Article pubs.acs.org/Langmuir

Mechanism of Chitosan Adsorption on Silica from Aqueous Solutions Alberto Tiraferri,* Plinio Maroni, Diana Caro Rodríguez, and Michal Borkovec Department of Inorganic and Analytical Chemistry, University of Geneva, Sciences II, Quai Ernest-Ansermet 30, 1205 Geneva, Switzerland S Supporting Information *

ABSTRACT: We present a study of the adsorption of chitosan on silica. The adsorption behavior and the resulting layer properties are investigated by combining optical reflectometry and the quartz crystal microbalance. Exactly the same surfaces are used to measure the amount of adsorbed chitosan with both techniques, allowing the systematic combination of the respective experimental results. This experimental protocol makes it possible to accurately determine the thickness of the layers and their water content for chitosan adsorbed on silica from aqueous solutions of varying composition. In particular, we study the effect of pH in 10 mM NaCl, and we focus on the influence of electrolyte type and concentration for two representative pH conditions. Adsorbed layers are stable, and their properties are directly dependent on the behavior of chitosan in solution. In mildly acidic solutions, chitosan behaves like a weakly charged polyelectrolyte, whereby electrostatic attraction is the main driving force for adsorption. Under these conditions, chitosan forms rigid and thin adsorption monolayers with an average thickness of approximately 0.5 nm and a water content of roughly 60%. In neutral solutions, on the other hand, chitosan forms large aggregates, and thus adsorption layers are significantly thicker (∼10 nm) as well as dissipative, resulting in a large maximum of adsorbed mass around the pK of chitosan. These films are also characterized by a substantial amount of water, up to 95% of their total mass. Our results imply the possibility to produce adsorption layers with tailored properties simply by adjusting the solution chemistry during adsorption.

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INTRODUCTION

The efficacy of chitosan-based materials frequently relies upon successful and stable adsorption of the polyelectrolyte on a substrate. The ability of chitosan to adsorb on mineral and metal oxide surfaces was shown in several studies.10−13 The hydroxyl groups of the solid substrate were suggested to play an important role in the interaction with the polyelectrolyte and in the formation of a stable adsorption layer.10,11 The structure of chitosan layers was investigated by adsorbing the polyelectrolyte on mica surfaces under acidic conditions.12,13 Atomic force microscopy (AFM) images of chitosan under liquid showed the presence of sparse or entangled elongated strands as well as aggregated bundles of these chains. The adsorbed films were observed to be relatively smooth and to have a flat conformation with a thickness of approximately 0.5 nm. Singlemolecule force spectroscopy of chitosan adsorbed on mica supported the strong affinity of the polyelectrolyte for the solid substrate.13 Studies of the swelling behavior of chitosan films suggested a relationship between DA and moisture uptake. The adsorbed mass of swollen chitosan films was found to be more than twice as large as for dry films in humid environments. In these investigations, swelling was rationalized with the interaction of chitosan amine moieties with phosphate groups in the buffer solution.14

Chitin and chitosan are polysaccharides composed of linked β(1 → 4) glucosamine and N-acetyl glucosamine residues.1,2 Specifically, chitosan is the polymer in which the proportion of acetylated uncharged units to the total number of units, also referred to as the degree of acetylation (DA), is lower than 50%. Being the only cationic biopolymer originating from natural sources, mostly seafood shells and fungi, it has attracted attention as a functional material in numerous fields such as food science, the cosmetics industry, and agriculture.2 For example, chitosan is used in the biomedical field to synthesize safe and effective delivery systems3 and in the environmental science area as both an adsorbent for the removal of contaminants from polluted water and as a flocculant to induce the precipitation of anionic compounds.4 The structural and physicochemical characteristics of chitosan in aqueous solution have been extensively studied.1,2,5−8 Chitosan properties, including water solubility, depend largely on the proportion and distribution of acetylated and nonacetylated residues. This polyelectrolyte is completely soluble only below pH 6, when most of its amino groups are protonated.2 Under these conditions, chitosan behaves as a weak cationic polyelectrolyte. However, at higher pH or for high DA, polymer chain association can be induced, leading to the formation of colloidal aggregates.9 Indeed, the possibility to alter the properties of chitosan by simple changes in the solution conditions renders this polymer attractive. © XXXX American Chemical Society

Received: February 28, 2014 Revised: April 11, 2014

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Substrates. Exactly the same substrates were used for both reflectometry and QCM-D experiments, making it possible to systematically combine results obtained by the two techniques. QCM-D quartz sensor crystals coated with a gold layer were purchased from Q-Sense (QSX 301, Gothenburg, Sweden). Following the deposition of a middle adhesion layer of titanium, the crystals were sputter coated with a silica layer of about 300 nm in thickness. The substrates produced by this procedure feature relatively high sensitivity in both analytical techniques used in this study. Silica immersed in aqueous solutions acquires a negative surface charge density, primarily through the dissociation of terminal silanol groups, and thus was chosen as representative negatively charged material for our investigation. These properties and the possibility of deployment in both reflectometry and QCM-D experiments are the main rationales for the choice of these substrates in our study. The precise thickness of the uppermost silica layer was determined by scanning-angle null ellipsometry (Multiskop, Optrel, Berlin, Germany), as discussed elsewhere.18 The obtained thicknesses are reported in Table 1.

While the numerous practical applications of chitosan prove that adsorption layers with desired properties can be produced, few studies address the fundamental question of its adsorption mechanism on negatively charged surfaces. However, an understanding of the adsorption behavior of chitosan is clearly important to the rational design and to the advancement of systems relying on the properties of chitosan layers. Research on the adsorption of polyelectrolytes on solid surfaces can be undertaken by applying numerous experimental techniques.15 In particular, reflectometry has been successfully employed to measure the mass of adsorbed polymer,16,17 while the quartz crystal microbalance with dissipation monitoring (QCM-D) has been extensively used to investigate the layer wet mass, which also includes the mass of the trapped and hydration solvent.18−21 By combining these two techniques, it is possible to obtain comprehensive information on the mass, layer thickness, and water content of adsorbed layers.18,22−25 Specifically, monitoring dissipation in QCM-D experiments has allowed the study of the viscoelastic properties of polysaccharides adsorbed on bare or functionalized silica.19−21 In this article, we investigate the adsorption behavior of chitosan on silica surfaces in aqueous solution. The chemical parameters during the adsorption step are systematically varied. In particular, we study the roles of pH and salt type and concentration on the chitosan adsorption mechanism and layer properties. We focus on two different pH conditions representing different protonation states of chitosan, which are relevant for the application of this biopolymer both in laboratory studies and in natural systems, such as wastewater and the human body.

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Table 1. Properties of the Substrates Used for Adsorption Experiments

a

quartz crystal sensor

thickness of silica layer (nm)

sensitivity, A (m2/mg)

RMS roughness (nm)a

Q1 Q2 Q3 Q4 Q5

260 261 260 259 258 260 ± 1

−0.0129 −0.0125 −0.0129 −0.0137 −0.0142

3.2 2.0 1.9 2.1 2.0 2.0 ± 0.1

Average of four random spots on each crystal.

Prior to use, crystals were treated using an oxygen-enriched UVozone cleaner for 10 min followed by immersion in 2% sodium dodecyl sulfate (SDS) for 30 min; they were then rinsed with Milli-Q water and finally dried in a flow of nitrogen before a 10 min cleaning cycle in the UV-ozone chamber. Before each experiment, the substrates were treated with cold piranha solution (a 3:1 volume mixture of H2SO4 98% and H2O2 30%) and washed thoroughly with water. No differences in thickness (> G″, and thus eq 5 reduces to the Sauerbrey equation (eq 3), with C = mq/f 0 and ΔD = 0. Here, the model was applied by fitting the overtones simultaneously and by using the layer thickness, shear modulus, and viscosity as regression parameters and fixing the film density, ρl, to 1 g/cm3. Protocol of Adsorption Experiments. In the beginning of each adsorption experiment, a solution of the desired electrolyte concentration and pH was pumped through the cell until equilibrium was reached. A baseline signal was thus recorded. Adsorption was then initiated by injecting a chitosan solution of the desired polyelectrolyte concentration having the same ionic composition and pH as the background solution. When the adsorption of chitosan reached saturation, as evidenced by a plateau in the signal, the solution was again substituted with a chitosan-free solution of the same composition. The last step was performed to assess any eventual desorption from the substrate. All adsorption experiments were conducted using 5 mg/L of chitosan, with the exception of reflectometry experiments performed to evaluate the adsorption kinetics and illustrated in the Supporting Information, in which this concentration was varied. The plateau value was not observed to depend on the chitosan concentration. Solutions of pH equal to or greater than 6 were degassed prior to experiments, and the pH of all of the inflowing solutions was monitored before the injection into the system throughout the entirety of the tests. When needed, the pH of these solutions was adjusted with NaOH. All experiments were conducted at room temperature (21 ± 2 °C). To test the reproducibility of our experiments and to verify the possibility to extend our conclusions to other types of silica surfaces, the mass of chitosan layers obtained on the sensor crystals from reflectometry was compared to that measured during representative experiments performed using a silicon wafer with a native uppermost silica layer. These runs were analogous to those described above, except for the choice of the adsorbing substrate. The difference between the mass of chitosan adsorbed on the sensor crystals and that adsorbed on the wafers was always less than 30%. Values obtained on the wafers were slightly lower, which is consistent with a smoother surface compared to that of the QCM-D crystals. Determination of Layer Thickness and Water Content. Layer properties were determined by a combination of reflectometry and QCM-D data or by application of a viscoelastic model, depending on the results obtained from QCM-D experiments. When the adsorbed layer was assumed to behave like an elastic rigid film, the Sauerbrey approximation was applied.33 In either case, the mass ratio of water with respect to chitosan in the layer, y, was estimated as18,22

(3)

where n is the overtone number and C is the mass sensitivity constant having a value of 0.177 mg Hz−1m−2 for the AT-cut, 5 MHz crystals used in this study. For this relationship to be valid, the adsorbed mass should be sufficiently rigid not to deform during oscillation.22 Therefore, for dissipative or viscoelastic films, the linear relationship between frequency and mass might not hold, as the layer does not fully couple to the crystal oscillation. The energy dissipation, D, was also monitored from the decay of the crystal oscillation D=

E′ 2πE

(4)

where E′ is the energy dissipated during one oscillation and E is the total energy stored in the quartz oscillator. Materials that form viscous adsorption layers on a crystal yield higher D factors due to their deformation during oscillation. Accordingly, the ΔD/Δf ratio gives information on how much energy is dissipated for a unit change in frequency: a low value relates to elastic and more compact layers, while a high ratio corresponds to nonrigid structures. In the case of dissipative films, the physical properties of the adsorbed layer were not obtained by means of the Sauerbrey approximation. Instead, the wet mass, layer thickness, and water content of the film were obtained by simultaneously fitting Δf and ΔD at multiple overtones using a Voigt-based viscoelastic model embedded in the Q-Tools software (Q-Sense, Gothenburg, Sweden).33,34 This model is a mechanical element analogy for a viscoelastic system that consists of a spring and dashpot in parallel, corresponding to shear rigidity and to the viscosity, respectively. The shifts in the resonance frequency, Δf, and dissipation factor, ΔD, due to the deposition of viscous material on the quartz surface can be calculated from the frequency and phase shift of the quartz oscillator overlaid by the viscoelastic film. Within this model, expressions for Δf and ΔD have been derived in terms of a Taylor expansion in the relevant case of a thin viscous layer in contact with water.34,35 The contribution to changes in frequency and dissipation of the viscoelastic film adsorbed on the quartz oscillator is

⎤ ⎛ η ⎞2 L l ρl ⎡ Δf G″ ⎥ ⎢1 − 2 ⎜ w ⎟ =− ρl ⎝ δw ⎠ G′2 + G″2 ⎥⎦ f mq ⎢⎣

ΔD =

2 4L ⎛ ηw ⎞ G′ ⎜ ⎟ 2 mq ⎝ δw ⎠ G′ + G″2

y=

Γwet − Γ Γ

(7)

The layer thickness was thus obtained from the relation

L=

Γwet ρ

(8)

In our case, as the density of water and that of dry chitosan were the same, namely, ρ = 1.0 g/cm3, this value also corresponded to that of the wet layer. All of these relations are approximate because they assume ideal mixing and a homogeneous film. When ΔD from QCMD experiments was larger than 2.0 × 10−6 at saturation, ΔD/Δf was larger than 0.2 × 10−6 Hz−1, and when the plots of Δf of the different overtones separated over time, the viscoelastic model discussed above was applied to estimate the layer thickness, the wet mass adsorbed, Γwet, and the water to chitosan mass ratio, y.

(5)

(6) C

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RESULTS AND DISCUSSION We present a set of data on the adsorption of chitosan on silica. The adsorbed polyelectrolyte dry mass was measured by reflectometry. The wet mass, which also includes the mass of water in the adsorption layer, was determined by QCM-D, which also provided information on the viscoelastic properties of the chitosan film. The thickness and the water content of the layers were obtained by combining dry and wet masses in the case of a thin rigid film, while a viscoelastic model was applied to study the properties of thick and viscous layers. Adsorption Behavior and Film Structure. Representative experimental results from reflectometry for the adsorption traces of chitosan onto silica under different pH conditions in 10 mM NaCl are shown in Figure 1. After reaching a constant

Figure 2. Representative QCM-D data for chitosan adsorbing on silica at different pH values and at a NaCl concentration of 10 mM. Time dependence of the average of the different overtones of the (a) dissipation change and (b) the corresponding observed frequency shift due to chitosan adsorption normalized by the overtone number. At time zero, a chitosan solution of 5 mg/L is introduced. The arrow indicates the time when the chitosan-free solution was introduced. All experiments were performed with 5 mg/L chitosan.

the plateau values of both frequency and dissipation obtained at pH 8 were significantly larger in magnitude compared to those observed in experiments performed at lower pH. These results are particularly telling when one considers the changes in dissipation, which reflect the viscoelastic properties of the adsorbed films. They suggest that layers of chitosan formed upon adsorption at higher pH were viscoelastic in nature, while those formed at low pH behaved like rigid films oscillating in tune with the underlying crystal. QCM-D experiments showed no significant desorption under any of the conditions employed in this study, corroborating the adsorption irreversibility also observed in reflectometry experiments. The values of dissipation change measured during QCM-D experiments are plotted versus pH and versus the corresponding shift in frequency in Figure 3a,b, respectively. The recorded

Figure 1. Representative reflectivity data for chitosan adsorbing on silica at different pH values and at a NaCl concentration of 10 mM. Time dependence of the (a) reflectivity and (b) corresponding dry adsorbed mass per unit area. At time zero, a chitosan solution of 5 mg/ L is introduced. The arrow indicates the time at which the chitosanfree solution was introduced. All experiments were performed with 5 mg/L chitosan.

baseline by flushing the cell with electrolyte solution, the inlet solution was changed to 5 mg/L chitosan at time zero. The adsorption of chitosan on the substrate is shown as a change in the reflectivity signal (Figure 1a), which is directly proportional to the dry mass adsorbed (Figure 1b). The mass increased linearly at first, reflecting the initial kinetics of the adsorption process. After a transient period, a plateau was observed, which corresponded to layer saturation. Following the adsorption plateau, the substrate was rinsed with the same chitosan-free electrolyte solution used to condition the system. In our experiments, the time needed to reach saturation as well as the total mass of chitosan adsorbed on the substrate increased with increasing pH. Signal shifts for both dissipation and frequency were especially pronounced for experiments conducted at around pH 8, where the transient portion of the curve was long and saturation was not reached until approximately 10 min. No significant change in mass was observed when the cell was flushed with the chitosan-free solution. This result indicates that the adsorption process was irreversible and that the layers formed on silica were always stable in the same solution used for adsorption. Similar results were obtained for QCM-D experiments. Typical data of the shift in frequency normalized by the overtone number, Δf/n, and of the dissipation change, ΔD, observed for three pH conditions in 10 mM NaCl are presented in Figure 2. Consistent with reflectometry, experiments conducted at higher pH produced profiles characterized by long transient times before they reached a final saturation plateau. In the three representative examples shown in Figure 2,

Figure 3. Dissipation change measured by QCM-D at adsorption saturation of chitosan on silica. The dissipation change is plotted against (a) the solution pH and (b) the corresponding shift in frequency (with opposite sign) normalized by the overtone number. Data refer to the average of the different overtones. A dissipation value of ΔD = 2.0 × 10−6 is usually defined as the threshold below which an adsorbed layer can be considered to behave as a rigid film. The average pK value of the charged groups of chitosan is approximately 6.8. All experiments were performed using 5 mg/L chitosan.

ΔD was lower than 2.0 × 10−6 for experiments conducted under pH conditions whereby chitosan is fully protonated, while a large increase in dissipation was measured above a pH of approximately 6.5. This result indicates that the viscoelastic properties of the adsorbed film changed across the pH range and that this transformation occurred roughly around the pK of D

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chitosan. The steepness of the ΔD versus Δf/n plot in Figure 3b is also a measure of the rigidity of the adsorbed layer. One can observe the presence of two regimes. The data profile is initially linear and relates to experiments performed at lower pH conditions. For values of dissipation change larger than 2.0 × 10−6, there is a clear increase in the slope, which changes from 0.16 × 10−6 Hz−1 for lower dissipation changes to roughly 0.23 × 10−6 Hz−1. These results show that the adsorbed layer became more dissipative due to a decrease in packing density at higher pH. Adsorbed Mass. Figure 4a presents the results of the chitosan dry mass of saturated layers adsorbed on silica in 10

adsorbed molecules by counterions in the diffuse layer.39 The same trends were observed earlier for the adsorption of poly-Llysine and poly(allylamine) on silica.18 With increasing pH, the mass of chitosan continued to increase steadily. Chitosan is very weakly charged around pH 8, as also supported by a measured electrophoretic mobility close to zero. In this pH range, chitosan still adsorbed irreversibly at the silica/aqueous solution interface through interactions with the hydroxylated species of the substrate. Previous studies have suggested that acidic surfaces such as silica can form hydrogen bonds with the acetyl groups of the polyelectrolyte backbone.10,40 However, at pH values above 9, adsorption was observed to decrease quickly, suggesting that other interaction forces, if present, were weak compared to electrostatic interactions. Under basic conditions, the electrophoretic mobility of chitosan had a negative sign (−0.35 × 10−8 m2 V−1 s−1 at pH 9), suggesting that the polyelectrolyte might adsorb negatively charged ionic species in water. In this case, the electrostatic repulsion between chitosan molecules and the surface prevents adsorption. Figure 5a presents the dependence of the chitosan adsorbed mass on the salt type and concentration at pH 4 and 8. NaCl

Figure 4. Adsorbed mass and layer properties for saturated layers of adsorbed chitosan on silica as a function of pH at 10 mM NaCl concentration. (a) Mass obtained from reflectivity experiments, (b) wet mass obtained from QCM-D experiments, (c) layer thicknesses, and (d) water/chitosan mass ratios. The average pK value of the charged groups of chitosan is approximately 6.8. Data points are the average of duplicate or triplicate experiments. All experiments were performed with 5 mg/L chitosan.

mM NaCl under varying pH conditions, calculated from reflectometry. Experiments were conducted for a wide range of pH from approximately 3 to 10. Low amounts of chitosan adsorbed on silica at the lowest pH. Increasing the pH of the adsorbing solution increased the adsorbed mass. Under these conditions, electrostatic attraction between the negatively charged silica and the positively charged chitosan, the release of counterions upon adsorption, and additional hydrophobic interactions are the main driving forces for chitosan deposition.15,36,37 The electrophoretic mobility of chitosan molecules was measured as approximately +1 × 10−8 m2 V−1 s−1 in the range of pH 4 to 5; see the Supporting Information. The observed trend is consistent with the adsorption of a weakly charged polyelectrolyte on an oppositely charged substrate,38 and it is due to two combined mechanisms. With increasing pH, chitosan becomes progressively neutral and the decreased repulsion between adsorbed molecules favors the accumulation of the polyelectrolyte on the surface. At the same time, the increase in silanol dissociation of the silica produces an increase in surface charge density, which provides an additional screening of the electrostatic potential between

Figure 5. Adsorbed mass and layer properties for saturated layers of adsorbed chitosan on silica as a function of ionic strength at (open circles) pH 8 and (solid symbols) pH 4. (a) Mass obtained from reflectivity experiments, (b) wet mass obtained from QCM-D experiments, (c) layer thicknesses, and (d) water/chitosan mass ratios. The experiments were performed at different NaCl concentrations (circles) or in CaCl2 solution with a total ionic strength of 10 mM (squares). Data points are the average of duplicate or triplicate experiments. All experiments were performed with 5 mg/L chitosan.

concentrations were varied between 0.1 and 100 mM. Solutions of CaCl2 with a total ionic strength of 10 mM were also employed to assess the possible effect of divalent cations that do not specifically interact with chitosan moieties. We will initially focus on the results obtained under acidic conditions, shown in the plots as solid data points. At pH 4, values of 20− 25 nm and +1 × 10−8 m2 V−1 s−1 were measured for the hydrodynamic radius and the electrophoretic mobility of chitosan molecules, respectively; see the Supporting InformaE

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that all chitosan molecules approaching the surface successfully attached, as expected due to electrostatic attraction between chitosan molecules and substrates at acidic pH. On the contrary, the long transient regime observed at pH 8 implies that the adsorption of approaching aggregates on the alreadyadsorbed layer under these conditions was slower compared to that on the bare surface.49 Our results did not quite follow firstorder kinetics: this discrepancy can be rationalized with a varying diffusion coefficient at increased chitosan concentration due to a stronger interaction between the polyelectrolyte molecules that resulted in a different aggregate size.50,51 These results suggest that chitosan aggregation is also induced by an increase in polyelectrolyte concentration in solution. This phenomenon can result in a disagreement from predictions of adsorption kinetics based solely on chitosan−silica interactions, whereby one assumes that chitosan molecules do not interact with each other; see the Supporting Information for further details. Layer Thickness and Water Content. From the dry mass measured by reflectivity and the wet mass measured by QCMD, one can estimate the thickness and water content of layers of chitosan on silica. The behavior of the adsorbed mass obtained by reflectometry and QCM-D in 10 mM NaCl as a function of pH was analogous; see Figure 4a,b. However, a noteworthy phenomenon was observed around the pK of chitosan. While the dry mass continued to increase steadily, a significant increase in wet mass was determined from the QCM-D data. This sharp increase in wet mass was due to a conformational alteration of the adsorption layer, as anticipated by the dissipation changes observed under conditions near and above the pK of chitosan; see Figure 3. These results suggest that layers deposited when chitosan is weakly charged or uncharged were viscous and contained a large amount of water. The properties of the saturated layers directly follow the results obtained for dry and wet masses. Figure 4c shows that chitosan formed flat monolayers at low pH, where films had a height between roughly 0.3 nm at pH 3 and 1.5 nm at around pH 6. This outcome is consistent with previous observations from AFM experiments of chitosan deposited on mica, where authors measured layer thicknesses in the range of 0.5 ± 0.1 nm for adsorption performed under acidic conditions.12,13 Chitosan was observed by AFM to form smooth and uniform monolayer structures of mostly elongated strands and some aggregate bundles. In our experiments, the water content of chitosan films was constant across the low-pH range and produced a water/ chitosan mass ratio of y = 2; see Figure 4d. This value corresponds to a water content of about 65%, which is typical of adsorption layers of weakly charged polyelectrolyte on oppositely charged substrates.15,18 Our observations indicate a significant increase in layer thickness and water content with increasing pH, in accordance with values of wet mass calculated from QCM-D experiments. Chitosan films reached a peak in thickness of 10 nm between pH 7 and 8. Values of the layer thickness estimated using the viscoelastic model were twice those that would be calculated with the Sauerbrey approximation. This result is related to a phenomenon referred to as missing mass effect, according to which the true mass adsorbed on the crystal is larger than the mass calculated with the Sauerbrey equation, due to the viscous loss of energy of the quartz oscillator coupled with the dissipative adsorption layer.35 The thickness of chitosan layers decreased sharply under basic conditions until no adsorption was measured at pH 10. Contrary to the peak observed for

tion. In our light-scattering experiments, these values were constant at varying ionic strength and composition and were consistent with reports in the literature.1,29,31 The mass measured at pH 4 was in the range of 0.05−0.2 mg/m2. These low values are typical for the adsorption of polyelectrolyte layers on oppositely charged substrates.15,24,39,41 In our study, the change in salt concentration over 3 orders of magnitude produced a change in adsorbed mass by a factor of 3 or less. A similar range was obtained earlier for the adsorption of other weakly charged polyelectrolytes on silica, namely, poly(L-lysine), polyacrylamide, and poly(dimethylamino ethyl methacrylate).42−44 At low salt concentrations, the charged chitosan molecules repel each other due to electrostatic repulsion, thus preventing dense packing of polyelectrolyte on the surface. The increase in adsorbed chitosan due to a higher salt concentration in solution was rationalized by the screening of electrostatic repulsion within the deposited molecules at increasing ionic strength, which allows higher adsorption densities.17,38,45,46 However, the increase in ionic strength also causes progressive screening of the chitosan−silica attraction at high salt concentrations, by compression of the electric double layer. In our study, this effect weakened the main driving force for adsorption, causing a decrease in the mass necessary to cause charge compensation in 100 mM NaCl. The change in polyelectrolyte adsorbed mass with addition of salt was recently described theoretically by including ion correlations in the density functional theory.47,48 This mass decrease is usually intensified due to the adsorption of counterions from the background solution that compete with chitosan molecules for adsorption sites. In our experiments, no effect of the change in ionic composition by substituting NaCl with CaCl2 was observed. This result is expected as neither silica nor chitosan has functional groups that interact specifically with calcium. Results obtained by varying the ionic strength and composition during the adsorption of chitosan on silica at pH 8 are shown as open points in Figure 5a. Under these conditions, chitosan is very weakly charged in aqueous solution and can give rise to colloidal aggregates with a hydrodynamic radius ranging between 250 and 450 nm, as shown by light scattering.1 The adsorbed mass was nearly constant at around 1 mg/m2 with varying salt concentration and then dropped to half this value in a solution of 100 mM NaCl. The significant decrease in adsorbed mass at the largest salt concentration could be the effect of two concomitant mechanisms, such as competition with background ions adsorbing onto silica and the shift in the adsorption cutoff induced by salt.42 Both theoretical and experimental studies suggested that in cases where electrostatic interactions are dominant for systems comprising weakly charged macromolecules and oppositely charged substrates, a sharp cutoff of adsorption exists at increasing pH or ionic strength, above which a significant decrease in adsorption occurs.17,38,42,43,47,48 In particular, a larger salt concentration induces a shift of this cutoff toward lower pH; that is, lower adsorption occurs with increasing ionic strength at fixed pH. Our system was characterized by a similar behavior, which explains the sudden reduction in adsorbed mass observed between 10 and 100 mM NaCl at pH 8. Once again, no specific effect of calcium and no desorption of chitosan were observed. Results on the kinetics of chitosan adsorption on silica are discussed in the Supporting Information. Data suggest that the adsorption process at pH 4 was transport-controlled, meaning F

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chitosan due to osmotic pressure exerted by the electrolytes in solution.28 The thickness data estimated at pH 8 show a decrease in layer thickness from roughly 11 to 8 nm with increasing ionic strength from 0.1 to 100 mM, consistent with the corresponding values of wet mass. The related reduction of water content shown in Figure 5d strengthens the hypothesis that chitosan layers might undergo compression as the osmotic pressure in solution increases. Only at the largest NaCl concentration does the water content increase, which is consistent with literature observations predicting the swelling of chitosan gels at high salt concentrations.52 The thickness of the saturation layers of chitosan observed at pH 8 was always relatively large, with films at least 20 times thicker than those observed at pH 4; see Figure 5c,d. The opposite dependence of thickness on salt concentration under the two different pH conditions produced a narrowing of the gap with increasing ionic strength. The most pronounced difference at 0.1 mM NaCl concentration corresponded to films approximately 50 times thicker at pH 8 than at pH 4. As discussed previously, the thick films obtained with deprotonated chitosan were the result of the deposition of aggregates from solution onto the previously adsorbed layer in a process similar to that of ripening observed in particle deposition.53 The corresponding change in dissipation of the layers formed under these conditions and recorded during QCM-D experiments suggests that all layers formed at pH 8 were viscous and dissipative, regardless of the amount of background electrolyte. These results substantiate once more the remarkable possibility to tune the properties of chitosan layers by simple changes in the composition of the adsorbing solution.

thickness, the water content of the layers kept increasing considerably at high pH; see Figure 4d. The large values of the water/chitosan mass ratio of around 16 corresponded to a water content of 95% in the adsorption film. These results are explained by considering the structure of chitosan aggregates that form when chitosan is fully deprotonated. Under these conditions, aggregates may resemble a gel with a loose structure characterized by a strongly swollen state.28 As mentioned above, chitosan molecules aggregate above pH 6.5. The presence of large and weakly charged aggregates produced thick adsorption layers on silica in a solution of 10 mM NaCl. The long transient times needed to reach saturation under these conditions as shown in Figure 2 suggest that adsorption proceeded by the deposition of molecules and aggregates from solution onto previously adsorbed molecules, before complete compensation of the surface charge. However, once a chitosan-free solution was introduced, layers were stable and no significant desorption was measured, implying that the film−substrate interactions were sufficiently strong. More importantly, the properties of chitosan layers could be tuned simply by adjusting the pH during adsorption. Flat, rigid, and thin films can be formed under acidic conditions, while stable, thick layers composed of viscous material are produced if chitosan is deprotonated. Below, we present results for the layer thickness and water content obtained at pH 4 and 8, illustrating how ionic composition influences the behavior of adsorption under conditions representative of the two regimes of aqueous chitosan. By directly combining reflectometry and QCM-D data, the thickness and water content of the layer can be calculated for adsorption experiments performed at pH 4 by varying the ionic composition in solution. The values of wet mass obtained from QCM-D are shown in Figure 5b as solid points. One observes that the wet mass was about 4 times larger than the dry mass reported in Figure 5a. Interestingly, while the dry mass went through a pronounced maximum across the NaCl concentration, the wet mass remained constant between 10 and 100 mM. The nearly constant wet mass calculated from QCM-D experiments suggests that the layers contained more trapped water under these conditions. Figure 5c shows that adsorption under acidic conditions gave rise to chitosan monolayers with thickness ranging from around 0.2 to 0.6 nm. As expected, layers were always thin and had a rigid structure, based on the small change in dissipation observed in QCM-D experiments. This observation is consistent with a strong chitosan−silica interaction, driving the conformation of polyelectrolyte on the substrate toward a flattened configuration. The layer thickness reached a plateau starting from 10 mM NaCl, while the water/ chitosan mass ratio, y, increased up to approximately 6 at the highest salt concentration, as reported in Figure 5d. These water/chitosan ratios corresponded to water contents ranging from 50 to 80% in the polyelectrolyte film. The increase in water content at increasing salt concentration explains the difference in wet and dry masses estimated at larger ionic strengths and also observed in previous studies.52 The presence of calcium did not significantly influence the outcome and only resulted in slightly lower water content of the film. Results obtained at pH 8 are shown as open points in Figure 5b−d. While the dry mass in Figure 5a remained constant between 0.1 and 10 mM NaCl, the wet mass decreased monotonically from approximately 12 to 8 mg/m2 with ionic strength; see Figure 5b. The reduction in wet mass may be rationalized by the compression of the adsorption layer of

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CONCLUSIONS The adsorption of chitosan onto silica surfaces was investigated by combining reflectometry and QCM-D. The influence of pH was studied in 10 mM NaCl. In mildly acidic solutions, chitosan formed rigid and thin adsorption monolayers with an average thickness of approximately 0.5 nm and a water content of roughly 60%. In neutral solutions, layers became significantly thicker (∼10 nm) as well as viscoelastic and were characterized by a highly hydrated state containing up to 95% water. In mildly basic solutions, no or little adsorption was observed. The effects of salt type and concentration were also investigated at two representative pH values. Chitosan behaved similarly to weakly charged polyelectrolytes, such as poly(dimethylamino ethyl methacrylate), at acidic pH, whereby electrostatic attraction and counterion release are the main driving forces for adsorption. The tendency of chitosan to form relatively large aggregates that trap a significant amount of water was observed at pH 8. Adsorption was observed to be practically irreversible in all cases. This investigation focuses on a typical chitosan sample with average DA and molecular weight. Changing these parameters will influence the quantitative results of the adsorption process. However, we expect that the discussed mechanisms will remain similar and that other chitosan samples will behave analogously. This study demonstrates that chitosan forms stable adsorption layers on oppositely charged silica surfaces under a wide range of conditions. The properties of the resulting adsorption layers are a direct consequence of the behavior of chitosan in aqueous solution. Significantly different films can be obtained by fine tuning the adsorption conditions. In particular, the possibility to produce tailored adsorption layers simply by adjusting the solution pH represents a remarkable opportunity G

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to use this polyelectrolyte in a variety of systems. These results, along with the availability and compatibility of chitosan, highlight its potential as a functional biomaterial.

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ASSOCIATED CONTENT

S Supporting Information *

Electrokinetic and sizing measurements of chitosan, roughness measurements of crystal surfaces, and kinetics of chitosan adsorption. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by a Marie Curie Intra-European Fellowship to A.T. within the Seventh European Community Framework Programme (PIEF-GA-2012-327977), by the Swiss National Science Foundation, and by the University of Geneva.

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