JPH107723A - Grafted ultrafine particles and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polyacrylamide-based ultrafine particle having a surface functional group having a larger surface area when compared with a unit weight, and to fix an object such as a ligand to the surface of the ultrafine particle with a solid. Provided is a method for producing ultrafine particles in which a molecular chain having a suitable length is introduced into the surface of the ultrafine particles so as not to cause an obstacle. SOLUTION: The method for producing grafted ultrafine particles of the present invention comprises a particle having a surface functional group in a solvent having a particle size of 0.05 to 0.5.
μm polyacrylamide-based ultrafine particles and terminal-functional oligomer are added, and mixed and stirred to disperse the polyacrylamide-based ultrafine particles and dissolve the terminal-functional oligomer, and then a condensing agent is added to the solvent to react. By performing a grafting reaction at a temperature of 0 to 50 ° C,
It is characterized in that the terminal functional oligomer is introduced into the polyacrylamide-based ultrafine particles.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to novel grafted ultrafine particles and a method for producing the same.

[0002]

2. Description of the Related Art Polyacrylamide gels are biologically inert and cannot be decomposed by microorganisms, and various salts, surfactants and the like used in ordinary affinity chromatography within a pH range of 2-10. Since it has the advantage of being stable in a solution such as guadinine hydrochloride, when a polyacrylamide gel is used for affinity chromatography or the like, a ligand can be immobilized using an amide group present in the gel. It has been practiced to make spherical gels with a porous structure and to bond functional groups on the gel surface and inside the pores with spacers or ligands.

[0003] However, the size of such a spherical gel having a porous structure obtained by a conventional manufacturing method is usually 0.8 to 30 µm, and by increasing the pore diameter as much as possible, a low molecular weight ligand or spacer can be formed inside the pore. The molecule is designed for immobilization as a functional group.
However, it is difficult for the ligand to penetrate into the inside of the pores of the gel, such as a glycoprotein having affinity (usually about 10 nm in size). This is especially true when a spacer of about 1 to 2 nm is inserted. Therefore, an adsorbent with a very high adsorption capacity should be formed in theory, but in reality, the adsorption capacity is limited by the concentration of functional groups on the gel surface, so that sufficient adsorption as an affinity adsorbent is required. There is a problem that performance cannot be obtained, and it is desired to develop a polyacrylamide gel in which a substrate having a surface functional group has a larger surface area as compared with a unit weight, and a spacer is suitably introduced on the surface. I was

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an effective functional group which can be used as an adsorbent having a substantially high adsorbing capacity by using a side chain of an oligomer such as a spacer (only with a molecular chain). The substrate (polyacrylamide-based ultrafine particles) having a surface functional group has a larger surface area when compared with a unit weight, and an immobilized body such as a ligand is not attached to the substrate. It is an object of the present invention to provide ultrafine particles obtained by introducing a molecular chain having a suitable length into the surface of a base material so as not to cause steric hindrance or the like with the surface of the material, and a method for producing the same.

[0005]

Means for Solving the Problems In order to achieve the above object, the present inventors have intensively studied ultrafine polyacrylamide (hereinafter, also simply referred to as PAA) particles having a molecular chain introduced therein and a method for producing the same. As a result, P
The AA-based ultrafine particles are polymerized in a state where they are atomized and dispersed as much as possible, and furthermore, the amide group, carboxyl group, and amino group existing as the surface functional group are used to react preferentially with the functional group. An end-functional molecular chain having a desired length with an amino group at one end and a functional group (carboxyl group, amino group, mercapto group or hydroxyl group) at the other end capable of reacting with an immobilized substance such as a ligand. It is possible to introduce the molecular chain having the performance as designed by grafting onto the surface of the substrate, thereby finding that it is possible to obtain the desired ultrafine particles. As a method of polymerizing in a state where PAA-based ultrafine particles are dispersed, in an organic solvent of alcohols,
In the precipitation polymerization of a monomer mixture containing an ethylenically unsaturated amide, preferably in a mixed solvent of water and an organic solvent of an alcohol, the amount of the crosslinking agent, the type of the solvent, the concentration of the initiator and the polymerization temperature are extremely limited. It was found that the primary particles obtained did not cause secondary agglomeration only under the combined conditions within the specified range, and as a result, the dispersion stabilizer (dispersion stabilizer polymer) was not used at the time of polymerization, so that Particle size 0.
PAA ultrafine particles of 5 μm or less can be produced, purification treatment after polymerization is easy, and as a method of introducing a molecular chain to the surface of the ultrafine particles, the PAA ultrafine particles having a desired length are introduced into a solvent. Add the terminal-functional oligomer, mix and stir to disperse the PAA-based ultrafine particles, dissolve the terminal-functional oligomer, and then add a condensing agent in the solvent and perform a graft reaction under mild temperature conditions. It has been found that the terminal functional oligomer can be introduced at a high concentration on the substrate, especially on the surface thereof, and that the purification treatment after the reaction is also simple, and the present invention has been completed based on these findings. is there.

That is, an object of the present invention is to provide (1) a particle having an amide group as a surface functional group in a solvent having a particle size of 0.05 to 0.05;
Add 0.5 μm polyacrylamide-based ultrafine particles and terminal-functional oligomer, mix and stir to disperse the polyacrylamide-based ultrafine particles and dissolve the terminal-functional oligomer, and then add a condensing agent to the solvent By performing the grafting reaction at a reaction temperature of 0 to 50 ° C., the method is achieved by a method for producing grafted ultrafine particles, which comprises introducing the terminal functional oligomer into the polyacrylamide-based ultrafine particles.

Another object of the present invention is that (2) the surface functional group is at least one selected from the group consisting of an amide group, a carboxyl group and an amino group. The method for producing grafted ultrafine particles described in (1).

Another object of the present invention is to provide (3) a method wherein the terminal functional oligomer is H ₂ N (CH ₂ ) _n COOH, H ₂ N
(CH ₂ ) _n NH ₂ , H ₂ N (CH ₂ ) _n SH and H
_{2 N (CH 2) n OH} , HOOC (CH 2) n COOH
(N in the formula is an integer of 7 or less), which is also achieved by the method for producing grafted ultrafine particles described in (1) or (2) above.

Another object of the present invention is to provide (4) polyacrylamide-based ultrafine particles having a specific gravity of 0.3 to 40 ° C. at a temperature of 0 to 40 ° C.
(1) to (3) above, characterized by being 1.2
This is also achieved by the method for producing grafted ultrafine particles described in any of the above.

[0010] Another object of the present invention is to provide (5) a grafted ultrafine particle obtained by the production method described in any of the above (1) to (4), wherein This is achieved by the grafted ultrafine particles having a group concentration of 0.001 mg equivalent or more.

Another object of the present invention is to provide (6) the above (1)
And (4) a grafted ultrafine particle obtained by the production method as described in any one of (1) to (4), wherein a terminal functional oligomer is mainly introduced into the surface of the ultrafine particle. Is done.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail based on embodiments.

First, the polyacrylamide (PAA) -based ultrafine particles used in the present invention can have a terminal functional oligomer grafted to the surface of the ultrafine particles by providing a large number of effective functional groups on the surface thereof. As a result, many substances such as ligands can be immobilized on the surface, and the target adsorbed substance can be adsorbed at a high concentration. Therefore, it is necessary to use only the surface functional groups of the ultrafine particles without using the functional groups inside the pores of the ultrafine particles. The surface area is large, the surface functional groups of the ultrafine particles are as large as possible, and when used as a filler for affinity chromatography, in addition to the above requirements, it is desirable that the spherical particles have a uniform particle size. The moisture absorption of the ultrafine particles is usually 10 to 400%, preferably 50 to 300%, more preferably 100 to 250%. If the moisture absorption is less than 10%, the particles are excessively cross-linked, resulting in extremely dense ultrafine particles, which have sufficient mechanical strength but large hydrophobic properties. Therefore, it is not preferable, and when it exceeds 400%,
Although it has a low degree of cross-linking and a porous structure with a relatively large pore size, it is not preferable because of insufficient mechanical strength.
Here, the “moisture absorption rate of PAA-based ultrafine particles” is (BA)
/ A × 100, where A and B in the formula are values determined by the following measurement procedure. The dry PAA-based ultrafine particles are weighed, and the weight is defined as Ag. A large excess of water is added to the dried PAA-based ultrafine particles, and the mixture is left for 24 hours. Thereafter, PAA-based ultrafine particles were added at 10,000 rpm, 17680
Centrifuge at G for 20 minutes to remove excess water. The PAA-based ultrafine particles that have absorbed water after centrifugation are weighed, and the weight is defined as Bg. All ~ are 25
C. and 1 atm.

Similarly, the gelation rate of the PAA-based ultrafine particles is usually 40 to 98%, preferably 50 to 90%, more preferably 70 to 85%. If the gelation ratio exceeds 98%, the particles are excessively crosslinked, resulting in extremely dense ultrafine particles, which have sufficient mechanical strength but large hydrophobic characteristics. If it is less than 40%, the degree of crosslinking is low and the porous structure has a relatively large pore size, but the mechanical strength is not sufficient, which is not preferable. Here, the “gelation rate of the PAA-based ultrafine particles” is defined as (B / A) × 100, and A
And B were values determined by the following measurement procedure.
The PAA-based ultrafine particles Ag are added to 100 ml of water. 50
C. and leave for 24 hours. The subsequent PAA-based ultrafine particles are centrifuged at 10,000 rpm and 17,680 G for 20 minutes to remove excess water. The water-absorbed PAA-based ultrafine particles after centrifugation are dehydrated and dried. The obtained dry PAA-based ultrafine particles are weighed, and the weight is measured in Bg.
And Note that all (except) are 25 ° C, 1
The test was performed under atmospheric pressure.

The PAA ultrafine particles have a particle size of usually 0.05 to 0.5 μm, preferably 0.08 to 0.5 μm.
It is in the range of 0.4 μm, more preferably 0.1 to 0.3 μm. When the particle diameter is less than 0.05 μm, it is ideally desirable that the particle diameter be as small as possible. However, it is equivalent to the size of the nucleus itself of the ultrafine particles formed at the initial stage of polymerization. This is because it is practically difficult to form ultrafine particles having a particle size smaller than the above. If the thickness exceeds 0.5 μm, a sufficient surface area cannot be obtained, and the surface functional group concentration becomes undesirably low.

The PAA-based ultrafine particles have a temperature of 0 to
At 40 ° C., its specific gravity is between 0.3 and 1.2, preferably between 0.1 and 1.2.
Desirably, it is 5 to 0.9. When the specific gravity is less than 0.3, all the particles float in the solvent, which is not preferable in that they react with the oligomer. If the specific gravity exceeds 1.2, it is not preferable because ultrafine particles precipitate and react with oligomers while being dispersed in a solution.

As described above, PAA-based ultrafine particles in which the moisture absorption rate, gelation rate, particle size, and specific gravity of the PAA-based ultrafine particles are within the above-mentioned ranges have high mechanical strength and various salts used in ordinary affinity chromatography. Effective surface functionality that has chemical stability in solutions such as surfactants and guanidine hydrochloride, can be applied in a wide pH range (range of pH 2 to 10), and can be used as an adsorbent with high adsorption capacity PAA-based ultrafine particles having a high concentration of groups can be obtained.

The above-mentioned PAA-based ultrafine particles can be obtained by the following method. That is, a monomer mixture containing an ethylenically unsaturated amide in an organic solvent of alcohols, and 100% by weight of a mixture of the monomer mixture and a crosslinking agent.
And a mixed solution in which a crosslinking agent in the range of 25 to 50% by weight is dissolved is maintained at a temperature of 30 to 95 ° C., and at least one selected from the group consisting of a peroxide initiator and an azo initiator is used. 1 × 10 ⁻⁵ to 8 × 10 ⁻² mol / initiator
By performing addition and polymerization in a range of 1 liter, PAA-based ultrafine particles having an amide group on the surface functional group can be obtained. More specifically, Japanese Patent Application No. Hei.
No. 325181, which will be described below.

First, as the organic solvent of alcohols used in the above-mentioned production method, for example, methanol, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, t-butyl alcohol, 1-pentanol, 2-pentanol , 3-pentanol, t-
Monohydric alcohols such as pentyl alcohol, 1-hexanol, 2-hexanol and 3-hexanol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, neopentyl glycol, pentamethylene glycol , Dihydric alcohols such as hexamethylene glycol, and 3 such as glycerol
In addition to alcohols having 1 to 6 carbon atoms such as polyhydric alcohols, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, and ethylene glycol monoisobutyl ether, polyethylene glycol having a molecular weight of 6000 or less, preferably 4000 or less, and the like. Can be These organic solvents can be used alone or in combination of two or more.

If necessary, water is usually added to the above-mentioned organic solvent of alcohols in an amount of 30% by weight with respect to 100% by weight of the total solvent (total solvent means water obtained by adding water to the organic solvent of alcohols). % By weight or less, preferably 10 to 25% by weight,
More preferably, it is desirable to add in the range of 15 to 20% by weight. If the addition amount exceeds 30% by weight, the obtained particles are not preferable because aggregation occurs and addition of water, which is a solvent having a high affinity for the polymer, increases the particle size. On the other hand, when water is added within the above range, without using a dispersion stabilizer during polymerization,
The generated particles can be kept in a state of being stably dispersed in a solvent as in dispersion polymerization without forming aggregates by precipitation polymerization, which is useful for forming monodisperse ultrafine particles.

Examples of the ethylenically unsaturated amide monomer that can be used in the above-mentioned production method include acrylamide, methacrylamide, diacetone acrylamide,
N-hydroxymethylacrylamide and the like can be mentioned, and these monomers can be used alone or in combination of two or more.

The monomer mixture containing the ethylenically unsaturated amide monomer may be any other monomer copolymerizable therewith, in addition to the above-mentioned ethylenically unsaturated amide monomer. Since it is preferable that the ligand can be adjusted by a functional group on the surface of the ultrafine particles so that the ligand fixed to the ultrafine particles can be selectively changed, the other copolymerizable monomer may have an -OH group, It is desirable that the monomer has at least one of —NH ₂ group and —SH group. Further, these other copolymerizable monomers can be used alone or in combination of two or more.

The content of the ethylenically unsaturated amide monomer contained in the above monomer mixture is usually 0.1 mol%.
The content is preferably at least 0.3 mol%, more preferably at least 0.5 mol%. If the content is less than 0.1 mol%, a good particulate polymer cannot be obtained.

The amount of the monomer mixture added to the organic solvent is usually 0.1 to 70% by weight, preferably 1 to 25% by weight, more preferably 2 to 25% by weight based on the organic solvent. It is in the range of 10% by weight. When the mixing amount of the amide monomer is less than 0.1% by weight, the polymerization reaction rate is slow because the concentration of the monomer is extremely low, and good ultrafine particles cannot be obtained. If it exceeds, the produced polymer has a large particle size and is liable to undergo secondary aggregation, so that there is a problem in using it as a filler for affinity chromatography. That is, it is desirable that the packing material for affinity chromatography be spherical particles as fine as possible and uniform in size because of the advantage that the diffusion of proteins and the like having a low diffusion rate becomes easy and the solute band becomes sharp.

Further, the crosslinking agent which can be used in the above-mentioned production method is a compound having at least two unsaturated double bonds in the molecule, for example, N, N'-methylenebisacrylamide, ethylenebisacrylamide, Various bisphenols such as alkylene bisacrylamides such as piperozine acrylamide, alkylene di (meth) acrylates such as ethylene di (meth) acrylate, ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, and bisphenol A di (meth) acrylate Di (meth) of A derivative
Acrylates, di-, tri-, tetra (meth) acrylates of polyhydric alcohols such as glycerin and pentaerythritol, di- or tri- (meth) acrylates of ethylene oxide-modified phosphoric acid, oligoesters (meth) acrylates, polyester (meth) acrylates, Urethane acrylate, di- or tri (meth) acrylate of isocyanurate or a derivative thereof, vinyl (meth) acrylate, acryl (meth) acrylate, poly-N-methylolacrylamide, N-methylolacrylamide, trimethylolpropane trimethacrylate, N -Methylol (meth) acrylamide, N-methoxymethylacrylamide, N-ethoxymethylacrylamide, Nn-butoxymethylacrylamide,
Nn-butoxymethyl (meth) acrylamide, N-
N-alkoxymethyl (meth) acrylamide derivatives such as isobutoxymethylacrylamide, Nt-octoxymethylacrylamide, divinylbenzene, vinylacetic acid, chloromethylstyrene, urea-formaldehyde resin, bisoxazoline compound, epichlorohydrin, Those usually used as a crosslinking agent, such as 1,3-dichloroisopropanol and glycerin diglycidyl ether, are exemplified.

Further, in the above-mentioned production method, since the purpose is to mainly use the surface functional groups of the obtained PAA-based ultrafine particles, it is possible to reduce the pore size of the porous particles by changing the addition amount of the crosslinking agent as in the prior art. There is little need to adjust the size and the like, but rather it is necessary to increase the bulk specific gravity so as to be dense particles to obtain the mechanical strength of the generated ultrafine particles. The amount of the crosslinking agent added is usually 1% based on 100% by weight of the mixture of the mixture and the crosslinking agent.
The range is from 0 to 70% by weight, preferably from 15 to 60% by weight, more preferably from 25 to 50% by weight. When the amount added is 1
If the amount is less than 0% by weight, the degree of crosslinking becomes insufficient, so that the mechanical strength of the obtained ultrafine particles is weak, and the thermal and chemical stability is not sufficient. )
Can not be repeatedly used in autoclaving, can not be used in a wide range of pH, and also can be applied to the stability to various organic solvents. It is not preferable because there is a problem. On the other hand, when the addition amount exceeds 70% by weight, the amount of the remaining crosslinking agent becomes too large, which is not economical. In addition, the above-mentioned problems are eliminated, but the degree of crosslinking is increased, and when used as an affinity adsorbent that is a packing material for affinity chromatography, it is not possible to obtain an expected adsorption capacity. It is not preferable because there is.

As the peroxide-based initiator and the azo-based initiator that can be used in the above-mentioned production method, conventionally known ones can be used. Among them, as the peroxide-based initiator, for example, benzoyl peroxide (BPO) and its substituted product, diacyl peroxide [RCOOCOR], dialkyl peroxide [ROOR], peroxyester [RCOOOO]
R '], peroxycarbonate [ROCOOOCOOO
R], hydroperoxide [ROOH] and redox initiators obtained by combining these with a reducing substance. Examples of the azo initiator include N-nitrosoacyl allylamine, azobisisobutyronitrile (AIBN). ), Azobisester [RR '(COO
R ″) CN = NC (COOR ″) RR ′], azotriphenylmethane [C ₆ H ₅ N = NC (C ₆ H ₅ ) ₃ ], diazoaminobenzene [C ₆ H ₅ N = NNHC ₆ H ₅ ] ,
Diazothioether [RN = NSR '], tetramethyltetrazen [(CH ₃ ) ₂ NN = NN (CH ₃ ) ₂ ],
Examples thereof include diazomethanes [RC = N = N] and sulfone azides [RSO ₂ N = N = N]. In the present invention, these may be used in an appropriate combination.

When the initiator is used, the concentration of the initiator is usually 1 × 1 to the mixed solution of the monomers.
0 ^-5 to 8 × 10 ^-2 mol / l, preferably 0.5 ×
It is in the range of 10 ^{−4 to} 5 × 10 ⁻² mol / l, more preferably 0.5 × 10 ⁻³ to 8 × 10 ⁻³ mol / l. When the concentration of the initiator is out of the range of 1 × 10 ⁻⁵ to 8 × 10 ⁻² mol / L, the resulting polymer is not preferable because it becomes non-spherical coarse particles.

In the above-mentioned production method, the mixed solution of the above-mentioned monomers is maintained at a temperature of usually 30 to 95 ° C., preferably 65 to 85 ° C., more preferably 70 to 80 ° C. The polymerization is carried out for 2 to 15 hours, preferably 3 to 10 hours, more preferably 3 to 7 hours.

When the temperature at the time of the above polymerization is lower than 30 ° C., the decomposition rate of the initiator becomes slow, and the generation of radicals due to the decomposition of the initiator becomes insufficient. Radical generation due to decomposition of the compound becomes too large, which is not preferable because there is a safety problem in combination with an increase in heat of polymerization reaction. If the polymerization reaction time is less than 3 hours, the production of the obtained polyacrylamide-based ultrafine particles is a production process, and the yield becomes extremely low.
If it exceeds 7 hours, the polymerization reaction has already been completed, and further extension of the polymerization time is useless.

After the completion of the polymerization, it is necessary to stop the radical polymerization reaction by, for example, rapidly lowering the reaction temperature with an ice bath or the like. Further, the stirring power (rotation speed), pressure, atmosphere, etc. of the solution during the polymerization are not particularly limited, and are appropriately determined in consideration of the above-mentioned set values, etc., but are preferably at normal pressure. In order to make the temperature distribution of the reaction system uniform in an inert gas atmosphere,
It is desirable that the process be performed in a range of up to 5000 rpm.

In the above method, no dispersion stabilizer is added during polymerization. By using such a dispersion stabilizer, there is an advantage that monodispersed particles can be obtained even under polymerization conditions in a relatively wide range as compared with the present production method, but on the other hand, purification treatment after polymerization is difficult and is not sufficiently performed. In the case of being unable to purify and further utilizing in biotechnology, there is a drawback that such a dispersion stabilizer adversely affects as an impurity,
Since the disadvantages are greater than the benefits of using the dispersion stabilizer, the use of such a dispersion stabilizer during polymerization must be avoided.

Next, a method for purifying the obtained PAA-based ultrafine particles is such that they do not contain impurities such as dispersion stabilizers.
It can be easily carried out based on a conventionally known method. For example, as in the method used in Examples described below, a centrifugal separator is used for the separation operation, and the ultrafine particles are reacted with a solution, a washing solution (alcohol) or a polymer. It is separated from the reprecipitation solvent (purified water and acetone, etc.) and separated by decantation or filter filtration.
Purification and finally drying by vacuum drying or the like can be used, for example, a purification method for obtaining the ultrafine particles of the present invention in the form of xerogel can be used.However, the present invention is not limited to the above-mentioned washing method, Other known purification methods can also be used.

Subsequently, according to the monomer mixture containing the ethylenic saturated amide monomer used in the polymerization, the PAA-based ultrafine particles obtained by the above-mentioned production method further include an amide group as the surface functional group and a carboxyl group. Groups or amino groups. Further, those prepared by modifying the surface functional groups of the PAA-based ultrafine particles to amide groups, carboxyl groups or amino groups can also be used. The preparation method is not particularly limited. For example, a PAA-based ultrafine particle whose surface functional group is only an amide group obtained by the above method is suspended in anhydrous ethylenediamine and heated at 90 ° C. In addition, aminoethylated polyacrylamide-based ultrafine particles having an amino group as a surface functional group can be prepared. Similarly, PAA whose surface functional group is only an amide group
Carboxyl-polyacrylamide-based ultrafine particles having a surface functional group as a carboxyl group can be prepared by alkali hydrolysis of the system-based ultrafine particles. In the following description, those including those obtained by these preparations are collectively referred to as “PAA-based ultrafine particles”.

Next, the PAA-based ultrafine particles and the terminal functional oligomer are added to a solvent, and the mixture is stirred and mixed.
When the terminal-functional oligomer is introduced into the PAA-based ultrafine particles by dispersing the A-based ultrafine particles and dissolving the terminal-functional oligomer, and then adding a condensing agent to the solvent to cause a grafting reaction. The reaction temperature is preferably 0 to 50 ° C., preferably 4 to 30 ° C., and more preferably a room temperature condition that does not require temperature adjustment. When the reaction temperature is lower than 0 ° C., the solvent may be solidified depending on the solvent, and the types that can be used are limited. In addition, the reaction speed is slow and the time required for the reaction is long, which is uneconomical. When the temperature exceeds 50 ° C., the grafting reaction proceeds, but when the obtained product of the present invention is used for affinity chromatography,
In the same reaction system, when immobilizing the ligand or adsorbing the target purified substance continuously, adverse effects such as denaturation or decomposition of the ligand or the target purified substance may occur if the temperature is kept high. It is not preferable because things are restricted. The reaction time is generally 1 to 96 hours, preferably 3 to 48 hours. When the reaction time is less than 1 hour, the grafting reaction is not completely completed, the terminal functional oligomer cannot be sufficiently introduced on the ultrafine particle surface, and when it exceeds 96 hours, This is undesirable because the grafting reaction has already been completed and the effect sufficient to continue the reaction cannot be obtained. Further, the solvent used in the grafting reaction is appropriately selected from the relationship with the actually used terminal-functional oligomer and the condensing agent.
Usually, neutral, acidic and alkaline aqueous solvents and organic solvents can be used. Therefore, when such an aqueous solvent is used, the pH is 2 to 13, and it can be used in a wide range.

As the condensing agent, those capable of immobilizing a terminal functional oligomer having a carboxyl group or an amino group by promoting a dehydration condensation reaction on the PAA-based ultrafine particles can be used. , Dicyclohexylcarbodiimide (DCC), and 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide-p-toluenemethsulfonic acid (CMC) and 1-ethyl-3- (3-dimethylaminopropyl)-
And water-soluble carbodiimide (WSC) such as carbodiimide / hydrochloric acid (EDC). In this case, DCC
Is the most inexpensive, but must be reacted in an organic solvent system because it is insoluble in water, and needs to be washed with a large amount of methanol or ethanol to remove the generated insoluble urea derivative. On the other hand, EDC and CMC, which are water-soluble carbodiimides (WSC), can be reacted in an aqueous solution, and the formed urea derivative is also water-soluble, so that washing after the reaction is easy.

The amount of the condensing agent added to the reaction system is as follows:
Usually 0.005 to 10 mol / liter, preferably 0.1 to 10 mol / liter.
The range is from 01 to 5 mol / liter. When the amount added is 0.
If the amount is less than 005 mol / liter, the reaction is not sufficiently promoted and it takes an extremely long time to complete the reaction, which is uneconomical. It is not preferable because a further effect corresponding to the addition cannot be expected.

The amount of the ultrafine PAA particles is usually 0.1 to 500 g / liter, preferably 5 to 500 g / liter.
~ 200 g / liter. The amount used is 0.1
When the amount is less than g / liter, a large amount of solvent is required, and the volume ratio of the whole reaction system is deteriorated, which is uneconomical. Therefore, it takes time for cleaning in the purification process after production, and it is uneconomical because the number of times of cleaning increases.

Further, the above-mentioned terminal functional oligomer includes:
For example, H ₂ N (CH ₂ ) _n COOH, H ₂ N (C
H ₂ ) _n NH ₂ , H ₂ N (CH ₂ ) _n SH and H ₂ N
(CH ₂ ) _n OH and HOOC (CH ₂ ) _n COOH (where n is an integer of 7 or less) can be used.
As a guide for determining the length of these terminal-functional oligomers, that is, the size of n in the above formula, for example, when the terminal-functional oligomer is used as a spacer of a carrier for affinity chromatography, It is desirable to select a length that allows the ligand to exhibit an optimum adsorption force without causing steric hindrance to the target substance to be purified.

The amount of the terminal functional oligomer to be used is generally 1 × 10 ^{−6 to} 5 × 10 ¹ mol / liter, preferably 1 × 10 ⁻³ to 1 × 10 ¹ mol / liter. When the used amount is less than 1 × 10 ⁻⁶ mol / liter, the concentration of the terminal functional oligomer is smaller than the surface functional group concentration of the ultrafine particles, so that the introduction rate into the ultrafine particles is not sufficient. If the amount used exceeds 5 × 10 ¹ mol / liter, it is not preferable because no further effect corresponding to excessive addition cannot be expected.

Next, as a method for purifying the grafted ultrafine particles formed by the grafting reaction, a conventionally known method is used so as to exclude organic solvents, reaction by-products, and impurities such as unreacted condensing agents and oligomers. It is desirable to simply carry out the method based on the method. For example, as in the method used in Examples described later, a centrifugal separator is used for the separation operation, and the ultrafine particles and the reacted solution, washing solution (alcohol) or reprecipitation solvent ( Purified water and acetone, etc.), and these are separated and purified by decantation, filter filtration, dialysis, etc., and finally dried by vacuum drying, freeze drying, etc., and the grafted ultrafine particles of the present invention are treated with xerogel. Although a purification method or the like obtained as a form can be used, the present invention is not limited to the above-mentioned washing method, and is conventionally known. Other purification methods can be used.

Next, in the grafted ultrafine particles obtained by purification, the functional group concentration per gram of the grafted ultrafine particles is 0.001 mg equivalent or more, preferably 0.05 mg equivalent or more, although it depends on the intended use. It is. If the concentration of the functional group is less than 0.001 mg equivalent, the subsequent immobilization rate of the ligand is small, so that it is necessary to prepare a large amount of the ultrafine particles, and the cost required for the subsequent reaction equipment increases, which is uneconomical. It is. The above-mentioned grafted ultrafine particles preferably have a functional group mainly grafted on the surface of the ultrafine particles.

[0043]

Embodiments of the present invention will be described below.

Example 1 Under a nitrogen atmosphere, 25 g of acrylamide was added to 1000 ml of ethanol and 20.5 g of N, N'-methylenebisacrylamide as a crosslinking agent (100% by weight of a mixture of a monomer mixture and a crosslinking agent was used for crosslinking. 45.1% by weight) was dissolved while stirring, and at the same time, the dissolved oxygen was sufficiently removed. Subsequently, at a temperature of 70 ° C., 0.821 g of 2,2′-azobisisobutylnitrile (initiator concentration 5 × 1) was used as an initiator.
0 ^-3 mol / l) was added, subjected to polymerization for 3 hours as pH 6.5, after completion of the reaction was quenched by the polymerization vessel in an ice bath, the radical polymerization was stopped, to obtain ultrafine particles of monodisperse spherical.

Next, the produced polyacrylamide-based ultrafine particles were separated by a centrifuge, and the supernatant was removed. Here, 1000 ml of ethanol was newly added, and the mixture was vigorously stirred for 1 hour, and then separated by a centrifuge. The same operation was performed three times, followed by filtration through a glass filter, followed by vacuum drying at 50 ° C. under reduced pressure.

The obtained ultrafine particles were converted into a 5% by weight polymer solvent using purified water as a solvent, and 20 times the amount of acetone was added as a precipitant thereto, followed by reprecipitation with good stirring. Thereafter, the mixture was separated by a centrifuge, and the supernatant was removed. The same operation was performed twice, and the mixture was filtered through a glass filter and dried under reduced pressure at 50 ° C. under reduced pressure to complete the purification.

The amide groups on the surface of the ultrafine particles have a pH of 10.
The reaction was carried out at 60 ° C. for 3 hours at a Na ₂ CO ₃ concentration of 0.04 mol / l so as to maintain an alkali state of 5 or more, and 2.0 g of a matrix converted to carboxyl groups (hereinafter, also simply referred to as CM) was purified. Add 120 ml of water and stir for 24 hours to fully disperse. The terminal-functional oligomer to be introduced is used as a spacer (hereinafter, also simply referred to as S) and diluted with 25 ml of purified water using 0.54 ml (0.01 mol) of ethylenediamine as a solvent. The mixture was adjusted to 5 to 5.5 and stirred until the matrix and the spacer were completely dispersed and dissolved.

Next, 1.610 g of CMC (condensation agent concentration: 0.025 M) as a condensing agent was added to the reaction flask, and at this time the reaction was started at 23 ° C. (room temperature), and the reaction was completed in 48 hours. .

Next, as a separation / purification step, a matrix (hereinafter simply referred to as C
M-S) in a centrifuge at 14500 rpm for 1
Separation was performed for 5 minutes, and the supernatant was removed. 100 ml of purified water is added thereto at 200 ° C. for 2 hours at 50 ° C.
The mixture was stirred at rpm, and centrifuged at 14,500 rpm for 20 minutes using a centrifuge. After the same operation was performed 5 times, freeze-drying was performed to complete the purification. The completion of the purification was confirmed by examining the supernatant liquid after centrifugation by a TNBS reaction, and the absence of diamine was confirmed.

Determination of Surface Amino Group 21.0 mg of CM-S was dispersed in 120 ml of purified water (agitated at 200 rpm at room temperature for 24 hours to sufficiently swell). Thereafter, the ultrasonic waves were completely dispersed for 2 hours. 6.0 ml of 0.01 N hydrochloric acid was added to this dispersion with a whole pipette, and titration was immediately performed with 0.01 N sodium hydroxide. As a result of measuring the electric conductivity at this time, 0.15 mg equivalent per 1 g of the ultrafine particles was measured. It was found that an amino group had been introduced.

Examples 2 to 15 Ultrafine particles were prepared in the same manner as in Example 1 except that the type and concentration of S, the type and concentration of the condensing agent, and the reaction time were set to the conditions shown in Table 1 below. Was prepared, and the amount of amino groups introduced on the surface was quantified. The results are shown in Table 1 below.

[0052]

[Table 1]

[0053]

According to the present invention, polyacrylamide-based ultrafine particles having a surface functional group and having a particle diameter of 0.05 to 0.5 μm can be easily purified after polymerization without using a dispersion stabilizer or a dispersion stabilizer polymer. Thus, it can be obtained in a monodispersed state with less contamination of impurities. Therefore, since the ultrafine particles and the terminal functional oligomer can be dispersed and dissolved in a solvent, the surface of the ultrafine particles can be maintained under mild temperature conditions (preferably at room temperature) by simply adding a condensing agent thereto and continuing stirring. The functional group reacts with the terminal functional group of the oligomer (acid amide bond), and the ultrafine particles, particularly the oligomer having an effective terminal functionality can be graft-introduced on the surface thereof at a high concentration. Furthermore, the ultrafine particles after the grafting can be obtained in a monodispersed state in which the purification treatment is simple and the contamination with impurities is extremely small. In the grafted ultrafine particles thus obtained, a spacer having a length that does not cause steric hindrance can be appropriately and selectively bound as a filler used in affinity chromatography according to a different target substance to be purified. By immobilization, it can be used as one kind of polyacrylamide gel as an optimal support capable of adsorbing a purification target substance that specifically binds to the ligand at a high concentration without causing steric hindrance. Furthermore, in the grafted ultrafine particles, since the terminal functional oligomer can be bonded to the surface in a high concentration, it is not limited to application to a filler used in affinity chromatography, and has a large reactivity directly to the terminal functional group of the oligomer. Since the target substance can be immobilized, it can be sufficiently applied to the production of materials for cosmetics, materials for diagnostics, developers (toners), paints, and the like.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Koichi Miura Inside Fujimori Kogyo Co., Ltd. 1-4-16 Nibashi Bakurocho, Chuo-ku, Tokyo

Claims (6)

[Claims] 1. A particle having a surface functional group in a solvent having a particle size of 0.0
5 to 0.5 μm of polyacrylamide-based ultrafine particles and a terminal functional oligomer are added, and mixed and stirred to disperse the polyacrylamide-based ultrafine particles and dissolve the terminal-functional oligomer, and thereafter, a condensing agent is added to the solvent. And a grafting reaction at a reaction temperature of 0 to 50 ° C. to introduce the terminal functional oligomer into the polyacrylamide-based ultrafine particles. 2. The method according to claim 1, wherein the surface functional group is at least one selected from the group consisting of an amide group, a carboxyl group, and an amino group. Method. 3. The method according to claim ₂ , wherein the terminal functional oligomer is H ₂ N
(CH ₂ ) _n COOH, H ₂ N (CH ₂ ) _n NH ₂ , H
₂ N (CH _{2) n} SH and _{H 2 N (CH 2) n} OH,
The method for producing grafted ultrafine particles according to claim 1, wherein the method is any one of HOOC (CH ₂ ) _n COOH (where n is an integer of 7 or less). 4. The grafted ultrafine particles according to claim 1, wherein the polyacrylamide ultrafine particles have a temperature of 0 to 40 ° C. and a specific gravity of 0.3 to 1.2. Production method. 5. Grafted ultrafine particles obtained by the method according to claim 1, wherein the functional group concentration per gram of the grafted ultrafine particles is 0.001.
Grafted ultrafine particles, which are not less than mg equivalent. 6. Grafted ultrafine particles obtained by the method according to claim 1, wherein a terminal functional oligomer is mainly introduced on the surface of the ultrafine particles. Grafted ultrafine particles.