bile salts form lyotropic liquid crystals

Bile salts form lyotropic liquid crystals

H. Amenitsch a,1, H. Edlund b,2, A. Khan c, E.F. Marques c,d, C. La Mesa e,*a Institute of Biophysics, Austrian Acad. Sci., University of Graz, Austria

b Department Chemistry Proc. Technol., Mid Sweden University, Sundsvall, Swedenc Department Phys. Chemistry I, University of Lund, Lund, Sweden

d Centro de Investigacao en Quimica, Dept. Chemistry, Faculty of Science, University of Porto, Porto, Portugale Department Chemistry, Universita Degli Studi di Roma La Sapienza, P. le Aldo Moro 5, Rome 00185, Italy

Received 28 January 2002; accepted 15 July 2002

Abstract

A reinvestigation of the phase diagrams relative to some conjugated and non-conjugated bile salts in water has

demonstrated the formation of lyotropic liquid crystalline phases, in contradiction with generally accepted statements.

The phase behaviour is complex and the phase diagrams are unusual, compared to most surfactants and lipids. In

particular, coexistence of liquid crystalline phases with crystals has been observed. The formation of liquid crystalline

phases requires very long equilibration times and the thermal stability of the lyotropic phases is moderate. The observed

structure is tentatively assumed to be of the reverse hexagonal type. Structural relations with currently accepted models

for the organisation of bile salts into micelles and solid form have been found.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Bile salts; Micelles; Liquid crystals; Phase diagrams; NMR; SAXS

1. Introduction

Bio-compatible surfactants are the subject of

widespread interest, because of their possible use

in the formulation of pharmaceuticals and other

topics. Among many such agents, interest is

devoted to surfactants that are metabolic by-

products. Bile salts (BS) are extremely interesting

in this regard, being the main metabolites of the

cholesterol pool in vivo. Interest toward such

compounds is reflected from studies on their

solution properties [1�/6] and on the solvent

capacity with respect to sterols, glycerides and

lipids [7,8].

The peculiarity of BS as surface-active agents is

due to their molecular structure. Surfactants and

lipids show a clear separation between the polar

region (containing the head-group) and the non-

polar one (consisting of flexible long alkyl chains)

[9]. The hydrophobic part of BS, conversely,

consists of a non-planar, rigid, steroid ring con-

taining up to three hydroxyl groups [10]. The OH

* Corresponding author. Tel.: �/39-6-4991-3707; fax: �/39-6-

490-631

E-mail address: [email protected] (C. La Mesa).1 Provisional address: Department Chem. Eng., University

of Delaware, USA.2 Provisional address: SAXS Beam-line at Elettra, Basoviza,

Trieste, Italy.

Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 79�/92

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groups lie in the same plane and the steroid ring,

thus, bears polar and non-polar sides, as indicated

in Fig. 1. In addition, naturally occurring bile

acids are conjugated with amino-acids such as

taurine and glycine, through a peptide bond; hence

the name tauro, or glyco, derivatives.

The rigidity and two-faced character of BS

imply that their packing into supra-molecular

structures does not follow the conventional fea-

tures peculiar to surfactant aggregation. Several

supra-molecular association models were pro-

posed [11�/13] and even the definition of BS

micelles was questioned for a long time [14,15].

BS aggregate and form micelles of small aggrega-

tion numbers, typically Nagg:/2�/10, which may

noticeably increase as a function of concentration

and/or added electrolyte [5,6,11,16]. In some caseslarge BS micelles are formed by a helical arrange-

ment of steroid units [17,18]. In the NaDC�/water

system, for instance, fibre-like structures can be

drawn from concentrated micellar solutions

[19,20].

BS are incorporated into liquid crystalline

phases [21�/23], but no liquid crystalline phases

in water have been observed up to now. This facthas been rationalised as an example of their

peculiar associative behaviour. According to an

authoritative scientist ‘‘. . .conjugated and free BS

form micelles, but not liquid crystals. . .’’ [24].

Indeed, BS form liquid crystalline phases in water

[25,26].

The occurrence, width and thermal stability of

such phases depend on: (i) the nature of the polarhead group; (ii) the number and position of OH

groups in the steroid skeleton; (iii) temperature;

(iv) concentration, and (v) ionic strength.

The formation of lyotropic mesophases in BS�/

water systems is peculiar. The structure of the

phases, the macroscopic phase appearance, the

observed phase sequence and the equilibrium

conditions between coexisting phases are signifi-cantly different from those observed in systems

containing surfactants and/or lipids [9,27].

The present investigation is focused on the

phase behaviour of binary mixtures containing

water and sodium tauro-cholate (NaTC), sodium

tauro-deoxycholate (NaTDC), sodium tauro-che-

nodeoxycholate (NaTCDC), sodium glyco-deox-

ycholate (NaGDC), and sodium deoxycholate(NaDC). The majority of such compounds have

two OH groups facing outwards from the steroid

nucleus. The cheno-deoxy derivative differs from

the deoxy ones in the position of OH groups: aC-3

and aC-7, compared to aC-3 and aC-12. Tauro-

cholate has three hydroxyl groups, in aC-3, aC-7

and aC-12 positions, respectively.

The packing of BS into organised lyotropicmesophases is the subject of this study. Some

results on the above systems, the ones relative to

optical polarising microscopy and preliminary

small angle X-ray scattering data (SAXS), have

been reported elsewhere. Here we report on more

recent findings and discuss some aspects which

have not yet been considered in detail. We focus

Fig. 1. Chemical structure of cholanic acids, indicating the

position of OH groups in 3a, 7a and 12a, in the top. The front

view and cross-section of BS anions are schematically reported

in the lower part of figure.

H. Amenitsch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 79�/9280

on the thermal stability of the phases and on linksbetween current models proposed for the forma-

tion of BS micelles and structure of the lyotropic

phases.

Apart from interest on a biologically relevant

subject, there are practical consequences of BS

organisation in mesomorphous phases, which

imply the use of such liquid crystalline matrices

in the topical administration of pharmaceuticals.

2. Experimental section

2.1. Materials

Basel, Switzerland, supplied D2O, 99.95% iso-

topic purity. Conductivity water (k close to 1 mS,at 25 8C) was used.

Mono-hydrate NaDC, nominal purity �/99%;

NaTC �/97%; emi-hydrate NaTDC �/97%;

NaTCDC, high purity; and NaGDC, containing

1.5 mol H2O/mol �/98%, were from Aldrich.

According to conductivity and surface tension

experiments, the samples are appreciably pure and

have critical micellar concentration values (cmc),very close to those reported in the literature [11].

The pH of BS solutions does change much with

composition. In particular, the pK of tauro (and

glyco) derivatives of bile acid salts is close to 2 [28].

In the case of NaDC, some evidence of hydrolysis

was observed at very low concentrations [29].

Perhaps, use of Tris, phosphate or other buffers

in studies on BS in solution has been questioned,because of the hydrophobic character of hydro-

xymethyl aminomethane ion and, more generally,

of the ionic strength effect on cmc values, micelle

aggregation numbers and counter-ion binding

[30�/33]. In other words, buffers stabilise the

hydrolysis, but have a non-negligible effect on

micelle size and shape, and, presumably, on the

phase diagram.

2.2. Sample preparation and phase diagram

determination

The samples were prepared by weight into glass

tubes, which were flame-sealed and mixed by

centrifugation. They were allowed to stay at

50 8C for, at least, 2 days. Thereafter they wereequilibrated at 20 8C for several weeks. Prelimin-

ary studies of the phase behaviour involved the

inspection between crossed Polaroids.

2.3. Polarised light microscopy

An Axioplan Universal polarising light micro-

scope (Zeiss) equipped with a SITC video-cameraand image processor Argus-20 (Hamamatsu

Photonics, Japan) was used. The microscope is

equipped with a hot stage, operating from 0 to

100 8C. Additional experiments were performed

with a Ceti optical polarising microscope,

equipped with an ME Super camera (Pentax)

and a Linkam unit, TP93, equipped with an HFS

91 heating stage (Linkam). Samples to be investi-gated as a function of temperature were sealed in

thin glass capillaries or between glass sheets for

optical microscopy.

2.4. Electrical conductance

A Wayne-Kerr bridge, mod.6425, was used. An

immersion-type conductivity cell was merged inthe sample, previously melted, and the system was

equilibrated at 20 8C for the required time. The

samples were located into a Hetofrig CB IIe

thermostatic unit and the thermal scan speed was

set at 0.1 (or 0.03) 8C min�1. Phase transitions

were inferred from significant changes in slope in

the conductivity versus temperature plots [34].

More precisely, the transition temperature is thepoint at which (@k /@T ) is a maximum and (@2k /

@T2)�/0.

From ionic conductivity the width of the two-

phase region can also be evaluated. It can be

demonstrated that such quantity is related to the

projection on the temperature axis of the segment

connecting the two branches of the curve. A

significant thermal hysteresis (over 10 8C, insome instances) was observed on cooling.

2.5. Adiabatic compressibility

Density, r , was measured by an Anton Paar

DMA 60 unit [35]. The uncertainty on such values

is to 5�/10�6 g cm�3 when the temperature is

H. Amenitsch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 79�/92 81

constant to 9/0.003 8C. Sound velocity, v (in cms�1), was measured at 5 MHz by a 2 cm3 home

built resonant cell, connected to a Matec 750 unit.

The uncertainty on sound velocity values is in the

range 0.5�/1 m s�1. The accuracy on adiabatic

compressibility values, b�/(rv2)�1, is better than

0.01% of the measured value. Details on the

experimental set-up and measuring procedures

are reported elsewhere [36].

2.6. Small angle X-ray scattering (SAXS)

Measurements were carried out in a Kratky

system, equipped with a position-sensitive detec-

tor, containing 1024 channels of width 53 mm. A

Cu�/Ka radiation of wavelength 0.1542 nm was

used and the sample-to-detector distance was fixedto 277 mm. The samples were located in a paste

holder between thin mica windows. The tempera-

ture in the camera was regulated by a Peltier

element (to 9/0.1 8C) and the chamber was kept

under vacuum, to minimise air scattering. Addi-

tional experiments, including those performed as a

function of temperature, were performed at the

SAXS Beam-line in Elettra, Trieste [37].

2.7. 2H (deuterium) quadrupolar splitting

2H has a spin quantum number I�/1 and

possesses an electric quadrupole moment. In

anisotropic liquid crystalline phases the interaction

of the quadrupole moment with the electric field

gradients is not averaged to zero and gives rise to a

splitting. The magnitude of the 2H quadrupolesplitting depends on the phase anisotropy. In poly-

phasic systems, the spectrum is a superposition of

those of the individual phases.

In lyotropic systems, water binds to the surfac-

tant head-group. In fast exchange regime between

the free state and the binding sites a linear relation

between splitting and solute volume fraction is

obtained, from which hydration and phase struc-ture are inferred [38].

Spectra were recorded at 41.45 MHz in a JEOL

FX 270 pulsed FT NMR spectrometer. A control

unit regulates the temperature to 9/0.2 8C. The

samples were equilibrated for long periods (usually

around 40 min), to avoid the occurrence of

temperature gradients. Spectra were repeated untilno changes were observed. In some instances up to

several weeks were required to get constant quad-

rupole splitting values. In all cases we have

investigated, the deuterium quadrupole splitting

does not give powder pattern spectra.

2.8. Pulsed field gradient (PFG) NMR self-

diffusion

The PFG technique is based on the combination

of a radio-frequency (rf) sequence and applied

magnetic field gradient pulses. The sequence con-

sists of two rf pulses (a 908 and a 1808 one) and

two gradient pulses with time duration d and

separation D between their leading edges. They are

located on either side of the 1808 rf pulse. The self-diffusion coefficient is determined by the decay of

a FT signal (at the centre of the echo attenuation)

as a function of the strength, or duration, of the

gradient pulse [39].

Measurements were performed by a Bruker

DMX200 spectrometer, at 200 MHz, with a probe

providing a maximum field gradient of 9 T m�1.

The accuracy on self-diffusion findings is to 9/2%of the measured value.

3. Results

3.1. Phase diagrams

The phase diagrams were constructed by visual

observation, optical polarising microscopy, NMRmethods, electrical conductance and SAXS. A

univocal assignment of the different phases and

the definition of the width of the two-phase

regions is not straightforward. In that sense, the

polymorphic character of BS is still elusive.

The kinetics of liquid crystal formation is slow.

Days to months are required to observe the

formation of liquid crystalline matter, in properexperimental conditions. The kinetics of meso-

phase nucleation depend on temperature. For

instance, in the water�/NaTDC system, the forma-

tion of anisotropic phases occurs in 3 days when

the samples are kept at 20 8C and about 2 days at

25 8C. The effect is reproducible.


An overview to the phase diagrams, reported in

Fig. 2(a�/e), indicates that the formation of a

solution region, as well as of anisotropic liquid

crystalline phases, is ubiquitous. Regions with

hydrated crystals are also observed in all systems.

Differences concern the region of existence of the

phases, the width of the two-phase regions, the

thermal stability and the kinetics of phase forma-

tion.

The two-phase region on the left-hand side of

the liquid crystalline phase in the water�/NaTDC

system has some remarkable features. Some sam-

ples are lyotropic dispersions, in others a weakly

anisotropic upper phase, forming in some days, is

Fig. 2. Temperature versus weight percent phase diagrams for different bile sodium salt�/water systems. In (a) the NaTCDC�/water,

(b) the NaTDC�/water one, (c) the NaGDC�/water system, (d) the NaDC�/water, and (e) the NaTC�/water system.


observed. From this phase, spherulitic crystals

grow and sediment. With time crystals begin

coming into the upper phase. Finally, the liquid

crystalline phase is formed. The kinetics of phase

transformation is sketched in Fig. 3. Each transi-

tion requires hours to days and the kinetic scheme

is reproducible.NaTC�/H2O and NaTDC�/H2O systems show

well-defined phase boundaries, with occurrence of

single and two-phase regions. NaTDC samples

display a monochromatic weak birefringence be-

tween crossed Polaroids. The NaDC�/H2O,

NaCDC�/H2O and NaGDC�/H2O systems, con-

versely, show a more complex phase behaviour,

with occurrence of a narrow liquid crystalline

region. In the case of the NaDC�/H2O system

some hydrolysis may occur [29]. This effect,

perhaps, is traceless in the phase behaviour. In

terms of the phase rule, in fact, the NaDC�/H2O

system is to be considered a two-component

system even in the presence of a reaction betweenthe components.

A wide two-phase region is present in all cases.

In some weeks, single-phase anisotropic samples

are formed. No such effects have ever been

reported in surfactants, or lipids. The coexistence

of crystals with micellar solution and/or lyotropic

phases and the slow kinetics of phase transforma-

tion are peculiar to all BS investigated here.Some comments need to be made:

(i) The formation of transient spherulitic crys-tals is common to all BS. It can be ascribed to

the occurrence of meta-stable liquid crystallites

or to nucleation processes, required for the

formation of the liquid crystalline phases.

(ii) The thermal stability of the mesophases is

moderate. The most stable liquid crystalline

phase, the one observed in the NaTDC�/water

system, disappears at temperatures close to35 8C (Fig. 4). In all systems, a significant

thermal hysteresis is observed on cooling.

(iii) The isotropic phase observed at concentra-

tions above the liquid crystal one is assumed to

be, in a first approximation, a micellar solution.

Such viscous isotropic phase (flowing under

gravity) is always in equilibrium with crystals.

Samples in this region resemble cubic liquidcrystalline phases in their consistency and

macroscopic phase behaviour. The possibility

of the occurrence of an isotropic liquid crystal-

line phase cannot be ruled out, but efforts to get

single-phase samples were unsuccessful.

(iv) Optical polarising microscopy textures in-

dicate that the size of the lyotropic crystallites

(domains) is small [25,26,40].

3.2. Self-diffusion

NMR self-diffusion measurements were per-

formed in the NaTC�/water and NaTDC�/water

systems, representative of tri-hydroxy and di-hydroxy salts, respectively. Water self-diffusion

in the two systems is nearly the same (Fig. 5) when

the surfactant self-diffusion is markedly different,

as indicated in the lower part of Fig. 5. Self-

diffusion values of NaTDC are systematically

lower than those of NaTC.

Fig. 3. The kinetics of phase transformation in BS�/water

systems, at :/20 8C. The kinetic processes reported in the

scheme can be long days, in NaTC�/H2O and NaTDC�/H2O

mixtures, or months, in the NaDC�/water system.


In the concentration range we have investigated

(well above the cmc) the monomer contribution to

the overall self-diffusion is negligible [41]. In fact,

the observed self-diffusion is a weight average

contribution of molecular and micellar species,according to [42,43]

Dexpt�Dmon(CMC=Ctot)

�Dmic(Ctot�CMC=Ctot) (1)

where Dmon and Dmic indicate the self-diffusion of

molecules and micelles, respectively.

[N.B. At low surfactant content, BS form

dimers, trimers, etc, in considerable amounts,

depending on the number and position of the

OH groups in the steroidal skeleton [18]. In that

case Dexpt�/(1/Ctot) Si�1DiCi , which reduces tothe two-site approximation in Eq. (1) at high

surfactant content.]

In the present experimental conditions, c �/cmc,

we can safely assume that the observed values are

ascribed to micelle diffusion. Self-diffusion values

of NaTDC micelles are systematically lower than

those of NaTC ones. Estimates of the solute

hydrodynamic radius were made through theStokes�/Einstein equation. At about 0.4 molal

BS, (approximately 20 wt% of the steroidal

surfactant), NaTDC micelles have hydrodynamic

radius of 39 nm, when NaTC ones are only 5.4.

Accordingly, di-hydroxy BS micelles grow much

more steeply than tri-hydroxy ones. Sodium cho-

late, for instance, forms smaller micelles than

deoxy- and chenodeoxycholate, and NaTDC

shows a pronounced micellar growth compared

to NaTC [11,16].

3.3. Liquid crystal formation

Optical anisotropic textures were observed bypolarising microscopy, as mentioned above. In

some instances, battonets and striated

non-geometric textures appear. The NaGDC�/

H2O system shows weakly birefringent and ill-

defined textures of the non-geometric, non-striated

type [40].

Attempts to separate the spherulitic crystals

occurring in biphasic samples were made and giantfibrous structures are observed when the bottom

phase is separated from the isotropic solution. In

some instances non-geometric and non-striated

textures are mixed with fibre-like ones. Blow and

Rich described optical textures indicating the

occurrence of helical-based fibres [19,20]. We do

not know, at this stage, whether the structure of

the liquid crystalline phase is directly related tosuch fibres.

A strong similarity has been observed by some

of us between such textures and those of fibres

drawn from concentrated micellar solutions [44].

Due to the small size of the lyotropic textures,

mentioned above [25,26], it is not easy to give a

Fig. 4. Electrical conductance, k (mS), versus temperature, in 8C, for a 46.7 wt. NaTDC�/water mixture (^), and for a 58.4 wt.

NaTC�/water one (k). The coexistence of solution and liquid crystalline phases occurs in the region where a steep decrease of

conductivity is observed. The slope of k of as a function of T is nearly the same in solution and in liquid crystalline phase. A significant

thermal hysteresis, indicated by the dotted line, is observed on cooling.


univocal phase assignment solely from optical

polarising microscopy. That is why many experi-

mental methods are required to characterise such

systems.

In two-phase regions the 2H NMR quadrupole

splitting superimposes isotropic signals. In single

phase regions most samples show broad lines (Fig.

6), but splitting is small, if any. This might be due

to the very small bond order parameter of hydra-

tion water [42,43,45], i.e. to a small anisotropy.

The signal width indicates also that fast exchange

conditions between free and bound water are not

fulfilled, presumably because the lyotropic micro-

domains are small. This hypothesis is in good

agreement with optical polarising microscopy

findings.

We do not consider, in this context, exchange

between OH (or NH) protons and water deuter-

ons. If present, they should give rise to large

quadrupole splitting effects, several kHz wide,

which are not observed in the present experimental

conditions. Please note that the very small quad-

Fig. 5. (a) Water self-diffusion, m2 s�1, as a function of BS weight percent in the NaTC�/water system (^), and in the NaTDC�/water

one (k), at 20 8C. (b) The BS self-diffusion, m2 s�1, in the NaTC�/water (k), and NaTDC�/water system (^), as a function of BS

weight percent, at 20 8C. Data are in logarithmic scale.


rupole splitting reported here (some hundred Hz

wide) rules out such possibility.

Structural length scales in lyotropic liquid

crystals are in the range 1�/50 nm and SAXS, or

SANS, can be used. Phases possessing long-range

order, (lamellar, hexagonal, cubic etc.), show well

defined Bragg peaks. A hexagonal phase, for

instance, is built up of long cylinders packed in a

2-D hexagonal array [46]. The Bragg peaks posi-

tion of the hexagonal phase is 1, �3, �4, �7, �9,

corresponding to the (10), (11), (20), (21) and (30)

(hk) reflections, respectively. The hexagonal cylin-

der radius and the interfacial area per molecule

can be calculated from the main spacing, taking

into account the solute volume fraction [27,47].

Diffraction patterns observed in the NaTDC�/

water system (1:1.73:1.89) are close to those

expected for hexagonal phases. Up to five reflec-

tions were observed, with peaks in the order

1:1.75:2.00:2.77:3.09. A selected example of

small-angle scattering findings is reported in Fig.

7.

A few SAXS results are reported in Table 1. As

can be seen there, the spacing is consistent, in a

first order approximation, with a hexagonal order,

or a tetragonal (distorted hexagonal) one. It has

been recently demonstrated that structures in solid

NaTDC and in the corresponding fibres are not

perfectly monoclinic [48]. The same behaviour

could also be observed in liquid crystalline phase

and could be partly responsible for departures

from the typical hexagonal structure. It is well

known, in this regard, that the structure of liquid

crystalline phases made up by rod-like aggregates

may show differences from the canonical hexago-

nal behaviour, depending on composition, struc-

ture of the surfactant, etc. [9,27].

Fig. 6. 2H NMR spectra of heavy water, at 15 8C, for NaTCDC�/D2O, (58.1, 63.2 and 65.2 wt., from the top), left, NaDC�/D2O,

(30.0, 32,8, 34.6 wt. from the top), centre, and NaGDC�/D2O systems, (39.2, 41,1, 40.8 and 43.2 wt., from the top). A bar 500 Hz wide

indicates the quadrupole splitting.

Fig. 7. SAXS patterns, reporting the signal intensity, I , in

arbitrary units, versus q , in A�1, for a 47.3 wt. NaTDC�/water

sample, at 25 8C. The patterns remain nearly constant up to the

phase transition temperature, about 35 8C.


Attempts to calculate area per molecule from X-

ray findings were made. The most reasonable

values were obtained by assuming the hexagonal

liquid crystalline phase to be of the reverse kind.

Strong support in favour of this tentative assign-

ment comes by the fact that no voids in the rods

are possible in the case of reverse hexagonal

phases. It must be kept in mind, in this regard,

that it is difficult to pack the steroidal skeleton of

BS into conventional micelles, either spheres or

rods.

The tentative hypothesis we suggest can be

considered quite exotic. Perhaps it finds support

in some models proposed by Giglio and co-work-

ers [49�/51]. According to his suggestions, long

rod-like aggregates based on a helical arrangement

of BS may form in solution. It is obvious to

forecast the formation of anisotropic structures

when the concentration of micellar surfactant

exceeds a certain value.

SAXS and NMR data, thus, indicate the

occurrence of non-conventional aggregates. The

observed spacing bears structural connections with

the long rod-like aggregates and fibres, for which a

helical arrangement has been proposed [19,20,49�/

51]. Keeping in mind the location in the phase

diagram, we assume the occurrence of reverse

phases, formed by water cylinders arranged in a

non-polar continuum (Fig. 8). This is in fair good

agreement with what is stated above. In addition,

recent findings by some of us show the occurrence

of some links between the size of micelles, crystals,

fibres and liquid crystalline rods [44].

According to X-ray diffraction, NaTC has the

smallest cylinder radius and the largest interfacial

area per molecule (0.65 nm2), as is to be expected

from the presence of three OH groups (Table 1).

NaTCDC has a large interfacial area compared to

other di-hydroxy salts, because the OH groups in

aC3 and aC7 do not pack as tightly as those

located in aC3 and aC12 positions. The trend

observed from interfacial areas, NaTC�/

NaTCDC�/NaTDC]/NaDC, is in agreement

with the salt solubility sequence.

Table 1

SAXS measurements in liquid crystalline samples of selected BS�/water systems, at 20 8C

Salt Weight percent Spacing (A)a Nearest-neighbour distance (A) Cylinder radius (A) Area per molecule (A2)

NaTC 59.9 37.0 42.7 16.3 65

NaTDC 40.0 51.5 59.5 26.3 40

NaTCDC 43.6 41.9 48.4 20.9 51

NaDC 39.1 46.9 54.2 23.4 45

N.B. Areas were calculated assuming a reverse hexagonal structure. The dependence on composition is moderate.a Obtained from the (100) reflection.

Fig. 8. Arrangement of BS into a reverse hexagonal structure.

In the top is reported the structure of the cylinders, below the

top view of the aggregates. Cylinders can be tilted with respect

to the z -axis or may be arranged in a tetragonal (distorted

hexagonal) structure. The area indicated by a dotted arrow

indicates the preferred orientation of the polar regions in the

aggregates.


Temperature has a puzzling effect on spacings.

According to Luzzati [46], the spacing regularly

decreases with T . In the NaTDC system, con-

versely, the spacing increases, or decreases, with

temperature, depending on composition, i.e. on

the sample location in the phase diagram. In other

words, liquid crystalline phases swell, or shrink,

depending on composition. In the case of the

NaTDC�/water system, for instance, swelling is

observed at relatively low concentration, close to

the micellar solution side.

The ionic conductivity of liquid crystalline

phases increases with temperature (Fig. 4). The

slope of k (T ) is nearly the same in liquid crystal

and in solution phase. This implies that ion

transport mechanisms in liquid crystalline and

solution phases are nearly the same. In the

water�/NaTDC system the transition temperature

from liquid crystalline to solution phase is 35 8C(Fig. 4) or lower. Presumably, such transition is

related to physiological requirements, to circum-

vent dis-metabolic diseases.

Adiabatic compressibility measurements, per-

formed in concentrated BS micellar solutions

(Fig. 9), indicate the occurrence of significant

changes in the solution structure at about 35 8C.

The effect may be ascribed to the collapse of

hydrogen bonding networks or to a significant

reduction of hydrophobic interactions at physio-logical temperatures.

3.4. Molecular packing

The formation of supra-molecular aggregates is

ubiquitous in aqueous BS systems. Very peculiar

packing modes into micelles, fibres, liquid crystalsand solid form have been observed and proposed

[12,14,18,44]. In water BS form micellar aggre-

gates, becoming progressively anisometric on in-

creasing the surfactant concentration and/or the

medium ionic strength. Generally, such aggregates

are formed by a helical arrangement of BS anions

and are stabilised by intermolecular hydrogen

bonding [49]. Significant differences have beenobserved between aggregates formed by di- and

tri-hydroxy BS, respectively. The latter, usually,

form small aggregates. It has been demonstrated

also that BS packing at polar�/apolar interfaces in

mixed micelles is perpendicular for di-hydroxy and

flat for tri-hydroxy salts, respectively [52].

If NaTDC micelles are larger and grow faster

with concentration than NaTC ones, the forma-tion of liquid crystalline phases should obey the

same rules, as, indeed, is observed. As to the

packing in liquid crystalline form, slight differ-

ences have been found between the different

Fig. 9. Dependence of adiabatic compressibility, b , in g�1 cm s2, on temperature, in 8C, for 35.7 wt. NaTC mixtures. The

discontinuity occurs at T values close to the phase transition temperature observed in the adjacent liquid crystalline phase.


systems. Similarities can also be found betweenpacking in solid and in liquid crystalline form.

4. Discussion and conclusions

BS form liquid crystalline phases in water.

Attempts to rationalise the observed behaviour

can be made on the basis of the salt solubility

sequence, in turn dependent on chemical structure.Tri-hydroxy salts are more soluble than di-hy-

droxy ones and taurine (or glycine) conjugates

more soluble than free salts. Cheno-deoxy deriva-

tives are slightly more soluble than deoxy ones. All

these observations are reflected in the cmc and in

the maximum solubility limit in micellar form. The

observed sequence is TC�/TCDC:/GDC�/

TDC�/DC. The width of the mesomorphousphase for the di-hydroxy salts, conversely, is in

the order TDC�/TCDC�/GDC:/DC. On these

grounds, an increasing head-group hydrophilicity

favours the formation of liquid crystalline phases.

The effect played by the number and position of

OH groups in the phase behaviour is considerable.

Changing the OH group position from aC-12 to

aC-7, for instance, implies a lower thermal stabi-lity of the lyotropic phase. In general, the chemical

structure of BS plays an intricate role in the

aggregation processes, since it is related to the

packing peculiarities of the different BS. This is

not surprising, since electrostatic, hydrophobic

and intermolecular hydrogen bond interactions

play a role in the structure of micelles and,

consequently, in the self-assembly of aggregatesforming liquid crystalline phases.

The thermal stability of such liquid crystalline

phases is small and a weak Gibbs energy difference

between the isotropic solution and the liquid

crystal phase is expected. The thermal stability of

the liquid crystalline phases increases with increas-

ing the hydrophilic character of the head-group.

This fact can be put in evidence by relating thebehaviour predicted by the above lyotropic scale

to the width of the liquid crystalline phases for the

different phase diagrams.

The kinetics of phase transformation can vary

significantly from system to system. Birefringent

textures form in 1�/3 days in the NaTDC�/water

system, but months are required to get the sameresults in the NaDC�/water one. The very long

time required to have equilibrium structures is a

qualitative indication that the difference in ther-

modynamic stability between the different states of

organisation is small.

The formation of anisotropic textures from the

dispersed solid and the occurrence of transient

spherulitic crystals is common in all BS�/watersystems. Such unconventional nucleation effects,

the long equilibration times and the puzzling

temporal sequence of phase transformations,

may have led most authors to circumvent the

occurrence of liquid crystalline phases.

The liquid crystalline phases are, presumably,

built up of reverse micellar aggregates. Structural

links between the reverse hexagonal phase and therod-like helical aggregates proposed for the iso-

tropic phase are reasonable. Above the liquid

crystal phase border extremely viscous phases

occur; it is not easy to ascertain whether they are

true solutions or else.

Some remarks need to be made on the coex-

istence of solid with liquid crystalline phases.

Lyotropic liquid crystals formed by soaps, surfac-tants and lipids require a liquid-like state of the

flexible chains [53]. Obviously, this statement does

not hold for BS. It is difficult to reconcile the

proposed organisation of bile acid salts in terms of

structures proposed for surfactants and lipids.

Comparison could be made with zwitterionic

surfactants having a steroidal nucleus as hydro-

phobic group. It is well known, in this regard, thatCHAPS and CHAPSO aggregate in stacks [54],

but no evidence of liquid crystal formation is

available.

Let us compare the behaviour of BS with a class

of planar molecules forming lyotropic phases, the

chromonics (a family of substances including

drugs, dyes and some antibiotics) [55]. They

consist of aromatic ring structures, with polargroups facing from the ring, i.e. polar and non-

polar regions are not clearly separated. In terms of

the lyotropic behaviour, thus, BS occupy an

intermediate position between chromonics and

flexible alkyl chain surfactants.

According to the present findings, liquid crystal

formation does not imply any particular physical


state for the molecule. It is the occurrence ofpositional order of (and in) the domains which

determines the occurrence of liquid crystalline

order for BS.

Acknowledgements

The Swedish Research Council for Engineering

Sciences (TFR) and PRAXIS XXI, Portugal

(project 2/2.1/QUI/411/94) are acknowledged for

financial support. C.L.M. thanks the Departmentof Physical Chemistry 1, at Lund, for a Visiting

Professorship. The administration of SAXS Beam-

line at Elettra is acknowledged for time machine

facilities. Thanks are due to Professor Edoardo

Giglio, La Sapienza University, for suggestions on

the structural relations between SAXS data of

liquid crystalline phases and X-ray diffraction

observed in solution, in solid form or in fibres.

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bile salts form lyotropic liquid crystals

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