di?%!” LA R LIQUIDS

ELSEVIER JoumalofMolecularLiquids69(1996) 161-181

Dynamica. a,nd structura,l properties 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 longer- lived 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

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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/n- butoxyethanol 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 sta- bility 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 af- fect 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 are reviewed and discussed in this paper.

2. SELF-ASSOCIATION 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 re- ported here. Experimental techniques have been selected in order to look for the inter- relations 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 - p.9 v2 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 0

E _’ ‘5 -200

3 E

0 -400

& -600 r b - -1000

0.0 0.2 04 06 0.6 1.0

x2

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” quan- tity 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. Re- garding 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 frac- tion 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” 13: I>’ -400 -

3 .-’ b ,

- -600 I I I I r 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 points can be described quite well by the functional form

(as .V7JE a = i

0 < x2 I x; a + b. (“2 - xg “20 5 x2 5 xi

“2 c 21 x; 5 x2 5 1

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; 102.Xb 2 lOl’.a* lO”.b* lolrc*

Methanol llfl - - 310fl 345&3 345f3 Ethanol 6.0f0.2 29f3 - 483&l (103f2)lO 340flO 2-propanol 3.5f0.2 20f3 - 723f3 (30f2).102 330420 t-butanol 2.5f0.2 13f2 - 963f2 (64&5).103 33Ok20 n-butoxyethanol l.lf0.2 4.0f0.5 - 818f6 (25k2).103 150f20

* (cm5 . dyn-’ m&-l)

7 -200 @ 0 B - 5 00 cl@ O 00

-0 0 00 O

“E -400 - .z

$0 O

N 00 -T 0 - w

-600

? I> & -800

&FT v -@o

Y) - -1000 I r ,

0.0 0.05 0.1 0.15

x2

Figure 3. n-butoxyethanol/water mixtures. Plots of the quantity (/3s. p,,,)E/3:2 at 25 (0) and 40 (0) ‘C as a function of cosolvent mole fraction.

It has been suggested [7] that (Ps.V,)~ as given by Eq. (3) can describe the forma-

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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 compress- ible 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 con- centration 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 hy- drophobic 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 de- termining 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 transi- tions in various surfactant systems (24-27). These studies show that monomer-micelle formation transitions are accompanied by large (4.. .8 cm-‘) shifts in the fundamen- tal 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 2800

v (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 indicates an increasing amount of aggregation of alcohol molecules as the concentration is increased.

2970

2960 ‘, I

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, (0): t-butanol/water, (0): n-butoxyethanol/water mixtures. (b) The water rich region.

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 ag- gregates. 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. This hehaviour is char- acteristic of surfactants in a concentration region encompassing the critical micelle con- centration (CMC).

7 o- ??0 a * -0

a (a) E

00

7 -200 - ??0’0

.@’ ??a

- -1.5 =. .

u e % -400 -

.’

c? 9 - -3.0 5

.9 N

-c -600 - w, .”

c-45 g /

3 I>’ - -6.0

$ -800 - v - -7.5 h

I / I , I 0 0.2 04 0.6 0.6 1.0

X2

7 0 5 E

7 5

-200

h? E -400

Y N

3 -600 9. I>’

0

-1.5

-3.0 5

-45 3 I

,’

-6 0

Figure 6. t-butanol/water mixtures. (a) Concentration dependence of: (0): fre- quency shift of the C - H stretching vibration peaked at highest frequency; (0): (/3s . Vm)E/z2 quantity. (b) The water rich region.

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 -

c 45 oc

5

930- :.:::‘_,

F- m’

20 I I -3 -2 -1

log x2

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

1

0 0.02 0.04 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 AH; = (9.6 * 0.4) kJ.mol-’ and AS: = (66 f 1) J.mol-‘.K-‘. Thus, the forma- tion of n-butoxyethanol microaggregates from aqueous monomers is entropy driven, the endothermal formation enthalpy counteracting aggregation. This result, commonly ob- tained 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 aggre- gates (hydrophobic effect) (28,291.

Though compressibility and IR data indicate formation of alcohol microaggre- gates 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

r

L

I I I I

0.06923 V I

(4

___ ____ _____------

x2

?r--

0.02885 0.04905 0.06923 v

I I I I

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 A n uences 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; + AA = d (E1 + Asr) . C1 + d. (e2 + Abz). c2 (5)

where d is the cell depth in cm. From Eq. (5) we obtain for the mean molar extinction coefficient of the mixture

A

E = (c, + C,) d

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, we should like to report succinctly the results obtained at 2 ‘C for t-butanol/water mixtures.

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

a’ ,’

P- 00 - ,’

d ,a,’

I’

60 - 3

,a

b 40-

_*_--.

20 - 00 o---*-- O__-Q-

0.000 0.02 0.04 0.06 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 occurs for increasing alcohol concentration. A two-state model has been proposed to 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 OF ALCOHOLS ON THERMAL STABILITY OF BIOMOLECULES AND MICELLES FORMATION

A common method of investigating the role of various kinds of non covalent inter- actions 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 prob- lems 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 0.05 0.10 0.15

x2

Figure 11. The CMC values of surfactant dodecyltrimethylammonium hromi- de as a function of mole fraction of alcohol at 25 ‘C. (0): methanol/water, (A\: ethanol/water, (*): 1-propanol/water mixtures.

176

5

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 exam- ple 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) so- lutions 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 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 com- position typical for each alcohol; thereafter an inverted trend is observed, i.e. the 7’,,, increases with increasing x2. The ~cg values 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 sys- tems 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 struc- ture 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

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of ordered macromolecular structures. Such interactions arise from the unique three- dimensional 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 u ions (Figure 12) to find enthalpy and entropy of 1 t 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) (9)

AHO = 7-2. a (AGk) m iW T

Aso = A%, - AG; m T

(10)

(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 ' , I I 0 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

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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 differ- ent 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 hyd and 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 en- tropy 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 Nazionale Fisica della Materia (INFM).

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