JPS6354287B2 - - Google Patents

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to a method for producing porous granular N-acylated chitosan which is extremely suitable for use as a carrier for affinity chromatography, a carrier for solidifying physiologically active substances, a filler for chromatography, and the like.

[Conventional technology]

Chitosan is a β-based compound of D-glucosamine obtained by treating chitin, which is a component of the outer skin of crustaceans such as crabs and shrimp, with an aqueous solution of caustic soda.
It is a polysaccharide with (1,4) bonds. A method for obtaining N-acetyl chitosan using chitosan as a raw material is disclosed in Japanese Patent Publication No. 59-30163, but this method involves dissolving chitosan in an acidic solution and using a nonionic surfactant as a particularly advantageous method. A method is mentioned in which an emulsion is obtained by adding an agent and dispersing and emulsifying the solution, which is made into powder by contacting it with an alkaline aqueous solution, which is then washed with water, dispersed in an inert solvent, and then subjected to N-acylation treatment with an acylating agent. There is. That is, as is clear from the description of the examples, the method for molding powdered chitosan before N-acylation treatment is to add a benzene solution containing a surfactant to an acidic aqueous solution of chitosan, and emulsify it by stirring thoroughly. This emulsion is then injected into an alkaline aqueous solution with vigorous stirring and filtered to obtain a coagulated regenerated product of chitosan powder and granule precipitates. In addition, the viscosity reduction of the high molecular weight chitosan solution is measured, and the chitosan concentration of the solution is only in the range of 0.5 to 2.0%. Since high molecular weight chitosan is used, due to the inherent properties of chitosan, when dissolved in an acidic aqueous solution such as acetic acid or formic acid, the viscosity of the solution becomes extremely high even at a concentration of a few percent, resulting in a gelatinous state.
Particularly in order to obtain granules, considerable dispersion becomes necessary in order to avoid gelling conditions. For this purpose, a low-concentration chitosan solution is obtained by dispersing the chitosan using a suspending agent, pore control agent, emulsifier, surfactant, etc. However, when such additives are used,
Depending on the proportion used, the particle size and the state of micropores of the porous granules obtained change, making it impossible to obtain a uniform viscosity distribution and making the porous granules brittle in terms of strength. Even if such granular chitosan is subjected to N-acylation treatment, the performance of the granular chitosan itself remains as a drawback, and when used as a carrier, filler, etc.
- It had the disadvantage of destroying the acylated chitosan and causing problems such as clogging.

Problems to be Solved by the Invention The present invention solves the drawbacks of the conventional methods described above, and provides porous granular chitosan with excellent strength, which has extremely uniform particle size and uniform micropores on the spherical cut surface. By N-acetylation using
The purpose of this method is to simplify the process without using various additives as in conventional methods, and to obtain porous granular N-acetylated chitosan of good quality.

[Means to solve the problem]

The present invention involves dissolving low molecular weight chitosan in an acidic aqueous solution, dropping the solution into a basic solution and solidifying it to form porous granular chitosan, which is then brought into contact reaction with an acylating agent in an organic solvent to form porous granular chitosan. Particulate N-acylated chitosan is obtained. High-molecular-weight chitosan can be made into low-molecular-weight chitosan by the method of Japanese Patent Application No. 161191/1989, which was previously filed by the present inventor, and also by the method of Japanese Patent Application No. 161191/1987, which was also filed by the present inventor. Porous granular chitosan can be obtained using low molecular weight chitosan by the method of No. That is, high molecular weight chitosan can be made into low molecular weight chitosan of any molecular weight by heating in an aqueous sodium perborate solution, and the average molecular weight of the low molecular weight chitosan used in the present invention is 10,000 to 230,000, preferably 30,000. ~200,000, and the degree of deacetylation is preferably 70% or more from the viewpoint of ease of dissolving in an acidic aqueous solution and increasing the degree of acylation by acylation treatment. Low molecular weight chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, formic acid, butyric acid, etc. alone or in a mixture. Since chitosan has a low molecular weight, it is possible to increase the concentration of the chitosan solution and keep the viscosity of the solution low.
It can be 20%. The acidic solution contains alkaline substances such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, and ethylenediamine, and is coagulated from a basic aqueous solution to which polar alcohol such as methanol and ethanol is added as necessary. drop it into the bath,
Porous granular chitosan is coagulated and regenerated. The porous particulate chitosan thus obtained is subjected to an N-acylation reaction using an acylating agent to obtain porous particulate N-acylated chitosan. In order to improve the degree of acylation, the specific surface area of the porous granular chitosan to be subjected to acylation is preferably about 15 to 90 m ² /g. As the acylating agent, acetic anhydride, monochloroacetic anhydride, dichloroacetic anhydride, propionic anhydride, benzoic anhydride, n-caproic anhydride, n-anhydride
-Butyric acid etc. are used. The organic solvent used during acylation is an inert solvent that is inert to the acylating agent and the reaction product and does not have any effect on the acylation reaction, such as dimethylformamide, dimethyl Acetamide, benzene, dioxane, methanol, ethanol, n-
Butanol etc. can be used alone or in combination.

【Example】

The specific surface area of porous granular chitosan and porous granular N-acylated chitosan in the method of the present invention was determined by rapidly freezing the sample in liquid nitrogen at 10 torr, -40°C,
After vacuum drying for 8 hours and degassing at 140°C for 40 minutes, automatic specific surface area measuring device (Shimadzu Micromeritics)
2200 model) using the BET method. The degree of substitution of acyl groups in the porous particulate N-acylated chitosan obtained by the method of the present invention is calculated by determining the composition ratio of nitrogen element to carbon element (N/C value) by elemental analysis. For example, the degree of acetylation (DS) for acetylated chitosan is DS={0.5831/(N/C)}
-3. The degree of acetylation (DS) of propionylated chitosan is calculated from DS {0.3889/(N/C)}-2, and the numerical value of the formula is determined by the acylating agent. In addition, the degree of 0-acylation of acylated chitosan can be confirmed using infrared absorption spectrum (D1750/
D1670) can be estimated from the value. Hereinafter, the present invention will be explained in detail by giving examples. ◇ Example 1 74 g of chitosan with an average molecular weight of 38,000 and a degree of deacetylation of 80% was dissolved in 926 g of water containing 37 g of acetic acid.
The viscosity of this solution was 2500 cp at 20°C. This solution was allowed to fall into a basic solution of 10% NaOH, 30% methanol, and 60% water through a nozzle of 0.25 m/mφ in a spherical shape, solidified and regenerated, and then washed with water until it became neutral. The average particle size of the obtained porous granular chitosan is 0.8
Its specific surface area in m/m was 78.6 m ² /g. This porous granular chitosan was mixed with 3 moles of acetic anhydride as an acylating agent per mole of glucosamine residue in chitosan in 50 ml of dimethylformamide, and N-acylation reaction was carried out at room temperature for 24 hours. This was washed three times with 50 ml of dimethylformamide and thoroughly washed with water to obtain porous granular acetylated chitosan. In a similar manner, porous granular N-acylated chitosan was obtained using 3 moles of propionic anhydride, n-butyric anhydride, benzoic anhydride, and monochloroacetic anhydride per mole of glucosamine residue as acylating agents. These acylation degrees, specific surface areas, (D1750/D1670)
The values are shown in Table 1.

[Table] ◇ Example 2 30 g of chitosan having an average molecular weight of 91000 and a degree of deacetylation of 95% was dissolved in 970 g of water containing 15 g of formic acid. The viscosity of this solution was 4000 cp. The solution was dropped into a basic solution of 10% NaOH, 50% ethanol, and 40% water through a nozzle of 0.25 m/mφ, solidified and regenerated, and washed with water until it became sufficiently neutral to obtain porous granular chitosan. . The particle size of the obtained porous granular chitosan was in the range of 250 to 194 μm, and the specific surface area was 62.0 m ² /g. This porous granular chitosan was reacted with 2 mol of n-caproic anhydride per 1 mol of glucosamine residue in 50 ml of dimethylformamide at room temperature for 24 hours, washed three times with 50 ml of dimethylformamide, and further washed with water. After washing thoroughly with water, porous granular caproylated chitosan was obtained. The degree of acylation is 0.75 and the specific surface area is 76.4m ² /
g, (D1750/D1670) value was 0. ◇ Comparative Example As a comparative example, porous granular caproylated chitosan was produced using high molecular weight chitosan with an average molecular weight of 230,000 and a degree of deacetylation of 78%, and chitosan with an average molecular weight of 28,000 obtained by reducing the molecular weight of this chitosan. compared. 13g of high molecular weight chitosan and 987g of water containing 6.5% acetic acid
The chitosan concentration was 1.3%. The viscosity at 20°C was 3400 cp. In addition, 65 g of low molecular weight chitosan was mixed with 935 g of water containing 32.5 g of acetic acid.
to make a solution with a chitosan concentration of 6.5%. The viscosity of this product at 20°C was 830 cp. Both solutions were mixed with 10% NaOH, 30% CHOH, and 60% water, respectively.
% through a nozzle, the granular chitosan was coagulated and regenerated, and thoroughly washed with water until it became neutral, yielding porous granular chitosan. The average particle size of these granular chitosan is 1.0 m/m, and the specific surface area of the one obtained from high molecular weight chitosan is 38.7.
m ² /g obtained from low molecular weight chitosan 67.3
m ² /g. Take 50ml of each of these granular chitosan,
Add 2 mol of n-caproic anhydride per 1 mol of glucosamine residue in 50 ml of dimethylformamide, react at room temperature for 24 hours, wash 3 times with 50 ml of dimethylformamide, and thoroughly wash with water to obtain each porous granular caproyl. Chemical chitosan was obtained. Their acylation degree and specific surface area (D1750/D1670) values were as shown in Table 2. In addition, 2 ml of granules were prepared using high molecular weight chitosan and low molecular weight chitosan.
To this, add α- to 2 ml of phosphoric acid buffer solution.
A solution adjusted to contain 38 mg of amylase as a protein was added, placed in a centrifuge tube, and allowed to react at room temperature for 2 hours.The amount of adsorption was determined from the degree of adsorption, and the results are also shown in Table 2.

【table】

【Effect of the invention】

In the method of the present invention, since porous granules are produced using low molecular weight chitosan, the concentration of the acidic chitosan solution can be increased, so the strength of the granules increases and N-acylated chitosan can be used as a carrier. When used as a filler, etc., the granules do not break during use. Furthermore, as described in the reference example, when N-acylated chitosan granules are produced using high molecular weight chitosan, the concentration of chitosan per unit volume is low, so naturally the degree of introduction of caproyl groups involved in adsorption is also low. Therefore, it can be seen that the amount of adsorption is also lower than that produced using low molecular weight chitosan at a high concentration. In order to produce such a high-performance carrier, it is advantageous to use chitosan with a low molecular weight and form porous particulate N-acylated chitosan with a high concentration. By the method of the present invention, reactive groups such as monochloroacetyl groups, dichloroacetyl groups, succinyl groups, or hydrophobic groups such as acetyl groups, propionyl groups, butyl groups, caproyl groups, etc., can be added to porous particulate chitosan. By selectively introducing an acylating agent suitable for this purpose, it can be used as a carrier for affinity chromatography, a carrier for immobilizing physiologically active substances, a packing material for chromatography, and the like.

Claims (1)

[Claims] 1 Dissolve low molecular weight chitosan in an acidic aqueous solution,
A method for producing porous granular N-acylated chitosan, which comprises dropping the solution into a basic solution and solidifying it to form porous granular chitosan, which is then subjected to a contact reaction with an acylating agent in an organic solvent.