Dissertation

Formation and Stabilization of

Vesi les in Mixed Surfa tant Systems

Nina Vla hy

University of Regensburg
Natural S ien es Fa ulty IV

Chemistry & Pharma y

Ph.D. Supervisor: Prof. Dr. Werner Kunz

Adjudi ators: Prof. Dr. Werner Kunz

Prof. Dr. Ksenija Kogej

Prof. Dr. Jörg Heilmann

Chair: Prof. em. Dr. Dr. Josef Barthel

July 2008
Promotionsgesu ht eingerei ht am: 20. April 2008

Promotionskolloquium am: 4. Juli 2008

For Katko: my sister, my toughest
ompetitor, my biggest fan
iv
Contents

Contents v

Prefa e xi

1 Introdu tion 1
1.1 Surfa e A tive Agents . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.2 Surfa tant Self-Assembly: Vesi les . . . . . . . . . . . . . . . . 3

1.1.3 Catanioni Surfa tant Mixtures . . . . . . . . . . . . . . . . . 4

1.1.4 Appli ation of Catanioni Vesi les in Cosmeti Formulation . . 7

1.2 Ion Ee ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2.1 Ions in Water . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2.2 Hofmeister Ee t . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.2.3 Ion Pairing in Water . . . . . . . . . . . . . . . . . . . . . . . 12

1.2.4 Collins' Theory of Mat hing Water Anities . . . . . . . . . . 13

1.2.5 Ion-Spe i Ee ts in Colloidal Systems . . . . . . . . . . . . 17

I Salt-Indu ed Morphologi al Transitions in Non-Equimolar

Catanioni Systems 19
2 Blastulae Aggregates: Spontaneous Formation of New Catanioni
Superstru tures 21
2.1 Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.2 Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

v
vi CONTENTS

2.3 Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

2.4.1 Chara terization of SDS / DTAB Mi ellar Solution . . . . . . 26

2.4.2 Salt-Indu ed Mi elle-to-Vesi le Transition . . . . . . . . . . . 27

2.5 Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2.5.1 Models of the Mi elle-to-Vesi le Transition . . . . . . . . . . . 32

2.5.2 Blastulae Vesi les . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.5.3 The O urren e of Convex-Con ave Patterns in Biologi al Sys-

tems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2.5.4 Raspberry Vesi les . . . . . . . . . . . . . . . . . . . . . . . . 38

2.5.5 Blastulae Vesi les: A General Trend in Catanioni Systems? . 38

2.6 Con lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3 Spe i Alkali Cation Ee ts in the Transition from Mi elles to

Vesi les Through Salt Addition 41
3.1 Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.2 Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3.3 Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.4.1 Phase Diagrams . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.4.2 Counterion Ee ts . . . . . . . . . . . . . . . . . . . . . . . . 45

3.4.3 Co-ion Ee ts . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.4.4 Nonioni Ee ts . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.4.5 Ee ts of `Hydrophobi Ions' . . . . . . . . . . . . . . . . . . 52

3.5 Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.5.1 Aggregation Behavior of Catanioni Systems . . . . . . . . . . 54

3.5.2 Counterion Properties . . . . . . . . . . . . . . . . . . . . . . 55

3.5.3 Collins' `Law of Mat hing Water Anities' . . . . . . . . . . . 56

3.5.4 Counterion Sele tivity of Alkyl Sulfates . . . . . . . . . . . . . 56

3.5.5 Counterion Sele tivity of Alkyl Carboxylates . . . . . . . . . . 57

3.5.6 Alkyl Sulfates vs. Alkyl Carboxylates . . . . . . . . . . . . . . 57

CONTENTS vii

3.5.7 Mole ular Dynami s Simulations . . . . . . . . . . . . . . . . 58

3.5.8 Generalization of the Con ept: Hofmeister Series of Headgroups 60

3.5.9 The Anioni Ee t . . . . . . . . . . . . . . . . . . . . . . . . 60

3.5.10 The Non-Ioni Ee t . . . . . . . . . . . . . . . . . . . . . . . 60

3.5.11 Ee ts of `Hydrophobi Ions' . . . . . . . . . . . . . . . . . . 62

3.6 Con lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4 Anion Spe i ity Inuen ing Morphology in Catanioni Surfa tant

Mixtures with an Ex ess of Cationi Surfa tant 65
4.1 Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

4.2 Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

4.3 Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 67

4.4 Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.4.1 Ion Binding in Catanioni Surfa tant Mixtures . . . . . . . . . 68

4.4.2 Phase Behavior upon Salt Addition . . . . . . . . . . . . . . . 70

4.4.3 Anion Spe i ity in Physi o-Chemi al Properties of Alkyl-

trimethylammonium Systems . . . . . . . . . . . . . . . . . . 72

4.4.4 Inuen e of Salt on the Aggregation Behavior of Surfa tants . 73

4.4.5 Explaining Counterion Spe i ity in Surfa tant Systems . . . 76

4.4.6 Dierent self-aggregation behavior of atanioni systems in

the atanioni - and anioni -ri h regions . . . . . . . . . . . . . 77

4.5 Con lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

II In reasing the Stability of Catanioni Systems 79

5 Inuen e of Additives and Cation Chain Length on the Kineti
Stability of Supersaturated Catanioni Systems 81
5.1 Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

5.2 Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

5.3 Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 83

5.4 Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . 85

5.4.1 Shift of Solubility Temperature with Time . . . . . . . . . . . 85

viii CONTENTS

5.4.2 Behavior of the Anioni -Ri h Region of the Phase Diagrams

Without Additives . . . . . . . . . . . . . . . . . . . . . . . . 88

5.4.3 Ee t of Additives on the Stability and the `Solubility Tem-

perature Depression' . . . . . . . . . . . . . . . . . . . . . . . 92

5.5 Con lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

III Toward Appli ation 103

6 Use of Surfa tants in Cosmeti Appli ation: Determining the Cy-
totoxi ity of Catanioni Surfa tant Mixtures on HeLa Cells 105
6.1 Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

6.2 Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

6.3 Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 107

6.3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

6.3.2 Growing HeLa Cell Cultures . . . . . . . . . . . . . . . . . . . 108

6.3.3 HeLa Toxi ity Test . . . . . . . . . . . . . . . . . . . . . . . . 109

6.3.4 Dete tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

6.3.5 Evaluation of spe tros opi al data . . . . . . . . . . . . . . . . 111

6.4 Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . 111

6.4.1 Single-Chain Surfa tants . . . . . . . . . . . . . . . . . . . . . 111

6.4.2 Catanioni Surfa tant Systems . . . . . . . . . . . . . . . . . . 113

6.5 Con lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

7 Spontaneous Formation of Bilayers and Vesi les in Mixtures of

Single-Chain Alkyl Carboxylates: Ee t of pH and Aging 117
7.1 Abstra t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

7.2 Introdu tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

7.3 Experimental Pro edures . . . . . . . . . . . . . . . . . . . . . . . . . 120

7.4 Results and Dis ussion . . . . . . . . . . . . . . . . . . . . . . . . . . 122

7.4.1 Lowering of the Solubility Temperature of Fatty A ids . . . . 122

7.4.2 The Ee t of pH on Vesi le Formation . . . . . . . . . . . . . 123

7.4.3 Cryo-TEM Study of Time-Dependent Vesi le Formation . . . 127

 7.4.4 Cytotoxi ity Potential of Surfa tant Mixtures . . . . . . . . . 130

7.5 Con lusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Con lusion 133

Bibliography 137

A knowledgements 161

List of Publi ations 163

List of Oral and Poster Presentations 165

ix
x
Prefa e

Surfa tants an self-assemble in dilute aqueous solutions into a variety of mi rostru -

tures, in luding mi elles, vesi les, and bilayers. In re ent years, there has been an

in reasing interest in unilamellar vesi les, whi h are omposed of a urved bilayer

that separates an inner aqueous ompartment from the outer aqueous environment.

This interest is motivated by their potential to be applied as vehi les for a tive

agents in osmeti s and pharma y. A tive mole ules an be en apsulated in the

bilayer membrane if they are lipophili or in the ore of the vesi le if they are

hydrophili . Furthermore atanioni systems an be used as models for biologi al

membranes.

When oppositely harged surfa tants are mixed, new properties appear due to the

strong ele trostati intera tions between the harged headgroups. These so- alled

atanioni mixtures exhibit low riti al mi elle on entration ( m ) values and a

non-monotoni hange in the surfa tant pa king parameter (P ) as the mixing ratio

is varied. One advantage of atanioni systems, as ompared with more robust gen-

uinely double hained surfa tants, are their greater sensitivity to parameters su h

as temperature or the presen e of salts. To optimize the appli ations it is important

to have a general understanding of the interplay of intera tions between the surfa -

tants and of the fa tors inuen ing the phase diagram of a mixed system. In this

thesis the formation and stabilization of atanioni vesi les was studied. The ee t

of al ohol and salt on the morphologi al transitions is des ribed, and the potential

of using su h systems for osmeti al and pharma euti al purposes was explored.

A short overview of the phase behavior of atanioni systems is given in the Intro-

du tion.

The morphologi al transitions o urring in mixed surfa tant systems upon the in-

xi
 rease of the ioni strength are reported in Chapter 2. A mi elle-to-vesi le transition

is investigated and an intriguing new intermediate stru ture, named blastulae vesi-

le, is des ribed.

Many phenomena in olloid s ien e that involve ele trolytes show pronoun ed ion

spe i ity. In order to elu idate the spe i ion ee ts in atanioni surfa tant sys-

tems with dierent surfa tant headgroups were ompared. Using various experimen-

tal te hniques and MD simulations, and employing a general on ept of `mat hing

water anities', a detailed study of ion spe i ity in atanioni systems is des ribed

in Chapters 3 and 4. A Hofmeister like series for lassifying surfa tant headgroups

is established.

Considering that ontrolling the pre ipitation phenomena is useful for a vast number

of industrial appli ations, the ee t of various additives on the stability of atanioni

systems was probed and is reported in Chapter 5.

Chapter 6 introdu es an important parameter that should be onsidered when for-

mulating new en apsulating systems for either osmeti or medi al use: the toxi ity

of the parti ipating surfa tant mole ules. We evaluated the ytotoxi ity of a number

of ommonly used surfa tants, as well as that of atanioni surfa tant mixtures.

Seeing that the presen e of only a small amount of a ationi surfa tant in the mix-

ture results in a large in rease in its toxi ity, we fo used on a mixture of two anioni

and in osmeti formulation already ommonly used surfa tants. In Chapter 7 we

present a way to form vesi les in su h mixtures at room temperature and at physi-

ologi al pH.

This thesis omprises dierent studies ranging from phase diagram determination to

ytotoxi ity studies. For this reason, the thesis was written so that ea h hapter lay-

out follows the usual onvention: Abstra t, Introdu tion, Experimental Pro edures,

Results, Dis ussion and Con lusions. The bibliography is given at the end of the

thesis in order to avoid repeating. The dierent studies led to several publi ations

whi h are already published, a epted or submitted, summarized in the following

table. A omplete list of publi ations and a list of oral and poster presentations,

whi h were presented at international ongresses, are also given at the end.

xii
Chapter Publi ation

2 N. Vla hy, A. Renon ourt, M. Dre hsler, J.-M. Verbavatz, D. Touraud,

W. Kunz, Blastulae aggregates: New intermediate stru tures in the

mi elle-to-vesi le transition of atanioni systems. J. Colloid Interf. S i.

2008 320, 360-363.
3 A. Renon ourt, N. Vla hy, P. Bauduin, M. Dre hsler, D. Touraud, J.-M.

Verbavatz, M. Dubois, W. Kunz, B. W. Ninham, Spe i alkali ation

ee ts in the transition from mi elles to vesi les through salt addition.

Langmuir 2007 23, 2376-2381.

3 N. Vla hy, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Role

of the surfa tant headgroup on the ounterion spe i ity in the mi elle-

to-vesi le transition through salt addition. J. Colloid Interf. S i. 2008

319, 542-548.

3 N. Vla hy, B. Jagoda-Cwiklik, R. Vá ha, D. Touraud, P. Jungwirth,

and W. Kunz, Hofmeister series of headgroups and spe i intera tion of

harged headgroups with ions. J. Phys. Chem. B 2008 (Submitted).

4 N. Vla hy, M. Dre hsler, D. Touraud, W. Kunz, Anion spe i ity in-

uen ing morphology in atanioni surfa tant mixtures with an ex ess

of ationi surfa tant. Comptes rendus Chimie A adémie des s ien es

2008 (Submitted).
5 N. Vla hy, A. F. Arteaga, A. Klaus, D. Touraud, M. Dre hsler, W. Kunz,

Inuen e of additives and ation hain length on the kineti stability of

supersaturated atanioni systems. Colloids Surf., A 2008 (A epted).

6 N. Vla hy, D. Touraud, J. Heilmann, W. Kunz, Determining the de-

layed ytotoxi ity of atanioni surfa tant mixtures on HeLa ells.

Colloids Surf., B 2008 (Submitted).

7 N. Vla hy, C. Merle, D. Touraud, J. S hmidt, Y. Talmon, J. Heilmann,

W. Kunz, Determining the delayed ytotoxi ity of atanioni surfa tant

mixtures on HeLa ells. Langmuir 2008 (Submitted).

xiii
xiv
Chapter 1

Introdu tion

1.1 Surfa e A tive Agents

1.1.1 General
Surfa e a tive agents (a.k.a. surfa tants) are mole ules with a hemi al stru ture

that makes them parti ularly favorable to reside at interfa es. All lassi al sur-

fa tant mole ules onsist of at least two parts, one whi h is soluble in water (the

hydrophili part) and the other whi h is insoluble in water (the hydrophobi part).

The hydrophili part (a polar or ioni group) is referred to as the head group and the

hydrophobi part (a long hydro arbon hain) as the tail (Figure 1.1). The hydropho-

bi part of a surfa tant may be bran hed or linear. The degree of hain bran hing,

the position of the polar head group and the length of the hain are parameters

1,2
that ae t the physi o- hemi al properties of the surfa tant . The primary lassi-

 ation of surfa tants is made on the basis of the harge of the polar head group:

anioni s, ationi s, non-ioni s and zwitterioni s.

Surfa tant mole ules adsorb at interfa es, thereby redu ing the free energy of the

3
system . When surfa tants are dissolved in water, the hydrophobi group disrupts

the hydrogen-bonded stru ture of water and therefore in reases the free energy of

the system. Surfa tant mole ules therefore on entrate at interfa es, so that their

hydrophobi groups are removed or dire ted away from the water and the free energy

of the solution is minimized. The distortion of the water stru ture an also be de-

1
Figure 1.1: S hemati representation of a surfa tant mole ule (with all the parameters involved
in the theory of the pa king parameter) and a unilamellar vesi le.

reased (and the free energy redu ed) by the aggregation of surfa e-a tive mole ules

into lusters (mi elles) with their hydrophobi groups dire ted toward the interior

of the luster and their hydrophili groups dire ted toward the water. The pro ess

1,3
of surfa tant lustering or mi ellization is primarily an entropy-driven pro ess .

However, the surfa tant mole ules transferred from the bulk solution to the mi elle

may experien e some loss of freedom from being onned to the mi elle. In addi-

tion, they may experien e an ele trostati repulsion from other similarly harged

surfa tant mole ules in the ase of surfa tants with ioni head groups. These for es

in rease the free energy of the system and oppose mi ellization. Hen e, mi elle

formation depends on the for e balan e between the fa tors favoring mi ellization

(van der Waals and hydrophobi for es) and those opposing it (kineti energy of the

mole ules and ele trostati repulsion).

The on entration at whi h mi elles rst appear in solution is alled the riti al

mi ellar on entration, abbreviated CMC, and an be determined from the dis on-

tinuity of the ine tion point in the plot of a physi al property of the solution as a

1,3
fun tion of the surfa tant on entration . Above this on entration, almost all of

the added surfa tant mole ules are onsumed in mi elle formation and the monomer

on entration does not in rease appre iably, regardless of the amount of surfa tant

added to the solution. Sin e only the surfa tant monomers adsorb at the interfa e,

the surfa e tension remains essentially onstant above the CMC. Thus, the surfa e

3
tension an be dire tly related to the a tivity of monomers in the solution . The

4,5
CMC is strongly ae ted by the hemi al stru ture of the surfa tant , by the tem-

2
 6 7 6
perature and by the presen e of osolutes su h as ele trolytes or al ohols . Mi elle

formation, or mi ellization, an be viewed as an alternative me hanism to adsorp-

tion at interfa es. In both ases, the driving for e of the pro ess is the tenden y of

8
the surfa tant to remove their hydrophobi groups from the onta t with water .

1.1.2 Surfa tant Self-Assembly: Vesi les

As was mentioned previously, surfa tants an self-assemble in dilute aqueous solu-

tions into a variety of mi rostru tures, in luding mi elles, vesi les, and bilayers. In

re ent years, there has been an in reasing interest in unilamellar vesi les, whi h are

omposed of a urved bilayer that separates an inner aqueous ompartment from

the outer aqueous environment (Figure 1.1). This is mainly be ause of their wide

appli ation in biology and medi ine as model ell membranes, as well as their strong

9
potential as drug arriers and other en apsulating agents of industrial relevan e .

Two major theoreti al approa hes have been pursued in the modeling of surfa tant

self-assembly: the urvature-elasti ity approa h and the mole ular approa h.

The urvature-elasti ity approa h des ribes the vesi le bilayer as a ontinuous

membrane hara terized by the spontaneous urvature and the elasti bending mod-

10,11
ulus . In this approa h, the formation of nite-sized vesi les thus depends on the

12,13
interplay between these two quantities . The theory provides an elegant, simple

way to des ribe the formation of vesi les, however, be ause this approa h is based

on a urvature expansion of the free energy of a membrane, it breaks down for small

vesi les, for whi h the urvature is quite pronoun ed.

1416
The mole ular approa h was pioneered by Israela hvili, Mit hell, and Ninham

who developed a geometri pa king argument that allows one to predi t the shape

of self-assembling mi rostru tures, in luding spheroidal, ylindri al, or dis oidal mi-

elles, vesi les, and bilayers. Whi h aggregates form, is determined primarily by

geometri pa king of amphiphiles, hydro arbon hain stiness, and the hydrophili -

hydrophobi balan e. For dilute solutions in whi h intera tions between aggregates

are not important, the ne essary (geometri ) onditions for formation of an aggre-

gate an be des ribed by a a surfa tant pa king parameter P = v/(lmax a) 1416 , where
v is the volume per hydro arbon hain, or the hydrophobi region of the surfa tant,

3
a is the a tual headgroup area in the lm, and lmax is an optimal hydro arbon hain

length related to about 90% of the maximum extended length (see Figure 1.1). The

optimal stability of the dierent aggregates o urs as follows: (1) spheri al mi elles

P ≤ 1/3; (2) globular or ylindri al mi elles 1/3 < P ≤ 1/2 (3) vesi les or bilayers

1/2 < P ≤ 1.
Low pa king parameters (around 1/3) are found for single hained surfa tants

with a strongly polar head group. An in rease in the pa king parameter an

be obtained by adding a se ond hain, therefore doubling the hydro arbon vol-

1722
ume. To rea h this value, double hain surfa tants , two surfa tants of opposite

2326 2733
harge , or the asso iation of a surfa tant and a o-surfa tant an be used.

In the latter two ases, a pseudo-double hain surfa tant is obtained by either an ion-

pair formation between the anioni and ationi surfa tant or due to an asso iation

of the two dierent mole ules via hydrogen bounds.

In many ases, the formation of vesi les requires the input of some form of en-

3439
ergy, for example, soni ation, inje tion or extrusion . However, vesi les have

been found to form spontaneously in some aqueous surfa tant systems, in lud-

40
ing solutions ontaining (i) mixtures of le ithin and lysole ithin , (ii) mixtures

41 42
of long- and short- hain le ithins , (iii) mixtures of AOT and holine hloride ,

20,21,4345
(iv) dialkyldimethylammonium hydroxide surfa tants , (v) ationi siloxane

46 24,4750
surfa tants , and (vi) mixtures of ationi and anioni surfa tants . These

spontaneously-forming vesi les oer advantages over the more traditional phospho-

lipid vesi les in being easier to generate and more stable, thus making them more

attra tive as en apsulating agents in diverse pra ti al appli ations, in luding the

ontrolled delivery of drugs, a tive substan es in osmeti s, and fun tional food

51,52
ingredients su h as enzymes .

1.1.3 Catanioni Surfa tant Mixtures

The main thermodynami driving for e for the asso iation of a ationi and an

anioni surfa tant mixture is the release of ounterions from the aggregate surfa es.

This results in a large entropy in rease. Sin e the two surfa tants are single- hained,

the resulting atanioni surfa tant an be onsidered as a pseudodouble- hained

4
surfa tant, in the sense that the two hains are not ovalently bound to the same

headgroup. For these systems a non-monotoni hange in P is observed, with a

1,3
pronoun ed maximum as the mixing ratio is varied . Due to the strong ele trostati

intera tion between the oppositely harged headgroups, atanioni mixtures exhibit

CMC values mu h lower than those of the surfa tants involved. The CMC is dire tly

orrelated to the leaning e ien y of surfa tants, pointing to yet another advantage

of su h systems.

Mixtures of anioni and ationi surfa tants exhibit ri h mi rostru ture phase

behavior in aqueous solutions. Aggregate stru tures su h as spheri al and rod-like

mi elles, vesi les, lamellar phases, and pre ipitates have all been observed depend-

24,26,48,5357
ing on the on entration and the ratio of the surfa tants in solutions .

Around equimolarity a zone of pre ipitation is observed. However, when one of the

surfa tants is present in a small ex ess, the ationi -anioni surfa tant bilayers usu-

ally spontaneously form losed vesi les. The phase behavior of atanioni mixtures

is represented in Figure1.2.

Figure 1.2: S hemati phase behavior en ountered in atanioni surfa tant systems. Phase
notations: V − and V + : regions of negatively and positively harged vesi les; 2φ: two-phase regions,
i.e. mostly demixing of phases between a vesi ular and a lamellar phase or a vesi le and a mi ellar
phase; L− and L+ : lamellar phase with an ex ess of respe tively anioni and ationi surfa tants;
P: pre ipitate region; I − and I + : mixed mi ellar solutions with an ex ess of respe tively anioni
and ationi surfa tants (reprodu ed from Khan 58 ).

5
 59
Yuet and Blanks htein presented a detailed mole ular thermodynami theory

to des ribe the formation of atanioni vesi les. Their theory reveals that (i) the dis-

tribution of surfa tant mole ules between the two vesi le leaets plays an essential

role in minimizing the vesi ulation free energies of nite-sized vesi les, (ii) the om-

position of mixed ationi /anioni vesi les is mainly determined by three fa tors: the

transfer free energy of the surfa tant tails, the ele trostati intera tions between the

harged surfa tant heads, and the entropi penalty asso iated with the lo alization

of the surfa tant mole ules upon aggregation, and (iii) the entropy asso iated with

mixing nite-sized vesi les an be an important me hanism of stabilizing vesi les in

solution. The present mole ular-thermodynami theory also has the ability to over

the entire range of vesi le sizes (or urvatures), thus enabling a des ription of small,

energeti ally stabilized, vesi les.

The free energy of vesi ulation, gves , an be viewed as omposed of the follow-

ing ve ontributions: (1) the transfer free energy, gtr , (2) the pa king free energy,

gpack , (3) the interfa ial free energy, gσ , (4) the steri free energy, gsteric , and (5) the

ele trostati free energy, gelec . These ve free-energy ontributions a ount for the

essential features that dierentiate a surfa tant mole ule in the vesi le and in the

monomeri state. The transfer free energy, gtr , ree ts the so- alled hydrophobi

60
ee t , whi h onstitutes the major driving for e for surfa tant self-assembly in wa-

ter. Indeed, the transfer free energy is the only favorable free-energy ontribution to

mole ular aggregation, with the other four free-energy ontributions working against

this pro ess. The hydrophobi region in a vesi le is dierent from bulk hydro arbon.

In a vesi le, the surfa tant tails are an hored at one end on either the outer or inner

interfa es, whi h restri ts the number of onformations that ea h surfa tant tail an

adopt while still maintaining a uniform liquid hydro arbon density in the vesi le

hydrophobi region. This subtle dieren e between a bulk hydro arbon phase and

the hydrophobi region in a vesi le is aptured by the pa king free energy, gpack . In

addition, free-energy penalties are imposed, upon aggregation, by the reation of

the outer and inner hydro arbon/water interfa es, aptured in gσ , and by the steri

repulsions and ele trostati intera tions between the surfa tant heads, aptured in

gsteric and gelec , respe tively.

6
 A distin tion is made in the literature between two types of atanioni sys-

tems: (1) In the `simple mixtures' of ationi and anioni surfa tants or atanioni

surfa tant systems with ex ess salt, both surfa tants still behold their own ounteri-

ons. (2) The `true atanioni s' (ion pair amphiphiles) onsist of surfa tant systems

where the original ounterions have been removed and repla ed by hydroxide and

hydronium ions. The ombination of the ounterions at equimolarity thus forms

water mole ules. Ea h surfa tant stands as ounterion for the surfa tant of opposite

harge. The present work will fo us on the rst type of atanioni systems (with

ounterions).

1.1.4 Appli ation of Catanioni Vesi les in Cosmeti Formu-

lation
Vesi les are ommonly used in osmeti s and pharma y as vehi les for a tive agents.

A tive mole ules an thus be en apsulated in the bilayer membrane if they are

lipophili or in the ore of the vesi le if they are hydrophili . En apsulation is useful

to prote t a tives in preventing any undesired rea tion. Vesi les an thus be used

as ve tors to deliver drugs to a spe i pla e, without being destroyed. The phar-

ma euti al appli ations ontinuously in rease and vesi les are used more and more

in the dermatology for prevention, prote tion and therapy.

The rst en apsulation experiments on atanioni systems were performed by Harg-

61
reaves and Deamer on the etyltrimethylammonium bromide / sodium dode ylsul-

fate system. They su essfully entrapped glu ose, however, the system was limited

24
to high temperatures (> 47◦ C). Ten years later, Kaler at al. pro eeded with sim-

ilar experiments using vesi les formed from etyltrimethylammonium tosylate and

sodium dode ylbenzenesulfonate (CTAT/SDBS) mixtures. A more omprehensive

62
study of the entrapment ability was made by Tondre et al. on the CTAT/SDBS

50
and by Kondo et al. on the didode yltrimethylammonium bromide / sodium do-

de ylsulfate (DDAB / SDS) system. The surfa tant on entration as well as the

ratio of the two parti ipating surfa tant have proved to have a big ee t on the

entrapment e ien y.

7
Another problem is the release of the entrapped a tive ingredient. In most ases a

50
non-ioni surfa tant is used (i. e. Triton X-100) . A more elegant solution for tar-

geted drug delivery is designing vesi les that be ome unstable at an easily tuned pH

value. It is known that, for example, tumors and inamed tissues exhibit a de reased

6367
extra ellular pH . For this reason a large number of groups have fo used their

6878
attention on the preparation of pH-sensitive liposomes as possible drug arrier

systems. Furthermore, improving the bio ompatibility of produ ts used in osmeti

formulation even further is always sought after. For this reason it is important to

identify the skin irritating properties of ommer ially used surfa tants. In addition

to their entrapment abilities, vesi les serve also as models for membranes of biolog-

7981 8285
i al ells and as templates for the synthesis of nanoparti les, extra tion of

86 87
rare earth metal ions , and as gene delivery systems .

1.2 Ion Ee ts

1.2.1 Ions in Water
It has long been known that the dissolution of ions brings about hanges in solvent

88
stru ture . The region of modied solvent surrounding an ion has been denoted

89
as a osphere of the ion . The degree and manner in whi h ospheres overlap in

the lose-range en ounter of two ions depends spe i ally on the nature of both

ions and the primary for es between them. Ion hydration has been studied exten-

88,9093 9498
sively experimentally as well as theoreti ally . Terms su h as ` onta t'

pairs and `solvent-separated' pairs have ome into use to distinguish the results of

omplete and partial elimination of solvent mole ules from between two intera ting

ions (see Se tion 1.2.3.). The basi features responsible for ion-spe i short-range

intera tions are, in the ase of monoatomi ions, their harge, their size, their po-

larizability, and availability of ele trons and/or orbitals for ovalent ontributions.

Additional features of polyatomi ions are the harge density distribution, and, in

99
some ases, the presen e of hydrophobi groups . The ease with whi h hydration

ee ts a ompanying asso iation-disso iation pro esses an be observed, depends

8
on the number of ion pairs existent at any instant. Thus the ee ts are observable

more readily with weak ele trolytes and with pairs of multivalent ions, than with

strong 1-1 ele trolytes.

Ions have long been lassied as being either kosmotropes (stru ture makers) or

haotropes (stru ture breakers) a ording to their relative abilities to indu e the

stru turing of water. The degree of water stru turing is determined mainly by two

quantities: the in rease or de rease of vis osity in water due to added salt, and

entropies of ion solvation. For example, the vis osity η of an aqueous salt solution

typi ally has the following dependen e on ion on entration c 91 :

η/η◦ = 1 + Ac1/2 + Bc + · · · (1.1)

where η◦ is the vis osity of pure water at the same temperature. A is a on-

stant independent of c; its orresponding term an be explained by Debye-Hü kel

theory as being due to ounterion s reening at low ion on entrations. The onstant

B, whi h is alled the Jones-Dole B oe ient, is the quantity that des ribes the

degree of water stru turing. B is positive for kosmotropi ions and negative for

haotropi ions. (One issue in interpreting experiments is how to separate the on-

tributions of the anion from the ation. The standard assumption is that K+ has

the same B oe ient as Cl− , be ause K+ and Cl− have approximately the same

100
ioni ondu tan e and be ause the value of B for KCl is approximately zero.)

Water stru turing is also ree ted in entropies of ion solvation. Again, the ef-

fe ts of the anion from the ation need to be separated (it is assumed that the

90
solvation entropies are additive ). Furthermore, the ion solvation entropy needs

to be divided into ion and hydration water ontributions. Subtra ting the former,

∆SII is obtained, whi h des ribes the hange in entropy of hydration water due to

90
the presen e of an ion . Ions whi h are kosmotropi in vis osity experiments tend

to have a negative hydration omponent to their solvation entropy, implying that

they order the nearby waters, while haotropi ions have a positive ∆SII . Figure

1.3 plots the entropy of water near monovalent ions as al ulated from the entropy

of hydration of the ion (from dissolving the ion in water) versus the ioni radius of

92
the ion . A negative ∆SII (upper portion of Figure 1.3) indi ates tightly bound

9
 15
-
F
+
Li
10

+
II
Na r (Å)
S

0
-
0.5 1.0 1.5 2.0 2.5
Cl
-

-5
-

+
Br
K

-10
+
Rb
-
+
Cs I

-15

Figure 1.3: The entropy of pure water minus the entropy of water near an ion in calK −1 mol−1 .
The rystal radii of the ions in angstroms are plotted along the abs issa. Positive values of ∆SII
(lower portion of gure) indi ate water that is more mobile than bulk water. Negative values of
∆SII (upper portion of gure) indi ate water that is less mobile than bulk water. Kosmotropes
are in the upper portion of the gure; haotropes are in the lower portion of the gure (Figure
reprodu ed from Collins 101 ).

water that is less mobile than bulk water, whereas a positive ∆SII (lower portion

of Figure 1.3) indi ates loosely held water that is more mobile than bulk water.

In reasing ion size (de reasing ion harge density) is asso iated with in reasing mo-

bility of nearby water mole ules. If this mobile, loosely held water is immediately

102
adja ent to the ion, as suggested by x-ray and neutron dira tion data , then the

horizontal line in Figure 1.3 indi ating ∆SII = 0, separates strongly hydrated ions

(above the line) from weakly hydrated ions (below the line). Sin e this transition

from weak to strong hydration o urs at a larger size for anions than for ations,

the anions must be more strongly hydrated than the ations, sin e anions begin to

immobilize adja ent water mole ules at a lower harge density than do ations. The

experiments show that water is ordered by small or multivalent ions and disordered

by large monovalent ions. Therefore, water ordering has generally been interpreted

90,103
in terms of ion harge densities . Charge densities are high on ions that have a

small radius and/or a large harge.

The water-ordering ee t of ions is also theoreti ally extensively studied. Contin-

10
 104
uum ele trostati s models su h as that of Debye and Hü kel utilize a ma ros opi

diele tri onstant and assume that all intera tions involving ions are stri tly ele -

trostati , implying the existen e of long range ele tri elds strong relative to the

strength of water-water intera tions. In these models, ions are often thought of as

point harges and water as a dipole whi h orients in the long-range ele tri eld.

Su h models are unable to a urately des ribe su h simple ion-spe i behaviors

as their tenden y to form onta t ion pairs, whi h is a major determinate of the

solubility of spe i salts and of the role of spe i ions in biologi al systems. For

example, models employing a ma ros opi diele tri onstant predi t that all ions

are strongly hydrated and will be repelled from nonpolar surfa es by image for es. In

fa t, weakly hydrated ions (e.g., ammonium, hloride, potassium, and the positively

105107
harged amino a id side hains) a tually adsorb to nonpolar surfa es and in-

108,109
terfa es . The driving for e for this adsorption has been to be the release of

110
weakly bound water to be ome strongly intera ting bulk , a pro ess not in luded

in the al ulations utilizing the ma ros opi diele tri onstant. Sophisti ated mi-

111,112
ros opi al ulations have indi ated a role for the polarizability of weakly

113
hydrated ions (as opposed to their dehydration energy) and dispersion for es in

driving them to neutral interfa es.

1.2.2 Hofmeister Ee t

114,115
A related property is the Hofmeister ee t . In 1888, Franz Hofmeister om-

pleted the rst systemati study on spe i -ion ee ts. He reported that salts ae t

the solubility of proteins in water. Certain ions pre ipitate proteins in water (`salt-

ing out') while others help solubilize them (`salting in'). This behavior has been

interpreted as a modulation of the hydrophobi ee t by salts due to the hanges in

the water stru ture brought about by ions. Su h salt ee ts orrelate with harge

densities of the salts. Small ions tend to ause `salting out', that is, to redu e

hydrophobi solubilities in water, whereas large ions tend to ause `salting-in', in-

reasing nonpolar solubilities. In Hofmeister's papers an ordering of salts, and later

an ordering of ions, usually denoted as `The Hofmeister series' was developed. `The

Hofmeister series' orders ions as a monotoni fun tion of their surfa e harge den-

11
sity and thus water anity, with the strength of water-water intera tions separating

strongly hydrated from weakly hydrated spe ies. It is most onvenient to generate

a separate series for anions and for ations ( . f. Figure 1.4).

Chaotropes
Cosmotropes Salting-in
Salting-out “Soft”
“Hard” Organic

SO42- > OH- > F- > Cl- > Br- > NO3- > I- > SCN- > ClO4-
NH4+ > K+ > Na+ > Cs+ > Li+ > Rb+ > Mg2+ > Ca2+ > Ba2+

Figure 1.4: A typi al Hofmeister series.

1.2.3 Ion Pairing in Water

Ion pairing des ribes the (partial) asso iation of oppositely harged ions in ele -

trolyte solutions to form distin t hemi al spe ies alled ion pairs. If the ion asso-

iation is reasonably strong (the value depends on the harges of the ions and the

relative permittivity of the solvent), there is usually little di ulty in separating the

properties of the ion pair from the long-range nonspe i ion-ion intera tions that

exist in all ele trolyte solutions. However, when the ion asso iation is weak, there

is a strong orrelation between these nonspe i ion-ion intera tions ( hara terized

in terms of a tivity oe ients) and ion pair formation ( hara terized in terms of

an asso iation onstant). Spe ies are generally des ribed as ion pairs if two oppo-

sitely harged ions in solution stay together at a separation r, whi h is smaller than
some spe ied uto distan e R. Ions further apart than R are onsidered `free'.

Various theories have been proposed for hoosing the value of R and for des ribing
the properties of the ion pairs and free ions that together produ e the observed be-

havior of ele trolyte solutions. It is generally a epted that ions an not approa h

ea h other more losely than some `distan e of losest approa h' a due to the strong
repulsive for es of the ele tron shells of the ions, even if polarizable. The distan e

a is understood to bear some relation to the sum of the ( rystal ioni ) radii of the

oppositely harged ions, generally a ≥ r+ + r− . In summary, two ions of opposite

sign are onsidered to form an ion pair if their distan e apart is between a and R for

12
 116
a time longer than the time needed to diuse over su h a distan e . On e ions are

paired, they are thought to have no tenden y to asso iate with other ions in dilute

solutions, although, at higher ele trolyte on entrations, ion triplets, quadruplets,

or larger aggregates may form. A major role in the asso iation of ions in solution

into pairs is thought to be played by long-range ele trostati for es between the ions,

usually modeled as a Coulomb's law attra tion, attenuated by the solvent permit-

tivity. Very short-range intera tions (hard or nearly-hard sphere repulsions) involve

the mutual ex lusion of ions at r < a. However, at distan es a < r < R, solvation of
the onstituent ions must be onsidered. On this basis an ion pair may be lassied

as a (double) solvent-separated ion pair (2SIP), when the primary solvation shells

of both ions remain essentially inta t, as a solvent-shared ion pair (SIP), if a single

solvent layer exists in the spa e between the ion partners of the pair, or as a onta t

ion pair (CIP), if no solvent exists between the partners and the ions are in dire t

onta t (Figure 1.5).

Figure 1.5: S hemati representation of ion-pair types: (a) solvent separated (2SIP), (b) solvent
shared (SIP), and (c) onta t (CIP). The omplete solvation shell around the ion pair is not
displayed. (Reprodu ed from Mar us and Hefter 117 )

1.2.4 Collins' Theory of Mat hing Water Anities

Taking into a ount experimental ndings a simple model for the ion-indu ed stru -

103 103
turing and disordering of water has been proposed . Collins proposed that ion

ee ts on water stru ture ould be explained by a ompetition between ion-water

intera tions, whi h are dominated by harge density ee ts, and water-water inter-

a tions, whi h are dominated by hydrogen bonding. For example, lithium is a small

ion with high harge density, so it intera ts strongly with the water dipole to strongly

13
orient the water mole ules in the ion's rst solvation shell. Larger ions having lower

harge density have a lower tenden y to orient water in the ion's rst solvation shell

( haotropes). A ordingly, ions with high harge density have a high propensity to

order water (kosmotropes). He suggested that anions are stronger than ations at

water ordering be ause of the asymmetry of harge in a water mole ule: the negative

end of water's dipole is nearer to the enter of the water mole ule than the positive

end. Therefore, anions feel a larger ele trostati potential at the surfa e of a water

118
mole ule than ations. The al ulations of Kalyuzhnyi et al. indi ate that the

solvation model of Collins yields qualitative agreement with the experimental data.

10

CsI

CHAOTROPE-CHAOTROPE
5
CsCl

KOSMOTROPE-KOSMOTROPE
NaCl
q (kcal mol )

0
-1

NaF NaBr

-5 KF

CHAOTROPE-KOSMOTROPE
LiCl

-10
CsF

LiBr

KOSMOTROPE-CHAOTROPE
-15 LiI

-60 -40 -20 0 20 40 60

-1
W - - W + (kcal mol )
A C

Figure 1.6: Relationship between the standard heat of solution of a rystalline alkali halide (at
innite dilution) in kcal · mol−1 and the dieren e between the absolute heats of hydration of the
orresponding gaseous anion and ation, also in kcal · mol−1 . The ions are assied as haotropes
(weakly hydrated) or kosmotropes (strongly hydrated). The enthalpy of solution of haotrope-
haotrope and kosmotrope-kosmotrope salts is positive (takes up heat), whereas the enthalpy of
solution of haotrope-kosmotrope and kosmotrope- haotrope salts is either negative (gives o heat)
or positive (takes up heat). Figures reprodu ed from Collins 101 .

Collins' law of mat hing water anities states that oppositely harged ions in

14
free solution form inner sphere ion pairs spontaneously only when they have equal

103
water anities . Experimental observations of the systemati dependen e of the

heats of solution of simple alkali halides on the water anity of the individual ions

(absolute free energies of hydration) and the dependen e of the solubilities of the

alkali halides on ion size ontributed to the realization that a simple law ontrolled

the tenden y of ions of opposite harge to form inner sphere ion pairs. In Figure 1.6,

the enthalpy of solution is plotted on the verti al axis: those salts learly above the

line at 0 produ e old solutions upon dissolution; those salts learly below the line

at 0 produ e hot solutions upon dissolution. Plotted on the horizontal axis is the

dieren e in absolute free energies of hydration (water anity) of the onstituent

ions of the salt. We see that when the onstituent ions of a salt are mat hed in water

anity (kosmotrope-kosmotrope and haotrope- haotrope salts), old solutions are

produ ed, suggesting that no strong intera tions with water have o urred (whi h

would release heat) and that the oppositely harged ions of the dissolved salt tend

to stay together. This is to be expe ted: the point harge at the enter of a (small)

kosmotropi ion an get loser to the point harge at the enter of an oppositely

harged (small) kosmotropi ion than it an to the point harge at the enter of

the oppositely harged portion of a medium size zwitterion (water mole ule); and,

the point harges at the enters of the two harges on the medium size zwitterions

an get loser to the harges on other water mole ules than it an to the point

harge at the enter of a (large) haotrope. In ontrast, when the onstituent ions

are mismat hed in water anity (kosmotrope- haotrope and haotrope-kosmotrope

salts), hot solutions are often produ ed, suggesting that a strong intera tion of

the small ion with water has o urred and that the oppositely harged ions of the

dissolved salt have separated. This is also to be expe ted, sin e the point harge

at the enter of a (small) kosmotropi ion an get loser to the point harge at the

enter of the oppositely harged portion of a medium size zwitterion than to the point

harge at the enter of the oppositely harged (large) haotrope. The requirement

of a haotrope-kosmotrope or kosmotrope- haotrope salt for an exothermi heat of

solution is a ne essary but not su ient ondition sin e when su h a salt is dissolved

the kosmotropi ion will generate heat as it goes from a (large) haotropi partner

15
to a (medium size zwitterioni ) water mole ule, and the haotropi ion will take up

heat as it goes from a (small) kosmotropi partner to a medium size zwitterioni )

water mole ule. The net ee t an be exothermi or endothermi .

+ +
_ + _
Small Small Small-Small

_
+ + _ +
Big Big Big-Big

+ + _ X
Small
Big

Figure 1.7: Ion size ontrols the tenden y of oppositely harged ions to form inner sphere ion
pairs. Small ions of opposite sign spontaneously form inner sphere ion pairs in aqueous solution;
large ions of opposite harge spontaneously form inner sphere ion pairs in aqueous solution; and
mismat hed ions of opposite harge do not spontaneously form inner sphere ion pairs in aqueous
solution.

This is s hemati ally represented in Figure 1.7. Small ions of opposite harge

will tend to ome together be ause the point harges at their enters an get loser

to ea h other than with the point harges at the enters of the medium size water

mole ules. Large ions of opposite harge will ome together be ause the released

water mole ules an form stronger medium - medium intera tions. And (small)

kosmotropi ions will not spontaneously dehydrate to form an inner sphere ion pair

with an oppositely harged (large) haotropi ion be ause the point harge at the

enter of the kosmotropi ion an get loser to the point harge at the enter of the

oppositely harged portion of a medium size zwitterions than to the point harge

at the enter of an oppositely harged (large) haotrope. Thus, it an be on luded

that oppositely harged ions in free solution spontaneously form inner sphere ion

pairs only when they have equal water anities.

16
1.2.5 Ion-Spe i Ee ts in Colloidal Systems
Ion-water intera tions are important throughout biology and hemistry. Ions af-

119123
fe t the onformations and a tivities of proteins and nu lei a ids . Ion om-

plexation in ells is ru ial for the a tivities of biomole ules su h as enzymes and

124,125
drugs . Ions regulate the ele trostati potentials, ondu tan es, and permeabil-

126,127
ities of ell membranes , the stru tures of mi elles, and the hydrophobi ee t,

107,128
whi h drives partitioning, permeation, and folding as well as binding pro esses .

129,130 131
In hemistry, ions ae t the rates of hemi al rea tions , rates of gelation

132
(widely used in food appli ations), ion-ex hange me hanisms (widely used for

hemi al separations), and the expansion and ontra tion of lays, responsible for

133,134
environmental pro esses su h as mudslides .

It has been observed that in asso iation olloids, the hanges in the balan e of

for es ontrolling the aggregate stru ture are ree ted in the hanges in interfa ial

135
on entrations of water and other omponents . In surfa tant systems, this hange

is ree ted in the dierent self-aggregation morphologies. This is espe ially true for

atanioni mixtures, due to their enhan ed sensitivity to outside parameters. For

this reason, atanioni surfa tant systems have been hosen to elu idate the ion-

spe i behavior and role of the surfa tant headgroups in olloidal hemistry.

17
18
 Part I

Salt-Indu ed Morphologi al
Transitions in Non-Equimolar
Catanioni Systems

19
Chapter 2

Blastulae Aggregates: Spontaneous

Formation of New Catanioni
Superstru tures

2.1 Abstra t

The transition of ioni mi elles to vesi les upon the addition of salt was explored.

The atanioni surfa tant solution was omprised of sodium dode ylsulfate (SDS)

and dode yltrimethylammonium bromide (DTAB) with an ex ess of SDS. The hange

in aggregate size an be a ommodated by the in rease of ounterion binding and

onsequent dehydration of the surfa tant headgroups. A new type of intermediate

stru ture was found: a symmetri ally shaped spheri al super-stru ture, whi h we

named blastulae vesi le. In ontrast to known raspberry-like or egg- arton stru -

tures, we believe that harge u tuations within the bilayers are responsible for

this spontaneous super-aggregation to o ur in the presen e of only a small amount

of sodium hloride. A possible me hanism for the observed pattern formation is

proposed.

21
2.2 Introdu tion

It is known for a long time that double hain surfa tants an spontaneously form

43
vesi les . A similar phenomenon an be observed with mixtures of ationi and

anioni surfa tants (here alled atanioni s). These systems display a wide variety

of phase behavior and stru tures su h as mi elles, vesi les, dis s and folded bilay-

24,136,137
ers an be observed . Re ently, new self-assembled stru tures, su h as onion

phases and i osahedra were found in `true' atanioni solutions with no other ions

138,139
than the surfa tant mole ules . Spontaneously formed vesi les are of appli a-

tional interest, espe ially in atanioni systems, sin e they an be tailored at will

by varying the anioni / ationi surfa tant ratio, the size of the hain length or the

26
nature of the polar heads .

For dilute systems, lo al aggregate urvature determined by geometri al onstraints

embodied in a surfa tant pa king parameter P = v/(lmax a), where v and lmax are the
volume and length of the hydrophobi part, respe tively, and a the area per mole ule
at the interfa e (headgroup) is a onvenient variable that hara terizes phase dia-

15
grams . A ne essary ondition for the formation of vesi les from either single or

mixed surfa tants an be shown to be that the pa king parameter is 1/2 < P < 1.
For a single surfa tant that ondition an be satised by hoosing a double hained

surfa tant, e.g. didode yldimethylammonium bromide (DDAB); for mixed surfa -

tant systems a pseudo-double hain surfa tant is obtained by ion-pair formation

18,45
between an anioni and a ationi surfa tant .

However, this ondition is not su ient. While the type of aggregate that forms

with ioni surfa tants an be broadly understood in terms of a balan e between

for es due to the pa king properties of surfa tant tails and those due to double-layer

ele trostati intera tions, onditions for the formation of single or few walled vesi les

16,140,141
are very subtle . For ee ts of global pa king onstraints and inter-aggregate

142
intera tions, see André et al. . As the surfa tant parameter (intrinsi lo al ur-

vature) varies in the range 1/2 < P <1 by e.g. varying head group area via salt

addition or temperature or mixing hain lengths, then, if the surfa tant hains are

exible, the system forms vesi les that grow as P in reases to form a lamellar phase

22
at P = 1.
If the hains are not exible, the system at rst forms multiwalled vesi les, then

vesi les, then lamellar phases again. At P =1 this urious phenomenon is due to

pa king onstraints that emerge be ause the interior and exterior surfa tants of a

urved bilayer experien e very dierent onstraints. Depending on those hain and

headgroup onstraints the system forms ubi phases at P ≈ 1. These are phases

of zero average urvature but varying Gaussian urvature. Again, with in rease in

surfa tant on entration equivalent to in reased repulsion between aggregates, the

system an form equilibrium states of supra aggregation. In these the interior an

be mi elles or ubi phases prote ted by few walled vesi les.

20,45,143149
The appearan e of so many dierent stru tures is known for a very long time .

But apart from the system of double hained didode yldimethylammonium salts

with dierent ounterions studied by Ninham and Evans, they have been little ex-

plored.

Vesi les from atanioni systems are easily prepared. There is an expe tation that

62,150,151
they might also be used as vehi les for ontrolled delivery of drugs or as

150152
templates for the synthesis of hollow parti les . One advantage of atanioni

vesi les as ompared with more robust genuinely double hained surfa tants is their

153 47
greater sensitivity to parameters su h as temperature or the presen e of salts

used to indu e transitions from vesi les to mi elles or to pre ipitation. Of parti ular

interest is the dire t transition from mi elles to vesi les. Su h a phenomenon oers

in prin iple an easy way of en apsulating a tive agents by dissolving them in the

mi ellar phase prior to vesi le formation. Mi elle-to-vesi le transition was already

26,154,155
observed when diluting a mi ellar solution with water , hanging the anioni

156158 159
/ ationi surfa tant ratio , in reasing temperature , or upon the addition of

160 161163
organi additives and salt .

The ee t of ioni strength on atanioni systems was previously studied experi-

47
mentally by Brasher et al. . Their results show that the addition of monovalent

salt hanges the phase behavior and aggregate properties of mixed surfa tant solu-

tions. Theoreti ally the ee t of salt on the atanioni s was des ribed by Yuet et

59
al. , however, due to the onstri tions of the model (smeared surfa e harges and

23
point-sized ions), they ould not reveal the spe i ity of dierent ions.

In the present hapter, we explore salt-mediated transition of mi elles to vesi les

47,48,164
in a well-studied system . We are on erned with the inuen e of salts on a

atanioni system omposed of sodium dode ylsulfate (SDS) and dode yltrimethy-

lammonium bromide (DTAB) in aqueous solution, with an ex ess of anioni surfa -

tant. In reasing amounts of sodium hloride was su essively added to a solution of

mixed SDS / DTAB mi elles. The mi ellar system was rst hara terized by rheo-

metri measurements and ryo-TEM. We report on the parti ular morphologies that

arise during the salt-indu ed mi elle to vesi le transition. The formation of irregu-

lar onvex- on ave patterns and a se ondary self-assembly of vesi le-like stru tures

upon the addition of sodium hloride is presented. In order to study this transition

in detail, the on entration of salt in the system was in reased in small in rements

and the ee t was studied using dynami light s attering, ryo- and freeze-fra ture

transmission ele tron mi ros opy. Two dierent mi ros opy te hniques were used in

order to ex lude the artifa ts that might arise during the preparation of samples.

FF-TEM provides a dire t visualization of the three-dimensional stru ture of parti-

les. The fra ture follows the path of least resistan e, and in olloidal dispersions,

the fra ture surfa e propagates along the interfa e of two phases. This makes FF-

TEM ideal to study membrane surfa es. To observe whether the vesi les are losed

and if the membranes are inta t ryo-TEM was employed. Furthermore, ryo-TEM

is very appropriate to study mi ellar solutions.

2.3 Experimental Pro edures

Materials Sodium dode yl sulphate (SDS) (purity: 99%) and sodium hloride

were pur hased from Mer k, Germany. The ationi surfa tant used was 99% do-

de yltrimethylammonium bromide (DTAB) pur hased from Aldri h, Germany. All

hemi als mentioned above were used as re eived without further puri ation. Mil-

lipore water was used as solvent in all ases.

24
Sample Preparation Surfa tant sto k solutions were prepared by dissolving weighed

amounts of dried substan es in Millipore water. The solutions were then left for 24

hours to equilibrate at 25◦ C. The atanioni solutions were prepared by mixing the

surfa tant sto k solutions to obtain a xed anioni / ationi surfa tant mass ratio

of 70/30 (this orresponds to a molar ratio of about 2.5/1). The total surfa tant

on entration was kept at 1 wt.% at all times. Salts were added to the mi ellar

solution at in reasing on entrations. The solutions were then stirred and left to

equilibrate for a week at 25◦ C before making measurements.

Dynami Light S attering (DLS) Measurements Parti le size analysis was

performed using a Zetasizer 3000 PCS (Malvern Instruments Ltd., England), equipped

with a 5 mW helium neon laser with a wavelength output of 633 nm. The s attering

angle was 90◦ and the intensity auto orrelation fun tions were analyzed using the

CONTIN software. All measurements were performed at 25◦ C.

Rheology Rheologi al experiments were performed on a Brookeld DV - III+ rate

ontrolled rheometer. A one-and-plate geometry of 48 mm diameter and with a 0.8

deg one angle was used (spindle model CP - 40).

Cryo-Transmission Ele tron Mi ros opy ( ryo-TEM) Spe imens for ryo-

TEM were prepared by pla ing a small drop ( a. 4µl) of the sample on a holey arbon
grid. Immediately after blotting with lter lm to obtain a thin liquid lm over the

grid, the sample is plunged into liquid ethane (at its melting temperature). The vit-

ried lm is then transferred under liquid nitrogen to the ele tron mi ros ope. The

grid was examined with a Zeiss EM922 EF Transmission Ele tron Mi ros ope (Zeiss

NTS mbH, Oberko hen, Germany). Examinations were arried out at temperatures

around 90 K. The TEM was operated at an a eleration voltage of 200 kV. Zero-loss
ltered images (DE = 0 eV) were taken under redu ed dose onditions (100 - 1000

2
e/nm ). Images were registered digitally by a bottom mounted CCD amera system

(Ultras an 1000, Gatan, Muni h, Germany) ombined and pro essed with a digital

imaging pro essing system (Digital Mi rograph 3.9 for GMS 1.4, Gatan, Muni h,

Germany).

25
Freeze-Fra ture Ele tron Mi ros opy Samples used for ryo-fra ture were

ryoprote ted by 30% gly erol and frozen in liquid N2 . Freeze-fra ture was per-

formed in a Balzers (Balzers, Switzerland) apparatus at −150◦ C under a va uum

of 10−6 Torrs. Metalli repli as were obtained by Pt and arbon shadowing of fra -

ture surfa es. The repli a were examined and photographed with a Philips CM 12

transmission ele tron mi ros ope.

2.4 Results
2.4.1 Chara terization of SDS / DTAB Mi ellar Solution
The system under study is a well-known mixture of ationi and anioni single- hain

surfa tants. The total surfa tant on entration (1 wt.%; ≈ 33 mM) and the anioni

/ ationi molar ratio (2.5 / 1) remained onstant throughout all the experiments.

The initial sample was olorless and isotropi , orresponding to the mi ellar region

of the phase diagram (Figure 2.1).

Figure 2.1: S hemati ternary phase diagram of the SDS / DTAB system at 25◦ C (the bla k
ross shows the starting point (referen e sample), to whi h sodium hloride was added)

DLS measurements onrm a mi ellar solution indi ating a hydrodynami radius

(RH ) of 10 nm and a relatively high polydispersity index (0.27). Figure 2.2 (left)

shows a ryo-TEM image of our referen e solution (without added salt), exhibiting

very long rod- or ribbon-like mi elles, in equilibrium with spheri al mi elles (hen e

26
explaining the high polydispersity). Long rod-like mi elles have already been ob-

165
served by SANS measurements in systems similar to ours . Results from rheometry

experiments show that the vis osity de reases with applied strain rate (Figure 2.2

(right)) therefore exhibiting properties of non-Newtonian shear-thinning uids. This

kind of behavior is ommon for solutions ontaining large non-spheri al mole ules in

a solvent with smaller mole ules. It is generally supposed that the large mole ular

hains tumble at random and ae t large volumes of uid under low shear, but that

they gradually align themselves in the dire tion of in reasing shear and produ e less

resistan e. This behavior onrms the presen e of rod-like mi elles. No enthalpy

hange ould be dete ted by dierential s anning alorimetry so that no information

ould be dedu ed about possible phase transitions o urring in the system between

10 and 80◦ C 166168 . Probably, the amount of surfa tant was too low for su h a

dete tion with our equipment.

13
12
11
10
9
/ Pas

8
7
6
5
4
3
200 400 600 800 1000
-1
/ s

Figure 2.2: Left: Cryo-TEM image of a SDS/DTAB aqueous solution at the molar ratio of 2.5/1
and a total surfa tant on entration of 1 wt.% (reprodu ed from ref. 169 ); right: vis osity of the
same sample as a fun tion of shear rate.

2.4.2 Salt-Indu ed Mi elle-to-Vesi le Transition

Upon the addition of sodium hloride, the solutions exhibited a transition from a

olorless to a blue solution, the blue olor being typi al of the presen e of large

obje ts. Samples with dierent salt on entrations were analyzed by dynami light

27
 90

80

70

60

50
/ nm

40
H
R

30

20

10

0
0.00 0.01 0.02 0.03 0.04

-1
c / molL
NaCl

Figure 2.3: In rease in the mean hydrodynami radius of atanioni aggregates upon the addition
of sodium hloride.

s attering. The addition of hloride salts auses an in rease in average parti le size

and a ertain turbidity of the solution (Figure 2.3). DLS indi ated a signi ant

in rease in the mean hydrodynami radius (RH ) of the mi elles from 10 to a. 70

nm. It an be expe ted that the salt s reens the ele trostati intera tions, whi h

leads to smaller headgroups and therefore a higher pa king parameter.

Freeze-fra ture and ryo-TEM onrm the formation of lamellar sheets in the

sample (Figure 2.4). However, by arefully observing the ryo-TEM images one an

see that at lowest on entration of added salt (10 mM) the long rod-like mi elles

present in the starting solution start to break-up and luster together, see Figure 2.5

(left). Other images from the same solution show that these lusters seem to form

small pie es of lamellae, whi h eventually lose to form round vesi les. The urving

of membranes is represented by the presen e of darker, sti-looking edges, due to

the higher ele tron density in these points. Figure 2.5 (right) shows some urved

pie es of lamellar sheets as well as some omplete vesi les. The vesi les represented

in ryo-TEM appear to be perforated; su h perforated vesi les have previously been

28
Figure 2.4: Cryo-TEM (left) and FF-TEM (right) photographs representing the formation of
multi-lamellar sheets upon the addition of 10mM NaCl. The arrows show regions where we an
observe the unraveling of ribbon-like mi elles into lamellar sheets. The molarity in the ase of
FF-TEM experiments orresponds to the on entration of the solutions prior to ryo-prote ting.

170174
observed in various surfa tant systems . Surprisingly, FF-TEM images do not

onrm su h perforations.

The addition of salt produ es dramati ee ts dete ted by freeze-fra ture. At

20 mM of NaCl large spheri al, highly undulated aggregates are observed. As men-

tioned previously, FF-TEM exploits the property that surfa es fra ture along the

area of least resistan e. In the ase of vesi les this is within the bilayer. There-

fore, only 3-dimensional obje ts an be observed. The size range of the parti les

is from 150 to 500 nm, see Figure 2.6 (left). This apparent polydispersity is most

likely due to the hara terization te hnique used; the measured size of the obje t

depends on the region where the samples are fra tured. Some of the aggregates are

fra tured lose to the middle; Figure 2.6 (right) shows a ring of vesi les. Sin e the

aggregates pi tured in Figure 2.6 are observed in high amounts, they most likely

represent the same obje t, fra tured in dierent pla es ( lose to the `poles' of the

blastulae vesi les, as opposed to the middle of the vesi le). The images suggest

that the inside of these parti les are hollow and lled with the same solvent as the

surrounding (water). Due to the similarities in appearan e we named the observed

stru ture blastulae, taking the name from biologi al origin; the blastulae are an early

29
Figure 2.5: Cryo-TEM photographs representing the breaking-up of ribbon-like mi elles and
onsequent lustering of the pie es. Clusters of elongated aggregates are indi ated by bla k (left),
whereas individual aggregates are designated by white arrows. The image on the right shows
lusters of smaller aggregates (bla k arrows) and vesi les with perforated surfa es (white arrows).

Figure 2.6: FF-TEM photographs representing the formation of blastulae-like lusters upon
the addition of 20mM NaCl ut near the surfa e (left); ut through the middle (right), learly
representing the solvent lled avity. The arrow shows an individual vesi le with its own membrane.

stage of embryoni development onsisting of a spheri al layer of ells surrounding

a uid-lled avity. On the basis of the present pi tures we annot say, whether the

blastula vesi le is an aggregate onsisting of one individual membrane or a luster

of elongated mi elles as observed in ryo-TEM. Both possibilities will be explored

30
further in the text. However, it is interesting to note that some unilamellar indi-

vidual vesi les are also present. Interestingly, the average diameter of the vesi les

( a. 60 nm) is of the same size as the bulges forming the blastulae stru ture. This

might speak for the possibility that the blastula is a tually a luster of individual

small vesi les. These, however, are not deformed in a way that it is usually observed

in aggregates. A me hanism for this type of lustering will be dis ussed later on,

pointing to the similarity with spe i -site (or ligand-re eptor) binding. It should

175,176
be noted that similar aggregates have been observed in another ontext and

will also be dis ussed later.

Figure 2.7: FF-TEM image of individual unilamellar vesi les upon the addition of 30 mM NaCl.
The bar represents 100 nm.

As more salt is added to the system the lusters begin to disaggregate. At sodium

hloride on entrations of 30 mM only individual unilamellar vesi les are observed

as an be seen in Figure 2.7.

Finally, at NaCl on entrations of 40 - 45 mM, a loose and unstru tured, randomly

pa ked aggregation of vesi les is observed, see Figure 2.8. These aggregates are

not spheri ally symmetri al and the individual vesi les forming the aggregates are

31
Figure 2.8: FF-TEM photograph representing the aggregation of vesi les upon the addition of
45 mM NaCl to the referen e SDS/DTAB mi ellar solution. The bar represents 200 nm.

deformed.

In summary, two features are new in this system: i) the appearan e of blastulae

stru tures, and ii) the series of dierent stru tures that are indu ed by salt addition

only, without any further additives.

2.5 Dis ussion

2.5.1 Models of the Mi elle-to-Vesi le Transition
In the following we propose a possible me hanism for the observed pattern formation.

The dierent steps are represented in Figure 2.9.

`Lamellar Model' It is well known that, as an ele trolyte is added to a mixed

mi ellar solution, the ee tive surfa e area of the surfa tant headgroups be omes

smaller. This ee t is mainly due to ounterion on entration and s reening as well

as onsequent dehydration of the neutralized heads. This ee t favors a lamellar

32
pa king (step a → b′ ). The formation of large multi-lamellar sheets is energeti ally

unfavorable, so these start urving and onsequently forming large spheri al obje ts.

13
A theoreti al model has been suggested by Safran et al. , showing that in ases of

mixed surfa tants vesi les are more stable than lamellar stru tures. This is due to

the formation of surfa tant bilayers, where the two monolayers onsist of dierent

surfa tant on entrations, whi h results in equal and opposite monolayer sponta-

neous urvatures. This model is in a ordan e with our result, where the large

spheri al aggregates are shown to form from mi elles via lamellar sheets. The main

fa tor governing the pa king arrangement in these phases is the degree of hydration

of the polar headgroup.

As the polar headgroups are dehydrated even more by the addition of salt, the pa k-

ing parameter in reases and the formation of individual vesi les is favored. In the

present atanioni system the outer layer of the vesi les is omposed of positively

and negatively harged groups with the latter being in ex ess. A redistribution of

harges may then take pla e in the highly exible vesi le membranes leading to lo al

pat hes of positive and negative harges. The lo ally positive harges of one vesi le

an intera t favorably with the negative harges of another vesi le and vi e versa

(Figure 2.9, stage f ). In other words, the vesi les an attra t ea h other due to the

+, − intera tions among the headgroups of surfa tants belonging to dierent vesi-

les. This is somewhat similar to harge u tuations in polarizable obje ts leading

to van der Waals intera tions. But the dieren e is that here the ion harge dis-

tribution of the headgroups u tuates, and not the ele tron distribution. The lo al

Coulomb attra tion may be strong enough to over ome the overall repulsive for e of

equally harged vesi les a ting at larger distan es. Sin e vesi les are spontaneously

formed from lamellar sheets, they are, at this starting point, in lose enough onta t

for this to happen. In this ase only lo alized parts of the membrane (pat hes) are

intera ting, similarly to spe i site binding.

As the ion strength in the solution is in reased the blastulae vesi les disaggre-

gate. We suppose this is due to the fa t that the intera tions between the oppositely

harged headgroups of neighboring vesi les are weaker than those within a vesi le

(one must onsider the additional strong van der Waals for es between the hydropho-

33
 f

+ NaCl + NaCl + NaCl + NaCl

a b d e
c

b’
c’

Figure 2.9: S hemati al representation of the observed stru tures that form through in reasing
salt on entration: From the starting mi ellar solution, whi h exhibits rod-like mi elles (a) two
possibilities are presented: In the ` lustering model', the rod-like mi elles (a) start breaking-up into
smaller pie es (b), whi h start lustering together to form spheri al aggregates. In the `lamellar
model', multi-lamellar sheets are formed upon addition of salt (b′ ). These start losing to form
undulated giant vesi les(c′). At higher ioni strength, the formation of blastulae lusters (c) is
observed, where the outer layer is omposed of individual vesi les, whi h en lose a uid-lled
avity. The vesi les intera t through oppositely harged headgroups of surfa tants belonging to
dierent vesi les (f ; the open and lled ir les represent headgroups of opposite harges). As salt
on entration is further in reased, the attra tion is s reened and the vesi les segregate (d). Still
higher on entrations of salt in the system produ e a non-spe i intera tion, where van der Waals
for es dominate, ausing the vesi les to loosely aggregate while deforming (e).

34
bi surfa tant tails). Therefore, as more salt is added and the harges on the vesi les

are s reened, the `bonds' between the vesi les are broken. The luster begins to dis-

aggregate due to the net negative harge of the individual vesi les, resulting in the

formation of individual unilamellar vesi les.

At still higher ioni strength the ele trostati repulsions are s reened and the ee t

of van der Waals for es be omes signi ant. A loose and unstru tured, randomly

pa ked aggregation of vesi les is observed. A similar behavior (o ulation of vesi-

les) was observed upon the addition of NaCl to the mi ellar solution of dode yl-

177
benzene sulfoni a id .

`Clustering Model' In this model, the ribbon-like mi elles start breaking up into

smaller ylindri al aggregates ( orresponding to step a → b). These ould be small

vesi les, whi h grow until retaining a radius at whi h they are most stable (small

vesi les are thermodynami ally unfavorable due to high urvature). Again these

aggregates start lustering together despite an overall net negative harge. Steps

c→e remain the same as in the `lamellar model'.

2.5.2 Blastulae Vesi les

Mi ros opy images learly show that the blastulae lusters are spheri al and hollow.

Figure 2.6 (right) pi tures the uid-lled avity and the astounding symmetry of the

aggregate. The interesting phenomenon is that in this luster the vesi les are not

deformed in a way that is usually observed in aggregates. Aggregation of vesi les

without a ompanying deformation of membrane had until now been observed only

in systems to whi h spe i ligands and re eptors were added. In su h ase no

deformations o ur when vesi les are brought together by a spe i site-binding

(ligand-re eptor) intera tion. This is be ause not the whole vesi le surfa e is involved

in the intera tion, but only a dis rete number of onta t points on ea h surfa e. This

kind of self-assembly of vesi les driven by ligand-re eptor oupling was reported by

178180
Chiruvolu et al. and Walker et al. .

It is interesting to translate this on ept to blastulae formation by proposing a

u tuation of the ele tron density in the membrane as was des ribed above. Su h

35
a formation of lo alized partitions of opposite harge has been previously reported

181
by Aranda-Espinoza et al. . A spontaneous partitioning of positively harged

mixed bilayer vesi les in the presen e of negatively harged parti les resulting in an

ele trostati repulsion between oppositely harged parti les was observed. In our

ase the ee t is reversed, ele trostati attra tion of vesi les of equal net harge are

found. Re ently, the asymmetry of harge in lipid bilayers indu ed by monovalent

182
salts was reported. A ording to Gurtovenko , the dieren e in the headgroup

orientation on both sides of the bilayer, oupled with salt-indu ed orientation of

water dipoles, leads to an asymmetry in the harge-density proles and ele trostati

potentials of the bilayer. In this report lipid vesi les with only one type of mole ules

are present. In our ase, where two oppositely harged surfa tants are present, the

salt-indu ed asymmetry may be the reason for the pat hes of the single harged

surfa tants of opposite sign. Finally, the overall shape of the blastulae formed by

the `atta hed' vesi les an be explained by the preferential lo al aggregate urvature

determined by geometri al onstraints, obeying the same rules as the formation of

vesi les from surfa tants. It should be noted that the attra tion of overall equally

183
harged obje ts is dis ussed from time to time in literature . The present ndings

and the model proposed here may help to understand su h phenomena.

As far as oexisten e of vesi les and mi elles are on erned, intermediate stru tures

have been reported in the literature: open vesi les, mesh phases, or even pat hes

56,170,184
and dis s . However, to our knowledge su h symmetri ally shaped hollow

stru tures as the blastulae vesi les have never been observed before in atanioni

systems. One of the possible explanations is that blastulae are the diluted ounter-

part of the `oyster phase' whi h has been observed for other harged bilayers in the

185
absen e of salt .

The rationale in the sequen e of observed shapes (as s hemati ally represented in

Figure 2.9), when spontaneous urvature is varied via omposition, is given by a

general me hanism: a o- rystallization o urs, followed by a segregation of ex ess

material. The amount of non-stoi hiometri omponent distributes between the

186
latti e and the edges and thus ontrols the shape of the rystallized olloids .

36
2.5.3 The O urren e of Convex-Con ave Patterns in Biolog-
i al Systems

Experimentally, the o urren e of onvex- on ave deformations of high regularity,

similar to our blastulae, was rst observed in a study of lipid extra ts from ba terial

187,188
membranes . The egg arton pattern is mostly found in omplex lipid mixtures

189
of biologi al origin su h as Streptomy es hygros opi us and brain sphingomyelin .

Later on, this kind of urved patterns has also been observed in a few examples with

a more simple omposition su h as in vesi les of DMPC mixed with a polymerized

190 191
amphiphile with butadiene groups , N-nervonoyl sphingosylphosphoryl holine

both in their gel state and in systems ontaining the lipid omplex soybean le ithin

192
and poly(diallyldimethylammonium hloride) . However, the repeating onvex-

on ave patterns in these so- alled `egg arton' stru tures are found on at sheets

and the obje ts are multilamellar vesi les with diameters largely ex eeding the diam-

eters of the bulges. By ontrast, FF-TEM images of the present atanioni system

show that the building blo ks forming the blastulae aggregate are of the same av-

erage diameter as the individual vesi les present ( a. 60 nm) in solution. The

origin of onvex- on ave bilayer deformation is believed to result from onstraints

imposed by limiting hydration of the headgroup and a frustration arising from the

193
spontaneous urvature of the bilayers . This ee t had already been dis ussed by

194
Gebhardt et al. . Undulation and the formation of so- alled egg arton stru ture

had been extensively studied theoreti ally. A `hat and saddle' model was proposed

195
by Helfri h to explain the existen e of orrugated membranes of biologi al origin.

Fournier has shown that anisotropi in lusions an indu e spontaneous bending in

196,197
at lipid membranes, whi h attra t ea h other to form an egg arton stru ture .

A model al ulation based on bilayer bending elasti ity yielding disordered egg ar-

198
ton textures was proposed by Goetz and Helfri h . However, all these al ulations

are not appropriate to explain the pe uliar vesi le patterns experimentally observed

in present work.

37
2.5.4 Raspberry Vesi les

For the same reason the blastulae are dierent from the so- alled raspberry vesi les.

The term `raspberry vesi les' was used previously to des ribe onvex- on ave vesi les

obtained after the indu tion of osmoti sho k on giant unilamellar vesi les in dierent

175
phospholipid systems . The `raspberry ee t' is related to the deating of the

liposome due to the volume redu tion, the onsequen e being the existen e of an

ex ess membrane. This ex ess membrane indu es the formation of inverted `daughter

176
vesi les'. Similar observations have been reported by Ménager and Cabuil for the

osmoti shrinkage of liposomes lled with a ferrouid and in the ase of liposomes

194
subje ted to a gradient of glu ose . However, the evolution of these vesi les is

dierent from the one observed in our ase. The initial membrane undulation of

giant vesi les seems to be a ommon step in all ases. In the aforementioned ases,

the osmoti shrinkage of vesi les was shown to be reversible whi h is proof of a

persistent membrane ne k onne tion between the mother and daughter vesi les. At

high enough osmoti strength the membrane ruptured. No vesi les with individual

membranes were observed. This is not the ase in our system, where the in rease

of ioni strength promotes the formation of individual membranes, resulting in a

spheri ally symmetri al luster of unilamellar vesi les, the blastulae. Higher ioni

strength results in a separation of the vesi les.

2.5.5 Blastulae Vesi les: A General Trend in Catanioni Sys-

tems?

Finally, we would like to report that similar images have been found also in samples

ontaining esium hloride and in a system omposed of sodium dode yl arboxylate

and DTAB in the presen e of sodium hloride ( . f. Figure 2.10). Further experi-

ments and theoreti al insights are ne essary in order to larify the exa t me hanism

of formation of su h stru tures and the reason for this intermediate stru ture to be

found only in atanioni systems ontaining salt.

38
 Figure 2.10: Blastulae vesi les in an SDS / DTAB system with the addition of 20 mM CsCl.

2.6 Con lusions

We have presented a way to make hollow regularly shaped stru tures by spontaneous

se ondary self-assembly of vesi les without additives ex ept salt. These spheri al,

symmetri al aggregates of individual vesi les were never observed previously, even

in systems with ligand-re eptor binding. A me hanism of formation for this type

of super-stru tures was proposed, showing the importan e of harge u tuation.

Finally, su h blastulae aggregates ould be onsidered as an intermediate step in the

formation of unilamellar vesi les from bilayers in non-equimolar atanioni systems.

39
40
Chapter 3

Spe i Alkali Cation Ee ts in the

Transition from Mi elles to Vesi les
Through Salt Addition

3.1 Abstra t
A transition from mi elles to vesi les is reported when salts are added to atan-

ioni mi ellar solutions omposed of sodium dode anoate (SL) / dode yltrimethy-

lammonium bromide (DTAB) and sodium dode ylsulfate (SDS) / DTAB, with an

ex ess of the anioni omponent. The ounterion binding and in rease in aggregate

size was monitored by mass spe trometry, rheology and dynami light s attering

measurements, whereas the vesi les were hara terized by freeze-fra tion and ryo-

transmission mi ros opy experiments. The ee t of ounterions on the formation of

vesi les in both systems was studied and ompared to evaluate the role of the surfa -

tant headgroups on the ounterion spe i ity. The hange in aggregate size an be

a ommodated by the in rease of ounterion binding and onsequent dehydration of

the surfa tant headgroups. A lassi ation of the ations ould be made a ording

to their ability to in rease the measured hydrodynami radii. It was observed that,

if the sulfate headgroup of the anioni surfa tant is repla ed by a arboxyli group,

the order of the ions was reversed.

41
3.2 Introdu tion

The investigation of spe i ion ee ts has engaged resear hers for de ades. Despite

that, ion properties and their intera tions with other mole ules are still not under-

stood in detail, and we are far away from being able to predi t their behavior. A

major di ulty in the study of salts presents the fa t that many phenomena involve

the a tion of ations and anions of the ele trolyte.

Mole ular self-assembly in surfa tant systems is largely dependent on the number

of water mole ules surrounding the headgroups. When ions are added to a solution,

3,59
they dehydrate the surfa tant headgroups . This auses a de rease in the value

a (ee tive area per mole ule at the interfa e) and onsequently an in rease of the

stru tural pa king parameter P, whi h may result in the formation of vesi les. The

ee t of salts on a harged system an dier mu h depending on their kosmotropi

or haotropi hara ter. It is well known that small ions of high harge density (e.g.

sulfate, arboxylate, sodium) are strongly hydrated (kosmotropes), whereas large

monovalent ions of low harge density (e.g. iodide, potassium) are weakly hydrated

101,199
( haotropes) . Ea h salt is therefore expe ted to have an individual inuen e

on vesi le formation, whether it tends to adsorb at the interfa e between mi elle and

water or remains strongly hydrated in the bulk.

101
Collins' on ept of `mat hing water anities' provides us with a simple model

of spe i ion-ion intera tions. From various experimental results (for an extensive

200
review see ref. ) Collins on luded that the dominant spe i for es on ions of

the same valen y in water are short-range for es of hemi al nature and that the

long range ele trostati for es generated by simple ions in water are weak relative

200
to the strength of water-water intera tions . Therefore, onta t ion-pair forma-

tion is a tually dominated by hydration-dehydration. A good agreement was also

found with re ent al ulations of water pairing, where expli it water mole ules were

201
modeled . It is hallenging to see if this simple on ept holds also for spe i

ion-headgroup intera tions.

A salt indu ed mi elle-to-vesi le transition was studied in two atanioni systems:

sodium dode anoate (SL) / dode yltrimethylammonium bromide (DTAB) and sodium

42
dode ylsulfate (SDS) / DTAB. An ex ess of the anioni surfa tant was present in

both systems. The two systems diered only in the headgroup of the anioni sur-

fa tant in order to elu idate the fa t that not the types of the ion alone, but rather

the spe i ation-surfa tant intera tions shape the surfa e behavior. Large ation

spe i ity was found, however the series experien ed a reversal when the arboxy-

late ion was ex hanged for a sulphate one. SDS / DTAB is a well-known throughly

47,48
studied system , whereas, by using a fatty a id based surfa tant, the system re-

sembled more to a biologi al membrane than a system ontaining SDS; it had been

determined that ellular membranes onsist of roughly 2 to 5 % of free long- hain

arboxyli a ids. The atanioni surfa tant trimethylammonium headgroup, in om-

202,203
parison, resembles the holine group that is often present in membranes . The

ee t of dierent anions and ations on the mi ellar solutions was studied by phase

diagrams, rheology, dynami light s attering and mass spe trometry, whereas the

vesi les were hara terized by freeze-fra ture and ryo-TEM imaging. The me ha-

nism of mi elle-to-vesi le transition was investigated and ompared.

3.3 Experimental Pro edures

Materials The surfa tants, sodium dode yl sulfate (SDS; Mer k, Germany; assay

> 99%), sodium dode anoate (SL; Sigma, Germany; grade: 99-100%) and dode-

yltrimethylammonium bromide (DTAB; Mer k, Germany; assay > 99%) were used
as re eived. All sodium and hloride salts used in the experiments were supplied by

Mer k, Germany. Millipore water was used as solvent in all ases.

Sample Preparation Surfa tant sto k solutions were prepared by dissolving weighed

amounts of dried substan es in Millipore water. The solutions were then left for 24

hours to equilibrate at 25◦ C. The anioni -ri h region of the phase diagrams was

used in both ases. The atanioni solutions were prepared by mixing the surfa -

tant sto k solutions to obtain a xed anioni / ationi surfa tant mass ratio: 60/40
(this orresponds to a molar ratio of about 2/1) and 70/30 (molar ratio 2.5/1) was

used for SL / DTAB and SDS / DTAB mixtures, respe tively. The starting ratio

43
was determined from phase diagrams (see Figures 2.1 and 3.1). The total surfa tant

on entration was kept at 1 wt.% at all times. Salts were added to the mi ellar

solution at in reasing on entrations up to 50 mM. The solutions were then stirred

and left to equilibrate for a day at 25◦ C before making measurements.

Ele trospray Mass Spe trometry (ES-MS) Cation anities for the vesi ular

interfa e/ arboxylate group were determined by ele trospray mass spe trometry.

ES-MS was arried out using a Thermoquest Finnigan TSQ 7000 (San Jose, CA,

USA) with a triple stage quadrupole mass spe trometer. The solutions were sprayed

through a stainless steel apillary held at 4 kV, generating multiple harged ions.

Data were olle ted using the X alibur software.

Other Methods Cryo- and freeze-fra ture transmission ele tron mi ros opy, dy-

nami light s attering and rheology were performed as des ribed in Chapter 2.

3.4 Results

3.4.1 Phase Diagrams

The addition of salts to the atanioni mixtures at a ertain ratio of the surfa tants

indu ed an easily observable aggregation, due to a formation of a bluish olor or tur-

bidity. Salts were added to samples from all parts of the phase diagrams, however

homogeneous vesi ular systems upon salt addition were observed only at a ertain

anioni / ationi surfa tant ratio (see Phase Diagrams, Figures 2.1 and 3.1). For

this reason, the starting solution was taken from the mi ellar region of the phase

diagram, a little way from the equimolarity line, with an ex ess of the anioni spe ies

(with a mass ratio of 60/40 and 70/30 for SL / DTAB and SDS / DTAB mixtures,

respe tively).

44
 4% SL
0.00
1.00

0.25
0.75
I_

0.50 equimolarity
0.50

0.75
0.25

I+
1.00
0.00
water 0.00 0.25 0.50 0.75 1.00 4% DTAB

Figure 3.1: S hemati ternary phase diagram of the SL / DTAB system (right) at 25◦ C (the bla k
arrow shows the starting point (referen e sample), to whi h dierent salts were added). Figure
reprodu ed with permission from the do toral dissertation of A. Renon ourt.

3.4.2 Counterion Ee ts

The ee t of various ations was studied by varying the on entration of dierent

alkali hloride salts (LiCl, NaCl, KCl, CsCl). As the on entration of salt in the

system was in reased, the solutions exhibited a transition from a olorless to a blue

solution, the blue olor being typi al of the presen e of large obje ts. This transition

ould be observed for all salts, however, the on entration at whi h vesi les appeared

was strongly dependent on the nature of the ation. Further in rease of added salt

on entration nally led to a phase separation, with one of the two phases being

blue and isotropi and the other turbid.

Samples with dierent salt on entrations were rst analyzed by dynami light s at-

tering. The addition of hloride salts auses an in rease in average parti le size and

a ertain turbidity of the solution (Figure 3.2). DLS indi ated a signi ant in rease

in the mean hydrodynami radius (RH ) of the mi elles from 10 up to a.170 nm. In

agreement with our visual observations the rate of in rease of the measured hydro-

dynami radius was dierent for ea h hloride salt. This highlights strong ation

spe i ity.

45
For instan e, in the SL / DTAB system (Figure 3.2, left) a small amount of LiCl (<

10 mM) was su ient to dramati ally in rease the hydrodynami radius (over 100
nm), whereas with CsCl, on entrations higher than 60 mM were needed to obtain

parti les of that size. A lassi ation of the ations an be made a ording to their

ability to in rease the measured hydrodynami radius of the aggregates forming in

+ + + +
the SL / DTAB surfa tant mixture: Cs < K < Na < Li .

200
60

150
(nm)

R (nm) 40
H

100
R

20
50

0 0
0 10 20 30 40 50 0 10 20 30 40 50
c (mmol/L) C (mmol/L)
SALT SALT

Figure 3.2: The ee t of various ations on the growth of the hydrodynami radii RH of the
atanioni aggregates in SL / DTAB (left) and SDS / DTAB (right) systems with an ex ess of
anioni surfa tant in both systems: LiCl (△), NaCl (N), KCl (), CsCl ().

However, if the arboxyli headgroup of the anioni surfa tant was repla ed by

a sulfate group, the order in whi h the ions assist the formation of vesi les was

reversed ( ompare with Figure 3.2, right). Salts ontaining big ations having a

smaller harge density (for instan e CsCl) more e iently indu ed vesi le/aggregate

formation than small highly harged and highly hydrated ations (su h as LiCl). A

+ + + +
reversed Hofmeister series was therefore observed: Cs > K > Na > Li .

Cation anities for the vesi ular interfa e / sulphate (or arboxylate) group

were determined by ele trospray ionization mass spe trometry. Although in this

method the ionization pro ess takes pla e in the gas phase, the hemi al nature of

surfa tant monomers and simple ions is the same in the liquid and in the gas phase.

204,205
Therefore, the preferential ion-surfa tant intera tions an easily be noted . Re-

46
sults show that most of the mass signals (peaks) remain un hanged independently

of the nature of the added salts. A typi al ES-MS spe trum of the omplete m/z re-

gion (m and z are the mass and harge of an ion, respe tively) is shown in Figure 3.3.

100
-
A

80
Relative Abundance

60

- + -
(2A + H )
40

20

- -
(2A +Na+) - + -
- + - - (2A +K )
(A + K + Cl )
0
200 300 400 500 600 700
m/z

Figure 3.3: ES-MS spe trum of 1 wt.% SL / DTAB mi ellar solution with 15mM NaCl; A− :
dode yl arboxylate anion (199 Da); C+ : dode yltrimethylammonium ation (228 Da).

Figure 3.4 represents the anion fragmentation patterns of the SL / DTAB atan-

ioni mixture upon the addition of various salts: for better visibility only a part

of the m/z region is presented. A loser look reveals that the position of the peak

+ −
representing the binding of sodium ions to the arboxylate (2A
−
+ Na ) remained

un hanged. Also the size of the peak was omparable in all spe tra with the ex-

eption of spe trum (A). When LiCl was added, two same-sized peaks appeared,

one representing the binding of sodium, the other of lithium ions to the arboxy-

+
late anion. This suggests that Li was able to ome loser to the vesi ular surfa e,

repla ing a part of the sodium ions at the interfa e. The exa tly opposite was ob-

served in the ase of CsCl (Figure 3.4D). The latter suggests a very small o urren e

+ −
of esium ions at the vesi le interfa e (very small (2A
−
+ Cs ) peak). A general

ordering of the ations ould be determined from the ES-MS spe tra, with lithium

showing the greatest anity for the anioni group and the other ations following:

+
Li >Na+ >K+ >Cs+ . As observed previously, this order exhibited a reversal if the

47
surfa tant mixture ontained a sulphate headgroup (SDS).

- + -
(2A + H )
- -
100
+
100
(2A + H )

80
80

Relative Abundance
Relative Abundance

60
60

40
40

- -
- - 20 (2A + Na+) - -
20 +
(2A + Na+) (2A + C )
- - - + -
(2A + Li+) - - -
(2A + C ) -
(A + C + Cl ) + - -
+
(A + C + Cl )
0
A 0
B
400 500 600 700 400 500 600
m/z m/z

- + -
- - (2A + H )
(2A + H+) 100
100

80
80
Relative Abundance

Relative Abundance

60 60

40 40

- + - - + -
20 - - - + - 20
(2A + Na ) (2A + C )
+
(2A + Na ) (2A + C )
- - - - - -
- -(A + C+ + Cl ) +
(A + C + Cl ) -
C
+
(2A + K ) + -

0 0
D (2A + Cs )
400 500 600 700 400 500 600
m/z m/z

Figure 3.4: Ion binding as determined by ES-MS: addition of 15mM of (A) LiCl, (B) NaCl, (C)
KCl and (D) CsCl to a SL / DTAB mi ellar solution; A− : dode yl arboxylate anion (199 Da);
C+ : dode yltrimethylammonium ation (228 Da).

Results from rheology experiments performed on atanioni solutions with vari-

ous salts showed that the vis osity de reases with applied strain rate ( . f. Figure

3.5). This behavior is ommon for solutions ontaining large non-spheri al mole ules,

whi h tumble at random under low shear, but align themselves in the dire tion of

3
in reasing shear and produ e less resistan e as the shear rate is in reased . This

behavior pointed to the presen e of rod-like mi elles in the solutions, whi h was

onrmed also by mi ros opy (see ahead). No hange in the rheologi al behavior of

the samples was observed however as the nature of the salt is varied. Probably, the

hange in the overall on entration of elongated mi elles present in the solution was

48
too low for su h dete tion to be possible with our equipment.

3
/ Pas

0
0 50 100 150 200 250 300
-1
/ s

Figure 3.5: Vis osity of 1 wt.% SL / DTAB mi ellar solution upon the addition of 15 mM LiCl
(△), NaCl (N), KCl (), CsCl () as a fun tion of shear rate.

Homogeneous samples exhibiting a bluish olor typi al for solutions ontaining

vesi les were further investigated by ryo- and FF-TEM. The images onrmed an

in rease in the size of the aggregates and high polydispersity ( ommon to non-

equimolar atanioni systems). Figure 3.6(A) shows a pi ture similar to what we

have reported in the previous hapter. Not only the SDS / DTAB system, but also

the SL / DTAB system onrmed the presen e of long ribbon-like mi elles unravel-

ing and forming sheets. It seems that the addition of salts produ ed a similar ee t

in both systems, hinting at a general me hanism of mi elle-to-vesi le transitions in

atanioni systems. Figure 3.6(B) shows many half- losed and some already fully-

losed vesi les.

Cryo-TEM images of the SDS / DTAB system onrm the high polydispersity

in su h mixtures. Vesi les of various sizes were seen to exist in equilibrium with

ribbon-like mi elles.The in rease in measured RH was therefore most likely due to

49
 A B

Figure 3.6: Cryo-TEM photographs of a SL/DTAB aqueous solution at the molar ratio of
approximately 2/1 and a total surfa tant on entration of 1wt.% upon the addition of 20 mM of
NaCl. The arrows in (A) show the presen e of long ribbon-like mi elles unravelling and forming
sheets. These sheets then urve and form spheri al aggregates (B).

the ombined ee ts: formation of vesi les as well as the elongation of rods. The

206,207
latter ee t is well known in literature .

FF-TEM, be ause of the advantageous fra ture ourse within hydrophobi zones,

provides us with the information regarding the membrane surfa e of the vesi les.

A pe uliar dieren e ould be observed. The addition of sodium ions to the two

systems assisted in the formation of very dierent vesi les. While the sulfate group

ontaining surfa tant mixture formed polyhedral (fa eted) vesi les with very sti

looking membranes, the arbooxylate system exhibited the presen e of individual

unilamellar vesi les with ompletely smooth membranes (Figure 3.7). Fa eted vesi-

les are present when the tails of the surfa tants are rather sti due to rystallization.

This is ommonly seen in atanioni systems. This stiness may also des ribe the

hydration of the surfa tant mole ule.

Similar behavior ould be found within one system, when the ee t of various

ations on the membrane stiness was ompared. The stronger preferential binding

of ions ould also be observed by the distortion of the SDS / DTAB vesi le membrane

+ +
as it be ame dehydrated when Cs and Li ions were ompared (see Figure 3.8).

50
 200nm

Figure 3.7: FF-TEM photographs representing the ee t of 30 mM NaCl on the SL / DTAB (left)
and the SDS / DTAB (right) mi ellar solution. The left image presents vesi les with ompletely
smooth membranes. When the arbooxylate group is ex hanged for a sulphate group, the vesi les
be ome sti-looking and fa eted.

3.4.3 Co-ion Ee ts

The binding of ounterions to a mixed atanioni mi elle/solution interfa e depends

1
on the surfa tant molar ratio , i.e. the harge of the aggregates. Zeta potential

measurements of the atanioni solutions with and without salt onrmed the pres-

en e of negatively harged aggregates. Therefore, no anion spe i ity was to be

expe ted. Samples were nevertheless he ked also in the presen e of various sodium

salts (NaCl, NaBr, Na2 CO3 , NaSCN, NaOA , NaNO3 ). All salts proved to have a

signi ant inuen e on the growth of the mi elles, however, no signi ant spe i ity

ould be found for the dierent anions (the urves overlap - . f. Figure 3.9).

3.4.4 Nonioni Ee ts

Two non ioni additives, glu ose and urea, having respe tively salting-out and

salting-in ee ts, were also added to the same mixed mi elles solutions. No ee t

whatsoever on the growth of the measured hydrodynami radius ould be noti ed.

Even though glu ose and urea were added at a on entration as high as 1 mol/L,

no in rease in the hydrodynami radius of the aggregates was found.

51
Figure 3.8: FF-TEM images the ee t of 45 mM CsCl (left) and LiCl (right) on the referen e
SDS / DTAB mi ellar solution. The bars represent 100 nm in both ases.

3.4.5 Ee ts of `Hydrophobi Ions'

Above observations on alkali salts have shown that all alkali ations aused an in-

rease in the measured hydrodynami radii. At high ele trolyte on entrations the

ele trostati repulsions are s reened and the vesi les aggregate. This ee t has not

been observed when so alled `hydrophobi ' ions are present. Tetramethylammo-

nium hloride (TMAC) and holine hloride (ChCl) were added to mixed mi ellar

solutions of SDS / DTAB and SL / DTAB. Results obtained by DLS show only a

slight in rease of the hydrodynami radius upon the addition of TMAC to a mi ellar

solutions (from 20 to 30 nm).

A larger dieren e in the ation anity for the aggregate surfa e ould be deter-

mined by ES-MS. Figure 3.10 shows anion fragmentation patterns upon the addition

of salt, tetramethylammonium hloride (for better visibility only a part of the m/z
+
region is presented). ES-MS spe tra hint that TMA was able to ome loser to

the vesi ular surfa e, repla ing a part of the sodium ions at the interfa e (Fig-

ure 3.10(A)). The exa t opposite was observed in the ase of arboxylates (Figure

52
 175
80
150

125
60

R [nm]
100
[nm]

H
H

40
75
R

50
20

25

0
0 10 20 30 40 50 0.00 0.01 0.02 0.03

c [mM] c [mM]

Figure 3.9: The ee t of various anions / sodium salts on the growth of the hydrodynami radii
RH of the atanioni aggregates in SL / DTAB (left) and SDS / DTAB (right) systems: NaCl
((•), NaSCN (), NaBr (), Na2 CO3 (△), NaOA (◦) and NaNO3 (N)

3.10(B)). The results obtained for holine hloride salts were omparable.

FF-TEM images of the SDS / DTAB solution showed the presen e of vesi les

at low TMAC on entrations ( .f. Figure 3.11, left), similarly to the ase of alkali

salts. However, the membranes of the vesi les were more exible and are therefore

more likely to fuse. Surprisingly, at higher on entration only small dis s and mi-

- + -
(2L + H )
(2DS- + K+)- 100
100 (2DS- + TMA+)-

80
80 - + -
(2L + Na )
Relative Abundance
Relative Abundance

(2DS- + Na+)- 60
60

40 (2DS- + H+)- 40 (2L- + K+)-

- + -
20
(L- + C+ + Cl - )- (2L + TMA )
20

0
A 0
B
500 600 700 800 400 500 600 700
m/z m/z

Figure 3.10: Ion binding as determined by ES-MS: addition of 40 mM of TMAC and to a (A)
SDS / DTAB and (B) SL / DTAB mi ellar solution. DS- : dode ylsulfate anion (265 Da); L- :
dode yl arboxylate anion (199 Da); K+ : dode yltrimethylammonium ation (228 Da).

53
 100 nm

Figure 3.11: FF-TEM photographs representing the ee t of 20 mM (left) and 40 mM (right)
TMAC on the referen e SDS/DTAB mi ellar solution.

elles ould be observed (Figure 3.11, right). In the ase of SL / DTAB, however,

no vesi les were observed. Small dis s and mi elles were present over the whole

on entration region. The same pi ture ould be found when 50 mM of ChCl was

added to SDS/DTAB and SL/DTAB.

3.5 Dis ussion

3.5.1 Aggregation Behavior of Catanioni Systems
The main thermodynami driving for e for the asso iation of a ationi and an an-

ioni surfa tant mixture is the release of ounterions from the aggregate surfa es.

This results in a large entropy in rease. For these systems a non-monotoni hange

1,3
in P is observed, with a pronoun ed maximum as the mixing ratio is varied . In

the system presented here vesi les ould only be formed when the anioni surfa tant

is in molar ex ess ompared to the ationi surfa tant. Therefore, only this ratio

was onsidered here.

The geometry of aggregates in olloidal systems is attributed to the pa king of the

amphiphili mole ules. The pa king parameter is dependent on the length and vol-

54
ume of the hydrophobi tail and the size of the hydrophobi head of the surfa tant

mole ule. These fa tors are often expressed in a pa king parameter P = v/(lmax a),
where v and lmax are the volume and length of the hydrophobi part, respe tively,

and a the area per mole ule at the interfa e. If the hydro arbon part of the surfa -

tant is kept onstant and only the headgroup is varied, then the dieren e in the

aggregation behavior an be attributed solely to the properties of the polar head-

group. Be ause a des ribes the ee tive headgroup size, whi h in the ase of ioni

surfa tants is largely determined by repulsive ele trostati for es, and not the ioni

radius, the hange of morphology in surfa tant systems is largely dependent on the

number of water mole ules surrounding the headgroups. It has been observed by

135
hemi al trapping method that in asso iation olloids, the hanges in the balan e

of for es ontrolling the aggregate stru ture are ree ted in the hanges in inter-

fa ial on entrations of water and other omponents. The ions, when added to a

mi ellar solution, an dehydrate the surfa tant headgroups. This auses a de rease

in the value a and onsequently an in rease of the stru tural pa king parameter

60,208
P. As a onsequen e, the riti al mi ellar on entration is redu ed and the

209
aggregate morphology hanged . The ee ts are aused by destru tion of the hy-

dration layer of the surfa tant, de reased ele trostati repulsions, and an in reased

60
ounterion binding . Consequently, the surfa tant monomers an be pa ked loser

together leading to a bilayer formation.

3.5.2 Counterion Properties

The ee t of salts on a harged system an dier mu h depending on the salting-in

107,115,210
or salting-out properties of their ions . Salting-out ions are usually small,

with relatively small polarizabilities. They have high ele tri elds at short distan es

211
and have tightly bound hydration water. NMR eviden e suggests that small ions

do not show spe i binding to mi elles and retain their mobility and their water

212
of hydration right up to the mi ellar `surfa e' although this result depend on the

type of surfa tants. Salting-in ions are usually large, with signi ant polarizabilities.

They have weak ele tri elds and a loose hydration sheath, whi h an be easily

removed. NMR proves that they perturb phospholipid (surfa tant) headgroups in a

55
 213
signi ant way, probably through ion pairing . Ea h salt is therefore expe ted to

have an individual inuen e on vesi le formation, whether it tends to adsorb at the

interfa e between mi elle and water or remains strongly hydrated in the solution.

3.5.3 Collins' `Law of Mat hing Water Anities'

101
Re ently, a simple `law of mat hing water anities' has been proposed . It is

based on the tenden y of oppositely harged ions to spontaneously asso iate in

aqueous solution. Only oppositely harged ions with mat hing absolute free energies

of hydration should spontaneously form inner sphere ion pairs. This is supposed

to be due to the fa t that the strength of intera tion between the ions and the

water mole ules is orrelated to the strength with whi h the ion intera ts with other

200
ions . Small osmotropi ions an ome lose together forming inner sphere ion

pairs without intermediate water mole ules. The same is supposed to be true for big

haotropi ions, whereas when a osmotropi ion approa hes a haotropi ounterion,

the ions should remain separated by at least one water mole ule. With this model

an impressive number of phenomena and properties an be des ribed.

3.5.4 Counterion Sele tivity of Alkyl Sulfates

In the present ase the headgroups in ex ess are alkylsulfates. In ontrast to double-

harged sulfate ions, su h headgroups have a more haotropi behavior. With this

assumption and a ording to Collins' on ept, alkylsulfates should ome in lose

onta t with haotropi ions like esium, whereas lithium ions remain further away.

+
Therefore it is expe ted that Cs s reen more e iently the negative harge ex ess

+
on the aggregates than Li , and this is pre isely what is observed, .f. Figure 3.2

(right). The same ordering of ations was found in systems ontaining pure SDS

214
mi elles and in the ase of another anioni surfa tant with a sulfate group, sodium

177
dode ylbenzenesulfonate . To make a more quantitative analysis we ompare the

salt on entrations to the ex ess on entration of SDS ( ompared to DTAB), whi h

is approximately 0.015 mol/L. From Figure 3.2 (right) it an be inferred that this

on entration roughly orresponds to the asymptoti limit for mi elle to vesi le

56
 + +
transition in the ase of K and Cs hlorides. Therefore it may be on luded

that these ations have a strong propensity to the sulfate groups and that sulfate

headgroup- ation pairs are formed; in this ase the ex ess negative harge of the

vesi les is neutralized. This nding is in agreement with the asso iation onstants

154
found for a similar surfa tant n-o tylbenzenesulfonate .

3.5.5 Counterion Sele tivity of Alkyl Carboxylates

We have seen that with the sulfate group the binding in reases with the de reasing

size of the hydrated alkali metal ions. The order is reversed when the headgroup

is ex hanged for a arboxylate. The arboxylate headgroup exhibits water-ordering

properties and the addition of an alkyl hain should not hange the kosmotropi

behavior of the headgroup. A ording to Collins' on ept, alkyl arboxylates should

then ome in lose onta t with kosmotropi ions like lithium, making it more possi-

ble to form inner-sphere ion pairs, whereas esium ions remain further away. There-

+
fore, it is expe ted that Li s reens more e iently the negative harge ex ess on

+
the aggregates than Cs , and this is pre isely what is observed in Figure 3.2 (left).

The same order of ounterion-binding to arboxylate headgroups that was found in

215218
our atanioni surfa tant system are found in polyele trolyte solutions , when

219,220
measuring the ation anity to ion-ex hange resins , in studies of membrane

220 221
potentials , of ele trophoreti mobility of olloids , and of ion-transport phenom-

222
ena . Our results are also in agreement with measurements of ounterion binding

81,212,223,224 222,223,225
to soap mi elles and long- hain fatty a ids . Re ent MD simu-

lations have shown that the arboxylate groups dominate the ounterion behavior

210
even in omplex systems su h as proteins .

3.5.6 Alkyl Sulfates vs. Alkyl Carboxylates

Similar ounterion spe i ity was observed in the ase of polyele trolytes. As in

the ase of surfa tants, the spe i -ion ee ts do not depend only on the individual

properties of the parti ipating ioni group and its ounterion ( harge, size, harge

distribution and polarizability), but also on the overall harge of the polyion, as well

57
as on the possible ooperating binding sites whi h might inuen e the binding site.

Furthermore, even at high dilution a substantial number of ounterions is for ed

226,227
into lose proximity to the polyion by the long-range ele trostati for es , so

that there always exist a large number of ion pairs for whi h solvation ee ts should

217,218
be observable. Strauss used the dilatometri method to measure the volume

hanges that o urr when polyele trolyte solutions are mixed with solutions ontain-

ing dierent spe i ally intera ting ounterions. Polyele trolytes ontaining sulfate

and arboxylate headgroups exhibit opposite series of ounterion binding, similar to

the series observed in our experiments on surfa tant mi elles. Dilatometri results

learly show spe i ities depending on both the polyion and on the metal ion whi h

would not be expe ted on the basis of long-range ele trostati density of the polyion.

The ee ts of the latter are governed predominantly by the linear harge density of

the polyion and the linear harge densities of the studied sulfonates and a rylates are

the same. It follows that the intera tions giving rise to the observed volume hanges

involves spe i sites on the polyions. Similar results of alkali ations to polya ry-

215,216,228230
lates and polysulfonates have been dis ussed also by other authors . The

reversal of the ation series in the presen e of polya rylates was attributed to a

ompetition between water of hydration and the anion for a given ation, whi h is

related to the harge distributions, polarizabilities, and the ee tive eld strengths

221,222
of the ions . The on ept was however not studied thoroughly.

The reversion of series was observed also when measuring the ation anity to ion-

219,220,231 222 232

ex hange resins , ion-transport phenomena , swelling of hydrogels and

212,215,223,224,233,234
ounter-ion binding to long- hain fatty a ids .

3.5.7 Mole ular Dynami s Simulations

235
Also MD simulations show similar results. Chialvo and Simonson modeled a

+
short- hain polystyrenesulfonate and found the intera tion with Li too weak to

230
ause desolvation. Similarly, Lipar et al. reported the binding to polyanethole-

+
sulfoni a id to be strongest for Cs . Re ently, an extended omputational study

of pairing of sodium and potassium with a broad set of biologi ally relevant anions

201,236
was des ribed . The monovalent sulfate and the arboxylate groups and their

58
pairing with sodium vs. potassium were examined. The stru tures of the four in-

vestigated onta t ion pairs, together with the ation - anion oxygen distan es are

shown in Figure 3.12.

Figure 3.12: Geometries of the onta t ion pairs of a) sodium-a etate, b) potassium-a etate, )
sodium-methylsulfate, and d) potassium-methylsulfate. The ations bind bidentally to two anioni
oxygens. Reprodu ed from Vla hy et al. 236 .

A ombination of ab initio al ulations with a polarizable ontinuum solvent

model and mole ular dynami s simulations were used to quantify the relative ation-

anion asso iation free energies, i.e., the values of ∆∆G onne ted with repla ing

236
potassium with sodium in a onta t ion pair with a etate or methylsulfate anion .

−
−
These two free energy dieren es for CH3 COO and CH3 SO4 are −2.50 and +0.37
k al/mol, respe tively. We see that while a etate strongly prefers sodium over potas-

sium (by about 2.5 k al/mol), methylsulfate weakly (by roughly 0.4 k al/mol) prefers

potassium over sodium. These ndings are onsistent with our experimental results

59
 101
and also with the empiri al Law of mat hing water anities .

3.5.8 Generalization of the Con ept: Hofmeister Series of

Headgroups
In the pre eding paragraphs a omparison of two headgroups is given. However, more

headgroups are of interest in olloidal hemistry and biology, espe ially sulfonates

and phosphates. Con erning hydrogen phosphate, both the harge density as well

201,214,237,238
as several experimental results suggest that it an be lassied between

the arboxylate and the sulfonate headgroup. Following omputational as well as

experimental results, we propose a way to lassify headgroups in a Hofmeister like

series, ordering them from osmotropi to haotropi headgroups: - arboxylate, -

hydrogen phosphate, -sulfonate, -sulfate. (presented in Figure 3.13). With this

series many spe i -ion phenomena in olloidal systems an now be explained.

3.5.9 The Anioni Ee t

The addition of sodium salts proved to have a signi ant ee t on the growth of the

mi elles (Figure 3.9). This in rease of parti le size is mainly due to a de rease of

the head group repulsions be ause of ele trostati s reening. Sin e the mi elles are

very probably negatively harged, the anions will not ome into lose onta t with

the mi ellar surfa es and the onset of vesi le formation o urs at approximately the

same on entration for all sodium salts. The small dieren es in radius size observed

are likely to be due to the dieren es in the hydration of the anions; in the ase of

−
strongly hydrated Cl larger vesi les were observed than in the presen e of other

less hydrated ions. A similar ee t was previously observed in studies of negatively

239
harged phospholipid vesi les .

3.5.10 The Non-Ioni Ee t

The fa t that the addition of glu ose and urea had no ee t on the growth of the

mi elles (no mi elle to vesi le transition was observed) suggests that ele trostati

intera tion is ne essary and is the rst order ee t. Hydration numbers show that

60
Figure 3.13: Ordering of anioni surfa tant headgroups and the respe tive ounterions regarding
their apabilities to form lose pairs. The green arrows represent strong intera tions ( lose ion
pairs).

glu ose dehydrates the headgroups as well as the salts (the hydration numbers for

+ 116 240
Cs and glu ose are 4.3 and 3.5 respe tively. Even mu h higher on entra-

tions of glu ose and urea (up to 1M) had no ee t on the growth of the surfa tant

aggregates. This onrms that dehydration of the headgroups alone is not su-

ient to trigger the mi elle to vesi le transition. The reason must be sought in the

ele trostati s. Even if these un harged mole ules modied the surrounding water

stru ture, this ee t is not strong enough to signi antly hange the urvature via

adsorption to the headgroups. If the mi elles were onsisted of just one type of

surfa tant (one harge), then the addition of salt would s reen the repulsion be-

tween the headgroups, ausing the surfa tant mole ules to arrange in a bilayer. The

opposite ase would be if the surfa tants were mixed in equimolar amounts. Then

61
the salts would s reen the attra tion between them, most likely ausing the distan e

between the headgroups to in rease. Sin e our mixed mi elles ontain an ex ess of

the anioni surfa tant, we an assume an asymmetri distribution of harges (as was

also proposed in the pre eding hapter). Therefore the addition of salt should have

a similar ee t as in the ase of `single- harged' mi elles.

3.5.11 Ee ts of `Hydrophobi Ions'

Ammonium ations have a salting-in hara ter and enhan e the solubility of other-

wise poorly water-soluble ompounds. They are well-known as phase transfer ata-

lysts. In water the ioni hara ter of these ions is dominant, whereas in hydrophobi

241
environments the organi hara ter prevails . However, there is a signi ant dif-

feren e between the properties of ammonium and tetramethylammonium ions. The

ammonium ation behaves mu h like a big alkali ation; it is weakly hydrated, its

+
hydration sphere being similar to the hydration sphere of the Cs ion.

By ontrast, the tetramethylammonium ation is an intermediate ase between `sim-

242
ple' ions and hydrotropes su h as xylene sulfate . It seems plausible to assume

that this `hydrophobi ' ation an penetrate into the mi ellar surfa e mu h as an-

243246
ions like tosylate and sali ylate in the ase of ationi surfa tants. If this is

true, the tetramethylammonium ion an a t as a o-surfa tant, perhaps even par-

tially repla ing the ationi surfa tant and perturbing lamellar stru tures - a feature

247
that is well known from hydrotropes . Interestingly, ES-MS suggest that a strong

binding of the hydrophobi ions to the mixed mi elles is present only in the ase of

sulfates, but not also when a arboxylate surfa tant is present. The initial formation

of the vesi les, observed in the SDS / DTAB system at low salt on entrations ould

be solely due to the in reased ioni strength. However, this does not explain the

observed hanges in membrane exibility. The destru tion of vesi les at higher salt

on entrations suggests the in orporation of the hydrophobi ion into the membrane

and a hange in the pa king of the surfa tants.

Although the information obtained from ele troni mi ros opy is more or less the

same for both systems at high salt on entrations, the reason for the presen e of

only mi elles and small dis s are most likely dierent. In the ase of SDS, the

62
strong binding of the `hydrophobi ions' should ause a disruption of the membrane,

destroying the large vesi ular stru tures. On the other hand, it is likely that the

+
TMA ion does not ome into lose onta t to the SL / DTAB mi ellar surfa e.

Therefore, it assists aggregate growth only by hanging the properties in the bulk.

As ammonium ions have salting-in properties, they do not indu e the dehydration

of the ioni surfa tant headgroups and therefore also not assist in vesi le formation.

Further dis ussion on alkyl trimethylammonium groups an be found in Chapter 4.

3.6 Con lusions

Through the addition of salts a transition from rod-like mi elles to vesi les was

observed in aqueous solutions omposed of DTAB and an ex ess of either SL or

SDS. Whereas no anion spe i ity for the added salts appeared in the formation

of vesi les, the nature of the ation was found to inuen e strongly the riti al salt

on entrations around whi h mi elles turn to vesi les. In the present ase of neg-

atively harged vesi les, ion-spe i ee ts were expe ted for ations, sin e they

a umulate in high on entration in the vi inity of the vesi le. The observed ation

spe i ity followed the lassi al Hofmeister series for ation adsorption to sulfate

headgroups. When alkyl arboxylates were present in solution, the ations followed

a reversed Hofmeister series. The ion spe i ity ould be qualitatively explained

a ording to Collins' on ept of mat hing water anities. To this purpose, the

headgroup of an alkylsulfate had to be regarded as a haotrope and the alkyl ar-

boxylate as a kosmotrope. The morphologies observed during the mi elle-to-vesi le

transition are analogous to those presented in the pre eding hapter, hinting at a

general me hanism ommon to atanioni systems.

63
64
Chapter 4

Anion Spe i ity Inuen ing

Morphology in Catanioni Surfa tant
Mixtures with an Ex ess of Cationi
Surfa tant

4.1 Abstra t

In the previous hapter we reported on an ion spe i mi elle-to-vesi le transition,

when salts were added to a atanioni mi ellar solution omposed of sodium do-

de yl arboxylate (sodium laureate, SL) and dode yltrimethylammonium bromide

(DTAB), with an ex ess of SL. In the present hapter, we illustrate the ion spe i-

 ity, when DTAB was in ex ess in the same system. In this ase, no transition to

vesi les was observed, but an elongation of mi elles upon salt addition. The oun-

terion binding and in rease in aggregate size was monitored by mass spe trometry,

dynami light s attering measurements, and ryo-transmission mi ros opy. The

me hanism was argued by employing the ability of the ounterion to dehydrate the

surfa tant headgroup. This theory was onrmed in phase with Collins' on ept of

mat hing water anities.

65
4.2 Introdu tion

Mixtures of surfa tants show enhan ed performan e in te hni al pro esses (e. g.

detergen y, tertiary oil re overy, drug arrier systems, otation), when ompared

to pure surfa tant systems. Surfa tant mixtures for spe i appli ations are of-

ten hosen based on empiri al eviden e and experien e. However, to optimize the

appli ations it is important to have a general understanding of the interplay of in-

tera tions between the surfa tants in a mixed system and of the fa tors inuen ing

the phase diagram. Therefore, it is of interest to study the self-aggregation and

mi ellization of su h mixtures.

When oppositely harged surfa tants are mixed, new properties appear. Aqueous

atanioni mixtures exhibit a wide range of unique properties that arise from the

strong ele trostati intera tions between the oppositely harged heads. They exhibit

low riti al mi elle on entration (CMC) values and a non-monotoni hange in the

2
surfa tant pa king parameter (P ) as the mixing ratio is varied . For this reason a

large number of aggregate stru tures su h as spheri al and rod-like mi elles, vesi les,

lamellar phases, and pre ipitate have all been observed depending on the on en-

tration, the size of the hain length or nature of the polar heads, and the ratios

24,26,48,5357
of the surfa tants in solutions . One advantage of atanioni systems as

ompared with more robust genuinely double hained surfa tants are their greater

153 47
sensitivity to parameters su h as temperature or the presen e of salts .

The ee t of salt type on various physi o- hemi al properties of a system was rst

observed over one hundred years ago by Franz Hofmeister who dis overed the de-

114
penden e of protein solubility on the type of added inorgani ele trolyte . Sin e

then, a wide range of ion-spe i phenomena in biology, pharma y, and hemistry

was observed. They have been re ently reviewed in a spe ial issue of `Current Opin-

115
ion in Colloids and Interfa e S ien e' . Extensive studies have shown that the

ounterion has a strong ee t on the thermodynami s and aggregation properties

248250
of surfa tants . Salt ee ts on the m , mi ellar size, and degree of disso i-

251
ation for a given surfa tant may follow a lyotropi (or Hofmeister) series . An

ion's position in the lyotropi series an be orrelated with its harge and hydrated

66
radius. Depending on the harge density of the anion, it an intera t more or less

strongly with the ationi headgroups of mi ellar surfa e. Su h a `binding' de reases

the ele trostati repulsion between the surfa tant headgroups and hen e favors ag-

gregation. This lowers for instan e the CMC and the degree of ionization of the

2− −
mi elles. A typi al Hofmeister series for anions is as follows: SO4 < C2 H3 O2 <
− − −
Cl
−
< NO3 < −
Br < ClO4 < I
−
< CNS
− −
(the positions of the NO3 and Br ions

are often swit hed in the lytropi series).

Single-tailed surfa tants usually form globular mi elles in aqueous solution above

4
their CMC . An in rease in surfa tant on entration may indu e the formation

252,253
of worm-like mi elles . Similarly, addition of organi and inorgani ounteri-

206,252254 255
ons , un harged ompounds like aromati hydro arbons , or an oppositely

26,158
harged surfa tant an transform spheri al mi elles into worm-like mi elles.

In the previous hapter, we have reported a way to lassify headgroups in a Hofmeis-

ter like series. We also were able to explain how salt-indu ed mi elle-to-vesi le

transitions in the anioni -ri h regions of the phase diagrams in atanioni systems

depend on both ion and headgroup spe i ities. In the present hapter, this study

is extended to the ationi -ri h region. We fo us on the inuen e of the ounterion

identity on the aggregation behavior of non-equimolar mixed surfa tant solutions,

omposed of sodium dode anoate (SL) and dode yltrimethylammonium bromide

(DTAB) with an ex ess of DTAB. The ee t of dierent anions and ations on the

mi ellar solutions will be shown by means of phase diagrams, ryo-TEM, mass spe -

trometry and dynami light s attering. Collins' on ept of mat hing water ani-

101
ties will be shown to be also very valuable for the omprehension of the found

series of salt sensitivity. And nally, the aggregation behavior will be ompared to

the one found in the anioni -ri h region of the orresponding phase diagram.

4.3 Experimental Pro edures

Materials The surfa tants, sodium dode anoate (SL; Sigma, Germany; grade: 99-

100%) and dode yltrimethylammonium bromide (DTAB; Mer k, Germany; assay >
99%) were used as re eived. All sodium and hloride salts used in the experiments

67
were supplied by Mer k, Germany. They were also used as re eived without further

puri ation. Millipore water was used as solvent in all ases.

Phase Diagrams Surfa tant sto k solutions were prepared by dissolving weighed

amounts of dried substan es in Millipore water. The solutions were then left for

24 hours to equilibrate at 25◦ C. The atanioni solutions were prepared by mixing

the surfa tant sto k solutions to obtain a xed anioni / ationi surfa tant mass

ratio of 30 / 70. The starting ratio was determined from phase diagrams. The total

surfa tant on entration was kept at 1 wt.% at all times. Salts were added to the

mi ellar solution at in reasing on entrations. The solutions were then stirred and

left to equilibrate for a day at 25◦ C before making measurements.

Other Methods Ele trospray mass spe trometry, ryo-transmission ele tron mi-

ros opy and dynami light s attering were performed as des ribed in the previous

hapters.

4.4 Results and Dis ussion

4.4.1 Ion Binding in Catanioni Surfa tant Mixtures
In the studied atanioni system, the ationi surfa tant DTAB was in ex ess. There-

fore, it was expe ted that the variation of the anions will produ es a larger ee t as

the variation of the type of ations. Figure 4.1 shows ele trospray ionization mass

spe tra of the SL / DTAB atanioni solutions (mass ratio 30 / 70) upon the addi-

tion of dierent salts. Although in this method the ionization pro ess takes pla e

in the gas phase, the hemi al nature of surfa tant monomers and of simple ions is

the same as in the liquid phase. Therefore, ounterion anities for the surfa tant

204,205
an easily be noted . Be ause our surfa tant mixtures already ontained oun-

terions, we added the same on entration of salts. In this way, we ould observe the

ompetition of the ounterions for the surfa e of the surfa tant aggregate.

+
Figure 4.1 represents the DTA fragmentation patterns upon the addition of

68
 100 + 100 +
K K

Relative Abundance 80 80

Relative Abundance
60 60

+ - +
(2K + SCN )

40 40 + - +
(2K + Br )

+ - +
20 (2K + Br ) 20

+ - +
+
(2K + A )
- + (2K + A )

A B
0 0
100 200 300 400 500 600 700 100 200 300 400 500 600 700

100 m/z m/z

+
K
Relative Abundance

80 80

60 60

Relative Abundance
+ - +
(2K + Br )
+ - +
(2K + Br )
40 40

+ - +
20 (2K + NO3 ) 20

+ - +
(2K + A ) + - +
(2K + A )
D
C
0 0
100 200 300 400 500 600 700 100 200 300 400 500 600 700

K
+
m/z m/z

Figure 4.1: Ion binding as determined by ES-MS: addition of 15mM of (A) NaSCN, (B) NaBr,
(C) NaNO3 and (D) Na2 SO4 to a SL / DTAB mi ellar solution; A− : dode yl arboxylate anion
(199 Da); K+ : dode yltrimethylammonium ation (228 Da); the peaks between 250 and 300 (m/z)
are solvent related.

various salts: for better visibility only a part of the m/z region is presented. It

turns out that most of the mass signals (peaks) remain un hanged, independently

of the nature of the added salts.

A loser look revealed that the position of the peak representing formation of ion

+ − +
pairs between the oppositely harged surfa tant ions (2K + A ) remained un-

hanged. Also the size of the peak was omparable in all spe tra. Similarly, the

peak orresponding to the binding of bromide ions to the dode yl trimethylammo-

+ − +
nium ation (2K + Br ) was present in all spe tra, regardless of the nature of

the added salt. The height of this peak, however, was less pronoun ed in spe trum

(A). When omparing the binding of anions to the dode yl trimethylammonium

−
ation, we observed that only the SCN ex hange the bromide anions at the mi-

− −
ellar surfa e. NO3 and Cl showed approximately the same anity for the alkyl

trimethylammonium group, whereas in spe trum (D), when Na2 SO4 was added to

69
the atanioni mixture, no peak representing the binding of sulfate ions was visible.

These results suggest that the strongly hydrated sulfate anion did not ome lose

to the mi ellar surfa e, whereas the loosely hydrated thio yanate anion was able

to ome loser to the mi ellar surfa e, repla ing a part of the bromide ions at the

interfa e. A general ordering of the ations ould be determined from the ES-MS

spe tra, with thio yanate showing the greatest anity for the anioni group and the

− 2−
other ations following: SCN
−
> Br
−
> NO3 > Cl
−
> CH3 COO
−
> SO4 . The

variation of the ation of the salt produ ed no ee t on the ation fragmentation

patterns, as an be expe ted in a system with an ex ess of ationi surfa tant. The

m/z peaks remained un hanged for all added hloride salts.

4.4.2 Phase Behavior upon Salt Addition

4% SL
0.00
1.00

0.25
0.75
I_

0.50 equimolarity
0.50

0.75
0.25

I+
1.00
0.00
water 0.00 0.25 0.50 0.75 1.00 4% DTAB

Figure 4.2: S hemati ternary phase diagram of the SL / DTAB system at 25◦ C (the arrow
represents our starting solution (referen e sample; mass ratio 30 / 70).

The inuen e of salts on the SL / DTAB atanioni mixture was further followed

by observing the phase behavior; the starting phase point in the phase diagram be-

ing a mi ellar solution, . f. diagram shown in Figure 4.2. Dierent salts ae ted

the system in various ways. While the addition of some salts produ ed no visible

ee t, others indu ed an easily observable aggregation (formation of a bluish olor

70
or turbidity). As the salt on entration was in reased the aggregation be ame more

pronoun ed. The ee ts are reported in Table 4.1.

Salt 25 mM 50 mM

NaSCN bluish/turbid turbid

NaBr bluish/turbid turbid

NaNO3 bluish turbid

NaOA lear lear

Na2 SO4 lear lear

NaCl lear lear/bluish

LiCl lear lear/bluish

KCl lear lear/bluish

CsCl lear lear/bluish

Choline Cl lear lear

Table 4.1: Visual observations of the ee t of various salts on the SL / DTAB (mass ratio: 30 /
70) atanioni solution. The referen e sample was a lear homogeneous solution.

The aggregation was he ked by light s attering; however problems were reported

by the apparatus due to the high s attering intensity and the high polydispersity of

the solutions. Despite that we are able to onrm an in rease in the hydrodynami

radius of the parti les in a ordan e with our visual observations ( . f. Figure 4.3).

We emphasize that the absolute values of the hydrodynami radii should be taken

with a grain of salt, be ause the aggregates formed in solutions are rods (as will be

shown with ryo-TEM further on), and the CONTIN software used by the Zeta-

sizer 3000 al ulates the radius of spheri al aggregates. However, the trend in the

in rease of RH is important. The degree of mi ellar growth varied in exa tly the

2− −
inverse order of the hydrated radius of the ounterion: SO4 ≈ C2 H3 O2 < Cl
−
<
−
NO3 < Br
−
< −
CNS .

−
Salts ontaining big anions having a weakly distributed harge (CNS ) indu ed

2−
more e iently the mi ellar growth than the divalent sulphate (SO4 ). Apparently

71
 130
120
110
100
90
80
70
[nm]

60
H

50
R

40
30
20
10
0
0 20 40 60 80 100 120 140 160

c [mM]
salt

Figure 4.3: The ee t of various ations / hloride salts on the growth of the hydrodynami
radii RH of the atanioni aggregates in SL / DTAB systems: NaOA (N), Na2 SO4 (), NaCl (◦),
NaNO3 (), NaBr (•) and NaSCN (△).

the large hydrated radius of the sulfate ion hinders its ability to bind to the mi elle

ontaining an ex ess of DTAB.

4.4.3 Anion Spe i ity in Physi o-Chemi al Properties of Alkyl-

trimethylammonium Systems
The ompetition of various anions for the binding sites on the surfa e of CTAB mi-

256
elles was he ked by Larsen and Magid . They found a strong binding of nitrate

to the mi elle, displa ing bromide. The positions of bromide and nitrate ions are

257
often ex hanged in the lyotropi series. As an example, Cohen and Vassiliades

− −
found that NO3 was less ee tive than Br at lowering the CMC of CTAB. The

lyotropi series was observed also when mi ellization of alkyltrimethylammonium

halides, heats of ounterion binding, surfa e tension, and the thermodynami and

72
 256,258261
mi roenvironmental properties of the mi elles were ompared . The on-

trolling fa tor in this ion-spe i ity seems to be the distan e of losest approa h of

the ion to the mi elle.

4.4.4 Inuen e of Salt on the Aggregation Behavior of Sur-

fa tants
The geometry of aggregates in olloidal systems is attributed to the pa king of

the amphiphili mole ules. The fa tors governing the shape of the aggregates are

expressed in its simplest form by a pa king parameter P = v/(lmax a), whi h is

dependent on the length (lmax ) and volume (v ) of the hydrophobi tail and the ef-

fe tive size of the hydrophili headgroup (a). The area per mole ule at the interfa e

depends on the hydration of the surfa tant headgroup, whi h in turn depends on

the ion harge density and the distan e to the small ounterions. Furthermore, it

an be inuen ed by the ioni strength of the solution. When ions ome lose to the

headgroups, the harges are more or less neutralized the value a (area per mole ule

at the interfa e) de reases, and onsequently the stru tural pa king parameter P in-

60
reases . Therefore, a de rease of the riti al mi ellar on entration and a hange

60,208,209
in the aggregate morphology an be observed .

As was mentioned above, the system under onsideration has an ex ess of DTAB.

Previous studies have shown that the self-aggregation behavior of alkyl tetramethy-

256
lammonium surfa tants is relatively independent of the hydro arbon hain length .

The phase behavior of DTAB (C12 ) and CTAB (C16 ) is therefore omparable. The

formation of rodlike mi elles in CTAB systems at high on entrations is widely

256,262266
known . Alkylammonium halides have also been reported to exhibit a tran-

sition from spheri al to rod-like mi elle shape with in reasing on entration of added

salt. At lower ioni strength only spheri al mi elles are formed, while at salt on-

entrations higher than a ertain threshold, larger rod-like mi elles are formed in

equilibrium with the spheri al mi elles. The length of the rod-like mi elle in reases

253,267278 277
strongly with in reasing ioni strength on entration . Ozeki, et al. re-

ported that the sphere-rod transitions of mi elles took pla e in aqueous NaBr solu-

73
tions of DTAB when the salt on entration ex eeded 1.8 M, whereas measurements

of Sudan red B in the same system indi ated that the minimum NaBr on entration

required to indu e the sphere-rod transition was 1.0-1.5 M. This may be due to a

de rease in intrami ellar repulsion by ele tri al shielding due to ioni atmosphere.

At higher salt on entrations, there is a dehydration of the spheri al mi elles due to

the salting-out ee t of NaBr.

By adding an anioni surfa tant, we lower the harge density of the previously

Figure 4.4: Left: spheri al and small rod-like mi elles in the SL / DTAB referen e solution
(before the salt was added); right: signi ant growth and networking of rods upon the addition of
salt (25 mM NaBr).

ationi mi elles. In this way, we also lower the on entration of salts ne essary

to produ e ylindri al mi elles. Similar ee ts are observed with the addition of

279281
anioni hydrotropes to ationi surfa tants . Hydrotropes bind strongly to op-

positely harged surfa tant ions and redu e the headgroup area of the surfa tant

by redu ing the headgroup repulsions. Thus, they are ee tive at promoting the

elongated mi elle formation.

The elongation of ylindri al mi elles was studied by ryo-transmission ele tron

mi ros opy. Figure 4.4 shows that our initial mi ellar solution (referen e sample)

exhibits the presen e of spheri al mi elles, as well as some short rods. An elongation

of the ylindri al mi elles was observed as salts were added to the solution. These

74
Figure 4.5: Cryo-TEM images representing the ylindri al growth as a fun tion of anion type;
the ee t of 25 mM NaNO3 (left) and NaBr (right).

mi elles were quite exible; small loops were observed, and the long mi elles formed

networks. This aused an in rease in the vis osity (observed at the preparation of

the solutions) and an in rease of the turbidity of the solutions. However, the growth

and the on entration in rease of the rods was salt spe i (Figure 4.5). The addi-

tion of NaBr produ ed a higher on entration of longer rods than the addition of

NaNO3 . These then started networking together and forming a net-like stru ture.

Surprisingly, we ould not see any formation of vesi les. Upon the addition of salts,

the system transformed from spheri al mi elles and small rods to long ylindri al

mi elles, and eventually to pre ipitation as the salt on entration was in reased fur-

ther. On the other hand, we have observed the transition from rod-like mi elles to

vesi les in the anioni -ri h region of the phase diagram of this sample (when SL was

282
in ex ess). A similar system to ours was studied by Sierra et al. . They inves-

tigated the phase behavior of atanioni mixtures omposed of DTAB and sodium

unde anoate. They found the presen e of spheri al mi elles on the anioni ri h side

of the phase diagrams and short rods on the ationi ri h side. The rods elongated

and aggregated as the omposition neared equimolarity. No hexagonal mesophase

was dete ted and the phase separation at equimolarity was pre eded by the forma-

tion of bundles of rod-like mi elles. The entanglement of long thread-like mi elles

75
 283
in CTAB mi elles upon salt addition was observed also by Aswal et al. .

4.4.5 Explaining Counterion Spe i ity in Surfa tant Sys-

tems
Sin e the ationi surfa tant was in ex ess in the atanioni system, and also in

the mixed mi elles, it is reasonable to suppose that anions were in average loser to

the mi ellar surfa e than ations. Consequently, the ee t of anion variation was

more pronoun ed than the ee t of ation variation. But what makes the dieren e

between anions of the same harge? It is their harge density and polarisability, in

line with the ordering from hard (high harge density) ions, su h as a etate, to soft

(low harge density and often highly polarizable) ions, su h as thio yanate.

Again Collins' on ept of so- alled `mat hing water anities' will be used to ex-

plain ounterion spe i ity. A ording to Collins' idea, soft ions should ome in

101
lose onta t with soft ions and hard ions in lose onta t with hard ions . By

ontrast, when hard and soft ions ome together, they do not approa h so far that

they loose their hydration sphere. Therefore, the intera tion between a hard and

a soft ion should be weak in water. Numerous phenomena in physi al hemistry

an be explained by this on ept. For quaternary ammonium ions, it is not sur-

prising that they behave like soft, low-density ions. A ording to Collins' on ept it

+
is therefore lear that DTA intera t more with softer ions than with harder ones,

−
and this is pre isely what was found in the present study. For example, SCN ions

ome in lose onta t to the ationi headgroup and they share a ommon hydra-

tion shell, whereas a etate anions will stay away from the headgroup and keeps its

own hydration shell. Consequently, the pa king parameter will in rease more, when

NaSCN is added to the mi ellar solution than when NaOA is added, and therefore

the tenden y to form rod-like mi elles is more pronoun ed in the ase of NaSCN.

This ee t was already dis ussed in detail in Chapter 3, where mole ular dynami s

(MD) simulation was used to lassify surfa tant headgroups from hard to soft, in the

236
same spirit as the ions . A similar ordering as was done in the previous hapter

for anioni headgroups an be done here to represent the ounterion binding to the

76
alkyl ammonium headgroup surfa tant ( . f. Figure 4.6).

Figure 4.6: Ordering of anioni ounterions regarding their anity for the alkali ammonium
headgroup.

4.4.6 Dierent self-aggregation behavior of atanioni sys-

tems in the atanioni - and anioni -ri h regions
Why we are able to get vesi les in the anioni -ri h region, but only thread-like

mi elles in the atanioni -ri h region is a di ult questions. This phenomenon was

already observed by other authors, but never really explained. If the hanges in

the pa king parameter are onsidered, the values of the hydrophobi part remain

the same, only the a value diers. However, a (as reported in literature, e. g.

282
referen e Sierra et al. ) does not dier enough to explain this signi ant dieren e

in the end pa king. One of the best explanations is the dierent hydration behavior

of the headgroups ompared to the hydration of the ounterions. With the help

284
of diele tri relaxation spe tros opy Bu hner et al. were able to observe that

the mi elle surfa e of the SDS surfa tant is strongly hydrophili and the adsorbed

ounterions are generally separated by a layer of water mole ules. On the other

hand, is the surfa e of the alkyltrimethylammonium bromides hydrophobi , with

284
the bound halide ions dire tly atta hed .

77
4.5 Con lusions
A study of the inuen e of added salt on the aggregation behavior of non-equimolar

mixed surfa tant solutions was ondu ted. The system was omposed of sodium

dode anoate (SL) and dode yltrimethylammonium bromide (DTAB) with an ex ess

of ationi surfa tant. The addition of salts produ ed a sphero ylindri al growth

of the mi elles, markedly dependent on the anion identity. The e ien y of the

anions to elongate the mi elles ould be explained by Collins' on ept of mat hing

water anities and the lassi ation of the ationi surfa tant headgroup as a soft,

polarizable entity.

78
 Part II

In reasing the Stability of Catanioni

Systems

79
Chapter 5

Inuen e of Additives and Cation

Chain Length on the Kineti
Stability of Supersaturated
Catanioni Systems

5.1 Abstra t
The stability of mixed surfa tant solutions of sodium dode ylsulfate (SDS) with

etyltrimethylammonium bromide (CTAB) and with dode yltrimethylammonium

bromide (DTAB) was studied as a fun tion of time. These spe i mixtures were

shown to have a solubility temperature below that of pure surfa tant solutions in the

anioni -ri h region. The stability of su h supersaturated solutions was studied with

and without dierent additives. Surfa tant mixtures without additives were shown

to destabilize with time varying between 3 and 28 days, depending on the surfa tant
ratio. Generally, the stability of solutions in reased by in reasing the per entage of

the anioni surfa tant. The variation of the hain length of the ationi surfa tant

produ ed a large ee t on the stability of su h mixed surfa tant systems. The pres-

en e of simple ele trolytes de reased, while the addition of middle- hain al ohols

in reased its stability. Bluish solutions orresponding to a vesi ular region were

81
observed at ratios lose to equimolarity in samples without salt, and in the anioni -

ri h region upon the addition of middle- hain al ohols. Fluores en e and dynami

light s attering measurements showed that the destabilization of the solutions is not

due to the formation of bigger aggregates, but rather due to a shift of the equilib-

rium between mi elles and monomers, leading to the liberation of monomers, whi h

pre ipitate. The lifetime of vesi les and mi elles ould therefore be ontrolled by

varying the omposition of the surfa tant solutions and by additives. Controlling the

pre ipitation phenomena is of importan e for a large number of industrial pro esses,

su h as oil / solute re overy pro esses after extra tions or hemi al rea tions.

5.2 Introdu tion

Mixtures of ationi and anioni surfa tants ( atanioni s) are onstituted of ve

types of spe ies, in luding two ounterions. In these systems a ompetition between

various mole ular intera tions due to van der Waals, hydrophobi , ele trostati and

/ or hydration for es may result in a wide variety of phase behaviors and mi rostru -

24
tures . Among others, atanioni mi elles, vesi les and bilayer stru tures, an be

more parti ularly found. In mixtures of anioni and ationi surfa tants a pre-

24,134
ipitation zone is observed around equimolarity . The pre ipitation zone an

be ontrolled by hanging the temperature or/and the ioni strength of the solu-

47,153
tion . In reasing temperature results in a general redu tion in the tenden y

of pre ipitation, whereas the in rease of ioni strength generally shows the oppo-

site ee t. The formation of nite stru tures in equimolar atanioni mixtures

is thus limited to high temperatures. It is therefore of obvious interest to study

the parameters whi h ould extend the temperature range in whi h atanioni sur-

fa tant self-aggregation an take pla e. Furthermore, ontrolling the pre ipitation

and mi ellization phenomena of atanioni mixtures are of pra ti al importan e in

many industrial pro esses, su h as oil / solute re overy pro esses after extra tions or

47,153,285287 288,289
hemi al rea tions , foaming pro esses and wetting time of textiles .

Mi elles and surfa tant monomers are in dynami equilibrium and individual surfa -

tant mole ules are onstantly being ex hanged between the bulk and the mi elles.

82
 290
Furthermore, the mi elles themselves disintegrate and reassemble ontinuously .

While the pro ess of surfa tant mi ellization is primarily entropi ally driven, mi elle

formation depends on the balan e of for es between the fa tors favoring mi elliza-

tion (van der Waals and hydrophobi for es) and those opposing it (ele trostati

60,291,292
repulsions and kineti energy of the mole ules) . The relaxation times of mi-

elles are dire tly related to the mi ellar stability and are mu h longer for nonioni

surfa tants than for ioni surfa tants, be ause of the absen e of ioni repulsions be-

290
tween the headgroups . For the same reason mi elle kineti s in atanioni systems

293,294
were also found to be very slow . Thus, mixed surfa tant solutions an remain

295
supersaturated for long periods of time before pre ipitation is omplete . As the

296
equilibrium states of su h systems are questionable , the time dependent stabil-

ity of ommon atanioni mixtures was studied in this paper. The ee t of ioni

strength and the inuen e of the variation of ounterions were investigated, and also

the inuen e of added al ohols as well as the importan e of a dierent hain length.

5.3 Experimental Pro edures

Materials The surfa tants, sodium dode yl sulfate (SDS: Mer k, Germany, grade:

99%), dode yltrimethylammonium bromide (DTAB: Mer k, Germany; assay > 99%)
and etyltrimethylammonium bromide (CTAB; Sigma, Germany; grade: 99%) were
used as re eived. All sodium and hloride salts used in the experiments were sup-

plied by Mer k, Germany. The al ohols: methanol, 1-propanol, 1-butanol, 1-de anol

(Mer k, Germany, > 99%), ethanol (J. T. Baker, Holland, 99.9%), 1-pentanol

(Aldri h, Germany, 99.5%), 1-hexanol (Fluka, Germany, 99%), 1-heptanol (Alfa

Aesar, Germany; 99%), 1-o tanol (A ros Organi s, U.S.A., 98%), and itronellol

(Mer k, Germany, 98%) were also used as re eived. Millipore water was used as

solvent in all ases.

Determination of the solubility temperature (This measurement was per-

formed by A. Arteaga during his graduate studies. Results are represented with his

permission.)The solubility temperature at dierent time intervals was determined

83
by visual observation. All systems were observed at 1 wt.% total surfa tant on-

entration. The ationi / anioni mixtures at dierent mass ratios were prepared

from 5 wt.% surfa tant sto k solutions. The samples were rst heated to 39◦ C, then

ooled down and left to equilibrate for 5 hours at 0◦ C, and then the temperature

was in reased by approximately 0.5◦ C per minute. The measured solubility tem-

peratures (between 0◦ C and 99◦ C) orrespond to the transition from pre ipitate to

isotropi phase; i. e. to a mi ellar or vesi ular solution. The pro edure was repeated

after a ertain time to analyze the kineti s of the system.

Phase Diagrams Phase diagrams of the systems were onstru ted a ording to

the pro edure des ribed above. The samples had to be heated after mixing in order

to obtain a homogeneous lear solution. Dierent surfa tant ratios exhibited dier-

ent initial solubilization temperatures, TI (more throughly explained in the Results

se tion). The ee t of additives was studied by adding an appropriate amount of

salt or al ohol to the SDS / CTAB mixture. Again, in some ases the sample had to

be heated in order to obtain a lear solution. The maximum temperature to whi h

the samples were heated was 39◦ C. The samples were then ooled down and kept

at 20◦ C. Phase diagrams were onstru ted by visual observation and by measuring

transmission using a Hita hi U-1000 spe trophotometer. The samples were also ob-

served at 0◦ C in order to shorten the destabilization times and to he k whether the

order of destabilization is the same at both temperatures.

Counterion Binding Cation anities for the surfa tant aggregate interfa e were

determined by ele trospray mass spe trometry. ES-MS was arried out using a Ther-

moquest Finnigan TSQ 7000 (San Jose, CA, USA) with a triple stage quadrupole

mass spe trometer. The solutions were sprayed through a stainless steel apillary

held at 4 kV, generating multiple harged ions. Data were olle ted using the X al-

ibur software. The surfa tant on entration was kept 1wt.% and the on entration

of the various hloride salts was 0.1 M in all ases.

System Kineti s The evolution of the surfa tant aggregates was further followed

using uores en e spe tros opy and dynami light s attering (DLS) measurements.

84
Pyrene (Aldri h, opti al grade) was used as the external uores en e probe to mon-

itor the hange in surfa tant self-assembly. The preparation of water saturated with

297,298
pyrene was as reported previously . The uores en e emission spe tra of pyrene

were re orded on a Cary E lipse uores en e spe trophotometer (Varian, In .) at

20◦ C. From the spe tra, the ratio of intensities of the rst and the third vibra-

tional peak of pyrene, I1 /I3 , was al ulated. Parti le size analysis was performed

using a Zetasizer 3000 PCS (Malvern Instruments Ltd., England), equipped with a

5 mW helium neon laser with a wavelength output of 633 nm. The s attering angle

was 90◦ and the intensity auto- orrelation fun tions were analyzed using the Contin

software.

Ee t of Al ohols on the System Morphology Spe imens for ryo-transmission

169
ele tron mi ros opy ( ryo-TEM) were prepared as des ribed before ; frozen sam-

ples were examined with a Zeiss EM922 EF Transmission Ele tron Mi ros ope (Zeiss

NTS mbH, Oberko hen, Germany), whereas the repli a were examined and pho-

tographed with a Philips CM 12 transmission ele tron mi ros ope.

5.4 Results and Dis ussion

5.4.1 Shift of Solubility Temperature with Time
The inuen e of the ationi surfa tant hain length on the solubility temperatures

of atanioni mixtures was investigated by omparing mixtures of sodium dode-

ylsulfate (SDS) with etyltrimethylammonium bromide (CTAB) and with dode-

yltrimethylammonium bromide (DTAB). CTAB and DTAB have the same quater-

nary ammonium polar headgroups, but CTAB has a longer hain (16 arbon atoms)

than DTAB (12 arbon atoms). Owing to stronger hydrophobi repulsions of its

hydro arbon hain, CTAB is therefore less soluble in water than DTAB. The Krat

point of pure CTAB in water at 1 wt.% is onsequently higher (≈ 25◦ C) 299 than

that of DTAB (< 0◦ C) 300 . The solubility temperatures (TS ) of the orresponding

atanoni mixtures, i. e. DTAB / SDS and CTAB / SDS with a total surfa tant

on entration of 1 wt.% and dierent anioni / ationi surfa tant (molar) ratios

85
were followed as a fun tion of time ( . f. Figures 5.1 and 5.2). Both systems dis-

played the same marked dieren es in their solubility temperatures over the whole

anioni / ationi ratios in the initial days after mixing. The TS in the ationi -ri h

regions were generally higher than the TK of the pure ationi surfa tant. The same

trend was observed for only a part of the phase diagram in the anioni -ri h region.

Around equimolarity, a maximum of solubility temperatures (TS ) was rea hed. This

maximum lay within the zone of pre ipitation usually observed at room tempera-

ture. A bluish zone, orresponding to a vesi ular region, was lo ated in both sides of

301,302
the entral area of insolubility . It was mu h more extended in the anioni -ri h

region (approximately between 0.6 - 0.7) than in the ationi -ri h region (around

0.35). The vesi ular regions ould be extended by raising the temperature. Harg-

61
reaves and Deamer noted the formation of liposomes in mixtures of CTAB and

SDS above ≈ 47◦ C. The sequen e of dierent phases resembled those observed in

24,49,303
similar atanioni systems . Obviously, the behaviour of two single- hain sur-

fa tants is mu h dierent in a atanioni system. The pa king between the ationi

and anioni surfa tants is quite dierent from the single ioni one and ontributes to

in rease the stability of the atanioni rystal, leading thus to higher solubility (and

Krat) temperatures. Interestingly, however, at a ertain ratio of anioni / ationi

surfa tants a `solubility temperature depression' exists. This depression was lo ated

in a range of a molar fra tion of the anioni surfa tant between 0.65 − 0.85 and

ould be observed immediately after mixing. With time, the mixtures undergo a

ontinuous evolution of TS toward higher temperatures. Surprisingly, the solubility

temperature was almost onstant over time in the ationi ri h region, whereas a

large time dependen y ould be observed in the anioni ri h region, . f. Figures 5.1

and 5.2. Furthermore, the shift in the solubility temperature was more pronoun ed

in systems ontaining a longer hain ationi surfa tant (Figure 5.2). For this reason

we fo used our attention on the anioni part of the phase diagram of the CTAB /

SDS system.

86
 100
90
80
70
Temperature (°C)

60
50
40
30
20
10
0
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0
Molar fraction SDS

Figure 5.1: Shift of solubility temperature in DTAB / SDS mixtures at dierent surfa tant ratios
as a fun tion of time (ctot = 1wt.%): observations after (N) 1 day, () 4 weeks and (•) 6 weeks.

100
90
80
Tempertarure (°C)

70
60
50
40
30
20
10
0
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0
Molar fraction SDS

Figure 5.2: Shift of solubility temperature in CTAB / SDS mixtures at dierent surfa tant ratios
as a fun tion of time (ctot = 1wt.%): observations after (N) 1 day, () 4 weeks and (•) 6 weeks.
Both Figures reprodu ed with permission from the do toral dissertation of A. Arteaga.

87
5.4.2 Behavior of the Anioni -Ri h Region of the Phase Di-
agrams Without Additives
The samples (with an ex ess of SDS) were prepared as des ribed in the Experi-

mental Se tion and heated in order to obtain a lear homogeneous solution. The

temperature needed an be des ribed by the equation TI (◦ C) = −0.43 · X + 64.9,

where TI (◦ C) is the initial solubility temperature expressed in Celsius and X is the

per entage of SDS in the solution ( . f. Figure 5.3 (left)). Then the samples were left

to ool down and observed at 20◦ C. The results from spe tros opy measurements

of the SDS / CTAB system at 20◦ C are presented in Figure 5.3 (right).

100
40

38 80
Transmission (%)

36 60
T (oC)

34
40
32
20
30

60 65 70 75 80 0
0 5 10 15 20 25
XSDS time (day)

Figure 5.3: Left: The temperature needed to obtain lear homogeneous solutions in the anioni -
ri h region an be des ribed by a straight line with a slope of k = −0.43(±0.03) and n = 64.9(±2.6);
right: Transmission of the SDS / CTAB atanioni system as a fun tion of time for the following
ratios: 65/35 (), 70/30 (•), 75/25 (N), 80/20 (), and 85/15 (◦)

We see that the higher the ratio of the anioni surfa tant, the more stable the

system is. Whereas the solution with 65% and 70% of SDS were pre ipitated after

10 days, solutions with 80% and 85% were still lear after 30 days. The ele tro-

stati intera tions and onsequent harge neutralization between the SDS mole ules

and the oppositely harged CTAB promote long relaxation times of atanioni mi-

elles. These, in turn, are a ountable for the supersaturation of our system and

the temporary solubility temperature depression observed. The anioni and ationi

88
surfa tant mole ules an be present in solution either as monomers, in orporated

into surfa tant aggregates (mi elles, vesi les, et .) or as pre ipitate (DSCTA(s) - a

salt formed from DS − CT A+ ions). Thus, there are two equilibria present in su h

mixtures: monomer-mi elle and monomer-pre ipitate. Two dierent types of phase

separation in su h systems an be observed. Phase separation in atanioni surfa -

tant mixtures may o ur due to the entanglement of rod-like mi elles, the formation

of lamellar phase, or the formation of densely pa ked vesi les. In some ases, how-

ever, there seems to be a ompetition between the kineti s of vesi le formation and

134,304
the kineti s of pre ipitation of ationi -anioni pairs . Earlier measurements on

atanioni systems have shown that pre ipitation is primarily enthalpi ally driven,

while mi ellar formation is primarily entropi ally driven. This is due to the ordering

of the surfa tant headgroups in the rystalline pre ipitate as a onsequen e of harge

153
neutralization .

In order to understand whether phase separation is a onsequen e of pre ipita-

tion of solid atanioni salt or the onsequen e of mi ellar (vesi ular) aggregation,

dynami light s attering and uores en e te hniques were employed. Parti le size

measurements showed a de rease in the size of the parti les with time, as an be

observed in Figure 5.4. After 3 days the solution was pre ipitated and the laser

beam passing through the upper ( lear) part of the solution dete ted only very

small parti les (RH ≈ 2.5nm).

This behavior is quite dierent from that observed in a previously studied SDS /

169
DTAB system . There, an in rease in turbidity was observed in a ordan e with

the in rease of the hydrodynami radius of the orresponding parti les. Although

the solutions were turbid, no pre ipitation took pla e, even after months of observa-

tion. The behavior of the SDS / CTAB was dierent. A pre ipitation was observed

without any dete tion of an intermediate turbidity. The reasons for the dierent be-

haviors of the two systems are most likely due to the dierent Krat temperatures of

the two ationi surfa tants. Pre ipitation is a ommon phenomenon in atanioni

surfa tant mixtures, whi h is regulated by two ompeting for es: the free energy of

305
the solid rystalline state and that of the mi ellar solution . The strong intera -

tions make pa king into a rystalline latti e energeti ally favorable for surfa tant

89
 12

10

8
% of particles

0
0 2 4 6 8 10 12 14 16 18 20
RH [nm]

Figure 5.4: Parti le size followed by DLS: SDS / CTAB atanioni system with a (molar) ratio
65/35 after 1 day () and after 3 days ().

monomers, parti ularly when the hydrophobi mat h is high. It has been shown

that by ex hanging the ounterion, we are able to inuen e the energeti state of

the mi ellar solution. A mu h bigger variation between systems is observed in the

free energies of the rystalline state. As we an see from the phase diagrams, the

solubility of the atanioni salt is dire tly orrelated to the solubility of the surfa -

tant monomers. The solubility (and Krat point) de reases with in reasing hain

length, and is therefore lower for the CTAB mixture in omparison to the DTAB

surfa tant mixture. We an not ex lude at this point the possibility that the pre ip-

itate was formed from an entanglement of rod-like mi elles or similar big parti les

282,283
as was already reported by other authors . However, su h transitions take a

longer time, suggesting the formation of larger parti les would be dete ted before

the a tual pre ipitation takes pla e. For this reason, we believe our system yields

90
a similarity to the SDS / DPLC (dode ylpyridinium hloride) mixture studied by

134
Stellner et al. . In this ase, the destabilization of solutions was not due to parti le

growth (and formation of big aggregates), rather was the observed pre ipitation due

to the liberation of monomers.

Pyrene uores en e emission spe tros opy was used to further investigate the

hanges in phase behavior. The low uores en e ratio, I1 /I3 , of pyrene on the rst

day after preparation of the solutions showed the presen e of highly hydrophobi

parti les in the solution (I1 /I3 = 1.1; . f. Figure 5.5). The I1 /I3 ratio in 1 week old
solutions (ma ros opi ally the solutions were two-phased: the lower part ontaining

the pre ipitant, whereas the upper part onsisted of a lear supernatant) showed

an in rease from a value of 1.1 to a value around 1.3. This value orresponds to

306
the polarity found for SDS mi elles . This suggests that as the atanioni salt

pre ipitated, the remaining pyrene was solubilized inside the mi elles formed by the

ex ess anioni surfa tant. In this way, a large amount of the surfa tant was removed

from the solution (as pre ipitate); only the ex ess amount remained in solution. Had

all of the SDS surfa tant pre ipitated as well, a mu h higher pyrene ratio would have

been observed (I1 /I3 (water) ≈ 1.8 307,308 ), whereas, if bigger aggregates had formed,

one would expe t the pyrene intensity ratio to remain un hanged. In the latter

ase, the pyrene probe would be solubilized either inside rod-like mi elles or the

hydrophobi interior of vesi ular membranes, whose environment mi ro-polarity is

the same as that in spheri al mi elles.

A higher stability of systems when the fra tion of one of the omponents was

in reased in omparison to the other omponent was already observed by Stellner

134
et al. . When the SDS on entration is in reased (and the CTAB on entration

held onstant), pre ipitate an not form due to a hange in the mi ellar (and onse-

quently monomer) omposition. The monomer on entration then does not ex eed

the monomer-pre ipitate boundary (for a more detailed explanation the reader is

134
referred to Stellner et al. ).

91
Figure 5.5: Pyrene uores en e emission spe trum of a SDS / CTAB atanioni system with a
(molar) ratio 70/30 after (left) 1 day, (right) 7 days.

5.4.3 Ee t of Additives on the Stability and the `Solubility

Temperature Depression'
Ee t of Ioni Strength Addition of ele trolytes to ioni surfa tant solutions

may modify both the inter- and intrami ellar intera tions. Therefore, the phase

behavior of surfa tant solutions is expe ted to be ae ted signi antly upon the

addition of salts. Sodium bromide was added to the atanioni mixture at dierent

ratios (in the anioni ri h region) to study the ee t of ioni strength on the phase

stability. The observed ee t was similar for all ratios. Generally, the in rease of

ioni strength de reased the destabilization time of the sample ( . f. Figure 5.6).

The higher the on entration of the added NaBr was, the shorter were the desta-

bilization times. Whereas the solution with 0.5 M of NaBr was pre ipitated after

only 2 days, the solution with 0.1 M NaBr started showing rst signs of turbidity

after 10 days. The destabilization times are shortened down to two hours when the

solutions are kept in an i e bath. A general in rease in the tenden y of pre ipita-

tion of atanioni mixtures upon the in rease of ioni strength was already observed

47,153
previously . When surfa tant monomers are salted out by the presen e of an

92
 2
ele trolyte, mi ellization is favored . Catanioni systems exhibit dierent behav-

iors upon salt addition. This is due to the ability of mixed surfa tant systems to

adjust the aggregate omposition to attain the lowest possible free energy state for

given onditions. In atanioni systems two salt-indu ed phenomena are observed,

47 169
depending on whether the equimolar (true) or non-equimolar atanioni sys-

tems are studied. To qui kly review, if the surfa tant mi elles onsist of just one

type of surfa tant (one harge) or one of the omponents is in ex ess, then the ad-

dition of salt s reens the repulsion between the headgroups, ausing the surfa tant

mole ules to arrange in a bilayer. The opposite ase is true, if the surfa tants are

present in equimolar amounts. The salts then s reen the attra tion between them,

most likely ausing the distan e between the headgroups to in rease. In these ases a

vesi le-to-mi elle transition was observed. Sin e we are dealing with non-equimolar

atanioni mixtures, where SDS is in ex ess, the addition of salt to our surfa tant

mixture is expe ted to have the same ee t as on pure SDS mi elles. Addition of

NaCl to SDS, for instan e, is known to de rease the CMC and in rease the mi ellar

6
size . Furthermore, by raising the ioni strength of the solution, the harges on the

surfa tant headgroups are s reened due to a stronger ounterion binding. This, in

ee t, de reases the solubility of the surfa tant monomers and therefore assist in

pre ipitation.

Ee t of Cation / Anion Variations A good orrelation has been observed

between the temperature to whi h it is required to heat the solution for omplete

solubilization to o ur (TI ) and the stability of the then isotropi solution. For this

reason, the heating temperatures of solutions ontaining various salts and al ohols

are reported in Table 5.1. The salt on entration was kept onstant in all ases at 0.1
M. The solubility temperatures for all sodium salts are 29(±0.5)◦ C. The variation of

the anion apparently has no ee t on the solubility of the atanioni mixture. This

was expe ted as the overall net harge was negative (we had an ex ess of the anioni

surfa tant SDS). A big variation of the temperature was observed when dierent

hloride salts were added to the mi ellar solution. Mass spe tros opy results (Figure

5.7) showed that at this relatively high on entration of added salt (0.05 M `new'

93
 100

80
Transmission (%)

60

40

20

0
0 5 10 15 20 25
time (day)

Figure 5.6: Transmission of the SDS / CTAB atanioni system: the ee t of ioni strength on
the stability of a SDS / CTAB solution (molar ratio 80/20) without salt (), with 0.1 (•) and 0.5
M (N) NaBr.

ounterions vs. ≈ 0.016 M of `original' sodium ounterions) all ounterions were in

the lose proximity of the mixed mi elle surfa e, regardless of the fa t whether they

normally exhibit salting-in or salting-out behavior.

Despite this, we saw a large dieren e in the stability of the solutions when the

solutions ontaining LiCl or NaCl were ompared with solutions ontaining KCl or

CsCl. After one week at 20◦ C, the rst two solutions were still lear and isotropi ,

while the later two were already pre ipitated (see Figure 5.8). This dieren e in TI
an be attributed to the stronger binding of less hydrated potassium and esium ions

309
to the ex ess dode yl sulfate ions . The use of KCl pre ipitation to remove SDS

310,311
is ommonly used in biology . The ee t of `hydrophobi ' ounterions ( holine

and T MA+ ) was found to be in the middle of both.

A stronger binding of organi ounterions in omparison to sodium to dode yl

312
sulfate mi elles was already observed using uores en e te hniques showing that

94
 Salt TI (0.1 M) Al ohol TI (0.1 M) TI (0.2 M) TI (0.05 M)

No salt 35.5 Methanol 35.5

LiCl 29 Ethanol 36 36
NaCl 29 n-Propanol 36 36
KCl 37 n-Butanol 35 34.5
CsCl 31.5 n-Pentanol 30.5 27
ChCl 37 n-Hexanol 21∗ 21∗
TMACl 37.5 n-Heptanol 21∗ 21∗
NaBr 29.5 n-O tanol 21∗ 21∗
NaSCN 29 n-De anol > 39∗∗ > 39∗∗
Na2 SO4 28.5 Citronellol > 39∗∗ > 39∗∗

Table 5.1: Ee t of additives on the initial solubility temperature (TI ); the initial sample is a
70/30 mixture (with an ex ess of SDS); ∗
the samples were isotropi and slightly bluish already at
room temperature (21 C);
◦ ∗∗
the samples were not lear even at 39◦ C.

the binding of alkylammonium ions is dominated by their hydrophobi ity. As was

pointed out previously, we are able to inuen e the energeti state of the mi ellar

solution by ex hanging the ounterion. This an explain the small dieren es in

313
the stability of the systems with dierent ounterions. Mukerjee et al. have

investigated the binding of ounterions to dode yl sulphate anions by ondu tivity.

+
They have reported that the Li ion is less rmly atta hed to the mi elle than the

+ +
Na ion and this one in turn less than Cs . The lower binding therefore promoted

the formation of ionized spe ies. The gain in free energy was however not large

enough to over ome the free energy of the rystallization, and thus all of the systems

eventually pre ipitated.

The anity of spe i ion- ounterion pairing has been previously extensively

169 31
studied on the vesi ular and two-phase regions of a similar system. The ordering

of ations was found to follow the Hofmeister series in all ases. The ee ts of the

ions were found to be strongly dependent on the ability of the ounterions to form

lose ion pairs with the surfa tant headgroups and the behavior was explained using

101
Collins' Law of Mat hing Water Anities . As was shown in Chapters 3 and 4,

95
 - + +
(2A +Na )
100
- + +
100
(2A +Li )

80
80

60
60

40
40 (2A-+M+)+

(2A-+H+)+
(2A-+Na+)+
20
20 (2A-+M+)+ (A-+M++Cl-)+
- + - +
(A +M +Cl )
A B
0
0 500 600 700 800 900
500 600 700 800 900 m/z
m/z
(2A-+ Cs+)+
100
(2A-+K+)+ 100

80
80

60
60
- + +
(2A +M )
- + +
(2A +M )
- + +
40 - + +
40 (2A +Na )
(2A +Na )
- + - +
(2A +H ) (A +M +Cl )
- + +
- + - +
20 20 (A +M +Cl )
(2A-+H+)+
C D
0 0
500 600 700 800 900 600 700 800
m/z
m/z

Figure 5.7: Ion binding as determined by ES-MS: addition of 0.1 M of (A) LiCl, (B) NaCl, (C)
KCl and (D) CsCl to an SDS / CTAB mi ellar solution. A− : dode ylsulphate anion (265 Da);
M + : etyltrimethylammonium ation (284 Da).

the alkyl sulfate headgroup behaves like a soft, haotropi ion.

The anions showed a destabilization time omparable to sodium hloride. The

variations of the anion produ ed no hange in the stabilization times and therefore

no spe i ity ( . f. Figure 5.9). This is most likely due to the fa t that the anions

did not a umulate near the same- harged mi ellar surfa e, but assisted purely by

in reasing the ioni strength of the medium.

Ee t of al ohols It is known that al ohol penetrates in the palisade layer

314 315317 318

of mi elles and depresses the surfa tant CMC . Nakayama et al. and

319
Kaneshina et al. have observed a depression of the CMC and the Krat point of

ioni surfa tant in the presen e of longer hain al ohols. The Krat point depression

was explained quantitatively by a model in whi h it is proposed that the melting

96
Figure 5.8: Ee t of additives on the stability of an SDS / CTAB atanioni system with a
(molar) ratio 70/30: (from left to right) LiCl, NaCl, KCl and CsCl (all salts 0.1 M) after 1 week
at 20◦ C

Figure 5.9: Ee t of additives on the stability of an SDS / CTAB atanioni system with a
(molar) ratio 70/30: (from left to right): without salt, with LiCl, NaCl, ChCl (Choline Chloride),
NaSCN, Na2 SO4 (all 0.1 M) after 3 hours on i e.

97
point of the hydrated solid agent is depressed owing to the formation of a mixed

318
mi elle of surfa tant and al ohol .

Figure 5.10: Ee t of additives on the stability of an SDS / CTAB atanioni system with
a (molar) ratio 70/30: (from left to right) methanol, ethanol, n-propanol, n-butanol n-pentanol,
n-hexanol, n-heptanol, n-o tanol, n-de anol, itronellol (all 0.1 M) after 1 week. Solutions from
methanol to butanol are slightly turbid / pre ipitated, pentanol is lear; hexanol to o tanol are
bluish and homogeneous, de anol and itronellol are turbid (due to the low al ohol solubility).

The heating temperatures of solutions ontaining al ohols of various hain length

and on entrations are reported Table 5.1. A ording to the results reported, the al-

ohols an be lassied into four groups: (i) short- hain al ohols (< C4 ), whi h have
no ee t on the TI or on the stability of the system; (ii) pentanol, whi h de reases

TI and the solubility somewhat; (iii) middle- hain al ohols (between C6 and C8 )
whi h lower TI and in rease the stability substantially; and (iv) long- hain al ohols

(> C10 ), whi h are inappropriate for use due to their low solubility ( . f. Figure

5.10). Variation of on entration generally produ ed little ee t. Middle- hain al-

ohols from hexanol to o tanol have provided best results. The initial solubility

temperature was redu ed to below room temperature (< 21◦ C) and the stability of

the solutions was in reased drasti ally. The samples were homogeneous and bluish,

hinting at the presen e of larger parti les. Best ee ts were observed when the al-

ohol on entration range was kept between 0.02 and 0.2 M. Lower on entrations

produ ed no ee t and larger amounts of al ohol resulted in an al ohol-surfa tant

gel.

98
Figure 5.11: Pyrene uores en e emission spe trum of a SDS / CTAB atanioni system with a
(molar) ratio 70/30 upon the addition of 0.05 M hexanol after (left) 1 day, (right) 1 week.

Fluores en e measurements reported a presen e of hydrophobi domains in the

solution ( . f. Figure 5.11). In fa t, the polarity of the aggregates sensed by

the pyrene probe was even lower when hexanol was added to the atanioni mix-

ture (I1 /I3 = 1.04); lose to that found in toluene (I1 /I3 = 1.03) 307,308 or in 2-

propanol (I1 /I3 = 1.07) 307 . This hints at the fa t that hexanol forms mixed mi-

elles/aggregates with the two surfa tants, thus redu ing the on entration of sur-

fa tant monomers. Solutions with dierent amounts of hexanol were observed at

room temperature for up to 6 months and still showed no sign of destabilization.

Also the I1 /I3 ratio in su h samples remained stable over time.

Furthermore, solutions ontaining small amounts of hexanol (0.05 M) were kept

in an i e bath for a period of up to 2 weeks and again no visible hanges appeared.

After 2 weeks, the sample was warmed to 20◦ C and he ked by ryo-transmission

mi ros opy. Solutions exhibited a high polydispersity. Spheri al and ribbon-like

mi elles, as well as vesi les were observed ( .f. Figure 5.12). This proves that it

99
Figure 5.12: Cryo-TEM photographs of a SDS / DTAB aqueous solution at a molar ratio of
70/30 and a total surfa tant on entration of 1 wt.% upon the addition of 0.05 M hexanol: spheri al
(bla k dots) and ribbon-like mi elles (top), as well as vesi les (bottom) are seen.

100
is possible to obtain vesi les stable for a long time in mixed surfa tant systems at

temperatures mu h lower than the Krat temperatures of the two orresponding

surfa tants.

5.5 Con lusions

The o urren e of supersaturation has been investigated in mixed surfa tant solu-

tions omposed of SDS / CTAB and SDS / DTAB. A temporary `solubility temper-

ature depression' has been observed in the anioni -ri h part of the phase diagram.

We have shown that the stability of su h supersaturated solutions an be tuned by

in reasing the ioni strength, by variation of ounterions, and by the addition of

al ohols. The presen e of simple ele trolytes generally de reased, while the addition

of middle- hained al ohols in reased its stability.

Destabilization and on urrent pre ipitation of the systems was shown not to o ur

due to the formation of bigger aggregates, but rather due to a shift of the equilibrium

between mi elles and monomers. The addition of low on entrations of middle- hain

al ohols resulted in the formation of vesi les in the atanioni systems at tempera-

tures signi antly lower than the Krat temperatures of the ioni surfa tants.

The lifetime of vesi les and mi elles ould therefore be ontrolled by varying the

omposition of the surfa tant solutions and by additives. Controlling the pre ipita-

tion phenomena is of importan e for a large number of industrial pro esses, where

formulations need to be tuned.

101
102
 Part III

Toward Appli ation

103
Chapter 6

Use of Surfa tants in Cosmeti

Appli ation: Determining the
Cytotoxi ity of Catanioni
Surfa tant Mixtures on HeLa Cells

6.1 Abstra t
The ytotoxi ity of ommonly used syntheti surfa tants and atanioni mixtures

of those was evaluated using MTT on HeLa ells. The 50% inhibition on entration

(IC50 ) for MTT redu tion was al ulated. The ee t on hain length in rease and

in lusion of polyoxyethylene groups on the toxi ity was tested on single surfa tant

systems. A general trend of in reasing toxi ity with in reasing hain length and

the presen e of polyoxyethylene groups was observed. The measured IC50 values

of atanioni systems lie between those of parti ipating surfa tants. The in rease

in toxi ity as the ationi surfa tant was added to the anioni one was however

not linear. A steep de rease of the IC50 values (and therefore in rease in the toxi

properties) was observed immediately already at low on entrations of the ationi

surfa tants. This behavior is analogous to the enzyme a tivity in atanoni mi-

roemulsions.

105
6.2 Introdu tion

Vesi les are ommonly used in osmeti s and pharma y as vehi les for a tive agents.

A tive mole ules an thus be en apsulated in the bilayer membrane, if they are

lipophili , or in the ore of the vesi le, if they are hydrophili . En apsulation is

useful to prote t a tives in preventing any undesired rea tion. Vesi les an thus be

used as ve tors to deliver drugs to a spe i pla e, without being destroyed.

Improving the bio ompatibility of produ ts used in osmeti formulation further is

sought after. For this reason it is important to identify the irritating properties of

ommer ially used surfa tants. Lately, atanioni systems have been investigated

as potential delivery systems due to their ability to spontaneously form vesi ular

phases. Furthermore, new surfa tants are being synthesized ontinuously. For this

reason, it is important to establish an easy way to estimate the potential toxi ity of

su h substan es.

Performing ytotoxi ity tests on established ell lines is a good alternative to ex-

pensive and morally questionable animal tests. In the present study, ytotoxi ity

of ommer ially used surfa tants and ommon atanioni surfa tant mixtures were

measured on HeLa ells in order to identify their toxi ity for use in pharma euti-

al and osmeti appli ations. HeLa ells are a well-known ell line that is used

320323
to asses the ytotoxi ity of hemi al ompounds and reportedly show good

reprodu ibility and a signi ant orrelation with in vivo results

324
. Cell viability

was evaluated by the tetrazolium MTT redu tion assay, based on the uptake and

the redu tion of the soluble yellow MTT tetrazolium salt by mito hondrial dehydro-

genase to a blue insoluble MTT formazan produ t. The IC50 value ( on entration

of test substan e that lowers MTT redu tion by 50% ompared with the untreated

ontrol) was al ulated from absorban e data. Previous tests have shown that the

mito hondrion-based MTT test is a very sensitive indi ator of surfa tant ytotoxi-

325
ity . Intra ellular organelles (su h as mito hondria) are ae ted already by low

surfa tant on entrations due to their high sensitivity to surfa tant membrane a -

tions. Furthermore, high orrelation between MTT redu tion assay tests and y-

tosoli la tate dehydrogenase leakage (LDH) in vitro and the Draize eye irritan y

106
data in vivo were reported
325
. In fa t, ytotoxi ity in vitro proved to be more sen-

326
sitive and quantitative than the Draize test .

A few reports on erning the toxi ity of surfa tants using dierent assays should be

325,327332
noted . The toxi ity of SDS is ommonly investigated as an example of an

ioni surfa tant. It has been established that ioni surfa tants show higher toxi ity

values than the non-ioni ones, and that ationi surfa tants are more potent than

328
their anioni ounterparts . However, reports on the toxi properties of atanioni

mixtures are very s ar e. The ytotoxi ity of a SDS/CTAB/ holesterol mixture was

333
tested on murine ma rophage-like ells by Kuo et al. . A dose-dependent apoptosis

was observed. The study did however not in lude the ee t aused by the variation

of the ratio of the surfa tants and their hain lengths.

6.3 Experimental Pro edures

6.3.1 Materials
HeLa Cell Line HeLa ells were distributed by the Ameri an Type Culture Col-

le tion (ATCC). Cells were ultured in Earle's minimum essential medium (MEM)

ontaining 0.85 g/L NaHCO3 supplemented with FCS (fetal alf serum)(10%), L-

Glutamin (2 mM), NEA (non-essential amino a ids)(1%), Amphoter in B (0.4µg/ml)

and Peni ilin G / Streptomy in sulfate (100 u/mL).

Surfa tant Solutions The surfa tants, sodium o tanoate (SO; Sigma, Germany;

grade: 99%), sodium dode anoate (SL; Sigma, Germany; grade: 99-100%), sodium

dode yl sulfate (SDS; Mer k, Germany; assay > 99%), sodium laurelth sulfate

+ −
(Texapon 70; Na C12 (EO)2.2 OSO3 , a tive assay: 70%, gift from Cognis, Germany)

+ −
and sodium dode ylether arboxylate (Akypo Soft 45NV; Na C11 (EO)4.5 OCH2 COO ,

a tive assay: 21%, gift from Kao Chemi als, Germany), o tyltrimethylammonium

bromide (OTAB; Mer k, Germany; assay > 99%), de yltrimethylammonium bro-

mide (DeTAB; Mer k, Germany; assay > 99%), dode yltrimethylammonium bro-

mide (DTAB; Mer k, Germany; assay > 99%), etyltrimethylammonium bromide

107
(CTAB; Sigma, Germany; grade: 99%) were used as re eived. Surfa tant sto k solu-

tions were prepared by dissolving weighed amounts of dried substan es in Millipore

water. The solutions were then left for 24 hours to equilibrate at 25◦ C. The atan-

ioni solutions were prepared by mixing the surfa tant sto k solutions to obtain

dierent anioni / ationi surfa tant mass ratios.

6.3.2 Growing HeLa Cell Cultures

Substan es: MEM - EARLE Medium, PBS - DUBECCO Buer, Trypsin / EDTA,

Flask ontaining HeLa ells

1. The old medium was removed from the ask ontaining HeLa ells that we wished

to transplant.

2. The ells were rinsed with approximately 4 ml of PBS buer solution. With this

we have removed all the free-swimming ells; only the ells stu k to the bottom of

the ask remained.

3. 3 ml of Trypsin / EDTA was added and the ells were in ubated at 37◦ C for

5 minutes. Trypsin disables the ell's ability to bind to surfa es ( onsequently, the

ells are swimming freely in the suspension).

4. The ell suspension was transferred into a new ask, 3 ml of new MEM medium

was added to stop the rea tion of Trypsin and the ells were entrifuged at 800/min
for 5 minutes (20 C).
◦

5. On e entrifuged, the supernatant was arefully removed.

6. 4 ml of new MEM medium was added and the solution was homogenized using a

pipette until ell- lusters disappeared.

7. A desired amount of ell suspension (depending on when we needed the ells

ready for the next experiments) was transferred into a new ask and MEM medium

was added to a olle tive volume of 20 ml.

8. The ells were left to in ubate at 37◦ C for minimum 3 days.

108
6.3.3 HeLa Toxi ity Test
1. The viability of the ells was he ked with Trypanblue (solution: 0.04 g Tryptan-
blue in 10 ml PBS buer); 10 µl ell suspension +10 µl Trypanblue => we ounted

the living ells under the mi ros ope (the defe ted ells were olored blue, the inta t

one's white).

2. Number of ells in the 16 squares: f.i. 330

330 · 2 = 660 dilution with Trypanblue

660 · 10 = 6.600 ells in 1µl

6.600 · 1.000 = 6.600.000 ells in 1 ml

3. We need 150.000 ells per experiment:

6.600.000 : 150.000 = 44
That means we need to dilute 1 ml of the ell suspension with 44 ml of medium.

For 1 plate (60 wells with 75 µl) 4.5 ml of ell suspension is needed (2 plates = 9
ml); we prepare 0.5 ml ell suspension + 22 ml of medium.

4. Con entration of substan es of whi h we wish to measure toxi ity: 1 mg/ml (1

wt.%); 0.1 mg/ml if the substan es are very potent ( ationi surfa tants).

5. Filling the plates:

o The wells at the borders were lled with 150µl of pure medium (36 · 150 µl = 5.4
ml)

o The middle 60 wells were lled with 75 µl medium

o 60 µl medium was added to wells 2 / B-G (the olumns are des ribed using num-

bers, and rows using letters)

o Wells 2B/2C were additionally lled with 15 µl of sample 1; 2D/2E with 15 µl of

sample 2; 2F/2G with 15 µl of sample 3.

6. With a 6- anal pipette the solutions were thoroughly mixed in wells 2B - 2G

and then 75 µl of the solutions was transfered from olumn 2 to olumn 3 (wells

3B - 3G). This pro edure was repeated until row 10 (10B - 10G). The last 75 µl of

solutions from row 10 was dis arded. Row 11 was reserved for our blind probe.

7. Finally, 75 µl of ell suspension was added to all 60 middle wells with (without

mixing)(in the end the whole plate should ontain 150 µl / well).

8. The ells were left to in ubate for 68 hours (±1 hour).

109
Figure 6.1: 96-well plates used for toxi ity studies immediately after preparation (top) and after
in ubation with MTT (bottom).

110
6.3.4 Dete tion
1. 15 µl of MTT solution (4 mg/ml of MTT in PBS buer) was added to all wells

ontaining ells (0.9 ml MTT solution per plate).

2. The plates were left to in ubate for 4 hours.

3. The ex ess MTT ontaining medium was arefully remove. Well 1A was emptied

as well.

4. The empty wells were lled with 150 µl 10 wt.% SDS solution (in water; 18.3 ml

for two plates).

5. The mi ro-plates were pla ed in laminar ow overnight, then the opti al density

of ea h plate was measured with a mi roplate reader at 560 nm.

A sample plate immediately after the preparation pro ess and after the MTT in u-

bation is presented in Figure 6.1. The wells at the rims are lled only with medium

(pink olor), the blue olor is a sign of inta t ells and the transparent solution of

ell apoptosis.

6.3.5 Evaluation of spe tros opi al data

The IC50 value (in µg /ml), whi h represents the on entration of test substan e that
lowers MTT redu tion by 50% ompared with the untreated ontrol, was al ulated

for ea h substan e from the on entration-response urve. A sample al ulation

sheet is presented in Figure 6.2. Experiments were repeated four times (n = 5),

and the average IC50 is reported. Maximal observed (absolute) standard deviation

was about 15%. Positive ontrol measurements were performed with xanthohumol

(HeLa ells: IC50 ≈ 7 µg/mL).

6.4 Results and Dis ussion

6.4.1 Single-Chain Surfa tants
Cell viability was determined by the tetrazolium MTT dye redu tion in the ell

ulture system. Evaluation of the results revealed a dose-dependent ytotoxi ity

after 68 hours of exposure. The IC50 values are reported in Table 6.1.

111
 HeLa -Cells
Testsubstance:SDS/DTAB 90/10
Konz[µg/ml] OD Average corr. ave. Restakt. StAbw
[%] [%]
Empty value 0.0428
0.043
Control 0.8590 0.8325 0.8739
0.8141 0.9065 0.8372 0.854 0.811
1000 0.0519 0.0532 0.053 0.0098 1.2 0.1
500 0.0511 0.0472 0.049 0.006 0.8 0.3
250 0.0457 0.0433 0.045 0.002 0.2 0.2
122.5 0.0661 0.0821 0.074 0.031 3.9 1.4
62.5 0.3885 0.3824 0.385 0.343 42.2 0.5
31.25 0.4840 0.5725 0.528 0.485 59.9 7.7
15.63 0.7098 0.7054 0.708 0.665 82.0 0.4
7.8 0.8245 0.8394 0.832 0.789 97.3 1.3
3.9 0.9018 0.9091 0.905 0.863 106.4 0.6

Conc. >50%: 31.25Value: 59.9µg/ml

Conc.<50%: 62.5 Value: 42.2µg/ml
IC 50 =48.73µg/ml

Figure 6.2: Cal ulation of the IC50 value from the spe tros opi al data.

Comparison of the IC50 values of the surfa tants revealed the following ytotoxi

properties:

1) Anioni surfa tants generally exhibited a lower ytotoxi ity than atanioni sur-

fa tants.

112
 Anioni surfa tants Cationi surfa tants

name IC50 [µg/mL℄ name IC50 [µg/mL℄

SO 530.5 ± 41.7 OTAB 23.3 ± 1.8

SL 115.8 ± 8.2 DeTAB 4.8 ± 0.6
SDS 177.1 ± 7.8 DTAB 5.2 ± 0.3
Texapon 70 69.2 ± 1.7 CTAB 1.6 ± 0.2
Akypo Soft 45NV 76.3 ± 4.8

Table 6.1: The IC50 values ( on entration of test substan e that lowers MTT redu tion by 50%
ompared with the untreated ontrol) of singe- hain ioni surfa tants.

2) Surfa tants ontaining sulphate headgroups showed higher IC50 values than those

bearing a arboxylate (having the same hain-length).

3) An in rease of the toxi ity of the surfa tant was observed as the length of the

hydro arbon hain was in reased ( . f. Figure 6.3). An exponential de rease in the

IC50 values was observed, whi h leveled o at a ertain hain length.

4) The presen e of the oxyethylene groups in reases the toxi properties of the sur-

fa tants. Two surfa tants ontaining 12 arbon atoms, and polyoxyethylene groups,

one with a sulphate (Texapon 70) and one with a arboxylate headgroup (Akypo

Soft 45 NV) were ompared to those without the EO groups. An in rease in the

toxi properties is observed for both surfa tants, however Akypo Soft 45 NV exhibits

a higher IC50 value, than its sulfate ontaining ounterpart, despite the fa t that

SDS shows higher IC50 values than SL.

6.4.2 Catanioni Surfa tant Systems

The toxi ity of atanioni surfa tant mixtures omposed of SDS / DTAB and SL

/ DTAB were analyzed for dierent surfa tant ratios. The results are presented in

Table 6.2 and Figure 6.4.

We an see that the presen e of only a small amount of a ationi surfa tant

in reased the toxi ity of the mixture signi antly. This was somewhat surprising,

as a linear in rease of the toxi ity was expe ted.

113
 600
500
400
300
200
100
[ g/mL]

30
50
IC

20

10

0
8 10 12 14 16
n (C)

Figure 6.3: Ee t of hydro arbon hain length on the ytotoxi ity of ioni surfa tants: n(C)
represents the number of arbons in ationi CnTAB () and anioni SCn () surfa tants.

SDS / DTAB SL / DTAB

xSDS IC50 [µg/mL℄ xSL IC50 [µg/mL℄

0 4.2 ± 0.2 0 4.2 ± 0.2

0.1 3.5 ± 0.1 0.1 5.1 ± 0.4
0.2 3.8 ± 0.3 0.2 4.6 ± 0.6
0.3 3.7 ± 0.2 0.3 6.3 ± 0.5
0.4 6.7 ± 0.5 0.4 7.1 ± 0.2
0.5 8.2 ± 0.8 0.5 7.2 ± 0.4
0.6 7.9 ± 0.6 0.6 13.0 ± 1.2
0.7 11.6 ± 1.0 0.7 16.4 ± 0.9
0.8 20.1 ± 1.6 0.8 28.6 ± 2.1
0.9 43.1 ± 3.2 0.9 49.7 ± 3.4
1.0 177.1 ± 7.8 1.0 115.8 ± 8.2

Table 6.2: The IC50 values of atanioni surfa tant mixtures.

114
 200

175

150

125
[ g/mL]

100
50

75
IC

50

25

0.0 0.2 0.4 0.6 0.8 1.0

X
SDS/SL

Figure 6.4: The mito hondrial redu tion of MTT after a 68-hour in ubation of SDS / DTAB
() and SL / DTAB () mixture at dierent surfa tant ratios with HeLa ells.

This behavior is similar to the one found when the enzymati a tivity in atan-

334
ioni emulsions was studied . It was observed that the presen e of DTAB exerts

an inhibiting ee t on the enzyme. At weight fra tions higher than 0.22, the enzyme
was ompletely inhibited. This is approximately the weight fra tion at whi h the

toxi ity of the mixtures equals that of a pure DTAB solution. It is highly proba-

ble that at weight fra tion lower than 0.22 DTAB is ompletely in orporated in the

atanioni mi elles and its toxi ity is redu ed by the strong ele trostati intera tions

with the oppositely harged anioni surfa tant.

Results suggest that in order to keep the toxi ity of potential drug delivery systems

low, a high fra tion of the anioni omponent is ne essary. So far, vesi les have been

shown to exist in the anioni -ri h region of the atanioni phase diagrams, however

at ratios lose to equimolarity. The vesi ular region was shifted to higher anioni

169,335
fra tions when salt was added to the system . A system of lower toxi ity om-

posed of a mixture of anioni surfa tants will be reported in the following hapter

115
(Chapter 7).

6.5 Con lusions

The ytotoxi ity of single- hain ioni surfa tants and atanioni mixtures was eval-

uated using MTT on HeLa ells in order to examine their potential use in osmeti

and pharma euti al formulation. It was onrmed that anioni surfa tants generally

exhibit higher IC50 values than atanioni ones. The toxi ity an further be inu-

en ed by the hydro arbon hain length and the presen e of polyoxyethylene groups.

A general trend of in reasing toxi ity with in reasing hain length and the pres-

en e of polyoxyethylene groups was observed. A non-linear in rease was observed

as ationi surfa tants were added to anioni ones. A steep de rease of the IC50

values was observed already at low fra tions of the ationi surfa tants, suggesting

that in potential drug delivery systems a high fra tion of the anioni omponent is

ne essary.

116
Chapter 7

Spontaneous Formation of Bilayers

and Vesi les in Mixtures of
Single-Chain Alkyl Carboxylates:
Ee t of pH and Aging

7.1 Abstra t
We report the observation of bilayer fragments, some of whi h lose to form vesi les,

over a large range of pH at room temperature from mixtures of single- hain bio om-

patible ommer ially available non-toxi alkyl arboxyli surfa tants after neutral-

ization with HCl. The pH at whi h the morphologi al transitions o ur, was varied

only by hanging the ratio between two surfa tants: the alkyl-oligoethyleneoxide-

arboxylate and sodium laureate. The ee t of aging of the mixed surfa tant sys-

tems in the pH region desired for dermatologi appli ation (4.5 < pH < 7) was also

studied.

7.2 Introdu tion

There is a growing interest in the eld of syntheti surfa tants used for dermatologi

purposes. These surfa tants exhibit a wide range of stru tures; parti ularly useful is

117
the formation of vesi les, whi h an be used as delivery systems. For osmeti rea-

sons, the surfa tants forming vesi les must be skin ompatible (non-irritant), easy to

manufa ture, and the vesi le region must be stable within the range of physiologi al

pH at room temperature.

A simple geometri al hara terization of hain pa king an be used to analyze trends

15,60
in surfa tant phase behavior . The geometri properties of surfa tants depend

on the ratio between the ross-se tional area of the hydro arbon part and that of

the headgroup. Low pa king parameters (around 1/3) are found for single hain

surfa tants with a strongly polar headgroup. These systems tend to form spheri-

al mi elles, whereas a pa king parameter value around one favors the formation

of lamellar stru tures. An in rease in the pa king parameter an be obtained by

adding a se ond hain, whi h doubles the hydro arbon volume. Double- hain sur-

18,21 2326
fa tants , two surfa tants of opposite harge , or the asso iation of a surfa -

2733
tant and a o-surfa tant , an be used. In the latter two ases a pseudo-double

hain surfa tant is obtained by either an ion-pair formation between the anioni and

ationi surfa tant, or due to asso iation of the two dierent mole ules via hydrogen

bonds.

As ationi surfa tants and short al ohols are undesirable in osmeti formulations

due to their toxi ity, and be ause long- hain al ohols (> C12 ) exhibit high melting

points, monoalkyl arboxylates were hosen for our study. Fatty a ids form a range

of aggregates depending on the a id on entration and the ionization degree of the

336,337
terminal arboxyli group . The formation of vesi les from mono arboxyli

338
a ids has long been known. Gebi ki and Hi ks rst observed the formation of

vesi les from unsaturated, long- hain fatty a ids. Later, Hargreaves and Deamer

61
showed that also saturated fatty a ids an form vesi les . A vesi le phase is spon-

taneously formed, when short or middle hain (< C12 ) fatty a ids are neutralized

61,81
with HCl ; two types of amphiphiles are then present in the solution, the proto-

nated and the ionized form. The ratio between the two determines the aggregation

morphology.

Despite the simpli ity of the me hanism of vesi le formation, possible appli ations

339,340
of fatty a id vesi les in osmeti s remain largely unexplored . This may be a

118
 onsequen e of two obsta les: (i) the high solubility temperature of the long- hain

arboxylates and, (ii) the generally too basi pH ne essary for the solubilization of

the arboxylate. The problem of the solubility temperature of the alkyl arboxylates

61
has been dis ussed by Hargreaves et al. Below 25◦ C only alkyl arboxylates with

short alkyl hains are water-soluble. However, these are inappropriate due to their

skin irritating properties. The solubility temperature of sodium laureate is reported

336
to be equal to or above room temperature, depending on the on entration . With

longer alkyl hains (n > 12) the solubility temperature be omes even higher.

It has previously been reported that the formation of vesi les o urs at pH values

at or near the pKa, where approximately half of the arboxyli groups are ion-

61,336,339
ized . For this reason, fatty a id vesi les are present only over a narrow pH

range. Designing vesi les that be ome unstable at an easily tuned pH value is of

great interest for targeted drug delivery. It is known that, for example, tumors and

6367
inamed tissues exhibit a de reased extra ellular pH . For this reason a large

number of groups have fo used their attention on the preparation of pH-sensitive

6878
liposomes as possible drug arrier systems.

A range of sugar-based gemini surfa tants has been re ently studied, be ause they

exhibit pH-dependent aggregation behavior. However, these surfa tants are ationi

341,342
and therefore vesi les are observed only at neutral and high pH values . The

343
same problem o urs also in solutions of bola-amphiphiles . Systems omposed of

mixed single- and double-short-tailed PEO ether phosphate esters might be promis-

ing for the formation of pH-sensitive vesi les. In that ase a vesi le phase was

observed at higher on entrations. The pH ee t, however, was only studied on

vesi ular solutions, so one an not onrm a mi elle-to-vesi le transition as a onse-

344
quen e of protonation .

For arboxylates with longer alkyl hains, desirable for osmeti formulations (i.e.

lauri a id; pKa ≈ 8 − 8.5 24,61 , depending on the on entration), the pH of vesi le

formation is also generally too basi . The addition of medium- and long- hain al o-

61,339
hols has been proven to expand the pH region of vesi le formation , but toward

even higher pH values, whi h is undesirable. One way to lower the pH range of

vesi le formation is to introdu e another arboxyli group to the fatty a id hain,

119
thus lowering the pKa of the a id. This has been previously done by de Groot et al.

345
by the formation of 2-(4-butylo tyl) maloni a id . However, to the best of our

knowledge, no one has tried to use a mixture of single- hain fatty a id soaps yet.

Re ently, spontaneous formation of vesi les below room temperature, at a idi pH

(between 2 and 4), by neutralization of a parti ular industrial single- hain alkyl ar-
346
boxylate surfa tant was reported . Our intent was to obtain spontaneously-formed

vesi les over a wide range of physiologi al pH at room temperature, by hanging the

ratio of two surfa tants: Akypo Soft 45NV (an alkyl-oligoethyleneoxide- arboxylate

already used in osmeti formulations; AS) and sodium laureate (SL). The ee t

of pH and aging on vesi le formation was studied by visual observation, dynami

light s attering and transmission ele tron mi ros opy. The bio ompatibility of the

surfa tant mixture was he ked by measuring the ell viability. Based on our results,

we propose a general method to obtain vesi les on a large range of physiologi al pH

using only non-toxi alkyl arboxylates.

7.3 Experimental Pro edures

Chemi als Sodium laureate (SL) was pur hased from Sigma-Aldri h at a purity of

+ −
99%. Sodium dode ylether arboxylate (Akypo Soft 45NV; Na C12 (EO)4.5 OCH2 COO )

was a gift from Kao Chemi als (Germany). A ording to the information given by

Kao Chemi als, AKYPO Soft 45NV ontains mole ules with roughly a Gaussian

distribution of the number of EO groups with an average value of 4.5. AKYPO Soft

45NV is supplied as an approximately 21 wt.% aqueous solution. Sto k solutions of

1wt.% were prepared by dissolving weighed amounts of sodium laurate in deionized

water and by dilution of the original Akypo Soft 45NV solution. Dierent solutions

were then prepared by mixing dierent ratios of the two sto k solutions.

Phase Diagrams The phase behavior as a fun tion of temperature was deter-

mined by visual observations. The samples were ooled down and left to equilibrate

at 0◦ C, then the temperature was raised by approximately 0.5◦ C per minute. The

measured solubility temperatures orrespond to the transition from pre ipitate to

120
isotropi phase, i.e., to a mi ellar or vesi ular solution. The samples were titrated

with 0.1 M HCl at 25◦ C. A Con ort, type C831 pH-meter, with a Bioblo k S ienti

glass ele trode (Consort ref. nr. SP02N), was used.

Dynami Light S attering Parti le size analysis was performed by a Zetasizer

3000 PCS (Malvern Instruments Ltd., England), equipped with a 5 mW helium-neon

laser with a wavelength output of 633 nm. The s attering angle was 90◦ and the

intensity auto orrelation fun tions were analyzed using the CONTIN software. All

measurements were performed at 25◦ C.

Cytotoxi ity Tests HeLa ells were provided by the ATCC (Ameri an Type

Culture Colle tion). The passage numbers of the HeLa ells used in the proje t

varied from 25 to 30. Cells were ultured in Earle's minimum essential medium

(MEM) ontaining 0.85 g/L NaHCO3 supplemented with FCS (10%), L-Glutamine

(2 mM), NEA (1%), Amphoter in B (0.4 µg/ml) and Peni illin G / Streptomy in

sulfate (100 u/mL). Keratyno ytes (SK-Mel-28) were provided by the ATCC. The

passage numbers varied from 15 to 20. Cells were ultured in Dulbe o's MEM

ontaining 3.7 g/L NaHCO3 supplemented with FCS (10%), L-Glutamine (2 mM),

NEA (1%), Amphoter in B (0.4 µg/ml) and Peni illin G / Streptomy in sulfate (100
u/mL). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay

347
pro edure was prepared a ording to Mosmann in 96-well mi ro-plates using the

doubling dilution method (as des ribed in the previous hapter). First, the wells of

the rst olumn were lled with 135µl of MEM medium. All subsequent olumns

were lled with 75 µl of medium. Then, 15µl of 1wt.% test solutions was added

to ea h well in the rst olumn and the solutions were thoroughly mixed with a

6- hannel pipette. 75µl of the test substan e / medium mixture was transferred

from the wells in the rst olumn to those in the se ond. Again the solutions were

mixed and the pro edure was repeated for the whole plate. Then, 75µl of ells

were seeded (2.5 · 103 ells / well). After a 68-hour in ubation at 37◦ C, 15µl of

MTT (5 mg /mL) were added to ea h well. After a 4-hour in ubation, the medium

ontaining MTT was removed and 150µl of 10% SDS solution was added. The

121
mi ro-plates were pla ed in laminar ow overnight, then the opti al density of ea h

plate was measured with a mi roplate reader at 560 nm. The IC50 value (µg /ml),

whi h represents the on entration of test substan e that lowers MTT redu tion by

50% ompared with the untreated ontrol, was al ulated for ea h substan e from

the on entration-response urve. Experiments were repeated four times (n = 5),

and the average IC50 is reported. Maximal observed (absolute) standard deviation

was about 15%. Positive ontrol measurements were performed with xanthohumol

(HeLa ells: IC50 ≈ 7 µg/mL).

Cryo-TEM We prepared vitried ryo-TEM spe imens in a ontrolled environ-

ment vitri ation system (CEVS), at a ontrolled temperature of 25◦ C and xed

100% relative humidity, followed by quen hing into liquid ethane at its freezing

348
point . We examined the spe imens, kept below −178◦ C, by an FEI T12 G2

transmission ele tron mi ros ope, operated at 120 kV, using a Gatan 626 ryo-

holder system. Images were re orded digitally in the minimal ele tron-dose mode

by a Gatan US1000 high-resolution ooled-CCD amera with the DigitalMi rograph

software pa kage.

7.4 Results and Dis ussion

7.4.1 Lowering of the Solubility Temperature of Fatty A ids
To form vesi les at room temperature, the solubility temperature TSol of the sur-

fa tant mixture has to be equal to or below this temperature. The use of sodium

laureate is therefore limited, be ause it is insoluble in water at temperatures below

24◦ C (for solutions of 1 wt.%). We were able to lower the solubility temperature by

mixing sodium laurate with a surfa tant that is soluble at lower temperatures. AS

was hosen be ause its TSol = 5◦ C. Figure 7.1 shows that the solubility temperature

de reased linearly with in reasing amounts of AS for a xed total surfa tant on en-

tration of 1wt.%, and ould be des ribed by the equation: TSol (◦ C) = −19.9·A+23.9,
where TSol represents the solubility temperature of the mixture, and A the amount

of AS, expressed in wt.%.

122
 25

20
[ C]

15
o

solubility

10
T

0
0.0 0.2 0.4 0.6 0.8 1.0

amount of Akypo Soft 45NV (wt.%)

Figure 7.1: Solubility temperature of mixed surfa tant solutions (AS and sodium laureate; ctot
S =

1 wt.%) as a fun tion of the amount of AS in the solution. A linear t des ribes the dependen e
with the equation: TSol (◦ C) = (−19.9 ± 0.1) · A + (23.9 ± 0.1), where TSol represents the solubility
temperature of the mixture and A the amount of Akypo expressed in wt.% (the total surfa tant
on entration was kept at 1 wt.%)

7.4.2 The Ee t of pH on Vesi le Formation

225
The titration of alkaline soaps with HCl has been des ribed by Rosano et al. ,

when investigating the ee ts of surfa e harge on lipid-water interfa es. In these

experiments, the appearan e of a plateau (buering apa ity) at a pre ise pH during

titration was oin ided with the formation of lipid liquid- rystals.We observed this

plateau in solutions of dominating quantities of sodium laureate. As more SL was

repla ed by AS, the plateau region be ame smaller, disappearing below 0.5 wt.%

SL (mass ratio 1 : 1). By redu ing the pH of the samples we observed a su es-

sion of two (or three) phases, depending on the mass ratio of the two surfa tants

(Figure 7.2). At high pH, all solutions were isotropi and olorless, orrespond-

ing to the mi ellar region of the phase diagram. By de reasing the pH, a bluish

olor appeared, attributed to a formation of vesi ular stru tures. In the presen e

123
of pure AS, only two phases appeared onse utively, while in SL / AS mixtures, a

se ond phase transition to pre ipitation was observed at low pH values. In 1 wt.%

SL solutions pre ipitation began at pH values below 7, whereas the addition of AS

lowered the pre ipitation boundary to pH = 3.5 (in solutions with SL / AS ratio

25/75). Pre ipitation when the surfa tant ratio nears equimolarity, had been ob-

served in previous studies and was attributed to the in reased on entration of the

169
onjugated a id . The titrations were performed one week after the preparation

of the samples. Ma ros opi ally, the pH values at whi h the phase transitions took

pla e remained the same with respe t to time. This is not true mi ros opi ally, as

des ribed below.

The formation of fatty a id vesi les is restri ted to a narrow pH region lose to the

61,336,339
pKa of the a idi omponents . The apparent shift in the pKa of fatty a id

anions (from the pKa = 4.67 of arboxyli group 349 to the pKa = 8 of lauri a id 61 )
61
has been attributed to the lo al de rease in pH at highly harged surfa es . Sin e

pure fatty a id/soap vesi les (without additional amphiphiles) ontain an amphiphile

that is not harged (the neutral form of fatty a id), the two bilayer-forming om-

350
ponents are asso iated by hydrogen bonds instead of ele trostati intera tions .

The formation of vesi les near the pKa of the a id an then be explained by the

formation of stable hydrogen bond networks between the ionized and neutral a id

forms.

It has been observed that the addition of al ohols to fatty a ids auses an in rease

339
in the pH of the region of vesi le formation . In that ase, vesi les are formed due

to stable hydrogen bonds between the al ohol headgroup and the ionized a id head-

group. The addition of the al ohol means larger presen e of the non-ionized spe ies

in the solution (due to the high pKa of the al ohols); a larger amount of arboxyli

a id will be in the ionized form to a hieve the same protonated/ionized headgroup

ratio needed for vesi le formation (1 : 1). An opposite ee t an then be expe ted

in the ase when the added omponent has a mu h lower pKa than the fatty a id,

as is the ase of AS. This will be ompletely ionized at a neutral pH, therefore more

protonated a id groups will be required for the formation of vesi les, resulting in a

de rease of the pH of vesi le formation. This was onrmed by our titrations, where

124
the pKa (and orresponding bluish region) de reased with in reasing amount of AS

in the mixture.

pH
5

1
0.00 0.25 0.50 0.75 1.00 1.25

V [mL]
HCl

Figure 7.2: Dierent phase transitions observed by titration of mixed surfa tant solutions
(sodium dode anoate and AS; ctot
S = 1 wt.%; Vo = 15 mL) ontaining (from bottom to top)
1; 0.75; 0.5; 0.25; and 0 wt.% of AS. The dashed lines represent a bluish solution (possibly vesi les);
the solid lines represent the lear (mi ellar) solution (to the left) and the turbid phase (to the right
of the vesi ular phase).

The hydrodynami radius of parti les in solutions at dierent surfa tant ratios

was measured by dynami light s attering. The results onrmed the presen e of

large aggregates (possibly vesi les) over a large pH region (Figure 7.3) in mixed

surfa tant solutions. Be ause solutions of industrial surfa tants are highly polydis-

169
perse (the polydispersity of AS has been previously reported ), only the average

radius ould be measured. The regions where RH was found to be 80 − 120 nm

(the average radius of vesi les) is plotted in Figure 7.3 for dierent surfa tant ratios.

In reasing the amount of AS lead to a de rease of the pH, at whi h vesi les were

formed. The width of the pH range depended strongly on the surfa tant ratio. This

range is the largest for the ratio AS / SL : 0.75 / 0.25, where it extends from pH 3.5

125
to 7.5. At this ratio the omplete range of skin pH or physiologi al pH is overed.

A redu tion of this range was observed when the amount of Akypo Soft 45NV was

either in reased or de reased.

10

8
Micelles

pH
4

Precipitation
2

0
0.0 0.2 0.4 0.6 0.8 1.0

AS [wt.%]

Figure 7.3: pH range where (after 3 weeks) the average hydrodynami radius RH was approx-
imately 100 nm (possible region of vesi le formation) in mixed surfa tant solutions (SL and AS)
S = 1 wt.%); the open squares () represent the solutions
as a fun tion of AS on entration (ctot
examined by ryo-TEM.

The (average) hydrodynami radius (RH ) of the obje ts present in the vesi le

ontaining solutions is shown in Figure 7.4 for dierent surfa tant ratios as a fun tion

of the pH. The obtained values suggest the presen e of vesi les, but large variations

exist. In presen e of 0.75 wt.% of AS only small vesi les are measured with a small

polydispersity index (around 0.2). At smaller ratios of AS, or in presen e of pure

sodium laurate, bigger obje ts of higher polydispersity are observed. Be ause light

s attering is insu ient to hara terize systems of high polydispersity, ryo-TEM

was used to analyze the system further.

126
 200

175

150

125
[nm]

100
H
R

75

50

25
2 3 4 5 6 7 8 9
pH

Figure 7.4: Average hydrodynami radius, RH , of the aggregates present in bluish solutions as
a fun tion of pH for dierent surfa tant ratios: (△) 0; (◦) 0.25; (•) 0.5; (N) 0.75; () 0.85; () 1
S = 1 wt.%).
(all expressed in wt.% of AS; ctot

7.4.3 Cryo-TEM Study of Time-Dependent Vesi le Forma-

tion
The AS / SL ratio (75/25), at whi h the largest pH interval of vesi le formation was

measured, was further investigated by ryo-transmission ele tron mi ros opy. The

evolution of self-assembly aggregates was followed over a period of two months at

three dierent pH values. The sequen e of observed morphologies is summarized in

Table 7.1.

We see from Table 7.1 that in the rst weeks after preparation mi elles and

dis s are the observed stru tures; these are presented in Figure 7.5. With time,

membranes started forming and eventually vesi les appeared (Figure 7.6). The

higher the pH of the solution, the faster this transition took pla e; in samples with

pH = 6.7 vesi les were observed already after 2 weeks, whereas in solution with

pH = 5.5, these were visible just after 4 weeks. After 2 months vesi les were still

127
 pH No. of days after preparation

5 10 17 28 60

4.5 D, MP M D MP

5.5 M, D M, D M, V M, MP

6.7 MP, V M, D, V (C) M, MP, V (I)

Table 7.1: Development of various surfa tant self-assembled aggregates with time; M - mi elles,
D - dis s, MP - membrane pie es, V - vesi les, V (C/I) - vesi les with either usps, or in omplete
vesi les.

not observed in solution with the lowest pH ( . f. Figure 7.7 a). However, the

two samples at higher pH values already showed in omplete vesi les and membrane

pie es ( . f. Figure 7.7 b). It seems that after a ertain time, the vesi les in

these systems begin to destabilize. We noted that despite the ri h morphologi al

development that ould be observed by mi ros opy, ma ros opi ally the solutions

appeared about the same during the period of observation. Furthermore, we an

see that not all regions where big obje ts were dete ted by DLS ontain vesi les.

These were only found at higher pH values (above 5.5), whereas at low pH, dis s

and membrane pie es were the dominating form.

The results suggest it is possible to obtain vesi les in the region dened by the

two pKa; at ertain surfa tant ratios an even broader pH range was observed. This

345
is in a ordan e with results obtained by de Groot et al. for a bran hed monoalkyl

surfa tant with a malonate headgroup. A bluish zone was observed between the two

pKa values (the pKas of the two arboxylate groups on maloni a id are 2.85 and

345
5.70, respe tively) . In that region o-existen e of small unilamellar (SUV) and

multilamellar (MLV) vesi les was found. Above pH = 5.8 the solutions were lear

345
and only SUVs were observed by TEM .

Su h a high polydispersity of morphologies, where at dis s oexist in equilibrium

with spheri al open and omplete unilamellar vesi les, was previously observed in

atanioni systems. A possible explanation for the oexisten e of these stru tures

was proposed by Jung et al. by evaluating the parameters ontributing to the

351
spontaneous urvature . In some systems however (namely mixtures of anioni

128
Figure 7.5: Cryo-TEM images of a SL / AS mixed surfa tant solution at (left) pH = 4.5, 10
days after mixing, and (right) pH = 5.5, 5 days after mixing. The bla k arrows point to dis s,
the double arrows to a partially folded dis , whereas the white arrowheads to globular stru tures
(possibly mi elles). The white arrow points to an elongated stru ture.

Figure 7.6: The formation of small vesi les after 2 weeks in samples with pH = 6.7 and large
multilamellar vesi les after 4 weeks (pH = 5.5); arrowheads point to very small perfe t vesi les,
while the arrow points to an in omplete two-lamellar vesi le (`F' indi ates a frost parti le).

129
Figure 7.7: Phase behavior after 2 months: (a) small membrane fragments at low pH (= 4.5);
(b) destabilization of vesi les at high pH (= 6.7).

and zwitterioni surfa tants), dis oidal stru tures are only short-lived intermediates

352
in the mi elle-to-vesi le transitions . The vesi ulation depends on the balan e

between the unfavorable edge energy of the disks and the bending energy required

to form spheri al stru tures. In the ase of atanioni surfa tant mixtures, the edges

of the dis s are stabilized by the ex ess ationi surfa tant. It is possible that the

relatively bulky EO groups stabilize the dis s observed in our system.

7.4.4 Cytotoxi ity Potential of Surfa tant Mixtures

Akypo Soft 45 NV is ommonly used in osmeti formulations, su h as hair on-

353357
ditioners and hair dyes . Improving the bio ompatibility of produ ts used in

osmeti formulation even further is always sought after. Cytotoxi ity of our mix-

tures on HeLa ells and Keratyno ytes was measured in order to identify the toxi

properties of the two surfa tants for use in pharma euti al and osmeti al appli a-

tions. Cell viability was evaluated by the tetrazolium MTT redu tion assay, based

on the uptake and the redu tion of the soluble yellow MTT tetrazolium salt by

mito hondrial dehydrogenase to a blue insoluble MTT formazan produ t. The IC50

value was al ulated from absorban e data. HeLa ells were used, be ause they

reportedly show good reprodu ibility and a signi ant orrelation with in vivo re-

130
 324
sults , whereas Keratyno ytes were hosen to he k the skin ompatibility of su h

surfa tant mixtures.

120

100
[ g/mL]

80
50
IC

60

40
0.0 0.2 0.4 0.6 0.8 1.0

Akypo / SL

Figure 7.8: The mito hondrial redu tion of MTT after a 3-day in ubation of SL / AS mixture
at dierent surfa tant ratios with HeLa ells () and Keratyno ytes ().

IC50 values reported in Figure 7.8 show a dose-dependent de rease in toxi ity

when AS is ex hanged for SL. Therefore any surfa tant mixture formed with SL is

bene ial. The order of the IC50 values was the same for both ell lines; however the

absolute values were slightly lower in the ase of the Keratyno ytes. The lowering

of the pH of the surfa tant test solutions showed no pronoun ed ee t. The volume

of the added surfa tant solution was probably too small to hange the pH value of

the ell medium signi antly and the buering apa ity of the medium too large for

any ee ts to be observed.

7.5 Con lusions

Two obsta les mentioned in the introdu tion, namely, the high solubility temper-

ature of alkyl arboxylates and limited pH region of vesi le formation have been

131
over ome by using a mixture of two alkyl arboxylates with two very dierent pKa

values. The presented results show that it is then possible to spontaneously form

bilayer stru tures, su h as vesi les, in a pH range between the two onsidered pKa.

Sodium laureate (with a pKa around 8.5) and Akypo Soft 45 NV (with a pKa = 4.67)
ould be thus used at dierent mixing ratios to obtain vesi ular solutions over the

entire range of pH omparable to skin or physiologi al pH. Furthermore, we have

used ommer ially available and bio ompatible surfa tants that are already used for

osmeti purposes and are inexpensive, whi h is of signi ant importan e for appli-

358
ation purposes . However, more resear h is required to improve their appli ation

potential by in reasing the olloidal stability of fatty a id vesi les and assure the

81,359
vesi les are ompletely losed. Both are ommon problems in su h systems .

132
Con lusion

In this thesis the formation and stabilization of atanioni vesi les was studied.

Catanioni surfa tant mixtures were hosen, be ause they were shown to form vesi-

le spontaneously under ertain onditions and exhibit an enhan ed sensitivity to

outside parameters, su h as temperature or the presen e of salts. Due to this sen-

sitivity, atanioni mixtures are a good system to study spe i -ion ee ts. The

interest in vesi les was further motivated by their potential as models for biologi al

membranes and as en apsulation systems. To optimize the appli ations it is impor-

tant to have a general understanding of the interplay of intera tions between the

surfa tants and of the fa tors inuen ing the phase diagram of a mixed system.

First, the morphologi al transitions o urring in mixed surfa tant systems upon the

in rease of the ioni strength were explored. The transition of mixed ioni mi elles

to vesi les in a atanioni surfa tant solution, omprised of sodium dode ylsulfate

and dode yltrimethylammonium bromide, with an ex ess of the anioni omponent,

upon the addition of salt is des ribed in Chapter 2. A new type of intermediate

stru ture was found: a symmetri ally shaped spheri al super-stru ture, whi h we

named blastulae vesi le. A me hanism of formation for this type of super-stru tures

was proposed, suggesting the importan e of harge u tuation in the vesi le mem-

brane.

The spe i -ion ee ts on this system was further studied and ompared to one

where the sulphate surfa tant was repla ed by a arboxylate surfa tant (sodium do-

de anoate). No anion spe i ity was found in the anioni -ri h region of the phase

diagram for the added salts, whereas the nature of the ation was found to strongly

inuen e the riti al salt on entrations around whi h mi elles turn to vesi les. This

was explained by taking into a ount that in the present ase of negatively harged

133
vesi les, the ations a umulate in high on entration in the vi inity of the vesi le.

The observed ation spe i ity followed the lassi al Hofmeister series for ation

adsorption to sulfate headgroups. When alkyl arboxylates were present in solution,

the ations followed a reversed Hofmeister series. The ion spe i ity was qualita-

tively explained a ording to Collins' on ept of mat hing water anities. To this

purpose, the headgroup of an alkylsulfate had to be regarded as a haotrope and

the alkyl arboxylate as a kosmotrope. Based on MD simulations performed on the

aforementioned headgroups, a general `Hofmeister series of surfa tant headgroups'

is established in Chapter 3.

In Chapter 4, the study of ion-spe i ity was extended to the ationi -ri h region

of the phase diagram of the atanioni system. No vesi les were found, however,

the addition of salts produ ed a sphero ylindri al growth of the mi elles, markedly

dependent on the anion identity. The e ien y of the anions to elongate the mi-

elles ould again be explained by the Collins' on ept and the lassi ation of the

trimethylammonium surfa tant headgroup as a soft, polarizable entity.

The ee t of various additives on the stability of mixed surfa tant solutions of

sodium dode ylsulfate with etyltrimethylammonium bromide and with dode yltrimethy-

lammonium bromide was studied as a fun tion of time and is reported in Chapter 5.

The lifetime of vesi les and mi elles in these systems ould be ontrolled by varying

the omposition of the surfa tant solutions and by additives. Controlling the pre-

ipitation phenomena is of importan e for a large number of industrial pro esses,

where formulations need to be tuned. These spe i mixtures were shown to have

a solubility temperature below that of pure surfa tant solutions in the anioni -ri h

region. The stability of su h supersaturated solutions was in reased by in reasing

the per entage of the anioni surfa tant and shortening the hain of the ationi

surfa tant. The presen e of simple ele trolytes de reased, while the addition of

middle- hain al ohols in reased their stability. Experimental results suggested that

the destabilization and on urrent pre ipitation of the systems was not due to the

formation of bigger aggregates, but rather to a shift of the equilibrium between mi-

elles and monomers. The addition of low on entrations of middle- hain al ohols

resulted in the formation of vesi les in the atanioni systems at temperatures sig-

134
ni antly lower than the Krat temperatures of the parti ipating ioni surfa tants.

In Chapter 6 we evaluated the ytotoxi ity of single- hain ioni surfa tants and

atanioni mixtures using MTT on HeLa ells. It is important to onsider the tox-

i ity of parti ipating surfa tants when formulating new en apsulating systems for

either osmeti or medi al use. It was onrmed that anioni surfa tants gener-

ally exhibit higher IC50 values (lower toxi ity) than atanioni ones. The toxi ity

ould further be inuen ed by the hydro arbon hain length and the presen e of

polyoxyethylene groups. A general trend of in reasing toxi ity with in reasing hain

length and in lusion of polyoxyethylene groups was observed. A non-linear in rease

was observed as ationi surfa tants are added to anioni ones. A steep de rease

of the IC50 values is observed already at low fra tions of the ationi surfa tants,

suggesting that in potential drug delivery systems a high fra tion of the anioni

omponent is ne essary.

Seeing that the presen e of only a small amount of a ationi surfa tant in the

mixture resulted in a large in rease in its toxi ity, we fo used on a mixture of two

anioni and in osmeti formulation already ommonly used surfa tants. In hapter

7 we present a way to form vesi les in a mixture of two alkyl arboxylates with two

very dierent pKa values. We have shown that it is then possible to spontaneously

form vesi les in a pH range between the two onsidered pKa. Sodium laureate and

Akypo Soft 45 NV ould be thus used at dierent mixing ratios to obtain vesi ular

solutions over the entire range of pH omparable to skin or physiologi al pH.

135
136
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160
A knowledgements

From experien e I an tell you that these last pages of a PhD thesis are the most

widely read pages of the entire publi ation. It is here where you supposedly nd out

to what extent you have inuen ed the life and work of the author. While this may

be true to some extent, you have to weigh my verdi t with the disturbingly low level

of sanity left in this PhD andidate after several years of studying toxi mole ules,

redening the olor blue and sear hing for `berries' in aqueous surfa tant solutions.

The work des ribed in this thesis has been arried out under the guidan e of Prof.

Dr. Werner Kunz at the Institute for Physi al and Theoreti al Chemistry, University

of Regensburg. I would like to thank him for the trust he showed by letting me

work independently and the opportunity to ollaborate with a number of other

groups around the world. Furthermore, thanks to all of his various personalities

(the arabi one I am espe ially fond of ) for keeping us amused during lun hes and

long onferen e rides.

Part of the experimental work was performed at the Department of Pharma eu-

ti al Biology, University of Regensburg. Thank you to Prof. Dr. Jörg Heilmann,

Dr. Birgit Kraus, and Gabi Brunner for their patien e and trust they showed by

letting this near-theoreti ian work in their sterile environment.

Spe ial thanks to Dr. Didier Touraud for de orating our lab in pink olor and

making ammonia the ommonly used air freshener. Furthermore, Didier, thank you

for your en ouragement through the years; for knowing when to push me further and

when to ba k o and, espe ially, for always taking the time to dis uss new (mostly

razy) ideas.

The good basi s were developed already during my undergraduate studies. For

that, I am thankful to Prof. Dr. Ksenija Kogej, for tea hing me to pay attention

161
to detail and work in an organized fashion. An indispensable skill that was proven

ne essary to survive in the sometimes he ti half-Fren h institute.

No s ienti work is done in solitude. Three mi ros opists are responsible for

my newly found appre iation for (abstra t) art: Dr. Jean-Mar Verbavatz (CEA,

Sa lay), Dr. Mar us Dre hsler (University of Bayreuth), and Prof. Dr. Ishi Talmon

(Te hnion-Israel Institute of Te hnology). Thank you for your wonderful pi tures

and informative dis ussions.

I thank my oworkers that transformed the `supposed-to-be' serious work en-

vironment into a fun pla e (although it sometimes resembled more a loony-bin):

Alina for showing me the ropes from the rst day on, Chloé for her ability to turn

ba k time and make me feel like a kid whenever I'm in her ompany, Geli for the

smile I was greeted with every morning, Jeremy for his onversations on and o the

OrbiTrek.

To my traveling ompanions: Moj a & Neza. After the adventures we had,

oming ba k to work seemed like va ation. I am looking forward to all the ities

that still await us.

Thank you to Jorge Cham, the reator of PhD omi s, for helping me ght

insanity with irony.

To Daniel: Thank you for reading my thesis, listening to my endless omplaining,

helping me pi k out my interview suit, wiping the tears away when the reviewers

(wrongfully, of ourse!) reje ted my manus ripts and nursing me after my eye oper-

ation. You are my endless sour e of motivation and I am lu ky to have you to open

my eyes (despite my newly-found 150% vision) when needed.

To my father: thank you for en ouraging me on all my paths. I am still waiting

for that Vla hy & Vla hy arti le however ...

162
List of Publi ations

Published:
1-N. Vla hy, J. Dolen , B. Jerman, K. Kogej, Inuen e of Stereoregularity of the

Polymer Chain on Intera tions with Surfa tants: Binding of Cetylpyridinium Chlo-

ride by Isota ti and Ata ti Poly(metha ryli a id). J. Phys. Chem. B 2006 110,
9061-9071.

2-A. Renon ourt, N. Vla hy, P. Bauduin, M. Dre hsler, D. Touraud, J.-M. Ver-

bavatz, M. Dubois, W. Kunz, B. W. Ninham, Spe i alkali ation ee ts in the

transition from mi elles to vesi les through salt addition. Langmuir 2007 23, 2376-
2381.

3-N. Vla hy, D. Touraud, K. Kogej, W. Kunz, Solubilization of metha ryli a id

based polymers by surfa tants in a idi solutions. J. Colloid Interf. S i. 2007 315,
445-455.

4-N. Vla hy, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz, Role of the sur-

fa tant headgroup on the ounterion spe i ity in the mi elle-to-vesi le transition

through salt addition. J. Colloid Interf. S i. 2008 319, 542-548.

5-N. Vla hy, A. Renon ourt, M. Dre hsler, J.-M. Verbavatz, D. Touraud, W. Kunz,

Blastulae aggregates: New intermediate stru tures in the mi elle-to-vesi le transi-

tion of atanioni systems. J. Colloid Interf. S i. 2008 320, 360-363.

A epted:
6-N. Vla hy, A. F. Arteaga, A. Klaus, D. Touraud, M. Dre hsler, W. Kunz, Inu-

en e of additives and ation hain length on the kineti stability of supersaturated

atanioni systems. Colloids Surf., A 2008.

7-N. Vla hy, C. Merle, D. Touraud, J. S hmidt, Y. Talmon, J. Heilmann, W. Kunz,

163
Determining the delayed ytotoxi ity of atanioni surfa tant mixtures on HeLa

ells. Langmuir 2008.

Submitted:
8-N. Vla hy, M. Dre hsler, D. Touraud, W. Kunz, Anion spe i ity inuen ing

morphology in atanioni surfa tant mixtures with an ex ess of ationi surfa tant.

Comptes rendus Chimie A adémie des s ien es 2008.

9-N. Vla hy, B. Jagoda-Cwiklik, R. Vá ha, D. Touraud, P. Jungwirth, and W. Kunz,

Hofmeister series of headgroups and spe i intera tion of harged headgroups with

ions. J. Phys. Chem. B 2008.

10-N. Vla hy, D. Touraud, J. Heilmann, W. Kunz, Determining the delayed yto-

toxi ity of atanioni surfa tant mixtures on HeLa ells. Colloids Surf., B 2008.

164
List of Oral and Poster Presentations

Oral Presentations:
09/2007 21 Conferen e of the European Colloid and Interfa e So iety, Genève,
th

Switzerland. Blastula vesi les: Formation of regular patterns through se ondary

self-assembly of atanioni vesi les.

Poster Presentations:
08/2005 29. International Conferen e of Solution Chemistry, Portoroz, Slove-

nia.

Mixed Solutions of Isota ti Poly(metha ryli a id) and Cetylpyridinium Chloride

in water.

08/2006 1st European Chemistry Congress, Budapest, Hungary.

Solubilization of Hydrophobi Polymers with Surfa tants.

09/2007 21th Conferen e of the European Colloid and Interfa e So iety, Genève,
Switzerland.

Ee t of hydrophobi  ations on vesi ular self-assembly.

11/2007 Formula V, Potsdam, Germany.

Inuen e of additives on the stability of (eute ti ) supersaturated atanioni sys-

tems.

165
Herewith I de lare that I have made this existing work single-handed. I have only

used the stated utilities.

Regensburg, June 2008

Nina Vla hy

166