Recyclable Universal Solvents for Chitin to Chitosan with Various

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Recyclable Universal Solvents for Chitin to Chitosan with Various Degree of Acetylation and Construction of Robust Hydrogels Yan Fang, Rongrong Zhang, Bo Duan, Maili Liu, Ang Lu, and Lina Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b03055 • Publication Date (Web): 20 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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ACS Sustainable Chemistry & Engineering

Recyclable Universal Solvents for Chitin to Chitosan with Various Degree of Acetylation and Construction of Robust Hydrogels

Yan Fang,a Rongrong Zhang,a Bo Duan,a Maili Liu*,c Ang Lu*,a Lina Zhang∗ab

a

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China b

School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China

c

State Key Laboratory of Magnetic Resonance and Molecular Physics, Wuhan

Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, China

∗

Correspondence to: Lina Zhang (E-mail: [email protected]) Ang Lu (E-mail: [email protected]) Maili Liu (E-mail: [email protected])

1

ACS Paragon Plus Environment


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ABSTRACT: Chitin and chitosan are enticing natural polymers derived from seafood wastes, and their applications mostly depend on the degree of acetylation (DA). For their efficient utilization, a series of universal solvents for the direct dissolution from chitin to chitosan with various DA ranged from 5 to 94% were designed, and robust hydrogels were constructed from their solution via a physical regeneration, for the first time. The NMR results demonstrated that K+ of KOH interacted easily with C=O group to break the NH…O=C intermolecular hydrogen bonds of chitin, whereas Li+ of LiOH could bound with NH2 group to promote the destruction of NH…O6 hydrogen bonds of chitosan. Thus, a series of LiOH/KOH/urea aqueous solutions with weight ratio of LiOH to KOH from 0 to 2.5 were developed to directly dissolve these biomacromolecules with DA ranged from 5 to 94%. Subsequently, a series of coagulants were also exploited for the regeneration of these chitin/chitosan solutions to construct the robust hydrogels with different DA. These chitin/chitosan hydrogels exhibited homogeneous network structure consisted of nanofibers with mean diameter of ~30 nm, as well as the excellent mechanical properties and high transparency. By adding fluorescent agent with strong affinity with only –NH2 group, the resulted fluorescent hydrogels with different DA could be visually recognized, because the intensity reflected the different content of the active amino group. Furthermore, the waste water after regeneration could be easily recycled via reduced pressure distillation to reuse, without consuming any chemicals or producing byproducts, leading to the thoroughly green process. This work opened a new avenue to dissolve biomacromolecules from chitin to chitosan via universal solvents and to construct 2


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new materials via green transformation, which would be beneficial to the sustainable development in the world.

Keywords chitin, chitosan, universal solvents, green transformation, recycling of waste water

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INTRODUCTION Faced with the depletion of the fossil resources and the pollution caused by the petroleum-based materials, the development of the natural polymers especially derived from agricultural and marine waste into functional materials has become global strategy. Marine life has higher bioactivity and more functions compared with organisms on land, but are less explored and investigated.1 Near 8 million tons of seafood wastes derived from crab, shrimp and lobster shells such as chitin are produced globally every year, however, some of which are dumped in landfill or the sea.2 As the second most abundant natural polymer, chitin and its deacetylative derivative chitosan, have been demonstrated to be great potentials in various fields, owing to the innately bioactivity, biodegradability, biocompatibility, and antimicrobial activity.3-8 Hence, it would bring great benefits if the seafood wastes could be utilized and transformed to high valued products. Degree of acetylation (DA) is a structural parameter which influences the biological and mechanical properties as well as the biodegradability etc of chitin/chitosan, leading to great demand to fabricate chitin/chitosan bulk materials with different DA.9-12 However, the process ability of chitin/chitosan with various DA was restricted by their poor solubility, which depends also on DA.13 To date, only chitin with extremely high DA and chitosan with low DA could be dissolved in few solvents. With DA higher than 90% the samples could be dissolved in solvents such as dimethylacetamide (DMAc)/LiCl,14 CaCl2/MeOH15 and ionic liquids,16 whereas chitosan with DA lower than 40% could be dissolved in traditional acidic media by 4


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protonation of the -NH2.17 Additionally, ionic liquid can also dissolve samples with DA below 15%.18 However, the dissolution of chitin/chitosan with moderate DA values in between was scarcely reported. The hindrances led to the heterogeneous techniques such as acetylation of chitosan and deacetylation of chitin are frequently applied, if the construction of bulk materials within the whole DA range especially the moderate DA was required, however, leading to time/energy consuming and poor mechanical properties.19-27 In that case, a direct dissolution/regeneration strategy will be highly evaluated. If chitin or chitosan samples with various DA from 0 to 100% can be directly dissolved and regenerated, the construction of various materials with assigned DA can be successfully achieved. Therefore, the seeking for such universal solvents and coagulants for the chitin/chitosan with various DA is essential; however, it has never been reported. In our laboratory, the solvent of alkali/urea aqueous solution has been demonstrated to completely dissolve the chitin with DA > 90% and chitosan with DA of 11%, and a numbers of functional materials have been constructed, showing excellent mechanical properties and bioactivities.28-35 Interestingly, the different alkali (LiOH, NaOH and KOH)/urea aqueous solutions displayed different dissolving ability, i.e., NaOH/urea dissolved the chitin samples with DA of 94%,30 whereas LiOH/KOH/urea can dissolve the chitosan with DA of 11%.31 Since the DA value only dominate the content between acetyl amine group and amino group, it is reasonable that such groups have different affinity with LiOH, NaOH and KOH, and thus it is possible to explore direct solvents for samples within the whole DA range. In the present work, 5



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the effects of alkali and DA on the dissolution of the samples, as well as the interactions between the alkalis and acetyl amine/amino group were investigated, for the first time. Moreover, a series of direct solvents and coagulants were rationally designed to realize the physical dissolution and regeneration of chitin/chitosan with the DA ranged from 5 to 94%. Furthermore, to protect the environment and reduce the waste of chemicals, the recycling of universal solvents and coagulants was carried out. Thus, water-based and eco-friendly solvents for the second abundant natural polymer would be developed to open a green pathway for the construction of functional materials, leading to the great improvement of the green processing technology for the biopolymers derived from seafood wastes. EXPERIMENTAL SECTION Materials. The raw chitin powder was purchased from Golden-Shell Biochemical (Zhejiang, China), which contained about 10% impurity such as protein and mineral substances. To remove protein and mineral substance, the chitin powder was purified by an established method in our laboratory.34 The weight-average molecular weight (Mw) of the purified chitin powder was determined to be 2.8×105 g/mol in 11 wt% NaOH/4 wt% urea at 5 oC by dynamic light scattering. To prepare a series of chitin with different DA, 50 g purified chitin was suspended in 1000 mL of a 50 wt% NaOH aqueous solution and reacted at 80 or 100 oC for 0.25 to 8 h. To avoid oxidation, the reaction was performed under nitrogen. After the reaction, the slurries were washed with deionized water until neutral and then dried in a vacuum oven at 60 oC for 48 h. The obtained powders were coded as S-1, S-2, S-3, S-4, S-5, S-6 and S-7, 6


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corresponding to the reaction time with 0, 0.25, 0.5, 3, 5, 7, 8 h, respectively. The degree of acetylation (DA) of the chitin powders was determined by two-abrupt-change potentiometric titration method and calculated using the following equation36 DA =

∆ν × c NaOH × 10 −3 × 16 × 100% m × 0.0994

(1)

where cNaOH and ∆ν represent the concentration and volume of NaOH consumption between the two abrupt changes of pH, respectively; m is the dry weight of chitin/chitosan power. The DA was determined to be 94, 82, 73, 58, 36, 12 and 5% for the samples of S-1, S-2, S-3, S-4, S-5, S-6 and S-7, respectively. To explore the effect of alkali species on the crystal structure of chitin and chitosan, S-1 with highest DA and S-7 with lowest DA were treated by the three kinds of alkalis as follows, respectively. S-7 was dispersed in 2 mol/L LiOH, NaOH and KOH aqueous solution, respectively, and then was frozen/thawed twice. The resultant solution was precipitated by ethanol. Subsequently, the products were separated by centrifugation, washed with water until salt-free and dried at 60 oC for 48 h. S-7 also underwent a similar process. Dissolution and regeneration. To dissolve chitin/chitosan, these samples were dispersed into the designated alkali/urea aqueous solvent and then were stored under -35 oC for 4 h until completely frozen. The frozen solid was then fully thawed and stirred extensively at room temperature. After removing air bubbles by centrifugation at 7000 rpm for 10 min at 5 oC, a transparent solution with the desirable concentration was obtained. To evaluate whether chitin/chitosan solutions were suitable for 7



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construction of chitin/chitosan based materials, the solutions were used to fabricate hydrogels. Firstly, the resultant solution was cast on a glass plate with a 0.5 mm thick layer and then immersed into various coagulants at 5

o

C, such as 45 wt%

dimethylacetamide (DMAc) aqueous solution, ethanol and ethylene glycol, respectively, for 2 h to form hydrogels. Subsequently, the resulted chitin/chitosan hydrogels were washed with deionized water until pH = 7. Recycling

of

the

Waste

Water.

The

waste

water

derived

from

the

dissolution/regeneration was handled with reduced pressure distillation to evaporate the coagulants and water out, and the rest solid was a mixture of LiOH, KOH and urea. The weighed mixture of LiOH/KOH/urea was dissolved in weighted water to form transparent solvent again. And the recovered solvent was used to re-dissolve the powder of chitin/chitosan via freeze/thaw. Then the resultant solution was regenerated in corresponding coagulants to form hydrogels at 5 oC. Preparation of fluorescent hydrogels. Chitin/chitosan hydrogels were immersed in an aqueous 1,1,2,2-Tetrakis[4-(3-sulfonatopropoxyl)phenyl]ethylene sodium salt (TSTPE) solution with 0.01 mg/mL for 24 hours and washed with deionized water, to obtain the fluorescent hydrogels. Characterization. In order to measure the solubility of the samples in the solvent, the weighted sample powder was added to 20 g solvent and frozen-thawed twice. The resultant solution was then centrifuged at 7000 rpm for 10 min. The remaining undissolved fractions were separated and washed with water to neutral, and then dried at 60 oC for 48 h in a vacuum oven. The solubility was calculated by 8


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S=

(W0 − W1 ) ( 20 + W0 − W1 )

(2)

where W0 is the weight of the original powder; W1 is the weight of the undissolved fractions and S is the solubility. The wide angle X-ray diffraction (XRD) pattern of the samples was carried out on an XRD diffractometer (D8-Advance, Bruker). The XRD patterns with Cu Kα radiation (λ = 0.15406 nm) at 40 kV and 30 mA were recorded in the region of 2θ from 5 to 45°. FT-IR of the samples was recorded on a Perkin-Elmer FT-IR spectrometer (model 1600, Perkin-Elmer Co. USA). The tested samples were prepared by the KBr disk method. The optical and fluorescent images of those hydrogels were observed with an Axioskop 200 microscope equipped with a Coolsnap MP3.3 camera (Carl Zeiss, Germany). To explore the interaction between the alkalis and chitin/chitosan, the liquid-state NMR measurements of the samples were carried out on an Avance Ⅲ 600 MHz NMR spectrometer (Bruker) at 5 oC.

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N-acetyl-D-glucosamine (GlcNAc) and

2-amino-2-deoxy-D-glucose (GlcN) were used as models to represent chitin and chitosan, respectively, as they could be dissolved in LiOH, NaOH and KOH. GlcNAc (5 wt%) and GlcN (5 wt%), were dissolved in 2 mol/L LiOH, NaOH or KOH aqueous solution, respectively. 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) and dioxane were used as an internal chemical-shift references (0 ppm/1H and 66.66 ppm/13C). Nitromethane (CH3NO2) was used as an external chemical-shift reference (382 ppm/15N). The 13C-NMR chemical shifts assignment was done referring to the report37 and confirmed by titration with variable concentrations of KOH (data not shown). 9



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The stability measurement of the chitin/chitosan aqueous solution was carried out on a rheometer (DHR-2T, A Instruments, USA) ranging from 0 to 60 oC at an angular frequency of 1 rad/s with heating rates of 1.0 oC/min. The temperature at the intersection of the storage modulus (G’) and loss modulus (G’’) was defined as the gelation temperature. The structure and morphology of the chitin/chitosan hydrogels as well as the powders were characterized by scanning electron microscopy (SEM) and UV-vis spectrometry. The optical transmittance (Tr) of the hydrogels were observed with a UV-vis spectrometer (UV-6, Shanghai Meipud an instrument Co., Ltd., Shanghai, China) at a wavelength from 200 to 800 nm. SEM observations of the inner structure of hydrogels were carried out on a Hitachi S-4000 microscope. The mechanical properties of hydrogels were measured on a universal testing machine (CMT6350, Shenzhen SANS, China) according to ISO527-3-1995 (E) at a speed of 1 mm/min. The size of the hydrogels specimens for tension was 60 mm× 10 mm × 1 mm. The gravimetric method was employed to measure the water content ( W H O ) of the 2

hydrogels, and W H O was calculated as 2

W H 2O =

Ws − Wd × 100 % Wd

(3)

where Ws is the weight of the swollen hydrogels and Wd is the weight of the dried gel. RESULTS AND DISCUSSION Interactions between alkali and chitin/chitosan. To clarify the effects of the alkali on the dissolution of chitin/chitosan, the solubility of chitin/chitosan with DA in a wide range from 5 to 94% in LiOH, NaOH and KOH was determined, respectively. 10


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The solubility of chitin/chitosan in both NaOH and KOH decreased dramatically with reducing DA, whereas an opposite trend was observed in LiOH (Figure 1), indicating DA dominated the chitin/chitosan dissolution in LiOH, NaOH and KOH. Structurally, the C=O of acetyl amine groups are linked to NH of the adjacent chains to form strong intra-sheet hydrogen bonds within the chitin crystal.38 During the deacetylation reaction, the acetyls of chitin were peeled off with exposing amino groups along the chains, leading to the modification of the intra-sheet hydrogen bonding, as the NH…O=C hydrogen bonds of chitin were replaced by NH…O6 hydrogen bonds of chitosan.39 It is not hard to imagine that LiOH, NaOH and KOH may exhibit different capacity of destroying NH…O=C of chitin and NH…O6 hydrogen bonds of chitosan, which played a key role in the dissolution of chitin/chitosan with various DA. For proofs, the effect of alkali species on the crystalline structure of chitin was studied. The FT-IR spectra of S-1 (DA = 94%) and alkali treated S-1 displayed significant differences at 1660 and 1623 cm-1 (Figure 2a), ascribed to amide I vibrations representing the C=O groups involved in the intra-sheet hydrogen bonds with NH group along the chitin. Specifically, compared with the native S-1 samples, the peak intensity at 1622 cm-1 decreased after treated with both KOH and NaOH, whereas that treated with LiOH changed hardly (Figure 2b), suggesting that the NH…O=C of intra-sheet hydrogen bonds of chitin were destroyed easily by NaOH and KOH at low temperature rather than LiOH, consistent with XRD (Fig. S1a). The crystallinity of the raw S-1 and LiOH-treated S-1 were 67 and 64%, respectively, whereas the crystallinity of NaOH and KOH treated S-1 decreased to 52 and 48%, 11



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respectively. The morphology of the alkali treated S-1 samples also varied, as shown in Fig. S1b. The LiOH treated S-1 sample retained as microfibrils similar to the native S-1, whereas smooth pattern was observed after NaOH and KOH treatment, suggesting the good dissolution of S-1 in NaOH and KOH. The results indicated that NaOH and KOH could break the NH…O=C intra-sheet hydrogen bonds of chitin, and led to dissolution, rather than LiOH. To further explore the interactions between the alkalis with chitin, we measured 1H-NMR,

13

C-NMR and

15

N-NMR spectra of

GlcNAc, monomer and representative of chitin, in aqueous (D2O) solutions with the absence and presence of LiOH, NaOH and KOH, respectively. As shown in Figure S2a, the chemical shifts in a narrow range of 1.2–5.0 ppm were ascribed to the backbone protons of GlcNAc and assigned accordingly. When alkalis were added, these signals in the alkaline solutions shifted to upfield, suggesting the interaction between GlcNAc and alkalis, leading to the weakening of the electron density of the backbone hydrogen. Meanwhile, the formation of stronger interaction between GlcNAc and alkalis also could influence the electron density of the backbone carbon, as shown in Figure S2b, c and d. When adding alkalis, the chemical shifts of C1-C6 and CH3 underwent down-field drift and the chemical shifts of those above carbons (C1- C6) in LiOH solution was more downfield shift than in NaOH and KOH solutions. For instance, the relative chemical shift changes (δalkali - δD2O) of C2β was 3.701, 3.497, 3.518 ppm for LiOH, NaOH and KOH. Two kinds of interactions occurred in the solutions, between OH- and proton of hydroxyl or amide and between oxygen and alkalis ions (Li+, Na+ and K+), two interactions both could affect the 12


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chemical shifts. At the same concentration of alkalis, the effect of interaction between OH- and hydroxyl or amide proton on the chemical shift may be similar. The difference in chemical shift changes among LiOH, NaOH and KOH may reflect mainly the interaction between oxygen or nitrogen and Li+, Na+ or K+ in the solutions. Therefore, the relative chemical shift changes of C1-C6 in LiOH was largest, suggesting that Li+ had larger effect on oxygen of hydroxyl and nitrogen of amide in chitosan than that of Na+ and K+, consistent with the results of 15N NMR. As shown in Figure 2c, the signal of 15N of GlcNAc moved to lower field in LiOH, compared with that in NaOH and KOH. Therefore, the above results showed that Li+ existed stronger effects on the oxygen of hydroxyl and nitrogen of amide than Na+ and K+. It should be noted, however, that the three kinds of alkalis displayed contrary influences on the signal of C=O. The chemical shifts of C=O in NaOH and KOH systems was more upfield shifted than in LiOH (Figure 2d), where the relative chemical shift changes (δD2O - δalkali) of β-C=O was 0.02, 0.56, 0.57 ppm for LiOH, NaOH and KOH, respectively, indicating that Na+ and K+ had stronger interaction with C=O. In fact, only KOH and NaOH could dissolve chitin, other than LiOH, suggesting that the interaction between alkali and C=O played a key role in the dissolution of chitin. Based on the above results, the stronger interaction between Na+ or K+ and the NH…O=C group promoted the destruction of the intermolecular hydrogen bonds of chitin, leading to its dissolution. On the other hand, for chitosan, the XRD profiles of S-7 (DA = 5%) and the S-7 sample treated with LiOH, NaOH and KOH was shown in Fig. S3a.The crystalline 13



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diffraction peaks at 2θ = 10.3, 19.9 and 29.5o were indexed as (020), (110) and (130) lattice diffraction of chitosan, respectively. Obviously, the intensity of the diffraction peaks corresponding to the (020) and (130) planes weakened after LiOH treatment, compared with that after NaOH and KOH, indicating that LiOH easily broken the chitosan crystal structure. From the SEM images of S-7 and the alkali treated S-7 sample (Fig. S3b), the S-7 sample treated with LiOH exhibited smooth pattern, indicating the chitosan dissolution occurred in LiOH, whereas those treated with NaOH and KOH remained unchanged. The results indicated that the hydrogen bonds of chitosan were more easily destroyed in LiOH than NaOH and KOH. To further investigate the interaction between chitosan with these three kinds of alkalis, GlcN was regarded as a model to represent chitosan. Figure 3 shows the 1H NMR and 13C NMR of GlcN in D2O, LiOH, NaOH and KOH. All GlcN solutions underwent mutarotation, leading to two sets of chemical shifts, corresponding to α- and β-conformation of GlcN. Obviously, the alkali solutions caused down-field drift for the chemical shifts of H1-H6 and C1-C6, as a result of the interaction between GlcN with alkalis. However, the chemical shifts of backbone protons and carbons in LiOH were more downfield shifted than that in NaOH and KOH, such as relative chemical shift changes (δalkali - δD2O) were 3.98, 3.87, 3.79 ppm for α-C2 and 1.35, 1.30 and 1.28 ppm for α-C6, in presence of LiOH, NaOH and KOH, respectively. It was revealed that Li+ had larger effect on oxygen/nitrogen of hydroxyl/amino than Na+ and K+, resulting in the destruction of the NH…O6 hydrogen bonds of chitosan, leading to the good dissolution of chitosan in LiOH system. This was consistent with our 14


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observation that LiOH solution has high solubility for chitosan than that of NaOH and KOH. Dissolution of chitin/chitosan via universal solvents. On the basis of the above mentioned analysis, a schematic model to qualitatively describe the interactions between alkali and chitin/chitosan was proposed in Figure 4a. KOH or NaOH prefer to interact with C=O, leading to the destruction of the NH…O=C intermolecular hydrogen bonds of chitin. However, LiOH exhibited stronger effects on the amino group, resulting in the destruction of NH…O6 hydrogen bonds of chitosan. Therefore, depending on the ratio of C=O/NH2 along the chitin/chitosan chain, i.e. the DA value, a series of universal solvents to dissolve chitin to chitosan with various DA were developed, as summarized in Table 1. When DA was in the range from 90 to 100%, the solvent was 8 wt% KOH/4 wt% urea；with a decrease of DA, LiOH was necessary for the emerging amino groups, thus the solvent was 3 wt% LiOH/8 wt% KOH/9.5 wt% urea for chitin with DA in the range from 70 to 90%; with further reducing DA, the solvent was regulated as 4.5 wt% LiOH/7 wt% KOH/8 wt% urea for the samples with DA in the range from 10 to 70%, as well as 5 wt% LiOH/2 wt% KOH/14 wt% urea for that with DA in the range from 0 to 10%, respectively. Summarily, a series of LiOH/KOH/urea aqueous solutions with weight ratio of LiOH to KOH from 0 to 2.5 were developed to directly dissolve the chitin/chitosan samples with DA ranged from 5 to 94% via freezing-thawing method. As expected, a series of transparent chitin/chitosan solutions were obtained, as shown in Figure 4b, indicating that these solvents were validated to dissolve successfully chitin/chitosan with various DA. 15



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To further clarify the dissolution of chitin/chitosan in alkali/urea aqueous solvents, the 1H NMR experiments were performed, as shown in Figure 4c. The chemical shifts of chitin/chitosan solutions with varying DA at 3.97 ppm, 3.27 ppm, 3.13 ppm, 3.06 ppm, 2.80 ppm, 2.09 ppm, 1.51 ppm can be attributed to H1, H6, H4, H3, H6, H5, H2 and CH3, respectively. No new peaks were identified for the chitin/chitosan derivatives in the 1H NMR spectra, suggesting the dissolution of these samples in the alkali/urea systems was a physical process. To determine the stability for further applications of the solvents and the resultant chitin/chitosan solutions, the gelation behavior of the concentrated chitin/chitosan solutions were investigated with dynamic viscoelastic measurements (Figure 5a and b). Apparently, the gelation temperature of those solutions with 5 wt% concentration ranked from 32 to 50 oC, indicating the resultant chitin/chitosan solutions were relatively stable at room temperature and suitable for their materials construction. Therefore, a series of LiOH/KOH/urea aqueous solvent could successfully dissolve the intransigent polysaccharide from chitin to chitosan with various DA, via tuning the ratio of KOH to LiOH in the solvents, to prepare transparent solution for further applications. Green transition of chitin/chitosan into robust hydrogels. For construction of materials, a series of coagulants were developed, to regenerate the above-mentioned solutions of chitin/chitosan with different DA. Several types of aqueous system such as 5 wt% Na2SO4, 5 wt% NH4Cl and 5 wt% CaCl2 and water-miscible organic solvents (ethanol, acetone, ethylene glycol and 45 wt% DMAc) were employed here. The hydrogels regenerated in organic solvents were relatively more transparent and 16


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mechanically stronger than that from the aqueous systems. This could be explained by the violent solidification process in the salt solution, which resulted in the phase separation and the formation of in homogeneous structure. Therefore, the organic solvents (ethanol, ethylene glycol and 45 wt% DMAc) were chosen as the coagulants for the physical regeneration of the chitin/chitosan. Interestingly, DA also dominated the selection of the organic solvents as coagulants. In general, the optical transmittance of the regenerated hydrogels is an auxiliary method to evaluate the homogeneity of the regenerated materials. As shown in Fig. S4, the S-1, S-2 and S-3 with high DA (from 70 to 100%) regenerated in ethanol exhibited higher optical transmittance (Tr) than that in 45 wt% DMAc and ethylene glycol (Table S1). However, the S-4 and S-5 with middle DA (from 30 to 70%) regenerated in 45 wt% DMAc exhibited excellent transparence with Tr values of 90 and 89% at 800 nm, respectively. Furthermore, the S-5 and S-5 with low DA (from 0 to 30%) regenerated in ethylene glycol were relatively more transparent. However, the S-6 and S-7 hydrogels regenerated in ethanol and 45 wt% DMAc emerged serious wrinkle and extremely white, suggesting phase separation. In view of the SEM images of cross-section of the regenerated chitin/chitosan hydrogels (Fig. S5). The transparent hydrogels, i.e. S-1, S-2 and S-3 regenerated in ethanol, S-4 and S-5 regenerated in 45 wt% DMAc, and S-6 and S-7 regenerated in ethylene glycol all displayed homogeneous network architecture, weaved by nanofibers with mean diameter of ~ 30 nm, as shown along the dotted line in Fig. S5. It has been demonstrated in our labs that chitin and chitosan existed as an extended wormlike chain in alkali/urea aqueous 17



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solutions and easily aggregated in a parallel manner to form homogeneous nanofibers.30-32 On the contrary, micro- and nano-porous structures would greatly contribute to reduce the optical scattering and reflecting, and thus improve the light transmittance.40, 41 Therefore, these hydrogels with low light transmittance displayed heterogeneous structure, as a result of the phase separation, such as the S-1, S-2, S-3 hydrogels regenerated in ethylene glycol, as well as S-6 and S-7 hydrogels regenerated in ethanol and 45 wt% DMAc. It is well-known that the polymer gelation with coagulants involves the ternary system (solvent, coagulant, polymer). The polarity of coagulation (H2O > ethylene glycol > DMAc > ethanol) and the diffusion of alkali in the coagulation as well as the hydrophilic-hydrophobic properties of macromolecular chain, are the main factors, resulting in the difference in the ability of the solvents to destroy the interaction between the solvent and macromolecules. As mentioned above, under a moderate coagulation condition the chitin/chitosan chains in the solution could be slowly self-aggregated in parallel to form nanofibers (Figure 6a), endowing the hydrogels with high optical transmittance and excellent mechanical properties. As shown in Figure 6b and c, the optical transmittance of the hydrogels at 800 nm were all over 80%, suggesting the uniform structure. Moreover, the values of σb of these transparent hydrogels at swelling equilibrium displayed high strength ranged from 1.33 to 2.19 MPa (Figure 6d), much higher than the reported chitosan hydrogels by neutralization with acidic solutions (σb ranging from 0.1 to 0.372 MPa).42,43 The tough hydrogels could be twisted and knotted, as shown in Figure 6b. Additionally, the transparent hydrogels exhibited more toughness with 18


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reducing DA, and the elongation at break (εb) values at swelling equilibrium state of S-1 (DA = 94%) hydrogel regenerated in ethanol were 45%, and increased to 87% for S-4 (DA = 58%) regenerated in 45 wt% DMAc. This could be explained that less contribution of acetyl group on hydrogen bonds in the network with reducing DA, accelerating the sliding of chain under tensile force. Therefore, it could be concluded that ethanol, 45 wt% DMAc and ethylene glycol, were suitable coagulants for chitin/chitosan with high, moderate and low DA values. Moreover, the regenerated hydrogels displayed excellent mechanical properties with high optical transparence, as a result of the formation of the homogeneous network structure consisted of chitin/chitosan nanofibers. These robust hydrogels would have great application in varied fields such as hygiene, agriculture, biomedical materials and pollutant absorbents. Additionally, due to those hydrogels containing abundant NH2 and C=O, they could be catalyst or ideal catalyst supports in chemical reactions.44 Moreover, this was a thoroughly green process for the construction of novel materials derived from renewable resource by using nontoxic and eco-friendly solvents via physical dissolution and regeneration, which would be beneficial to the sustainable chemistry and engineering. Recycling of the waste water. To protect the environment and reduce the waste of chemicals during the production process, a recycling technology was carried out to recover the universal solvents. After regeneration of chitin/chitosan to prepare hydrogels, the obtained waste water generally contained organic coagulants (ethanol, DMAc or ethylene glycol), LiOH, KOH, urea and water, where the organic coagulants 19



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and water could be easily removed by reduced pressure distillation, due to the evaporable properties, where the evaporating temperature was 60 oC, 60 oC and 68 oC for ethanol, DMAc or ethylene glycol, respectively. For example, in Figure 7 the waste water after the regeneration of S-3 solution contained ethanol, LiOH, KOH, urea and water (Figure 7a). Via the reduced pressure distillation at 60 oC, the ethanol and water were easily evaporated and the rest solid was a mixture of LiOH, KOH and urea (Figure 7b). The mixture could form transparent solvent again by adding H2O (Figure 7c), and as expected such recovered solvent was capable of dissolving the S-3 sample well again via freeze/thaw to obtain a transparent S-3 solution (Figure 7d). The resultant S-3 solution could be regenerated in ethanol to fabricate robust and transparent hydrogel (Figure 7e) again. Clearly, the recovery of the universal solvent was simple and facile, and without consuming any chemicals or producing byproducts. Therefore, the construction of new materials derived from chitin/chitosan was environmentally friendly process, and recycling of the waste water easily performed, showing great potentials in the green chemistry field. Fluorescent hydrogel based on chitin/chitosan. For visually identification of chitin and

chitosan

components

in

these

hydrogels,

1,1,2,2-Tetrakis[4-(3-sulfonatopropoxyl)phenyl]ethylene sodium salt (TSTPE) was selected as fluorescent agent to add into the hydrogels. TSTPE could interact with the amino groups of chitosan via electrostatic interaction, leading to the fluorescent emission. As shown in Figure 8, TSTPE was absorbed onto amino group on the surface of the chitosan nanofibers of the hydrogels to produce blue-colored 20


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fluorescent. Moreover, the fluorescent properties of the hydrogels were investigated with the excitation peak in 360 nm. Those fluorescent hydrogels exhibited an intense and narrow emission spectrum with a peak at 480 nm, where the fluorescence intensity increased with decreasing DA. Namely, the fluorescence intensity of the chitin/chitosan hydrogels depended on the content of the amino group. Therefore, the hydrogels consisted of chitin/chitosan with various DA could be visually recognized via the fluorescent emission method. Furthermore, they could be easily fabricated for wide applications such as photoluminescence device, fluorescent and biosensor. CONCLUSION A series of universal solvents for the dissolution from chitin to chitosan with various DA were developed successfully. It was demonstrated that KOH in the solvents preferred to destroy the NH…O=C intermolecular hydrogen bonds of chitin, whereas LiOH could easily break the NH…O6 hydrogen bonds of chitosan. Thus, by tuning the weight ratio of KOH to LiOH in the solvents, a series of LiOH/KOH/urea aqueous solutions were used as universal solvents for the dissolution of the chitin samples with various DA from 5 to 94% via freezing/thawing method. The dissolution of chitin/chitosan in LiOH/KOH/urea systems was a physical process. Depending on DA and the moderate interaction between alkali and coagulants, the resultant solutions with various DA were regenerated, respectively, in ethanol, 45 wt% DMAc and ethylene glycol to form robust hydrogels. The regenerated hydrogels were weaved with nanofibers, which greatly contributed to the reinforcement, leading to the excellent mechanical properties with high transparence. Moreover, by adding 21



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fluorescent agent, the hydrogels could be visually recognized, as a result of the dependence of fluorescent intensity on the content of amino group. Furthermore, the waste water after regeneration could be easily recycled by reduced pressure distillation, where the organic coagulants and water could be easily removed due to the evaporable properties. Moreover, the rest solid could be reused to dissolve chitin/chitosan, without consuming any chemicals or producing byproducts. Therefore, this work provided a physical process of dissolution and regeneration for the construction of novel materials from seafood waster by using nontoxic and eco-friendly solvents. This green transformation from renewable resource into new materials would be beneficial to the sustainable chemistry and engineering. ACKNOWLEDGMENTS This work was supported by the Major Program of National Natural Science Foundation of China (21334005), the Major International (Regional) Joint Research Project of National Natural Science Foundation of China (21620102004), the National Natural Science Foundation of China (51203122, 51573143 and 20874079), the Fundamental Research Funds for the Central Universities (2015203020202). SUPPORTING INFORMATION XRD patterns and SEM images of S-1 and S-1 treated with LiOH, NaOH and KOH, respectively, as well as S-7;

1

H NMR and

13

C NMR spectra of

15

N-acetyl-D-glucosamine dissolved in D2O, LiOH, NaOH, and KOH, respectively;

SEM images of the cross-sectional structures and photographs of the regenerated chitin/chitosan hydrogels fabricated from different coagulating baths; physical and mechanical properties of the chitin/chitosan hydrogels regenerated in different 22


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Table 1. Samples and solvents for chitin/chitosan with different DA. Samples No. S-1 S-2 S-3 S-4 S-5 S-6 S-7

DA (%) 94 82 73 58 36 12 5

Solvents 8 wt% KOH/4 wt% urea 3 wt% LiOH/8 wt% KOH/9.5 wt% urea 3 wt% LiOH/8 wt% KOH/9.5 wt% urea 4.5 wt% LiOH/7 wt% KOH/8 wt% urea 4.5 wt% LiOH/7 wt% KOH/8 wt% urea 4.5 wt% LiOH/7 wt% KOH/8 wt% urea 5 wt% LiOH/2 wt% KOH/14 wt% urea

Concentration (wt%) 5 5 5 5 5 6 6

Figure 1. Three-dimensional phase diagrams for solubility of chitin/chitosan with various DA in LiOH, NaOH and KOH aqueous solutions, respectively.

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Figure 2. FT Raman spectra of S-1 and S-1 treated with H2O, LiOH, NaOH and KOH, respectively (a, b); 15N NMR spectra of 15N-acetyl-D-glucosamine dissolved in D2O, LiOH, NaOH and KOH (c); Expansions of the C=O regions of 13C NMR of spectra of 15

N-acetyl-D-glucosamine dissolved in D2O, LiOH, NaOH and KOH (d).

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Figure 3. 1H NMR (a) and 13C NMR (b, c, d) spectra of 2-amino-2-deoxy-D-glucose dissolved in D2O, LiOH, NaOH and KOH, respectively.

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Figure 4. A schematic model to qualitatively describe the interactions between alkali and chitin/chitosan (a); Photographs of transparent chitin/chitosan aqueous solutions (b); 1H NMR spectra of chitin/chitosan aqueous solutions (c).

Figure 5. Temperature dependences of the storage modulus G′ (blue) and loss modulus G″ (red) of the chitin/chitosan solutions (a); Gelation temperature of chitin/chitosan solution as a function of DA (b), where the data were evaluated from (a). 32


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Figure 6. SEM images of cross-sectional structures of the regenerated chitin/chitosan hydrogels, where S-2 hydrogel was regenerated in ethanol; S-4 hydrogel was regenerated in 45 wt% DMAc; S-6 hydrogel was regenerated in ethylene glycol (a); photographs of the chitin/chitosan hydrogels knotted and twisted (b); the light transmittance values of hydrogels at 800 nm (c) and mechanical properties (d) of the 33



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chitin/chitosan hydrogels, where S-1, S-2 and S-3 were regenerated in ethanol; S-4 and S-5 were regenerated in 45 wt% DMAc; S-6 and S-7 were regenerated in ethylene glycol.

Figure 7. Graphical illustration of the recycling of the waste water: the waste water (ethanol, LiOH/KOH/urea and water) (a); the obtained solid mixture of LiOH, KOH and urea (b); the recovered solvent dissolved with water (c); the S-3 solution dissolved in the recovered solvent (d); the regenerated robust hydrogel (e).

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Figure 8. SEM images of the cross-sectional structures of S-5 fluorescent hydrogels (a); mechanism for the in-situ synthesis of the fluorescent hydrogel (b, c); photographs (d) and the fluorescence intensity (e) of fluorescent hydrogel based on chitin/chitosan.

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Table of Content Recyclable Universal Solvents for Chitin to Chitosan with Various Degree of Acetylation and Construction of Robust Hydrogels Yan Fang, Rongrong Zhang, Bo Duan, Maili Liu*, Ang Lu*, Lina Zhang∗

Synopsis: Recyclable universal solvents for chitin/chitosan with various degree of acetylation were developped, and robust hydrogels with fluorescence were constructed from their solutions via green pathway.

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Recyclable Universal Solvents for Chitin to Chitosan with Various

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