JP6220496B2 - Method for producing polymer particle, polymer particle, packing material for chromatography column, and chromatography column - Google Patents

Description

  The present invention relates to a method for producing polymer particles, polymer particles produced by the production method, a packing material for a chromatography column using the particles, and a chromatography column.

Conventionally, synthetic polymers such as poly (meth) acrylic esters and poly (meth) acrylamides and particles of natural polymers such as polysaccharides have been used as chromatographic carriers. Among these, the synthetic polymer particles are either a method of polymerizing a monomer hydrophobized with a protecting group or the like, or normal phase suspension polymerization (O / W type) or reverse phase suspension polymerization (W / O type). Manufactured by the method.
In the case of hydrophobization treatment, a secondary treatment for converting to a hydrophilic group must be performed after the polymerization, and the reverse phase suspension polymerization requires a large amount of an organic solvent, and the particle preparation process is complicated. However, since acrylamide monomers are generally poorly soluble in organic solvents, normal phase suspension polymerization using them has not been performed so far, and preparation of polyacrylamide particles using acrylamide monomers is not common. In particular, a method of polymerizing a monomer hydrophobized with a protecting group or the like, or a method of polymerizing by reverse phase suspension is employed (Patent Documents 1 to 3).

Japanese translation of PCT publication No. 2003-511659 Special table 2009-503203 JP 2006-111717 A

  Problems to be solved by the present invention include a method for producing polymer particles from an acrylamide monomer by a normal phase suspension polymerization method, a polymer particle produced by the production method, a packing for a chromatography column using the particles, and It is to provide a chromatography column.

  Therefore, as a result of intensive studies, the present inventors have dispersed a monomer composition containing a polyfunctional monomer having a plurality of N-substituted acrylamide groups in an aqueous medium, and polymerized the monomer composition, so that the acrylamide monomer can be polymerized. The present inventors have found that polymer particles can be produced by a normal phase suspension polymerization method.

  That is, the present invention includes a step of dispersing a monomer composition containing a polyfunctional monomer having a plurality of N-substituted acrylamide groups in an aqueous medium, and a step of polymerizing the dispersed monomer composition. The present invention provides a method for producing polymer particles.

Moreover, this invention provides the polymer particle manufactured by the said manufacturing method.
Furthermore, the present invention provides a packing material for a chromatography column using the polymer particles as a carrier.
Furthermore, the present invention provides a packing material for affinity chromatography in which a ligand is immobilized on the polymer particles.
Furthermore, this invention provides the chromatography column with which the said filler is packed.

  According to the present invention, polymer particles can be produced from an acrylamide monomer by a normal phase suspension polymerization method.

  The method for producing polymer particles of the present invention comprises a step of dispersing a monomer composition (also referred to as a polymerizable composition) containing a polyfunctional monomer having a plurality of N-substituted acrylamide groups in an aqueous medium and polymerizing the monomer composition. It is characterized by including. The said monomer composition means the composition containing a monomer.

The polyfunctional monomer used in the present invention has a plurality of N-substituted acrylamide groups. As the N-substituted acrylamide group possessed by the polyfunctional monomer, an acrylamide group in which an alkyl group having 1 to 3 carbon atoms is substituted with a nitrogen atom is preferable from the viewpoint of obtaining polymer particles with less gel adhesion.
Further, the number of N-substituted acrylamide groups is preferably 2 to 6, more preferably 2 to 4, and particularly preferably 2 from the viewpoint of obtaining polymer particles with less gel adhesion.

  Preferable specific examples of the polyfunctional monomer include those represented by the following formula (1). By using such a polyfunctional monomer, polymer particles can be efficiently obtained by a normal phase suspension polymerization method.

[In the formula (1), R ¹ represents an alkylene group having 2 to 8 carbon atoms, R ² and R ³ represent an alkyl group having 1 to ³ carbon atoms, and R ² and R ³ are adjacent together. A heterocycle may be formed together with the nitrogen atom and R ¹ . However, the total number of carbon atoms of R ¹ , R ² and R ³ is 5 or more. ]

Here, the alkylene group having 2 to 8 carbon atoms represented by R ¹ may be linear or branched. For example, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1 -Diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3-diyl group, butane -1,4-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7- A diyl group, an octane-1,8-diyl group, etc. are mentioned. Carbon number of an alkylene group becomes like this. Preferably it is 3-8, More preferably, it is 3-6.

The alkyl group having 1 to 3 carbon atoms represented by R ² and R ^3, preferably a methyl group or an ethyl group, more preferably R ² and R ³ are both methyl or ethyl, is ethyl group Is more preferable.

R ² and R ³ together may form a heterocyclic ring with the adjacent nitrogen atom and R ¹ . Such heterocyclic groups include 2-methylpiperazine-1,4-diyl group, 2-ethylpiperazine-1,4-diyl group, 2,5-dimethylpiperazine-1,4-diyl group, 2,5- Diethylpiperazine-1,4-diyl group, 1,4-perhydrodiazepine-1,4-diyl group, 6-methyl-1,4-perhydrodiazepine-1,4-diyl group, 6-ethyl- Examples include 1,4-perhydrodiazepine-1,4-diyl group.

The total number of carbon atoms of R ¹ , R ² and R ³ is 5 or more, but is preferably 5 to 15 and more preferably 5 from the viewpoint of efficiently obtaining polymer particles by the normal phase suspension polymerization method. It is -12, More preferably, it is 6-10, Most preferably, it is 7-9.

Moreover, as a suitable specific example of the polyfunctional monomer used in the present invention, N, N′-diethyl-1,3-bis (acrylamide) propane (DEAAP) [solubility parameter: 22.6 (MPa) ^1/2 ] N, N′-diethyl-1,6-bis (acrylamide) hexane (DEAAH) [21.7 (MPa) ^1/2 ], 1,4-bis (acryloyl) piperazine (BAP) [23.6 (MPa) ) ^1/2 ], N, N′-diethyl-1,4-bis (acrylamide) butane [22.3 (MPa) ^1/2 ], N, N′-diethyl-1,5-bis (acrylamide) pentane [22.0 (MPa) ^1/2 ], N, N′-dimethyl-1,3-bis (acrylamide) propane [23.4 (MPa) ^1/2 ], N, N′-dimethyl-1,6 -Bis (acrylamide) hexane [22.3 ( Pa) ^1/2], N, N'-dimethyl-1,4-bis (acrylamide) butane [23.0 (MPa) ^1/2], N, N'-dimethyl-1,5-bis (acrylamide) Pentane [22.6 (MPa) ^1/2 ] and the like can be mentioned, and these can be used alone or in admixture of two or more.

  Here, in this specification, the solubility parameter means the SP value calculated by the fedors method, and the solubility parameter δ of the compound is obtained by the following equation.

δ = (ΔE / ΔV) ^1/2 (MPa) ^1/2

(In the formula, ΔE represents the evaporation energy (J / mol), and ΔV represents the molar volume (cm ³ / mol) at 25 ° C.)

The solubility parameter δ of the polyfunctional monomer is preferably 10.0 (MPa) ^1/2 or more and 30.0 (MPa) ^1/2 or less from the viewpoint of obtaining polymer particles with little gel adhesion. (MPa) ^1/2 or more and 25.0 (MPa) ^1/2 or less are more preferable, 17.5 (MPa) ^1/2 or more and less than 24.0 (MPa) ^1/2 are more preferable, and 20.0 (MPa) ) ^1/2 or more and 23.5 (MPa) ^1/2 or less are more preferable, and 21.0 (MPa) ^1/2 or more and 23.0 (MPa) ^1/2 or less are more preferable.

  In addition, the amount of the polyfunctional monomer used is preferably 20 to 99% by mass, and preferably 50 to 90% by mass with respect to the total amount of monomers, from the viewpoint of mechanical strength of the polymer particles, particularly pressure performance when used as chromatographic column particles. % Is more preferable.

In addition, the monomer composition used in the present invention may contain another monomer copolymerizable with the polyfunctional monomer having a plurality of N-substituted acrylamide groups. As such other monomer, any of a non-crosslinkable (monofunctional) monomer and a crosslinkable (polyfunctional) monomer can be used, and these may be used in combination. The solubility parameter δ of the other monomer is preferably 10.0 (MPa) ^1/2 or more and 29.0 (MPa) ^1/2 or less, more preferably 15 from the viewpoint of obtaining polymer particles with less gel adhesion. 0.0 (MPa) ^1/2 or more and 27.5 (MPa) ^1/2 or less, more preferably 16.5 (MPa) ^1/2 or more and 26.0 (MPa) ^1/2 or less, more preferably Is 17.5 (MPa) ^1/2 or more and 24.0 (MPa) ^1/2 or less.

  As the non-crosslinkable monomer, (meth) acrylamide monomers, styrene monomers, N-vinylamide monomers, vinyl ketone monomers, and acrylonitrile monomers are preferable.

Examples of the (meth) acrylamide monomer include dimethylacrylamide (DMAAM) [23.9 (MPa) ^1/2 ], diethylacrylamide (DEAAM) [20.7 (MPa) ^1/2 ], and propylacrylamide [22.0. (MPa) ^1/2 ], isopropylacrylamide [21.6 (MPa) ^1/2 ], acryloylmorpholine [28.0 (MPa) ^1/2 ], acryloylpyrrolidine [27.9 (MPa) ^1/2 ], And acryloylpiperidine [25.8 (MPa) ^1/2 ].

  Among the above (meth) acrylamide monomers, those represented by the following formula (3) are preferable.

[In formula (3), R ⁴ represents a hydrogen atom or a methyl group, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms R ⁵ and R ⁶ independently. ]

R ⁴ is preferably a hydrogen atom. R ⁵ and R ⁶ are preferably an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, and an isopropyl group. The number of carbon atoms of the alkyl group is preferably 1 or 2.

Examples of the styrene monomer include styrene [18.9 (MPa) ^1/2 ], α-methylstyrene [18.8 (MPa) ^1/2 ], halogenated styrene [20.0-21.8 ( MPa) ^1/2 ] (fluorostyrene [20.0 (MPa) ^1/2 ], chlorostyrene [21.0 (MPa) ^1/2 ], bromostyrene [21.2 (MPa) ^1/2 ], iodine Styrene [21.8 (MPa) ^1/2 ]), 4-methoxystyrene [19.2 (MPa) ^1/2 ], 4-ethoxystyrene [19.0 (MPa) ^1/2 ], 1,2- And dihydroxy-3- (4-vinylbenzyloxy) propane [28.0 (MPa) ^1/2 ].
Examples of the N-vinylamide monomer include N-vinylformamide [26.1 (MPa) ^1/2 ], N-vinylacetamide [23.5 (MPa) ^1/2 ], and N-vinylpropamide [22. .6 (MPa) ^1/2 ], N-vinylbutamide [22.0 (MPa) ^1/2 ], N-methyl-N-vinylformamide [21.7 (MPa) ^1/2 ], N-methyl-N -Vinylacetamide [19.5 (MPa) ^1/2 ], N-methyl-N-vinylpropamide [19.3 (MPa) ^1/2 ], N-methyl-N-vinylbutamide [19.1 (MPa) ^1/2 ], N-ethyl-N-vinylformamide [21.1 (MPa) ^1/2 ], N-ethyl-N-vinylacetamide [19.3 (MPa) ^1/2 ], N-ethyl-N -Vinylpropamide [19.1 (MPa) ^1/2 ], N-eth Ru-N-vinylbutamide [18.9 (MPa) ^1/2 ], N-propyl-N-vinylformamide [20.7 (MPa) ^1/2 ], N-propyl-N-vinylacetamide [19.1 ( MPa) ^1/2 ], N-propyl-N-vinylpropamide [18.9 (MPa) ^1/2 ], N-propyl-N-vinylbutamide [18.8 (MPa) ^1/2 ], N-isopropyl -N-vinylformamide [20.3 (MPa) ^1/2 ], N-isopropyl-N-vinylacetamide [18.8 (MPa) ^1/2 ], N-isopropyl-N-vinylpropamide [18.7] (MPa) ^1/2 ], N-isopropyl-N-vinylbutamide [18.6 (MPa) ^1/2 ] and the like.

Examples of the vinyl ketone monomer include acrolein [21.6 (MPa) ^1/2 ], methyl vinyl ketone [17.7 (MPa) ^1/2 ], and ethyl vinyl ketone [17.7 (MPa) ^1/2]. ], Propyl vinyl ketone [17.7 (MPa) ^1/2 ], isopropyl vinyl ketone [17.2 (MPa) ^1/2 ], methacrylolein [21.0 (MPa) ^1/2 ], isopropenyl methyl ketone [17.8 (MPa) ^1/2 ], isopropenyl ethyl ketone [17.7 (MPa) ^1/2 ], isopropenyl propyl ketone [17.7 (MPa) ^1/2 ], isopropenyl isopropyl ketone [17 .3 (MPa) ^1/2 ] and the like.

Examples of the acrylonitrile-based monomer include acrylonitrile [22.7 (MPa) ^1/2 ], methacrylonitrile [22.0 (MPa) ^1/2 ], and the like.
Among these non-crosslinkable monomers, (meth) acrylamide monomers are preferable when improving alkali resistance.

Moreover, as said crosslinkable monomer, a bifunctional monomer is preferable and an epoxy group containing unsaturated monomer is more preferable. For example, glycidyl methacrylate (GMA) [19.5 (MPa) ^1/2 ], 3,4-epoxycyclohexylmethyl methacrylate (M100) [18.5 (MPa) ^1/2 ], (4-vinylbenzyl) glycidyl ether (VBGE) [19.9 (MPa) ^1/2 ], glycidyl acrylate [19.7 (MPa) ^1/2 ], allyl glycidyl ether [16.9 (MPa) ^1/2 ] and the like. As bifunctional monomers, hydroxyl-containing unsaturated monomers such as hydroxyethyl acrylamide [28.3 (MPa) ^1/2 ], hydroxypropyl acrylamide [28.1 (MPa) ^1/2 ]; diacetone acrylamide [21.9 ( MPa) ^1/2 ], 2-acetoacetoxyethyl methacrylate [21.1 (MPa) ^1/2 ] and other carbonyl group-containing unsaturated monomers; methacrylic acid [22.0 (MPa) ^1/2 ], 4-vinyl Carboxylic acid group-containing unsaturated monomers such as benzoic acid [23.6 (MPa) ^1/2 ]; crosslinkable unsaturated monomers such as divinylbenzene [19.0 (MPa) ^1/2 ] can also be used.

  In addition, when improving the alkali tolerance of particle | grains, the thing which does not have a carboxylic ester bond is preferable among the other monomers copolymerizable with the above polyfunctional monomers.

The combination of the monomers is preferably a combination of the polyfunctional monomer having a plurality of N-substituted acrylamide groups, the non-crosslinkable monomer, and the crosslinkable monomer. Above all, a combination of a polyfunctional monomer having a plurality of N-substituted acrylamide groups, a (meth) acrylamide monomer and an epoxy group-containing unsaturated monomer, a polyfunctional monomer having a plurality of N-substituted acrylamide groups, an N-vinylamide monomer and an epoxy. A combination of a group-containing unsaturated monomer, a combination of a polyfunctional monomer having a plurality of N-substituted acrylamide groups, a vinyl ketone monomer and an epoxy group-containing unsaturated monomer, and a polyfunctional monomer having a plurality of N-substituted acrylamide groups; A combination of a (meth) acrylamide monomer and an epoxy group-containing unsaturated monomer is more preferred.
Moreover, when using a non-crosslinkable monomer and a crosslinkable monomer together, the usage-amount of a noncrosslinkable monomer is 1-80 mass parts with respect to 100 mass parts of polyfunctional monomers which have two or more said N substituted acrylamide groups. Preferably, 1.5-50 mass parts is more preferable, and 10-40 mass parts is still more preferable.
On the other hand, the use amount of the crosslinkable monomer is preferably 1 to 80 parts by mass, more preferably 5 to 25 parts by mass, and 5 to 20 parts by mass with respect to 100 parts by mass of the polyfunctional monomer having a plurality of N-substituted acrylamide groups. Further preferred.

  Moreover, as said monomer composition, what contains the organic solvent is preferable. Any organic solvent may be used as long as it contains at least one polyfunctional monomer having a plurality of N-substituted acrylamide groups. By using an organic solvent that acts as a porous agent, porous polymer particles can be used. Can be manufactured.

Such organic solvents include hexane [solubility parameter: 15.0 (MPa) ^1/2 ], heptane [15.2 (MPa) ^1/2 ], octane [15.5 (MPa) ^1/2 ], Aliphatic hydrocarbons such as nonane [15.6 (MPa) ^1/2 ], decane [15.8 (MPa) ^1/2 ], undecane [15.9 (MPa) ^1/2 ]; cyclohexane [8. Alicyclic hydrocarbons such as 1 (MPa) ^1/2 ] and cyclopentane [8.1 (MPa) ^1/2 ]; benzene [21.2 (MPa) ^1/2 ], toluene [18.2 ( MPa) ^1/2 ], xylene [18.6 (MPa) ^1/2 ], naphthalene [21.2 (MPa) ^1/2 ], ethylbenzene [18.5 (MPa) ^1/2 ], etc. hydrogen like; carbon tetrachloride [24.9 (MPa) ^1/2], halogen such as tetrachloroethane [23.8 (MPa) ^1/2] Hydrocarbons; 1-butanol [23.2 (MPa) ^1/2], 1-pentanol [22.4 (MPa) ^1/2], 1-hexanol [21.9 (MPa) ^1/2] 1-heptanol [21.4 (MPa) ^1/2 ], 1-octanol [20.0 (MPa) ^1/2 ], 4-methyl-2-pentanol [18.9 (MPa) ^1/2 ] Aliphatic alcohols such as 2-ethyl-1-hexanol [18.8 (MPa) ^1/2 ]; alicyclic alcohols such as cyclohexanol [34.4 (MPa) ^1/2 ]; 2-phenyl Aromatic alcohols such as ethyl alcohol [22.5 (MPa) ^1/2 ] and benzyl alcohol [23.3 (MPa) ^1/2 ]; diethyl ketone [17.2 (MPa) ^1/2 ], methyl isobutyl ketone [16.9 (MPa) ^1/2], diisobutyl ketone [1 .8 (MPa) ^1/2], acetophenone [20.9 (MPa) ^1/2], 2-octanone [17.4 (MPa) ^1/2], cyclohexanone [20.1 (MPa) ^1/2] Ketones such as: dibutyl ether [15.6 (MPa) ^1/2 ], diisobutyl ether [15.2 (MPa) ^1/2 ], anisole [19.2 (MPa) ^1/2 ], ethoxybenzene [19 Ethers such as 0.0 (MPa) ^1/2 ]; butyl acetate [17.6 (MPa) ^1/2 ], isopentyl acetate [17.4 (MPa) ^1/2 ], 3-methoxybutyl acetate [19 .4 (MPa) ^1/2 ], esters such as diethyl malonate [19.5 (MPa) ^1/2 ], and linear polymers such as homopolymers of non-crosslinkable vinyl monomers. These organic solvents can be used alone or in admixture of two or more.
Among these, ketones, aromatic alcohols, and alicyclic hydrocarbons are preferable from the viewpoint of efficiently obtaining polymer particles by a normal phase suspension polymerization method.

The organic solvent is preferably one containing at least one organic solvent having a solubility parameter δ of 15 (MPa) ^1/2 or more from the viewpoint of obtaining polymer particles with little gel adhesion, and 17 (MPa) ^1/2. What contains 1 or more types of the organic solvent which is the above is more preferable, and what contains 1 or more types of the organic solvent which is 17-30 (MPa) ^1/2 is more preferable. Further, from the viewpoint of obtaining polymer particles with little gel adhesion as described above, an organic solvent having a solubility parameter δ of 19 (MPa) ^1/2 or more and an organic solvent having a solubility parameter δ of less than 19 (MPa) ^1/2 A mixed liquid of an organic solvent having a solubility parameter δ of 17 to 19 (MPa) ^1/2 or more and an organic solvent having a solubility parameter δ of less than 20 to 30 (MPa) ^1/2 is more preferable.

The solubility parameter of the organic solvent [delta] ([delta] _mix to be described later in the case of mixture), 15~30 (MPa) ^1/2 is preferable, 17~30 (MPa) ^1/2, more preferably, 17 to 26 (MPa ) ^1/2 is more preferable, and 17 to 22 (MPa) ^1/2 is more preferable.
In addition, the upper limit of the absolute value of the difference between the solubility parameter δ of a polyfunctional monomer having a plurality of N-substituted acrylamide groups and the solubility parameter δ of an organic solvent (δ _mix described later in the case of a mixed solution) is a porous material with little gel adhesion From the viewpoint of obtaining polymer particles, 5.8 (MPa) ^1/2 or less is more preferable, less than 5.2 (MPa) ^1/2 is more preferable, and less than 4.9 (MPa) ^1/2 is more preferable. .5 (MPa) ^1/2 or less is particularly preferable. On the other hand, the lower limit thereof is preferably 1.7 (MPa) ^1/2 or more, more preferably 2.5 (MPa) ^1/2 or more, from the viewpoint of obtaining porous particles.

The solubility parameter of the organic solvent can be calculated in the same manner as in the polyfunctional monomer. However, when the organic solvent is a mixed liquid composed of two or more kinds, the solubility parameter δ _mix is obtained by the following equation.

δ _mix = (Σδ _i ² φ _i ) ^1/2 (MPa) ^1/2

(Wherein, [delta] _i represents the solubility parameter of each organic solvent constituting the mixture, phi _i denotes the volume fraction of the organic solvent. In addition, phi _i is as the volume of solvent that is soluble additive The calculation was performed assuming that the total volume of the solvent = the sum of the volumes of the respective solvents was established.)

  When an organic solvent is used, the amount used is usually about 50 to 1000 parts by weight, preferably 100 to 600 parts by weight, and more preferably 150 to 400 parts by weight with respect to 100 parts by weight of the total amount of monomers. It is.

Moreover, the manufacturing method of this invention disperse | distributes the said monomer composition in an aqueous medium.
The aqueous medium is not particularly limited as long as it contains water, and examples thereof include a water-soluble polymer aqueous solution. Examples of the water-soluble polymer include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, starch, gelatin and the like.
The usage-amount of an aqueous medium is about 100-10000 mass parts normally with respect to 100 mass parts of monomer composition total amount, Preferably it is 500-6000 mass parts, More preferably, it is 500-3000 mass parts.

  Moreover, when using water as a dispersion medium of an aqueous medium, you may use dispersion stabilizers, such as a calcium carbonate, sodium sulfate, sodium nitrite, a calcium phosphate, sodium chloride, for example. The usage-amount of a dispersion stabilizer is about 0.1-5 mass parts normally with respect to 100 mass parts of water.

  Moreover, the manufacturing method of this invention superposes | polymerizes the monomer composition disperse | distributed above. “Polymerize the monomer composition” means that the monomer contained in the monomer composition is polymerized. Such polymerization is usually performed in the presence of a polymerization initiator.

  The polymerization initiator is preferably a radical polymerization initiator. Examples of radical polymerization initiators include azo initiators, peroxide initiators, redox initiators, and the like. Specifically, azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2, Examples include 4-dimethylvaleronitrile, benzoyl oxide, di-tert-butyl peroxide, benzoyl peroxide-dimethylaniline, and the like. The usage-amount of a polymerization initiator is about 0.1-5 mass parts normally with respect to 100 mass parts of monomer total amounts.

In addition, an anionic surfactant such as an alkyl sulfate ester salt, an alkylaryl sulfate ester salt, an alkyl phosphate ester salt, and a fatty acid salt as well as various surfactants can be added as an emulsifier in the reaction system.
It is also possible to add a polymerization inhibitor such as tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride or ferric chloride, and a polymerization aid such as dodecyl mercaptan.

  The polymerization temperature may be determined according to the polymerization initiator. A preferable polymerization temperature is preferably within 20 ° C in the upper and lower directions, more preferably within 10 ° C in the upper and lower directions around the 10-hour half-life temperature + 20 ° C. For example, when azobisisobutyronitrile is used as the polymerization initiator, the 10-hour half-life is about 60 ° C., and therefore, it is preferably 60 to 100 ° C. within 20 ° C. around 80 ° C. 90 ° C. is more preferable. In addition, when heating, a polymerization initiator may be added before a heating and may be added after a heating.

The polymerization time is usually 2 to 20 hours, preferably 3 to 10 hours.
In addition, the polymer particle produced | generated by making it superpose | polymerize can be obtained by removing a porous agent and an unreacted monomer by distillation, extraction, washing | cleaning, etc. as needed.

According to the present invention, polymer particles can be produced from an acrylamide monomer by a normal phase suspension polymerization method. That is, according to the production method of the present invention, the particle size and pore size of the polymer particles derived from the acrylamide monomer can be easily adjusted as compared with the case of polymerization by reverse phase suspension polymerization.
In addition, the porous polymer particles produced by such a production method have high hydrophilicity and are difficult to cause nonspecific adsorption of biopolymers and the like, and thus are useful as a carrier for chromatography.
In particular, the polymer particle of the present invention is extremely useful as a protein-binding particle to which a protein (for example, an immunoglobulin-binding protein such as protein A) as a primary probe such as an antigen or an antibody is bound.

The packing material for a chromatography column of the present invention uses the polymer particles as a carrier. Such fillers are suitable for use in affinity chromatography. Examples of such a filler include those in which a ligand is immobilized on the polymer particles. The type of the ligand is not particularly limited as long as it has an appropriate affinity for the target. For example, proteins such as protein A, protein G, and avidin; peptides such as insulin; antibodies such as monoclonal antibodies Enzyme; hormone; DNA; RNA; carbohydrates such as heparin, Lewis X, ganglioside; iminodiacetic acid, synthetic dye, 2-aminophenylboric acid, 4-aminobenzamidine, glutathione, biotin and derivatives thereof Compounds can be used. Although the ligand illustrated above may use the whole, the fragment and fusion obtained by a recombinant, an enzyme treatment, etc. may be used. Further, it may be an artificially synthesized peptide or peptide derivative. Suitable ligands for the separation or purification of immunoglobulins are, for example, protein A and protein G, and more preferably the immunoglobulin binding domain of protein A.
Moreover, the chromatography column of the present invention is filled with the above-mentioned packing material. The column is suitable for use in affinity chromatography.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

Each analysis condition in the examples is as follows.
<Average particle size, coefficient of variation>
The average particle size (volume average particle size) and coefficient of variation were measured using a flow type particle image analyzer (model number FPIA-3000) manufactured by Sysmex Corporation for a sample diluted with water so that the solid content concentration was about 2.5%. It was determined by setting the total count to 500.
<Specific surface area>
The specific surface area was measured on a sample obtained by vacuum drying in a tert-butyl alcohol solvent, using an autopore IV9520 manufactured by Shimadzu Corporation under conditions in which the pore diameter range was 10 to 5000 nm.

Example 1
0.9 g of (4-vinylbenzyl) glycidyl ether (manufactured by Toray Fine Chemical), 0.9 g of N, N-dimethylacrylamide (manufactured by Kojin), 0.9 g of N, N-diethylacrylamide (manufactured by Kojin) 6.1 g of N, N′-diethyl-1,3-bis (acrylamide) propane (manufactured by JSR), 7: 3 of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and acetophenone (manufactured by Inoue Fragrance Co., Ltd.) An organic monomer solution was prepared by dissolving in 18.1 g of a mixed solution (mass ratio).
Meanwhile, 2.5 g of polyvinyl alcohol (manufactured by Kuraray: PVA-217), 0.2 g of sodium dodecyl sulfate (manufactured by Kao Corporation: Emar 10G), 2.5 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and sodium nitrite ( 1.2 g of Wako Pure Chemical Industries, Ltd.) was added to 245 g of pure water and stirred to prepare an aqueous solution. This aqueous solution was put into a 500 mL separable flask, a thermometer, a stirring blade and a cooling tube were attached, set in a hot water bath, and stirring was started at 360 rpm in a nitrogen atmosphere. Next, the above organic monomer solution was added to a separable flask and heated with a hot water bath. When the liquid temperature reached 85 ° C., 2,2′-azobisisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) 0. 1 g was added and stirred for 3.5 hours. After the reaction product was cooled, it was transferred to a 500 mL polypropylene bottle, the polymer particles were allowed to settle, the supernatant was discarded by decantation, and the remaining polymer particles were washed with water and ethanol.
The obtained polymer particles were porous particles having an average particle size of 46 μm, a coefficient of variation of 15%, and a specific surface area of 94 m ² / g.

Example 2
Instead of N, N′-diethyl-1,3-bis (acrylamide) propane, N, N′-diethyl-1,6-bis (acrylamide) hexane (manufactured by JSR) was used, and 2-octanone and acetophenone Polymer particles were produced in the same manner as in Example 1 except that 2-octanone was used instead of the mixed solution and the stirring speed was changed to 280 rpm.
The obtained polymer particles were porous particles having an average particle size of 39 μm, a coefficient of variation of 14%, and a specific surface area of 62 m ² / g.

Example 3
Instead of (4-vinylbenzyl) glycidyl ether, 3,4-epoxycyclohexylmethyl methacrylate (manufactured by Daicel: Cyclomer M100) was used, and the mass ratio of 2-octanone and acetophenone mixture was changed from 7: 3 to 6: 4. In addition to the polyvinyl alcohol, sodium dodecyl sulfate, sodium sulfate and sodium nitrite, an aqueous solution is prepared using 0.5 g of polyvinyl alcohol (Kuraray Co., Ltd .: PVA-405), and an organic monomer solution is added. Polymer particles were produced in the same manner as in Example 1 except that the temperature was changed from room temperature to 85 ° C. and the stirring speed was changed to 375 rpm.
The obtained polymer particles were porous particles having an average particle size of 52 μm, a coefficient of variation of 15%, and a specific surface area of 94 m ² / g.

Example 4
Instead of 3,4-epoxycyclohexylmethyl methacrylate, 0.4 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.) was used, and the amount of N, N-dimethylacrylamide was changed to 1.1 g. The same procedure as in Example 3 except that the amount used was changed to 1.1 g, the mass ratio of the mixed liquid of 2-octanone and acetophenone was changed from 6: 4 to 4: 6, and the stirring speed was changed to 345 rpm. Polymer particles were produced.
The obtained polymer particles were porous particles having an average particle size of 56 μm, a coefficient of variation of 12%, and a specific surface area of 97 m ² / g.

Example 5
The monomer was changed to 0.9 g of (4-vinylbenzyl) glycidyl ether, 1.8 g of N, N-dimethylacrylamide, and 6.1 g of N, N′-diethyl-1,3-bis (acrylamide) propane. -Use acetophenone instead of the mixed solution of octanone and acetophenone, change the amount of polyvinyl alcohol used to 0.5 g, change the amount of sodium dodecyl sulfate and sodium sulfate to 0.1 g each, and stirrer speed to 330 rpm Polymer particles were produced in the same manner as in Example 1 except for changing to.
The obtained polymer particles were porous particles having an average particle size of 103 μm, a coefficient of variation of 39%, and a specific surface area of 21.3 m ² / g.

Example 6
0.6 g of (4-vinylbenzyl) glycidyl ether, 1.3 g of N, N-diethylacrylamide and 4.4 g of 1,4-bis (acryloyl) piperazine (manufactured by Bio-Rad) were dissolved in 26.0 g of acetophenone, An organic monomer solution was prepared.
On the other hand, 1.5 g of polyvinyl alcohol, 0.1 g of sodium dodecyl sulfate, 2.6 g of sodium sulfate and 1.3 g of sodium nitrite were added to 263 g of pure water and stirred to prepare an aqueous solution.
Polymer particles were produced in the same manner as in Example 1, except that the organic monomer solution and the aqueous solution were used, and the stirring speed was changed to 450 rpm.
The polymer particles obtained had an average particle size of 55 μm, a coefficient of variation of 26%, and a specific surface area of 100 m ² / g.

Example 7
N, N-dimethylacrylamide is used instead of N, N-dimethylacrylamide, and 8: 2 mixed solution of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and acetophenone (manufactured by Inoue Fragrance Co., Ltd.) instead of acetophenone ( Polymer particles were produced in the same manner as in Example 5 except that (mass ratio) was used.
The obtained polymer particles had an average particle size of 70 μm, a coefficient of variation of 38%, and a specific surface area of 100 m ² / g.

Example 8
Polymer particles were produced in the same manner as in Example 7, except that 2-octanone was used instead of the mixed liquid of 2-octanone and acetophenone.
The polymer particles obtained had an average particle size of 76 μm, a coefficient of variation of 48%, and a specific surface area of 27.5 m ² / g.

Example 9
Polymer particles were produced in the same manner as in Example 8, except that diisobutyl ketone was used instead of 2-octanone.
The obtained polymer particles had an average particle size of 90 μm, a coefficient of variation of 35%, and a specific surface area of 36.3 m ² / g.

Example 10
Polymer particles were produced in the same manner as in Example 8 except that isopentyl acetate was used instead of 2-octanone.
The obtained polymer particles had an average particle size of 79.0 μm, a coefficient of variation of 42%, and a specific surface area of 25.3 m ² / g.

Example 11
Polymer particles were produced in the same manner as in Example 2 except that diisobutyl ketone was used instead of 2-octanone.
The obtained polymer particles had an average particle size of 70.3 μm, a coefficient of variation of 29.1%, and a specific surface area of 8.5 m ² / g.

Test example 1
The surfaces of the polymer particles obtained in Examples 1 to 11 were observed with an SEM and evaluated according to the following criteria. The “absolute value” in the table means the absolute value of the difference between the solubility parameter of the polyfunctional monomer and the solubility parameter of the organic solvent.

(Evaluation criteria for polymer particles)
(Double-circle): The adhesion of gel is hardly seen and it is porous.
○: Almost no adhesion of gel, but almost no pores.
Δ: Gel adhesion is observed.

Comparative Example Instead of N, N′-diethyl-1,3-bis (acrylamide) propane, N, N′-dimethyl-bis (acrylamide) ethane (manufactured by JSR) was used, and a mixed liquid of 2-octanone and acetophenone. The organic monomer solution was prepared in the same manner as in Example 1 except that the mass ratio was changed from 7: 3 to 5: 5.
Next, the aqueous solution prepared in the same manner as in Example 1 was put into a 500 mL separable flask, a thermometer, a stirring blade and a cooling pipe were attached, set in a hot water bath, and stirring was started at 300 rpm in a nitrogen atmosphere. Then, the above organic monomer solution was added thereto, heated with a hot water bath, and when the liquid temperature reached 85 ° C., 0.1 g of 2,2′-azobisisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) was added. did.
As a result, N, N′-dimethyl-bis (acrylamide) ethane was dissolved without being dispersed in the aqueous solution, and normal phase suspension polymerization could not be performed.

Claims (15)

It has 2 to 6 N-substituted acrylamide groups in which an alkyl group having 1 to 3 carbon atoms is substituted with a nitrogen atom, and the solubility parameter is 20.0 (MPa) ^1/2 or more and 24.0 (MPa) ^1/2 the polyfunctional monomer is less than, the polyfunctional monomer contains an organic solvent at least one of dissolving, the absolute value of the difference between the solubility parameter of the solubility parameter and the organic solvent of the polyfunctional monomer is 5.8 ( the MPa) ^1/2 or less der Ru monomer composition, a step of dispersing in an aqueous medium,
And a step of subjecting the dispersed monomer composition to normal phase suspension polymerization. A method for producing polymer particles.   The production method according to claim 1, wherein the polyfunctional monomer has 2 to 4 N-substituted acrylamide groups.   The production method according to claim 1 or 2, wherein the polyfunctional monomer has two N-substituted acrylamide groups. The manufacturing method according to any one of claims 1 to 3, wherein the solubility parameter of the organic solvent is 17 (MPa) ^1/2 or more. Wherein the organic solvent is one solubility parameter of 19 (MPa) solubility parameter between the organic solvent is ^1/2 or higher 19 (MPa) is a mixture of an organic solvent is less than ^1/2 claims 1-4 1 The production method according to item. An organic compound containing a polyfunctional monomer represented by the following formula (1) and one or more organic solvents that dissolve the polyfunctional monomer , wherein the organic solvent has a solubility parameter of 19 (MPa) ^1/2 or more. the mixture der Ru monomer composition of an organic solvent solvent solubility parameter is 19 (MPa) of less than ^1/2, the step of dispersing in an aqueous medium,
And a step of subjecting the dispersed monomer composition to normal phase suspension polymerization. A method for producing polymer particles.

Wherein (1), R ¹ represents an alkylene group having 2 to 8 carbon atoms, R ² and R ³ shows the alkyl group having 1 to 3 carbon atoms. However, the total number of carbon atoms of R ¹ , R ² and R ³ is 5 or more. ] The monomer composition of claim 1 to 6 containing a monomer selected from (meth) acrylamide monomer, a styrene monomer, N- vinyl amide monomers, vinyl ketone monomers, acrylonitrile monomers and an epoxy group-containing unsaturated monomer The manufacturing method of any one of Claims. The production method according to claim 7 , wherein the (meth) acrylamide monomer is represented by the following formula (3).

[In formula (3), R ⁴ represents a hydrogen atom or a methyl group, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms R ⁵ and R ⁶ independently. ]   The packing material for chromatography columns which uses the polymer particle manufactured by the manufacturing method of Claim 1 or 2 as a support | carrier.   A packing material for affinity chromatography in which a ligand is immobilized on polymer particles produced by the production method according to claim 1. The packing material for affinity chromatography according to claim 10 , wherein the ligand is an immunoglobulin binding protein. A chromatography column packed with the packing material according to any one of claims 9 to 11 . The manufacturing method of the filler for chromatography columns including the process of manufacturing a polymer particle by the manufacturing method of any one of Claims 1-8 , and the process of fix | immobilizing a ligand to the obtained polymer particle. The production method according to claim 13 , wherein the ligand is an immunoglobulin binding protein. A method for producing a chromatography column, comprising a step of producing a packing material for a chromatography column by the production method according to claim 13 or 14 , and a step of filling the obtained packing material.