JPH02280833A - Compounding and separating agent and its preparation - Google Patents

Abstract

PURPOSE:To prepare a compounding and separating agent having high protein separation function by filling hydrophilic polymer-type separating agent having a specific swelling degree in water and a huge mesh structure in fine pores of an organic porous substrate having specific property values. CONSTITUTION:A solution containing uncrosslinked raw materials of a porous hydrophilic polymer for a hydrophilic polymer-type separating agent is filled in fine pores of an organic polymer substrate having swelling degree of 10ml/g- dry in water, crosslinked degree of >=4-100mol.%, and a porous structure. Then, a crosslinking agent is added and the raw materials of the hydrophilic polymer are crosslinked so that a hydrophilic polymer-type separating agent having swelling degree of 10-100ml/g-dry in water and a huge mesh structure is filled in the pores and a compounding and separating agent is thus prepared. A desirable porous polymer substrate to be used is one having pore volume of 0.5-3ml/g and pore diameter 200-100,000Angstrom .

Description

[Detailed Description of the Invention] (a) Object of the Invention (Field of Industrial Application) The present invention provides biopolymers such as proteins that have high mechanical strength and good liquid permeability when packed in a column. The present invention relates to a complex separation agent suitable for use in chromatographic separation and a method for producing the same.

(Prior art) Separation of biopolymers such as proteins differs from the separation of ordinary low-molecular-weight substances when the separation target is a biopolymer.
subject to various restrictions. That is, they are sensitive to heat, organic solvents, acids, alkalis, etc., and if they come into contact with organic solvents, acids, or alkalis, they will undergo deterioration or decomposition, so it is necessary to use mild conditions for separation.

For this reason, primary separation agents have conventionally been used in the separation of biopolymers such as proteins.

■ A separation agent that crosslinks polysaccharides such as dextran and agarose.

■ A separation agent made by crosslinking polyvinyl alcohol with epichlorohydrin.

■ A copolymer separating agent of an acrylic ester monomer and a crosslinking monomer, polyalkylene glycol I-reacrylic ester.

(2) A separating agent in which the surface of the porous separating agent (2) above is coated with a hydrophilic substance.

■ A separation agent filled with agarose glue in the pores of a porous diatomaceous (inorganic) carrier.

The above separation agents (1) and (2) are highly hydrophilic, prevent irreversible adsorption of proteins, and have a large network structure, making them excellent as 1% &l'le permeation chromatography separation agents. Has separation ability. However, these separating agents are soft and have poor mechanical strength, and in industrial-scale separations where columns are packed with a high bed height and separation is performed at high flow rates, one separating agent is compacted. The drawback is that fluid cannot flow through it.

The above separation agent (2) is hard and has excellent mechanical strength. However, since hydrophobic properties remain in the separation agent, irreversible adsorption of proteins tends to occur, and the separation agent has a porous structure.

Compared to the separation agent (2), the separation ability is inferior.

The above-mentioned separation agent ① is obtained by coating the separation agent 1 in Table I with a hydrophilic substance, which causes irreversible protein adsorption and is hard, so it has excellent mechanical strength. However, because it has a porous structure, it is inferior in resolution by 1 compared to the separating agents (1) and (2).

The above-mentioned separation agent (■) is a porous inorganic material.
It is filled with 7 galose, and its structure means that it is hard and has excellent mechanical strength. However, inorganic porous materials generally have the disadvantage that they are decomposed by acids or alkalis, and separation agents using them are subject to various restrictions in the washing process during separation, and there are problems with durability.

(Problems to be Solved by the Invention) The present invention has the advantage that, while maintaining the excellent separation ability for proteins and the like possessed by the hydrophilic Telimer separation agent having a large network structure, the present invention has the drawback that the same separation agent has poor mechanical strength. The present invention aims to provide a conventional composite separation agent that has been greatly improved and a method for producing the same.

佽) Structure of the invention (rounding means to solve the problems) The present inventors have conducted various studies to solve the above problems, and as a result 1. An organic polymer having a pore structure with excellent mechanical strength. By utilizing the pores of the substrate and filling the pores with a hydrophilic polymer separation agent that has poor mechanical strength but exhibits excellent performance in separating biopolymers such as proteins, this purpose can be achieved. was able to achieve this.

That is, 1. the composite separating agent of the present invention has a degree of swelling in water of 10-L' and a degree of cross-linking of 4 to 1 at 1 il-dry or less.
00 mol% and has a pore structure with a swelling degree of 10 ml/g in water.
This is a separating agent filled with a hydrophilic polymer-based separating agent that is ~100 d/9-dry and has a large network structure.

The swelling degree of the composite separating agent of the present invention in water is 10 ml.
/g~100+d/g-dry and has a giant network structure as a raw material hydrophilic polymer for a hydrophilic polymer separation agent.
Preferred are polysaccharide systems such as dextran and agarose, and hydrophilic synthetic polymer systems such as polyvinyl alcohol, which are highly hydrophilic and are unlikely to cause irreversible adsorption of biopolymers such as proteins.

These raw material hydrophilic polymers are subjected to a crosslinking reaction and used as a hydrophilic polymer separation agent having a large network structure.

The composite separation agent of the present invention has a swelling degree in water of 10 ml/9-rlry or less and a crosslinking degree of 4 to 1o.

K in the pores of an organic polymer base having a pore structure is impregnated with a hydrophilic 4 remer solution, a raw material before crosslinking for a hydrophilic polymer separation agent, and then a crosslinking agent is added to the pores. The raw material hydrophilic polymer is cross-linked in the pores, and the degree of swelling in water is 10 ml/g to 100 d.
/N-dry and can be easily produced by a method of producing a hydrophilic polymer-based separating agent having a large network structure.

The degree of swelling in water according to the present invention is 10 m! 7'9
-Dry or less, the degree of crosslinking is 4 to 100 mol %, and an organic polymer substrate having a pore structure (hereinafter sometimes simply referred to as a "porous polymer substrate") has a tendency to swell in water. It is necessary that the degree of crosslinking is 10/77-dry or less and the degree of crosslinking is 4 to 100 mol%. The reason is that when the degree of swelling exceeds 10 d/g-dry or the degree of crosslinking becomes less than 4 mol%, the mechanical strength decreases. It is from.

In addition to organic polymers, materials with pore structures include inorganic porous materials such as porous silicic acid.
Its use is also possible, but in general.

Inorganic porous materials have the disadvantage of being decomposed by acids or alkalis, and composite separating agents using them are not preferred because they are subject to various restrictions in the washing step during one separation.

The pores of the porous polymer substrate in the present invention are as follows.

It has a volume in which a sufficient amount of hydrophilic 1-limer separation agent can be formed and filled, and a substance to be separated from biopolymers such as proteins can diffuse into the pores. size (pore radius). but,
On the other hand, if the pore volume is too large or the pore radius is too large, the mechanical strength will decrease, so the preferable pore volume is 0.5 to 3 at/i, and the preferable pore radius is is 200 to 100,000X.

The organic polymer for producing the porous polymer substrate in the present invention includes a monounsaturated monomer having one vinyl group or 471-unit (nyl group) in the molecule, and a monounsaturated monomer having two vinyl groups or impropenyl groups in the molecule. A copolymer with the above-mentioned polyunsaturated monocapsule is preferred.

Examples of the monounsaturated monomer include aromatic monovinyl compounds such as styrene, ethylvinylbenzene, and p-methylstyrene; glycidyl (meth)acrylate (Note: This is a general term for glycidyl acrylate and glycidyl methacrylate. Hereinafter, the same shall apply.Esters having a polymerizable vinyl group or isopropenyl group and having a functional group such as allylglycidyl ether, 2-hydroxyethyl (meth)acrylate, polyethylene glycol (meth)acrylate, etc. Ether compounds (meth)acrylic acid and (meth)acrylic acid esters such as diacrylic acid, methacrylic acid, methyl acrylate, and methyl methacrylate; vinyl groups of carboxylic acids such as vinyl formate, vinyl acetate, allyl formate, allyl acetate, etc. Containing esters can be mentioned.

Examples of the polyunsaturated monomer include aromatic polyvinyl compounds such as divinylbenzene, trivinylbenzene, or substituted derivatives thereof; ethylene glycol diacrylate, ethylene glycol dimethacrylate,
Fully 7-(meth)acrylate compounds such as polyethylene glycol methacrylate; glycerin poly(meth)acrylate compounds such as glycerin triacrylate and glycerin dimethacrylate; and l-lyacrylic compounds having a heterocycle such as triallyl isocyanurate. It will be done.

The method for producing the porous/17-molecular substrate used in the present invention from such monounsaturated monomers and polyunsaturated monomers is known, and is described, for example, in JP-A-60-96605. Therefore, the porous polymer substrate of the present invention can be easily produced using such a known method. That is,
The above-mentioned monounsaturated monomer and the above-mentioned di- or more polyunsaturated monomer are subjected to suspension polymerization in water to which a lanocal polymerization initiator is added in the coexistence of a porosity-forming agent. If the softening agent is removed, the porous polymer substrate used in the present invention can be easily obtained.

Porous polymer substrates that satisfy the same conditions as those required for the porous polymer substrate of the present invention exist among those already commercially available as ion exchange resins, adsorbents, etc.; It is also possible to carry out using commercially available porous polymer substrates.

Next, K within the pores of such a porous polymer substrate.

Swelling degree in water is 10-100td/9-dry
Various methods are possible for producing the composite separating agent of the present invention by filling the hydrophilic polymer separating agent with a large network structure, but the simplest method is
As mentioned above, a preferred method is to introduce a raw hydrophilic polymer solution before crosslinking of a hydrophilic polymer separation agent into the pores of a porous polymer substrate that has the necessary conditions for the porous polymer substrate of the present invention. The material is impregnated, and then a crosslinking agent is added to cause the raw hydrophilic polymer to undergo a crosslinking reaction within the pores.

This is a method for producing a hydrophilic polymer-based separating agent that meets the conditions of the present invention.

As a hydrophilic polymer (i.e. raw material hydrophilic polymer) before crosslinking of a hydrophilic polymer separation agent.

Examples include polysaccharide-based materials such as dextran, agarose, and chitosan, and hydrophilic synthetic polymer-based materials such as /IJ vinyl alcohol and polyacrylamide.

Further, as a solvent for these starting hydrophilic polymers, any solvent can be used as long as it can dissolve the starting hydrophilic polymers, but water is usually the most common. The concentration of the raw hydrophilic polymer to be dissolved in the solvent is selected depending on the performance of the intended separating agent, etc.
It is usually in the range of 2 to 50% by weight, preferably 5 to 20% by weight. In general, a hydrophilic polymer separation agent with a high swelling degree is likely to be formed from a low concentration raw material/remer solution, and a hydrophilic polymer separation agent with a low swelling degree is easily formed from a high concentration raw material hydrophilic polymer solution. easy to form.

The solvent solution in which the raw material wood-loving polymer was dissolved is then
The pores of the porous polymer substrate are impregnated, and various methods can be used for the impregnation. - For boat fishing, the simplest impregnation method is to add a porous polymer substrate to the raw material hydrophilic polymer solution, impregnate it, and then remove excess solution by filtration or the like.

A crosslinking agent is then added to the raw hydrophilic polymer solution impregnated into the pores of the porous polymer substrate to cause a crosslinking reaction within the pores to form a hydrophilic polymer separation agent.

Examples of the crosslinking agent include compounds having two or more OH group-active functional groups, such as epihalohydrin such as epichlorohydrin, dialdehyde compounds such as glutaraldehyde, and diisocyanate compounds such as methylene diisocyanate. Furthermore, when a compound having more amino groups than chitosan is used as a raw material hydrophilic polymer, a cybaride such as 1,8-dichlorooctane can also be used as a crosslinking agent.

A crosslinking reaction using such a crosslinking agent is usually carried out by using a porous polymer substrate whose pores are impregnated with a raw material hydrophilic polymer solution.
This is carried out by adding a crosslinking agent to a system that is dispersed/suspended in a suitable medium. The amount of crosslinking agent added at this time is selected depending on the intended performance of the separating agent. Generally speaking, when the amount of the crosslinking agent added is increased, the degree of swelling of the resulting hydrophilic polymer separating agent decreases, and when the amount of the crosslinking agent added is decreased, the degree of swelling of the separating agent becomes large. If the amount of crosslinking agent added is too large, the properties of the raw material hydrophilic polymer will be impaired, so the amount added is usually adjusted to
The amount is within the range of 90, 1 to 2 mol per mol of the +7 mer structural unit.

If the crosslinking reaction between the crosslinking agent and the raw material hydrophilic polymer can be controlled by adding a catalyst, etc., the crosslinking reaction can be carried out using a porous material impregnated with the raw material hydrophilic 4 reamer solution mixed with the crosslinking agent in advance. Polymer base.

This can be carried out by adding the catalyst etc. to a system dispersed/suspended in a suitable medium. In addition, if the crosslinking reaction can be started by changing the crosslinking reaction conditions such as temperature, a porous polymer substrate impregnated with a solution containing the raw material polymer, crosslinking agent, catalyst, etc.
After dispersion/suspension in a suitable medium, the crosslinking reaction can be carried out by changing the crosslinking reaction conditions (for example, temperature) to conditions that allow the crosslinking reaction to proceed effectively.

The crosslinking reaction catalyst varies depending on the type of crosslinking agent.

For example, in the case of epichlorohydrin, an alkali such as sodium hydroxide or potassium hydroxide is effective, and in the case of a dialdehyde compound, a mineral acid such as hydrochloric acid or sulfuric acid is effective.

As a medium for dispersing and suspending a porous polymer substrate impregnated with a raw material hydrophilic polymer solution, any medium can be used.

When a hydrophilic porous polymer substrate such as a copolymer of (meth)acrylic acid or (meth)acrylic acid ester is used as the porous polymer substrate.

Specific examples of the dispersing and suspending medium include organic solvents such as toluene, dichlorobenzene, and nitromethane.

In addition to organic solvents, a raw material hydrophilic polymer solution for impregnation can also be used as a dispersing or suspending medium.

The crosslinking reaction is usually carried out at a temperature ranging from 5 to 90°C.

It will take 1 to 10 hours.

In such a crosslinking reaction, the degree of swelling in water of the hydrophilic polymer separating agent produced is 10 to 100
Pressure is applied to dry, preferably 10 to 50 m/#-dry, but the degree of swelling can be easily adjusted by adjusting the solution concentration of the raw hydrophilic polymer and the amount of crosslinking agent added, as described above. I can make you do it. Furthermore, it is already known that the hydrophilic polymer-based separating agent formed by the crosslinking reaction of such raw material hydrophilic primer solution usually has a giant network structure.

After the crosslinking reaction is completed, the resulting separation agent is separated and then washed with a hydrophilic organic solvent such as methanol or ethanol, water, etc. to remove unreacted raw material hydrophilic polymer and suspension medium. The composite separating agent of the present invention is obtained in which the pores of the polymer base are filled with the desired hydrophilic polymer separating agent.

Regarding the content ratio of the porous polymer base and the hydrophilic polymer-based separating agent in the composite separating agent of the present invention, a higher proportion of the hydrophilic 4-limer-based separating agent is desirable for the purpose of separation. Therefore, the volume ratio of the porous polymer substrate to the hydrophilic polymer separating agent is preferably in the range of 0.5 to 3.

In the present invention, the degree of crosslinking of a copolymer of a monounsaturated monomer and a polyunsaturated monomer (that is, a porous polymer substrate) is expressed as a weight ratio determined according to the following formula.

Moreover, the degree of swelling was measured by the following method.

After washing the resin and allowing it to swell sufficiently in water, place it in a 10-inch cylinder and tap the bottom to make it exactly 10
+d Weigh out.

This resin was dehydrated using a centrifuge and then heated to 50°C.

Dry thoroughly in a vacuum dryer at a rate of 10 ml/g, weigh the weight accurately, and calculate the swelling degree from the following.

(Example) The present invention will be described in more detail below with reference to Examples, but the present invention is not limited by the Examples.

Example 1 Hydrophilic spherical porous polymer (particle size 1
20μ, pore radius 3,0OOX, pore volume 1.75t
d/l, specific surface area 15.4 m”/fl, moisture content in water 4
．． 5 tptl/1-dry, degree of crosslinking 30% by weight) 5
Put dry g into a 100d beaker, add 4g of Textlan, 2.4g of NaOH, 40.6g of NaBH.
An aqueous solution of Ji' dissolved in water was added to the mixture while stirring at room temperature for 4 hours. After impregnating the same solution into the pores of the spherical porous polymer at room temperature for 12 hours, the solution adhering to the surface of the porous body was removed by centrifugation to obtain a dextran solution-impregnated porous body.

On the other hand, 230 dt - tL of toluene was placed in a 0.51 three-bottle flask equipped with a stirrer and a reflux condenser.

Ethyl cellulose (manufactured by Berkey Leeds, trade name EC-
T100) 2.9 was dissolved to prepare a dispersion bath. After dispersing the above dextran solution-impregnated porous material in the obtained dispersion solution, epichlorohydrin 20- as a crosslinking agent was added, the temperature was raised to 50 tl, and the mixture was stirred at this temperature for 8 hours into the pores of the porous chamber. The impregnated dextran was subjected to a crosslinking reaction.

After the completion of the reaction, a composite product consisting of a porous material and dextran produced using 1 kW of reactants was separated from a toluene solution, and washed in order of toluene, ethanol, and water to obtain a composite separation agent.

In addition, the types and additives of each component used in this Example 1
t is shown in Table 1.

Example 2 A composite separation agent of 1 ml was prepared in the same manner as in Example 1, except that the average molecular weight of the dextran used was changed as shown in Table 1.

Table 1 Note: *1... All manufactured by Sigma Ken Chemical *2...
・Tomo Berkey Leeds Co., Ltd., product name EC'-T100
Experimental Example 1 The composite separation agent obtained in Example 1 was subjected to a pressure loss measurement test in the following manner.

That is, a glass column (with a shut) having an inner diameter of 10 wφ was filled with the composite separating agent 4o- obtained in Example 1, which was sized to a particle size of 74 to 208 μm.

The bed height of the loaded composite separating agent was 50 ml.

The column was kept at 25°C with circulating water, and 0.0
5M, pH 7.0 phosphate buffer solution at 1-7 m/Hr
It was flowed at a constant flow rate. Once the packed bed is stable and the pointer of the pressure gauge attached to the top of the column is constant, read the scale of the pressure gauge and do not pack the composite separation agent based on that value.
If you perform the same operation as described above in a so-called aerodynamic ram state, subtract the reading from the pressure gauge and get the pressure loss (ΔP2 unit = 9/cryt" 150ty b@
d) was calculated.

When the pressure drop (ΔP) was measured by varying the flow velocity (LV, linear flow velocity unit: m/Hr), the results shown in Figure 1 were obtained.
(below), when the composite separation agent obtained in Example 1 is used, a linear relationship is established between ΔP and the LV button, and LV = 7
Even when the liquid was passed at a high flow rate of m/Hr, no deformation or crushing of the separating agent particles was observed.

For comparison, a similar test was conducted using a cross-linked agarose gel (manufactured by Pharmacia, trade name: Sepharose CL-6B) in place of the composite separation obtained in Example 1, and Δ
When the relationship between P and LV was determined, the results shown in FIG. 1 were obtained. In the case of cross-linked agarose glue, ΔP increased rapidly when the flow rate Lv exceeded 1 m/Hr, and it became difficult to pass the liquid when the LV exceeded 2.5 m/Hr.

Experimental Example 2 Example 1. The composite separating agent obtained in Example 2 and the spherical porous polymerized W used in the production of the composite separating agent in Example 1 were each packed into a 10 wmφ x 500 mm glass column, thoroughly washed with water, and then calibrated. Perform the following operations to create a curve.

The performance as a carrier for gel permeation chromatography was evaluated.

That is, 1 aqueous solution of dextran with known molecular weight (1 degree 5
Load 300 μl of 100 μi polyethylene glycol aqueous solution (concentration 2 w/v%) onto the above column, then flow distilled water at a flow rate of 0.4 pd/min to bathe dextran and polyethylene glycol, respectively. I let it out.

Dextran and polyethylene glycol in the eluate were each detected using a differential refractometer, and the holding container KaV value obtained from the elution position It (peak top position fit) using the following formula was plotted against the molecular weight. The results shown in Figure @2 were obtained as a calibration curve.

In the formula, vt: Total volume of separating agent (-) V6: Eluate aI (#!/) vo: Excluded volume (-) Note that vo is dextran with a molecular weight of about 2 million (manufactured by Pharmacia AB, trade name T2O00). This is the value obtained using

As is clear from the results shown in Figure 2, the spherical porous polymer used in the production of the composite separation agents of Examples 1 and 2 has pores through which dextran having a molecular weight of approximately 500,000 can diffuse. have. In contrast, the composite separation agent obtained in Example 1.

Polyethylene glycol with a molecular weight of approximately 10,000 cannot diffuse. From this, the composite separating agent obtained in Example 1 is
It can be seen that the hydrophilic polymer separation agent is formed in the pores of the porous polymer substrate. Also, this Example 1.
The calibration curve of the composite separation agent obtained in 2 shows good linearity and is suitable as a carrier for permeation chromatography.

Example 3 In a 100-beaker, add 2O-t- of 8mM-HCt solution.
After that, add powdered polyvinyl alcohol (Nippon Gosei Kagaku Kogyo Co., Ltd., trade name Ichi7nol NL-05) 2
9 was added and dispersed, heated at 98°C for 1 hour to dissolve, cooled to room temperature, added the same dry spherical porous polymer 5I as in Example 1 to the solution, and dissolved the fine particles of the porous body. The holes were allowed to soak for 4 hours at room temperature. Thereafter, the solution adhering to the surface of the porous body was removed by centrifugation to obtain a porous body impregnated with a polyvinyl alcohol solution.

Meanwhile, 150 ml of toluene solution having the same composition as in Example 1 was placed in a three-bottle flask, Nos. 0 and 51, equipped with a stirrer and a reflux condenser.
- to prepare 5 dispersion baths. After dispersing the polyvinyl alcohol solution-impregnated porous material in the obtained dispersion solution, geltaraldehyde as a crosslinking agent was mixed in advance with a toluene bath solution of 100-100% having the same composition as the dispersion solution, and the temperature was raised to 50°C. The mixture was heated and stirred at this temperature for 4 hours to cause a crosslinking reaction of the polyvinyl alcohol impregnated into the side pores of the porous body.

After the reaction was completed, the reactant was filtered through t-w to separate the generated composite from a toluene solution, and washed in order of toluene, ethanol, and water to obtain a composite separation agent.

The types and amounts of each component used in Example 3 are shown in Table 2. Further, Table 3 shows the water content and degree of swelling in water of the porous polymer before composite and the obtained composite separation agent.

Example 4 After putting 200mM acetic acid solution into a 100-beaker, the nine low-molecular-weight chitosan 2. shown in Table 2 was added. Add IJF,
The mixture was stirred at room temperature for 2 hours to dissolve. Next, 5 g of the same dried spherical porous polymer as in Example 1 was added to this, and in the same manner as in Example 3, a porous body impregnated with the low-molecular-weight chitosan solution was obtained.

Next, a composite separating agent was produced in the same manner as in Example 3, except that the amount of crosslinking agent was changed as shown in Table 2. The water content and degree of swelling in water of the obtained composite separation agent were
Shown in the table.

Notes to Table 2: *1...Nippon Gosei Chemical Co., Ltd., trade name Gohsenol NL-95 *2...Hakai Chemical Co., Ltd., trade name Chitosan 100
The above chitosan 20Jilli-, perboric acid) I
Add Jum 8.51 to a solution of 500 - of water,
Chitosan heat-treated at 50°C for 3 hours to reduce its molecular weight Table 3 Experimental Example 3 A calibration curve was created using the same method as Experimental Example 2 for the composite separation agents obtained in Examples 3 and 4. The results are shown in Figure 3.

polyethylene glycol cannot diffuse. On the other hand, the spherical porous polymer used in the production of the composite separating agent in Examples 3 and 4 has fine particles into which dextran with a molecular weight of about 500,000 can diffuse, as shown in Figure 2. It has holes. From this.

In the composite separating agents obtained in Examples 3 and 4, the hydrophilic polymer separating agent was formed in the pores of the porous polymer substrate, and the calibration curve showed good linearity. It is suitable as a carrier for glue permeation chromatography.

Example 5 7.0 g of a dried spherical porous composite consisting of styrene, ethylvinylbenzene and hinopinylbenzene was immersed in methanol and gradually replaced with water.

Let it be in a water-swollen state. Next, the water on the surface of the water-swollen porous body was removed by suction.

On the other hand, 3N sodium hydroxide (40) was placed in a three-bottle flask (0 and 21) equipped with a stirrer and a reflux condenser.
8 g of dextran was dissolved therein while stirring at room temperature for 4 hours. The above porous body was added to the solution, and the 7'xtran solution was impregnated into the pores of the porous body while stirring at room temperature for 12 hours. Thereafter, 3.9 d i of epichlorohydrin was added while stirring, the temperature was raised to 50° C., and while stirring at that temperature for 8 hours, the dextran impregnated into the pores of the porous body was subjected to a crosslinking reaction.

After the reaction was completed, the reaction product was transferred to a beaker containing 200 g of water, neutralized with IN hydrochloric acid, and then filtered.

The composite f and the solution were separated and washed with water to obtain a composite separation agent.

The moisture content and degree of swelling in water of the obtained composite separation agent were as shown in Table 6. For comparison, the values of porous body A before composite are also shown.

Table 4 shows the physical properties of the spherical porous bodies used.

Table 5 shows the types and amounts of each component used in Example 5.

Example 6 A composite separating agent was produced in the same manner as in Example 5, except that the dextran solution composition and the amount of epichlorohydrin were changed as shown in Table 5.

What is the water content and degree of swelling in water of the composite separation agent obtained?

This is the standard in Table 6.

Example 7 Except for changing the round spherical porous polymer (2) K.
A composite separating agent was produced in the same manner as in Example 5.

Table 4 shows the physical properties of the spherical porous body 0) used, and Table 6 shows the water content and degree of swelling in water of the composite separation agent obtained.

Table 5 Note) *1...All are manufactured by Sigma Chemical Company Average molecular weight: 71500 Experimental example 4 Calibration curves were prepared in the same manner as in Experimental example 2 for the composite separation agents obtained in Examples 5, 6, and 7. Create.

The results are shown in Figure 4.

The spherical porous polymer used in Examples 5, 6, and 7 itself adsorbs dextran and polyethylene glycol, so it cannot be used as a carrier for gel permeation chromatography of these substances. However, by compounding,
Dextran was no longer adsorbed, and a calibration curve as shown in FIG. 4 was obtained. Therefore, it can be used as a carrier for gel permeation chromatography such as dextran, oligomaltose, etc. having a molecular weight of 6a.

Experimental Example 5 The adsorption amount of bovine serum albumin on the complexed separation agent obtained in Example 5 and on the spherical porous body A before complexing was investigated for comparison.

That is, a 0.50 M Tris/HCl buffer solution (pH 7,50) containing 0.20 weight of bovine serum albumin was prepared in a 200-mL Erlenmeyer flask. on the other hand.

Each of the 5.00-separating agents was buffered with a buffer solution, the adhering water was removed by centrifugation i41@, and the mixture was added to the above prepared solution. These solutions were allowed to stand at 5° C. for 12 hours and then shaken using a shaker at 1θ° C. for 6 hours (100 spm, 4 crn strokes). After shaking, completely filter the solution.
The absorbance of the F solution was measured at 280 nm, and the weight of the protein adsorbed on the separation agent was calculated from the calibration curve. The results are shown in Table 7.

Table 7 (e) Effects of the Invention The composite separating agent of the present invention has the following properties: Because it has a structure filled with a hydrophilic polymer separation agent that has a large network structure that exhibits high performance, it has high mechanical strength and is less likely to become compacted when packed in a column.

It is highly commercially available9 and is excellent as a carrier (separating agent) for chromatographic separation of biopolymers such as proteins due to its hydrophilic polymer separation agent having a large network structure.

[Brief explanation of drawings]

FIG. 1 is a graph illustrating the measurement results of the relationship between flow rate LV and pressure drop ΔP when a column is packed with the composite separation agent of Example 1 and cross-linked agarose rkK for comparison. be. In addition, Figure 2 shows the dextran and polyethylene glycol calibration curves for the composite separating agent of Example 1.2 and the spherical porous polymer as the raw material used in the production of the composite separating agent of Examples 1 and 2. This is a drawing shown. Similarly, the third
The figure shows Examples 3 and 4. Figure 4 shows the dextrin and polyethylene glycol calibration curves of Examples 5, 6 and 7. Figure 2 Hoshoyo! bv Figure 3 holding 8M KαV

Claims (1)

[Scope of Claims] 1) The degree of swelling in water is 10 ml/g-dry or less, the degree of crosslinking is 4 to 100 mol%, and the swelling in water is in the pores of an organic polymer substrate having a pore structure. A composite separating agent filled with a hydrophilic polymer-based separating agent having a degree of dryness of 10 to 100 ml/g-dry and a large network structure. 2) The raw material hydrophilic polymer solution for the hydrophilic polymer separation agent according to the first claim before crosslinking is impregnated into the pores of the organic polymer substrate according to the first claim, and then the crosslinking agent is added to the organic polymer substrate. A method for producing a composite separating agent according to claim 1, wherein the hydrophilic polymer-based separating agent according to claim 1 is produced by subjecting a raw hydrophilic polymer to a crosslinking reaction within the pores.