Physicochemical Behavior of Homogeneous Series of Acetylated

Biomacromolecules 2005, 6, 131-142

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Physicochemical Behavior of Homogeneous Series of Acetylated Chitosans in Aqueous Solution: Role of Various Structural Parameters Guillaume Lamarque, Jean-Michel Lucas, Christophe Viton, and Alain Domard* Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riaux - UMR CNRS 5627, Domaine scientifique de la Doua, Baˆ timent ISTIL, 15, Bd. A. Latarjet, 69622 Villeurbanne Cedex (France) Received June 24, 2004; Revised Manuscript Received September 15, 2004

Physicochemical properties of four different homogeneous series of chitosans with degrees of acetylation (DA) and weight-average degrees of polymerization (DPw) ranging from 0 to 70% and 650 to 2600, respectively, were characterized in an ammonium acetate buffer (pH 4.5). Then, the intrinsic viscosity ([η]0), the root-mean-square z-average of the gyration radius (RG,z), and the second virial coefficient (A2) were studied by viscometry and static light scattering. The conformation of chitosan, according to DA and DPw, was highlighted through the variations of R and ν parameters, deduced from the scale laws [η]0 ) KwM Rw and RG,z ) K′M νw, respectively, and the total persistence length (Lp,tot). In relation with the different behaviors of chitosan in solution, the conformation varied according to two distinct domains versus DA with a transition range in between. Then, (i) for DA < 25%, chitosan exhibited a flexible conformation; (ii) a transition domain for 25 < DA < 50%, where the chitosan conformation became slightly stiffer and, (iii) for DA > 50%, on increasing DPw and DA, the participation of the excluded volume effect became preponderant and counterbalanced the depletion of the chains by steric effects and long-distance interactions. It was also highlighted that below and beyond a critical DPw,c (ranging from 1 300 to 1 800 for DAs from 70 to 0%, respectively) the flexibility of chitosan chains markedly increased then decreased (for DA > 50%) or became more or less constant (DA < 50%). All the conformations of chitosan with regards to DA and DPw were described in terms of short-distance interactions and excluded volume effect. Introduction Chitin and chitosan are linear copolymers of (1f4)-linked2-acetamido-2-deoxy-β-D-glucan (GlcNAc) and 2-amino-2deoxy-β-D-glucan (GlcN). If chitin is fully insoluble in both aqueous and usual organic solvents, as long as the copolymer is soluble in dilute acidic media, it is termed chitosan. Despite chitosan being much less widespread in biomass than chitin, it can be obtained from partial N-deacetylation of chitin under severe alkaline conditions.1-4 Chitosan finds numerous applications5,6 in agriculture,7 biomedicine8-10 (for example as drug delivery system11-14), paper-making, water treatment, or food-industry.15,16 Its properties strongly depend on the Degree of acetylation (DA),17 corresponding to the molar ratio of GlcNAc units within the chain, and the molecular weight,10,16 which influence not only its physicochemical behaviors18-20 but also its biological activity.21-23 The knowledge of the chitosan behavior and conformation in aqueous solution versus DA, the weight-average degree of polymerization (DPw), and the polydispersity index (Ip) is then of major interest, for example, in the production of nanoparticles.13,24 In dilute acidic media, thanks to the protonation of the amino functions, chitosan is fully soluble and behaves as a cationic polyelectrolyte. The value of the intrinsic pK (pK0) * To whom correspondence [email protected].

should

be

addressed.

E-mail:

was already demonstrated to increase from 6.46 to 7.32 as a function of DA.19,25 Thus, both electrostatic and steric effects must be taken into account when studying the influence of DA and DPw on the solution behavior of chitosan. Indeed, in the case of flexible polymer chains, the accumulation of charges would lead to a considerable expansion due to electrostatic repulsions. Among the analytical techniques allowing the investigation of chitosan behaviors, viscometry26-32 and static and dynamic light scattering18,30,33-36 are the most commonly used. Although the studies on the physicochemical behavior and conformation of chitosan in aqueous solution at high ionic strength are numerous, most of them only reported analysis on a very restricted range of chitosan in terms of DA28,31,32,35-37 and DPw.29 Commercial samples were generally used without further purification and degraded by means of sonication,31,38-41 nitrous deamination,42 or acidic hydrolysis, leading to relatively high and variable polydispersity indexes.33 Thus, preparation of the samples could have some important incidences on Ip39,43 and aggregate formation42 by self-association of chitosan chains leading to curved shapes of Zimm plots44 and rendering the determination of Mw (weight average molecular weight), RG,z45 (root-meansquare of the z-average gyration radius), and A242,46 (second virial coefficient) very tricky. In this work, the conformation of the copolymer was studied thanks to the calculations of the total persistence length Lp,tot, R and ν parameters (from

10.1021/bm0496357 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/16/2004

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the scale laws [η]0 ) KwM Rw and RG,z ) K′M νw), which were preferred to the determination of the empirical B31,47 parameter. Studying polyelectrolytes in aqueous solution required addition of salt to minimize the electrostatic contributions and screen ionic sites along the chains. If some authors traditionally used NaCl48 or NaOAc49 in the case of chitosan, it has been shown that these salts were unable to avoid aggregation of chitosan chains,26,42 especially at high DA. By breaking interchain hydrogen bonds in solution, ammonium salt, first proposed by Domard and co-worker50 then Anthonsen et al.,42 is now preferred by several authors as eluent for HP-SEC51 and for the characterization of chitosan in aqueous solution18 for its ability to prevent chitosan from aggregation. The choice of the solvent and salt,49,52 the ionic strength52 and pH34,52 of the solution, and dn/dc (refractive index increment) as a function of DA seemed to be preponderant in the consistency and reliability of the results published.53,54 Furthermore, some authors processed their chitosans either by deacetylation or reacetylation27,51 (in heterogeneous or homogeneous media, respectively) leading to a blockwise and statistical ordering of the GlcN and GlcNAc residues along the polymer chain.55 These compositions heterogeneities could also represent a major drawback for the study of the laws of behavior of chitosan in aqueous solutions and the calculation of the persistent lengths.44 Indeed, Brugnerroto et al.53,56 demonstrated by molecular modeling that a block, an alternate and a statistic ordering of the GlcNAc and GlcN units along the polymer chain, led to three different variations of Lp as a function of DA. This was attributed to the enhanced flexibility brought about by blocks of several screened GlcN units and could explain why several authors found different behaviors for chitosans having the same DA but prepared from different processes. Among the means to process chitosan samples, reacetylation in a hydro alcoholic medium at different DAs is a well-known reaction that allows us to reach precise DA values19 without degrading the copolymer.20 The relatively narrow Ip of the proceeded samples was also demonstrated to decrease as DA increased.18,20 Improving the clarification of chitosan solutions is also important to suppress aggregates that could induce wrong measurements and lead to curved Zimm plots or negative second virial coefficients.42 Even though the mechanism of the aggregate formation is still an open discussion, most of them can be removed either by centrifugation or filtration on decreasing the membrane pore size.29 Recently, the existence of a universal law of behavior for chitosan in aqueous solution according to DA was revealed by Sorlier et al.19 and then confirmed by Schatz et al.18 By means of homogeneous series of acetylated chitosans with similar Ip, they demonstrated that properties of chitosan deduced from: potentiometry, interferometry, SLS and viscometry measurements underscored a triple behavior depending on DA. When DA was increasing, properties of chitosan were changing through three distinct domains evolving from a polyelectrolyte state to that of isolated charges on polymer chains in a hydrophobic environment and, between, a transition range. Since their purpose was to check the influence of DA on the physicochemical properties

Lamarque et al.

of chitosan, their studies were restricted to a polymer series of DPw ) 2200 and 1000, respectively. The very narrow range of DPw studied did not allow them to determine the conformational parameters R and ν, or the persistence lengths versus DPw. Thus, it was of major importance to estimate the influence of DPw on the rigidity of chitosan to be then able to foresee its conformation, whatever DA and DPw, according to a theorical approach taking into account the excluded volume effect. To perform that, about thirty chitosans were homogeneously reacetylated (DAs ranging from 0 to 70%) from four different initial samples of low DA differing in their DPw (from 650 to 2600) and were either analyzed by capillary viscometry and static light scattering (SLS), in “batch mode”, or coupled with steric exclusion chromatography (SEC, “micro-batch mode”) in an ammonium acetate buffer (pH 4.5). The data resulting from these analyzes enabled us for the very first time to better understand the combined role of DPw and DA on the behavior of chitosan in aqueous solutions at high ionic strength. Experimental Section Purification and Acetylation of Chitosan. Four different batches of chitosan (batches 144, 154, 124, and 114) with DAs of 2.23, 1.2, 1.51, and 2.65%, respectively, were purchased from France Chitine (France). The chitosan powders were dissolved (5 g/L (w/v)) in an aqueous acetic acid solution, filtered successively through 5, 1.2, 0.8, and 0.45 µm pore size membranes (Millipore), and precipitated by means of a dilute aqueous solution of ammonia. After repeating washings with deionized water and centrifugations cycles (until the conductivity of the supernatant reached that of water), purified chitosans were then lyophilized. Acetylation was performed as follows. Purified chitosans were then dissolved in a 0.1% acetic acid/1,2-propanediol mixture. Different amounts of a solution of pure and fresh acetic anhydride in 1,2-propanediol were added, in stoechiometric conditions, to reach the desired DA. At the end of the reaction, reacetylated chitosans were fully precipitated by addition of dilute aqueous ammonia and washed several times with deionized water at pH 7.5 in order to preserve the -NH2 form. Reacetylated chitosans with the lowest DPw and highest DA were fully soluble whatever the pH. In the latter case, salts and propanediol were removed by ultrafiltration (Amicon) against deionized water (pH 7.5) through a YC05 membrane (MwCO 500, Millipore). In all cases, reacetylated chitosans were finally lyophilized. Characterization of Chitosans. Reacetylated samples were dissolved in dilute acidic D2O (at pD 3-4), and their DAs were analyzed by 1H NMR spectroscopy, as proposed by Hirai et al.57 Spectra were recorded on a Bru¨kerSpectrospin AM 300 spectrometer (300 MHz). About 200250 scans were acquired. The absence of propanediol within the reacetylated chitosans was confirmed for all samples. The water content was determined by thermo-gravimetric analysis (DuPont Instrument TGA 2000). Viscometry. Intrinsic viscosities were measured at 25 ( 0.1 °C using an Ubbelohde automatic capillary viscometer with a inner diameter of 0.53 mm (Viscologic TI 1

Biomacromolecules, Vol. 6, No. 1, 2005 133

Acetylated Chitosans in Aqueous Solution

Table 1. Molecular Characteristics of Chitosans of Various DAs and DPw Determined by SLS in Batch and Micro-Batch Modea

a

HP-SEC analysis Mw(105g/mol) DPw 6.024( 0.181 3716( 112 4.260( 0.128 2580( 79 4.521( 0.136 2608( 84 4.494( 0.135 2528( 84 4.578( 0.137 2518( 85 4.391( 0.132 2359( 82 4.619( 0.129 2447( 80 n.d. n.d. n.d. n.d.

batch mode Mw (105g/mol) 4.188( 0.125 3.899( 0.115 4.198( 0.119 4.276( 0.123 4.315( 0.124 6.012( 0.187 12.590( 0.210 4.406( 0.137 4.477( 0.213

DPw 2583( 77 2362( 70 2421( 69 2405( 69 2374( 68 3230( 100 6671( 113 2367( 70 2372( 69

Ip 1.44( 0.04 1.37( 0.03 1.27( 0.02 1.28( 0.02 1.29( 0.02 1.51( 0.07 2.13( 0.10 1.20( 0.04 1.18( 0.05

0.986( 0.023 0.900( 0.021 0.889( 0.023 0.873( 0.022 0.867( 0.024 0.857( 0.026 0.861( 0.028

609( 14 546( 13 522( 14 491( 12 481( 13 458( 14 453( 15

1.61( 0.13 1.43( 0.08 1.34( 0.09 1.31( 0.07 1.24( 0.13 1.17( 0.07 1.11( 0.06

2112( 55 2149( 64 2265( 68 2313( 56 2236( 60 2219( 63 2185 ( 44 n.d. n.d.

3.048( 0.110 3.105( 0.098 3.311( 0.112 3.373( 0.121 3.483( 0.119 5.823( 0.175 7.583 ( 0.196 3.565 ( 0.115 3.565 ( 0.126

1886( 68 1880( 59 1903( 64 1900( 68 1922( 66 3129( 94 3974 ( 103 1916 ( 62 1868 ( 55

1.65( 0.06 1.37( 0.06 1.29( 0.06 1.28( 0.05 1.29( 0.05 1.53( 0.05 2.14 ( 0.05 1.20 ( 0.05 1.12 ( 0.05

1652 ( 43 1701 ( 51 1632( 57 1675( 34 1596( 48 1597( 48 1654( 48

2.134 ( 0.079 2.250 ( 0.081 2.512( 0.082 2.371( 0.080 2.427( 0.085 2.455( 0.090 2.600( 0.086

1322 ( 49 1361 ( 49 1441( 47 1334( 45 1334( 47 1318( 48 1364( 45

1.57 ( 0.04 1.31 ( 0.03 1.20( 0.03 1.26( 0.02 1.27( 0.02 1.20( 0.02 1.16( 0.02

batch 114

DA(%) 2.7 9.8 29.4 40.0 49.5 59.9 66.0 59.9b 66.0b

144

2.2 8.6 22.3 40.1 45.9 62.1 69.4

1.232( 0.049 1.182( 0.035 1.211( 0.048 1.166( 0.035 1.154( 0.035 1.138( 0.046 1.301( 0.047

761( 30 718( 22 711( 28 656( 20 640( 19 608( 24 684( 25

124

1.5 9.8 30.9 39.4 48.2 59.7 71.0 59.7b 71.0b

3.413( 0.089 3.548( 0.106 3.940( 0.118 4.106( 0.099 4.052( 0.109 4.129( 0.121 4.170 ( 0.083 n.d. n.d.

154

1.2 10.4 31.6 39.8 49.9 60.1 70.5

2.668 ( 0.069 2.813 ( 0.084 2.844( 0.100 2.977( 0.060 2.904( 0.087 2.974( 0.089 3.153( 0.095

n.d., not determined. b Additional filtration on 0.22 µm.

SEMATech). Reacetylated chitosans were dissolved (0.10.3% (w/w)) in a degassed 0.2 M acetic acid/0.15 M ammonium buffer (pH 4.5). The critical concentration of chain entanglement C* was determined considering C*[η]0 ) 1. The following analyses were achieved with solutions of concentration at least 0.8 times lower than C*. Static Light Scattering (SLS), Batch Mode. Each chitosan was dissolved in the same degassed acetate buffer as the one used for viscometry measurements. All samples were filtered on 0.45 and then 0.22 µm pore size membranes, and analyses were performed 24 h after the last filtration so that the solutions could degas. Samples with the highest DAs and DPws were further filtered on a 0.22 µm pore size membrane before analysis to remove aggregates, if necessary. Multi-angle laser light scattering (MALLS) detection was used in batch (i.e., alone) and micro-batch mode (i.e., coupled with a HP-SEC device). Batch measurements were performed with a Dawn DSP-EOS (Wyatt) equipped with a 25 mW Ga/As laser operating at λ ) 690 nm. Solutions were analyzed in scattering glass cells at five different concentrations, below C*, and scattered light intensities were measured at 18 angles ranging from 23 to 147°. Light intensity measurements were derived following the Rayleigh-Debye equation, and Mw, the weight-average molecular weight, RG,z, the root-mean-square z-average of the gyration radius, and A2, the second virial coefficient, were deduced by means of Zimm plots. The refractive index increment dn/dc was chosen as a function of each DA, according to Schatz et al.18 High-Performance Size Exclusion Chromatography, Micro-Batch Mode. The polymer separation was performed on two serially connected columns (TSK G3000-PW and

TSK G6000-PW, i.d. ) 7.8 mm, l ) 300 mm). The detection was operated by a differential refractometer (Waters 410) coupled on line with a MALLS detector (Dawn DSP-F, Wyatt) equipped with a 5 mW He/Ne laser operating at λ ) 632.8 nm. Analyses were performed in micro-batch mode using the K5 flow cell. A degassed AcOH (0.2 M)/AcONH4 (0.15 M) buffer (pH 4.5) was used as eluent after two filtrations on a 0.22 µm pore size membrane (Millipore). The concentrations used ranged from 0.04 to 0.1% (w/w) before injection so that the results obtained for the different chitosans were independent of the starting solution concentrations. All chitosan solutions were filtered on 0.45 and then 0.22 µm pore size membranes. The flow rate was maintained at 0.5 mL/min, and the amount of sample injected was 200 µL. Results and Discussion Characterization of the Studied Samples. The main purpose of this work was to investigate laws of behavior related to the physicochemical properties of chitosan in aqueous solutions in relation with both DA and DPw. The ionic strength of the solution was fixed to µ ) 0.15 M with an ammonium salt to sufficiently screen the protonated amino groups, hinder electrostatic repulsions, and prevent chains from aggregation. Four purified chitosans with starting DA < 5% and differing in their initial degree of polymerization were reacetylated in mild conditions allowing us to produce four series of chitosans with DAs of 10, 30, 40, 50, 60, and 70% without degrading the polymer (Table 1). For more clarity, processed samples were named Cx,y, where x represents the average DPw of the batch, determined by HP-SEC (i.e., 650,

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Figure 1. Zimm plots (in negative scale representation) for various chitosans in solution in an AcOH (0.2M)/AcONH4 (0.15 M) buffer: (a) C2600,2.7; (b) C2600,66 before and (c) after further filtration on 0.22 µm pore size membranes.

1 600, 2 200, and 2 600 for the batches 144, 154, 124, and 114, respectively), and y the DA. All of the samples proceeded exhibited a random distribution of the GlcNAc/GlcN residues along the polymer chain by 1H NMR spectroscopy, according to the method of Vårum et al.58 HP-SEC measurements revealed a specially narrow range of DPw for each series. Thanks to the elimination of soluble oligomers during the repeated washing/centrifugation cycles of the reacetylation process,18,19 Ip monotonically decreased on increasing DA. Ip found by SLS in batch mode for the two highest DAs of the series 114 and 124 strongly suggested

that these four chitosan solutions underwent self-association. These aggregates were concentration dependent since Zimm plots revealed a positive curvature of the concentration function (Figure 1). Most of these aggregates were removed by means of an additional filtration through 0.22 µm pore size membrane (Figure 1, parts b and c) with a loss of matter 50%), [η]0 fell down to much lower values, and (iii) in between, hydrophilic and hydrophobic effects on chain dimensions were counterbalanced and a transition range was observed. Thus the variation of C*, defined as the reverse of [η]0 in the case of Gaussian coils, underscored the same behavior (data not shown) and varied from 0.63 to

Figure 3. Determination of R and log(K) versus DA. Mws were determined by SLS in (a) micro-batch and (b) batch measurements. Experimental points in dotted circles were those of Schatz et al.18

4 mg/mL. Considering these findings, concentrations we choose for the parent and fractionated solutions (0.4-1 mg/ mL and 0.05-0.4 mg/mL for SLS in micro-batch and batch mode, respectively) guaranteed analyses in dilute regime. A first insight in the study of chitosan conformation required the determination of the K and R set of constants of the Mark-Houwink-Kuhn-Sakurada (MHKS) equation [η]0 ) KM Rw, calculated by plotting log([η]0) versus log(Mw). Mw was determined either by SLS in batch or micro-batch mode (Figure 3). Variations of R and log(K) versus DA were represented in Figure 4 and followed the same trend as those of Figure 2. Unfortunately, R and log(K) values derived from Mw, determined either by HP-SEC or SLS, revealed some disparities. The highest Mw could be overestimated by HPSEC because of a nonoptimum column separation and/or polymer chain associations (Table 1). Values of the Huggins constant K, decreasing versus DA, were consistent with others,27,28 indicating that the solubility of chitosan in the buffer decreased as a function of DA, and were confirmed by the variation of A2 (see below). For batch measurements, chain conformation of chitosan varied from a random coil in a good solvent (R ) 0.65) to a wormlike chain in perturbed conditions (R ) 1.05).61 Thus, the increase of rigidity versus DA (i.e., the decreasing charge density) suggested that the ammonium salt sufficiently screened the amino groups to counterbalance the electrostatic repulsions responsible for the chain expansion. Chitosan was subsequently allowed to coil up and its apparent flexibility enhanced. Nevertheless, R values were largely over 0.5 (i.e., in θ conditions) whatever the DA within 0-25%, indicating that,

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Figure 4. Variations of (a) log(K) and (b) R from the MHKS equation versus DA. The curves were derived from Mw determined either by HP-SEC (s) or SLS (- - -) in batch mode.

despite the high ionic strength, we may assume that the electrostatic contributions to the excluded volume effect was not completely inhibited for very low DAs. As DA increased, Wang et al.28 proposed that intramolecular hydrogen bondings could prevent the acetamido group and the β-(1f4) linkage of the glucopyranose ring from free rotation and be responsible for an increase of excluded volume caused by the bulky acetamido groups. Even though R depends on the nature of the solvent, ionic strength and Ip, Wang et al.28 (0 < DA < 31%) and then Anthonsen et al.27 (0 < DA < 60%) found R linearly increasing with DA, but no one observed a transition range within DA ) [20;50], as we did. The plateau we observed could be the main consequence of the different solvents and dn/dc values we used to perform our studies. The ammonium salt used in this report could be responsible for particular interactions between the polymer and the solvent and explain the different results of Anthonsen et al.27 However, the same behavior was already observed by Sorlier et al.62 for the variation of dn/dc in an aqueous solvent containing KClO4 as added salt. The different solvent they used and the similar behaviors they obtained suggested that only the purity and the structure of the sample, and, on the other side, the pH and ionic strength of the solution are responsible for the results observed. Furthermore, compared to Wang et al.,28 the nonlinear decrease of the refractive index increment along with DA could also induce important variations in the determination of Mw and subsequently lead

Lamarque et al.

Figure 5. Variation of RG,z versus DA determined by SLS for the four series of reacetylated chitosans: DPw ) 650 ()), 1600 (4), 2200 (O), and 2600 (0). (a) With the HP-SEC device and (b) in batch mode. The results were compared to those of Schatz et al.18 (DPw ) 1000, 3). Solutions of chitosan were filtered on 0.45 and 0.22 µm pore size membranes before analysis. A further filtration on 0.22 µm was necessary for the four chitosans of highest DPw and DA.

to a quite different behavior of R. These two phenomena could explain the absence of a transition range in their results. Nevertheless, we may remind the reader that dn/dc is a parameter traducing the electric polarizability and a parameter largely influenced by the apparent charge density, then by DA and the screening of the protonated amino group. Static Light Scattering measurements. (A) Variations of RG,z and Lp,tot. Variations of RG,z were followed as a function of DA by SLS in batch and micro-batch mode. RG,z values determined by HP-SEC were somewhat dependent on the separation conditions of the samples, especially for low DA, and could explain the differences observed with those determined by SLS in batch mode (Figure 5). Depending on DPw of the chitosan series analyzed, two kinds of behavior were observed on increasing DA. For DPw > 1600, the variation of RG,z seemed to follow a different behavior as those observed until now and increased versus DA. On the contrary, for DPw < 1600, the decrease of RG,z versus DA, which followed the same trend as [η]0, was surprising when compared to the variation of R. The polyelectrolytic behavior was characterized by the relatively high values of RG,z, then, as the proportion of GlcNAc units increased, the hydrophobicity of the chains was responsible for their depletion and the fall of RG,z. On the opposite, the conformation change of chitosan from a flexible to a stiff molecule



should subsequently imply an increase of RG,z.34 The presence of a critical DPw,c for which we observed a transition of conformation suggested short- and long-distance interactions were involved, this latter being represented by the global excluded volume effect βtot(DA, DPw). As a first attempt to explain the different behaviors of chitosan in solution, we propose to share the global excluded volume effect into an electrostatic, βel, and a steric term, βst βtot(DA, DPw) ) βel(DA) + βst(DA,DPw) If the electrostatic contribution βel would mainly depend on the charge density, and subsequently to DA, on the opposite, both DPw and DA should have a strong influence on βst. Indeed, whatever the increase of the number of repetitive units or the bulky GlcNAc residue proportion, they both imply a loss of chain flexibility by steric hindrance or entropic effects. We are totally conscious of the approximations introduced by the split of βtot(DA, DPw) into two contributions. According to the Odijk and Houwaart63 relation, βtot(DA, DPw) ) 8πL2p,totλD, where λD is the Debye screening length, the Odijk-Skolnik-Fixman (OSF) theory predicted that the persistence length Lp,tot can be shared into Lp,i, the intrinsic persistence length characterizing the local stiffness of the chain, and Lp,e, the electrostatic persistence length related to the electrostatic repulsions between adjacent ionic sites: Lp,tot ) Lp,i + Lp,e. Introducing Lp,tot in the former equation results in a “coupled term” (∝ Lp,i × Lp,e) in the expression of βtot(DA, DPw) we must take into account. This condition was satisfied when studying the variation of βtot versus DA, since both βel(DA) and βst(DA,DPw) varied with DA. On the other hand, we will see below that the electrostatic contribution to βtot can be reasonably neglected when modeling the conformation of chitosan versus DPw. Considering this point of view, for DAs ranging from 50 to 70%, the continuous loss of charge density of chitosan chains leading to the decrease of βel could be counterbalanced (DPw ) 1600) or even overcome (DPw > 1600) by the continuous increase of βst, ascribable to the steric hindrance brought about by GlcNAc units. As a result, the increase of βtot reflected the extension of the chains prevented from depletion. This hypothesis will be comforted by the variations of Lp,tot and A2 (see below). However, the study on the variations of RG,z became trickier for very high values of DA because of self-association of chitosan, which led to the formation of aggregates (Figure 5b) and curved Zimm plots (Figure 1). An additional filtration of these solutions on 0.22 µm pore size membranes enabled us to decrease RG,z to more reliable values, but caused experimental uncertainties. A second empirical method to follow the chitosan conformation was to determine the K′ and ν parameters derived from the scale law RG,z ) K′M νw. These parameters were calculated as described previously for viscometric measurements, but a chromatographic method was also used to determine ν average values, as follows. Each set of the four chitosans with equivalent DAs were injected in HP-SEC. The average values of ν calculated from each eluted peaks by means of the software ASTRA 4.7 (Wyatt) were then compared to those derived from Figure 5. Except for the values determined by this last method, both variations of

Figure 6. Variations of log(K′) and ν as a function of DA, derived from Figure 5 (0, micro-batch and O, batch modes) and average ν values calculated from SEC-peaks of each set of the four chitosans with equivalent DA (4).

log(K′) and ν agreed with the previous observations and also revealed an increasing chain stiffness (Figure 6). ν was respective of DA and varied from 0.5 (Gaussian coils in θ conditions) to 0.65 (wormlike chains with excluded volume effect). The value of ν ) 0.46 obtained for DA < 5% in batch mode seemed to be therefore underestimated. This contradicts previous observations by coupled SLS measurement in batch mode33,45 and HP-SEC53 reporting ν as being constant on increasing DA. Only Wu et al.37 found ν > 0.6 in a 0.2 M AcOH/0.1 M AcONa buffer by means of dynamic light scattering, characteristic for stiffer polymers, but the DA of 9% they used was far from confirming our results. Determination of RG,z being more sensitive to experimental errors than [η]0, variations of ν and log(K′) on increasing DA were proportionally much lower than those of R and log(K) and definitely more difficult to underline. Despite all of the difficulties we met with in the determination of RG,z, the variation of the persistence length Lp,tot along with DA and DPw could be helpful to confirm or not the previous variations of conformation. Persistence lengths were calculated for each chitosan from the Benoit-Doty relation, valid for monodisperse wormlike chains in θ conditions64 RG,z2 )

LLp,tot 2Lp,tot3 2Lp,tot4 (1 - e-L/Lp,tot) - Lp,tot2 + 3 L L2

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Table 2. Persistence Lengths Determined from Each Eluted HP-SEC Peaka batch 114

DA(%) 2.7 9.8 29.4 40.0 49.5 59.9 66.0

Lp(nm) 12.0( 0.7 12.6( 0.8 13.4( 1.0 13.0( 1.0 13.0( 1.0 14.4( 1.2 10.6( 0.9

DPw 3716( 112 2580( 79 2608( 84 2528( 84 2518( 85 2359( 82 2447( 80

Ip 1.28( 0.02 1.28( 0.02 1.27( 0.02 1.25( 0.02 1.24( 0.02 1.13( 0.07 1.13( 0.10

144

2.2 8.6 22.3 40.1 45.9 62.1 69.4

7.8( 0.5 9.0( 0.6 6.8( 0.4 7.6( 0.5 6.0( 0.4 7.2( 0.5 8.6( 0.6

761( 30 718( 22 711( 28 656( 20 640( 19 608( 24 684( 25

1.27( 0.13 1.23( 0.08 1.24( 0.09 1.23( 0.07 1.19( 0.13 1.15( 0.07 1.10( 0.06

124

1.5 9.8 30.9 39.4 48.2 59.7 71.0

14.4( 1.2 12.0( 0.7 12.6( 0.8 13.2( 1.0 12.9( 1.0 11.2( 0.7 12.3( 0.8

2112( 55 2149( 64 2265( 68 2313( 56 2236( 60 2219( 63 2185( 44

1.43( 0.06 1.37( 0.06 1.22( 0.06 1.21( 0.05 1.14( 0.05 1.15( 0.05 1.18( 0.05

154

1.2 10.4 31.6 39.8 49.9 60.1 70.5

10.3( 0.9 10.6( 0.9 11.4( 0.7 12.8( 1.0 12.5( 0.8 10.9( 0.9 12.1( 0.7

1652( 43 1701( 51 1632( 57 1675( 34 1596( 48 1597( 48 1654( 48

1.24( 0.04 1.26( 0.03 1.15( 0.03 1.17( 0.02 1.18( 0.02 1.15( 0.02 1.09( 0.02

a The I were those derived from the linear dependence of the (y ) p log(Mw), x ) log(RG,z)) plots.

L corresponds to the contour length of the polymer chain, and the length per monomer unit was kept constant to 4.9 Å. Determining accurate Lp,tot implied the consideration of the polydispersity of the samples. It was evaluated by a normalized and logarithmic theorical function44 that fitted correctly our experimental weight distribution (by HP-SEC). βtot was estimated through the evaluation of the Flory expansion coefficient for the gyration radii RG and Lp,tot calculated thanks to the iterative method proposed by Berth et al.33 from RG,z and Mw determined by SLS in batch mode. Lp,tot was also determined by fitting a theorical line depending on Lp,tot to every plot (y ) log(Mw), x ) log(RG,z)) obtained from the HP-SEC peaks of each chitosan samples, thanks to the software Astra. Unfortunately, as pointed by Berth et al.,29 one can hardly highlight any systematic effect of both DA and DPw on the RG,z-Mw relationship, when derived from chromatographic peaks. This was also illustrated by the irrespective variation of ν versus DA by using this last method (Figure 6b). The difficulty to obtain well-Gaussian chromatographic peaks led us to estimate Lp,tot on a narrow distribution of Mw, where the radius of gyration dependence versus Mw was strictly linear, which was only partially representative of each chitosan samples. This could explain the inconsistent results observed when calculating Lp by means of the linear fitting (Table 2) and underlined that both RG,z and ν values derived from HP-SEC/MALLS peaks must be considered very cautiously when trying to study their dependence against DA, because they both depend on the quality of sample separation. In contrast, determination of Lp,tot by SLS in batch mode led to more reliable results (Figure 7). It is worth noting that the similarity between all the plots of Figures 4-7 confirm the consistency of our data. Thus, the universal law of behavior describing the variation

Figure 7. Variations of Lp,tot versus (a) DA and (b) DPw. Only samples C2600,59.9, C2600,66, C2200,59.7, and C2200,71 after their additional filtration on 0.22 µm pore size membrane were represented. The results of Schatz et al.18 were only represented on Figure 7(a).

of a given property of chitosan with DA seemed to be also valuable for the description of its conformation in aqueous solution for dilute systems. Even though this increase was theorically proposed by molecular modeling,56,65 this is the first experimental report of a significant rise of Lp,tot along with DA by SLS measurements in batch mode. Only Brugnerotto et al.53 observed the same trend by SEC/MALLS measurements in microbatch mode but their results relied on the same technique we used to determine Lp,tot by chromatographic fit, which led, in our case, to inconsistent results. Moreover, they used a constant dn/dc ratio when performing their analysis, despite its increase along with DA is now established. Nevertheless, in batch mode measurements, one can conclude that, for DPw ) 650, the increase of Lp,tot versus DA was inconsistent with the progressive fall of RG,z (Figures 5b and 7a). This unexpected result should rather be ascribable to the slight fall in the polydispersity index along with DA of this chitosan series (Table 1). Specifically, the logarithmic function we used to describe each mass distribution was very sensitive to the polydispersity index of the sample.44 The fact that Ip obviously decreased on increasing DA could lead to underand over-estimated Lp,tot values, for DA < 25% and DA > 50%, respectively, and may contribute to the described variations. The artifact in the calculation of Lp,tot resulting from the decrease of Ip on increasing DA could then take part of the increase of chain rigidity observed in Figure 7a,



Figure 8. Schematic representation of one possible variation of βst(DA,DP) and βel(DA) versus DA, leading to the global behavior observed for βtot and Lp,tot in the case of DPw > 1600.

for DPw ) 650. Thus, for DPw < 1 600, we strongly assumed that the variation of Lp,tot must decrease or, at least, be more or less constant on increasing DA, as that observed by Schatz et al.18 Compared to ours, the relatively high Ips of the samples analyzed by Schatz et al.18 also seemed to be responsible for the relatively low Lp,tot they calculated. Thus, their results could have fitted correctly our laws of behavior in the single case where the polydispersity indexes were more or less equal to those presented here. As a conclusion, it is worth noting that, for DPw < 1600, the excluded volume effect is negligible and only short-distance interactions were responsible for the conformation of chitosan. On the other hand, in the case of DPw > 1 600, variations of Lp,tot and RG,z versus DA were consistent with each other and could be explained by the theory of the excluded volume, in the case of long-distance interactions became preponderant to the detriment of any others. As DA increased, βst(DA,DP) was successively negligible (DA < 25%), equal (25% < DA < 50%), and higher (DA > 50%) than βel(DA). Besides, βel(DA) could keep on falling as the charge density along the polymer chains dropped (Figure 8). This simple representation could be used to explain the conformation of chitosan chains observed in solution as a function of DA. It is of major importance to realize this representation implies that βst(DA,DP) and βel(DA) are coupled each other (through DA), which is respective to the OSF theory. In contrast, the variation of Lp,tot versus DPw underlined quite different behaviors (Figure 7b). Even though Lp,tot could have been overestimated for the chitosan series C650, it seemed therefore that, whatever the DA, the increase of the chain length from DPw ) 650 to a critical chain length (illustrated by DPw,c) led to enhance the polymer flexibility. This strongly suggested that, in the range of DPw ) [650; DPw,c], the chain stiffness was mainly limited to shortdistance interactions, and neither βst(DA,DP) nor βel(DA) varied in a large extent, whatever the DA. On the other hand, for DPw > DPw,c, long-distance interactions became preponderant and two kinds of behavior were highlighted. (i) For DA below 50%, Lp,tot seemed to be nearly constant as the chains became longer, underlining that βst(DA,DP) = βst(DA) was almost constant versus DPw. Thus, for a nonnegligible charge density, where the electrostatic contribution to the excluded volume effect was superimposed to that of

Figure 9. Schematic representation of one possible variation of βst(DA,DP) and βel(DA) versus DPw leading to the global behavior observed for βtot and Lp,tot: (a) For DA < 50% and (b) for DA > 50%.

the steric hindrance, the influence of the chain length did not seem to be preponderant. (ii) In contrast, on increasing DA from 50% to 70%, the fall of the electrostatic contribution to the global excluded volume effect, due to the drop of charge density, was counterbalanced by the increase of βst(DA,DP) because of the addition of new GlcNAc residues along the chain, which emphasized the participation of steric hindrance to the excluded volume effect. As a consequence, for DA > 50%, chitosan chains tended to become stiffer as DPw increased (Figure 9). It is worth noting that previous studies from Tsaih et al.31 on a chitosan of DA ) 17% confirm our results. Thanks to viscometric measurements, they pointed out that chitosan chains were rather stiff and flexible for DPws below and beyond DPw,c ) 1300, respectively. Unfortunately, the highly polydispersed samples (Ip > 2.5) they used and the very narrow range of DA and DPw studied led to imprecise and too specific results. The present work decisively extends the study to a wide range of chitosans in terms of DA and DPw. It also underlines that a slight shift of the conformational transition from DPw ) 1800 to 1300 occurs for DAs ranging from 0 to 70%, respectively. This was ascribable to an increasing participation of βst(DA,DP) to the global excluded volume effect as DA increased (Figure 8). From these findings, it is now established that the whole conformations and behaviors of chitosan in a dilute aqueous system versus DPw can be simply explained by considering two participations, βst(DA,DPw) and βel(DA), to the global excluded volume effect. However, if splitting βtot in two terms can be considered as correct for describing a system versus DA, such an approximation could be applied versus DPw only if

140


Lp,e = 0. In this case, the coupled term of the Odijk-Houwaart relation can be neglected. For µ ) 0.15 M, Lp,e was determined as being lower than 0.2 nm for each degree of acetylation and then could be neglected in the calculation of Lp,tot. According to the Manning theory,66 ionic sites of a highly charged polyelectrolyte are importantly screened by the counterions of a solution containing a high concentration of salt. Thus, at high ionic strength, the theory predicted that some counterions of the solution could be condensed to the protonated sites of the polymer, leading to the decrease of its apparent charge density. This consequently implies that the solution behavior of chitosan versus DPw, at high ionic strength (µ ) 0.15 M), can be described more or less as a neutral copolymer. Nevertheless, for the highest DA values, self-association of chitosan chains could play a major role in the behavior observed and aggregation artificially emphasize the apparent stiffness of chitosan. This led us to be very cautious when considering the law of behavior on the variation of Lp,tot versus DA or DPw, but both variations of R and ν confirmed the results observed for Lp,tot. As a matter of fact, to our knowledge, the behavior of Lp,tot with DPw was never highlighted experimentally. Lp,tot covered the range of [4; 10] nm. These values were lower than those reported by Terbojevich et al.45 for two series of commercial chitosans of DA ) 15 (DPw ) [1200; 3750], Lp,tot ) [19; 21.5] nm) and 40% (DPw ) [950; 6700], Lp,tot ) [20; 23] nm), and Brugnerotto et al.53 for one single series of chitosan with DPw ≈ 680 of various DAs ()[0; 56] %, Lp,tot ) [11; 15] nm). In contrast, our values were more consistent with those reported by Berth et al.33 with Lp,tot ) 6 nm for a chitosan of DA ) 7% and a DPw ) 770. The various preparations and clarification methods of the chitosan solutions we used before analysis were closer to those of Berth et al.33 than the others, which could take part in the differences observed. Static Light Scattering Measurements. (B) Variation of A2. The variations of A2 versus DA and DPw depend on the intrinsic solubility of the copolymer and are linked to its affinity toward the solvent (Figure 10). The loss of solubility with DA was already attributed to the decrease of hydophilicity of the polymer chains in favor of hydrophobic interactions brought about by GlcNAc residues18 and agreed with that of log(K) (Figure 4). Despite the use of ammonium acetate as added salt, these measurements confirmed the formation of aggregates for C2600,59.9, C2600,66, C2200,59.7, and C2200,71, revealed by a drop of A2 to very low or negative values.42 On the other hand, the solubility of chitosan markedly decreased as DPw increased from 650 to 1600 and then leveled off for higher values. Both variations of A2 versus DA and DPw must be related to the variation of βel and βst. (i) According to Figure 8, on increasing DA, the fall of βel was counterbalanced by that of βst and consequently resulted in the decrease of A2. This led to the increase of segment/ segment interactions between chains to the detriment of the interactions between chains and the solvent and would explain the chain depletion observed for the smallest DPw (Figure 5). (ii) Second, whatever the DA, A2 drastically fell down to lower values on increasing Mw, proving again the

Lamarque et al.

Figure 10. Dependence of the second virial coefficient on: (a) DA and (b) Mw determined by SLS measurements in batch mode in AcOH (0.2 M)/AcONH4 (0.15 M) buffer.

preponderant influence of the steric contribution to the excluded volume effect on the chitosan solution behavior. More specifically, for DA > 50% (i.e., copolymer almost neutral) and DPw < 2600 (i.e., before aggregation), A2 was proportional to M2, as predicted by the Flory-Krigbaum expression A2 ) (NaNk2βtot/2M2)h(ztot), where Na is Avogadro’s number, Nk is the number of Kuhn segment, and ztot is the excluded volume parameter (h(ztot) ) (1/5.73ztot)ln(1 + 5.73ztot)). However, the experimental and predicted values were nonconsistent between each other, which could be the consequence of (a) the nonnegligible effect of DA on βst, (b) the slight contribution of βel on the global volume effect, even at high DA and DPw, and (c) a relation more complicated between βst and βel that the one we proposed. Conclusion Thanks to the systematic study by viscometric and SLS measurements of four homogeneously reacetylated chitosan series, differing in their DA and DPw, we extended all of the previous studies on this topic and highlighted different laws of behavior for the conformation of this polyelectrolyte versus DPw and DA. Conformations of chitosan were followed by the variation of the empirical conformational parameters R and ν, derived from the scale laws [η]0 ) Kw M Rw and RG,z ) K′Mνw, respectively. We particularly highlighted that they could be represented by the universal law of behavior of chitosan, valuable to describe the variation


of one of its physicochemical parameters versus DA. The results were then confirmed by SLS measurements and the variation of Lp,tot. If experiments achieved by coupling online a SEC device led to unreliable results, those obtained in batch mode allowed us to calculate accurate and precise persistence lengths. Their variation versus DA agreed with that obtained by viscometry measurement. Both conformation and solution behavior of chitosan at high ionic strength were explained by splitting the excluded volume effect into an electrostatic and a steric contribution. In our model, splitting the excluded volume effect was allowed because: (i) on varying DA, the contributions were both coupled and (ii) on varying DPw, Lp,e can be easily neglected with regards to Lp,i. The conformation of chitosan according to DA and DPw was thus explained as follows. (i) Below a critical DPw (ranging from 1300 to 1800, depending on DA) and for low DAs, the rigidity of the chain was essentially due to short interactions between adjacent protonated amino groups. On increasing DA, hydrophobic and steric interactions represented by βst(DA,DPw) first counterbalanced (25% < DA < 50%) and then overcame (DA > 50%) the polymer affinity with the solvent, leading to the depletion of the chain. (ii) On the opposite, beyond DPw,c, the excluded volume effect was assumed to lead to the different conformations of chitosan observed in all of the ranges of DA. On increasing DA, the influence of βel(DA) was rapidly overcome by the increase of βst(DA,DPw), because of the addition of new bulky GlcNAc units along the chain. Concomitantly, as DA increased, βst(DA,DPw) was also responsible for the slight shift of DPw,c to lower values, characterizing the change of flexibility of the chains versus their length. Thus, on increasing DPw from 650 to 2600, the enhanced flexibility which would have been the result of a chain length increase in the case of a Gaussian coil (DA < 25%) is then counterbalanced (25 < DA < 50%) or even overcome (DA > 50%) by the steric contribution of the global excluded volume effect. This subsequently led to the increase of the chain stiffness observed. To conclude, the continuous increases of βtot on increasing both DA and DPw were responsible for the self-association of chitosan chains revealed by curved shapes of Zimm plots, a drastic increase of RG,z and a drop of A2. Their presence could lead to a colloidal dispersion, far from the dilute system theory and models we used. The radii of gyration as well as the persistence lengths we determined could be then not wellrepresentative of isolated chains in solution and lead to an overestimated apparent rigidity. Even though the concentration of these aggregates is rather low ( 1300. For DPw < 1300, the electrostatic contribution to the excluded volume must be taken in consideration. (ii) For DA > 50%, the steric contribution to the global volume effect contributed to the chitosan chains rigidity. The chains become also mainly hydrophobic and a few of them undergo aggregation, which could change the theories involved to describe a local and semidilute system. Between, we observe a transition domain corresponding to a meta-stable system in which the whole interactions and the thermal agitation counterbalanced each other more or less. It is then preponderant to consider that the DA range generally studied ()[20; 50]) in previous published works corresponded approximately to the transition domain of our universal law of behavior. Added to the fact that the important Ips of the chitosan samples analyzed in the literature tended to level off the variations of the conformational parameters, this could explain why most of the previous studies reported R, ν, and Lp,tot as being constant according to DA. Thanks to this work, it is now worth considering the DPw when analyzing a solution property of chitosan. Acknowledgment. These studies are taking part of the CARAPAX project from the 5th European framework Program “Quality of Life and Management of Living Resources”. References and Notes (1) Sannan, T.; Kurita, K.; Iwakura, Y. Makromol. Chem. 1975, 176, 1191. (2) Kurita, K.; Tomita, K.; Tada, T.; Ishii, S. J. Polym. Sci. Part A 1993, 31, 485. (3) Tolaimate, A.; Desbrie`res, J.; Rhazi, M.; Alagui, A.; Vincendon, M.; Vottero, P. Polymer 2000, 41, 2463. (4) Chang, K. L. B.; Tsai, G.; Lee, J.; Fu, W.-R. Carbohydr. Res. 1997, 303, 327. (5) Hirano, S. 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BM0496357

Physicochemical Behavior of Homogeneous Series of Acetylated

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