Preparation of Chitosan Gel Beads by Ionotropic Molybdate Gelation

This process consisted in adding the beads to 2 mL of hydrogen peroxide (50% .... Beyond this time, the metal uptake continues at a much lower rate fo...
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Biomacromolecules 2001, 2, 1198-1205

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Preparation of Chitosan Gel Beads by Ionotropic Molybdate Gelation Laurent Dambies,† Thierry Vincent,† Alain Domard,‡ and Eric Guibal*,† Laboratoire Ge´ nie de l’Environnement Industriel, Ecole des Mines d’Ale` s, 6, avenue de Clavie` res, F-30319 Ale` s Cedex, France; and Lyon I, Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux, UMR CNRS 5627, Universite´ Claude Bernard, 43, Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France Received May 2, 2001; Revised Manuscript Received August 24, 2001

A new process is described for the preparation of chitosan gel beads using molybdate as the gelling agent. This new gelation technique leads to a structure different from that produced during alkaline coagulation of a chitosan solution. Instead of a morphology characterized by large open pores, gel beads produced in a molybdate solution, under optimum conditions (pH 6; molybdate concentration, 7 g‚L-1), have a double layer structure corresponding to a very compact 100-µm thick external layer and an internal structure of small pores. Experimental conditions, especially pH and molybdate concentration, were selected to optimize molybdate content and the stability of the bead shape. Introduction Chitosan is a biopolymer produced by N-deacetylation of chitin, the most abundant natural polymer, along with cellulose. Chitin is extracted from several sources including cuticles of arthropods1 and fungal biomass.2 Chitosan is a unique biopolymer because of its cationic character in acidic solutions, related to the protonation of its amine groups. This protonation is responsible for the solubility of chitosan in numerous dilute mineral and organic acids, an exception being sulfuric acid.3 Chitosan has been extensively studied because of its interesting metal ion uptake properties.4-6 One factor limiting this uptake is the low porosity of crude chitosan due to the poor accessibility of internal sorption sites, especially for metal ions with a large ionic radius. Metal ion sorption may therefore be limited only to the external layers of the sorbent particles.6 Several processes have been proposed in order to improve diffusion properties and thus enhance the sorption performances. They include decreasing the crystallinity through dissolution and reprecipitation under appropriate conditions, dissolution and freeze-drying,7 and gel formation by dissolution and coagulation of a viscous solution.4,8-9 The wide range of applications of the last technique explains the number of studies dedicated to the coagulation of chitosan solutions. Several processes have been tested. The most frequently used is alkaline coagulation in water, in the presence of either sodium hydroxide, ethylenediamine, ammonium hydroxide, or hydroalcoholic mixtures to decrease the surface tension.10 This coagulation is subject to a possible redissolution when the polymer comes into contact with acidic solutions. To avoid this problem, a cross-linking is often carried out, using for example dialdehydes (glutaral* Corresponding author. Telephone: +33 (0)4 66 78 27 34. Fax: +33 (0)4 66 78 27 01. E-mail: [email protected]. † Ecole des Mines d’Ale ` s. ‡ Universite ´ Claude Bernard.

dehyde) or polyaldehydes,8,11-14 epoxides, epichlorhydrine, hexamethylene diisocyanate,10 and ethylene glycol diglycidyl ether.4 However, the cross-linking occurring on amine groups reduces their availability. This chemical treatment can also cause a decrease in the reactivity of the polymer, due to a reduction in the diffusion properties. Alternative processes exist, though they have received less attention. The ionotropic gelation process has been proposed for the preparation of chitosan gel beads. Vorlop and Klein used this process for the entrapment of enzymes by means of ferrous and ferric cyanide, polyphosphates or organic compounds.15 More recently, Lee et al. extensively studied the ionotropic gelation of chitosan with polyphosphates as the gelling agent and they used the gel beads for the sorption of copper.16 These processes typically correspond to gelation techniques by heterogeneous cross-linking. Metal ions have also been used for the homogeneous coagulation of chitosan.17 Draget et al. have studied the homogeneous cross-linking of chitosan with molybdate polyoxoanions and have shown their considerable potential for the gelation of this biopolymer.18 The homogeneous and the heterogeneous techniques differ by the gelation mechanisms. The shrinking core model could describe the heterogeneous procedure: gelation progressively migrates from the surface of the bead to the center of the gel, while in the case of the homogeneous gelation, the process is expected to produce a uniform distribution of cross-links. The heterogeneous molybdate gelation of chitosan has been successfully tested for the preparation of hollow chitosan fibers, and these fibers have been used for the extraction of chromate ions from acidic solutions. Chromate anions are adsorbed at the surface of the fiber while the organic extractant flowing in the lumen of the fiber re-extracts metal anions from the fiber.19,20 The technique used for the preparation of hollow fibers makes profit of the heterogeneous structure resulting from the heterogeneous gelation process.

10.1021/bm010083r CCC: $20.00 © 2001 American Chemical Society Published on Web 10/09/2001

Preparation of Chitosan Gel Beads

This work deals with the preparation of chitosan gel beads using molybdate as the coagulating agent. Indeed, previous studies have shown that chitosan is very efficient at removing molybdate from acidic solutions: the optimum pH tends to 3 for a medium concentration range (i.e. 50-200 mg of Mo L-1).9,21 Previous studies have been focused on the sorption of molybdate on chitosan flakes and preformed chitosan gel beads, after glutaraldehyde cross-linking, and with raw materials. The interactions between molybdate and preformed beads are suspected to be different due to the different availability of amine groups in both systems. The structure of molybdate-gelled beads is compared to that of gel beads prepared by the conventional alkaline treatment. The first section deals with the optimization of the process, taking into account the role of several experimental parameters on the molybdate content in the beads, the stability of the molybdate, and the gelation kinetics. Parameters include the pH of the coagulation bath, the molybdate concentration, and the characteristics of the chitosan. The second part is dedicated to the comparison of the morphology of gel beads obtained under different conditions, using scanning electron microscopy (SEM) and X-ray diffraction. Material and Methods Materials. Four different chitosans from different sources and suppliers were tested. Three samples were produced from shrimp shells. Their references are as follows: ABER 90 (lot no. A17G28), from Aber Technologies (Plouvien, France), the fraction of acetylation was FA ) 0.13 and the weight-average molecular weight was Mw ) 191000; IND 342 and NIG 2332 from France Chitine (Marseille, France), characterized by their weight-average molecular weight Mw ) 200000 and 670000 g‚mol-1, and their FA ) 0.17 and 0.13, respectively. Molecular weight of chitosan samples was determined by size exclusion chromatography on TSK GW columns with an on-line double detection obtained with a Waters R410 differential refractometer and a Dawn-Wyatt multiangle laser light scattering photometer. In the case of an eluent at pH 4.3 (0.15 M acetic acid/ammonium acetate buffer) the refractive index increment was fixed at 0.172 cm3 g-1.7 The deacetylation percentage was determined by FTIR spectrophotometry on chitosan films (chitosan in solution in acetic acid 1% w/w) using a Perkin-Elmer 1760 FTIR spectrophotometer. The spectra were treated by the methods of Baxter et al. and Miya et al.9 Other reagents were supplied by Carlo Erba (Italy) for acetic, hydrochloric, and sulfuric acids. Preparation of Chitosan Gel Beads. Chitosan was dissolved in an acetic acid solution (0.3 M) in order to obtain a concentration of 4% (w/w). The solution was filtered to remove possible insoluble particles and let to stand for 2 days. Several molybdate solutions corresponding to various pH’s and concentrations of molybdate were prepared by dissolution of the exact amount of molybdate salt in demineralized water and used for the coagulation of the polymer solutions. The viscous chitosan solution was pumped then delivered dropwise (through a thin nozzlesdiameter 0.6 mm) into the continuously stirred coagulating bath. Aliquots of 100 beads were collected and left to stand for 3 days in the same coagulating solution, which was stirred with a

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reciprocal shaker. A total of 20 of these beads were mineralized to evaluate their total molybdate content. This process consisted in adding the beads to 2 mL of hydrogen peroxide (50% w/w) and 0.5 mL of nitric acid (65% w/w). The mixture was then heated at 80 °C for 3 h and demineralized water was added to give a final volume of 25 mL. The solution was filtered through a Millipore membrane (porosity: 1.2 µm) and the metal content of the filtrate was analyzed. Then, 20 other beads were dried at 105 °C for 72 h to determine the dry mass of the material (chitosan plus molybdate). Then, 20 beads were mixed with NaOH (1 M), rinsed and dried at 105 °C for 72 h to measure the dry mass of chitosan. Finally, 20 beads were mixed with 100 mL of phosphoric acid (0.1 M) for 72 h in a reciprocal shaker to deduce the amount of molybdate strongly bound to chitosan (the labile fraction of molybdate physically sorbed on the polymer network was easily released in phosphoric acid). Molybdate concentrations in solution were determined by inductively coupled plasma spectrometry of atomic emission using an ICP-AES Jobin-Yvon JY 36 (Jobin-Yvon). Coagulation kinetics were studied by measuring the bead diameters and their molybdate concentration during the coagulation step. Here, 10 beads were coagulated in 400 mL of an ammonium molybdate solution (initial concentration ) 7 g‚L-1) at pH 6. Beads were collected at pre-selected times and then rinsed with demineralized water. Their diameter was measured before they were mineralized to determine their molybdate content. Molybdate uptake did not exceed 0.1% of the initial metal amount, and the molybdate concentration in the solutions was therefore considered as constant during the kinetics. pH was not controlled during the experiments due to the buffering effect of the initial molybdate solution. Similar procedures were adopted for the preparation and characterization of chitosan beads coagulated in sodium hydroxide. The chitosan solution was introduced dropwise as described above into a sodium hydroxide solution (2.5 mol‚L-1), and the beads obtained were rinsed with water until a constant pH was obtained. The beads were then mixed for 72 h with a dilute sulfuric acid solution (pH 3) containing a sufficient initial concentration in molybdate, Co, to reach, at equilibrium, a final concentration, Ceq, above 200 mg‚L-1. Co was calculated according to the equation: Co )

(qeqm) + (VsolCeq) Vsol

where m is the mass of chitosan, Vsol represents the volume of the coagulating solution and qeq the concentration of molybdate in the beads in equilibrium with a solution of molybdate at the initial concentration Co. Assuming a value of 200 mg‚L-1 for Ceq, the sorption capacity qeq was calculated according to the Langmuir equation proposed by Milot for molybdate sorption on chitosan gel beads at pH 3:22 qeq )

qmbCeq 1145 × 0.232Ceq ) 1 + bCeq 1 + 0.232Ceq

where qm is the maximum sorption capacity (mg of Mo g-1), and b is the affinity coefficient (L‚mg-1).

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Figure 1. Variation of the total amount of molybdate (striped areas, mg‚Mo g-1) in the gel beads measured at equilibrium (qeq) as a function of the initial pH (pHi) of the coagulation solution (molybdate concentration 7 g‚L-1), pHf (-O-) is the pH measured at equilibrium, the numbers on the striped areas represent the percentage of strongly bound molybdate).

Characterization of the Materials. Wide-angle X-ray diffraction patterns were recorded, after the gel beads have been dried, by means of a Philips PW1729 X-ray diffractometer using the KR X-ray of copper. Scanning electron microscopy was obtained on freeze-dried samples after coating with carbon. Samples were observed with a scanning electron microscope, JEOL.JEM 35.CF. Results and Discussion Influence of the pH of the Coagulating Solution. Figure 1 shows the variation, at equilibrium, of the total molybdate content (qeq) in the chitosan gel beads as a function of the initial pH (pHi). The initial molybdate concentration in solution is maintained constant at 7 g‚L-1. This figure also gives the pH at equilibrium (pHeq) and the amount (%) of molybdate strongly bound to the polymer, measured after elimination of the weakly sorbed metal by a treatment with phosphoric acid (0.1 M). Increasing pH significantly lowers the amount of molybdate accumulated in the beads, from a maximum value of 750 mg of Mo/g of chitosan, at pHi ) 5.5, to only 72 mg for pHi ) 9. The maximum molybdate uptake remains considerably lower to that of chitosan gel beads, coagulated in sodium hydroxide, then subjected to molybdate sorption at pH 3.9,18 Therefore, the method of preparation influences the molybdate content and reveals a difference in the interaction mechanism. With preformed gel beads, the protonated amine groups are involved in an ion exchange mechanism, and the maximum sorption capacity of Mo is close to 1.2 g g-1, corresponding to a molar ratio Mo/-NH3+ of about 2. In this case, molybdate is considered to be sorbed under polynuclear forms. Indeed, heptamolybdate species predominate in solution. As a consequence the stoichiometry between these polynuclear species (7 Mo) and protonated amine groups tends toward 3-4 (to maintain the ratio Mo/-NH3+ of 2). In the case of beads prepared by coagulation in molybdate solutions, the maximum molybdate content is 750 mg of Mo g-1, corresponding to a molar ratio of 1.2. Under different experimental conditions, corresponding to the direct addition of molybdenum trioxide (1.9 g‚L-1) to a solution of chitosan (pH 5, 10 g‚L-1). Draget et al. observed in situ cross-linking.18 Depending on the calculation

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hypotheses, they found an average distance between the monomers in interaction with molybdate corresponding to 22 or 52 residues. Their results cannot be directly compared to ours since the two gelation processes are quite different. Then, homogeneous molybdate gelation with low concentration chitosan solutions provides a close and direct contact between polymer chains and gelling agent molecules, thus allowing a uniform distribution of cross-links. On the opposite hand, in the case of heterogeneous cross-linking, gelation proceeds through a progressive diffusion of the gelation mechanism from the surface to the core of the bead. In the latter case, another difference comes from that initial cross-links due to chain entanglements associated with low energy interactions (H bonds, hydrophobic interactions) are preexisting. At pHi ) 9, for an initial concentration of molybdate of 7 g‚L-1, the molybdate content in the beads and the interactions with the polymer become so weak that treatment with phosphoric acid almost completely removes the molybdate sorbed in the bead. At pH 5 and 5.5 an anisotropic shrinking mechanism at the beginning of coagulation leads to a decrease in the size of the sorbent, and the particles progressively lose their spherical form. Nevertheless, pH was shown not to influence the dry mass of the beads, so the depletion can only be attributed to a loss of water during the coagulating procedure, related both to the osmotic parameter and to syneresis. A similar shrinking was observed in the case of metal sorption onto modified gelatin beads.23 For chitosan gel beads prepared by coagulation in a solution of sodium hydroxide, the water content is 96.6% and decreases to 87.6% after molybdate sorption. The pH of the solutions was not controlled and was only measured at the end of the coagulation step (pHf, Figure 1). A decrease in pHf was observed, to 6 and 6.6, for pHi’s of 7 and 8, respectively. This decrease favors a greater affinity of the biopolymer for molybdate under selected experimental conditions. It explains the unexpectedly high molybdate content of the beads on these pHi’s. As Domard and Vachoud recently proposed, this behavior must be related to the formation of cross-links, involving, on the one hand, ionic interactions between ammonium groups on chitosan chains and the negative charges of molybdate ions and, on the other hand, hydrogen bonding between the OH groups or the numerous oxygen atoms, borne by the metal species and other chitosan chains.24 Depending on pHi, the labile fraction varies between 12% and 25%, but it remains minimal for pHi’s between 7 and 8. For further experiments, pHi ) 6 was selected as the optimum pHi. Figure 2a represents the relation between the amount of molybdate accumulated on the chitosan beads and the percentage of polynuclear hydrolyzed molybdate species (Mo7O246- + Mo7O23(OH)5-) in the coagulating solution. Previous studies on molybdate sorption by chitosan gel beads have shown that a direct relation can be observed between the efficacy of the sorption and the presence of polynuclear hydrolyzed species.25 The speciation of metal ions depends on both the total concentration of the metal and the pH of the solution (Figure 2b). For optimum pHi’s, the shape of

Preparation of Chitosan Gel Beads

Figure 2. (a) Relation between total molybdate content in the beads, the percentage of polynuclear molybdate species (O) and the content of strongly bound molybdate for various pHf (4) (value mentioned on each point) (molybdate concentration was 7 g‚L-1, pH varies). (b) Molybdate speciation as a function of pH for a concentration of metal of 7 g‚L-1.

the sorption isotherm (not shown) is very favorable (high initial slope and a plateau reached at a low residual concentration) while, for the other pH’s, the sorption curve begins with an unfavorable trend followed by an increase in sorption capacity. The concentration corresponding to the beginning of the increase depends on pH and can be related to the concentration of molybdate for which polynuclear hydrolyzed species appear. Figure 2a also shows the relation between the amount of molybdate strongly bound to the polymer network and the total amount of molybdate on the beads. In the concentration range investigated, molybdate release is a linear function of the total molybdate content in the gel beads. Thus, part of the Mo is strongly sorbed thanks to electrostatic interactions with the polymers while another part is clustered in the water surrounding the polymer network. When molybdate sorption exceeds the sorption capacity, the metal anions are then accumulated by physical sorption. This free fraction is easily removed by phosphoric acid, and the release is proportional to the excess of sorbed molybdate. In the case of the heterogeneous ionotropic gelation of chitosan with polyphosphate, Lee et al. also observed a change in the mechanism and structure of the gel on varying the pH of the gelling solution.16 At alkaline pH, they identified a two-step procedure: a deprotonation was accompanied with slightly ionic cross-linking, while in acidic solutions, the gel beads were completely ionically cross-

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Figure 3. (a) Influence of the molybdate concentration in the coagulating solution (pHi ) 6) on the metal content in the beads and the strongly bound fraction of molybdate (numbers in %). (b) Relation between the total molybdate content in the beads, the percentage of polynuclear species (O), and the strongly bound molybdate (4) for pHi ) 6. The different molybdate concentrations are mentioned on the curves.

linked. This change in the mechanism affects the stability of the beads. Thus, when they are gelled in acidic solutions, the beads are white and brittle, while when they are prepared under alkaline conditions, they become transparent and elastic. Influence of the Molybdate Concentration in the Coagulating Solution. Figure 3a shows the relation between the molybdate concentration in the coagulating solution, the molybdate content in the chitosan gel beads, and the fraction of molybdate strongly bound to the polymer network. Increasing the molybdate concentration to 10 g‚L-1 contributes to a linear increase of the content of molybdate in the beads from 710 to 1240 mg‚g-1. Above this concentration, the molybdate uptake does not vary significantly. The maximum corresponds to the levels reached in chitosan beads coagulated in sodium hydroxide solutions then saturated by a direct contact with a molybdate solution. However, to reach the same level of saturation, a higher initial molybdate concentration is necessary in the former case than in the latter. In agreement with the discussion above, it appears that increasing molybdate concentration in the coagulating solutions leads to a decrease in the fraction of molybdate strongly bound to the polymer network (Figure 3a). The total molybdate content per bead can be related to the speciation of molybdate and the predominance of polyoxoanions (Figure 3b). Experiments performed with lower molybdate concentrations in the coagulating solution (1 and 3 g‚L-1) preclude any coagulation of chitosan. This

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Figure 4. Variation of the total metal uptake (4, qeq in mg of Mo/ bead), and the diameter of the beads (b, in mm.) as a function of time, for a molybdate concentration in the coagulation bath of 7 g‚L-1 and pHi ) 6.

behavior confirms the necessity of a critical concentration of cross-linking agents for gelation to take place.24 On the other hand, with 20 g‚L-1 Mo, spherical beads can be obtained, but for the same reasons as discussed in the above paragraph, they progressively lose their shape, more rapidly than in the case of 7 g‚L-1 Mo at pH 5 and 5.5. The shrinking step also leads to the formation of additional cross-links that stabilize the shape achieved at the end of the depletion.24 Coagulation Kinetics. Gelation kinetics were studied from the variations of both the total molybdate uptake and the diameter of the beads. Figure 4 shows a typical kinetic curve obtained under optimal conditions for coagulation (pH 6, concentration of Mo ) 7 g‚L-1). The gelation kinetics are relatively fast. Two hours are sufficient to reach a quasi equilibrium corresponding to 95% of the total amount of molybdate immobilized on the beads. Beyond this time, the metal uptake continues at a much lower rate for 14 h. After that, it remains constant with a maximum level of 0.355 mg of Mo per bead. The diameter of the beads also becomes constant at this time, reaching 2.44 ( 0.07, as compared with 2.89 mm at the beginning of the experiment. It is noteworthy that molybdate transfer to chitosan gel beads is faster in this case than that observed during sorption of molybdate on chitosan gel beads coagulated in sodium hydroxide.9 In the latter case, 48 h of contact are necessary to reach equilibrium. This difference can be attributed to a difference in the interaction mechanism, related to differences in the stoichiometry between Mo and -NH3+ groups. Another possible factor is the enhanced diffusion of molybdate in the solution inside the gelling bead. The coagulation could proceed stepwise. Thus, during the first seconds of contact, very thin gel layers are formed at the periphery of the beads, although the center remains liquid. Then, rapidly, molybdate anions diffuse inside the bead, the solution is progressively gelled, and the beads become opaque to form white beads after 1 h. Several models were used for the description of sorption kinetics, especially the shrinking core model which, for example, was used for the modeling of copper sorption on alginate gel beads.26 However, our results do not fit the hypotheses of this model. This is related to the fact that, in our case, gelation occurs in the viscous solution, so the beads

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Figure 5. Influence of pHi on the gelation of chitosan with molybdate for different sources of chitosan (ABER 90 (0); NIG 2332 (]); IND 342 (O); closed symbols (and solid lines): total molybdate loading on the bead, open symbols (and dashed lines): fraction of molybdate strongly bound to the polymer network (%).

are not formed before contact with metal ions and their diameter varies during metal uptake. The decrease in the diameter of the beads can reach 15%, which corresponds to a decrease in volume of 50%. This is consistent with the discussions above and agrees with the observations of Martinsen et al. concerning alginate coagulation in calcium chloride solutions.27 They observed a reduction in the volume of the beads up to 50%, increasing with the concentration of calcium in the coagulation bath. Vorlop and Klein also observed a strong decrease in the diameter of chitosan beads gelled with sodium tripolyphosphate.16 When the beads are prepared at pHi 6 and 7, the shrinking is increased by the presence of phosphoric acid. The diameter is reduced from 2.89 mm to 2.25 mm (volume decrease: 53% compared to the volume of the drops at the outlet of the nozzle). This reduction in size can be explained by the formation of complementary linkages (complexation) between phosphate ions and molybdate moieties on the beads. This new shrinking is nonreversible. Indeed, a treatment with sodium hydroxide has no influence on the swelling of the beads. On the contrary, for pHi’s 8 and 9, due to lower molybdate content, the diameter of the beads increases. This is due to a weaker cross-linking at these pHi’s, and to the acidic treatment, which leads to a considerable decrease in the molybdate uptake. Change in the Gelation of Chitosan with Molybdate with Different Chitosan Samples. The preparation of chitosan beads by molybdate coagulation was tested on chitosans of different origins under the established optimal conditions (molybdate concentration: 7 g‚L-1 and various pHi’s). Figure 5 shows that the behaviors of two samples (ABER 90 and NIG 2332) are similar, concerning the total amount of molybdate in the polymer network, while the other chitosan (IND 342) gives a slightly lower molybdate sorption. ABER 90 and NIG 2332 gel beads have a similar chitosan mass per bead (0.470-0.480 mg, and 0.420-0.470 mg, respectively) while in the case of IND 342, it is 0.3600.395 mg (determined after Mo desorption). Chitosan mass in dried beads and molybdate content vary in the same way. Conversely, the comparison of molybdate uptake (mg of

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Figure 7. SEM microphotographs of chitosan gel beads prepared by sodium hydroxide coagulation: details of surface (left) and internal part (right) of the beads, for ABER 90 chitosan gel beads.

Figure 6. X-ray diffraction patterns of chitosan gel beads (air-dried and crushed) after different treatments.

molybdate/g of dry gelled material, not shown) demonstrates that the gelled materials have similar molybdate content (amount of molybdate/g of chitosan). If we consider the role of pH in Figure 5, we observe a similar trend whatever the chitosan sample with a fraction of molybdate strongly bound to the bead around 80%. The total amount of molybdate remains constant up to pHi ) 6 then decreases continuously. The pHf variations (not shown) reveal that whatever the chitosan source, pHf does not vary during the coagulation up to pHi ) 6, while, above pHi ) 6, pHf is strongly decreased (especially between pHi ) 6 and 8). The differences between the gelled beads can be attributed to differences in the molecular weight and purity of the materials. IND 342 is characterized by a higher degree of insoluble matter, and the solutions are more turbid than those obtained with ABER 90 and NIG 2332. Though a more complete study should be necessary to measure the influence of chitosan characteristics on the gelation mechanism, these preliminary results show that gelation is strongly influenced by the properties of the biopolymer. Characterization of Chitosan Gel Beads by X-ray Diffraction. Figure 6 gives the X-ray diffraction patterns obtained (after air-drying and crushing) for a raw chitosan (ABER 90), and for beads prepared from different ways: in a sodium hydroxide solution (BCNaOH); in a sodium hydroxide solution, then mixed with molybdate (BCNaO-

HMo); in a molybdate solution (BCMo), then desorbed with a sodium hydroxide solution (BCMoNaOH). While two wellresolved peaks characterize the diffraction pattern obtained with the raw material at 2θ ) 10° and 2θ ) 19.2° and a shoulder close to 2θ ) 21°, after dissolution and coagulation in sodium hydroxide, the peaks appear at 2θ ) 20° and 2θ ) 22.3°. The peak at 2θ ) 10° is much reduced, to a small shoulder. However, in the latter case, the crystallinity index is reduced with values of 40% and 30% for ABER 90 and BCNaOH, respectively. In the case of BCMoNaOH, although the small peak at 2θ ) 22.3° disappears, the crystallinity index remains close to 30%. More significant are the differences between the diffraction patterns of the samples containing molybdate (BCNaOHMo and BCMo). The presence of molybdate results in a marked decrease in the crystallinity and the presence of a unique peak at 2θ ) 8-8.2°. This change may be explained by the strong absorption of X-rays by molybdate (absorption coefficient higher than that of oxygen and carbon, the major elements in chitosan). This is confirmed by the reappearance of the crystalline structure when the samples brought into contact with molybdate are treated with 1 M sodium hydroxide (BCMoNaOH); the desorption of molybdate causes the original structure to reappear, though the peak at 2θ ) 22.3° is still absent. Thus, coagulation of chitosan with molybdate leads to an irreversible change in the crystalline structure of the polymer. The decrease in the crystallinity of the sorbent follows the variation: raw chitosan > chitosan dissolved and precipitated in NaOH > chitosan coagulated in molybdate solution and desorbed. This may be correlated to the SEM observations showing differences in the structure of the different materials. Characterization of Chitosan Gel Beads by Scanning Electron Microscopy. Preliminary experiments were performed on air-dried gel beads; however, their structure collapses during dehydration and it is impossible, even at high magnification, to observe the pore structure either in the bulk or at their surface. The beads were therefore freezedried. Figure 7 shows the structure of beads prepared in sodium hydroxide solutions with ABER 90. The former has an expanded structure with large internal pores, close to 100 µm in average size. A coagulation layer with pores smaller than 1 µm is observed on the surface of the beads. The thickness is about 30 µm. After their contact with the molybdate solution, the beads (BCNaOHMo) are also characterized by high internal porosity (Figure 8a), though the superficial layer does not appear (not shown). Therefore, the formation of the coagulation skin is closely related to

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Figure 8. (a) SEM microphotograph of a chitosan gel bead prepared by sodium hydroxide coagulation, then subjected to a molybdate solution (adsorption process): details of internal part of the bead. (b) SEM microphotograph of chitosan gel beads prepared by molybdate coagulation (ABER 90 chitosan).

Figure 9. SEM micrographs of chitosan gel beads prepared by molybdate coagulation, and subjected to an alkaline desorption (NaOH): details of surface (left) and internal part of the beads (right) for ABER 90 chitosan gel beads.

the molybdate coagulation mechanism (Figure 8b). Figure 8b shows the structure of the beads prepared by coagulation in a concentrated molybdate solution (pH 6, Mo ) 7 g‚L-1). The internal structure is very dense compared to the material coagulated in sodium hydroxide solutions. The size of the pores does not exceed 10 µm, and the thickness of the external wall is 100 µm. Figure 9 shows the structure of chitosan beads (ABER 90) prepared by molybdate gelation then treated with sodium hydroxide solutions to remove molybdate from the material. Scanning electron microscopy coupled with an X-ray dispersive energy analyzer demonstrates that molybdate is completely removed from both the external layers and the bulk of the beads (not shown). The external layer is as large as seen on BCMo, and no superficial porosity is detected. In the internal part of the beads, large holes were observed (diameter of 100 µm) which do not appear on BCMo. Nevertheless, the internal structure is more compact than in the beads directly produced by coagulation in sodium

Dambies et al.

hydroxide. These data are consistent with previous experiments on BCNaOH using size exclusion chromatography.22 This study shows that the internal structure of the material is made up of pores which range in size between 70 and 90 nm. Shinonaga et al. observed that the size of the pore in chitosan gel beads depends on the concentration of the sodium hydroxide solution.28 However, their experiments were performed on beads cross-linked with ethylene glycol diglycidyl ether. They found that the thickness of the external layer was 100 µm, while the diameter of the pores ranged between 50 and 100 µm. Kawamura et al. also obtained pore sizes of 100 µm with chitosan beads coagulated in sodium hydroxide solutions.4 The structure of the gel is significantly different to that obtained in the case of the wall-to-wall gelation,18 in which the material appears perfectly homogeneous. As a complementary evidence of the difference in the gelation mechanism between the homogeneous and the heterogeneous gelation techniques, it may be interesting to observe that a chitosan membrane cast on a glass surface, dried, and peeled exhibited a complex structure when treated with molybdate solutions (under similar experimental conditions: pH, molybdate concentration): a double solid layer (at each side) embedding a less dense/viscous layer appeared (not shown). Molybdate/chitosan interactions are controlled by metal speciation, and require the predominance of polynuclear anionic species. Heptamolybdate species have a high anionic charge, which enables polyoxyanions to react with protonated amine groups of the same and/or different chains. As mentioned above, the presence of a great number of oxygen atoms and one OH group also allows the formation of additional hydrogen bonds. This multiple reaction strengthens the structure of the polymer beads obtained by coagulation in molybdate solutions. A fast reaction occurs with the polymer first at the periphery of the bead leading to the formation of a strong and dense external layer with a low porosity, responsible for the shrinking of the particles and a considerable decrease in the concentration of polymer in the inner part. In a second stage, the diffusion of molybdate ions leads to a progressive coagulation of the inner part, with its lower polymer concentration and contributes to the formation of a small pore size internal structure. Conclusion Molybdate ions are very efficient gelling agents for chitosan. The stability of the gel beads obtained by this process depends on several parameters such as pH and concentration of the coagulating solution. Optimum conditions for the production of chitosan gel beads with a selected chitosan sample (FA, 0.13; Mw, 191000) are pH 6 and molybdate concentration of 7 g‚L-1. These conditions correspond to the predominance of molybdate polyoxoanions (polynuclear hydrolyzed species), identified as the most favorable molybdate species for both adsorption on chitosan and chitosan coagulation. Different interaction mechanisms are possible for these reactions since (a) the molar ratio between Mo and -NH3+ differs with the process; (b) the maximum molybdate uptake is lower for the coagulation process than for the sorption technique (except when a very

Preparation of Chitosan Gel Beads

high molybdate concentration is used (20 g‚L-1)); (c) the fraction of molybdate released from the gel beads (when treated with phosphoric acid) is significantly higher for beads saturated by sorption than for beads directly coagulated in a molybdate solution. These differences are confirmed by SEM observations. While freeze-dried beads produced by sodium hydroxide coagulation are characterized by a large openpore structure (with a thin external layer), in the case of molybdate coagulated gels, the structure is heterogeneous. The external layer is relatively large (100 µm) with no apparent pores, and the internal part of the beads is characterized by a more compact structure. This heterogeneity confirms the difference in the gelation mechanism between alkaline coagulation and ionotropic gelation but also between the heterogeneous and the homogeneous gelation techniques. Molybdate desorption (using sodium hydroxide solution) does not change the mechanism and the structure remains compact. It is interesting to observe that the treatment of molybdate impregnated chitosan beads (resulting from molybdate sorption on sodium hydroxide coagulated beads and from molybdate coagulated gel beads) with phosphoric acid removes the labile part of molybdate. This part is significantly more important for the “sorption” compared to the “coagulation” technique. These molybdate impregnated chitosan beads (MICB) have been successfully used for arsenic sorption.29 An increase in both the stability and the efficiency for As removal is observed for MICB produced by the coagulation technique.30 References and Notes (1) Roberts, G. A. F.; Wood, F. A. In AdVances in Chitin Science; Peter, M. G., Domard, A., Muzzarelli, R. A. A., Eds.; Universita¨t Potsdam: Potsdam, Germany, 2000; Vol. IV, pp 34-39. (2) Miyoshi, H.; Shimura, K.; Watanabe, K.; Onodera, K. Biosci. Biotechnol. Biochem. 1992, 56, 1901-1905. (3) Roberts, G. A. F. Chitin Chemistry; MacMillan: London, U.K., 1992; p 350. (4) Kawamura, Y.; Mitsuhashi, M.; Tanibe, H. Ind. Eng. Chem. Res. 1993, 32, 386-391. (5) Inoue, K.; Baba, Y.; Yoshizuka, K. Bull. Chem. Soc. Jpn. 1993, 66, 2915-2921.

Biomacromolecules, Vol. 2, No. 4, 2001 1205 (6) Guibal, E.; Jansson Charrier, M.; Roussy, J.; Le Cloirec, P. Langmuir 1995, 11, 591-598. (7) Piron, E.; Accominotti, M.; Domard, A. Langmuir 1997, 13, 16531658. (8) Rorrer, G. L.; Hsien, T.-Y.; Way, J. D. Ind. Eng. Chem. Res. 1993, 32, 2170-2178. (9) Guibal, E.; Milot, C.; Tobin, J. M. Ind. Eng. Chem. Res. 1998, 37, 1454-1463. (10) Fuji Spinning Co., Ltd., Process for Producing Granular Porous Chitosan, U.S. Patent 4 833 237, 1989-23-05, 1989. (11) Koyama, Y.; Taniguchi, A. J. Appl. Polym. Sci. 1986, 31, 19511954. (12) Roberts, G. A. F.; Taylor, K. E. Makromol. Chem. 1989, 190, 951960. (13) Crescenzi, V.; Paradossi, G.; Desideri, P.; Dentini, M.; Cavalieri, F.; Amici, E.; Lisi, R. Polym. Gels Network 1997, 3, 225-239. (14) Tual, C.; Espuche, E.; Escoubes, M.; Domard, A. J. Polym. Sci., B: Polym. Phys. 2000, 1521-1529. (15) Vorlop, K.-D.; Klein, J. Biotechnol. Lett. 1981, 3 (1), 9-14. (16) Lee, S.-T.; Mi, F.-L.; Shen, Y.-J.; Shyu, S.-S. Polymer 2001, 42, 1879-1892. (17) Brack, H. P.; Tirmizi, S. A.; Risen, W. M., Jr. Polymer 1997, 38, 2351-2362. (18) Draget, K. I.; Va˚rum, K. M.; Moen, E.; Gynnild H.; Smidsrød, O. Biomaterials 1992, 13, 635-638. (19) Guibal, E.; Vincent, T. SolVent Extr. Ion Exch. 2000, 18, 12411260. (20) Vincent, T.; Guibal, E. Ind. Eng. Chem. Res. 2001, 40, 1406-1411. (21) Guibal, E.; Milot, C.; Eterradossi, O.; Gauffier, C.; Domard, A. Int. J. Biol. Macromol. 1999, 24, 49-59. (22) Milot, C. Adsorption de Molybdate sur Billes de Gel de Chitosane. Application au Traitement d’Effluents Molybdife`res. Ph.D. Thesis. Universite´ de Montpellier II, 1998; 214 p. (23) Petersen, J. N.; Davison, B. H.; Scott, C. D.; Blankenship S. Biotechnol. Technol. 1990, 4, 435-440. (24) Domard, A.; Vachoud, L. Chitosan-Based Physical Gels, 8th International Chitin & Chitosan Conference, Yamaguchi, Japan, to be published in AdV. Chitin Sci., 2000. (25) Guibal, E.; Milot, C.; Roussy, J. Sep. Sci. Technol. 2000, 35, 1021. (26) Chen, D.; Lewandowski, Z.; Roe, Z. Biotechnol. Bioeng. 1993, 41, 755-760. (27) Martinsen, A.; Skjak-Braek, G.; Smidsrød, O. Biotechnol. Bioeng. 1989, 33, 79-89. (28) Shinonaga, M.-A.; Kawamura, Y.; Yamane T. J. Ferment. Bioeng. 1992, 74 (2), 90-94. (29) Dambies, L.; Roze, A.; Guibal, E. Colloids Surf., A: Physicochem. Eng. Asp. 2000, 170, 19-31. (30) Dambies, L.; Vincent, T.; Guibal, E. Arsenic Sorption on Specially Tailored Derivatives of Chitosan, submitted for publication in Water Res. 2001.

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