Physicochemical Properties of Physical Chitin Hydrogels: Modeling

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Biomacromolecules 2001, 2, 1294-1300

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Physicochemical Properties of Physical Chitin Hydrogels: Modeling and Relation with the Mechanical Properties Laurent Vachoud† and Alain Domard* Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux (UMR-CNRS 5627), ISTIL, Universite´ Claude Bernard, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France Received July 20, 2001

In this work, we were interested in the modeling of syneresis of physical chitin hydrogels by a mathematic law allowing us to predict the variation of the weight of the gel as a function of time. The variation of the weight of the gel during syneresis can be described by Wt/W0 ) (t1/2 + (W∞/W0)t)/(t1/2) + t) where W0, W∞, and Wt are the weights of the gel at the beginning of syneresis, for infinite time and for a time t, respectively. t1/2 corresponds to the half-time of syneresis. W∞/W0 and t1/2 were studied in relation with several parameters such as the ionic strength, pH, degree of acetylation of chitin and the initial concentration of polymer. The mechanical properties of chitin hydrogels maintained during syneresis in media of different pH’s and ionic strengths were also investigated. Introduction Chitin and chitosan are copolymers of β, (1f4) linked, 2-amino-2-deoxy-D-glucan and 2-acetamido-2-deoxy-D-glucan. Chitosan is usually prepared from N-deacetylation of chitin, a naturally occurring polysaccharide, largely widespread in biomass. The term chitosan is attributed to polymers of the series, soluble in dilute acidic media and then to those with a degree of acetylation (DA) below 60%. On the contrary, over 60%, we are in the presence of chitin. Chitin and chitosan can exist under the three physical forms corresponding to solutions, hydrogels and solids. In the gel state, chitin can find many applications in the fields of cosmetics, wound healing, wastewater treatment, sustained drug delivery systems, etc.... The formation of a chitin-based hydrogel in the course of acetylation of chitosan in an hydroalcoholic medium was described for the first time by Hirano and co-workers.1-5 The gelation mechanism has been extensively studied, especially the roles played by the nature of the solvent2 and cosolvent6,8 used, the chitin content,6 and the temperature.6,8 In a first paper on the formation of chitin gels, we proposed a relation between the stoichiometry of the reaction (molar ratio between acetic anhydride and glucosamine residues) and the acetylation degree of the network.8 More recently, we were interested in the study of the physicochemical properties of these gels, especially in the syneresis and the swelling/deswelling of the chitin network in different solvents.9 Syneresis is generated by a thermodynamic imbalance between the polymer (chitin) and the solvent created from the gel point. It is also related to a kinetic relaxation of the chain segments between two points * Author for correspondence. Telephone: 33-472-44-87-85. Fax: 33472-43-12-49. E-mail: [email protected]. † Present address: Laboratoire de Physique Mole ´ culaire et Structurale (UMR-CNRS 5094), Faculte´ de Pharmacie, 15, avenue Charles Flahault, 34060 Montpellier Cedex 2, France.

of cross-links. It leads to a progressive decrease of the gel volume in relation with a release of solvent. The weight of the gel decreases rapidly during the first 100 h following the gel point and then tends toward a plateau after approximately 200 h. The weight of the gel at the plateau corresponds to a situation in which an equilibrium between the various components is achieved. It was shown that different experimental parameters, corresponding to pH and ionic strength of the media in which the gel undergoes syneresis, temperature, chitin concentration, acetylation degree of the network, etc.,9 allow the regulation of the balance between attractive and repulsive interactions (syneresis/swelling) and, as a consequence, the control of the weight of the gel at the plateau. For various situations in which these gels can be involved, it is very important to predict with accuracy the syneresis. This is the reason that the first part of this paper is devoted to the modeling of experimental data of the weight loss of gel during syneresis. This modeling was performed by means of experimental results obtained in our previous paper9 and a theoretical law allowing us to predict the variation of the weight of the gel for long syneresis times (>200 h). Mechanical properties of a physical gel are among the most important characteristics. However, concerning chitin gels, no detailed study dealing with these properties is available in the literature. In this paper, the mechanical properties of the gel were investigated by means of the measurement of the storage modulus G′. This parameter was studied as a function of the gel weight at the plateau of syneresis, in relation with the variation of the different experimental parameters mentioned above. Materials The chitosan sample used to prepare the gels was from Aber Technologies (Plouguerneau, France, batch no.A32E03).

10.1021/bm0155874 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/01/2001

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The initial degree of acetylation (before reacetylation), determined by 1H NMR and DPw, obtained by SECMALLS were 2.4% and 810, respectively. Gel Formation. An aqueous/acetic acid (0.5% w/w) solution of chitosan (1% w/w) was successively filtered on Millipore membranes of porosity 3-0.22 µm. Then 100 g of this solution was mixed with propanediol (80 mL), and the mixture was left to stand overnight for degazing without stirring. A freshly prepared acetylating solution (20 mL of propanediol with the desired amount of acetic anhydride) was slowly added to the hydroalcoholic solution of chitosan. The mixture was then stirred during 30 s and transferred into a cylindrical mold. Modeling of Experimental Results. For the modeling of experimental data of syneresis,9 only t1/2 and W∞ were the variable parameters. The software used was KALEIDAGRAPH. It gives the values of t1/2 and W∞ for which the theoretical equation provides the best fitting with the experimental data. Vt, the rate of syneresis, is calculated from t1/2 and W∞ (see eq 7). The experimental details for the measurements of syneresis are mentioned in a previous paper.9 Mechanical Properties. Dynamic mechanical measurements were performed at room temperature on a RHEOMETRIX RMS 800 rheometer, with the parallel plate geometry, over a frequency range of 10-2-102 Hz. The diameter of the plates was 13 mm and the gap between the plates ranged from 1 to 2 mm. The strain applied was less than 5% in order to avoid the plastic behavior. Results and Discussion

(1)

and an exponential equation11 Wt ) (1 - e-kt) W∞

(2)

(3)

in the case of agar gels. In these equations, Wt is the weight of released solvent for a time t and W∞ is the weight of the released solvent for an infinite time. In the case of chitin gels, eq 4 gives the best fit with the experimental values (Figure 1). W∞ t Wt W0 ) W0 k+t

Wk ) W0

W∞ W∞ k 1+ W0 W0 ) 2k 2

k+

and (W0 + W∞) 2

(5)

As a consequence, k corresponds to the time for which the gel weight achieves the average value between the initial weight of the gel and that for an infinite time of syneresis. Therefore, k is the half-time of syneresis. We will note this time as t1/2. Finally, eq 4 becomes: W∞ t W0 Wt ) W0 t1/2 + t t1/2 +

were proposed to simulate the whey syneresis whereas Nagasaka and Taneya12 used the following relation Wt kt ) W∞ 1 + kt

0) and W∞, the gel weight at infinite time (when the thermodynamic equilibrium between the polymer and the solvent is achieved). Wt is the gel weight for a time t and k is a constant. The dimension of the parameter k involved in eq 4 is necessarily that of a time. If t ) k, then eq 4 becomes

Wk )

1. Modeling of Syneresis. Several mathematic laws describing the syneresis of various gels were published in the literature. Among them, a polynomial equation10 Wt ) a +btc W∞

Figure 1. Fitting of the evolution of the gel weight with time (in the solvent of gelation) for two different DA values: 92% (4) and 98% (O).

k+

(4)

with W0, the gel weight at the beginning of syneresis (t )

(6)

This equation is similar to that proposed by Nagasaka and Taneya12 for agar gels (eq 3). The only difference is that relation 6 describes the gel weight and not the weight of the released solvent. The use of eq 6 implies that the weight of the gel at the beginning of syneresis (W0) is known. Since syneresis is due to a thermodynamic imbalance between the polymer network and the solvent at the gel point, it allows the gel to reach a more stable state than initially. The use of eq 6 to fit our experimental data illustrates quite well this assumption because this theoretical law implies that the weight of the gel tends toward a value W∞ different from 0 (representing the thermodynamic equilibrium between the network and the solvent). If t1/2 ) 0 and t * 0, then Wt ) W∞, and whatever t may be, the gel weight Wt is equal to W∞, the weight of the gel corresponding to the thermodynamic equilibrium. Thus, in this case, syneresis is instantaneous. On the other hand, if

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Table 1. Role of DA on the Parameters Calculated from the Modeling of Experimental Curves Obtained in Ref 9a DA (%)

t1/2 (h)

W∞/W0 (%)

V0 (% h-1)

R

92 98

29.1 8.4

34.7 31.4

-2.2 -8.2

0.999 95 0.999 91

a t , half-time of syneresis; W , weight of the gel at the gel point; W , 1/2 0 ∞ weight of the gel at infinite time; V0, rate of weight loss for t ) 0; R, correlation coefficient.

t1/2 tends toward ∞, Wt ) W0 and then the gel weight is equal to the initial weight (W0). In this case, syneresis does not occur. The derivation of eq 6 leads to the rate of weight loss as a function of time:

[

t1/2

Vt )

W∞ -1 W0

1 dWt ) W0 dt [t1/2 + t]2

]

(7)

Table 2. Role of Chitin Concentration (Cchitin) at the Gel Point on the Parameters Calculated from the Modeling of Experimental Curves Obtained in Ref 9a

For t ) 0, the rate of weight loss is equal to W∞ -1 W0 V0 ) t1/2

Figure 2. Variation of the rate of weight loss with time (Vt) for two DA values: 92% (b) and 98% (9) (chitin concentration in the gel: 0.6%). Vt is calculated from eq 7.

(8)

It signifies that the rate of the weight loss at the beginning of syneresis depends on two parameters: the weight loss at infinite syneresis W∞/W0 and at half-syneresis time. In a recent paper, we published experimental data on the syneresis of chitin gels.9 Syneresis was measured in the solvent of formation of the gel or in a different media. We proposed to fit the experimental data obtained in every cases. Modeling of Syneresis in the Media of Gelation (Hydroalcoholic Mixture). Role of the Chemical Structure of the Network (DA). When we operate the modeling of the syneresis in the solvent of gelation, we consider the time t ) 0 as the moment where the acetylating mixture is added to the media. As a consequence, W0 can be considered as the weight of the gel at the gel point. The variations of the weight loss of chitin gels with DA ) 98 and DA ) 92% as a function of time are represented in Figure 1. Experimental values are represented by symbols and the solid lines correspond to the mathematical modeling according to eq 6. One can notice a very good agreement between the experimental and modeled results with coefficients of correlation close to 1 (see Table 1). The values of t1/2, W∞/W0, and V0 obtained for the two kinds of DA’s are reported in Table 1. In the latter case, the negative sign is related to a weight loss. V0 is calculated from t1/2 and W∞/W0 (see eq 8). As gelation, syneresis is a thermodynamic process which depends on the balance between attractive and repulsive interactions between polymer chain segments. |V0| is higher for the higher value of DA (98%), in relation with attractive interactions more important for this kind of DA. Then, the affinity of the polymer for the solvent is lower.9 From eq 7, it is possible to calculate |Vt| the rate of weight loss as a function of time. In fact, |Vt| exhibits two behaviors (Figure 2). In the first part, for times ranging between 0 and 17 h, syneresis is more

Cchitin (%)

t1/2 (h)

W∞/W0 (%)

V0 (% h-1)

R

0.24 0.35 0.47 0.59 0.81 1.13 1.63 2.15 2.46

21.9 12.4 11.0 10.3 12.0 10.9 8.1 8.9 8.6

20.1 24.8 26.1 27.3 29.8 33.4 39.4 41.7 43.8

-3.7 -6.05 -6.7 -7.1 -5.8 -6.1 -7.4 -6.5 -6.5

0.999 92 0.999 95 0.999 95 0.999 91 0.999 94 0.999 98 0.999 97 0.999 91 0.999 97

a DA ) 98% (t , half-time of syneresis; W , weight of the gel at the 1/2 0 gel point; W∞, the weight of the gel at infinite time; V0, rate of weight loss for t ) 0; R, correlation coefficient). Modeling with 10 or 11 points on a range of weight loss close to 5 g.

rapid for higher DA’s. In the second part (from 17 h to infinite time), the tendency is reversed. It can be concluded that the chitin gel with the higher DA tends more rapidly to the thermodynamic equilibrium W∞. Role of the Polymer Concentration. We also modeled the experimental curve of syneresis of gels made with different concentrations.9 The values of t1/2, W∞, and |V0| obtained for each concentration of chitin were reported in Table 2. They allow us to evidence several observations. |V0| increases on increasing chitin concentration whereas t1/2 decreases (Table 2). Moreover, as for the influence of DA, the role of the concentration on the rate |Vt| varies with time (Figure 3). The gelation time decreases on increasing the polymer concentration6 in relation with an increase of the probability to create more polymer/polymer interactions. As a consequence |Vt| increases with chitin concentration for t values below 6 h (Figure 3). After 6 h, the reverse situation is observed, and then the thermodynamic equilibrium, W∞, is achieved more rapidly when polymer concentration increases. This is confirmed from the comparison between the experimental values of syneresis (measured after a storage of 200 h) and theoretical values at equilibrium obtained by modeling (see Figure 4). Modeling of Syneresis in an Excess of Different Solvents. In a recent paper,9 we were interested in the study

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Table 3. Role of Ionic Strength (IS) on the Parameters Calculated from the Modeling of Experimental Curves Obtained in ref 9a IS (M) 10-6 0.01 0.02 0.04 0.1 10-6 0.02 0.1

Figure 3. Variation of the rate of weight loss with time (Vt) for two chitin concentrations: 0.24% (b) and 2.46% (9) (DA of the gel: 98%). Vt is calculated from eq 7.

Figure 4. Variation of syneresis as a function of chitin concentration: syneresis measured 200 h after the gel point, W200h/W0 (2) and syneresis at infinite time (thermodynamic equilibrium), W∞/W0 (9), calculated from the fitting of experimental curves.

of the change of the weight of the gel when, after gelation, it is immediately placed in an environment different from the medium of acetylation, and we studied the gel behavior in an excess of various solvents. Because the chitin network bears (-NH3+) functions with (CH3COO-) ions as counterions, the role of various parameters on syneresis such as pH and ionic strength were evaluated.9 Then, it seemed also interesting to model these experimental data, but it was necessary to take into account a few points before interpretation of the results. The values of W∞ and t1/2 are strongly sensitive to the number of experimental points and to the range of the weights used for the fitting. As a consequence, to obtain a good comparison between the parameters, W∞ and t1/2, the fitting was always carried out on the same number of experimental points and on the same range of weight (mentioned under each table). Moreover, contrarily to the modeling of the syneresis in the solvent of gelation, the origin of times t ) 0 was not kept when the acetylating mixture was added to the hydroalcoholic solution of chitosan but from the introduction of the gel into an excess of the considered media. In these conditions, W0 corresponds to the weight of the gel just before this introduction. This point

t1/2 (h)

W∞/W0 (%)

34.9 11.8 11.95 10.75 10.4

(a) DA ) 46.5 29.8 25.55 22.85 21.0

5.9 6.3 5.6

V0 (% h-1)

R

92%b -1.5 -6.0 -6.3 -7.25 -7.6

(b) DA ) 98%c 32.3 -11.5 30.6 -11.0 28.8 -12.6

0.998 99 0.998 42 0.999 76 0.998 73 0.998 72 0.999 95 0.999 93 0.999 94

a t , half-time of syneresis; W , weight of the gel at the gel point; W , 1/2 0 ∞ weight of the gel at infinite time; V0, rate of weight loss for t ) 0; R, correlation coefficient. b Modeling with five or seven points on a range of weight loss close to 5 g. c Modeling with six or seven points on a range of weight loss close to 2.5 g.

is very important, particularly for the study of the role of the pH operated after an extensive washing of the gel. Indeed, in this case, during the washing, gels lose an important part of their solvent. As a consequence, the theoretical data obtained in this case were not compared with those related to the role of ionic strength, the acetylation degree or the concentration of polymer for which no washing was carried out. The same point can be made for the comparison of the role of pH for a chitin gel with DA ) 92% and a chitin gel with DA ) 98% whose weight losses during washing are different. Role of Ionic Strength. In a previous study,9 the role of ionic strength on syneresis was studied by monitoring the weight of the gel (as a function of time) when it is placed in baths of different KCl concentrations. The values of t1/2, W∞/W0, and V0 obtained by fitting these experimental data for the two kinds of DA′ are reported in Table 3, parts a and b. An increase of the ionic strength leads to a reduction of the Debye screening length of the protonated amino functions but also to a dehydration of the polymer network thanks to the salting out effect produced by KCl.13,14 All these parameters favor the exclusion of the solvent and tend to increase |V0| (Table 3, parts a and b). From the comparison of parts a and b of Table 3, we notice that t1/2 decreases and |V0| increases on increasing the acetylation degree as observed above. However, this behavior is less pronounced for the highest values of KCl concentrations. It must be related to the fact that the apparent charge density of the network becomes equivalent for the two values of DA when salt concentration increases (to be published). As previously described,9 the behavior of the chitin gel with the lowest DA is more influenced by ionic strength. Nevertheless, the value of |V0| remains relatively constant for ionic strengths above 0.01 M. Role of pH. The modeling of experimental data9 allows us to evidence several behaviors. As shown in tables 4a and b, the value of W∞ is the highest for pH 4. Indeed, at this pH, for a given DA, the apparent charge density of the chitin segments and, then, the electrostatic repulsions are maximum. The free amino groups present on the polymer chains are at their maximum protonation, in a medium of relatively low ionic strength. As a consequence, the solvation of these ionic

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Table 4. Role of pH on the Parameters Calculated from the Modeling of Experimental Curves Obtained in Ref 9a pH

t1/2 (h)

W∞ (g)

V0 (% h-1)

R

(a) DA ) 34.5 66.5 26.45

-7.1 -1.05 -5.6

0.999 92 0.993 91 0.988 74

(b) DA ) 98% 82.3 71.8

-0.6 -1.35

0.9975 0.9993

W∞/W0 (%)

Table 5. Solubility of Lyophilized Chitin Gels in DMAc + LiCl (5%) As a Function of Time and As a Function of the Degree of Acetylation, at Room Temperature (Concentration Polymer/ Solvent: 1%(w/w))

92%b

2 4 10

9.0 31.95 13.3

6.0 11.5 4.0

4 10

30.1 21.0

6.4 5.6

a t , half-time of syneresis; W , the weight of the gel at the gel point; 1/2 0 W∞, the weight of the gel at infinite time; V0, rate of weight loss for t ) 0; R, correlation coefficient. b Modeling with 10 or 12 points on a range of weight loss close to 6.25 g. The pH of the bath was adjusted by means of either dilute NaOH or HCl, if necessary. c Modeling with six or seven points on a range of weight loss close to 2.5 g. The pH of the bath was adjusted by means of either dilute NaOH or dilute HCl, if necessary.

groups are maximum thus contributing to a maximum swelling. t1/2 decreases and |V0| increases when pH increases from 4 to 10, in relation with the deprotonation of the amino functions, but these variations are more important for the lowest DA due to the presence of a greater number of glucosamine residues (Table 4, parts a and b). At pH ) 2, the ionic strength of the media, due to the ammonium groups and their counterions, induces a reduction of the Debye screening length and then decreases the apparent charge density of the network.9 This reduction of the charge density leads to a more important and faster depletion compared to that observed at pH 4 (Table 4a). The depletion of the gel observed at pH 10 should be accompanied by a reorganization of the chain segments between two points of reticulation. This phenomenon is slower than the simple chain shrinking observed at pH 2, which is consecutive to the increase of the ionic strength. This certainly explains why the exclusion of the solvent observed at pH 2 is more rapid than at pH 10 (Table 4a). 2. Relation between Physicochemical and Mechanical Properties of the Gels. The gels are not soluble in the hydroalcoholic media. After a washing in water and a full drying, they behave as chitin in the solid state and then become soluble in the classical solvents of this polymer such as DMAc + 5% LiCl or NMP + 7% LiCl.15,16 This solubility must be related to the fact that the interactions responsible for gelation are physical interactions such as hydrogen bonding or hydrophobic interactions. As shown in Table 5, in agreement with previous results,9 the solubility characterized by the kinetics of solubilization increases on increasing DA. It is interesting to notice that a solution of chitin obtained in our conditions with a DA close to 98% gives rise to a gelation on contact with the hydroalcoholic mixture. The same result can be achieved with chitin obtained directly from deacetylation of chitosan. However, in this case, for the same polymer concentration, the gel is more rigid, certainly because chitin obtained from deacetylation of chitosan has a much higher molecular weight. As a consequence, the gel can be considered as indirectly solvoreversible. Nevertheless, it can be directly dissolved after several exchanges in a solvent of chitin; a method much more efficient for higher DA’s. Thus, the elimination of water becomes just as important as DA increases.

solubility

R

DA (%)

6 days

13 days

36 days

0.4 0.8 1 1.45 2 6 10 15

39 55 69 79 88 99.5 97.5 96

( ( + + +

( ( ( ( ( + + +

( ( ( ( + + + +

a R is the molar ratio anhydride/glucosamine residues. Key: (-) insoluble sample; (+) soluble sample.

Figure 5. Variation of the storage modulus ([) and dissipation modulus (9) as a function of the solicitation frequency (ω) for a gel stored for 200 h in 200 mL of deionized water (pH 4, DA ) 92%).

These chitin gels are thermostable even at 150 °C, in agreement with the results reported by Hirano et al.3 An increase of temperature increases the entropic parameter which favors the chain relaxation. It also reinforces the formation of hydrophobic interactions and other low energy interactions responsible for a densification of the cross-links and not of a dissolution or a melting. This result is very interesting since it allows an easy sterilization on heating the material with no important change of its properties, contrary to other classical methods which contribute either to reduce the molecular weight of the polymer (γ-irradiation) or to introduce highly toxic products (ethylene oxide). The physical cross-links between the polymer chains were also detected on Figure 5 which represents the evolution of the storage (G′) and loss moduli (G′′) as a function of the solicitation frequency, for the gel in a highly swelled state. Indeed, an important decrease of the storage modulus G′ was observed for frequency values below 1 rad‚s-1. For these values, the movements of the rheometer are low enough to observe changes in the low energy inter- and intramolecular cross-links and then changes in the physical entanglement between polymer chains. Nevertheless, we notice that the storage modulus is relatively constant over a wide range of frequencies located between 1 and 100 rad‚s-1. For the continuation of the study, the elastic modulus was chosen

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authors have introduced two types of elasticity: the entropic and the enthalpic energy type elasticities. In the case of the entropic energy type elasticity, they conclude that the relation between the elastic modulus E and the polymer concentration C is E ≈ C3ν/(3ν-1)

(10)

where ν - 1 is the fractal dimension between the junctions of the object. In the case of rods, ν ) 1, and eq 10 becomes E ≈ C3/2

Figure 6. Variation of the storage modulus, G′, as a function of chitin concentration (DA ) 98%): ([) experimental points; (s) mathematical law, G′ ) 8673.7C1.96 with C, the chitin concentration. The correlation coefficient R is found to be equal to 0.9979.

as the G′ value detected at the highest frequency studied, i.e., 100 rad‚s-1. After the study of the physical feature of the network, we were interested in the study of the mechanical properties of the gel. In particular, we investigated the shear modulus G′ as a function of the swelling/syneresis of the network. As discussed above, the swelling/syneresis of the gel can be controlled by playing either on molecular parameters (polymer content used for the gel elaboration) or on external parameters (pH and the ionic strength of the media in which the gels were stored). Thus, dynamic mechanical measurements were performed on samples prepared with different chitin concentrations and placed in baths with different pH’s and different ionic strengths. They were conducted on gels which are not so far from the thermodynamic equilibrium (syneresis time largely over 200 h). Role of Molecular Parameters. Role of Chitin Concentration. An increase of the plateau of the storage modulus with the polymer concentration was observed (not shown). This increase must be related to an increase of the crosslinking density of the network. The relation between the shear modulus G′ and the polymer concentration C has been investigated for many other hydrogels of biopolymers.17-19 It is typically described by the following kind of equation G′ ≈ Cn

(9)

with n being an exponent which depends on the conformation of the chain segments between two junction points and then on the gel morphology. For most biopolymer gels such as gelatin,17 agarose,18 or carragenans,19 n was found to be equal to 2. By fitting our experimental data on chitin gels, we observed the same situation in the range of concentrations located within 0.5-3.5%; i.e., n ) 2 (Figure 6). However, it is difficult to conclude about the structure of the gel. Indeed, the exponent 2 can be found for many different textures, such as assemblies of fibers or rigid cellular structures.20 To account for this exponent, another approach consists of the use of Jones and Marques’ relations.21 From the consideration of the concept of fractal dimension, these

(11)

If the elasticity of the gel is of enthalpic origin, the relation between the elastic moduli and the polymer concentration is E ≈ C(3ν+1)/(3ν-1)

(12)

For ν ) 1, relation 12 becomes E ≈ C2, which is consistent with our results if we consider chitin chains as rodlike polymers. Thus, we can conclude that the elasticity of chitin gels is mainly of enthalpic origin in the range of the concentrations studied. In Table 6, values of the shear modulus (G′) of chitin gels (our data) are compared with the shear modulus of other biopolymeric gels (found in the literature). We notice that G′ values of chitin gels are much greater than those of gelatin or other gels of polysaccharides. Among the polysaccharides, chitin gels exhibit the best mechanical properties. They are close to those of carrageenan.19 This can be attributed to the high stiffness of the chitin chains at the DA studied (98%). It is also interesting to point out that G′ values of chitin gels obtained by acetylation of chitosan exceed G′ values of chitosan chemical gels cross-linked with glutaraldehyde.22 The acetylation degree is necessarily lower for chemical gels since covalent cross-links are formed from the consumption of amino functions.22 As a consequence, the stiffness of the chain and the elastic modulus are weaker in the case of chemical gels. This result can also be ascribed to the conformation of the polymer chains which may be different between both types of gels. Role of Chitin Molecular Weight. The other structural parameter leading to a modification of the mechanical properties of the gel is the molecular weight of the polymer, in relation with the critical concentration of chain entanglement (C*). We observed an increase of the rigidity of the gel on increasing the molecular weight of the polymer. For a critical value (DP ) 7) a thixotropic gel was obtained. Role of External Parameters (pH and Ionic Strength). Swelling and, as a consequence, the mechanical properties of chitin gels are strongly influenced by the ionic strength and pH. In the former case, the apparent charge density is modified by the reduction of the Debye screening length13 whereas the latter parameter allows us to regulate the ratio between the charged and uncharged amine groups.27 Table 7 reveals an increase in the elastic modulus on decreasing the swelling defined as the weight of the gel after syneresis divided by the weight of the gel at the gel point. The modulus can be multiplied by a factor ranging between four and five

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Table 6. Storage Moduli G′ (Pa) of Various Hydrogels Based on Biopolymers, at Different Polymer Contents polymer content (%) 0.5 0.87 1 1.22 1.55 1.92 2 2.6 3 3.41

chitin gels

chitosan cross-linked with glutaraldehyde22

gelatin gels23

agar gels26

1666

carrageenan gels19

CMC gels25

pectin gels24

3333

6230 100 and 1000

2333

10 000

13 333

100

13 150 22 550 31 133 5000

50 000

1

1000

57 460 10 000

10 000

50

90 325

Table 7. Shear Modulus G′ Determined at 100 rad‚s-1 under Various Storage Conditions (Baths at Various pH’s and Ionic Strengths) swelling shear ratioa × [Wgel after syneresis/Wgel modulus at the gel G′ (Pa) (ω ) point] × 100 (%) 100 rad/s)

sample

storage conditions

1 2 3 4 5 6

pH4, DA ) 92% [KCl] ) 0M, DA ) 92% pH2, DA ) 92% [KCl] ) 0.04M, DA ) 92% pH 10, DA ) 92% [KCl] ) 0M, DA ) 98%

aCalculated

agarose gels18

69 51.5 33.8 26.9 24.3 28.1

5340 7485 9530 22 550 24 400 31 450

when measuring the shear modulus.

from the highest swelling state (pH 4, sample 1) up to the lowest (pH 10, sample 5). It is interesting to notice that the gel with DA ) 98% exhibits a higher shear modulus even if, under some conditions, its swelling is greater than the gel with DA ) 92% (see sample 6 in Table 7). This result could be ascribed to the hydrophobic interactions formed by the acetylated groups during syneresis which were responsible for associating phenomena between the polymer chains. They were recently evidenced in the case of covalently cross-linked chitosan gels by glutaraldehyde.22 In association with hydrogen bondings, they lead to the precipitation of chitin in aqueous systems when the DA reaches a value higher than 60%. This result can be related to the stiffness of the chain, which increases when DA increases. Conclusion This work gives some new information on the possibility to model the physicochemical behavior of chitin gels under different experimental conditions corresponding to either structural or environmental parameters. This information can be very useful to predict without any experiment the behavior of such gels. This is also particularly interesting to determine the initial conditions allowing a chosen final state. These gels are particularly useful for their biological properties. The studies we have in progress related to their use in the fields of tissue regeneration show that these modelings allow us to predict some behaviors for a given condition.

We also give the first results on the mechanical properties of these gels showing their dependence with various parameters. The most important result is that chitin gels can have the highest stiffness compared to other hydrogels of biopolymers. Thus, playing on various parameters, it is possible to provide these gels with a very large variety of mechanical properties. This is also particularly interesting when we consider the important applications of these gels in the field of biomaterials. References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16)

(17) (18) (19)

(20) (21) (22) (23) (24) (25) (26) (27)

Hirano, S.; Yamaguchi, R. Biopolymers 1976, 15, 1685. Hirano, S.; Ohe, Y. Agric. Biol. Chem. 1975, 39, 1337. Hirano, S.; Ohe, Y. Carbohydr. Res. 1975, 41, C1. Hirano, S.; Kondo, S.; Ohe, Y. Polymer 1975, 16, 622. Hirano, S.; Ohe, Y.; Ono, H. Carbohydr. Res. 1976, 47, 315. Moore, G. K.; Roberts, G. A. F. Int. J. Biol. Macromol. 1980, 2, 73. Moore, G. K.; Roberts, G. A. F. Int. J. Biol. Macromol. 1980, 2, 78. Vachoud, L.; Zydowicz, N.; Domard, A. Carbohydr. Res. 1997, 302, 169. Vachoud, L.; Zydowicz, N.; Domard, A. Carbohydr. Res. 2000, 326, 295. Pompei, C.; Casiraghi, E.; Lucisano, M. Milchwissenschaft 1994, 49, 562. St-Gelais, D.; Hache, S. Milchwissenschaft 1995, 50, 71. Nagasaka, K.; Taneya, S. Nippon Shokuhin Kagaku Kaishi 1996, 43, 1176. Debye, P.; Hu¨ckel, E. Physik. Z. 1923, 24, 185. Refojo, M. F. J. Polym. Sci. A-1, 1967, 5, 3103. Austin, P. R. German Patent DE 2 707 164, 1977. Austin, P. R.; Castle, J. E.; Albisetti, C. J. In Chitin and Chitosan; Skjåk-Bræk, G., Anthonsen, T., Sandford, P., Eds.; Elsevier Applied Science: London, 1989; p 749. Ferry, J. D. J. Am. Chem. Soc. 1948b, 70, 2244. Watase, M.; Nishinari, K.; Clark, A. H.; Ross-Murphy, S. B. Macromolecules 1989, 22, 1196. Rochas, C.; Landry, S. In Gums and Stabilizers for Food Industry; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, England, 1988; p 45. Guenet, J. M. ThermoreVersible gelation of polymers and biopolymers; Academic Press: London, 1992. Jones, J. L.; Marques, C. M. J. Phys. (Les Ulis, Fr.) 1990, 51, 1113. Draget, K. I. Polym. Gels Networks 1996, 4, 143. Henderson, G. V. S., Jr.; Campbell, D. O.; Kuzmicz, V.; Sperling, H. J. Chem. Educ. 1985, 62, 269. Clark, A. H.; Farrer, D. B. J. Rheol. 1995, 39, 1429. Hermans, J. J. Polym. Sci. A 1965, 3, 1858. Clark, A. H.; Ross-Murphy, S. B. Br. Polym. J. 1985, 17, 164. Domard, A. Int. J. Biol. Macromol. 1987, 9, 98.

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