Structure and dynamics of fluids in confinement: A case study of water, protein and ionic liquid in reverse micelles and microemulsions

A dissertation submitted for the degree of Dr. rer. nat. (Doctor rerum Naturalium) in the Faculty of Chemistry and Biochemistry at the Ruhr-University Bochum Germany

Sangeetha Balakrishnan 2007

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1st Examiner: Prof. Dr. Hermann Weingärtner 2nd Examiner: Prof. Dr. Roland Winter Thesis Committee Head: Prof. Dr. Dominik Marx Date of Defense: 10th December, 2007.

iii

iv Declaration

I hereby declare that the dissertation entitled “Structure and dynamics of fluids in confinement: A case study of water, protein and ionic liquid in reverse micelles and microemulsions” is my original work and has been written with no other sources and aids than quoted, and has not been submitted elsewhere for an examination, as thesis or for evaluation in a similar context.

Sangeetha Balakrishnan

7 November, 2007.

v Acknowledgements

I am indebted to many people who have been instrumental in the successful completion of this thesis. First among them is my advisor, Prof. Hermann Weingärtner, whom I would like to thank for the opportunity to pursue this research in the first place; and also for the freedom that he offered me to experiment my ideas. I owe a great deal of gratitude to Prof. Roland Winter, not just for graciously consenting to be my second referee; but more so for the many occasions when he so liberally gave of his time and guidance not just related to this thesis, but regarding research in general as well. I truly value those words of wisdom. For help with many administrative matters, I am grateful for the assistance of Mrs. Gundula Talbot, Mrs. Christel Tönnisen, and Mrs. Ursula Knieper. My colleagues deserve a special round of thanks for the congenial working atmosphere at the lab - Yathrib Ajaj, Mianmian Huang, Sasisanker Padmanabhan, and Peter Romahn - thanks to them all for helping create some wonderful memories. My thanks also to Dr. Holger Nadolny, my former colleague; for having been a good friend over the years. I am grateful to Nadeem Javid (Dortmund) for the successful SAXS collaboration with one of my projects. I would like to thank him and Dr. Sivakumar Sekharan for their help in obtaining some of the literature cited in the thesis. For help in proof-reading a part of the work, my thanks to Dr. Kaushik Chakrabarty. Special thanks to Nilesh Madhu for the many science and non-science discussions and for his support when things were at low ebb. My thanks to the DFG Graduiertenkolleg ‘Structure and Dynamics in Heterogeneous Systems’ for the financial support to carry out this work. Finally, and most importantly, my thanks to my family - my father, Wg. Cdr. A. M. Balakrishnan; my mother, Mrs. Vijayalakshmi Balakrishnan; and my sister, Ms. Revathi Balakrishnan - for all their love, support and the freedom they accorded me to pursue my dream.

vi vii Table of contents

Declaration iv Acknowledgements v Table of contents vii Preface xi Abbreviations and Symbols xiii

Chapter 1 Introduction 1 1.1 The hydrogen bond and its historical background 1 1.2 Classification of hydrogen bonds 2 1.3 Hydrogen bonding in water 4 1.3.1 Structure of liquid water 4 1.3.2 Water clusters 5 1.4 Water in confinement 6 1.5 Proteins in confinement 8 1.6 Ionic liquids in confinement 9

1.6.1 The C − HLF hydrogen bond 10 1.7 Self-assembly 10 1.7.1 The Hydrophobic effect and surfactants in solution 11

Chapter 2 State of the art 17 2.1 AOT – the surfactant 18 2.2 Infrared spectroscopy of AOT reverse micelles 20 2.2.1 The OH stretch region 20 2.2.2 The ‘n-water state’ conundrum 21 2.2.3 NIR spectroscopy in AOT reverse micelles 24 2.2.4 Other vibrational modes in AOT reverse micelles 25 2.3 Dielectric spectroscopy 27 2.4 Small angle x-ray scattering 29 2.5 Other techniques 32 2.5.1 The water libration band and terahertz spectroscopy 32 2.5.2 Non-linear infrared spectroscopy 32 2.5.3 NMR spectroscopy 33 2.5.4 Simulations 35 2.5.5 Molecular probes 36 2.5.6. Calorimetry 37 2.5.7 Other experimental techniques 38 2.6 Non-aqueous reverse micelle interior 38 2.7 Proteins in confinement 39 2.7.1 A first look at macromolecular crowding 40 2.7.2 The biological membrane 41 2.7.2.1 Reverse micelles as membrane mimics 42 2.7.2.2 Encapsulation of proteins in reverse micelles 42 2.7.3 Osmolytes 45 2.7.4 Hydration water 48 2.8 Ionic liquids in confinement 49 2.8.1 Research on confined ILs so far 50

2.8.2 [bmim][BF 4] and Triton X-100 53 2.8.3 Solvatochromism 54 2.8.4 Association constant 55

Chapter 3 Experimental techniques 57 3.1 Near-infrared spectroscopy 57 3.1.1 Principles of NIR spectroscopy 58 3.1.2 Anharmonic vibrations 60 3.1.3 Consequences of anharmonicity 61 3.1.4 Instrumentation 61 3.2 Dielectric relaxation spectroscopy 62 3.2.1 The dielectric constant and polarisation 63 3.2.2 Relaxation Times 64

ix 3.2.3 The Debye equation 65 3.2.4 Instrumentation 66 3.3 Small angle x-ray scattering 68 3.3.1 Theory 69 3.3.2 Instrumentation 71 3.4 Materials 72 3.4.1 Water in confinement 72 3.4.2 α-chymotrypsin in confinement 73

3.4.3 [bmim][BF 4] in confinement 74

Chapter 4 Results and discussion 75 4.1 NIR spectroscopy of bulk water 75 4.2 AOT reverse micelle — the anhydrous system 77 4.3 AOT reverse micelle — the hydrous system: Influence of hydration 78 4.3.1 NIR data 78 4.3.2 SAXS data 84 4.3.3 Dielectric relaxation data 86 4.4 Effect of hydrocarbon medium on AOT reverse micelles 89 4.4.1 NIR data 89 4.4.2 SAXS data 92 4.5 Temperature dependence of hydrogen bonding of micellar water 95 4.5.1 Hydration picture as a function of temperature 99 4.6 NIR analysis of α-CT hydration properties in confinement 99 4.6.1 The basis of the method 101 4.6.1.1 The spectra — origin and resolution 102 4.6.1.2 Resolution of Curve B from the difference spectrum 103 4.6.1.3 Resolution of Curve C from the difference spectrum 103 4.6.2 The kosmotropes 104 4.6.3 The chaotropes 104 4.7 IL in confinement 107

N 4.7.1 Polarity of the confined IL determined by E T(30) and ET solvent

x polarity scales 107 4.7.2 Structural changes in the confined ionic liquid 109 4.7.3 Determination of association constant 111

Chapter 5 Conclusions 114

Appendices A List of Figures 117 B List of Tables 120 C Spectral positions 121 D Derivation of the Benesi-Hildebrand equation 122 E List of References 125 F Curriculum Vitae 143

xi Preface

The study of fluids in confined geometries and close to interfaces has gained unprecedented momentum over the last decade owing to its applications in diverse fields ranging from geochemistry to astrobiology. A testament to this is the series of Confit workshops held triennially since the year 2000, serving as a confluence for the discussion of confinement effects on the microscopic dynamics of condensed matter. A variety of confined systems like simple organic and inorganic liquids, quantum liquids, water, polymers, biological systems, molecules and atoms; in conjunction with an equally impressive array of confining media like zeolites, carbon nanotubes, reverse micelles, clays, fullerenes, etc. make this a highly interesting field of study. Rapid advances in technology over the years have also contributed to significant developments that have lead to the expansion of the frontiers of confined-fluids research. The present thesis is an exposition of the investigation of the structure and dynamics of water, a protein -- α-chymotrypsin, and an ionic liquid -- 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim][BF 4]) confined within the nano pores of reverse micelles and microemulsions formed by two surfactants; Aerosol OT (AOT) and Triton X-100. Right from Lavoisier’s discovery of the composition of water to its detection on Mars and Europa, water has been the most widely investigated substance in laboratories around the world. In nature however, water often appears in interstices or is adsorbed on solid substrates like minerals; and the choice of the first experimental system in this thesis — water in confinement — is an attempt to study the properties of water in an environment that mimics this natural state. To this effect, the hydrogen bonding of water confined in AOT reverse micelles was chosen as the spectral tool to monitor the structure and dynamics of water in accordance with variation in extent of hydration, temperature, and the molecular make-up of the external solvent forming the reverse micelle. Most protein folding and other associated dynamic processes are studied in infinite dilution. In vivo , however, protein dynamics occur in a crowded cellular milieu and in confined spaces such as the chaperonin cavity, the proteosome etc. It thus makes sense to assume that proteins may experience different energy landscapes when folding in vivo than in

xii bulk, and this has been the driving force behind the studies involving the second experimental system — α-chymotrypsin in confinement . Several osmolytes were added to this confined protein system and an investigation was undertaken to elucidate the effect of macromolecular crowding on the hydration properties of the confined protein. The third system concerns ionic liquids, which are widely becoming popular in the modern day Green Chemistry movement. A model ionic liquid, [bmim][BF 4] was encapsulated in microemulsions formed by the surfactant Triton-X 100 and a comprehensive account of the solvatochromism, structure and association of the confined liquid in the nano domains of the colloidal aggregate is provided. The polarity of the microemulsions was determined using Reichardt’s dye. For the first time, the structural changes in a confined ionic liquid based on its C − HLF interaction are reported. Further, employing the Benesi- Hildebrand double reciprocal plot, pioneering insight into the association between the positively charged imidazolium ring and the hydroxyl groups of the oxyethylene units of the surfactant is presented. Given the industrial importance of ionic liquids, and by drawing parallels to their water/oil counterpart, it is hoped that the confined ionic liquid system may well throw open new avenues of research with the potential of novel applications. This justifies the choice of the third experimental system — ionic liquid in confinement — in the thesis.

xiii Abbreviations and Symbols

AOT, Aerosol OT Sodium bis(2-ethylhexyl)sulfosuccinate α -CT alpha-chymotrypsin

[bmim][BF 4] 1-butyl-3-methyl-imidazolium tetrafluoroborate FWHM Full width at half maximum IL Ionic liquid NIR Near-Infrared ni number of molecules of each water species per AOT molecule

Pi Fraction of individual water species in the total water in the reverse micelle

R [bmim][BF 4]]/[TX-100]

Rg Radius of gyration SAXS Small angle x-ray scattering TX-100 Triton X-100

Wo [H 2O]/[AOT] φ Volume fraction

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If there is one thing that I love about writing more than the rest, it’s that sudden flash of insight when you see how everything connects.

- Stephen King, in ‘ On Writing ’.

xv Chapter 1 Introduction

1.1 The hydrogen bond and its historical background The fundamental importance of the hydrogen bond lies in its role in driving molecular association. Since the energy of a hydrogen bond lies in between those of a covalent bond and the van der Waals interaction, hydrogen bonds associate and dissociate quickly at ambient temperatures. The earliest references to what may seemingly be called hydrogen bonds occurred about a century ago, when Werner [1] and Hantzsch [2] made use of the term Nebenvalenz to describe the bonding in ammonia salts. In 1913, Pfeiffer [3] introduced the phenomenon Innere Komplexsalzbildung to explain the reactivities of compounds with C=O and OH groups placed adjacently, with amines and hydroxides. In 1920, Latimer and Rodebush [4] in discussing the structure of ammonium hydroxide, suggested that ‘the hydrogen nucleus held between two octets constitutes a weak bond ’. It was however Pauling in 1935, who used the term hydrogen bond for the first time to account for the residual entropy of ice [5]. The mid-1930s saw a growing interest in the concept, with papers by Corey [6] on diketopiperazine and glycine explicitly mentioning the term ‘hydrogen bond’. But the subject of hydrogen bonding was formally ushered into the chemical mainstream by the chapter on hydrogen bonding in Pauling’s much cited monograph The nature of chemical bond . The two core ideas to emerge from Pauling’s work were that, in a configuration such as X − HLA , the H atom is considered to be the seat of hydrogen bonding and not the entity HLA ; and that the hydrogen bond is essentially an electrostatic interaction. Pauling’s ideas were developed and refined further, which eventually led to the practical definition of the hydrogen bond by Pimentel and McClellan [7]. According to them,

Chapter 1 Introduction

A hydrogen bond exists between a functional group X—H and an atom or a group of atoms A in the same or a different molecule when (a) there is evidence of bond formation (association or chelation), and (b) there is evidence that this new bond linking X—H and A specifically involves the hydrogen atom already bonded to X.

There are a host of experimental techniques that can affirm the formation of a bond (by forming large molecular aggregates), but it is the specific criterion (b) that sets a hydrogen bond apart from any ordinary molecular association. Many different spectroscopic and non- spectroscopic techniques have been successfully used to detect a hydrogen bond, and a brief synopsis on these is given in the forthcoming chapters, with special relevance to infra red and dielectric spectroscopies. It is worth mentioning that the hydrogen bond is strongly affected by weak environmental perturbations such as thermal motion and ionic or molecular interaction fields. These disturbances are so distinctive, in fact, that IR and Raman studies are the most commonly used experimental techniques to detect the presence of a hydrogen bond which satisfy both parts (a) and (b) of the definition of the H bond mentioned above.

1.2 Classification of hydrogen bonds The strength of hydrogen bonds can vary widely. An arbitrary classification of these bonds may accordingly be made into strong, moderate and weak bonds. This kind of a classification though disputable on account of the energies and therefore the properties of the hydrogen bonds falling in broad continuous ranges, nonetheless becomes important because of the different sensitivities and observation time scales of different probing techniques. Table 1.1 lists geometrical, energetic, thermodynamic and functional properties that aid in the classification of the hydrogen bond, and is entirely based on Jeffrey [8] and Desiraju & Steiner’s [9] description of the concept. The most important characteristic of the strong hydrogen bonds is their distinctive covalent character [10]. These bonds are formed by unusually activated donors and acceptors. Most often these happen to be between an acid and its conjugate base,

− + X − HLX , or between a base and its conjugate acid, X − HLX . The X—H and HLA

2 Chapter 1 Introduction distances in these bonds are comparable and may be studied by many of the techniques used to study covalent bonds.

Table 1.1 Some properties of strong, moderate and weak hydrogen bonds Strong Moderate Weak

X − HLA interaction mostly covalent mostly electrostatic electrostatic Bond energy 15 - 40 4 - 15 < 4 (kcal/mol) [ ]− − = − Examples FLHLF O HLO C C HLO [ ]+ − = − NLHLN N HLO C O HLπ P − OH LO = P O − HLO − H Os − HLO IR νs relative shift ~ 25% 10 - 25% < 10% Bond lengths H—A ≈ X—H HLA > X—H HLA >>X—H XLA range (Å) 2.2 - 2.5 2.5 - 3.2 3.2 - 4.0 HLA range (Å) ~1.2 - 1.5 ~1.5 - 2.2 2.2 - 3.2 Bond angles (°) 175-180 130-180 90-150 ( X − HLA )

The transition from strong to moderate hydrogen bonds brings in the concept of electrostatics into the picture. These are the most common hydrogen bonds in chemistry and nature, and are essential for the structure and functioning of biological macromolecules. The characteristic feature of this hydrogen bond is that the donor X atoms are electronegative in comparison to H and the acceptor A atoms have lone pair of unshared electrons. The weak hydrogen bond may be defined as an interaction X − HLA wherein a hydrogen atom forms a bond between two structural moieties X and A, of which one or even both are only of moderate to low electronegativity [9]. Desiraju and Steiner rightly point out the oxymoronic overtones underlying the term weak hydrogen bond based on Pauling’s concept of the hydrogen bond being a bond and being electrostatic in nature. They added that since bonding implies strength and requires a stronger electrostatic interaction in X − HLA , it would be better to see the hydrogen bond on phenomenological rather than energetic terms. This then satisfies Pimentel and McClellan’s criteria, and also shows that it is not necessary for a hydrogen bond to be strong to retain many of its characteristics. Weak hydrogen bonds though widespread, have only recently gained attention. These bonds are electrostatic but this characteristic is modified by variable dispersive and charge-

3 Chapter 1 Introduction transfer components that depend substantially on the nature of the donor and acceptor group. − The O HLPh bond is one of the strongest in this category and is comparable to a O − HLO − H bond; while the weakest in this category, formed by unactivated methyl groups are barely stronger than van der Waals attractions.

1.3 Hydrogen bonding in water The chemical constitution of water is very simple with an oxygen and two hydrogen atoms possessing a H–O–H bond angle of 104.5° completing the symmetrical picture. The structure of water determined by infrared spectroscopy shows an O-H bond length of 0.9572 Å [11]. The electrical dipole moment of the molecule has been found to be 1.83 x 10 -18 esu cm [12], with the moment pointing from the oxygen end of the molecule towards the hydrogen end, which also explains the solvating power of water. By virtue of its Lewis base character, water bears ionizing properties and can easily coordinate with Lewis acids. The high dielectric constant of water ( ε = 78.5) favours solvation and the dissociation of salts. The properties of liquid water are rationalized on the basis of the network formed by water molecules through hydrogen bonding, and this was first recognised by Wendell Latimer and Worth Rodebush [13]. Hydrogen bonding occurs when a hydrogen atom bonded to an electronegative oxygen atom associates with another oxygen atom. The hydrogen bond is highly directional and its three dimensional character makes water unique among other associative liquids. In this network, each water molecule can form up to four hydrogen bonds with the neighbouring molecules and these bonds are broken and formed on a picosecond timescale. It is this hydrogen bond network that makes water a profoundly anomalous liquid. High heat capacity, expansion on freezing, maximum density at 4 °C, high dielectric constant are all properties that discernibly set water apart from other liquids.

1.3.1 Structure of liquid water The structure of liquid water has been a subject of debate since many decades now. Even though it is the most abundant and most widely studied liquid on earth, the understanding of its structure and dynamics is yet to reach a unanimous consensus. Two schools of thought have emerged over the years in an attempt to delve deeper into water’s structure.

4 Chapter 1 Introduction

Probably one of the earliest attempts to develop a conceptual model to reproduce the observed behavior of water dates back to Röntgen [14] in 1892. He proposed that a shifting equilibrium between small ice crystallites suspended in a liquid of dissociated individual molecules was responsible for the density maximum of water. Thus Röntgen paved the way for what is now popularly known as the mixture model of water, which describes liquid water as an equilibrium mixture consisting of molecular species with different number of hydrogen bonds per molecule. That is, in an ensemble of water molecules, there are supposed to exist water molecules with 1, 2, 3 or more H bonds per water molecule. This description also implies that with variation in temperature, there occurs a change in the number of water molecules of the distinct sub-type. In contrast to this model is the continuum model in which water is envisaged to be a dielectric continuum. This model proposed by Pople [15] postulates that H bonds will be bent but not broken when water is formed from ice or when the temperature of water is raised.

1.3.2 Water clusters Studies of water clusters of different nuclearities and structure have garnered great attention since small water clusters are shown to be the perfect means to characterise structural changes and bonding mechanisms in passing from isolated molecules to bulk states. Several investigations to date have provided evidence that the water trimer [16], tetramer [17], and pentamers [18] have hydrogen bonded 2D cyclic minimum energy structures, while pioneering work by Saykally and others suggest 3D geometries for the larger water clusters with the hexameric forms representing the transition from 2D cyclic to the 3D geometries [19-21]. The chemical and physical environment of water clusters have been shown to influence their stucture [19,22], in addition to providing quantitative characterisation of the hydrogen bonded networks that exist in these clusters. Studies toward understanding the growth and properties of water clusters that are larger than hexamers are highly relevant in understanding solvation processes, although the development of large size water clusters has been a challenging scientific endeavour. In a pioneering demonstration of the presence of water clusters in solutions at room temperature, Köddermann et al. have reported on the presence of a dimer, a cyclic trimer, a cyclic tetramer, and a cage hexamer by FT-IR spectroscopy of water in CCl 4 [23]. They

5 Chapter 1 Introduction further demonstrate that with increase in temperature, while the monomer contributions slightly increase, the concentration of the hydrogen bonded cluster species decrease. This is because the larger hydrogen-bonded clusters successively breakdown into smaller species and finally into monomers. The contribution of the cyclic trimer decreases most rapidly with temperature on account of its unfavourable bent hydrogen bonds. Enormous progress in laser spectroscopy and ab-initio quantum mechanical studies has facilitated highly detailed studies of water clusters. The use of these tools to study water clusters with n ≥ 6 has proved very promising. For instance, water hexamer clusters with various isomers like ring, book, bag, cage, chair, and prism have been identified. These hexameric isomers and a variety of other clusters including heptamers, octamers, clathrate like structures (n = 12-26), up to icosahedral networks (n = 280) have been summarised by Ludwig in his review [24]. All these structures display a beautiful array of hydrogen bonding which allow the explanation of many of the anomalous properties of water.

1.4 Water in confinement Inspite of the conceptual differences in water models and the anomalies associated with it, water definitely plays a fundamental role in our lives. But, liquid water in nature appears in many confined regions or is attached to substrates. Examples include water in porous media like rocks and clays, water in biological molecules like cells or water attached to surfaces of macromolecules and membranes. The structure and dynamics of water in these confined regions is markedly different from bulk water. The characteristics of these nano water-pools are modified by the molecular motion of the water molecules, which is dependent on the distance of the latter from the confining walls [25]. In order to understand the properties of confined water, it is increasingly being encountered in many scientific disciplines. For instance, surface water has been found to be essential for electrochemistry, passivation and corrosion chemistry and in catalysis [26]. In environmental sciences, confined water plays a pivotal role in transport phenomena [27]. It wouldn’t be inappropriate to say that the chemistry of both organic and inorganic aerosols would be incomplete in the absence of confined water [28]. And perhaps the most important is the observation that the physicochemical properties of confined water influence the biological response of materials [29]. Hydration influences the equilibrium protein structure

6 Chapter 1 Introduction and hence its dynamics and biological function, which is exemplified by the observation that a dehydrated powder of lysozyme does not display any enzymatic activity [30]. Also, in bacteriorhodopsin, water molecules comprise the proton conduction pathways and mediate the switch by contributing the essential proton transfer from the chromophore to the aspartic acid residue, Asp85 [31]. Hydration is crucially important for the conformation and stability of nucleic acids as noted by Watson and Crick [32]. In our day-to-day life as well, confined water plays a fundamental role. For example, in electronics, the relative humidity and thereby the structure of the adsorbed water layers modifies the durability of hard disk drives by changing the media tribology [33]. With such wide ranging relevance, it hardly is surprising that confined water has emerged as a major research avenue today, with scientists from all over the world studying this liquid in an impressive array of confining media, employing various experimental and theoretical techniques [34]. Geometric confinement is known to induce modifications in both the structural [35] and dynamic [36] properties of bulk water. On confinement, water experiences a strong reduction of the tetrahedral order with a consequent distortion of the hydrogen bond network. Confinement also induces stratification in water. With the formation of distinct layers of water with markedly different characteristics close to the surface and away from it, there arises a bimodal dynamics in the nano water-pool [37]. The first component of this dynamics occurs in the sub picosecond regime and is similar to that observed in bulk water. On the contrary, the second, slow component is completely absent in bulk water and occurs in the hundreds to thousands of picosecond range. Not unexpectedly, experiments show a slow relaxation of water in contact with biomolecules like proteins. Another factor that comes into play here is the hydrophilic or hydrophobic nature of the confining interfaces. The interaction of water with these two types of interfaces is fundamental to explain the relationship between structure and biological function [30, 38- 43]. Also to be taken into consideration is the range till which the confining walls exert their influence on the confined water; i.e. how far from the surface is the water structure indistinguishable from bulk water. Based on extensive research by combining water absorption gravimetric measurements and immersion enthalpy determination at increasing water pre-coverage in confining media comprising of different clays and minerals [44-46], it

7 Chapter 1 Introduction has become evident that at most two to four water layers are influenced by the surface field of the solid, which implies an approximate thickness of around 10 Å. Molecular dynamic simulations however show that near smooth surfaces the first water monolayer forms a hydrogen bonded network with square-like and chain-like arrangement of molecules [47], whereas the second water layer connects the specific first layer with the rest of the water, which, starting from the third layer shows practically bulk-like behaviour even close to strongly hydrophilic surfaces [48].

1.5 Proteins in confinement The study of proteins in confinement is important not just from a physiological view-point, but also for potential practical applications, such as using entrapped proteins as biocatalysts and biosensors. For the latter purposes, enzymes have been encapsulated in sol-gel matrices, which have been shown to confer enhanced thermal stability to the enzymes [49-51]. Ping et al. have investigated the effects of cavity size on the thermal stability of silica-matrix- encapsulated acid phosphatase and horseradish peroxidase using the non-surfactant templating method and they find that the thermal stability of the entrapped protein increases as the pore size of the silica matrix decreases [52]. Bolis et al. examined the influence of protein stability upon confinement by characterising four proteins (titin I27, bacterial, yeast and human frataxin) in a polyacrylamide gel of nano-sized pores [53]. They found that all proteins were stabilised when confined in the gel, the most dramatic stabilisation however, being that of yeast frataxin, whose melting temperature increased by almost 5 °C in the gel. Ravindra et al . [54] demonstrated by means of pressure perturbation calorimetry and differential scanning calorimetry that the encapsulation of ribonuclease A in mesoporous silicates enhances its thermal stability. A high concentration of an inert synthetic or natural macromolecule, termed crowding agent, for example, a 1% agarose solution [55] and parallel zirconium layers [56] have also been shown to be suitable model experimental systems for studying the effect of confinement on proteins in vitro . Most globular proteins exist in two thermodynamic states at equilibrium; the native and the denatured states. It is observed experimentally that in the native state, the protein is folded and compact, with most of its potential surface area buried in its tightly packed interior. In the denatured state, the unfolded protein exposes the hitherto buried sites to the

8 Chapter 1 Introduction solvent and is biologically inactive. Molecular crowding serves to stabilise a protein because in a crowded situation, the lack of space favours the folded protein conformation over the unfolded state. Theoretical calculations are also shown to complement these experimental observations. Minton [57, 58] pioneered the application of statistical thermodynamics to study the effects of confinement on the structure and reactivity of proteins. Klimov et al. [59] studied the confinement effects on protein using all-atom Monte Carlo simulations; Friedel et al. [60] investigated confinement effects using the “Honeycutt-Thirumalai 46 bead BPN model”, while a recent simulation-based paper by Cheung et al. [61] reaffirms that excluded volume effects due to macromolecular crowding enhance the native state stability of a protein by directly linking the stabilisation to crowding-induced destabilisation of the unfolded states [57].

1.6 Ionic liquids in confinement In recent years researchers have expressed a burgeoning interest in room temperature ionic liquids (ILs) as substitutes for common organic solvents in many chemical processes. It wouldn’t be inappropriate to term ILs sui generis owing to their impressive properties like high thermal stability, high ionic conductivity, negligible vapour pressure, suitable polarity, electrochemical stability, non-flammable nature and easy recyclability, which in turn render them highly useful as environmentally benign solvents [62,63]. ILs are organic salts composed of cations and anions that are liquid at ambient conditions .§ These salts find wide applications in various processes like organic synthesis, catalysis, separations and polymerisation among a plethora of other uses. Inspite of the broad spectrum of research undertaken in ILs [64], the study of these neoteric solvents in confinement is still in its infancy and poses many intriguing questions; the answers to which could pave the way to enhance their potential applications. Expanding upon the ideas of confinement of water and other fluids, successful attempts have been made to confine ILs in microemulsions [65-70], micelles [71,72], controlled pore glasses [73], silica gels [74] and kaolinite [75].

§. Ionic liquids can be liquid at temperatures as low as -96 °C. In the patent and academic literature, the term ‘ionic liquid’ now refers to liquids composed entirely of ions that are fluid around or below 100 °C.

9 Chapter 1 Introduction

Not unexpectedly, encapsulation of ILs in controlled pore glasses lead to the lowering of their melting points, and in general ILs are shown to behave similar to conventional liquids under confinement.

− 1.6.1 The C H LF hydrogen bond − Experimental evidence for the existence of C HLX bonds, in general, is difficult to obtain as these bonds occur in tandem with other strong hydrogen bonds [76-78]. Given the weak − interactions of the C HLX bonds, they are often obscured by the stronger bonds, or even assigned to other phenomena. Nonetheless, there is unambiguous evidence from vibrational spectroscopy for the existence of these bonds in amide-functionalised imidazolium salts [79]. − For C HLX hydrogen bonds, it has been shown that hydrogen bonding has very little effect on the C—H bond length, and in some cases, it is even shortened, which leads to a blue-shift of the C—H bond stretching frequency [76,77]. To account for this observation, two disparate explanations have been invoked; the first, attributed to Hobza et al. suggests that the strengthened C—H bond arises due to a new mechanism called the anti-hydrogen bonding [76]; while Scheiner and Dannenberg consider the conventional hydrogen bonds and

C − HLX bonds to be similar, but assert that additional factors like anharmonicity and details of structurally mediated bond changes are essential to arrive at a correct explanation [77,78]. One of the reasons underlying this controversy is the weakness of the hydrogen bonds. Therefore, it becomes imperative while probing these bonds that one looks for methods to enhance the bond’s strength to arrive at a clear picture underlying this important phenomenon. Recently, it has been suggested that this can be achieved in molecular aggregates containing charges or by increasing the pressure of the system [80-82]. The − present work deals with the study of C HLF hydrogen bond in an IL confined in microemulsions formed by a non-ionic surfactant, which serves to stabilise the IL, thereby offering valuable insight into the structure of the IL in confinement.

1.7 Self-assembly Molecular self-assembly is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by non-covalent bonds [83]. Molecular self-assembly is ubiquitous in chemistry, materials science and biology, and

10 Chapter 1 Introduction is common throughout nature as well; as is evident by the numerous complex structures that nature exhibits around us [84]. The formation of molecular crystals, colloids, lipid-bilayers, phase-separated polymers, self-assembled monolayers, folding of polypeptide chains into proteins, and the folding of nucleic acids into their functional forms are some of the examples of molecular self-assembly. The study of molecular self-assembly is interesting for several reasons. Apart from the aesthetic appeal generated by the appearance of order from disorder, self-assembly has emerged as a means to understand the living-cell and the processes happening therein by employing colloidal aggregates like microemulsions, which are increasingly being referred to as membrane-mimics. The development of nanostructures also gets a boost as self-assembly has emerged as one of the practical strategies to generate their ensembles. Molecular self-assembly may generally be classified into two categories; static and dynamic [85]. Static self-assembly comprises of systems that are at global or local equilibrium and do not dissipate energy. These include molecular crystals, most folded proteins and all colloidal aggregates. Dynamic self-assembly, on the other hand, which includes in its purview, biological cells, comprises of systems that dissipate energy; the energy loss being a pre-requisite for the interactions between components that trigger self- assembly. The process of self-assembly occurs when molecules interact with one another through a balance of attractive and repulsive forces. These forces are generally weak, and include hydrogen bonds, van der Waals and Coulomb interactions. With special relevance to surfactants in solution, one may think of two guiding principles behind self-assembly. Like prefers like. We encounter this in hydrophobic and hydrophilic interactions that propel lipid molecules in solution to corral together to form an astounding array of geometries, as described in the next section. The second rule: Self assembly is governed by energetically favourable reactions simply means that when left unhindered, the right components will eventually evolve into complex ordered structures.

1.7.1 The hydrophobic effect and surfactants in solution The hydrophobic effect is probably the most important factor accounting for the self- assembly of both biotic and abiotic components. It was McBain, who first demonstrated the

11 Chapter 1 Introduction reversible formation of micellar aggregates in aqueous soap solutions [86], and credited this association to the attraction between the hydrocarbon chains of the soap molecules. Research since the days of McBain has now proved that it is the attraction between water molecules, as opposed to the attraction between the non-polar molecules that constitutes the hydrophobic effect. When a solute is dissolved in water, the attractive forces between the isotropically arranged water molecules are disrupted or destroyed. If the solute is ionic, it forms strong bonds with water molecules, thereby compensating for the disruption of the bonds existing in pure water, thus accounting for the solubility of polar molecules in water. If, on the other hand, the solute happens to be non-polar, its solubility in water is resisted on account of the absence of any similar compensation, as observed in polar solutes. Non-polar molecules in water, act like cavities, excluding water molecules from the volumes they occupy. In addition to this, weak van der Waals forces of attraction between water and the solute molecules also come into play. The interaction of hydrophobic solutes can be best understood by considering two cases; a small solute and a big solute, as explained by Chandler [87]. Figure1.1 illustrates these two cases with methane as a solute in water. In the small solute case depicted in part (a) of the figure, a small methane molecule (0.5 nm across) creates a cavity in the water continuum. But this excluded volume is so small that its presence doesn’t necessitate the breaking of any hydrogen bonds. Water molecules can adopt orientations that enable hydrogen bonding patterns to go around the solute. The water here, in essence resembles bulk water. In a large non-polar solute case, depicted by a cluster consisting of 135 methane-like particles hexagonally close packed to form a sphere of radius greater than 1nm, the situation however is different. The water molecules surrounding the solute no longer maintain the complete hydrogen bonding network. A fraction of the hydrogen bonding possibilities is therefore lost in the vicinity of an extended hydrophobic surface. In order to overcome this loss, less than one hydrogen bond per molecule is broken compared to the bulk liquid. As a result of this water tends to move away from the large non-polar solute. This concept of a dual nature of hydrogen bonding in the presence of small and large non-polar solutes was proposed by Stillinger [88] and provides the basis for understanding the hydrophobic effect.

12 Chapter 1 Introduction

Figure1.1 Configurations of liquid water molecules near hydrophobic cavities in molecular-dynamic simulations. The blue and white particles represent the oxygen (O) and hydrogen (H) atoms respectively in water. The red particle in (a) denotes the hydrophobic methane molecule and the hydrophobic cluster in (b) contains 135 methane-like particles. From [87].

The hydrophobic effect can be best illustrated by considering surfactants in solution. A surfactant (or surface active agent) is a substance that, when present at low concentration in a system, has the property of adsorbing onto the surfaces or interfaces of the system and of altering to a marked degree the surface or interfacial free energies of those surfaces (or interfaces) [89]. The term interface indicates a boundary between any two immiscible phases; the term surface denotes an interface where one phase is a gas, usually air. The surfactant presents itself as an interesting tool for investigation, owing to its amphiphilic nature. On account of this dual nature, amphiphiles align themselves in solution in such a way that the lyophobic moiety keeps away from solvent interactions while the lyophilic group remains in solution. Since water is the most common solvent, amphiphiles are described with respect to their hydrophilic and hydrophobic moieties. Accumulation of surfactants at interfaces (liquid/liquid or liquid/gas) is a spontaneous process as it results in a decrease of interfacial surface tension. In solution, a surfactant is characterised by its ability to form oriented monolayers at the interface and the ability to self- assemble in bulk solvent to lead to the formation of a myriad of mesoscopic fluid structures

13 Chapter 1 Introduction like micelles, vesicles, reverse micelles, microemulsions etc. The other hallmarks of a surfactant are its emulsification, wetting, dispersion and detergency properties. Though, the driving force behind the self-assembly of these structures is the hydrophobic effect, the size and shape of the aggregates are also known to depend on solution parameters, such as temperature, concentration, ionic strength, pH and the packing parameter [90]. If v is the surfactant molecular volume, A the area per polar head and l the length of the hydrophobic part, the number v/Al is called the packing parameter and gives a good idea of the shape of the aggregates that will form spontaneously: when, v/Al < 1/3 spherical micelles in water 1/3 < v/Al < 1/2 rod-like micelles in water 1/2 < v/Al < 2 lamellar phases in water/or oil 2 < v/Al < 3 rod-like micelles in oil 3 < v/Al spherical micelles in oil are formed. In the case of the presence of both oil and water, when v/Al <1 oil/water microemulsions v/Al > 1 water/oil microemulsions v/Al ~ 1 lamellar phases are preferred. In a surfactant, the hydrophobic group is usually a long-chain hydrocarbon residue, less often a halogenated or oxygenated hydrocarbon or siloxane chain and the hydrophilic group is an ionic or highly polar group. Depending on the nature of the hydrophilic group, surfactants may be classified as follows [89]: 1. Anionic : the surface active portion of the molecule bears a negative charge, for - + 3- + example, RCOO Na (soap), RC 6H4SO Na (alkybenzene sulfonate). + - 2. Cationic : the surface active portion bears a positive charge, for example, RNH 3 Cl + - (salt of a long chain amine), RN(CH 3) Cl (quaternary ammonium chloride) 3. Zwitterionic (or amphoteric): both positive and negative charges may be present in the + - surface active portion, for example, R NH 2CH 2COO (long chain amino acid) 4. Nonionic : the surface active portion bears no apparent ionic charge, for example,

RCOOCH 2CHOHCH 2OH (monoglyceride of a long chain fatty acid).

14 Chapter 1 Introduction

Figure 1.2 depicts various self-assemblies of surfactants in colloidal solution. Of the aggregates shown in the figure, reverse micelles and microemulsions are of relevance to this thesis, and a brief description of the two is therefore in order.

(a) (b)

(c) (d)

(f)

(e)

Figure 1.2 Surfactant shapes and various self-assemblies in colloidal solution. (a): single-tailed surfactant; (b): twin-tailed surfactant; (c): normal micelles; (d): reverse micelles; (e) interconnected cylinders; (f): planar lamellar phase.

Structurally, a reverse micelle is composed of an external shell made of the non-polar tails of the amphiphile, and the polar head groups along with the counter ions (if any) are sequestered in the interior of the aggregate, largely shielded from interaction with the bulk solvent [43]. Water and several other polar solvents are readily utilised as the polar solvent. Normally, the term reverse micelle is reserved for aggregates which are formed when the molar ratio of water to surfactant is less than ~15. This ratio is generally defined in terms of the parameter Wo as the ratio of molarity of water (polar solvent) to the molarity of the surfactant. For aggregates formed when Wo exceeds 15, the term water-in-oil microemulsion is used, indicating oil (non polar solvent) to be the dispersion medium. The ‘oils’ generally employed for this purpose are n-octane, iso-octane, heptane, cyclohexane, dodecane, pentane,

15 Chapter 1 Introduction benzene and halogenated alkanes like chloroform. In the present work however, the term reverse micelle is used to encompass the water-in-oil microemulsions as well for the sake of simplicity; although the differences between the two, that cross the realms of mere semantics, are addressed wherever appropriate.

16 Chapter 2 State of the art

Pierre-Gilles de Gennes was awarded a Nobel Prize in Physics in 1991 “for discovering that methods developed for studying order phenomena in simple systems can be generalised to more complex forms of matter” . He used the term ‘Soft Matter’ in his Nobel acceptance speech, referring to compounds which exhibit polymeric, colloidal or amphiphilic properties; and highlighted complexity and flexibility to be the hallmarks of this class of compounds [91]. The study of soft matter is concerned with understanding the properties of materials which have structural length scales in the range of few nanometres to several micrometres, and which are strongly affected by thermal fluctuations. Though the compounds comprising the soft matter class have been studied since a long time, it was only in the last two decades that it was realised that these systems share many properties, which prompted the unification of these subfields. Soft matter displays a wide range of interesting properties, and perhaps the chief among them is their ability to self-assemble into complex structures. The present thesis concerns two such soft matter components - the reverse micelle and the microemulsion - formed by two amphiphiles. The current chapter describes the relevant literature in this field and its organisation is as follows. Following a brief introduction on the surfactant AOT, Sections 2.2 through 2.6 present a detailed account on the structure and dynamics in AOT reverse micelles. Since the last review article dealing extensively with AOT reverse micelles was published in 1995, every effort has been made to cite the relevant papers published thereafter, rather than to reproduce the much sought-after review of De and Maitra [96]. The focus however is on infrared and dielectric spectroscopies, and small angle x-ray scattering, as these are the primary experimental techniques employed in this thesis. Section 2.7 introduces protein in confinement, beginning with a historical perspective on macromolecular crowding, and culminating in the much-debated concept of hydration water. Chapter 2 State of the art

Lastly, Section 2.8 serves as a complete compendium of research undertaken in confined ionic liquids thus far in a range of confining media.

2.1 AOT – the surfactant AOT or Aerosol OT (Sodium bis(2-ethylhexyl)sulfosuccinate) is a widely studied twin-tailed anionic surfactant (Figure 1). More than fifty years ago, AOT was recognised to form reverse micelles in non-polar solvents [92,93], and the reverse micellar solution thus formed was said to be “ water-clear and optically isotropic at rest ” [94].

O O

S O - N a + O O O

O

Figure 2.1 Structure of AOT

The preparation of AOT involves the diesterification of maleic or fumaric acid with 2-ethylhexanol, followed by the sulfonation of the diester with sodium bisulphite [95]. AOT is a white waxy solid producing a clear, colourless solution in alcohol. It has a molecular weight of 444.5 g mol -1, the linear length of the molecule is 11 Å and the maximum cross sectional area of the polar head part is 55 Å 2 [96]. AOT exhibits a remarkably rich phase behaviour, encompassing aqueous micellar, reverse micellar, vesicular, lamellar, bicontinuous cubic, and reverse hexagonal liquid crystalline phases. A typical phase diagram of the AOT/n-octane/water system is shown in Figure 2.2. One of the main advantages of studying AOT reverse micellar aggregates is that AOT does not require a co-surfactant to form reverse micelles, so that the system is limited to three components namely - water, AOT and oil. The simplest microstructure of water-AOT-oil is that of spherical water droplets of colloidal dimensions [97], possessing a small degree of polydispersity. The size of these reverse micelles is accurately controlled by their water content, denoted by the symbol Wo, which is the molar ratio of water to AOT ( Wo = [H 2O]/ [AOT]). The maximum water uptake in these reverse micelles, can however be controlled by

18 Chapter 2 State of the art the addition of organic and inorganic additives [98,99]. This maximum water uptake was calculated by Mitchell and Ninham [100] on the basis of the free energy variation of the total system due to changes in the molecular property of the surfactant.

Figure 2.2 Phase diagram of the ternary system AOT/n-octane/water at STP and schematic representation of some mesophase structures. (L 2 = reverse micelles, LC = liquid crystalline, L(D) = lamellar, H II (F) = reverse hexagonal, C = cubic phase. Adapted from [245].

A series of four papers over the last few years from the Eastoe group titled “What is so Special about Aerosol-OT?” [101-104] reports on the different molecular properties of this surfactant. The second article of this series [102] addresses the AOT microemulsion system. With the aim to identify what makes AOT an efficient surfactant for forming microemulsions, the authors investigated eleven AOT related compounds. These surfactants were from two separate homologous series, with either linear or branched hydrocarbon tails. This enabled the examination of the effect of chain structure on packing in curved interfacial films at the oil-water interface. The article reports that the linear di-chain surfactants formed microemulsion phases only in the presence of a co-surfactant, while the branched surfactants did not necessitate this requirement. Within the branched sulfosuccinates, variations in hydrophobicity were observed, induced by the different chain structures. For AOT however, its chain structure was found to give optimum aqueous phase solubility around room

19 Chapter 2 State of the art temperature; and it was deduced that its versatility as a microemulsifier is not dependent on its acclaimed “conelike molecular structure” as was previously believed. In addition to the findings of the Eastoe group, the speciality of AOT also lies in the numerous applications of its reverse micelles and microemulsions. Perhaps the most important of these applications from a physiological viewpoint is in the use of AOT reverse micelles as hosts for studying protein folding and other associated reactions, and as microreactors for enzyme catalysis. The literature on the incorporation of biomacromolecules like proteins, enzymes, and nucleic acids into reverse micelles is enormous, and there are many reviews that document the significant findings in this field [95,96,105-107]. The next most widely exploited use of these colloidal systems is their use as templates for nano- particle synthesis [108]. Again, some of the aforementioned reviews describe the various nano crystals produced using these soft colloidal templates [95, 107]. Some of the other interesting applications of AOT reverse micelles include their use in enhanced oil recovery [107], in the production of cosmetics and detergents [107], and in fabrication of sensors [109,110]. AOT has been classified as a food-grade surfactant [111] and hence offers the possibility of incorporating food ingredients like flavours, preservatives, vitamins etc. within its reverse micelles for carrying out reactions, thereby increasing the potential of the latter for extracting food components from a complex mixture. The ease of formation, excellent solubilisation capacity, bio-compatibility and the remarkable environment independent stability, favour AOT reverse micelles to be a better proposition over other compartmentalised systems for use in drug delivery [112].

2.4 Infrared spectroscopy of AOT reverse micelles 2.4.1 The OH stretch region IR spectroscopy has been found to be a particularly amenable technique to obtain information on the states of enclosed water in AOT reverse micelles. The fact that the technique is non-invasive, functional group selective, and sensitive to chemical environments makes it a perfect tool to probe the confined water. The suitability of the technique to detect different types of water at micellar interfaces stems from the very short observation times (10 -12 – 10 -14 s) which match the rapid time scales on which the water molecules are expected to interchange with one another (between 10 -7 and 10 -12 s). Thus, water molecules present in

20 Chapter 2 State of the art different environments are detected as separate bands provided the difference in vibrational energies between them is suitably large. According to accepted views the water pool can be divided into at least two populations: polar head hydration water (“bound” water) and, near the centre, bulk like water (“free” water) [113]. It is this bound water that is expected to exhibit anomalous properties, while with increase in hydration of the reverse micelles, the properties of free water approach those of neat water. The experimental plot of Wbound = f

(Wo) generally increases monotonically to a plateau with a slope almost equal to 1 at low Wo max W values [114]. The maximum value bound so defined is related to the number of hydration sites associated with one polar head. This parameter is vital in the understanding of the hydration shell of the polar head. Research over the years indicates the presence of between three and more than ten hydration sites per polar head group of AOT. IR studies have in general resorted to a deconvolution of the absorption band due to the OH stretching mode in the 3000-3800 cm -1 region to arrive at the microstructural characteristics of water in AOT reverse micelles.

2.2.2 The ‘n-water state’ conundrum A literature review indicates that in addition to the two state water model [115-120], AOT reverse micelles are shown to encapsulate three [114, 121-128], or four [129-133] distinct water types. Inspite of this disparity on the water states in reverse micelles, the main goal of most studies in this field has been to obtain the amount of bound water as a function of the total water content. There are also reports which show that although micellar water is different from neat water, it doesn’t seem to exist in different microenvironments [134-140], and that the uniform water structure is affected only by the overall ion concentration. All studies however find the IR spectrum of micellar water to be significantly different from that of neat water, and attribute this to the strong interactions of water with the head groups of the surfactant molecule as well as to an overall disruption of the three-dimensional hydrogen bonded network usually present in neat water. One of the earliest three-state water models was proposed by Jain et al. for the AOT/iso-octane/water system, where they consider the micellar water to be composed of bound, trapped and free water molecules [121]. They define bound water (~3490 cm -1) as composed of water molecules bonded to negatively charged polar head groups of AOT

21 Chapter 2 State of the art through sodium ions that are in the vicinity of the interface. The water molecules of hydrated sodium ions are hydrogen bonded with the polar head groups of AOT, and therefore constitute the bound water layer. The trapped water molecules (~3610 cm -1) are considered to be those that are located at the interface; unbonded to any other group or molecule, but trapped between the polar head groups of surfactant molecules at the interface. These water molecules are thought to behave as monomers or dimers and are thought to have penetrated the interfacial layer, consequently behaving like water molecules in a matrix. Free water (~3290 cm -1) is defined as the hydrogen bonded chains of water molecules occupying the central water pool of the reverse micelles. A closer look at the literature however indicates that there exists a discrepancy in the usage of terminology to denote the three water states. Different researchers use the terms trapped, interfacial and isolated; & bulk and free synonymously. Henceforth in the thesis, the terms free, bound, and trapped are used to denote the three water states, in keeping with the nomenclature adopted in the findings of the present work. As per Jain et al. [121] the total area of the broad and asymmetric OH bands corresponds to the sum of the peak areas of the different water states. They observe that with increase in water content in the reverse micelles, the bound water and trapped water fractions remain practically constant up to Wo = 10; thereafter gradually increase until Wo = 18, above which the values for both the fractions decrease. Further, the free water fraction was found to exhibit no significant change up to Wo = 10, but was found to decrease with increasing micelle size until Wo = 18, beyond which it increased. They rationalised these observations by considering that up to Wo = 10, there exists an equilibrium between the AOT monomers and the aggregated systems, and hence with the gradual addition of water, some water molecules are used up in hydrating the monomers while the others remain as bulk water inside the aqueous core in addition to the micellar bound water. This concept of equilibrium up to Wo = 10 was first proposed by Eicke [141]. The Wo range 10 to 18 is found to be interesting for the AOT reverse micellar system, and is often termed the micellar swollen region [141-143]. In this region, all the surfactant molecules are known to form micellar aggregates, leaving practically no surfactant monomer in solution. Beyond Wo = 18, all the added water is found to exist as free water, thereby causing a decrease in the bound water fraction. This study finds that the number of trapped water molecules per AOT molecule in

22 Chapter 2 State of the art

the entire Wo region studied is appreciably smaller than each of the free and bound water numbers. The trends observed in the number of free and bound water molecules per AOT molecule are found to be opposing prior to and beyond Wo = 18. There are many subsequent IR investigations on water in AOT reverse micelles, which are based upon the findings of Jain et al. Appendix C gives a list of these investigations, denoting the spectral positions of the different water species observed. Temsamani et al. [125] have investigated the water states of AOT reverse micelles as a function of counter ion, and have tested the entire alkali metal ion series. Their results indicate that the number of hydration sites associated with one polar head is independent of the counter ion nature and therefore, is only related to the hydration sites binding water molecules to the AOT molecule. Ikushima et al. [124] studied the AOT/water/ethane microemulsions under supercritical conditions by high pressure FT-IR spectroscopy. Upon Gaussian deconvolution of the observed broad OH peak, they found three water species with peak positions and full width at half maximum data in agreement with the values quoted by Jain et al. Accordingly, they propose the following figure for the water states in AOT reverse micelles.

Figure 2.3 Schematic representation of an AOT reverse micelle denoting the location of the three water species. Adapted from [34].

23 Chapter 2 State of the art

The relative areas of the three peaks of the spectrum were used to estimate the amounts of the free, bound and isolated water molecules. The picture of water hydration observed in this system is in contrast to the hydration observed in ordinary organic solvents. At 35 MPa, with increase in temperature (going from 306.1 K to 343.1 K), they found that the number of bound water molecules per AOT molecule increases, while the free water counterpart decreases for the Wo range studied. This indicates that the added water molecules are solubilised as bound water at small Wo; with increasing Wo, they are solubilised as trapped water as well as bound water; at much higher Wo however, the number of free water molecules becomes comparable to that of bound water molecules, while the number of trapped water molecules is relatively small. The four state water models [129-133] draw upon the ideas postulated by Jain et al. , the only difference however being that the bound water is considered to be of two types; water bound to the sulfonate groups, and water bound to the sodium counter ions.

2.2.3 NIR spectroscopy in AOT reverse micelles The NIR region (for details see Section 3.1) corresponds to overtones and combination bands of the OH, CH and NH bonds. In what can be considered the pioneering usage of NIR in the study of AOT reverse micelles, Sunamoto et al. [144] explored the combination band of the confined water, and arrived at two distinct water environments. The first, located at ~5263 cm -1 (1900 nm) was attributed to monomeric water, while the second band centered around 5208 - 4950 cm -1 (1920 - 2020 nm) was thought to be due to the bulk water. This paper however does not provide a comprehensive account of the AOT-water interactions, but provides an overview on the mode of surfactant interactions in a range of amphiphiles. In investigations of the AOT reverse micelles in the first OH stretch overtone region, Thompson et al. [145] detected ‘perturbed’ (interfacial) and bulk-like (free) water at 7142 cm -1 (1400 nm) and 5988 cm -1 (1670 nm) respectively, while Kawai et al. [146] endorsed the three-state water system by the location of water peaks at 7042 cm -1 (1420 nm), 6896 cm -1 (1450 nm) and 6060 cm -1 (1650 nm). Their observations on spectral behaviour are in agreement with those obtained in the mid-IR OH stretch investigations. Kise et al. recorded the OH stretching band of AOT in benzene, and compared it with the IR spectra of sodium ethane sulfonate [147]. A recent article [148] documented the near-infrared combination mode

24 Chapter 2 State of the art

(1800 - 2100 nm) of water in AOT/Brij-30/alkane/water reverse micellar system in two solvents, n-heptane and decalin. The percentage of bulk-water in the decalin based reverse micelle was found to be higher, which was explained based on the difference in penetrating properties of the two solvents.

2.2.4 Other vibrational modes in AOT reverse micelles In addition to the spectroscopic studies of the micellar OH stretch, some researchers have also investigated the vibrational modes associated with the carbonyl [115,121,124,133,139,149] and the sulfonate [115,139,149-152] moieties of the AOT head group. The carbonyl band in particular, has provided information on the rotational isomerism in the molecule. These investigations were aimed at studying the CO stretch between 1700 and 1790 cm -1, by resorting to deconvolution of the band into two, depicting the gauche and trans carbonyl conformations in AOT. These two conformations are thought to be exposed to different environments, as shown in Figure 2.4. If rotational isomerism exists in the molecule, then it can be seen that in the gauche conformation all the polar groups are directed towards the polar side of the interface while in the trans conformation a carbonyl group moves from the polar side toward the hydrocarbon part of the surfactant.

H H

H H H COOR Apolar side Polar side COOR - - COOR SO 3 SO 3 COO R H

Gauche Trans

Figure 2.4 The two carbonyl conformations in the AOT molecule.

It has been demonstrated that the proportions of the conformers vary with temperature and with the nature of the solvent environment [121]. In an apolar medium (low water content), the trans-like rotamer is expected to be favoured, because in this conformation the

25 Chapter 2 State of the art molecule is extended with the head group at the center. Here, the hydrocarbon chains are able to shield the head group from the apolar solvent. It has also been observed that with increase in hydration, there is an increase in the intensity of the low lying band at the expense of the other, and this has been thought to be due to the conformational changes about the acyl C-C bond associated with an increase in average area occupied by the head group. The symmetric sulfonate stretch band in AOT occurring around 1050 cm -1 gives one of the biggest evidences of the hydration picture in the reverse micelles. Moran et al . [149,150] found in the AOT/cyclohexane/water system, that the largest shifts in wavenumber occurred for Wo ≤ 3, which augmented the conclusion of Christopher et al. [139] that the first three water molecules added to the system interact very strongly with the sulfonate head group. The wavenumber shifts observed beyond this Wo value are found to be only minor (1 to 2 cm -1). The shifts observed are attributed to a weakening of the cation-anion interaction on hydration, which result in the removal of the sodium counterion from the sulfonate head group. The anytisymmetric sulfonate stretching mode exhibits a broad profile between 1100 and 1300 cm -1, consisting of at least four bands that change significantly with increasing hydration. Of these, the strong band at ca. 1161 cm -1, is found to contribute significantly to this region. As with the symmetric stretch counterpart, the most significant changes in the surfactant environment are found to occur for Wo ≤ 3, with no appreciable changes beyond

Wo = 15. This observation is in agreement with earlier results indicating that the water molecules in this region are located between the sodium cation and the three sulfonate oxygens [139]. The article also reports on the presence of two antisymmetric sulfonate stretch bands even beyond Wo = 20. This is taken to suggest that some sodium counterions interact with sulfonate head groups at this hydration, which is in agreement with the findings of Wong et al. [152]. A subsequent paper [153] on the interactions of water with polar head groups in AOT reports on the presence of two strong peaks at 1215 and 1245 cm -1 in the asymmetric sulfonate band, and shows an increase in the S 1215 /S 1245 ratio with the water loading. The data however are consistent with the earlier reports as they indicate that the greatest variations in the ratio are observed for Wo ≤ 6, with no noticeable changes beyond

Wo ≥ 16. The C-H stretch vibrations (2700-3000 cm -1) have also been used as indicators of micelle formation. Angelo et al. [32,154] in particular have reported based on this mode that

26 Chapter 2 State of the art

in the AOT/water/CCl 4 system, formation of micellar aggregates begins at a surfactant concentration as low as 10 -5 M.

2.5 Dielectric spectroscopy Dielectric relaxation spectroscopy has been shown to be a suitable technique to study the water confined in AOT reverse micelles. Some of the early studies in these colloids were carried out at relatively low frequencies (0.1 to 100 MHz) and at rather high volume fractions of the dispersed phase and the interpretation of data was chiefly done based on the phenomenon of percolation [155-158]. Experimentally, percolation is observed as a sharp increase in conductivity beyond a certain critical volume fraction of water [155,156,159], or upon increasing the temperature at constant water content [156]. Two approaches have been proposed to explain the mechanism underlying the percolation phenomenon. The first approach, the static percolation model, attributes percolation to the appearance of a bicontinuous oil and water structure [160,161]. Open water channels are thought to be responsible for the sharp increase in the electrical conductivity observed. These channels are formed when surfactant layers separating adjacent water cores open up during collisions or through the transient merging of droplets [162,163]. The second approach holds the attractive interaction between water globules responsible for the formation of percolation clusters [164]. This model takes into account the effect of cluster rearrangements due to Brownian motion. In this dynamic model, the charge transport is brought about by the hopping or diffusion of ions through globule clusters which rearrange with time [165]. Many studies [166,167] have shown that the data for the electrical conductivity below the percolation threshold can be interpreted in terms of the dynamic model. However, the complex permittivity data has caused controversy over the years. The experiments reported by Huang and coworkers [156] favour the static percolation model to describe the behaviour of the dielectric constant. On the other hand, dynamic percolation was found to give correct results for the dielectric constant measurements carried out by Chen, Delbos and Garti [166-168]. In a temperature dependent dielectric investigation of AOT reverse micelles, Dijk et al. [169] determined the low frequency permittivity of AOT/i-octane/water as a function of droplet volume fraction ( φ p) for Wo 7, 25 and 35 from 10 to 45 °C. They found that in all

27 Chapter 2 State of the art these cases, the permittivity value approached the permittivity of i-octane (1.94) in the limit

φ p → 0. In the reverse micelles with Wo = 25 and Wo = 35, the permittivity increased markedly with increasing temperature over the complete range of volume fractions studied, whereas for the system with Wo = 7, the permittivity exhibited a significant temperature dependence only for φ p > 0.5, where it decreased with increasing temperature. They further determined that for a given value of Wo, the polarisability of the reverse micelle droplet is temperature independent. An article by Tanaka and Shiromizu [170] describes the dielectric constant ( ε) determination of AOT reverse micelles in cylohexane, heptane, octane and decane, as a function of water loading. The authors argue that the topology of the solvent molecule is responsible for the shape of the assembly as well as the driving force for association. For the linear chain alkanes, there is a steep increase observed for the ε value as a function of water content, as water is initially introduced into the systems, and its slope is found to increase in the order of heptane < octane < decane. This is taken to suggest that the aggregates formed in decane are most unsymmetrical and consequently, highly strained. For the linear solvents, another interesting observation is that beyond a certain Wo value, a steep increase in the ε value occurs, which is thought to be due to a structural change in water domain induced by the formation of the ‘bulk’ water region. On the other hand the slope was found to be moderate in the cyclohexane system and there was no steep increase in ε evident, which implied that the reverse micelles formed in cyclohexane are nearly spherical and that their size is considerably small for a wide Wo range, in contrast to those formed in straight chain alkanes. The latest use of dielectric spectroscopy in AOT reverse micelles [171] employs five different hydrocarbons; cyclohexane, heptane, octane, decane, and dodecane. The authors show that the percolation onset (evidenced by the steep increase in ε) is shifted to lower Wo with increasing length of the solvent, which is explained on the basis of larger reverse micelles formed in higher alkanes. Within the dodecane reverse micelle series, the percolation onset (evidenced by the steep increase in conductivity) shifts to lower Wo as AOT concentration increases. A similar observation was obtained by Manabe et al. [172] which was explained by a charged-particle model by Eicke, [173] and improved later by Hall [174]. Peyrelasse and Boned [175] however, explain the phenomenon by a surface charge model.

28 Chapter 2 State of the art

Beginning from the mid nineties, the GHOST group has published many papers on water in AOT reverse micelles at microwave frequencies [118-120,126,176-181] where the dielectric response is dominated by the water component. Results from this group show that the dielectric spectrum consists of two relaxation regions; one at about 100 MHz and the other above 20 GHz. In the low frequency region at low Wo values, where the almost dehydrated reverse micelles exhibit a rigid structure, the dielectric relaxation is attributed to the rotational diffusion of the spherical micelles having a permanent dipole. The radii of the reverse micelles obtained from the observed relaxation time, from calculations based on the Stokes equation, are found to agree well with those reported earlier by other techniques. They further found that the relaxation time τ1 decreases with an increase in Wo upto ~ 15, beyond which it remains constant. This is found to be analogous with the trend observed for the mole fraction of bound water in the reverse micelles as a function of Wo as reported by the IR studies. This suggests that the progressive increase of mobility of AOT polar groups upon increase in water content, continues until the hydration structure around them is complete.

With their investigation of AOT reverse micelles in CCl 4 and heptane, Camardo et al.

[179] suggest that as Wo increases, an increasing number of AOT ion pairs could achieve sufficient mobility to reorient independently from the whole microaggregate. On this basis, the relaxation phenomenon observed at the highest Wo was attributed to the rotational diffusion of the ‘free’ AOT ion pairs at the water-surfactant interface. They further observed that at the highest Wo values, the investigated dielectric dispersion is located close to a relaxation process observed in concentrated electrolyte solutions and attributed to the rotational diffusion of dipolar solute species (ion pairs), as reported by Buchner [182,183].

2.4 Small angle x-ray scattering In the eighties and the early nineties, the results of small angle x-ray scattering (SAXS) studies on L1 and L2 microemulsion phases in the AOT system at low content of the dispersed phase was interpreted mainly within the framework of liquid state theories. The microemulsion droplets were characterised with respect to their size, polydispersity, form fluctuations and interaction parameters. The linear dependence of the droplet size on Wo was established. Average droplet radii were determined and their populations displayed a moderate (about 20 %) size polydispersity [184-188]. Increasing interaction between the

29 Chapter 2 State of the art droplets observed up on raising the temperature were described using Baxter’s model by introducing a temperature dependent stickiness parameter in the hard-sphere potential, leading to a phase transition at higher temperatures [189-191]. Evidence for the formation of cylinders at low Wo was found by SAXS for water-in-oil microemulsions stabilised by bis(2- ethylhexyl)sulfosuccinate with bi- or trivalent cations other than sodium [192-194]. In order to investigate the influence of the non-polar surrounding medium on the AOT reverse micelles, Hirai et al. have undertaken a SAXS study in four different solvents, namely n-hexane, n-heptane, n-octane, and iso-octane. Their results support the multi-state water theory in reverse micelles, and based on their plot of radius of gyration (R g) vs Wo, they endorse the existence of three different micellar water regions [195-197]. These regions are termed oligomeric (0 < Wo < ~8), transient (~8 < Wo < ~16), and momomeric (~16 < Wo).

In addition to the enlargement of the water pool with Wo evidenced by the increase in scattering intensities in the small q region; and the appearance of a bell-shaped profile for the p(r) function, the main finding of this paper is the dependence of micelle size on the nature of oil employed. As was predicted theoretically [198], the authors suggest that there is a penetration limit of apolar solvent which corresponds to the hydrocarbon chain length such that short chain apolar solvents penetrate easily into surfactant layers. This penetration enlarges the spontaneous curvature of the surfactant layer to reduce the micellar radius, as shown by their plot of slope of micellar radius and Wo against linear hydrocarbon chain length. The authors further propose that above a chain length of 9.5, the penetration of apolar solvent molecule in reverse micelles would hardly occur. Also, they find that within experimental error, the radius of dry AOT micelle does not show a hydrocarbon chain length dependence, which is attributed to the icosahedral packing of polar heads at Wo = 0. Amaral et al. have determined the structure of AOT/n-hexane/urea/water reverse micelles to seek an explanation for some of the observations of the percolation phenomenon [199,200]. It was previously shown that the percolation threshold in AOT reverse micelles can be altered by the presence of additives like gramicidin, acrylamide, urea and its derivatives. However these studies did not explain the formation of permanent bicontinuous structures above the percolation threshold. By using 3M and 5M urea solutions in AOT reverse micelles, the SAXS intensity plots in conjunction with their light scattering data show that the incorporation of urea enhances attractive interactions between reverse micelles. They

30 Chapter 2 State of the art show that for the system with 5M urea, the influence of attractive forces is more intense in the lowest studied concentration; φ = 0.06. Incidentally, this concentration corresponded to the φ value where a sudden increase in solution conductivity, associated to percolation was observed [201]. SAXS results indicate therefore that the structure of the discrete spherical droplets is retained after the percolation phenomenon. This suggests that the percolative transition does not occur from spherical droplets to bicontinuous phase but from non- interacting droplets ( φ = 0.06) to clustering ones due to inter droplet attractive interactions. Kotlarchyk, Shen, and Capel [202] investigated the phase behaviour in AOT/decane/water system with varying temperature by SAXS and demonstrated that the structure at room temperature for 0.60 ≤ φ < 0.75 is the droplet, the high temperature phase has the lamellar structure, and that the coexistence region of the droplet and lamellar structures covers a wide temperature range. The droplet (micelle) radius and the water core radius, both decreased with increasing φ . This can be explained if one allowed deviation of droplets from the spherical shape. With increasing droplet volume fraction, droplet-droplet distance decreases and the effect of the attractive forces of the hydrocarbon tails of surfactant molecules become more significant; two layers of neighbouring droplets adhere to each other with the possibility of droplet deformation. When droplet volume is kept constant, the mean radius of deformed droplets is smaller than that of the sphere. This is supported by the decrease in the width parameter with increasing droplet density due to increasing polydispersity. The calculated form factors for φ = 0.4, 0.5, and 0.6 reconfirm that the shape of the droplets depend on the droplet volume fraction, while the structure factor indicates that the gap between droplets becomes smaller with increasing φ , and that a close packed structure was formed at φ = 0.60. Pressure dependencies of the SAXS profile for φ = 0.40 and φ = 0.65 shows the presence of two peaks. The first broad peak gradually disappeared with increasing pressure. A new peak appeared at higher Q which increased in intensity with pressure. This indicates that the droplet structure at ambient pressure transforms to the lamellar structure with increasing pressure, at 0.40 ≤ φ ≤ 0.65, through the transient state with coexisting droplet and lamellar structures. However, the percolated droplet structure persisted up to about 20 MPa independent of φ . These results are consistent with earlier reports which state that the

31 Chapter 2 State of the art inter-droplet attractive forces with increasing pressure is the origin of the pressure induced phase transition.

2.5 Other techniques 2.5.1 The water libration band and terahertz spectroscopy In addition to the linear IR spectroscopy of the hydroxyl stretch, other optical experiments performed on AOT reverse micelles include the study of the libration motions and the terahertz frequency spectroscopy of the surface modes. The libration band occurring near 670 cm -1 was studied by Venables et al. [203] for water in AOT reverse micelles. They found that with increase in the micelle size, the band shifted to lower frequencies and gradually approached the shape of neat water. They further observed that the libration band at all compositions could be fit by a two-state water model based on the relative fractions of bound and free water molecules. With the recent advances in terahertz time domain spectroscopy, it has become possible to arrive at the properties of liquids below 100 cm -1. This far infrared spectroscopic technique provides sensitivity to the collective modes of liquid water arising from hydrogen bonding [204]. The dielectric function of neat water exhibits its primary relaxation features in the far IR region, between 1 and 100 cm -1. The modes pertaining to these relaxations are generally described in terms of a double Debye model [205]; but are nevertheless thought to be collective in nature. Boyd et al. [206] found that with increase in micelle size, the peak frequency of absorption moves from high frequency (~ 1THz at R = 15 Å) to lower frequency (~ 0.2 THz at R = 45Å) with a decrease in peak amplitude. They also showed that these absorptions are resonant in nature by the circular Cole-Cole plots of the spectra. This strong modification of the frequency-dependent dielectric function of water has been thought to arise from the restricted dimensions of the micelle cavity. They propose the surface oscillations of the confined droplet to be responsible for the observed spectral behaviour.

2.5.2 Non-linear infrared spectroscopy Non-linear infrared spectroscopy has been widely used of late, to study the confined water in AOT reverse micelles. Using this technique, many research groups, particularly those of Fayer and Bakker have reported on the presence of more than one component in the confined

32 Chapter 2 State of the art water vibrational lifetime decay, thereby suggesting the existence of more than one ensemble of hydrogen bonding environment. These groups are unanimous in the proposition of a ‘free’ and ‘interfacial’ water region in AOT reverse micelles [207-209]. Dokter et al. [209] in particular, observe using isotopically diluted water that the molecular mobilities of these two water species are different. They found that the core water reorients on a time scale close to that of free water (2-4 ps), while the interfacial water is highly immobile. In other studies using neat water confined in the reverse micelles, it was shown that the vibrational relaxation rate increases with increasing micelle size [210,211]. But owing to rapid intermolecular energy transfer, these authors could not ascertain the average dynamics of the water molecules. The observation of slow orientational dynamics for the interfacial water molecules is in agreement with recent molecular dynamic simulation on micellar systems [212]. These simulation studies have suggested that water molecules can remain bound to micellar surfaces for more than 100 ps. The inhomogeniety underlying the molecular reorientation of free and interfacial waters has been rationalised based on the difference in the activation energy for the reorientation process for the different water species. For core water molecules, the activation energy for reorientation is substantially lowered, because these molecules can break a hydrogen bond while simultaneously forming a new hydrogen bond with another water molecule, similar to neat water. For interfacial water molecules, this process is inhibited as they are hydrogen bonded to heavy immobile surfactant molecules. This causes a slow reorientation and slow hydrogen bond dynamics in interfacial water molecules.

2.5.3 NMR spectroscopy Using high resolution 1H, 2H, 13 C, 23 Na and very recently 51 V NMR spectroscopy, intermolecular interactions and structural rearrangement of AOT reverse micelles have been investigated. The 1H NMR chemical shifts ( δ) for water in AOT reverse micelles have been reported by a number of groups [152,213-218]. These studies are in agreement with the multi-state water model. At high Wo values, δ approached 4.8 ppm which is the same as that for neat water; while at lower hydrations, the smaller δ indicated a weak hydrogen bonding in the confined water.

33 Chapter 2 State of the art

Maitra [213] showed by 1H NMR spectroscopy that the effective surfactant length in the reverse micelle droplet is dependent on the water content as well as on the nature of the bulk solvent. He computed the thickness of the bound water layer to be 4.8 Å and concurred that with increasing micelle size, the bound water thickness reduced as the dissociated ions are distributed more to the free water core under these conditions. He obtained the size parameters for the water droplets in his two model systems; AOT/cyclohexane/water and AOT/i-octane/water from the chemical shifts of the water proton resonance, which agree well with those reported in the literature. By 2H NMR spectroscopy, Hauser et al. [219] showed that there are thirteen water molecules bound to AOT; of which two are more tightly bound than the rest. Wong et al. [152] showed by 1H NMR spectroscopy that the effect of increasing water in the micellar interior on 1H nuclear longitudinal and transverse relaxation rates demonstrates that the water is highly immobilised and the mobility increases with increasing water content. This paper also reports on the effect of water on the 23 Na NMR spectrum. Based on the observation that the line width in the largest water pool is about 10 times broader than those in saturated NaCl solutions, they suggest that the mobility of hydrated Na ions even in the largest AOT reverse micelles is still greatly reduced. They find that in their largest water pool (~6% water), about 25% of the Na + ions are dissociated from the sulfosuccinate head groups. 1H NMR studies on the water structure on AOT reverse micelles have been carried out by El Seoud [220,221]; who is one of the strongest proponents of the uniform-water structure in these aggregates. His method relies on the calculation of a so-called fractionation factor based on the observed chemical shifts of reverse micelle solubilised and neat waters. He argues that a unity fractionation factor for all aggregate solubilised water does not warrant the treatment of experimental data in terms of a co-existence of structurally different water layers. He concurs that the change in slope of certain physical parameters upon increase in

Wo may be attributed to the ‘expected’ decrease in water-AOT interactions after completion of the hydration of its head group, as opposed to the notion of formation of a second, bulk- like water. A recent paper from the Levinger group [222] probes the interior water pool of AOT reverse micelles using a highly charged decavanadate (V10) oligomer using 51 V NMR spectroscopy. The authors found that the negatively charged probe sequestered in the free

34 Chapter 2 State of the art water pool provides information on two vital properties: the local pH and the microviscosity of the intramicellar water pool. It is generally believed that at Wo values greater than 10, water in AOT reverse micelles approaches the properties of neat water. But the results presented in this paper, at Wo values all higher than 10 (12, 16, and 20) indicate otherwise. Furthermore, the V10 molecule shows that the core region of the reverse micelles remains at an apparent pH near neutral. This has been thought to be due to the migration of protons towards the reverse micelle interfacial regions, leaving the core region with counterions, but no excess protons. A follow-up paper from the same group [223] characterises the combined effects of intramicellar pH, microviscosity, ionic strength and temperature in the aqueous reverse micelles based on information from the 51 V NMR chemical shifts, line widths, relaxation times, and vanadate speciation. The authors found that when stock solutions were added to large reverse micelles ( Wo>12), the line widths of the vanadate monomer and dimer decreased, whereas the line widths increased in the smaller reverse micelles. The observed increasing line widths are thought to reflect an environment of decreased mobility and increased microviscosity. The authors further established changes occurring in the reverse micelles based on vanadate speciation. They found that at concentrations above 25 mM where the bulk aqueous solution contains only a monomer, a dimer was detected in the reverse micelle. This dimer formation in the reverse micelles was attributed to a decrease in pH in the reverse micelle along with an increase in ionic strength and viscosity.

2.5.4 Simulations The first comprehensive study of the interior of aqueous reverse micelles by molecular dynamic simulations was undertaken by Faeder and Ladanyi [224]. The simulations prior to this, based on both atomistic and reduced models had many shortcomings. The main focus of this work was to characterise the water interfacial structure at a molecular level. In addition to the identification of three distinct water regions, the paper provides new insight into water orientation and mobility in the reverse micelles. Based on the density profiles of the water oxygens the authors elucidate the fractions of the three water species, which are in good agreement with those reported by most IR studies. Further, they provide evidence for the water at the interface being tightly bound and rigid. The sodium ion density profiles indicate that at the smallest micelle size ( Wo = 1), nearly all of the counter ions reside on the surface

35 Chapter 2 State of the art and are triply coordinated by the sulfonate ions, forming a tightly packed rigid lattice which also includes a small number of water molecules. With increase in micelle size however, a small fraction of counterions (~10%) form solvent separated ion pairs or dissociate from the surface altogether. The water mobilities are quantified by the effective diffusion coefficient, which indicates that the water in the bound region is much more mobile than trapped water and has mobility closer to that of the free water, although the difference in mobilities between the bound and free water increases with

Wo. Mobility can also be characterised by the lifetime of molecules in the various regions, and it was found that the residence times of bound water in even the largest micelle are an order of magnitude longer than those of a typical hydrophobic cavity, which suggests that a larger portion of the mobility in the micellar systems occurs within the layers parallel to the interface. Faeder et al. [225] have also studied the effect of counterion size (Na + and K +) on the properties of the interior region of reverse micelles in the low Wo region. Abel et al. [226] present a theoretical investigation to provide for the first time, a fully atomic molecular model of the AOT reverse micelle. Their findings indicate that the reverse micelle and the confined water region are ellipsoidal in shape. Though the authors considered only monodisperse systems, they nevertheless find good agreement of their reverse micelle radius, and linear dependence of radius versus Wo data with some experimental findings. An atomic level molecular modelling of one AOT molecule and its interactions separately with water and carbon tetrachloride were reported by Derecskei et al. [227]. The phase diagram of AOT/water/oil has also been simulated via dissipative particle dynamics simulation method

[228]. With the exception of some differences in the sizes of the L2 and liquid crystal regions, the simulated phase diagram was found to be in good accord with the experimental one of Tamamushi et al. [229].

2.5.5 Molecular probes Frequently fluorescent probe molecules with sensitivity to specific properties of the system have been used to explore AOT reverse micelles. Of the many probes in use, the fluorescent coumarin dye C-343, has been found to be a particularly effective probe of solvation dynamics in reverse micelles [230-235]. In a recent publication employing this probe [235]

36 Chapter 2 State of the art differences in steady state spectra of C-343 in hydrogen bond donor solvents like water and polyols & non hydrogen bond donor solvents like dimethylacetamide and dimethylformamide in confinement were shown. In water containing reverse micelles, the C-

343 steady-state absorption and emission spectra shift to lower energy as Wo increases. Even at substantial water loading, the C-343 spectra maximize at a wavelength removed from those in neat water suggesting that the probe is located away from the intramicellar water and AOT head groups, where there are no effective hydrogen bonding sites. Among other probes, the molecule 8-anilino-1-naphthalene-sulfonate (ANS) has been successfully used for the time and space resolved studies of interfacial water in the AOT system [236]. The luminescence behaviour of 7-azaindole in AOT/heptane reverse micelles reveals a red-shift of the fluorescence emission along with a significant decrease in the quantum yield with increase in Wo. This was thought to be due to the location of the dye at the interfacial region of the micelle, such that its microenvironment became progressively polar upon increase in hydration [237].

2.5.6 Calorimetry Calorimetric studies have contributed a great deal to the data on water states in AOT reverse micelles, particularly in the late eighties and early nineties, when researchers were beginning to gather information from infrared spectroscopy. Aprano et al. [238] measured the molar enthalpy of AOT reverse micelles, and found that it was endothermic, and that it increased with a decrease in Wo, which was interpreted in terms of a semi-empirical model of water partitioning between two states. Goto and coworkers [239,240] later came up with a three- state water model based on their thermochemical measurements on this system. This proposition was based on their plot of relative molar enthalpy against Wo, which showed a maximum at Wo ca. 1, followed by a sharp fall and a successive gradual decrease from Wo 2 to 4. It then passed through a plateau between Wo 4 and 11, and then reached 0 at Wo above 15. These findings led to the conclusion that the dissolution states of water molecules change at low hydration from the immobilised to structured (ionic hydration) state of water, and that bulk water appeared above an approximate Wo 11 to 15. Their reasoning that the water molecules at ~ Wo = 1 are immobilised was found to be in agreement with the data presented by Martin and Magid [241], Eicke and Christensen [242], Ueda and Schelly [243], and

37 Chapter 2 State of the art

Hauser et al. [219]. It however is worth mentioning that the three-state water model considered here is different from the ones encountered in IR studies. According to Goto et al. , the three water regions include the water in the core of the pool; water located near the inner surface of the microemulsions; and the third region being located in between these two, thereby disregarding the trapped water.

2.5.7 Other experimental techniques In addition to the techniques mentioned above, various other experimental methods have also been used to elucidate the structure and dynamics of AOT reverse micelles. Small angle neutron scattering, for instance, was used to study the pressure induced phase transition in AOT/water/decane [244]. The pressure effect has also been studied by Köhling et al. in the AOT/n-octane/water system in the 0.1 – 3000 bar region [245]. At high AOT concentration and hydration level, pressure induced phase transition from the L 2 to a bicontinuous L 3 or a lamellar phase was observed. Kotlarchyk et al. have obtained information regarding the size, shape, aggregation number and internal structure of AOT/decane reverse micelles [246]. Shukla et al. report on the observation of two diffusive relaxation modes in a concentrated AOT reverse micelle due to density and polydispersity fluctuations [247]. Light scattering has often been used to derive the hydrodynamic radius of the reverse micelles [248-250]. ESR spin labelling was applied to characterise the polarity and the fluidity of the different compartments in the AOT system [251]. Acoustic spectroscopy [252] was used to characterize the droplet size distribution in the AOT reverse micellar system, and the technique along with small angle neutron and x-ray scattering measurements was found to yield the same linear dependence of the mean droplet size on the Wo value. Ultrasonic relaxation in AOT/heptane/water reverse micelles have provided insight into the shape fluctuation of the system, and the exchange of water molecules between the polar sheath of the micelles and the water pool [253].

2.6 Non-aqueous reverse micelle interior Some polar organic solvents, having high dielectric constants and negligible solubility in the surrounding non-polar medium can also be solubilised in reverse micelles. The most common polar solvents employed for this purpose include methanol, formamide,

38 Chapter 2 State of the art dimethylformamide, ethylene glycol, propylene glycol and glycerol [235 and references therein]. As in the case of water, these polar solvents are also confined to the nano core of the AOT reverse micelles, where they exhibit properties different from those of their bulk counterparts [230]. For example, FT-IR [230,234] and 1H NMR [255,256] spectroscopy have shown that glycerol and ethylene glycol interact with the AOT polar head through hydrogen bond interactions that maintain the typical spherical reverse micelle structure. Interestingly, these two solvents do not show any evidence for the properties of the neat liquid even at the highest solvent loading inside the reverse micelles. In contrast, formamide has been shown to retain the neat solvent properties within the confines of the AOT reverse micelles [256]. Not unexpectedly, the non-aqueous polar solvent containing reverse micelles have been found to swell much more rapidly than those containing water, attaining a size similar to Wo = 10

(aqueous) with W s ≈ 2 (non aqueous) [231,257].

2.7 Proteins in confinement A noteworthy characteristic of the interior of the cells is the high total concentration of macromolecules contained therein. Such an environment is usually called ‘crowded’ or ‘volume occupied’ rather than concentrated because no single macromolecule occurs at a high concentration, but put together, all the macromolecules account for a significant fraction (~20-30%) of the total volume. This fraction is thus physically unavailable to other molecules. However, most experiments in biochemistry are performed in dilute solutions where the total macromolecular concentration ranges from 1-10 g/L or less, wherein crowding is negligible. Overlooking this glaring physiological difference doesn’t help matters because crowding has both thermodynamic and kinetic effects on the properties of macromolecules. These effects are so pronounced that many estimates of reaction rates and equilibria obtained in dilute solutions differ greatly from those of the same reactions performed in crowded conditions [258,259]. Figure 2.5 shows a cross section of an Escherichia coli cell crowded with macromolecules. It has been determined that the total concentration of protein and RNA inside a cell of Escherichia coli is in the range of 300-400 g/L [260]. In such a crowded milieu, even if one assumes that there is no specific interaction between a protein and the other cellular components, the mere presence of other molecules in close proximity of a

39 Chapter 2 State of the art protein is enough to affect its stability and reactions [261-265]. This is commonly termed the excluded volume effect which may be manifested either as macromolecular crowding or confinement effects. The effects that arise from the presence of other soluble macromolecules are called macromolecular crowding, and the effects due to the presence of impenetrable boundaries are termed confinement effects [266].

Figure 2.5 Cross section of a small portion of Escherichia coli cell. The cell wall, two concentric membranes, transmembrane proteins and flagellum are shown in green. The cytoplasm is shown in blue and purple and nucleic acids are shown in yellow. [Reproduced from http://mgl.scripps.edu/ with the permission of David S. Goodsell, The Scripps Research Institute, La Jolla, CA]

2.7.1 A first look at macromolecular crowding The cellular cytoplasm is not only crowded, but also organised. The ubiquity and significance of molecular crowding in cells was first addressed by Ogston and Laurent more than four decades ago, and subsequently revived by the work of Arthur Kornberg. A. G. Ogston invoked the concept of macromolecular crowding to a steric model of spheres of bovine serum albumin excluded from a network of thin fibres of hyaluronic acid [267]. His approach to the concept was two-fold. He first looked upon it in a mechanistic way and calculated from first principles the excluded volume for a spherical particle with a given diameter in a random suspension of thin fibres. Later, from a thermodynamic point of view, by expressing the activity coefficients in algebraic forms, he determined the osmotic properties of the system [268]. In 1964, T. C. Laurent gave experimental basis and validated

40 Chapter 2 State of the art

Ogston’s calculations [269]. Laurent subsequently measured the solubility of various proteins in the presence of dextran and found that the larger the protein, the more its solubility was depressed in the presence of the polymer [270]. This observation was soon found to be of great use in increasing sensitivity in immunological analyses. Arthur Kornberg was among the first to grasp the physical significance of molecular crowding in his work on DNA replication. In their quest to make DNA replication work in vitro , he and his colleagues discovered that a high concentration of poly(ethyleneglycol) sets the system in motion. This polymer mimicked the crowding in cells and stabilised the binding of essential proteins to the origin of replication. The 1980’s saw a resurgent tide of interest in macromolecular crowding sweep over the scientific community. The most important work from this time came from Steven Zimmerman and Allen Minton. One of Zimmerman’s discoveries that the addition of poly(ethyleneglycol) increases the efficiency of DNA ligation has become the cornerstone of molecular biology today. The greatest contribution of Minton to molecular crowding comes from his development of a theoretical framework to describe its effects [271].

2.7.2 The biological membrane Biological membranes form the interface between the cell and its environment, and are key players in cellular homeostasis and metabolic-energy transduction. These membranes are however more complex than was thought when Singer and Nicholson’s widely celebrated fluid-mosaic model was first proposed in 1972 [272]. The model considers a lipid bilayer to be the main fabric of the membrane, into which are plugged a variety of membrane proteins. The bilayer, considered to be a pseudo two-dimensional liquid in which both lipids and membrane-associated proteins display sufficient lateral mobility imparts a certain degree of fluidity to the bilayer component. The fluid mosaic model which relies heavily on earlier models of membrane architecture (Danielli-Davson model, Robertson model), now faces some questions based on the generalisations it pre-supposes. The model posits that the membrane proteins occur at low concentrations and are dispersed such that they match the hydrophobic dimension of an unperturbed lipid bilayer with peripheral belts of exposed hydrophobic side chains. Further, the lipid bilayer is envisaged as a sea in which mainly monomeric proteins float unencumbered, such that the bilayer surface is directly in contact

41 Chapter 2 State of the art with the aqueous environment. Notwithstanding the insightfulness of the model, progress over the last three decades considers the generalisations propounded by the model misleading [273]. One of the models suggested to refine the Fluid-Mosaic model was by Israelachvili, who incorporated the need of membrane proteins and lipids to adjust to each other in his membrane structure. This model also includes membrane folding, pore formation, and thickness variations, as well as some degree of heterogeneity in its purview. Another elaboration of the fluid-mosaic model was developed by Erich Sackman, and it emphasises the importance of the cytoskeleton and the glycocalyx [274].

2.7.2.1 Reverse micelles as membrane mimics Reverse micelles are now being increasingly used as model systems that serve to mimic the membrane action in the study of confined proteins. Many surfactants including AOT, Cetyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) have been used for this purpose, and there exists a huge literature on AOT reverse micelles being employed as membrane mimics. The AOT monolayer in a reverse micelle has long been considered akin to a biological membrane, as the former surrounds and compartmentalises the polar micellar core -- which may well be alluded to the cellular cytoplasm. Extensive research has been done on the encapsulated alpha chymotrypsin ( α-CT) in AOT reverse micelles, probing its enzymatic catalytic activity, its secondary structure variation upon confinement, etc. and all these investigations point to a unified picture of a more stable protein inside the AOT reverse micelle. Some of the major findings from such research are presented in Table 2.1. In addition to α-CT, many other proteins like ribonuclease, lysozyme etc. have also been studied in confined geometries [95].

2.7.2.2 Encapsulation of proteins in reverse micelles Currently there are three methods to incorporate a protein in a reverse micelle. These are: injection of a concentrated aqueous protein solution, addition of dry lypophilised protein to a reverse-micellar solution, and phase transfer between aqueous and surfactant-containing organic phases [284]. The injection and dry-addition methods are commonly employed in biocatalytic application; the latter being particularly well suited to hydrophobic proteins. The phase transfer technique forms the basis for extraction of proteins from aqueous solutions.

42 Chapter 2 State of the art

Addition of a solute in reverse micelle could produce a small change in the volume V, denoted by dV , or in the interfacial area A, denoted by dA . Qualitatively speaking, the average localization of guest molecules in reverse micelles can be summarised as depicted in Figure 2.6 [285].

V R = A

(V + dV ) R = A

V R = ()A + dA

V R = ()A − dA

Figure 2.6 The localisation of guest molecules in reverse micelles.

(i) Guest molecules soluble in hydrocarbons remain in the outer bulk phase and have no discernible effect on the micellar structure and radius. (ii) The addition of solute in water pools can induce an increase in volume, dV , while the interfacial area remains constant. The radius of the water (V + dV ) pool in this case is given by R = . Here, the variation of the A micellar mass concomitant to the solubilisation of the hydrophilic component is equivalent to the perturbation observed upon addition of the same volume of water.

43 Chapter 2 State of the art

(iii) The addition of a solute anchored at the interface of the water pool induces an increase of the interfacial area, dS , whereas the volume of the water pool remains constant. Then the water pool radius is given by V R = . Hence an addition of a solute that gets located at the ()A + dA interphase, induces a reduction in the micellar radius. This scenario is synonymous with the increase of surfactant concentration at constant water content: more interface is available to trap the same quantity of polar compound. (iv) Addition of solutes larger than the water pool can lead to the formation of smaller aggregates surrounding the solute. Hence two micellar populations could be in equilibrium. If the solute is surrounded by a coat of surfactant, the volume of the water pool remains constant, whereas the interfacial area V of the empty micelles decreases, leading to R = . ()A − dA

α-CT, inside the AOT reverse micelles is located in the aqueous central core [285- 287], in accordance with the water shell model [288,289] of the uptake of guest molecules. According to this model, the protein is surrounded by one or more layers of water molecules that protect the macromolecule from the denaturing effects of the surfactant and organic solvents. α-CT is one of the widely studied digestive enzymes, whose catalytic mechanism and physicochemical properties are well established [290]. It is a 25 kDa serine protease consisting of three chains connected by five disulfide bonds and it catalyses the hydrolysis of peptide and ester bonds. The 3D structure of α -CT has been determined to be a compact ellipsoid of dimensions 40 x 40 x 51Å 3 and a molecular volume of 43,000 Å [291].

44 Chapter 2 State of the art

Figure 2.7 Structure of α-Chymotrypsin (E.C. 3.4.21.1) rendered by PyMol ( α helix: red, β- sheet: yellow, loop: green).

2.7.3 Osmolytes Osmolytes are small solute molecules that affect proteins by their osmoregulatory function, and are ubiquitous in nature. Depending on their action, they are commonly referred to as kosmotropes and chaotropes, the former being protein stabilisers and the latter acting to destabilise a protein. These terms are also used to refer to the apparent property of increasing, or decreasing respectively the structuring of water. Kosmotropes are also termed compensatory solutes [292] because of the physiological advantage they confer to the cell due to their presence, in that their concentration can be adjusted in response to stress. This enables the same cellular macromolecular structures to function over a wide range of external conditions. Kosmotropes are generally classified into two categories; the first consisting of polyols like glucose, sorbitol etc, while the other comprising of zwitterions containing a relatively hydrophobic cationic region; examples being trimethylamine oxide, glycine betaine, ectoine etc. The guanidinium ion and the thiocyanate ion are two of nature’s strongest denaturing agents. These ions and urea at high concentrations are considered excellent chaotropes. Although there exists a large literature on osmolyte action including both experimental and theoretical techniques, the exact mechanism by which osmolytes alter the protein stability still has room for ambiguity. There are in general two schools of thought that

45 Chapter 2 State of the art strive to explain the mode of action of the osmolytes. The preferential exclusion/preferential bonding model of Timasheff [293-298] advocates that in solution, kosmotropic molecules are preferentially excluded from the protein surface, thereby stabilising the folded state of the protein relative to the unfolded state because it exposes less surface area from which the osmolyte must be excluded. This altering of the hydration layer, rather than the kosmotrope contact with the protein is thought to stabilise proteins. Chaotropic molecules, on the other hand, bind directly to protein molecules thereby resulting in denaturation. Urea and GdHCl are known to preferentially bind to peptide groups on the unfolded protein. The other school of thought seeking to explain the osmolyte action on protein stability concurs that osmolytes alter the hydrogen bond network in water molecules surrounding the proteins which affects the protein stability. Kosmotropes are thought to be stabilising solutes that shift the local less dense water ⇔ more dense water equilibrium to the left by increasing the order of water, while chaotropes are believed to shift the above equilibrium to the right. [299,300] Chaotropes break down the hydrogen-bonded network of water, thereby allowing proteins more structural freedom, which encourages unfolding and denaturation.

46 Chapter 2 State of the art

Table 2.1 Properties of α-CT in AOT reverse micelles.

Hydrocarbon Major Result Technique Reference

i-octane micellar properties not affected by protein uptake, protein located in central water pool Synchrotron-SAXS 195 (and not at the interface)

i-octane enzyme obeys Michaelis-Menten Kinetics and K m values are higher than those in bulk water; UV, fluorescence ,CD 275 under conditions of low water content, enzyme's stability is greater than in bulk water.

n-heptane enzymatic activity depends on water content, and it may be restored at higher Wo. fluorescence 276 The hydrolysis of 2-naphthyl acetate catalysed by α-CT occurs at the micelle interface. spectroscopy

n-heptane protein in RM suffers no major conformational change CD 277

n-octane average polarity in the vicinity of the probe approaches that of bulk water at Wo>12 fluorescence 278 hydration picture: AOT head group hydration occurs first; protein spectroscopy hydration occurs next and is complete by Wo=10.

n-octane enzyme activity depends on the amount of water in contact with it and not on the total UV, fluorescence 279 bulk water in the system.

i-octane micellar properties not affected by protein uptake SAXS 280

i-octane changes observed in protein secondary structure in RM and bulk FT-IR 281 In RM: α-helix and β-sheet decrease; random and turn structures increase

i-octane protein incorporation affects structure and phase behaviour of the system, SANS 245 with pressure enhancing the changes.

n-octane pressure induced protein unfolding is different from that in bulk water FT-IR 282

n-octane enzyme stability decreases with increase in temperature (20-40°C) UV 283 catalytic activity and thermal stability maximum at Wo=10 High pressure (1-1500 bar) stabilises protein against thermal denaturation at all Wo.

47 Chapter 2 State of the art

2.7.4 Hydration water The unique physical and chemical properties of water have long been held responsible for life on earth. Biomolecules, in particular proteins, require water for folding and other biological functions. The amount of hydration water molecules adsorbed on to the protein surface, i.e. hydration levels of proteins, are critically related to the thermodynamic properties of the protein. Protein specific heats and thermal stabilities are greatly influenced by their hydration level, and a minimum monolayer hydration of protein seems inevitable for proper protein functioning. It has been estimated that about 0.34 – 0.39g of water/g of protein is essential for protein monolayer hydration in the crystalline state at cryogenic temperatures [301]. However the properties of this hydration water for proteins in solution at or close to physiological temperatures, has been a contentious issue. The hydration water molecules are generally classified into the inside, contact, the first layer and the second layer waters [302]. The ‘inside’ class comprises of water molecules buried in the protein cavities. The ‘contact’ class of molecules are located just outside the solvent accessible protein surface and mediate intermolecular interactions between adjoining molecules. The ‘first’ and ‘second layer’ water molecules are located outside the solvent accessible surface. While the first layer molecules interact with the protein surface through hydrogen bonds or van der Waals interactions, the second layer molecules display no interaction with the protein surface. There are many experimental techniques that detect and quantify the hydration water molecules accompanying protein in solution. SAXS [303], dielectric spectroscopy [304,305], magnetic relaxation dispersion [306] and computer simulations [307,308] have been found to provide valuable insight into the protein hydration structures and dynamics. McCabe and Fisher have demonstrated a method to determine the excluded volume of aqueous solutions of proteins and alkali halides using NIR Spectroscopy [309]. They show that the NIR difference spectra of aqueous solutions measured against water consist of three components: 1. a negative component consisting of an absolute spectrum of the amount of water excluded by the hydrated solute. 2. a positive component, contributed by the water of hydration of the solute, and 3. an additional component consisting of the absorption (if any) by the solute itself

48 Chapter 2 State of the art

This method was later successfully used to investigate the hydration of alkali halides [310] and 18-Crown-6 in aqueous solutions [311]. The basic characteristic of excluded volume effect is the mutual impenetrability of all solute molecules. This non-specific steric repulsion is ever present irrespective of the nature of interactions between the solute molecules. McCabe and Fisher’s concept has thus been adapted to yield the excluded volume and the hydration number of α-CT solution prepared in an osmolyte contained within the AOT/cyclohexane reverse micelles.

2.8 Ionic liquids in confinement ILs are highly solvating, non-coordinating media that allow the dissolution of a wide range of organic and inorganic solutes. The negligible vapour pressure of ILs and the possibility to alter the cation/anion combination to yield liquids with tunable properties like hydrophilicity/ hydrophobicity, acid/base character, viscosity, conductivity etc. has proved to be a boon to researchers in general, and synthetic chemists in particular, as these liquids are widely regarded as the ‘green solvents’ to replace many of the industrial solvents currently in vogue. The number of potential cation/anion combinations reputedly equate to 1 trillion (10 12 ) different ILs. [312] The Green Chemistry angle of interest in these solvents notwithstanding, the design of task specific ILs, such as chiral species have also stimulated a great deal of interest. The history of ILs dates back to the mid-nineteenth century when chemists reportedly discovered the first IL in an AlCl 3 catalysed Friedel Crafts alkylation. The ‘red oil’ that formed during the course of the reaction was eventually identified to be a stable intermediate of a carbocation and a tetrachloroaluminate anion [313,314]. The mid to late twentieth century saw some practical applications of imidazolium and pyridinium based salts as electrolytes in batteries and as solvents in electroplating [315]. It is thought that many viscous oils and unwanted by-products reported by chemists in their reactions during this time were infact organic liquid salts, which were unfortunately not analysed in detail beyond their initial identification. The early 1990s saw a rapid increase in interest in these salts with the synthesis and characterisation of organic salts of various anions like hexafluorophosphate, tetrafluoroborate, nitrate, methanesulfonate (mesylate), trifluormethanesulfonate (triflate) and bis(trifluoromethanesulfonyl)amide (TFSA). With the

49 Chapter 2 State of the art surging interest in ILs in both academia and industry, several excellent reviews on the subject have appeared that provide a comprehensive description of these neoteric solvents [64, 315- 319]. The first ever industrial process based on ILs, BASIL (Biphasic Acid Scavenging using Ionic Liquids), initiated by BASF a few years back, emphasises the indispensable applications of these solvents in our lives [63].

2.8.1 Research on confined ILs so far It is well known that water, proteins and other fluids when confined in nano-sized cavities, display anomalous properties, which are illustrated by melting point depression, boiling point elevation, changes in hydrogen bonding of the fluid, variation in the secondary structure in proteins etc. [121,282]. Expanding upon these ideas of confinement, microemulsions [65- 70,320,321], micelles [71,72,322-324], carbon nano-tubes [325], controlled pore glasses [73], and silica gels [74, 326-329] have been successfully used as confining media to study the properties of ILs in restricted geometries. The first study of IL confined by a microemulsion was reported by Gao et al. employing the IL 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim][BF 4]) [65]. They prepared [bmim][BF 4]/Triton X-100/cyclohexane microemulsions and characterised them by various experimental techniques. By means of conductivity measurements, they identified the oil/ionic liquid, ionic liquid/oil and the bicontinuous regions in this microemulsion at 35 °C. Their experiments based on freeze- fracture electron microscopy indicated a droplet structure for these microemulsions and with increasing weight ratios of [bmim][BF 4] to TX-100, they found that the size of the polar IL increases, akin to conventional microemulsions with water polar domains. Eastoe et al. studied the same microemulsions by small angle neutron scattering and arrived at the conclusion that these colloidal systems have nanometer-sized liquid domains similar to the widely studied AOT water/oil microemulsions [67]. The solvation dynamics and rotational relaxation of the probe Coumarin-153 in the same microemulsion system was explored by steady-state and pico-second time-resolved emission spectroscopy [66]. A recent article explores the effect of water addition on the microstructure of

[bmim][BF 4]/TX-100/benezene microemulsions [320]. The authors resorted to the addition of small amounts of water to the microemulsion and found by FT-IR analysis that the water molecules are solubilised in the polar outer shell of the microemulsion. Their 1H NMR

50 Chapter 2 State of the art investigation reveals that the water molecules interact with the oxygens of the surfactant through hydrogen bonding and the water oxygens interact with the imidazolium rings of the IL. This has lead the authors to postulate that water molecules in an IL microemulsion act as a glue to stick the IL and oxyethylene units of the surfactant more tightly, thereby rendering increased stability to the system. In a recent report on the study of the microstructural characteristics of

[bmim][BF 4]/Triton X-100/p-xylene microemulsions at 25 °C, the authors suggest that the interaction between the electronegative oxygen atoms of the oxyethylene units of Triton X- 100 and the electropositive imidazolium ring may be the driving force for the solubilisation of [bmim][BF 4] as the polar core of the nano-aggregates [69]. Gao et al. have also prepared and characterised 1-butyl-3-methyl-imidazolium hexaflurophosphate ([bmim][PF 6])/TX- 100/water containing ternary microemulsions, where they recognised three types of microstructures[70]. These are water in [bmim][PF 6], [bmim][PF 6] in water, and bicontinuous regions in the microemulsions. Extending up on this work, Seth et al. report on the solvent and rotational relaxation studies in the same system using Coumarin 153 and Coumarin 490 probes [321]. All their experiments were conducted at IL to surfactant weight ratio of 1.5 at different Wo, ranging from 2 to 10. The authors find a monotonic decrease of both solvent and rotational relaxation time with increase in Wo. This has been attributed to the gradual shifting of the probe molecules to the core of the microemulsions resulting in an exposure to free water-like environment with increase in Wo.

Anderson et al. reported micelle formation in the ILs [bmim][PF 6] and methylimidazolium chloride, with different surfactants such as Brij-35, Brij-700, etc. [71]. Fletcher and Pandey also investigated micelles formed by Brij-35, Brij-700, Tween-20, and Triton X-100 in 1-ethly-3-methyl-imidazolium bis (trifluoromethyl sulfonyl)imide[72].

Recently, Tang et al. reported on the self-aggregation of Brij-76 in [bmim][BF 4] [260]. The solvent and rotational relaxation of C-153 in [bmim][BF 4] confined in poly(oxyethyleneglycol)ethers [323] and Brij-35 [324] containing micelles has also been reported. Among non-surfactant media for IL confinement, Chen et al. encapsulated the IL

[bmim][PF 6] in multiwalled carbon nanotubes [325] and found that the confinement effect induced the formation of [bmim][PF 6] crystals which were more stable than the crystals

51 Chapter 2 State of the art formed in the bulk system. They further speculate that confinement could result in the enhancement of the weak interactions, such as hydrogen bonding, π-π stacking, van der Waals forces and electrostatic forces in the IL, contributing to the high stability of the crystals formed. Imidazolium-based ILs confined in controlled pore glasses [73] were shown to register lowered melting points, up to -30 °C, compared to the bulk ILs. Further, the melting point depression was found to be enhanced by a decrease in the pore size. FT-IR and FT-Raman analysis of various imidazolium based ILs encapsulated in silica gels [74] also shows significant differences in the vibrational properties of the IL in comparison to the bulk. Preparation of a hybrid class of compounds called ionogels involving the confinement of ILs in a porous silica matrix was reported by Vioux and coworkers [327-329]. The properties of 1,3-dimethylimidazolium chloride confined between two parallel walls have been reported by Pinnila et al . with relevance to their charge-transfer capacity for application in the production of dye-sensitised solar cells [330]. The test IL was confined between two parallel non-corrugated walls varying in distance from 2.5 to 4.5 nm and the changes in the structural and dynamical properties of the IL were studied in relation to the interwall distance. In an extension of this study, the same group reports on the changes in structural and kinetic effects induced by a uniform electric field perpendicular to the confining walls [331]. Kaolinite as a confining medium for the ionic liquid, 1-ethyl pyridinium chloride has been explored recently by Letaief et al. where they report on the successful intercalation of the ionic liquid into the interlamellar spaces of the clay [75]. It has been found that [bmim][PF 6] (volume: 5 L) adsorbed onto atomically flat mica has coexisting liquid and solid phases [332]. With a sufficiently high IL concentration the ‘drop-on-layer-phenomenon’ was observed from the atomic force micrograph images. As the IL concentration increased, the droplet number decreased while the layer underneath became clearer and showed multilayer structures. The thickness of each layer corresponded to multiples of 0.7-0.8 nm, which is comparable to the length of an imidazolium cation. Atkin and Warr [333] have studied the solvation force profiles for three ILs, ethylammonium nitrate, propylammonium nitrate, and 1-ethyl-3- methylimidazolium acetate confined between silicon nitride tips and mica, silica, and graphite using an atomic force microscope. Their experiments reveal that the formation of solvation layers in the ILs are dependent on surface charge, surface roughness, and orientation of the cations in the interfacial layer.

52 Chapter 2 State of the art

As opposed to the study of neat ILs, the study of ILs in confined geometries is only now emerging as a novel field of research. Within a very short span of a few years, some very interesting applications of confined ILs have been realised. Alklyimidazolium bromide based ILs confined to a silica surface through siloxane bonds have been shown to be a suitable stationary phase for high performance liquid chromatography by the Baker group

[334]. Using [bmim][PF 6] containing metallic precursors confined onto an electrode surface, Yu et al. have demonstrated a cost-effective method for the electro deposition of platinum nanoparticles [335]. Shi et al. [336] have synthesised a silica gel confined IL, containing a metal complex as heterogenised catalyst for the carbonylation of amines and nitrobenzene to ureas. In addition to being environment friendly, the process was found to provide an enhanced catalytic activity, along with the advantage of catalyst regeneration.

2.8.2 [bmim][BF 4] and Triton X-100

The IL [bmim][BF 4] is a clear, colourless, viscous liquid with a molecular weight of 226.02. The structure of the compound is shown in Figure 2.8; it is miscible with water, but insoluble in most common organic solvents.

CH3 + NCHN 3 (a) BF - 4

N+

(OCH CH ) OH H3CC(CH 3)2CH 2(CH 3)2C 2 2 x

O- (b) (c)

Figure 2.8 Structures of (a) [bmim][BF4], (b) Triton X-100, and (c) Reichardt’s dye.

It is prepared from its chloride salt following a standard procedure [337]. The latter is first prepared by treating a mixture of 1-methylimidazole and 1-chlorobutane (1:2 mole ratio) in

53 Chapter 2 State of the art

1,1,1-trichloroethane at 70 °C for 72 hours under nitrogen. Upon cooling to room temperature, the solution is washed repeatedly with dry ethyl acetate, and the halide salt is then recrystallised from ethylacetate:acetonotrile mixture. The final step in the synthesis involves stirring of a triply distilled acetone solution of a mixture of [bmim]Cl and NaBF 4 (in 1:1.2 mole ratio) for 24 hours at room temperature, which produces a solution of

[bmim][BF 4]. Triton-X 100 is a non-ionic surfactant with an average molecular weight of 647 (for x = 10). It is the most commonly used non-ionic detergent for solubilizing membrane proteins during isolation of membrane protein complexes.

2.8.3 Solvatochromism In the classical picture of solvatochromism, a molecular dipole experiences a change in its dipole moment upon photoexcitation. The solvent rearranges to solvate the dipole, resulting in a change of the reaction field and creating a difference in solvation energy of the molecule in the ground and excited states. This difference in solvation energy, manifested as the difference in absorption (or emission) energy, is called the solvatochromic shift. The polarity of ILs has been empirically determined by means of a variety of solvatochromic probe dyes, using their long-wavelength solvent dependent absorptions or emissions. However, the most widely used scale for measuring IL polarity is the E T(30) parameter determined on the basis of the large negative solvatochromism of the betaine dye [2,6-diphenyl-4-(2,4,6- triphenylpyridinium-1-yl)phenolate], known as Reichardt’s dye [338,339]. This dye has been shown to exhibit one of the largest solvatochromic effects; its charge transfer absorption wavelength shifts from 925 nm in hexane (non-polar solvent) to 453 nm in water (polar solvent). This extraordinary band shift stems from the differential solvation of the dye’s highly dipolar electronic ground state and it’s considerably less dipolar first excited state. With increasing solvent polarity, the dipolar ground state is better stabilised by solvation in comparison to the excited state, which may be even destabilised because its solvation shell is still equal to that of the ground state according to the Franck-Condon principle. Apart from Reichardt’s dye, the solvatochromic behaviour of several fluorescent probes in the IL [bmim][PF 6] has been investigated by Fletcher et al. [340], while Muldoon et al. [341] have studied the polarity and nucleophilicity of a range of ILs using Reichardt’s

54 Chapter 2 State of the art dye and the square planar [Cu(acac)(tmen)] + salt. Aki et al. established the polarity of four imidazolium- and pyridinium-based ILs employing UV-Vis and fluorescence spectroscopies [342]. Very recently the solvatochromic parameters of imidazolium-based IL with some protic molecular solvents have also been determined [343]. Carmichael and Seddon [344] determined the polarity of several imidazolium based ILs using nile red. In addition to solvatochromic probes, the polarity of ILs has also been determined based on their static dielectric constants ( ε) recorded in the microwave region. Wakai et al. in particular report the ε values of five imidazolium based ILs which classify them as moderately polar with their polarities corresponding to alcohols of intermediate chain length (C5 to C8) [345]. Another article documents the available information on the ε values of some widely used imidazolium, pyridinium, pyrrrolidinium, and alkyammonium based ILs [346]. This database clearly indicates the polarity of the IL to be greatly dependent on the nature of the anion.

2.8.4 Association constant A great deal of our knowledge on non-covalent interactions comes from association constant measurements. It is this value which affords us a means to draw a picture of the underlying science in the interaction under consideration. In principle, a broad range of spectroscopic techniques can be employed for the determination of association constant in host-guest complexes. For an accurate determination of this value, one requires to monitor the changes in the concentration of the components as a function of the composition of the mixture. Although UV-Visible spectroscopy has been successfully used for this purpose, it however is mandatory that at least one of the solutes possess a suitable extinction coefficient to facilitate a measurement. Standard NMR methods have been reported to suffer from a lack of sensitivity at low concentrations [347]. Infrared spectroscopy appears to be a useful alternative, as this technique can be used even at sub-millimolar concentrations, and more importantly, is responsive to conformational changes and bonding phenomena accompanying complexation. There exists a voluminous literature on the study of weak complexes formed by non- covalent interactions. Not infrequently, the properties of these weak complexes are studied by infrared spectroscopy, and the data analysis is often done by employing one of the

55 Chapter 2 State of the art variants of the Benesi-Hildebrand Equation. The equation first introduced by Benesi and Hildebrand to determine the interaction of iodine with aromatic hydrocarbons [348] has since been used to investigate the molecular association in a wide range of host-guest interactions. These range from complex formation in two liquids like that in chloroform-benzene [349], serotonin-HAS interactions in reverse micelles [350], to inclusion complex formation between cyclodextrins and aromatic compounds [351]. The present work makes use of the following form of the Benesi-Hildebrand equation to determine the association constant of the confined [bmim][BF 4] in TX-100/cyclohexane microemulsions, 1 1 1 = [] + A Ao K a TX Ao

Here, [TX] is the molar concentration of the surfactant in the microemulsions, A is the absorbance of the complexed IL, Ao is the absorbance of the complexed TX in solution and Ka is the association constant for the interaction between IL and TX molecules (The derivation of the equation is given as Appendix D). The plots 1/[A] against 1/[TX] drawn in this case, are analogous to the Lineweaver-Burk plot in enzyme kinetics. As described by earlier researchers, this microemulsion formation is driven by the electrostatic interaction between the positively charged imidazolium moiety and the negatively charged OH groups of the oxyethylene units of the surfactant.

56 Chapter 3 Experimental techniques

This chapter provides an introduction to the principles underlying the operation of the three major experimental tools employed in this thesis; namely, near-infrared spectroscopy, dielectric spectroscopy, and small angle x-ray scattering.

3.1 Near-Infrared spectroscopy Ever since the discovery of near-infrared energy by Herschel [352] in 1800, NIR spectroscopy has grown by leaps and bounds. From what started as the determination of fat and moisture in agricultural products in Ben-Gera and Norris’ seminal paper [353], NIR spectroscopy today finds applications in diverse fields like polymers, textiles, pharmaceuticals, petrochemicals and foodstuffs, just to name a few. The usefulness of the technique is largely due to the rapid and non-destructive analysis of bulk samples, coupled with the recent developments in chemometric analysis which afford quantitative and qualitative assays [354]. The NIR spectral region lies between 780 nm and 2500 nm, adjacent to the 2500-25,000 nm (4000–400 cm -1) region popularly known as the mid-infrared region. The predominant NIR spectral features include: the methyl C-H stretching vibrations, methylene C-H stretching vibrations, aromatic C-H stretching vibrations, and O-H stretching vibrations. Minor but still important spectral features include: methoxy C-H stretching, carbonyl associated C-H stretching; N-H from primary amides, secondary amides (both alkyl, and aryl group associations), N-H from primary, secondary, and tertiary amines, and N-H from amine salts. The occurrence and spectral properties (intensity, frequency) of these NIR bands are governed by the anharmonicity involving the light hydrogen atoms.

The advantages of NIR measurements over other vibrational techniques are as follows [355]: Chapter 3 Experimental techniques

• C-H associated vibrational information is repeated eight times from 690 nm to 3000 nm. This repetitive information gives a great deal of flexibility for pathlength selection and information content; • Low cost instruments with high signal-to-noise (SNR) are simple to make and typically exhibit signal-to-noise ratios (SNR) of 25000-100000:1; • High NIR throughput is possible, even when employing low cost fibre optics; • Variable pathlengths for industrial use are possible, typically 1 mm to 10 cm or more using different NIR spectral regions; and • NIR light penetrates plant and animal tissue easily for biomedical applications (when using 900 nm and longer).

3.1.1 Principles of NIR spectroscopy Molecular energy can assume many different forms like translational, rotational, electronic, and vibrational. Of these, only vibrational and electronic energies influence spectroscopy in the NIR range, although rotational energy can influence the NIR spectra of gas-phase samples. The predominant vibrational energy refers to the oscillations of atoms through their bonds in a molecule, given by

2/1 h  k  E =   (3.1) 2π  µ  where k is the force constant of the bond and is the reduced mass given by m m µ = 1 2 (3.2) + m1 m2

in which m1 and m2 are the masses of the two atoms as shown in Figure 3.1. Molecular vibrations can be effectively depicted using the harmonic oscillator model [356-358], which assumes that the potential energy of a vibrating system ( V) at any given time is a quadratic function of the displacement of the atoms involved in the vibration, as shown by 1 V = kx 2 (3.3) 2

58 Chapter 3 Experimental techniques in which x represents the displacement of the atoms from their equilibrium position and k is the restoring force constant. Figure 3.1 shows the potential energy as a function of displacement for a diatomic molecule m1-m2, which can be likened to two oscillating balls connected by a spring.

= m1

= m2

vibrational energy levels Potential Potential Energy (V) v = 3 determined by quantum chemistry v = 2 v = 1 v = 0

x

(m 1-m2 distance)

equilibrium position

Figure 3.1 Schematic representation of the harmonic oscillator model; poetential energy versus atomic displacement for a diatomic molecule m 1-m2.

Although the classical ball and spring model can be used to describe the concept of vibrational molecular energy, a quantum theory model is needed to determine the specific energy levels that are possible for a particular vibration. When this is accomplished, it becomes evident that the vibrating system does not have a continuum of vibrational energy levels but rather a set of discrete, quantised energy levels, defined by the equation  1  Eυ = ν + hν (3.4)  2 

where ν is the vibrational quantum number for the vibration, Eν is the energy of the νth quantum level of that particular vibration, and ν is the fundamental frequency of the vibration

59 Chapter 3 Experimental techniques

(equal to 1/2 π (k/ )1/2 ). In any case where the transition of an energy state is from 0 to 1 in any of the vibrational states ( ν1, ν2, ν3, …) the transition is considered a fundamental and is allowed by selection rules.

3.1.2 Anharmonic vibrations Vibrational spectroscopy made complicated is NIR made possible [359]. The whole existence of vibrational overtone and combination bands, and thus NIR spectroscopy depends on: (i) mechanical anharmonicity: most real molecules undergo anharmonic, rather than harmonic vibrations; and (ii) electrical anharmonicity: for all heteronuclear diatomic pairs in a molecule, the dipole moment is not exactly a linear function of the interatomic distance. Mechanical anharmonicity is a necessary consequence of the fact that atomic nuclei, when pressed sufficiently close together, experience a strong repelling force and when separated far enough, eventually dissociate. In this case, the potential energy of the molecule at any given time is not simply a quadratic function of the displacement, as in the harmonic oscillator model, but can be better approximated by using higher-order terms of displacement: V = k (1)x(2) + k (2)x(3) + k (3)x(4)+ … (3.5) in which x once again represents the displacement of the atoms from their equilibrium positions. According to the above equation, higher order terms become especially important for large displacements of the atoms from their equilibrium positions. Electrical anharmonicity is a reflection of the nonlinear relationship between dipole moment and atomic displacement. For a heteronuclear diatomic molecule, the dipole moment at the point of coincidence of the atoms should be zero and should also be zero when the atoms are infinitely separated. Between these two extremes, the dipole moment goes through a maximum value. In all known cases, the maximum in the dipole moment occurs at an atomic separation slightly smaller than the equilibrium atomic separation.

60 Chapter 3 Experimental techniques

3.1.3 Consequences of anharmonicity There are several important consequences of mechanical and electrical anharmonicity that make NIR possible [359]. • overtone transitions, which involve an increase in the vibrational quantum number of greater than one, are allowed to occur. • combination modes, which involve a simultaneous increase in the vibrational quantum numbers of two or more different vibrations from absorption of a single photon, are allowed to occur; and • the separation of the vibrational energy states of a given vibration are no longer equal, as in the harmonic oscillator case (Equation 3.4). As a result of this the frequencies of overtone bands are not exact integer multiples of the fundamental vibrational frequency, but must be adjusted due to anharmonicity.

It is important to note that vibrations involving hydrogen atoms (C-H, N-H, and O-H) tend to be very anharmonic, and asymmetric stretching vibrations tend to be more anharmonic than symmetric stretching vibrations of the same group. In general, the intensities of overtone and combination bands are at least one order of magnitude weaker than fundamental vibrations. Furthermore, these bands tend to decrease drastically in intensity as the order of the overtone or combination increases. This is because higher order approximations in the potential energy function (Equation 5) are required to get higher order overtone and combination bands.

3.1.4 Instrumentation The instrument used in this thesis is a Perkin-Elmer UV-VIS-NIR Lambda 9 spectrometer. It features an all-reflecting double-beam, double-monochromator optical system as shown in the following figure.

61 Chapter 3 Experimental techniques

Figure 3.2 Optical system of UV/VIS/NIR Lambda 9 spectrometer [360].

The optics of the system consists of two monochromators in series in Littrow configuration; each monochromator has two gratings (1440 lines/mm for UV-VIS range, 360 lines/mm for NIR range) with an automatic changing grating during monochromator slewing. The source is a prealigned deuterium lamp for the UV range and a prealigned tungsten-halogen lamp for VIS and NIR ranges (with automatic changing during monochromator slewing). The detectors are a side window photomultiplier for UV-VIS range and a PbS detector for NIR range. The range of the instrument is 185-3200 nm (54054-3125 cm-1), and enables acquisition of both transmittance and reflectance spectra. The cuvette-holders in the instrument are connected to a Lauda RM 6 thermostat by means of a circulating water bath for accurate temperature control.

3.2 Dielectric relaxation spectroscopy The dielectric relaxation experiments measure the collective polarisation of all the polar molecules in a sample under investigation. The dielectric relaxation time provides a measure of the time taken by a system to reach the final (equilibrium) polarisation after an electric

62 Chapter 3 Experimental techniques field is switched on (or off). The technique measures the phenomenological coefficient ε (* ω) (the complex dielectric function), which may be decomposed into the real and imaginary parts as, ε (* ω) = ε (' ω) − iε ('' ω) (3.6)

Molecular theories of dielectric relaxation provide microscopic understanding of the relaxation phenomena, and the technique is now used to study a great variety of model systems.

3.2.1 The dielectric constant and polarisation The static dielectric constant (relative permittivity) of a material is defined as [361], C ε = (3.7) Co where C is the capacitance of a parallel plate capacitor with the space between the plates filled with an isotropic substance, and Co is the capacitance with vacuum in between the plates. The permittivity of the material is thus dependent on the polarisability of the molecules in between the plates. In the case of an isotropic non-polar molecule, the total polarisability is a sum of electronic and atomic polarisations; while in an isotropic polar molecule, an additional orientation polarisation comes into the picture. Each of these three types of polarisabilty is a function of the frequency of the applied field. A material may have several polarisation effects or dielectric mechanisms that contribute to its overall permittivity. A dielectric material has an arrangement of electric charge distribution that can be displaced by an electric field. The charges become polarised to compensate for the electric field such that the positive and negative charges move in opposite directions. Each dielectric mechanism has a characteristic resonant or relaxation frequency, as shown in Figure 3.3. As the frequency is increased, the slower mechanisms drop off, which leaves only the faster processes to contribute to the storage. The dielectric loss factor correspondingly peaks at each critical frequency. Electronic polarisation occurs when an electric field causes a displacement of the electrons relative to the nucleus in each atom. Atomic polarisation occurs

63 Chapter 3 Experimental techniques when the neighbouring positive and negative ions are displaced with respect to each other under the influence of an electric field. In the absence of an electric field however, the permanent dipole moments of the molecules are distributed randomly in all directions and change direction constantly because of the thermal motion of the molecules. Under the influence of an electric field, there is a tendency for the permanent dipoles to align themselves parallel to it, and this is termed orientation polarisation.

Figure 3.3 Frequency responses of dielectric mechanisms [362].

3.2.2 Relaxation Times Dielectric relaxation is a consequence of the movement of dipoles or electric charges due to a changing electric field. This is a relatively slow process in comparison to the electronic transitions and molecular vibrations that occur above 10 12 Hz. Dielectric spectroscopy is sensitive to relaxation processes in a wide range of characteristic times (10 5 – 10 -12 s), and the latter is denoted by the following expression

64 Chapter 3 Experimental techniques

1 τ = (3.8) 2πν max

At frequencies below νmax , the alternating electric field is slow enough so that the dipoles are able to keep pace with the field variations. Because the polarisation is able to develop fully, the loss ( ε’) is directly proportional to the frequency. As the frequency increases, ε’’ continues to increase, but ε’ begins to decrease due to the phase lag between the dipole alignment and the electric field. Above the relaxation frequency both ε’ and ε’’ drop off as the electric field is too fast to influence the dipole rotation and the orientation polarisation disappears.

3.2.3 The Debye equation For the description of dielectric relaxation phenomena, a number of different equations have been developed over the years. A majority of spectra are best described by the sum of various relaxation processes, achieved by a combination of various equations. The dielectric spectra described in the present thesis are however best fit by the Debye equation, which is the simplest way of describing the dielectric behaviour of a liquid. It is assumed that the decrease of the polarisation in the absence of an outer electric filed is directly proportional to the polarisation itself. Therefore, it can be expressed by a time law of the first order [363], ∂ → 1 → P µ (t) = − Pµ (t) , (3.9) ∂t τ

Upon solving this differential equation one gets, → →  t  Pµ (t) = Pµ )0( exp −  , (3.10)  τ  from which the step response function,  t  F or (t) = exp −  , (3.11) P  τ  and the pulse response function,

65 Chapter 3 Experimental techniques

1  t  F or (t) = exp −  , (3.12) P τ  τ  can be calculated. It has already been shown that the complex permittivity, ε (* ω) , is given by

ε ω = ε ω − ε ω = ε + ε − ε L [ or ( )] (* ) (' ) i ('' ) ∞ ( ∞ ). iω f P t' (3.13)

L [ or ( )] where iω f P t' is the Laplace transformed pulse response function of the orientation polarisation. As per the above equation, a Fourier transform of the pulse response function gives the complex permittivity as   ε ω = ε + ε − ε 1 − t  (*) ∞ ( ∞ ). Liω  exp   (3.14) τ  τ 

The Debye equation can thus be written as

ε − ε ∞ ε (* ω) = ε ∞ + (3.15) 1+ iωτ which can be split in to the real part,

ε − ε ∞ ε (' ω) = ε ∞ + (3.15a) 1+ ω 2τ 2 and the imaginary part ε − ε ε ('' ω) = ωτ ∞ . (3.15b) 1+ ω 2τ 2

3.2.4 Instrumentation The dielectric spectra were recorded using two network analysers, the details of which are as follows.

66 Chapter 3 Experimental techniques

(i) High frequency (50 MHz to 20 GHz) The dielectric spectra in this region were measured using a computer controlled microwave network analyser — Hewlett Packard HP 8720 C and an open-ended coaxial probe — HP 8507B. The HP 8720 is a high performance microwave network analyser for measurements of reflection and transmission parameters. It integrates a synthesised source, signal separation device, a three-channel receiver for measurement of test-device characteristics, and a large- screen display. The following is a simplified block-diagram of the network analyser system [364].

PHASE LOCK

SYNTHESISED SIGNAL DISPLAY SOURCE 130 MHz SEPARATION R to 20 GHz A B RECEIVER

DIGITAL CONTROL

DUT POWER SUPPLY

Figure 3.4 Simplified system block diagram.

The built-in synthesised source of the HP 8720 generates a swept or CW (continuous wave) signal in the range of 50 MHz to 20 GHz. The source output power is levelled by an internal automatic levelling control circuit. A portion of the source signal is routed to the R sampler in the receiver, and fed back to the source for phase lock. The signal separation device separates the source signal into a reference path and a test path. The signal transmitted through or reflected from the DUT goes to the receiver for comparison with the reference signal. The receiver then converts the source signal to a 4 kHz IF (intermediate frequency) for signal processing, retaining both magnitude and phase characteristics. The IF is converted to digital signals, and finally routed to the CRT for display.

67 Chapter 3 Experimental techniques

The probe 8507 is made of stainless steel and has a working temperature range of - 40° C to 200 °C. During measurements this probe is fixed in a glass cell which contains the sample under study. This glass cell is immersed in a water bath connected to a Lauda RM 6 thermostat for temperature control. The system calibration is done by three known standards: open circuit, short circuit, and spectrophotometric grade toluene.

(ii) Low frequency (300 kHz to 1300 MHz) The dielectric spectra in this region were measured using a Agilent Technologies 8712ES RF network analyser. The analyser integrates a synthesised radio frequency source with built-in couplers for signal separation, a combination narrowband and broadband receiver, and a display. In the analyser the radio frequency signal is separated into reflectance, reflected, and transmitted signals in the receiver assembly. These inputs are processed as either narrowband or broadband signals and then multiplexed into analog to digital converters to be converted into digital signals, which are processed by the CPU assembly. For the measurements, a two-step calibration procedure was adopted. Firstly, the calibration of the network analyser was performed using an open and short circuit followed by a matched load (50 ). The second set of calibration was done with air and spectrophotometric grade toluene to calculate the reflection coefficient of the empty cell. The calibration was adopted for each measurement at different frequency regions. Each spectrum was measured with a total of 201 data points.

3.3 Small angle x-ray scattering (SAXS) SAXS is a fundamental method for structure analysis. Its applications cover various fields from metal alloys to biological macromolecules in solution. The first application of the technique dates back to the late nineteen thirties when the principles of SAXS were developed in the seminal work of Guinier in his studies with metal alloys. The scattering of x-rays at small angles (close to the primary beam) was found to provide structural information on inhomegeneities of the electron density with dimensions between one and a few hundred nanometres. The technique yields information not just on the shapes and sizes of the particles, but also on internal structure of disordered and partially ordered systems.

68 Chapter 3 Experimental techniques

Conceptually, a SAXS experiment involves irradiation of a sample with a collimated x-ray beam, measurement of the resultant intensity as a function of the angle between the incoming beam and the scattered beam, and finally the determination of the structure responsible for the observed pattern. Scattering patterns are caused by the interference of secondary waves that are emitted from electrons upon irradiation. A secondary spherical wave is generated when a plane monochromatic wave A0 exp( ik 0 r) is incident at a point scattering center. At some observation point, the resulting wave is then given by + A0 exp( ik 0 r) (A0b / r)exp( ikr ) (3.16)

where k0 and k are the incident and scattering wave vectors with | k0| = | k| = 2 π/λ, λ denoting the wavelength, A0 and A0b/r are the scattering amplitudes of the two waves, and r is the vector which determines an observation point corresponding to the scattering center. The stronger the interaction between the incident wave and the point center, the greater the constant b, which has the dimension of length and is called the “scattering length”. The incident wave interacts with all electrons, which become the source of secondary waves. The superposition of these waves gives the first approximation to the scattering wave. In turn, the scattering waves are scattered by all centers, and superposition of all these waves gives the second approximation and so on. Successive approximations of this type converge into the resultant wave when the interaction between the incident wave and separate centers is not large [365].

3.3.1 Theory It is a general principle of scattering theory that most of the information about the structure of a sample can be found in measurements over scattering angles ( θ) which satisfy the following condition, 0.1 ≤ Qa ≤ 10 where Q = 4π sin( θ /)2/ λ (3.17) and a is the dimension characterising the size of the scattering entities in the structure. For very small angles, it is reasonable to approximate sin θ/2 by θ/2, and hence Q is assumed to be directly proportional to the scattering angle. This relationship means that SAXS

69 Chapter 3 Experimental techniques measurements using Cu Kα X-rays ( λ = 0.154 nm) allow the characterisation of structures with sizes of the order of 1 to 100 nm. At low scattering angles, the scattering intensity I(Q) obeys Guinier’s law [366],  1  I(Q) = Nρ 2ν 2 exp − Q 2 R 2  (3.18)  3 g 

 1  = I )0( exp − Q 2 R 2  (3.19)  3 g  where N is the number of scatterers per unit volume, ρ the electron density difference between the scatterers and the matrix, ν is the scatterer volume and Rg is the radius of gyration of the scattering entity. This equation, however, applies in the absence of inter- particle interference in a dilute monodisperse system, if the scattering angle is sufficiently small such that QR g ≤ 1.0. The observed smeared intensity is then given by /1 2 π = 3 − 1 2 2  I(Q) I )0( Wi )0( exp  Q Rg  (3.20) Rg  3 

where Wi(0) is the slit length weighting function at Q = 0. Most real systems however are far from being monodisperse, and possess a range of scatterers of different sizes. This leads to a curvature in plots of the scattering intensity against Q -2, as equations 3.19 and 3.20 are not strictly obeyed. The limiting slopes of such plots lead to weight-averaged values of Rg and the determination of the distribution of scatterer sizes becomes difficult. At high angles the scattered intensity I(Q) depends only upon the surface area of the scatterers and can be approximated by the following equation 2πρ 2 S I(Q) = (3.21) Q 4 where S is the total surface area separating the two phases in the sample. This equation is known as Porod’s law . For a real case homodisperse, sufficiently dilute solution, the observed scattered intensity is a sum of the scattered intensities of the individual particles. Therefore it becomes

70 Chapter 3 Experimental techniques imperative to find a model particle, which is “equivalent in scattering” with the particle in solution, i.e. whose scattering curve agrees within experimental error with the experimental curve. A complete solution to the problem of finding a model equivalent in scattering frequently requires several cycles of approximation, sometimes by trial and error. It is, however, possible to obtain a great deal of quantitative information from the scattering curve without resorting to the ambiguity of trial and error. The parameters so obtained form the basis for an overall SAXS analysis [367]. One of the most important directly obtainable parameters is the distance distribution function p(r) which is obtained by Fourier inversion of the scattering curve.

∞ 1 p(r) = I(Q). Qr sin. Qr .dQ (3.22) π 2 ∫ 2 0

The other important parameter so obtained is the radius of gyration . Formally, it corresponds to the radius of inertia in mechanics; it is the root-mean square of the distances of all 2 electrons from their center of gravity. In a plot of ln I vs (2 θ) (Guinier plot), Rg is proportional to the square root of the slope of the tangent in the limit 2 θ → 0, and is given by

Dmax ∫ p(r)r 2 dr 2 = 0 Rg (3.23) Dmax 2 ∫ p(r)dr 0 where Dmax is the maximum diameter of the scattering particle.

3.3.2 Instrumentation SAXS measurements mentioned in the thesis were performed in a Kratky compact camera

(Anton Paar KG, Graz, Austria) with Cu K α radiation ( λ = 1.5 Å, Ni-filter). The body of the compact camera forms a tightly evacuated sealed space, where x-rays enter and leave through a 0.25 mm thick beryllium window. The instrument is in the group of Prof. R. Winter at the University of Dortmund. The scattering intensity was recorded by a scintillation counter in a step scanning mode at room temperature. The scattering profiles were corrected for back

71 Chapter 3 Experimental techniques ground scattering and desmeared. Standard analysis was executed using Guinier plots in the -1 range Q < 1.3 / Rg Å .

3.5 Materials This section provides an insight into the chemicals and the sample preparation methods adopted for the experimental analysis. This description is divided into three parts in accordance with the three confined media studied.

3.5.1 Water in confinement Aerosol OT (AOT) obtained from Fluka (purity >99%), was vacuum dried at 90 °C for 48 hours. It was then stored over P 2O5 in a desiccator and was used without further purification for the preparation of reverse micellar solutions. To this effect, four non-polar solvents were used, namely n-pentane, cyclohexane, n-octane, and n-dodecane (all of purity >99%). These hydrocarbons were purchased from J. T. Baker and stored over 0.4 nm molecular sieve (J. T. Baker) before use. A 0.1 M solution of AOT in each of these solvents was used as the stock solution. The reverse micellar samples were prepared by injecting calculated volumes of bi- distilled water to 5 mL aliquots of AOT in the desired solvent. Good mixing of the sample was obtained by mechanical disruption of the water droplets by hand shaking; and the solubilization time was found to be directly dependent on Wo. All measurements were done on single-phase systems. Samples prepared this way were used for the investigation of influence of hydration (Section 4.3), influence of solvent (Section 4.4), and influence of temperature (Section 4.5) on water confined in AOT reverse micelles. NIR spectra of all samples were recorded on a Perkin Elmer Lambda 9 UV/VIS/NIR spectrometer at 25 ± 0.2 °C except for Section 4.5. All measurements were done in matched quartz cuvettes of 1 cm pathlength, except wherever mentioned. Each spectrum was recorded at a scan speed of 30 nm/minute, and in each measurement the vibrational contributions from

AOT/hydrocarbon solution was subtracted using an appropriate reference solution. The ν1+ν3 combination band of water was the spectral region under consideration (for reasons refer Section 4.1). No smoothing was done and spectroscopic manipulation such as base line correction, curve-fitting, and Gaussian band deconvolution were performed using

72 Chapter 3 Experimental techniques

TableCurve 2D (version 4) Jandel Scientific Software (AISN Software Inc.). The statistical parameters obtained were used as a guide to determine the best fit. For each dielectric measurement (Section 4.3.3), the spectra from the high frequency (100 data points) and low frequency (201 data points) network analysers were combined and analysed using the curve fitting program TableCurve 2D. Calibration was done as mentioned in Section 3.2.4, using spectrophotometric grade toluene (Sigma Aldrich). The spectra were found to fit best to the Cole-Cole and Debye type relaxation process, as evidenced by the r2 values. Dielectric measurements were carried out in the AOT/cyclohexane/water system, and all the tested samples were prepared by weight, keeping constant the dispersed phase volume fraction (at φ = 0.1) and varying the Wo value in the range 2 to 18. The volume fraction of the dispersed particles was calculated assuming an ideal mixing behaviour.

3.5.2 α-chymotrypsin in confinement As with the water confinement studies, AOT and cyclohexane were given the same treatment of storage over P 2O5 and molecular sieves respectively. The method adopted to encapsulate α-chymotrypsin (Fluka) in AOT reverse micelles was as follows. A 0.1 M phosphate buffer (Na 2HPO 4 and NaH 2PO 4 from J. T. Baker) at pH 7.2 was used to prepare the complete range of osmolyte solutions. This resultant solution was then used to prepare a 13 M solution of α-CT. Reverse micellar solutions containing α-CT were prepared by injecting calculated volumes of this protein solution into 5 mL aliquots of

0.1M AOT/cyclohexane to yield the sample with the desired Wo [368]. Solutions for the reference cuvette were prepared by adding the corresponding amounts of osmolyte solution prepared in phosphate buffer in place of the protein solution. The concentration of the protein in the solutions was determined using a molar absorptivity coefficient of 51,000 M -1cm -1 at 282 nm. As osmolytes, sorbitol (Sigma), proline (Sigma), glycerol (J. T. Baker), urea (Merck), guanidine hydrochloride (Acros) and KSCN (Riedel-de Haen) solutions, each concentrated to the extent of 0.05 M were used. A relatively dilute concentration of the protein was employed to avoid protein aggregation. Further, the absorbance, if any by the protein was considered negligible in the region of interest. Spectra were recorded at a scan speed of 30

73 Chapter 3 Experimental techniques nm/minute at 1 cm pathlength. A detailed note on the analysis to obtain hydration water from the recorded micellar water spectrum follows in Section 4.6.

3.5.3 [bmim][BF4] in confinement

All analyses with the confined IL were done at 35 °C. The IL [bmim][BF 4] was obtained from Merck and vacuum dried at 70 °C for ~ 48 hours. Triton X-100 (TX-100) from Fluka and cyclohexane from J.T. Baker were used as received. For the investigation of solvatochromism (Section 4.7.1) and structure (Section 4.7.2), the weight fraction of TX-100 in the microemulsions was 0.45, and the IL/TX-100 molar ratios (R) were 0.2, 0.5, 1.0, 1.5 and 2.0 [65]. For the [bmim][BF 4] structural studies, the spectra of the microemulsion samples were recorded in the 1500-2500 nm region in matched quartz cuvettes of 2 mm pathlength. Appropriate TX-100/cyclohexane solution was used as the reference. For solvatochromic studies, Reichardt’s betaine dye from Sigma Aldrich was used as received. A ~350 M stock solution of the probe was prepared in ethanol and stored in an amber coloured bottle at 4 °C. Suitable aliquots of the probe solution were transferred to 1cm quartz cuvettes and vacuum evaporated. Thereafter 3 mL of the appropriate microemulsion solution was transferred to the cuvette, mixed thoroughly by mechanical shaking and allowed to equilibrate for about 4 hours. The spectra were then recorded in the 420-600 nm region. Solutions for association constant determination (Section 4.7.3) were prepared at IL/TX-100 molar ratios of 0.5 and 1.5, and the TX-100 fraction was varied from 0.19 to 0.83 between the two series of experimental samples. Spectra were recorded in the 1570-1680 nm region, attributed to the first overtone of the asymmetric C-H stretch.

All [bmim][BF 4] based spectra were recorded at 15 nm/minute, and a Savitzky-Golay smoothening was resorted to. Owing to the hygroscopicity of [bmim][BF 4], every precaution was taken to minimise atmospheric exposure of the microemulsion samples.

74 Chapter 4 Results and discussion

This chapter presents the results obtained in this work, accompanied by a discussion to explain the observations. As mentioned in the Preface, this thesis is an exploration of the structure and dynamics of three model systems in confinement. The results of the first system, water in confinement , are presented and explained in Sections 4.3 to 4.5, introduced by the consideration of bulk water and the anhydrous reverse micelle systems. Section 4.6 deals with the results of α-chymotrypsin in confinement and discusses the relevant crowding effects. Finally, Section 4.7 presents a comprehensive investigation of [bmim][BF 4] in confinement . A note on the spectral scale adopted in the thesis: In keeping with the convention that NIR spectra are presented in the wavelength scale, coupled with the necessity that the water combination bands be compared with the older mid-infrared results, the NIR spectra in Section 4.1 through 4.6 are represented in both the wavelength and wavenumber scales. NIR spectral representation of the ionic liquid is restricted to the wavelength scale for the sake of simplicity.

4.1 NIR spectroscopy of bulk water Over the past four decades, NIR spectroscopy has been extensively used to study hydrogen bonds, hydration, and self-association of a variety of compounds ranging from simple molecules like water and alcohols to complex ones like polymers and proteins. Of the different X-H bands observed in the NIR region, the bands due to the OH overtones and combinations appear strongly. It has been observed that the spectral shapes of the overtone and combination OH bands, in comparison to the fundamental bands are sensitive to the perturbations of the degree of hydrogen bonding [369]. It is also known that the OH bands of monomeric and polymeric species are better separated in the NIR region than in the fundamental [370,371]. Chapter 4 Results and Discussion

Water, a triatomic molecule, has three normal vibration modes; the symmetric stretch

(ν1), the asymmetric stretch ( ν3), and the bending mode ( ν2). The NIR spectrum of water looks simple, but is far from being so, with a great deal of dogma surrounding the spectral assignments owing to the changes in wavelengths and intensities of the bands because of hydrogen bonding, and the degree of hydration in a mixed system. It is generally believed that the 1450 and the 970 nm bands are the first and the second overtones respectively, of the OH asymmetric stretch [372], although this belief is not entirely true. These are in fact the combination modes involving the symmetric and asymmetric stretching modes of water. This conclusion was reached at upon comparing the liquid and vapour state spectra of water, which showed the combination modes to be stronger than the overtones of either the symmetric or asymmetric stretch [373]. A major reason for the ambiguity in this assignment is due to the accidental degeneracy of the first overtone of the asymmetric OH stretch with the sum of symmetric and asymmetric stretches in dilute solutions [374]. Falk and Ford showed by means of frequency differences between vapour and liquid phase water spectra that the strong bands observed in the spectrum of water below 2500 nm must be due to the combination bands, and not due to overtones [375]. Figure 4.1 shows the NIR spectrum of bulk water with the accepted band assignments.

Wavelength (nm)

00 00 00 00 00 00 00 0 25 20 18 16 14 12 10 80 4.0 ν +ν 2 3 3.5

3.0 ν +ν 1 3 2.5

A 2.0

1.5 R ν + 3 ν +

1.0 2 ν

0.5 ν +ν +ν 2ν +ν 1 2 3 1 3 0.0 4000 5000 6000 7000 8000 9000 10000 11000 12000

Wavenumber (cm -1 )

Figure 4.1 NIR spectrum of bulk water at 25 °C recorded at 2 mm pathlength.

76 Chapter 4 Results and Discussion

The largely overlooked and contentious band in the spectrum is the weak shoulder at ~ 1790 nm. It was first discovered by Ellis [376] and is attributed to the simultaneous excitations of -1 oscillation frequencies ν2, ν3 and the hindered rotation frequency νR (510 cm ) [377].

4.2 AOT reverse micelle — the anhydrous system

Figure 4.2 shows the NIR spectrum of the AOT/cyclohexane system without any added water. The spectral band assignments for the system are as follows.

Wavelength (nm)

0 0 0 0 00 25 200 180 1600 1400 120 100 800 5

4

3 A

2

1

0 4000 5000 6000 7000 8000 9000 10000 11000 12000 Wavenumber (cm -1 )

Figure 4.2 NIR spectrum of AOT/cyclohexane system at Wo = 0 at 25 °C recorded against a blank cuvette.

In keeping with the convention adopted for the spectral assignment of the NIR spectra of hydrocarbons, the peaks between 1700 and 1800 nm (5555-5882 cm -1) are thought to be the first overtone of C-H stretch. The second overtone of the same is believed to occur between 1150 and 1210 nm (8264-8696 cm -1), and the third overtone between 880 and 914 nm (11364-10292 cm-1) [378]. The two sharp peaks located around 5890 and 5810 cm -1 can be best assigned to the CH 3 asymmetric and symmetric stretches respectively of the AOT -1 backbone. The solitary peak around 5500 cm is due to the CH 2 stretch, from the AOT tails. The two sharp peaks around 7050 and 7200 cm -1 may well be assigned to the 2xC-H stretch

77 Chapter 4 Results and Discussion

and C-H bending of both CH 2 and CH 3 groups, again originating from the surfactant. The C- H combination bands are observed between 4000 and 5000 cm -1 and these exhibit complex spectral features with high absorbencies.

4.3 AOT reverse micelle — the hydrous system: Influence of hydration 4.3.1 NIR data The microstructural characteristics of the water in AOT reverse micelles were studied in the

~ 1400 nm region with the broad and asymmetric ν1+ν3 band. The experimental interpretations are based on the changes that become apparent in this band with increasing water content in the reverse micelle. Figure 4.3 shows the NIR spectrum of bulk water and micellar water at three different levels of hydration.

Wavelength (nm) 1700 1600 1500 1400 1300 0.5

0.4

0.3 A

0.2

0.1

0.0 6000 6500 7000 7500 8000 Wavenumber (cm -1 )

Figure 4.3 NIR spectra of the ν1+ν3 combination band of water in AOT/cyclohexane/water reverse micelles at Wo = 4, 12, and 20 (the three curves starting from the bottom). The topmost curve is the spectrum of bulk water (absorbence divided by 5).

The absorption band observed at each hydration value is very broad and asymmetric, indicating the presence of different states of solubilised water molecules. The conspicuous increase in the absorbance, increase in band-width, and the shift of the micellar spectra towards lower wavenumber with increasing hydration, all point towards a strengthened

78 Chapter 4 Results and Discussion

hydrogen bond interaction with increase in Wo, as would be normally expected. A closer observation of the figure also reveals that the micellar water spectral shape begins to resemble more and more that of the neat water spectrum as the water loading in the reverse micelle is increased. This is a strong testament to earlier observations by different researchers that although micellar water is different from bulk water, its properties nevertheless approach those of the latter with increasing water content [121]. To quantify the changes occurring within the water micellar domain, the band was subjected to a Gaussian deconvolution in keeping with the attempts of most earlier researchers. Attempts were made to fit this band as a sum of different constituent sub-bands; the best fit of the experimental band however was obtained when a three-Gaussian fit was resorted to. The three Gaussian peaks are thought to denote three different water species located within the reverse micelle. These three species differ in the extent of hydrogen bonding encountered in water. Figure 4.4 shows the deconvoluted spectrum of

AOT/cyclohexane/water at Wo = 12, and the three Gaussians observed are centered around 6740, 6920, and 7053 cm -1 with full width at half maximum (FWHM) values of 481, 244, and 99 cm -1 respectively.

Wavelength (nm) 1700 1600 1500 1400 1300

0.30

0.25

0.20 A

0.15

0.10

0.05

0.00 6000 6500 7000 7500 8000 Wavenumber (cm -1 )

Figure 4.4 The curve fitted (solid black) and the experimental (dotted black) spectra in the ______AOT/cyclohexane/water system at Wo =12. ( : free water; : bound water; : trapped water)

79 Chapter 4 Results and Discussion

These frequency positions are in good agreement with those reported previously by researchers in both the fundamental and NIR region [see Appendix C]. The first sub-band is thought to represent the central water pool, forming the core of the reverse micelles. These molecules should be strongly hydrogen bonded to one another and hence the appearance of this band at the lowest wavenumber with the highest FWHM. This band may well be likened to the component in vibrational spectrum of bulk water, interpreted according to the model proposed by Walrafen [379] that describes the collective in-phase stretching vibrations of large clusters of water molecules (up to ~ 20 molecules) with linear hydrogen bonds having tetrahedral arrangement. The second sub-band represents the bound water; molecules that are hydrogen bonded to the sulfonate head groups of AOT. The weakened OH bond strength accounts for the appearance of this band at the middle wavenumber. For an analogy with the bulk water counterpart, this can be considered to correspond to the not in-phase OH stretching between nearest neighbour water molecules that are not fully tetrabonded; thus resulting in the formation of hydrogen bridges, thereby generating a weak bifurcated hydrogen bond. The narrowest sub-band at the highest wavenumber is due to water molecules that are trapped at the micelle-solvent (cyclohexane in this case) interface; in between the AOT non-polar tails. These water molecules should behave as monomer-like isolated water analogous to the ‘very’ weak H bonds in bulk water that are not arranged in a supramolecular network. Figure 4.5 shows a schematic representation of the AOT reverse micelle including the three different types of water molecules as described above.

Trapped water

Bound water

Free water oil

Na + counterions

Figure 4.5 Schematic representation of the AOT reverse micelle with the locations of the three water species.

80 Chapter 4 Results and Discussion

Once the micellar water deconvolution was accomplished, the relative ‘amounts’ of each water species was determined based on some calculations. This was done on the assumption that the total peak area corresponding to the micellar water band is the sum of the peak areas of the different states of water in the reverse micelle. This means that if A be the total peak area of the OH stretching band and A1, A2, and A3 represent the peak areas corresponding to free water, bound water and trapped water respectively, then,

A1 + A 2 + A 3 = A (4.1)

Also, if P1, P 2, and P3 be the fractions of free, bound and trapped water respectively in the total water in the system, then,

A A A P = 1 , P = 2 , and P = 3 (4.2) 1 A 2 A 3 A and

P1 + P 2 + P 3 = 1 (4.3)

Further, if n1, n 2, and n3 represent the numbers of the free, bound, and trapped water molecules per AOT molecule, then they can be obtained from the following relations.

n1 = P 1Wo, n2 = P 2Wo, n3 = P 3Wo (4.4) where n1 + n 2 + n 3 = W o (4.5)

The above calculations have been applied to the AOT/cyclohexane/water system and the forthcoming discussion is based on the changes observed in these two characteristic parameters; P and n. But before going into any further details of the NIR results, the radii obtained from the SAXS analysis are shown below, so that the reverse micelle characterisation can be done as a function of its radius. As is evident, and would be expected, the size of the reverse micelle increases with hydration.

81 Chapter 4 Results and Discussion

38 36 34 32 30

28

) 26

24

Radius ( 22 20 18 16 14 12 0 2 4 6 8 10 12 14 16 18 20 22 24 W o

Figure 4.6 Radius of gyration, Rg ( ■) and estimated micelle size, R (▲) determined by SAXS in AOT/cyclohexane/water reverse micelles.

The computed values of the water fractions in the reverse micelle based on equation 4.2 are plotted in the following figure. It is seen that that with increase in hydration, the bound water fraction increases till Wo = 14 (R = 25.4 Å), and beyond that it decreases. The free water fraction decreases slightly prior to Wo = 14, beyond which there is a dramatic increase in this fraction. The trapped water fraction remains more or less constant through out the investigated hydration regime.

0.65

0.60 x 0.55

0.50

0.45

0.40

0.35 i P 0.30 x

0.25

0.20

0.15

0.10 x 0.05 15 20 25 30 35 40 R (Å )

Figure 4.7 Variation of different fractions of water with Wo: free ( ■), bound ( •) and trapped ( ▲). x denotes water fractions of bulk water.

82 Chapter 4 Results and Discussion

The hydration picture that emerges from this data indicates that till Wo = 14, the water added to the system is mainly used for hydrating the sulfonate head groups. Beyond Wo = 14, the added water mainly behaves as free water, and thus, P1 was found to increase with the resultant decrease of P2. It is known from the literature that up to Wo = 10, there exists an equilibrium between the monomers and the aggregated systems [141] and therefore, around this hydration range, a part of the added water is used for hydrating the monomers, while the other remains as free water inside the aqueous cores in addition to the micellar bound water. The figure also includes the corresponding water fraction values of neat water. It can be seen that with increase in hydration, the Pi values approach those of neat water, corroborating the notion that with increase in hydration, micellar water more and more begins to resemble bulk water ( vide Figure 4.3). The other parameter calculated from the spectroscopic investigation is n — the number of molecules of each water species per AOT molecule. These data are shown in Figure 4.8.

12

10

8

6 i

n

4

2

0

15 20 25 30 35 40 R ( ¡)

Figure 4.8 Variation of the number of water molecules per AOT molecule with Wo: free ( ■), bound (•) and trapped ( ▲).

It can be seen that n2 increases till Wo = 14, reaches a value of ~ 9; beyond which its number remains constant which indicates the saturation of the AOT head groups. n1 displays a minor increase till Wo = 14, but after this, there is a steep increase in this quantity and reaches ~ 12.

83 Chapter 4 Results and Discussion

This steep increase corresponds to the growth in reverse micelle size. n3 remains almost constant and is negligible in comparison to the other two water species. These data thus confirm the micelle hydration picture that emerged from the variation in water fractions.

4.3.2 SAXS data The NIR results augur well with the SAXS data. The data shown in Figure 4.6 can be divided into three regions: Wo less than 10, Wo lying between 10 and 16 and Wo greater than 16. The first hydration region shows a slight increase in reverse micelle size, which corresponds to the early stages of AOT head group hydration. As was mentioned in Chapter 2, Eicke proposed that up to Wo = 10, there exists an equilibrium between the AOT monomers and the aggregated systems, so that water molecules added around this hydration region serve a dual purpose of hydrating the monomers, as well as serving as ‘nuclei’ for the eventual formation of the aqueous micellar pool. The Wo range 10 to 16 (10 to 18 reported in literature [141- 143]) may be considered the micellar swollen region, where all surfactant molecules form micellar aggregates. This region (23 to 35 Å) displays the most steep increase in reverse micelle size (Figure 4.6), and also serves as the ‘precursor’ region for the growth of free water molecules (Figure 4.8). Beyond Wo = 16 all micellar changes are less conspicuous as the micellar water begins to resemble bulk water with increase in hydration. The scattered intensity curves I(Q) of the reversed micellar systems with increasing hydration are shown in Figure 4.9.

3000

2000

1000 ) (arbit. (arbit. units) ) Q ( I

0

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 -1

Q ( ¢ )

Figure 4.9 Scattering functions of 0.1 M AOT/cyclohexane/water reverse micellar solutions at different hydration. ■: Wo = 4; ●: Wo = 7; ▲: Wo = 10: ▼: Wo = 14; *: Wo = 16; x: Wo = 20.

84 Chapter 4 Results and Discussion

With increasing Wo, the scattering intensities in the small q region increase. These changes of the scattering curves reflect an enlargement of the micellar dimension with increasing water pool radius. The behavior of the scattering intensity at small Q values is directly related to the radius of gyration ( Rg), from which, assuming a spherical shape, the radius of the particle can be calculated. The radii of gyration for micellar systems at different hydration levels were calculated by Guinier’s plots (Ln I(Q) versus Q2) as shown in Figure 4.10. Radii of gyration and the particle radii increase gradually for increasing hydration levels from Rg 15 Å to 28 Å (plotted in Figure 4.11). At Wo = 14, a rather sharp increase in size of the micelle is observed, from

17.6 Å at Wo = 10 to 25.4 Å at Wo = 14. So, all these results indicate an enlargement (non- continuous at low hydration level) of the water pool and hence increase in the size of the reverse micelles.

8.5

28.2 £ 8.0

27.2 £ 7.5

25.4 £ 7.0

17.6 £

I(Q) 6.5 Ln

15.7 £ Figure 4.10 Guinier’s plots of the 6.0 AOT/cyclohexane/water 15.0 £ reverse micellear solutions 5.5 at different hydration. ■: W = 4; ●: W = 7; ▲: W = 10: ▼: 5.0 o o o

0.000 0.002 0.004 0.006 0.008 W = 14; *: W = 16; x: W = 20. ¥ o o o

2 ¦

Q ( £ ¤

The pair distribution functions p(r) obtained using Equation 3.22 are shown in Figure 4.11. They are bell-shaped, typical for sphere-like particles. The maxima of the pair distribution functions also increase gradually with increasing hydration level, reflecting the fact that particle structural changes lead to an increased inner water pool size with increasing particle size of the micellar system. The maxima here correspond to the distances expected from the

5 radii of gyration (Rg) given by R = R . 3 g

85 Chapter 4 Results and Discussion

7

6

5

4

3

2 ) (arbit. units) (arbit.) r ( p 1

0

-1

0 10 20 30 40 50 60 70 80 ¨

r ( §

Figure 4.11 Pair distribution functions of the AOT/cyclohexane/water reverse micellear solutions at different hydration. ■: Wo = 4; ●: Wo = 7; ▲: Wo = 10: ▼: Wo = 14; *: Wo = 16; x: Wo = 20.

4.3.3 Dielectric relaxation data NIR and SAXS studies thus far confirm that properties of the micellar water asymptotically approach those of neat water with increase in water loading. To gain insight into the dynamics of the AOT reverse micelle system, dielectric measurements were carried out in the frequency range 0.3 MHz to 20 GHz, at a fixed volume fraction of dispersed particles ( φ =

0.1), as a function of molar ratio, Wo. This study is focussed on the dilute region of the systems, where the model of dispersed droplets in a continuous medium is still valid. Most dielectric studies on reverse micelles are performed at relatively low frequencies (0.1 to 100 MHz) and at relatively high volume fraction of dispersed phase where the data are explained based on the dynamic percolation phenomenon [166-168]. The GHOST group [118-120,126,176-181] has carried out experimental studies at microwave frequencies where the dielectric response is dominated by the water component, and the current investigation as will be seen in this section, can largely be explained based on the relaxation model proposed by them. Figure 4.12 (a) shows the real parts of the dielectric spectrum of AOT reverse micellar solutions as a function of frequency at selected Wo values at 25 °C.

86 Chapter 4 Results and Discussion

2.75 2.55 2.55 (a) (b) 2.70 2.65 2.5 2.5 2.60 2.55 2.45 2.45 2.50 2.45 '

2.4 2.4 ε 2.40 ε‘ 2.35 2.30 2.35 2.35 2.25 2.20 2.3 2.3 2.15 2.10 5.00E+009 1.00E+010 1.50E+010 2.00E+010 2.25 2.25 1e+08 1e+09 1e+10 1e+11 Frequency (Hz) Frequency (Hz)

Figure 4.12 Real ( ε’) part of the dielectric constant of the AOT/cyclohexane/water reverse micelles vs frequency at Wo = 2, 6, 10, 14, and 18 (bottom to top) (a); and the best fit curve according to Equation 4.6 at Wo = 10 (b).

The frequency dependence of the complex dielectric constant [ ε * (ω) = ε ' (ω) − iε '' (ω) ] can be described in terms of a super-position of a Cole-Cole and Debye type relaxation process according to the equation, ∆ε ∆ε ε * (ω) = ε + 1 + 2 (4.6) ∞ + ωτ 1−α + ωτ 1 (i 1 ) 1 i 2

where ε ∞ is the high frequency dielectric constant, ω is the angular frequency of the applied ∆ε ∆ε τ field, 1 and 2 are the low- and high-frequency dielectric increments, respectively. 1 τ α and 2 are the relaxation times of the two processes, and is a parameter characterising the τ width of the relaxation time distribution around 1 . The recorded spectra were fit to this equation and the real part of the dielectric spectrum at Wo = 10 is shown in Figure 4.12 (b). τ Figure 4.13 shows that the values of 1 rapidly decrease with increasing Wo, up to 14, and become nearly constant at higher molar ratios. This relaxation is located in a frequency region about three decades higher than that extensively studied in the literature that is usually related to a dynamic percolation phenomenon. At low molar ratios when the water content inside the micelle is low, the observed dielectric dispersion may be interpreted based on the Debye model. According to previous suggestions, the observed dielectric dispersion has been attributed to the rotational diffusion of the whole micellar aggregates [176-181].

87 Chapter 4 Results and Discussion

1.00E-009

9.00E-010

8.00E-010

7.00E-010

(s) 1 τ 6.00E-010

5.00E-010

4.00E-010

0 2 4 6 8 10 12 14 16 18 20 W o

τ Figure 4.13 Plot of relaxation time ( 1 ) vs Wo for AOT/cyclohexane/water reverse micelles.

With increase in Wo, the radius of the reverse micelles increases almost linearly, and τ according to the Debye Stokes equation, the relaxation time 1 should approximately increase as W3. However the experimental observation is different. The observation of τ decreasing 1 can be explained by resorting to the NIR observations which indicate a strong change in confined water properties untill Wo = 14; particularly, the increase in bound water fraction untill this hydration value, and a constancy in the fraction beyond this Wo value. τ Thus the observed deviation of 1 from the Debye equation can be explained by the growth of water pool inside the micellar core. A similar result was obtained at φ = 0.05, where according to results from diffusion coefficient and viscosity mesurements, the authors confirmed that the role played by interactions between microaggregates in such dilute systems is not important [176]. Up on increasing the water content in the micellar core, an increasing number of polar groups of AOT molecules can achieve enough mobility to contribute separately to the τ relaxation process. The observation that the values of 1 and P2 ( vide Section 4.3.1) arrive to saturation at the same value of ~ Wo = 14, suggests that the progressive increase of mobility of AOT polar groups continues until the hydration structure around them is completed.

88 Chapter 4 Results and Discussion

Table 4.1 Dielectric parameters of AOT/cyclohexane/water system at 25 °C. ______τ ε τ ∆ε Wo 1 ε’ ∞ α 2 2 ______2 9.23E-10 2.33 1.85 0.30 4.05E-12 0.33 6 7.50E-10 2.59 1.94 0.36 4.03E-12 0.34 10 5.92E-10 2.63 1.96 0.39 4.80E-12 0.39 14 4.33E-10 2.77 1.97 0.38 5.21E-12 0.40 18 4.10E-10 2.89 1.99 0.41 5.80E-12 0.53 ______

The general trend in the parameters shown in Table 4.1 largely agree with those reported in the literature [176-181]. The second dispersion observed here is located in the frequency region typical of bulk water, and therefore the observed dispersion can be τ attributed to the reorientation of dipoles of water molecules within the micellar core. The 2 values, it is believed are beset with experimental errors; however these are lower than the value of the bulk water relaxation time (8.27 ps at 25 °C) in the whole range of Wo. This result can be interpreted in terms of an increasing of the reorientation rate of water molecules confined inside micellar core. Similar results are obtained by dielectric measurements on electrolyte solutions [380].

4.4 Effect of hydrocarbon medium on AOT reverse micelles

4.4.1 NIR data With the confirmation of the multi-component water model in the AOT reverse micelles, the next series of experiments undertaken that fitted the bigger picture of micellar water studies was concerning the hydrocarbon used to form these reverse micelles. Given the fact that the confined water systems can serve as hosts to a range of molecules, it becomes imperative to have a proper understanding of the behaviour of the water states in accordance with the molecular make-up of the external oil. This section aims to provide a convincing rationale on the behaviour of cyclic and aliphatic hydrocarbons and the effect of alkane number on the

89 Chapter 4 Results and Discussion compartmentalized nano-scale water. For this purpose, four hydrocarbons viz, n-pentane, cyclohexane, n-octane, and n-dodecane were used to form the reverse micelles. It was observed that as the alkane number of the non-polar solvent increased, the maximum water solubilisation capacity of the reverse micelle decreased. AOT reverse micelles were found to solubilise water untill a Wo value of about 75 in n-pentane, 22 in cyclohexane, 40 in n-octane, and 11 in n-dodecane. Beyond these Wo values, phase separation was found to set in. This difference in water solubilisation is due to the difference in the penetrating capabilities of the solvents employed (and consequently the interdroplet attraction), as will be seen here. As in the AOT/cyclohexane/water system, the spectra of AOT reverse micelles in the other non-polar solvents were also recorded in the ν1+ν3 region and these were also subjected to a Gaussian deconvolution. These spectra too displayed the same spectral shape as in the cyclohexane system. Interestingly enough, the three-component water model was found to fit the n-pentane, n-octane, and n-dodecane systems as well. The table below shows the positions of the three water species in all the four systems studied.

Table 4.2 Positions of water species in AOT reverse micelles.

Solvent Free Water (cm -1) Bound Water (cm -1) Trapped Water (cm -1)

n-pentane 6750 ± 5 6937 ± 20 7050 ± 5 cyclohexane 6740 ± 22 6920 ± 10 7053 ± 6 n-octane 6747 ± 5 6920 ± 12 7050 ± 5 n-dodecane 6740 ± 2 6900 ± 4 7050 ± 2

Again based on the areas of each of these water species, calculations were carried out to arrive at the number of water molecules of each type associated with each AOT molecule in the other three solvents. The data so obtained are shown in the figure on the next page. As in cyclohexane, the reverse micelles formed in n-pentane, n-octane, and n- dodecane too display the same ‘general’ hydration picture, i.e. first added water molecules are taken up to hydrate the AOT head groups, followed by the growth of the central water pool upon complete hydration of the former. But what is interesting here is that the reverse micelles formed in these four solvents differ not only in their total water uptake, but also in the amount of water needed for the saturation of the AOT head groups. It may be recalled that for cyclohexane based reverse micelles, this occurred at Wo = 14, corresponding to ~ 9

90 Chapter 4 Results and Discussion

water molecules bound to each AOT molecule ( n2). In the n-pentane based reverse micelles, this complete hydration occurs at Wo = 25, with ~ 16 n2 molecules. With octane as the continuous medium, ~ 13 n2 water molecules herald complete hydration at Wo = 18. And finally, with n-dodecane, complete hydration sets in at Wo = 9 with ~ 5 n2 water molecules. It thus implies that the free water pool exhibits appreciable growth only beyond these Wo values for all the solvents. This observation is in agreement with the results of Hirai et al. [197] who report that the free water region growth depends on the hydrocarbon chain length and find that a 8 < Wo < 16 range corresponds to the growth region for the four hydrocarbons they employed. The reason for this can be obtained from Figure 4.14 (d).

60 20

18 50 16

40 14

12 30

10

8 20 6 per AOT molecule per AOT per AOT molecule AOT per 4 10 2 Number of free water moleculesfree water Number of (b)

(a) water bound Number molecules of 0 0 0 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80 W W o o

0.22 3.5 0.20

3.0 0.18 0.16 2.5 0.14

2.0 0.12

0.10 1.5 0.08

1.0 0.06 per AOT molecule per AOT 0.04 (d) 0.5

(c) trappedofwater Fractionmolecules 0.02 Number of trappedofNumbermolecules water 0.0 0 10 20 30 40 50 60 70 80 0.00 0 10 20 30 40 50 60 70 80 W o W o

Figure 4.14 Variation in the number of each water species (a, b & c) and fraction of trapped water (d) with Wo. n-pentane: ■; cyclohexane: ▲ ; n-octane: * ; n-dodecane: x.

It is observed from Figure 4.14 (d) that the fraction of trapped water molecules in n- pentane is the least while it is the highest in n-dodecane. It is reasonable to state that of all the solvents used in this study, n-pentane would be the best at penetration into the surfactant

91 Chapter 4 Results and Discussion monolayer, owing to its small size and the weakest intermolecular attraction prevalent in it among the four solvents. Thus, n-pentane causes a decrease in the trapped water fraction, by easily and rapidly occupying the space between the AOT tails and thereby making the interfacial film rigid. Because of the relative non-abundance of sites for water molecules to enter in the interfacial region, their number is observed to be low. It has been observed that the interaction between reverse micelle droplets decreases with decreasing chain length. This diminished attraction is related to the penetration of oil molecules into the interfacial film, and the resultant change in interfacial curvature and fluidity. In this case, with the maximum n-pentane penetration, it makes sense to expect an increase in the interfacial mixing entropy, resulting in the stabilisation of the interfacial film. n-pentane penetration also swells the aliphatic layer of the surfactant film, which causes a higher spreading pressure at the surfactant tail/oil interface, consequently leading to a more curved interface to reduce the micellar radius. Further, a more rigid, hardened interfacial film can be expected in n-pentane based reverse micelles as enhanced oil penetration is known to enhance the trans conformation of surfactant chains, resulting in the straightening of the latter. On the contrary, less penetration of solvent into the AOT tails causes a more flexible interface, leading to a greater micellar radius than that of short chain oils. Based on the preceding explanation, one may consider the long chain hydrocarbons as ‘poor’ solvents for interfacial films, resulting in attractive steric forces between reverse micelle droplets, similar to that seen between polymer-coated particles. It can also be observed from the same figure that, this reasoning of penetration based on size, holds good for cyclohexane upto a Wo value of 14, beyond this value the trapped water fraction remains almost constant.

4.4.2 SAXS data Figure 4.15 shows the scattering curves I(Q) of the reverse micellar systems with the different non-polar solvents at Wo = 10 and 20. The figure shows a gradual increase in intensities at low Q values indicating an increase in the radii of water pool of micellar systems.

92 Chapter 4 Results and Discussion

3000 10000

2500 ( b) 8000 (a)

2000 6000

1500

4000 1000

I(Q) (arbit.units) I(Q) (arbit.units) I(Q) 2000 500

0 0

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Q ( Å-1 ) Q ( Å-1 )

Figure 4.15 Scattering functions of 0.1 M AOT reverse micellar solutions. (a) corresponds to Wo = 10 and (b) to Wo = 20. n-pentane: ■; cyclohexane: ▲ ; n-octane: * ; n-dodecane: x.

The distance distribution functions P(r) were obtained by the inverse transformation method (which gives a real space representation and information about particle shapes) and are plotted in Figure 4.16 for Wo = 10 and Wo = 20. It is seen from part (a) of the figure that the water/AOT/n-pentane reverse micellar system shows a bell-shaped P(r) function, typical for spherical particles, with a P(r) maximum, P(r) max , at a distance about one half of the rmax = 25 Å value indicating that the system contains essentially monodisperse globular particles of the same size. Reverse micelles formed in cyclohexane and n-octane also show bell-shaped P(r) functions with P(r) max at about half of the rmax value, indicating the presence of monodisperse globular micelles. The system AOT/n-dodecane/water displays a P(r) function with a rather broad maximum, and P(r) max is smaller than half of the rmax value, and the form of P(r) indicates that the reverse micellar solution is polydisperse with a high population of elongated particles with maximum extension r of about 82 Å, along with a large number of globular particles.

In part (b) of Figure 4.16, P(r) of the system AOT/n-pentane/water at Wo = 20 also shows a bell-shaped curve with a P(r) maximum at a distance of about half of the rmax = 39 Å, which, again, indicates that the reverse micelles are rather monodisperse. The systems AOT/cylohexane/water and AOT/n-octane/water show bell-shaped P(r) functions with broader maxima, having P(r) max at 35 Å and 44 Å respectively, which are approximately equal to the radius of the micellar particles, but at higher r values, the form of the curve

93 Chapter 4 Results and Discussion

indicates a small population of elongated particles with rmax extended up to 74.8 Å and 93.3 Å for cyclohexane and n-octane, respectively.

7 18 ( b) 6 (a) 16

14 5 12 4 10

3 8

6

arbit.units) ( P(r)

P(r) ( arbit.units) (arbit.units) P(r) 2 4

1 2

0 0 0 20 40 60 80 100 0 10 20 30 40 50 60 70 80 90 r ( Å ) r ( Å )

Figure 4.16 Distance distribution function p(r) of 0.1 M AOT reverse micellar solutions. (a) corresponds to Wo = 10 and (b) to Wo = 20. n-pentane: ■; cyclohexane: ▲ ; n-octane: * ; n-dodecane: x.

It is reasonable to state that the tendency to form reverse micelles depends on the affinity between water and AOT polar groups, and the hydrocarbon solvent, as well as the cohesive energy of the solvent. The radii data obtained from the SAXS analysis are tabulated below. SAXS experiments reveal the radius of the micelle to be 25.9 Å in n-pentane, 22.86 Å in cyclohexane, 29.74 Å in n-octane and 38.57 Å in n-dodecane respectively at a hydration level of 10. These values can only be rationalised based on the extent of solvent penetration. From a steric point of view, the shorter chain hydrocarbons and cyclohexane may be the most favourable for penetration into the surfactant monolayer, because they are similar in length to the AOT tail groups, and can therefore align themselves more easily than their higher homologues.

94 Chapter 4 Results and Discussion

Table 4.3 Radii of AOT reverse micelles in different non-polar solvents.

Size n-pentane cyclohexane n-octane n- Parameters dodecane

Wo = 10 Wo = 20 Wo = 10 Wo = 20 Wo = 10 Wo = 20 Wo = 10

Radius of 19.96±0.03 29.9±1.14 17.6±0.23 28.16±1.05 22.9±0.20 34.37±0.32 29.7±0.90 Gyration (Å)

Radius 25.9±0.09 38.8±2.09 22.86±0.4 36.57±1.9 29.74±0.37 44.62±0.60 38.57±1.65 (Å) not spherical

Similar results have also been reported for alkane adsorption into monoglyceride and phosphatidylcholine bilayers. Gruen et al. [381] found that for saturated, or nearly saturated, equilibrium alkane concentrations, the maximum thickening of the surfactant layer and adsorption occurred for the shorter homologues. And as the chain length increased, the thickening and the adsorption declined, reaching nearly zero for their large, branched chain squalene. Other researchers have also found that oil molecules with a small molecular volume or high aromaticity produce a strong solvation effect as a result of entropy of mixing and consequently lead to a greater penetration of oil molecules into the surfactant chain layer, thus increasing the rigidity and curvature of the interface [100,130,383]. Binks et al. have determined the bending elasticity constant of AOT monolayers at the oil-water interface in AOT reverse micelles in a range of solvents ranging from heptane to tetradecane [384]. They attribute the decrease of this constant with increasing alkane number to the differing degrees of oil penetration into the surfactant chain region.

4.5 Temperature dependence of hydrogen bonding of micellar water

It is seen in the preceding sections that the broad band due to the OH stretch is a good probe of the water state because a change of the relative populations of differently hydrogen bonded molecules is revealed by a change in its shape and position. Extending upon this utility of the OH band, the current section shows the results of the temperature dependence of the vibrational dynamics of the reverse micelle confined water. Though the subject literature is replete with occurrences of hydrogen bonding population characterisation of the reverse

95 Chapter 4 Results and Discussion micelle water at room temperature, the temperature dependence of this property has been a rather overlooked issue. As of the writing of this thesis, there exists only one previous report of this study with pentane as the solvent, albeit in a temperature range of 22 to -53 °C by the Vanderkooi group [128]. In order to study this micellar water hydrogen bonding at higher temperatures, the ν1+ν3 OH combination band in the AOT/cyclohexane/water system was recorded in the 25 to 75 °C range. In this region the samples remained homogeneous and transparent thereby affording spectral analysis. The following figure shows a set of representative spectra at Wo = 8. The spectra showed the same pattern for other hydration values as well.

Wavelength (nm) 1700 1600 1500 1400 0.20

25 °C

0.15

A 0.10 75 °C

0.05

0.00 6000 6500 7000 7500 8000 Wavenumber (cm -1 )

Figure 4.17 Temperature-dependent NIR spectra of water in AOT/cyclohexane/water reverse micelles at Wo = 8 from 25 to 75 °C.

The absorption spectra of confined water molecules exhibit a prominent blue shift as well as a decrease in absorption maximum as the temperature is increased. Also, with increasing thermal energy, the spectra become discernibly narrower especially in the low energy region.

Similar observations are obtained for the NIR spectra of bulk water in the ν1+ν3 [385], ν2+ν3

[386], and ν1+ν2+ν3 [373] bands with increase in temperature; and with increasing concentrations of aqueous NaClO 4 solution [369]. While these visible changes in the overtone spectra unarguably indicate that the confined water is affected by the temperature variations, it yet remains to be seen as to what dynamic processes are occurring inside the

96 Chapter 4 Results and Discussion reverse micelle. To get an answer to this, the stretching vibrations were analysed using deconvolution into three Gaussian populations as explained in the previous section. This analysis showed the population shift of micellar water between the different hydrogen bonding conformations with temperature; which makes this technique a sensitive and detailed means of examining water structure in confinement. The results obtained from such deconvolution are presented in Figure 4.18 in terms of the fraction of individual water molecules. There are two interesting sets of observations seen from the figure. One concerns the variation in the water fractions; and the other is related to the hydration picture in the micelle as a function of temperature.

0.24

0.20

0.16

0.12

TrappedWater 0.08

0.8

0.7

0.6

0.5 Bound Water Bound 0.4

0.5

0.4

0.3

0.2

Free Water 0.1

0.0 0 5 10 15 20 25 W o

Figure 4.18 Temperature dependence of the different water fractions. ■ : 25 °C; ● : 35 °C; ▲: 45 °C; ▼: 55 °C; X : 65 °C; and * : 75 °C

Figure 4.18 shows that an increase in temperature results in a decrease in the free water fraction, accompanied by a concomitant increase in the bound and trapped water fractions. It makes sense to rationalize this observation based on the hydrogen bonding of the water molecules, which favours restricted connectivity structures at elevated temperatures. It appears clear that the thermal motion, obviously enhanced by increasing the temperature will tend to destroy the structures with the highest number of coordination. What is more

97 Chapter 4 Results and Discussion interesting here is that the variation in water fractions hold good for the entire hydration range investigated, which implies that the temperature dependent hydrogen bonding population behaviour is independent of the confining pore size. A similar variation in water fractions is also observed in neat water, as shown by Walrafen [387], Freda et al. [388], and McCabe et al. [389], all of which validated the mixture model of water. A related investigation of the temperature dependence of the HOD spectra (2% HDO in D 2O) produced spectra shown in Figure 4.19. Four bands are clearly observed here with each representing various overtone modes of vibration of different species. The 1416 nm band is clearly the non-H bonded stretching mode of vibration and the two adjacent shoulders centered around 1525 and 1558 nm are thought to represent the hydrogen bonded fractions of water. Figure 4.19 clearly shows the effect to be expected for the temperature dependence of a system involving multiple equilibria between hydrogen bonded and non hydrogen bonded species [7]. As the temperature is increased there is a large increase in intensity of the non H-bonded OH band accompanied by decrease in intensities of the absorption bands of hydrogen bonded species. Also, the existence of the isosbestic point at 1470 nm lends strong support to the assumption that a chemical equilibrium involving various hydrogen-bonded species is present in this system.

Wavenumber (cm -1 ) 7500 7000 6500 6000 5500 5000

0.25 OD stretch

75 °C 0.20

25 °C 0.15 A

0.10 25 °C 75 °C 0.05

0.00 1300 1400 1500 1600 1700 1800 Wavelength (nm)

Figure 4.19 Temperature variation of HOD spectra.

98 Chapter 4 Results and Discussion

4.5.1 Hydration picture as a function of temperature

The AOT head group hydration is complete at Wo = 14 at 25 and 35 °C as evidenced by the sharp increase in bound water fraction untill this Wo, and a decline in this number beyond this value (Figure 4.18). For temperatures from 45 °C and above, however, this head group hydration is seen to be completed even at a Wo value of 12. This can be traced to the increase in bound water fraction with increase in temperature. In addition to this, the other striking feature obtained from this figure is the appreciable increase in the trapped water fraction with temperature. This can be explained based on the inter-micellar interactions. It is known that the adjacent reverse micellar droplets interact with one another by the overlapping of the branched hydrophobic tails [390]. This is because the short but branched hydrophobic tails can penetrate each other over small distances without suffering much entropy loss while lowering the total free energy of the system. This is possible because the surfactant tail-tail interaction is not much stronger than the surfactant-oil interaction. At high temperatures, however, the oil molecules are not properly oriented to pack favourably and interact with the surfactant tails. Hence it can be expected that at higher temperatures, the AOT non-polar tails host a lesser fraction of the oil than at room temperatures. This then explains the creation of place for the trapped water molecules whose number increases as a result of increase in temperature. It is thus seen that temperature is a significant factor affecting the stability of the reverse micellar water. Water structures are gradually changed by increasing temperature and were analysed from the point of view of the change in the amounts of the different water species over the temperature range investigated.

4.6 NIR analysis of α-CT hydration properties in confinement

It is common knowledge that the interior of a typical eukaryotic cell is largely made up of proteins, which constitute more than half of the dry weight of the cell. Their main function is to shape the cell, for example, as cytoskeleton fibres. In addition to proteins, the cellular interior contains several other kinds of macromolecules like sugars, nucleic acids, lipids, etc. As stated earlier in Section 2.7, such an environment is usually called “crowded” and the large volume taken up by the crowding agents is known to affect the function and stability of the proteins, which in turn affects the cellular machinery. Molecular crowding is considered a

99 Chapter 4 Results and Discussion source of non-specific interaction between cellular proteins. Steric repulsion is the most common of all interactions between cellular macromolecules and is always present in crowded environments, independent of the magnitude of the general electrostatic and hydrophobic interactions. Reverse micelles have long been used to mimic cellular interior, with the AOT monolayer surrounding the water pool functioning as the cell membrane. This section describes how macromolecular crowding effects the hydration properties of α-CT encapsulated within the AOT reverse micelles. For this purpose, dilute osmolyte solutions (sorbitol, proline, glycerol, urea, GdHCl, and KSCN) have been used as crowders in the reverse micelle, which also serve to simulate the inherent heterogeneity of the cellular cytoplasm. The osmolyte concentrations have been deliberately kept low as the interaction of these molecules with the proteins is in conjunction with the “confinement effect” brought about by the reverse micelle per se . This section thus shows that excluded volume effect, as described by Minton and others, can predict many aspects of molecular crowding in vivo , when considered along with other factors like the protein hydration water. The protein hydration water is a good sensor of the cellular crowding and plays an important role in protein stability. There are only a handful of publications based on NIR that document the investigation of water bound to intact proteins or in aqueous solutions. The last publication quantifying the hydration water of a protein in aqueous solution based on this technique was by Vandermeulen and Ressler in 1980 [391], where they described the development of a variable path length cell to determine the protein hydration in bovine serum albumin, ovalbumin and β-lactoglobulin, following some of their earlier efforts to overcome the technical challenges associated with this technique [392,393]. The method adopted in this thesis is an extension of this technique, and is based largely on McCabe and Fisher’s method for investigating the hydration of a solute in aqueous solution [309,310] and incidentally is a pioneering effort in the quantification of protein hydration water in confinement. In the late nineties, Galinski et al. however reported on the hydration number data of some osmolytes using NIR [394].

100 Chapter 4 Results and Discussion

4.6.1 The basis of the method As highlighted in Section 2.7.4, McCabe and Fisher’s method is based on the observation that the NIR spectra of aqueous solutions measured against water consists of three components; (i) a negative component, which is a measure of the amount of water excluded by the hydrated solute; (ii) a positive component, contributed by the water of hydration of the solute; and (iii) an additional component that could possibly arise from the solute itself. The possibility of the third component arising in the present case is negligible as the protein concentration is kept low. It should be noted, however, that the hydration spectrum comprises the sum of both OH vibrations from hydration water and CH, NH and OH overtones from the protein. As long as the water molecules are in excess and protein overtones well separated, the spectrum can de facto be seen as an approximation of the water surrounding the protein. The figure on the following page is the schematic of the experimental set-up adopted here. The sample consists of a micellar solution containing AOT/cyclohexane hydrated by a

α-CT solution prepared in an osmolyte at a Wo = 10 and the reference cuvette contains the corresponding micellar solution sans the enzyme at the same Wo. The resultant spectrum in the ν1+ν3 region of the OH stretch is a negative peak, as the absorption coefficient of the contents in the sample cuvette is lower than that of the reference cuvette contents.

101 Chapter 4 Results and Discussion

AOT/cyclohexane/ α-CT 3- 3- AOT/cyclohexane/PO 4 buffer in PO 4 buffer + osmolyte + osmolyte

: α-CT surrounded by hydration water : osmolyte molecule

Sample Spectrum Reference Spectrum Difference Spectrum

Figure 4.20 Schematic representation of the spectral components in a typical difference spectrum set- up.

The water of hydration here is denoted operationally as water within the reverse micelle whose spectroscopic properties are different from those of micellar water; in other words, water whose absorbance remains after the micellar water has been compensated for or blanked out, and is in accord with the definition proposed by Cooke and Kuntz [395].

4.6.1.1 The spectra — origin and resolution Figure 4.21 shows the NIR difference spectra of the AOT/cyclohexane/ α-CT reverse micelles containing the different osmolytes. Figure 4.22 shows the set of data (explanation follows) for AOT/cyclohexane/ α-CT+sorbitol (i) and AOT/cyclohexane/ α-CT+KSCN (ii) at a hydration value of 10. In both (i) and (ii), curve A is the NIR difference spectrum of α-CT in

102 Chapter 4 Results and Discussion osmolyte encapsulated in AOT reverse micelles measured against AOT/cyclohexane/osmolyte solution. This recorded difference spectrum represents absorption entirely due to various forms of water. The difference spectrum, Curve A, is then resolved into two arbitrary component spectra: Curve B - representing the net contribution of the absorption by the water in the reference cuvette, and Curve C - representing the net contribution of the absorption by the water in the sample cuvette. Thus Curve A equals Curve C minus Curve B.

4.6.1.2 Resolution of Curve B from the difference spectrum Curve B can be determined from Curve A and the micellar water curve. The latter was recorded in a separate run by blanking out the contribution from AOT and cyclohexane from a sample consisting of AOT/cyclohexane/osmolyte in phosphate buffer. This is shown as Curve D in Figure 4.22. Since the reference absorption cell contains the exact AOT/cyclohexane/osmolyte solution as used in the recording of the micellar water spectrum, Curve B (reference cell contribution) must be of the same shape as the micellar water spectrum. This Curve B is in essence the contribution to the differential water spectrum by the water molecules in the reference cell that are in excess over the water molecules in the sample cell, and can be obtained by normalising the micellar water spectrum (Curve D). The normalisation factor for this purpose is obtained by taking the ratio of the absolute values of the absorbance of Curve A to that of Curve D at a particular frequency. The choice of this frequency is governed by the condition that the normalised spectrum (Curve B) so obtained should have a shape similar to curve D, and more importantly, any Curve C obtained thereafter should be positive or zero at all frequencies. Consequently, Curve B was then obtained by multiplying the absorbance values of Curve D by the normalisation factor; and represents the minimum net contribution from the absorption by the water in the reference cuvette to the difference spectrum, Curve A.

4.6.1.3 Resolution of Curve C from the difference spectrum Once Curve B was obtained, arriving at Curve C (hydration water) was a straight forward process by adding the absorbencies of Curves A and B. This curve thus represents the

103 Chapter 4 Results and Discussion hydration spectrum as this arises from the sample absorption cell and denotes the water molecules that are directly hydrogen bonded to the protein along with those water molecules that are affected by the presence of the protein.

Wavelength (nm) 1700 1600 1500 1400 A

6000 6500 7000 7500 8000

Wavenumber (cm -1 )

Figure 4.21 Difference spectra recorded in the presence of different osmolytes. The curves from top to bottom represent glycerol, KSCN, GdHCl, proline, sorbitol, and urea; each at 0.05M in AOT/cyclohexane/ α-CT reverse micelles.

Wavelength (nm) Wavelength (nm) 1700 1600 1500 1400 1700 1600 1500 1400 0.24 0.24 0.22 D 0.22 D 0.20 (i) (ii) 0.20 0.18 0.18 0.16 B 0.16 0.14 0.14 C 0.12 A

A 0.12

0.10 0.10 0.08 0.08 0.06 0.06 0.04 0.04 B 0.02 0.02 A C 0.00 0.00 A -0.02 -0.02 6000 6500 7000 7500 8000 6000 6500 7000 7500 8000 -1 Wavenumber (cm ) Wavenumber (cm -1 )

Figure 4.22 Resolution of the difference spectrum in AOT/cyclohexane/ α-CT+sorbitol (i) and AOT/cyclohexane/ α-CT+KSCN (ii).

104 Chapter 4 Results and Discussion

The excluded volume effect can be quantified in terms of the volume occupied by the hydrated protein. It is that volume from which the micellar water is excluded, and can be calculated using the following expression: 1000 FV V = (4.7) ex C where C is the molar concentration of α-CT and FV is the fractional volume. The latter is obtained by taking the ratio of the absorbance values of Curve B to that of micellar water spectrum at the frequency of normalisation. It therefore indicates the fraction by which the amount of micellar water is decreased due to the presence of the hydrated solute. The other φ parameters calculated here include apparent molar volume ( v ) and hydration number (n) based on the following equations.

1000 (d − d ) φ = o + M v and (4.8) Cd o d o (V − φ ) n = ex v d (4.9) M o

where do = density of AOT/cyclohexane/osmolyte (reference cell components) [ ρsolvent ]

d = density of AOT/cyclohexane/ α-CT+osmolyte (sample cell components) [ ρsolution ] C = molar concentration of α-CT [solute] M = molecular weight of α-CT

The values of these parameters are collated in Table 4.4 and can be rationalised based on the mode of osmolyte action in the vicinity of a protein. Of the six osmolytes considered here, glycerol, proline and sorbitol are known kosmotropes and the remaining three are strong chaotropes. The origin of their influence on protein stability lies primarily on their effect on the surrounding water, rather than in direct interaction between the osmolyte and the protein.

105 Chapter 4 Results and Discussion

Table 4.4 The protein hydration parameters.

282 -1 -1 Osmolyte A α-CT [CT] FV Vex (L mol ) Φv (L mol ) n 0.05M + (ε=51,000 Osmolyte M-1cm -1) Glycerol 0.0492 9.66E-7 0.8777 9.0854E8 630219.50 29100.6 Proline 0.0511 1.00E-6 0.8406 8.3862E8 686839.06 26862.14 Sorbitol 0.0635 1.24E-6 0.6912 5.5501E8 682878.12 17771.44 Urea 0.0545 1.07E-6 0.2728 2.5493E8 713017.54 8150.14 GdHCl 0.0748 1.47E-6 0.2158 1.4710E8 177041.56 4707.85 KSCN 0.0656 1.28E-6 0.0943 7.3324E7 451846.09 2335.87

4.6.2 The kosmotropes Glycerol, proline and sorbitol are more polar than water and act to enhance its structure due to their ability to form hydrogen bonds. They therefore interact with the water molecules rather than with the confined protein, which leads to their effective preferential exclusion from the protein surface, which in turn serves to increase the protein hydration sphere. As a result of this, the folded state of the protein is stabilised relative to the unfolded state as it exposes less surface area from which the osmolyte must be excluded. This mechanism φ explains the higher values of Vex , v and n in the presence of kosmotropes in comparison to the chaotropes as shown in Table 4.4. Within the kosmotropic series however, glycerol is seen to confer the highest protein stability, followed by proline and then sorbitol. Though this trend is in accordance with some earlier publications that show glycerol to be the strongest compensatory solute owing to its poly-olic nature, it is suggested that these values be considered on an individual basis, rather than to compare the data within the kosmotropic series. To resort to a comparison one would need to repeat these experiments at a few more osmolyte concentrations.

4.6.3 The chaotropes The chaotropic molecules, urea, GdHCl and KSCN are less polar than water, so that their presence in solution would lead to an energetically unfavourable disruption of the water

106 Chapter 4 Results and Discussion structure. Thus water molecules are expected to be expelled from the solution, leading to a ‘preferential binding’ of the chaotrope to the protein molecule. This reduction in the water molecules from the protein’s hydration sphere owing to the presence of chaotropes is depicted in the decrease of protein hydration number in Table 4.4. Again, it is suggested that within the chaotropic series the ‘exactness’ of the values be taken with a grain of salt because even though the trend supports the fact that KSCN is nature’s strongest protein destabiliser, further experiments need to be done to comment on the robustness of the inter-series data.

4.7 IL in confinement

In this section, the effect of confinement of the IL [bmim][BF 4] in Triton X-100/cyclohexane microemulsions is described using NIR and UV-Vis spectroscopies. The goal was to arrive at a comprehensive picture of the IL in confinement by the study of its solvatochromism, structure and association. For the estimation of the polarity of the confined IL the solvatochromic pyridinium N-phenloate betaine dye, commonly referred to as Reichardt’s dye was used. To gain insight into the structure of the IL upon encapsulation in TX-100 − microemulsions, the focus was mainly on the C HLF stretch, and the analyses is based on the changes that become apparent in this stretch with increase in IL concentration. For the determination of the association constant between the IL and the surfactant, the application of Benesi-Hildebrand double reciprocal plot was resorted to.

N 4.7.1 Polarity of the confined IL determined by E T(30) and ET solvent polarity scales

The E T(30) parameter as described in Section 2.8.3 is determined on the basis of the large negative solvatochromism of the Reichardt’s dye. It is defined as the molar electronic transition energy (in kcal mol -1) of the betaine dye in a particular solvent as per the following equation

28591 E ()30 = (4.10) T λ () max nm

where λmax is the longest wavelength intramolecular charge transfer π to π* absorption band of the zwitterionic betaine molecule. The number 30 stems from the original publication

107 Chapter 4 Results and Discussion

where the dye had by chance the reference number 30 [396]. As the E T(30) values are

-1 N expressed in the non-SI unit kcal mol , a dimensionless normalised ET scale was introduced

N N in 1983, using water ( ET = 1.0) and tetramethylsilane (TMS) ( ET = 0.0) as the reference solvents [397] according to equation 4.11.

[E (solvent )− E (TMS )] E N = T T T []()()− ET water ET TMS

[E (solvent )− 30 7. ] E N = ´T (4.11) T 32 .4

E N Table 4.5 shows the observed λmax , and calculated E T(30) and T values for the neat

[bmim][BF 4] and in confinement at different R values.

Table 4.5 Solvatochromic parameters of the experimental solutions at 35 °C.

λ -1 N Solution max (nm) ET(30) (kcal mol ) ET R = 0.2 563 50.8 0.620 R = 0.5 556 51.4 0.639 R = 1.0 551 51.9 0.654 R = 1.5 549 52.1 0.660 R = 2.0 547 52.3 0.667

Neat [bmim][BF 4] 545 52.5 0.673

The E T(30) value of the neat liquid are in good agreement with those previously reported in E N the literature [341]. As can be seen from the data presented in Table 4.5, the T value of the microemulsion solution increases with increase in the ionic liquid content and approaches a value of 0.667 for the largest microemulsion.

108 Chapter 4 Results and Discussion

4.7.2 Structural changes in the confined ionic liquid

Figure 4.23 depicts the NIR spectra of the neat and confined [bmim][BF 4] at different R values at 35 °C. The spectra were recorded in the complete NIR region of 780 to 2500 nm; but the following figure shows only the spectral window depicting discernible changes in the bands observed.

0 .7 5

0 .6 4

3 0 .5 A

2 0 .4 1 A

0 .3 0 1600 1800 2000 2200 2400 W avelength (nm )

0 .2

0 .1

0 .0 1600 1800 2000 2200 2400 W avelength (nm)

Figure 4.23 NIR spectra of [bmim][BF 4] confined in TX-100/cyclohexane microemulsions at different R values. Spectra from bottom to top depict R = 0.2, 0.5, 1.0, 1.5 and 2.0. Inset: Spectrum of neat [bmim][BF 4].

In the neat and the confined IL, the peak at 1613 nm is attributed to the first overtone of the asymmetric C-H stretch of the imidazolium moiety [69,79,398]. The small, distinct peak at

~1665 nm in the neat and confined IL is best assigned to the first overtone of the C − HLF stretch of the IL. It has previously been reported that C − HLX hydrogen bond interactions are weak in nature, which make it difficult to observe these experimentally; and that the effects of this bond are often obscured by those of the other stronger bonds occurring in tandem with these [79]. Because of the very low intensity of this band in the confined medium, these are shown separately in Figure 4.24 to provide a better understanding of the atomic interactions underlying this stretch. Vibarational spectroscopy has long been successfully used in seeking evidence for hydrogen bond formation based on the red shift of the OH stretching frequency; with the

109 Chapter 4 Results and Discussion magnitude of the stretch reflecting the strength of the hydrogen bond. Based on this convention, a careful observation of Figure 4.24 reveals changes in stark contrast to the − traditional hydrogen bond. The C HLF stretch undergoes a blue shift from R = 0.2 ( λmax =

1665 nm) to R = 2.0 ( λmax = 1661 nm); the small shift of 4 nm being indicative of the weak nature of the bond. This kind of observation has been rationalised in the past based on the shortening of the C-H bond length. In an attempt to explain the physical basis behind this, two schools of thought have emerged over the years. Hobza et al. attribute the strengthened C-H bond to a new mechanism called the anti-hydrogen bonding [76]; while Scheiner [77] − and Dannenberg [78] consider the conventional hydrogen bonds and C HLX bonds to be similar, but assert that additional factors like anharmonicity and details of structurally mediated bond changes are essential to delve deeper into a correct explanation. In the present context however, it is believed that confinement is the driving force behind the strengthening of the hydrogen bonding displayed by the ILs. This strengthening of hydrogen bonds on confinement is indeed a strong complement to recent observations that IL/oil microemulsions are similar in behaviour to the conventional water/oil microemulsions.

0.05

0.04

0.03 A

0.02

0.01

0.00 1640 1645 1650 1655 1660 1665 1670 1675 1680 Wavelength (nm)

− Figure 4.24 Spectral changes in the C HLF stretch region of the confined IL. Spectra from bottom to top depict R = 0.2, 0.5, 1.0, 1.5 and 2.0.

The broad band centered at ~1706 nm in the IL and the weak peak around 1690 nm in the confined IL (Figure 4.23) are assigned to the first overtone of the aliphatic C–H stretch.

110 Chapter 4 Results and Discussion

The intensity loss of this band in the confined IL is due to solvent subtraction (by using appropriately matched TX/cyclohexane samples in the reference cuvette, which resulted in negative peaks in the region). The peak at 2105 nm with a small shoulder at 2120 nm and the band at ~2150 nm are assigned to the third overtone of the in-plane C-H deformation of the imidazolium ring. The prominent band at ~2250 nm is due to the in-plane C-N overtone stretch of the imidazolium ring and as can be seen from the figure, these are still relatively strong even after confinement. The bands observed beyond 2300 nm can be attributed to the overtones and combinations of the aliphatic C-H stretch. The peak at 1900 nm is the ν2+ ν3 combination band of water. Though the IL was dried at 70 °C for about 2 days and utmost care was taken to avoid exposure of the experimental samples to air, the hygroscopic nature of ILs inevitably produced this band. In a recent report on the absorption of water by - - imidazoium based ILs, it was shown that among BF 4 , PF 6 and Tf 2N [(bis((trifluoromethyl)sulfonyl)amide] anions, the tetrafluoroborate ion has the strongest interaction with water [399] and therefore displays the highest water absorption. The presence of the water combination band is therefore regarded as an outcome of the - hygroscopicity of the BF 4 ion, and not to any experimental inadequacy.

4.7.3 Determination of association constant

This section gives a description of the determination of the association constant for the interaction between the confined IL and the inner surface of the TX-100/cyclohexane microemulsions. As described by earlier researchers, this microemulsion formation is driven by the electrostatic interaction between the positively charged imidazolium moiety and the negatively charged OH groups of the oxyethylene units of the surfactant. It may also be noted that the organisation of the IL is such that each cation is hydrogen bonded and surrounded by three tetrafluoroborate anions and vice versa. In the past, one of the many variants of the Benesi-Hildebrand equation has been used to understand the nature of molecular interactions in various supramolecular complexes [348,349]. NIR Spectroscopy has been demonstrated to be a pertinent technique for the determination of the association constant for the binding of phenol and other aromatic compounds to cyclodextrins in an ionic liquid medium [351]. Extending the same concept here, the Benesi-Hildebrand plot has been used to arrive at the association constant between the confined [bmim][BF4] and the TX-100 molecules. The

111 Chapter 4 Results and Discussion association constants were determined by making use of the absorbance changes in the C–H stretches of the confined IL. The measurements were performed in 2 mm quartz cuvettes at R values of 0.5 and 1.5 by subtracting the contributions of TX-100 and cyclohexane from the solution of IL/TX-100/cyclohexane. The spectra thus obtained are shown in the following figure. These spectra in essence, reflect the absorbance changes in the confined liquid as a function of added host concentration and the dilution effect by the host.

0.055 0.12 0.050 (a) (b) 0.045 0.10

0.040 0.08 0.035

0.030 A A

0.06 0.025

0.020 0.04 0.015

0.010 0.02 0.005

0.000 0.00 1580 1590 1600 1610 1620 1630 1640 1650 1580 1590 1600 1610 1620 1630 1640 1650 Wavelength (nm) Wavelength (nm)

Figure 4.25 NIR spectrum in the C–H stretch region of the confined IL. (a) R = 0.5. TX-100 weight fraction from bottom to top: 0.45, 0.5430, 0.6237, 0.7019, 0.7777 and 0.8335. (b) R = 1.5. TX-100 weight fraction from bottom to top: 0.1932, 0.3738, 0.45, 0.5430, and 0.6237.

Figure 4.25 depicts the Benesi-Hildebrand plot for the two different sized microemulsions based on the following equation. 1 1 1 = [] + (4.12) A Ao K a TX Ao where [TX] is the molar concentration of the surfactant in the microemulsions, A is the absorbance of the complexed IL, Ao is the absorbance of the complexed TX-100 in solution and Ka is the association constant for the interaction between IL and TX-100 molecules. (The derivation of the equation is given as Appendix D). Based on equation 4.12 the association constants for the R = 0.5 and R = 1.5 microemulsions were found to be 1.3156 M -1 and 0.5904 M -1 respectively.

112 Chapter 4 Results and Discussion

25

40 20 35 30 15 25

20 1/Abs 10 1/Abs 15 10 5 5 (a) (b)(b) 0 0 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1/[TX] 1/[TX]

Figure 4.26 Double reciprocal plots for R = 0.5 (a) and R = 1.5 (b) microemulsions.

Early investigations reveal the size of R = 0.5 and R = 1.5 microemulsions to be 15 nm and 80 nm respectively [65]. The weakening of the interaction between the confined IL and the surfactant OH groups with increase in microemulsion size can be rationalised based on the fact that it is only an increase in the concentration of IL that contributes to the microemulsion growth. As indicated earlier, for the two series of microemulsions investigated, the TX-100 fraction has been maintained constant. The increase in the number of IL molecules in the polar core of the microemulsions, with the number of TX-100 molecules remaining constant, results in the weakening of the electrostatic interaction experienced by the oppositely charged species. It doesn’t seem unlikely that with increase in microemulsion diameter, the prominent interaction prevalent in the system would be the weak hydrogen bonding between the IL molecules at the expense of the interaction of the IL cation with the TX-100 molecules. This − possibility is incidentally confirmed by the results based on the C HLF interactions of the confined IL, which indicates an increase in hydrogen bonding between the IL molecules with increase in microemulsion size. These two observations taken together provide irrefutable evidence for ILs behaving similar to water under conditions of confinement.

113

Conclusions

______

Surfactant science and engineering have garnered global attention over the last few decades mainly due to their industrial applications. Special attention has been focussed on the underlying concept of ‘self-assembly’ partly due to the recent developments in the field of nano-technology. This bottom-up strategy of forming surfactant aggregates from individual monomers in solution is at the heart of this thesis. Two surfactants, AOT and Triton X-100 were the molecules chosen to form reverse micelles and microemulsions to host the three model systems under consideration. These are water, α-chymotrypsin, and the ionic liquid

[bmim][BF 4]. Water in confinement was studied in AOT reverse micelles formed by four different non-polar solvents: n-pentane, cyclohexane, n-octane, and n-dodecane. A Gaussian deconvolution of the ν1+ν3 NIR spectra of the confined water in all the systems, revealed the existence of three distinct water types. These are termed free, bound, and trapped waters depending on the region in the reverse micelle where they are located, which imparts different hydrogen bonding strength to each species. Based on the areas of each sub-band from the NIR spectra, the relative amounts of each water species in the reverse micelle were quantised. The general picture of reverse micelle hydration that emerged from this analysis was that the first added water molecules to the system form the bound water component, contributing to the hydration of the AOT head groups. This growth in bound water region goes on until the AOT head groups are completely hydrated, beyond which the added water molecules formed the central water pool, accounting for the growth of the reverse micelle size. This general picture of hydration was confirmed by SAXS analysis as well. The hydration however varied in accordance with the external hydrocarbon medium used. NIR spectroscopy revealed these changes in terms of the variation in the water fractions, which were rationalised based on the differing penetration ability of these solvents. n-pentane was found to be the best at penetrating the surfactant monolayer, which as a consequnce indicated a higher spreading pressure at the surfactant tail/oil interface, leading to a more curved interface to reduce the micellar radius. By the same token, n-dodecane was expected to have

114 Chapter 5 Conclusions the highest micellar radius. These conjectures were confirmed by the SAXS analysis. Further, it was revealed from the distance distribution functions of the reverse micelles that AOT/n-dodecane/water reverse micelles are rather polydisperse possessing a high population of elongated particles; in contrast to the monodisperse globular particles in the other three systems. Dielectric experiments revealed two relaxation processes. The first was attributed to the rotational diffusion of the whole micellar aggregate; while the second was thought to be due to the reverse micellar water, which originated as result of the increase in water content. A temperature dependent study of the hydrogen bonding of micellar water in the AOT/cyclohexane/water system revealed a behaviour similar to that seen in neat water — a decrease in the free water fraction at the expense of the other two weakly hydrogen bonded components. Further, at a temperature as high as 45 °C, the AOT head group hydration was found to be complete at a hydration value of 12, as opposed to the same being accomplished at a hydration value of 14 at ambient conditions. The α-CT in confinement study was initiated with the objective of investigating the hydration properties of the confined enzyme. Different kosmotropic and chaotropic solutes were added to the AOT reverse micellar system, to mimic a typical cellular environment. This study turned out to be the first to quantify a protein’s hydration parameters under conditions of confinement, and highlighted the usefulness of NIR Spectroscopy in analyses beyond the usual spectral finger-printing. It was found that in the presence of kosmotropes, the protein hydration water increased in comparison to the protein’s chaotrope-solute environment, which corroborated the widely popular preferential exclusion model proposed by Timasheff and co-workers to account for protein stability imparted by kosmotropes.

The investigation of the ionic liquid, [bmim][BF 4] in TX-100/cyclohexane microemulsions indicated that the confined IL behaved similar to confined water. It was shown that with increase in the confined IL concentration, its polarity and hydrogen bonding increased. These were found to be in accord with the observation that with increase in microemulsion size, the association between the imidazolium moiety and the oxyethylene groups of TX-100 weakened. It is these interactions which were largely thought to be responsible for the solubilisation of the IL in the cores of microemulsions. This decrease in

115 Chapter 5 Conclusions association constant with increase in microemulsion size, was attributed to the increase in the hydrogen bonding between the IL molecules.

116

Appendix A: List of Figures

Figure Title Page Number Number

Figure 1.1 Configurations of liquid water molecules near hydrophobic cavities in molecular-dynamic simulations. 13 Figure 1.2 Surfactant shapes and various self-assemblies in colloidal solution. 15

Figure 2.1 Structure of AOT. 18 Figure 2.2 Phase diagram of the ternary system AOT/n-octane/water at STP and schematic representation of some mesophase structures. 19 Figure 2.3 Schematic representation of an AOT reverse micelle denoting the location of the three water species. 23 Figure 2.4 The two carbonyl conformations in the AOT molecule. 25 Figure 2.5 Cross section of a small portion of Escherichia coli cell. 40 Figure 2.6 The localisation of guest molecules in reverse micelles. 43 Figure 2.7 Structure of α-Chymotrypsin. 45 Figure 2.8 Structures of [bmim][BF4], Triton X-100, and Reichardt’s dye. 53

Figure 3.1 Schematic representation of the harmonic oscillator model. 59 Figure 3.2 Optical system of UV/VIS/NIR Lambda 9 spectrometer. 62 Figure 3.3 Frequency responses of dielectric mechanisms. 64 Figure 3.4 Simplified system block diagram. 67

Figure 4.1 NIR spectrum of bulk water at 25 °C recorded at 2 mm pathlength. 76

Figure 4.2 NIR spectrum of AOT/cyclohexane system at Wo = 0 at 25 °C. 77

Figure 4.3 NIR spectra of the ν1+ν3 combination band of water in AOT/cyclohexane/water reverse micelles and in the bulk

117 Appendix A: List of Figures

counterpart. 78 Figure 4.4 The curve fitted and the experimental spectra in the

AOT/cyclohexane/water system at W o =12. 79 Figure 4.5 Schematic representation of the AOT reverse micelle with the locations of the three water species. 80 Figure 4.6 Radius of gyration and estimated micelle size determined by SAXS in AOT/cyclohexane/water reverse micelles. 82

Figure 4.7 Variation of different fractions of water with Wo. 82 Figure 4.8 Variation of the number of water molecules per AOT molecule

with Wo. 83 Figure 4.9 Scattering functions of 0.1 M AOT/cyclohexane/water reverse micellar solutions at different hydration. 84 Figure 4.10 Guinier’s plots of the AOT/cyclohexane/water reverse micellear solutions at different hydration. 85 Figure 4.11 Pair distribution functions of the AOT/cyclohexane/water reverse micellear solutions at different hydration. 86 Figure 4.12 Real ( ε’) part of the dielectric constant of the

AOT/cyclohexane/water reverse micelles vs frequency at Wo = 2, 6, 10, 14, and 18 (a); and the best fit curve according to Equation

4.6 at Wo = 10 (b). 87 τ Figure 4.13 Plot of relaxation time ( 1 ) vs Wo for AOT/cyclohexane/water reverse micelles. 88 Figure 4.14 Variation in the number of each water species (a, b & c) and

fraction of trapped water (d) with Wo. 91 Figure 4.15 Scattering functions of 0.1 M AOT reverse micellar solutions. 93 Figure 4.16 Distance distribution function p(r) of 0.1 M AOT reverse micellar solutions. 94 Figure 4.17 Temperature-dependent NIR spectra of water in

AOT/cyclohexane/water reverse micelles at Wo = 8 from 25 to 75 °C. 96 Figure 4.18 Temperature dependence of the different water fractions. 97

118 Appendix A: List of Figures

Figure 4.19 Temperature variation of HOD spectra. 98 Figure 4.20 Schematic representation of the spectral components in a typical difference spectrum set-up. 102 Figure 4.21 Difference spectra recorded in the presence of different osmolytes. 104 Figure 4.22 Resolution of the difference spectrum. 104

Figure 4.23 NIR spectra of [bmim][BF 4] confined in TX-100/cyclohexane microemulsions at different R values. 109 − Figure 4.24 Spectral changes in the C HLF stretch region of the confined IL. 110 Figure 4.25 NIR spectrum in the C–H stretch region of the confined IL. 112 Figure 4.26 Double reciprocal plots for R = 0.5 (a) and R = 1.5 (b) microemulsions. 113

119

Appendix B: List of Tables

Table Title Page Number Number Table 1.1 Some properties of strong, moderate and weak hydrogen bonds. 3

Table 2.1 Properties of α-CT in AOT reverse micelles. 47

Table 4.1 Dielectric parameters of AOT/cyclohexane/water system at 25 °C. 89 Table 4.2 Positions of water species in AOT reverse micelles. 90 Table 4.3 Radii of AOT reverse micelles in different non-polar solvents. 95 Table 4.4 The protein hydration parameters. 106 Table 4.5 Solvatochromic parameters of the experimental solutions at 35 °C. 108

120

Appendix C: Spectral positions

Spectral positions (in cm -1) of water species in AOT reverse micelles.

______Hydrocarbon Water 1 Water 2 Water 3 Water 4 Reference ______

(a) Mid-IR region

CCl 4 3330 3465 3603 - 114 i-octane 3290 3490 3610 - 121 CCl 4 3330 3465 3603 - 122 n-heptane ~3330 ~3465 ~3603 - 123 CCl 4 i-octane ethane 1 3330 3490 3620 - 124 i-octane 3314 3463 3595 - 125 n-pentane 2 3330 3465 3603 - 128 toluene 3321 3450 3550 3635 129

toluene 3250 3400 3550 3600 130 n-heptane 3250 3450 3520 3600 131 i-octane 3230 3420 3540 3610 133

(b) Near-IR region

CHCl 3 ~5200 - ~5263 - 144 n-heptane 5988 - 7142 - 145 cyclohexane 6060 6896 7042 - 146 n-pentane ~6750 ~6937 ~7050 - this thesis cyclohexane ~6740 ~6920 ~7053 - n-octane ~6747 ~6920 ~7050 - n-dodecane ~6740 ~6900 ~7050 -

All measurements at ambient conditions except 1 and 2. 1: supercritical conditions 2: 295 to 220 K

121

Appendix D: Derivation of Benesi- Hildebrand Equation

Consider the following association between the IL and TX molecules, in the presence of cyclohexane which acts as the inert solvent.

IL + TX ⇔ IL LTX (1)

For this equation, the association constant, K a, may be written as,

[IL TX ] K = L (2) a [][]IL TX where [IL …TX] is the molar concentration of the host-guest complex and [IL] and [TX] are the initial concentrations of 1 -butyl-3-methyl-imidazolium tetraflurorborate and Triton X-100 respectively.

The observed absorbance for the ionic liquid may be defined as,

A = N A + N A (3) obs IL LTX IL LTX IL IL where the letters N and A represent mole fraction and absorbance respectively.

[IL TX ] Also, N = L (4) IL LTX [][] IL + IL LTX

[IL ] and N = (5) IL [][] IL + IL LTX

This implies that, N + N = 1 (6) IL LTX IL

Now, substituting equation (4) and the value of NIL from equation (6) into equation (3) gives,

[IL TX ] A = A + L ()A − A (7) obs IL IL LTX IL [][]IL + IL LTX

Also, it makes sense to think of the total ionic liquid concentration ([IL ]T) as,

[ ] = [ ]+ [ ] IL T IL IL LTX (8)

122 Appendix D: Benesi-Hildebrand Equation

Substituting this in equation (7) gives,

[IL TX ] A = A + L ()A − A (9) obs IL [] IL LTX IL IL T

Solving for [IL LTX ],

(A − A ) [][]IL TX = IL obs IL (10) L T ()A − A IL LTX IL

… If A is the absorbance of the C–H F stretch of the complexed IL, and Ao is the absorbance of complexed TX in solution, then, = ( − ) A Aobs AIL (11) and A = (A − A ) (12) o IL LTX IL substituting these values and equation (10) in equation (8), we arrive at

  [][]= −  A  [] IL IL T   IL T (13)  Ao  rewriting equation (10),

[][]=  A  IL LTX IL T   (14)  A o  solving equation (2) for [ IL ] and substituting in equation (8),

[IL TX ] []IL = L + []IL TX T [] L K a TX

 1  or [][]IL = IL TX 1+  (15) T L  []  K a TX  substituting equation (15) into equation (14),

    [][]= + 1 A IL LTX IL LTX 1 []    K a TX   Ao  on rearranging,

123 Appendix D: Benesi-Hildebrand Equation

1 1 1 = [] + (16) A Ao K a TX Ao

This is the form of the Benesi-Hildebrand equation that has been used to determine the association constant in the present study.

124 Appendix E: References

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378. Weyer, L. G.; Lo, S.-C. in Handbook of Vibrational Spectroscopy, Chalmers, J. M.; Griffiths, P. R. Eds. John Wiley & Sons Ltd, Chichester, 2002. 379. Walrafen, G. E. in Structure of Water and Aqueous Solutions, Luck W. A. P. Ed. Weinheim, 1974. 380. Pottel, R. in Water, a Comprehensive Treatise, Franks, F. Ed. Plenum Press, NY, 1973, Vol. 3, p414. 381. Gruen, D. W. R.; Haydon, D. A. Pure Appl. Chem. 1980 , 52 , 1229. 382. Lueung, R.; Shah, D. O. J. Colloid Interface Sci. 1987 , 120 , 330. 383. Frank, S. G.; Zografi, G. J. Pharm. Sci. 1969 , 58 , 993. 384. Binks, B. P.; Kellay, H.; Meunier, J. Europhys. Lett. 1991 , 16 , 53. 385. Choppin, G. R.; Violante, M. R. J. Chem. Phys. 1972 , 56 , 5890. 386. Fones, V.; Chaussidon, J. J. Chem. Phys. 1978 , 68 , 4667. 387. Monosmith, W. B.; Walrafen, G. E. J. Chem. Phys. 1984 , 81 , 669. 388. Freda, M.; Piluso, A.; Santucci, A.; Sassi, P. Appl. Spectrosc. 2005 , 59 , 1155. 389. Fisher, H. F.; McCabee, W. C.; Subramanian, S. J. Phys. Chem. 1970 , 74 , 4360. 390. Huang, J. S.; Safran, S. A.; Kim, M. W.; Grest, G. S.; Kotlarchyk, M.; Quirke, N. Phys. Rev. Lett. 1984 , 53 , 592. 391. Vandermeulen, D. L.; Ressler, N. Arch. Biochem. Biophys. 1980 , 199 , 197. 392. Ressler, N.; Vandermeulen, D. L. Biochim. Biophys. Acta 1972 , 56 , 662. 393. Ressler, N.; Ziauddin, V. C.; Janzen, W.; Karachorlu, K. Appl. Spectrsoc. 1976 , 30 , 295. 394. Galinski, E. A.; Stein, M.; Amendt, B.; Kinder, M. Comp. Biochem. Physiol. 1997 , 117A , 357. 395. Cooke, R.; Kuntz, I. D. Annu. Rev. Biophys. Bioengg. 1974 , 3, 95. 396. Dimroth, K.; Reichardt, C.; Siepmann, T.; Bohlmann, F. Liebigs Ann. Chem. 1963 , 661 , 1. 397. Reichardt, C.; Harbusch-Görnert, E. Liebigs Ann. Chem. 1983 , 5, 721. 398. Elaiwi, A.; Hitchcock, P. B.; Seddon, K. R.; Srinivasan, N. M.; Tan, Y-M.; Welton, T.; Zoraa, J. A. J. Chem. Soc., Dalton Trans. 1995 , 3467. 399. Tran, C. D.; De Paoli Lacerda, S. H.; Oliveira, D. Appl. Spectrosc. 2003 , 57, 152.

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Appendix F: Curriculum Vitae

Sangeetha Balakrishnan

Institute of Physical Chemistry II [email protected] NC 6/32, Ruhr-University Bochum Telephone: +49 (0)234 32 25533 Universitätsstrasse, 150 D-44780 Bochum, Germany

Education

From June 2003 Ph.D student at the Institute of Physical Chemistry II, Ruhr- University Bochum Supervisor: Prof. Dr. Hermann Weingärtner

August 2000 – May M.Sc. Applied Chemistry 2002 Anna University, INDIA.

M.Sc. Project Title Catalytic Oxidation of Sulphonated Phenolics in Waste Water using Activated Carbon carried out at Central Leather Research Institute (CLRI), India.

June 1997 – May 2000 B.Sc. Chemistry in Madras Christian College, University of Madras, INDIA.

May 1996 – Mar 1997 AISSCE (XII), Kendriya Vidyalaya (CBSE), Chennai, INDIA.

Academic Awards • Recipient of DFG scholarship for Ph.D since June 2003 under the framework of the Graduiertenkolleg ‘Structure and Dynamics in Heterogeneous Systems’. • Recipient of travel grant from Graduate School of Chemistry and Biochemistry (GSCB), Ruhr-University Bochum to attend the 18 th European Colloid and Interface Society Conference in Spain from Sep 18-24, 2004. • Recipient of Indian Air Force Subroto Memorial scholarship in M.Sc. for the year 2001-2002. • T.T. Thomas Prize for Chemistry in the year 1999-2000 in B.Sc. • Pattamadal Sankaranarayan Prize for Chemistry in the year 1998-1999 in B.Sc. • Raghunath Rao Prize for Chemistry in the year 1997-98 in B.Sc.

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Conference • 43 rd Meeting of the German Colloid Society , Oct 8-10, 2007, Presentations Mainz, Germany. Oral Contribution:1-butyl-3-methyl-imidazolium tetraflurorborate in Triton X-100 Microemulsion: A Comprehensive Study of Solvatochromism, Structure and Association in Confinement. • 3rd International Workshop on Dynamics in Confinement , Mar 23-26, 2006, Grenoble, France. Poster: Influence of Temperature on Water in Confinement: A Case Study of AOT Reverse Micelles • 42 nd Meeting of the German Colloid Society , Sep 26-28, 2005, Aachen, Germany. Oral Contribution: Influence of Solvent on the Water States in AOT Reverse Micelles • 6th Liquid Matter Conference , July 2-6, 2005, Utrecht, The Netherlands. Poster: Influence of Solvent on the Water States in AOT Reverse Micelles • Graduiertenkolleg Workshop , Dec 14, 2004, Witten Bommerholz, Germany. Oral Contribution: A Near Infrared Investigation of the Structure and Dynamics of Water in AOT Reverse Micelles • 18 th Conference of the European Colloid and Interface Society , Sep 19-24, 2004, Almeria, Spain. Poster: Structure of AOT Reverse Micelles: Probed by Near- Infrared and Dielectric Relaxation Spectroscopies.

Workshop • Scientific Writing Workshop – “Towards Excellent Papers – Participation The Craft of Scientific Writing”, July 5-6 & 25-26, 2006. Bochum, Germany. • “Neutron and X-Ray Scattering Techniques”, Mar 22, 2006, Grenoble, France. • “CommUNIcate! – Communicative Competence and Presentation Techniques ”, Oct 28, 2005, Dortmund, Germany . • “Soft Skills I & II: Communicating with Audiences and Giving Presentations”, Feb 11-13, 2004, Bochum, Germany.

Membership in • European Colloid and Interface Society (ECIS) – student Professional Bodies member since 2005. • German Colloid Society – student member since 2005 .

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