JP3096097B2 - Crosslinked copolymer particles and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to crosslinked copolymer particles which swell remarkably in water and a method for producing the same.
The present invention also relates to a hydrophilic separating agent derived therefrom, an ion exchange resin, a method for producing the same, and a method for separating a protein using the ion exchange resin.

[0002]

2. Description of the Related Art It is often practiced to recover a specific polymer substance from an aqueous solution containing various polymer substances. One example is the recovery of proteins from aqueous solutions. For example, to recover enzymes and proteins having biological activity from a culture solution of microorganisms and cells. In addition, a specific protein such as albumin is also collected from blood. Further, the recovery of protein from whey is one example.

[0003] Several methods have been used for the recovery of high molecular substances, such as proteins, from aqueous solutions. Among them, there are molecular sieves and adsorbents (hereinafter, collectively referred to as "separating agents"). ). For example, a column is filled with gel-like particles produced by reacting a natural polysaccharide such as cellulose or dextran with a crosslinking agent and swelling without dissolving in water, and flowing an aqueous solution containing a plurality of types of proteins into the column. Separation and recovery of specific proteins have been performed. This method is called gel filtration chromatography. This utilizes the phenomenon that the time required for the solute molecules to diffuse through the gel-like particles varies depending on the size of the solute molecules in the aqueous solution, that is, the molecular sieve effect. In gel filtration chromatography, a desired protein can be separated and recovered from an aqueous solution containing a plurality of types of proteins by adjusting the size of the mesh of the gel-like particles.

In the case of separating and recovering an ionic polymer substance, gel particles having an ion exchange group therein are also used as a separating agent. For example, 3 inside
When an alkaline aqueous solution containing a polymer substance having a carboxyl group and a nonionic polymer substance flows through a column filled with gel particles having a secondary amino group, the polymer substance having a carboxyl group is contained in the gel particles. And is trapped by the amino group present there. On the other hand, even if the nonionic polymer substance diffuses into the gel-like particles, it escapes from the particles without being trapped therein. When the eluent is allowed to flow through the column after the nonionic polymer substance has flowed out of the column, the polymer substance having a carboxyl group trapped in the gel particles flows out and is collected. In order to separate such an ionic polymer, gel-like particles obtained by reacting the above-mentioned natural polysaccharide with a crosslinking agent and ion-exchange groups such as amino groups and sulfonic acid groups are introduced into the gel particles. Used as

However, such gel-like particles made of natural products generally have low physical strength. In addition, there is a defect that it is easily decomposed by an acid or an alkali and is easily decomposed by a microorganism. Instead of gel-like particles made of natural products, particles made of hydrophilic synthetic polymers have been proposed. For example, Japanese Patent Publication No. 58-58026 discloses that a mixture of glycidyl methacrylate and ethylene glycol methacrylate is subjected to suspension polymerization in an aqueous medium to obtain porous crosslinked copolymer particles having glycidyl groups, and then the glycidyl groups are hydrolyzed. What was decomposed into hydrophilic porous cross-linked copolymer particles having pendant glycerol groups as a separating agent,
It is disclosed to perform chromatography. This method is called gel permeation chromatography.
Because, unlike the above-mentioned separating agent made of a natural polymer, the porous cross-linked copolymer particles have a myriad of pores inside, and the solute molecules are not contained in the network of particles but in this network. This is because they diffuse into the pores. Therefore, research on a porous separating agent has generally been conducted in a direction in which the volume of pores contributing to diffusion of solute molecules is increased as much as possible. JP-A-53-90991 discloses that a porous cross-linked copolymer particle having a glycidyl group is reacted with dimethylamine or the like to obtain a porous separating agent having an amino group.

The major difference between these porous separating agents made of synthetic polymers and the above-mentioned separating agents made of natural products is that the former hardly swells or hardly swells in water, while the latter hardly swells in water. Swelling remarkably. In the dry state, the former particles are composed of a dense and rigid skeleton portion made of a crosslinked copolymer and a space portion or pore surrounded by the skeleton portion, and have a remarkably large specific volume (ml / g).
have. When this is put into water, the water penetrates into the space inside the particles. However, a rigid backbone prevents swelling of the particles.

On the other hand, the latter hydrophilic separating agent made of a natural product has neither pores nor a firm skeleton in a dry state. When this is put in water, the particles swell because the water penetrates into the network in the particles and expands the network.
In general, the lower the crosslink density, the better the swelling.

[0008]

SUMMARY OF THE INVENTION The present invention provides a separating agent composed of a synthetic polymer which swells remarkably in water, that is, similar to the above-mentioned separating agent based on natural products. . One of the objects of the present invention is to provide a separating agent which has pendant glycerol groups and swells remarkably in water and is therefore suitable for gel filtration chromatography. Another object of the present invention is to have a sulfonic acid group or an amino group together with a pendant glycerol group,
An object of the present invention is to provide a separating agent which swells remarkably in water and is therefore suitable for adsorptive separation of ionic polymer compounds.

It is still another object of the present invention to provide a method for separating a high molecular compound such as a protein by chromatography using these separating agents. Still another object of the present invention is to provide a method for adsorbing and separating a protein from an aqueous solution using the above-mentioned separating agent having a sulfonic acid group or an amino group. Another object of the present invention is to provide a method for producing these separating agents. Still another object of the present invention is to provide a crosslinked copolymer having a glycidyl group suitable for producing these separating agents and a method for producing the same.

[0010]

The separating agent according to the present invention comprises:
Crosslinked polymer particles having pendant glycerol groups. This may further have either an amino group or a sulfonic acid group. It has a relatively small specific volume of 2.0-3.0 ml / g in the dry state. However, in water, it has the property of absorbing water and swelling to twice or more, usually three times or more the volume of when dried.

In order to produce the separating agent of the present invention, first, glycidyl methacrylate is copolymerized with dimethacrylate as a crosslinking agent represented by the general formula (1) to obtain a crosslinked copolymer having a glycidyl group. _{CH 2 = C (CH 3)} -COO- (R-O) n -OC-
C (CH ₃ ) = CH ₂ (1) (wherein, R represents an alkylene group having 2 to 4 carbon atoms such as ethylene, n-propylene, i-propylene, and n represents 1 to 4)
Preferred as the crosslinking agent are dimethacrylates of ethylene glycol or ethylene glycol oligomers represented by the formula (2).

CH ₂ ＣC (CH ₃ ) —COO— (CH ₂ —CH ₂ —
O) _n -OC-C (CH ₃ ) = CH ₂ (2) A separating agent derived from a copolymer of glycidyl methacrylate and a crosslinking agent represented by the formula (2) generally has a swelling property in water. Since it is abundant and does not cause a specific interaction with the protein, it is suitable as a protein separating agent.

A particularly preferred crosslinking agent is diethylene glycol dimethacrylate. Because it has a reactivity close to that of glycidyl methacrylate, only one monomer reacts preferentially during copolymerization, and the composition of the copolymer formed at the beginning and end of the reaction is reduced. The unwanted phenomenon of change can be avoided.

The proportion of the crosslinking agent in the monomer mixture subjected to the copolymerization reaction is 3 to 15% by mol% of vinyl groups, but is generally 2.5 to 10 by weight%. When the ratio of the cross-linking agent increases, a porous cross-linked copolymer is easily generated. Accordingly, both the swelling property of the resulting crosslinked copolymer in 1,4-dioxane and the swelling property of a separating agent derived from the crosslinked copolymer in water tend to decrease.
Conversely, as the proportion of crosslinker in the monomer mixture decreases, the swellability of the product generally increases, but the physical strength of the finally obtained separating agent decreases. A preferred ratio of the crosslinking agent in the monomer mixture is 3 to 10% by weight, particularly 4 to 9% by weight.
%.

The copolymerization reaction can be carried out by various known methods. Usually, a method of suspension polymerization in an aqueous medium in which a dispersion stabilizer and inorganic salts are present is employed according to a conventional method. Gelatin, sodium polyacrylate, carboxymethyl cellulose, polyvinyl alcohol and the like are used as the dispersion stabilizer. As the inorganic salts, sodium chloride, calcium chloride, sodium sulfate and the like are used. Having these inorganic salts dissolved in the aqueous medium helps to prevent the monomer from dissolving in the aqueous medium.

The suspension polymerization includes glycidyl methacrylate,
An organic phase comprising a crosslinking agent, a polymerization initiator and an organic solvent which dissolves these components but does not dissolve in an aqueous medium is suspended in the above-mentioned aqueous medium and reacted at 50 to 90 ° C. for 6 to 20 hours. It is. Of course, longer reactions can be carried out at lower temperatures if desired. Examples of the polymerization initiator include organic peroxides such as benzoyl peroxide and t-butyl hydroperoxide, and organic compounds such as azobisisobutyronitrile and 2,2'-azobis (2,4-dimethylvaleronitrile). Conventional ones such as azobis compounds can be used. The polymerization initiator is generally used in an amount of 0.01 to 5% by weight based on the monomer mixture.

As the organic solvent, toluene, cyclohexanol, 1-octanol, dibutyl ether, n-butyl acetate, isooctane and the like are used. The amount of the organic solvent used is generally 0.5 to 1.5 times the volume ratio of the monomer mixture at room temperature. When the monomer mixture is dissolved in an organic solvent and subjected to suspension polymerization, spherical crosslinked copolymer particles can be easily produced. When the amount ratio of the organic solvent to the monomer mixture increases, the specific volume of the cross-linked copolymer generally produced in a dry state increases. This tendency is particularly remarkable in the case of a so-called poor solvent for a homopolymer of glycidyl methacrylate such as isooctane or 1-octanol. When such a poor solvent is used, the resulting crosslinked copolymer tends to be porous. Therefore, when the ratio of the cross-linking agent in the monomer mixture is large, it is preferable to use toluene, cyclohexanol, or the like instead of these poor solvents to avoid making the resulting cross-linked copolymer porous.

In the suspension polymerization, the ratio of the organic phase to the aqueous medium phase (polymerization ratio) is preferably from 1: 3 to 1:10. If the amount of the aqueous medium phase is too large, the amount of the monomer dissolved in the aqueous medium phase increases, and the yield of the crosslinked copolymer particles decreases. Conversely, if the amount of the aqueous medium phase is too small, the suspension state of the organic phase becomes unstable, and it becomes difficult to obtain spherical crosslinked copolymer particles. The properties of the resulting crosslinked copolymer particles having a glycidyl group are preferably in the following ranges in order to induce a separating agent having desired properties.

Specific volume in dry state 1.8-3.0 ml / g, especially 2.0-3. 0 ml / g Specific volume in water 1.8-3.0 ml / g, especially 2.0-3. 0 ml / g Specific volume in 1,4-dioxane 5.0-15.0 ml / g That is, the crosslinked copolymer particles having a glycidyl group are 1,1
Those which significantly swell in 4-dioxane are preferred.
The ratio of the specific volume in the dry state to the specific volume in 1,4-dioxane should be at least 2.5 or more.

In order to increase the specific volume in the dry state and in water, the ratio of the organic solvent to the monomer mixture may be increased in suspension polymerization, or a poor solvent may be used as the organic solvent. To increase the specific volume in dioxane,
What is necessary is just to make the ratio of the crosslinking agent in a monomer mixture small.
The particle size of the crosslinked copolymer having a glycidyl group is generally 5 μm.
m to 2000 μm, but is specifically determined according to the particle size of the separating agent to be finally obtained. For example, when trying to obtain a separating agent having a pendant glycerol group used for gel filtration chromatography, the particle size of the crosslinked copolymer having a glycidyl group is better because the separating agent having a smaller particle diameter has a higher separation efficiency. Generally 10 μm to 150 in dry state
μm, preferably 30 μm to 100 μm. Also,
When trying to obtain a separating agent having an amino group or a sulfonic acid group in addition to the pendant glycerol group, the particle size of the crosslinked copolymer having a glycidyl group is larger because the separating agent having a larger particle size is easier to handle. 70 μm to 300 μm in dry state
m is preferred.

When the glycidyl group is hydrolyzed by reacting the above-mentioned crosslinked copolymer having a glycidyl group with water, the separating agent having a pendant glycerol group according to the present invention is obtained. This hydrolysis is preferably carried out by swelling the crosslinked copolymer having a glycidyl group described above with a hydrophilic organic solvent, and adding water and an acid as a catalyst to the swollen crosslinked copolymer to cause a reaction. By doing so, a separating agent having a specific volume in a dry state of 2.0 to 3.0 ml / g and a specific volume of 6.0 to 13.0 ml / g in water is obtained, which swells significantly in water. On the other hand, the glycidyl group is hydrolyzed to a glycerol group even when an acid aqueous solution is added thereto and heated without swelling the above-mentioned crosslinked copolymer having a glycidyl group with an organic solvent. The used separating agents generally have poor swelling properties in water.

As an organic solvent for swelling the crosslinked copolymer having a glycidyl group, 1,4-dioxane is generally used, and in addition, tetrahydrofuran, N, N-
Dissolves in water such as dimethylformamide, dimethylsulfoxide, etc. at an arbitrary ratio, and reduces the volume of the crosslinked copolymer particles to 2
A hydrophilic organic solvent that can swell more than twice can be used. The crosslinked copolymer having a glycidyl group is placed in these organic solvents, and is heated at room temperature to a temperature of 1 to 5 under heating.
When held for a long time, the volume often swells more than twice. Usually, it is preferable to sufficiently swell until the volume does not increase. Next, in a state where the swollen crosslinked copolymer is suspended in a large amount of an organic solvent, a large excess of an aqueous acid solution is added to the glycidyl group, and the suspension is heated to 20 to 90 ° C. for 0.5 to 90 ° C.
After holding for 5 hours, the glycidyl groups are hydrolyzed and converted to glycerol groups. The complete hydrolysis of the glycidyl group can be confirmed by the absence of absorption near 900 cm ^-1 corresponding to the epoxy ring absorption in the infrared absorption spectrum of the hydrolyzed particles.

The hydrolysis is preferably carried out under mild conditions so that the ester bond of the crosslinked copolymer is not broken as much as possible. The presence of a carboxyl group generated by cleavage of an ester bond in the resulting separating agent has an unfavorable effect on the separation of a polymer compound having an ionic functional group such as a protein. The amount of carboxyl groups in the separating agent having pendant glycerol groups formed by hydrolysis is 0.1 meq / m when swollen in water.
1 or less, particularly preferably 0.05 meq / ml or less.

The water-swellable polymer particles having pendant glycerol groups thus obtained can be used as such as a separating agent for gel filtration chromatography. In this case, the size of the particles when swollen in water is generally 20 to 300 μm, preferably 50 to 150 μm. The separating agent according to the present invention may have a tertiary amino group or a sulfonic acid group in addition to the pendant glycerol group. Such a water-swellable separating agent having an amino group or a sulfonic acid group is particularly effective for separating and recovering a high molecular compound having an amino group or a carboxyl group such as certain proteins. These separating agents having an amino group or a sulfonic acid group also have the property of swelling remarkably in water, that is, a specific volume in a dry state of 2.0 to 3.0 ml / g and a specific volume in water of 6.0 to 13.0 ml. / G 2.

The ion exchange capacity of these separating agents in the state of swelling in water is usually 0.05 to 0.5 meq / ml, preferably 0.4 meq / ml or less per 1 ml of the resin. In general, proteins have extremely large amino group equivalents and carboxyl group equivalents (molecular weight per mole of amino group or carboxyl group). Therefore, unlike capturing a low molecular weight ionic compound, when capturing a protein, the separating agent may have a small ion exchange capacity per unit volume. Rather, a large ion exchange capacity is disadvantageous in that a large amount of eluent must be used to elute the adsorbed protein.

The separation and separation of proteins by ion exchange or ion exchange chromatography using the separating agent according to the present invention.
When recovery is performed, the preferred ion exchange capacity of the separating agent is 0,1 swelling in water per ml of resin.
1 to 0.3 meq. When the ion exchange action of the separating agent is used, the particle size of the separating agent is preferably relatively large. Therefore, the separating agent of the present invention having an amino group or a sulfonic acid group usually has a capacity of 1 when swollen in water.
It is preferably in the range of 00 to 500 μm.

To obtain a separating agent having a pendant glycerol group and an amino group and remarkably swelling in water,
An amino group may be introduced into the water-swellable polymer particles having a pendant glycerol group. The introduction of the amino group is performed by introducing the above-mentioned water-swellable polymer particles having a pendant glycerol group into a mixed solution of an aqueous alkali hydroxide solution and a hydrophilic organic solvent, and allowing the mixed solution to permeate the polymer particles. The mixture is swollen with the mixed solution, and the hydroxyl group of the glycerol group is activated with alkali hydroxide. Then, an amine having a haloalkyl group or a salt thereof is added thereto to react the haloalkyl group with the hydroxyl group while maintaining the swollen state. It is preferred to carry out in two stages. Thus, a basic anion exchange resin having a specific volume in a dry state substantially equal to that of the polymer particles having pendant glycerol groups of the raw material and swelling more than the raw material in water can be obtained.

On the contrary, if the polymer particles having a pendant glycerol group are put into an aqueous alkali hydroxide solution and swollen with water, and then reacted with an amine having a haloalkyl group or a salt thereof, the reaction proceeds during the reaction. When the particles shrink, the basic anion exchange resin having a high swelling property in water cannot be obtained. As the hydrophilic organic solvent, usually, 1
A monovalent or polyhydric alcohol is used. In particular, carbon number 2
Alcohol No. 4 is preferred because of good compatibility with the aqueous alkali hydroxide solution. Of these, alcohols having a secondary hydroxyl group, particularly isopropanol, are preferable because they are considered to be effective for activating the hydroxyl group of the glycerol group. The hydrophilic organic solvent is preferably used in an amount of 0.3 to 3 (by volume) times the aqueous alkali hydroxide solution. It is considered that the hydrophilic organic solvent maintains the swelling state of the polymer particles and promotes uniform diffusion of the haloalkylamine into the polymer particles during the reaction between the polymer particles and the haloalkylamine. Sodium hydroxide is usually used as the alkali hydroxide, but potassium hydroxide, lithium hydroxide and the like are also used. The concentration of the aqueous alkali hydroxide solution is 1 to 1
0 is appropriate. When the concentration of the aqueous solution is low, the effect of activating the hydroxyl group of the glycerol group is weak. Conversely, if the concentration of the aqueous solution is too high, the ester bond of the polymer may be cleaved.

The mixed solution of the aqueous alkali hydroxide solution and the hydrophilic organic solvent is preferably used in a large excess with respect to the polymer particles so that the polymer particles can exist in a slurry state even after swelling. A tertiary amine represented by the formula (3) is used as the amine having a haloalkyl group.

[0030]

Embedded image

Wherein X represents chlorine, bromine or iodine, R ₁ represents an alkylene group, and R ₂ and R ₃ represent an alkyl group or a hydroxyalkyl group. In the formula (3), a haloalkyl group (X Preferred as -R _1- ) are lower haloalkyl groups having 2 to 4 carbon atoms, such as a 2-chloroethyl group, a 2-bromoethyl group, a 3-chloropropyl group and a 4-chlorobutyl group. R ₂ and R ₃ are preferably a lower alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a 2-hydroxyethyl group, an n-propyl group, an n-butyl group, an i-butyl group or a carbon atom. Number 2-4
Is a hydroxy lower alkyl group. Particularly preferred are 1-halo-2 (N, N-diethylamino) ethane and 1-halo-2 (N, N-diethylamino) ethane.
Halo-2- (N, N-dimethylamino) ethane and the like. The amine is usually used in an amount of 0.1 to 3 mol times based on the glycerol group of the polymer particles. The amine may be used in a free form or in a salt form such as hydrochloride, carbonate, bicarbonate and the like.

The glycerol-containing polymer particles are charged into the above-mentioned mixed solution of the alkali hydroxide aqueous solution and the hydrophilic organic solvent, and the slurry is maintained at 20 to 90 ° C. for 1 to 5 hours to reduce the hydroxyl groups of the glycerol group. After activating and swelling to twice or more the volume of the dried one, the above-mentioned tertiary amine is added thereto to further add 20 to 90 times.
When the temperature is kept at 1 ° C. for 1 to 10 hours, the haloalkylamine reacts with the glycerol group to produce the basic anion exchange resin according to the present invention.

The thus obtained separating agent having an anion exchange capacity has a specific volume in a dry state substantially equal to that of the polymer particles having pendant glycerol groups used as the raw material and is equal to or better than the raw material. It has a specific volume in water. In order to obtain a separating agent having a pendant glycerol group and a sulfonic acid group and remarkably swelling in water, a sulfite or a bisulfite is reacted with the swellable crosslinked copolymer having the glycidyl group described above. It can be obtained by swelling the polymer particles with a hydrophilic solvent, and then adding an aqueous acid solution to hydrolyze the glycidyl group.

As the sulfite or bisulfite, sodium sulfite or sodium bisulfite is usually used. The polymer is put into a 0.1 mol / liter to saturated aqueous solution of these salts and reacted at 30 to 90 ° C. for 1 to 10 hours. Thereby, the swellable polymer having a glycidyl group becomes an acidic cation exchange resin having a glycidyl group and a sulfonic acid group. Next, the unreacted glycidyl group is hydrolyzed to be converted to a pendant glycerol group. The hydrolysis of the glycidyl group of the sulfonated polymer needs to be performed in a state where the polymer is swollen with a hydrophilic organic solvent, similarly to the hydrolysis of the glycidyl group of the crosslinked copolymer. Simply placing the polymer in an aqueous mineral acid solution to hydrolyze the glycidyl groups does not provide a product that is highly swellable in water.

The hydrolysis of the glycidyl group of the sulfonated polymer is carried out by 1,4-dioxane, tetrahydrofuran,
A polymer is placed in a hydrophilic organic solvent, such as N, N-dimethylformamide or dimethylsulfoxide, which dissolves in water at an arbitrary ratio and kept at room temperature for about 1 to 5 hours to double the volume of the polymer particles when dried. Swell above. Then, an acid aqueous solution is added thereto, and the mixture is kept at 20 to 90 ° C. for 0.5 to 5 hours. The glycidyl group is hydrolyzed to form a water-swellable acidic cation exchange resin having pendant glycerol groups and sulfonic acid groups. .

The separating agent according to the present invention is useful as a packing material for a chromatography column. For example, to a column filled with water and filled with the separating agent according to the present invention, a sample aqueous solution containing various proteins having different molecular weights is supplied from the upper part thereof to flow down the column, and then water is supplied from the upper part of the column. When the aqueous sample solution is extruded, the various proteins in the aqueous sample solution are separated from each other and flow out from the bottom of the column. Since the separating agent according to the present invention has higher mechanical strength than that using a natural product as a raw material, the packing height in a column can be increased. For example, the separating agent according to the present invention can be packed in a column at a height of 50 cm or more and subjected to chromatography.

Further, among the separating agents according to the present invention, those having an amino group or a sulfonic acid group are useful as ion exchange resins. For example, a sample aqueous solution containing various proteins is supplied to an upper portion of a column filled with the separating agent having an amino group according to the present invention, and is caused to flow down the column. At this time, the pH of the sample aqueous solution is adjusted to be higher than the isoelectric point of the protein to be captured. The effluent from the bottom of the column is monitored, and if the protein to be trapped therein leaks beyond an allowable amount (leak), the supply of the aqueous sample solution to the column is stopped, and water is supplied instead. To extrude the sample solution remaining in the column. Next, when a low-concentration aqueous acid solution, for example, a 0.1 mol / liter aqueous hydrochloric acid solution is supplied to the column, the protein adsorbed on the separating agent elutes. Instead of the acid aqueous solution, a sodium chloride aqueous solution of about 0.5 to 1 mol / liter can be used.

As another method, the separating agent having ion exchange ability according to the present invention is introduced into an aqueous sample solution to adsorb the target component. Then, the separating agent is recovered by filtration or centrifugation, and the collected agent is introduced into an aqueous acid solution or aqueous sodium chloride solution to elute the adsorbed components. For example, the separating agent having a sulfonic acid group according to the present invention is introduced into whey whose pH has been adjusted to about 3 to adsorb lactalbumin and lactoglobulin in the whey. Next, the separating agent is added to an alkaline aqueous solution of about pH 9 or 0.5 to
When the solution is introduced into a sodium chloride aqueous solution of about 1 mol / liter, the adsorbed protein elutes. The separating agent according to the present invention swells remarkably in water and allows the protein to easily diffuse into the separating agent, so that the adsorption capacity for the protein is remarkably large. For example, according to the present invention, 30 g per liter of the separating agent packed in the column is used.
Above, preferably more than 50 g of protein can be adsorbed.

[0039]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. <Method of measuring specific volume of water-swellable polymer particles having glycerol group> About 10 ml of polymer particles swelling in water are put into a 10 ml measuring cylinder together with water,
The volume (V ₁ ml) is measured. After the polymer particles are transferred to a beaker and tilted to remove water, 100 ml of acetone is added and held for 10 minutes, and the operation of tilting and removing acetone is repeated twice. Next, 100 ml of toluene was added, and the mixture was held for 10 minutes. The operation of removing the toluene by tilting was repeated twice, and the same operation was repeated twice, except that toluene was replaced with n-heptane. The polymer particles are recovered by filtration and dried under reduced pressure to evaporate n-heptane. The weight (Wg) of the dried resin is measured. 10 m of dried resin
Put in a 1-measuring cylinder and measure the volume (V ₂ ml).

Specific volume of polymer particles in dry state: V ₂ / W (ml / g) Specific volume of polymer particles in swelling state in water: V ₁ / W (ml / g) Amino group or sulfonic acid group In the case of polymer particles having the same, the above operation is performed after the amino group or the sulfonic acid group is released.

<Measurement Method of Ion Exchange Capacity of Polymer Particles Having Glycerol Group and Amino Group> The polymer particles are treated with a 0.1 N NaOH aqueous solution to make the amino group free, and then washed well with water. About 5 ml of these polymer particles
Was collected in a 10 ml measuring cylinder, and its volume (Vm
l) is measured. After removing water by centrifugation, the mixture is transferred to a flask, and 100 ml of 0.1N HCl is added thereto, and the mixture is kept for 12 hours. Take 10 ml of the supernatant and add 0.1N-NaO
Titrate with H aqueous solution. Assuming that the consumption of the 0.1N-NaOH aqueous solution is Mml, the ion exchange capacity of the polymer particles is given by (10-M) / Vmeq / ml.

<Measurement Method of Ion Exchange Capacity of Polymer Particles Having Glycerol Group or Glycerol Group and Sulfonate Group> The polymer particles are treated with an aqueous 0.1N HCl solution to release carboxyl groups or carboxyl groups and sulfonic acid groups. After shaping, wash with water. About 5m of these polymer particles
l was collected in a 10 ml measuring cylinder, and its volume (V
ml). After water was removed by centrifugation, the mixture was transferred to a flask, and 100 ml of 0.1 N NaOH was added to the mixture.
Hold for 2 hours. Take 10 ml of the supernatant and add 0.1N-H
Titrate with Cl. The consumption of 0.1N-HCl aqueous solution
ml, the ion exchange capacity of the polymer particles is (10
-M) / Vmeq / ml.

<Measurement Method of Sulfonic Acid Group of Polymer Particle Having Glycerol Group and Sulfonic Acid Group> The polymer particles are treated with a 0.1N HCl aqueous solution to form a free sulfonic acid group, and then washed well with water. About 5m of these polymer particles
1 to a 10 ml measuring cylinder, and the volume (Vm
After measuring l), the sample is dried in a vacuum dryer at 100 ° C. for 24 hours. The dried polymer particles are subjected to elemental analysis by a conventional method to measure the sulfur content (Wmg). The amount of sulfonic acid groups in the polymer particles is given by (W / 32) / Vmeq / ml.

<Measurement method of exclusion limit molecular weight> Inner diameter 8.2 m
m is filled with polymer particles having pendant glycerol groups (filling height 47.5 cm). Dextran is injected into the column while continuously flowing demineralized water at 0.5 ml / min, and the time until dextran appears in the effluent from the column is measured. The above-mentioned measurement is performed for dextran having various molecular weights, and the exclusion limit molecular weight of dextran is determined from the measurement results by a conventional method.

<Measurement Method of Albumin Adsorption Capacity> The polymer particles were treated with a buffer solution (tris-HCl buffer (pH 7.5) for polymer particles having an amino group, acetate buffer (pH 7.5) for polymer particles having a sulfonic acid group). pH 4.5)]
And pH with sodium hydroxide or sodium acetate
Adjust Collect 5 ml of particles with polymer and centrifuge to remove buffer. This is put into 200 ml of a 4 g / liter bovine serum albumin solution at 10 ° C., and kept at 10 ° C. for 6 hours with gentle stirring. 280 of supernatant
absorbance in nm and 280 nm of the original albumin solution
The amount of albumin adsorbed on the polymer particles is calculated from the difference in the absorbance at the step (1).

Example 1 Glycidyl methacrylate 9
5.7 g, diethylene glycol dimethacrylate
3 g and 1.0 g of 2,2-azobis (2,4-dimethylvaleronitrile) were dissolved in 87 g of toluene. 120 g of calcium chloride, 6 g of polyvinyl alcohol and 0.001 g of sodium nitrite were added to the demineralized water.
6 g of the monomer solution was added to 600 g of an aqueous solution, and the mixture was stirred to disperse the monomer solution in the aqueous solution. In this state, suspension polymerization was carried out at 70 ° C. for 6 hours.
Crosslinked copolymer particles of 0 to 300 μm were obtained (yield 9).
1%). The specific volume of the dried product is 2.7 ml /
g, and the specific volume when swollen in 1,4-dioxane was 10.8 ml / g. Further, even if the crosslinked copolymer particles were dried and then added to water, the volume thereof hardly changed.

300 g of 1,4-dioxane was added to 30 g of the crosslinked copolymer particles having a glycidyl group, and the mixture was kept at room temperature for 3 hours and swollen with dioxane. Then, 150 ml of 1,4-dioxane and 50 g / l of 50 g / l were added. 150 ml of an aqueous sulfuric acid solution was added, and the mixture was heated to 50 ° C. and maintained for 3 hours to hydrolyze glycidyl groups into glycerol groups.
The polymer particles having pendant glycerol groups thus obtained have a specific volume of 2.6 ml / g in a dry state and a specific volume of 8.5 m in a state of being swollen in water.
1 / g. Cation exchange capacity estimated at about 0.05
meq / liter. Exclusion limit molecular weight is about 100
It was ten thousand.

Example 2 A copolymerization reaction and a hydrolysis reaction were performed in the same manner as in Example 1 except that the amount of glycidyl methacrylate was changed to 91.3 g and the amount of diethylene glycol dimethacrylate was changed to 8.7 g. Was performed. The specific volume of the crosslinked copolymer having an epoxy group is
1.9 ml / g in a dry state and 7.1 ml / g in a state swelled in 1,4-dioxane. The specific volume of the polymer particles having pendant glycerol groups was 7.5 ml / g when swollen in water. The exclusion limit molecular weight was about 500,000.

Example 3 Swollen polymer particles 10 having pendant glycerol groups obtained in Example 1
0 ml was dehydrated by centrifugation, and then 5N-N
The mixture was placed in a mixed solution of 24 ml of an aOH aqueous solution and 40 ml of isopropanol, and kept at 50 ° C. for 1 hour with gentle stirring. The slurry containing the swollen polymer particles is added to 2-chloroethyldiethylamine hydrochloride (Cl-C
_{H 2 -CH 2 -N- (CH 2} CH 3) 2 -HCl) 31
g was dissolved in 10 g of water, and the mixture was kept at 50 ° C. for 5 hours. The specific volume of the white spherical anion exchange resin thus obtained was about 2.0 ml / g in a dry state and about 8.5 ml / g in a state of being swollen in water. The ion exchange capacity was 0.3 meq / ml. The albumin adsorption amount was 58 g / liter.

Example 4 About 10 g of the glycidyl group-containing crosslinked copolymer obtained in Example 1 was placed in 100 ml of a 1 mol / l aqueous solution of sodium sulfite, and kept at 50 ° C. for 3 hours with stirring. The polymer particles were collected by filtration, and then the polymer particles were added to 100 ml of tetrahydrofuran.
And kept at 25 ° C. for 3 hours to swell the polymer particles. Further add 50 ml of tetrahydrofuran and 50 ml
g / L aqueous solution of sulfuric acid (150 ml) was added, and the mixture was kept at 50 ° C. for 3 hours with stirring to hydrolyze glycidyl groups into glycerol groups. The obtained white spherical particles had a specific volume of 2.5 ml / g in a dry state and a specific volume of about 10 ml / g in a state of swelling in water. The ion exchange capacity was 0.15 meq / ml. The albumin adsorption amount was 85 g / liter.

Example 5 A suspension polymerization was carried out in the same manner as in Example 1 except that the amount of glycidyl methacrylate used was 93.1 g and the amount of diethylene glycol dimethacrylate used was 6.9 g. Done.
Dry state of the formed white spherical crosslinked copolymer and 1,4
-The specific volumes in the state swollen with dioxane were 2.1 ml / g and 8.3 ml / g, respectively. The particle size of the dried polymer was about 30 to 250 μm. 300 g of tetrahydrofuran is added to 30 g of the crosslinked copolymer particles.
Then, the mixture was kept at room temperature for 3 hours to swell the particles. Thereafter, 150 ml of tetrahydrofuran and 150 ml of a 50 g / l sulfuric acid aqueous solution were added, and the mixture was kept at 50 ° C. for 3 hours. The resulting polymer particles having pendant glycerol groups had a specific volume in a dry state and a specific volume in a state swelled with water of 2.4 ml / g and 7.7 ml / g, respectively. The exclusion limit molecular weight was about 1,000,000.

Example 6 About 1 swelled polymer particle having a pendant glycerol group obtained in Example 1
After dehydration of 00 ml by centrifugation,
24 ml of N-NaOH aqueous solution and 40 m of isopropanol
1 liter of the mixed aqueous solution at 50 ° C. with gentle stirring.
Hold for hours. Next, 62 g of a 50% aqueous solution of 2-chloroethyl-diethylamine hydrochloride was added to the slurry, and the mixture was kept at 50 ° C. for 5 hours. The specific volume of the white spherical anion exchange resin thus obtained in a state of being swollen in water is 11 ml / g, and the anion exchange capacity is 0.24 meq / g.
ml.

Example 7 100 ml of a swollen polymer particle having a pendant glycerol group obtained in exactly the same manner as in Example 1 was added to 24 ml of a 5N NaOH aqueous solution.
And kept at 50 ° C. for 1 hour with gentle stirring. A solution prepared by dissolving 31 g of 2-chloroethyl-diethylamine hydrochloride in 10 g of water was added to the slurry, and the solution was treated with 50 g
C. for 5 hours. The specific volume of the thus obtained colored and spherical anion exchange resin is about 2 ml / g in a dry state and about 5 ml / g in a state of being swollen in water, which is inferior in swellability to that of Example 3. . Further, the ion exchange capacity was 0.3 meq / ml. The albumin adsorption amount was 5 g / liter.

Example 8 100 g / liter% was added to 30 g of the crosslinked copolymer particles obtained in exactly the same manner as in Example 1.
150 ml of a sulfuric acid aqueous solution was added, and the mixture was heated to 50 ° C. and maintained for 5 hours to hydrolyze glycidyl groups. The white polymer particles having pendant glycerol groups thus obtained have a specific volume of 3.2 ml when swollen in water.
/ G, which is remarkably lower than that of Example 1 and inferior in swellability.

[0055]

According to the present invention, it is possible to provide a hydrophilic synthetic separating agent which can swell remarkably in water, so that the advantages of both natural and synthetic separating agents can be obtained. It is very useful because it can selectively separate high molecular substances such as proteins.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ Identification code FI G01N 30/48 G01N 30/48 P (C08F 220/32 220: 28) (56) References JP-A-53-1087 (JP, A) JP-A-58-24354 (JP, A) JP-A-59-232104 (JP, A) JP-A-3-255111 (JP, A) JP-A-53-90991 (JP, A) JP-A Sho 61 -272654 (JP, A) JP-A-62-74904 (JP, A) JP-A-62-153306 (JP, A) Angewante Macromol Chemie, 96 (1981), p69-84 Angewante Macromol Chemie, 63 (1977) ), P23-36 Angewante Macromol Chemie, 117 (1983), p117-129 (58) Fields investigated (Int. Cl. ⁷ , DB name) C08F 220/00-220/70

Claims (9)

(57) [Claims] 1. Glycidyl methacrylate and a crosslinking agent represented by the following general formula (1): CH ₂ ＣC (CH ₃ ) -COO- (RO) n -OC-C
_{(CH 3) = CH 2 (} 1) ( wherein, R represents an alkylene group having 2 to 4 carbon atoms, n is an integer of 1 to 4) a crosslinked copolymer consisting a dry state And the specific volume in water is 1.8-3.
Crosslinked copolymer particles having a specific volume of 5.0 to 15.0 ml / g when swelled with 1,4-dioxane at 0 ml / g . 2. Glycidyl methacrylate and a crosslinking agent represented by the following general formula (1): CH ₂ ＣC (CH ₃ ) —COO— (RO) _n —OC—
C (CH ₃ ) = CH ₂ (1) (wherein R represents an alkylene group having 2 to 4 carbon atoms and n represents an integer of 1 to 4), and is compatible with the monomer mixture. The suspension polymerization of a polymerizable mixture comprising an organic solvent having a viscosity and a polymerization initiator in an aqueous medium, wherein the weight ratio of glycidyl methacrylate to the crosslinking agent in the monomer mixture is 97.5: 2.5-9.
By selecting the volume ratio between the monomer mixture and the organic solvent from the range of 0:10, respectively, from the range of 1: 0.5 to 1: 1.5, the specific volume in the dry state and in water is 1.8 to 3 2.0 ml / g, and the specific volume in a state of being swollen with 1,4-dioxane is 5.0 to 15.0 ml.
/ G, producing a cross-linked copolymer 3. Glycidyl methacrylate and the following (1)
Crosslinking agent represented by the formula CH ₂ ＣC (CH ₃ ) —COO— (RO) _n —OC—
C (CH ₃ ) = CH ₂ (1) (wherein, R represents an alkylene group having 2 to 4 carbon atoms, and n represents an integer of 1 to 4). And the specific volume in a dry state is 2.0 to
A hydrophilic separating agent having a specific volume of 6.0 to 13.0 ml / g when swollen with water; 4. The addition of the crosslinked copolymer particles according to claim 1.
A hydrophilic crosslinked copolymer particles having pendant glycerol groups with water decomposition products, specific volume in a state in which the specific volume in the dry state was swollen 2.0~3.0ml / g, water 6.0 parents ~13.0ml / g, the cation exchange capacity is equal to or less than 0.05 meq / ml
Aqueous separating agent. 5. The method according to claim 1, wherein the crosslinked copolymer according to claim 1 is swollen with a hydrophilic organic solvent, and then the swollen crosslinked copolymer is reacted with water to hydrolyze a glycidyl group. Method for producing hydrophilic cross-linked copolymer particles swelling in water to at least twice the volume when dried 6. Addition of the crosslinked copolymer particles according to claim 1.
Hydrophilic rack having pendant glycerol groups with water hydrolyzate
A hydrophilic ion exchange resin in which an amino group or a sulfonic acid group is introduced into a bridge copolymer particle , which has a specific volume of 2.0 to 3.0 ml / g in a dry state and is swollen with water. A hydrophilic ion exchange resin having a specific volume in a state of 6.0 to 13.0 ml / g. 7. Addition of the crosslinked copolymer particles according to claim 1
Having pendant glycerol groups with water decomposition products, hydrophilic specific volume in a state in which the specific volume in the dry state was swollen 2.0~3.0ml / g, water is 6.0~13.0ml / g The crosslinked copolymer particles are treated with a mixed solution comprising an alkali hydroxide, water and a hydrophilic organic solvent to form swollen particles by infiltrating the mixed solution into the interior thereof, and the haloalkyl particles represented by the following general formula (2) Amine (Wherein X represents a halogen atom, R ₁ represents an alkylene group, R ₂ and R ₃ represent an alkyl group or a hydroxyalkyl group) or a salt thereof, when dried in water. A method for producing a basic anion exchange resin which swells to twice or more the volume of the basic anion exchange resin. 8. The crosslinked copolymer particles according to claim 1,
Reacting an aqueous solution containing at least one selected from the group consisting of sulfites and bisulfites, followed by treatment with a hydrophilic organic solvent so that the organic solvent has penetrated into the interior and is twice the dry volume; Production of a water-swellable acidic cation exchange resin having both pendant glycerol groups and sulfonic acid groups, characterized in that the swelled to the above volume, and water is reacted with this to hydrolyze the glycidyl ring. Law 9. Addition of the crosslinked copolymer particles according to claim 1
Hydrophilic rack having pendant glycerol groups with water hydrolyzate
A hydrophilic ion exchange resin having an amino group or a sulfonic acid group introduced into a bridge copolymer particle, having a specific volume in a dry state of 2.0 to 3.0 ml / g and being swollen with water. An aqueous solution containing a protein is passed through a column packed with a column having a specific volume in the state of 6.0 to 13.0 ml / g and an ion exchange capacity of 0.4 meq / ml or less, to obtain 30 g or more per liter of resin. A method for recovering a protein, comprising adsorbing the protein, and then passing the eluent through to elute the adsorbed protein.