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Monodisperse polymer nanospheres: Fabrication, chemical modifications and applications

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Monodisperse polymer nanospheres: Fabrication, chemical modifications and applications

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Content MONODISPERSE POLYMER NANOSPHERES: FABRICATION, CHEMICAL MODIFICATIONS AND APPLICATIONS Copyright 2004 by Ryan Desousa A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) August 2004 Ryan DeSousa Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedication To G’ma, Mum, Dad, Keith and Marika. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgements I will be ever grateful to Dr. Prakash, for allowing me to do research under his guidance, for his patience, enthusiasm and constant support. It was here, as a part of this large and very diverse group, that I was exposed to concepts, phenomena and research at the frontiers of scientific study; many of which I had no clue even existed. All my present research skills acquired and techniques learnt, are a result of working with this group, of talented and friendly people. Dr. Olah’s support, encouragement and keen scientific insights are gratefully acknowledged. I would like to thank all the members of the Olah-Prakash group, both past and present, with whom I had the pleasure of doing research. In particular, I would like to thank Dr. Mihir Mandal, Dr. Chulsung Bae, Dr. Jinbo H u , Dr. Thomas Mathew and Dr. Konstantine Koltunov for their help and support during my time here, especially when I first joined the group. I would like thank Carole Phillips, Jessy May and Ralph Pan for keeping the group running and making research a lot easier for us to do. I would also like to thank the many friends I have made at Loker and the Chemistry Department, during my time here; Alain S, Leila, Marko, Giovanni, Pierre, Alfonzo, Monika, Bjoener, Alain G, Roman, Jacques and Chiradeep, thank you all for the many great times spent. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I gratefully acknowledge the efforts of my coworkers, Mike Julian and Reem Song, who worked with me on different aspects of the Photonic Band Gap Project. I thank Mike for his help and insights, during our stint with the photonic crystals, and without whose efforts and contributions Chapter One would not have been possible. I would also like to thank Mike for helping me out with the all the fluorescence measurements that I required at various other times. I would also like to thank Thomas Gadda and Melissa Grunlan from Dr. Weber’s group for helping me with the TGA, DSC and GPC, and also the FTIR software. You guys were a big help. I thank all my friends back home in India, my roommates here and the wonderful friends I have made over the last five years here at USC. They helped maintain the sanity through many a trying time. Kaj, Leon, Jimmy, Nimmy, Sambo, Reuben , Kenneth, Merzi, Rakesh, Pawan, Savio, Alpa; Renato, Rommel, Anoob, Vivek, Gagan, Raghav, Saurabh, Hithesh, Gaurav, Junaid, JP, Nilesh, Jassi, Pramod, Madhurima, Vanya, Priya C, Priya B, Raechelle, Suraj, Mitesh, Dona, Devang; this wouldn’t be possible without you guys. Thanks! Alpa thanks for your help way back when and Priya thanks for storing the avidin samples at precisely 4 °C, till I could use them. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V And last but not least, I thank my Family back home in India, without whose help, guidance, love and support, I would not be where I am today. G’ma, Mum, Dad, Keith and Marika; this ones for you. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vi Table of Contents Dedication ii Acknowledgements iii List of Tables ix List of Figures x List of Schemes xviii Abstract xix Chapter 1 1 1.1 Introduction 1 1.2 Chapter 1: References 6 Chapter 2 Fabrication of polymer nanospheres 8 2.1 Introduction 8 2.2 Emulsion Polymerization 11 2.3 Emulsifier Free Emulsion Polymerization 14 2.4 Fabrication of Polystyrene Nanospheres 2.4.1 Introduction 17 2.4.2 Experimental 18 2.4.3 Results and Discussion 20 2.5 Conclusion 36 2.6 Chapter 2: References 36 Chapter 3 Fabrication of dye-doped colloidal core-shell polystyrene nanospheres for photonic crystals 39 3.1 Introduction 39 3.1.1 What are Photonic Crystals? 39 3.1.2 Strategies for fabrication of photonic crystals 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vii 3.2 Studies on the inhibition of spontaneous emission and energy transfer in a photonic crystal 46 3.2.1 Fabrication Strategy 47 3.2.1.1 Synthesis of Dye Doped polystyrene nanospheres 48 3.2.1.2 Assembly of the colloidal polymer nanospheres into photonic crystals 54 3.3 Experimental 56 3.4 Results and Discussions 64 3.5 Doping of polystyrene nanospheres with charged dyes 80 3.6 Conclusion and Outlook 84 3.7 Chapter 3: References 85 Chapter 4 Surface chemical functionalization of polystyrene nanospheres 89 4.1 Introduction 89 4.2 “In-situ” grafting of polystyrene nanospheres with monomers having high water solubility 91 4.2.1 Introduction 91 4.2.2 Experimental 95 4.2.3 Results and Discussions. 100 4.3 “In-situ” grafting for the synthesis of thiol functionalized nanospheres 107 4.3.1 Introduction 107 4.3.2 Experimental 110 4.3.3 Results and Discussions 119 4.3.4 Conclusions 126 4.4 Direct chemical functionalization of polystyrene nanospheres 128 4.4.1 Introduction 128 4.4.2 Experimental 131 4.4.3 Results and Discussion 148 4.4.4 Conclusions 159 4.5 Chapter 4: References 160 Chapter 5. Poly-pentafluorostyrene nanospheres and pentafluorostyrene grafted nanospheres 164 5.1 Introduction 164 5.2 Experimental 166 5.3 Results and Discussion 189 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.4 Conclusions 214 5.5 Chapter 5: References 215 Bibliography 217 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ix List of Tables Table 2.1 Variation of particle size with monomer amount. 21 Table 2.2 Variation of particle size with reaction time, using 7.0 ml styrene. 25 Table 2.3 Variation of particle size with initiator. 26 Table 2.4 Effect of ionic strength and salt used on particle size. 28 Table 2.5 Effect of different concentrations of CaCL on final nanoparticle size. 29 Table 2.6 Variation of particle size with monomer amount, in presence and absence of added salt. 30 Table 2.7 Variation of size of polymer particles at different percentages of crosslinking. 35 Table 3.1 Formulation of the solutions of the two dyes in monomer. 60 Table 3.2 Different sizes of polystyrene nanospheres obtained with varying amount of monomer, and color of the corresponding crystals. 65 Table 4.1 Experiment for determining the number of amino groups per gram of resin. 151 Table 4.2 Determination of the number of unreacted acid groups. 152 Table 4.3 Determination of the corresponding number of amine groups on M grams of resin. 152 Table 4.4 Determination of the number of amino groups per gram of resin. 152 Table 4.5 Determination of the number of millimoles of amino groups per nanosphere. 154 Table 4.6 List of coupling agents used and the resulting colors. 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. X List of Figures Figure 2.1 Particle size ranges obtainable using different polymerization techniques. 09 Figure 2.2 SEM image of polystyrene nanoparticles obtained using 1.5 ml styrene. 22 Figure 2.3 SEM image of polystyrene nanoparticles obtained using 2.0 ml styrene. 22 Figure 2.4 SEM image of the polystyrene nanoparticles obtained using 3.0 ml styrene. 23 Figure 2.5 SEM image of the polystyrene nanoparticles obtained using 4.0 ml styrene 23 Figure 2.6 SEM image of the polystyrene nanoparticles obtained using 5.0 ml styrene. 24 Figure 2.7 SEM image of the polystyrene nanoparticles obtained using 7.0 ml styrene. 24 Figure 2.8 Variation of particle size with reaction time. 25 Figure 2.9 SEM image of the polystyrene nanoparticles obtained using 2.0 ml styrene and p = 3.97 * 1 O '3 31 Figure 2.10 SEM image of the polystyrene nanoparticles obtained using 3.0 ml styrene and p = 3.97 * 10"3 31 Figure 2.11 SEM image of the polystyrene nanoparticles obtained using 4.0 ml styrene and p = 3.97 * 10"3 32 Figure 2.12 SEM image of the polystyrene nanoparticles obtained using 5.0 ml styrene and p = 3.97 * 10'3 32 Figure 2.13 SEM image of the polystyrene nanoparticles obtained using 7.0 ml styrene and p = 3.97 * 10'3 and 85.0 ml water. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xi Figure 2.14 Polymer nanospheres obtained at 70 °C 34 Figure 2.15 Polymer nanospheres obtained at 90 °C 34 Figure 3.1 1-D alternating crystal 43 Figure 3.2 2-D alternating crystal 43 Figure 3.3 3-D alternating crystal 44 Figure 3.4 Lanthanide dye chosen for nanosphere doping. Ln = europium or terbium. 49 Figure 3.5 Emission spectra of europium and terbium dyes in styrene. 50 Figure 3.6 Coumarin 334 51 Figure 3.7 Nile Red. 51 Figure 3.8 Normalized photoluminescence spectra of Coumarin 334 and Nile Red in polystyrene polymer nanospheres. 52 Figure 3.9 Cartoon of the dye containing polymer nanospheres. (A) central core, (B) dye containing layer, (C) overcoat. 53 Figure 3.10 Diagram showing the angular photoluminescence detection method used. 58 Figure 3.11 Bathochromic shift of the Bragg peak with increasing size of the nanospheres. 64 Figure 3.12 SEM image of the of the blue reflective surface. 66 Figure 3.13 SEM image of the of the green reflective surface. 66 Figure 3.14 SEM image of the of the red reflective surface. 67 Figure 3.15 CCP lattice surface and edge, of the assembled colloids (SEM image). 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xii Figure 3.16 Magnified view of the surface and edge showing the packing in the assembled CCP crystal. 68 Figure 3.17 SEM image of the Ln-dye doped polystyrene nanoparticles 70 Figure 3.18 SEM image of red reflective dye doped polystyrene nanospheres. 71 Figure 3.19 SEM image of blue reflective dye doped polystyrene nanospheres 72 Figure 3.20 Emission spectra of the red reflective crystal at 0° incidence. Exc. at 365 nm, normalized to 401 nm. 73 Figure 3.21 Normalized reflectance of the blue crystal showing the angular dependence of the stop band at 490 nm to 400 nm for angles 5-55°. 74 Figure 3.22 Normalized reflectance of the red crystal showing the angular dependence of the stop band at 610 nm to 565 nm for angles 5-60°. 75 Figure 3.23 Emission spectra of the blue crystal at 0 - 60° incidence detection. Exc. 365 nm, normalized at 401 nm. Suppression of emission from the stop band is in agreement with the reflectance measurements. 77 Figure 3.24 Normalized angular emission spectra of the blue crystal divided by the normalized spectra of the red crystal at 0° incidence. Minima represent the center of the suppression of emission of Coumarin 334 for each spectrum. Spectra taken with excitation:emission bandpass slits set at 3:lnm except for (b) 5:1 nm and (c) 5:3 77 Figure 3.25 Normalized (560 nm) emission spectra of red control crystal at 0°, blue crystal at 50°, and blue crystal at 60°. 79 Figure 3.26 Basic Fuchsin 81 Figure 3.27 PhloxineB 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xiii Figure 3.28 Polymer nanospheres doped with Basic Fuchsin 83 Figure 3.29 (A) UV-Vis absorption spectra of Basic Fuchsin in water. (B) Visible absorption spectra of Basic Fuchsin doped polystyrene nanospheres. 83 Figure 4.1 Functional monomers successfully used for “in-situ” grafting. 92 Figure 4.2 Monomers to be investigated for “in-situ” grafting. 94 Figure 4.3 Acrylonitrile grafted polymer nanospheres.(Nitrile 2236 cm-1) 101 Figure 4.4 Acrolein grafted polymer nanospheres. (C=0 1720 cm-1) 102 Figure 4.5 Ethyl acrylate grafted polymer nanospheres. (C=0 1728 cm-1) 103 Figure 4.6 N-isopropyl acrylamide grafted polymer nanospheres. (C=0: 1662 cm-1, N-H s t : 3500 cm-1 to 3300 cm-1) 104 Figure 4.7 4-vinyl-benzenesulfonamide grafted polymer nanospheres. (0=S=0: asym st 1330cm-l, sym. st. 1160 cm-1, N-H st: 3500 cm-1 to 3250 cm-1) 104 Figure 4.8 N,N -diethyl- 4-vinyl-benzenesulfonamide grafted polymer nanospheres. (0=S=0: asym. st 1333cm-l, sym. st. 1155 cm-1) 105 Figure 4.9 SEM of 4-vinyl-benzenesulfonamide grafted nanospheres. 106 Figure 4.10 (a) p-acetoxy styrene. (I) 4 - substituted thioester of styrene. 109 Figure 4.11 Retrosynthesis of thioester (I), (i) Wittig Reaction, (ii) Conversion to the sulfoxide, (iii) Pummerer rearrangement to the thiol, (iv) Protection of the thiol as the ester. 111 Figure 4.12 (i) triphenylphosphonium bromide, n-BuLi, THF. 0 °C (ii) 4- (methylthio) -benzaldehyde, 40 °C. (iii) m-CPBA, -20 °C, CHCI3 . (iv) Ca(OH)2 . (v)NaHCC>3, Trifluoroacetic anhydride, DCM, 40 °C. (vi) Et3N, CH3OH. (vii) DCM, Acetyl chloride, Et3N. 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xiv Figure 4.13 DRIFT spectra of thioester (I) grafted polystyrene nanospheres. (C=0 1706 cm-1) 122 Figure 4.14 EDS of thioester (I) grafted polystyrene nanospheres 123 Figure 4.15 SEM of thioester (I) grafted nanospheres. 123 Figure 4.16 DRIFT spectra of thiol functionalized polystyrene nanospheres, after hydrolysis. 124 Figure 4.17 EDS of thiol grafted polystyrene nanospheres 124 Figure 4.18 EDS of gold colloid coated thiolated polystyrene nanospheres 125 Figure 4.19 (A) TEM image of the gold colloids. (B) TEM image of the gold colloids covered polymer nanospheres 126 Figure 4.20 Diffuse reflectance UV-Vis spectrum of gold colloid coated polymer nanospheres showing the plasmon band at 530 nm. 127 Figure 4.21 Proposed mechanism for electrophilic amination reaction 129 Figure 4.22 Proposed mechanism for electrophilic hydroxylation. 130 Figure 4.23 (A) Polystyrene nanospheres before amination. (B) Aminated polystyrene nanospheres. 149 Figure 4.24 DRIFT spectra of aminated polystyrene nanospheres 3461 and 3368 (N-H st), 1618 (NHb) cm-1. 150 Figure 4.25 DRIFT spectra of hydroxylated polystyrene nanospheres 3500 to 3100 (O-H s t), 1251 (C-Ost) cm-1. 150 Figure 4.26 IR spectrum of diazotized nanospheres. (2263 cm-1 N 2+ ) 155 Figure 4.27 UV-Vis spectra for some of the diazo-coupled nanospheres. (viii 10a) yellow color via coupling with dimethylaniline, (viii06b) red color via coupling with 2-naphthol and (viii 117b) purple color via coupling with Chicago acid. 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4.28 Figure 4.29 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 Figure 5.9 Figure 5.10 Acid chloride functionalized nanospheres; 1772 and 1731 (Ar C=0 st) Acylated polystyrene nanospheres. 1681 cm-1 (C=0 st) Nucleophilic attack on perfluorinated systems. SEM of the polypentafluorostyrene nanospheres obtained with no organic cosolvent during emulsifier free polymerization. SEM of the polypentafluorostyrene nanospheres obtained with using isopropanol as organic cosolvent during emulsifier free polymerization. DRIFT spectrum of pentaflurostyrene grafted nanospheres. Ar C-F: 1519 cm-1, Ar C-F: 968 cm-1. SEM image of pentafluorostyrene grafted polystyrene nanospheres. Photoluminescence Emission spectra from C510 doped nanospheres Photoluminescence spectra from Nile Red doped nanospheres. IR of p-aminophenoxide grafted nanospheres 3461 to 3379 (N-H st, very weak), 1520 (Ar C-F), 965 (Ar C-F) cm-1. (6 hour reaction) Diffuse reflectance visible spectrum of p-aminophenyl functionalized nanospheres diazotized nanospheres coupled to 2-naphthol. Diffuse reflectance visible spectrum of 2-(4-aminophenyl)- ethoxide functionalized nanospheres, diazotized and coupled to 2-naphthol. xv 158 159 165 191 192 193 194 195 196 198 199 200 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xvi Figure 5.11 Figure 5.12 Figure 5.13 Figure 5.14 Figure 5.15 Figure 5.16 Figure 5.17 Figure 5.18 Figure 5.19 Figure 5.20 Figure 5.21 DRIFT of 2-(4-aminophenyl)ethoxide functionalized nanospheres. Three hour reaction time. IR (v, KBr): 3456 & 3374 (N-H st), 1618 (N-H b), 1517(Ar C-F.), 956 (Ar C-F.) cm-1. DRIFT of 2-aminoethoxide functionalized nanospheres. IR (v, KBr): 3384 and 3307 (N-H st), 1133 (C-O), 971 (ArC-F) cm-1. XPS of amine functionalized nanospheres DRIFT of hydroxyl functionalized nanospheres. IR (v, KBr): 3423 (O-H st), 1133 (C-O), 965 (Ar C-F) cm-1. XPS of hydroxy functionalized nanospheres. EDS of thiol functionalized nanospheres, showing the sulfur peak. DRIFT spectrum of carboxylate functionalized nanospheres. IR (v, KBr): 1681 and 1561 (C=0 asym st), 1520 (Ar C-F), 1410 (C=0 sym st), 968 (Ar C-F) cm-1. Diffuse reflectance UV-Visible spectra showing the gold plasmon band for amine functionalized nanospheres coated with gold colloids. Diffuse reflectance UV-Visible spectra showing the gold plasmon band for thiol functionalized nanospheres coated with gold colloids. EDS of gold coated amine functionalized nanospheres, showing the gold peaks. EDS of gold coated amine functionalized nanospheres, showing the gold peaks. 201 202 202 203 204 204 205 206 207 208 208 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xvii Figure 5.22 TEM images of (A) an amine functionalized, (B) a thiol functionalized nanospheres showing gold colloids on the polymer nanosphere Figure 5.23 EDS of hydroxyl functionalized nanospheres coated with silver colloids, showing silver peaks. Figure 5.24 TEM image of a hydroxyl functionalized nanospheres coated with silver colloids. Figure 5.25. Normalized fluorescence spectra. Series 1: Carboxylic acid functionalized pentafluorostyrene grafted polystyrene nanospheres after treatment with biotin-4-fluorescein. Series 2: Avidin functionalized pentafluorostyrene grafted polystyrene nanospheres. Series 3: Avidin functionalized pentafluorostyrene grafted polystyrene nanospheres after treatment with biotin-4-fluorescein. Excitation 365nm Figure 5.26 Normalized fluorescence spectra of pentafluorostyrene grafted polystyrene nanospheres. Excitation 365 nm. 209 209 210 213 214 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Schemes Scheme 2.1 Decomposition of Initiator Scheme 2.2 Propagation of polymerization Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xix Abstract In this dissertation, the effects of a photonic band gap, generated by photonic crystals fabricated from polystyrene nanospheres, on dye emissions incorporated in the polystyrene nanospheres are examined. The dyes are incorporated into the polystyrene colloids used to construct the photonic crystal. To this end, the synthesis of colloidal polystyrene nanospheres, synthesis of dye doped polystyrene nanospheres and assembly of the colloids into an FCC lattice exhibiting a stop band are investigated. Various methods for chemically modifying the nanosphere surface, in order to increase its chemical reactivity are also investigated. Chapter one is a short introduction to the area of nanotechnology, polymer nanoparticles and their applications. In chapter two, the ability to control the polymer nanosphere size is examined and the fabrication of highly monodisperse polystyrene nanospheres, in a range of sizes, from 200 nm to 800 nm, by emulsifier free emulsion polymerization, is discussed. Chapter three, examines the use of colloidal polymer nanoparticles in fabricating photonic crystals. The ability to synthesize highly monodisperse nanospheres in a range of sizes is exploited and a variety of different sized but highly monodisperse nanospheres are synthesized. These are then used to assemble photonic crystals. Nanospheres doped with dyes, fabricated using a core shell approach, are used to assemble photonic crystals and to study the effects of the stop Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. XX band on the dye emissions; such as inhibition of spontaneous emission and energy transfer. Chapter four and chapter five discuss novel methods for chemically modifying these polymer nanospheres, in order to incorporate chemical diversity at their surface. In chapter four, the “in-situ” grafting technique developed previously, is examined for its applicability to a wider range of monomers. Methods for thiolation, amination, hydroxylation and carboxylation of the nanosphere surface, and further modifications are examined. In chapter five, the emulsifier free emulsion polymerization technique is extended to the synthesis of poly-pentafluorostyrene nanospheres and pentafluorostyrene grafted polystyrene nanospheres. These pentafluorostyrene grafted polystyrene nanospheres are then used to incorporate other chemical functionalities such as, amine, hydroxyl, thiol and carboxyl groups on the nanosphere surface, via nucleophilic substitution reactions occurring on the nanosphere surface at the pentafluorostyrene aromatic rings. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 Chapter 1 1.1 Introduction The term “nanotechnology” is commonly used to describe many types of research where the characteristic dimensions are less than 1,000 nm (1.0 pm). Materials having these dimensions fall into the category of submicron materials or nanomaterials. Included, are monodisperse polymer nanoparticles and polymer colloids of both organic and inorganic materials. These nanoparticles posses unique structural, mechanical, chemical and opto-electronic properties and are being investigated for uses in many diverse areas. Research into readily recoverable and recyclable polymer supported heterogeneous catalysts has grown rapidly in recent years and monodisperse polymer nanoparticles with their well defined and uniform structures are receiving increased attention as supports for such catalysts. Nord and coworkers1 way back in the 1940’s, described the catalytic properties of noble metal colloids stabilized by synthetic polymers. The synthesis of catalytically active metal colloids by aqueous alcohol reduction of metal salts in the presence of protective polymer was first reported by Hirai and coworkers.2 The colloidal particles of one to three nanometers mean diameter and narrow size distributions showed high activity and selectivity for the hydrogenation of olefins and dienes, the hydration of acrylonitrile and the light induced generation of hydrogen from water.2 ,3 a ,3 b In 1994 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sherrington and coworkers reported the development of a polybenzimidazole (PBI) supported MoV I catalyst,4 that was used in the epoxidation of propene and cyclohexene.5 Sherrington also developed a Pd(II) catalyst supported on cyano- fimctionalized polyimide beads, that was used in the Wacker oxidation of oct-1 - ene.6 Akashi and coworkers, in 1998, developed a method for depositing platinum nanoparticles onto polystyrene microspheres, via the reduction of khPtCle by aqueous ethanol in the presence of polystyrene nanospheres with surface grafted poly-(N-isopropylacrylamide).7 a ,7 b These immobilized Pt colloids were found to be active and stable heterogeneous catalysts for the hydrogenation of allyl alcohol in water. The high activity of the catalyst was retained after many turnovers. Recently in 2002, Asahi and coworkers reported the synthesis of Au/Pt bimetallic colloids supported and polystyrene nanospheres. These Au/Pt bimetallic nanoparticles were synthesized on the microsphere surface by the in-situ reduction of gold and platinum ions by the radicals generated by the initiator. The use of these supported bimetallic catalysts in the hydrogenation of olefins and visible light-induced hydrogen generation from water is currently being investigated. In similar works, Prakash and coworkers9 have successfully coated the surface of poly-vinylpyridine nanospheres with colloidal Palladium by one step absorption from the colloidal Pd solution. These Pd coated polyvinylpyridine nanospheres were successfully used in Pd catalyzed C-C coupling reactions, like the Suzuki-, Stille- and Heck- reactions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Here too the high activity of the catalyst was retained after many turnovers. Mecking and coworkers reported the fabrication of Rh complex1 0 coated to core shell polystyrene nanospheres. The nanospheres generated by emulsion polymerization were coated with a polyelectrolyte layer of poly(diallyldimethylammonium chloride). These polyelectrolyte coated nanospheres were used to immobilize the complex [(H)Rh(CO)(NaTPPTS)3]. This supported Rh catalysts was used to investigate the hydroformylation of methyl acrylate. The results were comparable to reaction with non supported catalysts, with methyl-2 - formyl propionate obtained as the major product. More recently, polymeric microspheres have been used in the multicolor optical coding for biological assays.1 1 Zinc sulfide capped cadmium selenide quantum dots were embedded into the polymer microspheres at precisely controlled ratios to obtain a highly luminescent microspheres. The use of these quantum dot tagged beads for biological assays was demonstrated in a DNA hybridization system using oligonucleotides probes on triple color encoded microspheres. Target DNA molecules were conjugated to a fluorescent dye. Optical spectroscopy at the single bead level yielded both microsphere coding and target signals. The microsphere coding identified the DNA sequence, whereas the target signal indicated the presence and abundance of that sequence. Use of polymer nanospheres and microspheres as oligonucleotide supports, in order to study DNA hybridization and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 single nucleotide polymorphisms has become increasingly common. Whitten and coworkers1 2 used biotinylated oligonucleotide strands attached to streptavidin coated polymer microspheres containing anionic poly(phenylene ethynylene) (PPE). The target oligonucleotide strand contained the energy transfer quencher QSY-7. The binding of the receptor and target oligonucleotides was determined by the quenching of the PPE fluorescence by QSY-7. In similar works, Chatterji and coworkers1 3 used polystyrene microsphere immobilized oligonucleotide targets to study DNA hybridization. The success of hybridization was confirmed by using the fluorescent dye oxazole yellow (dimeric), which fluoresces only as the dye-DNA complex. Polymer microspheres and nanospheres have long been used for chromatographic separations.1 4 Techniques for separation, based on imprinted nanospheres have recently received great interest. Cormack and coworkers1 5 developed a simple method for synthesizing highly monodisperse imprinted methyl methacrylate microspheres by precipitation polymerization. These microspheres were molecularly imprinted using theophylline as template. When investigated in the HPLC elution of theophylline, they showed a much stronger retention of theophylline as compared to similar but non imprinted microspheres. Moleculary imprinted microspheres, using astaxanthin (3,3’ -dihydroxy-(3,(3-carotene-4,4 ’-dione) as template were synthesized using microsuspension polymerization. These Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 imprinted microspheres were successfully used to separate astaxanthin from the saponified samples of microalga H. pulivalis and yeast P. rhodozyma. Capillary electrophoresis is a field where the use of molecularly imprinted microspheres is beginning to generate interest, de Jong and coworkers1 6 synthesized polymer nanospheres using (+) ephedrine as template using precipitation polymerization. These nanospheres, when used as the pseudostationary phase in the capillary electrophoretic separation of enantiomers, ephedrine and salbutamol, led to full base line separation of the enantiomers within ten minutes. Polymer nanoparticles and microparticles have also been investigated for sustained release. Non biodegradable polymers pose problems of toxicity, difficulty in removal and irregular rate of drug release.1 7 To overcome these problems, in the early 1970s greater attention was paid to the development of biodegradable nanoparticles. Yolles and coworkers1 8 were one of the first to report the use of polypeptides in parenteral Drug Delivery Systems. In the last decade commercial developments using these polymers have taken place resulting in the investigation of promising treatments for cancer, viral and bacterial infections, birth control and AIDS 1 9 ’ 2 0 ’ 2 1 ’ 2 2 > 2 3 ’ 2 4 Colloidal polymer nanospheres are also finding widespread use in the fabrication of photonic band gap crystals.2 5 This is discussed in more detail in Chapter 3 of this thesis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 The following chapters in this thesis, examine the synthesis of monodisperse organic polymer nanospheres having particle sizes in the range of 200 nm to 800 nm. Different and novel methods for modifying these organic polymer nanospheres, and use of these monodisperse polymer nanospheres to build photonic band gap materials are also examined. 1.2 Chapter 1: References (1) Rampino, L. D.; Nord, F. F. J. Am. Chem. Soc. 1941, 63, 2745. (2) Hirai, H.; Nakao, Y.; Toshima, N. J. Macromol. Sci., Chem. 1978, A12, 1117. (3) (a)Hirai, H.; Chawanya, H.; Toshima, N. React. Polym. 1986,3,127. (b) Toshima, N.; Yonezawa, T.; Hirai, H. J. Macromol. Sci., Chem. 1988, A25 (5 - 7), 669. (4) Sherrington, D. C.; Miller, M. M.; Simpson, J. J. Chem. Soc., Perkin Trans., 1994, 2091. (5) Sherrington, D. C.; Ahn, J. Chem. Comm. 1996, 643. (6) Sherrington, D. C.; Ahn, J. Macromolecules, 1996, 29,4164. (7) (a) Chen, C. W.; Chen. M. Q.; Serizawa, T.; Akashi, M. Chem. Comm. 1998, 831. (b) Chen, C. W.; Serizawa, T.; Akashi, M. Chem. Mater. 1999, 11, 1381. (8) Chen, C. W.; Serizawa, T.; Akashi, M. Chem. Mater. 2002, 14,2232. (9) Pathak, S.; Greci, M. T.; Kwong, R. C.; Mercado, K.; SuryaPrakash, G. K.; Olah, G. A.; Thompson, M. E. Chem. Mater. 2000, 12, 1958. (10) Mecking, S.; Thomann, R.;Adv. Mater. 2000, 13,12. (11) Nie, S.; Su, J. Z.; Gao, X.; Han, M. Nature Biotechnology, 2001, 19, 631. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 (12) Whitten, D.; Kushon, S. A.; Ley, K., D., Bradford, K.; Jones, R. M.; McBranch, D. Langmuir, 2002,18(20), 7245. (13) Chatterji, D.; Rondelz, F.; Kundu, S.; Faure, N.; Gosh, D. Langmuir, 2003, 19, 5830. (14) Neckers, D. C. J. Chem. Educ. 1975, 52, 695. (15) Cormack A. G. P.; Jinfang, W.; Sherrington, D. C.; Khoshdel, E. Angew.Chem. Int. Ed. 2003,42,5336. (16) de Jong G. J.; de Boer, T.; de Zeeuw, R., R.; Sherrington, D. C.; Cormack, P. A. G.; Ensing, K. Electrophoresis 2003, 23, 1296. (17) Nixon, J. R.; Jalil, R. J. Microencapsul. 1990, 7, 297. (18) Yolles, S.; Eldridge, J. E.; Woodland, J. H. R. Polym. News. 1971,1, 9. (19) Sanders, L. M.; McRae, G. I.; Vitale, K. M.; Kell, B. A.; J. Controlled Release 1985,2,187. (20) Rogers, J. A; Ownsu-Ababio, G.; The 20th meeting o f the Controlled Release Society, Washington D.C., July, 1993, pp 24 -30. (21) Brannon-Peppas, L.; Grosvenor, A. L., Smith, B. S. The 21st meeting o f the Controlled Release Society, Nice, France, June, 1994, pp 27-30. (22) Fujita, S. M.; Sherman, J. M.; Godowski, K. C.; Tipton, A. J.; The 7th meeting o f the American Association o f Pharmaceutical Science, San Antonio, Texas, November, 1992, pp 15-20. (23) Cowsar, D. R.; Tice, T. R.; Giley, R. M.; English, J. P.; Methods Enzymol. 1985, 112, 101. (24) Eldridge, J. H.; Staas, J. K.; Gettic, A.; Marx, P. A.; Tice, T. R.; Gilley, R. M. Science 1993, 260,1323. (25) Park, S. H.; Xia, Y. Langmuir 1999,15, 266-273. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 2 Fabrication of the polymer nanospheres 8 2.1 Introduction A lot of effort has gone into the fabrication of organic polymer nanoparticles.la,lb,lc The polymerization techniques used in their synthesis, fall under a general category called heterogeneous polymerization and hence share some general features. These polymerizations are usually two-phase systems in which the starting monomer(s) and/or the resulting polymer are in the form of a fine dispersion in an immiscible liquid. The polymerization initiator may be soluble in the monomer or in the medium. In addition to the monomer(s), polymerization medium and initiators, one or more additives are also added to the polymerization medium to emulsify and stabilize the monomer droplets and the resulting polymer particles. Depending on the polymerization technique employed, different results, with respect to size and dispersity of the final polymer nanoparticles, are obtained. Figure 2.1 depicts this particle size dependence on the polymerization technique. In suspension polymerization,2 ,3 the initiator is soluble in the monomer and both the initiator and the monomer are insoluble in the polymerization medium. A suitable droplet stabilizer is added as suspension agent and the monomer is Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. suspended in the polymerization medium as small droplets, generated by a constant rate of stirring. Polymerization is then initiated at the desired temperature (20 °C to 100 °C). Under these conditions monomer droplets are directly converted into (a) <«) (b ) (c) <d) 1000 100 1.0 10 0.1 0.01 P article S iz e (tun) Figure 2.1 Particle size ranges obtainable using different polymerization techniques, (a) Emulsion polymerization. (b) Dispersion polymerization, (c) Precipitation polymerization. (d) Suspension polymerization, (e) Emulsifier free emulsion polymerization. polymer latex nanospheres of approximately the same size. Particle sizes generally obtained are between 20 pm to 2.0 mm. However, these latex particles tend to be of low monodispersity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 In emulsion polymerizations4 ,5 ,6 the monomer is insoluble in the polymerization medium, but is emulsified in it by the aid of a surfactant or emulsifier. The initiator, unlike in suspension polymerization, is soluble in the polymerization medium, and not in the monomer. The polymerization is generally carried out between 40 °C and 80 °C. Under these conditions, the monomer present in the mixture forms droplets 1.0 pm to 10.0 pm. The resulting polymer particles thus produced, range from 0.05 pm to 0.3 pm in size and are of high monodispersity. Using a technique called microemulsion polymerization, polymer particle sizes smaller than 0.05 pm can be obtained. Another specialized type of emulsion polymerization called emulsifier free emulsion polymerizations8 produces stable monodisperse colloidal particles in sizes from 0.1 pm to 1.0 pm. In dispersion polymerization,9 ,1 0 ,1 1 the monomer and the initiator are both soluble in the polymerization medium, but medium is a poor solvent for the resulting polymer. Depending on the solubility of the growing polymer particles in the medium, phase separation occurs at an early stage. This leads to nucleation and the formation of primary particles. These primary particles are swollen by the polymerization medium and/or the monomer and the polymerization proceeds largely within individual particles, leading to the formation of spherical particles in the region 0.1 pm to 10 pm. These are generally of high monodispersity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 In precipitation polymerization,1 2 the initial state of the reaction mixture is the same as that in dispersion polymerization, that is a homogeneous solution. However, the primary particles formed do not swell in the medium and the polymerization proceeds literally as a precipitation polymerization. By virtue of its nature, precipitation polymerization gives polydisperse and irregularly shaped polymers particles. Thus, only emulsion type polymerizations yield monodisperse particles of diameter smaller than 1.0 pm and that can be called nanoparticles. The other polymerization types yield particles of diameter larger than 1.0 pm and/or with poor monodispersity. 2.2 Emulsion polymerization The use of emulsion polymerization for synthesis of polymer lattices is well documented.1 3 ,1 4 The reaction systems for emulsion polymerization generally consist of the polymerization medium that is generally water, a monomer (M), an initiator (In), and a surfactant at a concentration above the critical micellization concentration (cmc). The system is stirred and the monomer and surfactant form an emulsion, where the droplets are polydisperse and of the order of microns in size. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 The polymerization reaction is initiated by heating to a suitable temperature between 50 °C to 80 °C. This decomposes the initiator. (Scheme 2.1) Some of the 50 °C - 80 °C In2 ---------------------- ► 2 In ' Scheme 2.1 Decomposition of initiator monomer dissolves into the aqueous phase. Styrene for example at 80 °C is soluble in water upto 4.0 g/1.1 5 The initiator radicals propagate with the rare monomer units they encounter in the aqueous phase to produce oligomeric radicals that are relatively soluble in water. (Scheme 2.2) M M + In ‘ ^ ' M-In ^ ' M2-In Scheme 2.2 Propagation of polymerization While the oligomeric radical is propagating in the aqueous phase, it may also undergo termination with another radical species. Subsequent aqueous phase propagation of oligomers which escape termination enables the oligomers to attain a degree of polymerization at which the species is now surface-active. The resulting species becomes a micelle, either by entering a pre-existing micelle or by forming a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 micelle by aggregation with surfactant molecules in the aqueous phase. The oligomeric radical, now enclosed in a micelle where the concentration of the monomer is much higher than in the aqueous phase, propagates rapidly. The micelle now contains a longer polymer chain: this is a young latex particle. Particle formation goes on simultaneously with particle growth, and thus the distribution of particle sizes is broad at this early stage of polymerization; the earlier formed particles being larger and newly formed ones being smaller. As the particles grow in size, there are eventually a sufficiently large number of particles of a sufficiently large size, that all the newly formed radicals enter pre-existing particles rather than nucleating new ones. The polymerization moves to a new stage, where particle growth occurs without the formation of new particles. The latex particles are swollen with monomer which is present both in the particles and as monomer droplets. Latex particles maintain an approximately constant monomer/polymer ratio on account of a balance arising between the free energy of mixing (the polymer particle seeks to become indefinitely dilute in monomer) and surface energy effects (limiting contact between swollen latex particles and the continuous phase). In order to maintain this approximately constant monomer/polymer ratio within the latex particles, monomer migrates from the emulsion droplets, through the aqueous phase and into the polymer latex where they are consumed through conversion to polymer. The Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 average size of the particle increases but also monodispersity increases. At this stage each growing particle contains only one growing polymer chain. Eventually, all the monomer in the droplets is consumed and the droplets disappear. The polymerization now moves into a third stage, where the only monomer present is in the polymer particles. As propagation continues, this monomer is consumed and the weight fraction of polymer in the particle increases. The viscosity inside the particle also increases thus decreasing the rate of termination. At this stage the growing particle can contain more than one growing chain. Termination now takes place between two radicals present on the same growing particle. As more and more monomer is consumed the rate of the polymerization slows down, eventually all the monomer is consumed, and the polymerization has effectively ceased. Emulsion polymerization yields highly monodisperse latex particles in the size range 50 nm to 300 nm. 2.3 Emulsifier free emulsion polymerization Emulsion polymerization is a convenient method to synthesize polymer nanoparticles and the route is well known.1 3 ,1 4 However, for many purposes there are certain disadvantages with this method. Latex particles generated by this method are stabilized by the absorption of surface active agents, limiting their final size.1 6 Another problem encountered is the presence of surfactants on the polymer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 nanospheres after the reaction, its removal is difficult and leads to loss of stability,1 7 ’1 8 and complete removal of the surfactant cannot be guaranteed.1 9 ,2 0 In the early 1970’s, it was realized that monodisperse latexes could be obtained even if the preparation was carried out below the critical micelle concentration ’ and a technique for preparing polymer nanoparticles without the above drawbacks was developed by Goodwin and coworkers. The process is called emulsifier free emulsion polymerization. The product of such a reaction is monodisperse latex. Particle diameters of 200 nm to 1000 nm are available via this technique. The reaction ingredients and conditions are the same as those for standard emulsion polymerization except that in this case no surfactant is added. Nanoparticle formation in emulsifier free emulsion polymerization can be divided into two stages, particle nucleation and particle growth. At the particle nucleation stage, the characteristics of surfactant-present and surfactant-free emulsion polymerizations reaction differ significantly as seen from the number and size of particles produced.2 4 Typically 101 5 particle/cm3 of average diameter 50 nm are produced by reactions in the presence of surfactant, while reactions in the absence of surfactant produce around 101 2 particles/cm3 of diameter 500 nm. Several Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 6 mechanisms have been proposed for these emulsifier free systems to help explain the initial particle nucleation stage: A F 0 * 7 (a) homogenous nucleation was proposed by Fitch ’ ’ in which a growing oligomeric free radical precipitates from the aqueous phase when it reaches a critical chain length to form a primary particle (b) growing free radicals, which undergo termination followed by particle nucleation through coagulation of these non-active species (c) growing free radicals, which achieve a size and concentration at which they 9 0 1 6 q 1 become surface active and undergo micellization. ’ ’ Munro and coworkers3 2 proposed that none of the above mechanism could completely explain particle nucleation for all monomers and that different mechanisms could operate for monomers of different water solubility. In general, for relatively water soluble monomers like methyl methacrylate and vinyl acetate, particles are nucleated through mechanism (a), that is, homogenous nucleation, while for sparingly water soluble monomers like styrene micellar type nucleation or mechanism (c) occurred. A two-stage model for particle nucleation and growth was -JO proposed by Goodall and coworkers. They carried out the aqueous phase polymerization of styrene in the absence of emulsifier using potassium persulfate as the initiator. Accordingly, the particle nucleation and growth in emulsifier free emulsion polymerization of styrene can be divided two stages. Stage one is short, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 producing a large number of oligomeric micellar particle nuclei. These particle nuclei consist mainly of oligomers of molecular weight approximately 1000 and are stable due to adequate surface charge. The particle nuclei capture free radicals from the aqueous phase and produce higher molecular weight oligomers and the particle size increases. As this occurs, the surface charge density decreases drastically. The particle nuclei lose their stability and begin coagulating. This marks the beginning of stage two, no new particles are formed and the particle size increases due to coagulation and polymerization, until the monomer is consumed and final particle size is obtained. Particle number decreases with time in stage two due to particle coagulation and monodispersity also increases. This stage of particle growth is similar in both emulsifier-present and emulsifier-free systems. 2.4 Fabrication of polystyrene nanospheres 2.4.1 Introduction Goodwin and coworkers2 3 investigated the polymerization of styrene under emulsifier free conditions. In the work, they studied conditions for nanoparticle synthesis and factors affecting nanoparticle size and monodispersity. Factors like monomer concentration, ionic strength, initiator concentration and temperature of reaction were found to affect the final nanosphere size. These parameters were found to be closely related and a combination of these variables could be used to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 obtain nanospheres of a particular size. Recently Salovey and coworkers3 4 developed a method for emulsifier free emulsion polymerization of styrene as a batch reaction. In previous work this method was used by Greci and coworkers.3 5 They optimized the reaction conditions and were able to produce 450 nm to 500 nm size polystyrene nanospheres in high monodispersity. They then extended this procedure to synthesize 500 nm size polyvinylpyridine nanospheres. This present work is an extension of the previous work by Greci and coworkers. The main objective of this work is to, apply the variables enumerated by Goodwin an others to the batch conditions developed by Greci and coworkers, and develop reliable methods to fabricate highly monodisperse colloidal polymer nanoparticles in a wide variety of sizes, spanning from 200 nm to 1000 nm. The other goal was to use these colloidal organic nanoparticles to build and study photonic band-gap crystals. 2.4.2 Experimental Styrene and divinylbenzene, were purchased from Aldrich Chemical Co. Styrene was vacuum distilled to remove the inhibitor. Divinylbenzene was extracted with aqueous sodium hydroxide to remove the inhibitor, then washed with water and dried over magnesium sulfate. All other reagents were used as received. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19 All polymer syntheses were carried out in a 150 ml reaction kettle. The kettle was fitted with a condenser, a nitrogen inlet valve and a high torque mechanical stirring apparatus.3 5 The model of the mechanical stirrer used was the IKA Eurostar power control-visc. HPLC grade water was used for all polymer synthesis. Polymer analyses were carried out on a Cambridge 360 Scanning Electron Microscope (SEM). SEM samples were prepared by placing a drop of sample diluted with water on a glass plate and allowing it to dry. The sample was then sputter coated with gold to obtain good contrast and to increase the electrical conductivity of the sample. General method for synthesis of polystyrene nanospheres. The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150 ml reaction kettle; equipped with a condenser, gas inlet and the mechanical stirrer, and containing the required amount of water, was heated to 80 °C, stirred at 300 rpm and flushed for one hour with nitrogen. After one hour the gas flow was turned off and a certain volume of styrene and required mole percent of divinylbenzene were added to the water. The reaction mixture was stirred for twenty minutes to bring the monomer and crosslinker to the polymerization temperature, followed by addition of potassium persulfate initiator. The reaction mixture was stirred at 300 rpm and 80 °C for the desired time and then stopped. The Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 reaction mixture was cooled to room temperature. A small sample of the aqueous colloidal suspension was taken and diluted with water. This suspension was used for obtaining SEM images. 2.4.3 Results and discussion The batch size for the polymerization reactions was reduced from 700 ml used by Greci and coworkers3 5 to 70 ml. This not only optimized monomer usage, but also led to a greater control over some of the parameters governing nanoparticle formation and growth, such as temperature and stirring. According to Goodwin et al,2 3 the factors affecting final particle size in emulsifier free emulsion polymerization are: a) amount of monomer used b) reaction time c) reaction temperature d) amount of initiator e) ionic strength of the reaction medium. In order to fabricate polymer nanoparticles of different sizes, we evaluated the effect these factors had on the particles size, using styrene monomer. The first variable evaluated was monomer amount. Table 2.1 shows the particle sizes obtained for different monomer amounts, on completion of polymerization. As Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 expected, the size of the final polymer nanospheres increased with the increase in the amount of monomer used. Different amounts of styrene were tried keeping other variables unchanged. Each of the polymerizations was run for a time of 4.0 hours, Monomer (ml) Diameter (nm) (+/- 6 %) A 1.5 191 B 2.0 273 C 3.0 331 D 4.0 384 E 5.0 403 F 7.0 510 Table 2.1 Variation of particle diameter with monomer amount. within which the polymerization reactions were complete as indicated by the absence of monomer. Figure 2.2 through Figure 2.7 show the SEM images of the nanospheres, obtained with different amounts of monomer. In the polymerization of 1.5 ml styrene, (Case A, Table 2.1) the final nanoparticles were not as monodisperse as in the other polymerizations reactions evaluated with larger amounts of starting styrene. Nanosphere batches were defined as monodisperse, when the nanosphere diameters had a deviation of less than +/- 6 % from the mean diameter of the sample being analyzed. Mean diameters and deviations from it, were measured by scanning electron microscopy. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.2 SEM image of polystyrene nanoparticles obtained using 1.5 ml styrene. Figure 2.3 SEM image of polystyrene nanoparticles obtained using 2.0 ml styrene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.4 SEM image of the polystyrene nanoparticles obtained using 3.0 ml styrene. Figure 2.5 SEM image of the polystyrene nanoparticles obtained using 4.0 ml styrene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.6 SEM image of the polystyrene nanoparticles obtained using 5.0 ml styrene. Figure 2.7 SEM image of the polystyrene nanoparticles obtained using 7.0 ml styrene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 The next variable that was evaluated was reaction time. Table 2.2 and Figure 2.8 show the variation of particle size with time. A single polymerization Time (hours) Diameter (nm) (+/- 6 %) A 1.0 332 B 1.5 377 C 2.0 418 D 3.0 503 E 4.0 517 Table 2.2 Variation of particle diameter with reaction time, using 7.0 ml styrene. Size v/s Time 550 500 - 450 - 400 - 350 - 300 Time (h) Figure 2.8 Variation of particle diameter with reaction time, using 7.0 ml styrene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 reaction was run, using 7.0 ml of styrene and a stirring rate of 300 rpm. The reaction temperature was 80 °C. Samples from the reaction were withdrawn at one hour, one and a half hours, two hours, three hours and four hours after the start of the reaction. The particle size grew from 332 nm after one hour to 517 nm after four hours. The rate of growth of the nanoparticles was steady and rapid initially, but falls off towards the latter part of the reaction. The next variable tried was the amount of initiator used. Initiator amount was varied. However, no discemable trend with respect to size of the nanospheres was seen in the polymerization of 5.0ml styrene with 4.0 mole percent divinylbenzene as crosslinker, at 80 °C and a stirring speed of 300 rpm. (Table 2.3) Initiator (gm) Initiator (g/1) Diameter (nm) A 0.03 0.425 347 (+/- 6 %) B 0.06 0.850 403 (+/- 6 %) C 0.12 1.700 408 (+/- 6 %) D 0.30 4.285 Polydisperse Table 2.3 Variation of particle diameter with initiator. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 An initiator concentration of 0.850 g/1, (Table 2.3, entry B) was found to give, repeatedly, the most reliable results, with respect to size and monodispersity of the final nanospheres, within a short reaction time frame of 4.0 hours. The next variable to be tried out was the ionic strength of the medium. Ionic strength (p) is defined as half the sum o f the terms obtained by multiplying the molality o f each ion in the solution by the square o f its valence. (Equation 1.1) where, m is the molality of the ionic species of type i and z is the charge carried by the ionic species. The ionic strength of the aqueous medium can be modified by simply adding salts. Initially a variety of salts were tried, at different concentrations to evaluate their effect on particle size, in the polymerization of 3.0 ml styrene using 4.0 mole percent divinylbenzene as crosslinker and 0.06 grams potassium persulfate as the initiator, in 70 ml water at 80 °C and a stirring rate of 300 rpm. As can be seen from Table 2.4, increasing the ionic strength of the polymerization medium increased the final particle size. Different salts do this to different extents and with differing impacts on monodispersity. Potassium chloride (Table 2.4; entries 4a, 4b) increased the particle size with respect to the blank (Table 2.4; (1.1) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 SALT Amount (g) Ionic Strength (f* ) (* io 3 ) Size (nm) (+/- 6 %) 1 none (blank) 0.000 334 2a NH4CI 0.047 12.7 427 2b NH4CI 0.054 14.4 506 2c NH4CI 0.071 19.2 362 2d NH4CI 0.089 24.0 polydisperse 3a NaCl 0.052 12.7 371 3b NaCl 0.069 14.4 420 3c NaCl 0.070 16.8 polydisperse 4a KC1 0.066 12.7 polydisperse 4b KC1 0.075 14.4 polydisperse 5a CaCl2 0.032 12.7 611 5b CaCl2 0.037 14.4 730 6a imidazolium triflate 0.192 12.7 polydisperse 6b imidazolium triflate 0.217 14.4 polydisperse 7a N,N-diethyl-imidazolium triflate 0.235 13.1 525 7b N,N-diethyl-imidazolium triflate 0.257 14.4 polydisperse 8 NaN0 3 0.075 12.7 372 9 NaHC0 3 0.074 12.7 324 10 Na2 S 0 4 0.0417 12.7 362 Table 2.4 Effect of ionic strength and salt used on particle diameter. entry 1), however the resulting polymer nanospheres were polydisperse. Similar results were obtained for the ionic liquids: the imidazolium triflates. Use of ammonium chloride and sodium chloride increased the size of the final polymer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 particle, with the monodispersity being compromised only at higher ionic strengths. At a given ionic strength, calcium chloride showed the largest increase in the size of the final polymer nanoparticle, compared to any of the other salts used, though its effect on monodispersity was variable. As calcium chloride gave the largest increase in particle size, we next tried to evaluate the effect of lower concentrations of calcium chloride on particle size and monodispersity. The polymerization of 2.0 ml styrene with 4.0 mole percent divinylbenzene as crosslinker and 0.06 grams potassium persulfate as initiator, in 70 ml water at 80 °C and a stirring rate of 300 rpm was used to evaluate this effect (Table 2.5). Amount of CaCl2 (g) Ionic Strength (* 10'3 ) Size (nm) Dispersity 1 0.00 — 273 Monodisperse (+/- 6 %) 2 0.01 3.97 408 Monodisperse (+/- 6 %) 3 0.02 7.95 435 Unpredictable 4 0.03 11.91 535 Unpredicatble Table 2.5 Effect of different concentrations of CaCh on final nanoparticle diameter. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 As can be seen from Table 2.5, calcium chloride at ionic strengths higher than 3.97 * 10 '3 negatively affected the monodispersity of the final particles. The most appropriate amount of calcium chloride was that which corresponded to an ionic strength of 3.97 * 10'3. Table 2.6 shows the variation of particle size with monomer amount, when the polymerization were carried out at an ionic strength of 3.97 * 10'3 , using calcium chloride. It also shows the corresponding particle size, for a given monomer amount, when the polymerizations were carried out without any added salt. 4.0 Mole percent divinylbenzene was used as crosslinker. Monomer Amount (ml) Size (nm) (+/- 6 %) H = 3.97 * 10‘3 Size (nm) (+/- 6 %) No added salt A 2.0 408 273 B 3.0 485 331 C 4.0 613 384 D 5.0 728 403 E* 7.0 800 510 Table 2.6 Variation of particle diameter with monomer amount, in the presence and absence of added salt (* 85.0 ml of water was used here, with all other cases 70.0 ml of water was used.) Figure 2.9 through to Figure 2.13 show the SEM images of these larger polymer nanoparticles obtained in the presence of calcium chloride with different amounts of monomer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 Figure 2.9 SEM image of the polystyrene nanoparticles obtained using 2.0 ml styrene and p = 3.97 * 10'3. Figure 2.10 SEM image of the polystyrene nanoparticles obtained using 3.0 ml styrene and p = 3.97 * 10'3 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 Figure 2.11 SEM image of the polystyrene nanoparticles obtained using 4.0 ml styrene and p = 3.97 * 10'3. Figure 2.12 SEM image of the polystyrene nanoparticles obtained using 5.0 ml styrene and p = 3.97 * 10'3. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33 Trm £. / 'w " ? - ’ 4Ik Figure 2.13 SEM of the polystyrene nanoparticles obtained using 7.0 ml styrene, 85.0 ml water and p = 3.97 * 10'3. The next variable tried was temperature. The temperature was varied between 60 °C and 90 °C to see the effect on particle size for a batch of 2.0 ml styrene and 4.0 mole percent crosslinking. At 60 °C, no polymerization was observed, while at 70 °C and 90 °C, the nanospheres obtained had low monodispersity. (Figure 2.14 and Figure 2.15 respectively) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.14 Polymer nanospheres obtained at 70 °C. i'Hi" ! ' . \ 0 t v i-ih-' l u v.OOpm I ................ Figure 2.15 Polymer nanospheres obtained at 90°C. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 Percentage crosslinking of a polymer affects a number of polymer properties such as hardness, flexibility etc. We next decided to study, the effect of changing the percentage crosslinking, would have on the polystyrene nanospheres. (Table 2.7) Percentage Crosslinking (mole %) Size (nm) A 0 388 (+/- 6% ) B 2 337 (+/- 6 %) C 4 408 (+1-6%) D 6 322 (+/- 6 %) E 10 Polydisperse Table 2.7 Variation of diameter of polymer particles at different percentages of crosslinking.. The obtained polymer nanospheres were generally monodisperse. However, at around ten mole percent crosslinking, polydispersity was seen to set in. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 2.5 Conclusion Monodisperse polystyrene nanospheres have been synthesized successfully. By varying certain reaction parameters like monomer amount, reaction time, reaction temperature, initiator concentration and/or ionic strength of the reaction the size of the nanospheres can be selectively tuned. Certain parameters like monomer amount, reaction time and ionic strength play a more significant role than others in controlling the particle size. The size of the polymer nanospheres can be tightly controlled in the range 200 to 800 nanometers. 2.6 Chapter 2: References (1) (a) Ladfester, K. Topics in Current Chemistry, (Colloid Chemistry II) 2003, 227, pp 75 - 123. (b) Arshady, R., Microspheres, Microcapsules and Liposomes 2002, 4, pp 1 - 37. (c) Biopolymer and polym er nanoparticles and their biomedical applications-, Nakache, E.; Poulain, N.; Canadu, F.; Orecchioni, A.; Irache, J.; Nalwa, H. S. (Ed.); Handbook o f Nanostructured Materials and Nanotechnology-, Academic Press : San Diego, California, 2000. (2) Arshady, R. A.; Ledwith, A. Reactive Polymers 1983, 1, 159. (3) Grulke, E. A. Encycl. Polym. Sci. Engg. 1989, 19, 443. (4) Zimehl, R.; Lagaly, G.; Ahrens, J. Colloid and Polymer Science-A.1969, 2, 835. (5) Vanderhoff, J. W. Journal o f Polymer Science Polymer Symposia. 1985, 72, 161. (6) Emulsion Polymerization-, Piirma, I ., Ed.; Academic Press: New York, 1982. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (7) Microemulsions, Fundamentals and Applied Aspects', Canadu, F.; Marcel Dekker: NewYork, 1998. (8) Chapter 2; Sec 2.3; this thesis. (9) Ober, C. K.; Lok, K. P.; Hair, M.L. Journal o f Polymer Science-A. 1985, 23,103. (10) Dispersion and Polymerization in organic media', Barret, K. E J.; John Wiley: London, 1975. (11) Almog, Y.; Reich, S.; Levy, M. Brit. Polym. J. 1982, 14, 131. (12) Stover, D. H., Li, K. Journal o f Polymer Science-A. 1993, 21, 3257. (13) Emulsion Polymerization', Bovey, F. A.; Kolthoff, M. I.; Medalia, A. I.; Meehan, E. J.; Interscience Publications: New York, 1955. (14) Grancio, M. R.; Williams, D. J. Journal o f Polymer Science-A. 1970, 8, 2617. (15) Arshady, R. Colloid and Polymer Science. 1992, 270, 717. (16) Bamnolker, G.; Margel, S. J. Polym. Sci. - A , 1996, 34, 1857. (17) Clean Surfaces, Their Preparation and Characterization fo r Interfacial Studies; Wanderhoff J. W., van der Hul, H. J.; Tausk, R. J. M.; Overbeck J. T. G.; Goldfinger G., (Ed.); M. Dekker: New York, 1970. (18) Ottewill, R. H.; Walker, T. KolloidZ. u Z. Polymere 1968, 227, 108. (19) Ottewill, R. H.; Walker, T. KolloidZ. u Z. Polymere 1967, 218, 34. (20) Shaw, J. N. J. Polym. Sci.-C. 1969, 27, 237. (21) Dunn A. S.; Chong, L. C. H. Brit. Polymer J. 1970,2, 49 (22) Kotera, A.; Takeda, F.; Takeda Y. K olloid Z. u Z. Polymere. 1970, 239, 677. (23) Goodwin, J. W.; Hearn, J.; Ho, C. C.; Ottewill, R. H. Colloid and Polymer Science. 1974, 252, 464. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 (24) Chainey, M.; Hearn, J.; Wilkinson, M. C. Journal o f Polymer Science-A. 1987, 25, 505. (25) Fitch, R. M.; Prenosil, M. B.; and Karen, J. S. J. Polym. Sci.- C. 1969, 27, 95 . (26) Polymer Colloids-, Fitch, R. M.; Tsai, C. H.; Plenum: New York, 1971. (27) Polymer Colloids Preprints-, Fitch, M.; NATO Advanced Study Institute, Trondheim, 1975. (28) Arai, M.; Arai, K.; Saito, S. J. Polym. Sci. Polym. Chem. Ed. 1979,17, 3655. (29) Advances in Chemistry Series; Van der Hoff, B. M. E.; No. 34, Amer. Chem. Soc: Washington DC, 1967. (30) Cox, R. A.; Wilkson, M. C.; Goodall, A. R.; Hearn, J.; Creasey, J. J. Polym. Sci. Polym. Chem. Ed. 1977,15, 2311. (31) Chen, C. Y.; Piirma, I. J. Polym. Sci. Polym. Chem. Ed. 1980,18, 1979. (32) Munro, D.; Godall, A. R.; Wilkinson, M., C.; Randle, K.; and Hearn, J. J. Colloid Interface Sci. 1979, 68, 1. (33) Polymer Colloids II; Goodall, A. R..; Wilkinson, M. C.; Fitch, R. M., (Ed.); Plenium: New York, 1975. (34) Salovey, R.; Aklonis, J. J.; Zou, D., Sun, L. J. Polym. Sci.- A. 1992, 270, 717. (35) Synthesis and Functionalization o f polymer nanospheres; Greci, M. T.; Ph. D. Dissertation, USC, Los Angeles, 2000. (36) Greci, M. T.; Pathak, S.; Merkado, K.; Prakash, G., K. S, Thompson, M. E.; Olah, G. A. J. Nanosci. Nanotech. 2001, 1(1), 3. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3 39 Fabrication of dye-doped colloidal core-shell polystyrene nanospheres for photonic crystals In this chapter, the use of colloidal polystyrene nanospheres for building photonic crystals is explored. The ability and techniques developed to synthesize polystyrene nanoparticles in a range of highly monodisperse sizes are extended to the development of dye-doped polystyrene nanospheres. These nanospheres were then used to assemble photonic crystals and to study some of the effects the photonic band gap (PBG) had on the incorporated dyes, such as suppression of spontaneous emission and energy transfer. 3.1 Introduction 3.1.1 What are photonic crystals? Over the last ten to fifteen years, there has emerged a new class of materials called photonic band gap materials or, more simply photonic crystals. The underlying concept behind these materials stems from early notions by Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 • 1 9 • • Yablonovitch and John. The basic idea is to design materials so they can affect the properties of photons in much the same way as ordinary solids or crystals affect the properties of electrons. According to Bloch’s,3 or Fluoquet’s 4 theorem, in a periodic medium, waves can be found that propagate without scattering. This accounts for why the electrons can propagate so far in a metallic conductor. This is because in a conductor like copper, the copper forms a periodic lattice of nuclei, of the right periodicity, that permits the electron wave to propagate with out scattering - the wave only scatters off impurities and imperfections in this crystal lattice, the same holds true for a semiconductor crystal. This theorem holds true for all types of waves, whether they are electron waves or light waves. Photonic band gap (PBG) materials are new materials, which stem from a branch of science that asks the question: “Can photons be made to ‘behave’ like electrons, and if so what are the consequences?” Localization is a property usually associated with massive particles such as electrons, that is they are found around the nucleus in an atom, while propagation is a property associated with photons, photons emitted by a source, travel away from it in all directions at the speed of light. The semiconductor owes its function to the fact that it enables the normally localized electrons to flow coherently, the PBG materials on the other hand exhibits novel functions by enabling the propagating photon to localize coherently. Photonic Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 band gap materials are the photonic analogues of semiconductors in the electronics industry. Rather than a periodic array of atoms which modifies the energy- momentum relations of electrons and generates an electronic band gap, photonic crystals consist of periodically modulated dielectrics, the scale of this periodicity is of the order of the wavelength of light. This modulation in dielectric is achieved by a simple alternation of refractive index of the material. The wavelength of the observed photonic band gap depends not only on the scale of this periodicity, but also on the refractive index difference. The properties of electrons are governed by the Schrdinger equation5 while the properties of photons are governed by Maxwell’s equation .6 (Equation 2.1) {V* (1/s (r)) V } H(r) = co2 H(r) (2.1) In Maxwell’s equation H(r) represents the photon’s magnetic fields and e (r) and is the dielectric function (the square of the refractive index) and C O is the angular frequency. Equation (2.1) and the Schrodinger equation are linear eigenvalue problems whose solutions are determined entirely by the potential V, or the dielectric s, respectively. Therefore, if one were to construct a crystal containing a periodic array of macroscopic uniform dielectric structures, then, light traveling Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 through such structures would experience a periodic variation of refractive index, analogous to the periodic potential energy of an electron in an atomic crystal. Therefore, the dispersion curves of light become organized into bands in a Brillouin Zone in reciprocal space and as in the case of electrons, the photons could be described in terms of a band structure. Variations of refractive index cause splitting of the bands at the edges of the Brillouin Zone, that are called stop gaps. No waves can propagate within these gaps. These gaps are called photonic band gaps. Increasing modulation of refractive index increases the gap width. A photonic band gap is a range of frequencies for which photons are forbidden to travel through the crystal in any direction. Thus, with a large photonic band gap, the photonic crystal is basically an optical insulator, similar to an electric insulator which has a very large electronic band gap and hence across which no electron can flow. The simplest photonic crystal that can be conceived consists of a multilayer film, of alternating dielectrics. (Figure 3.1). This is a one dimensional (1-D) PBG material and the photonic band gap exists only for one direction Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 Figure 3.1 1-D alternating crystal of propagation of light. If, however one were to include periodicity in two dimensions (Figure 3.2) and eventually three dimensions (Figure 3.3), one could create a two dimensional and a three dimensional photonic crystal, respectively. The three dimensional photonic crystal could have a complete photonic bang gap, that is, no light of the desired frequency can exist in the material irrespective of the direction of propagation. Figure 3.2 2-D alternating crystal Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44 Figure 3.3 3-D alternating crystal Such a 3-D PBG crystal can be used to manipulate photons in many interesting ways. By carving a tunnel through the material, one can have an optical “wire” or an optical waveguide, from which no light can deviate. These abilities to trap and guide light have many potential applications in optical communications and computing, where tiny optical circuits would manage the ever increasing communications traffic. Other applications possible due to this increase control over light include more efficient lasers and LED light sources. 3.1.2 Strategies for fabrication of photonic crystals To build up three dimensional photonic crystals, there are basically two methods. The first uses techniques known for the processing of semiconductors. Three dimensional structures prepared in this way, that is patterned in the micrometer region, allow for the manipulation of only infrared radiation. Yablonovitch followed this concept and introduced the so called “Yablonovites”, in Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 which the material is perforated with cylinders.7 The drilling angle is chosen to give a cubic closed pack (CCP) lattice structure in a high refractive index material. Another related technique is the “woodpile” structure that is formed via wafer- fusion and laser beam assisted alignment techniques. A complete photonic band gap for a near infrared wavelength was reported recently for a woodpile structure formed from silicon.8 ,9 The second concept uses the self assembly of sub micrometer sized or meso-sized particles.1 0 Mesoscale (~ 10 nm to ~ 10 pm) materials are some of the most commonly encountered forms of matter. They are widespread in chemistry and biology: typical examples include dendrimers, quantum dots, gold colloids, silica colloids, polymer particles, proteins, viruses and cells.1 1 The ability to assemble these particles into two-dimensional (2-D) and three dimensional (3-D) crystalline structures is directly useful in many areas. For example 2-D crystalline lattices of polystyrene beads can be used as arrays of microlenses in imaging,1 2 as reactive masks for evaporation or reactive ion etching to fabricate regular arrays of micro and nanostructures,1 3 and as templates to cast elastomeric stamps for use in microcontact printing.1 4 3-D crystalline lattices of colloidal particles can be used as templates to generate porous membranes,1 5 diffractive elements in fabricating sensors,1 6 or optical components such as gratings,1 7 filters,1 8 switches,1 9 and photonic band gap crystals.2 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46 In the case with Xia and coworkers,1 0 the photonic crystals are formed by the crystallization of monodisperse colloids. Crystallization occurs in a cubic closed packed structure and are called as artificial opals. Artificial opals reflect visible light. They are either made from silica,2 1 which is the classic case, or from polymers such as polystyrene,2 2 poly-methylmethacrylate,2 3 (PMMA), or other hydrocarbon backbone polymers.2 4 Compared to silica opals, polymers offer a larger chemical variability to adjust the refractive index, to perform surface chemistry or to incorporate dyes. The polymers opal can be used as a template, for preparing inverse opals with high refractive index materials, by back filling the voids in the opal and then burning or dissolving away the polymer.2 5 ,2 6 ,2 7 3.2 Studies on the inhibition of spontaneous emission and energy transfer in a photonic crystal Photonic crystals once fabricated can be used to manipulate light in many ways. One such effect we set out to study was the inhibition of spontaneous emission. This fascinating situation arises when an excited atom or molecule like a dye, with an emission wavelength within the band gap is placed inside the photonic crystal. Spontaneous emission is no longer possible, and the atom is trapped in the excited state. If however, another dye molecule, whose emission does not coincide with the band gap, and which can accept energy from the excited dye, is also Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 present in the same photonic crystal, energy is transferred from the first dye to the second dye, resulting in an emission from the second dye molecule and the first dye molecule returns to the ground state. Much research has gone into studying the effects of photonic crystals on the spontaneous emission of materials inside the crystal. While a three-dimensional photonic crystal has not been produced to fully realize the suppression of spontaneous emission, several groups have reported mixed results of incomplete Photonic Band Gap (PBG) materials. Megens and coworkers have used a core -shell approach to place a dye inside silica spheres.2 8 They showed very clear suppression of emission of the dye inside the crystal. Romanov and coworkers2 9 incorporated dye molecules into poly(methylmethacrylate) beads and used them to study the effects of the photonic crystals on the spontaneous emission. Their work focused, on the changes in the effects of the crystals on spontaneous emission with various excitation intensities. 3.2.1 Fabrication strategy Fabrication of the photonic crystals for studying inhibition of spontaneous emission and energy transfer began with the synthesis of dye doped monodisperse colloidal polystyrene nanospheres, of appropriate size, followed by assembly of the colloidal nanospheres into a crystalline CCP lattice, which exhibit a photonic band gap at the desired wavelength. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 3.2.1.1 Synthesis of dye doped polystyrene nanospheres We have been able to synthesize monodisperse polystyrene nanospheres in a range of sizes3 0 using emulsifier free emulsion polymerization. We used this technique to reliably and repeatedly synthesize three different sizes of monodisperse polymer nanospheres, which, when assembled3 1 into a photonic crystal had their band gaps in the blue, green and red regions of the electromagnetic spectrum. In order to study inhibition of spontaneous emission and/or energy transfer it was necessary to incorporate dyes into the polymer bead. Lanthanide dyes The dyes initially chosen for the studies were two lanthanide dyes. Lanthanide dyes were chosen as they have sharp emission spectra, with narrow full widths at half maximum (fwhm) and emission wavelengths extending from the visible all the way into the near infrared region (1.5 pm). The optical transitions resulting in these emissions occur in the 4f electronic shell that is shielded from the surroundings by two closed outer 5s and 5p shells. Hence the emission from these dyes are relatively insensitive to the environment and quenching, and thus high quantum efficiencies can be achieved.3 2 The lanthanide dyes used have the general structure shown in Figure 3.4. The dye emission properties are determined by the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49 lanthanide (Ln) atom which was either europium or terbium. The emission spectra for the two dyes are shown in Figure 3.5. Excitation is at 360nm. Emission wavelengths were measured in nanometres. Different approaches to dye (BTFA)3 Ln Phen Figure 3.4 Lanthanide dye chosen for nanosphere doping. Ln = europium or terbium. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 1.0 - 0.8 - 0 . 6 — S 5 0 . 4 - 0.2 - 0.0 - w A (BTFA)3Eu(Phen) In Styrene -0 _ emission Excitation 360 nm. (BTFA)3Tb(Phen) in Styrene emission Excitation 360 nm. -O------------O - i i i i i i i 30 0 T— J — I— I— I— I — |— I — T" 40 0 t — |—i ------ 1 —r — |— i—i i i- | 1 i i-----1 —i —i —r - |—i —i —i------ 1 i i i —i—| 5QQ BOD 700 BOQ W avelength Figure 3.5 Emission spectra of europium and terbium dyes in styrene. incorporation were tried. However, attempts at incorporation of these dyes into the polymer nanospheres were associated with many complications, such as loss of size control during nanosphere growth and quenching of fluorescence. Organic dyes The problems associated with lanthanide dyes shifted the focus back to organic dyes. In this case the two dyes chosen were Coumarin 334 (C-334) (Figure 3.6) and Nile red (Figure 3.7). Coumarin 334 and Nile Red were chosen as a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 Figure 3.6 Coumarin 334 Figure 3.7 Nile Red. donor-acceptor pair because of their less than perfect energy transfer in the absence of a photonic crystal with overlap of the stop band with the emission. As seen in Figure 3.8, there is some degree of overlap between the emission of the Coumarin Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 0.7 — C oum arin 334 Excitation - - C oum arin 334 Em ission 0.5 Nile Red Excitation 0.4 Nile R ed Em ission 0.3 0.2 0.1 750 450 500 550 600 650 700 250 300 350 400 Wavelength (nm) Figure 3.8 Normalized photoluminescence spectra of Coumarin 334 and Nile Red in polystyrene nanospheres 334 and the Nile Red absorption. This is a prerequisite for efficient energy transfer. Increasing that overlap increases the efficiency of energy transfer. The dyes were incorporated during the emulsifier free emulsion polymerization of the nanospheres. In order to avoid quenching and to maintain a high concentration of dye near the nanosphere surface, the dye doped nanospheres were synthesized using a slightly modified procedure as compared to the non-doped nanospheres. This procedure involved a core shell approach, (Figure 3.9) in which a non-doped polystyrene core (A) of the required size was first synthesized. On this core, a polymer layer containing the dye was grown (B), followed by a thin highly Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 crosslinked overcoat of non-doped polymer (C). The aim was, to synthesize two different sizes of polymer nanospheres, each doped with the same, known ratio of A Figure 3.9 Cartoon of the dye containing polymer nanospheres. (A) central core, (B) dye containing layer, (C) overcoat. the two dyes. The sizes of the nanospheres were chosen such that, when they were assembled into a CCP lattice they exhibited stop bands covering different regions of the electromagnetic spectrum. In one photonic crystal, the stop band overlapped with the emission of only one of the dyes, in this case Coumarin 334, while in the second photonic crystal no overlap between the stop band and the emission of the dyes is seen. The first crystal was used to study the inhibition of Coumarin 334 emission by the stop band and subsequent energy transfer to Nile Red. The second crystal acted as a reference. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 3.2.1.2 Assembly of the colloidal polymer nanospheres into photonic crystals A number of methods have been demonstrated for the crystallization of mesoscale colloidal particles. Methods based on solvent evaporation3 4 are very effective in generating 2-D assemblies of mesoscale particles. Crystal domain sizes usually are less than 0.01 mm. Another simple and useful method for fabricating crystals is based on gravity sedimentation.3 5 Procedures based on this method are relatively slow taking weeks to a month to completely sediment the colloidal particles. This approach also has very little control over the morphology of the surface and the number of layers in the crystalline assemblies. Methods based on electrostatic interactions seem to be very successful for generating multilayer assemblies of mesoscale colloidal particles. Using this technique 3-D crystal 3 3 A assemblies as large as 1.0 cm have been generated. This method, however, requires very strict control on the experimental conditions such as density of the charges on the surface of the particle, the concentration of particles and the concentration of free electrolyte molecules in the dispersion medium. Another technique showing good results uses the tendency of meso-scale particles to crystallize at the meniscus between a vertical substrate and a colloidal suspension.3 7 As the meniscus of the colloidal suspension is slowly swept across the vertical substrate by evaporation, it leads to a thin, well ordered, planar structures across the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55 substrate. Initially this process, however, was limited to particles smaller than 400 in nm. Recently Vlasov and coworkers overcame this problem by adding a temperature gradient. The convective flow combats sedimentation and provides a continuous flow of particles to the meniscus region. Using this technique Vlasov and coworkers have succeeded in ordering silica spheres, 860 nm in diameter, onto a silicon wafer. The best method to form crystalline assemblies of meso-scale colloids over large areas (~ 1.0 cm3 ), is the technique developed by Xia and coworkers.3 9 It involves the use of a cell, formed by two glass substrates and a square frame of photoresist that has been patterned on the surface of one of the substrates. An aqueous suspension of spherical colloidal particles is injected into the cell, and external gas pressure and sonication is used to drive the colloids into the CCP lattice. This process has many attractive features a) it is relatively fast, for example polystyrene polymer nanospheres of 480 nm 2 , diameter can be crystallized into a 25 layer assembly over an area over ~ 1.0 cm in about 48 hours. b) it has tight control over surface morphology and the number of layers of crystalline assemblies c) it works for aqueous dispersions of a wide variety of spherical particles regardless of the chemical compositions and/ or surface properties Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56 The method we use for crystal growth, was a modified version of the method based on solvent evaporation. This method has been successfully used to grow crystals for PBG studies.2 3 3 In short a few drops of the aqueous colloidal dispersion of polymer nanoparticles was placed on a glass slide. This was then evaporated by heating the slide gently in an oven at around 70 °C. All current PBG studies were carried out on crystals prepared by this technique. We are presently working on the technique of preparing photonic crystals using the cell method developed by Xia and coworkers, for future studies. 3.3 Experimental Styrene and divinylbenzene, and the dyes were purchased from Aldrich Chemical Co. Styrene was vacuum distilled to remove the inhibitor. Divinylbenzene was extracted with aqueous sodium hydroxide to remove the inhibitor then washed with water and dried over magnesium sulfate. All other reagents were used as received. All polymer syntheses were carried out in a 150 ml reaction kettle. The kettle was fitted with a condenser, a nitrogen inlet valve and a mechanical stirring apparatus.4 0 The mechanical stirrer employed was the IKA Eurostar power control- visc. Sparkletts distilled water was used for the polymerization. Polymer analysis were carried out on a Cambridge 360 Scanning Electron Microscope (SEM). SEM Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57 samples for microscopy were prepared by placing a drop of sample diluted with water on a glass plate or on an Aluminum SEM stub and allowing it to dry. The sample was then sputter coated with gold. Reflectivity measurements were taken on a Cary 14 Spectrophotometer OLIS (Modernized Cary 14) UV/Vis/NIR spectrophotometer equipped with deuterium and tungsten lamps for UV and Vis/NIR regions. A Vertex™ Specular Reflectance Accessory provided angular reflectance data. A front face mirror was used as a baseline, and a neutral density filter was placed in the reference beam pathway. Steady state emission spectra were recorded using a Flourolog-3 model FL3-21 with a 450W xenon lamp source, double grating excitation monochromator, single grating emission monochromator, and a room temperature R928 PMT serving as the detector. A front face emission detection accessory was used to collect the emission from solid samples. As seen in Figure 3.10, this accessory has a 22 0 angle between the excitation beam and the detector mirrors. The incidence angle of detection was changed by rotating the sample on a fixed stage with 0° defined as perpendicular to the sample face. UV- Vis. spectra of the charged-dye doped polystyrene nanospheres were taken on a HP - 8453 model spectrometer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58 Excitation Detector 0° Incidence Figure 3.10 Diagram showing the angular photoluminescence detection method used. The light source and detector mirrors were fixed 22 ° apart. Rotation of the sample provided angular measurements with 0 0 being perpendicular to the sample face. Synthesis of polystyrene nanospheres. The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique.4 1 Depending on the required properties of the final product nanospheres some changes in the polymerization process were undertaken which are specified below. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 59 Synthesis of polystyrene nanospheres not containing any dyes The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150 ml reaction kettle, equipped with a condenser, gas inlet and mechanical stirring apparatus, and containing 70 ml water, was heated to 80 °C, stirred at 300 rpm and degassed for 1 hour with nitrogen. After 1 hour the gas flow was turned off and a certain volume (upto 7.0 ml) of styrene and 4.0 mole percent of divinylbenzene (DVB) were added to the water. The reaction mixture was stirred for 20 minutes to bring the monomer and crosslinker to the polymerization temperature, followed by addition of 0.06 grams of (0.3 mmole) of potassium persulfate initiator. The reaction mixture was stirred at 300 rpm and 80 °C for 4 hours and then stopped. The reaction mixture was cooled to room temperature. By varying the monomer amount used the size of the nanospheres obtained, after 4 hours of reaction, can be varied. This can also be done by varying the reaction times. Preparation of the solutions of the dyes in the monomers The following dye solutions (Table 3.1) were prepared and used in the synthesis of the dye doped nanospheres. In between uses, the dye solutions were stored under nitrogen at -20 °C, away from light. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 Solution Coumarin 334 (mg) Nile Red (mg) 10 mole % DVB in Styrene (ml) A 5.2 0.0 10 B 0.0 5.5 10 C 20.2 23.7 10 D 0.0 0.0 10 Table 3.1 Formulation of the solutions of the two dyes in monomer. Core-shell synthesis of nanospheres (Red Stop Band) containing the dyes Coumarin-334 and Nile Red The polymerization was carried out in a glass vessel fitted with a condenser, a nitrogen purge inlet, and a mechanical stirrer with a Teflon blade. 70.0 ml distilled water (Sparkletts) was added to the reaction vessel. A stirring rate of 300 rpm was used. The vessel was immersed in an oil bath heated to 85 °C. A steady stream of nitrogen was bubbled gently through the water for an hour. Gentle nitrogen purging was continued, but not bubbled through water. Next 1.5 ml styrene monomer and 0.086 ml divinylbenzene monomer (4.0 mole %) were added to reaction vessel. Stirring was continued at 85 °C and 300 rpm. After 20 minutes 0.0615 g potassium persulfate was added to initiate the reaction. The reaction mixture was stirred at 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. °C and 300 rpm for an additional hour. Next 0.2 ml of the prepared solution of Nile Red and Coumarin 334 (Solution C) was then added to the reaction mixture while maintaining the rate of stirring and temperature. After an hour 0.2 ml of a 10 mole percent solution of divinylbenzene in styrene was added. Some of the dye entered into the monomer phase. After half an hour, the nanospheres showed green reflectivity. (The reflectivity of the growing colloid nanospheres were checked by withdrawing a small sample of polymer colloid from the reactor. A few drops of this sample were placed on a glass slide and this was gently evaporated in an oven at 70 °C). Next, 0.2 ml of 10 mole % DVB in styrene was added, no dye leached into the monomer. About 0.8 ml of 10 mole % DVB in styrene was added. After an hour the nanospheres showed a green - red band gap. The rate of stirring was raised to 350 rpm; temperature was maintained at 85 °C. After about an horn- the desired pink colored reflectivity was obtained. The stirring was stopped and the reaction mixture was allowed to cool to room temperature. The reaction mixture was transferred to a separatory funnel where any unreacted monomer was removed. Core-shell synthesis of nanospheres (Blue Stop Band) containing dyes the Coumarin-334 and Nile Red The polymerization was carried out in a glass vessel fitted with a condenser, a nitrogen purge inlet, and a mechanical stirrer with a Teflon blade. 70 ml distilled Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. water (Sparkletts) was added to the reaction vessel. A stirring rate of 300 rpm was used. The vessel was immersed in an oil bath heated to 85 °C. A steady stream of nitrogen was bubbled gently through the water for an hour. Gentle nitrogen purging continued and 1.5 ml styrene monomer and 0.086 ml divinylbenzene monomer (4.0 mole %) were added to reaction vessel. Stirring was continued at 85 °C and 300 rpm. After 20 minutes 0.0615 g potassium persulfate was added to initiate the reaction. The reaction mixture was stirred at 85 °C and 300 rpm for an additional hour. Next, 0.2 ml of the prepared solution of Nile Red and Coumarin 334 (Solution C) was added to the reaction while maintaining the rate of stirring and temperature. After an hour, 0.2 ml of a 10 mole % solution of divinylbenzene in styrene was added. Some of the dye entered into the monomer phase. After half an hour, the nanospheres showed blue reflectivity. Next 0.2 ml of 10 mole % DVB in styrene was added with some dye leaching into the monomer. Next, 0.0234 g of the initiator was added as the reaction seemed to be going slow. After half an hour, 0.4 ml of 10 mole % DVB in styrene was added. After another hour and a half, the nanospheres showed the required blue -green band reflectivity. The stirring was stopped and the reaction mixture was allowed to cool to room temperature. The reaction mixture was transferred to a separatory funnel where any unreacted monomer was removed. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63 Core-shell synthesis of nanospheres containing either Nile red or Coumarin 334. The nanospheres doped with either Nile Red or Coumarin 334 were synthesized as in the case for the nanospheres containing a mixture of both dyes. In the case of Coumarin 334 doped nanospheres, Solution A (Table 3.1) was used to incorporate the dye during the polymerization, while for the Nile Red doped nanospheres, Solution B (Table 3.1) was used. Synthesis of Lanthanide Dye (Ln-dye) doped polystyrene nanospheres. For the synthesis of Ln-dye doped polymer nanospheres, solutions of the lanthanide dye in styrene, 4-vinylpyridine and ethylacrylate were prepared by dissolving 9.0 mg of the Ln-dye, (BTFA) 3 Ln (Phen), in 25 ml of each of these different monomers. This was used during the polymerization. The lanthanide (Ln) could be Europium or Terbium. These solutions were used to carry out the synthesis of the Ln-dye dopes nanospheres, via one step polymerization or core-shell type polymerizations. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64 3.4 Results and Discussion Nondoped polystyrene nanospheres were prepared in a range of sizes, which when assembled into a cubic lattice, were intensely colored. The nanospheres were assembled into cubic lattices as follows: a few drops of the nanosphere colloid suspension were taken from the reactor and placed on a glass slide. This slide was then placed in an oven and the water was gently evaporated at 50 °C to 70 °C. The dried latex films show an intense color, which corresponds to their Bragg peak (Figure 3.11). The color varies with the size of the nanospheres. This process (U 0.04 0.02 700 600 500 400 Wavtength (nm) Figure 3.11 Bathochromic shift of the Bragg peak with increasing size of the nanospheres. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 65 of color determination was also used to monitor the growth in size of the polymer nanospheres during the polymerization. The size of the nanospheres can be controlled either by controlling the amount of monomer used or by stopping the reaction at the appropriate time or a combination of both. The data in Table 3.2, shows the amount of styrene required, Monomer amount (ml) Diameter (nm) (+/- 6 %) Color A 1.5 210 Blue B 2.0 250 Green C 3.0 306 Red Table 3.2 Different sizes of polystyrene nanospheres obtained with varying amount of monomer, and color of the corresponding crystals. to obtain blue, green and red reflective crystals, at 4.0 mole percent crosslinking with divinylbenzne, a reaction time of 4.0 hours at 80 °C and stirring rate of 300 rpm. The corresponding SEM’s of their surfaces are seen in Figure 3.12, Figure 3.13 and Figure 3.14, respectively. Figure 3.11 shows the corresponding Bragg peaks. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 66 Figure 3.12 SEM image of the of the blue reflective surface Figure 3.13 SEM image of the of the green reflective surface Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 67 Figure 3.14 SEM image of the of the red reflective surface. As mentioned earlier the nanospheres were assembled into cubic lattices by placing a few drops of the nanosphere colloid suspension taken from the reactor on a glass slide. This slide was then placed in an oven and the water was gently evaporated at 50 °C to 70 °C. The lower temperature yields better results with respect to FCC packing of the colloids into a crystal and size of the crystals obtained. The colloids slowly assemble into a CCP crystalline lattice (Figure 3.15 and Figure 3.16), which show an intense color, which corresponds to the Bragg peak. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.15 CCP lattice surface and edge, of the assembled colloids (SEM image) - W - : - - ' H ■ , £ . J Figure 3.16 Magnified view of the surface and edge showing the packing in the assembled CCP crystal. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69 and which varies with the size of the nanospheres. The slower the evaporation process, the better is the quality of the crystal. Some form of heating is required as it supplies the kinetic energy required for the crystal assembly. The Bragg peak maximum corresponds to the optical stop band of the photonic crystal.2 3 8 Lanthanide based dyes (Figure 3.4) were tried out in the synthesis of dye doped polystyrene nanospheres. Initially, a solution of the Ln-dye in styrene monomer was used to synthesize the dye nanoparticles, using 4 mole percent di vinylbenzene as crosslinker and standard polymerization conditions of 80 °C, 300 rpm stirring rate and 4.0 hour reaction time. This process ran into many problems; a) the final size of the bead was much larger than predicted for a given amount of styrene, and was not reproducible. However, the monodispersity was not affected. (Figure 3.17) b) about forty minutes into the polymerization the fluorescence was quenched in the monomer phase c) no fluorescence was ever seen in the polymer nanospheres To check for temperature sensitivity of the dye, a sample solution of the dye in the styrene was kept at the polymerization temperature of 80 °C for the reaction time of four hours. No quenching of the dye was seen. Thus the dye was stable at the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 70 reaction temperature for the time of the reaction, the presence of free radicals could play a role in the quenching. Figure 3.17 SEM image of the Ln-dye doped polystyrene nanoparticles In order to address the problems of size control and quenching, a core shell approach was tried. Here the polystyrene core was first grown, at this point a small amount of the Ln-dye solution in styrene was added and a dye-doped shell was grown around the core. This technique solved the problem of size control, however no dye was seen in the nanospheres and quenching of the dye in the styrene was still a problem. Solutions of the Ln-dye in other monomers like ethyl acrylate and 4- vinylpyridine were tried out for growing dye doped shells around the polystyrene core. In the case of ethyl acrylate the same problems as with styrene persisted. In Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the case using 4-vinylpyridine, the extent of quenching was reduced and some dye was incorporated into the polymer nanospheres. However these nanoparticles with poly-vinylpyridine shell, stuck to each other and it was impossible to order them into a crystalline cubic lattice. Organic dyes, Coumarin 334 (Figure 3.6) and Nile Red (Figure 3.7) were successfully incorporated, individually and simultaneously, into polystyrene nanospheres using a core shell approach. Figure 3.8 shows the emissions of these two dyes in polystyrene nanospheres. Figure 3.18 and Figure 3.19 show the SEM images of the doped polystyrene nanospheres containing both the dyes. The 272 nm nanospheres assemble to form a red reflective crystal while the 218 nm nanospheres ! • 1 i Figure 3.18 SEM image of red reflective dye doped polystyrene nanospheres Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72 assemble to form a blue reflective crystal. The polymer nanospheres are monodisperse with a ± 6% size variation and spherical in shape. Figure 3.19 SEM image of blue reflective dye doped polystyrene nanospheres The emission spectrum at 0 0 incidence is shown in Figure 3.20. The sample was excited at 365 nm. The large peak at 435 nm is due to Coumarin 334 and the peak at 560 nm is from Nile Red. The spectrum was corrected by division of the signal by a reference signal taken just before the sample compartment. The data was then normalized to 401 nm for convenience. Figure 3.21 shows the reflection measurements with varying angle (5-55 °) for the blue reflective crystal. The data is normalized for convenience. As predicted by Bragg’s Law, the reflection peak shifts Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73 with changes in angle. At 5 0 the stop band appears at 490 nm and is shifted to 400 nm. This overlaps well with the Coumarin 334 emission peak without overlapping with the Nile Red emission. This also allows excitation light at 365 nm to enter the crystal without significant reflection. 1.6 1.4 1.2 1 g 0.8 I 0.6 0.4 0.2 0 600 650 450 550 400 500 Wavelength (nm) Figure 3.20 Emission spectra of the red reflective crystal at 0 0 incidence. Exc. at 365 nm, normalized to 401 nm. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 74 1.5- b < £ > /— 20 650 550 600 400 450 500 Wavelength (nm) Figure 3.21 Normalized reflectance of the blue crystal showing the angular dependence of the stop band at 490 nm to 400 nm for angles 5-55° The angular dependence of the stop band position for the red crystal is shown in Figure 3.22. There is minimal overlap with the emission peaks at small angles. The data has been normalized for convenience. The difference in reflection strength between the blue and red crystals is mostly due to differences in the number of layers in the crystals. As the crystal increases in thickness the effect from the stop band is more pronounced as previously reported. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75 D i. « i, & o V 5 3 < u Q d 600 650 700 750 400 450 500 550 W avelength (nm) Figure 3.22 Normalized reflectance of the red crystal showing the angular dependence of the stop band at 610 nm to 565 nm for angles 5 - 60° The emission spectra of the crystals were taken at -22 to 60° incidence in 5° increments. Because of the experimental setup of the front face detection system, angles above 60° were unavailable because of interference with the excitation beam. The crystal position relative to the excitation beam was visually verified with 560 nm light before each measurement. Excitation light of 365 nm was used with 3 nm : 1 nm excitation : emission bandpass slit settings for 0-40°. To increase the signal the slits were opened to 5:1 nm for 40-45°, and 5:3 nm for 50-60°. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 76 The spectra were normalized at 401 nm for convenience of comparison and because the effects of the stop band were minimal at 401 nm. The normalized spectra for the angles at each slit change were overlaid to verify no line shape change other than better signal to noise ratios. The normalized emission spectra for the blue crystal are shown in Figure 3.23. The dips in the Coumarin 334 emission centered at 435 nm correspond to the stop band positions seen by the reflectance measurements To better visualize the suppression of emission caused by the overlap of the stop band with the Coumarin 334 emission in the blue sample, the normalized spectrum at each angle was divided by the normalized emission spectrum of the red crystal measured at 0° incidence. Dips of these ratios below one in the area of Coumarin 334 emission represent suppression of the emission. The results are shown in Figure 3.24 for angles 0 - 60°. The positions of the suppressed emission correlate to the reflectance measurements of the blue crystal. This suppression imparts an angular dependence on the perceived color of emission to the crystal with a range of over 80 nm. The strength of the suppression was found to be dependent upon the crystal thickness, with thicker crystals having a deeper dip in the emission with the same stop band center. Dilution of the suspension before evaporation led to uniform but thinner crystals. These crystals showed the same stop Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 77 550 600 650 400 450 500 Wavelength (nm) Figure 3.23 Emission spectra of the blue crystal at 0 - 60° incidence detection. Exc. 365 nm, normalized at 401 nm. Suppression of emission from the stop band is in agreement with the reflectance measurements. 1.00 — .< # 7 ‘ .V * /b 0 .9 5 - 0 .9 0 - 0 .8 5 - 0 .8 0 - 500 460 480 420 440 Wavelength (nm) A n g le ■ O '01 - □ • '5' - a - '10' - P - ' 15' - c - -20' • E '25 ' '30 ' -A- '40 b' • '45 b' ’50 c' ’55 c' - G h ’ 6 0 c' Figure 3.24 Normalized angular emission spectra of the blue crystal divided by the normalized spectra of the red crystal at 0° incidence. Minima represent the center of the suppression of emission of Coumarin 334 for each spectrum. Spectra taken with excitation:emission bandpass slits set at 3:lnm except for (b) 5:1 nm and (c) 5:3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 78 band positions by transmission and reflectance studies but showed very minor changes with angle on the emission spectra. The crystals obtained by the evaporation technique differed in thickness. Thicker crystals had higher stronger emission measured because of the increased dye in the excitation pathway. Rotation of the sample also led to changes in the spot size of the excitation beam on the sample. This was minimized by visually confirming alignment of the crystal with the excitation beam prior to each experiment, but still caused a decrease in intensity with increasing angle. These intensity differences prevent accurate comparisons of the spectra at various angles without normalization. The suppressions of emission by the stop band make the spectra sensitive to the wavelength chosen for normalization. Normalization at 401 nm avoids the effects of the stop band for most of the samples except at very large angles. Normalization at wavelengths higher than 620 nm was very dependent upon the signal to noise ratio of the spectra. Because of these difficulties, a qualitative search for energy transfer was performed by normalization at the Nile Red emission maximum of 560 nm. The emission spectrum of the red crystal at 0° was chosen as the control because of its lack of overlap with the stop band and the emission peaks. The same dye containing monomer solution was used for both the red and blue crystals. A quantitative micropipette was used to accurately add the same amount of this dye containing monomer to each reaction. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 79 Thus, the ratio of dyes physically present in the smaller polymer nanospheres should be the same as in the larger polymer nanospheres. Figure 3.25 shows an overlay of the red control with the normalized spectra of the blue crystal at 50° and 60°. The reduced intensity from the Coumarin dye is readily apparent even slightly beyond the area suppressed by the stop band. Additionally, there was an increase in 3.5 2.5 S B © g h i 0.5 650 450 500 550 600 400 Wavelength (nm) Figure 3.25 Normalized (560 nm) emission spectra of red control crystal at 0°, blue crystal at 50°, and blue crystal at 60° Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80 emission at longer wavelengths than the stop band 450 nm to 530 nm. These increases in the ratio of Nile Red emission to the Coumarin 334 emission in the large angles of the blue crystal could be indicative of increased energy transfer. 3.5 Doping of polystyrene nanospheres with charged dyes. . The infrared region of the electromagnetic spectrum between 1300 nm and 1550 nm, is the telecommunication wavelength region. Extension of studies from the present visible region to this region is the next logical step. Many of the dyes emissive in the infrared region are charged dyes, either positively or negatively charged. The polystyrene nanospheres generated by emulsifier free emulsion polymerization have a net negative charge on the surface which gives them their colloidal properties. We tried to see if it was possible to dope the polystyrene nanospheres using electrostatic interactions. For this purpose we chose two dyes, Basic Fuchsin,(Figure 3.26) a positively charged dye, and Phloxine B, (Figure 3.27) a negatively charged dye. The process for incorporation of the dyes followed a core shell approach similar to the other dye doped nanospheres. After the growth of the core of required size, the polymer suspension was transferred hot, to a separatory funnel and any excess monomer was removed. The dye solution in water and a little acetone was added to the polymerization chamber, and the colloidal polymer solution was returned hot to the polymerization vessel. This transfer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81 n h 2 n h 2 h 2n Figure 3.26 Basic Fuchsin C O O N a N aO Br Figure 3.27 Phloxine B Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 82 process was carried out as quickly as possible. The polymerization reaction was stirred at 80 °C for half an hour to allow the dye to electrostatically bind to the nanoparticles. An overcoat, of 10 mole percent divinylbenzene in styrene was then added and the reaction continued till the appropriate size was obtained. (Figure 3.28) It was found that for a batch of 4.0 ml styrene, at 4 mole percent crosslinking and after 3 hours of core formation, could bind 0.2 mg to 0.3 mg of dye without loss of dye via leaching into the monomer phase. In the case of Basic Fuchsin a color change from pink to purple was seen on adsorption onto the nanoparticle. (Figure 3.29 A and Figure 3.29 B). No color change is observed for Phloxine B. After the reaction, the obtained nanoparticles were filtered and dried. These nanoparticles were swollen in solvent and washed many times with water and organic solvents. Any Phloxine B present on the nanoparticles was washed away at this stage, Basic Fuchsin remained on the nanoparticles. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 83 Figure 3.28 Polymer nanospheres doped with Basic Fuchsin Figure 3.29 (A) UV-Vis absorption spectra of Basic Fuchsin in water. (B) Visible absorption spectra of Basic Fuchsin doped polystyrene nanospheres. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 84 3.6 Conclusion and Outlook Monodisperse polymer colloid nanoparticles have been successfully synthesized in a range of sizes. These nanoparticles were assembled into CCP lattices using a simple and quick technique. On assembly into the cubic structure, the crystals show a strong Bragg peak, that showed the predicted bathochromic shift with increasing size of the nanoparticles. Attempts to incorporate lanthanide dyes into the nanoparticles failed. However organic dyes like Nile Red and Coumarin 334 were successfully incorporated into the nanospheres using a core shell approach. Control over size and monodispersity of the nanospheres was retained. These dye-doped nanospheres were used successfully, to show suppression of spontaneous dye emission in the presence of an overlapping photonic band gap. Studies on energy transfer were carried out and look promising, but irreproducible nature of the photonic crystals with respect to, number of layers of colloidal particles and poly-crystallinity of sample, as a result of fabrication technique prevent obtaining quantitative results of this energy transfer. Ongoing work on producing photonic crystals reproducibly using a cell is underway and once perfected should permit further studies in this area. The infrared region of the electromagnetic spectrum between 1300 nm and 1550 nm, is the telecommunication wavelength region. Extension of studies, from the present visible region to this region, is the next logical step. Work is ongoing on developing larger 800 nm to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 85 1000 nm polystyrene nanospheres for use in these studies. Recent success with doping the negatively charged nanospheres with positively charged dyes could also prove useful. 3.7 Chapter 3: References (1) Yablonovitch, E.; Phys. Rev. Lett. 1987, 58, 2059. (2) John, S.; Phys. Rev. Lett. 1987, 58, 2486. (3) Bloch., F. Z. Physik, 1928, 52, 55. (4) Flouquet, G. Ann. Ecole. Norm. Sup. 1883,12,47. (5) Scrodinger, E. Ann. Phys. 1926, 79, 361. (6) Maxwell, C. J. Philosphical Transactions o f the Royal Society o f London 1865, 155,459. (7) Yablonovitch E. J. Mod. Opt. 1994, 41, 173. (8) Lin, S. Y.; Fleming, J. G.; Hetherington D. L.; Smith, B. K.; Biswas, R.; Ho, K. M.; Sigalas, M. M.; Zubrzycki, W.; Kurtz, S.; Bur, J. Nature 1998, 394, 251. (9) Noda, S.; Tomoda, K.; Yamamoto, N.; Chutinan, A. Science 2000, 289, 604. (10) Xia, Y.; Gates, B.; Li, Z. Y. Adv. Mater. 2001, 13, 409. (11) Kumar, A.; Abbott N. L.; Biebuyck, H; Whitesides G. M. Acc. Chem. Res. 1995, 28,219. (12) Matijevic, E.; Langmuir, 1994, 10, 8. (13) (a) Roxio, C. B.; Deckman, H. W.; Gland, J.; Cameron, S. D.; Chianelli, R. R. Science 1987, 235, 1629. (b) Buncick, M. C.; Warmack, R. J.; Ferrell T. L. J. Opt. Soc. Am.-B 1987, 4, 927. (c) Lenzmann, F.; Li, K.; Kitai, A. H.; Stover H. D. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86 H. Chem. Mater. 1994, 6,156. (d) Bummeister, F.; Schafle, C.; Matthes, T.; Bohmisch, M. Langmuir 1997,13, 2983. (e) Boneburg, J.; Bummeister, F.; Schafle, C.; Leiderer, P.; Rein, D.; Fery, A.; Herminghaus, S. Langmuir, 1997, 13, 7080. (f) Padeste, C.; Kossek, S.; Lehmann, H. W.; Musil, C. R.; Gobrecht, J.; Teifenaur, L., J. J. Electrochem. Soc. 1997, 143, 3890. (14 ) Xia, Y.; Tien, J.; Qui, D.; Whitesides, G. M. Langmuir 1996, 12, 4033. (15) (a) Velev, O. D.; Jede, T. A.; Lobo, R. F.; Lenhoff, A. M. Nature 1997, 389, 447. (b) Holland, B. T.; Blanford, C. F.; Stein, A. Science, 1998, 281, 538. (16) (a) Holtz, J., H., Asher, S., A.; Nature 1997, 389, 829. (b) Holtz., J. S. W.; Munro, C. H.; Asher, S. A. Anal Chem, 1998, 70, 780. (17) Weissman, J. M.; Sunkara, H. B.; Tse, A. S.; Asher S. A. Science 1996,274, 959. (18) (a) Flaugh P., L. O’Donnell S., E. Asher, S. A. Appl. Spectroscopy 1984, 38, 847. (b) Spry R. J.; Kosan, D. J. Appl. Spectrocopy 1986, 40, 782. (19) Chang, S. Y.; Liu, L.; Asher, S. A. J. Am. Chem. Soc. 1994, 116, 6739. (20) (a) Tahran, 1.1.; Watson, G. H. Phys. Rev. Lett. 1996, 76, 313. (b) Vos, W. L.; Megens, M.; van Tats, C. M.; Bosecke, J. J. Phys. Condens. Mater. 1996, 8, 9503 (c) Miguez, H.; Meseguer, F.; Lopex, C.; Blanco, A.; Moya, J. S.; Requena, J.; Mifsud, A. Adv. Mater. 1998,10, 480. (21) Stoeber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62. (22) Goodwin, J. W.; Heam, J.; Ho, C. C.; Ottewill, R. H. Colloid and Polymer Science 1974,252,464. (23) Muller, M.; Zentel, R.; Maka, T.; Romanov, S.G.; Sontomayor-Torres, C. Chem. Mater. 2000,12, 2508. (24) Xia, Y.; Gates, B.; Li, Z. Y.; Lu, Y. Adv. Mater. 2000,12, 693 (25) Vlasov, Y. A.; Luterova, K.; Pelant, I.; Honerlage, B.; Astraton, V. N. Appl. Phys. Lett. 1998, 71,1616. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 87 (26) Blanco, A.; Lopez, C.; Mayoral, R.; Miguez, H.; Meseguer, F.; Mifsud, A.; Herero, J. Appl. Phys. Lett. 1998, 73, 1781. (27) Romanov, S. G.; Johnson, N. P.; Yates, H. M.; Pemble, M. E.; Butko, V. Y.; Sontomayor-Torres, C. Appl. Phys. Lett. 1997, 70, 2091 (28) Megens, M.; Winjnhoven, J. E. G. J.; Lagendijk, A.; Vos, W. L. Phys Rev A, 59(6), 1999, 4727. (29) Romanov, S.G.; Maka, T.; Sotomayor Torres, C. M.; JApp Phys, 91 (11) 2002, 9426. (30) Chapter 2, Section 2.4, this thesis. (31) Chapter 3, Section 3.2.1.2, this thesis. (32) Vos, W. L.; Polman, A. MRS Bulletin, 2001,26(8), 642. (33) Chapter 3, Sec 3.4, this thesis (34) (a) Denkv, N. D.; Velev, O. D.; Kralchevsky, P. A.; Ivanov, I. B.; Yoshimura, H.; Nagayama, K. Langmuir 1992, 8, 3183. (b) Dushkin, C. D.; Magayama, K.; Miwa, T.; Kralchevscky, P. A. Langmuir 1993, 9, 3695. (c) Lazarov, D. S.; Denkov, N. D.; Velev, O. D.; Kralchenscky, P. A.; Nagayama, K. J. Chem. Soc., Faraday Trans. 1994, 90, 2077 (d) Dimitrov A. S.; Nagayama, K. Langmuir 1996, 12, 1303. (e) Rakers, S.; Chi, L. F. Langmuir 1997, 13, 7121. (35) (a) Mayoral, R.; Requena, J.; Moya, J. S.; Lopez, C.; Cintas, A.; Miguez, H.; Meseguer, F.; Vazquez, L.; Holgada, M.; Blanco, A. Adv. Mater. 1997,2, 257. (b) Donselaar, L. N.; Philipse, A., P.; Suurmond, J.; Langmuir 1997, 13, 6018. (c) Miguez, H.; Messeguer, F.; Lopez, C.; Mifsud, A.; Moya, J. S.; Vazquez, L. Langmuir 1997,13, 6009. (36) (a) Pieranski, P.; Strzelecki, L.; Pansu, B. Phys. Rev. Lett. 1983, 50, 900. (b) Van Winkle, D. H.; Murray, C. A. Phys. Rev. 1986, 34, 562. (c) Sunkara, H. B.; Jethmalani, J. M.; Ford, W. T. Chem Mater. 1994, 6, 362. (d) Larsen, A. E.; Grier, D. G. Nature 1997, 385, 230. (37) Jiang, P.; Bertone, J. F.; Hwang, K. S.; Colvin, V. L. Chem. Mater. 1999, 11, 2132. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 88 (38) Vlasov, Y. A.; Bo, X. -Z.; Strum, J. C.; Norris, D. J. Nature 2001,414, 289. (39) Park, S. H.; Xia, Y. Langmuir 1999, 15, 266. (40) Synthesis and Functionalization o f polymer nanospheres; Greci, M. T.; Ph. D. Dissertation, USC, Los Angeles, 2000. (41) Chapter 2, Section 2.4.2, this thesis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 89 Chapter 4 Surface chemical functionalization of polystyrene nanospheres 4.1 Introduction Monodisperse polymer nanospheres have a large surface areas1 and surface uniformity,2 however they have relatively inert surfaces. In order to make use of the nanoparticles for different applications, their surface must be chemically functionalized. Therefore, there has been a great effort directed toward the finding of new methods for the surface chemical functionalization of polystyrene nanospheres. These include the direct synthesis of monomers containing functional groups, copolymerization of styrene with other functionalized monomers, and chemical modification of preformed polystyrene nanospheres. Direct synthesis of monomers containing functional groups was first performed by Margel and coworkers.2 They synthesized polymeric nanospheres consisting of chloromethylstyrene, formylstyrene and styrene sulfonylchloride. Using classical emulsion polymerization, with surfactants, and some organic cosolvents, they were able to synthesize, spherical and stable nanospheres. As an extension of their work Margel and coworkers synthesized poly- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90 chloromethylstyrene nanospheres in ethanol using poly-vinylpyrrolidinone as stabilizer.3 They then used these poly-chloromethylstyrene nanospheres to attach amino groups and other ligands. Another technique for functionalization of polystyrene nanospheres is the copolymerization of styrene with other functional monomers as carried out by Okubo and coworkers.4 They synthesized monodisperse polymer nanospheres by copolymerizing styrene, chloromethylstyrene and divinylbenzene in the presence of acrylic acid as a surfactant. These methods incorporate the functional groups throughout the nanospheres. As the functional groups present on the surface are the most useful, it is better to concentrate the functional groups only on the surface of the nanospheres. Greci and coworkers,5 developed a technique for grafting functionalized styrene monomers onto the surface of the growing polymer nanospheres, synthesized by emulsifier free emulsion polymerization. They called this technique the “in-situ grafting” technique. Surface functionalized polymer nanospheres can also be synthesized by chemical modification of the surface of the preformed poly- chloromethylstyrene and polystyrene nanospheres. Most functional or reactive polymers are prepared from chloromethylated polystyrene through nucleophilic substitution of the benzylic chloride 6 ’ 7. Synthesis of potassium and lithium derivatives8 ® and Grignard derivatives8 b of chloromethylated polystyrene, which can be further modified under mild conditions, have also been reported. Methods for Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91 functionalizing polystyrene nanospheres have been reported such as, lithiation of jp-bromopolystyrene 9 a ,9 b and polystyrene,1 0 a ,1 0 b and modification of polystyrene using organocalcium reagents.1 1 Using these methodologies a variety of functional groups have been directly attached to the polymer surface. This chapter discusses the chemical modification of the surface of the polystyrene nanospheres. “In-situ grafting” technique, developed by Greci and coworkers,5 is explored and its applicability to monomers having a high water solubility is examined. The “in-situ grafting” technique is later used in synthesis of thiol functionalized nanospheres. Finally post synthetic chemical modification of the polystyrene nanospheres, with amino and hydroxyl and other groups is discussed. 4.2 “In-situ grafting” of polystyrene nanospheres with monomers having high water solubility 4.2.1 Introduction “In-situ grafting” technique was developed by Greci and coworkers,5 for the synthesis of surface functionalized polystyrene nanospheres. The polystyrene nanospheres of the desired size were synthesized using emulsifier free emulsion polymerization. The substituted monomer was added in the last thirty minutes of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92 this reaction. This substituted monomer competitively reacts with the remaining monomer, resulting in surface functionalization. Various substituted styrenes have been investigated (Figure 4.1). These are para-acetoxy styrene (1), kS kk ' Y CH, k l kk 0 H3c 2 ,CH3 c h 3 kS kk k % N kS kk kk kS kk n h 2 kS kk XI 5 6 7 8 Figure 4.1 Functional monomers successfully used for “in-situ” grafting. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93 para-t-butoxystyrene (2), para-fluoro styrene (3), r ,2 ’,2’-trifluorostyrene (4), 2-vinylpyridine (5), 4-vinylpyridine (6), 4-vinylaniline (7), para- chloromethylstyrene (8). The “in-situ” grafting technique is a very simple method to obtain surface functionalized polystyrene nanospheres. In order to increase the range of functional groups that can be incorporated using this technique and as monomers with a highly water solubility cannot be used to form polymer nanospheres using emulsifier free emulsion polymerization; we decided to study the applicability of the “in-situ” grafting technique for surface grafting of such monomers. The monomers used for this study are shown in Figure 4.2. They include acrylonitrile (1), acrolein (2), ethyl acrylate (3), acrylamide (4), N-isopropylacrylamide (5), 1-vinyl-2-pyrrolidinone (6), styrenesulfonamide (7), N,N’-diethylstyrenesulfonamide (8). Except for N,N’-diethylstyrenesulfonamide all the other monomers have a high solubility in water. Depending on the monomer used and the results obtained, different grafting strategies were tried. For some systems, addition of the grafting monomer all at once in the last one hour of the polymerization reaction, ensured successful grafting, while with others the monomer was added in the last one hour of the polymerization, in tiny aliquots at regular intervals over a fixed time period, following which the polymerization reaction was carried out for an hour. The grafted monomer concentration was varied Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94 from four tenths of a mole percent all the way to sixteen mole percent. As most of the monomers used were IR active, Diffuse Reflectance Infrared Fourier Transform (DRIFT) Spectroscopy was used to verify successful grafting. O C2H5 O ✓ o = s = o I n h 2 o— s = o I C 2H5^ c 2h 5 5 6 7 Figure 4.2 Monomers to be investigated for “in-situ” grafting. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 95 4.2.2 Experimental Styrene, divinylbenzene, oxalyl chloride, triethylamine, styrene sulfonic acid sodium salt, N,N’ diethylamine, and the monomers to be grafted were purchased from Aldrich Chemical Co. Styrene was vacuum distilled to remove the inhibitor. Divinylbenzene was extracted with aqueous sodium hydroxide to remove the inhibitor then washed with water and dried over magnesium sulfate. All other reagents were used as received. All polymer syntheses were carried out in a 150 ml reaction kettle. The kettle was fitted with a condenser, a nitrogen inlet valve and a mechanical stirrer. The mechanical stirrer employed was the IKA Eurostar power control-visc. Polymer analysis was carried out on a Cambridge 360 Scanning Electron Microscope. SEM samples for microscopy were prepared by placing a drop of sample diluted with water on a glass plate and allowing it to dry. The sample was then sputter coated with gold. For synthesis of 4-vinyl-benzenesulfonamides, reactions were carried out under nitrogen under standard Schelnk conditions. The 4-styrenesulfonic acid sodium salt used was dried under vacuum overnight at 50 °C. The dichloromethane used was dried by distillation over calcium hydride, while N,N’-dimethylformamide was dried using barium oxide. Diffuse reflectance Fourier Transform IR (DRIFT) spectroscopy was performed on a Perkin Elmer Spectrum 2000 IR spectrometer, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 96 NMR spectroscopy was performed on a Varian Unity 300 Spectrometer, and GCMS was performed on a Hewlett Packard 5890 Series II instrument. Synthesis of 4-vinyl-benzenesulfonamide (7) Dry styrene sulfonic acid sodium salt, (3.0 g, 0.0145 moles), were taken in a Schlenk flask. The flask was purged with nitrogen and then cooled to 0 °C. Dry dichloromethane, 15 ml, and 0.5 ml dry DMF were added to the flask and the contents stirred under nitrogen at 0 °C for about 15 minutes. Oxalyl chloride (1.27 ml, 0.0145 moles) was added slowly with stirring. The reaction mixture was stirred at 0 °C for fifteen minutes. The reaction flask was then brought to room temperature and maintained for an hour. The reaction flask was vented regularly, to let out the gaseous by-products. After an hour, the reaction mixture was cooled to 15 °C and filtered. The filtrate was mixed with cold excess of aqueous ammonium hydroxide kept at 0 °C, with stirring. The temperature was then raised to 30 °C and maintained for an hour. The resulting slurry was filtered and the residue washed with diethyl ether. The collected organics were washed with cold aqueous sodium hydroxide and then with water till the washings were no longer alkaline. The organics were dried over MgSC> 4 . The organics were concentrated under reduced pressure to yield pale yellow crystals of 4-vinylbenzenesulfonamide. The amide purified by recrystallization from hot aqueous ethanol was obtained as white crystals: mp 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 97 °C - 137 °C. (Lit. 138 °C -139 °C )1 2 Yield: 2.23 g (84.15%). MS (El); m/r. 183. *H NMR ( 8, CDC13 ,22 °C): 7.87 (d,J = 8.5 Hz, 2H, aromatic), 7.52 (d, J = 8.5 Hz, 2H, aromatic), 6.75 ( dd, J = 17.6 Hz, 10.9 Hz, 1H, -CH= ), 5.88 and 5.44 (d, J = 17.6 Hz, 1H and J = 10.9 Hz, 1H, CH2 = ), 4.79 (s, 2H, NH2); 1 3 C NMR (8, CDC13 > 22°C): 142.2 ( Ar Cl), 142.0 (Ar C4), 135.5 (vinyl, -CH=), 127.5 (Ar C2), 126.9 (Ar C3), 117.8 ( vinyl, =CH2 ). IR (v, KBr): 1302 (0=S=0 asym. S t.), 1164 (O=S=0 sym. S t.), 3340 and 3256 (N-H st) cm'1 . Synthesis of N,N-diethyl-4-vinyl-benzenesulfonamide (8) Dry styrene sulfonic acid sodium salt, (2.0 grams, 0.0096 moles), were taken in a Schlenk flask. The flask was purged with nitrogen and then cooled to 0 °C. Dry dichloromethane (10 ml) and 0.3 ml dry DMF and were added to the flask and the contents stirred under nitrogen at 0 °C for about 15 minutes. Oxalyl chloride (0.84ml, 0.0096 moles) was added slowly with stirring. The reaction mixture was stirred at 0 °C for fifteen minutes and subsequently warmed to room temperature and maintained for an hour. The reaction flask was vented regularly, to let out the gaseous byproducts. After an hour, the reaction mixture was cooled to 0 °C. Triethylamine (5 ml) was added, followed by a slight excess of N,N-diethylamine, at 0 °C with stirring. The temperature was then raised to 30 °C and maintained for an hour. The resulting slurry was filtered and the residue washed with diethyl ether. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 98 The collected organics were washed with cold water. The organics were dried over MgSC> 4 . The organics were concentrated under reduced pressure to yield a yellow solid, N,N-diethyl- 4-vinylbenzenesulfonamide. The amide was purified by recrystallization form hot aqueous ethanol as pale yellow crystals, mp 75 °C- 76 °C. (Lit.74.5 °C - 75 °C ).1 3 Yield: 2.01 g (85.53%). MS (El); m/z: 239. !H NMR ( 5, CDC13;22 °C): 7.5 (d, J= 8.4 Hz, 2H, aromatic), 7.50 (d, J = 8.4 Hz, 2H, aromatic), 6.74 ( dd, J = 17.7 Hz, 11.0 Hz, 1H, -C H = ), 5.86 and 5.41 (d, J = 17.7 Hz, 1H and J = 11.0 Hz, 1H, CH2 = ), 3.23 (q, 4H, NCH2 ), 1.13 (t, 6H, CH3 ) ; 1 3 C NMR (6, CDC13 ; 22 °C): 141.9 ( Ar Cl), 139.1 (Ar C4), 135.9 (vinyl, -CH=), 127.5 (Ar C2), 126.5 (Ar C3), 117.0 ( vinyl, =CH2 ), 42.5 (NCH2 ), 14.3 (CH3 ) ppm. IR (v, KBr): 1330 (0=S=0 asym. st.), 1153 (0=S=0 sym. st.) cm'1 . “In-situ” grafted polystyrene nanospheres The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150 ml reaction kettle, equipped with a condenser, gas inlet and mechanical stirring apparatus, and containing 70 ml water was heated to 80 °C, stirred at 300 rpm and degassed for one hour with nitrogen. After an hour the gas flow was turned off and a certain volume (upto 7.0 ml) of styrene and 4 mole percent of divinylbenzene (DVB) were added to the water. The reaction mixture was stirred for 20 minutes to bring the monomer and crosslinker to the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 99 polymerization temperature, followed by addition of 0.06 grams of (0.3 mmol) of potassium persulfate initiator. The reaction mixture was stirred at 300 rpm and 80 °C for 3 hours. At this point the monomer to be grafted was added. Monomers to be grafted were dissolved in the minimum amount of water or 1:1 v/v aqueous ethanol, required to effect the dissolution of the monomer. The monomers to be grafted were added to the polymerization reaction in two ways, a) all the monomer was added at once (single step grafting) or, b) the monomer addition was staggered over a period of half an hour, (staggered grafting) After monomer addition was complete, the polymerization was allowed to proceed for half an hour. The reaction was stopped by cooling the reaction mixture to room temperature. A small sample of the polymer is taken for SEM analysis. The colloidal polymer was then frozen at -20 °C overnight, followed by thawing at room temperature. The polymer nanoparticles were centrifuged and washed repeatedly with acetone and water. Samples of the washed nanoparticles were dried overnight at room temperature, under vacuum. The dried samples were used for DRIFT and EDS studies. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 4.2.3 Results and Discussion Grafting of highly water soluble monomers was successfully carried out for a range of such monomers (Figure 4.2). The amounts of grafted monomer used were varied. Amounts of grafted monomer used were, 0.4 mole percent, 1.0 mole percent, 4.0 mole percent and 16.0 mole percent, respectively. Two types of addition techniques were also used. In one, the single step grafting, the monomer to be grafted was added all at once, in the other, staggered grafting, the monomer to be grafted was added dropwise over a period of half an hour. The results of the grafting, as determined by DRIFT spectroscopy, were found to vary with amount of monomer used and the monomer addition technique. Acrylamide (4), N-vinyl-2-pyrrolidinone (6) Grafting of Acrylamide (4), and N-vinyl-2-pyrrolidinone (6) was unsuccessful. Irrespective of the type of monomer addition technique used or the concentrations of grafted monomer, no signal for the carbonyl moieties could be seen in the DRIFT spectra. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 101 Acrylonitrile (1) Grafting of acrylonitrile (1) was successful only when 16.0 mole percent of grafted monomer was used, under staggered addition of the monomer. The nitrile frequency was seen at 2236 cm"1 using DRIFT spectroscopy (Figure 4.3). All other conditions were unsuccessful. 9S.8 94 92 90 88 86 84 82 80 % T 78 76 74 72 70 68 66 64 62.8 600 450.0 1000 800 1400 1200 1800 1600 2400 2000 3600 3200 2800 4000.0 Figure 4.3 Acrylonitrile grafted polymer nanospheres.(nitrile 2236 cm'1 ) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 102 Acrolein (2) Acrolein was successfully grafted for monomer concentration of 4.0 mole percent and 16.0 mole percent, using the staggered grafting technique. Regardless of the amount of monomer used, no grafting was seen if the monomer was added all at once. The characteristic IR frequency, (at 4.0 mole percent grafting) for acrolein grafted nanospheres was at 1720 cm'1 (C=0) (Figure 4.4) 127.1 125 120 115 110 105 100 % T 95 90 85 80 75 70.9 1200 600 450.0 1800 1600 1400 1000 800 3600 3200 2800 2400 2000 4000.0 Figure 4.4 Acrolein grafted polymer nanospheres. (C=0 1720 cm'1 ) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 103 Ethyl acrylate (3), N-isopropylacrylamide (5), 4-vmyl-benzenesulfonamide (7), N,N-diethyl-4-vmyl-benzenesulfonamide (8) These monomers were each successfully grafted for monomer concentrations of 1.0 mole percent, 4.0 mole percent and 16.0 mole percent, using the staggered grafting technique. The single step grafting technique also works for higher concentrations of grafted monomer (4.0 mole percent up). Figure 4.5, Figure 4.6, Figure 4.7 and Figure 4.8 show the IR spectra obtained for polystyrene nanospheres grafted with ethyl acrylate, N-isopropyl acrylamide, 4-vinyl-benzene sulfonamide and N,N-diethyl-4-vinyl benzenesulfonamide respectively, at 1.0 mole percent of grafted monomer. Figure 4.5 Ethyl acrylate grafted polymer nanospheres. (C=0 1728 cm'1 ) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 104 103.2 100 95 90 85 80 H T 75 70 65 60 55 52.1 1200 800 450.0 4000.0 3200 2800 2000 1800 1600 1400 1000 600 3600 2400 •1 Figure 4.6 N-isopropyl acrylamide grafted polymer nanospheres. (C=0 1662 cm'1 , N-H st 3500 cm'1 to 3300 cm'1 ) 100.1 95 90 85 8 0 75 70 65 60 %T 55 50 45 40 35 30 25 20.0 1800 1400 1000 800 600 450.0 4000.0 3200 2800 2400 2000 1600 1200 3600 Figure 4.7 4-vinyl-benzenesulfonamide grafted polymer nanospheres. (0=S=0 asym st 1330cm'1 , sym st 1160 cm'1 , N-H st: 3500 cm'1 to 3250 cm'1 ) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 105 % T 60 600 1600 1400 1000 800 450.0 4000.0 3200 2800 2400 2000 1800 1200 3600 •1 Figure 4.8 N,N -diethyl- 4-vinyl-benzenesulfonamide grafted polymer nanospheres. (0=S=0: asym. st 1333cm"1 , sym. st. 1155 cm'1 ) The grafted polystyrene nanoparticles obtained, were examined by SEM to confirm the spherical monodisperse nature of the nanospheres. (Figure 4.9) 4.2.4 Conclusion Polystyrene nanospheres were successfully grafted with various water soluble monomers. The requirement for successful grafting in the emulsifier free polymerization, was the maintenance of a very low concentration of grafted monomer in the aqueous phase, or else the monomer will homopolymerize in the aqueous phase with out being grafted. Therefore the staggered technique worked better than the single step technique. Differences in the solubility of the monomers Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 106 affect their ability to be grafted. Monomers highly miscible with water, such as acrylamide and acrylonitrile, were difficult to graft. However, acrolein and 1 -vinyl- Figure 4.9 SEM of 4-vinyl-benzenesulfonamide grafted nanospheres. 2-pyrrolidinone were amenable to grafting. Comparatively less soluble monomers such as ethyl acrylate, N-isopropyl acrylamide and the sulfonamides were relatively easy to graft on, even at low concentrations, using the staggered technique. No grafting was observed for any of the monomers at 0.4 mole percent concentration. This could be due to inadequate sensitivity of the DRIFT spectroscopy technique used for the detection. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 107 4.3 “In-situ” grafting for the synthesis of thiol functionalized nanospheres 4.3.1 Introduction The thiol or sulfhydryl group is a very useful moiety to have on the polymer surface. Besides being a good nucleophile,1 4 it also exhibits characteristic reactivity. For example, its reactivity to radicals, the thiol is known to add across alkenes and vinyl moieties in an anti-Markonikov manner,1 5 to yield sulfides as products. Under radical conditions thiols have been shown, in a similar manner, to add across alkynes.1 6 Thiols undergo easy oxidation to disulfides under basic conditions in the presence of oxygen.1 7 One characteristic of the thiol group, that is gaining increasing importance in the field of materials and supramolecular chemistry, is its interaction with gold.1 8 It has been postulated, that in the interaction with gold, the 1 f t S-H bond is cleaved resulting in a loss of molecular hydrogen. Besides the above reactions, the thiols can also be converted quite easily to sulfonium salts, sulfoxides, sulfones, sulfonic acids, thioacetals and thioacids.1 4 Considerable effort had been devoted to the incorporation of the thiol moiety onto the polymer surface. One drawback in the handling of thiols is their ready oxidation to the disulfide in the presence of oxygen. One of the earliest attempts at synthesizing thiol functionalized polymers was reported by Frechet and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108 coworkers1 9 via the lithiation of polystyrene, followed by quenching of the lithiated polymer with sulfur and then reduction of the resulting poly-sulfide to the thiol functionalized polymer using lithium aluminum hydride. They also showed that polymer supported thiols have a low tendency to undergo oxidation to the disulfide.1 9 Warshawsky and coworkers synthesized polymer supported thiols, using different synthetic routes for modifying preformed polystyrene2 0 which include; (a) attachment of a molecule with an active halogen to the polymer, followed by halogen substitution by sulfur, (b) formation of a polymeric thioether, followed by thioether splitting using sulfuryl chloride to yield the sulfenyl chloride polymer, reaction of the sulfenyl chloride with an aliphatic thiol to yield a disulfide and reduction of the thioether to yield the thiol, (c) attachment of a tailor-made disulfide to an amino polymer followed by reduction. Using the reactivity of thiols with vinyl groups in the presence of radical initiators, thiol functionalized polystyrenes1 5 and poly-isobutylenes2 1 have been synthesized. The “in-situ” grafting technique has been successfully used for the incorporation of functionality on the nanosphere surface.5 Incorporated hydroxyl functionality was shown to be excellent anchors for silver and ruthenium nanoparticles. Some success has been achieved at incorporating amino functionality too, using this technique. We decided to extend the “in-situ”grafting technique to include thiolation. In Figure 4.10, monomer (a) has been successfully grafted onto Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 109 the polymer nanosphere surface using “in-situ” grafting technique. Base hydrolysis of the acetoxy functionality led to the incorporation of hydroxyl groups on the surface. Based on these observations, we decided to use a similar monomer (I), shown in Figure 4.10. This monomer is a 4-substituted thioester of styrene. The idea was to synthesize the thioester monomer (I) and graft this onto the polystyrene nanospheres via the “in-situ” grafting technique. The polymer supported thiol can then be realized by hydrolysis of the grafted thioester in the presence of a base.2 3 / / r*i .ch3 ,CH, (a) O (I) Figure 4.10 (a) p-acetoxy styrene. (I) 4- substituted thioester of styrene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110 The retrosynthetic approach to thioester (I) is shown in Figure 4.11. It begins with 4-(methylthio)benzaldehyde (V). This can be converted to the thioether, 4-(methylthio)styrene (IV) using the Wittig reaction. The thioether can be cleaved to the thiol (II) as follows; conversion of the thioether (IV) to the sulfoxide (III), followed by Pummerer reaction on the sulfoxide. The thiol is immediately protected as the thioester (I). Work on grafting the sulfoxide (III) to the polymer nanospheres, using “in-situ” grafting, followed by deprotection to the thiol, via the thioether, failed to yield positive results. 4.3.2 Experimental 4-(methylthio)benzaldehyde, n-butyl lithium, styrene, divinylbenzene, m- chloroperoxybenzoic acid, trifluoroacetic anhydride, hydrogen tetrachloroaurate(III) trihydrate (HAICI4 .3 H2O) and acetyl chloride were purchased from Aldrich Chemical Co. Methyltriphenylphosphonium bromide was purchased from Lancaster Synthesis Inc. Styrene was vacuum distilled to remove the inhibitor. Divinylbenzene was extracted with aqueous sodium hydroxide to remove the inhibitor then washed with water and dried over magnesium sulfate. All other reagents were used as received. Silica Gel 60 from EM Science (Mesh 230 -400) was used for column chromatography. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I l l IV ,c h 3 o (I) in) m y c h 3 (III) y r^i k k c h 3 y kk c h 3 (IV) Figure 4.11 Retrosynthesis of thioester (I), (i) Wittig Reaction, (ii) Conversion to the sulfoxide, (iii) Pummerer rearrangement to the thiol, (iv) Protection of the thiol as the ester. (V) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 112 All polymer syntheses were carried out in a 150 ml reaction kettle. The kettle was fitted with a condenser, a nitrogen inlet valve and a mechanical stirring apparatus. Distilled (Sparkletts) water was used for all polymer synthesis. The mechanical stirrer was used was the IKA Eurostar power control-visc. Polymer analysis were carried out on a Cambridge 360 Scanning Electron Microscope. SEM samples for microscopy were prepared by placing a drop of sample diluted with water on a glass plate and allowing it to dry. The sample was then sputter coated with gold. For Energy Dispersive Studies (EDS) studies, the samples were coated with carbon. Transmission Electron Microscopy (TEM) analysis was carried out on Philips EM 420 T electron microscope at 120 kY accelerating voltage. TEM specimens were prepared by placing a drop of diluted sample on a copper TEM sample grid and drying in an open atmosphere. The dichloromethane used was dried over calcium hydride under reflux. Dry tetrahydrofuran (THF) was obtained under reflux over sodium metal. Diffuse reflectance Fourier Transform IR ( DRIFT) spectroscopy was performed on a Perkin Elmer Spectrum 2000 IR spectrometer and UV-Vis spectroscopy on a HP - 8453 model spectrometer both fitted with diffuse reflectance accessories. NMR spectroscopy was performed on a Bruker AM 360 spectrometer, and GCMS a was performed on ThermoFinnigan Trace DSQ ™ instrument.. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 113 Synthesis of 4-(methylthio)styrene (IV) Methyltriphenylphosphonium bromide, (28.3 g, 79.2 mmoles), was taken in a 500 ml Schlenk Flask fitted with a rubber septum. The flask was evacuated and purged with nitrogen, thrice. 300ml dry THF was added to the flask and the mixture was cooled to 0 °C in an ice bath, n-butyl lithium, (32.19 ml, 80.0 mmoles), was added slowly with stirring, maintaining the temperature at 0 °C. The reaction mixture was stirred for an hour at 0 °C. A clear orange solution was obtained. To this solution at 0 °C, was added 4-(methylthio)benzaldehyde (V) (8.75ml, 65.6 mmoles). The temperature was raised to 40 °C and the reaction was stirred at this temperature for 10 hours. 150 ml water is then added to the reaction mixture. The organic phase was collected and extracted thrice with water. The organic phase was dried over magnesium sulfate and concentrated. The oily liquid obtained, was subjected to column chromatography using 1:1 mixture of ethyl acetate and hexane as mobile phase to yield 4-(methylthio)styrene (IV) as a pale orange liquid. Yield: 8.01g (81.23%). MS (El); m/z: 149.9 *H NMR (5, CDC13 ,22 °C): 7.29(d, 8.2 Hz, 2H, aromatic), 7.18 (d, J = 8.2 Hz, 2H, aromatic), 6.62 (dd, J= 17.0Hz, 11.3 Hz, 1H, -C H = ), 5.69 and 5.18 (d, J = 17.1 Hz, 1H andJ = 11.3 Hz, 1H, CH2 = ), 2.42 (s, 3H, SCH3 ); 1 3 C NMR (8, CDC13 ; 22 °C): 138.2 ( Ar C4), 136.3 (Ar Cl), 134.6 (vinyl, -CH=), 128.2 (Ar C3), 126.4 (Ar C2), 113.2 ( vinyl, =CH2 ), 15.7 (SCH3 ) ppm. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 114 Synthesis of Sulfoxide (III) 4-(methylthio)styrene (IV), (6.885 g, 44.53 mmoles), was taken in a one liter round bottom flask containing 600 ml chloroform. The mixture was cooled to -15 °C to -20 °C and stirred at that temperature for about half an hour, m- chloroperoxybenzoic acid (75% purity), 10.314 g (45 mmoles), was added. The reaction mixture was stirred for three and a half hours at -20 °C. After the allotted time, 4.954 g (1.5 equiv, 67.5 mmoles) calcium hydroxide were added to the reaction mixture and was stirred for an hour while allowing the solution to rise to room temperature. The mixture was then filtered through a Whatman No. 1 filter paper. The filtrate was collected and concentrated to yield sulfoxide (III) as a pale yellow solid. Yield: 6.942 g (84.27%). MS (El); m/z: 165.9. NMR (6, CDC13 , 22 °C): 7.62 (d, J - 8.1 Hz, 2H, aromatic), 7.56 (d, J = 8.2 Hz, 2H, aromatic), 6.76 ( dd, J = 17.5 Hz, 11.1 Hz, 1H, -C H = ), 5.86 and 5.39 (d, J = 17.5 Hz, 1H, and J = 11.0 Hz, 1H, CH2 = ), 2.74 (s, 3H, SCH3 ) ; 1 3 C NMR ( 5, CDC13 , 22 °C): 144.51 ( Ar C4), 140.5 (Ar Cl), 135.74 (vinyl, -CH=), 127.2 (Ar C3), 124.1 (Ar C2), 116.5 ( vinyl, =CH2 ), 43.9 (SCH3 ) ppm. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 115 Conversion of sulfoxide (III) to the thioester (I) Sodium bicarbonate, (13.44 g, 160 mmoles), and 1.66 g (10 mmoles) of the sulfoxide (III) were taken in a 250 ml Schlenk flask fitted with a reflux condenser stoppered at one end. This system was evacuated and purged with nitrogen. 150 ml of dry dichloromethane was added followed by 4.2 ml (30 mmoles, 3 equivalents) of trifluoroacetic anhydride. The reaction mixture was heated to 40 °C and stirred for 1 hour under nitrogen. Carbon dioxide evolved was vented out continuously. After an hour the reaction mixture was cooled to room temperature and the mixture was filtered through a Whatman No. 1 filter paper. The filtrate was taken in a 200 ml round bottom flask and concentrated under vacuum. The flask and the resulting residue were flushed with nitrogen. To this was added an excess of a 1:1 v/v solution of triethylamine and methanol. The flask was purged thoroughly with nitrogen, and the mixture stirred at room temperature for fifteen minutes. A small sample was taken for GCMS analysis. MS (El); m/z: 135.9 corresponds to the thiol (II) (M.W. 136). The mixture was concentrated under vacuum to remove all traces of methanol. When not under vacuum, an atmosphere of nitrogen was maintained in the flask. The concentrated organics under nitrogen, were dissolved in 25 ml dry dichloromethane. 10 ml triethylamine was added to the flask, which was then cooled to 0 °C in an ice bath. 0.71 ml (10 mmoles) of acetyl chloride were slowly added and the reaction mixture was stirred for fifteen minutes at 0 °C and for one hour at Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 116 room temperature. The reaction mixture was extracted five times with 10 ml water. The organic phase was collected and dried over magnesium sulfate. The organics were concentrated under vacuum and purified by column chromatography, using 4:1 hexane : ethyl acetate as mobile phase to yield thioester (I) as a yellow liquid. Yield: 11.528 g (80.06%). MS (El); m/z: 177.6. lH NMR (6, CDC13;22 °C): 7.37 (d, J= 8.5 Hz, 2H, aromatic), 7.30 (d, J = 8.5 Hz, 2H, aromatic), 6.65 ( dd, J = 17.6 Hz, 10.9 Hz, 1H, -C H = ), 5.73 and 5.25 (d, J = 17.6 Hz, 1H and J = 10.9 Hz, 1H, CH2 = ), 2.35 (s, 3H, CH3 ) ; 1 3 C NMR (5, CDC13 ,22 °C): 194.4 (C O ), 138.8 ( Ar C4), 136.1 (A rC l), 134.7 (vinyl, -CH=), 127.2 (ArC3), 126.9 (ArC2), 115.6 ( vinyl, =CH2 ), 30.1 (CH3 ) ppm. IR (v, KBr): 1706 (C=0 st). “In-situ” grafting of polystyrene nanospheres grafted with thioester (I) The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150 ml reaction kettle, equipped with a condenser, gas inlet and mechanical stirring apparatus, and containing 70 ml distilled (Sparkletts) water was heated to 80 °C, stirred at 300 rpm and degassed for 1 hour with nitrogen. After an hour, the gas flow was turned off and a certain volume (upto 7.0 ml) of styrene and 4 mole percent of divinylbenzene (DVB) were added to the water. The reaction mixture was stirred for 20 minutes to bring the monomer and crosslinker to the polymerization temperature, followed by addition of 0.06 grams Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 117 (0.3 mmole) of potassium persulfate initiator. The reaction mixture was stirred at 300 rpm and 80 °C for two and a half hours. At this point 5.0 ml isopropanol was added as the cosolvent. Three mole percent of thioester (I) was dissolved in minimum of acetone, and was added to the reaction mixture. After monomer addition was complete, the polymerization was allowed to proceed for an hour. The reaction was stopped by cooling to room temperature. A small sample of the polymer was taken for SEM analysis. The colloidal polymer was then frozen at -20 °C overnight, followed by thawing at room temperature. The polymer nanoparticles were centrifuged and washed repeatedly with acetone and water. The washed nanoparticles were dried overnight at room temperature, under vacuum. Hydrolysis of the grafted thioester to the thiol. The hydrolysis of the thioester moieties on the nanosphere surface were carried out as follows: thioester grafted nanospheres, 0.052 g (0.5 mmole based on styrene monomer units), were swelled in a 1:1 v/v tetrahydrofuran : chloroform solvent mixture. To this mixture was added 1.0 ml (~ 10 mmoles) of diethylamine. The mixture was maintained under reflux for 12 hours in a nitrogen atmosphere. The products were centrifuged and washed well with acetone and water. The nanoparticles were dried, overnight, under vacuum. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 118 Synthesis of gold colloids Gold colloids used were prepared using the citrate reduction technique.24 In short, 50 ml of a 1.0 mM aqueous solution of hydrogen tetrachloroaurate(III) trihydrate (HAUCI4 .3 H2O) was taken in a 100 ml round bottom flask under nitrogen fitted with a reflux condenser having a calcium chloride guard tube. HPLC grade water was used throughout this process. The reaction mixture was heated at reflux for half an hour, with vigorous stirring. 5.0 ml of a 3.9 mM aqueous solution of tris- sodium citrate was added to this boiling solution and reaction was stirred under reflux. The reaction solution turned pink. The reaction was continued for twenty minutes after the appearance of this pink color. The reaction mixture was cooled to room temperature. A small sample was taken for Transmission Electron Microscopy (TEM) analysis. Immobilization of gold colloids on the thiol functionalized nanospheres Thiol functionalized nanospheres, 0.01 g, were swollen in minimum amount of THF. The THF was then evaporated and replaced by an equal volume of ethanol. 1 . 0 ml of the prepared colloidal gold solution was added to this suspension of nanospheres. The mixture was sonicated for a minute and then allowed to stand for five minutes. The mixture was again sonicated for a minute and then centrifuged at around 2500 rpm. If the supernatant was colorless, the process was repeated till the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 119 supernatant obtained on centrifuging, had a pink color. At this point the no more gold colloids could be absorbed on the nanospheres. The nanospheres were rinsed with water, sonicated for a minute and centrifuged. If the supernatant was pink in color the process was repeated till the supernatant was colorless. This process was carried out to get rid of any loosely bound gold colloids. 4.3.3 Results and Discussion Thioester (I) was successfully synthesized from 4-methylthio)benzaldehyde (V) as shown in Figure 4.12. In the first step the aldehyde group of 4- (methylthio)benzaldehyde (V) was converted to the vinyl group using the Wittig reaction. The product 4-(methylthio)styrene (IV), was obtained in ~ 80% yield. The deprotection of the thioether to the thiol was carried out using a procedure developed by Young and coworers25 with some modification. The thioether (IV) was oxidized with weto-chloroperoxybenzoic acid (m-CPBA) to convert the thioether to the sulfoxide (III). In order to prevent the epoxidation of the vinyl moiety, the temperature of the reaction was maintained at -20 °C and it never rose above -15 °C. Sulfoxide formation was the fastest at this temperature range without any epoxidation of the double bond. The reaction mixture was quenched with calcium hydroxide. This sulfoxide (III) was then subjected to Pummerer rearrangement in the presence of trifluoroacetic anhydride. Sodium bicarbonate Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 r fi (V) (IV) Ul IV ri cm) V I fl / (ii) V ll y / s (I) Figure 4.12 (i) methyltriphenylphosphonium bromide, n-BuLi, THF. 0 °C (ii) 4- (methylthio)benzaldehyde, 40 °C. (iii) w-CPBA, -20 °C, CHCI3 . (iv) Ca(OH)2 . (v) NaHCC>3, Trifluoroacetic anhydride, DCM, 40 °C. (vi) Et3N, CH3OH. (vii) DCM, Acetyl chloride, Et3N. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 121 was used to trap the formed trifluoroacetic acid, which if left untreated, leads to the polymerization of the styrene moiety. Triethylamine was tried as a trapping agent, but the reaction was messy and gave significantly lower yields. Care was taken to exclude air from the reaction mixture by maintaining a blanket of nitrogen over the reaction mixture. The Pummerer product obtained was hydrolyzed to the thiol (II) which, to prevent any losses by formation of the disulfide, was converted directly to the thioester (I) without isolation. Formation the thiol (II) was, however, confirmed by GCMS analysis. The disulfide however, can be easily converted back to the thiol in good yields, using LiAlH4 . 26 The thioester is obtained from the sulfoxide in almost eighty percent yield. The thioester (I) was grafted onto polystyrene nanospheres, synthesized by emulsifier free emulsion polymerization, using a single stage “in-situ” grafting technique. A cosolvent, such as isopropanol, added to the polymerization reaction just before addition of the thioester, was found to improve the density of the grafting The monomer being a viscous oil, was added as a solution, in the minimum amount of acetone required to make the monomer solution flow easily. The success of the grafting was confirmed by DRIFT spectroscopy showing the carbonyl frequency at 1706 cm''and EDS which shows the Ka line of sulfur (Figure 4.13 and Figure 4.14 respectively). The spherical nature of the nanoparticles was confirmed by SEM (Figure 4.15). The thioester moiety on the nanosphere surface was converted to the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 122 thiol by base hydrolysis, using diethylamine. The conversion of thioester to thiol was confirmed by DRIFT spectroscopy (Figure 4.16) and EDS (Figure 4.17). The DRIFT spectra showed a clear loss of the carbonyl frequency at 1706 cm'1 . The presence of sulfur was confirmed by the presence Ka line of sulfur in the EDS. 92.6 %T 1705.53 56.2 4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0 Figure 4.13 DRIFT spectra of thioester (I) grafted polystyrene nanospheres. (C=0 1706 cm'1 ) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with pem, ission 124 67.6 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 •1 Figure 4.16 DRIFT spectra of thiol functionalized polystyrene nanospheres, after hydrolysis. X-RflV! Live* Real* 0 - 2 0 keU 3 2 s Preset-* 2 0 0 s Remaining* 163s 52s 29% Dead s S i J f * * r FS= HK M EM 1 * 2 .4 0 0 keU ch 130= 1 2 . 6 > 605 c t s Figure 4.17 EDS of thiol grafted polystyrene nanospheres Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 125 The presence of thiol groups on the surface was confirmed by the affinity of the polymer nanospheres for gold colloids. 18 The polymer nanoparticles when mixed with colloidal gold solution, irreversibly bound gold colloids on the surface. The presence of gold on the nanosphere surface was confirmed by EDS, (Figure 4.18) which shows a the La line of gold and Ka line of sulfur. TEM (Figure 4.19 B) analysis of the polymer nanoparticle surface, shows the presence of the gold colloids. The diffuse reflectance UV-Vis spectra of the gold colloid coated nanospheres (Figure 4.20) shows the presence of the gold plasmon band at around 530 nm. 0 - 2 0 keU 343 Presets 57 s s Remai ni ng* 166s Au 76 5 c t s f s = m MEM 1» Figure 4.18 EDS of gold colloid coated thiolated polystyrene nanospheres Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 126 Figure 4.19 (A) TEM image of the gold colloids. (B) TEM image of a gold colloid covered polymer nanosphere. 4.3.4 Conclusion Functionalization of polymer nanospheres via the “in-situ” grafting technique has been successfully extended to include thiolation. This was achieved by the synthesis of a polymerizable thioester (I), from commercially available 4-(methylthio)benzaldehyde in good yield. In situ grafting of this monomer onto the polymer nanospheres during emulsifier free emulsion polymerization, led to the incorporation of the thioester functionality on the nanosphere surface, confirmed Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.5 0.25 -5 Q E L JQQ. Figure 4.20 Diffuse reflectance UV-Vis spectrum of gold colloid coated polymer nanospheres showing the plasmon band at 530 nm. by infrared analysis and EDS. Base hydrolysis of the thioester groups resulted in formation of thiol grafted nanospheres. These thiol functionalized nanospheres showed an affinity for gold colloids. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 128 4.4 Direct chemical functionalization of polystyrene nanospheres 4.4.1 Introduction As part of our effort to develop different methodologies for functionalizing polystyrene nanospheres, we investigated the post synthetic modification of the polystyrene nanospheres, that is, the chemical functionalization of the polymer nanosphere surface after synthesis of the nanospheres, as opposed to “in-situ” modification, which was a part of the polymerization reaction. Among the reactions attempted, were electrophilic amination and hydroxylation, and Friedel Craft type modifications of the polymer nanospheres. Aromatic electrophilic amination and electrophilic hydroxylation reactions, pioneered by Olah and coworkers,2 7 3 ’2 7 b provide a one step convenient method to incorporate amino and hydroxyl functionality on aromatic rings. The proposed mechanisms for the two reactions are shown in Figure 4.21 and Figure 4.22, respectively. The reactions take place in the presence of a protic superacid, in this case triflic acid. Trimethylsilylazide and bis-trimethylsilylperoxide were used as the aminating and hydroxylating agents, respectively. As amino and hydroxyl functionalities are useful chemical handles to have on the polystyrene bead surface, we decided to examine these two reactions as methods for incorporating amine and hydroxyl functionality onto the polystyrene nanosphere surface. Amino groups can Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 129 be converted to many other useful moieties. Diazotization and coupling, and conversion of these surface amino groups to other useful functionalities like (C H 3)3S iN 3 + 2 F 3C S 0 3H N H 2N 2 0 S 0 2C F 3 + M e 3S i 0 S 0 2C F 3 R + N , y N H 3+ CF3S 0 3’ R H N H , F 3C S 0 3' Figure 4.21 Proposed mechanism for the electrophilic amination reaction azide, isocyanide and isothiocyanate were also examined. Success at these surface modifications would indicate that the incorporated amino or other functionality is Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 130 H (CH3)3SiO-OSi(CH3 )3 + F3 CS03 H a K (H3 C)3Si ) Si(CH3 )3 R R Y OSiCH3 )3 -H •OH + (CH3 )3Si03SCF3 (CH3 )3SiOSCF3 + HOSi(CH3 )3 ► (CH3 )3SiOSi(CH3 )3 + CF3S03 H Figure 4.22 Proposed mechanism for electrophilic hydroxylation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 131 easily accessible. The carboxylic acid functionality is another useful moiety to have at the polymer surface. Incorporation of carboxylic acid functionality onto the polymer nanosphere surface was attempted, via the carboxylation of aromatics using the traditional AICI3 - oxalyl chloride reagent system.28 Friedel Crafts acylation of the polymer nanospheres was also attempted. 4.4.2 Experimental Styrene, divinylbenzene, o-dichlorobenzene, isoamyl nitrite and potassium persulfate, oxalyl chloride and aluminum chloride were purchased from Aldrich Chemical Co., trimethylsilyl azide from Lancaster Synthesis Inc., bistrimethylsilyl peroxide from Gelest Inc., and triflic acid from 3M Speciality Chemicals Division. Styrene was vacuum distilled to remove the inhibitor. Divinylbenzene was extracted with aqueous sodium hydroxide to remove the inhibitor then washed with water and dried over magnesium sulfate. All other reagents were used as received. All polymer syntheses were carried out in a 150 ml reaction kettle. The kettle was fitted with a condenser, a nitrogen inlet valve and a mechanical stirring apparatus. The mechanical stirrer was used was the IKA Eurostar power control- visc. For all polymer synthesis distilled (Sparkletts) water was used. Polymer analysis were carried out on a Cambridge 360 Scanning Electron Microscope (SEM). SEM samples for microscopy were prepared by placing a drop of sample Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 132 diluted with water on a glass plate and allowing it to dry. The sample was then sputter coated with gold. All reactions on the polymer nanospheres were carried out under nitrogen under standard schelnk conditions. The dichloromethane used was dried by refluxing with calcium hydride, and tetrahydrofuran used was dried by refluxing with sodium metal. Diffuse reflectance Fourier Transform IR ( DRIFT) spectroscopy was performed on a Perkin Elmer Spectrum 2000 IR spectrometer and UV-Vis spectroscopy on a HP - 8453 model spectrometer both fitted with diffuse reflectance accessories Synthesis of the polystyrene nanospheres The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150ml reaction kettle, equipped with a condenser, gas inlet and mechanical stirring apparatus was heated to 80 °C, stirred at 300 rpm and degassed for 1 hour with nitrogen. After 1 hour, the gas flow was turned off and a certain volume (upto 7.0 ml) of styrene and 4 mole percent of divinylbenzene was added to the water. The reaction mixture was stirred for 20 minutes to bring the monomer and crosslinker to the polymerization temperature, followed by addition of 0.06 grams of (0.3 mmol) of potassium persulfate initiator. The reaction mixture was stirred at 300 rpm and 80 °C for 4 hours and then stopped. The reaction Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 133 mixture was cooled to room temperature. The aqueous colloidal suspension was frozen overnight and thawed. The nanospheres were then centrifuged down, followed by repeated washings with acetone and water. The polymer nanospheres were dried overnight under vacuum before further use. Electrophilic amination of the polystyrene nanospheres. Polystyrene nanospheres, 5.2 g (50 mmole based on styrene monomer), were taken in a Schlenk flask with a magnetic stirrer and fitted with a rubber septum. The flask was evacuated for an hour at room temperature and then purged with dry nitrogen. 175 ml dry dichloromethane was added to the flask and the nanospheres were stirred under nitrogen for an hour to facilitate their swelling. After an hour, 75 ml o-dichlorobenzene was added and the mixture was stirred for 15 minutes. Next 3.12 ml triflic acid (34.37 mmoles) was then added very slowly at room temperature with stirring. The mixture was stirred for an hour while raising the temperature to 55 °C to 60 °C. Meanwhile a solution of trimethylsilylazide in dichloromethane was prepared by dissolving 1.25 ml trimethylsilylazide (12.5 mmole) in 10 ml dry dichloromethane. The trimethylsilylazide-dichloromethane solution was added slowly to the reaction flask maintained between 55 °C to 60 °C, over an hour. The flask was kept under a positive pressure of nitrogen and was vented at regular intervals to let out the liberated nitrogen. The reaction mixture was stirred at 55 °C Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 134 to 60 °C for twelve hours. The reaction mixture was cooled to room temperature. 100 ml water was added followed by 3.0 grams of sodium hydroxide. The mixture was stirred for two hours at room temperature and then repeatedly centrifuged to remove the reaction solvents. The nanospheres were rinsed a few times with acetone and centrifuged and then washed with water and centrifuged, repeatedly till the centrifugate was no longer alkaline, followed by washing five times with 2 0 ml portions of acetone and then set in an oven to dry at 80 °C. These aminated nanospheres were evacuated at room temperature for six hours. IR (v, KBr): 3461 and 3368 (N-H s t), 1618 (NH b) cm'1 . Determination of the number of accessible amino groups per aminated polystyrene nanosphere. I) Determination of the number of millimoles of accessible amino groups per gram of aminated nanospheres. Preparation of 0.005N Standard NaOH solution. (Stock Solution A) Sodium hydroxide, 0.2070 g, was dissolved in 100 ml water (Sparkletts) to give a 0.05175 M NaOH solution. 10.0 ml of this solution were taken and diluted to 100 ml to yield a 0.005175 M NaOH solution. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 135 Reaction of amino groups on the resin with acid. (Main reaction) In a 100 ml r.b. flask a known amount (M g) of resin was taken (around one gram). To this was added 5.0 ml THF and the mixture stirred for an hour. The polymer nanospheres were allowed to swell and 7.0 ml ethanol was added, a known volume of cone. H2S0 4 j 0.2 ml to 0.8 ml, was added via a 1.0 ml pipette. The flask was stoppered and stirred gently at 30 °C for 48 hours. After 48 hours the flask was opened . The mixture was very carefully transferred to a centrifuge tube and centrifuged. The centrifugate was collected in a 100 ml volumetric flask. The round bottom flask was washed with minimum portions of aqueous ethanol till all the resin was transferred to the centrifuge tube. The mixture was centrifuged and the centrifugate collected. The resin was washed five times with 10.0 ml portions of aqueous ethanol, sonicating for a minute each time, and then centrifuged down. The centrifugate was collected, and transferred to a 100 ml volumetric flask. The flask was diluted upto the mark with water. It contained the unreacted acid groups. This solution was titrated against 0.005175 N standard base to obtain the millimoles of unreacted acid groups (A). The difference between the reading from this step and the corresponding reading from the blank reaction (B) or the control reaction (C) (if (B) and (C) differ) yielded the number of millimoles of H2 SO4 used up in the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 136 reaction which directly corresponded to the total number of amino groups present in a given weight of resin. From this the number of millimoles of amino groups per gram of resin were calculated. Blank Reaction. In the blank reaction the experiment was then carried out as in the case of the main reaction but no polymer nanospheres were added. The titration of this acid solution with standard base yields the total number of millimoles of acid taken (B). Control Reaction. In the control reaction instead of aminated polystyrene polymer nanospheres, unmodified polystyrene nanospheres were used, all other steps remained the same as the main reaction. This step was used to verify if any losses of acid occur due to reasons other the reaction with amino groups on the resin. This titration yields the corrected total number of millimoles of acid groups present (C). If reading (C) is the same as reading (B) of the blank reaction no such losses occur. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 137 I I ) Determination of the number of nanospheres per gram of resin and the number of millimoles of amino groups per nanosphere. Preparation of water : ethanol solvent mix(90.10 : 9.9 mole fraction). Water, 38.0470 g, and 10.8351 g ethanol were weighed out in a Nalgene bottle, stoppered tightly. The density of the solution is determined using a pycnometer ( Fisher brand, Gay-Lussac Specific Gravity Bottle). B) Determination of the number of resin nanospheres per gram of resin. A dry 5.0 ml volumetric flask was dried overnight it an oven at 80 °C, cooled and weighed. A 10 ml burette (least count 0.05 ml) was filled with the above water- ethanol solvent mix. Carefully, 5.0 ml of this solvent was transferred from the burette into the standard volumetric flask. When the upper meniscus of the liquid in the flask reached the 5.0 ml mark of the flask, addition of the solvent form the burette was stopped and the level of the lower meniscus of the liquid in the burette was noted and marked on the burette. Call this, Volume A. The burette was stoppered between uses. The flask was washed, dried and weighed. A known weight of dried aminated resin (Z g) was added very carefully to the flask which was then stoppered. The flask was tapped on a flat surface so that all the resin settled at the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 138 bottom. The weight the flask and the resin was noted. The unstoppered flask and resin were heated for an hour in an oven at 80 °C. After cooling to room temperature the flask was weighed and any losses in weight noted. This heat-cool- weigh cycle was continued till a constant weight was obtained, which was noted and from this, the weight of the resin was obtained. The burette was filled with solvent-mix. 1.5 ml of the water-ethanol solvent mix was carefully added from the burette into the flask which was then stoppered tightly and sonicated gently at 1 0 °C for thirty minutes. The flask was allowed to warm to room temperature. The exterior of the flask was wiped dry. The remaining solvent mixture from the burette was slowly let into the flask till the upper meniscus of the fluid in the flask reached the 5.0 ml mark of the flask. Call this, Volume B. The flask was stoppered and weighed, (X) g. More solvent mix from the burette was let into the flask till the lower meniscus of the liquid in the burette reaches mark corresponding to Volume A. The flask was stoppered and weighed (Y) g. The flask was washed and dried and the experiment repeated. From the difference (Y-X) g, the weight of solvent displaced by the Z g of the resin was calculated. Using the density of the solvent mix measured previously, the volume of the displaced solvent was calculated. This corresponded to the volume (V) occupied by (Z) g of resin. From the SEM the average volume (v) of a single bead was determined. Dividing (V) by (v) gave the number of nanospheres in (Z) g of resin. From this the number of nanospheres per Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 139 gram of resin was determined. From Part I, the number of millimoles of amino groups per gram of the resin was known, equating this with the number of nanospheres per gram the number of millimoles of amino groups per bead was determined. Electrophilic hydroxylation of polystyrene nanospheres. Polystyrene nanospheres, 1.04 g (10.0 mmole based on styrene monomer), were taken in a Schlenk flask with a magnetic stirrer and fitted with a rubber septum. The flask was evacuated for an hour at room temperature and then purged with dry nitrogen. 1 0 0 ml dry dichloromethane was added to the flask and the nanospheres were stirred under nitrogen for an hour to facilitate their swelling. 6 . 2 2 ml triflic acid (70.0 mmoles) was then added very slowly at room temperature with stirring. The mixture was then cooled to 0 °C and stirred at that temperature for an hour. Next 1.08 ml bis-trimethylsilylperoxide (5.0 mmole) was added very slowly over half an hour. The reaction mixture was stirred at 0 °C for 3 hours. The temperature was gradually raised to room temperature and the reaction mixture was stirred at this temperature for 10 hours. Saturated aqueous sodium bicarbonate solution was carefully added to the reaction mixture till just alkaline. The mixture was stirred for two hours at room temperature and then repeatedly centrifuged to remove the reaction solvents. The nanospheres were rinsed a few times with acetone Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 140 and centrifuged and then washed with water and centrifuged, repeatedly, till the centrifugate was no longer alkaline, followed by washing five times with 2 0 ml portions of acetone and then set in an oven to dry at 80 °C. A small sample of the hydroxylated nanospheres were evacuated at room temperature for 6 hours. IR (v, KBr): 3500 to 3100 (O-H st), 1251 (C-0 st) cm'1 . Synthesis of isothiocyanate functionalized nanospheres. Aminated polystyrene nanospheres, 0.208 g (2.0 mmoles based on styrene monomer), were taken in a Schlenk flask fitted with a septum. The flask was purged with nitrogen and 6 . 0 ml carbon disulfide, 6 . 0 ml dichloromethane and 2 . 0 ml triethylamine were added. The reaction mixture was cooled to around -10 °C. The reaction was stirred at -10 °C for 10 hours. At the end of the allotted time, carbon disulfide and dichloromethane were removed under vacuum at5.0°C tol0.0°C . The flask was purged with nitrogen. Around 4.0 ml dichloromethane was added to the reaction flask and the flask was cooled to 0 °C in an ice bath. Meanwhile, a solution consisting of 0.570 g (2.056 mmoles) dicylohexylcarbodiimide (DCC) in 1:1 v/v DCM:THF was prepared. The DCC solution was slowly added to the reaction flask at 0 °C. The reaction mixture was stirred at room temperature for two hours and the allowed to rise to room temperature. The reaction mixture was stirred Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 141 at room temperature for twelve hours and then centrifuged and repeatedly washed with a 1:1 v/v DCM : THF solution, followed by washings with acetone and cold water. The washed nanospheres were rinsed a final time with acetone, centrifuged and then dried under vacuum for twelve hours. IR (v, KBr): 2117 (N=C=S)cm"1 . Synthesis of isocyanide functionalized polystyrene nanospheres. Aminated polystyrene nanospheres, 0.0666 g (0.64 mmoles based on styrene monomer), were taken in a Schlenk flask, fitted with a rubber septum and purged with nitrogen. 10.0 ml of dry tetrahydrofuran was added to the flask. A solution consisting of 2.1 ml (22.0 mmoles) acetic anhydride and 0.85 ml (22.0 mmoles) formic acid was added to flask. The flask was stirred at 55 °C for twelve hours. The reaction mixture was centrifuged and washed thoroughly with tetrahydrofuran. The washed nanospheres were taken in a Schlenk flask. The flask was evacuated to remove all the tetrahydrofuran and then purged with nitrogen. Next, 10.0 ml dichloromethane was added to the flask, followed by 0.524 g (2.0 mmoles) triphenylphosphine, 0.22 ml (2.0 mmoles) of carbon tetrachloride, and 0.7 ml ( 5.0 mmoles) of triethylamine. The reaction mixture was stirred for thirty-six hours at 40 °C. The reaction mixture was centrifuged and the nanospheres washed with N,N-dimethylformamide, followed by with washing with methanol and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 142 dichloromethane. The resulting nanospheres were dried under vacuum for twelve hours. IR (v, KBr): 2119 (isocyanide) cm"1 . Diazotization of the aminated polystyrene nanospheres. Aminated polystyrene nanospheres, 0.052 g, (0.5 mmole on basis of styrene monomer) were taken in a Schlenk flask fitted with a rubber septum and a magnetic stirrer. To this was added 2.5 ml dichloromethane. The mixture was stirred for an hour to allow the polymer nanospheres to swell followed by the addition of 2.5 ml ethanol. The flask was purged with nitrogen. 0.053 ml (1.0 mmole) concentrated H2 SO4 was added dropwise with stirring. The flask was cooled to 0 °C in an ice bath. With stirring 0.067 ml isoamyl nitrite (0.5 mmole) was added dropwise and the reaction was stirred at 0 °C for an hour. The flask was covered with aluminum foil and the temperature raised to 30 °C. The reaction mixture was stirred in the dark at 30 °C for 10 hours. The diazotized nanospheres were centrifuged down and washed with cold ethanol and then with cold water till no longer acidic. The nanospheres were then washed with acetone and then dried under vacuum at room temperature for 6 hours. IR (v, KBr): 2263 (N2 + ) cm"1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 143 Coupling reaction of the diazotized polystyrene nanospheres. In a round bottom flask, NaOH (0.12 g, 3 mmoles) was dissolved in about 1 0 ml water or methanol or a mixture of both, depending on the solubility properties of the coupling agent (amine or phenol) used. 1 . 0 mmole of coupling agent was added and the mixture stirred for an hour to effect dissolution. The reaction mixture was cooled to 0 °C in an ice bath. With vigorous stirring the prepared diazotized nanospheres were added slowly to the reaction flask. The color of the nanospheres immediately changed. The colored obtained depended on the coupling agent. The reaction mixture was stirred continuously and the temperature was raised to 30 °C. The reaction mixture was stirred for two hours at that temperature. The reaction mixture was then centrifuged and washed repeatedly with tetrahydrofuran followed by washings with water. A variety of coupling agents were tried yielding differently colored nanospheres. Synthesis of azide functionalized nanospheres from the diazotized polymer nanospheres, using hydroxylamine. Diazotized polymer nanospheres, 0.075 g (0.72 mmoles based on styrene monomer), were taken in a flask and 1 . 0 ml dichloromethane was added and the mixture was stirred for an hour to swell the polymer nanospheres. The dichloromethane was removed under vacuum and replaced with 3.0 ml methanol. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 144 The flask was cooled to 0 °C. A solution consisting of 0.165 g (5.0 mmoles) hydroxylamine in 5.0 ml water was added to the flask at 0 °C. The reaction mixture was stirred for half an hour at this temperature and then allowed to come to room temperature. The reaction mixture was stirred overnight. The polystyrene nanospheres were centrifuged and washed well with water and methanol and then dried under vacuum overnight. IR (v, KBr): 2104 (N3 ) cm'1 . Synthesis of azide functionalized nanospheres from the diazotized polymer nanospheres using, sodium azide. Diazotized polymer nanospheres, 0.075 g (0.72 mmoles based on styrene monomer), were taken in a flask and 1 . 0 ml dichloromethane was added and the mixture was stirred for an hour to swell the polymer nanospheres. The flask was cooled to 0 °C. The dichloromethane was removed under vacuum. A solution consisting of 0.325 g ( 5.0 mmoles) sodium azide in 9.0 ml water and 1.0 ml methanol was added to the flask at 0 °C. The reaction mixture was stirred for half an hour at this temperature and then allowed to come to room temperature. The reaction mixture was stirred overnight. The polystyrene nanospheres were centrifuged and washed well with water and methanol and then dried under vacuum overnight. IR (v, KBr): 2104 (N3 ) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 145 Esteriflcation of hydroxy functionalized nanospheres. Hydroxylated nanospheres, 0.026 g (0.25 mmole based on styrene monomer), were taken in a Schlenk flask. The flask was purged with nitrogen. 10.0 ml dichloromethane and 2.0 ml triethylamine was added to the flask. The mixture was stirred for an hour to allow the nanospheres to swell. The flask was cooled to 0 °C and 0.04 ml (~ 0.5 mmoles) acetyl chloride was added slowly. The reaction mixture was stirred at 0 °C for an hour and subsequently for six hours at room temperature. The reaction mixture was centrifuged and washed with water and acetone. The washed nanospheres were dried under vacuum overnight. IR (v, KBr): 1764 (C=0 s t), 1209 (C-0 st) cm'1 . Reactions of polystyrene nanospheres with oxalyl chloride and AICI3 - acid chloride functionalization. Polystyrene nanospheres, 0.104 g (1.0 mmole based on styrene monomer), were taken in a Schlenk flask fitted with a septum. The flask purged with nitrogen. To this was added 5.0 ml dichloromethane and the mixture was stirred for an hour to allow the nanospheres to swell. Oxalyl chloride, 0.027 ml (3.0 mmoles), was added and the reaction mixture was cooled to 0 °C in an ice bath. After the required temperature was attained 0.027 g (0.206) aluminum chloride was added, keeping the flask under a positive pressure of nitrogen. The flask was sealed and the reaction Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 146 mixture was stirred for an hour at 0 °C under nitrogen, following which the reaction mixture was brought to room temperature. The flask was vented at regular interval to release the liberated gaseous products. The reaction mixture was stirred at room temperature overnight, under nitrogen. At the end of the reaction the nanospheres were poured into ice-cold water and immediately centrifuged cold. Ice cold conditions are required to minimize hydrolysis of the acid chloride. The resulting nanospheres were washed with ice cold water and centrifuged till the supernatant was neutral to pH paper. The acid chloride functionalized nanospheres were washed with a ice cold acetone and dried under vacuum overnight. IR (v, KBr): 1772 and 1723 (Ar.C=0 st) , Amidation and Esterification of the acid-chloride functionalized polystyrene nanospheres. The acid chloride functionalized nanospheres were swollen dry tetrahydrofuran under nitrogen. The reaction mixture was treated with excess triethylamine. To obtain the amide, the corresponding amine was added in excess, and for the ester the corresponding alcohol was added. The resulting mixture was stirred for six hours at 30 °C. The reaction mixture was centrifuged, and washed thoroughly with water and acetone and then rinsed with acetone and dried overnight under vacuum. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 147 Amide: IR (v, KBr): 3421 (N-H st broad), 1680 (C=0 st) cm'1 . Ester : IR (v, KBr): 1738 (C=0 st), 1211 (C-0 st) cm'1 . Hydrolysis of the acid chloride functionalized nanospheres. The acid chloride functionalized nanospheres were swollen in dry THF and then treated with an excess of sodium hydroxide in 1 : 1 aqueous ethanol and stirred for twelve hours. This yielded carboxylate functionalized polymer nanospheres. The nanospheres were centrifuged and washed thoroughly with aqueous acetone solution till the supernatant was no longer alkaline, then rinsed with water and dried under vacuum overnight. IR (v, KBr): 1603 and 1550 (C=0 asym st), 1394 (C=0 sym st), 1278 (C-0 st) cm'1 . These carboxylate functionalized nanospheres were treated with an excess of a solution of sulfuric acid in tetrahydrofuran for twelve hours. The obtained carboxylic acid functionalized nanospheres were centrifuged, washed thoroughly with aqueous acetone till the centrifugate was no longer acidic. They were then dried overnight under vacuum. IR (v, KBr): 1721 (C=0 st), 1277 (C-0 s t) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 148 Friedel Crafts acylation of polystyrene nanospheres. Polystyrene nanospheres, 1.50 g (14.4 mmole on basis of styrene monomer), were taken in a Schelnk flask under nitrogen. To this was added 20.0 ml dry dichloromethane. The polymer nanospheres were allowed to stir for an hour under nitrogen to allow for swelling and then aluminium trichloride (0.615 g, 4.62 mmoles) was added. The flask was cooled to 0 °C in an ice bath and acetyl chloride (0.28 ml, 3.6 mmole) was added very slowly. The reaction mixture was stirred at 0 °C for an hour, then allowed to rise to room temperature and maintained there for two hours The reaction temperature was raised to 70 °C and the reaction mixture was stirred under nitrogen for 6 hours at 70 °C. The reaction was then quenched with ice-water and then centrifuged. The residue was washed well with aqueous sodium hydroxide, and then water. The nanospheres obtained were dried under vacuum overnight. IR (v, KBr): 1681 (C=0 st.). 4.4.3 Results and Discussion Hydroxylation and amination under superacidic conditions. The amination and hydroxylation of aromatics under superacidic conditions was successfully extended to polystyrene nanospheres. Use of trimethylsilylazide and bis-trimethylsilyperoxide, in the presence of triflic acid, led to the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 149 functionalization of the bead-surface aromatic rings with amine and hydroxyl groups, respectively. The spherical integrity of the nanospheres was maintained even in the super-acidic medium. (Figure 4.23) The success of functionalization was confirmed by DRIFT spectroscopy, which showed clearly the N-H stretch and bend frequencies for the amine group and O-H stretch and C-0 stretch frequencies for the hydroxyl group. (Figure 4.24 and Figure 4.25) Figure 4.23 (A) Polystyrene nanospheres before amination. (B) Aminated polystyrene nanospheres. Determination of the number of amino groups per polymer nanosphere. The number of amino groups per gram of aminated polystyrene resin was determined by reacting a known weight (M grams) of the aminated resin with a known amount of acid. (Table 4.1) The amount of unreacted acid in solution was then determined by back titration with standard alkali. (Table 4.2) From the blank Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110 108 106 104 102 100. 98 % T 96 94 92 90. 88 8 6 83.S 1000 800 600 450.0 2000 1800 1600 1400 1200 4000.0 3600 3200 2800 2400 Figure 4.24 DRIFT spectra of aminated polystyrene nanospheres 3461 and 3368 (N-H s t), 1618 (NH b) cm'1 . 149.6 140 130 120 110 100 80 70 60 50 4 0. 36.6 450.0 1000 800 600 1800 1600 1400 1200 3600 3200 2800 2400 4000.0 Figure 4.25 DRIFT spectra of hydroxylated polystyrene nanospheres 3500 to 3100 (O-H st) ,1251 (C-0 st) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 151 or control reaction (if the two differ) the amount of total acid in the absence of any amino groups was obtained. This corresponds to the total amount of acid present considering experimental errors. Subtraction of the amount of unreacted acid in the presence of amine groups from this value yields the millimoles of acid that had reacted with the aminated resin. (Table 4.3) The millimoles of acid reacted, directly correspond to the number of millimoles of accessible amino groups present on the bead surface in that weight of resin.(Table 4.3) From this the number of accessible amino groups per gram was determined. (Table 4.4) The control reaction was run to evaluate any errors arising due to the physical presence of the Blank Control Main Weight of aminated nanospheres (M) (g) 0.9951 Weight of polystyrene nanospheres (g) 0.9820 Normality of Stock Solution A (N) 0.005175 0.005175 0.005175 Volume of C.H 2 SO4 used (ml) 0.2 0.2 0.2 Volume of Stock Solution A required to neutralize unreacted acid (ml) 1.4 1.4 1.25 Table 4.1 Experiment for determining the number of amino groups per gram of resin Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 152 nanospheres themselves. After repeatedly performing the above experiment, each gram of aminated resin was found to have an average of 0.191659 millimoles of amine groups. Main Blank Control Volume of Stock Solution A required to neutralize unreacted acid (ml) 1.25 1.4 1.4 Normality of unreacted acid solution (N) 0.0323438 0.036225 0.036225 mmoles of unreacted acid 1.6172 (A) 1.8112 (B) 1.8112 (C) Table 4.2 Determination of the number of unreacted acid groups. (B -A ) (C -A ) mmoles of amino groups in the given weight of resin. 0.19406 mmoles 0.19406 mmoles Table 4.3 Determination of the corresponding number of amine groups on M grams of resin (B-A)/ M (C - A)/M mmoles of amino groups in 1 g of resin 0.195018 mmoles 0.19501 mmoles Table 4.4 Determination of the number of amino groups per gram of resin. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 153 To determine the number of millimoles of amino groups per nanosphere (Table 4.5), the volume occupied by one gram of resin was determined indirectly from the volume of liquid displaced by the one gram of aminated resin.(Archimides Principle). From SEM pictutres of the aminated resin, the average diameter, and from that the average volume of the aminated nanospheres was obtained. The number of nanospheres in one gram of resin was obtained by dividing the total volume occupied by one gram of resin by the volume of a single nanosphere. The number of millimoles of amino groups per nanosphere was obtained by equating the number of nanospheres per gram of aminated resin with the number of millimoles of amino groups per gram of aminated resin. All experiments were run repeatedly, to check for reproducibility and the final result represents and average of the results of these experiments. The average number of millimoles of amino groups per nanospheres obtained was 1.2764*1 O ' 14 millimoles. Modification of the aminated and hydroxylated nanospheres. The aminated and hydroxylated nanospheres were chemically further modified to give a variety of functionalized polystyrenes. On treatment with isoamyl nitrite in concentrated sulfuric acid, the aminated nanospheres underwent diazotization. Figure 4.26 shows the DRIFT spectrum of the diazotized nanospheres. These diazotized nanospheres were treated with a variety of coupling Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 154 I II III Weight o f empty flask (g) 12.723 11.6522 11.6521 Weight o f flask and resin (g) 12.8224 11.7384 11.7088 Weight o f resin (R) (g) 0.0994 0.0862 0.0567 Weight o f resin, flask and added volume-B o f added liquid (X) (g) 17.4998 16.4195 16.4178 Weight o f resin, flask and added volume-A o f added liquid (Y) (g) 17.5944 16.5016 16.4720 Weight o f displaced solvent (Z g) Z = Y - X 0.0946 0.0821 0.0542 M ole fraction o f solvent mixture Ethanol 9.900410 9.902020 9.90202 Water 90.09975 90.09797 90.09797 Density o f Solvent Mixture (D) (g/cm3 ) (pycnometer) 0.97267 0.97315 0.97315 Volume o f solvent displaced (V). (V=Z/D) cm3 0.092758 0.084365 0.055694 nm3 9.7258 * 101 9 8.4365 * 10iy 5.5694*10 iy Density o f resin (d) ( g/cm3 ) (d =R/V) 1.02202 1.02174 1.02180 Average diameter o f the aminated nanospheres (nm) 249.715 249.715 249.715 Average volume o f a single nanosphere (v) (nm3 ) 65252517.09 65252517.09 65252517.09 Number o f nanospheres in R grams o f resin (=V/v) 1.49049*10^ 1.29290*10u 8.53537*101 2 Number o f nanospheres in one gram o f resin 1.49948* 10 1 3 1.49989* 10 1 J 1.50536* 101 3 mmoles o f amino groups in 1 gram o f resin 0.191659 0.191659 0.191659 mmoles o f amino groups per nanosphere 1.2782* 10'1 4 1.2778* 10*1 4 1.2732* 10'1 4 Table 4.5 Determination of the number of millimoles of amino groups per nanosphere. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 155 agents to yields highly colored nanospheres. This coloration was due the formation of azo dyes on the nanosphere surface. Table 4.6 shows a list of coupling agents 146.! 140 135 125 110 105 %T 60.0 1000 800 600 4 50.0 2400 2000 1800 1600 1400 1200 4000.0 3600 3200 2800 Figure 4.26 IR spectrum of diazotized nanospheres. (2263 cm' 1 N 2+ ) used for the process and the colors of the obtained nanospheres. Figure 4.27 is a UV-Vis spectra for yellow, (viiilOa) red, (viii06b) and purple, (viiil 17b) colored nanospheres obtained by this technique. The amino and diazo groups on the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 156 Coupling Agents Color Observed Phenol Yellow Phloroglucinol Dark Orange Aniline Yellow N N ’ diethyl aniline Yellow o-aminophenol Orange 2 -naphthol Red orange 1 -naphtyl amine Dark Brown 5-amino-1 -naphthol Maroon 1 ,6 -naphthalene diol Reddish brown 8 -hydroxy quinoline Orange Chicago Acid Purple Table 4.6 List of coupling agents used and the resulting colors. polystyrene nanosphere surface were, by known procedures transformed into, the isothiocyanate2 9, the azide using hydroxylamine 3 0 a or sodium azide 3 0 b and the 1 1 isocyanide . All these transformations involved longer reaction times due to the heterogeneous nature of the reaction. The success of these transformations was confirmed by DRIFT spectroscopy, wherein the frequency associated with the amine group or diazonium group was lost and the characteristic frequency of the newly formed group was seen. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 157 0.7 j 0.6 - viiil Oa viiil 17b viiiOfib 0.5 0.4 A 0.3 0.2 0.1 0 400 500 600 700 800 900 1000 Wavelength (nm) Figure 4.27 UV-Vis spectra for some of the diazo-coupled nanospheres. (viiilOa) yellow color via coupling with dimethylaniline, (viii06b) red color via coupling with 2-naphthol and (viiil 17b) purple color via coupling with Chicago acid. Carboxylation and acyaltion of polystyrene nanospheres. Treatment of the polystyrene nanospheres with oxalyl chloride- aluminum trichloride reagent in dry dichloromethane led to the incorporation of the acid chloride moiety on their surface. Figure 4.28 shows the DRIFT spectrum of these acid chloride functionalized nanospheres. The two frequencies at 1772 cm' 1 and 1723 cm'1 , are characteristic of an aromatic acid chloride. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 158 128.3 120 115 110 105 100 95 % T 90 85 80 75 70 65 58.2 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 Figure 4.28 Acid chloride functionalized nanospheres; 1772 and 1731 (Ar C=0 st) Attempts at carboxylating the nanospheres using the technique developed by Olah and coworkers, 3 2 for the carboxylation of aromatics, however, failed. The acid chloride functionality once introduced was easily converted through standard protocol to the ester and the amide. The change in the carbonyl band in the IR spectra was seen with the formation of the ester; IR: 1738 (C=0 st), 1211 (C-0 st) cm'1 , and the amide; IR: 3421 (N-H st, broad), 1680 (C=0 st) cm'1 . The acid chloride was also base hydrolyzed to give the carboxylate salt, which in turn was converted to the carboxylic acid by treating with a strong acid such as sulfuric acid in ethanol. These changes were verified by DRIFT spectroscopy. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 159 Finally the polystyrene nanospheres were subjected to Friedel Crafts acylation, which yielded acylated nanospheres. The DRIFT spectrum (Figure 4.29) clearly shows a carbonyl frequency at 1681 cm'1 . 110 % T 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 Figure 4.29 Acylated polystyrene nanospheres. 1681cm'1 (C=0 st.) 4.4.4 Conclusions Post synthetic chemical modification of the surface of polystyrene nanospheres has been successfully carried out and amino, hydroxyl and carboxyl groups have been introduced onto the surface. Though aminations and hydroxylations were carried out in superacidic media and the carboxylation in the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 160 presence of Lewis acid AICI3, the integrity of the nanospheres was not affected. An attempt to calculate the number of amino groups incorporated, yielded 1.2764 * 10' 14 millimoles of amino groups per aminated nanosphere. The aminated , and hydroxylated nanospheres are amenable to further modifications. The amino groups have been converted into other useful functionalities like the isothiocyanate, isocyanide and diazonium salt. The diazonium salt was coupled to various coupling agents to give highly colored nanospheres. Using the AICI3- oxalyl chloride reagent system the polystyrene nanospheres were functionalized with the acid chloride moiety. This acid chloride functionality was then easily transformed into other functionalities like the acid, ester and amide. The successful functionalization of polystyrene nanospheres with useful moieties like the amine, hydroxyl and the acid chloride, provides for a diverse chemistry at the nanosphere and also provides useful chemical handles for attaching and binding other moieties to the polymer nanosphere surface. 4.5 Chapter 4: References (1) Kawahashi, N.; Matijevic, E. J. Colloid Int. Sci. 1990,138, 534. (2) Shahar, M.; Meshulam, H.; Margel, S. J. Polym. Sci.-A 1986,24,203. (3) Margel, S.; Nov, E.; Fischer, I. J. Polym Sci.-A 1991, 29, 347. (4) Okubo, M.; Iwasaki, Y.; Yamamoto, Y. Colloid and Polym. Sci. 1992, 270, 733. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 161 (5) Synthesis and Functionalization o f polymer nanospheres; Greci, M. T.; Ph. D. Dissertation, USC, Los Angeles, 2000. (6 ) Synthesis and Separations using Functional Polymers; Sherrington, D. C.; Hodge, P., John Wiley and Sons: New York, 1988,43. (7) Polymers as Aids in Organic Chemistry; Mathur, N. K.; Narang, C. K.; Williams, R. E., Academic Press: New York, 1980. (8 )(a)Brix, B.; Clark, T. J. Org. Chem. 1988, 53, 3365. (b) Itsuno, S.; Darling, G. D.; Stover, H. D. H.; Frechet, J. M. J. J. Org. Chem. 1987, 52, 4644. (9) (a)Camps, F.; Castells, J.; Fernando, M. J.; Font, J. Tetrahedron Letters 1971, 27, 1713. (b) Farrall, M., J., Frechet, M., J. J. Org. Chem. 1976, 41, 3877 and references cited therein. (10)(a)Braun, D. Makromol Chem. 1959, 30, 85. (b) Chalk, A. J. J. Polym. Sci.- B 1968, 6 , 649. (11) O’Brien, A. R.; Chen, T.; Rieke, R. D. J. Org. Chem. 1992, 57,2667-2677. (12) Wiley, R. H.; Schmitt, J. M. J. Amer. Chem. Soc. 1956, 78, 2169. (13) Ishizone, T.; Tsichiya, J.; Hirao, A.; Nakahama, S. Macromolecules 1992, 25, 4840. (14) The Chemistry o f the Thiol Group-Part 2; Saul Patai (Ed); John Wiley and Sons: New York, 1974. (15) Stranix, B. R.; Gao, P. J.; Barghi, R.; Salha, J. J. Org. Chem., 1997, 62, 8987. (16) Montevecchi, C., P.; Navacchia, M., L.; J. Org. Chem. 1997, 62, 5600. (17) Schlenoff, J. B.; Dharia, J. R.; Xu, H.; Wen, L.; Ming, Li. Macromolecules 1995, 28, 4290. (18) Andreoni, W.; Curioni, A.; Groenbeck, H. J. Am. Chem. Soc. 2000, 122, 3839. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 162 (19) Frechet, J.M. J.; de Smet, M. D.; Farrall, M. J. Polymer 1979, 20, 675. (20) Warshawsky, A.; Fridkin, M.; Stem, M. Journal o f Polymer Science, Polymer Chem. 1982, 20,1469. (21) Stadermann, D.; Maenz, K.; Gorksi, U.; Die Angewandte Makromoleculare Chemie 1997,51 -6 4 , 253. (22) Greci, M. T.; Pathak, S.; Mercado, K.; Prakash, G. K. Surya; Thompson, M. E. Journal o f Nanoscience and Nanotechnology 2001,1(1), 3. (23) Sita, L. R.; Christopher, E. D. C.; Babcock, J. R.; Hsung, R. P. Tetrahedron Letters 1995, 36(26), 4525. (24) Schmitt, J.; Decher, G.; Dressick, W. J.; Brandow, S. L.; Geer, R. E.; Shashidhar, R.; Calvert, J. Adv. Mater. 1997, 9(1), 61. (25) Young, N. R.; Gauthier, J. Y.; Coombs, W. Tetrahedron Letters 1985,25(17) 1753. (26) Reda, T.; Schmidt, H.; Rominger, F.; Gleiter, R.; Ohlbach, Q. European Journal o f Organic Chemistry, 1998, 2409. (27) (a) Olah, G. A.; Ernst, T. J. Org. Chem. 1989, 54, 1203. (b). Olah, G. A.; Ernst, T. J. Org. Chem. 1989, 54, 1204. (28) Encyclopedia o f Organic Reagents in Organic Synthesis, Vol. 2; Paquette, L., A., (Ed.); John Wiley and Sons: England, 1995, 3817. (29) Kitano, K.; Matsubara, J.; Ohtani, T.; Otsubo, K.; Kawano, Y.; Morita, S.; and Uchida, M.; Tetrahedron Letters 1999, 40, 5235. (30)(a) The Chemistry o f Diazonium and Diazo Groups; Saul Patai (Ed); John Wiley and Sons: New York, 1974, pp 247-340. (b) Bliss, E. A.; Griffin, R. J.; Stevens, M. F. G. J. Chem. Soc. Perkin Trans.I 1987, 10, 2217. (31) Maiorana, S.; Baldoli, C.; Licandro, E.; Casiraghi, L.; de Magistris, E.; Paio, A.; Provera, S.; Seneci, P. Tetrahedron Letters 2000,41, 7271-7275. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 163 (32) Olah, G. A.; Torok, B.; Joshek, J. P.; Bucsi, I.; Esteves, P. M.; Golam, R.; Prakash, G. K. S. J. Am. Chem. Soc. 2002, 124, 11379. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 164 Chapter 5 Poly-pentafluorostyrene nanospheres and pentafluorostyrene grafted nanospheres 5.1 Introduction Nucleophilic substitution reactions at the electron deficient fluorinated aromatic nucleus, have been well studied and their examples are well documented in literature.1 Nucleophiles like amines, thiolates,laand alkoxides and phenoxides,2 show very good reactivity towards fluorinated aromatics. Alcohols, phenols and thiols react only as the anion. The reactivity being greatest for the thiolate anion, followed by the alkoxide, phenoxide and then the amine. Thus for the reaction of aminophenols with fluorinated aromatics, by controlling the basicity of the reaction, either hydroxyldiarylamines or aminodiaryl ethers can be obtained. It was also shown by Chambers and coworkers,3 that the fluorine atoms that are substituents at positions ortho and meta to the site of nucleophilic attack were slightly activating, whereas the para - fluorine was slightly deactivating with respect to hydrogen at the same position. Reactions of pentaflurobenzene with nucleophiles are generally much more facile than hexaflurobenzene. Pentafluoronitrobenzene and pentafluoropyridine have been found to better candidates for nucleophilic aromatic substitution than pentafluorobenzene. Similarly, as seen in Figure 5.1 (I), when Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 165 the R group is an alkyl, aryl or unsaturated group, nucleophilic attack at the perfluorinated nucleus has been shown to be possible4 a ,4 b ,4 c (II) R = C-F, C-CF3 i C-N02, N, C-alkyl Nu= NH2, RO\ RS' Figure 5.1 Nucleophilic attack on perfluorinated systems. Pentaflurostyrene, Figure 5.1 (II) is a similar to (I) with R group being an alkene. This system should undergo nucleophilic substitution. Our interest with poly- pentafluorostyrene nanospheres or better still pentafluorostyrene grafted polystyrene nanospheres, is that these are amenable to modification at the surface using via nucleophilic substitution at the surface via pentafluorostyrene moieties. This method opens up a new method of incorporating functionality at the nanosphere surface not tried before. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 166 Data on structurally related systems is known though limited and these systems undergo nucleophilic attack. Hence pentafluorostyrene containing systems should also show these reactions. In this chapter we have investigated the attack of nucleophiles on the pentaflurostyrene system. Initially, studies were done on pentafluorostyrene monomer in solution. We also studied the synthesis of poly- pentafluorostyrene nanospheres via emulsifier free emulsion polymerization and the use of “in situ” grafting for the synthesis of pentafluorostyrene grafted polystyrene nanospheres. Surface chemical modification of these grafted nanospheres via nucleophilic attack at the nanosphere surface perfluorinated aromatic rings was also examined. 5.2 Experimental 1,3-butanediol, ethanolamine, Coumarin 334, Nile Red, isoamyl nitrite, styrene and divinylbenzene were purchased from Aldrich Chemical Co. 2-(4- aminophenyl)-ethanol, 1,3-butanedithiol, pentafluorostyrene and hydrogen tetrachloroaurate(III) trihydrate (HAICI4 .3 H2O) were purchased from Lancaster Synthesis Inc. Avidin was purchased Rockland Immunochemicals Inc. and biotin-4- fluorescein was purchased form Biotium Inc., Hayward, CA. Styrene was vacuum distilled to remove the inhibitor. Divinylbenzene was extracted with aqueous Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 167 sodium hydroxide to remove the inhibitor then washed with water and dried over magnesium sulfate. In order to obtain dry reagents, ethanol amine, 1,3-butanediol and 1,3-butanedithiol were distilled with barium oxide and dimethylsulfoxide (DMSO) was distilled over calcium hydride, and then stored under nitrogen. The dichloromethane used, was dried over calcium hydride. Tetrahydrofuran (THF) under reflux was dried over sodium. Dry N,N-dimethylformamide was obtained from EMD. Silica Gel 60 (Mesh 230 -400) from EM Science was used for column chromatography. All other reagents were used as received. All polymer syntheses were carried out in a 150 ml reaction kettle. The kettle was fitted with a condenser, a nitrogen inlet valve and a mechanical stirring apparatus. The mechanical stirrer was used was the IKA Eurostar power control- visc. Polymer analysis were carried out on a Cambridge 360 Scanning Electron Microscope. SEM samples for microscopy were prepared by placing a drop of sample diluted with water on a glass plate and allowing it to dry. The sample was then sputter coated with gold. For Energy Dispersive Studies (EDS) studies, the samples were coated with carbon. TEM specimens were prepared by placing a drop of diluted sample on a copper TEM sample grid and drying in an open atmosphere. X-ray Photoelectron Spectroscopy (XPS) was performed on VG ESCALAB II instrument. Diffuse reflectance Fourier Transform IR ( DRIFT) spectroscopy was performed on a Perkin Elmer Spectrum 2000 IR spectrometer and UV-Vis Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 168 spectroscopy on a HP - 8453 model spectrometer both fitted with diffuse reflectance accessories. NMR spectroscopy was performed on a Bruker AM360 MHz or Bruker AC250 MHz instrument, and GCMS was performed Hewlett Packard 5890 Series II instrument or a Thermofinnigan Trace DSQ ™ instrument. Fluorescence Spectra were recorded on a Flourolog-3 model FL3-21 with a 450W xenon lamp source, double grating excitation monochromator, single grating emission monochromator, and a room temperature R928 PMT serving as the detector Reaction of pentafluorostyrene with potassium-t-butoxide. Potassium-t-butoxide, (0.492 g, 4.35 mmoles), was taken in a Schlenk flask, under nitrogen. To this were added, 4.0 ml of dry tetrahydrofuran and 6.0 ml of dry N,N-dimethylformamide, at room temperature. The reaction mixture was stirred for a half hour. Pentafluorostyrene, (0.3 ml, 2.172 mmoles), was added. The reaction temperature was raised to 50 °C. The reaction mixture was stirred at this temperature, for five hours under nitrogen. At the end of five hours the flask was cooled to room temperature and the reaction mixture quenched with water. The reaction mixture was extracted five times with 10 ml water or till the aqueous phase was no longer alkaline to litmus. Each time the organic phase was collected. The combined organics were dried over magnesium sulfate. TLC (hexane) showed two Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 169 spots: R f: 0.59 and Rf: 0.26. The reaction mixture was concentrated under vacuum. The two products separated using column chromatography using hexane as eluant, were identified as the monosubstituted perfluorostyrene (Rf 0.59) and the disubstituted perfluorostyrene (Rf 0.26) using GCMS , NMR. (a) Monosubstituted product; Yield(GCMS): 64.80% MS (El) (HP Series II); m/z: 248. *H NMR (6,250 MHz, CDC13 ,22 °C): 6.67 ( dd, J = 18.1 Hz, 12.0 Hz, 1H, -CH= ), 6.09 and 5.62 (d, J = 18.0 Hz, 1H and J = 11.9 Hz, 1H, CH2 = ), 1.41 (s, 9H, 4-CH3 ); 1 9 F NMR (5, 360 MHz, CDC13, 22 °C): -144.5 (m, F aromatic), -156.0 (d, F aromatic). (b) Disubstituted perfluorostyrene; Yield(GCMS): 35.20% MS (El) (HP Series II); m/z: 302. !H NMR (5, 360 MHz, CDC13 ) 22 °C): 6.75(dd, J = 18.2 Hz, 11.9 Hz, 1H, -C H = ), 5.97 and 5.5 (d, J = 18.3 Hz, 1H and J = 11.9 Hz, 1H, CH2 = ), 1.40 (s, 9H, 4 -CH3) ), 1.35 (s, 9H , 2 -CH3); 1 9 F NMR (5, 360 MHz, CDCI3, 22°C): 138.9 (d, F aromatic), -145.9 (m, F aromatic), -153.2 (d, F aromatic). Reaction of pentafluorostyrene with phenol. Phenol (0.633 g, 6.74 mmoles) was taken in a Schlenk flask, under nitrogen. To this was added, 8.0 ml of dry tetrahydrofuran and the mixture was stirred to dissolve the phenol. 8.0 ml of dry N,N-dimethylformamide and 0.2134 g (5.34 mmoles) sodium hydroxide were added. The reaction mixture was stirred for an Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 170 hour to convert the phenol to sodium phenoxide. Pentafluorostyrene, (0.2 ml, 1.448 mmoles) was added. The reaction temperature was raised to 60 °C. The reaction mixture was stirred at this temperature, for six hours under nitrogen. At the end of six hours the reaction was cooled to room temperature and then quenched with water. The reaction mixture was extracted five times with 10 ml water or till the aqueous phase was no longer alkaline to litmus. Each time the organic phase was collected. The combined organics were dried over magnesium sulfate. TLC (hexane) showed two spots: R f: 0.49 and Rf: 0.26. The reaction mixture was concentrated under vacuum. The two products separated using column chromatography using hexane as eluant, were identified as the monsubstituted perfluorostyrene (Rf 0.49) and the disubstituted perfluorostyrene (Rf 0.26) using GCMS , NMR. (a) Monosubstituted product; Yield(GCMS): 69.07 % MS (El) (HP Series II); m/z: 268. *H NMR (5, 360 MHz, CDC13 ,22 °C): 7.33 (t, 2H, aromatic), 7.11 (t, 1H, aromatic), 6.98 (d, 2H, aromatic), 6 . 6 8 (dd, J = 18.0 Hz, 12.3 Hz, 1H, -C H = ), 6.11 and 5.71 (d, J = 18.0 Hz, 1H and J = 11.8 Hz, 1H, CH2 = ); 1 9 F NMR (6,360 MHz, CDCI3, 22 °C): -144,5 (m, F aromatic), -155.9 (m, F aromatic). (b) Disubstituted perfluorostyrene; Yield(GCMS): 30.09% MS (El) (HP Series II); m/z: 342. JH NMR ( 5, 360 MHz, CDC13 , 22 °C): 7.29 (m, 4H, aromatic), 7.05 (m, 2H, aromatic), 6.95 (m, 2H, aromatic), 6.85 (m, 2H, aromatic), 6.63( m, -C II= ), Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 171 6.07 and 5.56 (d, J = 18.0 Hz, 1H and J = 11.7 Hz, 1H, CH2 = ); 1 9 F NMR (6 , 360 MHz, CDCI3, 22 °C): -143.5 (m, F aromatic), -145.6 (d, F aromatic), -153.7 (d, F aromatic). Reaction of pentafluorostyrene with ethanethiol Ethanethiol is a very volatile compound with a foul odor and must be handled very carefully. Sodium hydride (0.024 g, 1.0 mmoles) was taken in a Schlenk flask, under nitrogen. The flask was placed in a water bath to maintain a constant temperature. To this was added 2.5 ml (excess) ethanethiol slowly with stirring. The hydrogen liberated was vented. The mixture was stirred for half an hour, venting the flask regularly. After half an hour the excess ethanethiol was removed under vacuum. The flask was then purged with nitrogen. To this were added 3.0 ml of dry N,N-dimethylformamide followed by 0.1ml (0.724 mmoles) of pentaflurostyrene. The temperature of the reaction mixture was raised to 50 °C and the mixture was stirred for twelve hours under nitrogen. At the end of the allotted time, the reaction mixture was cooled to room temperature and then quenched with water. The reaction mixture was extracted five times with 10 ml water or till the aqueous phase was no longer alkaline. Each time the organic phase was collected. The combined organics were dried over magnesium sulfate. TLC (hexane) showed four spots: Rf: 0.509, R f: 0.320, R f: 0.151 and Rf: 0.0188. The reaction mixture Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 172 was concentrated under vacuum. The products were separated using column chromatography using hexane as eluant, and the major one was then identified as the monsubstituted perfluorostyrene (Rf 0.509) using GCMS , NMR. The other products were identified as the di and tri substituted perfluorostyrenes from GCMS. (a) Monosubstituted product; Yield(GCMS): 53.41% MS (El) (Trace DSQ); m/z: 235.4. *H NMR (5, 360 MHz, CDC13 j 22 °C): 6.70 (dd, J= 18.0 Hz, 11.9 Hz, 1H, -CH= ), 6.15 and 5.74 (d, J = 18.0 Hz, 1H and J = 11.9 Hz, 1H, CH2 = ), 2.95 (q, 2H, CH2 ), 1.27 (t, 3H, CH3 ); 1 9 F NMR (5, 360 MHz, CDC13, 22 °C): -136.3 (m, F aromatic), -144.4 (m, F aromatic.) (b) Disubstituted perfluorostyrene; Yield(GCMS): 11.19% MS (El) (Trace DSQ); m/z: 277.5 (b) Trisubstituted perfluorostyrene; Yield(GCMS): 35.4% MS (El) (Trace DSQ); m/z: 319.4 Synthesis of poly-pentafluorostyrene nanospheres The poly-pentafluorostyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150 ml reaction kettle, equipped with a condenser, gas inlet and mechanical stirring apparatus and containing 60 ml water and 17 ml isopropanol (22.0 volume percent), was heated to 85 °C, stirred at 300 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 173 rpm and flushed for an hour with nitrogen. After one hour the gas flow was turned off and 1.0 ml pentafluorostyrene and 15.0 mole percent of divinylbenzene were added to the water. The reaction mixture was stirred for 20 minutes to bring the monomer and crosslinker to the polymerization temperature, followed by addition of potassium persulfate initiator. The reaction mixture was stirred at 300 rpm and 85 °C for two hours. The reaction mixture was cooled to room temperature. A small sample of the aqueous colloidal suspension was diluted with water. This was used for obtaining SEM images. The colloidal polymer was then frozen overnight at -20 °C, followed by thawing at room temperature. The polymer nanoparticles were centrifuged and washed repeatedly with acetone and water. Samples of the washed nanoparticles were dried overnight at room temperature, under vacuum. IR (v, KBr): 1521 (Ar C-F.), 963 (Ar C-F.) cm'1 . Synthesis of pentafluorostyrene grafted polystyrene nanospheres. The polystyrene nanospheres were synthesized via emulsifier free emulsion polymerization technique. A 150 ml reaction kettle, equipped with a condenser, gas inlet and mechanical stirring apparatus, and containing 60 ml distilled (Sparkletts) water, was heated to 80 °C, stirred at 350 rpm and flushed for an hour with nitrogen. After one hour, the gas flow was turned off and a certain volume (upto 7 ml) of styrene and 4 mole percent of divinylbenzene (DVB) were added to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 174 the water. The reaction mixture was stirred for 20 minutes to bring the monomer and crosslinker to the polymerization temperature, followed by addition of 0.06 g (0.3 mmole) of potassium persulfate initiator. The reaction mixture was stirred at 350 rpm and 80 °C for two and a half hours. At this point 6.0 ml isopropanol was added as cosolvent, and then 5.0 mole percent of pentafluorostyrene was introduced into the reaction. After addition of the monomer, the polymerization was allowed to proceed for an hour and a half. The reaction was stopped by cooling to room temperature. A small sample of the polymer was taken for SEM analysis. The colloidal polymer was then frozen at -20 °C overnight, followed by thawing at room temperature. The polymer nanoparticles were centrifuged and washed repeatedly with acetone and water. Samples of the washed nanoparticles were dried overnight at room temperature, under vacuum. The samples were use for DRIFT studies. IR (v, KBr): 1521 (Ar C-F.), 963 (Ar C-F.) cm'1 . Synthesis of Nile Red dyed polystyrene nanospheres grafted with pentafluorostyrene The polymerization was carried out in a 150 ml polymerization kettle fitted with a condenser, a nitrogen purge inlet, and a mechanical stirrer with a Teflon blade. 70 ml distilled water (Sparkletts) was added to the reaction vessel. A stirring rate of 300 rpm was used. The vessel was immersed in an oil bath heated to 80 °C. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 175 A steady stream of nitrogen was bubbled gently through the water for an hour. Depending on the size of the core required, a certain volume (upto 4.0 ml) styrene monomer and 4.0 mole percent divinylbenzene monomer were added to reaction vessel. Stirring continued at 80 °C and 300 rpm. After 20 minutes 0.06 g potassium persulfate was added to initiate the reaction. The reaction mixture was stirred at 80 °C and 300 rpm for an hour and a half. Meanwhile, 1.1 mg Nile Red was dissolved in a 0.2 ml solution of styrene and 10 mole percent divinylbenzene. After an hour an a half, this dye solution was added to the reaction, maintaining the temperature at 80 °C. The stirring rate was raised to 350 rpm. After about half an hour, 0.2 ml of a 10 mole percent solution of divinylbenzene in styrene was added and the polymerization continued for another half hour. At this point the reaction mixture was transferred to a separatory funnel where any unreacted monomer and dye were removed. The polymerization reactor was washed with acetone to remove any dye, followed by rinsing with water. The reactor was returned to the oil bath set at 80 °C. The polymer reaction mixture was returned hot, to the reactor. About 0.015 g potassium persulfate initiator and 10 ml isopropanol were added to the reaction followed by 0.21 ml ( 4 mole percent) of pentafluorostyrene. The polymerization reaction was continued for an hour. The step of removal of the polymer mixture, washing the reaction vessel, separation of the unreacted dye and monomer, and return of the polymer mixture to the reactor must be done as quickly as possible to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 176 prevent too much of a drop in temperature. The reaction was stopped by cooling to room temperature. A small sample of the polymer was taken for SEM analysis. The colloidal polymer was then frozen at -20 °C overnight, followed by thawing at room temperature. The polymer nanoparticles were centrifuged and washed repeatedly with acetone and water. The washed nanoparticles were dried overnight at room temperature, under vacuum. The samples were use for further modifications, DRIFT studies and florescence measurements. IR (v, KBr): 1521 (Ar C-F), 963 (Ar C-F) cm'1 . Synthesis of Coumarin 510 dyed polystyrene nanospheres grafted with pentafluorostyrene The polymerization was carried out in a glass vessel fitted with a condenser, a nitrogen purge inlet, and a mechanical stirrer with a Teflon blade. 70 ml distilled water (Sparkletts) and 0.01 g of calcium chloride (p = 3.97 * 10'3 ) was added to the reaction vessel. A stirring rate of 300 rpm was used. The vessel was immersed in an oil bath heated to 80 °C. A steady stream of nitrogen was bubbled gently through the water for an hour. After an hour the nitrogen bubbling was stopped and depending on the size of the core required, a certain volume (upto 5.0 ml) styrene monomer and 4.0 mole percent divinylbenzene monomer were added to reaction Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 177 vessel. Stirring continued at 300 rpm. After 20 minutes 0.06 g potassium persulfate was added to initiate the reaction. The reaction mixture was stirred at 80 °C and 300 rpm for an hour and a half. Meanwhile, 3.0 mg Coumarin 510 was dissolved in a 0.2 ml solution of styrene and 10 mole percent divinylbenzene. After an hour and a half, this dye solution was added to the reaction mixture while maintaining temperature at 80 °C, the stirring rate was raised to 350 rpm. After about half an hour, 0.2 ml of a 10 mole percent solution of divinylbenzene in styrene was added and the polymerization continued for another half hour. At this point the reaction mixture was transferred to a separatory funnel where any unreacted monomer and dye were removed. The polymerization reactor was washed with acetone to remove any dye, followed by rinsing with water. The reactor was returned to the oil bath set at 80 °C. The polymer reaction mixture was returned hot, to the reactor. About 0.015 grams initiator and 10 ml isopropanol were added to the reaction followed by 4.5 mole percent of pentafluorostyrene. The polymerization reaction was continued for an hour. The step of removal of the polymer mixture, washing the reaction vessel, separation of the unreacted dye and monomer and return of the polymer mixture to the reactor must be done as quickly as possible to prevent too much of a drop in temperature. The reaction was stopped by cooling to room temperature. A small sample of the polymer was taken for SEM analysis. The colloidal polymer was then frozen at -20 °C overnight, followed by thawing at room temperature. The Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 178 polymer nanoparticles were centrifuged and washed repeatedly with acetone and water. The washed nanoparticles were dried overnight at room temperature, under vacuum. The samples were used for further modifications, DRIFT studies and florescence measurements. IR (v, KBr): 1521 (Ar C-F), 963 (Ar C-F) cm'1 . Reaction ofpara-aminophenol with pentafluorostyrene grafted polystyrene nanospheres Pentafluorstyrene grafted nanospheres (0.052 g, 0.5 mmole based on styrene monomer) were taken in a round bottom flask and swollen with 2.5 ml tetrahydrofuran. To this mixture were added, 0.054 g (0.5 mmoles) /7-aminophenol, 0.0315 g (0.8 mmoles) sodium hydroxide and 2.5 ml N,N-dimethylformamide. The mixture was stirred for half an hour. The temperature was raised to 80 °C for a minimum of two hours. After the desired reaction time, the reaction mixture was centrifuged and repeatedly washed with water, followed by washings with acetone. The washed nanospheres were dried overnight under vacuum. These nanospheres were used for spectral analyses and further studies. IR (v, KBr): 3461 to 3397 (N-H st, very weak), 1520 (Ar C-F), 965 (Ar C-F) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 179 Reaction of 2-(4-aminophenyl)ethanol with pentafluorostyrene grafted polystyrene nanospheres Sodium hydride (0.012 g, 0.5 mmoles) were taken in a Schlenk flask under nitrogen. To this was added 2.5 ml of dry N,N-dimethylformamide. The reaction mixture was cooled to 0 °C and 0.0689 g (0.5 mmoles) 2-(4-aminophenyl)ethanol was added to the flask under a positive nitrogen pressure. The reaction mixture was stirred at 0 °C for five minutes and then the temperature was raised to room temperature. The flask was constantly vented to release the liberated hydrogen. The reaction mixture was stirred at room temperature for half an hour under nitrogen. The mixture is labeled as Mixture-A. In another Schlenk flask, purged with nitrogen, 0.052 g (0.5 mmole based on styrene monomer) pentafluorstyrene grafted nanospheres, were taken and swollen with 2.5 ml tetrahydrofuran. The flask was heated to 60 °C. Mixture-A was added to this flask. The flask was stirred for at least an hour. After the desired reaction time, the reaction mixture was cooled and quenched with water. The reaction mixture was centrifuged and repeatedly washed with water, followed by washing with acetone. The washed nanospheres were dried overnight under vacuum. These nanospheres were used for spectral analyses and further studies. IR (v, KBr): 3456 to 3374 (N-H st,), 1618 (N-H b), 1517 (Ar C-F), 968 (Ar C-F) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 180 Diazotization of para-aminophenol or 2-(4-aminophenyl)ethanol functionalized pentafluorostyrene grafted polystyrene nanospheres. Aromatic amine functionalized nanospheres (0.052 g, 0.5 mmole on basis of styrene monomer) were taken in a Schlenk flask fitted with a rubber septum and a magnetic stirrer. To this was added 2.5 ml dichloromethane. The mixture was stirred for an hour to allow the polymer nanospheres to swell followed by the addition of 2.5 ml ethanol. The flask was purged with nitrogen. 0.053 ml (1.0 mmole) concentrated H2SO4 was added dropwise with stirring. The flask was cooled to 0 °C in an ice bath. Isoamylnitrite (0.067 ml, 0.5 mmole) was added dropwise and the reaction was stirred at 0 °C for an hour. The flask was covered with aluminum foil and the temperature raised to 30 °C. The reaction mixture was stirred in the dark at 30 °C for 10 hours. The diazotized nanospheres can be used directly for further reactions. Before carrying out coupling reactions, the dichloromethane was removed under vacuum. A small sample was centrifuged down and washed a bit with cold ethanol and then with cold water till no longer acidic. The nanospheres were then washed with acetone and then dried under vacuum at room temperature for 6 hours. IR (v, KBr): 2263 (N2 + ) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 181 Coupling reaction of the diazotized nanospheres with 2-naphthol. In a round bottom flask, 0.12 g of NaOH (3.0 mmoles) was dissolved in about 10 ml water or methanol or a mixture of both, depending on the solubility properties of the amine or phenol used. 0.144 g (1.0 mmole) of the phenol, in this case 2-naphthol, was added and the mixture stirred for an hour to effect a solution. The reaction mixture was cooled to 0 °C in an ice bath. With vigorous stirring the prepared polymer diazonium salt was added slowly to the reaction flask. The reaction mixture was stirred continuously and the temperature was raised to 30 °C. The reaction mixture was stirred for two hours at that temperature, then centrifuged and washed repeatedly with tetrahydrofuran followed by washing with water. These washed nanospheres were used to obtain UV-vis spectra of the colored nanospheres. Reaction of ethanolamine with pentafluorostyrene grafted polystyrene nanospheres. Sodium hydride (0.024 g, 1.0 mmole) were taken in a Schlenk flask under nitrogen. To this was added 2.5 ml of dry N,N-dimethylformamide. The reaction mixture was cooled to 0 °C with stirring. 0.06 ml (1.0 mmole) ethanolamine was added to the flask. The reaction mixture was stirred at 0 °C for five minutes and then brought to room temperature. The flask was constantly vented to release the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 182 liberated hydrogen. The reaction mixture was stirred at room temperature for half an hour under nitrogen (labeled as Mixture-B). In another Schlenk flask purged with nitrogen, 0.026 g (0.25 mmole based on styrene monomer) pentafluorstyrene grafted nanospheres, were taken and swollen with 2.5 ml tetrahydrofuran. The flask was heated to 60 °C. Mixture-B was added to this flask. The reaction mixture was stirred for at least an hour. After the desired reaction time, the reaction mixture was cooled and quenched with water. The mixture was centrifuged and repeatedly washed with water, followed by washing with acetone. The washed nanospheres were dried overnight under vacuum. IR(v, KBr): 3384 to 3379 (N-H st,), 1517 (Ar C-F.), 1133 (C-O, 971 (Ar C-F.) cm'1 . Reactions of 1,3-propanediol or 1,3-propanedithiol with pentafluorostyrene grafted polystyrene nanospheres. Sodium hydride (0.024 g, 1.0 mmole) was taken in a Schlenk flask under nitrogen. To this was added 2.5 ml of dry N,N-dimethylformamide. The reaction mixture was cooled to 0 °C with stirring (labeled as Mixture-C). 1,3-propanediol (0.14 ml, 2.0 mmoles) or 0.20 ml (2.0 mmoles) 1,3- propanedithiol were taken in another Schelnk flask, that had been purged with nitrogen. The flask was cooled to 0 °C. (labeled as Mixture-D). Subsequently Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 183 Mixture-C was mixed with Mixture-D at 0 °C The flask was constantly vented to release the liberated hydrogen. The reaction mixture was stirred at room temperature for half an hour under nitrogen, (labeled as Mixture-E). Meanwhile in another Schlenk flask, purged with nitrogen, 0.026 g (0.25 mmole based on styrene monomer) pentafluorstyrene grafted nanospheres, were taken and swollen with 2.5 ml tetrahydrofuran. The contents of the flask were heated to 60 °C. Mixture-E was added to this flask. The flask was stirred for at least an hour at this temperature. After the desired reaction time the reaction mixture was cooled and quenched with water. The reaction mixture was centrifuged and repeatedly washed with water, followed by washings with acetone. The washed nanospheres were dried overnight under vacuum. These nanospheres were used for spectral analyses and further studies. I R (v, KBr) (Hydroxy substituted): 3423 (O-H st), 1135 (C-O), 965 (Ar C-F) cm’1 . Reactions of sodium-4-hydroxybutyrate with pentafluorostyrene grafted polystyrene nanospheres. Sodium hydride (0.0096 g, 4.0 mmole) was taken in a Schlenk flask under nitrogen. The flask was placed in a water bath. To this was added 6.0 ml of dry dimethysulfoxide (DMSO) with vigorous stirring. The flask was regularly vented to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 184 release the liberated hydrogen. The mixture was stirred for an hour at 30 °C. (labeled, Mixture-F). Sodium-4-hydroxybutyrate (0.504 g, 4.0 mmole) was taken in another Schelnk flask, that had been purged with nitrogen. 10 ml dry DMSO were added to the flask and the mixture was stirred for an hour in a water bath. Mixture-F was slowly added to the sodium-4-hydroxybutyrate solution with stirring. The reaction mixture was stirred at 40 °C for an hour, under nitrogen, (labeled Mixture-G.) Meanwhile in another Schlenk flask, purged with nitrogen, 0.104 g (1.0 mmole based on styrene monomer) pentafluorstyrene grafted nanospheres, were taken and swollen with 6.0 ml of a 1:1 v/v DMSO : tetrahydrofuran solution. The tetrahydrofuran was removed under vacuum. The flask was heated to 60 °C and Mixture-G was added to this flask. The contents of the flask were stirred for at least an hour at 60 °C. After the desired reaction time the flask was cooled and quenched with water. The reaction mixture was centrifuged and repeatedly washed with water, followed by washings with water and acetone. The washed nanospheres were dried overnight under vacuum. These nanospheres were used for spectral analyses and further studies. IR (v, KBr): 1681 and 1561 (C=0 asym st), 1520 (Ar C-F), 1410 (C=0 sym st), 968 (Ar C-F) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 185 These sodium carboxylate goups were converted to the free carboxylic acids by stirring overnight in aqueous hydrochloric acid. IR (v, KBr): 1713 (C=0 st), 1518 (Ar C-F), 964 (Ar C-F) cm'1 . Reactions of avidin with carboxylic acid functionalized pentafluorostyrene grafted nanospheres. Carboxylic acid functionalized nanospheres (0.026 g, 0.25 mmole based on styrene monomer units) of was stirred overnight in 1.0 ml distilled water. To this mixture was added a solution of 0.024 g (0.125 mmole) of l-(3-diethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) in 1.0 ml water. The mixture is stirred vigorously for five hours. Meanwhile, 2.0 mg of avidin was dissolved in 4.0 ml of a 100 mM borate buffer solution (pH 9.23). This solution was added to the nanosphere mixture and the reaction mixture was stirred for forty-eight hours at 30 °C. The reaction mixture was centrifuged and the nanospheres were washed with Phosphate Buffered Saline (PBS) to remove excess avidin and then suspended in 2.0 ml distilled water. The nanospheres were then treated with 5.0 ml of a 10 mM ethanol amine solution for twelve hours, in order to quench unreacted EDAC activated carboxylic acid groups. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 186 The nanospheres were the centrifuged, washed well with distilled water and then suspended in 3.0 ml distilled water. When not in use, these nanospheres were stored at 0 X to 4 X . IR (v, KBr): 3284 (N-H st), 1690 (amide C=0 st), 1524 (Ar C-F) cm'1 . Preparation of phosphate buffered saline. Sodium chloride (4.75 g), 0.575 g dibasic sodium phosphate, 0.1 g potassium chloride and 0.1 g monobasic potassium phosphate were dissolved in distilled water and the solution made to 500 ml. pH: 7.33 Preparation of 100 mM borate buffer. 6.18 g boric acid and 2.00 g sodium hydroxide were dissolved in distilled water and the solution made to one liter. pH: 9.23 Synthesis of gold colloids. Gold colloids used were prepared using the citrate reduction technique.5 50 ml of a 1.0 mM aqueous solution of hydrogen tetrachloroaurate(III) trihydrate (HAuCl4.3H20 ) were taken in a 100 ml round bottom flask under nitrogen fitted with a reflux condenser having a calcium chloride guard tube. HPLC grade water was used throughout this process. The reaction mixture was heated at reflux for half Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 187 an hour, with vigorous stirring. 5.0 ml of a 3.9 mM aqueous solution of tris-sodium citrate was added to this boiling solution and the reaction mixture was stirred under reflux. The reaction solution turned pink. The reaction was continued for twenty minutes after the appearance of this pink color. The reaction mixture was cooled to room temperature. A small sample was taken for Transmission Electron Microscopy (TEM) analysis. Synthesis of silver colloids Silver colloids were synthesized using the ethanol reduction technique.6 Silver nitrate (8.5 mg, 500 mmole) was dissolved in 50 ml ethanol. To the solution was added 220 mg (2.0 mmole as monomer unit) polyvinylpyrrolidone (PVP-K30) and the solution was allowed to reflux overnight under argon atmosphere. The resulting intense yellow solution was characterized to be silver colloids. Immobilization of gold and silver colloids on the functionalized nanospheres. Thiol functionalized nanospheres (0.01 g) were swollen in minimum amount of THF. The THF was then evaporated and replaced by an equal volume of ethanol. 1.0 ml of the prepared collidal gold solution was added to this suspension of nanospheres. The mixture was sonicated for a minute and then allowed to stand for five minutes. The mixture was again sonicated for a minute and then centrifuged at Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 188 around 2500 rpm. If the supernatant was colorless, the process was repeated till the supernatant obtained on centrifuging, had a pink color in the case of gold colloids and yellow color in the case of silver colloids. At this point no more colloids could be absorbed on the nanospheres. The nanospheres were rinsed with water, sonicated for a minute and then centrifuged. If the supernatant was colored the process was repeated till the supernatant was colorless. This process was carried out to get rid of any loosely bound colloids. Tagging of avidin coated nanospheres with biotin-4-fluorescein A stock solution of biotin-4-fluorecein was prepared by dissolving 4.0 mg of biotin-4-fluorescein in 1.0 ml DMSO. In a centrifuge tube, 0.013 g of avidin functionalized nanospheres were suspended in 1.0 ml of a 1:1 DMSO : H2O solution. To this mixture was added two drops of the biotin-4-fluorescein stock solution. The mixture was sonicated and allowed to incubate at 30 °C for two hours. The resulting mixture was centrifuged to separate the tagged beads followed by repeated washings with a 1:1 DMSO : water solution. This was continued till the washings showed no fluorescence. The nanospheres were then suspended in 1.0 ml aqueous ethanol and stored at 0 °C. For checking for nonspecific binding, a blank Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 189 reaction was performed by treating carboxylic acid functionalized nanospheres with biotin-4-fluorescein, according to the above protocol. 5.3 Results and Discussion Nucleophilic substitution at the pentafluorostyrene nucleus In order to gauge the reactivity of pentafluorostyrene to nucleophiles, reactions of pentafluorostyrene with various nucleophiles like the /-butoxide anion, phenoxide anion, and the thiolate anion were carried out. The reactions were monitored by GCMS. The products wherever possible, were separated by column chromatography and characterized by ^ 1 9 F NMR spectroscopy. Pentafluorostyrene reacts with t-butoxide anion at 50 °C, to yield the monosubstituted and disubstituted products in 65 % and 35% yields, respectively, as characterized by GCMS, in about five hours of reaction time. The two products were separated by column chromatography using hexane as the mobile phase. The monosubstituted product showed only two peaks in the 1 9 F NMR, indicating that the /-butoxy group was at the 4 -position (para to the vinyl moiety). The disubstituted showed the expected 3 peaks in 1 9 F NMR. The reaction of the phenoxide anion with pentafluorostyrene was also very facile and proceeded to completion in about six hours at 60 °C. Here too, the only products seen from GCMS, were the monosubstituted and the disubstituted perfluorostyrenes obtained Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 190 in 69% and 39 %, yields, respectively. These two products were separated by column chromatography using hexane as the mobile phase. From the two peaks seen in the 1 9 F NMR, the monosubstituted product has a substitution pattern similar to the monosubstituted /-butoxy product. The disubstituted product showed three peaks in the 1 9 F NMR. The reaction with the ethanethiolate anion also worked well and in about four hours time at 50 °C, monosubstituted, disubstituted and trisubstituted products were obtained in 62%, 10% and 5% yields respectively. However, about 20% of pentafluorostyrene was found to be unreacted. Continued reaction for another eight hours lead to complete conversion. The final product distribution from GCMS was monosubstituted 53.4%, disubstituted 11.2 % and trisubstituted 35.4%. Unlike the oxygen nucleophiles, wherein only mono- and di substituted products were obtained, the thiolate anion yielded mono-, di-, and tri substituted products. The trisubstituted product was obtained in greater amounts than the disubstituted product. Synthesis of the polymer nanospheres with the surface pentaflurostyrene groups Having confirmed that pentafluorostyrene was succeptible to nucleophilic attack by alkoxide and thiolate anions, we decided to study the use of these reactions to functionalize pentafluorostyrene moieties at the polymer nanosphere Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 191 surface. Polypentaflurostyrene nanoparticles synthesized using emulsifier free emulsion polymerization were spherical but not very monodisperse. (Figure 5.2) It was found that by using a water soluble organic co-solvent the properties of the final nanoparticles were improved. Isopropanol is miscible with water and with a boiling point of around 85 °C was tried and found most suitable for this purpose. Thus, by using a medium consisting of 88% (by volume) water and 22% isopropanol, poly-pentafluorostyrene nanospheres were synthesized in relatively Figure 5.2 SEM of the polypentafluorostyrene nanospheres obtained with no organic cosolvent during emulsifier free polymerization. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 192 high monodispersity (Figure 5.3 ). The co-solvent seemed to have an affect on the size of the final nanosphere, as the size of the nanospheres synthesized in the presence of the organic co-solvent was almost one a half times those, obtained in absence of any cosolvent. However, this effect was not explored any further. Figure 5.3 SEM of the polypentafluorostyrene nanospheres obtained with using isopropanol as organic cosolvent during emulsifier free polymerization. As we were interested in nucleophilic substitutions of pentafluorostyrene at the nanosphere surface, we carried out the “in-situ” grafting7 of pentafluorstyrene onto polystyrene nanospheres. Here too, the incorporation of pentafluorostyrene moieties onto the polymer nanosphere surface was improved in the presence of an Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 193 organic cosolvent. Thus during the polymerization, just before addition of pentafluorostyrene monomer, about 10 percent isopropanol (by volume) was added to the reaction, followed by 5.0 mole percent pentafluorostyrene. The success of the grafting was confirmed by diffuse reflectance infra-red (DRIFT) spectroscopy. (Figure 5.4) % T 90. 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 Figure 5.4 DRIFT spectrum of pentaflurostyrene grafted nanospheres. 1519 cm'1 (Ar C-F), 968 cm'1 (Ar C-F). The spherical monodisperse nature of the nanospheres were confirmed by scanning electron microscopy (SEM). (Figure 5.5) We also investigated the synthesis of fluorescent-dye doped polystyrene nanospheres grafted on the surface with pentafluorostyrene. These nanospheres Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 194 had a core of polystyrene that contained no dye, onto this core was grown the dyed doped polystyrene layer. The dyed layer was grown by simply dissolving the dye in styrene and adding it the polymerization at the desired time. Around this dye layer, a protective shell of 10.0 mole percent crosslinked polystyrene was grown followed Figure 5.5 SEM image of pentafluorostyrene grafted polystyrene nanospheres. by the grafting of the pentafluorostyrene moieties. The dyes used in our study were Nile Red and Coumarin 510 (C510). Figure 5.6 and Figure 5.7 show the photoluminescence spectra of these dye doped nanospheres. In these two figures, the dashed lines curves represent the excitation curves for the dyed nanospheres, while the other curve is the emission signal. Figure 5.7 shows the emission spectra Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 195 for the Nile-Red doped pentafluorostyrene grafted nanospheres for an excitation wavelength of 370 nm. The broad peak at around 464 nm was due to polystyrene T3 0.4 240 340 640 Wavelength (nm) Figure 5.6 Photoluminescence Emission spectra from C510 doped nanospheres emissions while the narrower peak at around 560 nm was due to Nile-Red. Figure 5.6 shows the emission spectra for Coumarin 510 doped pentafluorostyrene grafted nanospheres excitation wavelength of 397 nm. The peak at 460 nm was due to Coumarin 510. This peak overlapped the polystyrene emission which occurred at around the same wavelengths and hence the polystyrene emission was not seen. The emission of the dyes in the nanospheres was shifted with respect to the dye in solution. The presence of surface pentafluorostyrene moieties was confirmed by Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 196 1 o 2 4 0 3 4 0 4 4 0 540 640 7 4 0 W avelength (nm) E m ission (370 nm) Excitation (460 nm) Figure 5.7 Photoluminescence spectra from Nile Red doped nanospheres. DRIFT spectroscopy. These dyed nanospheres could be envisaged as fluorescent polymer nano-probes, to whose surface, using the nucleophilic susceptibility of the pentafluorostyrene groups, various other moieties could be attached. The dye containing nanospheres were investigated along with the non doped nanopsheres for their ability to be further functionalized at the surface, due to the reactivity of their surface pentafluorostyrene groups to nucleophiles. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 197 Nucleophilic substitutions at the nanosphere surface In order to study the amenability of the synthesized pentafluorostyrene grafted nanospheres to modifications by nucleophilic substitution reactions, we decided to carry out nucleophilic substitutions on the pentafluorostyrene grafted nanospheres using nucleophiles generated from p-aminophenol and 2-(4-aminophenyl)ethanol. The success of the procedure could be verified not only by DRIFT spectroscopy, but also by a change in the nanosphere color obtained by diazotization and coupling of the aromatic amino group incorporated on the nanosphere surface as a result of this reaction. The treatment of the pentafluorostyrene grafted nanospheres with the para-aminophenoxide anion led to the successful incorporation of this moiety onto the nanosphere surface, via their reaction with the nanosphere surface pentafluorostyrene groups. Figure 5.8 , the DRIFT spectrum of the p-aminophenoxide modified nanospheres, showed very weak N-H frequecies. This reaction was carried for six hours. More conclusive evidence was obtained from the UV-Vis spectrum of these functionalized nanospheres, after diazotizing the surface aromatic-amino groups and coupling to 2-naphthol. A very sharp color change of the nanospheres, from white to orange-red, occurred. Figure 5.9, the diffuse reflectance visible spectra of these diazo-coupled nanospheres, showed strong absorption in the 400 nm to 450 nm region. Similar Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 198 treatment of the 2-(4-aminophenyl)ethoxide functionalized nanospheres yielded similar results. Figure 5.10 shows the visible spectra of 2-(4- 98.5 96 94 92 90 86 84 82 %T 80 78 76 74 72 70 68 66 65.1 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 800 600 450.0 1000 Figure 5.8 IR of/7-aminophenoxide grafted nanospheres 3461 to 3379 (N-H st, very weak), 1520 (Ar C-F), 965 (Ar C-F) cm'1 . (6 hour reaction) aminophenyl)ethoxide functionalized nanospheres after diazotization of the incorporated surface aromatic amines and coupling them to 2-naphthol. With these nanospheres a well defined signal in the 450 nm to 500 nm region was observed. Figure 5.11 shows the DRIFT spectrum of the 2-(4-aminophenyl)ethoxide functionalized nanospheres. The reaction took place within two hours. The N-H stretching and N-H bending frequencies are quite clearly seen. Even though the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 199 Figure 5.9 Diffuse reflectance visible spectrum of /7-aminophenyl functionalized nanospheres diazotized nanospheres coupled to 2-naphthol reaction between the poly-pentafluorostyrene grafted nanospheres and the 2-(4- aminophenyl)ethoxide anion was carried out for a shorter reaction time than with the /rara-aminophenoxide anion, the former was introduced onto the polymer nanospheres to a much greater extent as seen from the relatively stronger NH frequencies in the IR spectra. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 200 Figure 5.10 Diffuse reflectance visible spectrum of 2-(4-aminophenyl)ethoxide functionalized nanospheres, diazotized and coupled to 2-naphthol. Functionalization of the nanospheres surface via nucleophilic substitution reactions. Having confirmed that the pentafluorostyrene grafted polystyrene nanospheres could be modified by nucleophiles via nucleophilic substitution, we decide to use this reaction to further functionalize the nanosphere with other useful functionalities like amino, hydroxyl, carboxylic acid and thiol groups. We used Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 201 78.7 76 74 72 70 68 66 64 62 60 S8 56 54 52 50 48.4 4000.0 3200 2800 1600 3600 2400 2000 1800 1400 1200 1000 800 600 450.0 Figure 5.11 DRIFT of 2-(4-aminophenyl)ethoxide functionalized nanospheres. Three hour reaction time. IR (v, KBr): 3456 & 3374 (N-H st), 1618 (N-H b), 1517(Ar C-F), 956 (Ar C-F) cm'1 . ethanolamine, sodium-4-hydroxybutyrate, 1,3-propanediol and 1,3 propanedithiol as the nucleophile precursors. The dye-doped perfuorostyrene grafted nanospheres were also subjected to similar nucleophilic substitution reactions. All the reactions were carried out under anhydrous conditions. The treatment of pentaflurostyrene grafted nanospheres with sodium-2-aminoethoxide led to the incorporation of the 2-aminoethoxide moiety on the nanosphere surface. The success of the incorporation was confirmed by DRIFT spectroscopy (Figure 5.12) and X-ray Photoelectron Spectroscopy (XPS) (Figure 5.13). The IR spectra clearly shows the N-H frequencies. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 202 % T 65 3384.61 3307.69 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0 cm-1 Figure 5.12 DRIFT of 2-aminoethoxide functionalized nanospheres. IR (v, KBr): 3384 and 3307 (N-H st), 1133 (C-O), 971 (Ar C-F) cm'1 . 16000 10000 6000 1100 JO 6 0 0 5< Binding Energy <«V) 200 100 Figure 5.13 XPS of amine functionalized nanospheres. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 203 In the XP-spectrum, (Figure 5.13) the amine nitrogen was defined by a very small peak. The oxygen of the resulting ether linkages were clearly seen and so too were the flourines from the pentafluorostyrene moieties. Treatment of pentafluorinated nanospheres with 1,3-propanediol and sodium-4-hydroxybutyrate resulted in hydroxyl and carboxylate functionalized nanospheres. Figure 5.14 and Figure 5.15 show the DRIFT spectrum and the XP-Spectrum respectively, for the hydroxyl functionalized nanospheres. Similarly the pentafluorostyrene nanospheres were also functionalized with thiol groups using 1,3-propanedithiol. Figure 5.16, the EDS of the thiol functionalized nanospheres shows the Ka line due to sulfur. 4000.0 3600 3200 2800 2000 1800 1200 1000 2400 1600 1400 800 600 4S0.0 cm-1 Figure 5.14 DRIFT of hydroxyl functionalized nanospheres. IR (v, KBr): 3423 (O-H st), 1133 (C-O), 965 (Ar C-F) cm'1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 204 40000 35000 30000 25000 20000 10000 5000 1200 1100 1000 800 700 €00 400 300 200 0 500 100 Binding Energy (»V) Figure 5.15 XPS of hydroxy functionalized nanospheres. X-RfiVs 0 - 2 0 keU Live* 138s Presets 200s Remaining! 62s Real! 165s 16* Dead 1.800 keU 12.0 > FS= H K eh 100= 51? ets M EW ! Figure 5.16 EDS of thiol functionalized nanospheres, showing the sulfur peak. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 205 The DRIFT spectrum for the carboxylate functionalized nanospheres is seen in Figure 5.17, and it showed the symmetric and asymmetric stretch frequencies of the carboxylate group. 102.0 100 95 90 85 80 % T 75 70 65 60 54.8 3600 4000.0 3200 2800 2000 2400 1800 1600 1400 1200 1000 600 450.0 800 Figure 5.17 DRIFT spectrum of carboxylate functionalized nanospheres. IR (v, KBr): 1681 and 1561 (C=0 asym st), 1520 (Ar C-F), 1410 (C=0 sym st), 968 (Ar C-F) cm'1 . When treated with gold colloids, the amino and thiol functionalized nanosparticles strongly adsorbed the gold colloids on their surface.Figure 5.18 and Figure 5.19 show the Diffuse Reflectance Visible spectrum of the gold colloid coated amino and thiol nanospheres, respectively. The characteristic plasmon resonance for gold was seen at around 530 nm to 550nm. Figure 5.20 and Figure Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 206 5.21 show the EDS spectra of the amino functionalized and thiol functionalized nanospheres coated with gold colloids, clearly showing the presence of gold. Figure 5.22 A and Figure 5.22 B show the TEM images of these gold colloid coated nanospheres. 0.05 JaO £ L JQ Q . Figure 5.18 Diffuse reflectance UV-Visible spectra showing the gold plasmon band for amine functionalized nanospheres coated with gold colloids. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 207 0.22 0.2 0.18 0.12 .500. .520. 200. Figure 5.19 Diffuse reflectance UV-Visible spectra showing the gold plasmon band for thiol functionalized nanospheres coated with gold colloids. When the hydroxyl functionalized nanospheres were similarly treated with silver colloids, the hydroxyl functionalized nanospheres strongly adsorbed the silver colloids on their surface. Figure 5.23 shows the EDS for the hydroxyl functionalized nanospheres coated with silver colloids. Figure 5.24 shows their TEM image. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 208 Au : Figure 5.20 EDS of gold coated amine functionalized nanospheres, showing the gold peaks. f t i S i Figure 5.21 EDS of gold coated amine functionalized nanospheres, showing the gold peaks. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.22 TEM images of (A) an amine functionalized, (B) a thiol functionalized nanosphere showing gold colloids on the polymer nanosphere. Figure 5.23 EDS of hydroxyl functionalized nanospheres coated with silver colloids, showing silver peaks. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.24 TEM image of hydroxyl functionalized nanosphere coated with silver colloids. The dye doped nanospheres were also subjected to above nucleophilic aromatic substitution reactions. It was however found, that beyond reaction times of one hour, a definite degradation in the fluorescence signal from the dye-doped nanospheres was seen. For reaction times longer than two hours no fluorescence was seen from the nanospheres. The fluorescence quenching had something to do with the presence of the nucleophiles, as when the fluorescent nanospheres were Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 211 stirred for twelve hours in just the reaction solvents at the reaction temperature a minimal loss in fluorescence intensity was seen. Fluorescent tagging of avidin functionalized nanospheres In order to examine the ability to attach biomolecules to these functionalized nanospheres, we decided to functionalize the acid functionalized nanospheres with the protein avidin and then study its binding to biotin-4-fluorescein. Biotin-4- fluorecein is a fluorescent conjugate of biotin with an emission at around 523 nm at pH 9.00. Avidin is basic glycoprotein found in raw egg white, has a molecular weight of 680008 a and is composed of four essentially identical polypeptide chains. 8 a ,8 b It combines stoichiometrically with biotin. The best preparations of avidin, bind around 15.0 pg of biotin per milligram of protein. The dissociation constant for biotin is 10'15M.9 Carboxylate functionalized nanospheres, that resulted from the reaction of sodium-4-hydroxybutyrate with the pentafluorostyrene grafted nanospheres, were treated with aqueous alcoholic hydrochloric acid to obtain carboxylic acid functionalized nanospheres. These carboxylic acid functionalized nanospheres were then coupled to the protein avidin by a method used by Anderson and co workers.1 0 The DRIFT spectra of the avidin coupled nanospheres showed strong N-H stretch and carbonyl amide frequencies, that indicated successful coupling. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 212 This was confirmed by coupling of the avidin functionalized nanospheres to biotin- 4-fluorescein. The avidin functionalized nanospheres when incubated with biotin-4-fluorescein, bound the fluorescent biotin conjugate to their surface. These biotin tagged nanospheres, on excitation with 365 nm UV-light, showed a green fluorescence not present in the non-tagged nanospheres. The presence of biotin-4- fluorescein on the surface of the avidin coated nanospheres was confirmed by fluorescence spectroscopy.(Figure 5.26). Figure 5.25 shows the normalized spectra for avidin functionalized nanospheres before (Series 2) and after (Series 3) treatment with biotin-4-fluorescein. The broad peak, seen in both Series 2 and Series 3, in the region of 425 nm to 450 nm, represents the emission from the polymer nanospheres. The emission from pentafluorstyrene grafted polystyrene nanospheres at 512 nm in Series 3, is the emission from the biotin-4-fluorescein. In order to check for non-specific binding of the biotinylated dye to the nanosphere surface, carboxylic acid functionalized nanospheres were treated with biotin-4- fluorescein, in the manner similar to the avidin functionalized nanospheres. However, almost no biotinylated dye was attached to these nanospheres, as seen from the absence of the 512 nm peak in their fluorescence spectrum (Series 1). When compared to Series 2, Series 1 showed a slight increase in fluorescence, in the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 213 3 2, 0.8 v o g 0.6 o 350 450 550 650 750 Wavelength (nm) Series 1 Series2 Series3 Figure 5.25. Normalized fluorescence spectra. Series 1: Carboxylic acid functionalized pentafluorostyrene grafted polystyrene nanospheres after treatment with biotin-4-fluorescein. Series 2: Avidin functionalized pentafluorostyrene grafted polystyrene nanospheres. Series 3: Avidin functionalized pentafluorostyrene grafted polystyrene nanospheres after treatment with biotin-4-fluorescein. Excitation 365nm. region 485 nm to 645 nm, which could indicate that some biotinylated dye does, through non specific interactions, stick to the non avidin containing nanospheres. Figure 5.26, shows the fluorescence emission spectra for pentafluorostyrene grafted polystyrene nanospheres. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 214 3 « S ® o c ® o Series 1 o 0.4- 3 U L 350 450 550 650 750 Wavelength (nm) Figure 5.26 Normalized fluorescence spectra of pentafluorostyrene grafted polystyrene nanospheres. Excitation 365 nm. 5.4 Conclusions The susceptibility of pentafluorostyrene monomer to nucleophilic aromatic substitution reactions was examined. A variety of nucleophiles were tried and it was found that pentafluorostyrene underwent nucleophilic substitution reactions with alkoxide, phenoxide and thiolate anions. Poly-pentafluorostyrene nanospheres and pentafluorostyrene grafted polystyrene nanospheres were successfully synthesized using the emulsifier free emulsion polymerization technique. These polymer surface Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 215 pentafluorostyrene moieties successfully underwent nucleophilic substitutions just as in the case with the monomer. This susceptibility of the pentafluorostyrene ring to nucleophilic reactions, was used to chemically modify the nanosphere surface with a variety of functional groups like amine, thiol, alcohol and carboxylic acid. The presence of these groups was confirmed by infrared spectroscopy, EDS and/or XPS. Amine and thiol functionalized nanospheres showed affinities for colloidal gold nanoparticles, while alcohol functionalized nanospheres showed an affinity for silver nanoparticles. Avidin was attached to surface of the carboxylic acid functionalized nanoparticles. These avidin functionalized nanoparticles bound a biotinylated fluorescent conjugate to their surface via the strong bitotin-avidin interaction. Synthesis of fluorescent polymer nanospheres containing embedded fluorescent dyes and surface pentafluorostyrene moieties was successfully attempted. However, the nanospheres lost their fluorescence when subjected to nucleophilic substitution reactions. 5.5 Chapter 5: References (1) (a) Peach, M. E.; Crowell, T. R. J. Flourine Chem. 1982, 21, 469. (b) James, J. H.; Peach, M. E., Willliams, C. R. J. Fluorine Chem. 1985, 27, 91. (2) Grashimova, T. N.; Kolchina, E. F. J. Fluorine Chem. 1988, 41, 345. (3) Chambers, R. D.; Seabury, M. J.; Williams, D. L. J. Chem. Soc.-Perkin Trans.-I 1988, 255. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 216 (4) (a) Battioni, P.; Brigaud, 0.; Desvaux, H.; Mansuy, D.; Taylor, T. G. Tetrahedron Lett. 1991, 32(25), 2893 (b) Allen, M. D.; Batsanov, A. S.; Brooke, G. M.; Lockett, S. J. J. Fluorine Chem. 2001,108, 57. (c) Elgie, K. J.; Scobie, M.; Boyle, R. W. Tetrahedron Lett. 2000,41,2753. (5) Schmitt, J.; Decher, G.; Dressick, W. J.; Brandow, S. L.; Geer, R. E.; Shashidhar, R.; Calvert, J. Adv. Mater. 1997, 9(1), 61. (6) Metal nanoparticles on high surface area latex supports and Modified semiconductor quantum dots as luminescent labels for in-situ hybridizations', Pathak, S.; Ph. D. Dissertation, USC, Los Angeles, 2001. (7) Synthesis and Functionalization o f polymer nanospheres; Greci, M. T.; Ph. D. Dissertation, USC, Los Angeles, 2000. (8) (a) Green, N. Biochem. J. 1964, 92, 16c. (b) DeLange, R.; Huang, T. J. Biol. Chem. 1971,246, 698. (9) Green, N. 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Core Title Monodisperse polymer nanospheres: Fabrication, chemical modifications and applications

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