Mechanisms Involved During the Ultrasonically Induced

Biomacromolecules 2009, 10, 1203–1211

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Mechanisms Involved During the Ultrasonically Induced Depolymerization of Chitosan: Characterization and Control Simina Popa-Nita, Jean-Michel Lucas, Catherine Ladavie`re, Laurent David, and Alain Domard* Universite´ de Lyon, Universite´ Lyon 1, UMR CNRS 5223 IMP, Laboratoire des Mate´riaux Polyme`res et des Biomate´riaux, Baˆt. ISTIL, 15, bd. A. Latarjet, F-69622 Villeurbanne Cedex, France Received December 12, 2008; Revised Manuscript Received February 16, 2009

The existence of two mechanisms involved in the ultrasonically induced depolymerization of chitosan is evidenced. The first leads to a rapid scission of polymer chains and a lowering of the polydispersity, and the second is responsible for obtaining short polymer chains and oligomers with a polydispersity increase. A systematic experimental study allowed us to identify and quantify the main parameters influencing the chain scission kinetics. Consequently, using a “master curve” approach, a general law of variation of the molecular weight during the depolymerization is proposed. This law can be used in various experimental conditions to easily control the production of chitosan chains of precise length and low polydispersity or a collection of chito-oligosaccharides (COS). Characterization of the latter by 1H NMR and MALDI-TOF mass spectrometry shows their high purity and an unchanged primary structure.

1. Introduction Polysaccharide based multifunctional biomaterials find a continuous and growing interest.1,2 Among polysaccharides, glycosaminoglycans (GAGs) are particularly interesting for their bioactive properties.3,4 In this family, chitin and chitosan show a remarkable potential.5,6 Chitin is widespread in biomass:7 it is present in the cuticles of arthropods, the endoskeletons of cephalopods, the extra-cellular matrices of numerous mushrooms, some yeasts, and algae. Chitosan, a deacetylated derivative of chitin, is a linear copolymer constituted of randomly distributed 2-acetamido-2-deoxy-β-D-glucan (GlcNAc) and 2-amino-2-deoxy-β-D-glucan (GlcN) residues linked together via β,(1f4) glycosidic bonds. Its properties strongly depend on: the degree of acetylation (DA) corresponding to the molar fraction of GlcNAc moieties,8 the molecular weight,9-11 and the polydispersity.12 Therefore, to clearly identify their physical and biological properties, it is of great interest to produce tailor-made chitosans of varying DAs and degrees of polymerization (DP), with a low polydispersity index (Ip). In the biomedical field, chito-oligosaccharides (COS) proved to be more applicable than the corresponding polymer due to their water solubility at any pH and specific bioactivities including: triggering cancer cells apoptosis,13 inhibiting tumor growth,14 immuno-stimulation,15 antimicrobial,6 or antioxidative16 activities. Furthermore, COS are widely used in multicomponent supramolecular systems for drug17,18 or gene19 delivery or as active elicitors.6,20 Among the methods reported to produce chitosans of adjusted molecular weight down to oligomers, chemical hydrolysis and enzymatic treatments are frequently proposed. In the first case, two main routes are followed: a reaction at high temperature in presence of concentrated hydrochloric acid,21-23 or a nitrous deamination at room temperature.24,25 An enzymatic treatment can be carried out using either chitinases26 or chitosanases,27 depending on the initial DA of the polymer. However, all these * To whom correspondence should be addressed. E-mail: alain.domard@ univ-lyon1.fr.

methods require a systematic purification of the resulting products due to the presence of additives used to initiate the reactions. Moreover, the obtained low molecular weight polymers or oligomers show a rather large polydispersity and in the case of the nitrous deamination, a part of the reducing ends are altered.28 As a result, the depolymerization of chitosan by ultrasound treatment was considered as an interesting alternative. Indeed, it was found that such a treatment resulted in the only production of short polymers in the case of different polysaccharides such as xanthan,29 schizophyllan,30 sodium hyaluronate,31 or carboxymethylcellulose.32 Nevertheless, the identification of the precise mechanisms governing this depolymerization is still under debate. At the macroscopic scale, a first general issue is to have an accurate picture of the reaction environment in the reactor, which implies the analysis of the distribution of the ultrasound waves. Experimental approaches based on physical methods, such as calorimetry,33 aluminum foil erosion,34 and thermal probes,35 as well as numerical simulations34,36 have been used to thoroughly study the effects of ultrasounds in different reactor geometries. The general picture of the ultrasonic field shows that the most important information is located around the axial direction (parallel to the probe), with a distribution of separated “active zones”, while in the radial direction (perpendicular to the probe), the intensity of the ultrasound waves decreases steeply.37,38 The configuration of the reactor (liquid level, immersed probe height, probe and reactor diameter, etc.) is found to be a key factor as it determines the energy distribution and in very specific conditions, the whole reactor may behave like a resonator.33 Even if there are still considerable controversies concerning the microscopic mechanisms, it is generally accepted that the process of depolymerization mainly originates from pure mechanical effects,39,40 at least in the low frequency domain, that is, less than 100 kHz.41 Nevertheless, to our knowledge, no accurate demonstration of this statement has been proposed yet.

10.1021/bm8014472 CCC: $40.75  2009 American Chemical Society Published on Web 03/26/2009

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Table 1. Characteristics of the Ultrasound Generatorsa generator

frequency f (kHz)

maximal power Pmax (W)

probe diameter φ (mm)

1 2

20 39

750 100

3 and 25 25

a Frequency (f), maximal electrical input power of the piezoelectric source of ultrasounds (Pmax), and diameter of the probes (φ).

At the level of interatomic distances within the macromolecules, there is still some debate regarding the place where the bond breakage occurs. By analogy with the chemical degradation, it is expected to take place at the weakest links in the polymer backbone, but works42,43 suggested that it mainly occurs at the midpoint of the polymer chains and the existence of a final limiting molecular weight is predicted, below which ultrasounds have no more effect. Several studies propose a random chain breakage but still consider that some bonds are more resistant, in relation to the decrease in the scission rate constant as lower values of DP are reached.44 Concerning the molecular weight distribution, different mathematical models (ex: continuous-distribution kinetics45) were applied to describe its evolution with the sonication time, but generally, a firstorder kinetics equation allows a suitable evaluation of the initial depolymerization rate constants.44,46 In the present paper, we report on an experimental study of the ultrasound treatment of chitosan solutions, highlighting the main parameters influencing the depolymerization kinetics. First, the results enable the identification of two mechanisms involved during the reaction. Their control allows us to obtain chitosan chains of precise degree of polymerization and low polydispersity; or, alternately, a collection of chito-oligosaccharides with a large polydispersity. Moreover, using a “master curve” approach, a general law of the evolution of the molecular weight during the depolymerization is proposed. This law can be used for the production of polymers with controlled degrees of polymerization in various experimental conditions.

2. Materials and Methods Initial Chitosan and Solution Preparation. Chitosan, obtained from chitin of squid pens, was purchased from Mahtani Chitosan Pvt. Ltd. (India). The polymer was purified by dissolution at 0.5% (w/v) in a stoichiometric amount of aqueous acetic acid and then filtration of the resulting solution successively on membranes (Millipore) of porosity: 3, 1.2, 0.8, and 0.45 µm. The polymer was then precipitated with aqueous ammonia. After repeated washings with deionized water, the precipitate was freeze-dried. Unless otherwise specified, the initial sample characteristics are defined by the degree of acetylation: DA ) (1.5 ( 0.1)%, the number-average degree of polymerization, DPn ) (1720 ( 60), and the polydispersity index, Ip ) 1.6 ( 0.1. Chitosan was redissolved in acetic acid aqueous solutions with a stoichiometric protonation of -NH2 sites. For the study of the influence of pH and ionic strength, a 2.6 M acetic acid/2 M ammonium acetate buffer (pH ) 4.7) was used. Another chitosan solution with a pH adjusted to 1.9 by adding acetic acid in excess was also prepared. pHs were measured using a PHM210 pH meter (MeterLab, Radiometer Analytical, France). Experimental Ultrasound Setups. Two ultrasound generators were used: (1) Sonics Vibra Cell (Fisher Scientific Bioblock, France) and (2) Lixea formulator type BA (Sinaptec SA, France). Their characteristics are reported in Table 1. The depolymerization was performed in two glass reactors (except during the study of the influence of the reactor geometry): (I) a cylindrical reactor of diameter Φ ) 3.5 cm and a maximum liquid height Hmax ) 7 cm; (II) a frusto-conical reactor with base diameters of 4 and 8 cm and a height Hmax ) 5 cm.

Popa-Nita et al. Solutions were homogenized by magnetic stirring, and the reactor temperature was kept constant during experiments thanks to a water circuit. Analytical Procedure. The depolymerization kinetics was followed from the decrease in the number average degree of polymerization (DPn) as a function of time. The average molecular weights and polydispersity index were determined thanks to a system coupling on line: size exclusion chromatography (SEC) columns (Protein Pack Glass 200SW (Waters) and TSKgel G6000PW (Tosohaas)) with a differential refractometer (Waters R 410, from Waters-Milipore) and a multiangle laser-light scattering detector operating at 632.8 nm (Wyatt Dawn DSP). A 0.15 M ammonium acetate/0.2 M acetic acid buffer (pH ) 4.5) was used as eluent at a flow rate of 0.5 mL/min. This online setup allowed the simultaneous determination of the concentration and molecular weight on each eluting time. Thanks to light scattering measurements, the results were absolute and independent of any calibration or reference standards. The refractive index increment dn/dc necessary for the calculations was determined in a previous study8 at 0.198 mL/g. Data collection and processing were driven by the Wyatt Technology Corporation ASTRA 4.73.04 software. Taking into account that in a SEC separation, each elution volume contains molecules of a single molecular weight, or at least a very narrow distribution, and then processed data allowed the calculation of the molecular weight (number and weight averages) and polydispersity index of the polymers. In all cases, monomodal chromatograms were obtained and over a wide range of elution volumes, a linear relationship between the logarithm of the molecular weight and the elution volume was registered (see Supporting Information). Separation and Purification of Chito-oligosaccharides. After precipitation with aqueous ammonia of high molecular weight polymer chains still present in solution (DPn ∼ 25), oligomers (DPn < 12) were recovered by centrifugation. Remaining salts were removed by ultrafiltration against deionized water through a YC05 membrane (MwCO 500, Millipore), responsible for the full elimination of the monomer and a partial loss of the dimer. Finally, pure COS were recovered after freeze-drying. 1 H NMR Spectroscopy. A total of 10 mg of chito-oligosaccharides were dissolved in 1 mL of D2O, and the solution transferred to 5 mm diameter NMR tubes. Measurements were performed on a Bruker ALS 300 spectrometer (300 MHz) at 25 °C. All chemical shifts were determined relative to the signal of HOD (δ ) 4.80 ppm). MALDI-TOF Mass Spectroscopy. MALDI-TOF mass spectra were acquired with a Voyager-DE STR (Applied Biosystems, Framingham, MA) equipped with a nitrogen laser emitting at 337 nm with a 4 ns pulse. The instrument was operated in the linear mode. The positive ions were detected in all cases. An external mass calibration of mass analyzer was used (mixture of peptides from Sequazyme standards kit, Applied Biosystems, Framingham, MA). The matrix used for the experiments was 2,5-dihydroxybenzoic acid (DHB) purchased from Sigma-Aldrich (St Louis, MO); no further purification of the matrix was operated. The solid matrix and chito-oligosaccharide samples were dissolved at 10 g/L and 1 g/L in water, respectively. A 10 µL volume of the matrix solution was then mixed with 10 µL of chito-oligomer solution. An aliquot of 1 µL of the resulting solution was spotted onto the MALDI sample plate and air-dried at room temperature.

3. Results and Discussion 3.1. General Aspects of the Depolymerization Kinetics: Modeling. When ultrasound waves are applied to a chitosan solution of high initial DP (DPn(0) > 1000), a rapid decrease of the degree of polymerization is observed, followed by a slower kinetics (Figure 1a). The effect of ultrasounds on the chitosan molecular dimensions was expressed as the difference between the reciprocal number-average degree of polymerization at time t, 1/DPn(t), and at the beginning of the treatment, 1/DPn(0) (Figure 1b). By analogy with the results of Montroll47

Ultrasonically Induced Depolymerization of Chitosan


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Figure 2. DPn and corresponding Ip of ultrasonically depolymerized chitosans in different experimental conditions (Table 2). All reactions were conducted for 6 h. Solid and dotted lines are guides for the eye. The inset represents a closer view of the low DP range of the main plots. Table 2. Experimental Conditions of the Different Depolymerization Reactions Presented on Figure 2a plot

reactor

V (mL)

cp (% w/w)

φ (mm)

P (W)

f (kHz)

A B C D E

I II II II II

20 100 100 100 100

0.5 0.25 0.25 0.25 0.25

3 3 25 25 25

285 285 285 100 100

20 20 20 20 39

a V, solution volume; cp, polymer concentration; φ, probe diameter; P, ultrasound input power; f, ultrasound frequency.

Figure 1. Evolution of the chitosan number-average degree of polymerization, DPn (a), and 1/DPn (b) during ultrasound treatment. Solid curves represent the data fit using eq 1 with n0 ) 0.013 and k ) 0.33 h-1. The value of reduced χ2 was around 3 × 10-4 (The reaction was carried out using the ultrasound generator 1, with P ) 285 W and φ ) 25 mm, in the reactor II. The solution characteristics were V ) 100 mL and cp ) 0.25% (w/w)).

concerning chemical depolymerization reactions of polydisperse systems, one can write

1 1 ) n0(1 - e-kt) DPn(t) DPn(0)

(1)

where n0 is the pre-exponential factor representing the fraction of glycosidic bonds available for degradation48 and k is the depolymerization rate constant. From a chemical point of view, the glycosidic bonds present on chitosan chains could be considered as equivalent (only β,(1f4), in a perfectly linear copolymer). Nevertheless, this is not fully exact if we consider the role of the location of these bonds with four possible linkages between two successive residues: DD, DA, AD, and AA, with A and D corresponding to an acetylated and deacetylated residue, respectively. Moreover, from a physicochemical point of view, glycosidic bonds are not all similar because the studied solutions are initially over the critical concentration of chain entanglement and also because the local conformation of polymer chains is determined by their persistence length, and finally, the global conformation of chitosan chains is quite complex and can be viewed through the pearl-necklace model with a succession of stiff and soft domains.49 All these considerations justify that from a statistical point of view, we may consider that glycosidic linkages are not equivalent and then n0 should be different from 1, especially in the case of

sufficiently long chains. In addition, the pre-exponential factor n0 is related to a limit degree of polymerization as

n0 )

1 1 DPn(t f ∞) DPn(0)

(2)

This first-order mechanism (eq 1) allows the interpretation of the experimental data (Figure 1). At a sonication time close to 360 min, a value of 1/DPn(t) - 1/DPn(0) of about 1.1 × 10-2 is reached, representing a DPn of about 85. This mechanism is characterized by a rate constant of 0.33 h-1 and a preexponential factor, n0, of 0.013, corresponding to a limit DPn close to 80 (see the modeling in Figure 1). A more detailed analysis of the evolution of the molecular weight distribution during ultrasound treatment showed that for short sonication times, the rapid decrease of DPn is accompanied by a lowering of the polydispersity index Ip (see plots in Figure 2). However, a second regime characterized by a slower kinetics and an increase of Ip is subsequently observed (plots C-E in Figure 2). Short polymer chains and oligomers of high polydispersity are then obtained. Depending on experimental conditions, this second regime becomes predominant at different treatment times, and thus, the lowest value of Ip depends on experimental conditions. For more clarity, a complete description of the experimental conditions of reactions leading to the results reported in Figure 2 is shown in Table 2. Two regimes are distinctly observed in Figure 2 related to the existence of two depolymerization mechanisms. The first induces a rapid and specific scission of the polymer chains with a polydispersity lowering down to a limit DP (Figure 1) resulting from the value of n0 and eq 2. This has to be related to the fact that polymer chains respond to the ultrasound field through brittle glycosidic bonds corresponding to a specific size. The

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1 1 ) k2t DPn(t) DPn(0)

Figure 3. Variation of 1/DPn as a function of the sonication time: (9) experimental data, (dashed and dotted curves) depolymerization mechanisms following eq 1 with n10 ) 0.013 and k1 ) 0.9 h-1; and n20 ) 1 and k2 ) 3.2 × 10-3 h-1, respectively. The solid black curve represents the fit using eq 3 as a sum of the two depolymerization mechanisms. The reaction conditions are listed in Table 2, plot E.

further formation of chains of lower size is then explained by a second mechanism, for which all glycosidic bonds are equivalent in energy. This leads to the production of short polymer chains and oligomers of high polydispersity. Furthermore, results presented in Figure 2 clearly show that ultrasound effects on macromolecules depend strongly on intrinsic parameters like acoustic (ultrasound power and frequency or probe diameter), geometry (type of reactor), and conditional parameters (solution volume, polymer concentration). Thus, when a given probe of low diameter is used (plots A and B in Figure 2 obtained with φ ) 3 mm), the first mechanism is predominant for a long time, related to mild experimental conditions. Chitosans with very low polydispersity (Ip ∼ 1.1) can then be obtained. The reactions were stopped after 6 h of treatment and the second kinetics domain could not be seen in such conditions. When a large diameter probe is used (plots C-E in Figure 2 obtained with φ ) 25 mm), both regimes are observed, regardless other parameters. Moreover, if the ultrasound frequency is 39 kHz, the depolymerization is very fast, and for the same treatment time (6 h), a collection of polymers of low DPn (∼25) and oligomers is produced (plot E in Figure 2). In these conditions, the whole experimental data can no longer be interpreted using only the simple theoretical model proposed above (eq 1), as no limit DPn exists (the production of monomers is evidenced). A second exponential term taking in consideration the second mechanism must be added. During this regime, all glycosidic bonds are equally available for degradation and thus, n20 ) 1. The results can then be interpreted using the following equation

1 1 ) n01(1 - e-k1t) + n02(1 - e-k2t) (3) DPn(t) DPn(0) where k2 represents the rate constant of the second mechanism. For example, a combination of a first mechanism of preexponential factor n10 ) 0.013 and a rate constant k1 ) 0.9 h-1 (dashed curve in Figure 3) with a second mechanism considering all bonds equally accessible (n20 ) 1) and a rate constant k2 ) 3.2 × 10-3 h-1 (dotted curve in Figure 3) is suitable to fit the experimental results obtained at f ) 39 kHz. It is interesting to notice that k2 , k1 and thus for the treatment times considered, the second term of eq 3 can be simplified to yield the following kinetics equation

(4)

The two depolymerization mechanisms coexist from the beginning of the reaction but the first stays predominant. Once a limit DPn of about 80 is reached, this mechanism levels off and the second takes over. This induces the production of low DP polymer chains and oligomers of high polydispersity. When the depolymerization reactions are conducted using ultrasounds of frequency 20 kHz, the presence of the second mechanism is clearly evidenced by the increase of the sample polydispersity (see plots C and D in Figure 2). However, the corresponding rate constant k2 is rather low and the precision of the experimental results does not allow its accurate determination. Therefore, in order to interpret the obtained data for f ) 20 kHz, we neglected the contribution of the second mechanism and only used eq 1. 3.2. Sonication-Induced Depolymerization: A Purely Mechanical Process. Temperature was often thought to play a role in the ultrasound treatment of macromolecular solutions; some authors44,50 stated that sonication-induced depolymerization rate constants decrease when temperature increases but opposite or nonsystematic trends were also found between 0 and 50 °C.51 To our knowledge, no in-depth study of the activation energy of the process was carried out yet. We studied the influence of temperature at 5, 18, 45, and 85 °C. Thus, solutions of chitosan concentration cp ) 0.5% (w/w) were sonicated in the reactor I, using the ultrasound generator 1 working at an input power of 285 W with a 3 mm diameter probe. The values of k were calculated using eq 1 and the results are presented in Figure 4 (Arrhenius plot). Only slight differences are observed in the calculated apparent rate constants. The activation energy (Ea) of the depolymerization process was estimated from the Arrhenius equation

k ) Ae-Ea/RT

(5)

where A represents the pre-exponential factor and R, the gas constant. We obtained an activation energy close to zero and a value of A of about 9.6 × 10-5 s-1. The activation energies for chitin52 and chitosan21 acid hydrolysis in solution vary between 124 and 158 kJ mol-1, depending on DA and reaction conditions. Compared to these values, we conclude that the ultrasonic degradation of chitosan can be considered as not thermally activated and thus corresponds to a pure mechanical rupture of glycosidic bonds. In this demonstration we only considered the first mechanism; nevertheless, the second was negligible for all the temperatures studied and we may conclude that in the investigated conditions, the second mechanism is also not thermally activated. The physical interpretation of the pre-exponential factor is not straightforward. Indeed, it cannot represent the frequency of collisions between two molecules in the proper orientation necessary to the reaction. As illustrated below, taking into account the complexity of the sonication process and the systems studied here, an exact interpretation of the obtained value cannot be proposed yet. 3.3. Depolymerization Reaction in the Presence of Acetic Acid. Ultrasounds have already been used to amplify the chitosan acidic hydrolysis,42 but in such experiments the boundary between the two degradation processes was not clearly investigated. In the present study, chitosan ultrasound depolymerization reactions in acetic acid aqueous solutions of pH


Figure 4. Arrhenius plot displaying the dependence of the ultrasonic degradation rate constant k as a function of the reciprocal absolute temperature 1000/T. The concentration of chitosan acetate in solution was 0.5% (w/w). The reactions were conducted in the reactor I, using the ultrasound generator 1, with P ) 285 W and φ ) 3 mm.

Figure 5. Variation of 1/DPn as a function of the sonication time for chitosan solutions of different pHs and ionic strengths (I). Solid curves represent the data fit using eq 1: in all cases, n0 ) 0.006 and (b) k ) 0.38 h-1; (gray triangle) k ) 0.37 h-1; (gray square) k ) 0.32 h-1. In all cases, the value of reduced χ2 was below 10-3. The concentration of polymer in solution was 0.5% (w/w) and the reactions were conducted in the reactor I, using the ultrasound generator 1, with P ) 285 W and φ ) 3 mm.

1.9 and 5 (the latter corresponds to the stoichiometric protonation of -NH2 sites) were compared. Solutions of chitosan concentration cp ) 0.5% (w/w) were then sonicated in reactor I, using the ultrasound generator 1 working at an input power of 285 W with a 3 mm diameter probe. Figure 5 represents the results expressed as the variation of 1/DPn as a function of the sonication time. In all cases, a first-order kinetics equation (eq 1) fitted accurately the experimental data (solid curves in Figure 5). A pre-exponential factor of 0.006 was calculated from the fit of the experimental data in the whole pH range, standing for a limit DPn close to 170. As discussed in section 3.4, the vertical geometry of the reactor influences the limit degree of polymerization, which accounts for the difference between n0 values calculated for reactions taking place in reactors I and II. Adding acetic acid in excess in order to decrease pH was accompanied by a change of ionic strength. For pH ) 1.9, the contribution to the ionic strength, I, due to acetic acid was evaluated to 4.6 M. An acetic acid/ammonium acetate buffer of the same I but of higher pH (4.7) was then used as solvent for a depolymerization test (solid circles in Figure 5). The comparison between these series of experiments shows that the


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Figure 6. Schematic representation of the parameters influencing the depolymerization kinetics: P, ultrasound electrical input power; f, ultrasound frequency; φ, probe diameter; H, liquid depth; h, immersed probe height; D, distance between the end of the probe and the bottom of the reactor; Φ, reactor diameter; cp, polymer concentration in solution.

weak difference in kinetics measured for pH ) 1.9 (k ) 0.38 h-1) and 5 (k ) 0.33 h-1) is induced by the corresponding change in ionic strength while pH does not play any role in the range investigated here. Indeed, the treatment of solutions of the same ionic strength (4.6 M) but different pHs (1.9 and 4.7, respectively) did not show any significant variation. Hence, in the conditions chosen here, the acidic hydrolysis was insignificant compared to the ultrasound depolymerization. Moreover, on increasing the ionic strength from 0.03 to 4.6 M, the scission kinetics was only slightly faster, possibly due to a decrease of polymer chain stiffness. 3.4. Experimental Parameters Influencing the Depolymerization Kinetics: Master Curve Approach. Aiming at controlling the ultrasonic depolymerization of chitosan for a further generalization, we studied the role of experimental parameters acting on the reaction kinetics. As seen in section 3.3, pH and ionic strength, known to influence physicochemical properties of polyelectrolyte solutions, had no significant role. Contrarily, the reactor geometry and acoustic parameters largely influenced the mechanisms of depolymerization (Figure 2). The energy distribution of the ultrasonic field in the reactor depends on its geometry53 (Φ, reactor diameter) and the configuration33 (H, liquid level; h, immersed probe height; D ) H - h, distance between the probe end and the reactor bottom; see Figure 6 for a schematic illustration of the experimental setup). Moreover, the ultrasound electrical input power (P) and the probe diameter (φ) are important factors characterizing the ultrasonic field. The polymer concentration (cp) also influences the rate and extent of the ultrasonic degradation.39,51 The role of all these parameters on the depolymerization kinetics was then quantified. Reactor Geometry. In a first series of experiments, chitosan acetate solutions of constant concentration (0.25% (w/w)) were sonicated in cylindrical glass reactors with different internal diameters; the vertical geometry of the cell was constant with: D ) (2.3 ( 0.1) cm and h ) (1.7 ( 0.1) cm. The generator 1 was used with a 25 mm probe and a 285 W input power. The results were analyzed and compared from the variation of 1/DPn versus time (Figure 7). As observed in Figure 7, the larger the reactor diameter, the slower the depolymerization reaction. During these experiments, the vertical geometry of the reactor was kept constant. Then, to have the same distance between the probe end and the reactor bottom, different solution volumes were treated. The degradation kinetics is slower when the volume and implicitly the number of polymer chains increase. To build a master curve of the sonication kinetics, we considered a reference internal reactor diameter Φref of 3.9 cm

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Figure 7. Variation of 1/DPn as a function of the sonication time for treatments made in cylindrical reactors of different internal diameters (Φ). a(Φ1) ) 2.5 and a(Φ2) ) 7.9 represent time shift factors allowing the building of a “master curve” at a reference internal reactor diameter Φref of 3.9 cm. In the reference conditions, data fit using eq 1 gives n0 ) 0.013 and k ) 0.81 h-1. Other experimental conditions were f ) 20 kHz, P ) 285 W, φ ) 25 mm, D ) (2.3 ( 0.1) cm, h ) (1.7 ( 0.1) cm, and cp ) 0.25% (w/w).

and calculated the time shift factors a(Φ) necessary to shift the data obtained at Φ1 ) 6.7 cm or Φ2 ) 11 cm in correspondence with the reference data, in the normalized time scale t/a(Φ). We found

a(Φ) )

( ) Φref Φ

R

(6)

with R ) -1.9 ( 0.2. This expression can be justified assuming a rate constant of depolymerization proportional to VA/VT, with VA, the “active volume”, that is, the fraction of the reactor volume in which depolymerization occurs, and VT, the total solution volume. For all experiments compared here, VA is constant as the acoustic parameters and the solution properties are invariable; and VT is proportional to Φ2, as the liquid level was also constant. Using reduced time scales t/a(Φ) for data 1 and 2, we obtained the “master curve” (solid black curve in Figure 7) overlaying all the normalized and reference data. Here also a first-order kinetics equation (eq 1) fitted well the results (χ2 < 10-3). In all cases, a pre-exponential factor of 0.013 was calculated and the ratios between the different rate constants were in agreement with the corresponding shift factors. For the following studies, when possible, this approach of building a “master curve” was used and the quality of the fits was checked by the value of the regression coefficients χ2 that usually stayed below 10-3. Studies of the distribution of the ultrasonic field in different reactors showed that in the direction perpendicular to the probe axis, ultrasound effects decrease sharply.38 Consequently, changes of geometry in this direction do not significantly disturb the ultrasound field but influence the degradation kinetics with the change in solution volume. Furthermore, it was demonstrated that ultrasounds mainly propagate in the axial direction of the probe.37 Hence, we tested the influence of changing the reactor vertical geometry. For that, chitosan acetate solutions of constant concentration (0.25% (w/ w)) and of different volumes were treated in the same cylindrical reactor (diameter: Φ ) 6.7 cm). The probe-immersed height (h) was kept constant. The ultrasound generator 1 was used with a 25 mm probe and a 285 W input power. The variation

Figure 8. Variation of 1/DPn as a function of the sonication time for treatments performed in the same cylindrical reactor with different distances between the probe end and the bottom of the reactor (D). Black and gray curves represent data fits using eq 1 with: (1) n0 ) 0.013 and k ) 0.32 h-1; and (2) n0 ) 0.009 and k ) 0.33 h-1. Other experimental conditions were: f ) 20 kHz, P ) 285 W, φ ) 25 mm, Φ ) 6.7 cm, h ) (1.7 ( 0.1) cm, and cp ) 0.25% (w/w).

of 1/DPn with the reaction time for two values of the distance between the probe extremity and reactor bottom: D ) (2.3 ( 0.1) cm and (5.3 ( 0.1) cm, respectively, are presented in Figure 8. The modification of the vertical geometry induces a change in the ultrasound field distribution within the reactor. Hence, macromolecules respond differently to the new sonication field leading to a variation of the number of glycosidic bonds available for degradation. Indeed, experimental results could be fitted using eq 1 but the calculated accessibilities were close to 0.013 and 0.009 for D ) D1 and D ) D2, respectively (Figure 8). For the simplest reactor geometries filled with water, experimental studies and numerical simulations of the distribution and effects of ultrasounds appeared rather complex.34,36 Only a detailed knowledge of the ultrasound interference (including reflections and superimposition effects) and attenuation within the reactor could predict the intensity distribution, crucial for the calculation of global effects. In addition, small changes in the geometry (probe position, liquid level) induce a large variation of the ultrasound distribution.33 Here also, the kinetics is affected by the reactor geometry related to the variation of D. A “master curve” regrouping experimental data is therefore possible only if this parameter is constant. Consequently, in the present study, series of experiments were operated in the same configuration of the reactor (reactor shape, liquid depth, immersed probe height) and only results from comparable reactions were analyzed. Acoustic Parameters. The effect of the ultrasound input power (P) and probe diameter (φ) on the depolymerization kinetics was also investigated. A total of 100 mL of chitosan acetate solutions of concentration 0.25% (w/w) were treated using the generator 1 and the reactor II. Results were analyzed from the variation of 1/DPn as a function of the reaction time (Figure 9). Equation 1 was used to fit the experimental data and master curves were built as above. As observed in Figure 9, the depolymerization kinetics increases with P and φ. Reference experimental conditions were defined at P ) 675 W and φ ) 25 mm; time shift factors allowing to build “master curves” corresponding to the other depolymerization data were again calculated



a(P) )

( )

(7)

a(φ) )

( )

(8)

Pref P φref φ

β

δ

with β ) 1.4 ( 0.2 and δ ) 1.0 ( 0.1. The physical meaning of these mathematical forms is complex. Studies of the role of P on the distribution and effects of the ultrasound field are numerous.53 Observations vary according to the power interval investigated but also with the type and size of the probe. For a reactor with an immersed probe, as in our case, Contamine et al.37 found that at power inputs of about 200 W, the intensity diminishes with the distance from the probe, whereas at lower powers standing waves exist, thus giving an oscillatory profile of the intensity from the probe end down to the reactor bottom. For a probe of diameter comparable to ours and for P ≈ 250 W, a fluid turbulence near the radiating surface was described, while for lower powers, a cone-like bubble structure was visible.54 Hence, the “active volume” and consequently the degradation reaction depend strongly, in a complex way, on P and φ. For example, in the latter case,

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considering as above the ratio VA/VT, we could predict a time shift factor proportional to the probe surface and consequently, to φ2. But here, a roughly linear dependence is found, which remains unexplained. Nevertheless, experimental data can be phenomenologically used to build a “master curve”. Polymer Concentration in Solution. The rate and extent of ultrasonic degradation are closely related to the concentration of macromolecules in solution (cp).39,51 Experiments done for cp between 0.25 and 2% (w/w) clearly illustrated that the more concentrated the solution, the slower the depolymerization reaction (results not shown). As above, data were analyzed as the variation of 1/DPn as a function of the sonication time allowing the construction of a “master curve” using time shift factors given by

a(cp) )

( ) cp,ref cp

ε

(9)

with ) -0.7 ( 0.1. The interpretation of this result remains difficult because on increasing the solution concentration, different effects sum up. At a macroscopic scale, the homogenization of the solution becomes more difficult and ultrasound waves have a shorter outreach, thus leading to slower polymer degradation. At a nanoscopic scale, chain entanglements should also be considered. Indeed, polymers form a network with a density of entanglements increasing with the concentration and therefore their response to ultrasounds changes. Our results allowed the identification and quantification of the main parameters influencing the depolymerization kinetics. In the case of the first mechanism, the number of glycosidic bonds available for degradation (n10) was found to be characteristic of the considered polymer and to depend on the vertical geometry of the reactor only (liquid and probe level: h, D). The scission rate constant k1 increased with the ultrasound input power (P) and the probe diameter (φ) and decreased with the solution concentration (cp) and the reactor diameter (Φ). A second mechanism with an “accessibility” of 1 played a key role in the depolymerization reactions conducted at the highest frequency studied (39 kHz), but from a kinetic point of view, it could be neglected at lower frequency (20 kHz). These results (eqs 6-9) allow the building of a “master curve”, predicting the variation of the chitosan molecular weight during the depolymerization

1 1 ) n01 × (1 - e-k1t) + n02 × (1 - e-k2t) DPn(t) DPn(0) (10) where,

ki ) Figure 9. Variation of 1/DPn as a function of the sonication time for treatments using the ultrasound generator 1, in the reactor II on solutions of V ) 100 mL and cp ) 0.25% (w/w). Other experimental conditions were (a) φ ) 25 mm, different electrical input powers; (b) P ) 285 W, different probe diameters. a(P1) ) 3.6, a(P2) ) 11.1 and a(φ1) ) 9.2 represent time shift factors allowing the building of “master curves” at the reference power 675 W and φ ) 25 mm, respectively. In the reference conditions, data fit using eq 1 gives (a) n0 ) 0.013 and k ) 1.2 h-1 and (b) n0 ) 0.013 and k ) 0.33 h-1.

ki,ref ai(Φ, P, f, φ, cp)

) ki,ref

( )( )( )( )( ) Φ Φref

Ri

P Pref

βi

f

fref

γi

φ φref

δi

cp

cp,ref

εi

(11)

with i ) 1 or 2, n10, ki,ref ) f (polymer,D,h), n20 ) 1; Ri ) -1.9 ( 0.1; β1 ) 1.4 ( 0.2; γ1 ) 3.1 ( 0.2; δ1 ) 1.0 ( 0.1; ε1 ) -0.7 ( 0.1. Values of the exponents (R - ε) were determined for the first mechanism. Taking into account that for constant acoustic

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Popa-Nita et al.

Figure 10. 1H NMR spectra of fully deacetylated chito-oligosaccharides. R or β, reducing end of the R or β anomer residue; m, middle residue; and n, nonreducing end residue.

Figure 11. Positive ion MALDI-TOF mass spectrum of the mixture of deacetylated chito-oligosaccharides in linear mode (with DHB matrix). (*) Peak corresponding to an impurity from the analyses conditions. The assignments of peaks are described in the text.

parameters, the “active volume” is identical, we may assume that the effect of the reactor diameter quantified for the first mechanism (R1) follows the same variation for the second, thus, R1 ) R2. Further studies on the expression of k2 are ongoing. To use this general law (eqs 10 and 11), one has to perform a first reaction in well-defined reference conditions, allowing the determination of n10 and ki,ref. Subsequently, eqs 10 and 11 can be used to predict and control the scission reactions in various configurations of the reactor and for different ultrasound characteristics. The second term of eq 10, related to the second mechanism is significant only when high frequencies are used to produce chito-oligosaccharides. 3.5. Characterization of Chito-oligosaccharides (COS) Obtained by Ultrasound Treatment. The characterization of COS obtained by ultrasonic depolymerization of a fully deacetylated chitosan (i.e., DA ) 0%) dissolved in a hydrochloride aqueous solution is presented below. 1H NMR spectroscopy analysis was performed and as shown in Figure 10, the different chemical shifts are in good agreement with those of chitosan oligomers from the literature.55 The distribution of a COS mixture isolated as described in Materials and Methods was also analyzed by MALDI-TOF mass spectroscopy (Figure 11). The goal of this analysis was to

determine if the chemical structure of the residues was preserved. Chito-oligosaccharides with different DPs are clearly resolved and the mass difference between two neighboring peaks is 161 mass units, corresponding to one GlcN repeating unit (C6H11O4N). The end-groups, deduced from different masses, correspond well to the expected ones (H and OH groups). The mass spectrum proves the perfect preservation of the residues chemical structure after the sonication treatment. The DP distribution ranged from 2 to 11 with a maximum population at DP ) 3 (from calculation, 3 × 161.16 (C6H11O4N) + 1.00 (H) + 17.00 (OH) + 22.99 (Na, the cationization ion) ) 524.5 mass units; from experiment, 524.7 mass units). The calculated number-average degree of polymerization of the studied fraction was found to be equal to DPn ) 4.7 and the polydispersity index Ip ) 1.2.

Conclusion The ultrasound-assisted depolymerization of chitosan consists in two mechanisms not thermally activated. The first induces a rapid and specific scission of polymer chains and a lowering of their polydispersity, the second independent process, much


slower in the experimental conditions explored, is responsible for obtaining short polymer chains and oligomers of high polydispersity. A mathematical equation predicting the effect of these mechanisms on the chitosan molecular weight was proposed. Hence, this study allows us not only to enlighten but also to quantify the mechanisms involved during the ultrasound treatment of macromolecules. One can thus anticipate the production of sonicated chitosans and chito-oligosaccharides in various experimental conditions. In addition, the resulting molecules are shown to be pure with an unchanged chemical primary structure. Acknowledgment. We thank Laurence Marmuse and Stephane Trombotto for their helpful recommendations and discussions. We are grateful for the technical assistance of Agne`s Crepet. These studies are part of the NanoBioSaccharides project from the 6th European Framework Program. Supporting Information Available. Representative results of the method used to fit the logarithm of the weight-average molecular weight as a function of the eluted volume as determined by ASTRA software. This material is available free of charge via the Internet at http://pubs.acs.org.

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Mechanisms Involved During the Ultrasonically Induced

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