Crystalline behavior of chitosan organic acid salts.

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous "Annealed" polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated "Tendon" polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

Crystalline Behavior of Chitosan Organic Acid Salts

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous “Annealed” polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated “Tendon” polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

Conformational Behavior of Chitosan in the Acetate Salt: An X-Ray Study

A well-defined X-ray fiber pattern of chitosan acetate was obtained by immersing a tendon chitosan, prepared from a crab tendon chitin by a solid-state N-deacetylation, in an aqueous acetic acid-isopropanol solution at 110°C. This pattern was very similar to that of chitosan salts with some inorganic acids, such as HF, HCl, and H₂SO₄, in which chitosan chains form an 8/5 helix, indicating that chitosan acetate also take up this conformation. This information may give an influential clue to the chitosan conformation in the aqueous acetic acid solution, the most popular solvent for chitosan. However, after one month of storage of the chitosan acetate, the fiber pattern, the density and its IR spectrum changed to those of the anhydrous polymorph of chitosan, suggesting that the acetic acid was removed accompanied with water molecules from the crystal during storage and that the polymorph can be obtained not only by annealing chitosan, but also through the chitosan acetate.     

Three D structures of chitosan

Crystal structures of two polymorphs of chitosan, tendon (hydrated) and annealed (anhydrous) polymorphs, have been reported. In both crystals, chitosan molecule takes up similar conformation (Type I form) to each other, an extended two-fold helix stabilized by intramolecular O3-O5 hydrogen bond, which is also similar to the conformation of chitin or cellulose. Three chitosan conformations other than Type I form have been found in the crystals of chitosan-acid salts. In the salts with acetic and some other acids, called Type II salts, chitosan molecule takes up a relaxed two-fold helix composed of asymmetric unit of tetrasaccharide. This conformation seems to be unstable because no strong intramolecular hydrogen bond like Type I form. Type II crystal changes to the annealed polymorph of chitosan by a spontaneous water-removing action of the acid. Chitosan molecule in its hydrogen iodide salt prepared at low temperature takes a 4/1 helix with asymmetric unit of disaccharide. The fourth chitosan conformation was found to be a 5/3 helix in chitosan salts with medical organic acids having phenyl group such as salicylic or gentisic acids. Similar conformation of chitosan molecule in the aspirin (acetylsalicylic acid) salt was suggested by a solid-sate NMR measurement.     

Gel Permeation Chromatography of Polysaccharides: Universal Calibration Curve

Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200°C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by N-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv = 1,720–12,600) and N-acetylation (zero to 26%). It was found that the presence of hydrated polymorphs or anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of N-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan’s functionality, by heating.     

Effect of Explosion on the Crystalline Polymorphism of Chitin and Chitosan

For identification of how explosion increases the reactivity of chitin and chitosan, changes in the crystalline polymorphism of these polysaccharides were studied by X-ray diffraction measurements. The α-chitin form of chitin did not change after being exploded, but an X-ray diagram of chitosan showed a hydrated crystal of low crystallinity before the explosion, and increased crystallinity of the hydrated form plus a small amount of an anhydrous crystal after the explosion. The improvement of accessibility to both polysaccharides caused by the explosion seemed not to arise from changes in their crystalline polymorphism or crystallinity     

Structural study of anhydrous tendon chitosan obtained via chitosan/acetic acid complex

The molecular structure and packing arrangement of anhydrous tendon chitosan was determined by the X-ray fibre diffraction method together with the linked-atom least-squares refinement technique. The specimen was prepared from chitosan/acetic acid complex which was obtained by exposing tendon chitosan to acetic acid vapour at room temperature for several days. There is high degree of orientation and crystallinity compared with the specimen obtained by the annealing method. Two chitosan chains are present in an orthorhombic unit cell of dimensions a = 8.26(2), b = 8.50(1), c (fibre axis) = 10.43(2) A and space group P2(1)2(1)2(1). The 2-fold helical chain is stabilised by O3 triple bond O5 hydrogen bond with the gt orientation of O6. There are direct hydrogen bonds (N2 triple bond O6) between adjacent chains along the a-axis, which makes a sheet structure parallel to the ac-plane. On the other hand, no hydrogen bond is found between the sheets.     

Spinning of hydroalcoholic chitosan solutions

We investigated the spinning of hydroalcoholic chitosan solutions. The dope composition was optimized in order to obtain a continuous alcogel fiber by water evaporation on heating the extruded hydroalcoholic solution. This alcogel fiber was then neutralized in aqueous alkali baths and washed in water to eliminate the residual alcohol and salts before final drying. Depending on the alcohol content in the filament at the neutralization step, on specific alcohol–chitosan interactions and on the nature and concentration of the coagulation base, the process yielded semicrystalline chitosan fibers with different proportions of anhydrous and hydrated allomorphs. Contrarily to the classical annealing method, the formation of mainly anhydrous crystals was obtained without significant molecular weight decrease by neutralizing the polymer in hydrophobic conditions. The control of allomorph content was shown to be related to the hydrophobicity of the solvent (alcohol fraction) at the neutralization step.     

Studies on chitosan: 2. Solution stability and reactivity of partially N-acetylated chitosan derivatives in aqueous media

The stability of the solutions of partially N-acetylated chitosans was studied by two methods: (1) 1% solutions of the chitosan derivatives in 0.1 M aqueous acetic acid were added dropwise to buffer solutions with pH from 8.6 to 12 and to a 0.1 M NaOH solution; (2) to each 0.5% solution of the derivatives in 0.1 M acetic acid was added the desired amount of a 1 M NaOH solution. The stability data obtained were summarized with respect to the degree of N-acetylation. It was found that the solutions of the derivatives with more than 50% acetyl content were stable even in alkaline conditions and the gelation and precipitation of the solutions did not occur. The reactivity of the derivatives with the degree of N-acetylation of more than 50% was studied using methyl 4-azidobenzoimidate (MABI) and ethylene glycol diglycidyl ether in homogeneous states. It was found that MABI reacted with amino groups of the chitosans only at neutral pH and glycidyl groups reacted at neutral and alkaline pH. It seems that these unique properties of chitosans with a degree of N-acetylation of more than 50% will enable us to prepare new chitosan derivatives.     

Biodegradable polylactide/chitosan blend membranes

Biodegradable blend membranes based on polylactide and chitosan with various compositions were prepared via a two-step processing pathway. In the first step, solutions of each component were properly mixed and cast into a gelatinous membrane, and in the second step, the obtained membrane was immersed into a mixed solution for the solvent extraction followed by a drying procedure to finally generate a well-blended membrane. An acetic acid-acetone solvent system was selected for poly(DL-lactide)/chitosan membranes, and another solvent system for poly(L-lactide)/chitosan membranes consisted of acetic acid and dimethyl sulfoxide. Some processing parameters, such as the concentration of component solutions and the composition ratio of mixed solvents and extraction solvents, were optimized by primarily considering whether the directly visible phase separation occurred during the processing procedures. Morphologies of these blend membranes were viewed using SEM. It was found that the processing parameters exerted quite notable impacts on the morphology of the membranes. The hydrophilicity of membranes was examined by measuring their water contact angle and swelling index. These blend membranes were also investigated for their miscibility using IR spectra, X-ray diffractograms, TG, DSC, and dynamic mechanical analysis methods. Although the presence of phase separation at a microscopic level was detected for these membranes, pronounced interactions between components were confirmed. The obtained results shown that some membranes prepared under optimized processing conditions had a partially miscible structure.     

Crystallinity of Partially N-Acetylated Chitosans

In order to search for a chitosan with low crystallinity, partially N-deacetylated chitins (PDC) and partially N-acetylated chitosans (PAC) with a low degree of N-acetylation (DAc) were examined by X-ray powder diffraction measurements. Three PDC samples, having less than 30% DAc and prepared by solid-state deacetylation, gave X-ray powder patterns showing the presence of α-chitin, a hydrated crystal of chitosan, or their mixture, respectively. When these PDC samples were treated with an acid-alkali, however, reduced crystallinity was observed. By annealing in water at 160 or 200°C, the latter PDC having DAc less than approx. 22% gave powder patterns indicating the presence of an anhydrous crystal which may spoil the chitosan’s functionality. In contrast, PAC prepared by N-acetylating pure chitosan (DAc=0%) in a swollen state, which can be expected to have random copolymers in the chain, was always less crystallized than PDC, this crystallinity depending on the molecular weight. In the case of high-molecular-weight PAC samples, whose DAc was in the range of 5–21%, the effect of high molecular weight on reducing crystallinity was larger than that of the degree of N-acetylation.     

Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation

In this study, the depolymerization of chitosan was carried out in an acetic acid aqueous solution and was followed by viscometry for molecular weight determination. It was found that the depolymerization rate increased with elevated temperatures and with high acid concentrations. Based on FTIR analysis, the chitosan was depolymerized randomly along the backbone; no other structural change was observed during the acid depolymerization process. Revealed in the TGA study, the degradation temperature and char yield of LMWCs (low molecular weight chitosan) were molecular weight dependent. The blood compatibility of LMWCs was also investigated: rouleaux formation was observed when erythrocyte contacted with LMWCs, which showed that LMWCs are able to interfere with the negatively charged cell membrane through its polycationic properties. Furthermore, as regards a kinetics investigation, the values of M_n (number-average molecular weight) were obtained from an experimentally determined relationship. The kinetics study showed that the complex salt, formed by amine on chitosan and acetic acid, acted as catalyst. Finally, the activation energy for the hydrolysis of the glycosidic linkage on chitosan was calculated to be 40 kJ/mol; the mechanism of acid depolymerization is proposed. In summary, LMWCs could be easily and numerously generated with acid depolymerization for further biological applications.     

Chitosan bicomponent nanofibers and nanoporous fibers

Nanofibers with average diameters between 20 and 100nm have been prepared by electrospinning of 82.5% deacetylated chitosan (Mv=1600 kDa) mixed with poly(vinyl alcohol) (PVA, Mw=124-186 kDa) in 2% (v/v) aqueous acetic acid. The formation of bicomponent fibers was feasible with 3% concentration of solution containing up to an equal mass of chitosan. Finer fibers, fewer beaded structures and more efficient fiber formation were observed with increasing PVA contents. Nanoporous fibers could be generated by removing the PVA component in the 17/83 chitosan/PVA bicomponent fibers with 1M NaOH (12 h). Fiber formation efficiency and composition uniformity improved significantly when the molecular weight of chitosan was halved by alkaline hydrolysis (50 wt% aqueous NaOH, 95 degrees C, 48 h). The improved uniform distribution of chitosan and PVA in the bicomponent fibers was attributed to better mixing mostly due to the reduced molecular weight and to the increased deacetylation of the chitosan.     

Enzymatic hydrolysis of chitosan films in water and physiological solution

It was shown that the processes of enzymatic hydrolysis of chitosan in aqueous acetic acid and on the surface of chitosan films in a solution of hyaluronidase in acetic acid are described by uniform kinetic constants. Kinetic parameters of enzymatic hydrolysis of the chitosan film samples in water and in physiological solution (Ringer–Locke’s solution) were determined. It was found that the introduction of medicinal agents and low-molecular-weight electrolytes to a chitosan-based film material reduces the rate of enzymatic hydrolysis of the films, which may indicate a possible increase in their service life when used on the wound surface.     

A simple preparation of half N-acetylated chitosan highly soluble in water and aqueous organic solvents

A simple and improved method of preparing highly soluble chitosan (half N-acetylated chitosan) was developed using a series of chitosan samples of low molecular weights, and the solubility of the half N-acetylated chitosan in water and organic solvents was investigated in detail. To reduce the molecular weight, chitosan was treated with NaBO3 under the condition that chitosan was homogeneously dissolved in aqueous acetic acid. Weight-average molecular weights of the obtained chitosan samples were determined using a size-exclusion chromatography system equipped with a low-angle laser light-scattering photometer. Each chitosan sample was then N-acetylated with acetic anhydride under the condition that chitosan was homogeneously dissolved in aqueous acetic acid again. The water solubility of the half N-acetylated chitosan thus prepared increased with decreasing molecular weight. From 1H NMR spectroscopy, it was suggested that the sequence of N-acetylglucosamine and glucosamine residues was random. The solubility of the half N-acetylated chitosan of low molecular weight was rather high even in aqueous dimethylacetamide and dimethylsulfoxide.     

Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions

Further utilization of chitosan nanofibrous membranes that are electrospun from chitosan solutions in trifluoroacetic acid (TFA) with or without dichloromethane (DCM) as the modifying cosolvent is limited by the loss of the fibrous structure as soon as the membranes are in contact with neutral or weak basic aqueous solutions due to complete dissolution of the membranes. Dissolution occurs as a result of the high solubility in these aqueous media of -NH(3)(+)CF(3)COO(-) salt residues that are formed when chitosan is dissolved in TFA. Traditional neutralization with a NaOH aqueous solution only maintained partial fibrous structure. Much improvement in the neutralization method was achieved with the saturated Na(2)CO(3) aqueous solution with an excess amount of Na(2)CO(3)(s) in the solution. We showed that electrospun chitosan nanofibrous membranes, after neutralization in the Na(2)CO(3) aqueous solution, could maintain its fibrous structure even after continuous submersion in phosphate buffer saline (pH = 7.4) or distilled water for 12 weeks.     

Chitosan-silica complex membranes from sulfonic acid functionalized silica nanoparticles for pervaporation dehydration of ethanol-water solutions

Nanosized silica particles with sulfonic acid groups (ST-GPE-S) were utilized as a cross-linker for chitosan to form a chitosan-silica complex membranes, which were applied to pervaporation dehydration of ethanol-water solutions. ST-GPE-S was obtained from reacting nanoscale silica particles with glycidyl phenyl ether, and subsequent sulfonation onto the attached phenyl groups. The chemical structure of the functionalized silica was characterized with FTIR, (1)H NMR, and energy-dispersive X-ray. Homogeneous dispersion of the silica particles in chitosan was observed with electronic microscopies, and the membranes obtained were considered as nanocomposites. The silica nanoparticles in the membranes served as spacers for polymer chains to provide extra space for water permeation, so as to bring high permeation rates to the complex membranes. With addition of 5 parts per hundred of functionalized silica into chitosan, the resulting membrane exhibited a separation factor of 919 and permeation flux of 410 g/(m(2) h) in pervaporation dehydration of 90 wt % ethanol aqueous solution at 70 degrees C.     

Visualizing Solvent Mediated Phase Transformation Behavior of Carbamazepine Polymorphs by Principal Component Analysis

The purpose of this research is to gain a greater insight into the hydrate formation processes of different carbamazepine (CBZ) anhydrate forms in aqueous suspension, where principal component analysis (PCA) was applied for data analysis. The capability of PCA to visualize and to reveal simplified structures that often underlie large data sets are explored. Different CBZ polymorphs were dispersed separately in aqueous solution, and then recovered and measured by FT-Raman spectroscopy. PCA was employed for visualizing the dynamics of the phase transformation from each CBZ polymorph to the dihydrate (DH). As a comparison to PCA visualization, the transformation process of each CBZ polymorph was quantified using PLS modeling. The results demonstrated that PCA has advantages in presenting the original data in terms of the differences and similarities, and also directly identify the statistical patterns in the data even when the data set is large. These advantages provided greater insight into the measured Raman spectra as well as the phase transformation process of CBZ polymorphs to the DH in aqueous environment.     

Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system.

Chitosan dissolved in acetic acid reacted with glutaraldehyde solution, ranging in concentration from 0.10 to 25.0 x 10(-2) mol dm3. The modified polymers were characterized by means of carbon, hydrogen and nitrogen elemental analysis, scanning electron microscopy, X-ray diffractometry, 13C nuclear magnetic resonance (NMR), infrared and Raman spectroscopies. The uptake of metallic cations in aqueous medium was checked through copper. The obtained data from 13C NMR, infrared and Raman spectroscopies evidenced the formation of an ethylenic double bond in the chitosan glutaraldehyde interaction. These data suggest that free pendant amine groups of chitosan polymer interact with the aldehydic group of the glutaraldehyde to form stable imine bonds, due to the resonance established with adjacent double ethylenic bonds. The crosslinking is formed by the nonuniform length of chains and by terminal unities. The crosslinking formation can involve two chitosan unities belonging, or not, to the same polymeric chain. The sequence of reactions was established for a chitosan:glutaraldehyde molar proportion of 1:20. The degree of crystallinity and particle size decreased as the amount of glutaraldehyde was increased in the polymer. Physical and chemical properties are not just affected for the chitosan glutaraldehyde reaction, but are also affected strongly by the dissolution of the natural chitosan.     

Photoresponse of chlorophyll-containing bileaflet membranes and the effect of phycocyanin as extrinsic membrane protein

Summary Artificial bileaflet membranes were formed from extracts of chloroplasts. Gradients of a redox potential were created across the membranes by adding various concentrations of ceric-cerous ions, ferric-ferrous ions, and ascorbic acid to the aqueous solutions on either side of the membrane. When a membrane interposed between solutions of different redox potential was irradiated with light, a potential difference of up to 50 mV was recorded. Analysis of the photoresponse allowed its separation into two components: a photoelectromotive driving force dependent upon the redox potential gradient, and a photoconductive pathway dependent upon the amount of light absorbed by the membranes. There appeared to be a limit to the photocurrent that could be drawn from a membrane at a particular intensity of irradiation; i.e., it did not increase indefinitely with increase of the redox potential gradient. Conductance of the photoconductive pathway was independent of temperature. Phycocyanin added to the aqueous solution participated in the photoresponse in a unidirectional manner that suggested facilitation of electron transport from membrane to acceptors in the aqueous solution.