Preparation and Hydrogel Properties of pH-Sensitive Amphoteric

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Preparation and hydrogel properties of pHsensitive amphoteric chitin nanocrystals Jie Jiang, Juan Yu, Liang Liu, Zhiguo Wang, Yimin Fan, Tsuguyuki Saito, and Akira Isogai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02899 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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Journal of Agricultural and Food Chemistry

Preparation and hydrogel properties of pH-sensitive amphoteric chitin nanocrystals Jie Jiang a, Juan Yu a, Liang Liu a, Zhiguo Wang a, Yimin Fan a,*, Tsuguyuki Satio b and Akira Isogai b a

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources,

Jiangsu Key Lab of Biomass-based Green Fuel & Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China b

Department of Biomaterials Science, Graduate School of Agricultural and Life Sciences, The

University of Tokyo, Tokyo 113-8657, Japan

ABSTRACT Different from single charged or uncharged nanocrystals, amphoteric chitin nanocrystals (A-ChNCs)

with

both

amine

2,2,6,6-tetramethylpiperidine-1-oxy-radical

and

carboxylate

(TEMPO)-mediated

groups oxidation

prepared and

with partial

deacetylation were individually nanodispersed by sonicating in water at pH 3 and pH 11. The effects of the amount of NaClO2 added in TEMPO-oxidation, deacetylation time, and sequence of the two treatments on the weight recovery ratios of the A-ChNCs were investigated. The A-ChNCs prepared under optimum conditions had an average length of ~544 nm and an average width of ~10 nm. The A-ChNCs nanodispersed in water at pH 3 and pH 11 had absolute ζ-potentials of >30 mV; however, in neutral water, they formed aggregations, which were nanodispersed again when pH was adjusted to 3 or 11, showing pH sensitivity. Hydrogels of

ACS Paragon Plus Environment


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A-ChNC were prepared by adding saturated NaCl solution and adsorbed both anionic and cationic dyes. Freeze-dried A-ChNC aerogels had three-dimensional network structures containing abundant pores.

Keywords: amphoteric chitin nanocrystals, pH sensitivity, TEMPO oxidation, deacetylation

1. Introduction The

second

most

abundant

biopolymer

on

earth,

chitin

consisting

of

2-acetamido-2-deoxy-β-D-glucose units is primarily present in the exoskeletons of arthropods.1,2 Chitin has attracted increasing attention owing to its mechanical strength, biodegradation, and biocompatibility.3-6 Chitin forms semicrystalline nanofibers embedded in a protein matrix, and the tight structures result in its insolubility in water and common organic solvents, which significantly prevent the large-scale applications of chitin.7,8 In 2001, Paillet and Dufresne reported the potential application of chitin whiskers as reinforcing fillers in thermoplastic matrices.9 Since then, new and broad applications of chitin in electronics, medical materials, and cosmetics have been developed in recent years.10-12 Chitin nanocrystals (ChNCs) are isolated from α-chitin by mechanical disintegration in water13,14 and combined with acid hydrolysis.15,16 However, ChNCs easily form aggregates and are difficult to be nanodispersed in

water.

In

2008,

Fan

et

al.

applied

2,2,6,6-tetramethylpiperidine-1-oxy-radical

(TEMPO)-mediated oxidation of α-chitin to introduce sodium carboxylate groups (C6-COONa) into the crystalline surfaces, resulting in the formation of ChNCs that were well nanodispersed in


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water under neutral and alkaline conditions. Electrostatic repulsive forces efficiently work between the anionically charged ChNC surfaces in water.17 ChNCs with cationic charges from the C2-NH3+ groups were successfully prepared from α-chitin by partial deacetylation and subsequent mechanical treatment in acidic water.18 Partially deacetylated ChNC films and ChNC films composed of bacterial cellulose exhibit high mechanical strengths and bacteriostatic activities.19,20 Due to the positive and negative surface charges of ChNCs, their hydrogels are formed by gas-phase coagulation with ammonia or hydrochloric acid and adsorb to acidic or basic dyes, respectively.21 Recently, TEMPO-mediated oxidation and subsequent partial deacetylation of α-chitin were performed by Ifuku et al. to prepare amphoteric ChNCs (A-ChNCs), which contained both cationic (C2-NH3+) and anionic (C6-COO-) groups and were nanodispersed in water under both acidic

and

alkaline

conditions.22

However,

the

TEMPO-mediated

oxidation

(TEMPO/NaBr/NaClO oxidation) was performed in water at pH 10, which caused severe depolymerization of the chitin molecules, resulting in the low yield of A-ChNCs (~38%). Thus, the TEMPO/NaClO/NaClO2 oxidation system has been used under weakly acidic conditions, and the yields of A-ChNCs have been effectively improved.23 The combination of TEMPO-mediated oxidation and subsequent deacetylation was used to prepare nanodispersed A-ChNCs in water under both acidic and alkaline conditions. The influence of the preparation conditions of A-ChNCs on their morphologies, ionic charges, and charge densities, which are significant for the large-scale production and further applications of ChNCs, has not been discussed in detail. The contents of carboxylate and amine groups in the A-ChNCs significantly influence their nanodispersibility in water at different pH values. In this study, we investigated the changes in



carboxylate and amine contents after every separate treatment. Moreover, the influence of the sequence of TEMPO-mediated oxidation and deacetylation on the properties of A-chitins and A-ChNCs was also studied. The amphoteric properties of chitins and A-ChNCs depend on the balance between the C6-carboxylate and C2-amine groups in these samples.

2. Experimental 2.1. Materials The α-chitin powder was purchased from Aladdin Chemical (China) and was used after purification with 1 M NaOH and 1 M HCl to remove as much protein and mineral as possible according to a previous report.24 TEMPO and NaClO2 were purchased from Sigma Aldrich (USA). All other reagents were purchased from Aladdin Chemical (China) and were used without further purification. 2.2. Partial deacetylation of chitin Chitin (1 g) was added to a flask containing 30% NaOH (20 mL), and the mixture was heated at 90°C for a designated time under continuous stirring in an oil bath. After the deacetylation treatment, the mixture was cooled in icy water, and the insoluble fraction was washed repeatedly with distilled water until neutral via centrifugation at 10251 ×g for 10 min. The partially deacetylated chitin (DeAc-chitin) was obtained as a water-insoluble fraction and stored at 4°C in the wet state. 2.3. TEMPO-mediated oxidation of chitin Chitin (1 g) was suspended in a pH 6.8 phosphate buffer containing a designated amount of NaClO2, NaClO (1 mmol), and TEMPO (0.016 g, 0.1 mmol). The oxidation process was


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conducted at 60°C for 4 h. After oxidation, a small amount of ethanol was added to the mixture to quench the reaction. The water-insoluble fraction was washed with distilled water via continuous centrifugation at 10251 ×g for 8 min, and then the insoluble fraction was kept at 4°C in the wet state before use. 2.4. Determination of carboxylate and amine contents The carboxylate content of TEMPO-oxidized chitin (Tox-chitin) samples was determined by an electrical conductivity titration method.24 A freeze-dried sample (0.1 g) was added to a beaker containing 60 mL distilled water, and the pH of the suspension was first adjusted to 10 by adding a 0.5 M NaOH solution for sufficient swelling and dispersing. After stirring for 0.5 h, a 0.1 M HCl solution was added to set the pH in the range of 2.5 to 3.0. Then, 0.05 M NaOH was added at a rate of 0.1 mL/min by using an automatic titration system until the pH was 11. The content of the carboxylate groups was calculated from the pH and conductivity data recorded every half-minute. The amine contents of the original and DeAc-chitin samples were measured by the same titration method as that used for measuring the carboxylate content. 2.5. Preparation of A-ChNCs After either partial deacetylation and subsequent TEMPO-mediated oxidation or TEMPO-mediated oxidation and subsequent partial deacetylation, the treated chitins (i.e., Tox-DeAc-chitins or DeAc-Tox-chitins, respectively) containing both amine and carboxylate groups were suspended in water at 0.1% (w/v). The pH values of the suspensions of the treated chitins were adjusted to 3 or 11 with 0.5 M HCl or 0.5 M NaOH, respectively. The suspensions were then mechanically disintegrated for 10 min using an



ultrasonic homogenizer (Sonics & Materials, Inc., USA) at 500 W and 20 kHz with a homogenizer tip (ϕ=13 mm) in start/stop intervals in ice water to avoid heating. Then, the mixture was centrifuged at 10251 ×g for 8 min to remove the unfibrillated fraction, and the A-ChNC nanodispersion was obtained in the supernatant. 2.6. Preparation of A-ChNC hydrogels A saturated NaCl solution was added dropwise to a Petri dish containing a 1% (w/w) chitin nanocrystal dispersion. The addition of the saturated NaCl decreased the electrostatic repulsion among A-ChNC elements and formed the A-ChNC hydrogel by aggregation. The mixture was allowed to rest for 10 h for the hydrogel to form completely. The hydrogel thus obtained was removed from the mixture and washed thoroughly with distilled water. 2.7. Analyses The original chitin and chemically treated chitins were freeze-dried and converted to pellets using a tableting apparatus. The X-ray diffraction (XRD) patterns of the pellet samples were recorded at diffraction angles between 5° and 35° using an Ultima IV diffractometer (Rigaku, Japan) with Cu Kα radiation (λ=0.1548 nm) at 40 kV and 40 mA. The morphologies of the chitin and chemically treated chitins were observed using an optical microscope equipped with a camera (ZEISS, Germany) after the samples were immersed in a dilute aqueous solution of toluidine blue or Ponceau S. The light transmittance spectra of the 0.1% (w/v) A-ChNC dispersions were recorded from 400 nm to 700 nm and were determined using a UV-vis spectrophotometer (Rigaku, Japan). The A-ChNC dispersion (30 mL) was poured into a Petri dish and dried in an oven at 50°C for 4 days to prepare cast-dried A-ChNC films. The sodium carboxylate groups in the


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A-ChNCs were converted to protonated carboxyl groups by immersing the A-ChNC films in 0.01 M HCl for 1 h and subsequently washing with distilled water to remove excess HCl. The films were then dried at 50°C. Fourier transform infrared (FTIR) spectra of the A-ChNC films were obtained using a VERTEX 80 V spectrometer (Bruker, Germany) in transmittance mode from 400 to 4000 cm−1 at 4 cm−1 resolution. The A-ChNC dispersion was diluted to 0.001% (w/w) with distilled water, placed on a mica plate, and dried at room temperature. The morphologies of the A-ChNCs were observed using a Dimension Edge atomic force microscope (AFM) (Bruker, Germany) in tapping mode using a standard silicon cantilever. The A-ChNC dispersion was diluted to 0.01% (w/w) with distilled water, and the size distribution and zeta potential of each A-ChNC sample were measured using a Malvern Zetasizer instrument at 25°C. The dye adsorption behavior of the A-ChNC hydrogels was investigated by adding a hydrogel (5 g containing 0.05 g dry A-ChNC) into a toluidine blue solution at pH 9 or a Ponceau S solution at pH 4 (100 mg/L, 25 mL).

3. Results and discussion 3.1. Amphoteric chitin prepared in different orders In previous studies, the C6-primary hydroxyl groups on the crystalline chitin fibrils were selectively oxidized to sodium C6-carboxylate groups by TEMPO-mediated oxidation, and the C2-acetamide groups were partly deacetylated by treatment with ~30% NaOH.17,18 In this study, these two treatments were sequentially applied to the chitin sample in the opposite order. The results showed that the different treatment sequence led to different yields and properties of the obtained chitin samples.



Figure 1. Yields and amine contents of DeAc-chitins obtained as water-insoluble fractions after deacetylation with 30% NaOH at 90°C for different times (A), and yields of Tox-DeAc-chitins after TEMPO/NaClO/NaClO2 oxidation of DeAc-chitin (prepared with 30% NaOH for 6 h in A) in water at pH 6.8 and 60°C for 4 h with different amounts of NaClO2 (B).

The purified chitin was subjected to partial deacetylation with 30% NaOH at 90°C for 6 h, resulting in an increase in amine content to 1.18 mmol/g, which corresponds to a degree of deacetylation of 22.8%. Although some of the chitin molecules were removed during deacetylation from the solid crystalline fibril surface in the water-soluble fractions, the water-insoluble DeAc-chitin was obtained with a yield of 89%. Based on a previous study concerning TEMPO-mediated oxidation of cellulose, the TEMPO/NaClO/NaClO2 system at 60°C under neutral conditions was selected to oxidize the DeAc-chitin (prepared with 30% NaOH for 6 h, as shown in Figure 1A) to maximize the molar masses of the Tox-DeAc-chitins.25 In the TEMPO-mediated oxidation of noncrystalline and low-crystallinity chitosans in water at pH 10, the abundant presence of


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C2-amine groups in the chitosans led to decreases in molecular mass; consequently, nearly no water-insoluble fraction was recovered after oxidation.26,27 When α-chitin is oxidized with the TEMPO/NaBr/NaClO system in water at pH 10, the TEMPO-oxidized chitins are obtained as water-insoluble fractions.17 In that oxidation, the yield of TEMPO-oxidized chitin decreases to 80%), which is advantageous.

Figure

2.

Yields

and

carboxylate

contents

of

Tox-chitins

prepared

with

TEMPO/NaClO/NaClO2 in water at pH 6.8 and 60°C for 4 h with different amounts of NaClO2 (A), and yields of DeAc-Tox-chitins after partial deacetylation of Tox-chitin (with 10 mmol/g NaClO2) with 30% NaOH at 90°C for various times (B).

When the TEMPO/NaClO/NaClO2 oxidation was initially applied to the original chitin, the carboxylate content of Tox-chitin increased with NaClO2 amount up to 10 mmol/g and then reached maximum values of 0.54−0.58 mmol/g. The yields of the water-insoluble



fractions were more than 94% in all cases examined, which were higher than those prepared by TEMPO/NaBr/NaClO oxidation in water at pH 10.17 These results show that TEMPO-mediated oxidation in water at pH 6.8 can avoid the degradation of the chitin molecules to some extent. After TEMPO-mediated oxidation of chitin with 10 mmol/g NaClO2, the yield was still more than 95%. The Tox-chitin prepared with 10 mmol/g NaClO2 was treated with 30% NaOH, and the yield of DeAc-Tox-chitin decreased with increasing treatment time (Figure 2B). It is likely that sodium carboxylate-rich molecules, which formed during the TEMPO-mediated oxidation of the original chitin, were partly removed from the water-insoluble Tox-chitin as soluble-fractions in 30% NaOH.28

Figure 3. Yields of A-ChNCs prepared from DeAc-Tox-chitin (A) and Tox-DeAc-chitin (B) under various conditions after sonication in water at pH 3 and pH 11 for 5 min.

The A-chitins containing both carboxylate and amine groups were prepared from DeAc-Tox-chitin and Tox-DeAc-chitin. The surfaces of the chitin fibrils should have cationic charges owing to the protonated C2-amine groups in the A-chitins under acidic conditions. In contrast, the C6-carboxylate groups in the A-chitins had anionic charges and


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led to electrostatic repulsion among the negatively charged A-chitin fibrils under alkaline conditions. Therefore, the two A-chitins became chitin nanofibrils (A-ChNCs) by being sonicated in water; however, the variations in yield of the A-ChNCs were different between the two A-chitins. In the case of the Tox-DeAc-chitins (Figure 3A), the increase in the amount of NaClO2 added in oxidation likely increased the carboxylate content of the A-chitins, resulting in the high yields of A-ChNCs under alkaline conditions. In contrast, in the case of the DeAc-Tox-chitins (Figure 3B), the amount of C2-amine groups formed in the samples probably increased with treatment time in 30% NaOH, resulting in higher yields of A-ChNCs under acidic conditions. As a consequence, two A-chitins prepared by the opposite sequence of deacetylation and TEMPO-mediated oxidation resulted in different anionic/cationic charge balanced on the surfaces of the chitin nanofibril. The different charge balances of the Tox-DeAc-chitins and DeAc-Tox-chitins led to different variations in yield of the A-ChNCs under acidic and alkaline conditions, as shown in Figure 3.



Figure 4. Light transmittances at 600 nm of aqueous 0.1% (w/w) A-ChNC dispersions prepared from DeAc-Tox-chitin (A) and Tox-DeAc-chitin (B) in water at pH 3 and 11 and after the pH was adjusted from 3 to 11 or from 11 to 3.

Figure 4 shows the light transmittances at 600 nm of the original 0.1% A-ChNC dispersions prepared from DeAc-Tox-chitin and Tox-DeAc-chitin and those after the pH adjustment. Almost all A-ChNC dispersions had high light transmittances at both pH 3 and 11 and after pH changed from 3 to 11 or 11 to 3 except the following three samples. The Tox-DeAc-chitin prepared with 12 mmol/g NaClO2 had high transmittances at pH 3, 11, and 3→11 but had a low value at pH 11→3. The DeAc-Tox-chitin prepared with 30% NaOH for 0.25 h had high light transmittances at pH 3, 11, and 3→11 but had a low value at pH 11→3. The DeAc-Tox-chitin prepared with 30% NaOH for 3 h had a high light transmittance at pH 3 but had relatively low values at pH 11, 3→11, and 11→3. These different transmittances or aggregations were likely caused by different contents of C6-carboxylate and C2-amine groups and by the different balances between the anionic and cationic groups on the A-ChNC surfaces.

3.2. Characterization of amphoteric chitin Considering the yields of the A-ChNCs in Figure 3 and the transmittance changes in Figure 4, the amount of NaClO2 used for DeAc-chitin and the treatment time in 30% NaOH for Tox-chitin was set to 6 mmol/g and 1 h, respectively, in the following


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experiments. Therefore, the DeAc-Tox-chitin and Tox-DeAc-chitin below were regarded as the chitin samples prepared under adjusted conditions.

Figure 5. XRD patterns (A) and crystallinities and crystal sizes (B) of original chitin and various A-chitins prepared under various conditions. DeAc-chitin was prepared from the original chitin with 30% NaOH for 6 h, Tox-DeAc-chitin was prepared from DeAc-chitin through TEMPO-mediated oxidation with 6 mmol/g NaClO2, Tox-chitin was prepared from the original chitin through TEMPO-mediated oxidation with 10 mmol/g NaClO2, and DeAc-Tox-chitin was prepared from DeAc-chitin with 30% NaOH for 1 h.

The XRD patterns, crystallinities, and crystal sizes of the original chitin and representative chemically treated chitins are shown in Figure 5. All the samples had typical diffraction XRD patterns of α-chitin. The crystal sizes of both [020] and [110] planes calculated from the XRD patterns before and after the treatments were nearly identical. However, the crystallinity of α-chitin in the rightmost sample of Figure 5B was higher than that of the original chitin. These results indicate that the disordered regions present in the



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original chitin were partly removed during the deacetylation and TEMPO-mediated oxidation treatments. As a result, the crystalline regions remained in the water-insoluble fractions of the chemically treated chitins. In particular, the Tox-DeAc chitin prepared from Tox-chitin through the TEMPO-mediated oxidation with 10 mmol/g NaClO2 and subsequent deacetylation with 30% NaOH for 1 h had the highest crystallinity because a significant amount of disordered region was removed during the deacetylation process (Figure 2B). Most of the carboxylate-rich molecules present in the Tox-chitin may also have been removed during the 30% NaOH treatment.

Figure 6.

Possible

structural

changes of

chitin

molecules

TEMPO-oxidation, and a combination of these two treatments.


after

deacetylation,

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Based on the optical microscopy images obtained (Figure S1), the structural changes of chitin molecules in the microfibrils during the deacetylation, TEMPO-oxidation, and a combination of these two treatments in different sequences are illustrated in Figure 6. The visible structure observed through optical microscopy was the helicoidal stacking sequence of the fibrous chitin-protein layer.29 Deacetylation introduced amine groups to amorphous regions between the layers and dislocated regions, which contained high contents of amine groups that were removed after subsequent TEMPO/NaClO/NaClO2 oxidation and resulted in the decrease in amine content of Tox-DeAc-chitin (Table S1) and the increase in crystallinity (Figure 5B). On the other hand, TEMPO/NaClO/NaClO2 oxidation separated the layers of the original chitin, and the larger surfaces of the dislocated regions with high contents of carboxylate groups were removed in the subsequent deacetylation step (Table S1), which further increased crystallinity (Figure 5B).

3.3. pH sensitivity of A-ChNCs



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Figure 7. The AFM images of A-ChNCs prepared from DeAc-Tox-chitin (A) and Tox-DeAc-chitin (B) through sonication in water at pH 3.

The

morphologies

of

A-ChNCs

prepared

from

the

DeAc-Tox-chitin

and

Tox-DeAc-chitin through sonication in water at pH 3 are shown in their AFM images (Figure 7). Similar AFM images were also observed for A-ChNCs from the A-chitin/water dispersion at pH 11. The two A-ChNCs both had rod-like nanocrystal morphologies with average widths and lengths of 6-15 nm and 200-600 nm, respectively. These morphologies of A-ChNCs were similar to those prepared from partially deacetylated chitins.17,18 However, the A-ChNCs with nanocrystal morphologies prepared in this study have both carboxylate and amine groups; therefore, they are amphoteric and have pH-sensitive dispersibility in water, different from previous reports.17,18


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Figure 8. Changes in light transmittance at 600 nm of 0.1% A-ChNC dispersions prepared from DeAc-Tox-chitin (A) and Tox-DeAc-chitin (C) through sonication in water at pH 3 and 11 after pH adjustment. Changes in the ζ-potential of A-ChNCs prepared from DeAc-Tox-chitin (B) and Tox-DeAc-chitin (D) were measured in water at different pH values. Tox-DeAc-chitin and DeAc-Tox-chitin were prepared under the same optimized conditions as those shown in Figure 5.

Because the A-ChNCs prepared in this study have both C6-carboxylate and C2-amine groups and are therefore amphoteric, their isoelectric points should depend on the pH of the dispersions, as in the case of water-soluble proteins. The electrostatic repulsion forces



between the A-ChNC elements decrease in water when the pH is near the isoelectric points, and the A-ChNC elements may form polyionic complex-like structures under these conditions, resulting in the formation of A-ChNC aggregates. The A-ChNCs can be sufficiently dispersed in water under acidic conditions because of the formation of protonated C2-ammoium groups, whereas they can be dispersed in water under alkaline conditions because of the formation of dissociated C6-carboxylate groups. The anionic C6-carboxylate groups of A-ChNCs in water under alkaline conditions become uncharged protonated carboxyl groups under acidic conditions; similarly, the cationic C2-ammoium groups in water under acidic conditions become uncharged C2-amine groups under alkaline conditions. The transmittances of the 0.1% (w/w) A-ChNC dispersions prepared from DeAc-Tox-chitin according to the optimized conditions were measured (Figure 8A). The A-ChNCs dispersions prepared from DeAc-Tox-chitin had the lowest light transmittance at pH ~6.3. Most likely, each A-ChNC sample showing the lowest light transmittance had an isoelectric point in water at the pH value. The ζ-potentials of both A-ChNC samples decreased from +32 mV to -30 mV with increasing pH from 3 to 11 (Figure 8B). These ζ-potential patterns are characteristic of typical amphoteric particles with both carboxylate and amine groups. The A-ChNCs dispersions prepared from Tox-DeAc-chitin formed aggregates at pH ~7 with an absolute ζ-potential value of ~0 mV (Figure 8C and 8D).


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Figure 9. Photographs of A-ChNC dispersions in toluidine blue and Ponceau S solutions at pH 4 and pH 9, respectively, after centrifugation at 10251 ×g for 1 min (A), A-ChNC hydrogel before (B) and after dye adsorption with toluidine blue at pH 9 (C) and Ponceau S at pH 4 (D). SEM image and photograph of A-ChNC aerogel prepared by freeze-drying (E). The A-ChNC samples were prepared from DeAc-Tox-chitin.

The A-ChNCs contained both carboxylate and amine groups and showed adsorption properties of both acid and basic dyes. Figure 9A shows that Ponceau S (acid dye) adsorbed on the A-ChNC in water at pH 4 through the electrostatic attraction between the cationic A-ChNC elements and the anionic Ponceau S molecules. Moreover, the neutralization of the cationic C2-ammonium groups in the A-ChNC elements resulted in aggregation, which was separated from the supernatant through centrifugation. The A-ChNC elements were dispersed in water at pH 9 because the cationic C2-ammonium groups in the A-ChNC elements led to electrostatic repulsion among the A-ChNC elements. A similar but opposite phenomenon was observed when the A-ChNC was mixed with toluidine blue in water at pH 4 and 9 (Figure 9A).



When a saturated NaCl solution was added to the 1% (w/w) A-ChNC dispersion, the A-ChNC dispersion became a stiff hydrogel through the salting-out effect (Figure 9B). The dye treatments were applied to the hydrogel by immersing the hydrogel in Ponceau S and toluidine blue solutions at pH 4 and 9, respectively. After shaking the mixtures for 10 h, the hydrogels turned to blue and red through the adsorption of cationic and anionic dyes, respectively (Figure 9C and 9D). In general, the adsorption was influenced by the physical and chemical structures, the functional groups of the materials and the dye solution conditions. The corresponding SEM image of an A-ChNC aerogel showed that many pores with sizes below 1 µm existed in the inner structure (Figure 9E), which had a positive effect on the adsorption.

Conflicts of interest There is no conflict to declare.

Acknowledgements This research was supported by the National Natural Science Foundation of China (No. 31100426) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Supplementary Information

Figure S1 Optical microscopy images of original chitin and treated chitins in water.

Table S1 Elemental C/N ratios of original chitin and treated chitin samples. Sample

Chitin

DeAc-chitin

Tox-DeAc-chitin

Tox-chitin

DeAc-Tox-chitin

C/N ratio

7.815

7.463

7.784

7.842

7.791



TOC Graphic


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Figure 1. Yields and amine contents of DeAc-chitins obtained as water-insoluble fractions after deacetylation with 30% NaOH at 90C for different times (A), and yields of Tox-DeAc-chitins after TEMPO/NaClO/NaClO2 oxidation of DeAc-chitin (prepared with 30% NaOH for 6 h in A) in water at pH 6.8 and 60C for 4 h with different amounts of NaClO2 (B). 267x111mm (150 x 150 DPI)



Figure 2. Yields and carboxylate contents of Tox-chitins prepared with TEMPO/NaClO/NaClO2 in water at pH 6.8 and 60C for 4 h with different amounts of NaClO2 (A), and yields of DeAc-Tox-chitins after partial deacetylation of Tox-chitin (with 10 mmol/g NaClO2) with 30% NaOH at 90C for various times (B). 271x111mm (150 x 150 DPI)


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Figure 3. Yields of A-ChNCs prepared from DeAc-Tox-chitin (A) and Tox-DeAc-chitin (B) under various conditions after sonication in water at pH 3 and pH 11 for 5 min. 265x114mm (150 x 150 DPI)



Figure 4. Light transmittances at 600 nm of aqueous 0.1% (w/w) A-ChNC dispersions prepared from DeAcTox-chitin (A) and Tox-DeAc-chitin (B) in water at pH 3 and 11 and after the pH was adjusted from 3 to 11 or from 11 to 3. 264x113mm (150 x 150 DPI)


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Figure 5. XRD patterns (A) and crystallinities and crystal sizes (B) of original chitin and various A-chitins prepared under various conditions. DeAc-chitin was prepared from the original chitin with 30% NaOH for 6 h, Tox-DeAc-chitin was prepared from DeAc-chitin through TEMPO-mediated oxidation with 6 mmol/g NaClO2, Tox-chitin was prepared from the original chitin through TEMPO-mediated oxidation with 10 mmol/g NaClO2, and DeAc-Tox-chitin was prepared from DeAc-chitin with 30% NaOH for 1 h. 258x120mm (150 x 150 DPI)



Figure 6. Possible structural changes of chitin molecules after deacetylation, TEMPO-oxidation, and a combination of these two treatments. 214x145mm (150 x 150 DPI)


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Figure 7. The AFM images of A-ChNCs prepared from DeAc-Tox-chitin (A) and Tox-DeAc-chitin (B) through sonication in water at pH 3. 246x135mm (150 x 150 DPI)



Figure 8. Changes in light transmittance at 600 nm of 0.1% A-ChNC dispersions prepared from DeAc-Toxchitin (A) and Tox-DeAc-chitin (C) through sonication in water at pH 3 and 11 after pH adjustment. Changes in the ζ-potential of A-ChNCs prepared from DeAc-Tox-chitin (B) and Tox-DeAc-chitin (D) were measured in water at different pH values. Tox-DeAc-chitin and DeAc-Tox-chitin were prepared under the same optimized conditions as those shown in Figure 5. 250x221mm (150 x 150 DPI)


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Figure 9. Photographs of A-ChNC dispersions in toluidine blue and Ponceau S solutions at pH 4 and pH 9, respectively, after centrifugation at 10251 ×g for 1 min (A), A-ChNC hydrogel before (B) and after dye adsorption with toluidine blue at pH 9 (C) and Ponceau S at pH 4 (D). SEM image and photograph of A-ChNC aerogel prepared by freeze-drying (E). The A-ChNC samples were prepared from DeAc-Tox-chitin. 298x138mm (150 x 150 DPI)


Preparation and Hydrogel Properties of pH-Sensitive Amphoteric

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