Facile preparation of chitin gels with calcium bromide dihydrate/methanol media and their efficient conversion into porous chitins

Abstract

In this study, we found that a chitin gel was facilely obtained by stirring a mixture of chitin with a highly concentrated CaBr₂·2H₂O/methanol solution at room temperature. We also examined the conversion of the resulting gel into a porous chitin by a regeneration technique. When the mixtures of chitin (0.9–3.5% (w/v)) with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol solution were stirred at room temperature, chitin was gradually swollen, leading to a gelling form after 48 h. The mixtures of chitin (1.8% (w/v)) with CaBr₂·2H₂O/methanol solutions in different concentrations also turned into a gelling form by the same procedure. The dynamic viscoelastic measurement of the resulting chitin gels supported their gelling state. Then, porous chitins were efficiently prepared by removing methanol from the gels under reduced pressure, followed by washing with water and lyophilization. The α-chitin crystalline structure of the porous chitin was confirmed by powder X-ray diffraction measurement. The morphology of the porous chitins was evaluated by the SEM measurement. Consequently, it was found that the porosities were depending on contents of chitin in the gels. Furthermore, the mechanical properties of the porous chitins were investigated by compressive testing, which were affected by the pore sizes of the materials. On the other hand, the amounts of CaBr₂·2H₂O in the gels did not strongly affect the porosities and mechanical properties of the porous chitins.

---

1. Introduction

Natural polysaccharides are widely distributed in nature and have been regarded as structural materials and as suppliers of energy.¹ They have increasingly been important because of possessing unique structures and properties, which are often rather different from those of common synthetic polymers. Compared with synthetic polymeric materials, therefore, natural polysaccharide-based materials have many promising properties, for example, good biocompatibility, biodegradability, non-toxicity, non-immunogenic, and so on.² Of the many kinds of natural polysaccharides, chitin, which is an aminopolysaccharide composed of N-acetyl-D-glucosamine residues linked though β-(1 → 4)glycosidic bonds, is one of the most abundant polysaccharides in nature and occurs mainly in exoskeletons of crustaceans, shellfish, and insects.^3–5 Despite its huge annual production and easy accessibility, chitin still remains as an unutilized biomass resource primary because of its intractable bulk structure causing the limitation of solubility with solvents and thus, only limited application has been paid to chitin, principally from its biological properties.⁶ Accordingly, the development of efficient procedures for the production of chitin-based functional materials is one of the promising and attractive topics in the research areas of polysaccharides. Although there have not been many reports on the dissolution of chitin, for example, it was reported that a saturated CaCl₂·2H₂O/methanol solution dissolved chitin in a few % concentrations at refluxed temperature.^7–10

On the other hand, in the previous study, we found that an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved or swelled chitin to form weak gel-like materials (ion gels).^11,12 Furthermore, we also reported that self-assembled chitin nanofibers with ca. 20–60 nm in width and several hundred nm in length were facilely produced by regeneration from the ion gels using methanol, followed by sonication.¹³ Interestingly, nanofibers with higher aspect ratios were produced by the regeneration using CaBr₂·2H₂O/methanol solution with moderate concentrations (relatively low concentrations) compared with those regenerated using a sole methanol.¹⁴ This result indicated that CaBr₂·2H₂O/methanol solution affected the regeneration process of chitin from the ion gel, inspiring us its specific affinity for chitin.

On the basis of the above background, in this study, we found that gelation simply took place when a mixture of chitin with a highly concentrated CaBr₂·2H₂O/methanol solution was only stirred at room temperature. Because chitin has not been swollen by stirring a mixture of the aforementioned CaCl₂·2H₂O/methanol solution at room temperature, it can be noted that a highly concentrated CaBr₂·2H₂O/methanol solution exhibits specific behavior for chitin. The dynamic viscoelastic measurement was conducted to characterize the present gels prepared under the various conditions. Moreover, we also performed the conversion of the chitin gels into porous chitins by regeneration technique. Porous chitins have previously been produced by the appropriate regeneration approaches mostly via multiple steps through several days from chitin solutions with the aforementioned calcium chloride solvent and the other solvent systems.^15–18 Moreover, these studies have not mostly revealed the precise dependence of porous morphologies on the procedures and conditions. Our method reported herein provides the quite facile procedure for the gelation and relatively regulated porositization of chitin prior to the previous methods. The present approaches, therefore, have potentials for further practical applications in the various research fields in the future.

2. Experimental

Materials

Chitin powder from crab shells was purchased from Wako Pure Chemicals, Tokyo, Japan. The values of weight-average molecular weight and degree of deacetylation of the chitin sample were estimated by the viscometric and IR analyses^19,20 to be 5 × 10⁵ and less than 5%, respectively. A powdered CaBr₂·2H₂O was purchased from Kanto Chemical Co. Inc., Tokyo, Japan and used without further purification. All other reagents and solvents were used as received from commercial sources.

Preparation of chitin gels with CaBr₂·2H₂O/methanol media

A typical procedure for the preparation of chitin gels with CaBr₂·2H₂O/methanol media was as follows. A mixture of chitin powder (92 mg, 0.45 mmol per unit) with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol solution (5.1 mL) was stirred at room temperature for 48 h to produce a 1.8% (w/v) chitin gel with CaBr₂·2H₂O/methanol media.

Preparation of porous chitins

A typical experimental procedure for the preparation of porous chitins as follows. The aforementioned chitin gel was left under reduced pressure at 60 °C for 3 h. Then, the product was washed 20 times with water (30 mL each) and lyophilized to give a porous chitin.

Measurements

The dynamic viscoelastic measurement was conducted on a rheometer (Rheosol-G1000, UBM). The SEM images were obtained using Hitachi S-4100H electron microscope. The powder X-ray diffraction (XRD) measurements were conducted using a PANalytical X'Pert Pro MPD with Ni-filtered CuKα radiation (λ = 0.15418 nm). The energy dispersive X-ray spectroscopy was performed using a Philips XL30 CP scanning electron microscope (SEM-EDX). The stress–strain curves were measured using a tensile tester (Little Senster LSC-1/30, Tokyo Testing Machine).

3. Results and discussion

Preparation and characterizations of chitin gels with CaBr₂·2H₂O/methanol media

We found that chitin was gradually swollen when its mixtures with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol solution were stirred in several contents (0.9, 1.8, 2.4, and 3.5% (w/v)) at room temperature (Scheme 1). Consequently, the mixtures were totally turned into gelling form after 48 h. Chitin was not completely swollen in mixtures with higher contents. The resulting chitin gels with CaBr₂·2H₂O/methanol media were characterized by the dynamic viscoelastic measurement. The frequency dependence of storage and loss modulus (G′ and G′′, respectively) in Fig. 1 showed signature of typical viscoelastic material with predominance of storage modulus on the whole frequency range. These results supported the gelling state of the samples obtained under the above conditions. Furthermore, the differences between the G′ and G′′ values were smaller with decreasing the contents of chitin (Fig. 1(d) → (a)), indicating relative weak-gel nature of the lower content gels.

Scheme 1 Procedure for preparation of chitin gels with CaBr₂·2H₂O/methanol media and their conversion into porous chitins.

Fig. 1 Evaluation of storage modulus G′ (open symbols) and loss modulus (closed symbols) as a function of frequency for 0.9, 1.8, 2.4, and 3.5% (w/v) chitin gels with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media ((a), (b), (c), and (d), respectively).

Then, we also prepared the 1.8% (w/v) chitin gels with 3.49, 3.65, and 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media. Because the total volumes of the media were adjusted to consistence in these gels, the amounts of CaBr₂·2H₂O for chitin were different depending on the concentrations of the media. The experimental procedure was conducted as the aforementioned, resulting in totally gelling form. However, CaBr₂·2H₂O/methanol media in lower concentrations did not afford total gelation. The dynamic viscoelastic data of the resulting gels in Fig. 2 showed that the G′ values were always larger than the G′′ values, supporting the gelling state of the samples. However, the gel with the lower CaBr₂·2H₂O concentration such as that with 3.49 mol L⁻¹ CaBr₂·2H₂O/methanol media exhibited the weak-gel nature because of the smaller difference of the G′ and G′′ values (Fig. 2(a)) compared with the other gels (Fig. 2(b) and (c)). The above rheological results indicated that gel behaviors were affected by both the contents of chitin and the amounts of CaBr₂·2H₂O in the gels.

Fig. 2 Evaluation of storage modulus G′ (open symbols) and loss modulus (closed symbols) as a function of frequency for 1.8% (w/v) chitin gels with 3.49, 3.65, and 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media ((a), (b), and (c), respectively).

Conversion of chitin gels with CaBr₂·2H₂O/methanol media into porous chitins

We then attempted to convert the aforementioned chitin gels into porous chitins by regeneration technique (Scheme 1). For the production of porous morphology, the swollen chitin network structures in the gels should be maintained even after regeneration. Therefore, methanolic media were quickly removed from the gels under reduced pressure conditions at 60 °C. Then, the remaining inorganic salts were washed out by water and the residual materials were lyophilized to give the sponge-like regenerated chitins.

The crystalline structure of the regenerated chitin was confirmed by the XRD measurement. The XRD profile of the regenerated material obtained from the 1.8% (w/v) chitin gel with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media mainly showed six diffraction peaks at around 9.5°, 13.0°, 19.4°, 21.0°, 23.8°, and 26.5° (Fig. 3(b)), which were assignable to 020, 021, 110, 120, 130, and 013 planes, respectively.²¹ This diffraction pattern typically corresponds to the crystalline structure of α-chitin and was in good agreement with that of an original chitin powder (Fig. 3(a)). This result indicated the α-chitin crystalline structure was reconstructed during the aforementioned regeneration procedure. Furthermore, the XRD result in Fig 3(b) did not observe the crystalline peaks due to a CaBr₂·2H₂O powder as shown in Fig. 3(c), suggesting the absence of inorganic salts in the regenerated material. The absence of the salts was also supported by the SEM-EDX spectrum of the same material because signals assignable to Ca and Br elements were not detected.

Fig. 3 XRD profiles of commercial chitin powder (a), porous chitin obtained from 1.8% (w/v) chitin gel with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media (b), and CaBr₂·2H₂O powder (c).

The porosities of the regenerated materials were confirmed by the SEM measurement. Fig. 4(a)–(d) shows the SEM images of the four materials with same magnification obtained from the 0.9, 1.8, 2.4, and 3.5% (w/v) chitin gels with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media. The SEM pictures of the former two samples showed the porous morphologies, whereas such morphologies were not observed in the SEM images of the latter two samples. Furthermore, the pore sizes in Fig. 4(b) were relatively smaller than those in Fig. 4(a). When the SEM images of the latter two samples were taken with more magnification, the porous morphologies were obtained with decreasing the pore sizes in the order of Fig. 4(c′) to (d′). These SEM results supported that porous chitins were produced by the aforementioned regeneration procedure from the chitin gels with CaBr₂·2H₂O/methanol media. The observations also indicated the contents of chitin in the gels strongly affected the pore sizes of the porous chitins and the gels in the lower contents of chitin gave the larger pores via the regeneration procedure. Because looser network structure should form in the gel with lower content of chitin (e.g., 0.9% (w/v)) due to voluminously swollen chitin chains, the larger pores were formed by the above regeneration procedure, which was suitably performed as the network structure was maintained.

Fig. 4 SEM images of porous chitins obtained from 0.9, 1.8, 2.4, and 3.5% (w/v) chitin gels with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media ((a), (b), (c) and (c′), and (d) and (d′), respectively).

The SEM measurement of the regenerated chitin samples produced from the 1.8% (w/v) gels with 3.49–3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media was also performed. As shown in the SEM images of these samples with same magnification (Fig. 5), the pore sizes were not largely different. These results suggested that the amounts of CaBr₂·2H₂O in the gels did not strongly affect the porous morphologies. This is probably because that the network structures of the chitin chains were mainly regulated by the chitin contents in the gels, which were responsible for the porosity of the regenerated chitin materials.

Fig. 5 SEM images of porous chitins obtained from 1.8% (w/v) chitin gels with 3.49, 3.65, and 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media ((a), (b), and (c), respectively).

The mechanical properties of the porous chitins were evaluated by compressive testing as one of the necessity properties for practical materials. Fig. 6 shows the stress–strain curves of the four porous chitins obtained from the 0.9, 1.8, 2.4, and 3.5% (w/v) chitin gels with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media. The results indicated that the materials prepared from the gels with the lower contents of chitin exhibited the more elastic properties with the larger fracture strain values, owing to the larger pore sizes. On the other hand, the stress–strain curves of the porous chitins resulted from the 1.8% (w/v) gels with 3.49–3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media (Fig. 7) showed almost the same profiles because of their similar porosities.

Fig. 6 Stress–strain curves of porous chitins under compressive mode obtained from 0.9, 1.8, 2.4, and 3.5% (w/v) chitin gels with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media ((a), (b), (c), and (d), respectively).

Fig. 7 Stress–strain curves of porous chitins under compressive mode obtained from 1.8% (w/v) chitin gels with 3.49, 3.65, and 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media ((a), (b), and (c), respectively).

Because of the porous morphologies, the porous chitins exhibited water absorbent ability by immersing them in water. For example the porous chitin resulted from the 0.9% (w/v) chitin gel with 3.85 mol L⁻¹ CaBr₂·2H₂O/methanol media absorbed 42.5 times its own weight in water.

4. Conclusions

In this study, we facilely prepared the chitin gels with highly concentrated CaBr₂·2H₂O media by stirring the mixtures at room temperature. The dynamic viscoelastic measurement supported gelling state of the products. The gels were efficiently converted into the porous chitins by removing methanol under reduced pressure, followed by washing with water and lyophilization. The XRD result of the porous chitin indicated reconstruction of α-chitin crystalline structure. The porosities of the materials were evaluated by the SEM measurement, which were affected by the contents of chitin, but not strongly influenced by the amounts of CaBr₂·2H₂O in the gels. The porosities affected the mechanical properties of the porous chitins evaluated by compressive testing. This study has revealed that CaBr₂·2H₂O/methanol solution is the good media for chitin and can be used for the efficient production of new functional chitin-based materials such as the porous chitins with relatively regular morphologies. The present approaches, therefore, will be practically applied in the various research fields in the future, for example, chitin-based environmentally benign absorbent can be developed by our procedure. As the other application, because chitin exhibits biocompatibility,⁶ the porous chitins are considered as promising materials in tissue engineering.

Notes and references

1. C. Schuerch, Polysaccharides, in Encyclopedia of Polymer Science and Engineering, ed. H. F. Mark, N. Bilkales and C. G. OverbergerJohn Wiley & Sons, New York, 2nd edn, 1986, p. 87.
2. L. N. Hassani, F. Hendra and K. Bouchemal, Drug Discovery Today, 2012, 17, 608.
3. K. Kurita, Mar. Biotechnol., 2006, 8, 203.
4. M. Rinaudo, Prog. Polym. Sci., 2006, 31, 603.
5. C. K. S. Pillai, W. Paul and C. P. Sharma, Prog. Polym. Sci., 2009, 34, 641.
6. R. A. A. Muzzarelli, Chitin nanostructures in living organisms, in Chitin Formation and Diagenesis, ed. S. N. Gupta, Springer, New York, 2011, ch. 1.
7. S. Tokura, N. Nishi, K. Takahasshi, A. Shirai and Y. Uraki, Macromol. Symp., 1995, 99, 201.
8. S. Tokura, S. Nishimura, N. Sakairi and N. Nishi, Macromol. Symp., 1996, 101, 389.
9. H. Tamura, H. Nagahama and S. Tokura, Cellulose, 2006, 13, 357.
10. H. Nagahama, T. Higuchi, R. Jayakumar, T. Furuike and H. Tamura, Int. J. Biol. Macromol., 2008, 42, 309.
11. S. Yamazaki, A. Takegawa, Y. Kaneko, J. Kadokawa, M. Yamagata and M. Ishikawa, Electrochem. Commun., 2009, 11, 68.
12. K. Prasad, M. Murakami, Y. Kaneko, A. Takada, Y. Nakamura and J. Kadokawa, Int. J. Biol. Macromol., 2009, 45, 221.
13. J. Kadokawa, A. Takegawa, S. Mine and K. Prasad, Carbohydr. Polym., 2011, 84, 1408.
14. R. Tajiri, T. Setoguchi, S. Wakizono, K. Yamamoto and J. Kadokawa, J. Biobased Mater. Bioenergy, 2013, 7, 655.
15. K. S. Chow, W. Khor and A. C. A. Wan, J. Polym. Res., 2001, 8, 27.
16. Y. Maeda, R. Jayakumar, H. Nagahama, T. Furuike and H. Tamura, Int. J. Biol. Macromol., 2008, 42, 463.
17. H. Tamura, T. Furuike, S. V. Nair and R. Jayakumar, Carbohydr. Polym., 2011, 84, 820.
18. S. S. Silva, A. R. C. Duarte, A. P. Carvalho, J. F. Mano and R. L. Reis, Acta Biomater., 2011, 7, 1166.
19. M. Poirier and G. Charlet, Carbohydr. Polym., 2002, 50, 363.
20. Y. Shigemasa, H. Matsuura, H. Sashiwa and H. Saimoto, Int. J. Biol. Macromol., 1996, 18, 237.
21. J. D. Goodrich and W. T. Winter, Biomacromolecules, 2007, 8, 252.

---