di?%!

LA R

LIQUIDS
ELSEVIER

Dynamica.

JoumalofMolecularLiquids69(1996)

a,nd structura,l

properties

161-181

of wa.ter/a,lcohol mixtures

G. Onori and A. Santucci

Dipartimento di Fisica, Universitb di Perugia
I-06100 Perugia (Italy)
Abstract

Our recent studies related to the properties of alcohol/water mixtures, show the
occurrence of some kind of molecular aggregation in the water rich region of composition
beyond a threshold alcohol concentration xi. The observed behaviour suggests that
for 22 < zf the alcohol molecules are essentially dispersed and surrounded by water
cages where the short range order and microdynamic of water molecules are changed
with respect to those of pure water. Alcohol molecules are in mutual contact at higher
concentration,
only, when almost all water is involved in hydration shells of alcohol
molecules. In addition, these studies show that the stabilisation of micellar structure and
nucleic acids conformation in water/alcohol mixtures is closely linked to the properties
and anomalous behaviour of the solvent systems. Accordingly, these results support the
hypothesis that the dominant mechanism by which an alcohol affects these processes is
through its effect on the structure of water.
The main results of these investigations are reviewed and discussed in this paper.
1.

INTRODUCTION

Alcohol molecules contain both hydrophilic groups and hydrophobic tails. This
unique duality towards an aqueous environment leads to a complex self-association
behaviour which is not exhibited in non-aqueous solvents. The solution behaviour of
alcohol molecules in the water rich region is largely established by the phenomenon
of apolar or hydrophobic hydration. Evidence from a wide range of spectroscopic and
thermodynamic data on water alcohol mixtures at low alcohol concentration strongly
suggests the formation of low entropy structures or cages of fairly regular and longerlived H bonds located around hydrophobic groups [l-5]. It has been suggested that water
molecules in these structures tend to organise in a way similar to stable gas clathrate
hydrates.
A renewed interest for this phenomenon comes from Stillinger hypothesis
[S] that the anomalous properties of supercooled water is a result of a progressive and
cooperative growth of clathrate-like structures forming as water is cooled. On increasing
alcohol concentration a progressive interference among these structures is expected to
cause a progressive loss of the low-entropy, high connectivity character of cages. These
0167-7322/%/%15.00 0 19% Elsevier Science B.V. All rights reserved
PII SO167-7322 (%) 0093324

162

time-averaged complexes have a strong tendency to cluster, leading the well known
hydrophobic bonds and to the formation of molecular microaggregates. Quite often the
mixture properties show an anomalous behaviour that occurs in the water rich region
and, for each alcohol, single out the critical cosolvent mole fraction, x2, corresponding
to a change in the association state of the alcohol molecules.
Further evidence for
the water/methanol, water/ethanol, water/l- and 2-propanol, t-butanol and water/nbutoxyethanol systems has been recently collected from compressibility [7-91, UV [lo]
and IR (9,111 absorption spectra, surface tension 1121, dielectric [13,14], neutron and
X-ray [15] measurements.
The structural transition at x2f resembles, for some aspect,
the micellization process. The observed behaviour suggests that alcohol molecules are
essentially monomolecularly dispersed and surrounded by water molecules at low alcohol
concentrations 22 < zf; above this concentration, some kind of molecular aggregation
occurs.
These results are of interest not only in themselves but also for their relevance to
the effects of solvent perturbation on activation parameters of organic reactions (161,
the formation of micelles and biological membranes [17], and the conformational stability of proteins and nucleic acids [18]. On this regard, the effect of alcohol, often
considered merely as a means for lowering the dielectric constant of solvent and to affect the interactions among charged groups appears more complex. Recent results from
our laboratories show that the stabilisation of nucleic acids conformation [19,20] and
micellar structure [20- 221 in water/alcohol mixtures is closely linked to the properties
and anomalous behaviour of the solvent systems. Accordingly, these results support the
hypothesis that the dominant mechanism by which an alcohol affects these processes is
through its effect on the structure of water.
The main results of these investigations
2. SELF-ASSOCIATION

are reviewed and discussed in this paper.

OF MONOHYDRIC

ALCOHOLS

IN WATER

Aqueous solutions of monohydric alcohols display a variety of spectroscopic and

thermodynamic
properties which are qualitatively different from those of comparable
non aqueous systems.
Some results from our laboratories for aqueous mixtures of
methanol, ethanol, 2-propanol, t-butanol and n-butoxyethanol
will be succinctly reported here. Experimental techniques have been selected in order to look for the interrelations between bulk and interfacial properties (compressibility and surface tension)
and as capable of examining each component of the system selectively (infrared and
dielectric spectroscopy).
The ability of self-association of alcohol molecules in aqueous solutions is directly
related to the amphiphilic nature of these molecules; i.e., they contain both hydrophilic
and hydrophobic groups. In the methanol, ethanol, 2-propanol, t-butanol series an
hydrophobic group of increasing sizes is present and it can be regarded as obtained by
successive substitution of a -H atom by a -CHs group.
Great attention has been devoted to the water/n-butoxyethanol
mixtures, since
the observed change in their bulk and surface properties are remarkably similar to that
associated with the micellization in the case of surfactants and they give evidence of

microheterogeneities

like micelles in this binary system.

2.1 Adiabatic
compressibility
The adiabatic compressibility @Is) is a quantity which refers to a volume unit of
solution and which does not take into account the number of interacting molecules. We
have shown [7-91 that the compressibility data can be rationalised more easily if one
considers molar excess quantities such as
(bs

V,)E

ps

v,

Xl

PSI

Cl

x2

Ps2

c2

(1)

where /3~ and c,,, are the adiabatic compressibility and mean molar volume of the
mixture respectively. Water is designed as component 1 and cosolvent as component 2.
Thus, ~1, PSI and cr are the mole fraction, compressibility and molar volume of water
respectively, and x2, P.Q and v2 the corresponding quantities of cosolvent. Note that

(Ps V77dE
= @k -

v2

p.9

x2

where @k is the alcohol apparent molar compressibility and that for a two phase system
one expects that @k = P.Q. v2 or (p.s. V,,,)E/zr2 = 0.
Plots of (/3~ V,,,)E/rc2 at 25 C as a function of cosolvent molar fraction for the
methanol/, ethanol/, P-propanol/ and t-butanol/water systems are shown in Figure 1.

,^
5

-200

-400

&

-600

b
-

-1000
0.0

0.2

04

06
x2

0.6

1.0

0.0

01

C2

x2

Figure 1. (a) Plots of the quantity (0s . G,,,)E/x2 at 25 C as a function of cosolvent

mole fraction. (0): methanol/water, (A): ethanol/water, (*): 2-propanol/water,
(0):
t-butanol/water.
(b) The water rich region.

A similar behaviour is observed for all the systems examined. This excess quantity is nearly constant at low alcohol concentration but increases steeply as more alcohol
is added to the solution starting from a concentration z; peculiar for each alcohol. Regarding the methanol/water mixtures, one observes a linear increase of (Ps Vm)E/x2

164

as a function of 22 when 22 > x2. Concerning the remaining water/alcohols mixtures

studied, the behaviour of this quantity can be described by considering three mole fraction ranges defined by signpost mole fractions zz and z$. The experimental points
in these three regions fall close to three straight lines that intersect each other at the
alcohol mole fractions xz and xi. (See Figure-2).

x
I>
13:

-400

3
.-
b

-600

I
0

xl

0.2

xg

0.4

0.6

0.8

1.0

x2

Figure 2. Plot of the quantity (ps . p,,,)E/x2 for the ethanol/water mixtures at 25
%. The curves were obtained from Eq. (3) by using the parameter values reported in
Tab. I.

The experimental

(as .V7JE=
2

a + b.
c

21

points can be described quite well by the functional form

(2 -

xg

0 < x2 I x;
20 5 x2 5
x; 5 x2 5 1

xi

Small deviations between calculated and experimental values occur at values about
2; and ~8, because the transitions observed at these two molar fractions are smoother
than those expected from Eq. (3). The best fit constants a, b, c and the values xz and
xi for all the systems are reported in Table I. These parameters strongly depend on the
kind of alcohol we used. In the methanol, ethanol, 2-propanol and t-butanol series it
is found that x; and zi values shift to lower concentrations, the a value decreases and
b value increases as the hydrophobic group becomes larger. On the other hand, in the
z; . . 1 range the behaviour of these four alcohols is very similar, as it is evident from
Figure la and from the c value reported in Table I. Thus, the substitution of a -H by
a -CHz group affects scarcely the value (ps V,,,)E/z2 in the x$ ... 1 range.
The observed behaviour for the n-butoxyethanol/water
system (see Figure 3),
though similar, is in part different from the described trend; in fact, the width of the

165

28 . . . x$ concentration range where one can observe a strong increase in (Ps . c,,,)E/z~
is very narrow and a shift of the transition towards lower x2 values is observed with
increasing temperature.
A nearly constant value 21 0 of (ps p,,,)E/x2 is observed at
x2 > x;.

Table I
Values of signpost mole fractions and coefficients for the least squares representations
by Eq. (3) of isentropic compressibility of water/alcohol mixtures
102.x;

Methanol
Ethanol
2-propanol
t-butanol
n-butoxyethanol

lOl.a*

102.Xb2

llfl
6.0f0.2
3.5f0.2
2.5f0.2
l.lf0.2

29f3
20f3
13f2
4.0f0.5

310fl
483&l
723f3
963f2
818f6

lO.b*

lolrc*

345&3
(103f2)lO
(30f2).102
(64&5).103
(25k2).103

345f3
340flO
330420
33Ok20
150f20

m&-l)

* (cm5 . dyn-

75

-200

-400

00

-0
E
.z
N
-T

w
?
I>
v
&

cl@

00 @ 0

0 00 O
$0

00

-600 -

&FT
-800 -@o

Y)
- -1000

I
0.0

0.1

0.05

0.15

x2

Figure 3. n-butoxyethanol/water
mixtures. Plots of the quantity
at 25 (0) and 40 (0) C as a function of cosolvent mole fraction.

It has been suggested [7] that (Ps.V,)~

(/3s. p,,,)E/3:2

as given by Eq. (3) can describe the forma-

166

tion of clathrate-like structures at low concentration followed by some kind of molecular

aggregation of solute beyond a critical alcohol concentration. In fact, the second order
term (with respect to the concentration) in Eq. (3) (range z;.
.$,) might be related to
mutual interactions between the solutes molecules themselves. Such interactions thus
occur only for concentrations 22 > 2;.
The simultaneous existence of clathrate-like partial cage structure and OH-water
hydrogen bond is assumed in the model proposed in Ref. 7. One should thus expect
(/3s V,,,)E changes resulting from two independent types of solute-solvent interactions.
The relative balance of the two different types of interactions appears by comparing the
data from alcohols having hydrophobic groups of different sizes.
Our data are consistent with the hypothesis that the diminution in (Ps l/,)E is
mainly due to the hydration of the hydroxyl group with the formation of less compressible structures [8]. Consistently, the dissolution at very high dilution in water of the
alcohols used in the present work is always markedly exothermic and not sensitive to
the hydrophobic nature of the alcohols [23]. The hydration of hydrophobic groups has
instead, as a main effect, that of hindering alcohol-alcohol associations at low cosolvent
mole fraction (22 > ~2). In addition, the data (Figure la) suggest that the effects
associated with the interaction between water and non-polar alkyl-group of the alcohol
molecules are present only at low x2 values (~2 < z;), whereas at higher alcohol concentration values the compressibility changes are due only to the interaction with the
polar hydroxyl group of alcohol molecules.
The transition observed in x5 ...z$,
more pronounced as the size of the apolar
group increases, can be interpreted in terms of aggregation of alcohol molecules along
with strong modifications in the solvation of hydrophobic groups. Effects due to hydrophobic solvation become more and more smoother as 22 approaches xi. Finally,
in the z;. . .1 ran ge these effects become negligible and interactions of water with the
polar hydroxyl groups and associations of the alcohol molecules are predominant in determining the concentration dependence of the adiabatic compressibility of concentrated
solutions.
The value N 0 of (0s . ~m)E/z2 observed in the Z*2 . .l concentration range for the
n-butoxyethanol/water
system suggests a high tendency towards alcohol aggregation
in this system. It should be noted that this system separates in two liquid phases at a
lower critical solution temperature of N 49 OC for a critical mole fraction of 0.05 [22].
2.2 Infrared

spectra

More definitive informations on the self-association process of monohydric alcohols

in aqueous environment can be derived from IR experiments by means of which solvent
and solute species can be examined independently.
In recent years infrared spectroscopy has been used to investigate phase transitions in various surfactant systems (24-27). These studies show that monomer-micelle
formation transitions are accompanied by large (4.. .8 cm-) shifts in the fundamental frequencies of the asymmetric and symmetric alkyl stretching bands. It has been
found that similar effects accompany the association of the monhoydric alcohols in water
19,121.
Figure 4 shows the infrared spectra, at T = 25 C, in the C -H

stretching

region

167

of ethanol in water in a range of ethanol mole fraction encompassing zz(w 0.06). From
this figure the concentration dependence of the C - H stretching frequencies is evident.
Below x$ the frequencies and shapes of all bands are constant and shifts of the
bands to lower frequencies are observed for x2 > x20. A similar trend is found for all the
alcohols we examined. Figure 5 shows a plot of frequency fi vs. alcohol mole fraction
for the C - H stretching absorption peaked at highest frequency. The concentration
dependence of this quantity parallels that of -(/?,
c,,,)E/z2. As an example, in Figure 6
relative shifts in the C - H stretching frequency and the -(p, . qm)E/x2 quantity
adequately normalised are shown as a function of x2 for t-butanol/water mixtures. The
data superimpose within the experimental errors. Similar results are obtained for the
other mixtures we studied.

-I
3000

2900
v

2800

(cm-)

Figure 4. Infrared spectra in the C-H stretching region of water/ethanol mixtures

at three ethanol concentrations;
(---):
z2 = 0.013; (- - -): z2 = 0.13; (...):
22 z=
0.35. T=25 OC.

The observed trend of i; as a function of x2 (Figure 5) is consistent with the

description of self-association of alcohol molecules inferred from compressibility data.
At low concentration,
v does not change with the concentration
(similarly to other
parameters characterising the C - H stretching bands) and the spectra maintain the
characteristics of those of monomeric solutions in the whole 0. . x; range. The high
C-H
stretching frequencies in this concentration range can be attributed to an aqueous
local environment of the -CH3
groups. At high concentrations, xi < x2 < 1, the rate
of changes is also minimal and the spectra are those characteristic of pure liquids. In the
intermediate range, x$ < x2 5 zi, the frequency changes rapidly as a function of the

168

concentration
reaching the values typical of pure alcohol. This behaviour
increasing
amount of aggregation
of alcohol molecules as the concentration

indicates
an
is increased.

2970

2960

,
0.0

0.2

0.4

0.6

0.8

1.0

X2

Figure 5. (a) Concentration

dependence
of the C-H
stretching
absorption
peaked
at highest frequency.
(0):
methanol/water,
(A): ethanol/water,
(*): 2-propanol/water,
mixtures.
(b) The water rich region.
(0): t-butanol/water,
(0): n-butoxyethanol/water

The transition
observed in the z;. ..r~!j range resembles,
for some aspects,
the
micellization
process.
In fact, the observed behaviour
is very similar to that exhibited
by the concentration
dependence
of the infrared spectra in aqueous n-alkanoate
solutions
for concentration
ranges encompassing
the critical micelle concentration
[25]. On this
regard, it has to be noted that the width si - x20 of the concentration
range, within
which the onset of alcohol microaggregates
takes place, strongly depends on the system.
In the case of methanol/water
mixtures
a gradual change in the whole concentration
range is observed.
As the size of the hydrophobic
group increases the width x$ - z.$
of the transition
decreases,
In the case of n- butoxyethanol/water
mixtures this range
is very narrow and comparable
with that observed for the largest n-alkanoates
[9]. In
this case the transition
is closely approximated
by a phase separation
model and the
concept of critical micelle concentration
appears to be significant.
Micelle-like
microaggregates
are probably
present in this system although
the
present data do no give us any information
on the size and the structure
of the aggregates.
The almost constant value of v in the xi. . 1 range for this system indicates
that little or no hydrocarbon
chain-water
contact in the aggregates is present.
Above ~2
the hydrophobic
part of this alcohol is seeing practically
only other alcohol molecules.
Such a conclusion is consistent
with the results obtained from compressibility
data.
2.3 Surface

tension

The formation of micelle like microaggregates

in n-butoxyethanol/water
system is
also reflected in its surface tension (7) [12]. Experimental
7 values vs. logxz at 2 and
45 C are shown in Figure 7. Surface tension decreases steeply at low n-butoxyethanol

concentrations and becomes constant at higher concentrations.

acteristic of surfactants in a concentration region encompassing
centration (CMC).

o-

a
E

=.

u
%

-200

(a)

-400

-600

-800

.
c?9

.9

N
-c
w,

??a

??
00

00

??
0

.@

-0
- -1.5

- -3.0

c-45

I>
$
v

This hehaviour is charthe critical micelle con-

- -6.0
- -7.5

h
0

I
0.2

/
04

I
0.6

,
0.6

I
1.0

X2

7
5

E
7

-200

0
-1.5

h?

-400

N
3
9.
I>

-3.0
-45

-600

5
3

-6 0

Figure 6. t-butanol/water mixtures. (a) Concentration dependence of: (0):

quency shift of the C - H stretching vibration peaked at highest frequency;
(0): (/3s . Vm)E/z2 quantity. (b) The water rich region.

fre-

Critical concentrations 2;, as determined from the abrupt change in the 7 vs.
log22 plots, are reported in Figure 8. As for the CMC of non-ionic surfactants (28,291,
z$ decreases with increasing the temperature. The binary solution of n-butoxyethanol
and water is known to show a phase separation and to have a lower critical temperature
at 49 C for a critical mole fraction of 0.05. The values of the demixing temperature
from Ref. [30] are also reported in Figure 8. At concentrations lower z; (region I),

170

n-butoxyethanol exists in a monomeric state while above ~5 and below the consolution
curve (region II), alcohol monomers coexist with alcohol microaggregates with monomer
concentration near .r;.

50
2 oc

-f

40 45 oc

c
5
Fm
930-

:.:::_,

20

-3

-2
log

-1
x2

Figure 7. Surface tension vs. logarithm of n-butoxyethanol

mole fraction.

0.04

0.02

0.06

x2

Figure 8. Phase diagram of n-butoxyethanol in Hz0 at atmospheric pressure. (0):

critical n-butoxyethanol mole fraction x2f as determined by surface tension measurements
(see also Tab. I). (0): critical temperature of demixing from Ref. [30].

The x; obtained at 25 C nearly correspond to the midway of the transition shown

171

by IR and compressibility measurements. Thus, correlation between surface tension and

compressibility (or IR) data appears evident.
The temperature dependence of ~1 can be used to calculate the enthalpy (AH;)
and entropy (AS&) of microaggregate formation per mole of monomers.
According
to a phase separation model, Z; can be related to the standard free energy (AG;
AH; - TAS&) of microaggregate formation [29] by
xz = exp(AGk/RT)
The application of the best procedure to the 2; vs. T plot by using Eq. (4) gives
Thus, the formaAH;
= (9.6 * 0.4) kJ.mol- and AS:
= (66 f 1) J.mol-.K-.
tion of n-butoxyethanol microaggregates from aqueous monomers is entropy driven, the
endothermal formation enthalpy counteracting aggregation. This result, commonly obtained for the micellization process of non-ionic surfactants, is usually connected to the
release of structured water around the isolated chains on formation of micellar aggregates (hydrophobic effect) (28,291.
Though compressibility and IR data indicate formation of alcohol microaggregates only beyond a threshold concentration x$, no defined break in the surface tension
vs.logx2 plots is observed for the aqueous mixtures of the other examined alcohols.
2.4 Near-infrared

absorption

spectra

An approximate evaluation of the number of water molecules (S) that can be

located in the first hydration layer of an alcohol molecule shows that x$ N l/(S + l),
i.e. the aggregation starts when almost all water is involved in hydration structures.
This suggests that water plays a dominant role in the process of self-association of
alcohol molecules. This clearly results from detailed studies of the effect of alcohols
on the Near-IR absorption [II] and on the dielectric relaxation [13,14] (see par. 2.5) of
water itself.
Figure 9a shows the influence of temperature on the extinction coefficient, ~1, of
the 1450 nm band of water, assigned to VI+Y~ (~1 is the symmetric stretching and ~3 the
asymmetric stretching mode). As the temperature is decreased there is a large decrease
in the absorbance at 1412 nm while the absorbance at longer wavelengths increases.
McCabe et al. [31], in a study of the spectral changes induced by temperature on
the 1450 nm band of water, assigned the sharp band at 1412 n7n of water to the overtone
of the non-hydrogen-bonded
0 H stretching vibration. According to this interpretation
the appearance of a minimum at 1412 nm in the differential spectra shown in Figure 9a
should suggest the growth of water molecules with both OH groups bonded at the
expense of those with only one OH group bonded.
Some difference spectra, AA, of very diluted aqueous solutions of ethanol versus
water at the same temperature (25 C) are shown in Figure 9b. The negative absorption,
with a broad minimum at 1420 nm, is due to the fact that the molar concentration, Cl,
of water in the sample cell is lower than that, Cp, in the reference cell, additional minor
components in these differential spectra arise from the absorption of the alcohol in the
sample cell and from the variation AE, and AE~ in the molar extinction coefficient of

172

0.06923

V
I

(4

___

____

_____------

x2

0.02885
0.04905
?r--

0.06923
L

1300

1400

1500

1600

Figure 9. (a) Influence of temperature on the extinction coefficient of water in the

1250-1650 nm range. The differential spectra shown in (a) were recorded with water at
25 OC in the reference cell and water at the indicated temperature in the sample cell. To
obtain the Aer values, the spectra have been corrected for the density changes of water
with temperature. (b) Near-infrared differential spectra of aqueous ethanol solutions at
the indicated cosolvent molar fraction (sample cell) measured versus water (reference
cell) at the same temperature of 25 *C. ( c ) I n Auences of ethanol on the molar extinction
coefficient of water in the 1300-1500 nm range. The differential spectra shown in (c)
were calculated from Eqs. (5) and (8) by making use of the data relative to the difference
spectra in (b).

173

water and of alcohol in the mixture with respect to the corresponding values ~1, ~2 of
pure components. So, the absorbance A, of the sample may be given by the relation
A = El

c;

d (E1 + Asr)
.C1 + d.

+ AA =

(e2 + Abz). c2

where d is the cell depth in cm.

From Eq. (5) we obtain for the mean molar extinction

(5)

coefficient of the mixture

E= (c, + C,)

the following expression

E =

El.

2 1 +

E2

. x2

Asr

z1 + Ag2 . x2

(7)

The quantity
Ae=A~,.x,+Ae:!.x,

(8)

in Eq. (8) reflects the change in the hydrogen-bonding equilibrium in water and alcohol
in the mixture with respect to the two pure components. In the extremely dilute region
x2/x, < 1, thus AE z Asr.xr. This quantity, calculated from Eq. (5) in the 1250.. .1650
nm range by making use of the data relative to the difference spectra shown in Figure 9b,
is shown in Figure 9c. The values so obtained show a sharp minimum at 1412 nm whose
intensity, but not position, depends on ethanol concentration. Similar results have been
obtained for the other alcohol/water mixtures examined.
The shape and position of the spectra shown in Figs. 9a and 9c are very similar,
particularly in the short wavelength spectral region assigned to free -OH groups and
suggest that the effect of alcohol on the hydrogen bonding equilibrium of water is similar
to a decrease in temperature.
This is in line with nuclear magnetic resonance (NMR)
[32] and neutron scattering [33] measurements besides computer simulation results [34]
that show that the reorientation rate of water molecules is decreased by addition of
alcohol molecules.
2.5 Dielectric
relaxation
Information on the change of structure and dynamics of water in hydration shells
of alcohol molecules can be obtained from dielectric relaxation measurements.
The
dielectric relaxation of n-butoxyethanoljwater
113) and t-butanol/water [14] mixtures
has been investigated by means of a frequency domain technique in the 10 MHz.. .3 GHz
frequencies range. The first part of the main relaxation band of water is present in the
frequencies range analysed. Our results give evidence of higher values of the dielectric
relaxation time r and of the low frequency dielectric constant E, for hydration water,
reinforcing the notion that static and dynamic properties of water around hydrophobic
groups are comparable to those of supercooled water. Moreover, the presence for both
alcohols of an anomalous behaviour of T and E, in the water rich region further supports
the existence of an alcohol aggregation process only above a threshold concentration x;.

174

In this context,
t-butanol/water

we should like to report succinctly

mixtures.

the results obtained

at 2 C for

A set of systematic measurements taken in the water rich region allowed us to

investigate both the hydrophobic hydration and the aggregation phenomena occurring
in this concentration region. Different from water, water/t-butanol solutions cannot
be described by a single relaxation process. Experimental data give evidence of two
relaxation processes, characterised by relative amplitudes and relaxation times strongly
dependent on alcohol mole fraction. The values of the two dielectric relaxation times
~1 and 72 as a function of alcohol mole fraction are plotted in Figure 10.

100

/I

00 -

P-

,a,
I

60 -

,a
b

40_*_--.

O__-Q20 -

0.000

00 o---*--

0.02

0.06

0.04

0.06

0.1

x2

Figure 10. Experimental dielectric relaxation times 71 (CI)and ~2 (0) vs. t-butanol
mole fraction. (--):
calculated according to the model in Ref. [14]. The dashed lines
for 22 > 0.045 are drawn to guide the eye.

The appearance of a break in the behaviour of both ~1 and ~2 has been evidenced
at an alcohol concentration z2f N 0.045. This value compare well with the corresponding
ones obtained from IR and compressibility data and indicates self-association of alcohol
molecules.
We emphasise that this aggregation process has been evidenced looking
at the behaviour of water molecules rather than at that of alcohol molecules. At a
concentration of t-butanol ranging between 0 and x1, a considerable increase of 71
A two-state model has been proposed to
occurs for increasing alcohol concentration.
interpret these results, considering water molecules as divided in two main groups, i.e.
hydration water and bulk water, mutually exchanging at a rate k and characterised
by relaxation times which do not depend on concentration.
Although this model is
extremely crude, it appears in quantitative agreement with experimental results at low
alcohol concentrations, thus providing a value for the hydration water relaxation time
H = (65f2)p.s and for the exchange rate Ic = (0.026fO.O03)ps-.
The present results on

17s

the dynamics of hydration water support the generally accepted model of hydrophobic
hydration, that considers water molecules in the first hydration layer slowed down in
their rate of rotation. Moreover, it can be stated that hydration water molecules, in
the water/t-butanol system, exchange with bulk molecules at a rate that is comparable
with the rotation rate.
3. INFLUENCE
BIOMOLECULES

OF ALCOHOLS
ON THERMAL
AND MICELLES
FORMATION

STABILITY

OF

A common method of investigating the role of various kinds of non covalent interactions in the conformations of nucleic acids and proteins is to study the conformation
and thermal stability of these macromolecules in altered water structure by adding small
quantities of monohydric alcohols of varying chain length 1181. This approach is widely
used to influence the rates and yield of many chemical reactions [16] as well as the
micellization process [17].
The role played by the solvent and by the solute-solvent interactions in such problems are in many respects far from to be cleared and the molecular nature of the influence
of water on macromolecules or chemical reactions remains uncertain.
Recent results from our laboratories show that the effects due to alcohols on two
very different systems and processes, the thermal denaturation of transfer ribonucleic
acid (tRNA) molecules [19,20] and the micellization of several surfactant molecules
[20-221, are strikingly similar and are closely paralleled in simpler physico-chemical
properties of alcohol/water mixtures themselves.
Figs. 11 and 12 clearly show that alcohols affect the micellization process.

0.05

0.10

0.15

x2

Figure 11. The CMC values of surfactant dodecyltrimethylammonium

hromide as a function of mole fraction of alcohol at 25 C.
(0): methanol/water,
(A\:
ethanol/water, (*): 1-propanol/water mixtures.

176

XPrll

Figure 12. The CMC values of sodium dodecylsulphate (SDS) as a function of

mole fraction of ethanol at 10 and 40 C. The concentration ~2~ corresponding to a
minimum in the CMC shifts towards lower values with increasing temperature.
(see
inset)

Addition of low quantities of alcohol reduce the CMC, but high concentrations tend
to increase it. This behaviour depends on the carbon atoms number of the surfactant
and the nature of the counterion and head group [al]. Nevertheless, in all the cases it
can be observed that the CMC values decrease on increasing the mole fraction of alcohol
until a minimum value is reached at ~2 = ZZ,,,; thereafter, they increase. Similar results
can be found in the literature both for ionic and nonionic surfactants [17]. The 2zrn
concentration shifts to lower values with increasing alkyl group size (Figure 11) and
with increasing temperature (Figure 12) assuming values surprisingly close to those
at which a transition in the compressibility (or IR absorption) of plain water/alcohol
mixtures has been observed (Figs. 1 and 5). Thus, it appears that the stabilisation of
micellar structure in the water/alcohol mixtures is closely linked to the properties and
anomalous behaviour of the solvent system.
It is interesting to note that in the same concentration range at which one found
a minimum in the CMC one found also an inversion of trend in several processes of
chemical and biochemical interest studied in the water/alcohol solvent; this, for example can be observed on studying the effect of alcohols upon the stability of protein and
nucleic acid conformations [18-201 or the equilibria and rates of chemical reactions (161.

177

As an example, the melting temperature (T,,,) of transfer ribonucleic acid (ULNA) solutions containing various amounts of methanol, ethanol and 1-propanol are plotted in
Figure 13 as a function of the alcohol mole fraction 22 [20].

0.05

0.10

0.15

0.20

x2

Figure 13. Variation of the melting temperature, T,,,, of an aqueous solution of

transfer ribonucleic acid molecules containing various amount of methanol, ethanol,
and 1-propanol as a function of the alcohol mole fraction. (0): methanol/water, (A):
ethanol/water, (*): l-propanol/water mixtures.

Similar to that found for the CMC (Figure 11) the observed effects depend on
the size and concentration of the alcohol added. The T,,, of tRNA at low values of
2s decreases gradually on increasing the alcohol concentration, and the effect is more
pronounced on increasing hydrocarbon content of the alcohol. The observed decrement
of T, is roughly linear in 22 and continues up to a minimum at an intermediate composition typical for each alcohol; thereafter an inverted trend is observed, i.e. the 7,,,
increases with increasing x2. The ~cgvalues at which the melting temperatures of tRNA
change their trend are close to those at which we observed a minimum in the CMC
(Figure 11). The close similarity between the behaviour of these two very different systems and processes is striking. It is relevant that the only common feature is the same
solvent system.
These considerations support the contention that changes in the solvent structure due to addition of alcohol are an important factor in the formation of micellar
aggregates and in the conformational properties of biomolecules. The structure of the
solvent is strictly related to the hydrophobic interactions. The primary contribution to
the strength of these interactions derives from changes in the structure of water when
non polar groups interact with one another. Hydrophobic interactions are believed to
be closely related to the micellization process and to contribute to the maintenance

178

of ordered macromolecular structures. Such interactions arise from the unique threedimensional structure of water and should be changed considerably by variations in the
solvent structure due to addition of alcohol.
According to Benzinger 135,361 and as developed by Frank and Lumry [37,38] the
structuring of water around non-polar groups is largely a compensated process, so that
the entropy and enthalpy associated with such a structuring nearly compensate each
other and thus make only a small contribution to the free energy. As a consequence,
one would expect that enthalpy and entropy of micellization rather than free energy
are affected by alcohol addition.
We use the temperature dependence of CMC for
sodium dodecyl sulphate (SDS) so 1ut ions (Figure 12) to find enthalpy and entropy of
micellization as a function of ethanol mole fraction [22]. In particular, the standard state
value of the free energy, AG&, enthalpy, AH;, and entropy, AS&, of micellization per
mole of surfactant were calculated by the well-known relations
AG& = RT . ln(CMC)

AHO

Aso

a
iW

7-2.

(9)

(AGk)

(10)

A%, - AG;

(11)

were the critical micelle concentration was taken in mole fraction scale. The results at
35 C were plotted in Figure 14 as a function of 22.

-30 ' ,
0

I
0.05

0.10

0.15

x2

Figure 14. Micellization of SDS in ethanol/water mixtures at 35 C. Standard

state values of the free energy, enthalpy, and entropy, vs. mole fraction of ethanol.

Large but compensating

changes in the enthalpy and entropy appear from the

179

data. These thermodynamic quantities go to a minimum near the concentration ~8

where a maximum structuring in the water/ethanol mixtures is expected [8].
The overall enthalpy and entropy of micellization is probably due to many different effects. An important contribution, AHhvd and AShvd, to these thermodynamic
quantities is usually attributed to the release of structured water around the isolated
chains on formation of micellar aggregates (hydrophobic effect). It is usually accepted
that this process is endothermic (AHhvd > 0) and accompanied by a large entropy
change (AShyd > 0). Based on the discussion of evolution of water/alcohol structure
given above one would expects that AHhvd and AShvd are modulated by the alcohol
concentration.
As a matter of fact, the overall enthalpy and entropy of micellization
change with zz (Figure 14) in a way consistent with the suggested evolution in the
water- ethanol structure and with the hypothesis that the addition of ethanol affects
essentially the AH hydand AShvd contributes. The initial decrease of AH& and AS:
(Figure 14) on increasing ~2 can be related to a decrease in the local enthalpy and entropy due to cage formation by ethanol molecules; these thermodynamic quantities go
to a minimum for 22 = xi where the change in the degree of structure induced in the
water by the addition of new hydrophobic groups is presumably zero and then increases
for 22 > x; where a progressive distortion of cages is expected. Consistent with the
above suggestions, the observed changes in the enthalpy and entropy (Figure 14) nearly
compensate each other and thus give a net free energy which is almost independent of
alcohol concentration.
In conclusion, the present experiments support the assumption that the dominant
mechanism by which short chain alcohols affect the micellization process is trough their
effect on the structure of the solvent.
4. CONCLUSIONS
The present data clearly indicate the important role that water and solvation play
in the self-association of alcohol molecules in the conformational stability of nucleic
acids and in the micellization process. The data cannot be accounted for satisfactorily
by considering the solvent as a continuous characterised by bulk properties; instead the
structure of the solvent and structural changes that take place in the hydration regions
appear to play a dominant role in the observed process. Available literature data [IS-181
provide further support that the same conclusion could be valid for a number of other
systems of biological and chemical interest.
ACKNOWLEDGEMENT
This work was partially supported by the Istituto
(INFM).

Nazionale Fisica della Materia

REFERENCES
1

F. Franks and D.J. Ives, Rev. Chem. Sot., 20 (1966) 1

180

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

29
30
31
32

F. Franks in Water: A Comprehensive Treatise, F. Franks (ed.) Plenum, New

York, 1973, Vol. 2, Chap. 1 and references therein
F. Franks and D.S. Reid in Water: A Comprehensive Treatise, F. Franks (ed.)
Plenum, New York, 1973, Vol. 2, Chap. 5 and references therein
F. Franks in Water: A Comprehensive Treatise, F. Franks (ed.) Plenum, New
York, 1975, Vol. 4, Chap. 1 and references therein
M. Blandamer in Water: A Comprehensive l+eatise, F. Franks (ed.) Plenum, New
York, 1973, Vol. 2, Chap. 9 and references therein
F.H. Stillinger, Science, 209 (1980) 451
G. Onori, J. Chem. Phys., 87 (1987) 1251
G. Onori, J. Chem. Phys., 89 (1988) 4325
M. DAngelo, G. Onori and A. Santucci, J. Chem. Phys., 100 (1994) 3107
G. Onori, 11 Nuovo Cimento, D9 (1987) 507
G. Onori, Chem. Phys. Letters, 15 (1989) 212
M. DAngelo, G. Onori and A. Santucci, Chem. Phys. Letters, 220 (1994) 59
D. Fioretto, A. Marini, G. Onori, L. Palmieri, A. Santucci, G. Socino and L.
Verdini, Chem. Phys. Letters, 196 (1992) 583
D. Fioretto, A. Marini, M. Massarotti, G. Onori, L. Palmieri, A. Santucci and G.
Socino, J. Chem. Phys., 99 (1993) 8115
C. Petrillo, G. Onori and F. Sacchetti, Mol. Phys., 67 (1989) 697
M.J. Blandamer In: Advances in Physical Organic Chemistry,V. Gold (ed.) Academic Press, New York, 1977, Vol 14, pp 203-351
G.C. Kresheck In: Water: A Comprehensive Treatise, F. Franks (ed.), Plenum,
New York, 1975 Vol. 4 pp 95-167
D. Eagland In: Water: A Comprehensive Treatise, F. Franks (ed.), Plenum, New
York, 1975 Vol. 4, pp 305- 518
S. Beneventi and G. Onori, Biophys. Chem., 25 (1986) 181
G. Onori, S. Passeri and A. Cipiciani, J. Phys. Chem., 93 (1989) 4306
A. Cipiciani, G. Onori and G. Savelli, Chem. Phys. Letters, 143 (1988) 505
G. Onori and A. Santucci, Chem. Phys. Letters, 189 (1992) 598
D. Peeters and P. Huyskens, J. Mol. Structure, 300(1993) 539
J.H. Umemura, D.G. Cameron and H.H. Mantsch, J. Am. Chem. Sot., 84 (1980)
2272
J.H. Umemura, H.H. Mantsch and D.G. Cameron, J. Coll. Inter. Sci., 83 (1981)
558
P.W. Yang and H.H. Mantsch, J. Coll. Inter. Sci., 113 (1986) 218
W.M. Cross, J.J. Kellar and J.D. Millar, Applied Spectroscopy, 46 (1992) 701
V. Degiorgio in: Physics of Amphiphiles, Micelles, Vesicles and Microemulsions,
V. Degiorgio and M. Corti (eds.) North-Holland, Amsterdam, 1985 p. 303 and
reference therein
G.C. Kresheck, in : A Comprehensive Treatise, F. Franks (ed.) Plenum, New
York, 1975, Vol. 4, p. 95 and references therein
H.L. Cox and L.H. Cretcher, J. Am. Chem. Sot., 48 (1962) 451
W.C. MC Cabe, S. Subramanian and H.F. Fischer, J. Phys. Chem., 74 (1970) 4360
E. Goldamer and M.D. Zeidler, Ber. Bunsenges. Phys. Chem., 73 (1969) 4

181

33
34
35
36
37
38

F. Franks, J. Ravenhill, P.A. Egelstaff and D.I. Page, Proc. R. Sot. London, Ser.
A, 319 (1970) 189
D.A. Zichi and P.J. Rossky, J. Chem. Phys., 84 (1986) 2814 and references therein
T.H. Benzinger In: Thermodynamics of Life Growth, F. Held (ed.) AppletonCentury-Crofts, New York, 1969 chapter 14
T.H. Benzinger, Nature (London), 229 (1971) 100
R. Lumry and H. Frank, Proc. Int. Biophys. Congr. 6th VII-30, (1978) 554
R. Lumry In: Bioenergetics and Thermodynamics: Model Systems, A. Braibanti
A. (ed.), Reidel, Dordrecht, 1980 pp. 405-423