JP2528502B2 - Method for producing chromatography packing material - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a packing material for chromatography. More specifically, the present invention relates to a method for producing cellulose microspheres by alkaline hydrolysis of cellulose triacetate microspheres.

(B) Conventional technology The development of biotechnology is enabling large-scale production of biopolymers (proteins, polysaccharides, etc.) that are useful to humans, but methods for separating and purifying these useful substances are indispensable. This is a technology, and various studies have been made. Among them, column chromatography is the most effective method and various separation modes are used. Natural polymers such as agarose, dextran, and cellulose, and synthetic polymers such as polyvinyl alcohol, polystyrene, and polyacrylamide are used as the material for the column chromatography packing material. Has the advantages of low non-specific adsorption, good flow rate characteristics, high-speed processing, low cost compared with other materials, and suitable for large-scale purification. As a method for producing spherical cellulose particles, roughly classified, a method of solidifying spherically shaped droplets from a solution of cellulose, and a method of regenerating spherical cellulose by hydrolyzing a spherical cellulose derivative are known. The method of alkali-hydrolyzing spherical cellulose triacetate particles to obtain cellulose particles is the most practical method in view of the performance and economy of the obtained cellulose particles.

(C) Problems to be Solved by the Invention However, in this method, since hydrogen bonds are generated between the hydroxyl groups of the cellulose molecules generated when the cellulose triacetate is hydrolyzed, shrinkage of the particles occurs,
Along with this, there are disadvantages that the density of cellulose on the outer surface of the particles is increased, and as a result, the exclusion limit molecular weight is reduced and the cut-off molecular weight range is narrowed. In order to cover such a defect, a method of dissolving only the outer surface of the obtained cellulose particles or swelling with an alkali and then fixing the swollen state by adding a crosslinking agent has been tried, In addition to complicating the process and impairing the economical efficiency, which is a good property of the cellulose material, in each case, to obtain cellulose particles that sufficiently satisfy both the separation performance (exclusion limit molecular weight, fractionation molecular weight range, etc.) and flow rate characteristics. Was difficult. From the above viewpoints, the inventors of the present invention have earnestly studied, and as a result, arrived at the present invention.

(D) Means and Actions for Solving the Problems According to the present invention, when the cellulose triacetate microspherical particles are hydrolyzed with alkali to be converted into the cellulose microspherical particles, the reaction system reacts with a hydroxyl group to form an ether bond. There is provided a method for producing a packing material for chromatography, which comprises adding a compound having two or more functional groups capable of producing

The spherical cellulose triacetate particles that can be used in the present invention may be obtained by any method as long as the particle diameter is 10 to 500 μm. However, those having a uniform mesh structure are preferable.

The compound having two or more functional groups in the molecule capable of reacting with a hydroxyl group to form an ether bond in the present invention,
Any so-called cross-linking agent may be used as long as it has two or more functional groups capable of reacting with a hydroxyl group to form an ether bond, such as halogen, epoxy, and isocyanate in the same molecule. Specific examples of such a cross-linking agent include haloepoxides such as epichlorohydrin and epibromhydrin, 1,
3-dichloro-2-propanol, 1,3-dibromo-2-
Examples thereof include dihalo alcohols such as propanol and diepoxides such as diglycidyl ether and diglycidyl-1,2-ethanediol.

The hydrolysis reaction of the cellulose triacetate spherical particles into the cellulose spherical particles can be carried out, for example, by dispersing the raw material particles in a dilute alkaline aqueous solution and stirring at about room temperature.

The alkali used is preferably an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide. These alkalis are used as an aqueous solution, and the concentration thereof is usually preferably 0.5 to 5 N. The concentration of alkali will affect the degree of swelling of the raw material particles. The amount of alkali used is about 1.5 to 5 times the molar amount of the acetyl group content in the raw material particles.

In addition, in order to control the alkali concentration in the reaction system, add alkali when the hydrolysis has progressed to a certain extent, or add acid to neutralize some alkali to control the alkali concentration during the crosslinking reaction. Good.

The cross-linking agent is added so that the cross-linking reaction is carried out. The timing of addition may be any of the beginning, the middle and the end of the hydrolysis reaction. The addition amount is 1 to 50 per 100 g of dry weight of cellulose triacetate particles.
It is g. The cross-linking reaction is performed in
It can be carried out at -80 ° C, preferably 40-60 ° C. The reaction time is usually 1 to 3 hours.

The usefulness of the present invention is shown below with reference to Examples, but the present invention is not limited to these Examples.

(E) Example Production Example of Cellulose Triacetate Particles 300 g of cellulose triacetate (DS = 2.98, manufactured by Daicel Chemical Industries) was added to 2500 ml of methylene chloride and isopropanol (IP
A) Add dibutyl phthalate to a solution dissolved in 300 ml of mixed solution.
Add 600 ml slowly with stirring. The resulting solution contains 7.7% by weight of cellulose triacetate. This solution is dispersed in a 1% aqueous polyvinyl alcohol solution containing a small amount of antifoaming agent. Dibutyl phthalate
300 ml and 300 ml of water are mixed and stirred, and the cellulose triacetate droplets are gelled. After deliquoring, it was thoroughly washed with IPA to obtain about 3 IPA wet particles of cellulose triacetate.

Example 1 About 1 of cellulose triacetate particles (IPA wet) obtained in Production Example 1 was dispersed in 3N-NaOH aqueous solution 1,
The reaction was carried out at room temperature for 1 hour. Then epichlorohydrin
After adding 5 g, the temperature was raised to 40 ° C. and the reaction was carried out for 2 hours. After cooling, it was neutralized and washed thoroughly with water to obtain about 0.4 cellulose particles.

This product is classified by wet classification, 40-100 μm particles are packed in a glass column with an inner diameter of 10 mm and a length of 470 mm,
Polyethylene glycol of known molecular weight was passed through deionized water as a carrier and subjected to gel filtration chromatography. As a result, it was found that the exclusion limit molecular weight of this was about 20,000 in terms of polyethylene glycol. The calibration curve is shown in FIG.

In the figure, the vertical axis represents the ratio of Kav: stationary phase gel volume in which the sample can diffuse, and is represented by the following equation.

Ve: Elution volume of sample Vt: Total volume of column Vo: Exclusion volume The horizontal axis indicates the molecular weight of polyethylene glycol.

Comparative Example 1 Cellulose triacetate particles obtained in Production Example 1 (IPA
About 1 (wet) was dispersed in 3N-NaOH aqueous solution 1 and reacted at room temperature for 3 hours to obtain cellulose particles. After neutralization, the product was thoroughly washed with water and then classified to obtain particles of 40 to 100 μm, which were evaluated as a gel filtration chromatography packing material in the same manner as in Example 1. The exclusion limit molecular weight of the cellulose particles was 8500 for polyethylene glycol. The calibration curve is shown in FIG. 2, and it can be seen that the molecular weight cut-off range is clearly smaller than in Example 1. Further, the pressure loss with respect to the flow rate of the cellulose particles in Example 1 and the comparative example were almost the same.

Example 2 In the same manner as that carried out in Example 1, cellulose triacetate particles from a cellulose triacetate dope with a cellulose triacetate concentration of 2.4% by weight are obtained.
It was dispersed in N-NaOH aqueous solution 1 and subjected to hydrolysis and crosslinking in the same manner as in Example 1 to obtain cellulose particles. The cellulose particles having been classified and 40-100 μm were evaluated in the same manner as in Example 1. The exclusion limit molecular weight of the cellulose particles was 300,000 for polyethylene oxide (PEO).

Comparative Example 2 About 1 of the same cellulose triacetate particles (IPA wet) used in Example 2 was hydrolyzed with an aqueous 3N-NaOH solution at room temperature for 3 hours to give cellulose particles. After neutralization and washing, the particles were classified, and the particles of 40 to 100 μm were evaluated in the same manner as in Examples 1 and 2. The exclusion limit molecular weight of the particles was 150,000.

Example 3 A cellulose triacetate dope having a cellulose triacetate concentration of 5.6% by weight was prepared in the same manner as in Production Example 1. Cellulose triacetate particles 1
(IPA wet) was dispersed in 3N sodium hydroxide 1 containing 5 g of epichlorohydrin and reacted at room temperature for 1 hour and at 40 ° C. for 2 hours. After neutralization and washing, classification was carried out to obtain particles of 40-100 μm. When the same evaluation as in Example 1 was performed, the exclusion limit molecular weight of the cellulose particles was 50,000 for polyethylene oxide.

Comparative Example 3 The exclusion limit molecular weight of the cellulose particles obtained by treating the same cellulose triacetate as used in Example 3 only with a 3N-NaOH aqueous solution was 20,000 for polyethylene oxide.

Example 4 1,3-In place of epichlorohydrin used in Example 1
The reaction was carried out using 10 g of dibromo-2-propanol. The exclusion limit molecular weight of the obtained cellulose particles was 20,000 for polyethylene glycol, which was the same as that obtained in Example 1.

(F) Effect of the Invention When a cellulose triacetate spherical particle is hydrolyzed with an alkali, a compound having a bifunctional group capable of reacting with a hydroxyl group, that is, a cross-linking agent is allowed to coexist, thereby suppressing the formation of hydrogen bonds and generating it. It is possible to suppress the shrinkage of cellulose, and it is possible to obtain cellulose particles that satisfy both the separation performance and the flow rate characteristics. Further, in the method of the present invention, crosslinking can be carried out in various swelling states by controlling the alkali concentration in the system, and the properties of the produced cellulose particles can also be controlled.

[Brief description of drawings]

FIG. 1 shows a calibration curve of the cellulose triacetate particles obtained in Example 1. FIG. 2 shows a calibration curve of the cellulose triacetate particles obtained in Comparative Example 1.

Claims (1)

(57) [Claims] 1. When hydrolyzing cellulose triacetate fine spherical particles with an alkali to convert them into cellulose fine spherical particles, two or more functional groups in the molecule capable of reacting with a hydroxyl group to form an ether bond in the reaction system. A method for producing a packing material for chromatography, which comprises adding the compound having