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Articles Dynamic Rheology of Sodium Deoxycholate Gels Aida Jover, Francisco Meijide, Eugenio Rodrı´guez Nu´n˜ez, and Jose´ Va´zquez Tato* Universidad de Santiago de Compostela, Facultad de Ciencias, Departamentos de Quı´mica Fı´sica y Fı´sica Aplicada, Lugo, 27002 Spain Received July 25, 2001. In Final Form: November 20, 2001 A dynamic rheological study of sodium deoxycholate gels was carried out within the linear viscoelastic region. The effects of the oscillatory frequency f, sodium deoxycholate concentration, pH, temperature, and ionic strength (NaCl) were studied. The G′ ∝ f n relationship, the creep-recovery experiments, and the frequency-temperature superposition principle strongly suggest that the interactions in the gel are physical in nature and that its mechanical behavior can be explained by a simple relaxation mechanism. It is concluded that the hydrogen bonds are the molecular forces involved in the formation, relaxation, and strength of the gel.
Introduction Bile salts are key biological surfactants in vertebrates that contain a hydrophobic side, a hydrophilic side, and a short hydrophilic tail.1 Having a planar polarity,2 they form aggregates in aqueous solution above their critical micelle concentrations. Although many studies using different experimental techniques have been published, some contradictions about the aggregation process and the structure of the aggregates are still unresolved,3 and some differences between di- and trihydroxy bile salts (for instance, their aggregation numbers3,4 or complexation by β-cyclodextrin5) are not definitively understood on a molecular basis. Sodium deoxycholate (NaDC, a dihydroxy bile salt) presents a particular behavior in aqueous solution. The aggregation number increases from six (in the absence of added electrolytes and at high pH)3 to very large values near the gelation threshold. For example, at pH 7.3, Small has obtained a value of 552 for the aggregation number from ultracentrifugation measurements.6 Gels, first noticed by Sobotka and Czeczowiczka,7 are formed at pH values close to neutrality. This is not a general characteristic for other bile salts, particularly for trihydroxy ones. Sodium lithocholate,8 a monohydroxy bile salt, and chenodeoxycholic acid, another dihydroxy bile acid, also form gels.9 Several authors have focused their attention on the gelation of deoxycholate solutions. At pH values close to * Author to whom correspondence should be addressed. Phone: 34-982-223-996. Fax: 34-982-224-904. E-mail:
[email protected]. (1) Carey, M. C.; Small, D. M. Arch. Intern. Med. 1972, 130, 506. (2) Hofmann, A. F.; Small, D. M. Annu. Rev. Med. 1967, 18, 333. (3) Coello, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. J. Pharm. Sci. 1996, 85, 9. (4) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. Recent Res. Dev. Phys. Chem. 1999, 3, 323. (5) Ramos Cabrer, P.; Alvarez-Parrilla, E.; Meijide, F.; Seijas, J. A.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. Langmuir 1999, 15, 5489. (6) Small, D. M. Adv. Chem. Ser. 1968, 84, 31. (7) Sobotka, H.; Czeczowiczka, N. J. Colloid Sci. 1958, 13, 188. (8) Terech, P.; Smith, W. G.; Weiss, R. G. J. Chem. Soc., Faraday Trans. 1996, 92, 3157. (9) van Berge-Henegouwen, G. P.; Hofmann, A. F.; Gaginella, T. S. Gastroenterology 1977, 73, 291.
neutrality, Blow and Rich10,11 proposed that deoxycholate aggregates grow according to a helicoidal structure. They also noticed the importance of the adsorption of protons on the aggregates during the process and suggested the formation of intermolecular hydrogen bonds between deoxycholate molecules. Sugihara et al. reached this same conclusion, when studying the behavior of these systems under different experimental conditions12,13 and the solgel transition at high pressures.14 More recently, an unusual formation of pyrene excimers during the gelation process was observed.15 It was also concluded that the gel behavior of the solution is due to the entanglement of polymer-like aggregates, which must have a minimum critical size to make the entanglement possible.16 D’Archivio et al.17 also concluded that the interactions between large aggregates are responsible for gel formation. Reis and da Silva,18 from viscosity measurements, also noticed that NaDC form gels at pH 7.1 and concentrations above 5 mM. Dynamic rheology provides information on the viscoelastic behavior of materials through the determination of the storage and loss moduli. The dependence of these quantities on the oscillatory frequency gives rise to socalled mechanical spectroscopy,19 allowing for the complete viscoelastic characterization of the studied material. The literature cited above indicates a lack of rheological studies on gels formed by bile salts. In this paper, we (10) Rich, A.; Blow, D. M. Nature 1958, 182, 423. (11) Blow, D. M.; Rich, A. J. Am. Chem. Soc. 1960, 82, 3566. (12) Sugihara, G.; Tanaka, M. Bull. Chem. Soc. Jpn. 1976, 49, 3457. (13) Sugihara, G.; Tanaka, M.; Matuura, R. Bull. Chem. Soc. Jpn. 1977, 50, 2542. (14) Sugihara, G.; Ueda, T.; Kaneshina, S.; Tanaka, M. Bull. Chem. Soc. Jpn. 1977, 50, 604. (15) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J.; Mosquera, M.; Rodrı´guez Prieto, F. Langmuir 1996, 12, 1789. (16) (a) Jover, A.; Meijide, F.; Rodrı´guez Nu´n˜ez, E.; Va´zquez Tato, J. Langmuir 1998, 14, 4359. (b) Jover, A. Doctoral Thesis, Universidad de Santiago de Compostela, Lugo, Spain, 1994. (17) D’Archivio, A. A.; Galantini, L.; Giglio, E.; Jover, A. Langmuir 1998, 14, 4776. (18) Reis, M. S. H.; da Silva, A. M. R. Rev. Port. Farm. 1986, 36, 32. (19) Ross-Murphy, S. B. In Physical Techniques for the Study of Food Biopolymers; Ross-Murphy, S. B., Ed.; Blackie: London, 1994.
10.1021/la011178h CCC: $22.00 © 2002 American Chemical Society Published on Web 01/26/2002
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Figure 1. Dependence of the complex shear modulus on the deformation of the samples at an oscillation frequency of 3.16 Hz and a temperature of 293 K. [NaDC] ) 0.1158 mol dm-3, [phosphate] ) 0.02324 mol dm-3. pH values are shown in the corner of the plot.
present the results of a rheological study of NaDC gels prepared under different experimental conditions, paying particular attention to the influence of the oscillatory frequency, NaDC concentration, pH, temperature, and ionic strength on the mechanical moduli. Experimental Section NaDC (Sigma) was purified as described elsewhere.3 Other chemicals including NaH2PO4, NaOH, and NaCl (Merck) and HCl (Panreac) were used without further purification. Water was Milli-Q grade. Solutions of NaDC were added to the appropriate mixtures of NaH2PO4, NaOH or HCl, and NaCl to obtain the desired pH and ionic strength. The homogeneous samples were transferred to cylindrical vessels (having the same diameter as the measurement cone of the rheometer) and were kept at room temperature for at least 24 h. Rheological measurements were carried out in a Ha¨ake RS100 rheometer with a cone-and-plate geometry (diameter, 35 mm; angle, 4°). Each sample was transferred to the plate, and once in the measurement position, its contour surface was covered with a thin layer of paraffin oil to avoid dehydration. The temperature was kept constant by circulating water (through the bottom plate) from a Ha¨ake K15 thermocryostat. All dynamic measurements and creep-recovery experiments were performed in the linear viscoelastic region to ensure that the calculated parameters correspond to an intact network structure. In the dynamic experiments, the operating frequencies were in the range 0.2-60 s-1. Duplicate experiments showed excellent reproducibility and indicated that gel stability and curing were completed after 24 h. Densities were measured with an Anton Paar DMA 45 densimeter at the desired temperatures.
Results and Discussion All oscillatory experiments were performed with applied stresses lower than 1 Pa to ensure that we were working within the linear viscoelastic region. The upper limit of the corresponding strain domain of this linear viscoelastic region is ∼3% (Figure 1), a characteristic value for weak gels.19 The available range of pH’s at which the gelation can be studied is restricted by two factors. First, the gel is not formed at high pH values, and second, deoxycholic acid precipitates at low pH values.20 Therefore, the influence of the acidity on the mechanical nature of the gel can be studied only in the pH range 6.7-7.4. Representative curves are shown in Figure 2, where the storage modulus, G′, and the loss modulus, G′′, the two components of the complex shear modulus, are plotted against the oscillatory frequency, f. It must be noticed that the system does not exhibit terminal flow for G′ >
Jover et al.
Figure 2. Dependence of the storage and loss moduli on the operating frequency for samples prepared under the following conditions: [NaDC] ) 0.1737 mol dm-3, [phosphate] ) 0.02324 mol dm-3, pH ) 6.70 (open symbols) and 7.14 (solid symbols), and T ) 293 K. Table 1. Values of the Exponent (n) of the Power Law between G′ and f Obtained for Samples under Different Experimental Conditions of NaDC Concentration, pH, Temperature, and NaCl Concentration [NaDC] (mol dm-3)
pH
T (K)
0.1158 0.1158 0.1158 0.1158 0.1158 0.1158 0.1158 0.1158 0.1158 0.1158 0.1737 0.1737 0.1737 0.1737 0.2894 0.2894 0.2894 0.2894
6.72 6.85 6.88 6.90 6.96 7.02 7.12 7.19 7.27 7.33 6.80 6.92 7.05 7.14 6.85 6.91 6.99 7.09
293 293 293 293 293 293 293 293 293 293 293 293 293 293 293 293 293 293
-
0.31 0.31 0.32 0.33 0.33 0.34 0.33 0.34 0.34 0.34 0.31 0.31 0.31 0.30 0.32 0.30 0.31 0.33
0.1158 0.1158 0.1158 0.1158
7.27 7.27 7.27 7.27
283 288 298 303
-
0.31 0.32 0.36 0.40
0.1737 0.1737 0.1737 0.1737
6.92 6.92 6.92 6.92
283 288 298 303
-
0.30 0.30 0.30 0.34
0.1737 0.1737 0.1737 0.1737
6.92 6.92 6.92 6.92
293 293 293 293
0.15 0.30 0.45 0.64
0.28 0.27 0.24 0.23
[NaCl] (mol dm-3)
n
G′′, and therefore it regains its equilibrium configuration through Brownian motion within the time scale of the experiment.21 This figure also shows that the expected crossover point of G′ and G′′ will be located at frequencies below 0.2 s-1, yielding relaxation times greater than 5 s. Furthermore, the storage modulus obeys the scaling law
G′ ∝ f n
(1)
with the values obtained for the scaling exponent n being close to 0.30 (Table 1). All of these facts are characteristic of so-called weak gels19 and suggest that the interactions in the gel network (20) Hofmann, A. F.; Mysels, K. J. Colloids Surf. 1988, 30, 145.
Dynamic Rheology of Sodium Deoxycholate Gels
Langmuir, Vol. 18, No. 4, 2002 989 Table 2. Values of the Retardation Time (λ) Obtained from Creep-Recovery Experiments with Samples Prepared at the Indicated pH Valuesa pH
λ (s)
6.67 6.69 6.81 6.99 7.06 7.09 7.13
68 76 48 41 48 54 47
a [NaDC] ) 0.05789 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and T ) 293 K.
Figure 3. Variation of the storage modulus with the pH of samples prepared under the following conditions: [NaDC] ) 0.1158 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and T ) 293 K.
Figure 5. Variation of the storage modulus (at 4.64 s-1) with temperature for samples prepared with [NaDC] ) 0.1737 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and pH ) 6.92 (circles) or [NaDC] ) 0.1158 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and pH ) 7.27 (squares).
Figure 4. Results for creep-recovery experiments carried out under an applied stress of 2.0 Pa on samples prepared at 293 K with [NaDC] ) 0.05789 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and the pH values shown in the corner of the plot.
are physical in nature. This finding is in agreement with previous interpretations10-16 deduced from results obtained with other experimental techniques. At constant pH, the concentration of NaDC (from 0.116 to 0.289 M) has little effect on the storage modulus (results not shown) or on its dependence on the operating frequency (see some values of the exponent n in Table 1). On the other hand, Figure 3 shows that the storage modulus G′ increases when the pH decreases, the maximum increasing factor being 6 (these results were obtained at a constant frequency of f ) 3.18 s-1). This dependency of the storage modulus G′ on pH is parallel to the dependence of the aggregation number on pH. For instance, the aggregation number increases by ∼10% when the pH is reduced from 6.9 to 6.7.16 Figure 4 shows two typical creep-recovery curves obtained at pH 6.69 and 7.13 under an applied stress of τo ) 2.0 Pa. The experimental data perfectly fit the equation
γ(t) ) γM +
τ0 τ0 (1 - e-t/λ) + t GK ηM
(2)
which corresponds to Burgers’ constitutive model. Here γ(t) is the strain at time t; γM and ηΜ are the instantaneous strain and viscosity of the Maxwell element, respectively; (21) Ferry, J. D. In Viscoelastic Properties of Polymers; Wiley: New York, 1970.
and GΚ is the shear modulus of the Kelvin element in Burger’s model. It can be concluded that the mechanical behavior of the system is simple in nature in that only one retardation time, λ ) ηΚ/GΚ, is involved. Sometimes, individual elements can be qualitatively identified with definite processes at a molecular level.22 For example, it has been pointed out23 that the Kelvin element represents that part of the structure in which secondary bonds are breaking and reforming during the experiment. Thus, different retardation times should be related to different types of these bonds. Therefore, in this system, it seems that only one type of secondary bonds is involved in the strain relaxation during the experiment. This is also supported by the fact that λ is essentially constant with pH, with a slight increase occurring at lower pH values (Table 2). This is in agreement with the observed tendency of G′ noted above (see also Figure 3). At constant oscillatory frequency, the storage modulus decreases with temperature between 283.0 and 303.0 K (Figure 5). The scaling law exponent slowly increases with increasing temperature (Table 1). However, both effects are small. The effect of the temperature was also analyzed by the method of reduced variables21 through the reduced storage modulus, defined as
G′p ) G′(ToFo/TF)
(3)
where To is a chosen reference temperature (283.0 K in this case) and Fo is the corresponding density of the sample. The temperature superposition principle suggests that all of the contributions to the dynamic moduli are (22) Davis, S. S. J. Pharm. Sci. 1969, 58, 412. (23) Barry, B. W. J. Colloid Interface Sci. 1968, 28, 82.
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Figure 7. Variation of the storage modulus (at 4.64 s-1) with the concentration of added NaCl in samples prepared with [NaDC] ) 0.1737 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and pH ) 6.92 at 293 K. Table 3. Values of the Shift Factor Corresponding to the Master Curves in Figures 6a and 6b
Figure 6. Frequency-temperature superposition of the storage modulus for two samples prepared at [phosphate] ) 0.02324 mol dm-3 and (a) [NaDC] ) 0.1737 mol dm-3, pH ) 6.92 or (b) [NaDC] ) 0.1158 mol dm-3, pH ) 7.27. Master curves at the reference temperature, To, of 283 K.
proportional to FT and is based on the assumption of equal temperature dependence of all relaxation times. Equation 3 indicates that a change in temperature should cause a change in the modulus equivalent to multiplication by the temperature-density correction factor (ToFo/TF). Following this method, G′p is represented in a double logarithmic plot against the product faT, where aT is an empirically determined shift factor. The results obtained are shown in Figure 6, where it can be seen that all of the data are superposed in a master curve. Therefore, it can be concluded that only the relaxation rate and not the underlying mechanism is affected by the temperature change. The values obtained for aT are summarized in Table 3, and they range between normal limits, similar to those for other systems.24 The addition of an inert electrolyte to increase the ionic strength has a stronger influence on the storage modulus and the scaling law exponent than do the NaDC concentration, pH, and temperature. Figure 7 shows that the storage modulus increases by a factor of ∼100 when the concentration of NaCl is 0.64 M. This change is accompanied by a decrease in n (from 0.30 to 0.23), as can be seen in Table 1. All n values shown in Table 1 are typical of very lightly cross-linked systems.21,25 It is known that gels formed by covalent bonds have storage moduli that are practically independent of the oscillation frequency, whereas for physical gels, this dependence becomes more pronounced as the interactions become weaker. Therefore, the decrease in n with increasing ionic strength suggests stronger interactions in the gel, thus increasing its strength. The method of reduced variables was also used to analyze the effect of the ionic strength. Figure 8 shows that the principle is satisfied, although the product aIf extends over a very large range because of the great values achieved by the empirical factor aI (Table 4). Therefore, (24) Lopes da Silva, J. A.; Gonc¸ alves, M. P.; Rao, M. A. Carbohydr. Polym. 1994, 23, 77. (25) Egelandsdal, B.; Fretheim, K.; Harbitz, O. J. Sci. Food Agric. 1986, 37, 944.
T (K)
aT (a)
aT (b)
288 293 298 303
0.75 0.45
0.40 0.25 0.15
0.10
Figure 8. Master curve obtained for the effect of the addition of NaCl on samples with [NaDC] ) 0.1737 mol dm-3, [phosphate] ) 0.02324 mol dm-3, and pH ) 6.92 at 293 K. The reference values correspond to the samples with no added NaCl. Table 4. Values of the Shift Factor Corresponding to the Master Curve in Figure 8 [NaCl] (mol dm-3)
aI
0.15 0.30 0.45 0.64
4 15 45 10 000
it can be concluded that there is no change in the nature of the relaxation mechanism, although the ionic strength appreciably affects the strength of the gel. The obvious increase in the shift factor aI, accompanied by the decrease in the exponent n discussed above, also corresponds to the formation of stronger gels when the ionic strength increases. All of the present experimental results indicate that the mechanical behavior of the NaDC gels implies only one simple relaxation mechanism, with the involvement of only one type of secondary bonds. Several authors13,16,26 have concluded that the formation of intermolecular (26) Murata, Y.; Sugihara, G.; Fukushima, K.; Tanaka, M.; Matsushita, K. J. Phys. Chem. 1982, 86, 4690.
Dynamic Rheology of Sodium Deoxycholate Gels
hydrogen bonds is the main driving force responsible of the formation of polymer-like structures in NaDC solutions at acid pH values close to neutrality. Therefore, the simplest explanation for the dynamic behavior of the gel (revealed through the power law, the creep-recovery experiments, and the frequency-temperature superposition principle) is that hydrogen bonds are the most important molecular forces involved in the formation, relaxation, and strength of the gel. This is also consistent with (i) the decrease of G′ with temperature (Figure 5), as the hydrogen bonds are weakened with increasing temperature, and (ii) the ionic strength effects. An increase
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in the NaCl concentration facilitates the formation of larger aggregates,27 leading to a more effective interaction or entanglement of these aggregates, which would be responsible for the gel behavior of the solution.17 Acknowledgment. Financial support from the Xunta de Galicia (Project XUGA PGIDT99PXI26201B) is gratefully acknowledged. LA011178H (27) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356.
