Macro Molecules 5

8/4/2019 Macro Molecules 5

http://slidepdf.com/reader/full/macro-molecules-5 1/11

Kinetics Study of the Solid-State Acid Hydrolysis of Chitosan:Evolution of the Crystallinity and Macromolecular Structure

Anayancy Osorio-Madrazo,†,‡,§ Laurent David,† Stephane Trombotto,† Jean-Michel Lucas,†

Carlos Peniche-Covas,‡ and Alain Domard*,†

Laboratoire des Materiaux Polymeres et des Biomateriaux, UMR CNRS 5223 IMP, Universite de Lyon,

Universite Lyon 1, Batiment ISTIL, 15 Boulevard Andre Latarjet, F-69622 Villeurbanne Cedex, France,Centro de Biomateriales, Universidad de La Habana, Ave. Universidad s/n, 10600 Ciudad de La Habana,

Cuba, Facultad de Química-Farmacia, Universidad Central de Las Villas, Km 5 1/2 Carretera a Camajuanı ,

54830 Santa Clara, Cuba

Received February 11, 2010; Revised Manuscript Received April 6, 2010

The heterogeneous hydrolysis of fully deacetylated chitosan solid samples was carried out with concentratedHCl. The hydrolysis kinetics was studied at different temperatures and HCl concentrations. From 5 to 50 °C inthe hydrolysis time range up to 50 h, a monomodal distribution of molecular weights was observed connected tothe only degradation of amorphous domains. Between 70 and 90 °C and for the hydrolysis longest times, amultimodal distribution appeared with the additional hydrolysis of the crystalline phase. The crystallinity indexincreased from 57 to 73% with the elimination and partial recrystallization of amorphous regions. X-ray diffractionpatterns revealed the presence of the anhydrous polymorph, absent in the starting materials only containing thehydrated polymorph. The apparent crystallite width (from the Scherrer equation) of both the anhydrous and hydratedallomorphs did not vary significantly with time despite the increase in the fraction of anhydrous allomorph.Therefore, the hydrolysis in the solid state was complex, revealing several regimes. The activation energy parameterswere deduced, and the mechanisms were discussed.

Introduction

Chitosan is a linear natural copolysaccharide of (1f4)-linked-2-acetamido-2-deoxy- β-D-glucan (GlcNAc) and 2-amino-2-deoxy- β-D-glucan (GlcN). Because of its weak occurrence inbiomass, it is produced from the N-deacetylation of chitin,mainly extracted from crustacean cuticles. When this copolymeris soluble in dilute acidic media, it is termed chitosan. If it isobtained by heterogeneous deacetylation, then it has a degreeof acetylation (DA) below 40%, although a statistic reacetylationof low DA chitosans yields soluble copolymers up to DAs of ∼70%.1

Recent studies were devoted to microcrystals obtained fromnatural polymers as an attractive way to increase mechanicalperformances of composite materials.2-4 Therefore, a numberof investigations concerning the preparation of cellulose whis-kers were reported. These whiskers were used as model fillersin several kinds of matrixes, including synthetic and naturalpolymers.2-4 Native cellulose (Cellulose I) exhibits amorphousregions and usually displays ∼70% crystallinity. For example,in the cellulose from Halocynthia roretzi, called tunicin,

microfibrils are highly crystalline.5 Chitin microfibrils are presentin arthropod cuticles, where they are organized into helicalstructures displaying a cholesteric pattern in electron micro-graphs of oblique sections.6 In arthropods cuticles, the diametersof chitin crystallites are around 3-5 nm,7 and, as for cellulose,3,4

chitin whiskers have been used as reinforcing phase in polymermatrixes. Suspensions of chitin crystallites were prepared byacid hydrolysis with the objective of dissolving regions of low

lateral order so that the insoluble highly crystalline residue wasconverted to a stable suspension by mechanical shearing.8 Chitinwhiskers could be obtained from squid pens,9 Riftia tubes,10

crab,8 and shrimp shells.11 Chitosan could also be used as anatural reinforcing phase in polymeric matrixes, taking advan-tage of its biological properties.12 In the field of tissueengineering/repair, chitosan whiskers could be incorporated into

polymeric matrixes to provide the necessary short-term me-chanical resistance in addition to the bioactivity. As for other β,(1f4) linked polysaccharides, acid hydrolysis of chitosancould remove amorphous domains, and the crystalline residuecould be converted to a stable suspension mixed with otherpolymers to obtain bioactive nanocomposite films.

In this work, crystalline particles of chitosan were preparedby solid-state acid hydrolysis from a highly deacetylatedsemicrystalline chitosan so that the crystallinity index of thematerial was increased during the reaction. The chitosanhydrolysis has been mostly described in solution where theoriginal crystallinity is necessarily lost. Vårum et al.13 undertooka study of the depolymerization kinetics of chitosans with DAs

of 47 and 62% at temperatures between 20 and 80 °C in solutionwith 0.4 M HCl and for highly deacetylated chitosans with DAs) 0.2 and <0.03% at higher temperatures between 75 and120 °C. Their results showed the specificity of the acid-catalyzedcleavage of the four possible glycosidic linkages. Domard andCartier14 also studied the acid hydrolysis of fully deacetylatedchitosan in solution with 12 M HCl at 72 °C with the purposeto find optimal conditions for the preparation of D-glucosamineoligomers.

In fact, relatively little is known about the hydrolysis of chitosan in the solid state. Belamie et al.15 studied this hydrolysiswith gaseous HCl at different temperatures and molar ratios of H2O per glucosamine residue (r H2O). They provided a method

* To whom correspondenceshould be addressed. E-mail: [email protected].

† Universite de Lyon.‡ Universidad de La Habana.§ Universidad Central de Las Villas.

Biomacromolecules 2010, 11, 1376–13861376

10.1021/bm1001685 2010 American Chemical Society

Published on Web 04/19/2010



for the direct preparation of high-molecular-weight oligomerswith a relatively low polydispersity. An alternative method stillin the solid state, using a concentrated aqueous solution of HCl,was studied at 65 °C to test the role of HCl concentration onthe hydrolysis evolution.15

This article is the first reporting on a new method of acidhydrolysis of chitosan in the solid state under mild conditions.It should allow a better understanding of the hydrolysis kineticsand crystallization mechanisms revealing the formation of the

anhydrous allomorph. In a first step, we prepared a fullydeacetylated sample with a relatively high crystallinity indexby heterogeneous deacetylation.16 Then, the solid-state acidhydrolysis kinetics was studied under different HCl concentra-tions and temperatures, following the evolution of crystallineand macromolecular structures during hydrolysis.

Materials and Methods

The chitosan, Ch1, from shrimp shell chitin, was supplied by MahtaniChitosan (India, batch no. 2224). The chitin was deacetylated underheterogeneous conditions, in which the product was never dissolved.The Ch1 molecular characteristics are: number-average molecularweight ( M n) ) (1.48 ( 0.07) × 105 g/mol, (DA) ) (22.7 ( 0.2) %,crystallinity index (CrI) ) (56.7 ( 0.5) %, water content (9.5 ( 0.5)% w/w, and ash content ∼0.1% w/w.

Preparation of Fully Deacetylated Chitosan. N-acetyl groups canconstitute structural defects in chitosan crystals.17 Therefore, to obtaina sample of high crystallinity, we had first to achieve a full deacety-lation. The process was carried out under heterogeneous conditions topreserve the crystalline domains. Flakes of Ch1 were deacetylated withNaOH 40% w/w (1/40 w/v: solid/liquid ratio) using a multistep processdescribed by Lamarque et al.16 combining freeze-pump out-thaw(FPT) cycles. FPT cycles allow a better accessibility of reagents toN-acetyl groups including those of crystalline regions. They alsoeliminate oxygen likely to degrade the polysaccharide chain underalkaline conditions. After eight FPT cycles, the deacetylation was carriedout by heating at 100 °C the degassed suspension of chitosan during40 or 120 min. Then, the dispersion was neutralized with HCl 12 Mand washed several times with distilled water for the salts elimination.Finally, chitosan was lyophilized. The procedure was repeated two orthree times to achieve the full deacetylation.

Characterization of Materials. Water Content. The water contentof samples (5-15 mg) was determined with a TGA 2950 thermogravi-metric analyzer (Dupont instruments). A temperature ramp of 2 °C/ min was maintained up to 200 °C, operating under a constant flow of Helium. The water content was calculated from the weight loss of thesample up to 160 °C.

Ash Content. The ash content from a ∼1.5 g sample was calculatedfrom the residual weight after thermal treatment for 4 h up to 800 °Cin a muffle oven Nabertherm Program Controler S27.

Molecular Weight. Weight- and number-average molecular weights

were evaluated by size exclusion chromatography (SEC). Chitosansolutions at 0.1% (w/v) were prepared in a AcOH (0.2 M)/AcONH4

(0.15 M) (pH 4.5) buffer, used as eluent, then filtered through 0.22 µm pore size membranes (Millipore). The chromatographic equip-ment was composed of an IsoChrom LC pump (Spectra-Physics)connected to a Protein Pack 200 SW (WATERS) column and a TSKgel G6000 PWXL. A multiangle laser light scattering (MALLS)detector DAWN DSP (Wyatt) operating at 632.8 nm was coupledon line to a WATERS 410 differential refractometer. Dependingon DA,18 the refractive index increment dn /dc ranged from 0.183to 0.198 cm3 g-1.

Degree of Acetylation. 1H NMR spectroscopy was used to determinethe DA. Polymer (10 mg) was dissolved in 1 mL of D2O acidifiedwith 5 µL of concentrated HCl (12 M). Spectra were recorded on aBruker ALS 300 spectrometer (300 MHz for 1H) at 298 K. The DAwas calculated, as proposed by Hirai et al.,19 from the ratio of the methyl

proton signal of the (1f4)-2-acetamido-2-deoxy- β-D-glucan residuesto the whole H2 to H6 proton signals. 1H chemical shifts were expressedfrom the signal of HOD as reference (δ 4.78).

Wide-Angle X-ray Scattering Measurements. Pellets of 1 mmthickness made from chitosan flakes were analyzed by X-ray diffractionfor estimating CrI. Wide-angle X-ray scattering (WAXS) patterns wererecorded in reflection mode with a Siemens D 500 diffractometeroperating at 35 kV and 30 mA with the Cu KR radiation. The pelletswere deposited on a rotating neutral support. The diffraction angle 2θvaried between 7 and 130° by steps of 0.04° for the first experimentsand between 7 and 70° by steps of 0.06° for the followings. CrI wasdetermined from the ratio of the crystalline contribution estimated fromthe crystalline peaks to the total area of the diffractograms, discussedin the Crystalline Microstructure section.

Solid-State Hydrolysis of Chitosan by Impregnation with

Concentrated HCl. Preceding the hydrolysis of the fully deacetylatedchitosan, the latter was prehydrated to a desired level to favor a betteraccessibility to the reactive. Then, HCl with a concentration necessaryto protonate the amine groups (given by the DA) was added to allowthe hydrolysis kinetics studies. The added volume of HCl (V HCl) wascalculated from eq 1 to obtain a total molar ratio (prehydration plusaddition of concentrated aqueous HCl) of water to glucosamine residuer H2O ) 60 mol/mol;

where CHCl is the concentration of added HCl (mol L-1), V HCl is thevolume of concentrated HCl added (L), m is the weight of chitosan(g), w is the water content (weight fraction) in the chitosan, Fc is thevolumic mass of the HCl solution (g L-1) at a concentration C HCl, and

M GlcN, M GlcNAc, M HCl, and M H2O are the molecular weights (g mol-1) of glucosamine and N -acetyl-glucosamine residues, HCl, and H2O,respectively.

Washing of Hydrolyzed Chitosan. Hydrolyzates were successivelywashed with 1.5 M HCl, 1.0 M ammonia, and distilled water untilneutrality, and the resulting solid was freeze-dried. Washings ensuredthe dissolution and elimination of DP e 15 oligomers coming mainlyfrom the hydrolysis of the amorphous phase,15 allowing a highlycrystalline chitosan to be obtained.

Results and Discussion

Preparation and Molecular Characterization of the

Starting Samples. Water Content. Water can act as solvent,reactive, and plasticizer. The raw original (Ch1) and N-deacetylated samples contained (Table 1) between 7.7 and 9.8%

(w/w) of water, as expected for chitosans in the free amine form.This corresponds to r H2O between 0.75 and 0.97, respectively,and to about one molecule of water per glucosamine residue.

Degree of Acetylation. N-acetyl groups can constitute struc-tural defects in chitosan crystals, and a limit DA near 42% exists,where the lowest value of crystallinity is measured.17 Therefore,to obtain chitosan with a high crystallinity, we first operated afull deacetylation, carried out under heterogeneous conditionsto preserve the original crystalline domains. DAs of the preparedsamples are listed in Table 1. After a first 2 h of deacetylation16

of Ch1, DA decreased appreciably from 22.7 to 2.9% (Ch1-D1in Table 1), and a second 2 h step led to a fully deacetylatedchitosan (Ch1-D2 and Ch1-D2′ for the results obtained withtwo different sample series). For the sample obtained by

deacetylation with a 40 min multistep process, a third step hadto be performed (Ch1-D3′′) to achieve a full deacetylation.

r H2O )

V HCl[Fc - C HCl M HCl

M H2O ] + mw

M H2O

(1 - DA)(1 - w)m[1 - DA

M GlcN+

DA M GlcNAc]

(1)

Solid-State Acid Hydrolysis of Chitosan Biomacromolecules, Vol. 11, No. 5, 2010 1377



Molecular Weight E Volution during Deacetylation. Table 1also shows the number-average molecular weight ( M n), thepolymerization degree (DPn) and the polydispersity index ( I p)for all samples. After a first 2 h step16 for Ch1, M n decreasedappreciably (Ch1-D1). With the second 2 h step16 (Ch1-D2 andCh1-D2′), it did not vary significantly as well as for the third40 min step16 (sample Ch1-D3′′). I p varied in the first step fromabout 3.8 to 1.9, and for the subsequent steps, this variationwas negligible. The deacetylation under heterogeneous condi-tions is first operated in the amorphous domains and then, inthe crystallites,17 in a second step, with a much lowerreactivity.17,20 The crystalline network limits the chain degrada-tion during deacetylation by a barrier effect yielding a highmolecular weight fully deacetylated chitosan.17,20 Besides, thefractions of low molecular weights produced during degradation

are soluble in the reaction media. Therefore, the molecularweight distribution ( M n, I p) remains practically unchanged inthe residual polymer after the different deacetylation steps.

Crystalline Microstructure. Clark and Smith21 established forthe first time the X-ray fiber diffraction pattern of the hydrated(tendon) polymorph of chitosan. They proposed an orthorhombicunit cell with dimensions similar to those recently proposed byOkuyama et al.:22 a ) 8.95, b ) 16.97, and c (fiber axis) )10.34 Å. In this crystalline form, the molecule has a 21 helicalsymmetry, and the conformation in the unit cell is stabilizedby O3 · · ·O5 hydrogen bonds. The number of water moleculesin the unit cell is eight, that is, two molecules in an asymmetricunit (1 H2O/monosaccharide).

The anhydrous polymorph, the second crystalline form of chitosan, has been obtained by annealing the hydrated chitosanat∼240 °C in water.23-25 This form is produced when polymerchains are broken and hydrophobic interactions are favored,which is the case by heating. The diffraction pattern of annealedchitosan displays a strong (110) reflection at 2θ ≈ 15° (Cu KRradiation), which was not observed in the tendon form. Ogawaet al.23 proposed that the new polymorph was energetically morestable because of additional interchain hydrogen bondingsformed upon removal of loosely bound water molecules betweenchains along the [010] direction in the tendon chitosan.23,24 Saitoet al.24 prepared annealed samples by heating chitosans in waterat 200-220 °C and confirmed that the relative proportion of the different polymorphs in the annealed samples depended on

temperature, the origin, and the molecular weight.23-25 Ogawa25also examined the crystallinity changes in aqueous suspensions.

More recently, Okuyama et al.26 obtained the anhydrouschitosan by keeping a chitosan/acetic acid complex in 100%RH for several days at room temperature. Because of mildconditions, the X-ray diffraction pattern showed a well-preservedorientation and crystallinity compared with those obtained byannealing. Thus, the 3D structure of the new allomorph wasbetter described.26 In the anhydrous polymorph, there is no directinteraction between successive sheets of polymer chains alongthe a axis. Chitosan molecules have a two-fold helical symmetryand the conformation in the unit cell is stabilized by strongO3 · · ·O5 and weak O3 · · ·O6 hydrogen bonds within a repeatingperiod of 10.43 Å.

The main peaks observed in WAXS diffraction patterns of deacetylated samples before hydrolysis (Figure 1a) are locatedat 2θ ) 10.80° for the (020)h planes of the hydrated crystalline

structure and around 20°, corresponding to the contribution of two peaks at 20.16 and 21.80°, related to the reflections of (200)h

and (220)h planes of the hydrated polymorph. This is thecharacteristic spectrum of the hydrated chitosan described byClark and Smith.21

The X-ray diffractogram of Ch1-D2 allowed us to calculatea crystallinity index (CrI) of 69% as estimated by the methodof Focher et al.27 using the following equation

whereI

200 is the maximum intensity (at 2θ )20°

) of thereflection (200)h for the hydrated allomorph and I am is theintensity of the amorphous halo at 2θ ) 16°. This method failswhen a significant crystalline contribution of the anhydrousallomorph is observed, with the (110)a reflection near 14.95°.

We calculated the values of CrI from the separated crystallinepeak areas of the Ch1-D2 diffractogram. We first estimated thediffractogram of the amorphous contribution directly on the Ch1-D2 diffractogram, as indicated in Figure 1-I. Then, the crystallinecontribution area ( Acrist) between 7 and 70° (2θ) was obtainedfrom the subtraction of this amorphous contribution to the totaldiffractogram of Ch1-D2 ( Atotal). This allowed us to calculateCrI ) Acrist / Atotal of 36% for Ch1-D2 by integration of thecrystalline peaks and the total diffraction pattern between 7 and

70° (2θ) (Figure 1-I). Because of an overestimation of thecontribution of the amorphous phase, CrI is underestimated.

Table 1. Characterizations of the Original Ch1 and Deacetylated Chitosans Prepared from the Ch1 Sample by a One or Multi-StepHeterogeneous Deacetylation16

chitosan heterogeneous water ash DA Mn/105 DPn I p CrIdeacetylation (%, w/w) (%, w/w) (%, n/n) (g/mol) (%)

Ch1 commercial 9.52 0.1 22.7 1.48 ( 0.07 876 ( 4 3.81 ( 0.03 56.7 ( 0.5sample ofchitosan

Ch1-D1 one step 9.75 n.d.a 2.9 1.12 ( 0.03 691 ( 2 1.91 ( 0.02 57.0 ( 0.4(1st batch)(120 min)

Ch1-D2 two steps 8.12 n.d. 0.4 1.10 ( 0.03 680 ( 2 1.81 ( 0.02 67.1 ( 0.4(from 1st batch)(2 × 120 min)

Ch1-D2′ two steps 7.73 n.d. 0.1 1.16 ( 0.03 720 ( 2 1.55 ( 0.01 68.5 ( 0.2(from the 2nd batch)(2 × 120 min)

Ch1-D2′′ two steps n.d. n.d. 1.1 1.19 ( 0.04 737 ( 3 1.50 ( 0.01 n.d.(from the 3rd batch)(2 × 40 min)

Ch1-D3′′ three steps 8.21 n.d. 0.1 1.17 ( 0.03 727 ( 2 1.41 ( 0.01 67.5 ( 0.2(from the 3rd batch)(3 × 40 min)

a n.d.: not determined.

CrI ) ( I 200 - I am)/ I 200 (2)

1378 Biomacromolecules, Vol. 11, No. 5, 2010 Osorio-Madrazo et al.



An improved method for evaluating CrI, is based on the useof the diffractogram of an amorphous sample (AmCh) (Figure1-II). To obtain this sample, Ch1-D2 was dissolved at 2% (w/ v) in a stoichiometric amount of HCl and then lyophilized. Thecollected chitosan hydrochloride was left under an atmosphereof 28% (w/w) ammonia for several days for neutralizing andrecovering a quasi amorphous sample with a low amount of NH4Cl. Despite the difficulties to produce the amorphous system(the lyophilization of a chitosan hydrochloride solution caninduce a partial crystallization), the diffractogram of AmChpresented in Figure 1-IId practically did not exhibit the peak at2θ ) 10.80° for the (020)h planes of the hydrated allomorphand should be close to that of the amorphous state. To evaluatethe value of CrI (Figure 1-II), the diffractogram of AmCh was

adjusted by a multiplicative coefficient with the diffractogramobtained for the semicrystalline samples prepared in our work,

in the scattering angle range from 27 to 29° (2θ). Then, theamorphous contribution was smoothed by interpolation with acubic spline. The calculation of CrI was performed from theratio of the amorphous contribution area ( Aam) to the total area( Atotal) of the Ch1-D2 diffractogram in the scattering angle range(2θ) from 7 to 70°. (See Figure 1-II.) CrI obtained from thismethod for Ch1-D2 (CrI ) 1 - [ Aam / Atotal]) was close to 67%.

As shown in Figure 1, the shape of the amorphous contribu-tion is difficult to estimate only considering the semicrystallinesamples. Therefore, in the following, for all of our samples, weestimated CrI considering the diffractogram of the amorphoussample (AmCh). This probably also underestimates CrI butshows a closer agreement than from the method of Focher et

al.27 used for samples exhibiting the only hydrated allomorph.All diffraction patterns for the initial and deacetylated chitosans

Figure 1. Procedures for the calculation of crystallinity indexes from WAXS diffraction patterns of the fully deacetylated chitosan Ch1-D2 preparedby multistep heterogeneous deacetylation. (Ia,IIa) Diffraction diagram of the original chitosan Ch1-D2. (Ib) Estimated cubic spline curve for theamorphous contribution of Ch1-D2 in the WAXS diffraction pattern a. (Ic) Estimated crystalline contribution of Ch1-D2 after subtraction of theamorphous estimation b in the total diffraction pattern a. (IId) Diffraction diagram of the amorphous chitosan sample (AmCh) prepared fromCh1-D2. (IIe) Estimated crystalline contribution of Ch1-D2 after subtraction of the area of the amorphous diffractogram d. (a,b,d) Curves wereshifted of 165 units vertically for clarity.

Table 2. Characteristics of the Fully Deacetylated Chitosan Ch1-D2 and Hydrolyzed Chitosans Obtained by Solid-State Hydrolysis ofCh1-D2 with 3 M HCl at Room Temperature (∼22 °C)

starting andhydrolyzedchitosans time (h) M n /105 (g / mol) DPn I p

L020 (nm) hydratedpeak 2θ ≈ 11°

L110 (nm) anhydrouspeak 2θ ≈ 15°

anhydr.allomorph (%) CrI (%)

yield(%)

Ch1-D2 0 1.10 ( 0.03 680 ( 2 1.81 ( 0.02 4.5 ∼0 ∼0 67.1 ( 0.4H1_22 2.0 1.09 ( 0.02 677 ( 1 2.07 ( 0.01 3.5 7.4 29.7 68.8 ( 0.3 94.6H2_22 3.5 1.08 ( 0.02 669 ( 1 2.31 ( 0.01 3.5 5.9 31.4 69.7 ( 0.4 94.0H3_22 6.4 1.06 ( 0.02 660 ( 1 2.22 ( 0.02 n.d.a 5.9 32.2 70.8 ( 0.9 93.1H4_22 14.0 1.05 ( 0.03 655 ( 2 2.83 ( 0.02 n.d. 5.9 32.5 72.0 ( 0.6 92.3H5_22 25.0 1.03 ( 0.02 637 ( 1 2.22 ( 0.02 n.d. 6.3 32.3 72.1 ( 0.3 91.4H6_22 49.0 0.97 ( 0.02 602 ( 1 2.18 ( 0.02 n.d. 7.4 31.9 72.8 ( 0.5 90.0


Table 3. Characteristics of the Fully Deacetylated Chitosan Ch1-D2′ and Hydrolyzed Chitosans Obtained by Solid-State Hydrolysis ofCh1-D2′ with 3 M HCl at 5 °C

starting andhydrolyzedchitosans time (h) M n /105 (g / mol) DPn I p



anhyd.allomorph (%) CrI (%)

yield(%)

Ch1-D2′ 0 1.16 ( 0.03 720 ( 2 1.55 ( 0.01 4.3 ∼0 ∼0 68.5 ( 0.2H1_5 1.0 1.15 ( 0.02 716 ( 1 2.43 ( 0.02 3.9 7.4 30.0 69.9 ( 0.2 95.0H2_5 3.0 1.15 ( 0.03 715 ( 2 2.33 ( 0.02 3.5 5.9 29.6 70.7 ( 0.4 94.7H3_5 6.0 1.14 ( 0.03 710 ( 2 2.30 ( 0.02 3.5 5.9 32.0 71.9 ( 0.4 94.1H4_5 15.8 1.14 ( 0.06 707 ( 4 2.46 ( 0.02 3.5 5.9 29.8 72.1 ( 0.4 93.5H5_5 25.8 1.13 ( 0.05 702 ( 3 2.07 ( 0.01 3.5 6.3 30.9 71.9 ( 0.3 92.9H6_5 48.5 1.12 ( 0.03 695 ( 2 2.16 ( 0.01 3.5 7.4 32.0 72.1 ( 0.4 92.2


http://pubs.acs.org/action/showImage?doi=10.1021/bm1001685&iName=master.img-000.jpg&w=476&h=185



(Table 1) were very similar and characteristic of the hydratedallomorph (Figure 1a). A small increase in CrI was noticed upondeacetylation.

Kinetics of the Solid-State Hydrolysis of Fully Deacetyl-

ated Chitosan with HCl. The evolution of CrI and molecularweight during the acid hydrolysis of the semicrystalline chitosanwas followed for different reaction temperatures and HClconcentrations.

Chitosan Hydrolysis with 3 M HCl at Low Tempera-

ture. The results obtained with 3 M HCl for the fullydeacetylated Ch1-D2 at room temperature (∼22 °C) and Ch1-D2′ at 5 °C are reported in Tables 2 and 3, respectively.

Considering the initial water content of the samples and theamount of water used in the prehydration, the effectiveconcentration of HCl for the hydrolysis was 1.5 M. For clarity,we will report the concentration of the added HCl, that is, 3 M,without considering the prehydration and the natural watercontent.

Molecular Weight E Volution during Hydrolysis. When hy-drolysis was performed at room temperature or at 5 °C, thedecrease in M n with the hydrolysis time was weak (Tables 2and 3 and Figure 2), a monomodal molecular weight distributionwas observed at all studied times (Figure 3), and the productswere recovered above 90%. Under these conditions, the mac-romolecular structure was almost preserved after hydrolysis.

From a chemical point of view, water in excess (the reactant)was not a limiting factor (r H2O ) 60 mol/mol). It allowed alarge hydration of the amorphous parts and then their depo-lymerization. HCl was also in excess, and no competitionoccurred between its use for catalyzing the hydrolysis of glycosidic bonds and the protonation of the free amine functions.

Crystalline Microstructure E Volution during Hydrolysis. Atlow temperatures, the increase in CrI was very weak, and aplateau was reached at ∼72% (Figure 4). Therefore, the progressof the hydrolysis in the amorphous regions seemed to be limited.X-ray diffraction patterns of the hydrolyzates differed from thoseof the starting samples where only the peaks of the hydratedpolymorph were observed (Figure 5). After hydrolysis andwashing, a well-defined peak appeared near 2θ ) 15°, thus

revealing the presence of the anhydrous polymorph. In the solidstate, hydrolysis should occur preferably in the amorphous

domains, yielding oligomers, the majority of which are solubleand eliminated by washing, but also chain ends remainingbonded to the crystalline bundles, which are likely to crystallizein the anhydrous form. We estimated the contribution of eachpolymorph to the diffractograms introducing the coordinates of atoms in the unit cell, as proposed by Okuyama et al.22,26 foranhydrous26 and hydrated polymorphs22 using the PowderCell2.4 software by W. Kraus and G. Nolze.28 The refinement of the powder diffraction patterns of our hydrolyzates was carriedout in the 2θ range within 8 and 27°, where the contribution of the amorphous phase is weak compared with the crystallinecontribution (Figure 1). The unit cell parameters were optimizedfor both crystalline phases, maintaining constant the relative

coordinates of the atoms.22,26 The background was set to aconstant value as well as the full-width at half-maximum (fwhm)

Figure 2. Decrease in the number-average molecular weight duringthe solid-state hydrolysis with 3 M HCl of Ch1-D2′ at 5 °C (-2-)and of Ch1-D2 at room temperature (∼22 °C) (-9-), 50 (-b-), 70(-4-), 80 (-0-), and 90 °C (-O-); and with 12 M HCl at roomtemperature (-[-).

Figure 3. SEC/MALLS chromatograms (refractive index detector).Dashed lines: non hydrolyzed fully deacetylated chitosans Ch1-D2(-·- ·-) and Ch1-D2′(---). Solid lines: hydrolyzed chitosans preparedby hydrolysis during 49 h with 3 M HCl of (a) Ch1-D2′ at 5 °C and (b)Ch1-D2 at room temperature (∼22 °C); (c) 50; (d), 70; (e), 80; and

(f), 90 °C. Inset A: Chromatograms of hydrolyzed chitosans preparedby hydrolysis of Ch1-D2 with 3 M HCl at 70 °C for different times ofhydrolysis. The chromatograms in the inset A at 16 and 49 h wereshifted vertically of 0.04 and 0.08 units, respectively, for clarity.

Figure 4. Evolution of the crystallinity index CrI during the solid-statehydrolysis with 3 M HCl of Ch1-D2 ′ at 5 °C (-2-) and Ch1-D2 atroom temperature (∼22 °C) (-9-), 50 (-b-), and 80 °C (-0-).


http://pubs.acs.org/action/showImage?doi=10.1021/bm1001685&iName=master.img-003.png&w=233&h=198





of the peaks for each allomorph. The refinement yielded thescaling factor for each phase and the corresponding fraction of the two polymorphs. The recrystallization into the anhydrousallomorph was favored by a relatively higher molecular mobilityand conditions allowing hydrophobic interactions25 (in a highlyacidic medium) during hydrolysis. Besides, a partial transitiontook place from the initial hydrated polymorph to the anhydrousbecause the proportion of the latter increased by ∼30% afterhydrolysis (Tables 2 and 3), although CrI only changed of ∼2%.

The apparent width, Lhkl, of anhydrous and hydrated crystalswas deduced using the Scherrer equation. The fwhm of the(110)a reflection at around 2θ ) 15° and of the (020)h reflectionat near 2θ ) 11° were used to estimate L110 of anhydrous and

L020 of hydrated crystals respectively (Table 2 and 3). The newanhydrous crystals showed an apparent width higher than thatof the initial hydrated crystals. For high-molecular-weightpolysaccharides, the flexibility of chain ends could allow acrystallization by chain folding,29-31 resulting in crystal sizeson the order of 10-20 nm.29 Chanzy et al.30,31 obtainedmonocrystals of cellulose and mannan with low molecularweight using polymer chains of dimensions close to the size of the crystals. For chitosan, the ideal chain length corresponds to

a DP between 20 and 6014

related to contour lengths of about10 to 30 nm, respectively. Cartier et al.32 obtained lamellar single

crystals with a square-like shape using a fully deacetylatedchitosan of DPn ) 35. In our work, such oligomers could formand crystallize in the HCl impregnated amorphous phase. Wecould also consider the addition of anhydrous crystals onto theinitially existing hydrated ones, possibly coming from therecrystallization of chain segments entrapped in the crystallites.The L110 and L020 dimensions of the anhydrous and hydratedpolymorphs, respectively, could be an indication of the equato-rial development (cell parameter c: fiber axis) of the anhydrous

crystals with respect to the original hydrated ones duringhydrolysis. This mechanism would yield complex structurescomposed of hydrated crystal cores surrounded laterally byanhydrous crystalline blocks.

Chitosan Hydrolysis with 3 M HCl at High Tempera-

tures (50, 70, 80, and 90 °C). To accelerate the hydrolysiskinetics and thus increase the crystallinity, we investigatedstudies at higher temperatures. Tables 4 and 5 show thecharacterizations of the hydrolyzed samples at 50 and 80 °C,respectively.

Molecular Weight E Volution during Hydrolysis. The increasein the hydrolysis temperature induced a more important decreasein the molecular weight ( M n) (Tables 4 and 5 and Figures 2

and 3), and above 50 °C, the evolution of M n reached a plateaufor hydrolysis times above 10 h (Figure 2, empty symbols).Obviously, smaller percentages of hydrolyzates were recovered(Tables 4 and 5).

Distribution of Molecular Weights as a Function of

Temperature. The distribution of molecular weights (Figure 3)was dependent on the temperature. As mentioned above, it wasmonomodal for all studied times at the lowest temperatures(Figure 3a-c) and for short times at the highest temperatures(70, 80, and 90 °C), but in the latter case, an asymmetric oreven multimodal distribution was obtained for longer times withan increase in I p. (See Figure 3d,e,f and 3A.) Below 6 h of hydrolysis at 70 and 80 °C and below 3 h at 90 °C, the

distribution remained monomodal. Beyond, it was multimodalfor times of 12 h at 70 and 80 °C and 6 h at 90 °C, when theplateaus in the curve of evolution of M n with time were reached(Figure 2). This multimodal distribution centered on DPs 90,50, 16 (Figure 3d,e,f and 3A) was attributed to the specifichydrolysis of the crystalline fraction. (See below.)

Crystalline Microstructure E Volution during Hydrolysis. Forthe shortest times and still at 50 °C, the variation of CrI wassimilar to that at room temperature (Figure 4). Then, CrIdecreased to ∼65% after 49 h. The increase in temperatureinduced a degradation of crystalline domains evidenced by apronounced decrease in CrI when exceeding 50 °C (Figure 4).In the diffractograms of hydrolyzates, the intensity of the

anhydrous allomorph peak at 2θ) 15°, after appearing for shorttimes, decreased for the longest times, revealing a preferential

Figure 5. WAXS diffraction patterns obtained for (a) Ch1-D2 preparedby heterogeneous deacetylation (hydrated polymorph preponderant)and (b) hydrolyzed chitosans prepared by solid-state hydrolysis during6 h with 3 M HCl of (b) Ch1-D2′ at an hydrolysis temperature of 5 °C,(c) Ch1-D2 at room temperature (∼22 °C), (d) Ch1-D2 at 50 °C, and(e) Ch1-D2 at 80 °C. These diffraction diagrams allowed us to

estimate an anhydrous polymorph proportion of 32% at 5 and 22 °C,33% at 50 °C, and 37% at 80 °C. The curves b-e were shiftedvertically 100, 150, 220, and 290 units, respectively, for clarity.

Table 4. Characteristics of the Fully Deacetylated Chitosan Ch1-D2 and Hydrolyzed Chitosans Obtained by Solid-State Hydrolysis ofCh1-D2 with 3 M HCl at 50 °C

starting andhydrolyzedchitosans time (h) M n /105 (g/mol) DPn I p




yield(%)

Ch1-D2 0 1.10 ( 0.03 680 ( 2 1.81 ( 0.02 4.5 ∼0 ∼0 67.1 ( 0.4H1_50 1.4 1.03 ( 0.02 638 ( 1 1.63 ( 0.01 3.5 6.8 31.9 69.3 ( 0.3 90.7H2_50 3.2 0.97 ( 0.02 600 ( 1 1.85 ( 0.01 3.5 6.1 32.0 69.8 ( 0.5 89.5H3_50 6.1 0.90 ( 0.03 560 ( 2 2.06 ( 0.01 n.d.a 6.1 33.1 67.4 ( 0.5 87.4H4_50 11.0 0.64 ( 0.01 397 ( 1 2.10 ( 0.02 n.d. 6.7 33.7 65.7 ( 0.6 85.4H5_50 25.1 0.61 ( 0.02 381 ( 1 1.70 ( 0.02 n.d. 5.8 33.4 66.7 ( 0.5 82.0

H6_50 49.0 0.39 ( 0.02 243 ( 1 2.00 ( 0.02 n.d. 6.1 34.0 65.3 ( 0.4 78.2a n.d.: not determined.





degradation of this phase (Table 4 and Figure 7). Figures 5eand 7 illustrate the X-ray pattern of Ch1-D2 hydrolyzed for 6 hat 80 °C with the highest proportion of the anhydrous polymorphobtained in this work, close to 37%. This must be related tooptimal conditions in terms of molecular weight of the producedoligomers and the strong hydrophobic context. Therefore, theformation of the anhydrous form resulted from the balancebetween the recrystallization of mobile species produced byhydrolysis and the preferential degradation of their crystals,

favored by the rupture of hydrogen bonds above 50 °C. Thepreferential degradation could originate from the lower densityof hydrogen bonding in the anhydrous form or from topologicalconstraints if the anhydrous crystals develop at the peripheryof pre-existing hydrated crystals.

Depolymerization Rates as a Function of Temperature

and Associated Activation Energies. To study quantitativelythe kinetics of hydrolysis in the solid state, we estimated thefraction of hydrolyzed glycosidic bonds S for the differenthydrolysis times at the studied temperatures. S is related to thenumber-average degree of polymerization (DP) accordingly toS ) 1/DP - 1/DP0, where DP0 is the initial number-averagedegree of polymerization.

The degradation kinetics at low temperatures (5 and∼

22 °C)showed two different regimes (Figure 6a). At higher tempera-tures (70, 80, and 90 °C), it was complicated by the presenceof three kinetics regimes (Figure 6c). The pseudo-zero-orderkinetics equation33

traditionally used for the carbohydrate hydrolysis, like chito-san,13 which is an approximation of the first-order equation34

where n0 is the fraction of available glycosidic bonds (theaccessibility35), but eq 4 could not be used to model theexperimental data of the hydrolysis in the solid state.

In the literature, eq 3 appears largely satisfied because S couldcorrespond to a few units, that is, S , n0 or kt , 1. Equations3 and 4 were thus successfully used for the degradation insolution, where all glycosidic bonds are available for thedegradation.36,37 In the solid state, the carbohydrate hydrolysiskinetics was frequently treated in the same way as in solution,but the use of these equations was debated.38

By analogy to cellulose studies, where the observed down-

ward curvature in the kinetics plots neither followed the zero-order eq 3 nor the first-order eq 4,39 the sum of parallel first-

order processes ranked in two or three main groups wasproposed, for which glycosidic bonds degrade approximatelyat the same rate40

With cellulose, a global deviation was explained invokingthe presence of the so-called weak links (w) or “highdefects”.39,41,42 Two distinct stages during the degradation werehypothesized: a fast initial attack of the weak links, followedby a slower degradation of the rest of the amorphous fraction(a).42 The presence of weak links was also proposed to explainthe differences observed on the initial rate of cellulose degrada-tion in heterogeneous conditions.43 In the view of eq 5, cellulosewas composed of amorphous domains (a) with some weak links(w) and crystalline regions (c).44

Deviations from eq 4 are clearly observed in the hydrolysiskinetics plots of chitosan in the solid state, which is governedby different regimes (Figure 6), as expected for semicrystalline

chitosans with a high DP0.44

In the following, we will use eq 5as a guideline for the interpretation of the kinetics.At low temperatures, for the first 6 h (regime I), the access

to weak links or “high defects” was relatively easy and thusresponsible for a fast depolymerization. After 6 h (regime II),only the amorphous phase was hydroly zed, but the depolym-erization became slower because of topological constraintsconstituted by crystallites as proposed for the cellulose degrada-tion.38 At the earlier times of degradation (Figure 2) with kt ,

1, the so-called Ekenstam’s approximation could be applied tothe “weak links” degradation kinetics. Therefore, eq 5 wasrewritten as eq 6

These interpretations allowed us to calculate the hydrolysispseudo rate constants (k I ) n0

wk w) of the weak links at lowtemperatures, that is, 5 and 22 °C, from the slopes of thedegradation kinetics curves in the first regime (I) below 6 h(Figure 6a).

After the weak links hydrolysis in regime I, we assume thatthe parallel hydrolysis of initially less accessible bonds triggersregime II. This should mainly concern amorphous parts (na

0;k a),but a moderate hydrolysis of the crystalline domains (anhydrous:nc

0; k c or nanh0 ; k anh) could also be considered, accounting for the

plateau evolution of CrI and the anhydrous (anh) allomorphfraction with the hydrolysis time. Then, eq 5 became

Table 5. Characteristics of the Fully Deacetylated Chitosan Ch1-D2 and Hydrolyzed Chitosans Obtained by Solid-State Hydrolysis ofCh1-D2 with 3 M HCl at 80 °C





yield(%)

Ch1-D2 0 1.10 ( 0.03 680 ( 2 1.81 ( 0.02 4.5 ∼0 ∼0 67.1 ( 0.4H1_80 1.4 1.02 ( 0.02 632 ( 1 2.33 ( 0.02 3.1 5.4 32.0 65.9 ( 0.5 77.6H2_80 3.4 0.64 ( 0.02 396 ( 1 2.52 ( 0.03 n.d.a 5.4 34.7 63.4 ( 0.7 75.5H3_80 6.2 0.39 ( 0.02 242 ( 1 2.65 ( 0.03 n.d. 5.4 37.2 63.6 ( 0.4 73.7H4_80 13.8 0.11 ( 0.01 68 ( 1 3.37 ( 0.03 3.1 5.4 32.0 63.5 ( 0.4 71.5

H5_80 25.8 0.10 ( 0.01 62 ( 1 4.57 ( 0.03 2.9 5.4 19.7 61.3 ( 0.5 68.2H6_80 49.2 0.09 ( 0.01 56 ( 1 5.13 ( 0.03 2.9 5.4 19.5 59.9 ( 0.7 65.6


1DP

-1

DP0) kt (k : apparent rate constant) (3)

S ) n0(1 - e-kt ) (4)

S ) nw0 (1 - e-k wt ) + na

0(1 - e-k at ) + nc0(1 - e-k ct )

(5)

S ) k It ) nw0 k wt at t < 6 h (6)




The pseudo rate constants of the parallel process (k II ) na0k a

+ nc0k c ) na

0k a + nanh0 k anh) at low temperatures were obtained

from the slopes of the plots in regime II (Figure 6a).An alternative modeling (model 2) with S ) nw

0 (1 - e-k wt )in the low time range and eq 7 for the larger t values was used.This yielded the same quantitative results as with eqs 6 and 7(Figure 6b) but could better account for experimental data, witha regime I up to 15 and 26 h at 22 and 5 °C, respectively. Thereis a significant variation of the accessibility nw

0 with temperaturefrom 5 to 22 °C related to a variation of accessible links (Figure6b) with increasing the molecular mobility of chitosan chains,bound water molecules, or both.

The kinetics analysis of the three regimes (Figure 6c) at hightemperatures (70, 80, and 90 °C) was also performed. In regimeI, the initial slopes of the plots agreed with the values of thepseudo rate constants of weak links deduced at low temperatures,that is, 5 and 22 °C (Figure 8), and the interpretation of thehydrolysis mechanism in regime I should be still valid.Unexpectedly, the slopes in regime II at high temperatures are

higher than those of regimes I and III and should be interpretedas resulting from different processes, that is, amorphous andcrystalline degradation (mainly the anhydrous allomorph deg-radation as previously described) or several mechanisms (ran-dom hydrolysis in parallel with a cooperative hydrolysis). In asimple analysis, the kinetics equation could be separated intoseveral parallel processes as in eq 5. This hypothesis should beconsidered as a simplification because the degradation in regimeIII possibly occurs only after a significant degradation andrecrystallization of the amorphous phase (in regimes I and II).

As discussed above, the multimodal distribution of molecularweights (Figure 3) only obtained in regime III, that is, afterlonger times at high temperatures (70, 80, and 90 °C), is the

fingerprint of the degradation of the crystalline phase. Besidesthe increasing contribution with time of the anhydrous allomorphin the first step of hydrolysis (Figure 7), for longer times, athigh temperatures, it decreased (peak at 2θ ≈ 15° in thediffractograms of Figure 7c). Therefore, the anhydrous crystalswere preferentially hydrolyzed. Indeed, the global content of hydrated allomorph (CrI × (100 - % anhyd. allomorph)) wasalmost constant, that is, the hydrated allomorph initially presentwas mostly preserved during hydrolysis. Therefore, regime III(Figure 6c) mainly represents the hydrolysis of the anhydrousallomorph, and the pseudo rate constants (k III ) nanh

0 k anh) of thehydrolysis of this allomorph could be estimated from the slopesof plots in regime III observed at high temperatures.

In brief, we deduced the pseudo rate constant of the weaklinks hydrolysis k I ) nw0 k w from the initial slopes (regime I) of

kinetics plots at the different temperatures studied. The apparentpseudo rate constant of the anhydrous allomorph hydrolysis k III

) nanh0 k anh was estimated from regime III at high temperatures.

In the hypothesis of parallel degradations occurring in thecrystalline and amorphous phases, the sum of the pseudo rateconstants in the amorphous and anhydrous allomorphs: k II )

(na0k a + nanh

0 k anh) was calculated from slopes in regime II atdifferent temperatures. From the values of the pseudo rateconstant k II and the pseudo rate constant of the anhydrousallomorph, k III, the values for the amorphous degradation alonecould be estimated: na

0k a ) k ′II ) k II - k III. Values of na0k a were

both over those of nanh0 k anh and nw

0 k w. This faster kinetics could

be understood as a result of a higher “accessibility” value ( na0 >

nanh0 and na

0 . nw0 ).

Figure 6. Evolution of the fraction of hydrolyzed glycosidic bonds S (×1000) with the hydrolysis time during solid-state hydrolysis: (a) at lowtemperatures with two degradation regimes (I and II) with 3 M HCl forfully deacetylated chitosans: Ch1-D2′ (-2-) at 5 °C; Ch1-D2 at: roomtemperature (∼22 °C) (-9-) and 50 °C (-b-); and with 12 M HCl forCh1-D2 at room temperature (∼22 °C) (-]-); (b) at low temperaturesconsidering an alternative model_2 using eq 4 for the calculation of the

hydrolysis rate constants (see text); (c) at high temperatures, displayingthree degradation regimes (I, II, and III) with 3 M HCl for fully deacetylatedchitosan Ch1-D2 at 70 (-4-), 80 (-0-), and 90 °C (-O-).

S ) nw0+ k IIt ) nw

0+ na

0k at + nc

0k ct (7)





The Arrhenius plot of n0k is reported in Figure 8 for thedifferent hydrolysis regimes. The activation energy calculatedin regime I was E I ) 50 ( 2.5 kJ/mol, and considering eq 4 inmodel 2 for the rate constant calculation at low temperatures,it was E I-m2 ) 45 ( 2.2 kJ/mol. In regime II, E II ) 83 ( 16kJ/mol. In regime III, E III ) 80 ( 19 kJ/mol. These valuesshould represent the apparent activation energies for weakglycosidic linkages (regime I), parallel hydrolysis of amorphousand anhydrous forms (regime II), and crystalline domains of the anhydrous allomorph (regime III), respectively. They weremuch lower than 152.2 kJ/mol reported by Vårum et al.13 for

the hydrolysis of chitosan (DA ) 2%) in solution with 0.4 MHCl and were lower than 94.1 kJ/mol reported by Rupley45 for

the hydrolysis of chitin in solution with 11 M HCl. In the solidstate, the macromolecular network induces stresses on thehydrogen bonds and also chain entanglements during hydrolysis,which play a major role in the hydrolysis dynamics bydecreasing the activation energy. In addition, alteration of thisnetwork is largely affected by temperature so that the apparentactivation energies of the degradation of amorphous andcrystalline phases should reflect the evolution of the H-bondnetwork structure together with molecular events yielding

accessibility and ultimately chain scissions. Therefore, the lowest E value corresponded to the hydrolysis of the particularly

Figure 7. Evolution with time of the WAXS diffraction patterns of hydrolyzates prepared by solid-state hydrolysis with 3 M HCl of Ch1-D2 at (a)room temperature (∼22 °C) and (b) 80 °C. The curves were shifted 100 units for clarity. The shift of one curve (25.8 h) in part b was different:150 units for clarity.

Figure 8. Arrhenius plots for the degradation pseudo rate constantsof fully deacetylated chitosan in the solid state, determined from theslopes of the time course of degradation in Figure 6a,c. It allowed usto calculate the activation energies and pre-exponential factors: inregime I (-2- weak links), in regime II (-[- hydrolysis ofamorphous domains, and anhydrous allomorph, see text), and inregime III (-9- anhydrous allomorph). The values of the pseudorate constants of amorphous regions deduced from the differencebetween those obtained in regimes II and III at high temperaturesare also shown (-3-). The pseudo rate constants obtained for lowtemperatures when the alternative model_2 (involving eq 4, see textand Figure 6b) was used revealed similar results (-O- regime I,-0-

regime II).

Figure 9. Scheme proposed for the solid-state acid hydrolysis of fullydeacetylated chitosan in amorphous and crystalline phases showingthe crystallization changes. The number of total chain scissions isthe sum of several contributions: S ) S w + S am + S anh (+S hyd).At the beginning, the hydrolysis concerned weak links of theamorphous regions (S w in regime I). Then, the hydrolysis of theremaining amorphous domains initially less accessible and the in-growing anhydrous allomorph occurred (S am and S anh in regime II).Finally, after an exhaustive hydrolysis of the amorphous regions, thereaction concerned the depolymerization of the crystalline phases,mainly of the anhydrous allomorph (S anh in regime III). The latter

behavior was emphasized at the highest temperatures: 70, 80, and90 °C, absent at 5 °C and room temperature, and less pronouncedat 50 °C for the studied hydrolysis times.







stressed links in the network, namely, the “weak links”. Ourresults allow us to propose a scheme (Figure 9) where the

various degradation regimes are summarized.Role of HCl Concentration: Hydrolysis with 12 M HCl

at Room Temperature. To increase the hydrolyzate crystal-linity, studies at higher concentration in catalyst were performed.Table 6 shows characteristics of Ch1-D2 hydrolyzed with 12M HCl at room temperature. As explained above, the use of 12M HCl actually corresponds to 6.79 M. Results at thisconcentration had similarities with those at room temperaturewith 3 M HCl (Table 2), but the decrease in molecular weightoccurred faster (Figure 2). A monomodal molecular weightdistribution was observed for all hydrolyzates. Again, anhydrouscrystals appeared at the beginning of the hydrolysis. Theirproportion increased up to 32.5% after ∼7 h and then remained

almost constant. Figure 10 shows the variation of CrI duringthe hydrolysis of Ch1-D2 with 3 and 12 M HCl at roomtemperature. For 12 M HCl, CrI increased more rapidly thanfor 3 M HCl. In addition, CrI at the plateau was slightly higher(∼73%). Working with 3 M HCl, r HCl was 1.65 mol/mol andworking with 12 M HCl, r HCl was 8.45 mol/mol, whereas r H2O

was 60 mol/mol for both experiments. The hydrolysis kineticsthus depended mainly on r HCl. In particular, k I increased from7.1 × 10-6 h-1 at [HCl] ) 1.5 M to about 40.3 × 10-6 h-1 at[HCl] ) 6.79 M. This evolution is in agreement with a changein the pre-exponential factor with the HCl concentration.

Conclusions

The solid-state hydrolysis of fully deacetylated chitosan byimpregnating with HCl constitutes an efficient method for

increasing the crystallinity. The kinetics and the evolution of CrI depend both on temperature and HCl concentration.

WAXS of initial raw and deacetylated chitosans only showedpeaks corresponding to the hydrated polymorph. In contrast,after hydrolysis and washings of the fully deacetylated polymer,a peak characteristic of the anhydrous polymorph appeared dueto the recrystallization of mobile chain segments producedduring the hydrolysis of amorphous regions. With 3 M HCl,the highest proportions of anhydrous polymorph were near 32%

at 5 and ∼22 °C, 34% at 50 °C, and 37% at 70, 80, and 90 °C. Alimit range for the apparent crystallite width of this allomorphseemed to be around 5.1 and 7.6 nm, whatever the HClconcentration and temperature used.

At low temperatures (5 and ∼22 °C), using 3 M HCl, it waspossible to increase CrI from 67 to 73%. However, this increasereached a plateau. When the hydrolysis was performed with 12M HCl at room temperature, CrI increased more rapidly, butthe plateau was reached earlier. At 50 °C, with 3 M HCl, CrIincreased only in the earliest times of reaction (up to ∼70%)and then decreased to 65% at 49 h. At temperatures over 50 °C,the decrease was already observed after 1 h. Only CrI valueslower than in the non hydrolyzed chitosan (CrI ) 67%) were

obtained, and the preservation of the crystalline parts seemedto be difficult.In the hydrolysis range from 0 to 50 h, the product obtained

after washings always exhibited a monomodal distribution of molecular weights for the kinetics at 5 °C, room temperature,and 50 °C. In contrast, at higher temperatures (70, 80, and90 °C), a multimodal distribution centered on DPs 90, 50, and16 appeared for the longest times. This was attributed to thehydrolysis of the crystalline parts. Therefore, three phenomenacontributed to the hydrolysis. At the beginning, it concernedweak links or “high defects” of amorphous domains. Then, theremaining amorphous phase and the crystalline phase (mainlythe in-growing anhydrous polymorph) were hydrolyzed inparallel. Then, anhydrous crystallites were mainly degraded at

the highest temperatures and times.To better know the crystallization changes of chitosan and

increase even more its crystallinity, a second and third solid-state acid hydrolysis in a multistep process will be studied in anext paper as the second part of this work.

Acknowledgment. We thank the EC R2 Programme “Polylife”(project II-0259-FA-FDC) for financial support; R. Vera for theWAXS experiments carried out at the Centre de Diffractometrie

Henri Longchambon at the UniVersite Claude B ernard Lyon 1

(http://cdalpha.univ-lyon1.fr/); and P. Calvini (Ministero BeniCulturali, Soprintendenza ai Monumenti, Via Balbi 10, 16126Genova, Italy) for helpful discussions.

Supporting Information Available. Details of the experi-mental conditions choice to perform the hydrolysis of chitosan

Figure 10. Evolution of the crystallinity index CrI during the solid-state hydrolysis of Ch1-D2 with 3 M HCl (-9-) and 12 M HCl (-[-)at room temperature (∼ 22 °C).

Table 6. Characteristics of the Fully Deacetylated Chitosan Ch1-D2 and Hydrolyzed Chitosans Obtained by Solid-State Hydrolysis ofCh1-D2 with 12 M HCl at Room Temperature (∼22 °C)





yield(%)

Ch1-D2 0 1.10 ( 0.03 680 ( 2 1.81 ( 0.02 4.5 ∼0 ∼0 67.1 ( 0.4H1_C 1.4 1.09 ( 0.04 675 ( 3 2.01 ( 0.02 3.5 7.6 29.4 69.9 ( 0.5 94.2H2_C 3.4 1.08 ( 0.04 637 ( 3 2.26 ( 0.02 3.5 6.3 31.0 72.0 ( 0.4 93.3H3_C 6.7 0.93 ( 0.04 578 ( 3 2.13 ( 0.02 n.d.a 5.1 32.5 72.8 ( 0.5 92.0H4_C 11.2 0.77 ( 0.05 479 ( 3 2.40 ( 0.01 n.d. 6.7 32.5 73.0 ( 0.7 90.8

H5_C 25.8 0.70 ( 0.02 435 ( 1 2.55 ( 0.02 n.d. 6.7 33.2 72.9 ( 0.5 88.7H6_C 50.9 0.71 ( 0.02 442 ( 1 2.40 ( 0.02 n.d. 5.4 32.8 73.0 ( 0.4 87.5






in the solid state. This material is available free of charge viathe Internet at http://pubs.acs.org.

References and Notes

(1) Varum, K. M.; Ottoy, M. H.; Smidsrod, O. Water-solubility of partiallyN-acetylated chitosans as a function of pH: effect of chemicalcomposition and depolymerisation. Carbohydr. Polym. 1994, 25, 65–70.

(2) Dubief, D.; Samain, E.; Dufresne, A. Polysaccharide microcrystals

reinforced amorphous poly( β-hydroxyoctanoate) nanocomposite ma-terials. Macromolecules 1999, 32, 5765–5771.(3) Angles, M. N.; Dufresne, A. Plasticized starch/tunicin whiskers

nanocomposite materials. 2. Mechanical behavior. Macromolecules

2001, 34, 2921–2931.(4) Mathew, A. P.; Dufresne, A. Morphological investigation of nano-

composites from sorbitol plasticized starch and tunicin whiskers. Biomacromolecules 2002, 3, 609–617.

(5) Nishiyama, Y.; Langan, P.; Chanzy, H. Preparation of tunicin celluloseI β samples for X-ray and neutron diffraction. Fibre Diffr. ReV. 2003,11, 75–78.

(6) Neville, A. C. Biology of Fibrous Composites; DeVelopment Beyond

the Cell Membrane; Cambridge University Press: New York, 1993; p224.

(7) Revol, J. F.; Marchessault, R. H. In vitro chiral nematic ordering of chitin crystallites. Int. J. Biol. Macromol. 1993, 15, 329–335.

(8) Gopalan Nair, K.; Dufresne, A. Crab shell chitin whisker reinforcednatural rubber nanocomposites. 1. Processing and swelling behavior. Biomacromolecules 2003, 4, 657–665.

(9) Paillet, M.; Dufresne, A. Chitin whisker reinforced thermoplasticnanocomposites. Macromolecules 2001, 34, 6527–6530.

(10) Morin, A.; Dufresne, A. Nanocomposites of chitin whiskers from Riftia

tubes and poly(caprolactone). Macromolecules 2002, 35, 2190–2199.(11) Phongying, S.; Aiba, S. I.; Chirachanchai, S. Direct chitosan nanos-

caffold formation via chitin whiskers. Polymer 2007, 48, 393–400.(12) Struszczyk, M. H. Global requirements for medical applications of

chitin and its derivatives. Pol. Chitin Soc. 2006, 95–102, monographXI.

(13) Vårum, K. M.; Ottoy, M. H.; Smidsrod, O. Acid hydrolysis of chitosans. Carbohydr. Polym. 2001, 46 , 89–98.

(14) Domard, A.; Cartier, N. Glucosamine oligomers: 4. Solid state-crystallization and sustained dissolution. Int. J. Biol. Macromol. 1992,14, 100–106.

(15) Belamie, E.; Domard, A.; Giraud-Guille, M. M. Study of the solid-state hydrolysis of chitosan in presence of HCl. J. Polym. Sci., Part

A: Polym. Chem. 1997, 35, 3181–3191.(16) Lamarque, G.; Cretenet, M.; Viton, C.; Domard, A. New route of

deacetylation of chitins by means of freeze-pump out-thaw cycles. Biomacromolecules 2005, 6 , 1380–1388.

(17) Lamarque, G.; Viton, C.; Domard, A. Comparative study of the firstheterogeneous deacetylation of chitins in a multistep process. Biom-

acromolecules 2004, 5, 992–1001.(18) Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A. Typical

physicochemical behaviors of chitosan in aqueous solution. Biomac-

romolecules 2003, 4, 641–648.(19) Hirai, A.; Odani, H.; Nakajima, A. Determination of degree of

deacetylation of chitosan by 1H NMR spectroscopy. Polym. Bull. 1991,26 , 87–94.

(20) Lamarque, G.; Viton, C.; Domard, A. Comparative study of the secondand third heterogeneous deacetylations of R- and β-chitins in amultistep process. Biomacromolecules 2004, 5, 1899–1907.

(21) Clark, G. L.; Smith, A. F. X-ray diffraction studies of chitin, chitosan,and derivatives. J. Phys. Chem. 1936, 40, 863–879.

(22) Okuyama, K.; Noguchi, K.; Miyazawa, T.; Yui, T.; Ogawa, K.Molecular and crystal structure of hydrated chitosan. Macromolecules

1997, 30, 5849–5855.

(23) Ogawa, K.; Hirano, S.; Miyanishi, T.; Yui, T.; Watanabe, T. A newpolymorph of chitosan. Macromolecules 1984, 17 , 973–975.

(24) Saito, H.; Tabeta, R.; Ogawa, K. High-resolution solid-state 13C NMRstudy of chitosan and its salts with acids: conformational characteriza-tion of polymorphs and helical structures as viewed from theconformation-dependent 13C chemical shifts. Macromolecules 1987,20, 2424–2430.

(25) Ogawa, K. Effect of heating an aqueous suspension of chitosan onthe crystallinity and polymorphs. Agric. Biol. Chem. 1991, 55, 2375–2379.

(26) Okuyama, K.; Noguchi, K.; Hanafusa, Y.; Osawa, K.; Ogawa, K.

Structural study of anhydrous tendon chitosan obtained via chitosan/ acetic acid complex. Int. J. Biol. Macromol. 1999, 26 , 285–93.(27) Focher, B.; Beltrame, P. L.; Naggi, A.; Torri, G. Alkaline N-

deacetylation of chitin enhanced by flash treatments. Reaction kineticsand structure modifications. Carb. Polym. 1990, 12, 405–418.

(28) Federal Institute for Materials Research and Testing (BAM) in Unterden Eichen 87, D-12205 Berlin. http://www.ccp14.ac.uk/ccp/ web-mirrors/powdcell/a_v/v_1/powder/e_cell.html.

(29) Wunderlich, B. Macromolecules Physics; Academic Press: New York,1973; Vol. 1.

(30) Buleon, A.; Chanzy, H.; Froment, P. Single crystals of cellulose IVII:influence of the cellulose molecular weight. J. Polym. Sci.: Polym.

Phys. Ed. 1982, 20, 1081–1088.(31) Chanzy, H.; Dube, M.; Marchessault, R. H.; Revol, J. F. Single crystals

and oriented crystallization of ivory nut mannan. Biopolymers 1979,18, 887–898.

(32) Cartier, N.; Domard, A.; Chanzy, H. Single crystals of chitosan. Int.

J. Biol. Macromol. 1990, 12, 289–294.(33) Tanford, C., Physical Chemistry of Macromolecules; Wiley: New York,

1961.(34) Montroll, E. Molecular size distributions and depolymerization reac-

tions in polydisperse systems. J. Am. Chem. Soc. 1941, 63, 1215–1220.

(35) Sharples, A. The hydrolysis of cellulose. Part 1. The fine structure of Egyptian cotton. J. Polym. Sci. 1954, 13, 393–401.

(36) Daruwalla, E. H.; Narsian, M. G. Detection and identification of acid-sensitive linkages in cellulose fiber substances. Tappi 1966, 49, 106–111.

(37) Sharples, A. Acid Hydrolysis in Alcoholysis. In Cellulose and

Cellulose DeriVatiVes, 2nd ed.; Bikales, N. M., Segal, L., Eds.; Wiley-Intersciences: New York, 1971; Vol. V, pp 991-1006.

(38) Calvini, P. The influence of levelling-off degree of polymerisation onthe kinetics of cellulose degradation. Cellulose 2005, 12, 445–447.

(39) Emsley, A. M.; Stevens, G. C. Kinetics and mechanism of the low-temperature degradation of cellulose. Cellulose 1994, 1, 26–56.

(40) Philipp, H. J.; Nelson, M. L.; Ziifle, H. M. Crystallinity of cellulosefibers as determined by acid hydrolysis. Text. Res. J. 1947, 17 , 585–596.

(41) Yachi, T.; Hayashi, J.; Takai, M.; Shimizu, Y. Supermolecular structureof cellulose: stepwise decrease in LODP and particle size of cellulosehydrolyzed after chemical treatement. J. Appl. Polym. Sci.: Appl. Polym

Sym. 1983, 37 , 325–343.(42) Feller, R. L.; Lee, S. B.; Bogaard, J. The Kinetics of Cellulose

Deterioration. In Historic Textile and Paper Materials: Conser Vation

and Characterization.; Needles, H. L., Zeronian, S. H., Eds.; AmericanChemical Society: Washington, DC, 1986; pp 329-346.

(43) Calvini, P.; Gorassini, A. On the rate of paper degradation: lessonsfrom the past. Restaurator 2006, 27 , 275–290.

(44) Calvini, P.; Gorassini, A.; Merlani, A. On the kinetics of cellulose

degradation: looking beyond the pseudo zero order rate equation.Cellulose 2008, 15, 193–203.(45) Rupley, J. A. The hydrolysis of chitin by concentrated hydrochloric

acid, and the preparation of low-molecular-weight substrate forlysozyme. Biochim. Biophys. Acta, Spec. Sect. Mucoproteins Muco-

polysaccharides 1964, 83, 245–255.

BM1001685


Documents

Macro Molecules 5