JP3301628B2 - Cation exchange resin having phosphoric acid group and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing a cation exchange resin and its having useful phosphoric acid group as such a liquid chromatographic column packing material.

[0002]

2. Description of the Related Art Cation-exchange resin-based packing materials for liquid chromatography having a phosphate group require that the characteristics of the exchange group be changed by the hydrogen ion concentration because the exchange group is a weakly acidic cation exchange resin. In addition, it has a feature that it can be used at a higher hydrogen ion concentration than a carboxyl group and has an affinity for a nucleic acid or the like, so that it is effective for separating proteins and the like.

Conventionally used packing materials for liquid chromatography having a phosphate group include (1) hydroxyapatite (JP-A-2-18707),
(2) Natural polymer gels such as dextran, agarose, cellulose, etc., into which phosphate groups have been introduced [SELEX P; trade name of BIORAD], (3) Polystyrene gels with phosphate groups introduced Things [RE Ferre
l, HS Olcott, H. Frenkel-Conrat, J. Am. Chem. S
oc., 70, 2101 (1948)], and (4) those produced by reacting a gel having an epoxy group with aminophosphoric acid (JP-A-1-113654).

[0004] It is more difficult for hydroxyapatite to adjust the amount of phosphoric acid groups, the particle size, the pore volume, and the particle strength as compared with organic polymer compounds. In addition, dextran gels, agarose gels, cellulose gels, etc., in which natural saccharides are cross-linked with epichlorohydrin, etc., take in molecules by a network structure due to cross-linking. The storage capacity can be adjusted relatively easily. However, particularly when large molecules such as proteins are incorporated, the particles are soft and easily crushed because the degree of crosslinking is reduced and the network structure of the gel is widened. Therefore, there is a problem that the chromatography under pressure (high-performance liquid chromatography) operation causes deformation of particles and pressure loss. Further, a styrene-based gel in which a phosphate group is introduced has a high hydrophobicity, and an enzyme or the like may cause hydrophobic adsorption or deterioration.

On the other hand, a gel prepared by reacting a gel having an epoxy group with aminophosphoric acid also acts as an exchange group with an amino group. But not an amphoteric ion exchange resin.

[0006]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned drawbacks of the prior art, and can easily adjust the amount of phosphoric acid groups, particle size and pore volume, has high strength, and to provide a method which can produce a suitable cation exchange resin and then to the separation adsorption of substances.

[0007]

The present invention achieves the above object by synthesizing a crosslinked polymer having a glycidyl group and then reacting it with phosphoric acid to quantitatively introduce a phosphate group. It was done.

That is, the present invention relates to aqueous suspension polymerization in the presence of an organic solvent which is incompatible with water, using 90 to 30 parts by weight of glycidyl monovinyl ester and 10 to 70 parts by weight of a polyvinyl ester of a polyhydric alcohol in a total amount of 100 parts by weight. The present invention relates to a method for producing a cation exchange resin having a phosphate group, wherein the obtained crosslinked polymer particles are reacted with phosphoric acid. Further, the present invention provides the cation exchange resin
It relates to a cation exchange resin obtained by a production method.

Hereinafter, the present invention will be described in more detail. In the present invention, first, 90 to 30 parts by weight of glycidyl monovinyl ester and 10 to 70 parts by weight of a polyvinyl ester of a polyhydric alcohol are used in a total amount of 100 parts by weight in the presence of an organic solvent that is incompatible with water. Kaisei 53
Aqueous suspension polymerization is carried out according to the method described in JP-A-10-1087 to produce a porous crosslinked polymer having a glycidyl group. Here, as glycidyl monovinyl ester,
Glycidyl acrylate, glycidyl methacrylate,
Glycidyl crotonate, glycidyl itaconate, diglycidyl fumarate and the like can be mentioned.

On the other hand, polyvinyl esters of polyhydric alcohols include alkylene glycols having 2 to 3 carbon atoms or esters of these glycols with acrylic acid or methacrylic acid, for example, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate. Acrylate, propylene glycol dimethacrylate, propylene glycol diacrylate, polyethylene glycol dimethacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, etc.
Alternatively, an acrylate or methacrylate such as propanetriol triacrylate or propanetriol trimethacrylate can be used.

In the present invention, glycidyl monovinyl ester is useful for introducing a phosphate group (exchange group) by reacting with phosphoric acid. Therefore, if the glycidyl monovinyl ester is reduced, the amount of the introduced phosphate group is reduced, and the ion exchange capacity is reduced. descend. Further, when the amount of glycidyl monovinyl ester is too large, the amount of the polyvinyl ester of the polyhydric alcohol is reduced, resulting in particles having low mechanical strength. That is,
The polyvinyl ester of a polyhydric alcohol is necessary as a crosslinking agent to maintain the physical strength of the crosslinked polymer. Therefore, it is preferable to appropriately determine the mixing ratio within the range of 90 to 30 parts by weight of glycidyl monovinyl ester and 10 to 70 parts by weight of polyvinyl ester of polyhydric alcohol according to the desired ion exchange capacity and mechanical strength.

The organic solvent incompatible with water used in the present invention acts as a pore regulator and is necessary for making the formed particles porous and increasing the surface area of the particles, and dissolves the polymerizable monomer. Then, a material which is inert to the radical polymerization reaction is used. Further, since the polymerization is carried out by an aqueous suspension polymerization method, it is necessary that the pore control agent is insoluble or hardly soluble in water as a polymerization dispersion medium.

Examples of such pore regulators include aliphatic halogenated hydrocarbons such as dichloromethane, dichloroethane, and trichloroethane; aliphatic or aromatic esters such as ethyl acetate, butyl acetate, dimethyl phthalate, and diethyl phthalate; Hardly soluble alcohols such as alcohol, heptyl alcohol and octyl alcohol are exemplified. If these pore regulators are too small, the pores are not sufficiently opened, and the amount of protein or the like adsorbed is reduced, and if too large, the pore volume becomes too large, resulting in poor mechanical strength. Since it is insufficient, it is used in an amount of 30 to 300 parts by weight, preferably 100 to 200 parts by weight, based on the monomer.

The polymerization initiator is usually a peroxide such as benzoyl peroxide, lauroyl peroxide, t-butyl hydroperoxide, cumene hydroperoxide, methyl ethyl ketone peroxide, or azobisisobutyronitrile, azobis α, α- Dimethylvaleronitrile,
An azo-based polymerization initiator such as azobisisobutylamide is used. The amount used is 0.1 to 10 of the polymerizable monomer.
% By weight. At the time of polymerization, salts such as sodium chloride and calcium chloride may be dissolved in water in order to prevent the polymerizable monomer from dissolving in water as a polymerization dispersion medium. In addition, a dispersant such as gelatin, polyvinyl alcohol, carboxymethyl cellulose, hydroxyethyl cellulose, or hydroxyapatite is used to stabilize the monomer in the dispersion medium.

The amount of the aqueous phase is substantially equal to or more than that of the organic solvent.
Used up to about 20 times. The polymerization reaction is usually 50 to 9
This is performed at 5 ° C. for about 3 to 10 hours. After the completion of the polymerization, the porous crosslinked polymer particles having a glycidyl group are filtered, washed, dried and classified as necessary.

Thereafter, the obtained porous crosslinked polymer particles having a glycidyl group are reacted with phosphoric acid to introduce a phosphate group. For this reaction, an aqueous phosphoric acid solution was added to 1 part by weight of the porous crosslinked polymer particles having a glycidyl group.
Use 5 parts by weight or more. The reaction solvent is not particularly limited as long as it dissolves phosphoric acid and does not introduce an anion exchange group by reacting with a glycidyl group. The concentration of the phosphoric acid aqueous solution can be used in a range of 0.01% by weight or more, preferably 1% by weight or more and up to 100% by weight (concentrated phosphoric acid).

The reaction is carried out at 30-200 ° C., preferably at 50 ° C.
Perform at ９０90 ° C. for several minutes to 10 hours. When the reaction is carried out under the above reaction conditions, the phosphate group introduction reaction and the hydrolysis reaction occur competitively. Therefore, by adjusting the concentration of the phosphoric acid aqueous solution, this competitive reaction can be controlled, and quantitative phosphate group formation can be achieved. It can be easily introduced.

The resulting crosslinked polymer particles into which phosphate groups have been introduced are washed with methanol, water and, if necessary, an aqueous potassium hydroxide solution or an aqueous sodium hydroxide solution. Crosslinked polymer particles suitable as a filler can be obtained.

[0019]

Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.

Example 1 (a) Preparation of Crosslinked Polymer Particles Having Glycidyl Ester Groups A mixture of 140 g of glycidyl methacrylate, 60 g of tetramethylolmethane methacrylate, 180 g of butyl acetate, 120 g of isoamyl alcohol and 0.7 g of azobisisobutyronitrile was prepared. 1000 ml of ion-exchanged water is added, and while stirring, the pH is adjusted to 7 using an aqueous sodium hydroxide solution.
Adjusted to ~ 8. Thereafter, a polymerization reaction was performed at 70 ° C. for 6 hours. After cooling the reaction product, the produced copolymer particles were collected by filtration and washed sequentially with methanol and water. Next, it was air-dried for one day and further placed in a vacuum dryer at 80 ° C. for 6 hours. The dried particles were classified to obtain 80 g of porous crosslinked polymer particles having a size of 355 to 425 µm.

(B) Phosphoric acid group introduction reaction 20 g of the porous crosslinked polymer particles obtained in (a) were placed in 300 ml of concentrated phosphoric acid and stirred well. This was heated at 60 ° C. and stirred for 3 hours to carry out a reaction. After the reaction, cool,
The obtained porous crosslinked polymer particles were washed with ion-exchanged water until the particles became neutral. Thereafter, decantation was performed five times using ion-exchanged water to remove fine particles.

(C) Measurement of the amount of exchanged groups The porous crosslinked polymer particles obtained in (b) were placed in a vacuum drier, dried at 80 ° C. for 6 hours, and 1 g thereof was packed in a column. And This was washed with a 0.1 hydrochloric acid aqueous solution, and further washed with ion-exchanged water until the washing solution became neutral. Next, the mixture was washed with 20 ml of a 0.1N aqueous sodium hydroxide solution, and the filtrate was recovered. The filtrate is 0.1
Titration was carried out with an aqueous solution of N hydrochloric acid, and the amount of exchanged groups was determined by the following formula.

[0023]

X = (20−Y) × 0.1 / Z wherein X is the amount of exchange groups (meq / g), Y is the titer (ml),
Z indicates the amount (g) of column packing material].

(D) Measurement of Protein Adsorption Amount of γ-protein was added to 100 ml of 3 mM phosphate buffer adjusted to pH 4.0.
15 mg of globulin was dissolved. Next, Example 1 (b)
1 g of the porous cross-linked polymer synthesized in the above was added and stirred for 40 minutes. The absorbance of the supernatant was measured using a phosphate buffer as a control, and the amount of adsorption was determined by the following equation.

[0025]

## EQU2 ## where a represents the absorbance of the supernatant, and b represents the absorbance when 15 mg of γ-globulin is dissolved in 100 ml of phosphate buffer. Represents). As a result, the amount of adsorption was 2.2 mg. Table 1 shows the results.
It was shown to.

Example 2 Production of crosslinked polymer particles having a glycidyl ester group was carried out in the same manner as in Example 1 (a).

20 g of the obtained porous crosslinked polymer particles were put into a 50% by weight aqueous solution of phosphoric acid consisting of 100 ml of ion-exchanged water and 100 g of concentrated phosphoric acid, and stirred. The mixture was heated at 60 ° C. and reacted while stirring for 3 hours. After the reaction, the mixture is cooled and the obtained porous crosslinked polymer particles (column filler)
Was repeatedly washed with ion-exchanged water until neutral. Thereafter, decantation was performed five times using ion-exchanged water to remove fine particles. For the obtained porous cross-linked polymer particles, the amount of exchange groups was measured by the same operation as in Example 1 (c), and the amount of adsorbed protein was measured by the same operation as in Example 1 (d). The results are shown in Table 1.

Example 3 Production of crosslinked polymer particles having a glycidyl ester group was carried out in the same manner as in Example 1 (a).

40 g of the obtained porous cross-linked polymer particles were placed in an 11% by weight aqueous phosphoric acid solution comprising 400 ml of ion-exchanged water and 50 g of concentrated phosphoric acid, and stirred. The mixture was heated at 60 ° C. and reacted while stirring for 3 hours. After the reaction, the system was cooled, and the obtained porous crosslinked polymer particles (column filler) were repeatedly washed with ion-exchanged water until the particles became neutral. Thereafter, decantation was performed five times using ion-exchanged water to remove fine particles. For the obtained porous cross-linked polymer particles, the amount of exchange groups was measured by the same operation as in Example 1 (c), and the amount of adsorbed protein was measured by the same operation as in Example 1 (d). The results are shown in Table 1.

Comparative Example 1 To a mixture of 140 g of glycidyl methacrylate, 60 g of tetramethylolmethane methacrylate and 0.7 g of azobisisobutyronitrile was added 1,000 ml of ion-exchanged water, and the mixture was stirred at pH 7 using an aqueous sodium hydroxide solution with stirring.
Adjusted to ~ 8. Thereafter, a polymerization reaction was performed at 70 ° C. for 6 hours. After cooling the reaction product, the produced copolymer particles were collected by filtration and washed sequentially with methanol and water. Next, it was air-dried for one day and further placed in a vacuum dryer at 80 ° C. for 6 hours. The dried particles were classified to obtain 80 g of non-porous crosslinked polymer particles of 355 to 425 µm.

Example 1 of the obtained polymer particles
When the amount of exchange groups was measured by the same operation as (c),
It was found that only 0.1 meq / g could be introduced. Further, the amount of adsorbed protein was measured by the same operation as in Example 1 (d), and it was 0.1 mg. The results are shown in Table 1.

[0032]

[Table 1]

As is clear from the results shown in Table 1, according to the present invention, the amount of the exchange group can be easily adjusted, and a cation exchange resin having a high adsorptivity can be easily produced.

[0034]

According to the present invention, it is possible to easily produce a cation exchange resin having a phosphate group in which the amount of exchange groups and the pore volume can be easily adjusted. Is suitable for separating and adsorbing biological substances and has high strength, so that it can be used under pressure.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C08F 220/20 C08F 220/20 G01N 30/48 G01N 30/48 P

Claims (2)

(57) [Claims] 1. Glycidyl monovinyl ester 90-3
0 parts by weight and polyvinyl ester of polyhydric alcohol 10
Aqueous suspension polymerization is carried out in the presence of an organic solvent incompatible with water with a total amount of ~ 70 parts by weight being 100 parts by weight, and the obtained crosslinked polymer particles are reacted with phosphoric acid. Production method of cation exchange resin. 2. Obtained by the production method according to claim 1.
Cation exchange resin.