Amphiphilic Molecules in Aqueous Solution144973/FULLTEXT01.pdf · part of the L 1 phase area, but...

Amphiphilic Molecules in Aqueous SolutionEffects of Some Different Counterions

The Monoolein/Octylglucoside/Water System

Gerd Persson 2003

Department of ChemistryBiophysical Chemistry

Umeå UniversitySweden

Department of Natural and Environmental SciencesMid Sweden University

Sweden



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.




Sweden



AbstractThe 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(MO)/n-octyl-β-D-glucoside (OG)/2H2O-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 counterions 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 anomalous behavior of the 2H2O quadrupolar 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 OG-rich structures (micelles, hexagonal and cubic phase of space group Ia3d) 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 L1 phase were found to remain rather small and discrete in the larger part of the L1 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 Pn3m structure to an Ia3d structure, and upon further addition of OG a lamellar (Lα) phase formed. Since the larger part of the phase diagram contains a lamellar structure (present either as a single Lα 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



Gerd Persson




Sweden

ii

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: “Seiryoku Zenyo”, meaning “Maximum efficiency”.

Copyright © 2003 by Gerd Persson

ISBN 91-7305-501-8 Printed by Kaltes Grafiska AB, Sundsvall, 2003

iii

Till Gabriel

och Görgen

iv

v

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

(MO)/n-octyl-β-D-glucoside (OG)/2H2O-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 counterions 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

anomalous behavior of the 2H2O quadrupolar 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 OG-rich structures (micelles, hexagonal and

cubic phase of space group Ia3d) 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 L1 phase were found to remain rather small and discrete in the larger

part of the L1 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 Pn3m structure to an Ia3d structure, and upon further addition of OG a lamellar (Lα)

phase formed. Since the larger part of the phase diagram contains a lamellar structure (present

either as a single Lα 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.

vi

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-ββββ-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-rac-glycerol in the

ternary system 1-monooleoyl-rac-glycerol/n-octyl-ββββ-D-glucoside/water. Persson,

G.; Edlund, H.; Lindblom, G. Eur. J. Biochem., 2003, 270, 56.

vii

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 L3 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. Presentation 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

viii

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

5.2. The 1-monooleoyl-rac-glycerol/n-octyl- ββββ-D-glucoside/2H2O system 29

Paper III 30

Paper IV 31

6. Ideas for future work 32

Acknowledgements 34

References 35

Papers I - IV 39

1

Introduction

In our daily life the utilization of amphiphilic molecules is very important. In many 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 structures, 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 well.

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, Biophysical Chemistry at Umeå University.

Gerd Persson, August 11, 2003

2

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 terms

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 water1,

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-assembled 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 without forming regular micelles. These are called hydrotropes.

3

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 chain2, 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 two 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 ionic 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

sulfates7, alkyl sulfonates8, alkyl phosphates9,10, 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 S O

O

O

Na+

N+

Br

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 the 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 surfactant

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 only 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 temperature22 and the critical micelle concentration (cmc) towards lower

4

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

SO

OO

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 micelles24, 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 additives, 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 polyoxyethylene28,29 or a polyhydroxy30,31 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 surfactant 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 polyoxyethylene

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 the alkylglucosides, since the

glucoside moiety is rather rigid.

5

OO

OO

OOH

a)

OO

OH

OH

OHOH

b)

Figure 1.4 Nonionic surfactants.

a)Pentaoxyethylene dodecyl ether (C12E5).

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 connection 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.

OH O

OH

O

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

6

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 crystalline phases38-40,

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,

usually 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 L1 (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 molecules. 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, more complex micellar structures also exist. They can

7

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 referred to as L2 (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, into 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. Vesicles

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

8

hexagonal, lamellar and cubic as well as intermediate structures. The phases appear in a

certain order according to the Fontell schedule52, 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 cylinders packed in a hexagonal lattice. These

cylinders can either be normal (tails in), denoted HI, or reversed (aqueous core), denoted HII.

The lamellar phase, Lα, 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 structures 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 type 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.

9

Micelles (L1)

Normal hexagonal

(HI)

Lamellar (Lα)

Reverse hexagonal

(HII)

Reverse micelles

(L2)

cpp 0.33 0.5 1 >1Increasing

H + 0 - C or T

Micellar cubic

Normal bicontinuous

cubic

Reverse bicontinuous

cubic

Reverse micellar

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 L3 phase is excluded for the same

reason. In this schedule it appears in the vicinity of the lamellar and the

bicontinuous cubic phases.

10

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

l*a

vcpp = 2:1

where v is the volume of the hydrocarbon chain, a the area per headgroup and l the length of

the fully extended hydrocarbon chain.60 v (nm3) and l (nm) can be estimated from

)nn(027.0 Mec +=v and cn127.015.0 +=l 2:2 a and b

where nc is the total number of carbons per chain and nMe is the number of methyl groups.61

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:

+=21 R

1

R

1

2

1H 2:3

where R1 and R2 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, H0, corresponding to a minimum in

the free energy. Thus, as soon as H deviates from H0, 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

11

the possibility to determine the energy required bending a surfactant film. This offers an

explanation to the formation of bicontinuous cubic as well as L3 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 solubility 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 the 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 modifying the surfactant, this problem can be solved. Modifications include changing the

headgroup, introducing unsaturations and branches in the hydrocarbon chain63, as well as

mixing the surfactant with additives and other surfactants.

3.2. Micelle formation

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.

n3

S

2 S....SSS2 ⇔⇔ 3:1

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 separation 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.

12

Gibbs phase rule According to Gibbs phase rule, the number of coexisting phases depends

on the number of components and the degrees of freedom for the system.

2+=+ cpF 3:2

In eq. 3:2 F is the number of degrees of freedom (T, P and X1, X2….), 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 of 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:

βα = TT (thermal equilibrium)

βα = PP (mechanical equilibrium)

βα = ii µµ (chemical equilibrium)

where α and β indicate two different phases and i refers to component i.

The interpretation of two first equalities is that 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

Binary 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 still have two phases

in coexistence. This results in two-phase areas.

13

wt %

Τα βα + β

a bc

Figure 3.1 The lever rule.

The fraction of each phase can be determined from the mass balance.

For a given total concentration, the composition 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 connection 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 wα and wβ and can be determined by measuring the

distance from point c to the respective single-phase boundaries, according to:

( )( )ab

cbw −

−=α and ( )( )ab

acw −

−=β3:3

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 triangle, 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 the 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 determined from the distance from that point to

14

the respective corner of the triangle, similar to the determination of total composition in the

ternary system.65,66

B0 50 100

C

0

50

100

A

0

50

100P(B) = 10 %

P(C) = 60 %

P(A) = 30 %

Figure 3.2 A ternary diagram.

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 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 appear 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 polarization 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

15

independent of direction (the ordinary), and one for 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 when 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:

16

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 resulting 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: L1 – isotropic micellar solution, HI – normal hexagonal

phase, Lα – 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 necessary 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 depleted 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

17

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

ionic surfactants is:

ClnRT2

1

lnRT2

12 ∂

γ∂⋅−≈∂γ∂⋅−=Γ

a4:1

where R is a constant, T is the temperature and a is the activity of the solute.

aln∂γ∂

can be obtained from the first derivative of a polynomial fitted to γ vs. ln a at

concentrations below cmc.70 From Γ2 (given in mmol/m2) the area per molecule at the surface,

σ, (given in Å2) can be estimated using

2AN

1020

Γ=σ 4:2

where NA is Avogadro´s constant.

For a surfactant solution, the surface tension decreases with increasing concentration until

micelles form.65 Thereafter the surface tension becomes 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.

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

γ (mN

/m)

20

30

40

50

60

70

80

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

18

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

κ (m

S/cm

)

0,0

0,2

0,4

0,6

0,8

1,0

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 α−=β 1 . α can roughly be estimated from the ratio of the slopes before

and after cmc.

cmcbelowcmcabove CC

∆κ∆

∆κ∆=α 4:3

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 behavior 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

19

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 magnetic 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, H1δ , defined as ( ) erspectrometTMSH1 νν−ν , is a field independent

number characteristic for a proton in a certain type of environment. Upon micellization the

magnetic environment changes and consequently also H1δ .73

Pulsed Gradient (PG) NMR If the magnetic field contains inhomogeneities, this will affect

the relaxation processes. This is utilized in the self-diffusion 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 larger 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.

{ })3/(D)G(T20

22 eeII δ−∆δγ−τ−= 4:4

20

In eq. 4:4 I denotes the observed echo intensity, I0 is the echo intensity in the absence of field

gradient pulses, τ is the time between the 90° and 180° pulses, T2 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 T1 = T2

then SE is appropriate, while for systems with T1>>T2 STE is the preferred pulse

sequence.74,75

For STE the echo attenuation is given by

{ })3/(D)G(T2TT0

221 eeeI

2

1I δ−∆δγ−τ−−= 4:5

where τ is the time between the first and the second 90° pulses, T is the time between the

second and third 90° pulses, T1 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.

( ) 0.surfmic

obs.surf DP1PDD −+= 4:6

where obs.surfD is the observed diffusion coefficient, micD is the micellar diffusion coefficient

and 0.surfD is the diffusion coefficient of the free surfactant, usually measured at a

concentration well below cmc. P is the fraction of micellized surfactant given by

21

P=(Ctot – cmc)/Ctot 4:7

where Ctot is the total surfactant concentration and cmc the critical micelle concentration. Dmic

is measured by adding a hydrophobic probe that will 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:

mic0

.surf

obs.surf

0.surf

mic0counterion

obscounterion

0counterion

DD

DD

DD

DD

−−

−−=β 4:8

where obscounterionD is the observed diffusion coefficient, and 0

counterionD 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:

H

B0mic R6

TkD πη= 4:9

where 0micD is the micellar diffusion coefficient at infinite dilution, Bk is the Boltzmann

constant, T is the temperature, η is the viscosity of the medium and HR 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

)1(DD agg0micmic φ⋅−= k 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 included in HR 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 accounted for by introducing a shape factor. In

22

this work we have assumed either prolate or oblate aggregates. The shape factors for

prolate69,79,80 and oblate69,80 micelles are

1B

1BBlnprol

2

2

−

−+= and

1B

1Barctanobl

2

2

−

−= 4:11 a and b

where B is the ratio between the short and long axis.

The corresponding expressions for the micellar diffusion coefficient at infinite dilution are

prolr6

TkD B0

mic.prol πη= and oblr6

TkD B0

mic.obl πη= 4:12 a and b

where r is the length of a fully extended surfactant molecule including the tail, headgroup and

the corresponding hydration. The obstruction effects are taken into account by means of

⋅−⋅=

3

agg0mic.prolmic

prolB1D.prolD

φk 4:13 a

and

⋅−⋅=

32

agg0mic.oblmic

prolB1D.oblD

φk 4:13 b

Eq. 4:10, 4:13 a, and 4:13 b were evaluated for three different 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 resulting in a quadrupolar splitting, ∆. Among

others 2H (deuterium) and 23Na can be mentioned. In the present work, deuterium has mainly

been utilized, and the rest of this section is therefore concentrated on deuterium.

Deuterium is not a commonly occurring nucleus, and has to be introduced either as 2H2O 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

23

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

between the laboratory frame and the director coordinate system. When the 2H2O molecules

are subjected to fast chemical exchange between different sites, having different values of νQ,

the magnitude of the resulting splitting is obtained by

∑ −θν=∆i

ii Spi

)1cos3( LD2

Q 4:14

where pi refers to the fraction of 2H2O in site i. In most cases we deal with samples in which

the microcrystallites are randomly distributed, i.e. powder samples. Thus, all values of cos θLD

are equally probable, and the observed quadrupolar splitting corresponds to that for θLD = 90o.

In isotropic phases, the motion and orientation of the aggregates are such that the quadrupole

interaction averages to zero 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

studied are macroscopically oriented so that θLD = 54,7o, 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 between -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

24

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 appropriate,

dividing the water molecules into bound and free water, according to

)(X

XnPP fbfbbff ∆−∆+∆=∆+∆=∆

W

Surf.4:15

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 XSurf. and XW 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 crystalline phase, equation 4:15 reduces to

W

Surf.S

X

Xn Qν=∆ 4:16

From eq. 4:16 we see that ∆ depends linearly on the ratio XSurf./XW 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.

XSurf./XW is linear over a wide range of water concentrations, indicating ideal swelling.83-85

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 of 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

θ=λ sind2n (Bragg´s law) 4:17

25

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

AB

C

θ

d

θ

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 indices 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:

2h

hd

a= 4:18

If the volume fraction of the hydrocarbon part, φhc, is known then the thickness of the

hydrocarbon layer, 2rhc, and the area per molecule at the hydrocarbon/headgroup interface,

αLA , can be obtained from:

hchcr2 φa= 4:19

and

hcL r

VA =α 4:20

where V is the volume per molecule. For an Lα phase 2rhc ≈ 1.8 l50 (eq. 2:2 b).

For a two-dimensional hexagonal lattice, the Bragg reflections are related to the unit cell

dimension, a, by the relation

( )hkkh2

3d

22hk

++= a 4:21

26

The radius of the hydrocarbon core, rhc, and the area per molecule at the

hydrocarbon/headgroup interface, ΙHA , can be obtained from:

π=2

3r hchc

φa 4:22

and

hcH r

V2A

1= 4:23

For a cubic lattice

( )222hkl

lkhd

++= a

4:24

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

electrostatic 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 modifications

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 Ca2+ and Mg2+. Other examples are the increase in counterion

binding90, and the changes in stability of the liquid crystalline phases91 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

27

experimental techniques used for the Na+/Ca2+ 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 surfactant 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 monovalent and divalent

counterions should decrease even at very small fractions of divalent counterion.

The results obtained both for the Na+/Ca2+ octyl sulfate system and the

piperidine+/piperazine2+ octanesulfonate system show that this is indeed the case. 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 counterion. The fraction Ca2+

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 counterion, 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 Ca2+ and this effect was interpreted as originating from the displacement of Na+ by

Ca2+ 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 (nc) in the alkyl chain of the alkylpyridinium

28

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

APOS/2H2O 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.

NaOS

0 50 1000

50

100

L1

HI

S

MPOS

0 50 1000

50

100

L1

C

HI

S

EPOS

0 50 1000

50

100

L1

C

HI

S

PrPOS

0 50 1000

50

100

L1

HI S

BPOS

0 50 1000

50

100

L1 HI

S

PePOS

0 50 1000

50

100

L1

HI S

HexPOS

0 50 1000

50

100

L1

HI

S

Lαααα

HepPOS

0 50 1000

50

100

L1

HI S

LααααOPOS

0 50 1000

50

100

L1 S

Lαααα

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

HepPOS and OPOS Lα phases indicate the location of the apparent

singlets.

Abbreviations: L1, isotropic solution phase; C, cubic phase; HI, normal

hexagonal phase; Lα, lamellar liquid crystalline phase; S, solid; NaOS,

Sodium octanesulfonate; MPOS, Methylpyridinium octanesulfonate;

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

29

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 the 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 binding

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.

Further, the 2H2O quadrupolar splittings obtained in the lamellar phases (and also to some

extent in the hexagonal phases) showed anomalous behavior. In neither of the three Lα 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 L1-

Lα two-phase area, while for HepPOS and OPOS the apparent singlets appeared well away

from the phase boundaries.

The anomalous behavior of the 2H2O 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

each site, which results in the observed 2H2O quadrupolar splittings.

5.2. The 1-monooleoyl-rac-glycerol/n-octyl-ββββ-D-glucoside/2H2O 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

Rosenbusch43 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 mechanism raises a number of questions.92 From

my point of view, questions concerning phase structure and phase equilibria are of major

30

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

binary OG/2H2O phase diagram30,93 consists of a large micellar solution phase followed by a

hexagonal (HI), a bicontinuous cubic phase of space group Ia3d, and a lamellar liquid

crystalline (Lα) phase with increasing OG concentration. However, the HI has a low melting

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 Lα phase, a cubic phase of space group Ia3d, and a cubic phase of space group

Pn3m, forms at 25 °C. At higher temperatures a reversed hexagonal phase, as well as reversed

micelles are found.38-40

0 50 1000

50

1000

50

100

L1

H1

C1

Lαααα

.........

............

...............

OG

MO2H2O

eq. mol.

Figure 5.2 The MO/OG/water system. The dotted area in the L1 phase indicates the

area where flow-birefringency was observed. Abbreviations: L1, isotropic

solution phase; HI, normal hexagonal phase; Lα, lamellar liquid

crystalline phase.Paper III

31

When mixing MO and OG it was found that OG-rich structures (micelles, HI and cubic

phase of space group Ia3d) 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 L1 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 Pn3m structure to an

Ia3d structure, and upon further addition of OG an Lα phase formed. Comparing with the

Fontell schedule (figure 2.2) this means that the Pn3m structure has the most negative

curvature, followed by the Ia3d structure.

Since the larger part of the phase diagram contains a lamellar structure (present either as a

single Lα 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 revealed 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 Pn3m structure is close to be converted to the Ia3d

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. It 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 Pn3m structure to a less curved structure. The observed

phase sequence upon heating is Lα→ Ia3d and Ia3d → Pn3m. Again, this is in agreement with

the Fontell schedule, if the Pn3m structure has the most negative curvature. When comparing

the MO/OG/water-system with a similar one, the MO/dodecylmaltoside (DM)/water-

system94, 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 the two cubic phases required very little energy. In fact,

32

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.

MO50 60 70

OG

0

10

2H2O

50

ABC

DE

FGH

Pn3m

Ia3d

Lα + Ia3d Lα

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:

Pn3m, cubic phase of space group Pn3m; Ia3d, cubic phase of space

group Ia3d; Lα, lamellar liquid crystalline phase. Paper IV

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 the notorious metastability shown by especially bicontinuous

cubic phases.

It has been suggested that the cubic structure present during the protein crystallization is

the Pn3m, even though the MO concentration indicates that the structure should be Ia3d. The

results presented here show that the Pn3m 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 that 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.

33

In the APOS systems, the effects of excess AP+, OS- and salt could be of interest to

investigate. In a preliminary study, no liquid crystalline 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.

34

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 of 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 especially, 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 acknowledged 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.

35

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