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Influence of pH, Ionic Strength, and Temperature on Self-Association and Interactions of Sodium Dodecyl

Sulfate in the Absence and Presence of Chitosan

Masubon Thongngam and D. Julian McClements*

Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003

Received May 25, 2004. In Final Form: September 30, 2004

Chitosan is a cationic biopolymer that has many potential applications in the food industry because of its unique nutritional and physicochemical properties. Many of these properties depend on its ability to interact with anionic surface-active molecules, such as surfactants, phospholipids, and bile acids. The purpose of this study was to examine the influence of pH (3 and 7), ionic strength (0-200 mM NaCl), and temperature (10-50 °C) on the interactions between a model anionic surfactant (sodium dodecyl sulfate, SDS) and chitosan using isothermal titration calorimetry, selective surfactant electrode, and turbidity measurements. At pH 3 and 30 °C, SDS bound strongly to chitosan to form an insoluble complex that contained about 4-5 mmol of SDS/1 g of chitosan at saturation. When SDS and chitosan were mixed at pH 7 they did not interact strongly, presumably because the biopolymer had lost most of its positive charge at this pH. However, when SDS and chitosan were mixed at pH 3 and then the solution was adjusted to pH 7, the SDS remained bound to the chitosan. The presence of NaCl (0-200 mM) in the solutions decreased the critical micelle concentration (cmc) of SDS (in both the absence and the presence of chitosan) but had little influence on the amount of SDS bound to chitosan at saturation. The cmc of SDS and the amount of SDS bound to the chitosan at saturation were largely independent of the holding temperature (10-40

°C). Nevertheless, the enthalpy changes associated with micelle dissociation were highly temperature- dependent, indicating the importance of hydrophobic interactions, whereas the enthalpy changes associated with SDS-chitosan binding were almost temperature-independent, indicating the dominant contribution of electrostatic interactions. This study provides information that may lead to the rational design of chitosan- based ingredients or products with specific nutritional and functional characteristics, for example, cholesterol lowering.

Introduction

Chitosan is a cationic biopolymer that has many potential biological and industrial applications, including cholesterol lowering, heavy metal chelation, wastewater treatment, texture modification, encapsulation, and emul- sion stabilization.1-8Many of these applications depend on the interactions between chitosan and anionic surface- active substances, for example, small molecule surfactants, phospholipids, or bile acids. The rational application of chitosan for these applications, therefore, depends on a better understanding of the origin and nature of chitosan- anionic surfactant interactions. The purpose of this study was to examine the influence of solution and environ- mental conditions (pH, ionic strength, and temperature) on chitosan-sodium dodecyl sulfate (SDS) interactions to provide a better understanding of the characteristics of chitosan-anionic surfactant interactions.

Chitosan is a (1f4)-linked 2-amino-2-deoxy-β-D-glucan derived from fully or partially deacetylated chitin.1It has three types of reactive functional groups: an amino group at the C-2 position (pKa ∼ 6.3-7) and primary and secondary hydroxyl groups at the C-3 and C-6 positions, respectively.1,3,9At relatively low pH (<6.5), chitosan is highly positively charged because of protonation of the amino groups. Chitosan, therefore, tends to be highly soluble in acidic aqueous solutions because of its high degree of hydration and the strong electrostatic repulsion between the molecules, provided that there is not a sufficiently high concentration of multivalent anions present to promote chitosan cross-linking and aggregation.

On the other hand, at higher pH values (g6.5), the amino groups become deprotonated, which means that chitosan loses its positive charge and tends to precipitate from solution.3,9In general, the properties of chitosan in aqueous solution depend on its molecular weight and degree of deacetylation, as well as the prevailing solution condi- tions.3,4Previous studies have shown that chitosan can interact with anionic surfactants to form either soluble or insoluble complexes depending on the solution condi- tions.4,10-12It has been proposed that these complexes are stabilized by a combination of electrostatic, ion-dipole, and hydrophobic interactions and can be formed even when

* To whom correspondence should be addressed. E-mail:

mcclements@foodsci.umass.edu.

(1) Shahidi, F.; Arachchi, J. K. V.; Jeon, Y. J. Trends Food Sci. Technol.

1999, 10, 37-51.

(2) Rinaudo, M.; Domard, A. In Chitin and chitosan; Skjak-Braek, G., Anthonsen, T., Sandford, P., Eds.; Elsevier Applied Science: London, 1989.

(3) Claesson, P. M.; Ninham, B. W. Langmuir 1992, 8, 1406-1412.

(4) Kubota, N.; Kikuchi, Y. Macromolecular complexes of chitosan.

In Polysaccharides: structural, diversity and functional versatility;

Dumitriu, S., Ed.; Marcel Dekker: New York, 1998.

(5) Ravi Kumar, M. N. V. Bull. Mater. Sci. 1999, 22, 905-915.

(6) Jeuniaux, C.; Voss-Foucart, M. F.; Poalicek, M.; Bussers, J. C. In Chitin and Chitosan; Skjak-Braek, G., Anthonsen, T., Sandford, P., Eds.; Elsevier Applied Science: London, 1989.

(7) Herrera, F. P.; Mata-Segreda, J. F. Rev. Biol. Trop. 1996-1997, 44/45 (3/1), 613-614.

(8) Schulz, P. C.; Rodriguez, M. S.; Del Blanco, L. F.; Pistonesi, M.;

Agullo, E. Colloid Polym. Sci. 1998, 276, 1159-1165.

(9) Skjak-Braek, G.; Anthonsen, T.; Sandford, P. Chitin and chitosan;

Elsevier Applied Science: London, 1989.

(10) Wei, Y. C.; Hudson, S. M. Macromolecules 1993, 26, 4151-4154.

(11) Vikhoreva, G. A.; Babak, V. G.; Galich, E. F.; Gal’braikh, L. S.

Polym. Sci., Ser. A 1997, 39, 617-622.

(12) Thongngam, M.; McClements, D. J. J. Agric. Food Chem. 2004, 52, 987-991.

10.1021/la048711o CCC: $30.25 © 2005 American Chemical Society Published on Web 12/02/2004

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the surfactant concentration is below its critical micelle concentration (cmc).10-17

In a previous study, we showed that SDS-chitosan interactions could be conveniently studied using a com- bination of surfactant selective electrode (SSE), isothermal titration calorimetry (ITC), and turbidity measurements.12 The SSE technique provides information about the amount of surfactant bound to the chitosan, the ITC technique provides information about the enthalpy changes associ- ated with surfactant-chitosan interactions and surfactant micelle dissociation, and the turbidity technique provides information about the formation of insoluble complexes.

In the present study, we use the same techniques to examine the influence of pH, ionic strength, and tem- perature on SDS-chitosan interactions to provide a more detailed understanding of the origin and nature of the interactions involved.

Materials and Methods

Materials. Analytical grade SDS, sodium chloride, sodium acetate, acetic acid, hydrochloric acid, and sodium hydroxide were purchased from the Sigma Chemical Co. (St. Louis, MO).

Medium molecular weight chitosan (75-85% deacetylation) was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI).

Double-distilled water was used for the preparation of all solutions.

Sample Preparation. Flaked chitosan and SDS were dis- solved in acetate buffer solutions (100 mM acetic acid/sodium acetate) of various pHs (pH 3, 5, and 7) and NaCl concentrations (0-200 mM). These solutions were stirred overnight before each experiment to ensure complete dissolution and dispersion of the biopolymer. At pH 3 and 5 the chitosan dissolved completely, but at pH 7 the chitosan remained as insoluble particles.

ITC. An isothermal titration calorimeter (VP-ITC, Microcal, Inc., Northampton, MA) was used to measure the enthalpy changes resulting from titration of SDS into either buffer solution or chitosan solution. Ten microliter aliquots of SDS solution were injected sequentially into a 1.48-mL titration cell initially containing either acetate buffer solution or chitosan in acetate buffer. Each injection lasted 20 s, and there was an interval of 300 s between successive injections. For most of the experiments the temperature of the solution in the titration cell was 30.0 °C, but in some experiments a number of temperatures were used:

10.0, 20.0, 30.0, and 40.0 °C. The solutions were stirred at 315 rev min-1 throughout the experiments. Measurements were carried out in duplicate and were highly reproducible, with the standard deviation in the enthalpy measurements being less than 5% of the mean value.

SSE. SSE was used to follow the binding interaction between SDS and chitosan by measuring changes in the electromotive force (EMF) due to changes in the free SDS concentration in solution. The SSE cell consisted of a SDS-selective electrode (Thermo Orion, Beverly, MA), a double junction reference electrode (Thermo Orion, Beverly, MA), and a pH meter (420A+, Thermo Orion, Beverly, MA). The SSE experiments were designed to mimic the ITC experiments, that is, have a similar total surfactant concentration range and surfactant-to-chitosan ratio.

Consequently, 100-µL aliquots of SDS solutions were added into test tubes containing 14.8 mL of either acetate buffer or chitosan solution. The mixtures were then vortexed and incubated overnight before the EMF signals were measured. Most of the solutions were incubated and analyzed at 30 °C, but in some experiments a number of temperatures were used: 20, 30, and 40 °C. Measurements were carried out in duplicate, and the

results were reported as the mean and standard deviation. In chitosan-free buffer solutions, the EMF signal decreased as the free SDS concentration was increased. In most studies, the SSE data is presented as the change in EMF due to the addition of SDS to the solutions: ∆EMF ) EMF0- EMF, where EMF0and EMF are the signals measured in the absence and presence of surfactant, respectively. The value of ∆EMF, therefore, increases with increasing free SDS concentration. However, some of the results are also plotted as EMF versus pH, where EMF is the value measured directly in the sample being analyzed.

Turbidity Measurements. Turbidity measurements were used to provide information about the formation of insoluble SDS-chitosan aggregates. The turbidity experiments were also designed to mimic the ITC experiments, that is, using a similar total surfactant concentration range and surfactant-to-chitosan ratio. Hence, 100 µL aliquots of SDS solution were added into test tubes containing 14.8 mL of either acetate buffer or chitosan solution. The resulting solutions were thoroughly mixed using a Vortex mixer and then incubated overnight to ensure equi- librium, and then the turbidity was measured at a wavelength of 600 nm using a 1-cm-path-length cuvette (Spectronic 21D, Milton Roy, Rochester, NY). Most of the solutions were incubated and analyzed at 30 °C, but in some experiments a number of temperatures were used: 20, 30, and 40 °C. Measurements were carried out in duplicate, and the results are reported as the mean and standard deviation. Turbidity measurements were normal- ized with respect to chitosan concentration in the solution (τ/

cchitosan) to take into account dilution effects associated with the titration of surfactant solution into the chitosan solution.

Typically, the turbidity measurements were reproducible to better than 5%.

Results and Discussion

Influence of NaCl on SDS-Chitosan Interactions.

ITC Measurements. Electrostatic forces are believed to play a major role in determining the interactions between anionic SDS and cationic chitosan.12For this reason, we studied the influence of NaCl on SDS-chitosan interac- tions at pH 3 and 30 °C, because salt is known to screen electrostatic interactions.18The enthalpy changes result- ing from sequential injection of 100 mM SDS into either buffer solution or chitosan solution containing either 0 or 150 mM NaCl were recorded by ITC (Figure 1). The SDS concentration in the injector was well above the cmc of SDS;12 hence, the surfactant was initially present pre- dominantly as micelles when injected into the reaction cell.

In the absence of chitosan and NaCl, a broad endo- thermic peak was observed between 0 and 6 mM SDS, followed by a steep decrease in enthalpy from 6 to 9 mM (13) Bakeev, K. N.; Ponomarenko, E. A.; Shishkanova, T. V.; Tirrell,

D. A.; Zezin, A. B.; Kabanov, V. A. Macromolecules 1995, 28, 2886- 2892.

(14) No, H. K.; Lee, K. S.; Meyers, S. P. J. Food Sci. 2000, 65, 1134- 1137.

(15) Li, X.; Tushima, Y.; Morimoto, M.; Saimoto, H.; Okamoto, Y.;

Minami, S.; Shigemasa, Y. Polym. Adv. Technol. 2000, 11, 176-179.

(16) Lima, C. F.; Nome, F.; Zanette, D. J. Colloid Interface Sci. 1997, 187, 396-400.

(17) Goddard, E. D. Colloids Surf. 1986, 19, 301-329.

(18) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992.

Figure 1. Enthalpy change per mole of surfactant (∆H) injected into the reaction cell versus the total surfactant concentration present in the reaction cell for 100 mM SDS solution injected into either buffer solution or 0.1 wt % chitosan solution (pH 3.0, 100 mM acetate buffer, 30 °C) in the absence or presence of salt (0 or 150 mM NaCl).

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SDS, followed by a region where the enthalpy change was relatively small and endothermic (Figure 1). At the beginning of the experiment the SDS concentration in the injector was well above the cmc, whereas there was no SDS in the reaction cell. Hence, the large endothermic peak observed at low surfactant concentrations could be attributed to the dissociation of surfactant micelles when they were titrated into the reaction cell, because the exposure of nonpolar surfactant tails to the surrounding aqueous phase is endothermic at this temperature.19-23 Once the total surfactant concentration in the reaction cell exceeded the cmc of the surfactant, micelle dissociation no longer occurred, and the endothermic enthalpy change decreased appreciably. At surfactant concentrations greatly exceeding the cmc, the enthalpy changes are primarily due to micelle dilution effects.12The dependence of the enthalpy change on the surfactant concentration can be used to determine the cmc of the surfactant, that is, from the inflection point in the ∆H versus surfactant concen- tration curve.24Using this method we determined the cmc of the surfactant to be ∼6.6 ( 0.2 mM at 0 mM NaCl, which is in close agreement with previous studies in the absence of salt.25In the presence of 150 mM NaCl (but absence of chitosan), the width of the endothermic peak associated with micelle dissociation decreased appreciably, which can be attributed to the fact that salts suppress the cmc of ionic surfactants.25The influence of NaCl on the cmc of the SDS in the absence of chitosan is plotted in Figure 2a, which clearly shows that there is a steep decrease in the cmc with increasing salt concentration from 0 to 50 mM NaCl, followed by a more gradual decrease at higher salt concentrations. The maximum enthalpy change associated with micelle dissociation was relatively independent of salt concentration (Figure 2b).

In the presence of chitosan, the dependence of the enthalpy change on the surfactant was both qualitatively and quantitatively different from that observed in the absence of chitosan (Figure 1). As the surfactant concen- tration was increased in the system containing 0 mM NaCl, a broad exothermic peak was observed from 0 to∼5 mM SDS, followed by a broad endothermic peak from∼5 to

∼10 mM SDS, followed by a relatively steep decrease in enthalpy from ∼10 to ∼12 mM SDS, followed by a relatively constant and low endothermic enthalpy change at higher surfactant concentrations. The broad exothermic peak observed at low surfactant concentrations can be attributed to binding of surfactant molecules to the chitosan. Presumably, there is a large exothermic reaction between surfactant and chitosan when binding occurs, which is observed until the chitosan has become saturated with surfactant. Once the chitosan is saturated, any additional surfactant goes into the aqueous solution surrounding the chitosan-SDS complexes. The broad endothermic peak observed at higher surfactant concen-

trations can, therefore, be attributed to the dissociation of surfactant micelles when they are titrated into the reaction cell because the free (nonbound) surfactant concentration in the aqueous phase is initially below the cmc. Once the free surfactant concentration exceeds the cmc then the micelles no longer dissociate when they are titrated into the reaction cell and the enthalpy change becomes appreciably less endothermic. At higher surfac- tant concentrations, only a small endothermic change is observed because of micelle dilution effects, as observed in the absence of chitosan.

The effective cmc (cmc*) of the SDS in the presence of chitosan was determined from the enthalpy versus sur- factant concentration measurements as described previ- ously, that is, from the inflection point in the ∆H versus surfactant concentration curves.12 Provided that SDS- chitosan complexes do not interfere with the behavior of free SDS in the surrounding aqueous phase, then cmc* ) cmc + Csat, where Csatis the amount of surfactant bound to the chitosan. The value of cmc* determined in the presence of 0.1 wt % chitosan (0 mM NaCl) was 11.0 mM, which suggests that 4.4 mM SDS was bound to the chitosan, because the cmc of SDS in the absence of chitosan is 6.6 mM (see above). As would be expected, the value of Csatdetermined using this method is very similar to the width of the broad exothermic peak associated with SDS- chitosan binding, that is, 4.5-5 mM SDS (Figure 1).

When the SDS was titrated into the solution containing chitosan in the presence of 150 mM NaCl, there was a slight decrease in the width of the exothermic enthalpy change associated with SDS-chitosan binding, as well as an appreciable decrease in the height and width of the endothermic peak associated with micelle breakup (Figure 1). The relatively small influence of salt on the exothermic

“binding” peak suggests that the addition of salt only weakened the SDS-chitosan interaction slightly, whereas the relatively large influence of the salt on the endothermic

“micelle dissociation” peak can be attributed to the ability of NaCl to reduce the cmc of the ionic surfactant as (19) Tanford, C. The hydrophobic effect: formation of micelles and

biological membranes; Krieger Publishing Co.: Malabar, FL, 1991.

(20) Wang, Y.; Han, B.; Yan, H.; Kwak, J. C. T. Langmuir 1997, 13, 3119-3123.

(21) McClements, D. J. J. Agric. Food. Chem. 2000, 48, 5604-5611.

(22) Olofsson, G.; Wang, G. Isothermal titration and temperature scanning calorimetric studies of polymer-surfactant systems. In Polymer- surfactant systems; Kwak, J. C. T., Ed.; Marcel Dekker: New York, 1998.

(23) Ananthapadmanabhan, K. P. Surfactant solutions: adsorption and aggregation properties. In Interactions of surfactants with polymers and proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993.

(24) Wangsakarn, A.; Chinachoti, P.; McClements, D. J. J. Agric.

Food Chem. 2001, 49, 5039-5045.

(25) Jonsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactant and polymers in aqueous solution; John Wiley and Sons, Inc.: New York, 1998.

Figure 2. Influence of NaCl concentration on SDS micellization and binding behavior measured by ITC in the absence and presence of 0.1 wt % chitosan (pH 3.0, 100 mM acetate buffer, 30 °C): (a) cmc (no chitosan) and cmc* (+ chitosan) and (b) maximum enthalpy change associated with micelle breakup and SDS-chitosan binding.

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discussed earlier. The influence of NaCl on cmc* deter- mined in the presence of chitosan was compared with the value of cmc determined in the absence of chitosan (Figure 2a). cmc* and cmc had similar dependencies on the salt concentration, decreasing fairly steeply from 0 to 50 mM NaCl, followed by a more gradual decrease at higher salt concentrations. On the other hand, the values of Csat

calculated from these measurements were fairly inde- pendent of NaCl, varying from around 4.4 to 4.7 mM SDS.

The influence of NaCl on the maximum depth of the exothermic SDS-chitosan binding peak and the maximum height of the endothermic SDS dissociation peak is shown in Figure 2b. With increasing salt concentration, there was a slight decrease in the magnitude of the enthalpy change associated with micelle dissociation in the presence of the chitosan-SDS complex but little change in the magnitude of the enthalpy change associated with SDS- chitosan binding. The ITC results, therefore, suggest that NaCl only had a small influence on the binding of SDS to chitosan.

SSE and Turbidity Measurements. SSE measurements were also used to study the influence of NaCl on the binding of SDS to chitosan, because the EMF of the SSE probe decreased as the surfactant concentration in the aqueous phase increased. The SSE data was presented as the change in EMF due to the addition of SDS to the solutions: ∆EMF ) EMF0- EMF, where EMF0and EMF are the signals measured in the absence and presence of surfactant, respectively. The value of ∆EMF, therefore, increased with increasing free SDS concentration in the samples. ∆EMF was measured in the absence or presence of 0.1 wt % chitosan in aqueous solutions containing either 0 or 150 mM NaCl (pH 3, 30 °C; Figure 3). In the absence of chitosan and salt, there was a relatively steep linear increase in ∆EMF with increasing log [SDS] at relatively low surfactant concentrations, followed by a more gradual increase at higher surfactant concentrations. The initial linear increase can be attributed to the increase in free surfactant monomer concentration in the solutions, whereas the more gradual increase at higher concentra- tions can be attributed to the fact that micelles are formed so that the monomer concentration does not increase as dramatically above the cmc.26The surfactant concentra- tion where this transition in slope occurred was around 6-7 mM SDS, which is in close agreement with the cmc of SDS determined by ITC (see above). In the presence of 150 mM NaCl, the transition in the slope of ∆EMF with increasing log [SDS] was observed at a lower surfactant

concentration (1-2 mM SDS), because the cmc of the surfactant was decreased by the presence of the salt (see above).

In the presence of chitosan, the dependence of ∆EMF on log [SDS] is very different from that observed in the absence of chitosan (Figure 3). At relatively low surfactant concentrations, the value of ∆EMF in the presence of chitosan is close to zero and much smaller than that measured in the absence of chitosan. This suggests that there is little free surfactant in the system, which can be attributed to strong binding of the surfactant to the chitosan molecules. However, once a certain SDS con- centration is exceeded, then the value of ∆EMF increases steeply, suggesting that the chitosan became saturated with surfactant and any additional surfactant added to the solution was free in the aqueous phase. At higher surfactant concentrations, the slope of the curve changed, which suggested that micelles were being formed in the aqueous phase. The amount of surfactant bound to the chitosan at saturation (Csat) could be estimated from the EMF measurements by determining the surfactant con- centration where the ∆EMF value first began to increase steeply (Figure 3). The value of Csatdetermined using this method was around 4-4.5 mM SDS, which is in close agreement with the values determined by the ITC method discussed above.

Turbidity measurements were used to study the influ- ence of the NaCl and SDS concentrations on the formation of insoluble complexes in the SDS-chitosan solutions (Figure 4). In the absence of chitosan, the solutions remained transparent at all SDS concentrations used, indicating that no aggregates large enough to scatter light were formed. In the presence of chitosan, there was a steady increase in the normalized turbidity (measured turbidity divided by chitosan concentration in the reaction cell) from 0 to 6.7 mM SDS until a maximum value was reached, after which the normalized turbidity either remained fairly constant or decreased slightly. The initial increase in turbidity with increasing surfactant concen- tration indicates that an increasing amount of insoluble SDS-chitosan complexes were being formed that were large enough to scatter light. The fact that the normalized turbidity reached a relatively constant level or decreased at higher surfactant concentrations suggests that the chitosan was saturated with SDS and that the complexes began to either partially dissociate or change their dimensions with a further increase in surfactant concen- tration. The change in turbidity with increasing surfactant concentration was lower in the absence of salt than in its presence, which suggested that either the concentration or the size of the insoluble complexes formed was different.

The ITC and SSE measurements indicated that the (26) Mokus, M.; Kragh-Hansen, U.; Letellier, P.; le Maire, M.; Mu¨ ller,

J. V. Anal. Biochem. 1998, 264, 34-40.

Figure 3. Change in EMF (∆EMF) measured by SSE in SDS solutions in the absence and presence of 0.1 wt % chitosan (pH 3.0, 100 mM acetate buffer, 30 °C) containing 0 or 150 mM NaCl.

Figure 4. Influence of SDS concentration on the normalized turbidity (τ/cchitosan) of SDS-chitosan solutions (pH 3.0, 100 mM acetate buffer, 30 °C) containing 0 or 150 mM NaCl. The solutions initially contained 0.1 wt % chitosan. No turbidity was observed in the absence of chitosan.

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chitosan became saturated with surfactant somewhere around 4.5 mM SDS, which suggests that some aggregate formation or restructuring occurred even when the chi- tosan was saturated with SDS. We postulate that the anionic SDS may have acted as a cross-linking agent between cationic chitosan molecules, but further work is needed to establish this.

Influence of pH on SDS-Chitosan Interactions.

The purpose of these experiments was to examine the influence of pH on the interaction between chitosan and SDS. Samples of SDS and chitosan were, therefore, prepared using 100 mM acetate buffers of different pHs (3 and 7). The chitosan formed clear solutions at pH 3 indicating that it was fully dissolved, but it formed visible particles at pH 7 indicating it was at least partly insoluble.

Different pH values were obtained by varying the ratio between 100 mM acetic acid and 100 mM sodium acetate.

ITC Measurements. ITC measurements of the enthalpy changes resulting from titration of SDS into buffer and chitosan solutions at pH 3 and 7 are compared in Figure 5. At pH 3, the enthalpy versus surfactant concentration curves were qualitatively different in the presence and absence of 0.1 wt % chitosan because of the large exothermic change associated with the SDS-chitosan interaction at relatively low surfactant concentrations (see above). At pH 7, the enthalpy versus surfactant concen- tration curves were fairly similar in the presence and absence of 0.1 wt % chitosan, which suggested that there was little interaction between the surfactant and polysac- charide at this pH. This might have been expected because the chitosan was not fully soluble at pH 7, with the chitosan forming a cloudy suspension rather than a transparent solution.

SSE and Turbidity Measurements. The SSE technique was used to monitor the influence of pH on the binding of SDS to chitosan. The EMF of the solutions was measured as a function of total SDS concentration using a SDS- sensitive electrode (Figure 6). At pH 3, there was a large difference between the EMF versus SDS concentration profiles in the presence and absence of chitosan, indicating that there was strong binding of the added surfactant to the chitosan until the biopolymer became saturated at

∼4.5 mM SDS (see above). On the other hand, at pH 7, the EMF versus SDS profiles were fairly similar in the presence and absence of chitosan, indicating that little surfactant bound to the chitosan. We could not make reliable turbidity measurements at pH 7 because the chitosan itself was highly insoluble and formed large particles that quickly settled to the bottom of the cuvettes used to make the UV-visible spectrophotometry mea-

surements. The chitosan is insoluble at this pH because it loses its positive charge due to deprotonation of its amino groups, which reduces its hydration and the electrostatic repulsion between the molecules, thereby promoting aggregation.

In certain practical applications, it is important to know whether anionic surfactants that bind to chitosan at pH 3 are released when the solution is adjusted to pH 7, for example, binding of bile acids to dietary fibers in vivo, where the pH changes from highly acidic to neutral when the food is transferred from the stomach to the small intestine. For this reason, we examined the influence of mixing conditions on the binding interaction using SSE:

(i) 5 mM SDS and 0.1 wt % chitosan were mixed at pH 7, and then a series of solutions of lower pH were made by adding HCl; (ii) 5 mM SDS and 0.1 wt % chitosan were mixed at pH 3, and then a series of solutions of higher pH were made by adding NaOH (Figure 7). In this case the EMF (rather than ∆EMF) of the samples was measured as a function of pH, so that an increase in EMF cor- responded to a decrease in free SDS. In the SDS-chitosan solution that was mixed at pH 3, the EMF did not change appreciably as the pH was increased to 7, which suggested that SDS molecules were not released from the surfac- tant-chitosan complex. On the other hand, in the SDS- chitosan solution that was mixed at pH 7, the EMF of the solutions remained fairly constant when the pH was decreased from pH 7 to pH 6 and then increased steeply from pH 6 to pH 5, after which it gradually increased, suggesting that the SDS molecules only bound to the chitosan once it became sufficiently positively charged at lower pH values. This study suggests that an irreversible (at least on the experimental time scale) insoluble SDS- Figure 5. Enthalpy change per mole of surfactant (∆H) injected

into the reaction cell versus the total surfactant concentration present in the reaction cell for 100 mM SDS solution injected into either buffer solution or 0.1 wt % chitosan solution at pH 3 and 7 (0 mM NaCl, 100 mM acetate buffer, 30 °C).

Figure 6. Change in EMF (∆EMF) measured by SSE in SDS solutions in the absence and presence of 0.1 wt % chitosan at pH 3 and 7 (0 mM NaCl, 100 mM acetate buffer, 30 °C).

Figure 7. Influence of pH and solution history on the change in EMF (∆EMF) measured by SSE in SDS-chitosan solutions containing 5 mM SDS and 0.1 wt % chitosan initially prepared at either pH 3 or pH 7 (0 mM NaCl, 100 mM acetate buffer, 30 °C).

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chitosan complex formed at low pH as a result of an electrostatic association between the negatively charged SDS micelles and the positively charged chitosan and that this complex did not dissociate at higher pH where the chitosan would normally be expected to lose its charge.

One possible explanation of this phenomenon is that the effective pKa of the positively charged groups on the chitosan was increased when they were bound to SDS molecules.

Influence of Temperature on SDS-Chitosan In- teractions. ITC Measurements. The purpose of these experiments was to examine the influence of temperature on the interaction between chitosan and SDS at pH 3 (100 mM acetate buffer). The influence of temperature on the enthalpy versus surfactant concentration profiles was measured using ITC when SDS micelles were titrated into either buffer solution or chitosan solution (Figure 8).

In the absence of chitosan, the enthalpy change associated with micelle dissociation changed from exothermic to endothermic as the temperature increased from 10 to 40

°C (Figure 8a). Nevertheless, the width of this enthalpy change (which is closely related to the position of the inflection point used to calculate the cmc) remained fairly constant at 6.8 ( 0.3 mM from 10 to 40 °C, which suggested that the cmc of the surfactant was relatively insensitive to temperature. In the presence of chitosan (Figure 8b), the enthalpy change was strongly exothermic at all holding temperatures at relatively low SDS concentrations (<5 mM), which we postulated was due to the fact that the SDS bound to the chitosan as micelles rather than as monomers (see later). The fact that the width of this exothermic trough was fairly similar at all temperatures suggested that the amount of SDS bound to the chitosan at saturation was not strongly influenced by temperature.

Indeed, the value of Csat, determined from cmc and cmc*

measurements, was 5.0 ( 0.5 mM for holding tempera- tures ranging from 10 to 40 °C. At intermediate SDS concentrations (5-10 mM), the mixing enthalpy showed a broad endothermic peak at high holding temperatures but a broad exothermic peak at low holding temperatures.

These enthalpy changes can be attributed to the saturation of the chitosan with SDS, followed by dissociation of additional surfactant micelles added to the reaction cell (see above). Hence, it seemed that SDS-chitosan binding (Figure 8b) was relatively insensitive to the holding temperature but that the enthalpy change associated with micelle dissociation (Figure 8a,b) was highly temperature- dependent.

The maximum enthalpy changes associated with micelle dissociation (in the absence and presence of chitosan) and for surfactant binding to chitosan are shown in Figure 9.

These values were calculated from the enthalpy versus surfactant concentration profiles measured by ITC in the regions where micelle dissociation and surfactant binding dominated (Figure 8). The enthalpy changes associated with micelle breakup go from exothermic to endothermic as the temperature is increased, both in the presence and absence of chitosan. Even so, the presence of chitosan in the reaction cell does tend to reduce the maximum enthalpy change observed at the higher holding temper- atures. On the other hand, the binding interaction between SDS and chitosan is strongly exothermic, and relatively insensitive to temperature from 10 to 50 °C. The difference in the temperature dependencies of the enthalpy changes associated with micelle disruption and binding can be attributed to the different types of molecular interaction that dominate. Micelle dissociation involves the exposure of nonpolar surfactant tails that were originally located in the nonpolar interior of the surfactant micelles to the surrounding water molecules, thus, increasing the contact area between hydrophobic groups and water. The hydro- phobic effect is known to be strongly temperature- dependent, going from exothermic at relatively low temperatures (<25 °C) to endothermic at relatively high temperatures.19,27Thus, the strong temperature depen- dence of the hydrophobic interaction accounts for the change in enthalpy with temperature associated with SDS micelle dissociation (Figure 8a,b). On the other hand, the binding of anionic SDS to cationic chitosan is largely driven by electrostatic interactions, which presumably have a lower dependence on temperature (Figure 8b).

We postulated that SDS was bound to the chitosan in the form of surfactant micelles, rather than as individual monomers, because this would account for the fact that

(27) Creighton, T. E. Physical interaction that determine the proper- ties of proteins. Proteins: structures and molecular properties; W. H.

Freeman and Company: New York, 1993.

Figure 8. Influence of temperature on the enthalpy change per mole of surfactant (∆H) injected into the reaction cell versus the total surfactant concentration present in the reaction cell for 100 mM SDS solution injected into either (a) buffer solution or (b) 0.1 wt % chitosan solution (pH 3.0, 0 mM NaCl, 100 mM acetate buffer).

Figure 9. Influence of temperature on maximum enthalpy change associated with micelle breakup and SDS-chitosan binding measured by ITC in the absence and presence of 0.1 wt % chitosan (pH 3.0, 0 mM NaCl, 100 mM acetate buffer).

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we did not observe a large temperature dependence for the enthalpy change associated with SDS-chitosan bind- ing (Figures 8b and 9). We postulate that either (i) the micelles titrated into the reaction cell reacted directly with the chitosan molecules without dissociating or (ii) the micelles titrated into the reaction cell first dissociated into monomers and then reformed into micelles after binding to the chitosan. Nevertheless, it is not possible to distinguish between these two cases using ITC measure- ments, because they only determine the overall enthalpy change. In the former case there would be no enthalpy change associated with micelle dissociation, whereas in the latter case there would be an enthalpy change associated with micelle dissociation that would be almost exactly compensated for by an opposite enthalpy change associated with micelle reformation.

To provide further information about whether the SDS molecules were bound to the chitosan molecules as monomers or micelles, we measured the dependence of the enthalpy changes associated with the SDS-chitosan interaction when SDS monomers (rather than micelles) were titrated into a 0.01 wt % chitosan solution (Figure 10). We found a relatively high exothermic interaction between SDS and chitosan at low concentrations, which became more highly exothermic as the holding temper- ature was increased from 10 to 40 °C. These results suggested that there was a strong interaction between SDS and chitosan, until the chitosan became saturated with surfactant at about 0.45 mM SDS per 0.01 wt % chitosan, which is similar to the 4.5 mM SDS per 0.1 wt

% observed earlier. The fact that the interaction became increasingly exothermic as the temperature was increased suggested that it involved a hydrophobic interaction where nonpolar groups were moving from a polar environment to a nonpolar environment. We, therefore, postulated that the surfactant monomers formed micelles when they interacted with the chitosan molecules, because this process involves movement of the nonpolar tails of the surfactant molecules from an environment where they are surrounded by water molecules to an environment where they are in contact with other nonpolar groups. In summary, these measurements suggest that the surfac- tant molecules bind as micelles rather than as monomers to the chitosan.

SSE and Turbidity Measurements. SSE and turbidity measurements were also used to provide further informa- tion about the influence of holding temperature (20-40

°C) on surfactant binding and insoluble complex formation (Figures 11 and 12). The general trends in ∆EMF and

normalized turbidity versus surfactant concentration were similar at all the holding temperatures studied, which suggested that temperature did not have a strong influence on the binding of surfactant to chitosan or on the nature of the complexes formed.

Conclusions

This study has used ITC, SSE, and turbidity measure- ments to examine some of the major factors expected to influence the binding of SDS to chitosan. The main results of this study are the following:

(1) When SDS and chitosan were mixed at pH 3, the SDS bound strongly to the chitosan to form an insoluble complex that contained about 4-5 mmol of SDS/1 g of chitosan at saturation.

(2) When SDS and chitosan were mixed at pH 7, we found no evidence of strong binding between them, presumably because the chitosan lost most of its positive charge at this pH and, therefore, the electrostatic attrac- tion between SDS and chitosan was weak. However, when SDS and chitosan were mixed at pH 3 and then the solution was adjusted to pH 7, the SDS remained bound to the chitosan, suggesting that an irreversible complex was formed (at least on the experimental time scale).

(3) The addition of NaCl (0-200 mM) decreased the cmc of SDS (in both the absence and the presence of chitosan) but had little influence on the amount of SDS bound to chitosan at saturation.

(4) The cmc of SDS and the amount of SDS bound to the chitosan at saturation were largely independent of the holding temperature (10-50 °C).

(5) The enthalpy changes associated with micelle breakup were highly dependent on temperature, indicat- ing the importance of hydrophobic interactions, whereas Figure 10. Influence of temperature on the enthalpy change

per mole of surfactant (∆H) injected into the reaction cell versus the total surfactant concentration present in the reaction cell for the 1.4 mM SDS solution injected into 0.01 wt % chitosan solution (pH 3.0, 0 mM NaCl, 100 mM acetate buffer). In this experiment the surfactant was injected into the reaction cell in the form of monomers.

Figure 11. Influence of temperature on the change in EMF (∆EMF) versus total surfactant concentration dependence for SDS-chitosan solutions initially containing either 0 or 0.1 wt

% chitosan (pH 3.0, 0 mM NaCl, 100 mM acetate buffer).

Figure 12. Influence of temperature on the normalized turbidity versus total surfactant concentration dependence for SDS-chitosan solutions initially containing 0.1 wt % chitosan (pH 3.0, 0 mM NaCl, 100 mM acetate buffer).

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the enthalpy changes associated with SDS binding to chitosan were largely independent of temperature, indi- cating the dominant nature of electrostatic interactions.

The results of this study have provided a more detailed understanding of the origin and characteristics of the interactions between anionic surfactants and chitosan, which may prove useful in the design of industrial products or processes, for example, foods that can reduce cholesterol levels.

Acknowledgment. This material is based upon work supported by the Cooperative State Research, Extension, Education Service, United States Department of Agri- culture, Massachusetts Agricultural Experiment Station (Project No. 831), the National Research Initiative, Competitive Grants Program, United States Department of Agriculture, and the CREES.

LA048711O

References

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