase in deactivator concentration and deviation from linearity. Complete monomer conversion can induce loss of end groups so, for polymers with high end-group functionality, it is best to avoid conversions exceeding 95% although in many cases complete conversion have been reported. The number average degree of polymerisation (DPn) is defined by DPn = Mn Eq3 where Mn is the number moleculat weight, and Mo is the monomer molecular weight. DPn is independent of the concentration of the transition metal. The loss of initiators or propagating chains in these early stages does lead to a loss of direct targeting of Mn and is variable across different monomers, solvent conditions, temperatures and structure of initiators. The initiator efficiency, the number of initiators molecules that form polymer chains, is a factor that often requires optimisation and is studied during this thesis. Mo 38

39 2.4 Components of ATRP ATRP has been developed to use a halogenated organic initiator with an appropriately designed catalyst comprising a transition metal and an organic ligand. Traditionally, the ATRP equilibrium is controlled by the catalyst complex which is used in a stoichiometric ratio to the initiating alkyl halide species. The optimum ratio of ligand to metal halide to initiator has been reported as 2:1:1 for copper based systems but other ratios have been used. Below this ratio the polymerisation rate is usually slower, and above this ratio, the polymerisation rate remains constant. It should be noted that the optimum ratio could vary with regard to changes in the monomer, counter ion, ligand, and temperature. The monomer to initiator ratio provides the targeted molecular weight Metal Catalyst The most important component of ATRP is the catalyst, a transition metal halide. It s the key to ATRP since it determines the dynamics of exchange between the dormant and active species which in turn defines the ATRP equilibrium. The catalyst is used to remove terminal halogens to generate propagating radicals, which are quick to react to form a growing chain which itself is then reversibly capped with the halogen. The catalyst is susceptible to oxidation during the polymerisation but this can be restricted by operating under inert (nitrogen) conditions. An efficient ATRP transition metal catalyst, such as Cu, Rh, Ni, Pd and Fe, must have an affinity toward a halogen, and a metal centre which has at least two readily accessible oxidation states separated by one electron which results in an expandable coordination sphere, which permits the oxidized transition metal, to accommodate a (pseudo)-halogen. It is important that the halogen can migrate rapidly between the growing chain and the catalyst, and the bonds formed with both species can be broken homolytically, as such the halogen is usually bromine or chlorine. Fluorine is normally too electronegative forming a strong C-F bond preventing a homolytic cleavage although a secondary fluorine has been used for ATRP, while iodine has also been utilized in some cases. (12) The metal should complex the ligand relatively strongly and most catalysts used consist of copper bound to nitrogen donor ligands, due to the low cost of copper and its versatility compared to other transition metals. 39

40 2.4.2 Ligands The primary function of the ligand is to ensure solubilisation of the metal catalyst. The reactivity of the metal catalyst is therefore influenced by the steric and electronic properties of the ligand. Bulky side groups on the ligand can sterically hinder bond formation with the halogen, while electron withdrawing groups can prevent homolytic cleavage of the halogen-metal bond. The first ligand used by Matyjaszewski and Wang (4) was 2, 2 -bipyridyl (bipy), a nitrogen bidentate ligand. This was quickly followed by other multidentate nitrogen ligands with more complex structures which in some cases have proved to be superior in controlling radical formation. While amino-based ligands are used for copper-based ATRP, phosphorus-based ligands are used for most other transition catalysts, used in ATRP Initiator In ATRP, alkyl halides (RX) are typically used as the initiator. R is typically an alkyl group which forms the free radical which can be stabilized by adjacent chemical groups, X is usually bromine or chlorine, which can rapidly and selectively migrate between the growing chain and the transition-metal catalyst. Iodine works well for acrylate polymerisations in copper-mediated ATRP and has been found to lead to controlled polymerisation of styrene in ruthenium based ATRP. The main role of the initiator is to produce a known number of growing polymer chains. If initiation is fast and termination is negligible, then the number of chains capable of propagation is ideally constant and equal to the initiator concentration. The monomer to initiator ratio provides the targeted number average degree of polymerisation (DPn) (number of monomer units per chain) or number average molecular weight (Mn). The theoretical Mn (and therefore DPn) will decrease with an increase in the initial concentration of initiator. The initiator is normally chosen so that the structure mimics the structure of the monomer, with the aim of making the rate of initiation and propagation equivalent; for example a benzyl halide could be used with styrene. Many different types of halogenated compounds including alkanes, aromatic esters, ketones, nitriles and sulfonyl halides can be used as initiators. Specially designed initiators can be used to synthesize polymers with advanced architectures such as macro-initiators for copolymer synthesis and multifunctional initiators for star polymer formation. 40

41 2.4.4 Monomer A variety of monomers such as styrenes, acrylonitrile,(13,14,15) (meth)acrylates, (16,17,18,19) and (meth)acrylamides, (20,21) have been successfully polymerised using ATRP. Under the same conditions using the same catalyst, each individual monomer has its own unique rate of activation (kact) and deactivation (kdeact), and therefore equilibrium constant (Keq=kact/kdeact) leading to a specific rate of radical propagation kp. For a combination of monomers the equilibrium constant Keq and radical propagation rate kp, determine the polymerisation rate. However a number of monomer functional groups are poorly tolerated in ATRP reactions, including carboxylic acids and some ionic groups, which may react with the catalyst. Carboxylic acid groups can be introduced by polymerisation of their salts. This was first reported by Armes, Billingham, O Dell and Ashford (22) who polymerised sodium methacrylate in aqueous media at ph 9 using a poly(ethylene oxide) macroinitiator. Other monomers such as vinyl acetate and halogenated alkenes cannot be polymerised by ATRP due to insufficient stabilization of the radical formed Solvents ATRP can be carried out in solution, bulk or in a heterogeneous system (emulsion, or suspension). A solvent is often required to ensure dissolution of the catalyst/ligand complex and reduce viscosity at high conversions, particularly necessary when the polymeric product is insoluble in its monomer. Traditionally solvents such as toluene, acetone, diphenylether, N,N-dimethyl formamide and various alcohols can be used for ATRP. The use of the correct solvent is important to avoid solvent-based chain transfer reactions, and prevent poisoning or restructuring of the catalyst. Solvents reduce propagation rates, but polar solvents can increase and in fact are used to accelerate the polymerisation rate, in some cases providing better control over the product Additives Sometimes additives are required for a successful ATRP. Deactivator Cu(II)X2 can be added at the start of the polymerisation to speed up the development of the dynamic equilibrium reducing the initial termination rate. The Lewis acid, aluminium alkoxide can activate a polymerisation by stabilizing the catalyst in the higher oxidation state. This is needed for the controlled polymerisation of MMA catalyzed by RuCl2- (PPh3)3. (17) 41

42 2.5 Aqueous and Protic ATRP Initial ATRP reactions involved the use of organic solvents, which were predominantly aprotic. In 1997 Nishikawa, Ando, Kamigaito, and Sawamoto (17) first reported the effects of the use of water and methanol used in living radical polymerizations. They concluded that the presence of water (ten equivalents to the initiator) and methanol (at high concentrations) had little or no effect on the molecular weight dispersity or kinetics of the polymerisation of methyl methacrylate in the presence of a ruthenium complex. In 1998 the first successful aqueous ATRP of 2-hydroxyethyl acrylate was carried out by Coca, Jasieczek, Beers, and Matyjaszewski. (23) This was quickly followed by the publication by Armes, Billingham, O Dell and Ashford (22) who polymerised sodium methacrylate. Since then the Armes research group has published many papers relating to aqueous ATRP which investigated the use of different catalysts, initiators and monomers polymerised at differing temperatures. As the rate of polymerisation is fast in water it leads to a lack of control, broad dispersity and low initiator efficiency. To gain control over the polymerisation, the aqueous systems were modified by the addition of a water miscible solvent such as methanol or DMF leading to greater control over the polymerisations. Initially these studies involved the polymerisation of hydrophilic monomers. Polymerisations of hydrophobic polymers were usually carried out using emulsion or dispersion polymerisations. This was the case until Robinson, Khan, D Paz Banez Wang and Armes (16) reported the homogenous polymerisation of 2-hydroxyethyl methacrylate in an alcohol/water system and then McDonald and Rannard (24) reported the homogenous polymerisation of n-butyl methacrylate using a water/isopropanol mixture. Replacing organic solvents with either water or an alcohol/water system helps to dissipate the heat of polymerisation and produce relatively high molecular weight polymers, allowing ATRP to be used as an environmentally friendly industrial process Homogenous aqueous ATRP For homogenous aqueous ATRP (25) the monomer and the polymer produced are soluble in water or a mixture of water and organic solvent. ATRP proceeds as if it were in a single organic solvent but the rate of polymerisation can be increased by a factor of as the volume of water used increases, especially if the monomer has ionizable 42

43 pendant groups. The increase in the rate of polymerisation is due to a number of factors: 1. Solvation of the monomer molecules via hydrogen bonding leads to a shift in electron density which may in turn, increase the reactivity of the monomer s double bond. 2. Monomers with ionic pendant groups dissociate, resulting in a greater electrostatic repulsion between two growing radicals, reducing the possibility of termination by protecting the propagating radical centre via a strongly bound hydration shell formed between the water and growing polymer. 3. Deactivators dehalogenate in protic media which, in turn, affects the equilibrium, reducing the deactivation of the radicals and leading to a reduction in the effective dormant state. This effect can be reduced by the addition of extra Cu (II) halide salts. Additionally, homogenous aqueous ATRP is highly sensitive to changes in ph and ionic strength, and the number of water soluble monomers which can be used in homogenous polymerizations is limited Heterogeneous ATRP To use aqueos ATRP to polymerise, water insoluble monomers requires the use of a specialized aqueous heterogeneous ATRP (25) method which will allow a wide range of monomers to be polymerized on an industrial scale. A number of different methods can be used including suspension, emulsion, mini- and micro-emulsion and dispersion polymerisation techniques. The methods differ in the initial state of their polymerisation mixtures, mechanism of particle formation and the size of the product particles. 2.6 Limitations of ATRP The main limitation of ATRP, industrial or otherwise, is the removal of the catalyst which can be present in the order of 10 4 ppm. Catalysts have low activities in ATRP, therefore an ATRP requires high levels of catalyst (catalyst ratio is 1:1, one catalyst molecule mediates one polymer chain) to drive the polymerization. As the transition 43

44 metal is toxic and can induce polymer ageing or discolouration the catalyst needs to be removed from the final polymer but purification of the final polymers is both difficult and costly. Methods of purification include immobilization of the catalyst during the polymerisation by attaching it to solid supports (this can lead to the loss of control of the polymerization: reducing mobility of the catalyst), passing polymers down ion exchange, silica, or alumina columns, precipitation using an appropriate mixture of solvents, or removal using water-solvent extraction with a complexing metal agent. However post-process purification has a draw back as it can fractionate the final polymer, add significantly to production costs, and is a considerable hurdle in commercial ATRP. Additionally these methods do not take into account the possibility that free initiator may still be present: little consideration is given to this as ATRP theory dictates that all the initiator is activated simultaneously so the chain grows evenly to give a dispersity approaching one. 2.7 ATRP Polymer Architecture ATRP has been used to gain unprecedented control over polymer architecture using many commercially available monomers (26) enabling the composition, topology, dispersity and polymer functionality to be controlled. The types of polymers that can be prepared are homo- or co-, statistical, gradient, periodic, segmented block or graft polymers with architectures that are linear, network, gel, comb, brush, branched (hyperbranched), star, cyclic and dendritic. Architectures can vary from dimers to polymers with low to very high molecular weights. ATRP is tolerant of a number of chemical groups, present on monomers, initiators or macro initiators, such as hydroxy, cyano, amino, amido, esters and carboxylic salts. Therefore ATRP can be used to prepare polymers with a range of different functional groups, and substitution of terminal halogens. ATRP can control the functional groups, position, which can dictate the chemical and physical properties of the polymers, and can facilitate preparation of polymers which are cross-linked, have chain extensions are a part of a supramolecular assembly. 44

45 2.8 Applications of ATRP Polymers An in depth analysis of the applications of polymers prepared using ATRP is beyond the scope of the thesis however the applications of ATRP polymers are many and varied, some include the preparation of sealants, (27) lubricants, (28) thermos plastic elastomers, (29) blend compatiblisers, (30) pigment dispersants, (31) surfactants, (32) polar thermoplastic elastomers, ionic conductors, antibacterial/antifouling agents, (33) nano gels and delivery scaffolds, (34,35,36) coronary stents, (37) and drug delivery agents. (36,38,39) 2.9 Conclusion In summary ATRP critically involves the transfer of the halogen atom which is responsible for the control of the polymerisation, uniform growth of the polymeric chains, virtual removal of termination and chain transfer reactions and increases the possibility of a mono-disperse polymer. The ATRP mechanism relies on the instantaneous initiation of chains at the start of the polymerization to control the polymerization and ensure that the dispersity approaches one. During the initiation, the formation of a low level of terminated product, the deactivator, is required to ensure the ATRP equilibrium favours the deactivated state. In this PhD the initiation mechanism has been investigated studing the fate of initiators used in the ATRP of 2- hydroxypropyl methacrylate (2-HPMA). 45

46 References 1. M. Szwarc, Nature., 1956, 178, A.D. Jenkins, R.G. Jones, G. Moad., Pure Appl. Chem., 2010, 82, M. Kato, M. Kamigaito, M. Sawamoto, T. Higashimura, Macromolecules., 1995, 28, J.S. Wang, K. Matyjaszewski, J. Am. Chem. Soc., 1995, 117, T. Pintauer, K. Matyjaszewski, Chem. Soc. Rev., 2008, 37, M.S. Kharasch, E.V. Jensen, W.H. Urry, Science., 1945, 102, M.S. Kharasch, E.V. Jensen, W.H. Urry, J. Am. Chem. Soc., 1947, 69, M.S. Kharasch, B.M. Kuderna, W.H. Urry, J Org Chem., 1948, 13, H. Fischer, Chem Rev., 2001, 101, K. Matyjaszewski, Macromolecules., 2012, 45, K. Matyjaszewski, Isr. J. Chem., 2012, 52, G. David, C. Boyer, J. Tonnar, B. Ameduri, P. Lacroix-Desmazes, B. Boutevin, Chem. Rev., 2006, 106, M. Al-Harthi, A. Sarashti, J.B.P. Soares, L.C. Simon, Polymer., 2007, 48, K. Matyjaszewski, P.J. Miller, N. Shukla, B. Immaraporn, A. Gelman, B.B. Luokala, T.M. Siclovan, G. Kickelbick, T. Vallant, H. Hoffmann,T. Pakula, Macromolecules., 1999, 32, C. Tang, T. Kowalewski, K. Matyjaszewski, Macromolecules., 2003, 36, K.L. Robinson, M.A. Khan, M.V. de Paz Banez, X.S. Wang, S.P. Armes, Macromolecules., 2001, 34, T. Nishikawa, T. Ando, M. Kamigaito, M Sawamoto, Macromolecules., 1997, 30, N. Chan, M.F. Cuningham, R.A. Hutchinson, Macromol Chem Physic., 2008, 209, D.A. Shipp, J.L. Wang, K. Matyjaszewski, Macromolecules., 1998, 31, D.A.Z. Wever, P. Raffa, F. Picchioni, A.A. Broekhuis, Macromolecules., 2012, 45, M. Teodorescu, K. Matyjaszewski, Macromolecules., 1999, 32, E.J. Ashford,V. Naldi, R. O Dell, N.C. Billingham, S.P. Armes, Polymer Prep., 1999, 40,

47 23. S.C. Coca, C.B. Jasieczek, K.L. Beers, K. Matyjaszewski, J Polym Sci Pol Chem., 1998, 36, S. McDonald, S.P. Rannard, Macromolecules., 2001, 34, J. Qui. B. Charleux, K. Matyjaszewski, Prog. Polym. Sci., 2001, 26, K. Matyjaszewski, N.V. Tsarevsky, J. Am. Chem. Soc., 2014, 136, K. Matyjaszewski, Prog. Polym. Sci., 2005, 30, M. Chen, W.H. Briscoe, S.P. Armes, H.Cohen, J.Klein, Eur. Polym. J., 2011, 47, K. Matyjaszewski, D.A.Shipp, G.P. McMurtry, S.G. Gaynor, T. Pakula, J. Polym. Sci. Part A., 2000, 38, E.G. Koulouri, J.K. Kallitsis, G. Hadziioannou, Macromolecules., 1999, 32, C.Auschra, E. Eckstein, A. Muhlebach, M-O. Zink, F. Rime, Prog. Org. Coat., 2002, 45, W. Jakubowski, K. Matyjaszewski, Macromol. Symp., 2006, 240, T. Matsugi, J. Saito, N. Kawahara, S. Matsuo, H. Kaneko, N.Kashiwa, M. Kobayashi, A.Takahara, Polym. J., 2009, 41, J.K. Oh, R. Drumright, D.J. Siegwart, K. Matyjaszewski, Prog. Polym. Sci., 2008, 33, P.L. Golas, K. Matyjaszewski, Chem. Soc. Rev., 2010, 39, S.A.Bencherif, N.R.Washburn, K. Matyjaszewski, Biomacromolecules., 2009, 10, R.E. Richard, M. Schwarz, S Ranade, A.K. Chan, K. Matyjaszewski, B. Sumerlin, ACS. Symp. Ser., 2006, E.J. Lobb, I. Ma, N.C. Billingham, S.P. Armes, A.L. Lewis, J. Am. Chem. Soc., 2001, 123, D.J. Siegwart, J.K. Oh, K. Matyjaszewski, Prog. Polym. Sci., 2012, 37,

48 CHAPTER 3 APPLICATION OF FLUORESCENCE SPECTROSCOPY TO POLYMERS 48

49 CHAPTER 3 APPLICATION OF FLUORESCENCE SPECTROSCOPY TO POLYMERS 3.0 Fluorescent Chemistry Fluorescence is the phenomenon of photon emission following absorption of photon energy by a molecule or material that returns to its ground state. Fluorescence was first observed by Herschel in 1845 (1) when he was working with quinine sulphate which emitted a vivid blue light. The concept of fluorescence was further studied by Stokes who identified the main characteristics for a single photon excitation and in 1852 (2) used the term fluorescent to describe his findings. In the last twenty years there has been a rise in the use of fluorescent molecules, or fluorophores, used to follow the behaviour of compounds and biological processes. The use of fluorescence has proven to be a highly sensitive technique and in many cases replaces the need to use radiotracer technology. There are, however, questions as to whether the presence of a fluorophore, which tends to be a poly aromatic, heterocyclic or an aromatic dye, affects the physical and chemical properties of the compound or system that is being studied. Here we shall look at the fundamentals of fluorescence chemistry, how it is used in polymer chemistry and the potential draw backs of fluorescent labells. 3.1 Fluorescence Process Fluorescence consists of a three stage energy transfer process which is represented by a Jablonski (3) diagram showing the initial final and intermediate electronic states of a molecule. Fig 1 Jablonski Diagram 49

50 3.1.1 Excitation When a fluorophore absorbs (A) a photon of electromagnetic radiation, the ground state (S0) electrons are promoted to an excited electronic singlet state (S2) Excited State The excited state S2 has a life time of 1-10ns, during which the fluorophore can undergo internal conversion (conformational changes and interactions with its environment). This results in the energy of the excited state S2 being partially dissipated yielding a relaxed singlet excited state S1. It is from the excited state S2 that the fluorescence (F) photon is emitted Fluorescence Emissions Once the photon is emitted the fluorophore returns to its ground state E0. As some of the energy of the initial photon has been dissipated, the emitted photon s energy is lower and it therefore has a longer wavelength than the wavelength of the energy of the initial photons. This difference in energy or wavelength is called the Stokes shift. This shift is important to the sensitivity of the fluorescence technique as it allows the emission photon to be detected against a low background Fluorescence can be quantified by calculating the fluorescence quantum yield Q which is the ratio of the number of fluorescence photons emitted to the number of photons absorbed and is a measure of the extent to which environmental interactions have an effect. Q= Number of photons emitted Eq 4 Number of photons absorbed Q will have a fixed value for a certain set of conditions with a maximum value of one. Each fluorophore has a characteristic fluorescence spectrum as well as a characteristic absorption spectrum. Normally the same fluorophore can be repeatably excited and detected, via a cyclical process. This cyclical process is the key to the high sensitivity of the fluorescence technique. 50

51 3.2 Fluorescent Labelling of Polymers Fluorescent labels or fluorophores are often large aromatic, heterocyclic, dye molecules; proteins conjugated to polymers, dendrimers, and semiconductor crystals. Fluorophores usually have molecular weights >>100, which when used to label molecules can lead to a considerable increase in the overall molecular weight of the molecule. This is in contrast to the organic radioisotopes 14 C or tritium which have atomic weights of 14 and 3 respectively will only increase the molecular weight by 2 for 14 C or, for example in the case of a labelled methyl group having 3 3 H isotopes, by six. Although the details of the methodologies used to add fluorescent labells to compounds is beyond the scope of this PhD, the general methodology for the fluorescent labelling of polymers is very similar to that used for the radiolabelling of polymers. The labells can be introduced either pre-or post- polymerization. For pre-polymerization the fluorescently labelled molecules are produced using either a fluorescent initiator or monomer. By using a fluorescent initiator, the position and number of fluorescent labells, and increases in molecular weight, will be known and controlled if the polymer is prepared using ATRP. Copolymerization of a fluorescent with a non fluorescent monomer is used to produce a fluorescently labelled polymers. However the number and position of the fluorescent labels per polymer chain is more difficult to control, as is the calculation of the increase in the molecular weight. Breul and Hager in their review (4) of the labelling of polymers, evaluated the fluorescent labelling of polymers using a wide range of fluorescent monomers to prepare polymeric materials. For post polymerization the fluorescent labelled molecules can be introduced using either a random or targeted derivatisation of the polymer functional groups e.g. end group or a group with selective reactivity on the polymer backbone. 51

52 3.3 Application of Fluorescence Spectroscopy to Polymers Fluorescent labelling of polymeric compounds has been used on many occasions to trace a molecule s behaviour in a wide range of applications. A detailed review of the applications is beyond the scope of this PhD although an overview of some of the more common uses will be given, in the context of polymer science. Fluorescent modification is a common approach to study polymers in a solution, or as solids. Their use allows the rapid identification and characterisation of a polymer for studies into polymerization mechanisms, polymer-surfactant systems, and diffusion of polymer solutions, gels, solids, and polymer properties in solution Polymer Dynamics By using a number of fluorescent spectroscopic techniques inclusive of: excimer emission, non radiative energy transfer between donor and acceptor labelled molecules and changes in emission intensities, the dynamics of polymer chains in solution can be studied. The nature of dynamic studies allows investigation of the rate of change in the conformation for high molecular weight chains compared to low molecular weight chains in dilute solutions at equilibrium (5,6,7,8) and agitation solutions (9,10) to determine whether the conformational mobility depends on solvent or polymer structure. Additionally fluorophores can be used to investigate the conformational equilibrium of polymers heated above their glass transition temperature, (11,12,13) or when stress is applied to the conformation mobility of cross linked polymers. Intramolecular excimer formation shows conformational transitions that take place in polymer backbones. For polymers that have donor and acceptor fluorophores at the chain ends, as the degree of separation between these fluorophores increases, the efficiency of the non radiative energy transfer decreases. This decrease can be used to monitor chain relaxation. Additionally double labelled polymers can be used to follow the mixing of two polymers. (13) 52

53 3.3.2 Free Radical Polymerization Fluorophores have been used to follow the free radical polymerization of monomers by monitoring the changes in fluorescence and equating this to the degree of monomer conversion. A number of stilbazolium salts (14) were used to investigate the free radical polymerization of thermally initiated methyl methacrylate, and a photo initiated mixture of 2 ethyl-2-(hydroxymethyl)propane 1,3 diol triacrylate and (TMPTA)-1- methylpyrrolidine-2-one. The studies showed that stilbazolium salts were good fluorophores for monitoring the progress of free radical polymerization via changes in their emission spectra. The degree of polymerization was found to be linear for the range of monomer conversions in photo initiated polymerizations. For thermally initiated polymerizations, the relationship is still linear below the gelling point, but shows a rapid increase once the polymer forms a rigid gel Polymer Surfactant Systems By chemically modifying commercial polymers, fluorescent labells can be incorporated allowing them to be used to determine the properties of polymer surfactant systems such as the critical aggregation concentration (CAC), and to characterise the microenvironment of the polymer surfactant complexes. (15) The fluorophore measurements of the of polymer surfactant systems rely on quenching the emissions from a fluorophore using a quencher added in increasing amounts to a solution containing a known amount of surfactant and fluorophore. Both the fluorophore and quencher are chosen so they have an affinity to the micelles. The decay of the fluorescence in the presence of different concentrations of quencher is recorded and analysed by kinetic models based on surfactant micelles and assuming that the distribution of probes and quenchers in polymer-surfactant micelles behave similarly. There are many examples of fluorescent labells used to monitor polymer surfactants and polymer-ionic surfactant systems some of which are: Polymer-ionic surfactant systems: poly(ethylene glycol) (PEO) or poly(vinyl pyrrolidone) with the surfactant sodium dodecyl sulphate (SDS) studied by Maltsh and Somasundaran (16) and the interactions between SDS and fully hydrolysed poly(vinyl alcohol) or PEO. (17) 53

54 Neutral polymer-non-ionic surfactant systems: The strength of interactions with nonionic surfactants was demonstrated by monitoring the interactions between modified hydrophobically poly(n-iso-propylacrylamide) and n-octyl-β-d-thioglucopyranoside in the presence of SDS. (18) Non and hydrophobic poly electrolytes and charged surfactant systems: Abuin and Scaiano (19) used fluorescence quenching in the study of poly electrolytes with oppositely charged surfactants. They determined the aggregation number of bound micelles in the system poly(styrene sulphonate) and dodecyl trimethyl ammonium bromide. 3.4 Problems associated the use of Fluorescent Labells with respect to Polymers The draw backs or problems of fluorescence spectroscopy can be split into those relating to the fluorescence microscopy technique and those associated with fluorescent labels. Those relating to fluorescence microscopy are beyond the scope of the PhD but those relating to fluorescent labells are within scope. There is an implicit assumption that fluorophores do not significantly affect the properties of polymers. However as the molecular weight of the fluorophore can be large, possibly many times that of the target molecule and fluorophores are normally hydrophobic, it is clear the presence of the fluorophore may affects or even dictates the behaviour of the labelled species. There are a number of examples in the literature that support the previous statement some of which are detailed below. Souter and Swanson, (20) studied the conformational behaviour of PMA in dilute aqueous solutions where the PMA had been copolymerized with two separate distinct, fluorescent labels. The introduction of aromatic fluorophores could be considered as a hydrophobic modification leading to an amphiphilic polymer which may in turn affect polymer solubility, association and surface interactions. The measurements were carried out using a range of ph values, and only over ph=4 did the results obtained from PMA labelled with the two fluorescent labels agree. Souter and Swanson concluded that the differences may be due to the labels hindering the motion of the PMA backbone, or the labels themselves which may have greater mobility at a ph of 4 54

55 or greater. The hydrophobic fluorescent labels may well alter the labels interactions with the PMMA and hence the relaxation of the polymer. Morawetz (21) studied the behaviour of fluorescently labelled PMA in an aqueous system and concluded that models based on fluorescent labelling are valid for the labelled polymer only, the results cannot be used to infer the state of the unlabelled PMA. Additionally, Morawetz (22) studied the conformational mobility of polymer chains by measuring the fluorescence depolarization of fluorescent labels fixed to polymers. He concluded that the fluorescent technique could only be used for high molecular weight polymers. It could not be used to compare the mobility of polymers to their low molecular weight analogues, because the rotational diffusion of labels attached to small molecules will be dominated by the rotation of the entire molecule. Egan and Winnik (23) found that increasing the number of fluorophores on poly(2-ethyl hexyl) methacrylate, used as a stabilizer in the dispersion polymerization of poly(vinyl acetate), resulted in changes to the mean particle size, distribution, composition and molecular weights of the colloidal polymer particles. Increasing the levels of fluorophores in the stabilizer resulted in small mean particle sizes and large amounts of irreversibly attached poly(2-ethyl hexyl) methacrylate. An average of one fluorophore per stabilizer molecule results in an ultra high molecular weight polymer. Finally it has benn noted that fluorolabelled biopolymer (protien) has diferent properties from the unlabelled equivalent biopolymer These examples do suggest that the presence of a fluorophore can have a substantial effect on the physical and chemical properties of polymers. The methodology of fluorescence spectroscopy has limits when the distribution of fluorophore is not homogeneous across the sample, or the fluorophore is subject to self quenching or quenching. During Winnik and Regismond s (15) investigations into study of polymer surfactant systems using fluorescence labels, the authors commented that there will always be a concern about whether or not the presence of the label on the polymer affects the system under investigation. Aromatic fluorophores are naturally hydrophobic and the attachment of these fluorophores to water soluble polymers can in essence convert the polymer into having amphiphilic properties. They go on to say that 55

56 even the presence of minute amounts of fluorophore can and does affect the properties of water soluble polymers. Best and Silescu (13) concluded, from fluorescence densitometry at polymer-polymer interfaces for the inter diffusion measurements of poly(styrene) poly(cyclohexyl methacrylate) blends, that the effects of the addition of a fluorophore on inter diffusion could be reduced by ensuring that the labelled polymers contained at least 200 styrene monomer units per fluorescent label. Breul, Hager and Schubert (4) reviewed the synthesis of fluorescent labelled polymers produced by copolymerizing fluorescent labelled monomers with non fluorescent labelled monomers. The review reported that the incorporation of fluorophores led to a modification of the thermal properties of the polymers. The majority of all investigations showed that an increase in the fluorophore content resulted in an enhanced Tg value and a decrease of the thermal stability. 3.5 Conclusion It is clear from the literature examples presented that the inclusion of a fluorophore can have substantial effects on the physical and chemical properties of polymers and assumptions of identical or near-identical behaviour of modified and unmodified materials are not necessarily valid. It is also well-established that the addition of even small numbers of hydrophobic groups, fluorescent or non-fluorescent, to water-soluble polymers can affect behaviour at interfaces. 56

57 References 1. J.F.W. Hershel, Phil. Trans. Roy. Soc. (London)., 1845, 135, G.G. Stokes, Phil. Trans. Roy. Soc. (London)., 1852, 142, A. Jablonski, Z. Phys., 1935, 94, A. M. Breul, M. D. Hager, U. S. Schubert, Chem. Soc. Rev., 2013, 42, G. Weber, Biochem. J., 1952, 51, T. Breechbuhler, M. Magat, J. Chim. Phys., 1950, 47, S. Kitamura, H.Yunokawa, T. Kuge, Polym. J., 1982, 14, C. Cuniberti, A. Perico, Eur. Polym. J., 1977, 13, T.S. Chen, J.K.Thomas, J. Polym. Sci. A., 1979, 17, B. Bednar, H. Morawetz, J.A.Shafer, Macromolecules., 1985, 18, E.V. Anufrieva, M.V. Volkenstein, T.V. Rozgovarova, Opt. Spectrosc., 1959, 7, G. Osterr, Y. Nishijima, Advan. Polym. Sci., 1964, 3, M. Best, H. Silescu, Polymer., 1992, 33, S. Wroblewski, K. Trzebiatowska, B. Jedrzejewska, M. P. R. Gawinecki J.Paczkowski, J. Chem. Soc. Perkin. Trans. 2,. 1999, F. M. Winnik, S. T.A. Regismond, Colloids. Surfaces. A., 1996, 118, C. Maltesh, P. Somasundaran, J. Colloid. Interface. Sci. 157, 1993, J. van Stam, N. Wittouk, M. Almgren, F.C. DeSchyrver, M. Da Graca Miguel, Can. J. Chem., 1995, 73, F. M. Winnik, H. Ringsdorf, J. Venzmer, Langmiur., 1991, 7, E. B. Abuin, J.C. Scaiano, J. Am.Chem. Soc., 1984, 106, I. Souter, L. Swanson, Macromolecules., 1994, 27, H. Morawetz, Macromolecules., 1996, 29, H. Morawetz, J. of Luminenescence., 1989, 43, L. S. Eagan, M. A. Winnik, M. D. Croucher J. Poly. Sci. A., 1986, 24,

58 CHAPTER 4 APPLICATION OF 14 C AND RADIOISOTOPES TO POLYMER CHEMISTRY 58

59 CHAPTER 4 APPLICATION OF 14 C AND RADIOISOTOPES TO POLYMER CHEMISTRY 1. Introduction The disciplines of radio and polymer chemistry have evolved considerably post war and, as a consequence, radioisotopes have frequently been used to assist with the development of polymer chemistry. The value of using radioisotopes lies in the high sensitivity and specificity that can be achieved in studies under virtually any experimental condition. The use of radioisotopes in polymer chemistry allow aspects of polymerization mechanisms to be studied and the quantification of groups down to picomole levels, using non-destructive methodologies. Labeling at specific positions within the polymer using radiolabelled initiators or monomers, allows the mechanisms of a polymerization and a polymer s chemical and physical properties to be studied. Using radiochemically and chemically pure compounds, the accuracy of radiochemical measurements can be as good as ± 0.5%. This chapter reviews the applications of radiochemistry to polymer chemistry, in particular to studying the mechanism of free radical polymerization and a polymer s stability, purification and degradation. 2. Use of Radioisotopes in Polymer Chemistry. The first use of radioisotopes to study a polymerisation mechanism was reported in 1944 by Pfann, Salley and Mark, (1) who irradiated bromine present in brominated polystyrene to produce 85 Br-labelled polystyrene. The presence of the 85 Br was used to quantify the level of bromine present in the polymer and assist with the understanding of the initiation mechanism using p,p-dibromo dibenzoyl peroxide. Landler and Magat (2,3) used the radiolabelled Grignard reagent 82 Br butyl magnesium bromide to initiate the polymerization of methyl methacrylate, allowing the initiation mechanism to be identified. Prior to 1950, the availability of radioisotopes was limited to relatively short lived radioisotopes such as 35 S, which was used to study the mechanistic aspects of polymer chemistry in a number of papers. For example Smith and Campbell used 35 S-labelled persulphate to determine the number of sulphur atoms per polymer chain of low and high molecular weight polystyrene to give an insight into the termination processes. (4,5), Mochel and Peterson used 35 S labelled thiols to investigated their use in 59

60 free radical polymerizations as chain transfer agents, to determine if sulphur atoms were incorporated into the polymer chains and enable the value of the chain transfer constant to be calculated. (6) From the early 1950 s, the commercial production of 14 C increased in significant quantities from reactors both in the UK and USA, allowing 14 C to be used extensively to study the mechanisms of organic and polymeric reactions which resulted in an avalanche of reports into the use of radioisotopes for determining mechanistic aspects of polymer chemistry, most notably by Bevington and his coworkers. (7) 3. Synthetic Strategy A number of factors have to be considered before the synthesis of a radiolabelled compound can proceed. In particular, an understanding of why the compound is required, under what conditions it will be used and the aims of the study. These parameters are essential since they define the optimum properties and design of the radiolabelled compound i.e. the radioisotope to use, position of the radiolabel, the levels of radioactivity and specific activity required. In the case of organic polymeric molecules, the labels will predominantly be the low energy beta-emitting radioisotopes 14 C or tritium ( 3 H) as detailed in Table 1. Property Tritium ( 3 H) 14 C Half Life (Years) Maximum Atomic Specific 3.59 x Bq g x Bq g -1 Activity Maximum Beta Energy(keV) Mean Beta Energy (kev) Nuclear Preparation 6 Li(n,α) 14 N(n,p) Decay Sequence Products 3 H 3 He + + β - + ν 14 C 14 N + + β - + ν Penetration Range In Air 6 mm 20 cm Penetration Range In Water/Tissue 6 microns 250 microns Table 1 Physical Properties of Tritium ( 3 H) and 14 C 60

61 As carbon and hydrogen are ubiquitous in organic compounds, they can be replaced with 14 C and 3 H respectively without changing the compound s chemical structure. Usually the chemical and physical properties of the radiolabelled compounds are virtually the same as those for their non-labelled counterparts, although isotope effects may occur since the C- 3 H and C- 14 C bonds are slightly stronger than the corresponding non-labelled bonds. Additional it must be noted that there is a small increase in mass ie 14 C has a increased atomic mass of 2 compared to 12 C and 3 H an increase of 1 compared to 1 H when a compund is radiolabelled. The molecular positioning of the radiolabel depends on the information sought and the proposed use of the radiolabelled compound. For example, when studying the deposition of a 14 C labelled fatty acid, the acid can be labelled anywhere within the molecule. However, if it is possible that the acid could decarboxylate, then the 14 C label should not be on the carbonyl group. Once the radioisotope and its position have been decided, the synthesis of the molecule has to be planned. The primary sources of 3 H and 14 C are 3 H gas and Ba 14 CO3 from which a number of important labelled precursors can be synthesized. (8) In planning the synthesis, the number of synthetic steps, availability of labelled and non-labelled precursors, reaction scales, methods of analysis and purification and the final specifications of the radiolabelled compound need to be considered. A radioisotope can be introduced precisely during the preparation of polymers by the use of 3 H or 14 C labeled initiators or monomers. Alternatively a radioisotope can be introduced into an existing polymer by modifying its structure, for example a 14 C methylation or 3 H reduction. Strategies for preparing radiolabelled polymers are well illustrated by Shemilt. (9) In special circumstances, polymers can be prepared using biosynthetic methodologies, by either growing a plant in an atmosphere of 14 CO2 which it photosynthesises, or feeding it with a radiolabelled nutrient. The polymers are then extracted from the plant. If a polymer cannot be labelled using 14 C it may still be radiolabelled by tritiation. The use of tritium usually involves the replacement of the hydrogen atoms with an equivalent number of tritium atoms. Tritiation results in a higher specific activity compared to labelled molecules having one 14 C atom. The higher specific activity enables polymers to be studied at low concentrations where a high degree of sensitivity is required. The range of methods and reagents used to tritiate 61

62 organic and polymeric molecules is vast and is beyond the scope of this PhD. For the purpose of the thesis focus will be on the use of 14 C. 4. Radio Analytical Techniques for Analysis of Radiolabelled Polymers The value of using radioisotopes lies in the high sensitivity and specificity that can be achieved in studies under virtually any experimental condition. In polymer chemistry, they allow aspects of polymerization mechanisms to be studied and the quantification of groups down to pico mole levels, using non-destructive methodologies. By using radiochemically and chemically pure precursors, the accuracy of radiochemical analyses can be as good as +/- 0.5%. A number of radiochemical analytical methods are available and their application to polymer chemistry is outlined below. 5. Radioisotope Dilution Analysis Radioisotope dilution analysis, is a method where by a radiochemical is used to determine the purity of a non-radiolabelled version of the radiochemical which exists in a mixture of closely related compounds, without the need to purify and separate the entire mixture. The analysis is dependent on knowing the identity and structure of the non radiolabelled chemical. To the mixture is added x1 mg of the pure radiochemical with a specific activity, a1 (Bq/mg). The mixure is thoroughly mixed a sample removed and purified to obtain a pure sample of the labelled and non labelled compound. A sample x2 is used to determine the specific activity a2 (Bq/mg) measured. The ratio of the active to inactive compounds depends on the mass of active substance (x1) and the mass (x2) of the inactive compound present in the pure sample. As radioactivity is conserved, then: a1 x1= a2(x1+ x2) (Eq5) By knowing a1, a2 and x1, the value of x2, the mass of the minor component hence impurity, can be calculated. Adapting this method, the purity of the minor component can be determined simply using the initial and final specific activities: Purity = a2/a1 x 100 (Eq6) 62

63 6. End Group Analysis Initiation should be the attachment of initiator or fragments across the monomer double bond with no initiator or fragments entering the polymer by abstraction or other side reactions. (10,11) The initiator usually forms the end groups in polymers which can act as reactive centres, a thorough knowledge of these centres can provide accurate information on the mechanisms of initiation, termination, chain transfer, retardation and chemical reactivity. Their concentration is usually low and accounts for only a small fraction of the polymer s total weight, so a sensitive and specific technique is required for their detection. Provided the end groups are stable and remain attached during the recovery and purification of the polymer, the use of radiolabelled initiators provides this sensitivity and specificity. By comparing the specific activities of the radiolabelled initiators or fragments with those of the final polymer, the number of radiolabelled initiators or fragments present can be determined. 6.1 Kinetic Chain Length Kinetic chain length is the average number of units of monomer consumed per initiator radical which is a development of the average degree of polymerization (DPn) defined as the number of monomer units in a polymer, macromolecule or oligonomer. End group analysis can be used to determine the kinetic chain length (ν) which is independent of termination or transfer. The kinetic chain length (12,13) is given by: ν = No. Monomeric units in Polymer = Rate of Polymerization No. Initiator Fragments in Polymer Rate of Initiation (Eq7) 6.2 Termination. By using end group analysis the termination method can be ascertained. Termination by combination will have twice the level of radioactivity compared to termination by disproportionation. Termination via combination DP=2ν (Eq8) Termination via disproportionation DP= ν (Eq9) 63

64 7. Specific Activity Measurements Using 14 C labelled monomers the reactivity ratio of monomers in a copolymer can be determined by comparing the specific activities of a 14 C monomer, with the specific activity of the final copolymer. For copolymers prepared using monomers at very low concentrations or conversions, monomer reactivity ratios are calculated (14,15,16,17,18,19) using: r = Specfic Activity of Copolymer (Eq10) Specfic Activity of Homopolymer r is the reactivity ratio of the 14 C monomer. By comparing the total levels of radioactivity with the specific activity of either a radiolabelled monomer or a homopolymer, prepared using the same conditions as the copolymer, the mass of monomer or homopolymer in the copolymer to be calculated. The levels of ester hydrolysis occurring in polymers and copolymers can also be determined by using monomers with 14 C ester groups. The degree of hydrolysis of the 14 C ester groups is given by: Degree of Hydrolysis = Specific Activity of the Polymer before Hydrolysis Specific Activity of the Polymer after Hydrolysis (Eq11) 8. Mechanistic Studies into Free Radical Polymerization In the late 1950s, John Bevington foresaw the potential of 14 C radioactive compounds for studying polymerization processes. As Bevington and co-workers started to report their findings, other polymer chemists, most notably Geoffrey Ayrey and his coworkers, also carried out investigations into polymer chemistry using radioisotopes. Both groups showed that the use of radioisotopes enabled the identification and measurements of minor fragments incorporated into polymers during synthesis and revealed hitherto inaccessible details of polymerization mechanisms. All aspects of free radical polymerization were studied using radiolabelled precursors, including reverse, co- and graft polymerizations, initiator efficiencies, propagation, termination, chain transfer, branching, retardation, inhibition and polymer stability. In 1963, Ayrey reviewed the use of radioisotopes in free radical polymerization. (7) 64

65 9. Use of 14 C Radiolabelled Initiators 14 C labelled azo-iso-butyronitrile (AIBN), dibenzoyl peroxide and associated peroxide initiators such as di t butyl peroxide, (20,21) bis para- and meta-(p-methoxybenzoyl) peroxides, (22,23) p,p dimethoxy dibenzoyl peroxide, (22,24,25) m,m dibromo dibenzoyl peroxide, (26) and 3,5 dibromo 4 methoxy dibenzoyloxy peroxide, (26,27) have been used to follow their decomposition and use in polymerizations. 9.1 Azo-iso-butyronitrile Initiator Decomposition The first use of 14 C AIBN was reported in 1952 by Arnett and Peterson (28) who determined that the efficiency of the AIBN, for the polymerization of a range of vinyl monomers, was %. Subsequently Bevington Melville and Taylor used 14 C AIBN to determine the number of AIBN-derived end groups (29) and the termination mechanism for the polymerization of styrene and methyl methacrylate at 25 C. Poly(styrene) terminated by combination, giving two 14 C end groups per molecule, poly(methy methacrylate) terminated by disproprtionationation giving only one 14 C end group per molecule. By using 14 C AIBN and measuring the number of 14 C initiator fragments per polymer chain Bevington, Bradburry and Burnett showed for a given monomer mixture, the rate of initiation was proportional to the initiator concentration, provided each decomposed initiator promoted the growth of one polymer chain. (11) Further studies were undertaken into the thermal and photochemical decomposition of AIBN to yield cyano-2-propyl radicals as shown in Scheme 1. (30,31,32) Step 1 Decomposition of Azoisobutyronitrile Azo-iso-butyronitrile Cyano-2-propyl radicals 65

66 Step 2 Self Reaction of Cyano-2-Propyl Radicals: Formation of Tetramethyl succinodinitrile Cyano-2-Propyl Radicals Tetramethylsuccinodinitrile Step 3 Self Reaction of Cyano-2-Propyl Radicals: Formation of Intermediate Dimethyl- N-(2-cyano-2-propyl)-ketenimine Cyano-2-Propyl Radicals Intermediate Dimethyl-N-(2-cyano-2-propyl)-ketenimine Step 4 Conversion of Intermediate Dimethyl-N-(2-cyano-2-propyl)-ketenimine to Tetramethylsuccinodinitrile Intermediate Tetramethyl succinodinitrile Dimethyl-N-(2-cyano-2-propyl)-ketenimine 66

67 Step 5 Reaction of Cyano-2-Propyl Radicals: Iso-butyronitrile and Metacrylonitrile Formation Cyano-2-Propyl Radicals Iso-butyronitrile Methacrylonitrile Scheme 1 Decomposition of 1, 1 Azo-iso-butyronitrile Ayrey, Evans and Wong (33) quantified the wasted radicals (31,32,34) formed by the decomposition of AIBN and the resulting disproportionation and combination of these wasted radicals using isotopic dilution analysis. The decomposition of azoisobutyronitrile, scheme 1 step 1, forms two cyano-2-propyl radicals, only 60% of which go on to initiate a polymerization. As a result of the cage effect of solvent molecules the remaining 40% wasted radicals react to give a number of other possible products. 84% of the wasted radicals form tetramethyl succinodinitrile, either via the self-reaction of the cyano-2-propyl radicals (step 2) or the formation of the intermediate dimethyl-n-(2-cyano-2-propyl)-ketenimine (step 3) which can form radicals and take part in polymerizations. Once the concentration of dimethyl-n-(2- cyano-2-propyl)-ketenimine reaches its maximum it can break down to form tetramethyl succinodinitrile (step 4) which doesn t take part in the polymerization process. A small proportion of the primary radicals are thought to form iso-butyronitrile (31,32,34,35) and methacrylonitrile (step 5). Scheme 2 details the reaction of methacrylonitrile with cyano-2-propyl and it s radical to give tetramethyl succinodinitrile. 67

68 Iso-butyronitrile and Methacrylonitrile: Tetramethylsuccinodinitrile Radical Formation Iso-butyronitrile Methacrylonitrile Tetramethylsuccinodinitrile Radical Scheme 2 Conversion of Isobutyronitrile with Methacrylonitrile to Tetramethyl succinodinitrile in the presence of Cyano-2-Propyl Radical The decomposition of 14 C AIBN in the presence and absence of oxygen was reported by Bevington. (12) In the presence of oxygen, (36) a peroxide is formed as shown in Scheme 3. For a near complete decomposition of AIBN, 23% tetramethyl succinodinitrile and 62% acetone cyanhydrin results. Step 1 Reaction of Azoisobutyronitrile with Oxygen Azo-iso-butyronitrile Cyano-2-Propyl Peroxide Iso-butyronitrile Radicals Iso-butyronitrile Radical Step 2 Formation of the Azo-iso-butyronitrile Peroxide Derivitative Peroxide Iso- Iso-butyronitrile Azo-iso-butyronitrile butyronitrile Radical Peroxide Scheme 3 Decomposition of Azo-iso-butyronitrile in the Presence of Oxygen 68

69 Without oxygen (37) the intermediate, dimethyl ketene cyano isopropyl imine, is formed. 9.2 Benzyl Peroxide: Peroxide formation The relative importance of the different decomposition pathways depends on monomer concentration, temperature, pressure, solvent and the nature of the substituents on the original peroxide. For symmetrical alkyl or aryl peroxides, such as benzoyl peroxide (BPO), thermal or photochemical decomposition results in two identical benzoyloxy free radicals which can decompose further, forming CO2 and phenyl free radicals. Two 14 C radiolabelled forms of BPO, labeled in either the ring or the carbonyl group, were used to demonstrate the generation of benzoyloxy and phenyl free radicals which reacted as shown in scheme 4. A control experiment (38) was carried out using low concentrations of 14 C carbonyl labelled BPO dissolved in pure benzene. Over time the peroxide dissociated to yield carbon dioxide, (39) quantified using radiochemical methodologies. In other solvents, BPO formed benzoyloxy free radicals which were found to initiate efficiently. (41) Step 1 Decomposition of Benzoyl Peroxide Benzoyl Peroxide Benzoyloxy Free Radical Step 2 Decomposition of Benzoyloxy Free Radical Benzoyloxy Free Radical Phenyl Free Radical 69

70 Step 3 Initiation via Benzoyloxy Free Radical Step 4 Initiation via Benzoyloxy Free Radical Scheme 4 Decomposition of Benzyl Peroxide and Subsequent Initiation Each labelled initiator was used to polymerize a monomer (M) such as methyl methacrylate at 60 o C. Phenyl radicals accounted for 43% of initiation showing the importance of competition between steps 3 and 4 in scheme 4. An equation relating the competition (C) between steps 2 and 3 in scheme 4 with the number and types of end groups present on the final polymer was derived as: C = No of Benzoate End Groups (Eq12) No s of Benzoate and Phenyl End Groups The relationship was explored for the benzoyloxy (41) and phenyl (42) free radicals. Under photochemical conditions, the benzoyl peroxide can decompose to form excited benzoyloxy radicals which decompose further to give the phenyl radicals, or decompose directly to phenyl radicals. End group analysis showed that the benzoyloxy radicals formed are not in an excited state, and phenyl radicals are formed directly from an excited peroxide molecule. (25,27,43,44,45) Similar studies looked at the effects of the structures of initiators and monomers on the stability of initiator free radicals and kinetics of polymerization. A variety of peroxide 70

71 initiators, specifically bis (p-methoxy) benzoyl peroxide, (22) bis (m-methoxy) benzoyl peroxide, (23) bis p,p (dimethoxy) benzoyl peroxide, (22,24) m,m (dibromo methoxybenzoyl) peroxide, (26) and (3,5 dibromo-4-methoxy benzoyloxy) peroxide, (26,27) were used to polymerize a series of esters of methacrylic acid, methyl, phenyl, benzyl, and cyclohexyl methacrylates, where the peroxide was labelled either on the benzene ring or carbonyl. Results indicated that electron donating groups on benzoyloxy free radicals stabilized the free radicals, which gave high levels of initiation, whilst electron withdrawing substituents decreased stability, resulting in 14 C phenyl free radical initiation. At low monomer concentrations more of the benzoyloxy radicals decomposed forming the phenyl radicals. (46) Dual labelled versions of benzoyl peroxide were used to quantify the number of benzoloxy and phenyl end groups present for the polymerization of naphthalene, 2-vinylnaphthalene and 4-vinylbiphenyl (47) and methacrylonitrile and acrylonitrile. (48) If the peroxide contains alkyl rather than phenyl groups, for example tert-butyl peroxide, decomposition will form alkyloxy free radicals which can undergo further decomposition to form CO2 and alkyl free radicals. Using 14 C methyl or di t butyl peroxide (20,21) to polymerize styrene at 80 C and 130 C, initiation is via the tertiary t butoxy radicals, at low monomer concentrations and high temperatures, the t butoxy radicals decompose further to give methyl and butoxy radicals. The number of labelled end groups can be determined by comparing the specific activities of the polymer before and after trifluoroacetic acid mediated hydrolysis of the t butoxy ester end groups. AIBN is preferred to benzoyl peroxide as an initiator as it doesn t induce decomposition and results in end groups of one type. 10. Initiator Efficiencies The efficiency of AIBN and benzoyl peroxide was originally believed to be approximately 100%. Using 14 C versions of the initiators and isotope dilution analysis, recalculation of the efficiencies showed that AIBN (9,32,35) had efficiencies of between 40 80%, dependent on the system investigated, whilst dibenzoyl peroxide gave values of approximately 100%. AIBN has a low efficiency because the free radicals can preferentially react with themselves, rather than with monomer, resulting in a reduction of the concentration of the AIBN free radicals. This is called the cage effect, which is thought not to exist for dibenzoyl peroxide based initiators. (43,49) 71

72 11. Initiator Co-Catalysts A co-catalyst is a component in a system, such as a solvent, which acts as an additional source of free radicals. Using a 14 C version of the co-catalyst, the degree to which it participates in the polymerization can be determined. Scheme 5 illustrates the use of 14 C methyl chloride as a solvent for the polymerization of iso-butene initiated by aluminium trichloride. (50) The synthesis showed that one in four polymer molecules had a 14 C methyl group, indicating the solvent acts as a co catalyst; and termination is by chain transfer. Step 1 Formation of 14 CH3 Aluminium Salt Step 2 Reaction of 14 CH3 Aluminium Salt with Isobutene Scheme 5 Polymerization of Iso-butene initiated by Aluminium Trichloride The polymerization of iso-butylvinyl ether initiated using boron trifluoride 14 C etherate, Scheme 6 established that 0.7% of the polymer contained a labelled end group, indicating very little termination is due to chain transfer. Step 1 Dissociation of boron trifluoride 14 C etherate 72

73 Step2 Propagation of the Polymerization of iso-butylvinyl ether Scheme 6 Polymerization of Iso-butyl Vinyl Ether Initiated using Boron Trifluoride 14 C Etherate Finally, the use of 14 C benzene for the polymerization of vinyl acetate, benzene molecules were found to be incorporated per polymer chain. (52,53) The incorporation was thought to be due to the presence of 14 C impurities. Using radio-chemically pure (54, 55) benzene, the incorporation decreased to one benzene molecule per polymer chain. 12. Polymerization: Free Radical By using 14 C monomers, the mechanisms of cross linking reverse, co-, graft and branched polymerizations and the physical behaviour and properties of the polymers could be studied. 13. Polymerization: Reversible Propagation Compared to propagation, the activation energy for reverse propagation is higher. At high temperatures the probability of reverse propagation increases, to the point when the ceiling temperature is reached where the rate of polymerization equals the rate of de-polymerisation. For the polymerization of methyl methacrylate (56) the ceiling temperature is 100 o C, at 125 o C (57) reversible polymerization occurs. The reversible propagation can be proven by preparing non-labelled poly(methyl methacrylate) in the presence of a highly pure 14 C labelled poly methyl methacrylate. After purification both the polymers were found to be radioactive, indicating a reversible propagation of either 73

74 a non-terminated chain or depolymerisation of the 14 C poly(methyl methacrylate). The reversible propagation can be quantified by measuring the specific activities of each polymer. 14. Polymerization: Copolymerization Radioisotope techniques have been used in attempts to rationalise statistical copolymerisation. (58) By comparing the specific activities of a 14 C monomer, used at the lowest concentration in a copolymerization, with the specific activity of the final copolymer, the reactivity ratios and velocity constants of two monomers can be calculated. (15,16,17,18) This method is important for copolymers consisting of similar monomers, such as methacrylic acid, methyl methacrylate and methyl acrylate (59,60) as the analysis of these copolymers and identification of the individual monomers can be difficult by normal chemical techniques. (58) The copolymerization of non-labelled vinyl acetate and methyl methacrylate with 14 C vinyl acetate or 14 C methyl methacrylate using AIBN gave the values of the reaction ratios and velocity constants, that were found to be in good agreement with published values. In studies of the copolymerization of 14 C methyl methacrylate and vinyl esters with benzyl peroxide velocity constants were different than published values. The monomer reactivity ratios of the dual labelled copolymerization of 3 H methyl methacrylate with a series of 14 C labelled methacrylates (methyl methacrylate, ethyl methacrylate, benzyl methacrylate phenyl methacrylate) were determined. Compared to the methyl ester the higher molecular weight esters had greater reactivities. For the polymerization of stilbene, it is thought that stilbene is highly reactive towards a benzyloxy radical (61) but unreactive with AIBN. By copolymerizing 14 C trans stilbene or 14 C p-fluorostilbene, (61,62) with methyl methacrylate and 14 C trans stilbene with methacrylonitrile, acrylonitrile or styrene, the reactivity ratios and velocity constants indicated that trans stilbene and p-fluorostilbene had very low reactivity towards AIBN compared to benzoyl peroxide. Stilbene and p-fluorostilbene reacted less with poly(methyl methacrylate) compared to the poly(methyl acrylate) (62) suggesting the presence of the methyl group may sterically hinder copolymerization. Stilbene is less reactive towards the poly(methacrylonitrile), whilst methacrylonitrile and acrylonitrile had similar reactivities to the AIBN and benzoyloxy peroxide radicals. 74

75 For the copolymerization of styrene with 14 C cinnamic acid, the low levels of cinnamic acid incorporated were measured and found to be one cinnamic acid monomer per 100,000 styrene units. Using 14 C radiolabelled isopropene or 14 C styrene, the levels of unsaturation present in poly(isoprene) (63) or styrenated oils, acids and esters could be quantified. (64) 15. Polymerization: Polymer Branching 14 C labelled polymers and monomers have been used to investigate how polymer chains can act as chain transfer agents leading to either branching or cross linking. The method involves either the polymerization of 14 C labelled monomer with an unlabelled high molecular weight polymer, or the polymerization of an unlabelled monomer with a 14 C labelled high molecular weight polymer. By comparing the specific activities of the starting 14 C monomer or 14 C labelled high molecular weight polymer with the 14 C polymer products, the levels of branching or cross linking could be determined. This methodology was used to determine the degree of chain transfer and subsequent branching occurring in the polymerization of styrene. 14 C labelled styrene was polymerized in the presence of unlabelled high molecular weight poly(styrene). (65) The 14 C styrene polymerization resulted in a 14 C-labelled high molecular weight poly(styrene), a low molecular weight 14 C poly(styrene) and a non labelled high molecular weight poly(styrene) as shown in scheme 7. The 14 C-labelled high molecular weight poly(styrene) formed due to chain transfer and branching, between the non labelled high molecular weight poly(styrene) and 14 C styrene. The polymers were fractionated and their specific activities indicated that the degree of branching was 1 branched chain per 10 linear chains for a 75% monomer conversion. At higher conversions a higher degree of branching occurred. 75

76 Scheme 7 Polymerization of 14 C Styrene with High Molecular Weight Poly Styrene Similar studies were carried out by polymerizing vinyl acetate in the presence of 14 C high molecular weight poly(vinyl acetate). At low conversions, the degree of branching was virtually negligible, whereas, for high conversions, there were several branches (66,67) By measuring the levels of radioactivity and specific activity, the method of termination was identified as being by combination. (68) In photolysis of brominated poly(styrene) in the presence of 14 C styrene and carbon tetrachloride, the number of branches, chain lengths and the transfer constant for carbon tetrachloride were calculated by measuring specific activities. (69) 76

77 A linear, high molecular weight vinyl acetate with 14 C acetate pendant groups was partially hydrolyzed and the number of free OH groups and therefore level of hydrolysis determined by the reduction in specific activity. This polymer was reacted with a low molecular weight linear polymer with acid chloride end groups, which formed branches. The number of branched chains was determined from changes in specific activity. (71) 16. Cross link Analysis 14 C-labelled cross linking agents such as diacid chlorides can be used to quantify the number of links introduced by comparing the specific activity of the cross linker to that of the cross linked polymer. This approach has been applied to studies of rubber elasticity and the cross linking of polyesters in the copolymerization of styrene and 14 C maleic anhydride. (71) 17. Polymerization: Graft Polymerization The mechanism of grafting can depend on the type of initiator used. Poly(methyl methacrylate)-poly(styrene) and poly(methyl methacrylate)-poly(isoprene) grafts can be obtained using benzoyl peroxide but not AIBN. (72,73,74) Grafting of poly(methyl methacrylate) with poly(isoprene) has been studied using 14 C benzoyl peroxide. Grafting occurs as a result of the initiation of poly(isoprene) by a 14 C phenyl or benzoyloxy radical, via addition to the double bond. This forms grafting points on the poly(isoprene) backbone where 60% of the methyl methacrylate can polymerize via benzoyloxy radicals. Additionally, the irradiation of some polymers using gamma radiation from a 60 Co source can form free radicals which can be used to initiate monomers forming graft polymers. For example, on mixing the products of irradiation of non-labelled poly(acrylonitrile) with 14 C acrylonitrile, the resulting polymer was radiolabelled. By contrast, reaction of unirradiated, non-labelled poly(acrylonitrile) with 14 C acrylonitrile yielded a non labelled product. (75) 77

78 18. Chain Transfer Transfer agents promote chain transfer, resulting in the termination of the original growing polymer and the formation of a new active polymer chain where the transfer agents become incorporated into the final polymer. (76,77) Using 14 C transfer agents the levels incorporated into the polymers could be determined. As shown in scheme 8, the addition of the transfer agent zinc 14 C diethyl during the polymerization of propene resulted in the incorporation of 14 C ethyl groups, and the transfer of a free radical. This caused a threefold drop in the final molecular weight of the polypropene. Scheme 8 Chain Transfer using Zinc 14 C Diethyl Chain transfer agents such as carbon tetrabromide have also been investigated for the polymerization of styrene using 14 C AIBN. Isotope dilution analysis showed the initiator radicals are terminated by the carbon tetrabromide, forming a carbon dibromide free radical which initiates the styrene polymerisation, resulting in a low specific activity polymer. (19) The transfer constant for styrene/carbon tetrabromide were calculated and found to differ from those published. Differences were attributed to the presence of very low molecular weight species formed by non-radical mechanisms. (13,78) Similar studies have been carried out using degenerative transfer agents, (79) for example diphenyl-2-picrylhydrazyl which was used in the polymerization of styrene and methyl methacrylate with 14 C AIBN at 60 C. Transfer constants, showed that diphenyl-2-picrylhydrazyl was a powerful retarder of methyl methacrylate polymerisation but has less effect on styrene. 14 C diphenyl-2-picrylhydrazyl could capture initiating radicals or deactivate polymer radicals early in chain growth enabling it to act as both a retarder and an inhibitor. (87,88,89) Using the same experimental model, the efficacy of a range of transfer agents were investigated. (80) 78

79 19. Inhibition and Retardation By using 14 C-labelled inhibitors or retarders, the levels incorporated into polymers can be quantified and by knowing the polymers molecular weights, inhibition and retardation mechanisms can be defined. The use of 14 C p-benzoquinone as an inhibitor was investigated, in conjunction with AIBN for the polymerization of styrene. (81) 90% of the quinone reacted with low molecular weight chains, rather than the initiator s free radicals, proven by removing the quinone groups with trifluoro acetic acid which left most chains free of radioactivity. This indicated the quinone was bonded via an ester linkage. The cleaved 14 C products were confirmed as esters of quinol. Additionally 14 C p-benzoquinone could act as a retarder and partial inhibitor in the same polymerization. This was illustrated when used in the polymerization of methyl and ethyl methacrylate in conjunction with dibenzoyl peroxide. (82) The change from retarder to inhibitor occurred at a critical quinone concentration. Below this concentration the 14 C p-benzoquinone acted as an inhibitor and above a retarder. (83,84,85) In the copolymerization of both monomers, p-benzoquinone acted as both an inhibitor and retarder depending on the initiator and conditions used. (83) Using 14 C p- benzoquinone with 14 C dibenzoyl peroxide at 25 C and 60 C, the quinone and benzoyloxy free radicals reacted with polymer free radicals via a combination mechanism. Replacing 14 C dibenzoyl peroxide with 14 C AIBN, at low p-benzoquinone concentrations, it acted as a retarder and at high concentrations as an inhibitor. (86) The inhibitor 14 C N-(3-N-hydroxyanilino-1,3-dimethyl butylidene), once oxidized, acts as a retarder for the polymerization of styrene and methyl methacrylate. (90) 20. Termination Excluding chain transfer processes, scheme 9 shows polymers can terminate by either combination or disproportionation. In the presence of 14 C labelled initiators, termination by combination results in polymers having two 14 C initiator fragments, and a specific activity twice that of a polymer terminated by disproportionation which has one 14 C initiator fragment. The termination method can be determined using end group analysis. 79

80 Initiation/Propagation of Ethene using 14 C labelled Initiator ( 14 CI) Termination: Combination Resulting in a Polymer with Two 14 C Fragments Termination: Disproportionation Results in Two Polymers each having a 14 C Fragment Scheme 9 Termination Mechanism Combination and Disproportionation The termination mechanisms for the polymerizations of methacrylic acid, its esters and styrene derivatives polymerized at 60 and 80 C (91,92) and methyl acrylate at 25, 45 and 60 C, (93) were studied using 14 C AIBN and benzene. The esters of methacrylic acid terminated by disproportionation (80-90%). The polarity of the substituents for styrene, p-methoxy styrene and p-chloro styrene, affected the method of termination. Styrene and p-chloro styrene terminated by combination, whilst 19% of p-methoxy styrene at 60 C, terminated by disproportionation and at 80 C it had risen to 47%. For the polymerization of methyl acrylate at 45 and 60 C the method of termination was found to be dependant on the rate of polymerization. At these temperatures the polymerization rate increased as did the level of chain transfer which resulted poly(methyl acrylate) having <2 initiator fragments. Once the affect of the chain transfer had been corrected, the number of initiator fragments was calculated to be approximately 2 indicating termination was by combination. At 25 C less chain transfere took place as a result the number of initiator fragments in the polymer was 2 indicating termination was by combination. Bevington and Eaves (94) showed that, for the polymerization of acrylonitrile at 60 0 C in N,N-dimethylforamide, termination was by combination. 80

81 21. Polymer Degradation: Pyrolysis, Photolysis, Irradiation and Hydrolysis 21.1 Pyrolysis The identification and quantification of products resulting from the pyrolysis of the 14 C styrene and methyl styrene (meta or para) copolymers, (95) showed that 14 C exchange had occurred. The ratio of the labelled monomeric fragments to non-labelled fragments, was 3:1, which was approximatly the theoretical ratio of monomers used in the randomly constructed copolymer. This proved the degradation of the chain produced odd and even fragments with equal probability with no reverse copolymerization as shown in scheme 10. Co-Polymerization of Styrene and Methyl Styrene Modes of pyrolytic degradation Exchange of 14 C from Styrene to Methyl Styrene Scheme 10 Pyrolysis of Poly( 14 C Styrene)- Poly(Methyl Styrene) Copolymer 81

82 21.2 Photolysis Photo degradation of 14 C poly(methyl methacrylate), with 14 C labelled ester groups, resulted in the release of 14 C methyl formate indicating the removal of the esters groups. For 14 C poly(methyl methacrylate) with the 14 C labelled polymer back bone little degradation of the polymer was observed. (47,40) 21.3 Irradiation Using 14 C poly(methyl methacrylate) and 14 C poly(methyl acrylate), the loss of labelled ester side groups during high energy ( 60 Co) gamma irradiation of the polymers was determined by Todd. (98) She measured the activity, and specific activity of the polymers before and after irradiation. During the irradiation the 14 C poly(methyl methacrylate) degraded whilst 14 C poly(methyl acrylate) cross linked. These results indicated that the level of loss of the number of methyl ester groups from 14 C (poly methyl methacrylate) was between % depending on the strength and length of the irradiation time. For 14 C poly(methyl acrylate) there was no significant loss of methyl ester groups. (75) 21.4 Hydrolysis The effect of tacticity on the degree and rate hydrolysis of 14 C ester groups can be determined by comparing specific activities of the polymer before and after hydrolysis. The tacticity had a major effect on the rate of the hydrolysis of poly(methyl methacrylate). Ester groups in a isotactic triadic structure were times more susceptible to hydrolysis than ester groups in either a hetrerotactic or syndiotatctic structures. (99,100) 14 C dibenzoyl peroxide labelled either in the ring or on the carbonyl groups was used to determine the degree and rate of hydrolysis of the benzoloxyl or phenyl radicals for poly(styrene). (51) For 14 C labelled carbonyl groups, there was almost 100% hydrolysis of the benzoyloxyl end groups, confirming the presence of ester linkages. The hydrolysis of ring-labelled benzoyloxy groups resulted in some of the polymer retaining its radioactivity, proving that some end groups were phenyl groups which where not bonded via ester linkages. These results supported the hypothesis that, at low monomer concentrations, the phenyl end group concentration increases because there is a greater chance that the benzoyloxyl radical will decompose rather than initiate a polymerization. For high monomer concentrations, initiation via benzoyloxyl free radicals, which form ester linkages and are easily hydrolysed. (101) 82

83 The esterification of poly(vinyl chloride) (PVC) and associated loss of labile chlorine was studed by Frye and Horst. (102) They prepared salts of 14 C 2-ethylhexanoic acid which where milled with samples of the PVC, heat treated, then precipitated from tetrahydrofuran using methanol. The precipitate showed a substantial level of radioactivity associated with the PVC indicating the displacment of the chloride as hydrochloric acid, by 14 C 2-ethylhexanoic acid. The study also showed that the degree of esterification was dependent on the salt of the ethylhexanoic acid used, the temperature and duration of the treatment. 22. Polymer Purification A polymer s solubility is highly dependant on its molecular weight and chain length, so purification of a polymer by precipitation can cause fractionation of the polymer. Low molecular weight chains are more soluble than higher molecular weight chains. Using 14 C labelled initiators, monomers or polymers, the efficiency of the purification/fractionation of a polymer can be assessed. Baines and Bevington (103) carried out a number of studies into the precipitation and fractionation of polymers, and copolymers which had been prepared using either ring labelled 14 C benzoyl peroxide or 14 C methyl methacrylate. The fractionation and subsequent hydrolysis of a 14 C initiated low molecular weight poly(styrene) showed that the polymer fractionated in accordance with its molecular weight with 95% of the polymer mass and 97% of the radioactivity being recovered. The fraction of benzoyloxy groups present was >0.95 and termination was by combination, with 2 initiator fragments per polymer chain. A mixture of equal masses of non-labelled polystyrene (Mn g mol -1 ) and 14 C poly(methyl methycrylate) (Mn gmol -1 ) were separated by precipitation, which was found to be highly efficient, with 94% to 96% recovery of 14 C methyl methacrylate. 83

84 23. Applications of Radiolabelled Polymers: Personal Products and Home Care Industries. As a 14 C radioisotope is unaffected by the physical or chemical properties of a system. It can be used to detect compounds at extremely low concentrations, the personal and home care industries have used radioisotopes to quantify the deposition, substantivity and penetration of ingredients present in their formulations Hair Applications The deposition and substantivity onto human hair of two samples of 14 C poly(dimethyl dialkyl ammonium chloride) (104) with molecular weights of gmol -1 (MERQUAT 100) and gmol -1 (MERQUAT 500) were determined. At 22 C and 40 C for a 5 minute application both molecular weights were found to be substantive to hair. MERQUAT 100 and 500 had substantivities of 1.32 and 1.80 and 5.44 and 7.4 micro grams per milli gram of hair. Similar studies were carried out using 14 C poly(ethylene imine) which showed the polymer was also highly substantive. (105) 23.2 Laundry Applications The substantivity of 70/30 14 C poly(methacrylic acid/ethyl acrylate) or 14 C poly(methacrylic acid), on to cotton and polyester fabrics, which where either free from or had been treated with dimethylol dihydroxyethyleneurea, was determined. The polymer substantivity on cotton and polyester was higher in the presence of dimethylol dihydroxyethyleneurea than in its absence. It was believed that the dimethylol dihydroxyethyleneurea acted as a coupling agent which assisted the polymers to bind to the cotton and polyester. (106) 23.3 Applications of Radiolabelled Polymers: Medical Applications Polymers used for the manufacture of implants it is important to know how these polymers behave both physically and chemically within the body. A number of radiolabel studies have been carried out to ascertain polymer biodegradability. Biodegradability studies of 3 H poly(lactic acid) (PLA) and 14 C poly(glycolic acid) (PGA) and their copolymers, were carried out using rats which had polymer pellets implanted into their soft and bone tissues. Over eleven months, groups of five rats were sacrificed and the activity levels in and around the implant areas, organs, muscle, as 84

85 well as in urine and faeces were determined. From the results the polymers biodegradability, half-life and sites within the body where polymer accumulated were assessed. For 100% PGA the half-life was 5 months, decreasing to 1 week for a 50:50 copolymer. The half life then increased to 6 months for 100% PLA. Tissue type had no effect on the rate of biodegradation and no significant levels of radioactivity were detected in excreta. (107) Similar studies were carried out for 14 C polyester amides and 14 C monomers. They showed the polymer biodegraded over a 12 month period, whilst the monomers took between 3-16 days. In both cases, the rate of biodegradation was dependent on water solubility. Samples of urine and exhaled 14 CO2 were monitored/analysed during the course of the study to quantify and identify the metabolites. (108) The elimination of sterilized 14 C poly(2-hydroxypropyl methacrylamide) (109) from the blood stream of rabbits could be determined over a period of 10 days by taking urine and blood samples and measuring the radioactivity. Over time, the levels in blood decreased, but those in urine increased. The rate at which the polymers were removed was dependent on the molecular weights and limits of the kidneys filtration system. The limiting molecular weight/molecule size of the kidneys is g mol -1. If the molecular weights were below these limits they were removed naturally. Polymers with molecular weights greater than the kidney limits stayed in the reticulo-endothelial system until they biodegraded to lower molecular weight species Applications of Radiolabelled Polymers: Pharmacological One use of polymers or polymer nanoparticles is to deliver drugs. They are designed so they can deliver a drug to a targeted tissue or organ, or enable the drug to be retained by the body increasing the drugs delivery time. To ensure the safety of the polymers or nano particles, Absorption, Distribution, Metabolism and Excretion (ADME) pharmacokinetics and toxicity studies have to be carried out. One method is to carry out the ADME studies using 14 C monomers, polymers or nanoparticles. 85

86 Polymers Mice were injected with 14 C malic and poly(malic) acids having various molecular weights. The biodegradability and metabolic elimination were quantified by monitoring the levels of radioactivity present in CO2 emissions, urine and blood over a 24 hour period. After five minutes, blood samples indicated there was a rapid increase in radioactivity levels which then levelled off to a constant value. Within an hour the monomer led to rapid 14 CO2 exhalation and negligible renal excretion. For the polymer, 70% of the injected radioactivity was recovered via renal excretion but no 14 CO2 was emitted. The mice where sacrificed at set time intervals over 24 hours to determine the levels and distribution of radioactivity in the body s tissues. The pharmacokinetics of poly(malic acid) where found to be dependent on the polymer molecular weight. Polymers with molecular weights lower than the limits of kidney filtration of g mol -1 are metabolised and eliminated in urine. The polymers with molecular weights higher than the limits of kidney filtration are circulated around the body by the blood system either to be incorporated into the body s tissues or pass out via the kidneys once the polymer had biodegraded to a lower molecular weight. (110/111) The breakdown of polymeric micelles comprising copolymers of poly(ethylene oxide), poly(aspartic acid) and covalently bound Adriamycin, (112) was investigated by using a micelle labelled with 14 C benzylamine, which formed a stable amide bond with free carboxyl groups which mimicked that between the micelle and the drug. ADME experiments were carried out to quantify levels of radioactivity in the blood, liver, spleen and other mice organs. The micelles were found to have a long circulation time, 68% persisting for 4 hours and 10% for 24hours, which was thought to be due to micelles retaining their structure and showing drugs can be delivered over a 24 hour period. Polymers that were not retained were excreted rapidly via urine Nanoparticles Nanoparticles are designed to carry and distribute active agents and are currently used in the pharmaceutical, medical, and cosmetic industries. The application of nanoparticles is relatively new, as such, their potential impact on human health is generally unknown. The use of highly sensitive radiotracer methodologies is well suited to study the ADME of these materials. Reviews (113,114) have been published on the application of radiotracer techniques to pharmacology and toxicology studies of 86

87 nanomaterials such as fullerene, endohedral fullerene and carbon nanotubes, and of the use of nanoparticles in nuclear medicine. A number of papers have also been published in which radiochemical methods were utilised to ascertain the physical behaviour of a number of different polymer derived nanoparticles. The bio distribution of 14 C fullerenes (115) and their ammonium salts where studied by injecting the compounds directly into the blood stream of rats. Within a minute, clearance from the blood was seen, accumulating (90-95%) in the liver which was still present after 120 hours. This indicated that these materials were not metabolized and no passage across the blood brain barrier was observed. The hydrophilic ammonium derivative remained in the blood stream, enabling it to deposit in various tissues, though not in the brain. However, for hydrophilic 14 C derivatives (116), most of the material was retained in the rat s body, and there was evidence that the hydrophilic 14 C derivatives had crossed the blood brain barrier, suggesting the solubility of these derivatives affected circulation and retention within the body. 14 C multiwalled carbon nanotubes (117) were found to clear rapidly from the blood system, with high levels of accumulation in the lungs, spleen and kidneys but none in the brain. A number of papers report the ADME, biodistribution and blood brain barrier penetration of nano particles. A study of the biodistribution and accumulation of 14 C poly(methyl methacrylate) (118) in rats introduced by either intravenous injection or subcutaneous injection. Within 30min of the intravenous injection of 14 C poly(methyl methacrylate) into rats, it was found to have distributed throughout the body with the highest accumulation (22%) in the lungs. After seven days, with the exception of liver, spleen and bone marrow, the distribution levels of 14 C poly(methyl methacrylate) had dropped, decreasing to 13% for the lungs. For the liver, spleen and bone marrow the levels increased, since these organs belong to the reticuloendothelial system responsible for the clearance of colloids and particulate matter from the blood. Excretory products (faeces, urine and breath) accounted for 5.3% of the 14 C poly(methyl methacrylate). For subcutaneous injection the majority (50-70%) of the 14 C poly(methyl methacrylate) was retained around the injection site and, for the rest of the body, again the highest levels were found in the liver, spleen and bone marrow, renal fatty tissue, skin and lymph nodes. The activity in the lymph nodes was particularly high. This result was also observed for the intraperitoneal injection of 14 C poly(methyl methacrylate) (119) where 87

88 the levels in the lymph nodes were times higher than the levels found for intravenous injection. The accumulation of 14 C poly(methyl methacrylate) in the lymphatic tissues may prove valuable in designing drug carriers for targeting tumour laden lymph nodes. Similar results were found for 14 C poly(hexyl cyanoacrylate) (119) The bio degradation and body distribution of human albumin covered poly(lactic acid) (120) and poly(methylidene malonate) (121) were determined via the intravenous injection into rats of the equivalent 14 C labelled polymers. The polymers where found in the liver, spleen, bone marrow, lymph nodes and also in excretory products. 14 C labelled versions of the nanoparticles of poly(cyanoacrylate) (122) with and without poly(ethylene glycol) and poly(butyl cyanoacrylate) (123) and with and without poly(sorbate) 80, were used to determine passage of the nanoparticles through the blood brain barrier into the central nervous system. In both cases the addition of the poly(ethylene glycol) and poly(sorbate) 80 enhances the passage of the poly(cyanoacrylate) and poly(butyl cyanoacrylate) across the blood brain barrier into the central nervous system compared to the poly(cyanoacrylate) and poly(butyl cyanoacrylate) without a poly(ethylene glycol) or poly(sorbate) Applications of Radiolabelled Polymers: Environmental Studies of Labelled Polymers As polymers are used for a wide range of applications, their environmental impact and disposal have been investigated using 14 C and 3 H labelled polymers. 14 C labelled polymers which undergo environmental monitoring are subjected to water distribution, adsorption and desorption tests. The biodegradation of the polymers via SCAS (semicontinuous activated sludge), Sturm and Bioaccumulation tests can be determined. SCAS tests involves the aeration of a mixture of a radiolabelled compound and activated sludge the the 14 CO2, liberated trapped and the levels quantified. 14 C labelled components in the liquors are also quantified and analysed. A Sturm test involves the mixing of a radiolabelled compound with soil which is then incubated and the 14 CO2, liberated trapped and the levels quantified. Bioaccumulation test involves feeding biological organisms such as fish with a radiolabelled 14 C test material compound then analyze the organism to quantify the uptake of the radiolabelled 14 C test material. 88

89 These tests can be run separately or in combination to assess the complete environmental impact of the polymer. Malik and Letey (124) investigated the adsorption of poly(acrylamide) and poly(saccharide) onto soils. They carried out sixteen hour distribution, adsorption and desorption tests of samples of 3 H poly(acrylamide) and 3 H poly(saccharide) (guar) polymers using a number of soil types. Results indicated that both sets of polymers did not penetrate soils and therefore the deposition for each soil type was approximately the same. However the adsorption was affected by the polymer molecular weight, and conformation. As the molecular weight and chain length increased, so did their level of deposition. Poly(ɛ-caprolactone) is a commercial polymer used for many applications, therefore knowledge of the biodegradability of the polymer is important. SCAS tests were carried out for 3 H/ 14 C poly(ɛ-caprolactone), (125,126) and 14 C poly(3-hydroxybutyrate-c-3- hydroxyoctanoate). (126) Poly(ɛ-caprolactone) was completely biodegradable, but due to its long lag time, and limited rate of hydrolysis, the rate of biodegradation was very slow. After 122 days, relatively little of the polymer had biodegraded. The poly(3- hydroxybutyrate-c-3-hydroxyoctanoate) was biodegradable in major anaerobic habitats. To determine the biodegradation of poly(ethylene) soil incubation studies were carried out using two sets of tritiated polyethylene disks. Set 1 was made up of 100% poly(ethylene) and set 2 15% oxidized poly(ethylene) /10% starch. (127) All plastic disks where incubated using sterile and non-sterile soils. On completion, biodegradability was determined by measuring the levels of 3 H in water and soluble metabolites. The soil was oxidized to quantify 3 H particulates. Both polymers were found to degrade slowly although the disks containing 10% starch degraded more quickly and to a greater extent compared to 100% poly(ethylene). To help assess the biodegradation of polymeric materials within the world s oceans, Sturm tests were carried to determine the biodegradation of 14 C poly(hydroxy butyrate) using a sea water and sediment system with and without the addition of nutrients. After 550 hours 70% of the 14 C poly(hydroxy butyrate) was found to biodegrade with 89

90 nutrients whereas only 10% of the 14 C poly(hydroxy butyrate) biodegraded after 300 hours without nutrients. (128) With the increasing use of polymers for medical, agricultural and environmental applications, it is important to design and produce polymers that can be biodegraded or biorecycled by bioassimilation to protect the environment. Therefore bio assimilation studies of 14 C uniformly labelled poly(lactic acid) were carried out over 12 days using earthworms. The 14 CO2 liberated was trapped and counted to determine the extent of the accumulation and digestion of the polymer. The presence of bioaccumulated polymer degradation products was shown by autoradiographing sacrificed earthworms. (129) SCAS, Sturm and bioaccumulation tests were carried out using fungi, soil invertebrates variety of wastes: sludges, manure and general household waste to determine the biodegradation of 14 C-labelled poly(methylmethacrylate), poly(phenol), poly(formaldehyde) and poly(styrene). (130) 35 day SCAS and 11 week Sturm tests indicated the degradation of the polymers was 0 to 0.29%, or 0.04 to 0.57% respectively for poly(methyl methacrylate), 0 to 0.17% or 0 to 0.15% for poly(phenol) and poly(formaldehyde) and 0 to 0.24% or % for poly(styrene), showing these polymers were not biodegradable. Bioaccumulation was zero as the soil invertebrates were unable to digest the polymers. (130) 14 C labelled poly(carboxylates), one an emulsion polymer with a molecular weight of gmol -1, the other a resin polymer with a molecular weight of gmol -1, were studied using all three tests. (131,132) The deposition study indicated that, within 2 hours, 95% of the emulsion polymer and 74% of the resin polymer adsorbed to the sludge from waste water. Desorption was low and reached equilibrium over 48 hours for both polymers. SCAS tests showed that 1% of the polymers were in the aqueous supernatant and 96-99% adsorbed to the sludge. A small percentage, ranging from 2.4% (day 1) - 4.5% (day 60), remained in the suspended solid phase. The Sturm test showed there was little biodegradation, recovery of 14 CO2 being at 0.85% for the emulsion polymer and 2.64% for the resin polymer, based on the original polymer concentrations. 90

91 24. Conclusion Between 1950 and the early 1970s, due to the great accuracy and sensitivity that could be attained, and relatively easy acess and availiability, radioisotopes were used in the understanding and development of free radical polymer chemistry. By using radiolabelled initiators, monomers or polymers, the mechanisms of the three steps of free radical polymerization (initiation, propagation and termination), the chemical and physical properties of polymers, their stability, and purification have all been studied. With the advent of computerization, new analytical techniques and the increases in cost and legislative requirements relating to the use of radioisotopes, there has been a steady decline in the use of radioisotopes not only in polymer chemistry but in many different areas of science. Consequently, radioisotopes have been used sparingly especially in the growth areas of polymer science, such as controlled polymerization. However as will be shown in this thesis, radioisotopes are uniquely suited to investigate the mechanistic aspects of ATRP, the mode of action of polymeric ingredients, and the chemical and physical properties of products/by products. The application of radiochemical methods can be applied to the investigation of other controlled polymerization techniques such as Nitroxide Mediated Polymerization (NMP) Reversible Addition Fragmentation chain Transfer (RAFT) polymerization and Iodine-Transfer Polymerization (ITP). 91

92 References 1. H.F. Pfann, D.J. Salley, H. Mark, J. Am. Chem. Soc., 1944, 66, Y. Landler, Rec. Trav. Chim., 1949, 68, Y. Landler, M. Magat, B. Soc. Chim. Belg., 1948, 57, W.V. Smith, H.N. Campbell, J. Chem. Phys., 1947, 15, W.V. Smith, J. Am. Chem. Soc., 1949, 71, W.E. Mochel, J.H. Peterson, J. Am. Chem. Soc., 1949, 71, G. Ayrey, Chem. Rev., 1963, 63, Preparation of Compounds Labelled with Tritium & Carbon 14, R. Voges, J.R.Heys, T. Moenius, Wiley, G. Shemilt, M. Newby, S.L. Kitson, J. Labeled Compd. Rad., 2007, 50, G. Ayrey, C.G. Moore, J. Polym. Sci., 1959, 36, J.C. Bevington, J.H. Bradburry, G.M. Burnett, J. Polym. Sci., 1954, 12, J.C. Bevington, J. Polym. Mater., 1993, 10, J.C. Bevington, B.J. Hunt, C.A. Barson, Macromol. Symp., 1996, 111, J.K. Allen J.C. Bevington T. Faraday Soc.,., 1960, 56, D.C. Blackley, H.W.Melville, Makromolekul. Chem., 1956, 18, R.H. Wiley, E.E. Sale, J. Polym. Sci., 1960, 42, R.H. Wiley, B. Davis, J. Polym. Sci., 1962, 62, S J.C. Bevington, M. Johnson, Eur. Polym. J., 1968, 4, 6, J.C. Bevington, Makromol. Chem-M. Symp., 1988, 20/21, J.K. Allen, J.C. Bevington, Proce. Roy. Soc. Lon. Ser. A., 1961, 262, J.C. Bevington, Makromolekul. Chem., 1959, 34, J.C. Bevington, J. Toole, L. Trossarelli, T. Faraday Soc., 1958, 54, JC Bevington J Toole L Trossarelli Makromolekul. Chem., 1958, 28, J.K. Allen, J.C. Bevington, Polymer, 1961, 2, J.C. Bevington, T.D. Lewis, Faraday Soc., 1958, 54, J.C. Bevington, J. Toole, L. Trossarelli, Makromolekul. Chem., 1959, 32, J.C. Bevington, T.D. Lewis, Polymer, 1960, 1, L.M. Arnett, J.H. Peterson, J. Am. Chem. Soc., 1952, 74, J.C. Bevington, H.W. Melville, R.P. Taylor, J. Polym. Sci., 1954, 12, J.C. Bevington H.W. Melville R.P. Taylor J. Polym. Sci., 1954, 14, J.C. Bevington, J. Chem. Soc., 1954, J.C. Bevington, Simp. Intern. Chem. Macromol. (Milan)., 1954, 186, 3. 92

93 33. G. Ayrey, K.L. Evans, D.J.D. Wong, Eur. Polym. J., 1973, 9, J.C. Bevington, H.G. Troth, T. Faraday Soc., 1962, 58, J.C. Bevington, T. Faraday Soc., 1955, 51, K. Ziegler, W. Deparade, W. Meye, Anal. Chem., 1950, 567, M. Talat-Erben, S. Bywater, J. Am. Chem. Soc., 1955, 77, J.C. Bevington, J. Polym. Sci., 1958, 29, J.P. Kennedy, J. Polym. Sci., 1959, 38, C.A. Barson, J.C. Bevington, J. Polym. Sci. Pol. Chem. 1997, 35, J.C. Bevington, D.O.Harris, M. Johnson, Eur. Polym. J., 1965, 1, J.C. Bevington, T. Ito, T. Faraday Soc.; 1968, 64, J.C. Bevington, Proce. Roy. Soc. Lon. Ser. A., 1957, 239, J.C. Bevington, J. Toole, J. Polym. Sci., 1958, 28, J.C. Bevington, T.D. Lewis, T. Faraday Soc., 1958, 54, J.C. Bevington, B.W. Malpass, J. Polym. Sci. Part A., 1964, 2, J.C. Bevington, G. Ayrey, Eur. Polym. J., 1987, 23, C.A. Barson, J.C. Bevington, T.N. Huckerby, Polym. Bull., 1986, 16, C.A. Barson, J.C. Bevington, Tetrahedron, 1958, 4, J.P. Kennedy, R.M. Thomas, J. Polym. Sci., 1960, 45, B.L. Funt, W. Pasika, Canadian J. Chem., 1960, W.H. Stockmayer, L.H. Peebles, J. Am. Chem. Soc., 1953, 75, L.H. Peebles, J.T. Clarke, W.H. Stockmayer, J. Am. Chem. Soc., 1960, 82, J.W. Breitenbach, G. Billek, E. Faltlhansl, E. Weber, Monatsh. Chem., 1961, 92, G. Henrici-Olive, S. Olive Makromolekul. Chem., 1962, 51, S. Bywater, T. Faraday Soc., 1955, 51, P.I. Levin, V.B. Miller, I.V. Nevskii, M.B. Neiman, V.A. Kargin, E.E. Rylov,T.R. Khim. Khim. Tekhnol., 1959, 2, G. Ayrey, Adv. Polym. Sci., 1969, 6, J.C. Bevington, B.W. Malpass, Eur. Polym. J., 1965, 1, J.C. Bevington, B.W. Malpass, Eur. Polym. J., 1965, 1, J.C. Bevington, C.S. Brooks, Makromolekul. Chem., 1958, 28, C.A. Barson, J.C. Bevington, Eur. Polym. J., 1987, 23,

94 63. L.R. McNall, L.T. Eby, Anal. Chem., 1957, 29, E.G. Bobalek, J.R. Bradford, F. Leutner, R. Akiyama, Anal. Chem., 1956, 28, J.C. Bevington G.M. Guzmann H.W. Melville Proc. Roy. Soc. Lon. Ser. A., 1954, 221, J.C. Bevington, G.M. Guzmann, H.W. Melville, Nature, 1952, 170, J.C. Bevington, G.M. Guzmann, H.W. Melville, Proc Roy. Soc. Lon. Ser. A.,1954, 221, H.W. Melville, P.R. Sewell, Makromolekul. Chem., 1959, 32, M.H. Jones, H.W. Melville, W.G.P. Robertson, Ricerca. Sci. Suppl. Simp. Init.Chim., 1954, 25, H.W. Melville, F.W. Peaker, R.L. Vale, Makromolekul. Chem., 1958, 28, N.A. Ghnem, Makromolekul. Chem., 1960, 36, F.M. Merret, T. Faraday Soc., 1954, 50, P.W. Allen, G. Ayrey, C.G. Moore, J. Polym. Sci., 1959, 36, G. Smets, J. Rooves, W.J. Van Humbeek, Appl. Polym Sci., 1961, 5, J.C. Bevington, D.E. Eaves, Nature, 1956, 178, G. Natta, I. Pasquon, Adv. Catal. 1959, Acedemic Press New York. 77. G. Natta, I. Pasquon, L. Guiffre, Chem. Ind. - Milan, 1961, 43, C.A. Barson, J.C. Bevington, B.J. Hunt, J. Polym. Sci. Pol. Chem., 1995, 33, C.A. Barson, J.C. Bevington, B.J. Hunt, J. Polym. Sci. Pol. Chem., 1996, 34, C.A. Barson, J.C. Bevington, B.J. Hunt, Polymer, 1998, 39, J.C. Bevington, N.A. Ghanem, H.W. Melville, J. Chem. Soc., 1955, J.C. Bevington, N.A. Ghanem H.W. Melville, T. Faraday Soc., 1955, J.C. Bevington, N.A. Ghanem, Makromolekul. Chem., 1961, 43, J.C. Bevington, N.A. Ghanem J. Chem. Soc., 1959, B.L. Funt, F.D. Williams, J. Polym. Sci., 1962, 57, B.L. Funt, F.D. Williams, J. Polym. Sci., 1960, 46, J.C. Bevington, N.A. Ghanem J. Chem. Soc., 1958, J.C. Bevington, Nature, 1955, 175,

95 89. J.C. Bevington, J. Chem. Soc., 1956, J.C. Bevington, N.A. Ghanem, J. Chem. Soc., 1956, G. Ayrey, A.C. Haynes, Eur. Polym. J. 1973, 9, G. Ayrey, F.G. Levitt, R.J. Mazza, Polym. J., 1965, 6, G. Ayrey, M.J. Humphrey, R.C. Poller, Polymer, 1977, 18, J.C. Bevington, D.E. Eaves, T. Faraday Soc., 1959, 55, F.W. Morthland, W.G. Brown, J. Am. Chem. Soc., 1956, 78, B.E. Bailey, A.D. Jenkins, T. Faraday Soc., 1960, 56, G. Ayrey, B.C. Head, D.J.D. Wong, Eur. Polym J., 1974, 10, A. Todd, J. Polym. Sci., 1960, 42, A.B. Robertson, H.J. Harwood, Polymer, Preprints, ACS., 1971, 12, H.J. Harwood, A.B. Robertson, Int. Symp. Macromol. Chem. Prep., 1969, 5, J.C. Bevington, C.S. Brooks, J. Polym. Sci., 1956, 22, A.H. Fry, R.W. Horst, J. Polym. Sci. 1960, 45, F.C. Baines, J.C. Bevington, Eur. J., 1967, 3, A.R. Sykes, Drug, Cosmet. Ind., 1980, J. Woodward, J. Soc. Cosmet. Chem., 1972, 23, C.E. Warburton, Jr. L.T. Flynn, J. Appl. Polym. Sci., 1972, 16, R.A. Miller, J.M. Brady, D.E. Cutright, J. Biomed. Mater. Res., 1977, 11, T.H. Burrows, G.J. Quarfoth, P.E. Slegen, R.L. McQuinn, Pol. Mater. Sci., and Eng., 1988, 59, J. Kopecek, L. Sprincl, D. Lim, J. Biomed. Mater. Res P. Fourine, D. Domurado, J. Bioact. Compat. Pol., 1992, 7, D. Domurado, P. Fourine, C. Braud, M.Vert, J. Bioact. Compat. Pol., 2003, 18, G.S. Kwon, M. Yokoyama, T. Okano, Y. Sakurai, Pharm. Res., 1993, 10, Z. Zhiyong, Z. Yuliang, Z. YuLiang, C. ZhiFang, Chinese, Sci. Bull., 2009, 54, M. V. Korsakov, F. Bechthold, Radiochemistry, 2000, 42, R. Bullard- Dillard, K.E. Creek, W.A. Scrivens, J.M. Tour, Top. Curr. Chem., 1996, 24,

96 116. S. Yamago, H. Tokuyama, E. Nakamura, K. Kikuchi, S. Kananishi, K. Sueki, H. Nakahara, S. Enomoto, F. Ambe, Chem. Biol., 1995, 2, D. Georgin, B.Czarny, M. Botquin, M.L. Hermite, M. Pinault, B. Bouchet-Fabre, M.Carriere, J.L. Poncy, Q.Chau, R. Maximillen, V. Dive, F. Taran, J Am. Chem. Soc., 2009, 131, J. Kreuter, Pharm. Acta. Helv., 1983, 58, P. Maincent, P. Thouvenot, C. Amicabile, M. Hoffman, J. Kreuter, P. Couvreur, Pharm. Res. 1992, 9, D.V. Bazile, C. Ropert, P. Huve, T. Verrecchia, M. Marland, A. Fryman, M. Veillard, G. Spenlehauer, Biomaterials, 1992, 13, F. Lescure, C. Seguin, P. Breton, P. Bourrinet, D. Roy, P.Couvreur, Pharm. Res., 1994, 11, P. Calvo, B. Gouritin, H. Villarroya, F. Eclancher, C. Giannavola, C. Klein, J.P. Andreux, P. Couvreur, Eur. J. Neurosci., 2002, 15, A. Ambruosi, H. Yamamoto, J. Kreuter, J. Drug, Target., 2005, 13, M. Malik, J. Letey, Soil, Sci. Soc. Am. J., 1991, 55, S. Ponsart, J. Coudane, B. Saulnier, J.L. Morgat, M.Vert, Biomacromolecules., 2001, 2, T.W. Federle, M.A. Barlaz, C.A. Pettigrew, K.M. Kerr, J.J. Kemper, B.A. Nuck, L.A. Schechtman, Biomacromolecules., 2002, 3, T.A. Anderson, D.M. Scherubel, R. Tsao, A.W. Schwabacher, J.R. Coats, J. Environ. Polym. Degr. 1997, 5, J. A. Ratto, J. Russo, A. Allen, J. Herbert,C. Wirsen, Am. Chem. Soc. Symposium, S., 2001, 786, M. Vert, I. Dos. Santos, S. Ponsart, N. Alauzet, J.L. Morgat, J. Coudane, H. Garreau, Polym. Int., 2002, 51, D.L. Kaplan, R. Hartenstein, J. Sutter, Appl. Environ. Microb., 1979, 38, K.M. Jop, P.D. Guioney, K.P.Christensen, E.M. Silberhorn, Ecotox. Environ. Safe., 1997, 37, M.B. Freeman, T.H. Bender, Environ. Tech. 1993, 14,

97 CHAPTER 5 UTLIZING 14 C-RADIOLABELLED ATOM TRANSFER RADICAL POLYMERIZATION INITIATORS 97

98 Utilising 14 C-radiolabelled atom transfer radical polymerisation initiators Chemical Communications 2009, DOI: /B913294E, Communication Contribution Mark Long: Student Principle Author First Author Executed All Practical Work David W Thornthwaite: Unilever PhD Supervisor: Organic Chemistry Suzanne H Rogers: Advised on the Practical ATRP Methodology: Taught Mark Long to carry out ATRP Polymerizations Gwénaëlle Bonzi: Advised on the Measurements of the Kinetics of ATRP Francis R Livens: Manchester PhD Supervisor: Radiochemistry Steve P Rannard: Unilever PhD Supervisor: Polymer Chemistry Communication Abstract 14 C-radio-initiated atom transfer radical polymerisations allow direct monitoring of the fate of initiating species The successful synthesis of radiolabelled ATRP initiator 14 C benzyl 2-bromoisobutyrate, using either 14 C benzyl alcohol or 14 C bromoisobutyric acid and their subsequent use to prepare 14 C radiolabelled samples of poly 2-hydroxy propyl methacrylate (poly(2- HPMA)) with degree of polymerization DP 10, 25, 50 was reported. Radio thin layer chromatography (R-TLC), liquid scintillation counting of fractions collected from gel permeation chromatography (GPC), were used to determine the fate of the initiating species during the ATRP of 14 C 2-HPMA. GPC and R-TLC data showed that there is an under-utilisation of the initiator. For the first time the use of radiolabelled initiators has enabled, the detection and quantification of terminated and low molecular species produced during an alcoholic ambient ATRP showing the reduced efficiency of the initiators. 98

99 Citations Unimolecular ligand initiator dual functional systems (ULIS) for low copper ATRP of vinyl monomers including acrylic/methacrylic acids Satyasankar Jana, Anbanandam Parthiban and Foo Ming Choo Chem. Commun., 2012, 48, 4256 DOI: /c2cc16663a Exploring the homogeneous controlled radical polymerisation of hydrophobic monomers in anti-solvents for their polymers: RAFT and ATRP of various alkyl methacrylates in anhydrous methanol to high conversion and low dispersity A. B. Dwyer, P. Chambon, A. Town, F. L. Hatton, J. Ford and S. P. Rannard Polym. Chem., 2015, 6, 7286 DOI: /C5PY00791G Selective Isotope Labelling of Leucine Residues by Using α-ketoacid Precursor Compounds Roman J. Lichtenecker, Nicolas Coudevylle, Robert Konrat and Walther Schmid ChemBioChem, 2013, 14, 818 DOI: /cbic Note: Cross Citations between the papers published for this thesis have been excluded Prior Art As stated in the communication the use of 14 C radiolabel to determine the fate if ATRP initiators is unique in the field of ATRP and calls into question the ATRP theory that states all initiator is used simultaneously at the start of a polymerization. 99

100 View Article Online / Journal Homepage / Table of Contents for this issue COMMUNICATION ChemComm Published on 28 September Downloaded by UNILEVER RESEARCH PORT SUNLIGHT on 29/01/ :51:27. Utilising 14 C-radiolabelled atom transfer radical polymerisation initiatorsw Mark Long, a David W. Thornthwaite, a Suzanne H. Rogers, a Gwe naëlle Bonzi, b Francis R. Livens c and Steve P. Rannard* b Received (in Cambridge, UK) 3rd July 2009, Accepted 10th September 2009 First published as an Advance Article on the web 28th September 2009 DOI: /b913294e 14 C-radio-initiated atom transfer radical polymerisations allow direct monitoring of the fate of initiating species. Since its earliest reports, 1a c atom transfer radical polymerisation (ATRP) has grown to become a leading controlled radical polymerisation technique in many academic and industrial research groups. The advent of aqueous and alcoholic ATRP 1d f significantly broadened the scope of the technique, allowing water-soluble polymers to be controllably synthesised at ambient temperatures under protic solvent conditions. Mechanistically, ATRP has been studied to determine reaction pathways, 2a enabling many of the complex architectures that have been reported. 2b f The almost ubiquitous 2-bromoisobutyryl bromide, 1, may be reacted with any hydroxyl to generate an ATRP initiating site, 3a allowing control of polymer chain-end structure. Together with controlled polymer synthesis, this has allowed systematic changes of structure and architecture and enabled the fundamental study of factors affecting specific polymer behaviour; for example, stimuli-responsive chain assembly. 3b,c Many investigations of polymer deposition, drug delivery and polymer nanoparticle formation have also been reported 4 although accurately quantifying the action, fate or even the presence of polymer in these studies remains a challenge. In several cases, the polymer has been dye-labelled 5 to aid observation but, by the very nature of the labelling, this technique modifies the polymer composition, solubility, response to stimuli and surface interactions. Radiolabels 6 have been utilised to aid polymer detection in studies such as mucoadhesion 6a or pharmaceutical delivery. This is often a post-treatment of pre-formed polymers, for example tritiation of labile protons, 6b,c chelation of heavy metal radioisotopes (e.g. 64 Cu), 6d methylation of amines 6e using 14 C-methyl iodide or iodination 6f,g using 125 I. These have the potential to modify polymer properties and behaviour. In contrast, substitution of 14 C for existing carbons 7 within the polymer building blocks (monomers/initiators) is probably the least intrusive labelling strategy. Selective labelling of the polymer end-groups also avoids statistical label incorporation, i.e. all labelled chains carry equal radioactivity. Here, we describe the first radiolabelled ATRP via the generation of 14 C-labelled 2-bromoisobutyric acid; the synthesis of two variants of an ATRP initiator, each with 14 C-labels at a different site; the utilisation of this initiator in ambient alcoholic-atrp and new observations, revealed through the ability to monitor selectively the fate of initiating groups. There are two potential strategies to 14 C-label ATRP initiators; the reaction of 1 with 14 C-labelled alcohols, Scheme 1A, and the reaction of 14 C-labelled 2-bromoisobutyric acid with any hydroxyl compound, Scheme 1C. The reaction of 1 with benzyl alcohol to form benzyl 2-bromoisobutyrate was conducted under conventional, unlabelled conditions to establish the optimum experimental techniques for high recovered yields (93%). To minimise the use of 14 C-labelled benzyl alcohol (0.02 moles), 2, the reaction was repeated (Scheme 1A, see ESIw) at a smaller scale (20%) a Unilever Research and Development Port Sunlight Laboratories, Quarry Road East, Bebington, Wirral, UK CH63 3JW b Department of Chemistry, University of Liverpool, Crown Street, Liverpool, UK L69 7ZD. srannard@liv.ac.uk; Fax: +44 (0) ; Tel: +44 (0) c School of Chemistry, The University of Manchester, Oxford Road, Manchester, UK M13 9PL w Electronic supplementary information (ESI) available: Experimental methodology, GPC analysis, NMR spectra, R-TLC data. See DOI: /b913294e Scheme 1 Strategies for synthesising 14 C-labelled ATRP initiators, (A) esterification of 14 C-labelled benzyl alcohol, (B) synthesis of 14 C-labelled 2-bromoisobutyric acid, (C) reaction of 14 C-labelled 2-bromoisobutyric acid with benzyl alcohol Chem. Commun., 2009, This journal is c The Royal Society of Chemistry 2009

101 View Article Online Published on 28 September Downloaded by UNILEVER RESEARCH PORT SUNLIGHT on 29/01/ :51:27. to form 3 (87%) as confirmed by nuclear magnetic spectroscopy (NMR) and radio thin-layer chromatography, R-TLC (Fig. 1A) (97% radiochemical yield, mci mg 1 specific activity, 96% radiochemical purity). 14 C-labelled 2-bromoisobutyric acid is required to allow a more general approach to forming labelled ATRP initiators. In order to avoid placing the radiolabel directly near a labile ester, through the use of 14 CO 2 and Grignard approaches, we chose to introduce the label via 14 C-methylation of diethyl 2-methylmalonate, 4, using 14 C-methyl iodide to form 5, Scheme 1B. As is typical within radiochemical reactions at this scale, a proportion of unlabelled methyl iodide was also used in order to ensure the optimum reaction of the 14 C-labelled material (see ESIw). Hydrolysis and subsequent decarboxylation of 5 led to the synthesis of 14 C-labelled potassium isobutyrate, 7, which underwent bromination to form the 14 C-labelled 2-bromoisobutyric acid, 8, in an overall 35% recovered chemical yield based on diethyl 2-methylmalonate (54% radiochemical yield, 15.6 mci mg 1 specific activity, 99% radiochemical purity). All steps were conducted under non-labelled conditions prior to radiochemical synthesis (see ESIw). The reaction of 8 with 1,1 0 -carbonyl diimidazole, 9, formed the bromoacid imidazolide, 10, which was reacted with benzyl alcohol to form the 14 C-labelled benzyl 2-bromoisobutyrate, 11 (56% radiochemical yield, 0.36 mci mg 1 specific activity, 98% radiochemical purity) (Fig. 1B). Comparison of the R-TLCs of 3 and 11 showed identical behaviour under optimised TLC conditions (see ESIw, Fig. S6/S16). 3 exhibited 98.3% of the total measured radioactivity within a single region (region 2, Fig. 1A), 0.9% within the baseline and 0.8% within a small decomposition peak; 11 showed 98.2% radioactivity in a single region (0.9% baseline; 0.9% decomposition). Fig. 1 Radio thin layer chromatography of 14 C labelled 2-bromoisobutyrate. (A) methylene-labelled initiator 3; (B) methyl-labelled initiator 11. Insets show background radioactivity. (Eluent: Et 2 O CH 3 CO 2 H 90/10 v/v.) Fig. 2 Monomer and polymer structures formed during this study: 2-hydroxypropyl methacrylate (HPMA), 12; unlabelled poly(hpma), 13; 14 C methylene-labelled poly(hpma), 14; 14 C methyl-labelled poly(hpma), 15. The benzyl alcohol derived, radiolabelled initiators (3, 11) and an analogous unlabelled initiator were used to initiate the ambient temperature methanolic ATRP of 2-hydroxypropyl methacrylate (HPMA), 12, producing polymers 13, 14 and 15 (Fig. 2). Several polymerisations using each initiator under identical conditions with targeted number average degree of polymerisation (DP n ) of 50 monomer units were conducted. Near identical kinetics, conversion, DP n and polydispersity were obtained for all polymers using each initiator variant (see ESIw, Fig. S17), confirming the absence of any observable impact on polymerisation from either radiolabel position. Gel permeation chromatography (see ESIw, Fig. S18) suggested a DP n of approximately 65 monomer units (relative to PMMA standards); ATRP often yields higher chain lengths than targeted. 1 Several reports suggest the loss of initiator, or the presence of termination reactions, in the initial stages of the polymerisation 2 when the concentration of Cu(II) species is particularly low and prior to established equilibrium between the dormant and active radical centres. 1 H NMR analyses gave an observed DP n of approximately 40 monomer units suggesting residual unreacted initiator within the purified sample. Conversions 495% were achieved for all polymerisations suggesting an actual DP n 4 47 units. The presence of a radiolabelled initiator residue allows a high sensitivity analysis of the resulting polymers and study of the fate of the initiator. A purely radioactivity-based detection method renders all unlabelled chemical species invisible to the analysis, so all signals from a radio-initiated ATRP must be related to reactions of the initiator. Initiators labelled at different sites (3 and 11) allow verification that degradation reactions, such as hydrolysis of the initiator, are not leading to observed radio species. R-TLC typically measures radiochemical purity (Fig. 1) but here it has been utilised to study polymers without normal purification techniques and at high monomer conversion (496%; target DP n = 10, 25 and 50 monomer units). When the samples were eluted with THF, no separation was observed (see ESIw, Fig. S19). A mixture of diethyl ether and acetic acid (90/10) led to an excellent separation of unreacted radio-initiator from the bulk sample (Fig. 3). The resulting R-TLCs can be divided into three clear regions: initiator, polymer and intermediate. The boundaries chosen for each region were derived from the pure initiator R-TLCs (Fig. 1A and B) and the apparent limits of the polymer separation at DP n = 50 (Fig. 3A and B). This journal is c The Royal Society of Chemistry 2009 Chem. Commun., 2009,

102 View Article Online Published on 28 September Downloaded by UNILEVER RESEARCH PORT SUNLIGHT on 29/01/ :51:27. Fig. 3 Radio thin layer chromatography of poly(hpma) with target DP n = 50 using (A) initiator 3; (B) initiator 11; (C) DP n = 25; initiator 11; (D) DP n = 10; initiator 11. Insets show intermediate region radioactivity. (Eluent: Et 2 O CH 3 CO 2 H 90/10 v/v.) Surprisingly, the intermediate region has significant radioactivity suggesting terminated radio-species, including oligomers or the restricted elution of some initiator during the R-TLC experiment. This is not observed for the initiators alone ( % activity). The combined radioactivity in the initiator and intermediate regions increases as lower DP n polymers are targeted. For DP n = 50, the average combined activity across both initiators (6 repeats) is 12.7% (3 = 13.3%; 11 = 12.2%) of the total activity, predicting chains of DP n = 57 monomer units. Lower target DP n polymers have considerably higher activity not associated with the polymer; DP n = 25, average 19.1%; DP n = 10, average 50.7%. The initiator region also follows these trends; DP n = 50 average 8.0%; DP n =25 average 11.9%; DP n = 10 average 36.1%. We recognise that a benzyl ester initiator is not ideal for the polymerisation of 12 in methanol, probably leading to the observed poor initiator efficiencies, however the ability to quantify the apparently unreacted initiator and species derived from the initiator is of value within mechanism studies. It is important to note that radiopolymers have a limited lifetime for study due to their physical degradation however we intend to develop this approach to derive new quantitative insight across various polymerisation techniques. We thank the Royal Society for an Industry Fellowship (SR) and Unilever for financial support. Notes and references 1(a) S. Wang and K. Matyjaszewski, J. Am. Chem. Soc., 1995, 117, 5614; (b) M. Kato, M. Kamigaito, M. Sawamoto and T. Higashimura, Macromolecules, 1995, 28, 1721; (c) T. Pintauer and K. Matyjaszewski, Chem. Soc. Rev., 2008, 37, 1087; (d) X.-S. Wang, S. F. Lascelles, R. A. Jackson and S. P. Armes, Chem. Commun., 1999, 1817; (e) I. Y. Ma, E. J. Lobb, N. C. Billingham, S. P. Armes, A. L. Lewis, A. W. Lloyd and J. Salvage, Macromolecules, 2002, 35, 9306; (f) S. McDonald and S. P. Rannard, Macromolecules, 2001, 34, (a) W. A. Braunecker and K. Matyjaszewski, Prog. Polym. Sci., 2007, 32, 93; (b) O. G. Schramm, G. M. Pavlov, H. P. van Erp, M. A. R. Meier, R. Hoogenboom and U. S. Schubert, Macromolecules, 2009, 42, 1808;(c) K. Ishizu, S. Takano, T. Murakami, S. Uchida and M. Ozawa, J. Appl. Polym. Sci., 2008, 109, 3554;(d) K.V.Bernaerts, C.-A. Fustin, C. Bomal-D Haese, J.-F. Gohy, J. C. Martins and F. E. Du Prez, Macromolecules, 2008, 41, 2593; (e) Y. Li, Y. Tang, R. Narain, A. L. Lewis and S. P. Armes, Langmuir, 2005, 21, 9946;(f) C. Ott, R. Hoogenboom and U. S. Schubert, Chem. Commun., 2008, (a) S. Jana, S. P. Rannard and A. I. Cooper, Chem. Commun., 2007, 2962; (b) Y. Li, R. Narain, Y. Ma, A. L. Lewis and S. P. Armes, Chem. Commun., 2004, 2746; (c) S. Liu and S. P. Armes, J. Am. Chem. Soc., 2001, 123, (a) M. L. Bruening, D. M. Dotzauer, P. Jain, O. Lu and G. L. Baker, Langmuir, 2008, 24, 7663; (b) D. M. Lynn, Adv. Mater., 2007, 19, 4118; (c) C. Alexander, Nat. Mater., 2008, 7, 767; (d) A. K. Bajpai, S. K. Shukla, S. Bhanu and S. Kankane, Prog. Polym. Sci., 2008, 33, 1088; (e) K. Stashevskaya, E. Markvicheva, S. Strukova, I. Prudchenko, V. Zubov and C. Grandfils, J. Microencapsulation, 2007,24, 129;(f) H.Liu,X.Hu,Y.Wang, X. Li, C. Yi and Z. Xu, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 2892; (g) T. He, D. J. Adams, M. F. Butler, A. I. Cooper and S. P. Rannard, J. Am. Chem. Soc., 2009, 131, 1495; (h) T. He, D. J. Adams, M. F. Butler, C. T. Yeoh, A. I. Cooper and S. P. Rannard, Angew. Chem., Int. Ed., 2007, 46, (a) M. Spiniello, A. Blencowe and G. G. Qiao, J. Polym. Sci., Part A: Polym. Chem., 2008, 46, 2422; (b) M. Mertoglu, A. Laschewsky, K. Skrabania and C. Wieland, Macromolecules, 2005, 38, (a) M. E. A. McGirr, S. M. McAllister, E. E. Peters, A. W. Vickers, A. F. Parr and A. W. Basit, Eur. J. Pharm. Sci., 2009, 36, 386; (b) J. C. Russell, J. R. Jones, T. A. Vick and P. W. Stratford, J. Labelled Compd. Radiopharm., 1993, 33, 957; (c) C. Postolache, L. Matei and R. Georgescu, J. Radioanal. Nucl. Chem., 2009, 280, 251; (d) G. Sun, J. Xu, A. Hagooly, R. Rossin, Z. Li, D. A. Moore, C. J. Hawker, M. J. Welch and K. L. Wooley, Adv. Mater., 2007, 19, 3157; (e) O. Oulanti, J. Widmaier, E. Pefferkorn, S. Champ and H. Auweter, J. Colloid Interface Sci., 2005, 291, 98; (f) N. Nasongkla, B. Chen, N. Macaraeg, M. E. Fox, J. M. J. Frechet and F. C. Szoka, J. Am. Chem. Soc., 2009, 131, 3842; (g) E. R. Gillies, E. Dy, J. M. J. Frechet and F. C. Szoka, Mol. Pharmaceutics, 2005, 2, (a) R. K. Zeidan, S. A. Kalovidouris, T. Schluep, R. Fazio, R. Andresini and M. E. Davis, Bioconjugate Chem., 2006, 17, 1624; (b) G. Shemilt, M. Newby and S. L. Kitson, J. Labelled Compd. Radiopharm., 2007, 50, 515; (c) R. G. Riley, J. D. Smart, J. Tsibouklis, J. A. Davis, G. Kelly, F. Hampson, P. W. Dettmar and W. R. Wilber, J. Biomed. Mater. Res., 2001, 58, Chem. Commun., 2009, This journal is c The Royal Society of Chemistry 2009

103 Page 1 of 15 Utilising 14 C-Radiolabelled Atom Transfer Radical Polymerisation Initiators Mark Long, David W Thornthwaite, Suzanne H Rogers, Gwénaëlle Bonzi, Francis R Livens and Steve P Rannard Supporting Information Instrumentation Analysis and Sample Preparation NMR All 1 H NMR and 13 C NMR spectroscopy was conducted using a Bruker Advance DRX500 spectrometer. Sample preparation for 1 H NMR involved approximately 2mg of the sample dissolved in 1ml of CDCl3, 13 C NMR involved approximately 50mg of the sample dissolved in 1ml of CDCl3 Liquid Scintillation Counter The analysis of all radioactive samples was conducted using a Packard Tri Carb 3100 TRC Liquid Scintillation Counter. The results are presented as Disintegrations Per Minute (DPM) All Liquid Scintillation samples comprised a 20ml scintillation vial containing a known mass of radioactive compound dissolved in an appropiate solvent and 10ml of Prosafe Liquid Scintillation Cocktail containing: 60-75% Phenyl Xylyl Ethane 20-40% Alcohol Ethoxylate 2-8% Alcohol Ether Phophate Ester % 2,5-Diphenyloxazole % 1,4-Bis(4-methyl-alpha-styryl)benzene Gel Permeation Chromatography (GPC) The GPC analyses were conducted in THF using instrumentation comprising: Agilent Series RID with a response time of < 0.2min 40 0 C Eppendorf Oven Jasco PU 1580 Pump Jasco AS 590 Auto Samplers Polymer Laboratories PLgel 5 µm mixed-c and PLgel 5 µm mixed-d columns with a PL guard column The eluent flow rate was 0.8 ml/min. Calibration was conducted using poly methyl methacrylate standards with molecular weights ranging between 1000 and 1.5 million. Radio TLC (R-TLC) R-TLC analysis was conducted using an AR 2000 BIOSCAN Radio TLC Imaging Scanner utilising a gas filled proportional counter filled with 90/10 Argon/Methane to detect the beta radioactive emissions from thin layer chromatography (TLC) plates. The TLC samples were (1.0% w/w) prepared in an appropriate solvent and eluted using Whatman TLC plates (Partisil LK6DF silica Gel 60A with FL indicator) with a silica thickness of 250um and 3cm pre adsorbent zone The plates were eluted using an appropriate solvent to a standard 15cm solvent front. After drying the plates were scanned using the AR 2000 BIOSCAN LSC. Mass Spectrometry Mass spectrometry was conducted using a Micromass LCT Mass Spectrometer using a cone voltage of 50 Volts. Samples were prepared in methanol at concentrations of approximately 1 mg ml -1

104 Synthetic Procedures Page 2 of 15 A) Synthesis of Benzyl 2-bromo isobutyrate To a 250ml one neck round bottomed flask containing a magnetic stirrer flushed with nitrogen was added dry dichloromethane (150ml), benzyl alchohol (0.1 mol), triethylamine (1.1eq) and dimethyl amino pyridine (0.013eq). The reaction mixture was cooled to 0 0 C using an ice water bath. With stirring, bromo isobutyryl bromide (1.1eq) was added dropwise to the reaction mixture using a pressure equalizing dropping funnel. Once this addition was complete, the reaction flask was left to stir for twenty four hours initially at 0 0 C but allowed to warm to room temperature. The reaction mixture was evaporated to remove the solvent to yield a crude yellow oil and a cream precipitate. Dilute hydrochloric acid and diethyl ether were added and the product was washed 4 times. Finally the mixture was washed with dilute sodium carbonate The combined diethyl ether layers were dried over sodium sulphate, filtered and evaporated to yield a bronze coloured oil product.the structure and purity of the final product was confirm by 1 H NMR, 13 C NMR and Mass Spectrometery. Chemical purity > 95%. Chemical Yield 23.9g (93%). Figure S1: 1 H NMR spectrum of Benzyl 2-bromo isobutyrate

105 Page 3 of 15 Figure S2: 13 C NMR spectrum of Benzyl 2-bromo isobutyrate ( p p m ) Figure S3: Mass Spectrum of Benzyl 2-bromo isobutyrate Theoretical Mass = 274 Daltons

106 Page 4 of 15 B) Synthesis of 3 The synthesis of 3 utlised benzyl ( 14 CH 2 ) alcohol ( moles, 13.56mCi) and followed the procedure described above. The radiochemical purity was determined by R-TLC using 3 eluents 90/10, Petroleum ether/ diethyl ether, 95/ Petroleum ether/diethyl ether and 100% dichloromethane. Total activity and specific activity were determined. Chemical Yield 4.5g (87%), Radiochemical Yield 97% Total Activity mci Specific Activity uci/mg Chemical Purity > 95% Radiochemical Purity 96% Figure S4: 1 H NMR spectrum of ( p p m )

107 Page 5 of 15 Figure S5: 13 C NMR spectrum of ( p p m )

108 Figure S6: R-TLC of 3 in various solvent mixtures Page 6 of 15 90/10, Petroleum ether: diethyl ether Counts Region mm 95/5, Petroleum ether:diethyl ether Counts Region mm 100% Dichloromethane Counts Region Bkg 1 Region mm

109 Page 7 of 15 C) Synthesis of Non Labeled 2-bromo isobutyric acid Step 1 Methylation of Diethyl Methyl Malonate To a 50ml one necked round bottom flask containing a magnetic stirrer was added of diethyl methyl malonate (5.9 mmol), sodium ethoxide (21% solution, 1.1eq) and ethanol (10ml) under nitrogen. The reaction mixture was stirred, and warmed to 50 0 C for 30 minutes. The reaction mixture was then cooled to room temperature. Methyl iodide (8.38 mmol, 1.4 eq) was added. The flask was flushed with nitrogen, sealed and warmed to 50 0 C for 4 hours. After cooling, the ethanol and unreacted methyl iodide were removed using a rotary evaporator. Dry diethyl ether was added to the residue and the sodium iodide was filtered to yield a clear crude solution of diethyl dimethyl malonate. The diethyl ether was washed with water to remove residual sodium iodide. The diethyl ether layer was dried over magnesium sulphate, filtered and evaporated to yield a clear bronze coloured liquid. The structure and chemical purity of the final product was confirmed by 1 H NMR Chemical Yield 0.81g (73%), Chemical Purity > 95% Figure S7: 1 H NMR spectrum of diethyl dimethyl malonate Step 2 Hydrolysis of diethyl dimethyl malonate To the diethyl dimethyl malonate, was added 2.4eq of an aqueous solution of Potassium Hydroxide (10ml). The solution was warmed to 80 0 C with stirring until the oil was completely dissolved. Step 3 Decarboxylation of the di-potassium salt of dimethyl malonate To the aqueous solution of the di-potassium salt of dimethyl malonate was added an aqueous solution of sulphuric acid(2.4 eq, 20ml). The mixture was refluxed for five hours. The resulting isobutyric acid was recovered via distillation (azeotroped with water). To the water/isobutyric acid mixture potassium hydroxide was added (1.2eq). The solution was then freeze dried to yield potassium isobutyrate Step 4 Bromination of Potassium Isobutyrate Dry dichloroethane (30ml) and hydrochloric acid (2 eq) were added to the 50ml round bottomed flask containing the potassium isobutyrate. The reaction mixture was stirred for one hour at ambient temperature and purged with nitrogen. Chlorosulphonic acid (0.75 eq) and bromine (1 eq) were added. The reaction mixture was refluxed under nitrogen for six hours, then evaporated to remove solvent, excess hydrochloric acid, and residual bromine. The oil was redissolved in diethyl ether and washed with water to remove residual bromine and free propanoic acid. The ether was dried over magnesium sulphate, filtered and evaporated. The structure and purity of the final product, 2-bromo isobutyric acid, was confirmed by 1 H NMR and 13 C NMR. Overall Chemical Yield 550mg (56%).

110 Figure S8: 1 H NMR of 2-Bromo isobutyric acid Page 8 of 15 Figure S9: 13 C NMR of 2-Bromo isobutyric acid ( p p m ) D) Synthesis of 2-Bromo 14 C (CH 3 ) isobutyric acid (11) 14 C (CH 3 ) Methylation of diethyl methyl malonate To a 50ml one necked round bottom flask containing a magnetic stirrer was added of diethyl methyl malonate (5.9 mmol), sodium ethoxide (21% solution, 1.1eq) and ethanol (10ml) under nitrogen. The reaction mixture was stirred under a nitrogen atmosphere, and warmed to 50 0 C for 30 minutes. The reaction mixture was left to cool to room temperature. Methyl iodide was added in two aliquots (initial addition 10 mci/0.18mmoles of 14 C-metyhyl iodide (8.38 mmol, 1.4 eq) from a sealed ampoule followed by non-labelled methyl iodide (8.2mmoles)). The flask was flushed with nitrogen and warmed to 50 0 C for 4 hours. Solvent and unreacted methyl iodide were removed via evaporation. Dry diethyl ether was added to the residue and the sodium iodide was filtered to yield a clear crude solution of diethyl 14 C (CH 3 ) dimethyl malonate. The diethyl ether was washed with water to remove any residual sodium iodide. The collected diethyl ether layers were dried over magnesium sulphate, filtered and evaporated to yield a clear bronze coloured liquid. Chemical Yield 76%, Radiochemical Yield 81% Total Activity 8.13 mci Specific Activity 9.64 uci/mg Chemical Purity > 95%

111 Page 9 of 15 Figure S10: 1 H NMR of Diethyl 14 C (CH 3 ) dimethyl malonate (5) Subsequent steps were repeated as above to produce 2-bromo 14 C (CH 3 ) isobutyric acid, as confirm by NMR and R-TLC. The total activity and specific activity were also determined. Chemical Yield 35%, Radiochemical Yield 54% Total Activity 5.4 mci Specific Activity 15.6 uci/mg Chemical Purity > 95% Radiochemical Purity 99% Figure S11: 1 H NMR of 2-Bromo 14 C (CH 3 ) isobutyric acid (8) ( p p m )

112 Page 10 of 15 Figure S12: 13 C NMR of 2-Bromo 14 C (CH 3 ) isobutyric acid (8) ( p p m ) Figure S13: Radioanalytical TLC of Bromo 14 C (CH 3 ) isobutyric acid (8) Counts Region Bkg mm Bkg 2

113 Page 11 of 15 Synthesis of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) To a 2 necked 50ml round bottomed flask was added 2-bromo isobutyric acid (2-bromo ( 14 CH 3 ) isobutyric acid ( mol) and of non labelled 2-bromo isobutyric acid ( mol)) and dry Tetrahydrofuran (25ml). The reaction mixture was warmed under nitrogen to 60 0 C then 1,1 -carbonyldimidazole (1.0eq) was added. The mixture was stirred and heated under nitrogen at 60 0 C until the 1,1 -carbonyldimidazole was totally dissolved and the effervescence (liberation of CO 2 ) ceased. At this point, benzyl alcohol (1.0eq) was added and the reaction mixture left to reflux for 4 hours. The reaction mixture was evaporated and the residue was redissloved in diethyl ether. The crude product was first washed with dilute hydrochloric acid followed by washing with aqueous sodium carbonate. The water layers were further extracted with diethyl ether. All diethyl ether layers were combined and dried over sodium sulphate, filtered and evaporated to yield a water clear oil. The structure of the final product, Benzyl 2-bromo ( 14 CH 3 ) isobutyrate was confirmed by 1 H NMR and 13 C NMR and the chemical and radiochemical purities determined by 1 H NMR and R-TLC using 3 eluents 90/10, Pet Ether: Ether, 95/ Pet Ether;Ether and 100% Dichloromethane The total activity and specific activity determined. Chemical Yield 1.1g (55%), Radiochemical Yield 56%, Total Activity 0.397mCi, Specific Activity 0.362uCi/mg, Chemical Purity 97%, Radiochemical Purity 98% Figure S14: 1 H NMR of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) ( p p m )

114 Page 12 of 15 Figure S15: 13 C NMR of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) ( p p m )

115 Page 13 of 15 Figure S16: R-TLC Trace of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) 90/10, Pet Ether: Ether Counts Region mm 95/5, Pet Ether: Ether Counts Region mm 100% Dichloromethane Counts Region mm Rf values Elution Solvent Observed Rf value 100% 40/60 PET ETHER /5 40/60 PET ETHER/DIETHYL ETHER 100% DICHLOROMETHANE 0.914

116 Page 14 of 15 Figure S17: Kinetic analysis of ATRP polymerisation of 2-HPMA using radiolabelled and non-labelled initiators. DP50 HPMA Polymerisation using non-labelled benzyl 2-bromo isobutyrate initiator % Conversion Ln[M] o /[M] Time (min) 100 DP50 HPMA Polymerisation using benzyl 2-bromoisobutyrate 14 CH3 labelled initiator % Conversion Ln[M] o /[M] Time (min) 100 DP50 HPMA Polymerisation using benzyl 2-bromoisobutyrate 14 CH2 labelled initiator 4 % Conversion Ln[M] o /[M] Time (min)

117 Page 15 of 15 Figure S18: Overlaid GPC chromatograms of samples taken during the ATRP polymerisations of 2- HPMA. a) Benzyl Bromo Isobutyrate b) Benzyl ( 14 CH 2 ) Bromo Isobutyrate c) Benzyl Bromo ( 14 CH 3 ) Isobutyrate NMR & GPC data of 2-HPMA ATRP polymerisation with a target DP n = 50 monomer units. Initiator type 1 H NMR (calc) Observed GPC DP n M n DP n M n M n PDI Poly(2-HPMA) (Target DP=50) Non-labelled (Target DP=50) 2 labelled Poly(2-HPMA) CH Poly(2-HPMA) CH 3 - (Target DP=50) labelled Figure S19: R-TLC of Poly (2-Hydroxy propyl methacrylate) using THF as eluent 1.36 Counts mm

118 CHAPTER 6 MONITORING ATOM TRANSFER RADICAL POLYMERIZATION USING 14 C RADIOLABELLED INITIATORS 118

119 Monitoring Atom Transfer Radical Polymerisation using 14 C-radiolabelled initiators Polym. Chem., 2011,2, DOI: /C0PY00275E Contribution Mark Long: Student Principle Author First Author Executed All Practical Work David W Thornthwaite: Unilever PhD Supervisor: Organic Chemistry Suzanne H Rogers: Advised on the Practical ATRP Methodology: Taught Mark Long to carry out ATRP Polymerizations Francis R Livens: Manchester PhD Supervisor: Radiochemistry Steve P Rannard: Unilever PhD Supervisor: Polymer Chemistry Paper Abstract This paper expands on the work published Chemical Communications by describing in greater detail the synthesis of the ATRP 14 C labelled initiators and the use of Radio- TLC and liquid scintillation counting, of fractions collected from conventional gel permeation chromatography (GPC), to study the fate of 14 C-labelled initiators in the ambient methanolic ATRP of 2-hydroxypropyl methacrylate at different targeted number average degrees of polymerisation. Benzyl 2-bromoisobutyrate was synthesised in unlabelled form and with 14 C-labels at different locations to establish no adverse effects of the radiolabel. Target chain lengths of 10, 25 and 50 monomer units were synthesised and comparison of GPC and R-TLC showed a significant under-utilisation of the initiator with approximately 16% clearly observable at high monomer conversion (>97%). New chains appeared to be initiated at monomer conversions >90% and as late as 300 minutes after polymerisation had commenced. Purification by repeated precipitation was shown to be superior to flash chromatography for the ability to remove residual unreacted or terminated initiator although increased fractionation could be seen with each repeat. 130

120 Citations Unimolecular ligand initiator dual functional systems (ULIS) for low copper ATRP of vinyl monomers including acrylic/methacrylic acids Satyasankar Jana, Anbanandam Parthiban and Foo Ming Choo Chem. Commun., 2012, 48, 4256 DOI: /c2cc16663a Exploring the homogeneous controlled radical polymerisation of hydrophobic monomers in anti-solvents for their polymers: RAFT and ATRP of various alkyl methacrylates in anhydrous methanol to high conversion and low dispersity A. B. Dwyer, P. Chambon, A. Town, F. L. Hatton, J. Ford and S. P. Rannard Polym. Chem., 2015, 6, 7286 DOI: /C5PY00791G Coupled UV Vis/FT NIR Spectroscopy for Kinetic Analysis of Multiple Reaction Steps in Polymerizations Alan Aguirre-Soto, Albert T. Hwang, David Glugla, James W. Wydra, Robert R. McLeod, Christopher N. Bowman and Jeffrey W. Stansbury Macromolecules, 2015, 48, 6781 DOI: /acs.macromol.5b01685 Kinetic modeling of miniemulsion nitroxide mediated polymerization of styrene: Effect of particle diameter and nitroxide partitioning up to high conversion L. Bentein, D.R. D hooge, M.-F. Reyniers and G.B. Marin Polymer, 2012, 53, 681 DOI: /j.polymer Note: Cross Citations between the papers published for this thesis have been excluded Prior Art As stated in the paper the use of 14 C radiolabel to determine the fate if ATRP initiators is unique in the field of ATRP and calls into question the ATRP theory that states all initiator is used simultaneously at the start of a polymerization. 131

121 Polymer Chemistry Volume 2 Number 3 March 2011 Pages ISSN PAPER Steve P. Rannard et al. Monitoring Atom Transfer Radical Polymerisation using 14 C-radiolabelled initiators

122 PAPER Polymer Chemistry Monitoring Atom Transfer Radical Polymerisation using 14 C-radiolabelled initiators Mark Long, a Suzanne H. Rogers, a David W. Thornthwaite, a Francis R. Livens b and Steve P. Rannard* c Received 27th August 2010, Accepted 23rd September 2010 DOI: /c0py00275e Published on 16 October Downloaded on 29/01/ :40:37. Radio thin layer chromatography (R-TLC) and liquid scintillation counting, of fractions collected from conventional gel permeation chromatography (GPC), have been used to study the fate of 14 C- labelled initiators in the ambient methanolic ATRP of 2-hydroxypropyl methacrylate at different targeted number average degrees of polymerisation. Benzyl 2-bromoisobutyrate was synthesised in unlabelled form and with 14 C-labels at different locations to establish no adverse effects of the radiolabel. Target chain lengths of 10, 25 and 50 monomer units were synthesised and comparison of GPC and R-TLC showed a significant under-utilisation of the initiator with approximately 16% clearly observable at high monomer conversion (>97%). New chains appeared to be initiated at monomer conversions >90% and as late as 300 minutes after polymerisation had commenced. Purification by repeated precipitation was shown to be superior to flash chromatography for the ability to remove residual unreacted or terminated initiator although increased fractionation could be seen with each repeat. Introduction Controlled polymerisations offer many advantages to the experimental polymer chemist including the determination of polymer chain length, low polydispersity, end group chemistry and polymer architecture including block copolymer, star polymer and graft polymer synthesis. 1 To the polymer end-user, the accurate description and purity of polymer properties, including the chain length and chemical nature of the macromolecule, are important for structure property relationships 2 and descriptors for model generation. 3 The last thirty years have seen the introduction of a range of controlled syntheses including Group-Transfer Polymerisation (GTP), 4 Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation, 5 Nitroxide-Mediated Polymerisation (NMP), 6 click chemistry, 7 dendrimer synthesis, 8 immortal polymerisation 9 and Atom Transfer Radical Polymerisation (ATRP). 10 Arguably, one of the most successful of these controlled polymerisation techniques is ATRP with reports of successful synthesis of branched, 11 block, 12 and star polymers, 13 polymerisation in emulsion conditions, 14 polymerisation using ionic liquids, 15 hydrophobic solvents and aqueous environments, 16 heterogeneous polymerisation from surfaces 17 and modification of natural polymers, 18 all using a range of monomer types. Many studies of ATRP mechanism have been reported 19 but our recent report of utilising 14 C-labelled initiators 20 for ATRP remains the only direct radiochemical study of the fate of initiator during polymerisation. Radiolabels have been utilised for many years to aid polymer detection in studies such as mucoadhesion 21 or pharmaceutical delivery. Often polymer-labelling is achieved by a treatment of pre-formed polymers, for example tritiation of polymer protons, 22 chelation of heavy metal radioisotopes 23 (e.g. 64 Cu), methylation of amines 24 using 14 C-methyl iodide or iodination 25 using 125 I. Each of these treatments is, by necessity, a chemical reaction that has the potential to degrade the polymer or, at least, to modify polymer properties and behaviour. In comparison, substitution of 14 C for existing carbons 26 within the polymer building blocks (monomers/initiators) is probably the least intrusive labelling strategy. Selective labelling of polymer endgroups also avoids statistical label incorporation along the polymer chain, i.e. all labelled chains carry a single radioactive site. Often, the radiolabelling of polymers is conducted to allow detection of very low concentrations of material in complex environments 27 (e.g. body fluids). The activity of the polymer allows a clear signal to be measured without interference from other chemical species and without the need for lengthy purification procedures that may alter the study sample. Herein, we describe our utilisation of 14 C-radiolabelled ATRP initiators and demonstrate the insights achieved by accurate detection and monitoring of both polymer and residual initiator during purification and analysis. a Unilever Research and Development Port Sunlight Laboratories, Quarry Road East, Bebington, Wirral, UK CH63 3JW b School of Chemistry, The University of Manchester, Oxford Road, Manchester, UK M13 9PL c Department of Chemistry, University of Liverpool, Crown Street, Liverpool, L69 7ZD, UK. srannard@liv.ac.uk; Fax: +44 (0) ; Tel: +44 (0) Electronic supplementary information (ESI) available: Synthetic methodology, NMR spectra, mass spectra and R-TLC data. See DOI: /c0py00275e Experimental Reagents and suppliers All reagents and solvents were purchased from the Sigma Aldrich Group unless otherwise stated. Prosafe Liquid Scintillation Cocktail was purchased from Meridian Biotechnologies; PMMA GPC Standards were purchased from Polymer Laboratories; This journal is ª The Royal Society of Chemistry 2011 Polym. Chem., 2011, 2,

123 Published on 16 October Downloaded on 29/01/ :40:37. Partisil LK6DF Silica Gel 60A TLC Plates were purchased from BDH; 14 C methyl iodide (98.4%) was purchased from Amersham Biosciences; 14 CH 2 benzyl alcohol (95%) was kindly supplied by Unilever. Material synthesis Synthesis of benzyl 2-bromoisobutyrate. The radiolabelled and non-labelled initiators were synthesised either through conventional esterification procedures utilising bromoisobutyryl bromide and benzyl alcohol reagents (non-labelled initiator and initiator 3) in dichloromethane solvent or through the synthesis of 14 C methyl-labelled 2-bromoisobutyric acid (8) followed by coupling to benzyl alcohol using 1,1 0 -carbonyl imidazole (initiator 11) in THF. 20 General procedure for the ATRP synthesis of poly(2-hydroxypropyl methacrylate). The polymers were synthesised using previously reported techniques 11c and employing Cu(I)Br/2,2 0 - bipyridine (bipy) catalyst at ambient temperature in methanol solvent using 5 g of 2-hydroxypropyl methacrylate monomer. DP n was modified by varying the concentration of initiator used in each polymerisation and a constant initiator : Cu(I)Br : bipy ratio of 1 : 1 : 2 was used throughout. Identical polymerisations for each initiator were performed in triplicate to confirm reproducibility, demonstrate consistent polymerisation across labelled and non-labelled initiators and allow accurate radioactivity measurements. Kinetic experiments involved removal of 150 ml of the reaction mixture and aeration to quench the polymerisation at regular intervals prior to analysis without additional purification. Polymer purification Silica flash chromatography. After 24 hours polymerisation, two 150 ml samples of the reaction mixture were removed for analysis without purification. 150 ml aliquots of the remaining reaction mixture were dried to remove methanol solvent and unreacted monomer, redissolved in THF and passed through a Kieselgel Merck Type Mesh silica column. Precipitation. 500 ml of the reaction mixture were added to dry methanol (2 ml). The mixture was then slowly added to deionized water (30 ml) whereupon the polymer formed a white precipitate. After sedimentation of the polymer, samples of the supernatant were removed for analysis prior to separation of the precipitate and two further repetitions of the process. Polymer characterisation and analysis Gel permeation chromatography, GPC. GPC analysis was conducted using an Agilent series refractive index detector, 400C Eppendorf oven, Jasco PU 1580 pump, Jasco AS 590 auto sampler, Polymer Laboratories PLgel 5 mm mixed-c and PLgel 5 mm mixed-d columns with a PL guard column. THF was used as the elution solvent at a flow rate of 0.8 ml min 1. Fractions of eluted solvent were collected over 1 minute intervals to enable radioactivity levels to be determined using liquid scintillation counting. Liquid scintillation counting. Liquid scintillation counting was conducted using a Packard Tri Carb 3100 TRC liquid scintillation counter and utilised a 20 ml scintillation vial containing a known concentration of radioactive compound and 10 ml of Prosafe Liquid Scintillation Cocktail containing 60 75% phenyl xylyl ethane, 20 40% alcohol ethoxylate, 2 8% alcohol ether phosphate ester, % 2,5-diphenyloxazole and % 1,4-bis(4-methyl-a-styryl)benzene. Results are presented as Disintegrations Per Minute (DPM). Radio thin layer chromatography (R-TLC). R-TLC analysis was conducted using an AR 2000 BIOSCAN Radio TLC imaging scanner utilising a gas filled proportional counter filled with 90/10 argon/methane to detect the b emissions from thin layer chromatography (TLC) plates. TLC samples (1.0% w/w) were eluted using Whatman TLC plates (Partisil LK6DF silica Gel 60A with indicator), with a silica thickness of 250 mm and 3 cm pre-adsorbent zone, to a standard distance. Nuclear magnetic resonance (NMR) spectroscopy. 1 H NMR and 13 C NMR spectroscopy was conducted using a Br uker Avance DRX500 spectrometer. 1 H NMR utilised approximately 2 mg of each sample dissolved in 1 ml of CDCl 3 ; 13 C NMR utilised approximately 50 mg of sample dissolved in 1 ml of CDCl 3. Results and discussion When choosing to radiolabel a polymer via an ATRP initiator, two strategies are immediately obvious; the reaction of 2- bromoisobutyric acid, 1, with a 14 C-labelled alcohol, Scheme 1A, Scheme 1 Synthesis of 14 C-labelled ATRP initiators: (A) esterification of 14 C-labelled benzyl alcohol, (B) synthesis of 14 C-labelled 2-bromoisobutyric acid, (C) reaction of 14 C-labelled 2-bromoisobutyric acid with benzyl alcohol. 582 Polym. Chem., 2011, 2, This journal is ª The Royal Society of Chemistry 2011

124 Published on 16 October Downloaded on 29/01/ :40:37. or the synthesis of 14 C-labelled 2-bromoisobutyric acid, 8, for esterification with hydroxyl-containing compounds, Scheme 1C. Radiolabelled materials are inherently energetic and undergo radioactive degradation via radiolysis. Although the 14 C isotope has a half-life of years, b-decay results in conversion of 14 C atoms to 14 N atoms. 26d As such, the formation of complementary initiators labelled in different positions allows confirmation of results and confidence in the integrity of the radiolabel during the timescale of the experiment. Utilisation of analogous non-labelled initiators allows comparison of polymerisations in the presence and absence of radiolabel to ensure no adverse impact of the radiolabel on the course of the polymerisation. We have previously described our approach to the production of 14 C-labelled initiators, 20 3 and 11, via the reaction of 2- bromoisobutyric acid, 1, with 14 C-labelled benzyl alcohol, 2, and, secondly, the synthesis of 14 C-labelled 2-bromoisobutyric acid, 8, and subsequent reaction with benzyl alcohol, as summarised in Scheme 1. These initiators were shown to initiate the ATRP of 2- hydroxypropyl methacrylate (2-HPMA) to produce near identical polymers. Gel permeation chromatography (GPC) and 1 H nuclear magnetic resonance (NMR) spectroscopy of polymers with three target number average degrees of polymerisation (DP n ) showed high conversion (>95%) with similar kinetics. The polydispersity (PDI) of each polymer was higher than expected, possibly due to the restricted solubility of the initiator in the methanol solvent chosen for the polymerisation but the values are within the reported range for ATRP conducted in protic solvents. Comparison with non-labelled initiator analogues, Table 1, showed near identical behaviour hence confirming the lack of impact on the ATRP mechanism of the radiolabels. additional monitoring of the progress of the polymerisation through the radioanalysis of the samples taken for kinetic studies. Each sample utilised for kinetic analysis was conventionally analysed using the GPC refractive index (RI) detector to establish number average molecular weight (M n ), weight average molecular weight (M w ) and PDI. Additionally, fractions were collected during GPC analysis from the eluted solvent, in one minute time portions, and measured using liquid scintillation counting. As such, the total radioactivity for each 60 second time slice can be determined for the eluted solvent. The resolution of such a measurement is restricted and is clearly not as accurate as the almost constant sampling by the RI detector, however, this analysis allows the detection and direct correlation of the radioactivity of the sample with the polymer concentration detection by RI. Fig. 1 shows the combined scintillation/ri GPC analysis of the 14 C CH 2 -labelled initiator, 3. An amount of broadening has occurred during transit through the GPC column although the peak is sharp and corresponds well with the radioactivity trace. 60 second sampling was adopted to ensure appreciable levels of detectable radioactivity in each time slice. The resolution of such a measurement could be enhanced by increasing the specific activity of the radio-initiator, allowing higher activity within smaller volumes of eluted solvent. When polymer samples were analysed using this combined approach, the initiator radioactivity was overlayed with both the radioactivity of the polymer sample and the RI trace from the GPC. After polymerising for just 30 minutes at ambient temperature, using initiator 3, with a target DP n ¼ 50 monomer Radio-analysis of Atom Transfer Radical Polymerisation Experimentally, polymerisation via ATRP can be considered as comprising three key steps: (1) initiator choice and synthesis, (2) polymer synthesis and (3) polymer purification. The presence of radiolabels allows a novel examination of these steps through focussing purely on the radiolabelled material within the complex mixtures that are present. Step 1 of initiator synthesis has been described above (Scheme 1) and will be discussed again later. During this study we chose to place the 14 C-label at either the methyl group of the tertiary carbon, 11, or the benzylic methylene group, 3, Scheme 1, to allow for comparative studies and validation of results through the elimination of false data from initiator fragmentation. The synthesis of the initiator and the comparison of polymerisation with unlabelled initiators have been described previously, 20 however, the radiolabel allows Fig. 1 GPC analysis of benzyl 2-bromoisobutyrate (initiator 3) studied using refractive index detection (green trace; concentration detection) and scintillation counting of fractions from the GPC eluent (red bars; radioactivity detection). Table 1 Summary of ATRP of 2-hydroxypropyl methacrylate with labelled and non-labelled initiators to target DP n ¼ 50 monomer units 1 H NMR (calc.) Observed GPC Initiator type DP n M n DP n M n M n PDI Poly(2-HPMA) (target DP n ¼ 50) Non-labelled Poly(2-HPMA) (target DP n ¼ 50) 14 CH 2 -labelled Poly(2-HPMA) (target DP n ¼ 50) 14 CH 3 -labelled This journal is ª The Royal Society of Chemistry 2011 Polym. Chem., 2011, 2,

125 Published on 16 October Downloaded on 29/01/ :40:37. units, a sample was taken and injected directly into the GPC with subsequent fractions taken from the eluent, Fig. 2A. The GPC RI chromatogram shows a distinct polymer peak with a retention time of ca. 19 minutes. Additional features include a peak at ca minutes, corresponding to unreacted monomer, and a clear peak at ca. 22 minutes. The monomer peak and the additional peak at 22 minutes were also clearly visible in the 30 minute sample taken from the polymerisation using initiator 11, with a 14 C CH 3 -label. Radioactivity measurement of the eluting solvent shows a strong signal corresponding to the main polymer RI peak and a second peak in the radioactivity corresponding to the RI chromatogram signal at 22 minutes. This also corresponds directly with the radioactivity trace derived from the unreacted initiator 3, Fig. 1, showing a significant concentration of material that appears to be unreacted or terminated initiator. Throughout the polymerisation the radioactivity of the initiator signal decreases progressively relative to polymer as new chains are formed. Fig. 2B shows the final polymer sample which had been left to polymerise for 24 hours. The peak corresponding to unreacted monomer (23.5 minutes) also reduced considerably, confirmed by our 1 H NMR study of conversion (97.2%). However, although both the radioactivity and RI initiator signals corresponding to initiator have reduced, they did not fully disappear during the reaction, suggesting either that a portion of the initiator added at the outset of the polymerisation did not initiate chains or that the initiation led to rapid termination before appreciable propagation. Radio thin-layer chromatography Radio thin-layer chromatography (R-TLC) is an excellent tool for determining the radio-purity of labelled materials without separating non-labelled impurities. 28 A conventional TLC experiment is conducted but the TLC plate is imaged using a proportional counter that is scanned along the plate, detecting radioactivity levels. As such, unlabelled materials are invisible to the scan and only the radiolabelled materials are detected after appropriate/conventional separation with solvent or solvent mixtures. Comparison to conventionally imaged unlabelled TLC under identical conditions confirms the compound assignment by reference to retardation factor (R f ) values. 20 The R-TLC of 14 CH 3 -labelled 2-bromoisobutyric acid is shown in Fig. 3A. Fig. 2 GPC analysis of ATRP of 2-HPMA using initiator 3. (A) Sample taken after 30 minutes polymerisation; (B) sample after 24 hours polymerisation. Refractive index (green trace; concentration detection) and scintillation counting of fractions collected from the GPC eluent (blue bars; radioactivity detection) are shown, overlayed with scintillation of fractions for initiator 3 (red bars) for comparison. Fig. 3 Radio thin layer chromatogram of: (A) 14 C methyl-labelled 2- bromoisobutyrate (initiator 11; red trace); (B) 14 C methylene-labelled 2-bromoisobutyrate (initiator 3; green trace) overlayed with poly- (2-HPMA) synthesised from 3 with a target DP n ¼ 50 monomer units (dark red trace), after 24 hours polymerisation time (>97% conversion); (eluent: Et 2 O CH 3 CO 2 H 90/10 v/v.). 584 Polym. Chem., 2011, 2, This journal is ª The Royal Society of Chemistry 2011

126 Published on 16 October Downloaded on 29/01/ :40:37. The ability to monitor the radio-species without purification of the sample allows direct measurement without concern that sample purification has removed a significant amount of the material under investigation. The polymer samples taken for GPC kinetic evaluation and fraction collection/scintillation counting were also subject to R-TLC. Fig. 3B shows the R-TLC of the unpurified polymer after polymerisation for 24 hours and an overlayed R-TLC of the 14 C CH 2 -labelled initiator 3 for comparison. It is clear that the initiator peak has been adequately separated from the polymer under these conditions. As previously described, the activity of the signal from the unreacted/terminated initiator can be compared to the total activity of the R-TLC and the proportion of initiator not leading to significant propagation can be measured directly. The R-TLC measurements of unreacted/terminated initiator can be directly compared to the radioactivity observed within the GPC fraction analysis. R-TLC leads to a clear separation of the initiator from the polymer although the GPC columns used during this study were not able to separate completely the low molecular weight tail of the polymer from the initiator and the resolution of the fractions did not adequately allow a baseline activity to be determined between the polymer and initiator. Nonetheless, by selecting regions of elution defined by the fraction collection of the initiator (GPC) or the detected peak definition (R-TLC), it was possible to estimate the amount of unreacted/terminated initiator throughout the polymerisation indicated by each method. Fig. 4 shows a comparison of polymerisations using both initiator 3, Fig. 4A, and 11, Fig. 4B, with a target DP n ¼ 50 monomer units. Both polymerisations reach very similar conversions (>95% by 1 H NMR) and PDIs (approximately M w / M n ¼ 1.4). Interestingly, both reactions consume monomer and initiator at similar rates but neither utilises all of the available initiator; indeed after 30 minutes, approximately 30% of the initiator appears to be unreacted or terminated. Although the GPC appears to estimate a higher amount of unused initiator in both polymerisations, probably reflecting the limited resolution of GPC fraction collection and inadequate separation, the trends follow the R-TLC results with good correlation. When averaged across the polymerisations and the analytical techniques, the percentage of initiator not utilised in polymer chain formation during the polymerisation is approximately 16%, consistent with our earlier report. Surprisingly, it is also evident from this analysis that initiator continues to be consumed at least up to 200 minutes after the polymerisation has nominally commenced. There is also evidence to suggest that new chains are being initiated after 300 minutes of polymerisation and monomer conversions of >90%. The results shown here for the monitoring of the ambient methanolic ATRP of 2-HPMA initiated by benzyl 2-bromoisobutyrate are clearly specific to this monomer within this particular solvent and using this initiator. The analysis does, however, suggest that, within an ATRP, the accurate targeting of DP n, with low PDI at high monomer conversion, through control of the ratio of monomer and initiator is not without complication, as seen in many reports. The poor utilisation of initiator will inevitably lead to the formation of polymers with higher M n than targeted but the continual initiation of new chains in the latter stages of monomer conversion will also lead to broad PDIs. It Fig. 4 Comparison of R-TLC (red trace), GPC/liquid scintillation (green trace), 1 H NMR (conversion; blue trace) and GPC/RI (PDI; black trace) analysis of ambient ATRP of 2-HPMA (target DP n ¼ 50) with benzyl 2-bromoisobutyrate in methanol. (A) Methylene labelled initiator 3 and (B) methyl labelled initiator 11. has become common to report PDIs > 1.2 as near monodisperse within controlled radical polymerisations but such values would indicate considerable loss of polymerisation control in classical living polymerisations such as anionic polymerisation. The radiolabelled material that we have quantified by R-TLC and GPC fractionation/liquid scintillation counting has been cautiously assigned as unreacted or terminated initiator as we have no chemical analysis of the structure of the compounds involved. The R-TLC shows R f values that correspond directly to the initiators 3 and 11, Fig. 3 and 5, and terminated initiator should have considerably different R f values determined by the different polarities and solubilities derived from their different chemical structures. Combined with the GPC/scintillation counting it is compelling evidence that the signals derive directly from unreacted initiator but at this stage we cannot be definitive that this is the case. Radio-analysis of polymer purification As discussed earlier, a key stage in the synthesis of polymers is purification to remove unreacted monomer, initiator and catalyst residues. This is especially critical in ATRP as organometallic This journal is ª The Royal Society of Chemistry 2011 Polym. Chem., 2011, 2,

127 Published on 16 October Downloaded on 29/01/ :40:37. Fig. 5 Radio thin layer chromatograms of: (A) poly(2-hpma) synthesised from 3 with a target DP n ¼ 50 monomer units (initiator shown as green trace for comparison): polymer sample at 24 hours polymerisation time (>97% monomer conversion) (dark red trace), polymer sample after silica flash chromatography (red trace), polymer sample after three precipitations (blue trace); (B) overlayed supernatant of repeated precipitations: precipitation supernatant 1 (red trace), precipitation supernatant 2 (blue trace), precipitation supernatant 3 (green trace) R- TLC data have been normalised to trace initiator peak to show comparative peak height with removed polymer. (Eluent: Et 2 O CH 3 CO 2 H 90/10 v/v.) catalysts are often employed. Very commonly, copper is a contaminant that is readily observed in ATRP samples as the polymers may be slightly green or blue in colour. Residual copper and monomer may lead to issues of toxicity if the often complex block copolymers are designed for use in biomedical applications and several techniques to minimise or completely remove metal-based catalysts have been reported. 29 Sufficient but incomplete removal of metal catalyst residues is often achieved by passing an ATRP polymer through a column of silica or alumina. The metal is retained on the column and ideally a clear/ colourless solution yielding a pure white polymer sample after solvent removal. Alternatively polymers may be precipitated into non-solvents to remove monomer residues and metal contaminants. It would also be expected that other contaminants such as unreacted initiator or terminated oligomers would also be removed during these procedures. The polymers produced with radiolabelled initiators were subjected to both flash chromatography through silica columns and subsequent repeated precipitation from methanol solution into deionised water. R-TLC was used at each stage to study the ability of each step to remove unreacted/terminated initiator. As can be seen from Fig. 5A, flash chromatography led to a limited increase in the radiopurity of the polymer with respect to unreacted/terminated initiator within the target DP n ¼ 50 polymer samples. Quantification of the activity attributable to unreacted/terminated initiator is shown in Table 2. The R-TLC suggests that reliance on simple flash chromatography for purification may provide benefits for catalyst removal but non-polymeric species related to initiator are not significantly removed and therefore will contribute to inaccuracies in DP n confirmation by 1 H NMR end-group analysis. Indeed, the lower chain length samples exhibited a small but noticeable increase in radioactivity from the initiator impurity after flash chromatography, probably due to a higher removal of oligomeric species by the column relative to the initiator removal at these lower chain lengths, Table 2. Characterisation of the polymers by 1 H NMR after passage through the silica column was compared with data obtained prior to flash chromatography, Table 2. The removal of initiator residues that was observed through R-TLC of the DP n ¼ 50 polymer sample indeed led to a considerable increase in the calculated DP n to approximately 55 monomer units. The observed relative increase in initiator residue for both the polymers with target DP n ¼ 25 and 10 monomer units led to a subsequent proportional decrease in calculated DP n by endgroup analysis, presumably through the weighted removal of oligomers relative to initiator and the resulting discrepancy through the inability to discriminate between end-groups and initiator contaminants using 1 H NMR. Precipitation of the polymer samples after flash chromatography led to a distinct removal of non-polymeric radiolabelled material and the reduction of initiator residues, Fig. 5A, but full removal was not seen even after three precipitations, Table 3. Table 2 Summary of the impact of silica flash chromatography on unreacted/terminated initiator removal (shown as percentage of total activity of sample) from poly(2-hpma) with different target chain lengths. Calculated DP n ( 1 H NMR end group analysis) is also shown before and after flash chromatography Initial initiator radioactivity (average, %) Initiator radioactivity post-silica column (average, %) Initial DP n ( 1 H NMR calc. monomer units) DP n post-silica column ( 1 HNMR calc. monomer units) Poly(2-HPMA) (target DP n ¼ 50) Poly(2-HPMA) (target DP n ¼ 25) Poly(2-HPMA) (target DP n ¼ 10) Polym. Chem., 2011, 2, This journal is ª The Royal Society of Chemistry 2011

128 Table 3 Impact of repeat precipitation on the removal of unreacted/terminated initiator (shown as percentage of total activity of sample) from poly(2- HPMA) with different target chain lengths. Calculated DP n ( 1 H NMR end group analysis) is also shown before and after precipitation Initiator radioactivity post-silica column (average, %) Initiator radioactivity post-precipitation (average, %) DP n post-silica column ( 1 H NMR calc. monomer units) DP n postprecipitation ( 1 H NMR calc. monomer units) Poly(2-HPMA) (target DP n ¼ 50) Poly(2-HPMA) (target DP n ¼ 25) Poly(2-HPMA) (target DP n ¼ 10) Published on 16 October Downloaded on 29/01/ :40:37. Increased fractionation of the polymer sample was clearly observed as the R-TLC of the supernatant after each precipitation showed an increase in the presence of polymer relative to removed initiator. In combination with initiator removal, fractionation would be expected to have a profound effect on the determination of DP n by 1 H NMR spectroscopy as the removal of both oligomers and unreacted/terminated initiator leads to an assessment of higher average polymer chain lengths. Table 3 shows the effect of precipitation on each of the different target DP n polymers within our study. Conclusions 14 C-Radiolabelling of initiators allows the controlled introduction of radioactivity into polymer molecules and the monitoring of initiator fate during the course of a polymerisation without the need to purify or extract the initiator from the reaction mixture. Within the ATRP investigation detailed here, we have shown that the benzyl 2-isobutyrate ATRP initiator (3 and 11) continues to be consumed throughout the first minutes of the polymerisation of 2-HPMA in methanol at ambient temperature. The initiator is not fully utilised leading to a residue identifiable by radiometric methods within both the R-TLC and the GPC eluent fractions. Progressive and slow initiator consumption throughout the polymerisation leads to the continued initiation of new chains and suggests a plausible explanation for the difficulty in achieving very low PDIs at high monomer conversion and for the often reported attainment of higher DP n than targeted using ATRP. Although similar slow consumption of ATRP initiators has been reported for polyethylene glycol (PEG) macroinitiators, 30 the previous studies showed complete reaction of each initiator and related initiator efficiency to increasing steric hinderance derived from increased PEG chain length. Purification techniques such as silica column flash chromatography and polymer precipitation have variable impact on the final sample, dependent on the DP n of the synthesised polymer. Flash chromatography does little to remove residual initiator but may impact the lower molecular weight fractions of the polymer distribution, generating misleading molecular weight information through NMR end-group analysis. Precipitation is an excellent technique for initiator residue removal but the expected fractionation of low molecular weight species impacts the final recovered molecular weight distribution. Further work is required to study different ATRP initiator/ monomer/solvent combinations to establish the general nature of our findings. The strategies for radiolabelling ATRP polymers presented here have application in the monitoring and study of other new polymerisation approaches such as RAFT and NMP. The final radiolabelled materials also allow the detection of polymers in complex environments and further work will endeavour to establish new insights of the behaviour of well defined polymers in a range of applications. Acknowledgements The authors wish to thank the Royal Society for an Industry Fellowship (SR) and Unilever for financial support (ML). Megan E. M. Rannard is also warmly thanked for help compiling graphical data. References 1(a) M. Szwarc, ACS Symp. Ser., 1981, 166, 1;(b) K. Matyjaszewski, ACS Symp. Ser., 2009, 1023, 3;(c) J. Jagur-Grodzinski, J. Polym. Sci., Part A: Polym. Chem., 2002, 40, 2116; (d) A. Hirao, S. Loykulnant and T. Ishizone, Prog. Polym. Sci., 2002, 27, 1399; (e) R. Godoy Lopez, F. D Agosto and C. Boisson, Prog. Polym. Sci., 2007, 32, 419; (f) H. S. Bisht and A. K. Chatterjee, J. Macromol. Sci., Polym. Rev., 2001, 41, (a) K.-I. Izutsu and K. Shigeo, Phys. Chem. Chem. Phys., 2000, 2, 123; (b) N. Hadjichristidis, H. Iatrou, M. Pitsikalis and J. Mays, Prog. Polym. Sci., 2006, 31, 1068; (c) S. Jana, S. P. Rannard and A. I. Cooper, Chem. Commun., 2007, For example: D. N. Theodorou, Mol. Phys., 2004, 102, (a) O. W. Webster, Adv. Polym. Sci., 2004, 167, 1;(b) O. W. Webster, W. R. Hertler, D. Y. Sogah, W. B. Farnham and T. V. RajanBabu, J. Am. Chem. Soc., 1983, 105, (a) R. T. A. Mayadunne, E. Rizzardo, J. Chiefari, Y. K. Chong, G. Moad and S. H. Thang, Macromolecules, 1999, 32, 6977; (b) C. Boyer, V. Bulmus, T. P. Davis, V. Ladmiral, J. Liu and S. Perrier, Chem. Rev., 2009, 109, 5402; (c) S. L. Brown, C. M. Rayner, S. Graham, A. Cooper, S. Rannard and S. Perrier, Chem. Commun., 2007, (a) B. Charleux, ACS Symp. Ser., 2003, 854, 438; (b) N. R. Cameron, C. A. Bacon and A. J. Reid, ACS Symp. Ser., 2003, 854, (a) B. Helms, J. L. Mynar, C. J. Hawker and J. M. J. Frechet, J. Am. Chem. Soc., 2004, 126, 15020; (b) M. J. Kade, D. J. Burke and C. J. Hawker, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, (a) D. A. Tomalia and J. M. J. Frechet, Prog. Polym. Sci., 2005, 30, 217; (b) F. Aulenta, W. Hayes and S. Rannard, Eur. Polym. J., 2003, 39, 1741; (c) W. J. Feast, S. P. Rannard and A. Stoddart, Macromolecules, 2003, 36, (a) S. Inoue, J. Polym. Sci., Part A: Polym. Chem., 2000, 38, 2861; (b) M.-L. Hsueh, B.-H. Huang and C.-C. Lin, Macromolecules, 2002, 35, (a) T. Pintauer and K. Matyjaszewski, Chem. Soc. Rev., 2008, 37, 1087; (b) S. Wang and K. Matyjaszewski, J. Am. Chem. Soc., 1995, 117, 5614; (c) M. Kato, M. Kamigaito, M. Sawamoto and T. Higashimura, Macromolecules, 1995, 28, (a) T. He, D. J. Adams, M. F. Butler, C. T. Yeoh, A. I. Cooper and S. P. Rannard, Angew. Chem., Int. Ed., 2007, 46, 9243; (b) T. He, D. J. Adams, M. F. Butler, A. I. Cooper and S. P. Rannard, J. Am. Chem. Soc., 2009, 131, 1495; (c) I. Bannister, N. C. Billingham, S. P. Armes, S. P. Rannard and P. Findlay, Macromolecules, 2006, 39, (a) J. V. M. Weaver, Y. Tang, S. Liu, P. D. Iddon, R. Grigg, N. C. Billingham, S. P. Armes, R. Hunter and S. P. Rannard, This journal is ª The Royal Society of Chemistry 2011 Polym. Chem., 2011, 2,

129 Published on 16 October Downloaded on 29/01/ :40:37. Angew. Chem., Int. Ed., 2004, 43, 1389; (b) E. S. Read, K. L. Thompson and S. P. Armes, Polym. Chem., 2010, 1, (a) P. T. Hammond, Macromolecules, 2009, 42, 368; (b) H. Gao and K. Matyjaszewski, Macromolecules, 2006, 39, (a) K. Min, H. Gao, J. A. Yoon, W. Wu, T. Kowalewski and K. Matyjaszewski, Macromolecules, 2009, 42, 1597; (b) G. Chambard, P. De Man and B. Klumperman, Macromol. Symp., 2000, 150, (a) M. Xie, Y. Kong, H. Han, J. Shi, L. Ding, C. Song and Y. Zhang, React. Funct. Polym., 2008, 68, 1601; (b) T. Biedron and P. Kubisa, J. Polym. Sci., Part A: Polym. Chem., 2002, 40, (a) X.-S. Wang, S. F. Lascelles, R. A. Jackson and S. P. Armes, Chem. Commun., 1999, 1817; (b) S. McDonald and S. P. Rannard, Macromolecules, 2001, 34, (a) C. Perruchot, M. A. Khan, A. Kamitsi, S. P. Armes, T. von Werne and T. E. Patten, Langmuir, 2001, 17, 4479; (b) C.-D. Vo, A. Schmid, S. P. Armes, K. Sakai and S. Biggs, Langmuir, 2007, 23, (a) J. Lindqvist and E. Malmstr om, J. Appl. Polym. Sci., 2006, 100, 4155; (b) D. Bontempo, G. Masci, P. De Leonardis, L. Mannina, D. Capitani and V. Crescenzi, Biomacromolecules, 2006, 7, 2154; (c) S. P. Rannard, S. H. Rogers and R. Hunter, Chem. Commun., 2007, (a) T. Pintauer and K. Matyjaszewski, Top. Organomet. Chem., 2009, 26, 221; (b) P. A. Gurr, M. F. Mills, G. G. Qiao and D. H. Solomon, Polymer, 2005, 46, 2097; (c) F. Stoffelbach, R. Poli, S. Maria and P. Richard, J. Organomet. Chem., 2007, 692, M. Long, D. W. Thornthwaite, S. H. Rogers, G. Bonzi, F. R. Livens and S. P. Rannard, Chem. Commun., 2009, M. E. A. McGirr, S. M. McAllister, E. E. Peters, A. W. Vickers, A. F. Parr and A. W. Basit, Eur. J. Pharm. Sci., 2009, 36, J. C. Russell, J. R. Jones, T. A. Vick and P. W. Stratford, J. Labelled Compd. Radiopharm., 1993, 33, 957; C. Postolache, L. Matei and R. Georgescu, J. Radioanal. Nucl. Chem., 2009, 280, G. Sun, J. Xu, A. Hagooly, R. Rossin, Z. Li, D. A. Moore, C. J. Hawker, M. J. Welch and K. L. Wooley, Adv. Mater., 2007, 19, O. Oulanti, J. Widmaier, E. Pefferkorn, S. Champ and H. Auweter, J. Colloid Interface Sci., 2005, 291, (a) N. Nasongkla, B. Chen, N. Macaraeg, M. E. Fox, J. M. J. Frechet and F. C. Szoka, J. Am. Chem. Soc., 2009, 131, 3842; (b) E. R. Gillies, E. Dy, J. M. J. Frechet and F. C. Szoka, Mol. Pharmaceutics, 2005, 2, (a) R. K. Zeidan, S. A. Kalovidouris, T. Schluep, R. Fazio, R. Andresini and M. E. Davis, Bioconjugate Chem., 2006, 17, 1624; (b) G. Shemilt, M. Newby and S. L. Kitson, J. Labelled Compd. Radiopharm., 2007, 50, 515; (c) R. G. Riley, J. D. Smart, J. Tsibouklis, J. A. Davis, G. Kelly, F. Hampson, P. W. Dettmar and W. R. Wilber, J. Biomed. Mater. Res., 2001, 58, 102; (d) J. N. Huffaker, Nucl. Phys. A, 1971, 171, (a) J. Kucka, M. Hruby and O. Lebeda, Appl. Radiat. Isot., 2010, 68, 1073; (b) K. Saatchi and U. O. Hafeli, Bioconjugate Chem., 2009, 20, E. Gattavecchia, D. Tonelli, A. Breccia, A. Fini and E. Ferri, J. Radioanal. Nucl. Chem., 1994, 181, For example (a) L. Mueller and K. Matyjaszewski, Macromol. React. Eng., 2010, 4, 180; (b) M. E. Honigfort, W. J. Brittain, T. Bosanac and C. S. Wilcox, Macromolecules, 2002, 35, 4849; (c) Y. Shen, H. Tang and S. Ding, Prog. Polym. Sci., 2004, 29, S. Perrier and D. M. Haddleton, Eur. Polym. J., 2004, 40, Polym. Chem., 2011, 2, This journal is ª The Royal Society of Chemistry 2011

130 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 1 of 10 Monitoring Atom Transfer Radical Polymerisation using 14 C- Radiolabelled Initiators Mark Long, Suzanne H. Rogers, David. W. Thornthwaite, Francis R. Livens, and Steve P. Rannard Supporting Information Synthetic Procedures A) Synthesis of Benzyl 2-bromo isobutyrate To a 250ml one neck round bottomed flask containing a magnetic stirrer flushed with nitrogen was added dry dichloromethane (150ml), benzyl alchohol (0.1 mol), triethylamine (1.1eq) and dimethyl amino pyridine (0.013eq). The reaction mixture was cooled to 0 0 C using an ice water bath. With stirring, bromo isobutyryl bromide (1.1eq) was added dropwise to the reaction mixture using a pressure equalizing dropping funnel. Once this addition was complete, the reaction flask was left to stir for twenty four hours initially at 0 0 C but allowed to warm to room temperature. The reaction mixture was evaporated to remove the solvent to yield a crude yellow oil and a cream precipitate. Dilute hydrochloric acid and diethyl ether were added and the product was washed 4 times. Finally the mixture was washed with dilute sodium carbonate The combined diethyl ether layers were dried over sodium sulphate, filtered and evaporated to yield a bronze coloured oil product.the structure and purity of the final product was confirm by 1 H NMR, 13 C NMR and Mass Spectrometery. Chemical purity > 95%. Chemical Yield 23.9g (93%).

131 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 2 of 10 Figure S1: 1 H NMR spectrum of Benzyl 2-bromo isobutyrate Figure S2: 13 C NMR spectrum of Benzyl 2-bromo isobutyrate ( p p m)

132 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 3 of 10 Figure S3: Mass Spectrum of Benzyl 2-bromo isobutyrate Theoretical Mass = 274 Daltons B) Synthesis of 3 The synthesis of 3 utlised benzyl ( 14 CH 2 ) alcohol ( moles, 13.56mCi) and followed the procedure described above. The radiochemical purity was determined by R-TLC using 3 eluents 90/10, Petroleum ether/ diethyl ether, 95/ Petroleum ether/diethyl ether and 100% dichloromethane. Total activity and specific activity were determined. Chemical Yield 4.5g (87%), Radiochemical Yield 97% Total Activity mci Specific Activity uci/mg Chemical Purity > 95% Radiochemical Purity 96%

133 Figure S4: 1 H NMR spectrum of 3 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 4 of ( p p m) Figure S5: 13 C NMR spectrum of ( p p m)

134 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 5 of 10 C) Synthesis of Non Labeled 2-bromo isobutyric acid Step 1 Methylation of Diethyl Methyl Malonate To a 50ml one necked round bottom flask containing a magnetic stirrer was added of diethyl methyl malonate (5.9 mmol), sodium ethoxide (21% solution, 1.1eq) and ethanol (10ml) under nitrogen. The reaction mixture was stirred, and warmed to 50 0 C for 30 minutes. The reaction mixture was then cooled to room temperature. Methyl iodide (8.38 mmol, 1.4 eq) was added. The flask was flushed with nitrogen, sealed and warmed to 50 0 C for 4 hours. After cooling, the ethanol and unreacted methyl iodide were removed using a rotary evaporator. Dry diethyl ether was added to the residue and the sodium iodide was filtered to yield a clear crude solution of diethyl dimethyl malonate. The diethyl ether was washed with water to remove residual sodium iodide. The diethyl ether layer was dried over magnesium sulphate, filtered and evaporated to yield a clear bronze coloured liquid. The structure and chemical purity of the final product was confirmed by 1 H NMR Chemical Yield 0.81g (73%), Chemical Purity > 95% Figure S6: 1 H NMR spectrum of diethyl dimethyl malonate Step 2 Hydrolysis of diethyl dimethyl malonate To the diethyl dimethyl malonate, was added 2.4eq of an aqueous solution of Potassium Hydroxide (10ml). The solution was warmed to 80 0 C with stirring until the oil was completely dissolved. Step 3 Decarboxylation of the di-potassium salt of dimethyl malonate To the aqueous solution of the di-potassium salt of dimethyl malonate was added an aqueous solution of sulphuric acid(2.4 eq, 20ml). The mixture was refluxed for five hours. The resulting isobutyric acid was recovered via distillation (azeotroped with water). To the water/isobutyric acid mixture potassium hydroxide was added (1.2eq). The solution was then freeze dried to yield potassium isobutyrate Step 4 Bromination of Potassium Isobutyrate Dry dichloroethane (30ml) and hydrochloric acid (2 eq) were added to the 50ml round bottomed flask containing the potassium isobutyrate. The reaction mixture was stirred for one hour at ambient temperature and purged with nitrogen. Chlorosulphonic acid (0.75 eq) and bromine (1 eq) were added. The reaction mixture was refluxed under nitrogen for six hours, then evaporated to remove solvent, excess hydrochloric acid, and residual bromine. The oil was redissolved in diethyl ether and washed with water to remove residual bromine and free propanoic acid. The ether was dried over magnesium sulphate, filtered and evaporated. The structure and purity of the final product, 2-bromo isobutyric acid, was confirmed by 1 H NMR and 13 C NMR. Overall Chemical Yield 550mg (56%).

135 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 6 of 10 Figure S7: 1 H NMR of 2-Bromo isobutyric acid Figure S8: 13 C NMR of 2-Bromo isobutyric acid ( p p m) D) Synthesis of 2-Bromo 14 C (CH 3 ) isobutyric acid (11) 14 C (CH 3 ) Methylation of diethyl methyl malonate To a 50ml one necked round bottom flask containing a magnetic stirrer was added of diethyl methyl malonate (5.9 mmol), sodium ethoxide (21% solution, 1.1eq) and ethanol (10ml) under nitrogen. The reaction mixture was stirred under a nitrogen atmosphere, and warmed to 50 0 C for 30 minutes. The reaction mixture was left to cool to room temperature. Methyl iodide was added in two aliquots (initial addition 10 mci/0.18mmoles of 14 C-metyhyl iodide (8.38 mmol, 1.4 eq) from a sealed ampoule followed by non-labelled methyl iodide (8.2mmoles)). The flask was flushed with nitrogen and warmed to 50 0 C for 4 hours. Solvent and unreacted methyl iodide were removed via evaporation. Dry diethyl ether was added to the residue and the sodium iodide was filtered to yield a clear crude solution of diethyl 14 C (CH 3 ) dimethyl malonate. The diethyl ether was washed with water to remove any residual sodium iodide. The collected diethyl ether layers were dried over magnesium sulphate, filtered and evaporated to yield a clear bronze coloured liquid. Chemical Yield 76%, Radiochemical Yield 81% Total Activity 8.13 mci Specific Activity 9.64 uci/mg Chemical Purity > 95%

136 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 7 of 10 Figure S9: 1 H NMR of Diethyl 14 C (CH 3 ) dimethyl malonate (5) Subsequent steps were repeated as above to produce 2-bromo 14 C (CH 3 ) isobutyric acid, as confirm by NMR and R-TLC. The total activity and specific activity were also determined. Chemical Yield 35%, Radiochemical Yield 54% Total Activity 5.4 mci Specific Activity 15.6 uci/mg Chemical Purity > 95% Radiochemical Purity 99% Figure S10: 1 H NMR of 2-Bromo 14 C (CH 3 ) isobutyric acid (8) ( p p m)

137 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 8 of 10 Figure S11: 13 C NMR of 2-Bromo 14 C (CH 3 ) isobutyric acid (8) ( p p m) Figure S12: Radioanalytical TLC of Bromo 14 C (CH 3 ) isobutyric acid (8) Counts Region Bkg mm Bkg 2

138 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 9 of 10 Synthesis of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) To a 2 necked 50ml round bottomed flask was added 2-bromo isobutyric acid (2-bromo ( 14 CH 3 ) isobutyric acid ( mol) and of non labelled 2-bromo isobutyric acid ( mol)) and dry Tetrahydrofuran (25ml). The reaction mixture was warmed under nitrogen to 60 0 C then 1,1 -carbonyldimidazole (1.0eq) was added. The mixture was stirred and heated under nitrogen at 60 0 C until the 1,1 -carbonyldimidazole was totally dissolved and the effervescence (liberation of CO 2 ) ceased. At this point, benzyl alcohol (1.0eq) was added and the reaction mixture left to reflux for 4 hours. The reaction mixture was evaporated and the residue was redissloved in diethyl ether. The crude product was first washed with dilute hydrochloric acid followed by washing with aqueous sodium carbonate. The water layers were further extracted with diethyl ether. All diethyl ether layers were combined and dried over sodium sulphate, filtered and evaporated to yield a water clear oil. The structure of the final product, Benzyl 2-bromo ( 14 CH 3 ) isobutyrate was confirmed by 1 H NMR and 13 C NMR and the chemical and radiochemical purities determined by 1 H NMR and R-TLC using 3 eluents 90/10, Pet Ether: Ether, 95/ Pet Ether;Ether and 100% Dichloromethane The total activity and specific activity determined. Chemical Yield 1.1g (55%), Radiochemical Yield 56%, Total Activity 0.397mCi, Specific Activity 0.362uCi/mg, Chemical Purity 97%, Radiochemical Purity 98% Figure S13: 1 H NMR of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) ( p p m)

139 Supplementary Material (ESI) for Polymer Chemistry This journal is (c) The Royal Society of Chemistry 2010 Page 10 of 10 Figure S14: 13 C NMR of Benzyl 2-bromo ( 14 CH 3 ) isobutyrate (11) ( p p m)

140 CHAPTER 7 CONTROLLED SYNTHESIS OF RADIOLABELLED AMINE METHACRYLATE WATER-SOLUBLE POLYMERS WITH VARYING END GROUPS AND STUDIES OF ADSORPTION BEHAVIOUR 140

141 Controlled synthesis of radiolabelled amine methacrylate watersolublepolymers with end-groups of varying hydrophobicity and studies of adsorption behaviour Polym. Chem., 2012,3, DOI: /C1PY00397F Contribution Mark Long: Student Principle Author First Author Executed All Practical Work David W Thornthwaite: Unilever PhD Supervisor: Organic Chemistry Suzanne H Rogers: Advised on the Practical ATRP Methodology: Taught Mark Long to carry out ATRP Polymerizations Francis R Livens: Manchester PhD Supervisor: Radiochemistry Steve P Rannard: Unilever PhD Supervisor: Polymer Chemistry Paper Abstract The effect of chain end chemistry on the behaviour of poly(2-(diethylamino)ethyl methacrylate) has been studied through the placement of various radioactive groups with increasing hydrophobicity at the chain end of the polymer. Controlled radical polymerisation with 14 C-labelled initiators was used to form polymers of very similar chain length including doubly-labelled, fluorescent-radio polymers. Monitoring and quantification of behaviour was conducted purely using radio techniques, without the need for fluorescence measurement, showing a clear impact on solid surface adsorption across a range of surface types (filter paper, photographic paper and hair). The studies presented had clear implications for the study of polymer behaviour through low level fluorescent modification of water-soluble polymers. 134

142 Citations How does a tiny terminal alkynyl end group drive fully hydrophilic homopolymers to self-assemble into multicompartment vesicles and flower-like complex particles? Tingting Liu, Wei Tian, Yunqing Zhu, Yang Bai, Hongxia Yan and Jianzhong Du Polym. Chem., 2014, 5, 5077 DOI: /C4PY00501E Fluorescent and chemico-fluorescent responsive polymers from dithiomaleimide and dibromomaleimide functional monomers Mathew P. Robin and Rachel K. O'Reilly Chem. Sci., 2014, 5, 2717 DOI: /c4sc00753k Exploring the homogeneous controlled radical polymerisation of hydrophobic monomers in anti-solvents for their polymers: RAFT and ATRP of various alkyl methacrylates in anhydrous methanol to high conversion and low dispersity A. B. Dwyer, P. Chambon, A. Town, F. L. Hatton, J. Ford and S. P. Rannard Polym. Chem., 2015, 6, 7286 DOI: /C5PY00791G Prior Art The use of dual labelling, 14 C radiolabel and fluorescent label to determine the effects of the presence of a fluorescent label on polymers has been rarely used and certainly never with diethyl amino ethyl methacrylate. It has proved to have been a successful method in determining the effect of the fluorescent label and increasing hydrophobicity of a group at a chain end and as stated the results have clear implications for the study of polymer behaviour through low level fluorescent modification of watersoluble polymers which has not been investigated before. 135

143 Polymer Chemistry View Online / Journal Homepage / Table of Contents for this issue Dynamic Article Links C < Cite this: Polym. Chem., 2012, 3, PAPER Controlled synthesis of radiolabelled amine methacrylate water-soluble polymers with end-groups of varying hydrophobicity and studies of adsorption behaviour Downloaded on 19 October 2012 Published on 04 November 2011 on doi: /c1py00397f Mark Long, a David W. Thornthwaite, a Suzanne H. Rogers, a Francis R. Livens b and Steve P. Rannard* c Received 7th September 2011, Accepted 20th October 2011 DOI: /c1py00397f The effect of chain end chemistry on the behaviour of poly(2-(diethylamino)ethyl methacrylate) has been studied through the placement of various radioactive groups with increasing hydrophobicity at the chain end of the polymer. Controlled radical polymerisation with 14 C-labelled initiators was used to form polymers of very similar chain length including doubly-labelled, fluorescent-radio polymers. Monitoring and quantification of behaviour was conducted purely using radiotechniques, without the need for fluorescence measurement, showing a clear impact on solid surface adsorption across a range of surface types (filter paper, photographic paper and hair). The studies presented here have clear implications for the study of polymer behaviour through low level fluorescent modification of watersoluble polymers. Introduction Radioisotopes have been used for many years to study chemical, biological and physical processes and elucidate reaction mechanisms. 1 8 Predominantly for reasons of cost and safety, alternative methods to label and monitor materials and molecules have been developed with the use of fluorophores and fluorescence spectroscopy being especially common. 9,10 In the context of polymer science, fluorescence studies have added mechanistic and physical insight to mixed polymer-surfactant systems, 11 diffusion in gels and solids, 12 polymerisation mechanism, and polymer behaviour in solution When fluorophores are chemically bound to a polymer to aid detection and monitor behaviour, there is an implicit assumption that the fluorophore does not significantly affect the properties of the polymer and that the observations effectively report the behaviour of the unlabelled materials. As the molecular weight and relative dimensions of the labels can be significant and the fluorophores are often hydrophobic, the validity of this assumption in the case of water-soluble polymers requires careful a Unilever Research and Development Port Sunlight Laboratories, Quarry Road East, Bebington, Wirral, UK CH63 3JW b School of Chemistry, The University of Manchester, Oxford Road, Manchester, UK M13 9PL c Department of Chemistry, University of Liverpool, Crown Street, Liverpool, L69 7ZD, UK. srannard@liv.ac.uk; Fax: ; Tel: Electronic Supplementary Information (ESI) available: Experimental details, radio-characterisation, 1 H NMR spectra of initiators, GPC characterisation of p(deaema), R-TLC of polymers, regression curves derived from surface tension measurements. See DOI: /c1py00397f investigation. The introduction of aromatic fluorophores can be considered as a hydrophobic modification leading to an amphiphilic polymer 11 with subsequent modification of behaviour such as solubility, association and surface interactions. In a 1996 review of his own work and published data from other groups regarding the behaviour of fluorescently labelled poly(methacrylic acid) in aqueous environments, Morawetz 19 concluded that the published behavioural models based on fluorescent labelling were valid for the labelled polymer only and cannot be used to infer the state of the unlabeled poly(methacrylic acid). Best and Silescu 29 concluded, from fluorescence densitometry at polymer-polymer interfaces, that the effects of fluorophore addition in polymer blend studies could be reduced by ensuring that the labelled polymers contained at least 200 monomer units per fluorescent label. Egan et al. 16 found that increasing the number of fluorophores on poly((2-ethyl hexyl) methacrylate) chains used as stabilizers in the dispersion polymerization of poly (vinyl acetate) resulted in changes to the mean particle size, distribution, composition and molecular weights of the final colloidal polymer particles when compared to stabilisers without fluorescent modification. Increased fluorophore addition within the stabilizer resulted in reduced mean particle sizes and large amounts of irreversibly attached poly((2-ethyl hexyl) methacrylate). The particle size distribution was also affected with the addition of an average of just one fluorophore per stabilizer molecule leading to the appearance of an ultra-high molecular weight polymer fraction. It is clear from the literature examples presented that the inclusion of a fluorophore can have substantial effects on the physical and chemical properties of polymers and assumptions of identical or near-identical behaviour of modified and unmodified 154 Polym. Chem., 2012, 3, This journal is ª The Royal Society of Chemistry 2012

144 View Online Downloaded on 19 October 2012 Published on 04 November 2011 on doi: /c1py00397f materials are not necessarily valid. It is also well-established that the addition of even small numbers of hydrophobic groups, fluorescent or non-fluorescent, to water-soluble polymers can affect behaviour at interfaces, 30a in blends and in solution and this has been utilised in many industrial applications such as associative thickeners. 30b The use of radiolabels in combination with fluorescent labels uniquely provides an independent measure of effects arising from the presence of a fluorophore without relying on fluorescence measurement. As already mentioned, radioisotopes have been widely used to monitor chemical processes and early reports of their use in polymer science date to the 1950s. The Bevington and Ayrey groups pioneered radioisotope application predominantly in the investigation of free radical polymerization mechanism; however, aspects of polymer stability, including bio-stability of polyurethanes, 36 poly(methyl methacrylate) hydrolysis, 37 and stabiliser mobility within polymers 38 have also been investigated. In medicine, radiolabelled materials have been used to investigate the in vivo behaviour of polymers used to deliver drugs and assist in the treatment of cancer The environmental biodegradability of synthetic aliphatic polymers such as poly(acrylic acid), poly(methacrylic acid), poly(sodium acrylate), poly(ethyl acrylate-co-methacrylic acid), poly(3- hydroxybutyrate-co-3-hydroxyoctanoate) and poly(3-caprolactone) have also been studied Physical properties such as the kinetics of adsorption at liquid liquid interfaces 51 and the adsorption/desorption and distribution of polyelectrolytes and uncharged polymers at solid-liquid interfaces such as glass, 50 silica, 52 leather, 53 poly(tetrafluoroethylene), PTFE, 54 and surface modified polystyrene latex particles 55 have previously been explored using radiolabelling techniques. Although polymer deposition/adsorption has been determined using either radio or fluorescent 55,58,59 labels, very few studies have directly compared radiolabelled material with identical fluorescently labelled materials 55 or controllably combined both labels within one polymer chain, thereby employing doublylabelled materials. Using a 14 C radioisotope to investigate the physicochemical properties of a polymer does not change the hydrophilicity/ hydrophobicity, charge or molecular structure and has a negligible impact on molecular weight. Providing such labelled polymers are radiochemically stable on the timescale of the experiment, combining fluorescent labels with 14 C radiolabels in a systematic manner allows the investigation of the effect of hydrophobic and fluorophore modification on the polymer chemical and physical behaviour without the inherent issues of fluorescer quenching. 60 Our recent reports have highlighted the synthesis of 14 C-labelled 2-bromoisobutyric acid 61a,b and its use in monitoring radiolabelled initiators during and after Atom Transfer Radical Polymerisation (ATRP). We have also drawn attention to the importance of small changes in chain end chemistry (even a single methyl group) in controlling the solution behaviour of water-soluble amine methacrylates, specifically the lower critical solution temperature of poly(2-(dimethylamino)ethyl methacrylate). 61c Herein we report a strategy to controllably introduce radioactivity at the chain end of a water-soluble polymer generated through ambient temperature ATRP using an 2-propanol/water solvent mixture and Cu(I)Br/2,2 0 -bipyridine catalyst system. 62 We utilise 14 C radiolabelled initiators with increasing hydrophobicity and ultimately generate a combined radio/fluorescentlabelled polymer containing one radioactive site and a single fluorophore (9-hydroxyfluorene) 63 at the chain end, Fig. 1. The adsorption of poly(2-(diethylamino)ethyl methacrylate) (pdeaema) from aqueous solution onto various substrates is monitored and the effect of the varying chain-end hydrophobicity is studied. Results and discussion 1. Radio and fluorescent-labelled initiator and polymer design, synthesis and characterisation Three initiators were synthesised using two approaches; either conventional ester synthesis utilising the reaction of 2-bromoisobutyryl bromide, 1, with 14 C-labelled primary alcohols, 2 or 3, or the reaction of 14 C-labelled 2-bromoisobutyric acid, 4, with 1,1-carbonyl diimidazole, 5, to generate an acid imidazolide which is capable of esterification with hydroxyl functional materials. In this second approach 9-hydroxyfluorene, 6, was utilised thereby generating a doubly-labelled radio-fluorescent initiator, 9. The syntheses are summarised in Scheme 1. The synthesis of radio-labelled materials requires assessment of both chemical and radio purity as labelled impurities will considerably hamper the accurate identification and quantification of the target material. The initiators were therefore subjected to radio thin layer chromatography (R-TLC) 60a,b under different solvent conditions. Each initiator was accurately assessed using different ratios of petroleum ether and diethyl ether; 7 utilised a 60 : 40 v/v ratio whilst 8 and 9 were analysed using a 90 : 10 v/v solvent mixture, Fig. 2. Analysis of the initiators using 1 H and 13 C nuclear magnetic resonance spectroscopy (NMR) (see Electronic Supporting Information, ESI) and UVvis spectrophotometry was comparable to the analysis of unlabelled analogue materials. Collectively the analysis demonstrated a high chemical and radiochemical purity for the initiators. The non-radiolabelled analogue initiators were used to demonstrate the ability of each material to polymerise the amine containing monomer 2-(N,N-diethylamino) ethyl methacrylate (DEAEMA) under identical ambient ATRP conditions using a 90/10 v/v 2-propanol/water solvent mixture. The formation of Fig. 1 Radiolabelled polymers with different end groups; A) methyl, B) benzyl and C) 9-hydroxyfluorene terminated. D) Labelled polymers in aqueous solution and E) adsorption onto surfaces. This journal is ª The Royal Society of Chemistry 2012 Polym. Chem., 2012, 3,

145 View Online Downloaded on 19 October 2012 Published on 04 November 2011 on doi: /c1py00397f Scheme 1 Synthesis of 14 C-radiolabelled ATRP initiators: i) reaction with 14 C-labelled primary alcohols and ii) reaction of 14 C-labelled 2- bromoisobutyric acid with 9-hydroxyfluorene. p(deaema) was compared with the identical polymerisations utilising the 14 C-labelled initiators, Table 1. The presence of the radiolabel had no appreciable effect on the outcome of the polymerisation or the number average degree of polymerisation (DP n ) as measured by 1 H NMR or gel permeation chromatography (GPC). Although there are differences in the observed M n and DP n, as measured by either GPC or 1 H NMR, the GPC results were determined using only refractive index and a standard calibration curve generated from poly(methyl methacrylate) standards, therefore the GPC data is reported as poly(methyl methacrylate) equivalent molecular weights. The three radiopolymers were considered sufficiently comparable within each measurement to utilise in studies of end-group effects. An evaluation of the kinetics of the polymerisations showed slightly different rates of reaction when using each initiator and greater than 96% conversion of DEAEMA in each case, Fig. 3. Although the polymerisations are not ideal, the methyl-functional initiator, 7, led to a slower polymerisation than the benzylfunctional initiator 8, which in turn was slower than the 9-hydroxyfluorenyl-functional initiator 9. Residual unreacted initiator was quantified by R-TLC through the elution of the final polymer sample with a 90/10 v/v mixture of petroleum ether and acetic acid (see ESI) as previously described. 60a,b Radioactivity associated with unreacted initiator varied from 3.72% (initiator 7) to 7.02% (initiator 8) and 14.59% (initiator 9) suggesting a decreasing initiator efficiency but also following the trend of increasing polymerisation rate i.e. faster Fig. 2 Radio thin layer chromatography of 14 C-labelled initiators: A) methyl 2-bromoisobutyrate, 7 (eluent: 60 : 40 petroleum ether 40 60/ Et 2 O v/v), B) benzyl 2-bromoisobutyrate, 8 (eluent: 90 : 10 petroleum ether 40 60/Et 2 O v/v), and C) 9-hydroxyfluorenyl 2-bromoisobutyrate, 9 (eluent: 90 : 10 petroleum ether 40 60/Et 2 O v/v). polymerisation resulting in higher levels of unreacted initiator. Although this seems counterintuitive (fewer radicals should maintain a slower polymerisation rate), these findings agree with our previously reported examination of initiator consumption under ATRP conditions, 60b and the identified continual formation of new chains at monomer conversions >90% and polymerisation times >200 min. The rapid consumption of monomer would result in a higher unreacted initiator concentration within the final polymer sample if relatively slow initiation (i.e. initial addition of monomer to unreacted initiator) relative to Table 1 Summary of the ATRP polymerisation of 2-(N,N-diethylamino)ethyl methacrylate with labelled and non-labelled initiators (target: DP n ¼ 50 monomer units; M n ¼ initiator). GPC data calculated from poly(methyl methacrylate) standards using THF as eluent Initiator M n ( 1 H NMR) DP n ( 1 H NMR) M n (GPC) DP n (GPC) PDI (GPC) ( 14 C) ( 14 C) ( 14 C) Polym. Chem., 2012, 3, This journal is ª The Royal Society of Chemistry 2012

146 View Online chromatography to remove catalyst and ligand residues, dried to remove all traces of reaction solvent, and subsequently used to prepare a 0.14% w/w solution at ph 2. The radioactivity of the solution was determined by liquid scintillation counting to provide initial activity levels. The papers were cut into 2 cm 2 samples whilst standard 5 cm virgin hair swatch samples were used as received. Two radioanalytical techniques, storage phosphor imaging, Fig. 4, and liquid scintillation counting were employed, with multiple repeats, to study each sample. Downloaded on 19 October 2012 Published on 04 November 2011 on doi: /c1py00397f Fig. 3 Comparative kinetic evaluation of the ambient polymerisation of 2-(N,N-diethylamino)ethyl methacrylate with initiators 7 (red data), 8 (green data) and 9 (blue data). Conversion is shown as coloured circles and ln[m 0 ]/[M] is given as triangles. Guidelines are shown to emphasise the differences of initiation and propagation. propagation is present during the polymerisation, possibly related to initiator structure or solubility. For truly living polymerisation to be observed, the rate of initiation must be considerably higher than the rate of propagation 64 (and termination). ATRP is a controlled radical process and not a living polymerisation (mistakenly described as such in many reports). Within the ATRP polymerisations that we have studied with radiolabelled initiators, truly living conditions have not been observed as expected; however, linear semi-logarithm plots suggest the attainment of a steady propagating radical concentrations during the main stages of propagation, even with considerable unreacted initiator present. Further work will focus on additional kinetic studies, however this is not the focus of this report. 2. Quantification of polymer adsorption onto hair, cellulose filter paper and photographic paper As discussed previously, fluorescent modification is a common approach to study the solution, solid state and adsorption behaviour of polymers. Fluorescent labels allow the rapid identification and quantification of modified polymers to elucidate temporal, gravimetric and spatial information. Difficulties arise when the distribution of fluorophore is not homogeneous across the sample, i.e. some polymer chains have high fluorophore modification, or the fluorophore interferes with the analysis technique, e.g. self-quenching. The polymers of the current study have been designed to vary only in chain-end modification, from methyl to benzyl to 9-hydroxyfluorenyl groups, and specifically to carry a single radioactive carbon at each chain-end to allow quantification of the behaviour of each polymer irrespective of fluorescence. Three substrates were chosen to establish variability of adsorption under identical controlled conditions using commercially relevant and non-ideal surface chemistry; untreated virgin hair, filter paper and photographic paper were used as models of biomaterials (keratin), hydrophilic cellulosic and hydrophobically modified substrates respectively (see ESI). To model a conventional experimental procedure, the radiolabelled polymers were purified by flash column Fig. 4 Autoradiography (storage phosphor imaging) images of radiolabelled poly(2-(diethylamino)ethyl methacrylate) adsorbed onto A) hair, B) filter paper and C) photographic paper. Poly(2-(diethylamino)ethyl methacrylate) has varying initiator-derived end-groups: i) methyl, ii) benzyl and iii) 9-hydroxyfluorenyl. Experiment-relevant autoradiography standards are shown for comparison. This journal is ª The Royal Society of Chemistry 2012 Polym. Chem., 2012, 3,