Infrared Study of the Hydrophobic Hydration and Hydrophobic

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J. Phys. Chem. B 2001, 105, 12714-12718

Infrared Study of the Hydrophobic Hydration and Hydrophobic Interactions in Aqueous Solutions of tert-Butyl Alcohol and Trimethylamine-n-oxide M. Freda, G. Onori,* and A. Santucci Istituto per la Fisica della Materia (INFM) Unita` di Perugia and Dipartimento di Fisica, UniVersita` di Perugia, Via Pascoli, I-06100 Perugia, Italy ReceiVed: May 3, 2001; In Final Form: September 18, 2001

This work concerns a comparison of the hydration properties and self-association behavior in aqueous solution of two biologically relevant simple molecules: the trimethyl-amine-N-oxide (TMAO) and the tert-butyl alcohol (TBA). These molecules are geometrically very similar, having the same hydrophobic moiety and different polar groups. Both molecules were used as a model to study hydrophobic behavior in water solution. In particular, water perturbation induced by TBA and TMAO molecules was studied as a function of the solute molar fraction X2 (0 < X2 < 0.05) by using the IR absorption bands due to the vibrational modes of water in the 4000-2600 cm-1 frequency region. Furthermore, possible clustering effects in aqueous solution of the TBA and TMAO hydrophobic groups were investigated by studying the behavior of the alkyl CH stretching band in the 3200-2800 cm-1 frequency region as a function of X2. The OH stretching absorption data show, in agreement with molecular dynamics simulation results and other suggestion found in the literature, that the interaction of the TBA and the TMAO with water are remarkably different. In fact, water molecules are more coordinated by TMAO than by TBA. Significant differences are also evident in the CH stretching data for the two molecules. For TBA, the data can be interpreted in terms of a self-aggregation process of the alcohol molecules occurring beyond a threshold value of the alcohol molar fraction (X2* ) 0.025). This phenomenon seems to be absent in the TMAO samples.

Introduction The physicochemical quantities associated with the introduction of hydrophobic species in water exhibit a number of uncommon features, which are related either to the hydration of these solutes (hydrophobic hydration) or to their mutual interaction (hydrophobic interaction). Since water forms the basis of all biologically important fluids, such hydrophobic effects are of wide interest. Among the systems in which hydrophobic hydration effects are found, the most commonly investigated is the series of monohydric alcohols. In the literature, the general description proposed for these systems states that at low concentrations (0-0.1 mole fraction range) the macroscopic properties of the water/alcohol solutions are mainly influenced by the alkyl residues of the solute molecules rather than by the hydrogen bonding between their polar moieties. In other words, the hydrophobic hydration and the hydrophobic interaction are supposed to dominate the physicochemical properties of these mixtures.1-4 Within the class of water-soluble monohydric alcohols, the tert-butyl alcohol (TBA) is the molecule with the largest hydrophobic group, i.e., it is the most hydrophobic molecule among the low-weight water-soluble alcohols. For these reasons, TBA appears to be the ideal candidate to investigate possible variations in the structural and dynamical properties of water near apolar solutes. Many of the physical properties of TBA/water mixtures were studied in great detail. Of particular interest are the extreme values of many physical properties exhibited by such mixtures * To whom correspondence should be addressed. Dipartimento di Fisica, Universita` di Perugia, Via A. Pascoli, I-06100 Perugia, Italy. E-mail: [email protected].

at the water rich end of composition scale. Examples include the minimum of the partial volume,2,5 the maximum of the excess heat capacity,6 the large ultrasonic absorption,7 a remarkable increase in light scattering,8 etc. All these anomalies occur in a composition range of about 3÷5 mol % in the TBA content. Our recent study on the properties of water/alcohol mixtures shows that this anomalous behavior can be associated with some kind of “hydrophobic clustering” of alcohol molecules in the water rich region of composition, beyond a threshold value X2* ∼ 0.025 of the alcohol molar fraction.9 The observed behavior suggests that alcohol molecules are essentially dispersed as monomers and surrounded by water molecules at low alcohol concentrations (X < X2*); above X2*, some clustering phenomena occur. Recent results indicate that such behavior of water-alcohol mixtures, including the presence of clustering effects, can have a dramatic influence on a large variety of macroscopic molecular processes occurring in water-alcohol mixtures. Example are the thermal unfolding of nucleic acids10 and proteins11 and the micellization process of surfactant molecules.12 In the present work, the hydration properties and the hydrophobic clustering effect of TBA in water are compared with the behavior in aqueous solution of a biologically relevant molecule: the trimethylamine-n-oxide (TMAO). The TMAO belongs to a class of small organic molecules (osmolytes) present in organisms living under condition of water stress, where it acts primarily as a regulator of the osmotic pressure in the intracellular fluids.13-15 In vitro, osmolytes typically stabilize the native state of globular proteins against thermal denaturation.15 TBA and other monohydric alcohol cause an opposite effect, since it is well-known that these solutes show a destabilizing effect upon the native conformation of proteins.1-3,11

10.1021/jp011673m CCC: $20.00 © 2001 American Chemical Society Published on Web 11/30/2001

Hydrophobic Hydration of TMAO and TBA Solutions

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Figure 1. Structural forms of TBA and TMAO.

However, both TMAO15 and TBA9 are strongly excluded from the protein domain, thus making unfavorable their direct binding with the protein. Therefore, the opposite action of TMAO and TBA on proteins must be correlated to differences in the hydration properties of the two molecules. An important effort to characterize the hydration properties of these two solutes comes from a recent ab initio and molecular dynamics simulation work.16 In this paper it is shown that water molecules are more tightly coordinated by TMAO than TBA. This result appears to be consistent with the relative strength of their dipole moments, being µTMAO ≈ 3µTBA. Similar considerations prompted us to compare the hydration characteristics of these two molecules by performing an infrared study of water/TBA and water/TMAO mixtures at selected values of solute mole fraction X2 in the water-rich region of composition. The IR spectroscopy is a noninvasive, selective technique, capable of distinguishing among populations of different hydrogen-bonded molecules. Therefore, this technique appears to be suitable to characterize the structure of the hydration shells as well as solute-solute interaction. Experimental Section Samples of H2O/TBA and H2O/TMAO were prepared by weight, and by using bi-distilled and deionized water. CH stretching band is recorded for D2O/TBA and D2O/TMAO samples. D2O (99.9% Aldrich product), TBA alcohol (Aldrich product) and anhydrous TMAO (Aldrich product) were used without any further purification. IR spectra were taken at room temperature by means of a IR 470 Shimadzu spectrophotometer, in the range 4000-2600 cm-1 (OH stretching band) and ca. 3200-2800 cm-1 for the CH bands. The spectroscopic cell has CaF2 windows and optical path length of 2µm and 6µm for detecting OH stretching band and CH stretching band, respectively. Results and Discussions OH Stretching Absorption Band in Water/TBA and Water/TMAO Samples. TBA and TMAO are geometrically very similar, having the same hydrophobic moiety and different polar groups (Figure 1). To investigate the effect of dissolving TBA or TMAO on the properties of solvent water, we performed a series of IR measurements on the OH stretching absorption band for water/ TBA and water/TMAO samples. In both cases, the molar fraction X2 of the solute was varied from 0 to 0.05. In Figure 2 the OH stretching absorption band for H2O/TBA (Figure 2a) and H2O/TMAO (Figure 2b) mixtures at X2 ) 0.05 are shown and compared with the corresponding OH stretching band of pure water. The changes observed with respect to the pure H2O band are small, indicating a distribution of hydrogen bond energies only slightly perturbed by the presence of the two solutes. However, the higher frequency edge of the OH absorption band decreases; this effect is more evident for

Figure 2. Normalized OH absorption spectra for pure H2O (s) and (a) H2O/TBA (- - ); (b) H2O/TMAO (- - ) at X2 ) 0.05.

TMAO/water solutions. The frequency shift of the OH oscillators is more clearly observed by analyzing the difference spectra (Figure 3a,b), obtained by subtracting the OH spectrum of pure water from the spectra of mixtures. A sharp peak is clearly visible in the low-frequency region of the spectrum (∼2985 cm-1) of TBA/H2O samples. This peak, whose entity increases with the molar fraction of TBA, can be attributed to the CH stretching absorption bands of the methylic groups of TBA. The same band is located for the TMAO/H2O samples at a frequency of about 2970 cm-1; however, its intensity is much less pronounced, and its contribution in the region of the OH stretching in TMAO sample is negligible. Looking in more detail at the difference spectra in Figure 3a,b, it is evident their approximate inversion symmetry. This behavior essentially indicate that, with increasing solute concentration, the OH spectra undergo a frequency, shift toward lowest frequencies instead of a distortion of their band line shape. In other words, adding TBA or TMAO to pure water causes a transfer of OH oscillators from the highest to the lowest frequency region of the spectrum. For TBA samples, the frequency edge of the OH band shifts toward intermediate frequencies. Furthermore, it appears less pronounced than that of TMAO samples, where the shift is visible at lower frequencies (3200÷2800 cm-1). These variation in the OH band profile are quite similar to those observed by decreasing the temperature of a pure water sample. (a) H2O/TMAO Spectra. In the literature, the presence of populations of water molecules involved in different hydrogenbond structures was quantitatively taken into account by decomposing the OH spectrum in a sum of three Gaussian

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Figure 3. Normalized difference spectra for (a) H2O/TBA and (b) H2O/ TMAO mixtures at X2 ) 0.03 (‚‚) and 0.05 (s).

bands.17-19 In Figure 4a, the OH stretching absorption band for pure water is reported together with its decomposition in Gaussian bands. These bands are centered respectively at (3603 ( 6) cm-1, (3465 (5) cm-1, and (3330 (20) cm-1, in good agreement with the frequency values reported in the literature. The highest frequency component at 3603 cm-1 is only a small part (about 6%) of the total absorption band and it is usually assigned to non-H-bonded or weakly H-bonded OH groups.20,21 The lowest frequency component at 3330 cm-1 (about 68% of the total band) has been related to H2O molecules involved in more regular structures of fairly unstrained H-bonds, and the intermediate component at 3465 cm-1 (about 26% of the total band) to water molecules trapped into more irregular structures of energetically unfavorable H-bonds.18-20,22,23 The increase of regular H-bonded structures in water, occurring, for example, by decreasing the temperature, is accompanied by a shift toward lower frequencies of the OH stretching absorption band, with a corresponding decrease of the force constant of the bonds. A similar situation is observed also analyzing the evolution of the OH stretching band for water encapsulated into the core of a reverse micelle.17 Starting from these experimental evidences, we have applied the same interpretative scheme to the TMAO/H2O OH stretching band to interpret the pronounced lower frequency shift of this spectrum. In this procedure the frequency and the width of the three Gaussian components are fixed to the values reported in the literature for the pure H2O spectrum. Therefore, the relative amounts of the different Gaussian bands within the total OH band are determined by variations of their amplitudes. It can be observed that a fitting procedure which employs a sum of

Freda et al.

Figure 4. (a) OH stretching band for pure water. (s) Experimental data. (- - ) Gaussian components from least-squares fitting (‚‚): fitted spectrum. (b) OH stretching band for H2O/TMAO system at X2 ) 0.05. (s) Experimental data. (- - ) Gaussian components from least-squares fitting; (‚‚): fitted spectrum.

only three Gaussian components cannot adequately reproduce the OH stretching absorption band, since there is an evident failure of the fit located in the low-frequency region. To adequately reproduce the band we need to introduce a further small Gaussian component centered at about 2973 cm-1. In Figure 4b the experimental OH stretching band is reported for the TMAO/H2O sample at X2 ) 0.05 together with the curve resulting from the fitting procedure. It is evident from the figure that the fitted curve is virtually indistinguishable from the measured one. Starting from this result, the same fit with four Gaussian components was performed for all the examined H2O/TMAO samples with molar fraction X2 ranging from 0.005 to 0.05. The contribution of A4 to the total area A (A4/A) (Figure 5) increases linearly with X2 in all range of molar fractions examined. A linear fit was performed to the data. The angular coefficient of the line is related to the number of OH oscillators per each TMAO molecule, which oscillate at the lower frequency (2973 cm-1). A value of two OH oscillators per TMAO molecule is found from the fitting procedure. Note that in the crystalline dihydrate TMAO the hydrogen bonds of the two N-O coordinated water molecules are shorter than those occurring between water molecules.24 These facts suggest that the abovementioned OH oscillators can be attributed to the two water molecule tightly bonded to the N-O group of the TMAO molecule, i.e., to the H2O-TMAO OH‚‚‚O bonds, which are stronger than the mean water-water hydrogen bonds. Little variations with the molar fraction X2 were also found for the

Hydrophobic Hydration of TMAO and TBA Solutions

Figure 5. Ratio between the area of the fourth Gaussian component A4 to the total area A as a function of TMAO molar fraction. (9) Experimental data. (s) Linear fit.

Figure 6. Ratio of the ith Gaussian area Ai to the total peak area A as a function of TMAO molar fraction. Component centered at (b) 3603, (9) 3465, and (2) 3330 cm-1.

ratio Ai/A of the other three Gaussian components (Figure 6). The contribution A3/A of the component at 3330 cm-1, assigned to water in regions characterized by a regular tetrahedral connectivity, slightly increases with X2: in fact, it ranges from 68% (pure water) up to 77% at X2 ) 0.05. The increase in intensity of the 3330 cm-1 component is accompanied by a corresponding decrease of the intermediate contribution A2/A from 26% (pure water) to 20% (X2 ) 0.05) and of the A1/A contribution (6% to about 3%). Again, this result shows that in the presence of TMAO molecules, water tends to reorganize itself around the solute in a more regular and unstrained structure. (b) TBA/H2O Samples. As it can be seen from Figures 2a and 3a, the presence of TBA molecule alters the structure and the band shape of OH stretching band in a less significant way than TMAO does. The reconstruction of the OH spectrum in terms of Gaussian bands, already discussed for the H2O/TMAO samples, cannot be easily done in the case of TBA, owing to the non negligible absorption in the 4000-2600 cm-1 region coming from the OH and the methylic groups of the alcohol. In particular, it seems arbitrary to abruptly subtract and isolate the last contribution, well visible in the difference spectra, because the methylic absorption band shifts toward lower frequency with respect to the pure TBA spectrum, with increasing the alcohol concentration in water (see Figure 8 in the next paragraph). However, if

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Figure 7. Normalized difference absorbance at 3600 cm-1 for (9) H2O/TBA and (b) H2O/TMAO samples. (s) Lines are guide for eyes.

we compare the difference spectra of H2O/TBA solutions (Figure 3a) with the spectrum of pure TBA (not shown), one can consider that the frequency range between ∼4000 and 3600 cm-1 is relatively free from pure TBA spectrum contribution. Therefore, some information can be extracted in this region of TBA/H2O spectra. We choose for example the minimum of the difference spectra, which is located at about 3600 cm-1. The absolute values of the minima are plotted in Figure 7 as a function of the molar fraction X2 of the solute; for the sake of comparison, the same quantity is plotted in Figure 7 for the H2O/TMAO samples. In both cases, the height of the minimum increases with the concentration of solutes. This increase is less pronounced for TBA than in the TMAO case; in addition, the trend observed for TBA solutions shows a break at a molar fraction X2* ∼ 0.025, while for TMAO solutions an almost linear trend with X2 is observed. Furthermore, the strength of the minima, which are strictly related to the amount of change in the OH stretching band, are much smaller in the case of TBA than that in the case of the TMAO molecule. The presence of a break for the H2O/TBA samples at a molar fraction of X2* ) 0.025 can be linked, as briefly reported in the Introduction, to the aggregation phenomena involving the alcohol molecules beyond a critical concentration.9 These effects are explained in further details in the next paragraph. We emphasize that the clustering process is detected here looking at the behavior of water molecules rather than at that of alcohol molecules. C-H Stretching Absorption Band. In recent years infrared spectroscopy has been used to investigate phase transitions in various surfactant systems.25-28 These studies show that monomermicelle formation transitions are accompanied by large (4-8 cm-1) 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 monohydric alcohols in water.17 Figure 8 shows the trend vs X2 of the spectroscopic frequency νj relative to the higher frequency CH stretching mode of the TBA methyl moiety. At low concentrations of the TBA, νj (as well as other parameters of the same absorption band) appears to be constant, in the whole 0 < X2 < X2* range (X2* ) 0.025). High values of νj in this molar fraction range mean that an aqueous environment essentially surrounds the CH3 groups of the TBA molecule. For X2 > X2*, the frequency decreases rapidly as a function of X2, reaching a value typical of the pure alcohol CH stretching band at higher molar fraction values. This

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Freda et al. Significant differences are also evident in the CH stretching data for the two molecules. For TBA, the data can be interpreted in terms of a self-aggregation process of the alcohol molecules occurring beyond a threshold value of the alcohol molar fraction (X2* ) 0.025). This phenomenon seems to be absent in the TMAO samples. References and Notes

Figure 8. Plot of the frequency maximum of the CH stretching band for TBA/H2O samples as a function of X2. (s) Lines are guide for eyes.

behavior indicates a progressive hydrophobic clustering of the alcohol molecules. On the contrary, no variation in frequency or in any other parameter of the CH band is observed for TMAO solutions. Here the CH stretching band maintains the features proper of a monomeric dispersion in water. In other words, the data seem indicate that the aqueous environment of the TMAO methyl groups remains unaltered in the whole X2 range examined. Conclusions A detailed IR study was performed on H2O/TBA and H2O/ TMAO samples as a function of the molar fraction X2 of the solutes. The aim was to investigate the modification induced by TBA and TMAO molecules on the properties of the water, and possible aggregation processes of these molecules in the aqueous environment. The picture emerging from the collected data shows that the behavior of TMAO and TBA molecules in aqueous solutions is quite different. According to previous suggestions found in the literature, the TMAO molecule tends to significantly reorganize the surrounding water into more regular structures, whereas the alcohol molecule seems to alter less significantly the properties of the aqueous environment. In addition, nonadditive perturbations of the H-bonded networks of solvent water are observed in the case of TBA solutions, but are absent in the TMAO case.

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