VOL. 16, 1987-1992 (1977)
BIOPOLYMERS
Architecture of Chitin Gel as Examined by Scanning Electron Microscopy SHIGEHIRO HIRANO, RYUJI YAMAGUCHI, and NORIAKI MATSUDA, Department of Agricultural Biochemistry, Tottori University, Tottori 680, J a p a n
Synopsis An architecture of the solid phase of chitin gel was examined by scanning electron microscopy. The ultrastructure of the xerogel was microporous with parallel channels surrounded with membranous walls. The pore shapes a t cross section were polyhedral with three walls a t the junctions. The pore was 30-50 pm in diameter and 80-300.pm in length, and the thickness of the walls was less than 1.5 pm. The gel is considered to be a polyphasic gel, consisting of small droplets of water held up in these pores.
INTRODUCTION Biological gels are of importance for supporting systems in tissue, which involve fibrous materials embedded in matrix materia1s.l There appear to be various fibrous materials in nature, e.g., cellulose [(l 4)-P-D-glucan] 4)2in higher plants, collagen (a glycoprotein) in animals, chitin[(l acetamido-2-deoxy-~-~-glucan] a t lower evolutionary levels in both, etc. 4)2-amino-2We have recently prepared chitin gel from chitosan[(l deoxy-P-D-glucan] by a facile, chemical N - a c e t y l a t i ~ n . ~ , ~ We wish to report a characteristic ultrastructure of the solid phase of chitin gel as examined by scanning electron microscopy.
- -
EXPERIMENTAL Materials Chitin gel was prepared from chitosan(Mr ca. 400,000; [a]&5 -3.1" (c 1.3, 50% formic acid)) by the action of acetic anhydride in aqueous acetic acid/methanol solutions (chitosan concentration = 1.0%)a t room temperature as r e p ~ r t e d .The ~ gel was dialyzed against running water for one day and in distilled water for three days to afford chitin gel[(CsH13NO5.ca. 600 H ~ O ) ~ ZTo ] . preserve ~ the three-dimensional shape of the gel, small cubes removed with a knife from the gel without any deformation were placed in a small vial, frozen rapidly in a mixture of acetone and dry ice (ca. -6O"C), and lyophilized. The xerogel thus produced was further dried over PzO5 in uacuo at 110°C for 5 hr. In some cases, the xerogel was further 1987 01917 by John Wiley & Sons, Inc.
1988
HIRANO, YAMAGUCHI, AND MATSUDA
Fig. 1. A surface view of the xerogel being deformed morphologicallyin part (see Materials section in the text). It shows long parallel channels. The bar at the lower right equals 500
w. sliced to afford xerogels being morphologically deformed in part (Fig. 1). These samples were used for scanning electron microscopic observation in the present study. The xerogels thus prepared showed ir absorptions (KBr) of N-acetyl group at 1630 and 1540 cm-' (C=O and NH), but no 0-acetyl group at ca. 1700 and 1240 cm-' (C=O and C-0). The ir spectrum was indistinguishable from that of chitin. A degree of N-substitution = 1.00 per hexosaminide residue was confirmed by the ratio of (N-acetyl-methyl)/ (methines and methylenes of sugar) in the nmr spectrum (DCOOD) and by the elemental analysis calculated for [ (CsH13NOs)n].*
Methods A Hitachi scanning electron microscope (S-500)was used by operating at an accelerating voltage of 20 kV. The other methods used were described in our previous paper.*
RESULTS AND DISCUSSION Transparent chitin gel become cloudy on freezing due to formation of ice crystallites in the cellular structure of gel, and its lyophilization and drying gave a microporous xerogel. No significant changes in the total
ARCHITECTURE OF CHITIN GEL
(a)
1989
(b)
Fig. 2. (a) and (b) General surface views of the xerogel. These show a polyphasic, microporous structure. The pore size is not uniform. The bar a t the lower right equals 500 bm.
volume were observed during the course of these treatments. When the xerogel was further sliced with a knife, a surface-tension deformation was accompanied as shown in Fig. 1. Figures 2(a) and (b) show scanning electron micrographs representing surface views of the xerogel. The structure had continuous, solid, membranous walls with individual pores separated by membranous walls. Its surface view was microporous with long parallel channels (see Fig. 1)which were surrounded with membranous walls. As can be seen in Figs. 3(a) and (b),the pores had membranous walls. The pore size was not uniform and was 30-50 pm in diameter and 80-300 pm in length. Four to six membranous walls attached to the pore wall at intervals of 20-50 pm in the cross section of the pore. The pore structure was associated with its preparative technique, more specifically the chitosan concentration. Higher concentrations of chitosan brought about the formation of rigid structure with smaller pores. A characteristic feature was that three membranous walls always made the junctions in the pore assemblies (Figs. 3 and 4). The thickness of the walls was not uniform either, but was less than 1.5 pm. These data indicate that chitin gel is microporous and polyphasic, and it consists of small droplets of water held up in these pores. The surface structure of the solid phase of chitin gel may be compared with those of some other gels as shown in Table I. The present gel is polyphasic and honeycomblike, and those of polyacryl-amide gel5 and agarose gel6 are
1990
HIRANO, YAMAGUCHI, AND MATSUDA
(a)
(b 1
Fig. 3. (a) and (b) Assemblies of polyhedral pores. The bar at the lower right equals 50 pm.
Fig. 4. A junction (as indicated by an arrow) consisting of three membranous wdls in a cross section of a deformed pore. The bar at the lower right equals 5 pm.
ARCHITECTURE OF CHITIN GEL
1991
TABLE I Ultrastructure of the Solid Phase of Chitin Gel in Comparison with Some Other Gels
Gel
Preparation of Sample
Surface View
N-Acetyl- Freezechitosan dried (Chitin)
Honeycomblike
Gelatin
Spongelike Spongelike
Freezedried Polyacryla- Freezemide dried Agarose
SpongeFreezedried like Embedded in Epon
Method of Pore Structure Wall AnalDimension Thickness ysisa 30-50 pm in diam 80-300 pm in height
1-40 pm in diam 2-10 pm in diam
-
0.3 pm in diam
Ref.
Less than SEM Present 1.5 pm work
SEM
7
0.3-1.0 pm SEM
5
TEM SEM
8 6
TEM
9
-
-
-208,
a SEM, scanning electron microscopic analysis; TEM, transmission electron microscopic analysis.
spongelike. The diameter of the pore size of chitin gel (30-50 pm) is much bigger than those of polyacrylamide gel (2-10 ~ m and ) agarose ~ gel (0.3
m).6 Some morphological changes are considered to occur during freezing: a possible artifact is due to the formation of ice crystallites with chitin chains being forced to the crystallite boundaries. Actually, the xerogel thus prepared held no more water in it and could not regenerate the original gel on soaking in water and in aqueous acetic acid. As can be seen in Figs. 3(a) and (b), the membranous walls are broken in part. The destruction is considered to occur during freezing. Only small changes in the morphology may occur during lyophilization and drying to preserve the characteristic morphology. The present gel is of interest not only for a model of fibrous materials by embedding it in various matrix materials such as proteoglycans, pectin, proteins, and so on,' but also for a new phase material of gel chromatography. The authors wish to thank Dr. Mitsuhiko Yamada, the Hitachi Co., Ltd., Tokyo, for taking these scanning electron micrographs.
References 1. Kirkwood, S. (1974) Ann. Reu. Biochern. 43,401-417. 2. Hirano, S., Kondo, S. & Ohe, Y. (1975) Polymer 16,622.
1992
HIRANO, YAMAGUCHI, AND MATSUDA
3. Hirano, S. & Ohe, Y. (1975) Agric. Biol. Chem. 39,1337-1338. 4. Hirano, S. & Yamaguchi, R. (1976) Biopolymers 15,1685-1692. 5. Blank, Z. & Reimschuessel, A. C. (1974) J. Mat. Sci. 9,1815-1822. 6. Malmquist, M. & Hafster, B. V. (1975) J. Gen. Microbial. 87,167-169. 7. Halberstadt, E. S., Henisch, H. K., Nickl, J. & White, E. W. (1969) J . Coll. Interf. Sci. 29,496-471. 8. Ruchel, R. & Brager, M. D. (1975) Anal. Biochem. 68,415-428. 9. Amsterdam, A., Er-el, Z. & Shaltiel, S. (1975) Arch. Biochem. Biophys. 171,673-677.
Received December 1,1976 Accepted February 9,1977
BIOPOLYMERS
Architecture of Chitin Gel as Examined by Scanning Electron Microscopy SHIGEHIRO HIRANO, RYUJI YAMAGUCHI, and NORIAKI MATSUDA, Department of Agricultural Biochemistry, Tottori University, Tottori 680, J a p a n
Synopsis An architecture of the solid phase of chitin gel was examined by scanning electron microscopy. The ultrastructure of the xerogel was microporous with parallel channels surrounded with membranous walls. The pore shapes a t cross section were polyhedral with three walls a t the junctions. The pore was 30-50 pm in diameter and 80-300.pm in length, and the thickness of the walls was less than 1.5 pm. The gel is considered to be a polyphasic gel, consisting of small droplets of water held up in these pores.
INTRODUCTION Biological gels are of importance for supporting systems in tissue, which involve fibrous materials embedded in matrix materia1s.l There appear to be various fibrous materials in nature, e.g., cellulose [(l 4)-P-D-glucan] 4)2in higher plants, collagen (a glycoprotein) in animals, chitin[(l acetamido-2-deoxy-~-~-glucan] a t lower evolutionary levels in both, etc. 4)2-amino-2We have recently prepared chitin gel from chitosan[(l deoxy-P-D-glucan] by a facile, chemical N - a c e t y l a t i ~ n . ~ , ~ We wish to report a characteristic ultrastructure of the solid phase of chitin gel as examined by scanning electron microscopy.
- -
EXPERIMENTAL Materials Chitin gel was prepared from chitosan(Mr ca. 400,000; [a]&5 -3.1" (c 1.3, 50% formic acid)) by the action of acetic anhydride in aqueous acetic acid/methanol solutions (chitosan concentration = 1.0%)a t room temperature as r e p ~ r t e d .The ~ gel was dialyzed against running water for one day and in distilled water for three days to afford chitin gel[(CsH13NO5.ca. 600 H ~ O ) ~ ZTo ] . preserve ~ the three-dimensional shape of the gel, small cubes removed with a knife from the gel without any deformation were placed in a small vial, frozen rapidly in a mixture of acetone and dry ice (ca. -6O"C), and lyophilized. The xerogel thus produced was further dried over PzO5 in uacuo at 110°C for 5 hr. In some cases, the xerogel was further 1987 01917 by John Wiley & Sons, Inc.
1988
HIRANO, YAMAGUCHI, AND MATSUDA
Fig. 1. A surface view of the xerogel being deformed morphologicallyin part (see Materials section in the text). It shows long parallel channels. The bar at the lower right equals 500
w. sliced to afford xerogels being morphologically deformed in part (Fig. 1). These samples were used for scanning electron microscopic observation in the present study. The xerogels thus prepared showed ir absorptions (KBr) of N-acetyl group at 1630 and 1540 cm-' (C=O and NH), but no 0-acetyl group at ca. 1700 and 1240 cm-' (C=O and C-0). The ir spectrum was indistinguishable from that of chitin. A degree of N-substitution = 1.00 per hexosaminide residue was confirmed by the ratio of (N-acetyl-methyl)/ (methines and methylenes of sugar) in the nmr spectrum (DCOOD) and by the elemental analysis calculated for [ (CsH13NOs)n].*
Methods A Hitachi scanning electron microscope (S-500)was used by operating at an accelerating voltage of 20 kV. The other methods used were described in our previous paper.*
RESULTS AND DISCUSSION Transparent chitin gel become cloudy on freezing due to formation of ice crystallites in the cellular structure of gel, and its lyophilization and drying gave a microporous xerogel. No significant changes in the total
ARCHITECTURE OF CHITIN GEL
(a)
1989
(b)
Fig. 2. (a) and (b) General surface views of the xerogel. These show a polyphasic, microporous structure. The pore size is not uniform. The bar a t the lower right equals 500 bm.
volume were observed during the course of these treatments. When the xerogel was further sliced with a knife, a surface-tension deformation was accompanied as shown in Fig. 1. Figures 2(a) and (b) show scanning electron micrographs representing surface views of the xerogel. The structure had continuous, solid, membranous walls with individual pores separated by membranous walls. Its surface view was microporous with long parallel channels (see Fig. 1)which were surrounded with membranous walls. As can be seen in Figs. 3(a) and (b),the pores had membranous walls. The pore size was not uniform and was 30-50 pm in diameter and 80-300 pm in length. Four to six membranous walls attached to the pore wall at intervals of 20-50 pm in the cross section of the pore. The pore structure was associated with its preparative technique, more specifically the chitosan concentration. Higher concentrations of chitosan brought about the formation of rigid structure with smaller pores. A characteristic feature was that three membranous walls always made the junctions in the pore assemblies (Figs. 3 and 4). The thickness of the walls was not uniform either, but was less than 1.5 pm. These data indicate that chitin gel is microporous and polyphasic, and it consists of small droplets of water held up in these pores. The surface structure of the solid phase of chitin gel may be compared with those of some other gels as shown in Table I. The present gel is polyphasic and honeycomblike, and those of polyacryl-amide gel5 and agarose gel6 are
1990
HIRANO, YAMAGUCHI, AND MATSUDA
(a)
(b 1
Fig. 3. (a) and (b) Assemblies of polyhedral pores. The bar at the lower right equals 50 pm.
Fig. 4. A junction (as indicated by an arrow) consisting of three membranous wdls in a cross section of a deformed pore. The bar at the lower right equals 5 pm.
ARCHITECTURE OF CHITIN GEL
1991
TABLE I Ultrastructure of the Solid Phase of Chitin Gel in Comparison with Some Other Gels
Gel
Preparation of Sample
Surface View
N-Acetyl- Freezechitosan dried (Chitin)
Honeycomblike
Gelatin
Spongelike Spongelike
Freezedried Polyacryla- Freezemide dried Agarose
SpongeFreezedried like Embedded in Epon
Method of Pore Structure Wall AnalDimension Thickness ysisa 30-50 pm in diam 80-300 pm in height
1-40 pm in diam 2-10 pm in diam
-
0.3 pm in diam
Ref.
Less than SEM Present 1.5 pm work
SEM
7
0.3-1.0 pm SEM
5
TEM SEM
8 6
TEM
9
-
-
-208,
a SEM, scanning electron microscopic analysis; TEM, transmission electron microscopic analysis.
spongelike. The diameter of the pore size of chitin gel (30-50 pm) is much bigger than those of polyacrylamide gel (2-10 ~ m and ) agarose ~ gel (0.3
m).6 Some morphological changes are considered to occur during freezing: a possible artifact is due to the formation of ice crystallites with chitin chains being forced to the crystallite boundaries. Actually, the xerogel thus prepared held no more water in it and could not regenerate the original gel on soaking in water and in aqueous acetic acid. As can be seen in Figs. 3(a) and (b), the membranous walls are broken in part. The destruction is considered to occur during freezing. Only small changes in the morphology may occur during lyophilization and drying to preserve the characteristic morphology. The present gel is of interest not only for a model of fibrous materials by embedding it in various matrix materials such as proteoglycans, pectin, proteins, and so on,' but also for a new phase material of gel chromatography. The authors wish to thank Dr. Mitsuhiko Yamada, the Hitachi Co., Ltd., Tokyo, for taking these scanning electron micrographs.
References 1. Kirkwood, S. (1974) Ann. Reu. Biochern. 43,401-417. 2. Hirano, S., Kondo, S. & Ohe, Y. (1975) Polymer 16,622.
1992
HIRANO, YAMAGUCHI, AND MATSUDA
3. Hirano, S. & Ohe, Y. (1975) Agric. Biol. Chem. 39,1337-1338. 4. Hirano, S. & Yamaguchi, R. (1976) Biopolymers 15,1685-1692. 5. Blank, Z. & Reimschuessel, A. C. (1974) J. Mat. Sci. 9,1815-1822. 6. Malmquist, M. & Hafster, B. V. (1975) J. Gen. Microbial. 87,167-169. 7. Halberstadt, E. S., Henisch, H. K., Nickl, J. & White, E. W. (1969) J . Coll. Interf. Sci. 29,496-471. 8. Ruchel, R. & Brager, M. D. (1975) Anal. Biochem. 68,415-428. 9. Amsterdam, A., Er-el, Z. & Shaltiel, S. (1975) Arch. Biochem. Biophys. 171,673-677.
Received December 1,1976 Accepted February 9,1977
