PHYSICOCHEMICAL INVESTIGATIONS ON MICROHETEROGENOUS SYSTEMS WITH SPECIAL REFERENCE TO SPECTROSCOPIC STUDIES A Thesis submitted to the University of North Bengal For the Award of Doctor of Philosophy in Chemistry BY MOUMITA CHAKRABORTY GUIDE DR. AMIYA KUMAR PANDA Department of Chemistry University of North Bengal 2013 DECLARATION I declare that the thesis entitled “PHYSICOCHEMICAL INVESTIGATIONS ON MICROHETEROGENOUS SYSTEMS WITH SPECIAL REFERENCE TO SPECTROSCOPIC STUDIES”, has been prepared by me under the guidance of Dr. Amiya Kumar Panda, Associate Professor, Department of Chemistry, University of North Bengal. No part of this thesis has formed the basis for the award of any degree or fellowship previously. Moumita Chakraborty Department of Chemistry University of North Bengal Darjeeling-734013 West Bengal, India CERTIFICATE FROM THE GUIDE I certify that Ms. Moumita Chakraborty has prepared the thesis entitled “PHYSICOCHEMICAL INVESTIGATIONS ON MICROHETEROGENOUS SYSTEMS WITH SPECIAL REFERENCE TO SPECTROSCOPIC STUDIES”, for the award of Ph.D. degree of the University of North Bengal, under my guidance. She has carried out the work at the Department of chemistry, University of North Bengal. Amiya Kumar Panda DDeeddiiccaatteedd ttoo mmyy PPaarreennttss ACKNOWLEDGEMENT The research work, as presented in the dissertation entitled “PHYSICOCHEMICAL INVESTIGATIONS ON MICROHETEROGENOUS SYSTEMS WITH SPECIAL REFERENCE TO SPECTROSCOPIC STUDIES”, has been carried out under the supervision of Dr. Amiya Kumar Panda, Department of Chemistry, University of North Bengal. I am immensely grateful to him for his constant inspiration and guidance during the tenure of my PhD work. I bid my gratitude to the authority of University of North Bengal for providing the facilities and accessibilities related to the research work. Financial supports from the University Grants Commission (UGC), Govt. of India, and Department of Science and Technology, Govt. of India (INSPIRE Fellowship) are gratefully acknowledged. I convey my sincere appreciation to all the faculty and staff members of the Department of Chemistry, University of North Bengal. Valued guidance, advice and suggestions from Prof. Chien-Hsiang Chang, Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan is gratefully acknowledged. I acknowledge them who have directly and indirectly inspired me to make this work happen. My heartiest thanks go to my fellow colleagues Kausik, Banita, Sujoy, Sudarshana, Pritam, Biplab, Prasant and Gourav for their cooperation and constant support. The work would not have materialized without the blessings and support of my parents and encouragement of my sister. Finally, I am ever grateful to my husband Mr. Ratul Chowdhury for his unconditional love, support and patience. ABSTRACT The thesis entitled “PHYSICOCHEMICAL INVESTIGATIONS ON MICROHETEROGENOUS SYSTEMS WITH SPECIAL REFERENCE TO SPECTROSCOPIC STUDIES” describes the spectroscopic investigation of dye / probe / aggregates comprising three specific systems viz., soft, flexible and rigid. The soft system describes the studies on the interaction between an anionic xanthene dye, eosinY with cationic surfactants in aqueous medium. The second category, i.e., the flexible system describes the interaction of eosinY with three different cationic polymers. This was done in order to understand the nature of interaction involved between the dye and macromolecules. The third category is associated with two different rigid systems, viz.,colloidal silica and surfactant stabilized colloidal dispersions of silver bromide in aqueous medium. This set of work has been categorized into two: (a) UV-visible absorption and emission spectral studies in order to identify the characteristics of the excited state H-bonding between colloidal silica and 7- hydroxycoumarin in aqueous medium. (b) The second category surfactant stabilized silver bromide nanoparticles were synthesized and characterized using different techniques. Photophysical behaviour of the anionic xanthene dye, eosin Y (EY) was investigated in solvents of different polarity as well as in the presence of aqueous cationic surfactants. By suitably analyzing the spectral data different solvatochromic parameters were analyzed and subsequently interpreted. Effects of aqueous surfactant solution on the spectral behaviour of dye were compared with that of the solvent induced properties of EY. A red shift, both in the absorption and steady state fluorescence spectra, for EY was observed with decreasing solvent polarity. EY tends to dimerize in aqueous medium specially in the higher concentration range (> 10µM). Extent of EY dimerisation depended on solvent polarity. Cationic surfactants hindered the dimerisation process of EY, as evident from the lower dimerisation constant (KD) values. Dye-surfactant interaction constants were evluated at different temperatures (298 – 318K) and subsequently the thermodynamic parameters of the interaction process, viz., changes in standard free energy (ΔG0), enthalpy (ΔH0) and entropy (ΔS0) were derived from the spectral data. Stokes shifts were calculated and correlated with the polarity of the medium. A red shift in the fluorescence spectra occurred with the increasing surfactant concentration. Fluorescence of EY was initially quenched by the cationic surfactants in their pre-micellar region. Fluorescence quenching was found to be of Stern-Volmer type where the excited state lifetime of EY remained unchanged in different surfactant media. However, upon further addition of surfactant intensity of the red shifted band increased which eventually attained a continuum. Fluorescence anisotropy value of EY increased non linearly in the post micellar region of surfactants. In the second part of the thesis absorption and emission spectral behaviour of eosinY was investigated to perceive the nature and extent of interaction with three different cationic polyelectrolytes, viz., poly[diallyldimethyl ammonium] chloride (PDMDAAC), N,N-dimethyl-N-dodecyl derivative of hydroxyl ethyl cellulose (LM200) and N,N-dimethyl-N-methyl derivative of hydroxyl ethyl cellulose (JR400). Both the electrostatic and hydrophobic forces were associated in the interaction processes. Interaction constant and stoichiometry of the dye- polymer aggregates in their ground states were evaluated by analyzing the absorption spectra at different concentration of the polymers (with a fixed dye concentration). Also, the thermodynamic parameters for the interaction process were evaluated. Extent of dye-polymer interaction followed the sequence: PDMDAAC>JR400> LM200. Excited state interaction phenomena were investigated by steady state fluorescence spectroscopy, anisotropy and excited state lifetime measurements. There was no significant change in the excited state lifetime of EY for all the three added polymers. Stern-Volmer quenching constants, also the excited state interaction constant of the dye-polymer aggregates, were calculated using standard method. Orientation of the dye molecule around the polymer matrix could have been predicted from the anisotropy measurements. Third part of the work describes the absorption and emission spectroscopic investigation, combined with FTIR studies, on the interaction of 7- hydroxycoumarin (7HC) and aqueous nanocolloidal dispersion of silica. Attempts were made to identify the characteristics of excited state H-bond formed between colloidal silica and 7HC. Both the absorption and emission spectra of 7HC depended on the concentration of silica. In the lower concentration range of silica, absorbance of 7HC decreased with increasing silica concentration. On the contrary, in the higher concentration range, there occurred a bathochromic shift in the absorption spectra of 7HC. Fluorescence behaviour followed an opposite trend compared to the absorption spectra. It is proposed that in the lower concentration range, excited state H-bond was formed between 7HC and colloidal silica. In the higher concentration range, decrease in fluorescence intensity was due to the self quenching of adsorbed dye molecules over silica surface following the mechanism of Homo Förster resonance energy transfer (HFRET). Results were correlated with the size and surface charge of colloidal silica as measured by dynamic light scattering and zeta potential studies. Last part of the work describes the synthesis and characterization of colloidal dispersions of silver bromide (AgBr) in aqueous surfactant medium. AgBr nanoparticles were prepared using a surfactant-assisted synthesis approach with hexadecyltrimethylammonium bromide (CTAB). The surfactant acted both as source of bromide ion as well as the stabilizing agent. Upon progressive addition of silver nitrate to aqueous CTAB solution, stable AgBr dispersions were obtained. Formation of surfactant cation (CTA+) stabilized AgBr was confirmed by way of XRD, FTIR and NMR studies. Thermal behaviour of the isolated nanoparticles was investigated by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), where the occurrence of phase transition in the surfactant stabilized nanoparticles was observed. Kinetics of the particle growth was investigated by dynamic light scattering measurements, which predicted the formation of surfactant bilayered structures associated with the nanoparticles of AgBr. Band gap of the nanoparticles was determined by suitably analysing the UV-visible spectral data, which concluded that the particles behaved like insulators. Morphology of the particles, studied by TEM measurements, was found to be spherical. Finally, enthalpy of formation of surfactant stabilized AgBr, determined calorimetrically, was found to be dependent on the concentration of the precursors. PREFACE The present dissertation deals with the physicochemical investigations on microheterogenous systems with special reference to spectroscopic studies. The dissertation begins with introduction and corresponding literature survey. The reported information of different microheterogenous systems in a general way have been reviewed followed by a description of the scope and perspective. Although many reports on the dye-surfactant interactions are available in the literature, however, systematic investigations on the photophysics of an anionic xanthene dye eosinY (EY) in the presence of different cationic surfactants and also in the solvents of different polarity are not common. Therefore, detailed investigations on the spectral behaviour of EY with reference to the above were considered to be important. Studies involving oppositely charged dye-polymer aggregates can shed light in understanding the interaction mechanism and thus explore the yet to be known aspects of different physicochemical decolourisation processes. Several reports are available in literature describing the interaction between silica and a number of dyes. However, studies on the interaction of colloidal silica with 7-hydroxycoumarin are not common. Therefore, a detailed spectral investigation on the absorption and fluorescence spectra of dye-silica aggregates are warranted for better technological applications as well as from the fundamental understanding point of view. Despite manifold applications of AgBr nanoparticles it is difficult to obtain stable colloidal dispersions of AgBr especially in the aqueous medium. A simple one–pot synthesis approach in obtaining stable AgBr nanoparticles in aqueous media in the presence of a cationic surfactant (hexadecyltrimethylammonium bromide) was adopted. Synthesized nanoparticles were characterized by different techniques, viz., XRD, FTIR, NMR, conductometric titration, UV-visible spectroscopy, dynamic light scattering, DSC-TGA, isothermal titration calorimetry and TEM measurements. The obtained results and the related observations have been summarized followed by conclusions. The dissertation then follows the basic data and off-prints of the published papers. TABLE OF CONTENTS Item Page No. INTRODUCTION 1- 42 AIMS AND SCOPES OF THE PRESENT STUDY 43 - 44 CHAPTER 1 45 - 64 Spectral behaviour of Eosin Y in different solvents and aqueous surfactant media. Spectrochim. Acta A 2011, 81, 458-465. CHAPTER 2 65 - 79 Molecular Basis of the Binding of Dye to Polycations: Absorption and Emission Spectral Studies. Communicated to Indian Journal of Chemistry, Section A. CHAPTER 3 80 - 92 Effect of Colloidal Silica on the Spectral Behaviour of 7-Hyroxycoumarin in Aqueous Medium. Spectrochim. Acta A 2012, 97, 722-727. CHAPTER 4 93- 115 Surfactant-assisted Synthesis and Characterization of Stable Silver Bromide Nanoparticles in Aqueous Media. Langmuir 2012, 28, 7282-7290 SUMMARY AND CONCLUSION 116 – 119 REFERENCES 120 - 136 APPENDIX (BASIC DATA) 137 - 157  BASIC DATA OF CHAPTER 1 137 - 140  BASIC DATA OF CHAPTER 2 141 - 146  BASIC DATA OF CHAPTER 3 147 - 151  BASIC DATA OF CHAPTER 4 152 - 157 REPRINTS LIST OF TABLES Item Page No. INTRODUCTION Table 1. 9 Classification of coumarin dyes with features and examples. CHAPTER 1 Table 1 53 Spectral parameters of EY in presence of different solvents and surfactants at 298K. Table 2 56 Thermodynamic parameters for the interaction of EY with different surfactants. Table 3 60 Fluorescence data of aqueous EY solution in the presence of different cationic surfactants at 298K. CHAPTER 2 Table 1 75 Thermodynamic parameters for the interaction of EY with different polymers. Table 2 77 Excited state interaction constant of 10 M EY-polymer aggregates at 298K. CHAPTER 3 Table 1 85 Hydrodynamic diameter and zeta potential data for thermally treated colloidal silica nanoparticles, Ludox®, in the absence and presence of 10 M 7HC at 2λ8 K. CHAPTER 4 Table 1 102 1HNMR data of CTAB and CTA+ coated AgBr nanoparticles. LIST OF FIGURES AND SCHEMES Item Page No. INTRODUCTION Scheme 1. 3 Schematic representation of xanthene dye. Scheme 2. 3 General formula of pyronin subgroup of fluorene dye. Scheme 3. 3 General formula of rhodamine subgroup of fluorene dye. Scheme 4. 4 General formula of fluorone dyes. Figure 1. 4 Deconvoluted absorption spectra of 10.0 mol dm−3 PIN in water obtained by fitting the absorbance data with Gaussian multi-peaks function. Three overlapping spectral components were found at 600 nm (for monomer), at 552 nm (for dimer) and at 515 nm (for higher aggregates of PIN). Scheme 5. 5 H-aggregates of merocyanine dye in acetonitrile solvent depending on the type of counterions. Scheme 6. 6 Merocyanine dye molecules are connected by intermolecular hydrogen bonds (a) and then stacked side-by-side by electrostatic force (b), giving rise to formation of ribbon-shaped J-aggregates. Figure 2. 7 Linear absorption (left) and single-photon fluorescence spectra (right) of trans-4- [p-(N,N-hydroxyethyl) aminino-styryl]-N-methylpyridinium iodide in different solvents at d0 = 1 × 10−5 mol/l. Scheme 7. 7 Structure of eosinY. Figure 3. 8 Absorption (A) and steady state emission spectra (B) of eosin Y in ([bmim][MS] ± water)/(Tween 20 + n-pentanol)/n-heptane polar domain-in-oil microemulsion system along with the spectra in pure water and IL + water mixture. Scheme 8. 10 Schematic representation for the arrangement of surfactant molecules at the air– water interface. Scheme 9. 11 Schematic representations of normal micelle of (A) double-tailed and (B)single- tailed surfactant in aqueous solution. Scheme 10. 13 Schematic representation of different types of surfactants: (a) single-tailed (b) double- tailed (c) and (d) gemini and (e) bolaform. Figure 4. 14 Effect of CTAB on the absorption spectrum of Alizarin Yellow R in aqueous solution at 25°C; I without surfactant; (1) 0.7 mM; (2) 0.8 mM; (3) 0.9 mM, (4) 1.0 mM, (5) 2.0 mM, (6) 3.0 mM, (7) 4.0 mM, and (8) 8.0 mM. Figure 5. 14 Visible absorption spectra 10 mol dm−3 of PIN in the presence of varying concentration of NaDC in aqueous medium, at 298 K. Concentration of NaDC (mmol dm−3): 1, 0; 2, 1; 3, 2; 4, 3; 5, 5; 6, 8 and 7, 10. Figure 6. 15 Determination of CMC by absorption spectroscopic method. Figure 7. 16 Determination of CMC by fluorescence spectroscopic method. Figure 8. 16 The visible absorption spectra of erythrosin B in water (1), in aqueous solution of anionic surfactant SDS (2), cationic surfactant CTAB (3) and neutral surfactant TX-100 (4) at 298K. Scheme 11. 19 Schematic representation of (A) poly(diallyldimethyl ammonium) chloride and (B) N,N-dimethyl-N-dodecyl derivative of hydroxyl ethyl cellulose(LM200) / N,N-dimethyl-N-methyl derivative of hydroxyl ethyl cellulose(JR400)[R = -CH3 : JR400, R = -C12H25 : LM200]. Figure 9. 20 (A) Absorption spectrum of Azure B–NaAlg system at various P/D ratios1 and (B) Emission spectra of 10− 5 mol dm− 3 acridine orange at 303 K in presence of varying amounts of Klebsiella K18 capsular polysaccharide. Figure 10. 22 Emission spectra of (a) PSF, and (b) ST, in the presence of different ctDNA concentrations. ( exc = 520 nm). Curves correspond to (a) 0, 6, 9, 13, 21, 37, 47, 71, and λ2 M of DNA; (b) 0, 4.6, 8.5, 12, 20, 36.5, 46, 60.5, 75, λ0, 102.5, and 114 M of DNArespectively. Scheme 12. 25 Schematic representation of a silica nanoparticle-based biosensor fabricated via (a) the entrapment technique and (b) covalent conjugation. Scheme 13. 26 Various preparation methods for colloidal silica: (a) Ion exchange from sodium silicate; (b) Hydrolysis and condensation from TEOS; (c) Milling or dispersion fromfumed silica; (d) Direct oxidation from silicon. Scheme 14. 27 Schematic representation of preparation of colloidal silica from TEOS. Scheme 15. 30 Schematic of the preparation of Janus nanoparticles based on the Langmuir- Blodgett technique. Scheme 16. 32 Proposed mechanism for the formation of metal particles by the microemulsion approach. Scheme 17. 33 Schematic representation of surfactant-capped nanoparticles in micellar medium. Figure 11. 36 XRD pattern of the textiles coated with silver particles. Figure 12. 36 FTIR spectra for the pure and modified silver nanoparticles. Figure 13. 37 1H NMR spectra of: (a) chitosan and (b) Cholesterol-modified chitosan conjugate with succinyl linkages. Figure 14. 37 Conductometric titration of oxidative-acid-treated diamonds (nominal size ∼ 100 nm). In this titration, an excessive amount of NaOH was first added into the nanodiamond suspension and then neutralized with 0.1 N HCl. The first region in the plot corresponds to neutralization of solution OH− groups and the second region corresponds to neutralization of surface –COO− group. Figure 15. 38 UV–vis diffuse reflectance spectra of (a) 0.02AgCl/Ag/MCM-41, (b) 0.1AgCl/Ag/MCM-41 and (c) 0.2AgCl/Ag/MCM-41. Figure 16. 38 DLS size distribution graph of AuNPs synthesized using chitosan as reductant. Figure 17. 39 (a) TGA/DSC curves for as-synthesized Mn oxide precipitated in ethanol (heating rate = 10 °C/min under a stream of air). Figure 18. 40 Plot of ΔHf vs ω-1 for the formation of Cu2[Fe(CN)6] in H2O/AOT/heptane w/o microemulsion at 303 K. Figure 19. 41 TEM images of silver nanoparticles synthesized using different molar concentration of tannic acid (a) 2.9 µM of tannic acid, average particle size 19.5 ± 3 nm, (b) 5.9 µM of tannic acid, average particle size 25.4 ± 2.8 nm, (c) 23.5 µM of tannic acid, average particle size 29.7 ± 4.3 nm. Figure 20. 41 (a) and (b) CTFs and diffractograms obtained based on two different TEM systems. CHAPTER 1 Figure 1. 49 (A) Absorption spectra of EY in water at different concentrations (in M, mentioned inside the figure). (B) Resolved absorption spectra of 10 M EY in H2O (purple and blue), the red and black lines represent the reconstituted and original spectra of EY respectively. (C) Absorption spectra of 10 M EY in different solvents (as mentioned in the figure). Temperature 298K. Figure 2. 52 Kosower Z vs. ET(30) profile for 10 M EY in (A) solvents of different polarity and (B) surfactants at 298K. Solvents and surfactants are mentioned inside the figure. Figure 3. 54 Absorption spectra of 10 M EY in water in the presence of different concentrations of surfactants at 298K. Surfactants and their concentrations (in M) are mentioned in the figure. Figure 4. 58 (A) Emission spectra of EY in water at different concentrations (in M, as mentioned in the figure). (B) Emission spectra of 10 M EY in different solvents (mentioned inside the figure). Temperatureμ 2λ8K; ex = 500nm. Figure 5. 59 Fluorescence spectra of 10 M EY in the presence of different concentrations of (A), CTAB; (B), CPC and (C), DDAB at 298K. Surfactant concentrations (in mM) have been mentioned in the figure. Figure 6. 60 Stern-Volmer Plot for the fluorescence quenching of 10 M EY in water by (O), CTAB and (Δ), DDAB at 2λ8K. ex = 500nm. Figure 7. 61 Fluorescence decay curves of 10 M EY in water and different surfactants (mentioned in the figure). ex = 500nm. Figure 8. 62 Variation of fluorescence anisotropy (r) of EY with the concentration of surfactant [surfactant] in water at 2λ8K. A 10 M EY in water was used. ex=500nm and em=540nm. Inset: anisotropy – concentration profile of EY in glycerol-water mixture is shown for comparison. Surfactants are mentioned inside the figure. CHAPTER 2 Scheme1. 68 Structures of A, eosinY; B, PDMDAAC and C, JR400 / LM200 (R = -CH3 : JR400, R = -C12H25 : LM200). Figure 1. 70 Absorption spectra of 10 M EY in presence of polymers at different polymer / dye ([P] / [D]) molar ratio. (A) PDMDAAC, (B) LM 200 and (C) JR 400. [P] / [D] values: (A) 1, 0; 2, 0.2; 3, 0.4; 4, 0.7 and 5, 0.9. For (B) and (C) 1, 0; 2, 0.4; 3, 1.1; 4, 1.4 and 5, 1.8. Temperature: 298K. Scheme 2. 71 Proposed model for the stacking of dye at the positive charge centres of polymer. Figure 2. 71 Absorption spectra of 10 M EY in presence of polymers at different polymer / dye ([P] / [D]) molar ratio in the higher range. (A) PDMDAAC (B) LM 200 (C) JR 400. [P] / [D] values are mentioned in the figure. Temperature: 298K. Figure 3. 73 Plot of CD / (A0-A) vs. 1 / CP according to Benesi-Hildebrand formalism to determine the interaction constant of EY (10 M) - polymer aggregates. Polymers: (A) PDMDAAC, (B) LM 200, and (C) JR 400. Temp (in K): □, 2λ8; ο, 303; Δ, 308; , 313 and ●, 318. Figure 4. 76 Fluorescence spectra of 10 M EY in presence of polymers at different polymer / dye ([P] / [D]) molar ratio. A, PDMDAAC; B, LM 200 and C, JR 400. [P] / [D] values: (A) 1, 0; 2, 0.2; 3, 0.4; 4, 0.7 and 5, 0.9 (B) and (C) 1, 0; 2, 0.4; 3, 1.1; 4, 1.4 and 5, 1.8. Temperatureμ 2λ8K. ex = 500nm. Figure 5. 77 Fluorescence spectra of 10 M EY in presence of polymers at different polymer / dye ([P] / [D]) molar ratio in the higher range. (A) PDMDAAC (B) LM 200 (C) JR 400. [P] / [D] values are mentioned in the figure. Temperatureμ 2λ8K. ex = 500nm. Figure 6. 78 Variation in the fluorescence anisotropy (r) of 10 M EY with polymer concentration ([P]) in aqueous medium at 298K. Polymers: □, PDMDAAC; Δ, JR 400 and ο, LM 200. ex=500nm and em=535nm. CHAPTER 3 Figure 1. 86 (A) Absorption and (B) emission spectra of 7HC in water at different concentrations at 2λ8K. Concentration of 7HC/ Mμ 1,2; 2,4; 3,6; 4,10; 5,12; 6,14; 7,16 and 8,20μ ex = 375 nm. Figure 2. 87 Absorption spectra 10µM 7HC in the presence of varying amount of Ludox® at 298 K. Ludox® was used in the two different concentration (wt.%) ranges. A: 1, 0; 2, 0.004; 3, 0.008; 4, 0.012; 5, 0.016 and 6, 0.02. In panel B spectra of 7HC is shown in the presence of 7, 0.0; 8, 2; and 9, 3 wt.% of Ludox®. Note the differences in the units of absorbance axis in two panels. Figure 3. 88 Emission spectra of 2 wt.% Ludox® in water. Temperatureμ 2λ8K; ex = 375 nm. Figure 4. 88 Emission spectra of 10µM 7HC in the presence of (A) lower wt.% (1, 0; 2, 0.004; 3, 0.008; 4, 0.012; 5, 0.016 and 6, 0.02) and (B) higher wt.% (2, 3, 4, 5, 6, 7, 8 and 9 wt.%) of Ludox® at 298K. Note: in the lower concentration range of Ludox®, fluorescence of 7HC increased (Panel A) while the fluorescence of 7HC was quenched in the presence of higher amount of Ludox® (Panel B). Scheme1. 89 H-bond formation between the imperfect silica and 7HC. Figure 5. 91 (a) IR spectra of 7-HC (1.0M) and H2O (1.0M) in acetonitrile, (b) IR spectra of SiO2 (1.0M) and H2O (1.0M) in acetonitrile, (c) IR spectra of 7-HC (1.0M), H2O (1.0M) and SiO2 (1.0M) in acetonitrile and Gaussian components from least squares fitting. CHAPTER 4 Figure 1. 99 XRD patterns of (A) CTAB, (B) silver nitrate, (C) CTA+ coated silver bromide nanoparticles isolated from water and (D) CTA+ coated silver bromide nanoparticles isolated using chloroform. Figure 2: 100 Expanded X-ray diffractograms of (MA) aqueous and (MB) chloroform extract of CTAB stabilized AgBr nanoparticles. Figure 3. 101 FTIR spectra of (A) pure CTAB and (B) aqueous extract of CTAB stabilized AgBr nanoparticles. Figure 4: 102 - 103 NMR spectra of (A) pure CTAB, (B) aqueous and (C) chloroform extract of CTAB stabilized AgBr nanoparticles. Figure 5: 104 Conductance profile of (Δ) CTAB and (ο) KBr against concentration of AgNO3 (in µM). Initial concentrations of both CTAB and KBr = 0.4mM. Figure 6. 105 UV-visible absorption spectra of CTA+ coated AgBr nanoparticles in aqueous medium at 25ºC. Concentrations of AgBr [in M]μ (1) 20, (2) 30 and (3) 50. A 0.4mM CTAB solution was used as blank. Figure 7. 106 Plot of (εhυ)2 vs. hυ for the determination of the band gap of silver bromide nanoparticles. Concentrations of AgBr (µM): O, 20;, 30; □, 40 and Δ, 50. Insetμ Band gap - silver bromide concentration profile. Figure 8. 107 dh vs. time profiles for silver bromide nanoparticles. Inset: dh vs. [AgBr] profile after52 hours of the particle formation. Scheme 1. 108 Schematic diagrams for (A) surfactant monolayer protected AgBr NPs and (B) AgBr NPs stabilized by CTA+ bilayer. C Figure 9. 111 (A) TGA data of pure CTAB (ο), CTA+ coated AgBr nanoparticles isolated from water (Δ) and chloroform (□). (B) DSC curves of pure CTAB (a), CTA+ coated AgBr nanoparticles isolated from water (b) and chloroform (c). Upper curves indicate heating while the lower curves were obtained during cooling. Figure 10. 112 Plot of ∆Hf vs. [AgNO3] for the formation of AgBr in presence of 0.4mM CTAB solution. Figure 11. 114 TEM images of different concentrations: A=10µM, B=20 µM, C=30 µM, D=40 µM and E=50 µM of silver bromide nanoparticles. Scale bars: 100 nm. IINNTTRROODDUUCCTTIIOONN 1 INTRODUCTION 1. General overview of dye A dye is a coloured substance with an affinity to bind on to a substrate on which it is applied. An aqueous medium is usually preferred as dispersion medium for dye formulation and the dyes require mordant to improve the fastness of their adsorption / binding onto the substrate of interest. Dyes can be defined as coloured ionising and aromatic organic compounds with affinity towards the substrate to which they are applied. Dyes are applied to numerous substrates, e.g., textile, leather, plastic, paper, etc. One of the main characteristics of dye is that it must get completely or at least partially soluble in which it is being put to. There are different ways of classification for dye molecules. Some of them are mentioned below: i. Organic / inorganic. ii. Natural / synthetic. iii. By area and method of application. iv. Chemical classification: based on the nature of their respective chromophores. v. By nature of the electronic excitation (i.e., energy transfer contents, absorption colorants and fluorescent colorants). vi. According to the method of dyeing. By the nature of their chromophore, dyes can be chemically classified into different categories: i. Acridine dyes and its derivates ii. Anthraquinone dyes and their derivates iii. Arylmethane dyes iv. Azo dyes: based on -N=N- azo structure v. Diazonium dyes: based on diazonium salts vi. Nitro dyes: based on a -NO2 (nitro) functional group vii. Nitroso dyes: based on a -N=O (nitroso) functional group viii. Phthalocyanine dyes and derivatives of phthalocyanine http://en.wikipedia.org/wiki/Color http://en.wikipedia.org/wiki/Chemical_affinity http://en.wiktionary.org/wiki/substrate http://en.wikipedia.org/wiki/Mordant http://en.wikipedia.org/wiki/Chromophore http://en.wikipedia.org/wiki/Category:Acridine_dyes http://en.wikipedia.org/wiki/Category:Anthraquinone_dyes http://en.wikipedia.org/wiki/Category:Azo_dyes http://en.wikipedia.org/wiki/Azo_compound http://en.wikipedia.org/wiki/Diazonium http://en.wikipedia.org/wiki/Nitro_functional_group http://en.wikipedia.org/wiki/Nitroso http://en.wikipedia.org/wiki/Phthalocyanine 2 ix. Quinone-imine dyes and derivatives of quinone x. Thiazole dyes and derivatives of thiazole xi. Xanthene dyes, derived from xanthene Dyes have become one of the indispensable ingredients for a variety of applications. From acting as colorants for plastics, textile dyeing industries and the highly sophisticated biotechnology industry, dyes are touching our life everywhere. Dyes are also used by industries for inks and tinting 2. Other industries where dyes are used in a variety of products include paper and pulp3, adhesives, art supplies, beverages4, construction, cosmetics, food5, glass, ceramics, paints6, polymers, soap, wax and biomedicine7, etc. Dyes are also used as probes to understand the microheterogeneity of compartmentalized systems, viz., micelles8, reverse micelles9, microemulsion10, polymers11, nanoparticles12, etc. Dye aggregates are useful in developing light-harvesting arrays for artificial photosynthetic systems. Use of such dye aggregates as light-harvesting antennas as well as photosensitizers in photoelectrochemical cells are available in literature13,14. Basic understanding of dye aggregation over nanoparticle surface as well as the excited-state interaction with the semiconductor support is important in developing efficient photoelectrochemical solar cells15. Xanthene dyes are those containing xanthylium as the chromophore with amino or hydroxyl groups meta to the oxygen as the usual auxochromes. Xanthene dyes are most commonly used synthetic dyes. Xanthene derivatives are used as sensitizers for organic photochemical reactions and in photochemical cells 16. They are important because of their brilliant hues and shades. They are generally very strong, with much higher oscillator strengths 17. As a consequence of their rigid chromophoric nucleus, xanthenes are often fluorescent. They are used for the direct dyeing of wool and silk and mordant dyeing of cotton. Paper, leather, woods, food, drugs and cosmetics are dyed with xanthene dyes 18. Xanthene is the parent heterocycle of fluorescein and its derivatives. http://en.wikipedia.org/wiki/Quinone http://en.wikipedia.org/wiki/Category:Thiazole_dyes http://en.wikipedia.org/wiki/Thiazole http://en.wikipedia.org/wiki/Xanthene 3 Scheme 1. Schematic representation of xanthene dye. Xanthene dyes are divided into three subgroups: fluorenes, fluorones and rhodols. Fluorenes and fluorones contain dyes of importance in histotechnology, the rhodols do not. i. Fluorene dyes: The pyronin subgroup of fluorenes has the general formula shown below. The dyes pyroninY and pyronin B belong to this category of dyes. Scheme 2. General formula of pyronin subgroup of fluorene dye. The rhodamine subgroup of fluorenes (e.g., rhodamine B) has the following general formula: Scheme 3. General formula of rhodamine subgroup of fluorene dye. ii. Fluorone dyes: Fluorones have the general formula as shown below. The fluorones are sometimes referred to as the eosins, and include many dyes used as counterstains to alum hematoxylin. Their general formula shown above is actually that of fluorescein, from which they are derived. http://stainsfile.info/StainsFile/dyes/45005.htm http://stainsfile.info/StainsFile/dyes/45010.htm http://stainsfile.info/StainsFile/dyes/45170.htm http://stainsfile.info/StainsFile/dyes/eosins.htm http://stainsfile.info/StainsFile/dyes/45350.htm 4 Scheme 4. General formula of fluorone dyes. 1.1. Aggregation of xanthene dye Aggregation is one of the features of dyes in solution, affecting their colouristic and photophysical properties and is therefore of special interest. Dye molecules possess strong intermolecular van der Waals like attractive forces between them, as a result they generally undergo the process of self-association in solution or at the solid-liquid interface, which is a frequently encountered phenomenon in dye chemistry. The aggregates in solution exhibit distinct changes in the absorption band compared to that of the monomeric species. By suitably analyzing the spectral data formation of different types of aggregates has been proposed. The bathochromically shifted bands are known as J-bands after the name of E.E. Jelley 19. On the other hand, hypsochromically shifted bands are commonly known as H-bands20. Aggregation behaviour of the cyanine dyes have extensively been studied as they are the best known self-aggregating systems. It is Figure 1. Deconvoluted absorption spectra of 10.0 mol dm−3 PIN in water obtained by fitting the absorbance data with Gaussian multi-peaks function. Three overlapping spectral components were found at 600 nm (for monomer), at 552 nm (for dimer) and at 515 nm (for higher aggregates of PIN) 22. 5 well known that the amphipathic dyes tend to aggregate even in the dilute regime, leading to the formation of dimer, and sometimes even higher aggregates21-23. Dimers as the simplest aggregates are the subject of many studies concerned with the thermodynamics of monomer–dimer equilibrium and photo-physical properties, and therefore being of special interest21,23-26. Xanthene dyes are also known to aggregate in aqueous medium when present even in lower concentration (as low as 10µM). Aggregation of these dyes has fundamental consequences in photographic technology, tunable lasers, fluorescence depolarization diagnostics devices and photomedicine23. Hence, studies on the aggregation behaviour of xanthene dyes are considered to be significant from the application as well as the fundamental understanding point of view. Geometry of the aggregates can restrict the transition of dye molecules from ground state to excited state. The aggregates in which monomers are stacked in parallel are known as H-aggregates. In this case the transition to the topmost level is allowed. Scheme 5. H-aggregates of merocyanine dye in acetonitrile solvent depending on the type of counterions, adapted from Kolev et al. 27 The aggregates in which the monomers are arranged in a head-to-tail manner are known as J-aggregates. In this case the only allowed transition is to the lowest split level. In case of aggregates having intermediate geometry, both these transitions are partially allowed and band splitting is observed26. 6 Scheme 6. Merocyanine dye molecules are connected by intermolecular hydrogen bonds (a) and then stacked side-by-side by electrostatic force (b), giving rise to formation of ribbon-shaped J- aggregates 28. There is always an equilibrium between dimer and monomer. Curve- fitting techniques can be employed to obtain the dimerization constant value of the xanthene dye and to decompose and analyze the absorption spectrum in terms of component bands21. At low concentrations, the dye is primarily in the form of monomers and dimers. If we neglect the effect of higher aggregates, the equilibrium between monomer and dimer (2M D) is described by the dimerization constant KD, which is given by the ratio between the molar concentrations of dimers, CD, and monomers, CM, at equilibrium: KD = CD/CM 2 (1) Thus, one needs to known values of CD and CM, which, in turn, can be determined from the molar absorptivity obtained from the spectral bands of monomeric and dimeric species. The total absorbance of a dye solution per unity of optical length at a given wavelength (A( )) is: A( ) = M( ).CM + D( ).CD (2) where, M and D represent the molar absorption coefficients of monomeric and dimeric species, respectively, of any band at a wavelength . The monomer and dimer concentrations can be calculated from eq 1 considering the mass balance of dye in the volume dispersion C = CM + 2 CD (3) where, C is the total analytical concentration of dye. Insertion of eq 1 and 3 into eq 2 affords the following expression: A ( ) = D ( ) (� − − ±√ +8. ��.�8.�� ) + M ( ) (− ±√ +8. ��.�4.�� ) (4) 7 By plotting the measured absorbances as a function of dye concentration at any wavelength, the molar absorptivity of monomers, M ( ), and dimers, D ( ), as well as the dimerization constant, KD, can be calculated using a nonlinear least-squares fitting routine29. Solvent effects are important in determining the photophysical properties of dye molecules in solution. Figure 2. Linear absorption (left) and single-photon fluorescence spectra (right) of trans-4-[p- (N,N-hydroxyethyl) aminino-styryl] -N-methylpyridinium iodide in different solvents at d0 = 1 × 10−5 mol/l30. Aggregation of xanthene dyes is promoted by polar protic solvents while it is impeded by polar aprotic solvents. The hydrogen bonding of solvents also play important role in inducing dye aggregation. Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It is hydroxyl xanthene and can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic. Scheme 7. Structure of eosinY. http://en.wikipedia.org/wiki/Fluorescent http://en.wikipedia.org/wiki/Dye http://en.wikipedia.org/wiki/Bromine http://en.wikipedia.org/wiki/Fluorescein http://en.wikipedia.org/wiki/Cytoplasm http://en.wikipedia.org/wiki/Collagen http://en.wikipedia.org/wiki/Muscle#Muscular_Composition http://en.wikipedia.org/wiki/Microscope http://en.wikipedia.org/wiki/Eosinophilic 8 There are actually two very closely related compounds commonly referred to as eosin. Most often used is eosin Y. The other eosin compound is eosin B . Eosin Y is a tetrabromo derivative of fluorescein. The spectral behaviour of EY is affected by the solvent polarity, pH of the aqueous medium, surfactant, polymers, colloidal particles, etc. Figure 3. Absorption (A) and steady state emission spectra (B) of eosin Y in ([bmim][MS] ± water)/(Tween 20 + n-pentanol)/n-heptane polar domain-in-oil microemulsion system along with the spectra in pure water and IL + water mixture. Spectra in pure water are shown through the black lines while the green lines correspond to the spectra in IL+ water in µE comprising 50mol% IL and ϕd = 0.057 The red lines represent the spectra of eosinY in IL+ water mixture31. 1.2. Brief study on coumarin dyes Coumarin is a naturally occurring dye, found naturally in many plants. Coumarins owe their class name to ‘Coumarou’, the vernacular name of the tonka bean (Dipteryx odorata Willd., Fabaceae), from which coumarin itself was isolated back in 182032. Coumarin is classified as a member of the benzopyrone family of compounds, all of which consist of a benzene ring joined to a pyrone ring33. There are four main coumarin sub-types: the simple coumarins, furanocoumarins, pyranocoumarins and the pyrone-substituted coumarins. The simple coumarins (e.g. coumarin, 7-hydroxycoumarin and 6,7- dihydroxycoumarin), are the hydroxylated, alkoxylated and alkylated derivatives of the parent compound, coumarin along with their glycosides. As a group, coumarins exhibit interesting fluorescence properties, which include a high degree of sensitivity to their local environment, including polarity and viscosity. This sensitivity has led to their widespread application as sensitive fluorescent probes of a wide range of systems, including homogeneous solvents and mixtures, and heterogeneous materials 34. Coumarins substituted at position 7 with an electron- donating group are known to http://en.wikipedia.org/wiki/Eosin_Y http://en.wikipedia.org/wiki/Eosin_B http://en.wikipedia.org/wiki/Eosin_Y http://en.wikipedia.org/wiki/Eosin_Y http://en.wikipedia.org/wiki/Fluorescein 9 Table 1. Classification of coumarin dyes with features and examples. Classification Features Example SIMPLE COUMARINS Hydroxylated, alkoxylated or alkylated on benzene ring. 7-Hydroxycoumarin FURANOCOUMARINS 5-membered furan ring attached to benzene ring. Linear or Angular Psoralen PYRANOCOUMARINS 6-membered pyran ring attached to benzene ring. Linear or Angular Xanthyletin PYRONE- SUBSTITUTED COUMARINS Substitution on pyrone ring, often at 3-C or 4-C positions Warfarin exhibit strong fluorescence 35. The use of coumarin dimers as photocontrolled molecules opening and closing a silica pore, wherefrom guest molecules are released or in photodegradable polymers has been reported in literature 36. Coumarin is used in the pharmaceutical industry as a precursor molecule in the synthesis of a number of synthetic anticoagulant pharmaceuticals similar to dicoumarol, the notable ones being warfarin and some even more potent rodenticides that work by the same anticoagulant mechanism. 7-hydroxy coumarin is a widespread natural product of the coumarin family37. It is also known as ‘Umbelliferone’. Umbelliferone is the parent compound for a large number of natural products. Herniarin (7-O-methylumbelliferone or 7- methoxycoumarin) occurs in the leaves of water hemp (Eupatorium ayapana) and rupturewort. O-Glycosylated derivatives such as skimmin (7-O- -D- glucopyranosylumbelliferone) occur naturally and are used for the fluorimetric determination of glycoside hydrolase enzymes38. There are great similarities between hydroxy derivatives and their ethers. Thus, 7-hydroxy-, 7- methoxy-, and 7-glucoxycoumarin give a family of nearly identical curves39. The ultraviolet activity of umbelliferone led to its use as a sunscreen agent, and http://en.wikipedia.org/wiki/Herniarin http://en.wikipedia.org/wiki/Water_hemp http://en.wikipedia.org/wiki/Eupatorium_ayapana http://en.wikipedia.org/wiki/Rupturewort http://en.wikipedia.org/wiki/Skimmin http://en.wikipedia.org/wiki/Fluorimetry http://en.wikipedia.org/wiki/Sunscreen 10 an optical brightener for textiles 40. It has also been used as a gain medium for dye lasers 41. Umbelliferone can be used as a fluorescence indicator for metal ions 42. In the majority of human subjects studied, coumarin is extensively metabolised to 7-hydroxycoumarin. The measurement of urinary 7-hydroxycoumarin following an oral dose of coumarin has been employed as a biomarker of human hepatic CYP2A6, the cytochrome P-450 (CYP) isoform which is responsible for coumarin 7 hydroxylation in human liver 43. 2. Surfactant and its classification The word ‘SURFACTANT’ is a diminutive form of the phrase “SURface ACTive Agent. Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups and hydrophilic groups. Therefore, a surfactant contains both a water insoluble component and a water soluble component. Surfactants diffuse in water and adsorb at interfaces between air and water or at the interface between oil and water, in the case where water is mixed with oil. They not only tend to accumulate on the surfaces,but they also alter the properties of the surfaces, being Scheme 8. Schematic representation for the arrangement of surfactant molecules at the air–water interface. active at the interfaces. In addition, because of their amphipathic structures, they can congregate to form a stabilized entity, called micelle, after the attainment of critical micelle concentration (CMC). http://en.wikipedia.org/wiki/Optical_brightener http://en.wikipedia.org/wiki/Textiles http://en.wikipedia.org/wiki/Gain_medium http://en.wikipedia.org/wiki/Dye_laser http://en.wikipedia.org/wiki/Dye_laser http://en.wikipedia.org/wiki/Fluorescence http://en.wikipedia.org/wiki/Organic_compound http://en.wikipedia.org/wiki/Amphiphilic http://en.wikipedia.org/wiki/Hydrophobic http://en.wikipedia.org/wiki/Hydrophilic http://en.wikipedia.org/wiki/Adsorption 11 Scheme 9. Schematic representations of normal micelle of (A) double-tailed and (B)single-tailed surfactant in aqueous solution. Based on the origin, surfactants are classified as follows: i. Soap. Soaps are naturally occurring substances, usually water-soluble sodium or potassium salts of fatty acids. Soaps are made from fats and oils, or their fatty acids, by treating them chemically with a strong alkali 44,45. They are less soluble in hard water. Examples include sodium palmitate, sodium oleate, sodium cholate, etc. ii. Detergent. Detergents refer to the synthetically prepared surfactants 46. Alkylbenzenesulfonates, a family of compounds that are similar to soap but are more soluble in hard water, because the polar sulfonate (of detergents) is less likely than the polar carboxyl (of soap) to bind to calcium and other ions found in hard water. Most of the detergents are synthetic. Examples of detergent include sodium dodecylbenzenesulphonate, sodium dodecylsulfate, cetyltrimethylammonium bromide, cetylpyridinium chloride, polyoxyethylene sorbitan monolaurate (Tween 20, Polysorbate 20), etc. Depending on the chemical constituents attached to the hydrophobic moiety, surfactants are classified as: i. Anionic: The hydrophilic portion of the molecule bears a negative charge. Alkylbenzene sulfonates (detergents), sodium or potassium salt of fatty acid (soaps), lauryl sulfate (foaming agent), di-alkyl sulfosuccinate (wetting agent), lignosulfonates (dispersants), etc., belong to this category. OSO3 Na A B 12 ii. Cationic: In this category the surface active portion of the molecule bears a positive charge. Long chain alkyltrimethylammonium bromide, cetylpyridinium chloride, double tailed alkyldimethylammonium bromide etc., are some of the examples of cationic surfactants. iii. Zwitterionic: If a surfactant contains a head with two oppositely charged groups, it is termed as zwitterionic or amphoteric. 1,2-diacyl-sn-glycero-3- hosphatidylcholine(lecithin), 3(ethyldimethylammonio)propane-1-sulfonate (NDSB-195) are some of the examples of this type of surfactant. iv. Nonionic: Nonionic surfactants do not have ionisable groups. Examples include polyoxyethylene sorbitan monolaurate (Tween 20), t- octylphenoxypolyethoxyethanol (Triton X100), polyethylene glycol lauryl ether (Brij35), etc. The typical surfactant molecules are composed of a single or double tail connected to a single head group; there are other types of surfactants, which have been developed 47. 13 Scheme 10. Schematic representation of different types of surfactants: (a) single-tailed (b) double- tailed (c) and (d) gemini and (e) bolaform. 2.1. Dye-surfactant interaction Although many studies have been undertaken on the interaction of oppositely charged dyes and surfactants, this area is still important and interesting from the theoretical, ecological and technological point of view. The enhanced energy transfer between dyes in dye-surfactant systems made them good model membrane systems of chloroplast 48,49. The deformation of the absorption and emission spectra of ionic dyes in the presence of oppositely charged ionic surfactants in aqueous solution proposes the formation of higher aggregates in the surfactant micelle 50-52. When surfactant interacts with dye, there occurs spectral shift in the bands of the dye with variations in the intensity. The effect becomes significant for oppositely charged dye-surfactant systems. Depending on the nature of aggregates a red or blue shift may occur in the absorption maxima of dye. Red shift in the absorption band of surfactant-dye aggregates with respect to the absorption band of the dye in aqueous solution is attributed to head-to-tail aggregation, i.e., formation of J-aggregates, whereas blue shift occurs due to parallel aggregation, i.e., formation of H-aggregates53. 14 Figure 4. Effect of CTAB on the absorption spectrum of Alizarin Yellow R in aqueous solution at 25°C; I without surfactant; (1) 0.7 mM; (2) 0.8 mM; (3) 0.9 mM, (4) 1.0 mM, (5) 2.0 mM,(6) 3.0 mM, (7) 4.0 mM, and (8) 8.0 mM 53. Figure 5. Visible absorption spectra 10 μmol dm−3 of PIN in the presence of varying concentration of NaDC in aqueous medium, at 298 K. Concentration of NaDC (mmol dm−3): 1, 0; 2, 1; 3, 2; 4, 3; 5, 5; 6, 8 and 7, 10 22. Also, the investigations on the behaviour of different dyes in aqueous surfactant solution can provide useful information about the mechanisms according to which surfactants operate as levelling agents and provide information on the thermodynamics and kinetics of dyeing process. This may directly affect the quality of dyeing, which is one of the goals of textile finishing. Surfactants are also used as solubilizers for water insoluble dyes, to break down dye aggregates in order to accelerate adsorption processes on fiber, as auxiliaries for improving dye adsorption and as dispersing agents54-56. The interactions of binary dye-surfactant systems find applications in other scientific fields, including analytical chemistry, photography, luminescence, and lasers, etc. Amphiphilic dyes, for which the concepts of color and surface-active properties coexist within the same molecular framework, are of particular interest 57. Besides, dye- surfactant interaction studies can effectively determine the critical micelle concentration of surfactants58,59. Determination of CMC by spectroscopic probing technique: CMC is one of the most important solution properties exhibited by surfactant molecules. At a fixed environmental condition, most of the physicochemical properties of a surfactant 15 solution show remarkable changes at the onset of micellization. It is, therefore, worthy to determine the CMC of a surfactant or a mixture of surfactants precisely. i. Absorption spectroscopy. The process is mostly applicable to the surfactants which give one or more absorption peak in the absorption spectrum. In a typical experiment, surfactant solutions of different concentrations are prepared and the absorption values are recorded. Figure 6. Determination of CMC by absorption spectroscopic method, adapted from Basu Ray et al. 60 The absorption values are then plotted against [surfactant] in the solution. The break point appeared in the profile is the CMC. For non-absorbing surfactants CMC is determined by absorption spectroscopy by adding a probe into the surfactant solution. ii. Fluorescence spectroscopy. The method is based on the fluorescence spectra of a dye which varies substantially with its environment. In fluorimetry, in the pre-micellar region, the dye (with its negligible aqueous solubility) accommodates itself in water (polar environment) and after micellization it partitions between water and the nonpolar micellar core or the dye may also reside at the palisade layer of the micelle 61. In a typical experiment surfactant solutions of different concentrations are prepared which varies from below CMC to above CMC having fixed amount of the probe and then from the plot of fluorescence intensity vs. [surfactant] the CMC can be determined. 16 Figure 7. Determination of CMC by fluorescence spectroscopic method, adapted from Basu Ray et al. 60 Among the various forms of environment, the micellar media can be the best choice to reach a deeper understanding of dye-media interactions as compared to the other solvation systems. According to Göktürk et al. 62, a dye molecule exhibits spectral changes in presence of varying amount of surfactants consistently and there exist sequential equilibria between surfactant monomers, micelles, dye aggregates and pre-micellar dye-surfactant complex, etc. From practical and scientific aspects, the studies in this area are still important and interesting. The interaction of eosin Y with cationic detergents has also been applied to the assay of such detergent63. The eosin Y-surfactant complexes is a good model for studying dye adsorption and absorption64 by surfactants, both of Figure 8. The visible absorption spectra of erythrosin B in water (1), in aqueous solution of anionic surfactant SDS (2), cationic surfactant CTAB (3) and neutral surfactant TX-100 (4) at 298 K, adapted from Bhowmik et al. 24 17 which are important in the dyeing industry. Studies of the influence of various nonionic, anionic and cationic surfactants on the fluorescence and absorption spectra of eosin yellow have been reported earlier65. The optimum solubilization showing dye–surfactant interaction can be utilized as separation of dyes from waste dye-stuffs of different textile, paper and pulp industries. The spectral behaviour of some anionic xanthene dyes namely erythrosin B, rose bengal and eosin have been studied in micellar solution of TX-100 (neutral), SDS (anionic) and CTAB (cationic) and the studies have been correlated with the photoelectrochemical studies of these dyes in aqueous solution of these surfactants24. In the present work systematic studies on the interaction between EY and different cationic surfactants have been reported. The surfactant head group, chain length, as well as the number of hydrocarbon chains (single or double tailed) have been varied. The surfactants used were cetyltrimethylammonium bromide, cetylpyridinium chloride, didodecyldimethylammonium bromide and didecyldimethylammonium bromide. Dye-surfactant interaction studies are believed to shed information on the polarity of the medium (governed by added surfactants), nature and extent of interactions between dye and surfactant molecules as well as the self-aggregation behaviour of dye in presence of the surfactants. 3. Cursory glance on polymers The word polymer literally means “many parts”. A polymeric material may be considered to be one that contains many chemically bonded parts or units which themselves are bonded together to form a solid. In other way a polymer is a large molecule composed of repeating structural units or chains typically connected by covalent chemical bonds. While polymer in popular usage suggests plastics, the term actually refers to a large class of natural and synthetic materials with a variety of properties and purposes. Hence, the terms polymer and polymeric material encompass very large, broad classes of compounds, both natural and synthetic, with a wide variety of properties. Because of the 18 extraordinary range of properties of polymeric materials 66, they play an essential and ubiquitous roles in everyday life67, from those of familiar synthetic plastics and other materials of day-to-day work and home life, to the natural biopolymers that are fundamental to biological structure and function. 3.1. Classification of polymer: Polymers are classified in different ways: 1. On the basis of arrangement of monomer unit. a) Homo polymers, e.g. polyvinyl chloride. b) Co-polymers, e.g. ethylene-vinyl acetate. 2. On the basis of their mode of formation. a) Condensation polymer, e.g. polyamide. b) Addition polymer, e.g. Saran (plastic) wrap. 3. On the basis of chemical composition. a) Organic polymer, e.g. deoxyribonucleotides. b) Hetero-organic polymer, e.g. polysiloxanes. c) In-organic polymer, e.g. polydimethylsiloxane. 4. On the basis of their occurrence. a) Natural polymer, e.g. cellulose. b) Synthetic polymer, e.g. polyethylene. 3.2. Polymer properties Polymer properties are broadly divided into several classes based on the scale at which the property is defined as well as upon its physical basis 68. The most basic property of a polymer is the identity of its constituent monomers. A second set of properties, known as microstructure, essentially describe the arrangement of these monomers within the polymer at the scale of a single chain. These basic structural properties play a major role in determining bulk physical http://en.wikipedia.org/wiki/Plastic http://en.wikipedia.org/wiki/Biopolymers http://en.wikipedia.org/wiki/Ethylene-vinyl_acetate 19 properties of the polymer, which describe how the polymer behaves as a continuous macroscopic material. Chemical properties, at the nano-scale, describe how the chains interact through various physical forces. At the macro-scale, they describe how the bulk polymer interacts with other chemicals and solvents. 3.3. Polyelectrolytes Polymer molecules containing ionizable subunits are known as polyelectrolytes or ionomers. Electrochemically active polymers can be classified into several categories based on the mode of charge propagation. The mode of charge propagation is linked to the chemical structure of the polymer. Polyelectrolytes are polymers whose repeating units bear an electrolyte group. These groups will dissociate in aqueous solutions (water), making the polymers charged. Polyelectrolyte properties are similar to both electrolytes (salts) and polymers (high molecular weight compounds), and are sometimes called polysalts. The adsorption behavior of polyelectrolytes has attracted a lot of attention recently because of the widespread industrial use of these substances for example in papermaking, in mineral processing, in waste water treatments, in paints and in cosmetics69,70. In papermaking, polyelectrolytes are used as retention aids to promote retention by flocculation and to improve drainage, as fixing agents to promote aggregation of colloidal material to the fibers and as wet and Scheme 11. Schematic representation of (A) poly(diallyldimethyl ammonium) chloride and (B) N,N-dimethyl-N-dodecyl derivative of hydroxyl ethyl cellulose(LM200) / N,N-dimethyl-N-methyl derivative of hydroxyl ethyl cellulose(JR400)[R = -CH3 : JR400, R = -C12H25 : LM200] . dry strength additives to promote the strength of paper71. In particular, cationic polyelectrolytes have found widespread industrial application as flocculants in A B http://en.wikipedia.org/wiki/Polymers http://en.wikipedia.org/wiki/Electrolyte http://en.wikipedia.org/wiki/Dissociation_(chemistry) http://en.wikipedia.org/wiki/Aqueous http://en.wikipedia.org/wiki/Charge_(physics) http://en.wikipedia.org/wiki/Salts http://en.wikipedia.org/wiki/Molecular_weight 20 branches of industry such as mining, water resource management, and papermaking70,72. Chemically, these polyelectrolytes are a group of macromolecules where the charges are localized pendant to the hydrophobic carbon backbone, most frequently as quaternary ammonium substituents, and which have molar masses in the order of 106 to 107 g/mol. Cationic polymers play important role in gene delivery for their competency of aggregating the DNA molecules73. Cationic polymers have also proven to be successful in the condensation of DNA74. Nowadays, hydrophobically modified biocompatible polymers are recognized as an important and attractive class of drug carriers, especially for intravenous administration of hydrophobic drugs75,76. The hydrophobically modified cationic polyelectrolytes proved to provide a hydrophobic environment favourable to Rutin partitioning/binding. Further, they allowed discrimination of factors responsible for polymer / Rutin interaction, as the effect of alkyl side chain length and microdomain compactness on the interactions could be ascertained by Bai et al.77. 3.4. Importance of dye-polymer interaction study Dye-polymer interaction has several importance. The functional group of a polymer can be detected by using dye-polymer interaction 78. Different complexes can be formed through different polyelectrolytes assembled with different Figure 9. (A) Absorption spectrum of Azure B–NaAlg system at various P/D ratios1 and (B) Emission spectra of 10− 5 mol dm− 3 acridine orange at 303 K in presence of varying amounts of Klebsiella K18 capsular polysaccharide81. A B 21 surfactants or dyes. The complexes can be used in dyeing, liquid crystal displays (LCDs), nonlinear optics, food, medicine, chromatograms, and other new technical fields79,80. To identify the molecular geometry of a polymer-dye complex, dye-polymer interaction study is used 82. The assembly of dyes molecules on metal-polymer complexes is of interest due to their potential applications in photovoltaic cell, separation, and wastewater treatment83. Several physiochemical parameters such as molecular weight of each repeating unit, stoichiometry of the dye-polymer complex, binding constant, and other related thermodynamic parameters like free energy, enthalpy, and entropy changes can be evaluated using polymer-dye interactions. Also, the equivalent weight of a polymer can be determined from the dye-polymer interaction studies84,85. The rate of isomerisation of azo-dye in some polymer matrices as well as in some solvents depend on dye-polymer interactions86 i.e., one can control the reaction rate by controlling the properties of the medium. Many reports are available in the literature on dye-polymer interaction studies in controlling the kinetics of photobleaching reactions87-89. Information about the dynamics of polymer chains during phase transition could be obtained by investigating the fluorescence properties of the dye embedded into polymer both at the bulk and single molecule levels90. Organic dye-doped polymer materials have numerous optoelectronic applications, including frequency conversion of light, waveguiding, optical signal processing, and optical data storage91,92and are also established as potential laser media93. Dye-polymer interaction is also a suitable tool for the removal of organic compounds form waste water by the method of adsorption94,95. Studies on binding of oppositely charged dye to different synthetic and natural polyions have been reported by many researchers 96-99. The encaging property of polymer highly influences the excited state properties of many xanthenes dyes 100 leading to variations in the spectra. Fluorescence intensity of a dye can get either enhanced or quenched. The usually encountered fluorescence quenching processes are collisional (dynamic) quenching and static (complex formation). i. Collisional quenching occurs when the excited fluorophore experiences contact with an atom or molecule that can facilitate non-radiative 22 transitions to the ground state. In the simplest case of collisional quenching, the following relation called the Stern-Volmer equation holds 26: (3) where, F0 and F are the fluorescence intensities of EY in the absence and presence of quencher, Q; [Q] = Quencher concentration. KSV = Stern-Volmer quenching constant. Plot of F0 / F vs. [Q] yields a straight line with a slope equal to KSV. ii. In case of static quenching a fluorophore form a stable complex with another molecule in the ground state and consequently the number of fluorophores emitting get reduced thus leading to the decrease in the fluorescence intensity. In such case the dependence of the fluorescence as a function of the quencher concentration follows the relation101: (4) where Ka is the association constant of the complex. Figure 10. Emission spectra of (a) PSF, and (b) ST, in the presence of different ctDNA concentrations. ( exc = 520 nm). Curves correspond to (a) 0, 6, 9, 13, 21, 37, 47, 71, and λ2 M of DNA; (b) 0, 4.6, 8.5, 12, 20, 36.5, 46, 60.5, 75, λ0, 102.5, and 114 M of DNArespectively102. In the present dissertation efforts were made to investigate the interaction of EY with oppositely charged aqueous polyelectrolytes. Also, the variation in the absorption and emission spectral bands as function of polyelectrolyte ][10 QK F F sv ][10 QK F F a 23 concentration was studied. Subsequently, the binding constant of the dye-polymer complex and related thermodynamic parameters were evaluated. The polymers chosen for the interaction studies are structurally different (as shown in scheme 11) so different types of interactions are likely to occur. 4. Colloidal dispersion Colloidal solutions (also called colloidal suspensions) are the subject of interface and colloid science. This field of study was introduced in 1861 by Scottish scientist Thomas Graham. A colloidal system consists of two separate phases: a dispersed phase (or internal phase) and a continuous phase (or dispersion medium) in which the colloid is dispersed. A colloidal system may be solid, liquid, or gas. One property of colloid systems that distinguishes them from true solutions is that colloidal particles scatter light. If a beam of light, such as that from a flashlight, passes through a colloid, the light is scattered by the colloidal particles and the path of the light can therefore be observed. The dispersed particles have a diameter of between approximately 1 and 1000 nanometers103 . The stability of a colloidal system is the capability of the system to remain as it is. Stability is hindered by aggregation and sedimentation phenomena, which are driven by the colloids tendency to reduce surface energy. Reducing the interfacial tension will stabilize the colloidal system by reducing this driving force. Nanoparticles are the clusters of 10-1000 atoms with size dependent physical, chemical, electronic, and optical properties104. In nanotechnology a particle is defined as a small object that behaves as a whole unit in terms of its transport and properties. Ultrafine particles or nanoparticles are sized between 100 and 1nm. Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials and atomic or molecular structures105. A bulk material should have constant physical properties regardless of its size, but at the nano-scale size-dependent properties are often observed. Thus, the properties of materials change as their size approaches the nanoscale and as the percentage of atoms at the surface of a material becomes significant. Nanoparticles often http://en.wikipedia.org/wiki/Interface_and_colloid_science http://en.wikipedia.org/wiki/Scotland http://en.wikipedia.org/wiki/Thomas_Graham_(chemist) http://en.wikipedia.org/wiki/Solid http://en.wikipedia.org/wiki/Liquid http://en.wikipedia.org/wiki/Gas http://en.wikipedia.org/wiki/Nanometre http://en.wikipedia.org/wiki/Nanometre http://en.wikipedia.org/wiki/Atom http://en.wikipedia.org/wiki/Molecular 24 possess unexpected optical properties as they are small enough to confine their electrons and produce quantum effects106. For example gold nanoparticles appear deep red to black in solution. Nanoparticles of usually yellow gold and grey silicon are red in color107. Other size-dependent property changes include quantum confinement in semiconductor particles108, surface plasmon resonance in some metal particles109 and superparamagnetism in magnetic materials110. Ironically, the changes in physical properties are not always desirable. Ferromagnetic materials smaller than 10 nm can switch their magnetisation direction using room temperature thermal energy, thus making them unsuitable for memory storage111. The reasons for the outstanding properties of nanoparticles may be due to their particle size, large specific surface area, strong surface area and change of electronic properties which is supported by the quantum effects of particles less than 10 nm. Because of a number of special properties exhibited by nanoparticles, they find multifarious applications. Nanoparticles are versatile enough to be used in many types of technological applications from delicate electronics 112 to revolutionary medical procedures 113. Among the wide range of applications the timeliest one is perhaps in medical applications. Their pathogen sized proportions make them useful in fighting viruses and bacteria in human body and in the production of anti-cancer drugs 114. Studies of nanoscale noble metal materials are additionally important because these materials have potential as optical filters 115, plasmonic waveguides 116, bio-chemo-sensors 117, and substrates for surface- enhanced spectroscopies 118. Nanoparticles of magnetic metals find applications as catalysts, nucleators for growth of high-aspect-ratio nanomaterials and toxic waste remediation. Nanomaterials find numerous applications in biology or medicine. They are used as fluorescent biological labels 119, in drug and gene delivery 120, in bio detection of pathogens 121, in detection of proteins 122, in probing of DNA structure 123, in tissue engineering 124, in separation and purification of biological molecules and cells 125, in MRI contrast enhancement 126, in phagokinetic studies 127, etc. http://en.wikipedia.org/wiki/Gold http://en.wikipedia.org/wiki/Surface_plasmon_resonance http://en.wikipedia.org/wiki/Superparamagnetism http://en.wikipedia.org/wiki/Magnetic 25 4.1. Colloidal silica Colloidal silica is a dispersion of fine amorphous, nonporous, and typically spherical silica particles in a liquid phase. Among different semiconductor nanocrystals, silica has got special importance because it can be used in different forms for solute pre-concentration and immobilization of analytical reagents 128. Besides, silica has got special attention for other specific properties like optical transparency 129, high hydrophilicity 130 and negative surface charge in aqueous media 131. According to Bonacchi et al. 132, silica nanoparticles have some fascinating properties compared to other conventional Scheme 12. Schematic representation of a silica nanoparticle-based biosensor fabricated via (a) the entrapment technique and (b) covalent conjugation133. nanosystems which include its photophysical inertness, absence of intrinsic toxicity, capability to host a large number of photochemically active species and also to protect the active material segregated inside its pore. These specific properties have additional advantages for silica nanoparticles in the field of bioanalysis and disease diagnostics 134. They can be used as templates for the formation of ordered mesoporous polymers of tunable pore size135. Colloidal silica is mainly used in catalysis, ceramics, paper, textile applications, strength enhancement in rubber, tobacco treatment, and medicine 136. Also, they find applications in high-temperature bonding, investment casting, refractory fiber bonding, and silicon wafer polishing 137. Several optimizations can lead to the binding of SiO2 particles with each other or with other substrates 138. Ludox® is commercially available spherical shaped silica particles suspended in aqueous medium which find different applications for their specific properties like optical transparency, high hydrophilicity and negative surface charge139-142. Therefore, http://en.wikipedia.org/wiki/Amorphous http://en.wikipedia.org/wiki/Silica 26 colloidal silica in the form of Ludox® can be considered as an appropriate model system in investigating its capability to alter (both enhancement and quenching) the absorbance and fluorescence intensity of fluorophores in aqueous medium. 4.1.1. Preparation of colloidal silica Colloidal silica can be prepared by various methods and starting materials143. These methods include ion exchange 144, neutralization or electrodialysis of aqueous silicates, hydrolysis and condensation of silane 145, peptization or milling of silica gel or powder 146, and direct oxidation of silicon 147. There are four most important methods to prepare colloidal silica from natural ore 148. The first among them is the preparation of colloidal silica from liquid sodium silicate by the ion exchange process. The sodium silicate is obtained from naturally available silica by melting the silica ore in the presence of alkali flux; subsequently, it is dissolved by heating under pressure to produce liquid sodium silicate, which is commonly known as water glass. The liquid sodium silicate has high viscosity, and therefore, it is diluted to a concentration of 3-5 wt%. Next, it is passed through an ion-exchange resin, and then fed into the alkali solution to form Scheme 13. Various preparation methods for colloidal silica: (a) Ion exchange from sodium silicate; (b) Hydrolysis and condensation from TEOS; (c) Milling or dispersion fromfumed silica; (d) Direct oxidation from silicon148. 27 a silica seed, which is then used to grow silica particles. The product is concentrated to 30 wt% to obtain the commercial product. The second method for preparing colloidal silica is from tetraethoxysilane (TEOS), which is well known as the Stöber method 145. TEOS is a silane monomer prepared from tetrachlorosilane, which in turn is derived from metallurgical grade silicon. The silicon itself is obtained by the reduction of naturally occurring silica ore at temperature over 1900°C in the presence of carbon. Scheme 14. Schematic representation of preparation of colloidal silica from TEOS. The third method is that of direct oxidation of silicon wherein colloidal silica is prepared by the direct oxidation of metallurgical grade silicon without using TEOS. The silicon is treated with water in the presence of alkali catalysts to produce silica along with the evolution of hydrogen and heat. The fourth method for preparing colloidal silica is by milling and peptization of silica that can be found either in the form of silica gel or fumed silica, which consists of preferentially coaccervated or aggregated primary silica particles. The properties of the colloidal silica prepared by this route largely depend not only on the milling and peptization process but also on the properties of the starting silica source such as purity, shape, and aggregation. Ludox® colloidal silica is an aqueous colloidal dispersion of silica particles. It is an opalescent liquid with a slight-to-moderate bluish cast. Because of their colloidal nature, particles of ludox® have high specific surface area which accounts for the novel properties and wide variety of uses. Du Pont offers seven general grades of Ludox®: HS-40%, HS-30%, TM, SM, AM, AS and LS. Du 28 Pont also offers a special grade Ludox® CL-X for packaging frictionizing. The particles in Ludox® are discrete uniform spheres of silica which have no internal surface area or detectable crystallinity. They are dispersed in an alkaline medium which reacts with the silica surface to produce a negative charge. Because of the negative charge, the particles repel one another resulting in a stable product.The stabilizing counter ion in Ludox® HS-30%, HS-40%, LS, SM, TM, AM and CL- X is sodium whereas in Ludox® AS the counter ion is ammonium. Most applications of Ludox® colloidal silica depend on the high surface area and reactivity of the suspended particles. The use of sodium aluminate instead of sodium hydroxide in stabilizing Ludox® AM gives it broader stability against variation of pH. In applications where a colloidal silica needs to be incorporated at a neutral or acid pH, Ludox® AM has been the grade of choice 149. 4.1.2. Aggregation between dye and colloidal silica dispersions Incorporation of dyes in small solid particles instead of bulk solid material is gaining interests150,151 for many reasons. Small particles reveal interesting optical scattering phenomena, especially in combination with interference effects. Dye-colloidal aggregates are important in the field of waste water treatment 152, lasing property 153, dye-sensitized solar cells 154, photocatalytic reactions 155, etc. Van Blaaderen et al. 150,151 synthesized dye incorporated silica colloids. Such dyed particles enable measurements of particle diffusion by fluorescence recovery after photobleaching156, and direct imaging of dense colloidal structures by confocal fluorescence microscopy157. Among the different fluorescent labels, dye-doped silica nanoparticles show distinct advantages over quantum dots, fluorescent dyes, up-converting phosphors and plasmon resonant particles because of their high quantum yield, photostability, water dispersibility and ease of surface modification with different functional groups for subsequent bioconjugation, due to well-known silica chemistry158. According to Ha et al. 159 and Bringley 160 in some cases, fluorescence could even be quenched, compared to the free dye molecules. Therefore, a detailed spectral investigation on the absorption and fluorescence behaviour of dye-silica aggregates is warranted for better technological applications as well as from the fundamental understanding point of 29 view. Towards this initiative spectroscopic investigations on colloidal silica-7HC aggregates in a wide concentration range of silica were undertaken. Although several reports are available in the literature which include the interaction between silica and a number of dyes 25,161-166, however, reports involving the interaction of Ludox® with 7-hydroxycoumarin are not common in literature. The occurrence of excited state H-bonding between silica and 7-hydroxycoumarin is supposed to be understood in a better way through fluorescence spectroscopic analysis, which could further be investigated via FTIR measurements. 4.2. Synthesis of nanoparticles 4.2.1. In different phases Methods for preparation of nanoparticles can be divided into physical and chemical methods based on whether there exist chemical reactions. On the other hand, these methods can be classified into gas phase, liquid phase and solid phase methods based on the state of the reaction system.  Gas phase: The gas phase method includes gas-phase evaporation method (resistance heating, high frequency induction heating, plasma heating, electron beam heating, laser heating, electric heating evaporation method, vacuum deposition on the surface of flowing oil and exploding wire method), chemical vapor reaction (heating heat pipe gas reaction, laser induced chemical vapor reaction, plasma enhanced chemical vapor reaction), chemical vapor condensation and sputtering method.  Solid phase: This method includes thermal decomposition, solid state reaction, spark discharge, stripping and milling method. The thermal decomposition method is one of the chemical methods that can synthesize well-dispersed NPs with good crystallization. The solventless thermal decomposition method by capping agents like oleate can be used for the preparation pure metal nanoparticles in regulated conditions either spontaneously or in the presence of a reducing gas.  Liquid phase: Liquid phase method for synthesizing nanoparticles mainly includes chemical reduction, sol gel, hot-soap, solvothermal method, 30 pyrolysis, and spray pyrolysis methods and methods of using templates like micelles and reverse micelles. 4.2.2. Synthesis of nanocolloidal dispersion in aqueous media There are different preparative routes for the synthesis of nanoparticles in aqueous media: 1. Monolayer and Langmuir-Blodgett techniques are more effective in controlling the molecular orientation and packing at a molecular level 167. The Langmuir–Blodgett (LB) technique allows the transfer of nanoparticles from solutions into thin films. Compression of nanoparticles at water interface using monolayer film as the surrounding matrix is one of the commonly used method for preparation of composite films 168. Not only can Langmuir-Blodgett films fabricate the packing and the thickness of the molecular films in a controlled manner, but also they provide possible templates to control the nucleation and growth of organized inorganic nanoparticles under mild conditions 170. However, although this method is useful in obtaining a thin film but this is not suitable for obtaining nano-colloidal distributions. Scheme 15. Schematic of the preparation of Janus nanoparticles based on the Langmuir -Blodgett technique 169. 2. Preparation of nanoparticles in microemulsion media: Synthesis of nanoparticles in microemulsions is an area of considerable current interest. 31 Among all chemical methods the microemulsion has been demonstrated as a very versatile and reproducible method that allows to control over the nanoparticle size and yields nanoparticles with a narrow size distribution 171. Depending on the proportion of various components and hydrophilic– lipophilic balance (HLB) value of the used surfactant microemulsions can be classified as water-in-oil (W/O), oil-in-water (O/W) and intermediate bicontinuous structural types that can turn reversibly from one type to the other. Water-in-oil microemulsions have been used to prepare nanoparticles for more than two decades, and a wide variety of materials has been synthesised by these methods 172-174. The preparation procedure of metallic nanoparticles in W/O microemulsion commonly consists of mixing of two microemulsions containing metal salt and a reducing agent respectively. After mixing two microemulsions, the exchange of reactants between micelles takes place during the collisions of water droplets result of Brownian motion, the attractive van der Waals forces and repulsive osmotic and elastic forces between reverse micelles. Successful collisions lead to coalescence, fusion, and efficient mixing of the reactants. The reaction between solubilizates results in the formation of metal nuclei. Growth then occurs around this nucleation point where successful collision occurs between a reverse micelle carrying a nucleus and another one carrying the product monomers with the arrival of more reactants due to intermicellar exchange. The nucleation reaction and particle growth take place within the micelles and the size and morphology of as-prepared nanoparticles depend on the size and shape of the nanodroplets and the type of the surfactant, whose molecules are attached on the surface of the particles to stabilize and protect them against further growth 172. However, this method also has limitations. Particles as synthesized cannot be directly used for practical applications as they are confined within the oil medium. They need to be isolated for further use which is not always feasible. 32 Scheme 16. Proposed mechanism for the formation of metal particles by the microemulsion approach174. 3. Preparation of nanoparticles in micellar media: In aqueous media, the nanosized particles are primarily separated by the ionic repulsion forces produced due to adsorption on their surface. In this perspective, stabilization of the nanoparticles by surfactants in aqueous solution has been proved to be one of the most effectual methods. The size, shape and other surface properties of the nanoparticles can be altered by different surfactants depending on the latter’s molecular structure, i.e., nature of head group, length of hydrophobic tail and type of counterions. Diffusion and attachment rates of surfactants on the nanoparticle surface control the termination of the particle growth 175. The preparation method consists of simple addition of aqueous metal salt solution and aqueous surfactant solution by controlling the concentration of the surfactant and the molar ratio of surfactant to metal salt in the reaction solution 176. 33 Scheme 17. Schematic representation of surfactant-capped nanoparticles in micellar medium. 4.3. Advantages of nanoparticle synthesis in aqueous surfactant solution In comparison to their bare counterparts, nanoparticles capped by surfactants stay well dispersed in solution for a longer time. Surfactant stabilized aqueous nano entities offer a unique environment for inorganic reactions, i.e., they act not only as a micro-reactor for hosting the reaction but also as a steric stabilizer to inhibit aggregation. The main advantages of this method are that (i) it is a soft technique, i.e., it does not require extreme conditions of temperature and pressure, (ii) the particle size, shape, and distribution can be controlled by simply varying the composition and dynamics, and (iii) these surfactant stabilized nanoreactors are capable of compartmentalization which in turn alters the ground transition, product states and reduces the reaction dimensionalities 177,178. 4.4. Importance of silver bromide nanoparticles Nanoparticles of silver bromide serve as model material to study the quantum confinement effects of indirect band gap semiconductors 179, sensitive photographic material for high-speed photography 180, and efficient photocatalyst for hydrogen generation from a solution of methanol and water 181. Silver bromide nanoparticles serve as a source of strongly biocidal but nontoxic Ag+ ions182. On the other hand, the importance of silver halides in photochemistry has shown how these materials are unique. This compound can absorb photons in the visible and shorter wavelengths to generate photoelectron and photohole pairs. The electrons subsequently combine with interstitial silver ions to form silver atoms under continuous irradiation 183. 34 Synthesis and characterization of silver halide nanoparticles have gained huge research interest over past few decades181,184-187. They have been widely used in optoelectronics 188,189, catalysis 190,191, surface-enhanced Raman scattering 192 and more recently surface enhanced fluorescence193. Jeunieau et al.194 prepared silver bromide nanoparticles by typically carrying out a reaction upon mixing two identical microemulsions, each containing one of the reactants forming the precipitate. Rosseti et al.195 explained the particle size dependence of the excited state electronic properties of silver iodide and silver bromide as a consequence of electron and hole localization in the small crystallites. Ohde et al.196 have synthesized silver halide nanoparticles in super critical carbon dioxide utilizing a water-in-CO2 microemulsion. Koh et al.197 synthesized silver haide nanocomposites using amphiphilic graft copolymer as the template. Silver halide fibers are flexible, water insoluble, non-toxic, and have good transmission in the mid-infrared region, therefore they can be used in many applications such as fiber-optic infrared spectroscopy, radiometry, and transmission of CO2 laser power for medical purposes198. The water-insoluble cationic polymer/silver bromide nanoparticle composites form good coatings on glass and exhibited long- lasting antibacterial properties toward both airborne and waterborne bacteria 184. Preparation of AgBr nanoparticles has also been reported in water-in-oil microemulsion media by Husein et al.186. They have prepared AgBr nanoparticles in water-in-oil microemulsion where the existence of bulk water was possible for which the nanoparticles got precipitated in that water pool. Nanoparticles have inherent tendency to coalesce in solution199. Hence, to form stable dispersions of nanoparticles, stabilizing agents that bind to the nanoparticle surface are essential. It has been reported earlier that capping agents comprising a reactive head and a hydrophobic tail acts as a good stabilizing agent for the synthesized silver bromide nanoparticles200. Many researchers have used cetyltrimethylammonium bromide (CTAB) as a capping agent to form stable dispersions of nanoparticles176,201-203. CTAB adsorbs on the surface of the nanoparticles and forms a bilayer structure which is favourable to enhance the stability of the nanoparticles176. Bai et al.204 have prepared and characterized AgBr nanoparticles in poly(vinylpyrrolidone) matrix. CTAB acts as a stabilizer in seed mediated 35 growth of silver nanodisks in aqueous media 205. He et al.187 have prepared novel layered AgBr based nanocomposite stabilized by CTAB. Sui et al.201,202 have used CTAB as a stabilizer in preparing positively charged silver nanoparticles. In the present dissertation, attempts have been made to synthesize nanocolloidal dispersions of silver bromide (AgBr) by way of simply adding silver nitrate to aqueous cetyltrimethylammonium bromide (CTAB) solution. CTAB served both as stabilizing agent as well as the source for bromide ion. AgBr nanoparticles are proposed to get stabilized by the layered structured surfactant cation assemblies. Although there are previous reports on the synthesis and characterization of AgBr nanoparticles176,206,207, however, studies on such a system in solution phase are not so common in the literature. Keeping in mind about the multifaceted application potentials, AgBr were prepared and characterized in aqueous CTAB solution in the concentration range (10-6-10-5M), well above its solubility limit. 4.5. Characterization of nanoparticle Nanoparticles can be characterized by a number of techniques for example XRD, FTIR, NMR, conductometry, UV-vis spectroscopy, dynamic light scattering studies, DSC, TGA, isothermal titration calorimetry, electron microscopy, etc. XRD: X-ray diffraction method is a useful tool for characterization of nanoparticles. From the XRD data, one can obtain the material composition, structure (three-dimensional coordinates of atoms, chemical bonding, molecular conformation and three-dimensional conformation, the electron density value, etc.) and the information on the interaction between molecules. 36 Figure 11: XRD pattern of the textiles coated with silver particles 208. FTIR: From the IR spectrum, one can observe the absorption and emission due to the molecular vibration and rotation in the electromagnetic wave infrared region (15000~10 cm-1). It reveals the unknown composition qualitatively according to the bands characteristic frequency, determines a component content of the sample (quantification) according to band intensity. It can also reveal the molecular structure (such as functional group, bond), identify isomer, and determine structures of compounds. Figure 12: FTIR spectra for the pure and modified silver nanoparticles 209. NMR: It can be a very selective technique, distinguishing among many atoms within a molecule or collection of molecules of the same type but which differ only in terms of their local chemical environment. NMR spectroscopy is used to unambiguously identify known and novel compounds, and as such, is usually 37 required by scientific journals for identity confirmation of synthesized new compounds. Figure 13. 1H NMR spectra of: (a) chitosan and (b) Cholesterol-modified chitosan conjugate with succinyl linkages 210. Conductometry: Conductometry has notable application in analytical chemistry, where conductometric titration is a standard technique. Conductometric titration is a type of titration in which the electrical conductivity of the reaction mixture is continuously monitored as one reactant is added. The equivalence point is the point at which the conductivity undergoes a sudden change. Figure 14. Conductometric titration of oxidative-acid-treated diamonds (nominal size ∼ 100 nm). In this titration, an excessive amount of NaOH was first added into the nanodiamond suspension and then neutralized with 0.1 N HCl. The first region in the plot corresponds to neutralization of solution OH− groups and the second region corresponds to neutralization of surface – COO− group 211. http://en.wikipedia.org/wiki/Analytical_chemistry http://en.wikipedia.org/wiki/Titration http://en.wikipedia.org/wiki/Electrical_conductivity http://en.wikipedia.org/wiki/Reaction_mixture http://en.wikipedia.org/wiki/Reactant http://en.wikipedia.org/wiki/Equivalence_point 38 .UV-Visible spectroscopy is one of the most widely used techniques for the characterization of nanoparticles. This technique is used in the study of nanomaterials as a diagnostic of nanoparticle formation. Used in conjunction with affinity labeling, UV-visible spectroscopy often provides the means of choice to gauge response in an analysis using nanoparticles. It has been further suggested that the spectroscopic properties of nanoparticles can provide an indicator of their size distribution by fitting the position of the surface plasmon resonance (SPR) to a simple wavelength function212. Figure 15: UV–vis diffuse reflectance spectra of (a) 0.02AgCl/Ag/MCM-41, (b) 0.1AgCl/Ag/MCM-41 and (c) 0.2AgCl/Ag/MCM-41213. Dynamic light scattering (DLS) studies: The size and surface charge (zeta potential) of the nanoparticles can be measured by DLS measurements. DLS is used to measure particle and molecule size. This technique measures the diffusion of particles moving under Brownian motion, and converts this to size and a size Figure 16. DLS size distribution graph of AuNPs synthesized using chitosan as reductant 215. 39 distribution using the Stokes-Einstein relationship. The technique is suitable for the characterization of colloidal particles over a wide range of sizes from a few nanometers to several micrometers. If the system is monodisperse, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, particle concentration, and the type of ions in the medium 214. Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA): DSC profiles of heating and cooling of the systems can also help in confirming the monolayer or bilayer structure of the surfactant protected nanoparticles. DSC is a thermal analysis apparatus measuring how physical properties of a sample change, along with temperature against time. In other words, the device is a thermal analysis instrument that determines the temperature and heat flow associated with material transitions as a function of time and temperature. During a change in temperature, DSC measures a heat quantity, which is radiated or absorbed excessively by the sample on the basis of a temperature difference between the sample and the reference material216. In TGA the % weight losses of the components are found out between a wide range of temperatures. The information regarding the thermal decompositon of the components helps in predicting the structure of the aggregates. Figure 17. (a) TGA/DSC curves for as-synthesized Mn oxide precipitated in ethanol (heating rate = 10 °C/min under a stream of air) 217. 40 Isothermal Titration Calorimetry can provide information about the standard enthalpy of formation of the nanoparticles and hence various thermodynamic parameters can be calculated. From such a plot one can determine the enthalpy of formation of a reaction involved in precipitation which is otherwise impossible. Similarly, when such a parameter is estimated in aqueous micellar media at different concentrations the extrapolated value can provide the absolute enthalpy of formation for a reaction associated with precipitation. Figure 18. Plot of ΔHf vs ω-1 for the formation of Cu2[Fe(CN)6] in H2O/AOT/heptane w/o microemulsion at 303 K 218. Transmission Electronic microscopy is a useful technique in the characterization of nanoparticles. It is one of the most impo