Amphiphilic Molecules in Aqueous Solution

Effects of Some Different Counterions The Monoolein/Octylglucoside/Water System

Gerd Persson 2003

Department of Chemistry Department of Natural and Biophysical Chemistry Environmental Sci ences Umeå University Mid Sweden University Sweden Sweden

Amphiphilic Molecules in Aqueous Solution

Effects of Some Different Counterions The Monoolein/Octylglucoside/Water System

Doctoral Thesis

Gerd Persson

Sundsvall and Umeå 2003

Avhandling som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för avläggande av filosofie doktorsexamen vid teknisk -naturvetenskapliga fakulteten vid Umeå Universitet kommer att försvaras vid en offentlig disputation i SCA -salen (O102), Kornboden, Mitthögskolan, Sundsvall fredagen den 3 oktober 2003, kl. 13.00.

Fakultetsopponent: Prof. Björn Lindman, Lunds Universitet, Lund, Sverige.

Department of Chemistry Department of Natural and Biophysical Chemist ry Environmental Sciences Umeå University Mid Sweden University Sweden Sweden Amphiphilic Molecules in Aqueous Solution Effects of Some Different Counterions The Monoolein/Octylglucoside/Water System

Abstract The aim of this thesis was to investigate amphiphilic molecules in aqueous solution. The work was divided into two parts. In the first part the effects of different counterions on phase behavior was investigated, while the second part concerns the 1 -monooleoyl -rac -glycerol 2 (MO)/n -octyl -β-D-glucoside (OG)/ H2O-system. The effects of mixing monovalent and divalent counterions were studied for two surfactant systems, sodium/calcium octyl sulfate, and piperidine/piperazine octanesulfonate. It was found that mixing monovalent and divalent co unterions resulted in a large decrease in cmc already at very low fractions of the divalent counterion. Moreover, the degree of counterion binding for piperidine in the piperidine/piperazine octanesulfonate system was much higher than predicted, probably due to the larger hydrophobic moiety of piperidine. The effects of hydrophobic counterions were studied for eight alkylpyridinium octanesulfonates (APOS). The results were discussed in terms of packing constraints. The 2 anomalous behavior of the H2O quadru polar splittings in the lamellar phases was explained by the presence of two or more binding sites at the lamellae surface. The MO/OG/water system was studied in general and the MO -rich cubic phases in particular. When mixing MO and OG it was found that O G-rich structures (micelles, hexagonal and cubic phase of space group Ia 3d) could solubilize quite large amounts of MO, while the MO - rich cubic structures where considerable less tolerant towards the addition of OG. The micelles in the OG -rich L 1 phase wer e found to remain rather small and discrete in the larger part of the L 1 phase area, but at low water concentration and high MO content a bicontinuous structure was indicated. Only small fractions of OG was necessary to convert the MO -rich cubic Pn 3m structure to an Ia 3d structure, and upon further addition of OG a lamellar (L α) phase formed. Since the larger part of the phase diagram contains a lamellar structure (present either as a single L α phase or as a dispersion of lamellar particles together with other phases), the conclusion was that introducing OG in the MO structures , forces the MO bilayer to become more flat. Upon heating the cubic phases, structures with more negative curvature were formed. The transformation between the cubic structures required very little energy, and this resulted in the appearance of additional peaks in the diffractograms.

Key words: liquid crystal, phase diagrams, counterions, alkylpyridinium octanesulfonates, 1- monooleoyl -rac-glycerol, n-octyl- β-D-glucoside, cubic phases

Language: English ISBN: 91-7305-501-8

Signature: Date: 11 August 2003 Amphiphilic Molecules in Aqueous Solution

Effects of Some Different Counterions The Monoolein/Octylglucoside/Water System

Gerd Persson

Department of Chemistry Department of Natural and Biophysical Chemistry Environ mental Sciences Umeå University Mid Sweden University Sweden Sweden Front cover: The two main principles of Judo. The left column reads in Japanese: “Jita Kyoei”, meaning “ Mutual welfare and benefit”. The right column reads in Japanese: “Sei ryoku Zenyo”, meaning “Maximum efficiency”.

Copyright © 2003 by Gerd Persson

ISBN 91-7305-501-8

Printed by Kaltes Grafiska AB, Sundsvall, 2003

ii Till Gabriel och Görgen

iii iv Abstract The aim of this thesis was to investigate amphiphilic molecules in aqueous solution. The work was divided into two parts. In the first part the effects of different counterions on phase behavior was investigated, while the second part concerns the 1 -monooleoyl -rac -glycerol 2 (MO)/n -octyl -β-D-glucoside (OG)/ H2O-system. The effects of mixing monovalent and divalent counterions were studied for two surfactant systems, sodium/calcium octyl sulfate, and piperidine/piperazine octanesulfonate. It was found that mixing monovalent and divalent co unterions resulted in a large decrease in cmc already at very low fractions of the divalent counterion. Moreover, the degree of counterion binding for piperidine in the piperidine/piperazine octanesulfonate system was much higher than predicted, probably due to the larger hydrophobic moiety of piperidine. The effects of hydrophobic counterions were studied for eight alkylpyridinium octanesulfonates (APOS). The results were discussed in terms of packing constraints. The 2 anomalous behavior of the H2O quadru polar splittings in the lamellar phases was explained by the presence of two or more binding sites at the lamellae surface. The MO/OG/water system was studied in general and the MO -rich cubic phases in particular. When mixing MO and OG it was found that O G-rich structures (micelles, hexagonal and cubic phase of space group Ia 3d) could solubilize quite large amounts of MO, while the MO - rich cubic structures where considerable less tolerant towards the addition of OG. The micelles in the OG -rich L 1 phase wer e found to remain rather small and discrete in the larger part of the L 1 phase area, but at low water concentration and high MO content a bicontinuous structure was indicated. Only small fractions of OG was necessary to convert the MO -rich cubic Pn 3m structure to an Ia 3d structure, and upon further addition of OG a lamellar (L α) phase formed. Since the larger part of the phase diagram contains a lamellar structure (present either as a single L α phase or as a dispersion of lamellar particles together with other phases), the conclusion was that introducing OG in the MO structures , forces the MO bilayer to become more flat. Upon heating the cubic phases, structures with more negative curvature were formed. The transformation between the cubic structures required very little energy, and this resulted in the appearance of additional peaks in the diffractograms.

v List of papers

The thesis is based on the following papers, which are referred to in the text by their roman numerals:

I. Competition between Monovalent and Divalent Counterions in Surfactant Systems. Carlsson, I.; Edlund, H.; Persson, G.; Lindström, B. J. Colloid Interface Sci ., 1996 , 180, 598.

II. Phase Behavior of N-Alkylpyridinium Octanesulfonates. Effect of Alkylpyridinium Counterion Size. Gerd Persson, Håkan Edlund, Erik Hedenström and Göran Lindblom submitted to Langmuir

III. The 1-Monooleoyl -rac-Glycerol /n -Octyl -βββ-D-Glucoside/Water – System. Phase Diagram and Phase Structures Determined by NMR and X-ray Diffraction. Persson, G.; Edlund, H.; Amenitsch, H.; Laggner, P.; Lindblom, G. Langmuir , 2003 , 19, 5813.

IV. Thermal behaviour of cubic phases rich in 1-monooleoyl -ra c-glycerol in the ternary system 1-monooleoyl -rac-glycerol/ n-octyl -βββ -D-glucoside/water. Persson, G.; Edlund, H.; Lindblom, G. Eur. J. Biochem ., 2003 , 270, 56.

vi Table of contents

Abstract v

List of papers vi

Introduction 1 1. Surfactants 2

1.1. General structure and criteria for surfactants 2 1.2. Different types of surfactants 3 Ionic surfactants 3 Non-ionic surfactants 4 1.3. Nonsoluble amphiphilic molecules 5 2. Phase structures 6

2.1. Isotropic solution phases: L1, L2 and L 3 6 2.2. Vesicles 7 2.3. Liquid crystalline phases 7 2.4. Packing parameter and curvature 8 Cpp 10 Spontaneous curvature 10 3. Phase equilibria in surfactant systems 11

3.1. Krafft temperature 11 3.2. Micelle formation 11 3.3. Presentati on of surfactant systems -phase diagrams 11 Gibbs phase rule 12 Binary phase diagrams and the lever rule 12 Ternary phase diagrams 13 4. Methods for characterization of surfactant systems 14

4.1. Polarizing microscopy 14 4.2. Surface tension 16 4.3. Conductivity 17 4.4. DSC 18

vii 4.5. NMR 18 1H NMR 19 Pulsed Gradient NMR 19 Quadrupolar splitting 22 4.6. SAXD 24 5. Results 26

5.1. Effects of different counterions 26 Paper I 26 Paper II 27 2 5.2. The 1-monooleoyl -rac -glyc erol/ n-octyl- βββ-D-glucoside/ H2O system 29 Paper III 30 Paper IV 31 6. Ideas for future work 32 Acknowledgements 34

References 35

Papers I - IV 39

viii Introduction In our daily life the utilization of amphiphilic molecules is very important. In man y areas including such diverse fields as cleaning products, food, paint, medicine, cosmetics and industrial as well as biological processes, this type of molecules play a crucial role. The self - organization of these molecules results in a diversity of stru ctures, among which micelles and bilayers can be mentioned. Now, the question is what exactly is an amphiphilic molecule? The word amphiphile is derived from the Greek words αµϕι (amphi) = both and ϕιλιοζ (philios) = friend. Thus, the word itself means something that likes, or rather is friendly to, both. In chemistry this is generally considered to mean two things that are immiscible, such as oil and water. So, an amphiphilic molecule likes both oil and water. Now, what does that mean and what can we use it for? Basically, this is the solution to a lot of problems involving oil and water. The use of amphiphilic molecules creates a means to dissolve water into oil and oil into water, which is something necessary for many applications. A few practical examples are washing dirty clothes, margarine, milk, and digestion of fat. What does an amphiphilic molecule look like? This will be discussed in detail later on, but as a short introduction it can be described as a frog tadpole, with a water -loving ”head” and an oil -loving ”tail”. This is a crude generalization, though, because not all molecules that fit this description are suitable for the job. Thus, there are other criteria as we ll. Amphiphiles can be categorized in a number of different ways. The most obvious concerns the nature of the ”head”. Anyone who have paid any attention to the declaration of contents of a laundry detergent have seen the words ”nonionic” and ”ionic” which refers to the nature of the ”heads” of the amphiphiles used in that product. This thesis consists of two parts. In the first part the effects of some different counterions on the behavior of ionic amphiphiles in aqueous solution is investigated. Paper I focus on the competition between mono- and divalent counterions at the micellar surface, while paper II concerns the effects of increasing counterion hydrophobicity/size. In the second part (paper III and IV) the behavior of a specific system consisting of two nonionic amphiphiles, n -octyl -β- D-glucoside (OG) and 1-monooleoyl -rac-glycerol (monoolein, MO) is investigated . The work that this thesis is based on has been done at Mid Sweden University in Sundsvall, jointly with Department of Chemistry, Biophysic al Chemistry at Umeå University.

Gerd Persson, August 11, 2003

1 1. Surfactants Amphiphilic molecules were shortly presented in the Introduction. In this section the structure and criteria will be presented and discussed. There are a number of different t erms that are used to describe this class of molecules. Detergent, tenside, surfactant and soap are among the more familiar.

1.1. General structure and criteria for surfactants Figure 1.1 shows a schematic picture of a surfactant. Any molecule that fits this description is amphiphilic, but not all amphiphilic molecules are water -soluble or will produce self - assembled structures. In order to get a water -soluble amphiphilic molecule that can form self - assembled aggregates the molecule must fulfill certain criteria.

tail headgroup

Figure 1.1 Schematic picture of a surfactant molecule a) The headgroup must be hydrophilic (water -loving) enough. Comparing the behavior of decanol and sodium decanoate shows this point. Decanol is almost insoluble in water 1, and adsorbs on any available surface or interface while the excess decanol forms a separate phase. Sodium decanoate, on the other hand, is soluble in water . Further, when a certain concentration, called cmc, is exceeded self -assemble d aggregates, micelles, are formed. Ionizable groups such as carboxylate are clearly hydrophilic enough, while the hydroxyl group is not. b) The hydrophobic (water -hating) part must be of the right size. Sodium acetate behaves more like an ordinary inorganic salt, while sodium decanoate form micelles. Generally, an alkyl chain of eight or more carbons is necessary, but if the hydrocarbon chain is too long the molecule will not be soluble at all. There is a category of molecules that resembles surfactants wit hout forming regular micelles. These are called hydrotropes.

2 1.2. Different types of surfactants Surfactants can be constructed in many different ways. There are several different types of headgroups, as well as tails, to choose between. The tail can be a flexible hydrocarbon chain of a stiff fluorocarbon chain 2, and the bile salts constitute a very different type of surfactant. 3 Moreover, there can be one or two tails bound to one headgroup4, and the tails can be branched.5 A bolaform surfactant6 has tw o headgroups, one in each end of the hydrocarbon chain, and a gemini surfactant6 consists of two surfactants joined by a spacer. One common way to classify surfactants is to look at the properties of the headgroup.

Ionic surfactants The headgroup of an i onic surfactant can be ionized in aqueous solution. Depending on the outcome of this, there are a number of subcategories. If the charge on the headgroup is negative, the surfactant is said to be anionic. Among those we find alkyl sulfates 7, alkyl sulfonat es 8, alkyl phosphates 9,10 , and fatty acid salts. 11 -13 If the residual charge is positive, the surfactant is cationic, and some examples of these are alkyltrimethylammonium14,15 and alkylpyridinium halides. 16

O + + O S O Na N Br O a) b)

Figure 1.2 Molecular structures of two common ionic surfactants. a) Sodium dodecyl sulfate (SDS). b)Hexadecyltrimethylammonium bromide (CTAB)

In some molecules the ionization leads to two separate, charged groups of opposite sign attached to th e hydrophobic part. 17,18 Such surfactants are called zwitterionic, and are common in biological systems. 18 This type of surfactant can also be regarded as nonionic, since the total charge is zero. Catanionic surfactants consists of oppositely charged surfa ctant ions, i.e. one surfactant acts as counterion to the other. 19 -21 Catanionic surfactants can be either symmetric (both alkyl chains are of equal length) or asymmetric (one chain is shorter than the other.) The properties of ionic surfactants are not o nly depending on the surfactant ion itself but also on the counterion. The counterion is often a monovalent, inorganic ion, such as sodium or chloride. Divalent ions, such as calcium and magnesium, usually shift the Krafft point (see sect. 3.1.) to a higher temperature 22 and the critical micelle concentration (cmc) towards lower

3 concentrationsPaper I , compared to the monovalent homologues. Differences can also be found between ions of the same valence due to differences in chemical properties. 23

O S O O + N

Figure 1.3 A symmetric catanionic surfactant, octylpyridinium octanesulfonate (OPOS)

Hydrophobic counterions may alter the association behavior more or less depending on the actual structure of the ion.Paper II Thus, CTACl form small mi celles 24 , while the presence of the tosylate ion promotes extensive micellar growth.25 Other examples are the changes in phase behavior observed for a series of alkylpyridinium octanesulfonates. Paper II Ionic surfactants are often sensitive towards ionic a dditives, resulting in lowering of the cmc due to the screening of charges. 26 High ionic strength is also known to promote micellar growth.27

Nonionic surfactants As the name implies, nonionic surfactant lacks groups that can easily be ionized, such as a polyoxyethylene 28,29 or a polyhydroxy 30,31 moiety. Compared to ionic surfactants of comparable size, non-ionic surfactants have lower cmc:s and the aggregation behavior is less sensitive to salt due to the lack of repelling charges in the headgroups. The solubility of polyoxyethylene surfactants usually change with temperature, and at a certain temperature (called the cloud point) a phase separation occurs. A similar behavior is also observed for some sugarbased surfactants. 32,33 For polyoxyethylene surfac tant systems, the cloud point depends on the number of oxyethylene units, and to a lesser extent the length of the alkyl chain.34 For alkylglucoside/water systems the phase separation temperature depends strongly on the length of the alkyl chain.33,35 Thus, for the n-nonyl -β-D-glucoside no separation was observed within the studied temperature range, while for the n-decyl -β-D- glucoside the phase separation occurs at all temperatures studied.33 There are several possible explanations for the cloud point phenomena for polyoxye thylene surfactants. According to one theory, a conformational change occur in the polyoxyethylene chain with increasing temperature, leading to a lower polarity of the headgroup and, thus, a lower solubility. 34,36 This is not a plausible explanation for t he alkylglucosides, since the glucoside moiety is rather rigid.

4 O O OH O O O

a)

HO OH

O OH O

HO b)

Figure 1.4 Nonionic surfactants.

a) Pentaoxyethylene dodecyl ether (C 12 E5). b)n-octyl- β-D-glucoside (OG)

Instead a mechanism based on the formation of a micellar network has been proposed.33 According to this theory, a network of entangled, wormlike micelles forms at low water content. Upon dilution of this network, the distance between c onnection points must increase. This leads to a more positive curvature and the free energy of the system increases. To counteract this, a dilute solution is expelled from the network, resulting in a phase separation. Micellar networks may form at high concentrations for n-octyl- β-D-glucoside (OG) 30 and n- nonyl -β-D-glucoside33 as well, but for these chain lengths it is possible for the network to rearrange to discrete micelles upon dilution. Phase separations can be induced in these systems, though, as can be seen in reference 37 and paper III.

1.3. Nonsoluble surfactants Even though the solubility of a surfactant in water may be very low, the solubility of water in the surfactant may be substantial.

O

HO O OH

Figure 1.5 1-monooleoyl -rac -glycerol (monoolein, MO)

5 1-monooleoyl -rac -glycerol (monoolein, MO) is an example of this type of substance. Monoolein has rendered a great deal of interest in recent years. The binary MO/water phase diagram has been extensively studied and consists of several liquid crystallin e phases 38 -40 , among which the two cubic structures have attained special interest. 41 -44

2. Phase structures

2.1. Isotropic solution phases, L1, L2 and L3 Micelles were mentioned previously. In aqueous solution a micelle is a cluster of surfactants, usual ly pictured as a spherical particle with a water -free core containing all the tails, and an outer shell containing the headgroups, some water and some of the counterions (Figure 2.1).

The normal micellar solution phase is also referred to as L 1 (liquid 1). Hydrophobic molecules can be solubilized in the micelle.

Figure 2.1 A schematic picture of a micelle showing the hydrocarbon core (inner circle) and the headgroup layer (outer circle) including associated counterions and water mo lecules. In reality a micelle is a dynamic system which forms and decomposes at a rather rapid rate. 45

Depending on the structure of the hydrophobic molecule, it may be solubilized in the water - free core or parallel to the surfactants. Micelles form at a certain concentration, called cmc or critical micelle concentration. Below the cmc, surfactants are present as monomers or dimers. Short -chain surfactants can behave like hydrotropes and form less well -defined micellar aggregates. A number of different, m ore complex micellar structures also exist. They can

6 grow, usually length -wise, forming rod-like46 , thread -like or worm -like micelles. 47 These micelles can in turn get entangled, forming networks. 47,48

The reversed or inversed micellar solution phase is r eferred to as L 2 (liquid 2). In the reversed micelles, the surfactant headgroups are directed towards the center of the aggregate and the tails are pointing outwards. In this type of micelle water droplets constitutes the core and the surrounding media, in to which the tails are protruding, is normally an oil.

The bicontinuous liquid, L3, also called the sponge phase, is isotropic, low-viscous, slightly turbid and flow-birefringent. It can be visualized as a molten bicontinuous cubic structure. 48,49

2.2. Ve sicles Another type of aggregate that can be found in dilute solutions is a vesicle.

Figure 2.2 A schematic picture of a vesicle.

A vesicle is a shell, consisting of a surfactant bilayer, which encapsulates an aqueous interior ( see figure 2.2). These structures can be very large (> 100 nm). Vesicles can form inside each other, similar to a Russian doll. Such a particle is called an onion or a multilamellar vesicle. Solutions containing vesicles are often bluish. This is due to the large particles, which scatters light. Due to the bilayer structure, a vesicle is more related to the lamellar phase than to micelles. In fact, vesicles can be formed by agitating a lamellar phase. 50 Reversed vesicles have also been found in a phospholipid/triolein/water system. 51

2.3. Liquid crystalline phases A liquid crystal is a material that has some order, but lacks the order of a solid crystal. Liquid crystals can either be thermotropic (they form upon heating) or lyotropic (they form when a solvent is added). When increasing the surfactant concentration beyond the solution phase a series of lyotropic liquid crystalline structures are usually found. These include

7 hexagonal, lamellar and cubic as well as intermediate structures. The phases appear in a certain order according to the Fontell schedule 52 , even though some structures may be absent in a specific system. Schematic pictures of some different liquid crystalline structures are shown in figure 2.3. The hexagonal phase consists of infinite cy linders packed in a hexagonal lattice. These cylinders can either be normal (tails in), denoted H I, or reversed (aqueous core), denoted HII .

The lamellar phase, L α, is built from infinite bilayers stacked on top of each other and separated by water layers . The cubic phases can be divided into two groups, discrete and bicontinuous. Within each group both normal and reversed structures are found. The discrete cubic phases are built from micellar aggregates arranged in a cubic lattice, while bicontinuous str uctures are based on infinite minimal periodic surfaces. 53 There are also a number of phases that are called intermediate. The cubic phases were thought of as intermediate phases when they first were discovered, but nowadays structures that index to other space groups than the cubic, hexagonal and lamellar are considered as intermediate. Among these phases of trigonal, tetragonal and rhombohedral space groups are found.54 -57 In defect lamellar phases the lamellae are pierced by uncorrelated holes. This ty pe is identified by the thin hydrocarbon-layer and large headgroup area obtained from x-ray diffraction when using the simple relations that usually applies to a lamellar phase. 58,59 (See sect. 4.6.)

2.4. Packing parameter and curvature Over the years a large number of amphiphilic systems have been studied, and one major conclusion is the phase sequence, the order in which phases appear. This is summarized in figure 2.3. The shape of the aggregate formed is determined by the surfactant ”need” to keep its hydrophilic and hydrophobic parts in the most favorable environment possible. There are two comparable approaches to analyze the observed phase sequence. The first considers the shape of the molecular ”building blocks”, while the second is based on the spontaneous curvature of the aggregate surface.

8 Normal Reverse Reverse Micelles Lamellar hexagonal hexagonal micelle s (L 1) (L α) (H I) (H II ) (L 2) cpp 0.33 0.5 1 >1 Increasing

H + 0 - C or T

Normal Reverse Reverse Micellar bicontinuous bicontinuous micellar cubic cubic cubic cubic

Figure 2.3 An idealized sequence of the structures found in surfactant systems as a function of surfactant concentration or temperature. The cpp and H parameters are defined in sect. 2.4. An increase in either C or T generally results in the formation of structures of higher cpp or less positive curvature. The cubic structures shown are just examples of possible structures. There are a number of different cubic structures, and most of them are

very difficult to visualize. The L 3 phase is excluded for the same reason. In this schedule it appears in the vicinity of the lamellar and the bicontinuous cubic phases.

9 Packing parameter (cpp) The packing parameter is defined as:

v cpp = 2:1 a* l where v is the volume of the hydrocarbon chain, a t he area per headgroup and l the length of the fully extended hydrocarbon chain.60 v (nm3) and l (nm) can be estimated from

v = 0.027(nc + nMe ) and l = 0.15 + 0.127nc 2:2 a and b

61 where nc is the total number of carbons per chain and nMe is the number of methyl groups. a is more difficult to estimate since the area per headgroup since, especially for ionic surfactants, a vary with the conditions in the solution. The packing parameter is useful for discussing trends due to a specific change in a system, such as change in salt concentration, temperature or the effects of an additive. From geometrical considerations one can easily determine theoretical values for different geometries (figure 2.3). When the packing parameter falls between the values specified for micelles, hexagonal and lamellar structures, the system is frustrated. It can respond by either a phase separation, or the formation of another structure that better correspond to the packing parameter, hence the term “intermediate”. 50

Spontaneous curvature Instead of considering the apparent shape of a single molecule, an aggregate can be thought of as being constructed by bending a surfactant film. 62 The mean curvature (H) at a point on a surface is defined as:

1  1 1  H =  +  2:3 2  R1 R2  where R 1 and R 2 are the radii of curvature in two perpendicular directions. The curvature is defined as positive when it curves around the hydrophobic part and negative when it curves towards the hydrophilic part. The curvature can be related to the energy required to bend the surfactant film. We then assume that there is a curvature, H 0, corresponding to a minimum in the free energy. Thus, as soon as H deviates from H 0, the system is no longer at equilibrium and the system is frustrated. Again, the response can either be phase separation or a structure of a “better” curvature, similar to the results for cpp. The main advantage with this model is

10 the possibility to determine the energy required bending a surfactant film. This offers an explanation to the formation of bicontinuous cubic as well as L 3 phases.

3. Phase equilibria in surfactant systems 3.1. Krafft temperature The Krafft phenomenon is important in studying surfactants in solution. At temperatures below the Krafft temperature, the aqueous s olubility of the surfactant is relatively low. Since all self -assembled structures are concentration -dependent, this means that there can be no aggregation if the temperature is too low, because then the concentration is too low. One notorious example is t he precipitation of calcium and magnesium soaps in hard water. The Krafft temperature depends on the stability of the surfactant crystal, and the calcium and magnesium soap crystals are more stable than the corresponding sodium and potassium soaps. By modi fying the surfactant, this problem can be solved. Modifications include changing the headgroup, introducing unsaturations and branches in the hydrocarbon chain 63 , as well as mixing the surfactant with additives and other surfactants.

3.2. Micelle formati on There are at least two fundamentally different approaches to model the micelle formation. The first one is based on the assumption that micelle formation is a kind of chemical equilibrium (the mass action law, eq. 3:1), while the second assumes a pseudo -phase separation.

S 3:1 2S ⇔ S2 ⇔ S3....Sn

The law of mass action predicts a continuous distribution of aggregation numbers from 1 to infinity. Further, increasing the surfactant concentration would shift the equilibria to the right. The phase sepa ration model involves a solubility limit, constituting the cmc. Once this limit is exceeded surfactants are separated out of the solution in the form of micelles. Neither of these models is perfect, but they do describe certain aspects of the micellization .64

3.3. Presentation of surfactant systems – phase diagrams The phase diagram is a convenient way of presenting a large body of data. To understand a phase diagram one must know how it was constructed and which constraints were applied.

11 Gibbs phase ru le According to Gibbs phase rule, the number of coexisting phases depends on the number of components and the degrees of freedom for the system.

F + p = c + 2 3:2

In eq. 3:2 F is the number of degrees of freedom (T, P and X 1, X 2….), p is the number of coexisting phases, and c is the number of components. Thus, for a system consisting of one component (for example water) we can have up to 3 coexisting phases (ice, liquid water and water vapor). When 3 phases are present, the number of degrees o f freedom is 0, which means that this occurs only at a certain temperature and pressure (for water the triple point is 0.01 °C, 6.11 mbar). 65 When interpreting a phase diagram it is important to keep in mind that the criteria for equilibrium are: α β T = T (thermal equilibrium)

Pα = Pβ (mechanical equilibrium) α β µi = µi (chemical equilibrium) where α and β indicate two different phases and i refers to component i. The interpretation of two first equalities is tha t at equilibrium there must not be any net transportation of either heat or mass. The third equality does not imply that component i is uniformly distributed in the entire system, only that the chemical potential of component i is the same in all parts of the system. Thus, the Gibbs free energy, G, is different in each phase, and by varying the amount of each phase, the system can minimize the total free energy. This is the origin of the appearance of multi -phase areas in multi -component systems.65,66

Bina ry phase diagrams and the lever rule Binary systems contain two components, usually the surfactant and water. In a binary system, the number of components is 2, giving a maximum number of degrees of freedom of 3 represented by pressure, temperature and one composition. If the pressure is kept constant, the binary system can be presented in a T -X diagram. When two phases coexist we still have one degree of freedom (two if we consider pressure as well). Thus, we are allowed to vary one variable, T or C, and s till have two phases in coexistence. This results in two -phase areas.

12 Τ α + β α β

a c b

wt %

Figure 3.1 The lever rule. The fraction of each phase can be determined from the mass balance.

For a given total concentration, the compositio n of each of the coexisting phases is determined by drawing an isothermal (horizontal) line connecting the two single phase areas, a tie -line. When moving along a tie -line the composition of each phase is constant and equal to the composition of the connec tion point, and the only thing changing is the amount of each phase. The exact amount of each phase can be determined by the lever rule. 65,66 The weight fractions of each phase in point c are w α and w β and can be determined by measuring the distance from point c to the respective single -phase boundaries, according to:

α (b − c) β (c − a) w = and w = 3:3 ()b − a ()b − a

Ternary phase diagrams When adding a third component to the system we increase the total number of degrees of freedom to 4. In order to make a two -dimensional representation of such a system we now must keep two variables constant, usually temperature and pressure, and the three -component system can be presented as a ternary diagram. Along each of the sides in the triangl e, an isothermal two -component diagram is drawn. Each corner represents a pure substance. The fraction of each component in a given point within the ternary diagram is proportional to the perpendicular distance from the point to the baseline opposite to th e corner of that component. In three -component systems we have three -phase triangles, as well as two -phase areas. Within the two -phase areas tie -lines can be determined. In the three -phase triangles the amount of each phase in a certain point can be deter mined from the distance from that point to

13 the respective corner of the triangle, similar to the determination of total composition in the ternary system. 65,66

C 0 100

P(A) = 30 % P(C) = 60 % 50 50

100 0 P(B) = 10 % A 0 50 100 B

Figure 3.2 A ternary diagram. The fraction of each comp onent in a given point within the ternary diagram is proportional to the perpendicular distance from the point to the baseline opposite to the corner of that component.

4. Methods for characterization of surfactant systems The complete determination of a phase diagram is tedious and time-consuming work that requires a number of different methods.

4.1. Polarizing microscopy By observing the sample between crossed polarizers, one can obtain information about the anisotropy of the sample. Phases that appe ar dark when viewed between crossed polarizers are described as isotropic, while those that appear bright are anisotropic. These expressions refer to the optical properties, but the same words are used to describe the phase structures as well. Isotropic structures are identical along any three orthogonal directions in space, while anisotropic are not. The optical activity of anisotropic phases is due to the fact that the refractive indices of anisotropic phases vary depending on the direction of polarizatio n of the incident light, relative to the structure of the phase. When light passes through an anisotropic phase, the emergent ray is split into two parallel rays, one for which the refractive index, n, is

14 independent of direction (the ordinary), and one fo r which n vary with direction (the extraordinary).

a) b)

Figure 4.1 The effect of anisotropic phases on polarized light. a) Crossed polarizers, no sample. No light is transmitted. The same effect is obtained whe n an isotropic sample is inserted between the polarizers b) Crossed polarizers, anisotropic sample. Light is transmitted.

The result is that a nonzero vector component of the emerging light is transmitted by the analyzer, and such mixtures appear bright. Depending on the structure of the observed liquid crystal different patterns are produced. 66 -68

a) b) c)

Figure 4.2 Examples of textures observed for different types of phases. a) Non -geometric striated; MPOS hexagonal phase b) Fanlike; HexPOS hexagonal phase. c) Maltese crosses and oily streaks; OPOS lamellar phase.

Shear induces readily observable anisotropy, or birefringency, in all cubic phases (both crystals and liquid crystals) and in micellar solutions with long cylindrical or entangled threadlike micelles. This is described as shear or flow birefringency. 66 The so-called ”penetration experiment” provides a simple and fast way of determine the phase behavior at a specific temperature. Basically, the experiment is performed as follows:

15 1 2 3

1. Put the dry surfactant on a microscope slide and cover with a cover glass. 2. Melt the surfactant, then let it cool to obtain a homogeneous crystal with sharp edges. 3. Add a drop of water.

As the water diffuses into the surfactant crystal, the re sulting concentration gradient produces all phases possible for that surfactant at that temperature. 66,69

L1 HI L1 Lα Crystal

Figure 4.3 Penetration scan performed on HexPOS at 25 °C.

Abbreviations: L 1 – isotropic micellar sol ution, HI – normal hexagonal

phase, L α – lamellar liquid crystalline phase.

Anisotropic phases are readily identified from their typical textures, while isotropic ones are more difficult. The major advantages of this experiment are the small amounts nece ssary and the rapidness with which an entire temperature -composition phase diagram can be mapped. The drawback is that concentrations are unknown.

4.2. Surface tension The surface tension, γ, of a pure liquid is determined by the interactions between the molecules in the liquid. It is a measure of the amount of work required to enlarge the surface. Adding a solute may either decrease or increase the surface tension of the resulting solution, depending on whether the solute is adsorbed to the surface or dep leted from it. The difference between the surface concentration and the bulk concentration is called the surface excess, Γ2, and can be both positive (adsorption) and negative (depletion). 65 The decrease in surface

16 tension with solute concentration ( ∂γ/∂C) is related to Γ2 by the Gibbs equation, which for ionic surfactants is:

1 ∂γ 1 ∂γ Γ = − ⋅ ≈ − ⋅ 4:1 2 2RT ∂ ln a 2RT ∂ ln C where R is a con stant, T is the temperature and a is the activity of the solute. ∂γ can be obtained from the first derivative of a polynomial fitted to γ vs. ln a at ∂ ln a 70 2 concentrations below cmc. From Γ2 (given in mmol/m ) the area per molecule at the surface, σ, (given in Å2) can be estimated using

10 20 σ = 4:2 N A Γ2 where N A is Avogadro´s constant. For a surfactant solution, the surface tension decreases with increasing concentration until micelles form.65 Thereafter the surface tension be comes more or less constant because once micelles form, all new surfactants will form micelles. Cmc can be determined from the break point in a γ vs. C (or ln(C)) graph.

80

70

60

50 (mN/m)

γ 40

30

20 -10 -8 -6 -4 -2 0 ln C

Figure 4.4 Surface tension vs. ln C for OPOS. The break in the curve indicates the onset of micellization.

4.3. Conductivity The conductivity, κ, of a solution depends on the transport of charges. Species with a small hydrodynamic radius conduct better than large ones since they can move faster. Ions of higher charge usually attract more water (hydration), resulting in a larger hydrodynamic radius. For ionic species at low concentrations, the conductivity is linearly dependent of concentration. 65

17 1,0

0,8

0,6 (mS/cm)

κ 0,4

0,2

0,0 0,00 0,05 0,10 0,15 0,20 0,25 C (M)

Figure 4.5 κ vs. C for OPOS. The break in the curve indicates the onset of micellization.

In solutions of micelle -forming amphiphiles a break in the κ vs. C-curve can be found at cmc, indicating a change in structure. The degree of counterion binding, β, is defined as the number of counterions close to a micelle divided with the aggregation number of that micelle and can be determined from β =1− α . α can roughly be estimated from the ratio of the slopes before and after cmc.

 ∆κ   ∆κ  α =     4:3  ∆C above cmc  ∆C below cmc

4.4. DSC Differential Scanning Calorimetry, DSC, measures the difference in thermal behavior between the sample and a reference. For example, when a sample melts, more energy is required to keep the temperature than for the reference. The shape and size of the resulting peak contains information about the type of transition involved and how much energy the transition requires.71 DSC is used to determine melting points, polymorphism and purity for one -component systems. For two -component systems, the behavi or may be more complex. Several types of discontinuities such as eutectics, peritectics and polytectics are found. 66 ”Peaks” originating from two -phase areas are broad and may be difficult to observe since the transition energy is smeared out over a large temperature interval. 72

4.5. NMR Nuclear Magnetic Resonance, NMR, is based on the splitting of energy levels of the nuclear magnetic spin, which occurs when an NMR active nucleus is placed in a static

18 magnetic field. The NMR signal is generated when the system returns to equilibrium after being disturbed. In the modern Fourier Transform (FT) NMR spectrometer a short pulse containing a large portion of the radio frequency (RF) area is used to cause this disturbance. Depending on the nucleus and on its mag netic and electric environment, the resulting signal appears at different frequencies. The process when the magnetization of a nucleus returns to its initial state is called relaxation and is described by two relaxation times, T1 and T2. The simplest NMR experiment consists of one pulse followed by acquisition. By using consecutive RF pulses of varying strength and length prior to acquisition, we can perform a number of different experiments. One common type of sequence is the spin -echo sequence (SE). It consists of a 90° pulse followed by a 180° pulse. 73

Figure 4.6 The Hahn spin echo pulse sequence

1H Protons have a spin quantum number = 1/2, resulting in two energy levels. This nucleus is the most common naturally occurring NMR active nucleus and proton NMR is widely used for structural determination of molecules. The chemical shift, δ , defined as ν − ν ν , is a field independent 1H ( 1H TMS ) spectromet er number characteristic for a proton in a certain type of environ ment. Upon micellization the magnetic environment changes and consequently also δ .73 1H

Pulsed Gradient (PG) NMR If the magnetic field contains inhomogeneities, this will affect the relaxation processes. This is utilized in the self -dif fusion experiment. When a magnetic field gradient is introduced between the two pulses in the spin -echo sequence, the refocusing of the signals will be dependent on the motion of the nuclei in this gradient. By repeating the experiment for successive large r gradients, the intensity of the observed peak will decrease and from this decay the self -diffusion coefficients can be obtained by fitting the data to eq. 4:4.

2 −2τ T2 {−(γGδ) D(∆−δ / 3)} I = I0e e 4:4

19 In eq. 4:4 I denotes the observed echo intensity, I 0 is the e cho intensity in the absence of field gradient pulses, τ is the time between the 90° and 180° pulses, T 2 is the transverse relaxation time, γ is the magnetogyric ratio, G is the field gradient strength, δ is the duration of the gradient pulse and ∆ is the time between the leading edges of the gradient pulses. At least two pulse sequences are commonly used for diffusion experiments, the common SE and the stimulated spin -echo sequence (STE).

Figure 4.7 The pulsed gradient stimulated spin echo sequence

Which sequence to use is determined by the relaxation of the system studied. If T 1 = T 2 then SE is appropriate, while for systems with T 1>>T 2 STE is the preferred pulse sequence. 74,75 For STE the echo attenuation is given by

1 2 I = I e−T T1e−2τ T2 e{−(γGδ) D(∆−δ / 3)} 4:5 2 0 where τ is the time between the first and the second 90° pulses, T is the time between the second and third 90° pulses, T 1 is the longitudinal relaxation time and all other variables and constants are as previously described. Not all surfactant molecules participate in micelles and the observed self -diffusion coefficient is a sum of the coefficients for free and micellized surfactants. A simple two -site model is usually applied to account for the presence of free surfactant.

obs 0 Dsurf . = PD mic + ()1− P Dsurf . 4:6

obs where Dsurf . is the observed diffusion coefficient, Dmic is the micellar diffusion coefficient

0 and Dsurf . is the diffusion coefficient of the free surfactant, usually measured at a conc entration well below cmc. P is the fraction of micellized surfactant given by

20 P=(C tot – cmc)/C tot 4:7 where Ctot is the total surfactant concentration and cmc the critical micelle concentration. Dmic is measured by adding a hydrophobic probe that wil l be solubilized in the micelles only. If both the surfactant and the counterion can be detected the degree of counterion binding can be calculated from:

D0 − Dobs D0 − Dobs β = counterion counterion surf . surf . 0 0 4:8 Dcounterion − Dmic Dsurf . − Dmic

obs 0 where Dcounterion is the observed diffusion coefficient, and Dcounterion is the diffusion coefficient of the counterion at concentrations well below cmc. Information about the size of the diffusing aggregate can be obtained from the Stoke -Einstein equation:

0 kBT Dmic = 4:9 6πηRH

0 where Dmic is the micellar diffusion coefficient at infinite dilution, kB is the Boltzmann constant, T is the temperature, η is the viscosity of the medium and R H is the hydrodynamic radius of the aggregate. At finite aggregate concentrations, aggregate obstruction effects have to be accounted for. For spherical aggregates this can be accomplished by

0 Dmic = Dmic (1− k ⋅φagg ) 4:10 where φagg is the volume fraction of aggregates and k is an interaction parameter. For hard spheres, according to theoretical predictions, the value of k is between 1 and 2.5. 76 However, the hydration layer should also be include d in RH and if the hydration is unknown, this can be accounted for by using a different k-value. 77 Often the shape of the diffusing aggregate deviates substantially from a sphere, which modifies the diffusion behavior. This can be acco unted for by introducing a shape factor. In

21 this work we have assumed either prolate or oblate aggregates. The shape factors for prolate 69,79,80 and oblate 69,80 micelles are

 2   2  ln B + B −1 arctan B −1 prol =   and obl =   4:11 a and b 2 B −1 B2 −1 where B is the ratio between the short and long axis. The corresponding expressions for the micellar diffusion coefficient at infinite dilution are

0 kBT 0 kBT D = prol and Dobl .mic = obl 4:12 a and b prol .mic 6πηr 6πηr where r is the length of a fully extended surfactant mo lecule including the tail, headgroup and the corresponding hydration. The obstruction effects are taken into account by means of

 φagg  D = prol .⋅D0 1− k  4:13 a mic prol .mic 3  B⋅prol 

and

 φagg  D = obl .⋅D0 1− k  4:13 b mic obl .mic 2 3  B ⋅prol 

Eq. 4:10, 4:13 a, and 4:13 b were evaluated for three di fferent k-values: 1.7, 2.0 and 3.4. It should be mentioned that the prolate geometry is not a very good approximation for a nonspherical micelle, since surfactants have a finite size. The ends of a prolate body are too “pointy” and the packing constraints are not ideal. A hemisphere -capped cylinder or a dumbbell -shaped aggregate provides better approximations.

Quadrupolar splitting There are several nuclei that possess an electric quadrupole moment, which will interact with electric field gradients resulti ng in a quadrupolar splitting, ∆. Among others 2H (deuterium) and 23 Na can be mentioned. In the present work, deuterium has mainly been utilized, and the rest of this section is therefore concentrated on deuterium. 2 Deuterium is not a commonly occurring nuc leus, and has to be introduced either as H2O or attached to the surfactant. When two or more phases are present in a sample, the resulting spectrum is a superposition of the individual spectra, since the exchange between the different

22 phases is slow. This is the main advantage with this method since it does not require macroscopical separation of the constituent phases.

The magnitude of ∆ depends on the effective quadrupolar coupling constant, νQ, the fraction water associated to the surfactant aggregate and an order parameter, S.

Figure 4.8 2H quadrupolar splitting, EPOS. Observe the dip in the middle. 80,81

If the aggregates are aligned macroscopically, ∆ also depends on θLD , which is the angle 2 between the laboratory frame and the director coordinate system. When the H2O molecules are subjected to fast chemical exchange between different sites, having different values of νQ, the magnitude of the resulting splitting is obtained by

∆ = p ν S (3cos 2 θ −1) 4:14 ∑ i Qi i LD i

2 where pi refers to the fraction of H2O in site i. In most cases we deal with samples in which the microcrystallites are randomly distributed, i.e. powd er samples. Thus, all values of cos θLD o are equally probable, and the observed quadrupolar splitting corresponds to that for θLD = 90 . In isotropic phases, the motion and orientation of the aggregates are such that the quadrupole interaction averages to z ero and the resulting signal is a singlet. It should be noted that there are situations where anisotropic phases yield singlets, as well. If the aggregates in the phase o studied are macroscopically oriented so that θLD = 54,7 , then the resulting signal is a singlet, even if the phase itself is anisotropic. Further, the order parameter, S, depends on the angle between the director coordinate system and the molecular reference frame, θDM . For a lyotropic liquid crystalline phase the value of S can vary betwee n -1/2 and 1, and when θDM = 54,7o S is equal to zero, resulting in that the signal again is a singlet. Yet another situation, where the observed quadrupolar splitting may be infinite small, occurs when either of the factors in the term νQi Si has opposite signs in different sites, causing partial cancellation of

23 the terms in equation 4:14. Finally, if the microcrystallites are too small, the resulting signal may also be a singlet. 82 ∆ is also concentration -dependent. Usually, a simple two -site model is app ropriate, dividing the water molecules into bound and free water, according to

XSurf. ∆ = Pf ∆f + Pb∆b = ∆f + n (∆b − ∆f ) 4:15 XW where P refers to the fraction of deuterons in each site, ∆f and ∆b are the magnitudes of the splitting for free and bound water, respectively, n is the average hydration number of the amphiphile and X Surf. and X W are the mole fractions of amphiphile and water, respectively. By assuming ordering of free water to be negligible, thus, giving no contribution to the quadrupolar splitting in liquid crys talline phase, equation 4:15 reduces to

XSurf. ∆ = nSνQ 4:16 XW

From eq. 4:16 we see that ∆ depends linearly on the ratio X Surf. /X W if the n, S and νQ remains constant, and this is called ”ideal swelling” indicating that when water is added it will join the structure as free water. It is indeed observed for a number of systems that a plot of ∆ vs . 83 -85 XSurf. /X W is linear over a wide range of water concentrations, indicating ideal swelling. Equation 4:15 and 4:16 fails at high surfactant concentrations, when there is too little water present to fill all ”bound” sites, if more that two sites are present, or if the aggregate rearranges. The latter causes a change in the order parameter. Examples of this behavior have also been reported for a limited number of systems.81,86 -89,Paper II

4.6. SAXD Small -Angle X -ray Diffraction, SAXD, is in most cases the only method, which gives information about the spatial arrangement of a liquid crystalline structure. The method is similar to ordinary X -ray crystallography, with the difference that liquid crystalline phases usually lack short -range order. Because o f this only a few Bragg reflections can be detected, and these occur close to the primary beam, hence the name small -angle. Constructive interference is observed when

nλ = 2dsin θ (Bragg´s law) 4:17

24 where n is an integer, λ is the wavelength of the radiation, d is the distance between two lattice planes in the crystal, and θ is the angle between the incident ray and the diffracting planes.68

θ θ

A C d B

Figure 4.9 Constructive interference occurs when the distance AB+BC is equal to an integer multiple of the incident wavelength.

The different planes in a crystal can be described by the Miller indices (h, k, l) For a lamellar structure the Miller indi ces are (h=n, k=l=0) and the distance between the lattice planes is simply the interlayer spacing (or the lattice parameter), a, described by:

a dh = 4:18 h2

If the volume fraction of the hydrocarbon part, φhc , is known then the thickness of the hydrocarbon layer, 2r hc , and the area per molecule at the hydrocarbon/headgroup interface, A , can be obtained from: Lα

2rhc = aφhc 4:19

and

V A = Lα 4:20 rhc

50 where V is the volume per molecule. For an L α phase 2r hc ≈ 1.8 l (eq. 2:2 b). For a two -dimensional hexagonal lattice, the Bragg reflections are related to the unit cell dimension, a, by the relation

3 dhk = a 4:21 2 ()h2 + k2 + hk

25 The radius of the hydrocarbon core, r hc , and the area per molecule at the hydrocarbon/headgroup interface, A , can be obtained from: HΙ

3φ r = a hc 4:22 hc 2π

and

2V A = H1 4:23 rhc

For a cubic lattice

a dhkl = 4:24 ()h2 + k2 + l2

Depending on the space group of the structure, different (h, k, l) values are allowed, and this give rise to characteristic patterns for each space group. Unfortunately, an unambiguous structural determination may require more peaks than can be obtained from a liquid crystal.

5. Results 5.1. Effects of different counterions The effects of different types of counterions on the micellization behavior have been studied extensively since the discovery of micelles. In the absence of other interactions than electrostati c ones, the balance between the electrostatic attraction and the loss in entropy determines the counterion association. Specific interactions such as polarizability and physical size result in differences between ions of the same valency. 23 Different modif ications to the counterion may result in dramatic changes in the association behavior of a given surfactant. One notorious example is the increase in Krafft temperature observed for fatty acid salts in the presence of Ca 2+ and Mg2+ . Other examples are the increase in counterion binding90 , and the changes in stability of the liquid crystalline phases 91 that occurs for hydrophobic counterions.

Paper I In this paper the effects of mixing monovalent and divalent counterions in the micellar phase was studied both experimentally and theoretically. Two surfactant systems were studied, sodium/calcium octyl sulfate, and piperidine/piperazine octanesulfonate. The

26 experimental techniques used for the Na +/Ca 2+ octyl sulfate system were conductivity measurements and Na + quadrupolar splittings, while the piperidine+/piperazine2+ octanesulfonate system was chosen specifically for the PGSE -NMR method. The Poisson- Boltzmann cell model was used to compare experimental observations with electrostatic theory. For a given surfac tant the cmc is lower for a divalent counterion than for a monovalent in the absence of other interactions than electrostatic ones. This is mainly caused by the decrease in entropy of mixing due to the ordering of counterions at the aggregate surface. The number of divalent counterions is half as large for a given number of surfactant molecules, compared to the same number of surfactant molecules with monovalent ones. Therefore divalent counterion association can be higher, which results in a larger screening of surfactant headgroups leading to a lower cmc. Moreover, when both monovalent and divalent counterions are present, the one with the higher valency will accumulate at the aggregates. Thus, the cmc of a surfactant in the presence of a mixture of monova lent and divalent counterions should decrease even at very small fractions of divalent counterion. The results obtained both for the Na +/Ca 2+ octyl sulfate system and the piperidine+/piperazine2+ octanesulfonate system show that this is indeed the cas e. The difference in cmc when changing from monovalent to divalent counterion is about 60 mM for both surfactants. Moreover, mixing monovalent and divalent counterions resulted in a large decrease in cmc already at very low fractions of the divalent counte rion. The fraction Ca 2+ necessary to achieve a decrease in cmc half of this, was about 0.1, while the corresponding fraction piperazine2+ in the piperidine+/piperazine2+ octanesulfonate system was 0.05. The degrees of counterion binding for each counterio n, β(i), were determined for the piperidine+/piperazine2+ octanesulfonate system. It was found that β(piperidine+) was much higher than predicted. This indicates an additional interaction, probably due to the larger hydrophobic moiety of piperidine. Na + quadrupolar splittings were measured in the hexagonal phase formed at higher concentrations of Na octyl sulfate. This quantity was found to decrease with increasing fraction Ca 2+ and this effect was interpreted as originating from the displacement of Na + by Ca 2+ at the aggregate surface.

Paper II The stability of the different liquid crystalline phases may change due to the counterion present in the system. In this paper a systematic study of these changes is presented. By varying the number of carbons (n c) in the alkyl chain of the alkylpyridinium

27 ion, eight different alkylpyridinium octanesulfonates (APOS) were synthesized. The names and corresponding abbreviations are given in the figure legend to figure 5.1. The binary 2 APOS/ H2O systems were prepared and the different phases were studied in detail by the appropriate methods. Since the common ion in the APOS is the octanesulfonate ion, the binary sodium octanesulfonate (NaOS) was also studied, as a reference.

100 100 100 NaOS MPOS EPOS HI L L H L 50 1 S 50 1 I 50 1 HI C S C S 0 0 0 0 50 100 0 50 100 0 50 100

100 100 100 PrPOS BPOS PePOS

L L L 50 1 50 1 HI 50 1

H H I S S I S 0 0 0 0 50 100 0 50 100 0 50 100

100 100 100 HexPOS HepPOS OPOS Lααα Lααα L L ααα L L 50 1 50 1 50 1 S

S HI S HI 0 0 0 0 50 100 0 50 100 0 50 100

Figure 5.1 NaOS and the eight APOS phase diagrams. Paper II The lines inside the

HepPOS and OPOS L α phases indicate the location of the apparent singlets.

Abbreviations: L 1, isotropic solution phase; C, cubic phase; HI, normal

hexagonal phase; L α, lamellar liquid crystalline phase; S, solid; NaOS , Sodium octanesulfonate; MPOS , Methylpyridinium octanesu lfonate; EPOS , Ethylpyridinium octanesulfonate; PrPOS , Propylpyridinium octanesulfonate; BPOS , Butylpyridinium octanesulfonate; PePOS , Pentylpyridinium octanesulfonate; HexPOS , Hexylpyridinium octanesulfonate; HepPOS , Heptylpyridinium octanesulfonate; OPOS , Octylpyridinium octanesulfonate

In systems like these, the packing parameter can not be calculated as a simple mean value of the two oppositely charged ions, since the degree of counterion binding vary, as well as the

28 contribution from the hydrophobic interaction. If the packing parameter ( cpp) would change linearly with increasing nc, this would result in a continuous change in the appearance of the phase diagrams. My results show that this is not the case in these systems. Instead the appearance of th e phase diagrams change in three steps. Increasing nc from 1 to 3 results in a decrease in stability of both the cubic and the hexagonal phases, but for nc = 3, 4 and 5 there appears as if there are no changes at all. For nc > 5 the continuous changes appear again, and this coincide with the occurrence of lamellar phases. The increasing stability of the lamellar phases with nc is in agreement with a cpp closer to unity. The origin of the initial decrease was attributed to the increase in counterion bindin g observed in the micellar phase, while the second decrease was explained by an actual increase in cpp. This increase in cpp was, however, rather small for each added methylene unit, resulting in the formation of defects in the lamellar phases at lower surfactant concentrations. 2 Further, the H2O quadrupolar splittings obtained in the lamellar phases (and also to some extent in the hexagonal phases) showed anomalous behavior. In neither of the three L α phases were the ideal swelling behavior found. Instead, the magnitude of the splittings were found to pass through a minimum. In the HexPOS system, the singlets occurred very close to the L 1-

Lα two -phase area, while for HepPOS and OPOS the apparent singlets appeared well away from the phase boundaries. 2 The anomalous behavior of the H2O quadrupolar splittings in the lamellar phases was explained by the presence of two or more binding sites at the lamellae surface. Upon dilution or heating, the development of holes in the lamellae causes changes in the order parameter for 2 each site, which results in the observed H2O quadrupolar splittings.

2 5.2. The 1-monooleoyl -rac-glycerol/ n-octyl -βββ-D-glucoside/ H2O system As mentioned earlier, 1-monooleoyl -rac-glycerol (MO) is a molecule that has attained a lot of attention over the years (see the reference list in paper III). It has been suggested that by mimic the native environment for membrane proteins it could be possible to obtain high -quality crystals, a major problem in structural determination of proteins. Thus, by introducing bacteriorhodopsin into a MO cubic phase Landau and Rosenbusch 43 were able to obtain crystals of very high quality. Unfortunately, the initial promising results have not been repeated with other proteins, and the origin of this lack of success remains unknown. The exact mechanism by which the crystallization took place has not yet been determined and the proposed mecha nism raises a number of questions. 92 From my point of view, questions concerning phase structure and phase equilibria are of major

29 interest. Since membrane proteins have to be solubilized by a surfactant in order to get them out of the membrane, it is important to know the effects of the surfactant on the MO phases. One common surfactant used for solubilizing membrane proteins is n -octyl -β-D-glucoside (OG). Thus, it seemed logical to investigate the ternary MO/OG/water system in general and the effect of OG on the MO-rich cubic phases in particular.

Paper III The entire ternary MO/OG/water phase diagram was determined at 25 °C. The 2 30,93 binary OG/ H2O phase diagram consists of a large micellar solution phase followed by a hexagonal (H I), a bicontinuous cubic phase of space group Ia 3d, and a lamellar liquid crystalline (L α) phase with increasing OG concentration. However, the H I has a low m elting point and disappears at temperatures above 23 °C. MO is an amphiphilic lipid that is almost insoluble in water. The solubility of water in MO is rather large, though, and with increasing hydration an L α phase, a cubic phase of space group Ia 3d, and a cubic phase of space group Pn 3m, forms at 25 °C. At higher temperatures a reversed hexagonal phase, as well as reversed micelles are found.38 -40

0OG 100

C1

50 50 ol. H . m ...... 1 eq L ...... 1......

Lααα 100 0 2 0 50 100 H2O MO

Figure 5.2 The MO/OG/water system. The dotted area in the L 1 phase i ndicates the

area where flow-birefringency was observed. Abbreviations: L 1, isotropic

solution phase; H I, normal hexagonal phase; L α, lamellar liquid crystalline phase. Paper III

30 When mixing MO and OG it was found that OG -rich structures (micelles, H I and cubic phase of space group Ia 3d) could solubilize quite large amounts of MO, while the MO-rich cubic structures where considerable less tolerant towards the addition of OG. The micelles in the OG -rich L 1 phase were found to remain rather small and discrete in the larger part of the

L1 phase area. At low water concentration and high MO content, the solution showed indications of a bicontinuous structure, though. (Dotted area in figure 5.2.) It was found that only small fractions of OG was necessary to convert the MO-rich cubic Pn 3m structure to an

Ia 3d structure, and upon further addition of OG an L α phase formed. Comparing with the Fontell schedule (figure 2.2) this means that the Pn 3m structure has the most negative curvature, followed by the Ia 3d structure. Since the larger part of the phase diagram contains a lamellar structure (present either a s a single L α phase or as a dispersion of lamellar particles together with other phases), the conclusion was that introducing OG in the MO structures, forces the MO bilayer to become more flat. Simple geometrical considerations of the two molecules reveale d that the resulting structure should have cpp ≈ 1. Since only a few of these units are necessary to change the entire phase, the conclusion is that the Pn 3m structure is close to be converted to the Ia 3d structure already in the binary system. The lack of vesicles in the dilute region was attributed to stiff bilayers, which resulted in the formation of lamellar particles instead. Adsorption of OG on the edges of these particles would prevent the fusion of these particles, thus stabilizing the dispersion. I t should be mentioned that the presence of these large lamellar particles might hide smaller vesicles. Therefore vesicles can not be excluded, even though no evidence of their presence was found.

Paper IV In this paper the effects of temperature on the MO-rich cubic phases in the MO/OG/water system was studied. As mentioned in the previous section, only small fractions of OG were necessary to convert the Pn 3m structure to a less curved structure. The observed phase sequence upon heating is L α→ Ia 3d and Ia 3d → Pn 3m. Again, this is in agreement with the Fontell schedule, if the Pn 3m structure has the most negative curvature. When comparing the MO/OG/water -system with a similar one, the MO/dodecylmaltoside (DM)/water - system 94 , it was found that the MO-rich cubic phases in the MO/OG/water -system have higher thermal and compositional stability than the corresponding phases in the MO/DM/water -system. According to these results, OG would therefore be the preferred surfactant. The conversion between t he two cubic phases required very little energy. In fact,

31 no transitions could be determined by the DSC unit used as the heating device. This indicates that the transformation between these two structures does not require a major rearrangement of molecules . From figure 3 in paper IV, one can see that the (2,2,0) peak of the Ia3d structure and the (1,1,1) peak of the Pn3m structure seem to coincide, but the possible epitaxial relationship was not investigated further.

OG 10 50 L Lα + Ia3d α E Pn 3m D F C G B H A Ia 3d 0 2 50 60 70 H2O MO

Figure 5.3 The MO-rich cubic phases in the MO/OG/water system. The letters A – H refers to samples. For more information, see paper IV. Abbreviations: Pn 3m, cubic phase of space group Pn 3m; Ia 3d, cubic phase of space Paper IV group Ia 3d; L α, lamellar liquid crystalline phase.

During heating, additional peaks appeared in certain temperature intervals. Further, differences were observed between the results obtained during heating and cooling. Both these effects were explained by th e notorious metastability shown by especially bicontinuous cubic phases. It has been suggested that the cubic structure present during the protein crystallization is the Pn 3m, even though the MO concentration indicates that the structure should be Ia 3d. T he results presented here show that the Pn 3m structure is sensitive towards the addition of OG, but it is possible to “undercool” the structure.

6. Ideas for future work There are several aspects of the results obtained during the work with this thesis t hat calls for a closer investigation. In part 1, where the effects of different counterion were investigated, the specific contributions from the hydrophobicity of the piperidine counterion on the obtained results may be of interest. Further, the phase behavior at higher surfactant concentrations of the piperidine and piperazine octanesulfonates was not investigated.

32 In the APOS systems, the effects of excess AP +, OS - and salt could be of interest to investigate. In a preliminary study, no liquid crystall ine phases were found in some of the systems, and the origin of these results may have been due to the presence of excess AP +, OS - or salt. Further, the exact structure of the MPOS and EPOS cubic phases should be determined. In part 2, the next step would be to investigate the effects on the MO-rich cubic phases of the different buffers, and other additives used during the crystallization experiment, as well as the proteins.

33 Acknowledgements It is said that the journey is more important than the destination. It is indeed true. My journey has been long and started well before I even became a student. Along the way I have met a lot of persons that have been more or less important for the joy of traveling, as well as for showing me the way. To mention all o f them by name would result in a very long list, and I probably would forget someone.

Thus, to my family, who supported me (more or less eagerly) during my early experiments at home (sorry for the chlorine gas); to my teachers, who sometimes have had more than a handful of work; to my friends and fellow judokas (it is always nice to throw someone after a hard day at the lab); and to former and present colleagues at the chemistry department: thanks for being there.

Some persons deserve to be mentioned es pecially, though. Therefore I say a special thanks to Malin for sharing (almost) everything, to my supervisors Håkan Edlund and Göran Lindblom for the support, and finally to my husband Hans - Göran and my sons Gabriel and Görgen for surviving this last year .

Without money it is difficult to do anything nowadays. Thus, Mid Sweden University is acknowledged for financial support.

Professor Peter Laggner is acknowledged for granting the beam time at the ELETTRA synchrotron, and Heinz Amenitsch and the others at ELETTRA are acknowledged for helping me with my experiments.

Academic Press is acknowledged for the permission to reprint the following article: ”Competition between Monovalent and Divalent Counterions in Surfactant Systems” by Carlsson, I.; Edlund, H. ; Persson, G. and Lindström, B. reprinted from Journal of Colloid and Interface Science , Volume 180, 598-604 copyright 1996, Elsevier Science, reprinted with permission from the publisher.

The Federation of European Biochemical Societies is acknowledged for the permission to reprint the following article: ”Thermal behaviour of cubic phases rich in 1 -monooleoyl -rac -glycerol in the ternary system 1-monooleoyl -rac -glycerol/ n-octyl -β-D-glucoside/water” by Persson, G.; Edlund, H. and Lindblom, G. reprinted from European Journal of Biochemistry , Volume 270, 56-65 copyright 2003, Blackwell Publishing, reprinted with permission from the publisher.

American Chemical Society is acknowled ged for the permission to reprint the following articles: ”The 1-Monooleoyl -rac-Glycerol/n -Octyl -β-D-Glucoside/Water – System. Phase Diagram and Phase Structures Determined by NMR and X -ray Diffraction” by Persson, G.; Edlund, H.; Amenitsch, H.; Laggner, P. and Lindblom, G. reprinted from Langmuir , Volume 19, 5813-5822 copyright 2003, American Chemical Society, reprinted with permission from the publisher. ”Phase Behavior of N -Alkylpyridinium Octanesulfonates. Effect of Alkylpyridinium Counterion Size” by Gerd Persson, Håkan Edlund, Erik Hedenström and Göran Lindblom, submitted to Langmuir2003.

34 References 1. Mandell, L.; Ekwall, P. Acta Polytech. Scand., 1968 , 74, 5 2. La Mesa, C.; Sesta, B. J. Phys. Chem. , 1987 , 91, 1450 3. Carey, M. C.; Small, D. M. Arch. Intern. Med ., 1972 , 130, 506 4. Warr, G. G.; Sen, R.; Evans, D. F.; Trend, J. E. J. Phys. Chem ., 1988 , 92, 774 5. Wormuth, K. R.; Zushma, S. Langmuir , 1991 , 7, 2048 6. Zana, R. J. Phys. Chem. B , 1999 , 103, 9117 7. Kékicheff, P.; Grabielle -Madelmont, C.; Ollivon, M. J. Colloid Interface Sci . 1989 , 131, 112 8. Tartar, H. V.; Wrigth, K. A. J. Am. Chem. Soc . 1939 , 61, 539 9. Jeon, J. S.; Sperline, R. P.; Raghavan, S.; Hiskey, J. B. Colloids Surf. A 1996 , 111, 29 10. Fröba, M.; Tiemann, M. Chem. Mater ., 1998 , 10, 3475 11. McBain, J. W.; Langdon, G. M. J. Chem. Soc., 1925 , 127, 852 12. Ekwall, P. In Advances in Liquid Crystal; Brown, G. H., (ed.); Academic Press, New York, 1975 13. Rendall, K.; Tiddy, G. J. T.; Trevethan, M. A. J. Chem. Soc., Farraday Trans. 1 1983, 79, 637 14. Hertel, G.; Ho ffmann, H. Prog. Colloid Polym. Sci ., 1988 , 76, 123 15. Laughlin, R. G. Cationic Surfactants 2 nd ed.; Vol. 37, pp. 1 -40. Marcel Decker, New York, 1990 16. Edlund, H.; Lindholm, A.; Carlsson, I.; Lindström, B.; Hedenström, E.; Khan, A. Prog. Colloid Polym. Sci ., 1994 , 97, 134 17. Nilsson, P. G.; Lindman, B.; Laughlin, R. G. J. Phys. Chem ., 1984 , 88, 6357 18. Mathews, C. K.; van Holde, K. E. Biochemistry, 2 nd ed .; The Benjamin/Cummings Publishing Company, Menlo Park, CA, 1996 19. Scott, A. B.; Tartar, H. V.; Lingafelter, E. C. J. Am. Chem. Soc., 1943 , 65, 698 20. Jokela, P.; Jönsson, B.; Khan, A. J. Phys. Chem ., 1987, 91, 3291 21. Jokela, P.; Jönsson, B.; Eichmüller, B.; Fontell, K. Langmuir , 1988, 4,187 22. van Os, N. M.; Haak, J. R.; Rupert, L. A. M. Physico -chemical properties of selected Anionic, Cationic and Nonionic surfactants ; Elsevier Science Publishers B. V.: Amsterdam, 1993 23. Ropers, M. H.; Czichocki, G.; Brezesinski, G. J. Phys. Chem. B, 2003 , 107, 5281 24. Reiss -Husson, F.; Luzzati, V. J. Phys. Chem ., 1964 , 68, 3504

35 25. Narayanan, J.; Manohar, C.; Langevin, D.; Urbach, W. Langmuir , 1997, 13, 398 26. Gunnarsson, G.; Jönsson, B.; Wennerström, H. J. Phys. Chem ., 1980, 84, 3114 27. Turro, N. J.; Yekta, A. J. Am. Chem. Soc. , 1978 , 100, 5951 28. Clunie, J. S.; Goodman, J. F. Symons, P. C. Trans. Faraday Soc., 1969 , 65, 287 29. Mitchell, D. J.; Tiddy, G. J. T.; Warin, L.; Bostock, T.; MacDonald, M. P. J. Chem. Soc. Faraday Trans. I, 1983 , 79, 975 30. Nilsson, F.; Söderman, O.; Johansson, I. Langmuir , 1996, 12, 902 31. Warr, G. G.; Drummond, C. J.; Griese r, F.; Ninham, B. W.; Evans, D. F. J. Phys. Chem ., 1986 , 90, 4581 32. Baltzer, D. Langmuir , 1993 , 9, 3375 33. Nilsson, F.; Söderman, O.; Hansson, P.; Johansson, I. Langmuir , 1998 , 14, 4050 34. Jönsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Po lymers in Aqueous Solution 2nd ed ; 2003 John Wiley & Sons, Chichester, England 35. Ryan, L. D.; Kaler, E. W. Colloids and Surfaces A, 2001 , 176, 69 36. Andersson, M.; Karlström, G. J. Phys. Chem ., 1985 , 89, 4957 37. Nilsson, F.; Söderman, O.; Reimer, J. Langmuir , 19 98 , 14, 6396 38. Lutton, E. S. J. Am. Oil Chem. Soc., 1965 , 42, 1068 39. Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr ., 1984 , 168, 213 40. Qiu, H.; Caffrey, M. Biomaterials , 2000 , 21, 223 41. Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K. ; Forsén, S. J. Am. Chem. Soc., 1979 , 101, 5465 42. Razumas, V.; Kanapieniené, J.; Nylander, T.; Engström, S.; Larsson, K. Anal. Chim. Acta , 1994 , 289, 155 43. Landau, E. and Rosenbusch, J. Proc. Natl. Acad. Sci. USA, 1996 , 93, 14535 44. Ganem -Quintanar, A.; Quintanar -Guerrero, D.; Buri, P. Drug Dev. Ind. Pharm. , 2000 , 26, 809 45. Aniansson, E. A. G.; Wall, S. N.; Almgren, M.; Hoffmann, H.; Kielmann, I. Ulbricht, W.; Zana, R.; Lang, J.; Tondre, C. J. Phys. Chem. , 1976 ; 80, 905 46. Mazer, N. A.; Benedek, G. B.; Carey, M. C. J. Phys. Chem. , 1976 , 80, 1075 47. Clausen, T. M.; Vinson, P. K.; Minter, J. R.; Davis, H. T.; Talmon, Y.; Miller, W. G. J. Phys. Chem. , 1992 , 96, 474 48. Monduzzi, M.; Olsson, U.; Söderman, O. Langmuir , 1993 , 9, 2914 49. Anderson, D.; Wennerström, H.; Olsson, U. J. Phys. Chem. , 1989 , 93, 4243 50. Evans, D. F.; Wennerström, H.; The Colloidal Domain , VCH Publishers, New York, 1994

36 51. Mays, H.; Almgren, M.; Dedinaite, A.; Claesson, P. Langmuir , 1999 , 15, 8072 52. Fontell, K. Colloid Polym. Sci. , 1990 , 268, 264 53. Lindblom, G.; Ril fors, L. Biochim. Biophys. Acta , 1989 , 988, 221 54. Funari, S. S.; Holmes, M. C.; Tiddy, G. J. T. J. Phys. Chem. , 1992 , 96, 11029. 55. Gustavsson, S; Quist, P. -O. J. Colloid Interface Sci. , 1996 , 180, 564 56. Fairhurst, C. E.; Holmes, M. C.; Leaver, M. S. Langmuir, 1996 , 12, 6336 57. Wang, K.; Orädd, G.; Almgren, M. ; Asakawa, T.; Bergenståhl, B. Langmuir, 2000 , 16, 1042. 58. Holmes, M. C.; Leaver, M. S.; Smith, A. M. Langmuir , 1995 , 11, 356. 59. Gustafsson, J.; Orädd, G.; Lindblom, G.; Olsson, U.; Almgren, M. Langmuir , 1997 , 13, 852 60. Israelachvili, J. N. Intermolecular and Surface Forces, 2 nd ed.; Academic Press, San Diego, 1992 61. Tanford, C. J. Phys. Chem. , 1972 , 76, 3020 62. Helfrich, W. Z. Naturforsch., 1973 , 28 C, 693 63. Bydén -Sjöbom, M.; Edlund, H.; Lindström, B. Langmuir , 1999 , 15, 2654 64. Hiemenz, P. C. Principles of Colloid and Surface Chemistry 2 nd ed .; Marcel Dekker, New York, 1986 65. Atkins, P. W. Physical Chemistry, 5th ed.; Oxford University Press, New York, 1994 66. Laughlin, R. G. The aqueous phase behavior of surfactants, 1st ed.; Academic Press: San Diego, 1996 67. Rosevear, F. B. J. Am. Oil Chem. Soc. 1954 , 31, 628 68. Bueche, F. J. Introduction to Physics for Scientists and Engineers 4 th ed .; McGraw -Hill Book Company, NewYork, 1986 69. Nilsson, F. Doctoral thesis, Lund University, 1998 “Alkylglucosides, physical -chemical properties” 70. Simister, E. A.; Thomas, R. K.; Penfold, J.; Aveyard, R.; Binks, B. P.; Cooper, P.; Fletcher, P. D. I.; Lu, J. R.; Sokolowski, A. J. Phys. Chem. , 1992 , 96, 1383 71. Physical Methods of Chemistry, 2 nd ed, Vol. 6: Determination of Thermodynamic Properties Ed. Rossiter, B. W.; Baetzold, R. C. John Wiley&Sons Inc. 1992 72. Chernik, G. C. Thermochim. Acta , 1993 , 220, 37 73. Günther, H. NMR Spectroscopy 2 nd ed. Basic principles, concepts and applications in chemistry; John Wile y & Sons, Chichester, England, 1995

37 74. Stilbs, P. Prog. Nuc. Mag. Res. Spect. , 1987 , 19, 1 75. Lindblom, G.; Orädd, G. Prog. Nuc. Mag. Res. Spect. 1994 , 26, 483 76. Evans, G. T.; James, C. P. J. Chem. Phys ., 1983 , 79 , 5553 77. Orädd, G. Lindblom, G.; Johansson, L. B. -Å.; Wikander G. J. Phys. Chem ., 1992 , 96, 5170 78. Nydén, M.; Söderman, O. Langmuir , 1995 , 11, 1537 79. Nilsson, F.; Söderman, O.; Johansson, I. J. Coll. Interface Sci. , 1998 , 203, 131 80. Westlund, P. O. J. Magn. Reson., 2002 , 145, 364 81. Sparrman, T.; Westlund, P. -O. Phys. Chem. Chem. Phys. , 2003 , 5, 2114 82. Lindblom, G. In Advances in lipid methodology; Christie, W.W., ed.; Oily Press: Dundee, Scotland, 1996; Vol. 3, p. 133 83. Arvidson, G.; Brentel, I.; Khan, A.; Lindblom, G.; Fontell, K. Eur. J. Biochem. , 1985 , 152, 753 84. Lindblom, G.; Rilfors, L.; Hauksson, J. B.; Brentel, I.; Sjölund, M.; Bergenståhl, B. Biochemistry , 1991, 30, 10938. 85. Rilfors, L.; Hauksson, J. B.; Lindblom, G. Biochemistry , 1994, 33, 6110 86. Hakala, M. R.; Wong, T. C. Langmuir , 1986, 2, 83 87. Guo, W.; Wong, T. C. Langmuir , 1986, 3, 537 88. Ikeda, K.; Khan, A.; Meguro, K.; Lindman, B. J. Colloid Interface Sci. , 1989, 133, 192 89. Edlund, H.; Sadaghiani, A.; Khan, A. Langmuir 1997, 13, 4953 90. Jansson, M.; Jönsson, B. J. Phys. Chem. , 1989 , 93, 1451 91. Jansson, M.; Jönsson, A.; Li, P.; Stilbs, P. Colloid Surf ., 1991 , 59, 387 92. Caffrey, M. Curr. Opin. Struc. Biol. , 2000 , 10, 486 93. Sakya, P.; Seddon, J. M.; Templer, R. H. J. Phys. II Fr , 1994 , 4, 1311 94. Ai, X.; Caffrey, M. Biophys. J. , 2000 , 79, 394

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