Hierarchical Structure and Physicochemical Properties of Plasticized

Article pubs.acs.org/Biomac

Hierarchical Structure and Physicochemical Properties of Plasticized Chitosan Qingkai Meng, Marie-Claude Heuzey,* and Pierre J. Carreau Department of Chemical Engineering and Research Center for High Performance Polymer and Composite Systems (CREPEC), Polytechnique Montreal, C.P. 6079, succ. Centre-Ville, Montreal, QC H3C 3A7, Canada ABSTRACT: Plasticized chitosan with hierarchical structure, including multiple length scale structural units, was prepared by a “melt”-based method, that is, thermomechanical mixing, as opposed to the usual casting-evaporation procedure. Chitosan was successfully plasticized by thermomechanical mixing in the presence of concentrated lactic acid and glycerol using a batch mixer. Different plasticization formulations were compared in this study, in which concentrated lactic acid was used as protonation agent as well as plasticizer. The microstructure of thermomechanically plasticized chitosan was investigated by X-ray diffraction, scanning electron microscopy, and optical microscopy. With increasing amount of additional plasticizers (glycerol or water), the crystallinity of the plasticized chitosan decreased from 63.7% for the original chitosan powder to almost zero for the sample plasticized with additional water. Salt linkage between lactic acid molecules and amino side chains of chitosan was confirmed by FTIR spectroscopy: the lactic acid molecules expanded the space between the chitosan molecules of the crystalline phase. In the presence of other plasticizers (glycerol and water), various levels of structural units including an amorphous phase, nanofibrils, nanofibril clusters, and microfibers were produced under mechanical shear and thermal energy and identified for the first time. The thermal and thermomechanical properties of the plasticized chitosan were measured by thermogravimetric analysis, differential scanning calorimetric, and DMA. These properties were correlated with the different levels of microstructure, including multiple structural units.

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INTRODUCTION Chitosan, a linear copolymer of β-(1-4) linked 2-acetamido-2deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glycopyranose, is derived from chitin, which is the second most abundant polysaccharide in nature and widely distributed in natural sources, such as crustaceans, insects, marine diatoms, algae, fungi, and yeasts.1,2 Chitosan is a nontoxic, biodegradable, and biocompatible pseudonatural cationic polymer with antibacterial activity and can be used in a wide range of applications in the areas of biomedicine, drug delivery systems, hydrogels, water treatment, membranes, food packaging, and so on.3,4 Chitosan shows antimicrobial activity because of the positive charge on the C2 of the glucosamine monomer.5 Chitosan films and coatings for food packaging applications have been reported to show antimicrobial activity against a wide variety of microorganisms including fungi and some bacteria.6 The use of plastics such as polyethylene (PE), polyvinyl chloride (PVC), polyvinyl alcohol (PVA), polylactide (PLA), nylon, and others in food packaging has continued to increase due to the low cost of materials and functional advantages, such as thermosealability, microwavability, optical properties, and unlimited sizes and shapes.7 Chitosan can be used as an antimicrobial additive in synthetic polymers to prepare antimicrobial food packaging materials. Composite films, made from chitosan and PLA by solution mixing and film casting, showed a great advantage in preventing the surface © 2014 American Chemical Society

growth of mycotoxinogen strains; however, because the composite films obtained by solution mixing were heterogeneous and highly water sensitive, the physicochemical properties of the films were inferior to the original PLA films.8 In another study, chitosan was blended with nylon-6 by solution casting and the chitosan endowed the nylon-6 with antibacterial properties.9 Nowadays, solution mixing and casting procedure is the most popular method to prepare plasticized chitosan and chitosan/ synthetic polymer blends. Chitosan acidic aqueous solution with or without plasticizers can be directly cast into chitosan films or chitosan/synthetic polymer blends when another synthetic polymer is mixed in. Without plasticizers, solutioncast chitosan forms crystals in different polymorphs (hydrated, anhydrous, or combined hydrated and anhydrous polymorphs) and crystallinity depending on acid type and preparation procedure.10 Because chitosan films are rigid and brittle in nature, plasticizers, for example, glycerol and sorbitol, are generally used during solution casting to improve the flexibility and processability of the chitosan films. The tensile strength and elongation at break generally decrease and increase, respectively, with increasing plasticizer concentration.11,12 Received: December 6, 2013 Revised: February 23, 2014 Published: February 24, 2014 1216

dx.doi.org/10.1021/bm401792u | Biomacromolecules 2014, 15, 1216−1224

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Table 1. Samples Designation and Corresponding Compositions designation

chitosan (g)a

C−La (g)b

glycerol (g)

water (g)

dry chitosan mass percentage (%)c

O-CS 2CS-1CLA CS-1CLA CS-2CLA CS-1CLA-3GC CS-1CLA-6GC CS-1CLA-3GC-1W CS-1CLA-3GC-2W CS-1CLA-3GC-3W CS-1CLA-18W

original chitosan 9 9 9 9 9 9 9 9 9

0 4.5 9 18 9 9 9 9 9 9

0 0 0 0 3 6 3 3 3 0

0 0 0 0 0 0 1 2 3 18

92.6 61.7 46.3 30.9 39.7 34.7 37.9 36.2 34.7 23.2

a Moisture content and ash content at 740 °C are 7.4 and 30.2 wt %, respectively, which were obtained by thermogravimetric analysis. bConcentrated lactic acid (>85%). cDry chitosan percentage based on the mass of unprocessed precursor mixture.

ical mixing, chitosan was manually mixed with desired amounts of concentrated lactic acid, glycerol, and deionized water, as summarized in Table 1. The precursor mixtures were subsequently thermomechanically mixed at 80 °C, 100 rpm for 15 min in the Brabender. The plasticized chitosan was compression-molded via a hot press (Carver Laboratory Press, model 3912) into sheets (thickness of 1 to 2 mm), disks (diameter of 25 mm and thickness of ∼1.5 mm), and rectangular (50 mm × 12.5 mm × 3 mm) shaped samples immediately after being discharged from the Brabender. The compression molding temperature and maximum pressure were 120 °C and 4 t, respectively, and the total compression procedure lasted 15 min beginning with a 3 min preheating. The variables for compression molding were chosen according to the processability and weight loss of the plasticized chitosan as well as the limiting pressure of the press. The molded samples were stored in sealed PE bags at room temperature. Characterization. The intrinsic viscosity was measured at 25 °C using a size 1 C40 Cannon-Ubbelohde viscometer over a concentration range of 0.025 to 0.1 g/dL. The solvent used was 0.02 M acetate buffer/0.1 M NaCl (pH 4.5). The weight-average molecular weight was calculated from the intrinsic viscosity using the Mark−Houwink relation with the parameters K = 8.43 × 10−3 mL/g and a = 0.92.17 X-ray diffraction (XRD) patterns of chitosan powder and diskshaped samples were recorded at room temperature with a Philips X’Pert X-ray diffractometer using Cu−Kα radiation (λ = 0.1542 nm) operating at 50 kV and 40 mA. The scattering range was 2θ = 5−35° with a step size of 0.02° and a scan speed of 0.02°/s. The disk-shaped samples were tested immediately after compression molding, which prevented aging effect. The percentage relative crystallinity Xc was determined from Xc = [Ac/(Ac + Aa)] × 100, where Ac and Aa represent the areas under the crystalline peaks and amorphous baseline, respectively. A Nikon Optiphot-2 microscope was used to observe the morphology of strings released from sample CS-2CLA after swelling 48 h in deionized water. Scanning electron microscopy (SEM) observations were carried out with a high-resolution Hitachi S-4700 microscope operated at 2 kV accelerating voltage. The observed surfaces were cut by an ultramicrotome (Leica, Ultracut FC) equipped with a diamond knife at −30 °C and then coated with platinum vapor. A Perkin-Elmer FT-IR spectrometer was used to record Fourier transmission infrared spectroscopy (FTIR) spectra of the original chitosan powder, concentrated lactic acid, as well as plasticized chitosans. The FTIR spectroscope could analyze powders as well as film samples. The FTIR spectra were recorded in an IR range from 4000 to 600 cm−1 at a spectral resolution of 4 cm−1 and a scanning speed of 32 kHz. To remove lactic acid or salts formed during the plasticization, sample CS-2CLA was extracted with methanol in a Soxhlet apparatus for 24 h. Differential scanning calorimetry (DSC) measurements were carried out under a helium flow of 25 mL/min using a TA Instruments DSC Q1000 calibrated with indium. The samples (∼10 mg) were

Solution casting is a batch method, which requires the evaporation of a large quantity of solvent. Recently, a mechanical kneading method has been developed as a possible alternative route to solution casting. Epure et al. confirmed the plasticization of chitosan in the presence of glycerol and water through mechanical kneading and they further investigated the effects of storage and relative humidity on the water uptake, crystallinity evolution, and mechanical and dynamic-mechanical thermal properties of the plasticized chitosan.13 In another study, different nonvolatile polyol plasticizers (glycerol, xylitol, and sorbitol) were used during thermomechanical plasticization, and the microstructure and morphology were determined to understand the plasticization mechanism.14 Thermomechanical mixing is a more promising method of plasticizing chitosan than solution casting, which could be carried out on already-existing polymer processing equipments and increase plasticization efficiency. Plasticized chitosan/ synthetic polymer blends can even be prepared in one single continuous process similar, as has been done for thermoplastic starch.15,16 There are only few reports on the thermomechanical plasticization of chitosan. In this work, we developed a new formulation for chitosan thermomechanical plasticization in which we reduced the amount of water by using concentrated lactic acid. Lactic acid acts as both plasticizer and protonation agent for chitosan; moreover, extra lactic acid in plasticized chitosan could also plasticize PLA when blending with PLA. Glycerol was also used as the primary plasticizer due to its good plasticization efficiency, large availability, and low exudation.13 The physicochemical properties of thermomechanically plasticized chitosan are discussed in this paper. The hierarchical microstructure of plasticized chitosan is also examined with the purpose of elucidating the processing−structure−property relationship.

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EXPERIMENTAL SECTION

Materials. Chitosan appearing as a white powder was purchased from Primex under the trade name of ChitoClear. The dry material content (94%), viscosity of 1 wt % chitosan solution (87 mPa·s), solubility (99.8%), degree of deacetylation (95%), and particle size of the chitosan (95% through 100 mesh) are reported by the manufacturer datasheet. The intrinsic viscosity was measured to be 664 mL/g, from which the weight-average molecular weight (Mw) was estimated to be ∼210 kg/mol (under conditions explained later). Glycerol (99.8%, Fisher Scientific) and lactic acid (>85%, Laboratoire MAT) were used as received. Deionized water was used if needed. Material Processing. Thermomechanical plasticization of chitosan was carried out via a Brabender batch mixer (Plasti-Corder Digisystem from C.W. Brabender Instruments). Prior to thermomechan1217


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encapsulated in aluminum standard pans and were heated from 20 to 200 °C at a heating rate of 10 °C/min; after the samples were cooled at the same rate, the second heating scan from 20 to 200 °C was also conducted at a rate of 10 °C/min. A TA Instruments TGA Q500 was used to measure the weight-loss behavior of plasticized chitosans from 30 to 750 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. A TA Instruments DMA 2980 was used to perform dynamic mechanical analysis on rectangular bars of plasticized samples with dual cantilever bending from −120 to 100 °C at a heating rate of 2 °C/min, a frequency of 1 Hz, and a 0.5% strain.

the same time, the new peak at 2θ = 18.3° becomes sharper, especially in the 3 g glycerol case (pattern C), and the diffraction peak at 2θ = 19.9° exhibits a slight intensity reduction compared to that of the original chitosan powder. The crystallinity of the plasticized chitosan decreases from 63.7% for the original chitosan powder (pattern A) to 54.5% (pattern B) by using concentrated lactic acid and further decreases to 46.8 and 43.0% (patterns C and D) with the addition of 3 and 6 g of glycerol, respectively. Glycerol molecules tend to further increase the spacing between chitosan chains in each sheet rather than separate the sheets from each other, most probably because glycerol molecules, which have an effective hydrodynamic radius of 0.31 nm,22 are quite large and cannot easily infiltrate the space (0.447 nm) between sheets. When substituting half of the glycerol with water, as shown in pattern E, the diffraction peak at around 2θ = 20° becomes even lower and broader, and the use of a large amount of water along with lactic acid results in no diffraction peak at all, indicating an amorphous structure (pattern F). Water molecules are more likely to penetrate into the space between chitosan sheets than glycerol molecules. Plasticized chitosan, which was prepared by solution casting and mechanical kneading in the presence of dilute acetic acid aqueous solution, showed a similar amorphous structure.13,23 Assuming that the crystallinity of sample CS-1CLA-18W is zero, the crystalline contents of patterns A−E indicated in Figure 1 were calculated using pattern F as the amorphous baseline. The crystallinity decreased from 63.7 to 35.9% (between patterns A and E), indicating the progressive breakdown of the chitosan crystalline structure by thermomechanical plasticization in the presence of lactic acid, glycerol, and water. Note that a favorable interaction between lactic acid and chitosan must exist to explain the effect of concentrated lactic acid on the chitosan crystalline structure during thermomechanical plasticization. Qu et al. reported that when they dried chitosan films, which were prepared by lactic acid aqueous solution casting in a vacuum oven at 80 °C for 3 h, D,L-lactic acid was grafted onto amino groups of chitosan in the absence of catalyst.24 They proposed a reaction mechanism where amino groups of chitosan were first conjoined with carboxyl groups in lactic acid by salt linkage; then, the salt linkage was transferred to an amide link by a dehydration reaction under vacuum. In this work, FTIR has been used to investigate the possible interactions between lactic acid and chitosan in the plasticized samples. The FTIR spectra of original chitosan, plasticized chitosans with various chitosan/lactic acid ratios, and concentrated lactic acid are compared in Figure 2. In the IR spectrum of the original chitosan (spectrum A), several absorption peaks, which are related to skeletal C−C vibrations as well as primary and secondary alcohol C−O stretching, overlap and form a peak centered around 1026 cm−1. Two peaks at 893 and 1151 cm−1 are assigned to the saccharide structure.24 An absorption band around 1313 cm−1 is due to the C−N stretching in secondary amides.24,25 This band broadens and shifts to lower frequency in spectrum (B), indicating a possible interaction due to hydrogen bonding between secondary amine groups of chitosan and lactic acid. However, with increasing lactic acid content, the peak around 1313 cm−1 overlaps with the peaks from lactic acid. Two peaks at 1588 and 1652 cm−1 are more important in the IR spectrum of the original chitosan because they represent the N−H bend in primary amines. From spectra A to C, the two peaks combine into a broad peak, which shifts to lower frequency (1573 cm−1)

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RESULTS AND DISCUSSION Morphology and Microstructure of Plasticized Chitosan. Figure 1 reports the XRD patterns of the original chitosan

Figure 1. XRD patterns of original chitosan powder and thermomechanically plasticized chitosan with different compositions: (A) O-CS, (B) CS-1CLA, (C) CS-1CLA-3GC, (D) CS-1CLA-6GC, (E) CS-1CLA-3GC-3W, and (F) CS-1CLA-18W.

powder and plasticized chitosans with different plasticization compositions. A tendon (hydrated) polymorph is observed for the original chitosan powder (pattern A) with two characteristic diffraction peaks at 2θ = 10.6° (d spacing = 0.834 nm) and 19.9° (d spacing = 0.447 nm) corresponding to the (020) and (100) reflections in hydrated chitosan crystals, respectively.18−21 The unit cell of chitosan hydrated crystal is orthorhombic with dimensions a = 0.895 nm, b = 1.697 nm, and c = 1.034 nm and classified to a space group P212121.19 In the hydrated chitosan crystals, adjacent chitosan chains are antiparallelly aligned and bonded with intermolecular hydrogen bonding, forming a sheet structure. Furthermore several sheets stacked together with hydrogen bonding, via water molecules present between them, form 3-D crystals. Plasticizing chitosan in the presence of an equivalent amount of concentrated lactic acid (pattern B) shifts the (020) reflection to 2θ = 6.2° (d spacing =1.42 nm) as well as generates a new diffraction peak on the left side of the original (100) reflection at around 2θ = 18.3° (d spacing = 0.485 nm). The lactic acid molecules substantially expand the spacing between chitosan chains in each sheet, which may be due to possible interaction between the amino groups of chitosan and the carboxyl groups in lactic acid. The effect of lactic acid on the change of chitosan crystalline structure will be discussed in detail later. With increasing glycerol amount from 0 to 6 g, the intensity of the diffraction peak at 2θ = 6.2° decreases as shown in pattern D; at 1218


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between (020) planes after compounding with concentrated lactic acid is ∼0.288 nm, which is approximately equal to the length of a lactic acid molecule. The reaction of lactic acid with the amino groups of chitosan chains deteriorates the intermolecular hydrogen bonding, which can eventually lead to the tear of chitosan crystalline sheets under thermomechanical mixing. Similar to chitin,27 chitosan is also believed to possess a hierarchical organization, which reveals various structural levels. We define the structural levels referring to the observations made in this study. Chitosan consists of polysaccharide chains with an average length of ∼670 nm for the chitosan used in this study. The chain length of the chitosan molecules was estimated from the average molecular weight obtained via the intrinsic viscosity measurement and the high DDA. The next higher structural level should be narrow, long crystalline nanofibrils of about 3−5 nm in diameter composed of 18−25 chitosan molecules (assuming analogy to chitin hierarchical structure27), which were not observed in our study. Nanofibrils bundle up to form nanofibril clusters with observable dimensions under atomic force microscope (AFM) and SEM. For example, a rodlike structure formed by chitosan chains with average length and diameter of around 400 and 25 nm, respectively, was observed by AFM.28 In our work, chitosan nanofibril clusters of about 1−4 μm in length and 40−70 nm in diameter were observed, as shown in Figure 4. The Mw of the chitosan used in our study (210 kg/mol) is much larger than the one used by Liang et al. (44 kg/mol).28 This implies the possibility of longer chitosan nanofibrils in our samples. Moreover, chitosan nanofibrils can be crystalline nuclei for each others to form nanofibril clusters.29 Therefore, the nanofibril clusters observed in our study may be much longer than the ones reported by Liang et al. Figure 4 compares the SEM images of cryo-cut surfaces of three plasticized chitosan samples: CS-1CLA, CS-1CLA-3GC, and CS-1CLA-6GC. The CS-1CLA sample exhibits a surface composed of compact nanofibril clusters (Figure 4A). With increasing glycerol content, the population of nanofibril clusters on the surface decreases, and more smooth areas are observed. In the CS1CLA-6GC sample (Figure 4C), sparse nanofibrils are separated by large smooth areas. Glycerol, which interacts with chitosan chains through hydrogen bonding, could split the nanofibril clusters into smaller nanofibrils. The smooth areas in the SEM images are believed to be composed of nanofibrils and amorphous chitosan chains, which cannot be distinguished under SEM. Considering that the mass percentage of chitosan in the CS-1CLA-3GC and CS-1CLA-6GC samples only decreases from 39.7 to 34.7% and many distinctive morphological differences are identified in their SEM images (Figure 4B,C), the dilution effect of glycerol may be negligible. The next higher structural level consists of the formation of bundles of some of these nanofibril clusters into long microfibers of about 1 to 3 μm in diameter, as illustrated in Figure 5. To visualize the hierarchical structure, we immersed a compression-molded small disk of sample CS-2CLA in deionized water for 48 h. During the immersion, the sample got swollen and some entangled strings, which are long microfibers, as shown in Figure 5, were released from the surface of the sample. The chitosan amorphous chains, nanofibrils, and nanofibril clusters between those microfibers were likely dissolved or dispersed in water. String bundles consisting of entangled long microfibers are clearly observed in Figure 5B. These microfibers, which were detached from the surface of the water-swelled CS-2CLA

Figure 2. FTIR spectra of (A) O−CS, (B) CS:CLA = 2:1, (C) CS:CLA = 1:1, (D) CS:CLA = 1:2, (E) CS:CLA = 1:2 extracted in methanol, and (F) concentrated lactic acid.

while its intensity increases. In amino salts, strong hydrogen bonding is experienced, where a corresponding broadening and frequency lowering of the associated N−H absorptions is observed.26 When lactic acid content continues increasing (spectrum D), the peak (1573 cm−1) intensity decreases and finally disappears in the IR spectrum of the concentrated lactic acid (spectrum F). After sample CS-2CLA was extracted in methanol (spectrum E), two distinct peaks at 1588 and 1652 cm−1 show up again and the spectrum is consistent with that of the original chitosan, which is indicative of the dominant presence of salt linkages between the lactic acid and the amino groups of chitosan. As shown in Figure 3A, the principal

Figure 3. Possible reaction between chitosan and lactic acid (A) and the resulting change in the crystal structure after the reaction (B).

reaction between lactic acid and chitosan is the formation of amino-carboxyl salt linkages during thermomechanical plasticization, and these salt linkages can be removed by methanol extraction. Figure 3B schematically depicts the increase in the distance between chitosan chains in a sheet due to the infiltration of salt-linked lactic acid molecules. Confirmed by the XRD patterns (Figure 1), the increase in the d spacing 1219


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physical (thermal and dynamic mechanical) properties of the plasticized chitosan samples. Thermal Properties. The effect of plasticization on the thermogravimetric behavior of the original chitosan powder and thermomechanically plasticized chitosans is presented in terms of weight loss in Figure 6A and of its temperature derivative in Figure 6B. The original chitosan powder, which was not pretreated, had a moisture content of ∼7.3 wt %; the evaporation of the moisture resulted in a small shoulder in Figure 6A and a peak at about 62 °C in Figure 6B. Following moisture evaporation, a plateau is evidenced in Figure 6A, ranging from ∼100 to 250 °C. After the plateau, a sharp weight loss happens due to the decomposition of chitosan, and the maximum thermal decomposition rate occurs around 315 °C. The plasticized chitosans show a different thermal decomposition behavior compared with the original chitosan powder: the weight loss is progressive with increasing temperature because of the continuing evaporation of the plasticizers including water, lactic acid, or glycerol. The CS-1CLA sample exhibits the best thermal stability among the plasticized chitosan samples. Because lactic acid molecules can disrupt the chitosan crystalline structure by interacting with amino groups during thermomechanical blending, two major peaks are observed for the CS-1CLA sample at 209 and 302 °C in Figure 6B, which are attributed to the decomposition of the amorphous and crystalline chitosan structures, respectively. Because the lactic acid molecules not only result in creating an amorphous phase but also deteriorate the chitosan crystalline phase, the temperature at maximum thermal decomposition rate for the CS-1CLA sample is ∼13 °C lower than that of the original chitosan powder. The presence of glycerol in the CS1CLA-6GC sample leads to a more significant weight loss at the same temperature, as compared with the CS-1CLA sample, and the derivative weight loss peak around 204 °C becomes broader because of the loss of glycerol. However, the double-peak pattern is still obvious: the first peak at ∼204 °C is assigned to the loss of glycerol and the thermal decomposition of the chitosan amorphous phase, and the other peak around 301 °C is ascribed to the thermal decomposition of the chitosan crystalline structure. When replacing some glycerol with water or increasing the water content as in samples CS-1CLA-3GC3W and CS-1CLA-18W, the weight-loss rate increases, especially below 150 °C due to the evaporation of water. The CS-1CLA-18W sample loses ∼30% of its original weight as the temperature approaches 150 °C and also shows two derivative weight loss peaks: the first peak at ∼101 °C is from the loss of water and lactic acid; the second one around 302 °C is from the thermal decomposition of the expanded chitosan crystals. Because water and glycerol were both used in sample CS1CLA-3GC-3W, it exhibits a triple-peak pattern, where the first peak around 122 °C, which is rather a shoulder in Figure 6B, is mainly due to the evaporation of water and the second and third ones at about 205 and 308 °C are due to the loss of glycerol and the amorphous phase and the thermal decomposition of the expanded chitosan crystals, respectively. The TGA results imply that glycerol is a more stable plasticizer than water, as expected. Figure 7 shows the DSC thermograms of the chitosan powder and the plasticized samples from the first heating scan. Thermogram (A), representing the original chitosan powder, exhibits a broad endothermic peak at ∼112 °C, which is due to the evaporation of moisture in the original chitosan powder. The broad endothermic peak disappeared in the second heating

Figure 4. SEM images of thermomechanical plasticized chitosan samples of different compositions: (A) CS-1CLA, (B) CS-1CLA-3GC, and (C) CS-1CLA-6GC.

sample, are the residual microfibers that have not been subdivided during plasticization. Our plasticized chitosan samples are composed of elements of these hierarchical structural levels, including individual chitosan molecular chains, nanofibrils, nanofibril clusters, and microfibers. The decrease in crystallinity and the disappearance of nanofibril clusters in the SEM images indicate the breakdown of the larger structural levels toward chitosan nanofibrils and molecular chains; the remaining crystallinity of the CS-1CLA-3GC-3W sample (Figure 1, pattern E) is believed to be mainly due to chitosan nanofibrils. In the following sections, we will discuss the 1220


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Figure 5. Optical microscopy images of the strings released from sample CS-2CLA after swelling in deionized water for 48 h for two different magnifications.

Figure 6. Thermogravimetric graphs of original chitosan powder and thermomechanically plasticized chitosan with various compositions: (A) weight percentage and (B) derivative weight.

of temperature on the mobility of these components is limited in this temperature range. The CS-1CLA-3GC and CS-1CLA6GC samples show the same heat-flow−temperature trend as the CS-1CLA sample. Although glycerol can help to divide nanofibril clusters into nanofibrils, the components in the plasticized chitosan samples still have limited mobility in the temperature range of 20−200 °C. When water is used, a broad endothermic peak is observed around 180 °C, as shown in curve E. With increasing water content (curves F and G), the endothermic peak shifts to lower temperature around 150 °C. The endothermic peaks observed in curves E−G disappeared in the second heating scan (not shown), which indicates that they were mainly due to the loss of water molecules that have been hydrogen bonded with chitosan and glycerol molecules. The average strength of hydrogen bonds in an amorphous polyamide decreases with increasing temperature.30 In other words, the plasticized chitosan samples are still distinct from conventional molten thermoplastic polymers because they consist of rigid molecules and several levels of crystalline structures. Dynamic Mechanical Properties. The loss of plasticizers obstructs the investigation of the phase-transition behavior of plasticized chitosan via DSC analysis, as seen in Figure 7. Therefore, to investigate the phase transition and viscoelastic behavior of these systems, DMA spectra were recorded from −120 to 100 °C for the plasticized chitosan samples of different

Figure 7. DSC thermograms (first heating scan) of original and thermomechanically plasticized chitosan samples of various compositions: (A) O−CS, (B) CS-1CLA, (C) CS-1CLA-3GC, (D) CS-1CLA6GC, (E) CS-1CLA-3GC-1W, (F) CS-1CLA-3GC-2W, and (G) CS1CLA-3GC-3W.

(not shown in the Figure). The heat-flow value of the CS1CLA sample decreases with temperature owing to the loss of plasticizer, and no apparent endothermic peak is observed. Because the plasticized chitosan is composed of rigid chitosan molecular chains, nanofibrils, and nanofibril clusters, the effect 1221


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Figure 8. DMTA spectra of thermomechanically plasticized chitosans with different compositions: (A) storage modulus (E′) and (B) loss tangent (tan δ).

crystalline units to smaller ones and even to amorphous molecules during thermomechanical plasticization. The increased content of amorphous chitosan molecules, along with the increased amount of glycerol, results in an E′ decrease at lower temperatures. It is interesting to note that when water is used, such as in the CS-1CLA-3GC-3W sample, the E′ decreases in a two-step pattern: the first reduction starts at about −90 °C and the second one starts around −60 °C. The first E′ decrease is believed to be related to the presence of water. Water molecules are smaller than glycerol molecules and can easily penetrate into the chitosan crystalline structure, resulting in a lower crystallinity, which means a more important amorphous phase. As discussed in the work of Quijada-Garrido et al.,31,32 the β-relaxation process corresponds to the motion of the chitosan lateral groups (−NH2, −CH2OH, and −NH−OCCH3) linked to glycerol by hydrogen bonding, while the αrelaxation, which is approximately treated as a glass transition, is interpreted as torsional oscillations between two glucopyranose rings across a glucosidic oxygen, and a cooperative hydrogen bond reordering. Increased amorphous phase and higher mobility of water molecules may favor the motion of lateral groups of chitosan at lower temperatures, leading to the appearance of the first E′ decrease stage. Figure 8B illustrates the variation of tan δ as a function of temperature for the plasticized chitosan samples. The CS-1CLA sample shows two primary relaxation peaks: the one at 11 °C is assigned to the β-relaxation representing the motion of the lateral groups of chitosan and the corresponding lactic acid molecules interacting with them;37,38 the other one at 52 °C is the α-relaxation, which originates from the motion of rigid segments of chitosan molecules in the amorphous phase as well as the corresponding reordering of hydrogen bonds.13,31 Note that the two primary relaxations occur only in the amorphous phase, where the dominant components are amorphous chitosan molecules, plasticizer molecules, and small crystalline units (e.g., nanofibrils). A shift of the two primary relaxation peaks to lower temperatures is observed as the glycerol content increases: Tg decreases from 52 °C for the CS-1CLA sample to 29 and 21 °C for the plasticized chitosan samples containing glycerol (CS-1CLA-3GC and CS-1CLA-6GC). The Tg value of 52 °C for the CS-1CLA sample is much lower than the Tg values reported in the literature for the neat chitosan, indicating indeed a plasticizing effect of concentrated lactic acid. The tan δ

compositions and reported in Figure 8. Because the loss of plasticizers occurs at high temperatures and the storage and loss moduli approach 0 MPa when temperature exceeds 100 °C, the DMA measurements were only conducted up to 100 °C. The DMA spectrum of the original chitosan could not be obtained because unlike the plasticized chitosan samples the chitosan powder cannot be directly compression molded into a rectangular sample shape because its melting point is higher than the thermal degradation temperature. However, the DMA spectrum for chitosan can be found elsewhere.31,32 Those chitosan DMA samples were prepared by solution casting using acetic acid aqueous solution as solvent. The glass-transition temperature (Tg) of neat chitosan has been reported by different researchers, but the values do not agree: for example, 85 °C has been reported by Quijada-Garrido et al., while higher Tg values such as 103,33 140−150,34 and 195 °C35,36 have been stated. Figure 8A illustrates the variation of the storage modulus (E′) as a function of temperature for plasticized chitosan samples of different compositions. The E′ value at −120 °C is defined as the initial storage modulus, and this property considerably decreases from 6800 MPa for CS-1CLA to about 2400 MPa for CS-1CLA-6GC as a result of dilution and plasticizing effect with increasing glycerol content. As previously discussed, different plasticizer content and composition (glycerol/water ratio) could lead to different crystallinities and microstructures; however, the decrease in the initial E′ seems to depend only on the total level of plasticizers, as the CS-1CLA-6GC (Xc = 43.0%) and CS-1CLA-3GC-3W (Xc = 35.9%) samples have a similar initial E′. With increasing temperature, the plasticized chitosan samples exhibit a different E′-temperature dependence mainly due to their different microstructures. The CS-1CLA sample has the highest crystallinity and the more intact and larger size crystalline components among all samples; therefore, it shows the highest E′ before the value goes toward 0 MPa after a sharp decrease. The E′ sharp decreases for the other samples shift to lower temperatures as the glycerol content increases and water is used. The sharp E′ decrease is attributed to the relaxation of the lateral groups in our plasticized chitosan samples (−CH2OH and −NH+3−OOC−CH(OH)-CH3) and molecular segments of chitosan molecules in the amorphous phase. In this study, glycerol is found to promote the breakdown of large chitosan 1222


Biomacromolecules peak value of the α-relaxation process increases with glycerol content, which indicates improved liquid-like behavior for the samples containing more glycerol. It is worth noting that for the CS-1CLA-3GC-3W sample, the left shoulder at about −40 °C should be assigned to the β-relaxation process of the amorphous chitosan molecules interacting with water molecules, and the maximum peak at ∼2 °C is due to the overlap of β- and α-relaxation processes of the rest of the amorphous phase; a third peak at 59 °C could be due to the relaxation of larger chitosan segments interacting with plasticizer molecules.

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CONCLUDING REMARKS

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AUTHOR INFORMATION

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ACKNOWLEDGMENTS

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REFERENCES

Article

We acknowledge the financial support for this project from the Network for Innovative Plastic Materials and Manufacturing Processes (NIPMMP) Canada. We are also grateful to Mrs. Weawkamol Leeapornpisit for her help with the microscopic analysis.

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In this work, a thermomechanical plasticization method was proposed and tested for different chitosan formulations. The experimental results confirmed that chitosan was partially plasticized in the presence of concentrated lactic acid and glycerol using a batch mixer. The microstructure of the thermomechanically plasticized chitosan was investigated by XRD, SEM, and optical microscopy, revealing a complex hierarchical structure with various levels of structural units; these included an amorphous phase, nanofibrils, nanofibril clusters, and microfibers, produced under the effects of mechanical shear and thermal energy. The thermomechanically plasticized chitosan obtained was actually a composite material in which the amorphous phase was reinforced by nanofibrils, nanofibril clusters, and microfibers. With an increase in plasticizer content (glycerol or water), the crystallinity of the plasticized chitosan decreased from 63.7% for the original chitosan powder to 43.0% for sample CS-1CLA-6GC and then further to almost a completely amorphous phase for the sample plasticized with extra water (CS-1CLA-18W). Differing from only one single thermal degradation peak at 315 °C, which was mainly due to the crystalline phase of the original chitosan powder, two degradation peaks attributed to the amorphous and deteriorated crystalline phases were observed in DTGA for the plasticized chitosans. In DSC, the plasticized chitosans showed endothermic peaks mainly due to the loss of plasticizers, and no endothermic peaks were observed during the second heating scan. In DMA testing, sample CS-1CLA showed the highest storage modulus of ∼6800 MPa at −120 °C and still possessed about 1060 MPa at 30 °C. With an increase in plasticizer content, the storage modulus decreased to about 2000 MPa at −120 °C, and the glass-transition temperature decreased from 52 °C for sample CS-1CLA to ∼2 °C for sample CS-1CLA-3GC-3W. Thermomechanical plasticization can be a promising way to plasticize chitosan on an industrial scale. The next important aspect will be to look for more efficient and thermally stable plasticizers as well as to modify the thermomechanical plasticization procedure (including more robust shearing) to divide finely the microfibers, nanofibril clusters, and nanofibrils into chitosan molecular chains.

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The authors declare no competing financial interest. 1223


Biomacromolecules

Article

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Hierarchical Structure and Physicochemical Properties of Plasticized

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