Effect of ethylene glycol on the thermodynamic and micellar properties of Tween 20

Transcription

1 Colloid Polym Sci (2003) 281: DOI /s ORIGINAL CONTRIBUTION C. Carnero Ruiz J.A. Molina-Bolı var J. Aguiar G. MacIsaac S. Moroze R. Palepu Effect of ethylene glycol on the thermodynamic and micellar properties of Tween 20 Received: 30 May 2002 Accepted: 23 September 2002 Published online: 5 December 2002 Ó Springer-Verlag 2002 C. Carnero Ruiz (&) Æ J.A. Molina-Bolı var J. Aguiar Departamento de Física Aplicada II, Escuela Universitaria Polite cnica, Universidad de Ma laga, Campus de El Ejido, E Ma laga, Spain ccarnero@uma.es G. MacIsaac Æ S. Moroze Æ R. Palepu (&) Department of Chemistry, St. Francis Xavier University, Antigonish, Nova Scotia B2G 2W5, Canada rpalepu@stfx.ca Abstract The aggregation behaviour of Tween 20 in ethylene glycol water mixed solvents has been investigated using surface tension, density, static and dynamic light scattering, and fluorescence measurements. Micellar and surface thermodynamics data were obtained from the temperature dependence of critical micelle concentrations in various aqueous mixtures of ethylene glycol. In order to evaluate the influence of the cosolvent, the differences in the Gibbs energies of micellization of Tween 20 between water and binary solvents were determined. This study allowed us to conclude that the ability of ethylene glycol to act as a structure breaker and its interaction with the surfactant hydrophilic group are the controlling factors of the micellization process. From the evaluation of the thermodynamics of adsorption at the solution air interface, it was determined that the surface activity of the surfactant decreases slightly with increasing concentration of ethylene glycol at a given temperature. Partial specific volume data, obtained by density measurements, indicate that the fraction of solvent molecules interacting with the micelle, via hydrogen bonds, remained roughly constant. The effect of cosolvent on the size and solvation of the aggregates was analysed by means of static and dynamic light scattering measurements. It was found that the aggregation number decreased, whereas the whole micellar solvation increased with the ethylene glycol content. Micellar micropolarity was examined using two different probes, pyrene and 8- anilinonaphthelene-1-sulfonic acid, and was found to increase with ethylene glycol addition, accompanied by an enhanced solvation. Fluorescence polarization measurements found by using coumarin 6 as a hydrophobic probe revealed an increase in the micellar microviscosity. The observed trends in these microenvironmental properties were ascribed to a participation by ethylene glycol in the micellar solvation layer. Keywords Thermodynamics Æ Micellization Æ Tween 20 Æ Ethylene glycol water mixtures Æ Light scattering Æ Fluorescence polarization Introduction The different association behaviours of surfactants in water and other solvents have stimulated the interest to elucidate how the solvent properties influence aggregation, and many studies have been performed to gain information on the role of the solvent in the aggregation phenomenon of amphiphiles. These investigations have focused mainly on two essential aspects: namely the nature of the interactions implied in

2 532 the process of micellar formation, and the structure of the aggregates formed. Evans et al. [1] established that the specific properties of water are not indispensable to promote surfactant self-assembly. Investigations of micellization in nonaqueous polar solvents, such as ethylene glycol, glycerol and formamide, which have properties resembling those of water, have shown that a solvent requires three conditions to induce surfactant aggregation [2]: (i) a high cohesive energy, (ii) a high dielectric constant, and (iii) a high hydrogen-bonding ability. It is important to point out that it has been proposed [3] that the capability of hydrogen bond formation is a necessary condition for the self-assembly of surfactants. To clear up the details of the interactions involved in the so-called solvophobic effect, many investigations have been carried out where water is partially replaced with another polar solvent. This reasonable approach permits an exploration of a range of compositions where the solvent characteristics change in a gradual manner. The micellar aggregation of surfactants in polar solvents other than water has been mainly studied by using ionic surfactants [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. These studies suggest that the driving force for the solvophobic effect has essentially the same origin as the hydrophobic effect, i.e. the large cohesive energy of the solvent. Micellization of nonionic surfactants in polar solvents has been much less studied, and often the results that appear in the literature are contradictory [19, 20, 21, 22, 23, 24, 25]. Penfold et al. [23] studied the micelle formation of monododecyl octaethylene glycol (C 12 E 8 ) and monododecyl hexaethylene glycol (C 12 E 6 ) in water mixed with ethylene glycol, sorbitol or glycerol, using small angle neutron scattering. They found that the size of the micelles increased with the addition of cosolvent. This behaviour was attributed to a decreasing hydration of the polyoxyethylene groups due to the interaction between water and the cosolvent, causing a reduced curvature in the micellar aggregate. On the other hand, Cantu el al. [21] observed that the micellar size of C 12 E 8 decreased with the addition of glycerol in the mixed solvent. They ascribed this fact to a reduction of the micelle solvation in the solvent system. Alexandridis and Yang [24] have recently reported a study on the effect of several cosolvents, including formamide, ethanol, and glycerol, on the micellar structure of Pluronic P105. This investigation showed that the micelle association numbers become smaller in the presence of formamide or ethanol. This behaviour was interpreted on the basis of a reduction of the interfacial tension between the hydrophobic chains and the solvent. As a consequence, the formation of smaller micelles becomes more favourable energetically. Recently, we have reported an investigation on the micellar properties of the nonionic surfactant, Triton X-100 in ethylene glycol water mixtures [25]. In this study, we observed a reduction in the hydrodynamic micelle radius, which was attributed to a decrease in the aggregation number rather than a change in micellar solvation. We also found evidence for the formation of a thicker solvation layer, consistent with a certain participation of the cosolvent in this region of the micelle. In this paper, we study the effect of ethylene glycol (EG) on micellization of Tween 20 (TW-20). This is another representative nonionic surfactant, with important structural differences in relation to Triton X-100, and frequently used in several applications, including emulsification, pharmaceutical preparations, and pre-extraction of membranes. However, very few studies appear in the literature on the characterization of its micellar formation. The outline of this paper is as follows: (i) we use surface tension measurements to examine the thermodynamic conditions under which micelles of TW-20 form in different solvent systems with different EG content, as well as the effect of the EG addition on the thermodynamics of adsorption of TW- 20 in the air-liquid interface; (ii) from both dynamic and static light scattering experiments, and density measurements we examine the changes in the size and solvation of the aggregates; and, (iii) we explore the change in the micellar microstructure through micropolarity and microviscosity changes upon EG addition. Experimental Materials The surfactant Tween 20 (polyoxyethylenesorbitan monolaurate), whose chemical structure is shown in Scheme 1, was purchased from Sigma (SigmaUltra) and used without further purification. Ethylene glycol (99%+, spectrophotometric grade) was from Aldrich. The fluorescence probes, pyrene from Sigma, coumarin 6 (C6) from Aldrich and 8-anilinonaphthalene-1-sulfonic acid (ANS) from Fluka were used as received. Doubly distilled water was used to prepare all micellar solutions. All experiments were carried out with freshly prepared solutions. Methods Surface tension measurements Surface tension measurements were performed according to the du Nouy method on a Fisher Surface Tensiomat, model 21, Scheme 1 Molecular structure of Tween 20 (with w+x+ y+z=20)

3 533 equipped with a 13-mm-diameter platinum iridium ring. The solutions were placed in a thermostatted beaker at a constant temperature. The adsorption process at the air aqueous solution interface was generally completed in about 10 min, and the repetition time for individual readings was 15 min. This procedure improved the accuracy on an individual surface tension reading by up to ±0.2 mn m 1. The surface tension values, c, were corrected as described in the instrument manual. The surface tension value of pure water was measured periodically to ensure consistency. Density measurements To determine the partial specific volume of micellar TW-20 in each solvent system, we performed density measurements with an Anton-Paar DMA 5000 density meter. The instrument has an accuracy of gcm 3 and it was calibrated with air and water at 25 C. The temperature was controlled within ±0.01 C. Light scattering measurements Light scattering experiments were made on a Malvern 4700 photon correlation spectroscopy (PCS) system. The instrument is equipped with a 75 mw argon ion laser operating at 488 nm with vertically polarized light. All measurements were carried out at 25.0±0.1 C using a circulating water bath. Cylindrical quartz cells of 10-mm diameter were used in all the light scattering measurements. These cells were soaked in nitric acid, rinsed with distilled water, and finally rinsed with freshly distilled acetone before use. Surfactant solutions were filtered once through a 0.1-lm Millipore filter directly into the cell and sealed until used. Dynamic light scattering (DLS) measurements were carried out to obtain the translational diffusion coefficients and the associated hydrodynamic radius of the micelles. The dependence of scattered light fluctuations on the translational diffusion coefficients of the micelles can be obtained from the autocorrelation function, G(s). This function is calculated from the product of two photon counts at time t and t+s such that GðÞ¼ s hiðtþ Iðt þ sþi. The normalized intensity autocorrelations function is given by [26]: g int IðtÞ Iðt þ sþ ðsþ ¼ h i IðtÞ IðtÞi This function, g int (s), is related to the normalized field autocorrelation function g field (s) by the Siegert relationship [26]: g field ðsþ ¼1 þ Cg int ðsþ 2 ð2þ where s is the delay time and C is the Siegert constant, an experimental fitting parameter of the measuring device. For a dilute dispersion of macromolecules formed by monosize and spherical particles, the application of the cumulant method [27] provides g int (s): g int ðsþ ¼e Cs where G is the mean decay rate, which describes the diffusion of the particles under study and is related to the so-called collective diffusion coefficient (D c ) by: D c ¼ C q 2 Here q is the scattering wave vector, which is related to the scattering angle h by q ¼ ð4pn=kþsinðh=2þ. The diffusion coefficient was measured at least three times for each sample. The average error in these experiments was estimated to be 4%. ð1þ ð3þ ð4þ The viscosities of all solvent systems were measured at 25 C with a Haake Rotovisco VT550 concentric-cylinder rotational viscometer. The viscosity measurements were performed with a NV sensor system and an outer cylinder using a shear rate of s 1, with a temperature control of ±0.1 C. In the static light experiments the Rayleigh ratio, R h, of the sample solutions was determined using toluene as a standard according to the relationship: R h ¼ I h R tol ð5þ I tol where I h and I tol are the scattered intensity of the sample solution and the toluene, respectively, and R tol is the Rayleigh ratio of toluene. For R tol at k=488 nm a value of m 1 was assumed [28]. The excess of scattering, DR h, is defined as the difference in the Rayleigh ratio between the micellar solution, R h, and the solvent solution in the absence of micelles, R 0 h, that is: DR h ¼ R h R 0 h All light scattering measurements were made at a scattering angle, h, equal to 90, after initial studies indicated that the micelles were too small to exhibit significant angular dependence of the scattered light within the experimental error. The intensity of scattered light was measured at least four times for each sample. The average error in these repeated measurements was approximately 2%. The refractive index values of solvents and TW-20 micellar solutions, required to compute the static light scattering data, were measured at 25.0±0.1 C using a digital Abbe refractometer (WYA-1S). Fluorescence measurements A Spex FluoroMax-2 spectrofluorometer was used for all fluorescence measurements. This instrument is equipped with a thermostatted cell housing that allowed temperature control to ±0.1 C. To estimate the micellar micropolarity, the fluorescence emission spectra of pyrene (1 lm) in TW-20 solutions (50 g/l) were recorded employing the S mode with band-passes for excitation and emission monochromators of 1.05 nm, and an excitation wavelength of 335 nm. From these spectra, the intensities I 1 and I 3 were measured at the wavelength corresponding to the first and the third vibronic band located near 372 and 384 nm. The ratio I 1 /I 3 is the so-called pyrene 1:3 ratio. Similarly, the emission spectra of ANS (5 lm) in micellar TW-20 solutions were obtained by using the same recording mode, band-passes of 1.05 and 2.00 nm for excitation and emission monochromators, respectively, and an excitation wavelength of 346 nm. Fluorescence anisotropy measurements were collected with the same apparatus and a polarization accessory, which uses the L- format configuration [29] and an automated wheel with Glan- Thompson polarizers. The fluorescence anisotropy, r, was determined as: r ¼ I V GI H ð7þ I V þ 2 GI H where the subscript of the fluorescence intensity values (I) refer to vertical (V) and horizontal (H) polarizer orientation. The software supplied by the manufacturer automatically determined the instrumental configuration factor G, required for the L-format configuration. The anisotropy values were averaged over an integration time of 20 s and a maximum number of three measurements were made for each sample. The anisotropy values of the probe in micellar media presented in this work are the mean value of three individual determinations. All fluorescence measurements were made at 25.0±0.1 C. ð6þ

4 534 Results and discussion Critical micelle concentration and thermodynamic study Critical micelle concentration (cmc) values were determined by using surface tension measurements. Representative plots of the surface tension (c) of a solution of TW-20 in various binary mixtures of EG plus water versus the log of the bulk concentration of the surfactant at 298 K are presented in Fig. 1. The cmc was taken as the concentration at the point of intersection of the two linear portions of the plots. Experimental cmc values are listed in Table 1. The cmc values of TW-20 were found to be in agreement with the published data [30] and decrease with temperature within the temperature range adopted in the present study. This behaviour is in line with the characteristics of nonionic surfactants [31]. The micelle formation of nonionic surfactants is assumed to be due to hydrophobic interactions [32]. Moreover, it has been established [33] that London dispersion forces are the main attractive forces in the formation of micelles. In the case of TW-20, the cmc values are increased in the presence of EG. Thus, EG is acting as a cosolvent and also as a structure-breaking solute. In micellar solutions, structure-breaking solutes are known to lower the hydrophobic effect, which is considered to be the driving force of micellization [10, 11, 12, 13, 14, 15, 16]. The increase in cmc values is larger than for ionic surfactants and can be attributed to the increase in solubilization of the nonpolar chains of the surfactant in EG. The temperature effect on the cmc values in the presence of EG is opposite to that in water and is attributed to the disruption of the solvent structure with the increase in temperature. Table 1 Critical micelle concentrations of Tween 20 in various ethylene glycol+water mixtures at different temperatures EG T cmc 10 4 (wt%) (K) (mol L 1 ) Thermodynamics of micellization The temperature dependence of the cmc of nonionic surfactants can be used to obtain the thermodynamic parameters of micellization. In accordance with the mass action model, the standard free energy of micelle formation per mole of monomer, DG 0 mic, is given by [34]: DG 0 mic ¼ RT ln x cmc ð8þ where x cmc is the mole fraction of surfactant at the cmc. The enthalpy of micellization can be obtained by applying the Gibbs-Helmholtz equation to Eq. 8: DHmic ln x cmc ¼ RT2 P The entropy in the micellization process can be estimated from the calculated enthalpy and free energy values as: DSmic 0 ¼ DH mic 0 DG0 mic ð10þ T Finally, it is possible to evaluate the effect of the cosolvent on the micelle aggregation process by means of the so-called free energy of transfer, DG 0 M, which is defined by [35]: DG 0 M ¼ DG0 mic EGþH 2 O DG0 mic H 2 ð11þ O Fig. 1 Surface tension of Tween 20 at 298 K in varying concentrations of ethylene glycol: filled circles, water; open circles, 20% EG; filled triangles, 40% EG The thermodynamic parameters of micellization that we have obtained by applying the above procedure are reported in Table 2. The free energy of micellization is negative and becomes less negative as the EG content in the mixed solvent system increases (Table 2), indicating that the formation of micelles becomes less spontaneous at higher EG content. The equation

5 535 Table 2 Thermodynamics of micellization of Tween 20 in various ethylene glycol + water mixtures EG T DG 0 mic DH 0 mic DS 0 mic DG 0 M (wt%) (K) (kj mol 1 ) (kj mol 1 ) (J K 1 mol 1 ) (kj mol 1 ) employed in the evaluation of DG 0 mic strictly applies when the mean aggregation number is large, which is not likely to be the case at higher EG contents [36]. Also, the values of enthalpy, if directly measured, will not depend on the choice of the standard state [37]. Therefore, the method employed in evaluating DHmic 0 and DSmic 0 should be viewed only as approximate. Some generalities can be postulated from the analysis of the present data. DHmic 0 goes from positive to negative and DSmic 0 decreases dramatically going from water to the mixtures with EG. However, these two effects cancel out in DG 0 mic in the end. DG0 mic is more or less in the same order of magnitude for water and the mixtures. Thus, in water the entropic contribution to DG 0 mic favours aggregation but in the mixtures with EG the enthalpic contribution predominates. Micelle formation is a structure formation from the monomeric surfactant molecules and hence the entropy change was expected to be negative. However, its positive value indicated that the hydrophobically structured water in monomeric surfactant solution becomes free or destructured in the micellar solution and the whole process is a combination of these two effects. It is apparent that the relative contribution from the latter is reduced in the presence of the additive EG. This same behaviour has been also found for surfactants in for example hydrazine and the effect for the structure of water has been previously discussed [38, 39]. The DG 0 M values were found to be positive in all cases and can be explained on the basis of reduction of the hydrophobic interactions aided by improved solvation. The values of the enthalpy of transfer function (not presented) were found to be exothermic in all cases. These values were obtained in a similar procedure employed for the evaluation of DG 0 M. The overall exothermic values for DHM 0 in the present study indicate that the exothermicity due to the structure-breaking ability of EG and its interactions with the oxyethylene groups of the surfactants is dominant over the endothermic effects due to transfer of hydrophobic groups from water to an aqueous EG mixture [40, 41]. Thermodynamics of adsorption For dilute solutions, the Gibbs equation can be written as [42]: C max ¼ ð12þ ln c T ;P where G max is the surface excess concentration of surfactant, R and T have their usual meaning, c is the corrected surface tension, and c is the concentration of surfactant. The minimum area per surfactant molecule, A min, at the air solvent interface was obtained from the surface tension measurements using the following equation: 1 A min ¼ ð13þ N A C max where N A is Avogadro s number. The values of the surface pressure at the cmc, P cmc, were obtained from the relationship: P cmc ¼ c 0 c cmc ð14þ Here c 0 and c cmc are the surface tension of pure solvent and of the micellar solution at the cmc, respectively. Finally, the free energy of adsorption, DG 0 ads, was determined using the equation: DG 0 ads ¼ DG0 mic P cmc C max ð15þ

6 536 where the standard state in the surface phase is defined as the surface covered with a monolayer of surfactant at a surface pressure equal to zero. Table 3 presents the adsorption parameters obtained for our system. The values of the G max and P cmc decrease with the ethylene glycol content, indicating that the hydrocarbon moiety of the surfactant interacts with EG, thereby leading to a shift of the surfactant molecules from the interface to the bulk solution. The temperature effect is due to an increase in molecular motion; resulting in poor packing at the liquid air interface [43]. The free energy of adsorption, DG 0 ads, represents the free energy of transfer of 1 mole of surfactant to the surface at unit pressure. The values of DG 0 ads are more negative than DG0 mic values at all EG concentrations, suggesting that when micelles are formed, work has to be performed to transfer the surfactant molecules from the monomeric form at the surface to the micellar state through the mixed solvent media. The values of DG 0 mic are found to be very slightly dependent on the temperature. The partial specific volume, m, of micellar TW-20 was calculated from [44]: t ¼ 1 1 dq ð16þ q 0 dc where q 0 and q are the densities of solvent and micellar solutions, respectively, and c is the surfactant concentration in g/ml. With the aim of evaluating the effect of the EG addition on the partial specific volume of TW- 20, the density of the surfactant solutions of varying concentrations was investigated as a function of the EG content in the solution. Figure 2 shows representative plots of density as a function of the surfactant concentration in different EG media. An acceptable linear dependence (r>0.99) was observed in all the cases. The Density studies We have investigated the effect of the addition of EG on the partial specific volume of micellar TW-20 by means of density measurements. The partial specific volume, m, represents the volume occupied per gram of dry micelle. Micelles, particularly nonionic ones, are strongly hydrated species; however, the volume of the water of hydration does not become part of the micellar partial specific volume unless water or cosolvent molecules are interacting thermodynamically with the micelle. In such a case, a reduction of the solvation of the hydrophilic group of the surfactant should be reflected in a decrease in the micellar partial specific volume. Fig. 2 Density of Tween 20 micellar solutions as a function of the surfactant concentration at 25 C in: filled circles, water; open circles, 30% EG Table 3 Surface excess concentration (G max ) minimum area per molecule (A min ) surface pressure at the cmc P cmc and Gibbs energy of Adsorption for Tween 20 in aqueous mixtures of ethylene glycol Composition T Gmax Amin Pcmc DG 0 ads (wt% of EG) (K) (10 6 /mol m 2 ) (10 20 /m 2 ) (mn m 1 ) (kj mol 1 )

7 537 Table 4 Density values of the solvent systems q 0 density increment of micellar solutions of Tween 20 dq/dc and the corresponding partial specific volumes m in different media at 25 C EG q 0 dq/dc m (wt%) (g cm 3 ) (ml g 1 ) corresponding results from these experiments, together with the partial specific volume as obtained from Eq. 16, are presented in Table 4. As can be seen, the partial specific volume of TW-20 was not affected, within the experimental error, by the EG addition. It is well known that nonionic surfactants with polyoxyethylene groups as hydrophilic moieties can incorporate water or solvent molecules into their structure through two different mechanisms: (i) by hydrogen bonding or, (ii) by mechanically trapping the molecules in the outer shell of the micelle [44]. It is expected that density measurements depend on the water or solvent molecules with a significant thermodynamic interaction with surfactant molecules via hydrogen bonds. Therefore, the observed behaviour for m (Table 4) can be interpreted in the sense that the portion of micellar solvation that interacts with TW-20 micelles, via hydrogen bonding, is not affected by the EG addition in the composition range studied. This fact can be due to three possible reasons: (a) that the micellar solvation remains unaffected with the EG addition, (b) that, by increasing the whole micellar solvation (EG+water), the amount of solvent molecules interacting via hydrogen bonding with TW-20 micelles remains roughly constant as a consequence of the minor ability of EG to form hydrogen bonds compared with water, and (c) it should also be noted that there could be two effects that cancel out each other. For example, (i) the micelle volume without solvation decreases with increasing EG content and (ii) the solvation volume increase with increasing EG content. Note that the second and third possibilities should imply a change in the composition of the micellar solvation layer. Micellar size: light scattering studies For dilute solutions, where the intermicellar interactions can be considered negligible, the collective diffusion coefficient is given by a linear function of the surfactant concentration [45, 46]: D c ¼ D 0 ½1 þ k D ðc cmcþš ð17þ where D 0 is the actual diffusion coefficient at infinite dilution, k D is a constant related to the second virial coefficient and c is the surfactant concentration. The hydrodynamic radius of micelles, R h, was calculated from D 0 by applying the Stokes-Einstein relation: R h ¼ k B T ð18þ 6pg 0 D 0 where k B T is the thermal energy factor and g 0 is the solvent viscosity. Light scattering studies were carried out at surfactant concentrations ranging from 5 to 25 g/l. It was found that the collective diffusion coefficients (data not presented) showed a negligible dependence on the surfactant concentration in all the studied media, i.e. for EG content ranging from 0% to 40%. This is an unexpected behaviour, and in disagreement with some results obtained by other authors [47] and even for us in the case of the Triton X-100 [25]. We think that this fact is a consequence of the small size of the micelles of TW-20, and it indicates the absence of intermicellar interactions in the systems for the micellar concentration range studied, as previously assumed. The actual diffusion coefficients, D 0, obtained were used to determine the hydrodynamic radii according to Eq. 18. The values of both parameters are presented in Table 5. Our results indicate a large reduction in D 0 with increasing EG concentration. However, the R h values show a maximum value at 10% EG, and a continuous decrease at higher EG content. This means that a considerable part of the reduction in D 0 value is attributable to the increase in solvent viscosity (see Table 5). On the other hand, since both the mean aggregation number and the entire micellar solvation determine the observed behaviour of R h, we need to gain information on the effect of EG addition on these factors. In order to examine the evolution of the aggregation number of TW-20 micelles upon EG addi- Table 5 Actual diffusion coefficients (D 0 ), solvent viscosities (g 0 ) and structural parameters of Tween 20 micelles as a function of ethylene glycol content in the mixed solvent at 25 C EG D g 0 R h M w N agg R 0 d a 0 (wt%) (cm 2 s 1 ) (cp) (nm) (Da) (nm) ( A 2 ) , , , , ,

8 538 tion, we decided to carry out static light scattering (SLS) measurements. The excess of scattering, DR h, is given by the expression: DR h ¼ Kc ð cmcþm w PðqÞ Sðq; cþ ð19þ where M w is the molecular mass of the micelles, P(q) the micelle form factor, S(q) the structure factor, and K is an optical constant given by: K ¼ 4p2 n 2 0 ðdn=dcþ2 N A k 4 ð20þ 0 where n 0 is the solvent refractive index, dn/dc is the refractive index increment of the sample solution, and k 0 is the wavelength of incident light in a vacuum. The micelle form factor characterizes the intramicelle interference and essentially describes the angular dependence of the scattering intensity. It is usually expressed in terms of the scattering wave vector, q. If the particle size is much smaller than the wavelength of light, as we expect for the micelles formed by TW-20 surfactant, then the scattering intensity generally shows a very low angle dependence, and the contribution of P(q) can be neglected, i.e. P(q)=1 [48]. The structure factor, S(q), reflects the intermicellar interactions and its value oscillates in a damped way around unity, reaching this value when interactions are negligible. In addition, as the total surfactant concentration, c, is much larger than the cmc value, we can assume that c cmc c. Lastly, because the conditions described above fall in the case of a nonionic surfactant at relatively low surfactant concentration [49, 50], we can express Eq. 19 as: DR h ¼ KM w c ð21þ Fig. 3 Excess scattering ratio DR 90 as a function of Tween 20 concentration at 25 C in various media: filled circles, water; open circles, 10% EG; filled triangles, 20% EG; open triangles, 30% EG; asterisks, 40% EG. Solid lines are the best fit of data to Eq. 21 In Fig. 3 the excess scattering ratios for TW-20 solutions at an angle of 90, DR 90, are displayed versus surfactant concentration in various media with different EG content. Data in Fig. 3 show a good linearity, indicating that the intermicellar interactions are negligible, as previously assumed, and, on the other hand, that the micellar molecular weights are essentially independent of the total surfactant concentration in the range studied. The micellar molecular weights, M w, of TW-20 in the respective media were estimated from the slopes of the plots in Figure 3. Subsequently, we obtained the aggregation number values, N agg, and the dry micellar volumes, V 0,by: V 0 ¼ tm w ð22þ N A By assuming a spherical geometry for TW-20 micelles, we estimated the micellar radii, R 0. The values so obtained were used to evaluate the solvation micellar factor, d, defined as grams of water or solvent mixture associated with 1 g of dry micellar surfactant, and which can be determined by [44]: " # R 3 h d ¼ 1 tq R 0 ð23þ 0 The values obtained for, M w, N agg, R 0, and d are listed in Table 5. In order to compare the N agg value of TW-20 in water that we obtained in the present study, we carried out an extensive review of the literature over the past ten years. We found only one paper [51] concerned with the determination of the aggregation number of TW-20, among other nonionic surfactants. In the paper, the aggregation number was evaluated on the basis of the photophysical behaviour of the dye safranine T in micellar media. The authors found that the aggregation number of TW-20 micelles ranges from 22 to 150, depending on the surfactant concentration range considered. However, judging by the results obtained in the same work for Triton X-100, which is extensively reported in the literature, it seems that the safranine T method tends to overestimate the value of the aggregation number when high concentrations of surfactant are used. Data in Table 5 shows that whereas the aggregation number of TW-20 micelles decreases, the solvation factor increases with the EG content in the solvent system. It is important to point out that the information given by the solvation factor, d, is of a hydrodynamic nature, reflecting not only that the solvent molecules are thermodynamically interacting with the micelle, but also that they are travelling with the micelle as it diffuses. Whereas the partial specific volume, m, provides information only on the solvent thermodynamically interacting with micelles, the solvation factor, d, provides

9 539 information on the entire solvent content associated with the micelles. In Table 5, we have also included the surface area per headgroup, a 0, as obtained from the dry radius and the aggregation number by assuming a spherical geometry. This is an important structural parameter that plays a decisive role in the geometric or packing properties of micelles, as it controls the magnitude of steric repulsions between the headgroups [52]. As can be seen in Table 5, the surface area per headgroup of TW-20 increases with the EG content in the solvent system. This behaviour is consistent with a participation of EG molecules in the solvation layer of the micelles. Note that substitution of several water molecules by larger EG molecules will lead to the formation of a thicker solvation layer. It is interesting to notice that this fact would produce an increase in the local viscosity of this region of the micelle. As a consequence, the effect of the EG addition on the microenvironmental properties of TW-20 micelles was also studied. Microenvironmental properties Changes in the microstructure of micelles are often analysed by exploring the behaviour of two important microenvironmental properties, namely micropolarity and microviscosity. In order to examine any possible modification in micellar micropolarity on EG addition, we used two fluorescent probes, pyrene and ANS, both of which are good indicators of the polarity of the probe solubilization site [53]. Pyrene is solubilized in the micellar palisade layer, close to the micellar surface. Thus the polarity sensed by this probe could reflect alterations in the degree of solvation of the micelle [53]. Figure 4 shows the change of the pyrene 1:3 ratio index on EG addition. It is seen that the pyrene 1:3 ratio increases with EG content, indicating that pyrene senses a more polar microenvironmental. However, this result is not necessarily due to an increase of micropolarity in the micellar palisade layer. Note that an increase of the surface area per headgroup, as observed in the preceding section, could force a more outward localization of the probe in the micelle, which would contribute to a more polar microenvironment. Figure 5 shows various emission spectra of ANS in TW-20 micellar solutions with several different EG concentrations. ANS is a surface probe whose emission properties are very sensitive not only to the polarity of the medium, but also to the viscosity in the probe environment [54]. It is well known that ANS is almost nonfluorescent in aqueous solutions, but exhibits a large fluorescence enhancement (increase of the emission quantum yield and lifetime) on binding to the micellar surface. This behaviour has been interpreted on the basis of the polarity-dependent twisted intramolecular charge transfer concept [55]. Consequently, results in Figure 5 can be explained either in terms of increasing polarity at the micellar surface or due to the transfer of some ANS molecules from the micellar surface to the bulk. This second possibility would be associated with a less rigid environment sensed by the probe. Although we can not extract definitive conclusions about changes in micropolarity from the results obtained by pyrene and ANS, these results in connection with the structural parameters in Table 5 seem to indicate that the presence of EG in the solvent system causes the formation of micelles with a more open structure where the solvent penetration is favoured and, therefore, with an increased solvation. Fig. 4 Variation of pyrene 1:3 ratio versus ethylene glycol concentration in Tween 20 micellar solutions at 25 C Fig. 5 Fluorescence emission spectra of 8-anilinonaphthelene-1- sulfonic acid in Tween 20 micellar solutions at 25 C: 1, water; 2, 40% EG; 3, 50% EG; 4, 60% EG

10 540 that depolarization of light emitted from probes associated with micelles can be produced by two different rotational processes [57]: (i) diffusion of the probe in the micelle, and (ii) rotation of the micelle itself. In our case, as the size of TW-20 micelles becomes smaller with the addition of EG, the second rotational process should increase. However, we must also take into account the increase of viscosity of the medium as a consequence of the EG addition. Probably, this effect could compensate to a certain extent the effect of the reduction in the micellar size as the EG content increases. Therefore, we postulate that the increase in the fluorescence anisotropy seen in Fig. 6 must be related with a greater rigidity of the microenvironment around the probe. Fig. 6 Fluorescence anisotropy of coumarin 6 in micellar solutions of Tween 20 as a function of the ethylene glycol content. The error bars in the fluorescence anisotropy plots indicate the standard deviation from triplicate measurements A common procedure to gain information about rotational diffusion of molecular probes is through the study of the degree of fluorescence polarization. Steadystate fluorescence anisotropy, r, is related to the viscosity around the probe, g, by Perrin s equation: r 0 r ¼ 1 þ k B T s ð24þ V g where r 0 is the anisotropy measured in a solvent of extremely high viscosity, k B is Boltzmann s constant, T is the absolute temperature, and V and s are the effective volume and the fluorescence lifetime of the probe, respectively. Therefore, larger anisotropy values correspond to a rigid environment at a fixed temperature. We carried out fluorescence polarization studies by using coumarin 6 (C6) as a probe. C6 is a hydrophobic probe whose fluorescent properties in homogeneous and micellar media have been reported [56]. Taking into account its spectral characteristics, it can be inferred that this probe is solubilized in the palisade layer of TW-20 micelles, where it senses a relatively polar microenvironment similar to that of ethanol. We have previously used C6 to study changes in the microviscosity of Triton X-100 micelles, and the fluorescence anisotropy of this probe correlates well with the viscosity values of its solubilization site [25]. Figure 6 shows the results obtained in our present experiments with C6. As can be seen, the anisotropy of the probe increases with the EG content of the solvent mixture. It is important to remark Conclusions From the study of the temperature dependence of the cmc of TW-20 in various media with different EG content, we have observed that the micellization process is less favourable in the cosolvent-water mixture and worsens as the cosolvent content increases. This effect has been ascribed to the structure-breaking ability of EG and the interaction of the cosolvent with the polyoxyethylene groups of the surfactant. Thermodynamic adsorption data indicate that the surface activity of the surfactant is reduced with EG addition at a fixed temperature. Light scattering and density measurements of TW-20 micellar solutions with varying concentrations of EG, provided information on both micellar size and solvation. It was found that the presence of the cosolvent produces a reduction in the micellar aggregation number and an increase in the whole micellar solvation. However, the fraction of solvent molecules interacting with micelles via hydrogen bonding seems not to change with the EG content. Microenvironmental studies revealed that the addition of cosolvent causes an increase in both micropolarity and microviscosity. These effects are consistent with the structural evolution followed by the aggregates, and can be rationalized if we assume a participation by EG in the micellar solvation of TW-20 micelles. Acknowledgements The authors wish to thank Prof. R. Hidalgo- A lvarez, Group of Fluid Physics and Biocolloids of the University of Granada, for providing light scattering research facilities. C. C. R., J. A. M.-B., and J. A. acknowledge financial support from the Spanish Science and Technology Ministry (Project MAT ). R.P. acknowledges the support received from the Natural Science and Engineering Research Council (NSERC) in the form of an operating grant.

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