Preparation and Characterization of Chitosan by a Novel

Research Article pubs.acs.org/journal/ascecg

Preparation and Characterization of Chitosan by a Novel Deacetylation Approach Using Glycerol as Green Reaction Solvent Cuiyun Liu,† Guanhua Wang,*,†,‡,§ Wenjie Sui,∥ Liangliang An,† and Chuanling Si*,†,§,⊥ †

Tianjin Key Laboratory of Pulp and Paper, College of Paper Making Science and Technology, Tianjin University of Science and Technology, Tianjin 300457, China ‡ Key Laboratory of Recycling and Eco-treatment of Waste Biomass of Zhejiang Province, Zhejiang University of Science and Technology, Hangzhou 310023, China § State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China ∥ Key Laboratory of Food Nutrition and Safety Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, China ⊥ State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China ABSTRACT: Traditional methods for chitin deacetylation require aqueous solution with high alkali content as reaction solvent, which generate huge environmental pressure for the treatment of vast alkaline wastewater. In this work, glycerol, a nontoxic, biodegradable, and sustainable liquid from biodiesel production, is employed as a novel reaction solvent for chitin deacetylation at elevated temperature. The results showed that the chitosan deacetylation degree (DD) was up to 85.36 ± 1.04% and the corresponding viscosity-average molecular weight (MV) was 30 874 ± 1123 g mol−1 under the optimized conditions of this process. The subsequent Fourier transform infrared (FTIR) characterization structurally demonstrated the occurrence of deacetylation and the fracture of glucosidic bond. Thermogravimetric analysis (TGA) and X-ray diffraction (XRD) analysis further revealed that the DD presented a negative effect on the thermal stability and crystallinity of chitosan. Consequently, the study successfully indicated that using glycerol as a reaction solvent could realize the deacetylation of chitin with lower NaOH concentration and provide an efficient and green process for chitosan preparation from chitin. KEYWORDS: Chitin, Deacetylation, Glycerol, Process optimization, Chitosan characterization

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INTRODUCTION Chitin is the most abundant of all natural polymers after cellulose and primarily found in three sources, namely, crustaceans, insects, and microorganisms.1−3 Organisms generate about 100 billion tons of chitin every year.4 Structurally, chitin is a linear polymer whose monomer is N-acetyl-Dglucosamine linked by β-1,4-glycosidic bonds and may be regarded as cellulose with hydroxyl at position C-2 replaced by an acetamido group.5 As the dominant N-containing renewable polymer on earth, chitin shows greatly potential application in sustainable chemicals and materials preparation.5,6 However, the highly insoluble property of chitin in nearly all common solvents has severely restricted its application in many fields.7,8 Chitosan, a derivative of chitin after deacetylation, has much better solubility in water than chitin.7,9,10 As the only alkaline amino polysaccharide in nature, chitosan has been extensively applied as a promising and versatile biopolymer. Since chitosan shows several unique physicochemical properties and biological functions, including nontoxicity, antimicrobial/antioxidant activities, biodegradability, and biocompatibility, it has been © 2017 American Chemical Society

widely applied in medicine, food, and environmental areas, such as drug delivery, food and cosmetics preservation, beverage/ juice purification, and metal chelation from wastewater.5,6 The traditional methods of chitosan preparation from chitin, which require aqueous solution with high alkali concentration (40−60%), generate severe pollution to the environment and increase the production cost, thereby limiting the widespread application of chitosan.11 Thus, a green and efficient process for chitosan preparation without generation of massive alkaline wastewater is significant to the extensive application of this abundant and renewable marine resource. Biochemical conversion of chitin to chitosan by enzymatic catalyst seems to be a possible option due to the mild reaction conditions without use of harsh chemicals.9 Kim et al. used extracellular chitin deacetylase to prepare chitosan at optimum temperature 60 °C and pH 5.5 and concluded that the isolated enzyme was Received: January 6, 2017 Revised: April 16, 2017 Published: April 25, 2017 4690

DOI: 10.1021/acssuschemeng.7b00050 ACS Sustainable Chem. Eng. 2017, 5, 4690−4698

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Reaction Conditions of Chitosan Preparation Process by UGARS temperature/°C

reaction time/h

alkaline concentration/%

solid−liquid ratio (chitin−glycerol, w/w)

120 140 160 180 180 180 180 180 180 180 180 180 180

12 12 12 12 12 12 12 4 8 16 12 12 12

30 30 30 10 20 30 40 30 30 30 30 30 30

1:40 1:40 1:40 1:40 1:40 1:40 1:40 1:40 1:40 1:40 1:30 1:50 1:60

Therefore, in this work, glycerol was innovatively applied as a reaction solvent to achieve an efficient and green deacetylation process of chitin. The deacetylation degree (DD) and viscosityaverage molecular weight (MV) of chitosan prepared by the proposed method were evaluated. The obtained chitosan products were further analyzed by Fourier transform infrared (FTIR), thermogravimetric analysis (TGA), and X-ray diffraction (XRD) to investigate their chemical structure, thermal stability, and crystallinity, respectively. In addition, the utilized glycerol and NaOH were recovered by a straightforward method.

valid in deacetylating chitin polymers, with up to 71−88% deacetylation degree.12 However, the high production cost of deacetylase and long reaction time restrict the commercial application of this enzymatic deacetylation of chitin.13 In order to shorten reaction time and improve deacetylation efficiency, some auxiliary steps are applied to accelerate the process. Sagheer et al. applied microwave radiation to prepare chitosan in 45% sodium hydroxide aqueous solution and found that the microwave heating could significantly reduce the deacetylation time without loss of deacetylation degree.14 Rashid et al. autoclaved γ-irradiated prawn shell to prepare chitosan at 150−160 °C with 50% (w/w) NaOH solution and suggested that the chitin deacetylation could be highly facilitated by elevating reaction temperature.7 Nevertheless, a large amount of wastewater is still produced inevitably by these ways since high alkali content solution was employed as reaction solvent. Recently, ionic liquids as green reaction solvents have been used for deacetylation reaction. Ishii et al. utilized imidazolium-based ionic liquids to improve deacetylation by hydrothermal treatment and the deacetylation degree of chitosan was increased from 77% to 86%.15 However, the valuableness and toxicity characters of ionic liquids as solvent are adverse to the wide application of this method and this dilemma promotes the exploration of new inexpensive and innoxious solvents for chitin deacetylation. Glycerol, as a high boiling (290 °C) solvent possesses many properties, such as nonvolatile, easily recyclable, highly inert, and stable and has been proven to be an environmentally benign reaction medium.16,17 Besides, compared with other promising green solvents such as ionic liquids, glycerol as the byproduct of biodiesel production is readily acquired, innoxious, inexpensive, and biodegradable.18−20 These properties conform to the requirements of the most ideal green solvent and the current sustainable chemical processes.16 Perin et al. used glycerol as an efficient and recyclable solvent to replace toxic chlorinated organic solvents for the thioacetalization of aldehydes and their results demonstrated that the reaction could proceed readily with satisfied product yields.21 Cabrera et al. presented a clean oxidative synthesis of disulfides using glycerol under microwave and found the process could obtain the corresponding products in good yields.22 In these reactions, glycerol was found to be stable and capable of facilitating the dominant reaction and product isolation. Because of the favorable properties of glycerol as a reaction solvent, this work attempts to evaluate the novel process using glycerol as a reaction solvent for the deacetylation of chitin.

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MATERIALS AND METHODS

Materials. Chitin from shrimp shells (poly-(1-4)-β-N-acetyl-Dglucosamine, CAS 1398-61-4) was purchased from Sigma-Aldrich. All of the reagents are chemically pure. Chitosan Preparation Using Glycerol As Reaction Solvent (UGARS). Chitosan was prepared from chitin in a heterogeneous deacetylation process where glycerol was used as the reaction solvent and chitin kept insoluble. The chitin was deacetylated for 4−16 h at different temperatures (120−180 °C) with 10−40% (w/w) NaOH solution at a ratio of 1:30−1:60 (chitin−glycerol, w/w). The 1% water (water/glycerol, w/w) was added to promote the ionization of NaOH and inhibit the polymerization of glycerol catalyzed by NaOH at high temperature. After reaction, the deacetylation product was obtained by centrifugation (7000 r min−1 for 5 min) and washed thoroughly with distilled water to remove the remaining NaOH and glycerol completely. Finally, the chitosan was dried by freeze-drying and stored in an enclosed bottle at ambient temperature. Table 1 lists the detailed experiment conditions of chitosan preparation process by UGARS. All the experiments were done in triplicate. The chitin deacetylation by conventional process was conducted according to the previous literature.1,6 The chitin was deacetylated by heating at 95 °C with 50% (w/w) NaOH solution with a ratio of 1:50 (w/v) for 12 h. The procedure of glycerol recovery was operated as following. The resultant after chitin deacetylation was first diluted by adding deionized water (1:1, w/w) to reduce the viscosity. The mixture was then centrifugated (7000 r min−1 for 5 min) to get the supernatant involving glycerol, NaOH, and water. The supernatant was concentrated by rotary evaporation at 40 °C. The recovered glycerol and NaOH were weighted and used for the next chitin deacetylation reaction. Because of the loss of glycerol and NaOH during the chitosan isolation process, fresh glycerol and NaOH was compensated according to the recovery rate. The schematic of chitosan preparation by UGARS and solvent recycling is presented in Figure 1. Characterization of Chitosan Prepared by UGARS. Determination of Deacetylation Degree (DD). Chitosan (0.3 g) was added in 30 mL of 0.1 M HCl aqueous solution and stirred until complete dissolution at ambient temperature. After 2−3 drops of indicator (methyl orange and aniline blue mixture) were added, 0.1 M NaOH 4691


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ACS Sustainable Chemistry & Engineering

where NH2% is amino content in the sample; c1 and c2 are the concentration of the standard solution of HCl and NaOH (M), respectively; v1 is the volume of HCl added (mL); v2 is the volume of NaOH at the end point of titration (mL); m is the chitosan sample mass (g); w is the water content of chitosan sample (%); 0.016 is the amount of amino corresponding to 1 mL of 1 M HCl aqueous solution (g), 9.94 is the ideal −NH2% content of chitin (16/161). Determination of Viscosity Average Molecular Weight (Mv). In total, 0.1 g chitosan samples were dissolved in 50 mL of 0.2 M CH3COOH/0.1 M CH3COONa solution (v/v, 1:1). The Ubbelohde viscometer was used to measure the passage time of the solution flowing through the capillary in a water bath at 25 ± 0.1 °C.8 The intrinsic viscosity was obtained by extrapolating the reduced viscosity versus concentration. The chitosan MV was calculated using the wellknown Mark−Houwink equation.7,24

[η] = K · MV α Figure 1. Schematic of chitosan preparation by UGARS and solvent recovery.

where [η] is the intrinsic viscosity, K and α are constants which depend on the nature of the polymer and solvent as well as temperature. In the work, K and α were taken as 1.81 × 10−3 and 0.93, respectively.7,25 FTIR Characterization. About 2 mg of dried chitosan powder and 200 mg of KBr were blended and triturated by an agate mortar. Then the mixture was compacted using an IR hydraulic press at a pressure 10 MPa for 60 s. The chitosan spectra (in the form of KBr discs) were gained by FTIR 650 (Gangdong Sci. & Tech. Co., Ltd., China) with a wavenumber range of 500−4000 cm−1. TGA Characterization. TGA was carried out using a thermogravimetric analyzer (TGA-Q50, Shimadzu, Japan). About 10 mg samples

aqueous solution was used as the titrant until the solution color changed from purple to blue-green (approximately pH 4.3). The chitosan DD was calculated by the following eqs 1 and 2:23

NH 2(%) = DD(%) =

(C1V1 − C 2V2) × 0.016 × 100% m(100 − w) NH 2 × 100% 9.94

(3)

(1) (2)

Figure 2. Effect of temperature (a), NaOH concentration (b), reaction time (c), and solid−liquid ratio (d) on DD and MV of chitosan prepared by UGARS. 4692


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ACS Sustainable Chemistry & Engineering were heated from ambient temperature to 700 °C with a heating rate of 10 °C min−1 under flow nitrogen atmosphere (20 mL min−1). XRD Characterization. The XRD patterns of chitosan samples were measured by a Shimadzu Lab XRD-6100 diffractometer (2θ = 5−40°) and with a Ni-filtered Cu Kα-radiation at 40 kV and 50 mA at ambient temperature. The relative crystallinity of the polymers was calculated by dividing the area of the crystalline peaks by the total area under the curve.26

Figure 3. Deacetylation reaction of chitin.

was beneficial to obtain excellent DD under the same reaction conditions for chitin from squid28 and shrimp shell.29 However, the chitosan MV showed a contrary tendency since its value decreased with the increment of temperature. As shown in Figure 2a, the MV of chitosan deacetylated at 120 °C was roughly 80 538 g mol−1 and that value was dropped drastically to about 30 874 g mol−1 at the highest temperature of 180 °C. According to Prashanth et al., the acetyl groups of chitin could not be completely removed without the degradation of polysaccharide chains, while in the presence of alkali.30 The MV reduction was primarily caused by the chain scission of chitosan molecules at glucosidic linkages (Figure 4). Tolaimate et al. demonstrated that an increase of temperature from 85 to 120 °C resulted in a decrease of MV from 490 kDa to 150 kDa, thereby confirming the influence of temperature on molecular weight.31 Besides, the degradation of the chitosan or chitin chain at high temperature would provide more reactive groups which could accelerate the deacetylation velocity.23 However, when the temperature continued to rise to over 200 °C, glycerol might be converted to polyglycerol by NaOH induced polymerization, which enhanced the viscosity of reaction solvent and inevitably decreased the recovery rate of glycerol after reaction.32 Therefore, in order to avoid the side reaction of glycerol, the reaction temperature was optimized at 180 °C. NaOH Concentration. As catalyst, NaOH plays an important role in the chitin deacetylation process. To investigate the effect of NaOH concentration on the properties of deacetylated products, the chitin was heated in a solid−liquid ratio 1:40 (chitin−glycerol, w/w) at 180 °C for 12 h with different NaOH concentrations (10−40%). As shown in Figure 2b, with the increase of NaOH concentration from 10% to 40%, the DD of chitosan increased gradually from 59.38% to 83.59%. When the NaOH concentration was 30%, the obtained chitosan had a value of 85.36% as the highest DD value, since it was decreased slightly to 83.59% when the final NaOH concentration was 40%. The deacetylation reaction of chitin is greatly affected by the steric hindrance from the compact structure of natural chitin, which precludes the attack of OH− to the amino group.28 Also, the diffusion rate of OH− from bulk solution to the surface or the inside of chitin particle would be closely related to the alkali concentration.29 Thus, the increase of NaOH concentration facilitated OH− to overcome the steric hindrance and realize the deacetylation of chitin.23,29 It seemed that with low concentration of NaOH (i.e., 10% and 20%), there were no significant changes in DD even though the reaction temperature and time were increased up to 180 °C and 12 h, respectively. However, the continuous increment of NaOH concentration to 40% resulted in the obvious raising of glycerol solvent viscosity, which negatively affected mass transfer of OH− and induced the decrease of chitosan DD. Therefore, the 30% NaOH concentration was chosen as the optimal catalyst concentration due to the preferred DD of obtained chitosan. Figure 2b also provides the change of chitosan MV with the rise of catalyst concentration. In the presence of NaOH, polysaccharide chains were found to be degraded, owing to the

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RESULTS AND DISCUSSION Deacetylation Process Using Glycerol As Reaction Solvent. In the previous alkali deacetylation of chitin, water is mostly used as a reaction solvent, which is always criticized due to the generation of vast wastewater with high alkali concentration.12,13 Here, the work shows that glycerol, which is a nontoxic, biodegradable, and recyclable liquid manufactured as the byproduct of biodiesel production, has great potential to serve as an alternative green solvent for the chitin deacetylation. However, several variations in this novel approach would have impacts on the chitosan properties. To optimize the deacetylation process, the effects of four crucial reaction parameters including reaction temperature, NaOH concentration, reaction time, and solid−liquor ratio were investigated. The DD and MV were chosen as the two main evaluation goals for the optimization process, since DD stands for the free amino group content of polymer chain and MV indicates the molecular weight of chitosan, both of which have significant effects on the subsequent applications.5,27 Reaction Temperature. Deacetylation temperature is one of the most important factors that affect the DD of chitosan.24 According to the previous studies, when deacetylation was performed at elevated temperature, the DD value of chitosan could be obviously improved after a shorter reaction time24 and when the deacetylation temperature was below 60 °C, the DD could not exceed 60% even though the NaOH concentration was above 50%.28 Thus, the choosing of temperature is of prime importance for this novel deacetylation process. In the preliminary experiments of this study, the chitin deacetylated at the temperature below 120 °C was found to be insoluble in 1% acetic acid solution. This phenomenon could be ascribed to the higher viscosity of glycerol compared with water, which impeded the accessibility of catalyst to chitin and decreased the deacetylation degree of chitosan. However, the chitosan DD raised remarkably when the reaction temperature was risen to over 120 °C. Therefore, in order to study the effect of the reaction temperature on chitin deacetylation by UGARS, the experiments at elevated temperature (120−180 °C) were performed and the results are illustrated in Figure 2a. The other reaction conditions of chitin deacetylation were constant at NaOH concentration of 30%, solid−liquid ratio (chitin: glycerol, w/w) of 1:40 and reaction time of 12 h. As expected, the temperature played a dominant role on chitin deacetylation process when the reactions were carried out with 30% NaOH catalyst concentration. It could be seen from Figure 2a that the DD of obtained chitosan increased significantly as the reaction temperature was raised in the selected range. When the reaction temperature was 120 °C, the DD of the obtained chitosan was about 55.43% and it rose to approximately 85.36% under the reaction temperature at 180 °C. The increase of reaction temperature facilitated the hydrolysis of acetamide to amino during the deacetylation process (shown in Figure 3) which resulted in the increased DD. Previous studies had also reported that higher temperature 4693


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Figure 4. Degradation reaction of chitin and chitosan.

susceptibility of the glucosidic bond to alkali.33 With the increase of NaOH concentration from 10% to 40%, the molecular weight of chitosan decreased from 83 037 g mol−1 to 28 693 g mol−1 due to the intensified breakage of the glucosidic bond. It was notable that the MV of chitosan obtained from deacetylation under 40% NaOH was lower than that under 30% NaOH, which suggested that compared with deacetylation of the amino group, the cleavage of the glucosidic bond was less affected by the increased viscosity of the reaction solvent. Reaction Time. In order to investigate the effect of reaction time on the DD and MV of chitosan, the chitin was deacetylated in a solid−liquid ratio 1:40 (chitin−glycerol, w/w) at 180 °C with 30% NaOH for 4 h, 6 h, 8 h, and 12 h, respectively, and the results are shown in Figure 2c. It could be seen that the chitosan DD elevated as the reaction time prolonged, while the MV showed a contrary tendency. It was suggested that the heterogeneous deacetylation took place preferentially in the amorphous region of chitin, then proceeded from the surface layer to the inside of the crystalline region.29 Thus, with the extension of reaction time, the DD increased due to the sufficient deacetylation of the inner layer chitin. As expected, the molecular weight of obtained chitosan reduced with the prolongation of reaction time, which was mainly attributed to the further breakage of the unit linkage of the chitosan chain. Moreover, the cracking of the chitosan chain also enhanced the deacetylation reaction due to more reactive sites exposed and facilitated the increase of chitosan DD.23 Although the impact of reaction time on chitin deacetylation was cumulative, excessive extension of reaction time caused a decline in deacetylation efficiency. When the reaction time was 12 h, the obtained chitosan showed preferable DD value exceeding 80% and further prolonging reaction time had a minor role on the continuous decrease of DD (shown in Figure 2c). Therefore, 12 h was chosen as the optimized reaction time. Solid−Liquid Ratio. Figure 2d illustrates the influence of the solid−liquid ratio (chitin−glycerol, w/w) on the DD and MV of chitosan obtained at 180 °C for 12 h under 30% NaOH concentration. Compared with other variables, the solid−liquid ratio showed minimal effect on the DD, which was also proved by previous reports when the deacetylation processes of chitin from pink shrimp29 and squid pens28 were studied. Nevertheless, after detailed comparison of DD under different solid− liquid ratios, it revealed that with the increase of glycerol ratio from 1:30 to 1:40, the DD of chitosan increased from 78.42% to 85.36% and the further addition of glycerol would not lead to apparent improvement of chitosan DD. This phenomenon indicated that the glycerol content with the solid−liquid 1:40 was enough for the sufficient deacetylation reaction. Besides,

the increase of glycerol used in the deacetylation process has little effect on the molecular weight of chitosan as shown in Figure 2d. Thus, it could be deduced that even the glycerol content was raised and the system was more advantageous to sufficient reaction, the breakage of chitosan chain would not be intensified since the reaction was fully completed under the lower glycerol content. As the solid−liquid ratio showed less effect on the chitosan MV and positive influence on the DD, higher glycerol content was preferable for this deacetylation process. However, vast glycerol use resulted in the dramatic increase of cost for glycerol recycling. Therefore, after meeting the requirement of chitosan DD, the solid−liquid ratio 1:40 with less glycerol content was preferred. Consequently, the variables tested in this work including reaction temperature, NaOH concentration, and reaction time, accelerated the reactions during chitin deacetylation process by UGARS, which promoted the increase of DD and the reduction of MV. However, over-raising these variables also caused side effects including chemical wasting and efficiency decreasing. Thus, the parameters for chitin deacetylation by the proposed approach were optimized at 180 °C, 30% NaOH concentration, 12 h, and 1:40 solid−liquid ratio. It was notable that in the previous literature which investigated the deacetylation process using aqueous solutions, the NaOH concentrations used were generally over 40% due to the highly recalcitrant structure of chitin.7,14,23 The apparently lower NaOH concentration employed in this approach was primarily ascribed to the higher reaction temperature which accelerated the transfer of NaOH from solution to the surface and the inside of chitin particle, thus realizing the effective deacetylation of chitin. The DD and MV of chitosan obtained at optimized conditions were about 85.36% and 30 847 g mol−1, respectively. The chitosan obtained by the traditional method had nearly an equal value of DD (83.54%), but much higher value of molecular weight (159 017 g mol−1). It is well-known that chitin degradation occurred during the deacetylation reaction, which induces the reduction of chitosan molecular weight.30,33 Weska et al. investigated the deacetylation process of chitin from shrimp shell and found that the viscosity molecular weight of chitosan was more than 150 kDa.24 Chang et al. also demonstrated that the chitosan obtained from pink shrimp shell possessed a molecular weight over 250 kDa.29 However, the MV of chitosan prepared by UGARS was from 30 kDa to 80 kDa and distinctly lower than that of chitosan, which was also prepared from shrimp shell α-chitin. The substantially lower molecular weight was mainly caused by the higher temperature applied in this novel approach. Recently, the lower molecular weight chitosan has been paid more attention in terms of 4694


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band at 1662 cm−1 corresponded to the stretching of CO in amide bond decreased obviously, which indicated the effective deacetylation of chitin.11 At the same time, the remarkable reduction of band at 1310 cm−1, which was assigned to the CO−NH bending vibration, also demonstrated the successful deacetylation.11 Furthermore, the bands assigned to the stretching vibrations of glucosidic bond of chitosan polysaccharide structure at 1151, 1098, and 1021 cm−1 weakened distinctly after the deacetylation process, which also confirmed the depolymerization of chitosan36 However, the three peaks (1151, 1098, and 1021 cm−1) of parts b, c, and d revealed weaker intensity than these peaks of e. It might be explained by the depolymerization of chitosan in this novel process had higher intensity than that in the traditional method. This also corresponded to the reduction of chitosan molecular weight. It was noteworthy that the bands nearly disappeared at 3260 and 3107 cm−1 originating from N−H stretching after deacetylation indicated that the reaction disturbed the regular hydrogen bond of N−H in the unreacted chitin, which also could be testified by the XRD characterization. TGA. Figure 6a,b shows the thermogravimetric analysis curves (TGA) and differential thermogravimetric (DTG) curves of representative chitin and chitosan samples, respectively, which reveal the weight loss of the samples when they are heated in an oxygen-free atmosphere. From the TGA curves it could be briefly found that all the obtained chitosan samples and chitin showed two decomposition steps, which accorded with the previous reports.11,14,26,35 The first decomposition occurred in the range of 70−120 °C corresponding to the moisture vaporization. The second decomposition emerged at 350−400 °C for chitosan samples and at 400−410 °C for the corresponding chitin as seen in Figure 6a. The second decomposition was attributed to the degradation of chitin and chitosan structure, including the dehydration of saccharide rings and the decomposition of the acetylated and deacetylated units of chitin.11 The percentage of residual mass after heating at 700 °C was about 30−40% and the higher residual mass of chitosan compared with that of chitin might be on account of minerals from the neutralization process during chitosan recovery. As shown in Figure 6b, the peak in the DTG curves of chitin and all chitosan samples signified the temperature at the maximum rate of weight loss whose quantitative value is given in Table 2. It could be observed that the decomposition temperature of chitin was apparently higher than obtained chitosan, which indicated the chitin had higher thermal stability than its deacetylated products. This observation was attributable to the destruction of chitin compact structure during the reaction, which weakened the bonding of polysaccharide chains and made the polymer more sensitive to high temperature. Moreover, after further comparison of DTG curves about pyrolyzed chitosan with different DD, it was found that higher DD chitosan degraded at lower temperature, which suggested that the N-acylation provided the thermal stability to samples.37 This result was also supported by other scholars who studied the thermal properties of chitosan extracted from silkworm chrysalides, shrimp, and crab shells.11,26 Besides, the decrease of chitosan molecular weight also contributed to the reduction of decomposition temperature. XRD. XRD analysis was applied to detect the crystallinity of chitosan prepared by UGARS. Figure 7 shows a comparison among the XRD patterns of the representative chitosan samples with different DD as well as chitin. It could be noticed that the

medicinal applications due to its nontoxic and high solubility properties as well as the positive physiological effects.34 Hence, the production of lower molecular weight chitosan is potentially beneficial for relevant applications. Characterization of Chitosan. FTIR. Besides the DD and MV, the obtained chitosan were further characterized by using FTIR, TGA, and XRD analysis to investigate the effects of deacetylation process on the chemical structure, thermal properties, and crystallinity of chitosan. FTIR is a convenient method to nondestructively characterize chitosan, which reflects the chemical structure of chitosan including the common features as well as particular vibrations. The FTIR spectra of representative chitosan samples obtained under different conditions as well as chitin are illustrated in Figure 5.

Figure 5. FTIR spectra of chitin and chitosan prepared by UGARS (a) chitin; (b) 1:40, 30%, 180 °C, 4 h; (c) 1:40, 20%, 180 °C, 12 h; (d) 1:40, 30%, 180 °C, 12 h; (e) chitosan obtained by conventional method).

As could be seen from the spectra, spectrum a showed basic bands of typical chitin: the broad band at about 3445 cm−1 corresponded to the vibrational OH stretching, the band at 1662 cm−1 assigned to the amide I stretching of CO, the band at 1623 cm−1 attributing to the stretching of C−N vibration with the superimposed CO group, the intense peak at 1556 cm−1 arising from the N−H deformation of amide II, and the band at 1310 cm−1 originating from amide III due to the formation of the CO−NH group.7,11,18,35 The bands at 3260 and 3107 cm−1, which suggested the characteristic stretching of N−H originated from hydrogen bond, demonstrated that the chitin used in this study was α-chitin as these bands were weak or not easily observed in β-chitin.14 The bands of b, c, and d corresponded to the spectra of representative chitosan obtained under different conditions. Figure 5e spectrum represented the chitosan prepared by the conventional method. The spectra of parts b, c, d, and e showed that the chitosan had the similarly characteristic absorption peaks. Therefore, it could be deduced that the chitosan prepared by UGARS had similar chemical structure to the chitosan produced using the traditional method. It could be seen from Figure 5 that after deacetylation, the intensity of 4695


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Figure 6. TGA curves (a) and DTG curves (b) of chitin and chitosan prepared by UGARS: (a)chitin; (b) 1:40, 30%, 180 °C, 4 h; (c) 1:40, 20%, 180 °C, 12 h; (d) 1:40, 30%, 180 °C, 12 h.

Table 2. Decomposition Temperature and Crystallinity of Chitin and Representative Chitosan Samples Prepared by UGARS deacetylation conditions samples a (chitin) b c d

temperature (°C) 180 180 180

NaOH concentration (%) 30 20 30

time (h) 4 12 12

DD% 62.27 ± 1.10 70.62 ± 1.28 85.36 ± 1.04

XRD patterns of chitin and chitosan had two characteristic crystalline peaks at 9−10° and 19−20°, which were in good agreement with previous studies.14,26,38 After deacetylation, the diffraction peaks of chitosan were found to shift slightly to a lower degree, which indicated that the crystal structure of chitin underwent perceptible changes during the reaction. This result also conformed to the FTIR spectra analysis. As shown in Table 2, the crystallinity of chitin was 66.49%, while the crystallinity of produced chitosan was 49.59−55.96%. This observation was also reported by other studies where the

MV (Da)

decomposition temperature (°C)

crystallinity (%)

67995 ± 2000 46920 ± 1360 30847 ± 1123

403 387 370 355

66.49 54.81 53.80 49.59

crystallinity of chitin and chitosan from crab shell and prawn shell was compared.14,38 It was suggested that the reduction of chitin crystallinity after reaction was primarily caused by the damage of crystalline area during the deacetylation reaction.26,30,38 In addition, it could be found that the crystallinity of chitosan reduced with the increase of chitosan DD. This result could be potentially attributed to the negative effect of N−H on the formation of crystallinity structure by the ordered polysaccharide chain. Besides, the lower crystallinity of chitosan with higher DD could also be a result from the generation of 4696


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Figure 7. X-ray diffraction patterns of chitin and chitosan prepared by UGARS: (a) chitin; (b) 1:40, 30%, 180 °C, 4 h; (c) 1:40, 20%, 180 °C, 12 h; (d) 1:40, 30%, 180 °C, 12 h.

presented a negative effect on the thermal stability and crystallinity. What’s more, the glycerol and base are recycled in this study. Finally, the study provides an effective approach for chitin deacetylation and potentially promotes the green utilization of chitin as a preparation source for chitosan.

lower molecular weight chitosan due to the main chain fracture during the deacetylation reaction.30 Recovery of Glycerol and NaOH. The recovery rates of glycerol and NaOH after different usage times are revealed in Table 3. The deacetylation process was conducted under the Table 3. Yield and Usage Time of Recovered Glycerol and NaOH

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usage time of glycerol and NaOH

recovery rate of glycerol and NaOH /%

DD of chitosan/%

1 2 3

90.79 89.48 90.03

85.36 ± 1.04 82.42 ± 1.13 80.36 ± 0.89

*Phone: +86 02260601313. E-mail: [email protected]. *Phone: +86 02260601313. E-mail: [email protected].

AUTHOR INFORMATION

Corresponding Authors

ORCID

Guanhua Wang: 0000-0003-1667-9698 Notes

The authors declare no competing financial interest.

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optimized conditions. The results showed that the recovery rates of glycerol and NaOH were kept stable at around 90% in the first three usage times. About 10% loss of recovery rate was possibly caused by the adsorption of a small amount of glycerol and NaOH to chitosan after centrifugation. With the increase of glycerol and NaOH usage times, the chitosan DD showed a small reduction from 85.36% to 80.36%, which might be attributed to the decrease of NaOH content in the reaction system due to formation of sodium acetate after deacetylation. Nonetheless, the recovery of glycerol and NaOH is feasible for the chitin deacetylation reaction due to the satisfied chitosan DD shown in Table 3.

ACKNOWLEDGMENTS Financial support for this study was kindly provided by Natural Science Foundation of Tianjin City (Grant 16JCQNJC05900), Foundation of Key Laboratory of Recycling and Eco-treatment of Waste Biomass of Zhejiang Province (Grant 2016REWB21), Foundation of State Key Laboratory of Pulp and Paper Engineering (Grants 201503, 201611), and Foundation of State Key Laboratory of Tree Genetics and Breeding (Chinese Academy of Forestry) (Grant TGB2016002).

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CONCLUSIONS This work innovatively proposed glycerol as green reaction solvent for the chitosan preparation from chitin at elevated temperature, which resulted in the successful deacetylation of chitin. The novel deacetylation conditions were optimized as reaction temperature of 180 °C, reaction time of 12 h, NaOH concentration of 30% and solid−liquid ratio of 1:40. Under the optimized conditions, the chitosan with high DD and low MV could be gained. The subsequent characterization of chitosan suggested that the deacetylation and degradation of chitin proceeded concurrently during the reaction and the DD

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Preparation and Characterization of Chitosan by a Novel

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