JP2000088826A - Manufacture of bulking agent for liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method for a bulking agent for a liquid chromatography free from swelling, shrinkage and prolongation of an equilibration time, excellent in production reproducibility, and having a reduced difference among production lots. SOLUTION: This bulking agent for a liquid chromatography is manufactured by (i) the first process for dispersing into an aqueous dispersive medium a polymerizable mixture containing a hydrohobic cross-linking monomer and a polymerization initiator, (ii) the second process for ploymerizing the dispersion for 20-240 minutes at 50-100 deg.C, (iii) the third process for cooling the reactionsystem of the polymerization to 10-40 deg.C, (iv) the fourth process for adding a cation exchange group containing monomer into the reaction system, (v) the fifth process for stirring the reaction system for 0.5-24 hours at 10-40 deg.C after the addition, (vi) the 6th process for conducting polymerization reaction for the reaction system at 50-100 deg.C, and for finishing polymerization within 20-240 minutes after initiation of tire polymerization at a stage having 0.1-20% of polymerization rate with respect to an addition amount of the cation exchange group containing monomer, and (iiv) the 7th process for washing a resulting polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a packing material for liquid chromatography, and more particularly to a method for producing a packing material for cation exchange liquid chromatography.

[0002]

2. Description of the Related Art Liquid chromatography analysis using a packing material having a cation exchange group such as a carboxyl group, a phosphate group or a sulfonic acid group as an ion exchange group is useful for various bio-related methods, including analysis of glycated hemoglobins. This is a very useful method for analyzing substances.

The following four methods have been disclosed as methods for producing the above-mentioned filler having a cation exchange group. (1) A method of reacting a compound having a cation exchange group with the crosslinkable particles. That is, the inorganic or organic particles having a reactive functional group are reacted with a compound having a group that reacts with the reactive functional group and a cation exchange group, so that the inorganic or organic particles have a cation exchange group. How to introduce. Specifically, for example, JP-A-1-262468 discloses a method in which a carboxyl group-containing compound or a sulfonic acid group-containing compound reacts with an epoxy group in a particle.

(2) A monomer having a cation exchange group is
A method of mixing and copolymerizing with a crosslinkable monomer. For example, Japanese Patent Publication No. 63-59463 discloses a carboxyl group-containing monomer of 5 to 90% by weight, a crosslinkable monomer of 10 to 90% by weight.
A method is disclosed in which 95% by weight and 0 to 85% by weight of a non-crosslinkable monomer are mixed and polymerized.

(3) A method in which a monomer having a cation exchange group is added during the polymerization of the hydrophobic crosslinkable monomer, and the polymerization is further continued. For example, the literature of Y. Ohtsuka et al [J. Applied Polym. S
ci., 27 (1982) 3279-3288] is not a method for producing an ion exchange resin, but ethylene glycol dimethacrylate was used as a hydrophobic crosslinkable monomer, and the monomer was polymerized for 10 to 60 minutes. Thereafter, a cation exchange group-containing monomer such as acrylic acid or methacrylic acid is added to the reaction system, and the polymerization is further carried out for 24 hours.
A time-continuous method is disclosed.

(4) A method in which a cation exchange group-containing monomer is polymerized near the surface of the hydrophobic crosslinked polymer particles. Japanese Patent Publication No. 8-7197 discloses that after a hydrophobic cross-linked polymer particle impregnated with a polymerization initiator is dispersed in an aqueous dispersion medium, a cation exchange group-containing monomer is added and polymerized. A method for polymerizing the monomer is disclosed.

[0007]

The following problems remain in the methods (1) to (4). Method (1) above: In the method of introducing an ionic group by post-treatment,
It is generally known that it is difficult to quantitatively introduce a compound having an ion exchange group, and it is difficult to introduce a monomer-derived ion exchange group as in the methods (2) and (4) above. In comparison, it is inferior in the reproducibility of production (Yoshimaki, Hosoya, Kizen, Tanaka: Chromatography, 16 (1) 7-12 (1995)). -The post-processing method is extremely complicated and requires a long time. As the functional group for reacting with the cation exchange group-containing compound, there are a hydroxyl group, a glycidyl group, an amino group and the like, all of which are hydrophilic groups, and use a large amount of a monomer having these hydrophilic groups. In order to prepare particles by polymerization, the pressure resistance and swelling resistance are poor.

[0008] Method (2):-Compared to the method (1), it is possible to quantitatively contain a cation exchange group in the filler, and the operation is simple. Since it is also present inside, the pressure resistance is inferior, and it takes a long time to equilibrate against a change in the external environment due to a change in eluent, etc., resulting in a drawback that the measurement time is prolonged.

Method (3): In the method of adding a hydrophilic monomer during polymerization, even if polymerization is carried out under the same conditions, reproducibility is poor, which is extremely inconvenient in production. That is, during the polymerization, a polymer mainly derived from a hydrophilic monomer is often aggregated and becomes unusable; when used as a filler for liquid chromatography, performance such as retention time and column pressure is high. Large variation; fine-tuning (eg: optimizing particle size)
Although it is possible, it is very complicated and costly; it has disadvantages such as easy aggregation due to a long polymerization time (24 hours).

Method (4): The procedure is complicated because it cannot be performed in a series of polymerization operations. -It is difficult to impregnate the hydrophobic cross-linked polymer with the polymerization initiator quantitatively, so that the reproducibility of the performance as a filler for liquid chromatography is poor. A continuous method of adding a cation exchange group-containing monomer in the course of polymerization is also described, but since the added monomer is not uniformly dispersed, polymerization is immediately started, The difference is large.

[0011]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above problems, and has as its object to use a method of introducing a cation exchange group by a post-treatment which is complicated and lowers reproducibility, There is no swelling / shrinking due to the presence of many hydrophilic groups inside the particles, no prolongation of the equilibration time, furthermore, without long or complicated operations, and excellent in production reproducibility, the difference between production lots An object of the present invention is to provide a method for producing a small amount of a packing material for liquid chromatography.

[0012]

The process for producing a packing material for liquid chromatography according to the present invention comprises the following steps. (I) a first step of dispersing a polymerizable mixture containing a hydrophobic crosslinkable monomer and a polymerization initiator in an aqueous dispersion medium, and (ii) raising the dispersion obtained in the first step to 50 to 100 ° C. Warm up, 20
(Iii) a third step of cooling the reaction system to 10 to 40 ° C., and (iv)
A fourth step of adding a cation-exchange group-containing monomer to the reaction system;
A fifth step of stirring for 5 to 24 hours, (vi)
The polymerization reaction was carried out by raising the temperature to
A sixth step of terminating the polymerization reaction at a stage of a polymerization rate of 0.1 to 20% of the added amount of the cation exchange group-containing monomer for up to 240 minutes, (vii) adding the obtained polymer to water and And / or a seventh step of washing with an organic solvent.

The details of the present invention will be described below step by step. The first step is a step of dispersing a polymerizable mixture containing a hydrophobic crosslinkable monomer and a polymerization initiator in an aqueous dispersion medium.

(Hydrophobic crosslinkable monomer) The above hydrophobic crosslinkable monomer is a monomer having no ion exchange group or having a very small amount even if it has an ion exchange group. 2 vinyl groups
And more hydrophobic than the cation exchange group-containing monomer used in the fourth step. Examples of such a hydrophobic crosslinkable monomer include styrene derivatives such as divinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, and divinylnaphthalene; ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate 1,3-butylene glycol di (meth) acrylate, 1,6-hexane glycol di (meth) acrylate, neopentyl glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, trimethylolethane tri (meth) acrylate , Trimethylolpropane tri (meth) acrylate, tetramethylolpropane tri (meth) acrylate, tetramethylol methane tri (meth) acrylate, tetramethylol methane tetra Meth) derivatives of (meth) acrylic esters such as acrylate; 1,3-butadiene, isoprene, 1,4
Aliphatic diene compounds such as hexanediene; and derivatives of the above monomers. These may be used as a mixture of two or more.

(Polymerization initiator) As the above-mentioned polymerization initiator,
There is no particular limitation, and a known water-soluble or oil-soluble radical polymerization initiator is used. Specific examples of the polymerization initiator include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; cumene hydroperoxide, benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, o-chlorobenzoyl. Peroxide, acetyl peroxide, t-
Butyl hydroperoxide, t-butyl peroxyacetate, t-butyl peroxyisobutyrate,
3,5,5-trimethylhexanoyl peroxide,
organic peroxides such as t-butylperoxy-2-ethylhexanoate and di-t-butyl peroxide;
2'-azobisisobutyronitrile, 2,2'-azobis (2,4-dimethylvaleronitrile), 4,4'-azobis (4-cyanopentanoic acid), 2,2'-azobis (2-methyl Azo compounds such as butyronitrile) and azobiscyclohexanecarbonitrile; and derivatives of the above-mentioned polymerization initiators.

The amount of the polymerization initiator used is preferably 0.05 to 5 parts by weight based on 100 parts by weight of the hydrophobic crosslinkable monomer. If the amount of the polymerization initiator is less than 0.05 part by weight, the polymerization reaction may be insufficient or the polymerization may take a long time. Things may occur. This polymerization initiator is used by dissolving it in the hydrophobic crosslinkable monomer.

(Polymerizable Mixture) The polymerizable mixture contains a hydrophobic crosslinkable monomer and a polymerization initiator as essential components. If necessary, other monomer (a) and other monomers may be used. The additive (b) and the like may be mixed.

The other monomer (a) is, for example, a non-crosslinkable monomer. Specifically, styrene, α-
Styrene derivatives such as methylstyrene, p-methylstyrene and chloromethylstyrene; vinyl halides such as vinyl chloride; vinyl esters such as vinyl acetate, vinyl propionate and vinyl stearate; methyl acrylate, methyl methacrylate; Ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, 2-ethylhexyl acrylate, 2-methacrylic acid 2-
(Meth) acrylic esters such as ethylhexyl, stearyl acrylate and stearyl methacrylate are exemplified.

The amount of the non-crosslinkable monomer to be used is preferably from 0 to 50 based on 100 parts by weight of the hydrophobic crosslinkable monomer.
Parts by weight.

However, among the non-crosslinkable monomers, hydrophilic monomers such as 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, and glycerol (meth) acrylate having a hydroxyl group; Glycidyl (meth) acrylate having a group;
A cation exchange group-containing monomer used in the step; an anion exchange group-containing monomer and the like are not used. This is because when these are used as materials for composing the filler particles, the pressure resistance and swelling resistance of the filler particles are reduced due to these hydrophilic groups present inside the particles.

Examples of the other additives (b) include a porosifying agent (b-1) and hydrophobic polymer particles (b-
2) and the like, but are not limited thereto, and known additives can be added.

Examples of the above-mentioned porous agent (b-1) include an organic solvent which dissolves the monomer but does not dissolve the polymer. By adding this, the resulting polymer becomes porous. can do. As the porosifying agent (b-1), for example, toluene, xylene, diethylbenzene,
Aromatic hydrocarbons such as dodecylbenzene; saturated hydrocarbons such as hexane, heptane, octane and decane; alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol.

The amount of the above-mentioned porous agent (b-1) to be used is from 0 to 10 based on 100 parts by weight of the above-mentioned hydrophobic crosslinkable monomer.
0 parts by weight is preferred.

The above-mentioned hydrophobic polymer particles (b-2) refer to hydrophobic polymer particles having a uniform particle size distribution, into which the above-mentioned hydrophobic crosslinking monomer and polymerization initiator are absorbed. After that, by performing polymerization, a filler having a uniform particle size distribution can be obtained. Examples of the polymer particles (b-2) include, for example, non-crosslinked polymer particles composed of a homopolymer or a copolymer of the monomers shown in the above other monomers (a). Styrene polymer, styrene-divinylbenzene copolymer, methyl (meth) acrylate polymer, ethyl (meth) acrylate polymer, and the like. As the polymer particles (b-2), crosslinked copolymer particles which are copolymers of the other monomer (a) and the hydrophobic crosslinkable monomer can also be used. In this case, ,
Low crosslinked polymer particles obtained by copolymerizing the ratio of the hydrophobic crosslinkable monomer to 10% by weight or less are preferable.

The method for producing the hydrophobic polymer particles (b-2) may be a known polymerization method, and examples thereof include emulsion polymerization, soap-free polymerization, dispersion polymerization, and suspension polymerization.

The average particle size of the hydrophobic polymer particles (b-2) is preferably from 0.1 to 10 μm, and the variation of the particle size is represented by a coefficient of variation (CV) (= standard deviation ÷ average particle size × 100). It is preferably 15% or less.

The amount of the hydrophobic polymer particles (b-2) used is 0.5 to 100 parts by weight of the hydrophobic crosslinkable monomer.
-100 parts by weight are preferred.

(Aqueous dispersion medium) The aqueous dispersion medium includes
Water or a mixture of water and an appropriate water-soluble organic solvent soluble in water is exemplified. Examples of the water-soluble organic solvent include alcohols such as methanol, ethanol, and isopropanol; acetone; acetonitrile; and the like, but are not particularly limited thereto. In the case of a mixture of a water-soluble organic solvent and water, the organic solvent is 20
% By weight or less is preferred.

The aqueous dispersion medium may contain a dispersant (c) and other additives (d), if necessary. Examples of the dispersant (c) include polymer compounds such as polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, starch, hydroxycellulose, and polyvinyl ether; inorganic salts such as calcium phosphate; sodium lauryl sulfate, sodium lauryl benzene sulfonate;
Ionic surfactants such as sodium polyoxyethylene lauryl sulfate and sodium dialkyl sulfosuccinate; and nonionic surfactants such as polyoxyethylene nonyl phenyl ether, polyethylene glycol monostearate, and sorbitan monostearate. The amount used is from 0.01 to 0.01% of the aqueous dispersion medium.
It is preferably used as a 20% by weight solution.

Examples of the other additives (d) include a pH adjuster; a substance for adjusting the solubility of the monomer used; and an antifoaming agent.

Examples of the pH adjuster include hydrochloric acid,
Inorganic acids such as nitric acid and phosphoric acid; organic acids such as acetic acid; bases such as sodium hydroxide and potassium hydroxide; various inorganic and organic buffers such as phosphate buffer and citrate buffer; Can be

Examples of the substance for adjusting the solubility of the monomer used include salts such as sodium acetate.

Examples of the above-mentioned antifoaming agent include known antifoaming agents such as various surfactants.

The amount of the aqueous dispersion medium to be used is preferably 200 to 10000 parts by weight based on 100 parts by weight of the polymerizable mixture.

The method of dispersing the polymerizable mixture in the aqueous dispersion medium in the first step is not particularly limited. For example, the polymerizable mixture is added to the aqueous dispersion medium and the dispersion is added at 10 to 40 ° C. And a method of stirring in the state described above.
It is preferable that an inert gas is filled or aerated in the dispersion.

The second step is a step in which the temperature of the dispersion obtained in the first step is raised to 50 to 100 ° C. and a polymerization reaction is carried out for 20 to 240 minutes. Hereinafter, this polymerization reaction is referred to as primary polymerization, and the polymerization reaction in the sixth step is referred to as secondary polymerization. The polymerization temperature of the primary polymerization is limited to 50 to 100 ° C. If the temperature is lower than 50 ° C., the polymerization rate is too slow and the polymerization takes too much time. If the temperature is higher than 100 ° C., the polymerization rate is too fast and aggregation tends to occur. The polymerization time of the primary polymerization is 20 to
Limited to 240 minutes. If the polymerization time is less than 20 minutes, the polymerization rate is too low, and when used as a filler for liquid chromatography, the reproducibility of its performance deteriorates. If the time exceeds 240 minutes, the polymerization rate is too high, and the possibility of causing a decrease in the polymerization rate of the secondary polymerization in the sixth step or causing aggregation in the secondary polymerization increases.

In the third step, the polymerization reaction system in the second step is
This is a step of cooling to ４０40 ° C. By cooling to 10 to 40 ° C., the primary polymerization reaction is interrupted before the polymerization is completely completed. "Before the polymerization is completely completed" means that the polymerization rate of the monomer containing the hydrophobic crosslinked polymer is preferably 98% or less, and the polymerization initiator is theoretically
A state in which 5% or more of the added amount remains. In this step, cooling to less than 10 ° C is not preferable because the work becomes complicated and the working time is prolonged, and depending on the contents, precipitation may occur due to a large decrease in solubility. Since the polymerization proceeds gradually, it becomes difficult to control the polymerization rate, and the reproducibility is reduced. The time required for cooling from the temperature at which the polymerization is performed to 10 to 40 ° C. is preferably within 120 minutes, more preferably within 60 minutes.

The fourth step is a step of adding a cation exchange group-containing monomer to the reaction system. The cation exchange group refers to a functional group that exhibits cation exchange ability at a certain pH, and includes, for example, a carboxyl group, a phosphate group, and a sulfonic acid group. The cation exchange group-containing monomer is
A monomer having one or more cation exchange groups and one or more polymerizable functional groups in one molecule. For example, monomers having a carboxyl group include (meth) acrylic acid, crotonic acid, Itaconic acid, citraconic acid, mesaconic acid, maleic acid, fumaric acid and derivatives thereof; examples of the monomer having a phosphate group include ((meth) acryloyloxyethyl) acid phosphate, (2
-(Meth) acryloyloxyethyl) acid phosphate, (3- (meth) acryloyloxypropyl)
Acid phosphate and derivatives thereof; and monomers having a sulfonic acid group include, for example, styrenesulfonic acid, allylsulfonic acid, 2- (meth) acrylamido-2-methylpropanesulfonic acid, (3-sulfopropyl)- Examples include itaconic acid, 3-sulfopropyl (meth) acrylic acid, and derivatives thereof; and salts of the above monomers, such as sodium salts and potassium salts.

In addition to the above-mentioned cation exchange group-containing monomer, the same cation can also be obtained by using a monomer having a functional group that can be converted into a cation exchange group by a chemical reaction and performing the chemical reaction after polymerization. An exchange group containing polymer may be prepared. Examples of the chemical reaction include a hydrolysis reaction and a transfer reaction. Examples of the functional group that can be converted by the chemical reaction include a group that can be converted to a cation exchange group by a hydrolysis reaction, and examples include an ester group. As an example of this method, for example, by using methyl methacrylate as a monomer, after polymerization, by heating under alkalinity to decompose an ester bond and change it into a carboxyl group, a cation exchange group-containing filler is obtained. Can be prepared.

The monomers used in the fourth step may be used as a mixture of plural kinds.

The amount of the monomer used in the fourth step is as follows:
The amount is preferably 10 to 200 parts by weight based on 100 parts by weight of the polymerizable mixture.

The monomer used in the fourth step is added at a reaction system temperature of 10 to 40 ° C. The addition may be performed all at once or may be performed dropwise. In the case of dropping, the addition is preferably completed within 60 minutes.

The fifth step is a step of stirring the reaction system at 10 to 40 ° C. for 0.5 to 24 hours after the completion of the above addition. This step is performed to uniformly disperse the cation exchange group-containing monomer in the aqueous dispersion medium and to stabilize the distribution equilibrium between the primary polymerized particles or the remaining monomers and the aqueous dispersion medium. The stirring time varies depending on the composition of the cation-exchange group-containing monomer or the aqueous dispersion medium to be used.
Limited to 5 to 24 hours. When the time is less than 0.5 hour, the cation exchange group-containing monomer is difficult to be uniform in the reaction system, and as a result, the reproducibility of the polymerization is reduced, and the effect is further improved even if stirring is performed for more than 24 hours. Not only do not
Depending on the type of the monomer, the polymerization proceeds even at 10 to 40 ° C., and there is a possibility that the reproducibility of the production may decrease due to the variation of the progress.

The reason why the temperature at the time of stirring is limited to 10 to 40 ° C. is that cooling to a temperature lower than 10 ° C. complicates the operation, prolongs the operation time, and, depending on the contents, has a large solubility. If the temperature exceeds 40 ° C., the polymerization proceeds gradually, so that the control of the polymerization rate becomes difficult, and the reproducibility decreases.

In the sixth step, the reaction system is heated to 50 to 100 ° C. to carry out a polymerization reaction. The polymerization is carried out for 20 to 240 minutes after the initiation of the polymerization, and the addition amount of the cation exchange group-containing monomer is 0.1%. ~
This is a step of terminating the polymerization reaction at a stage of a polymerization rate of 20%. The polymerization temperature of this secondary polymerization is limited to 50-100 ° C, but this temperature may be different from the temperature of the primary polymerization. When the temperature is lower than 50 ° C., the polymerization rate is too slow and the polymerization takes too much time. When the temperature is higher than 100 ° C., the polymerization rate is too fast and aggregation tends to occur. The polymerization time of the secondary polymerization is limited to 20 to 240 minutes. The above time is 2
If the time is less than 0 minutes, the polymerization rate of the cation exchange group-containing monomer is too low, and when used as a packing material for liquid chromatography, the ion exchange capacity is too small to perform a sufficient ion exchange reaction. Insufficient separation. Also,
If the above time exceeds 240 minutes, aggregation of the cation exchange group-containing monomer by the homopolymer is likely to occur, which is not preferable.

In the above step, the polymerization is terminated at a stage of a polymerization rate of 0.1 to 20% of the amount of the cation exchange group-containing monomer added. When the polymerization rate is less than 0.1%, the polymerization rate is too low, and when used as a filler for liquid chromatography, the ion exchange capacity is too small, and a sufficient ion exchange reaction with the substance to be measured is not performed. , Resulting in insufficient separation. When the polymerization rate is more than 20%, aggregation is likely to occur and reproducibility is greatly reduced. In addition, when used as a filler for liquid chromatography, the column pressure increases, which is extremely inconvenient in use. Here, the polymerization rate indicates the amount of the cation exchange group-containing monomer in the finally obtained polymerization product with respect to the amount added, and can be removed by washing in the following seventh step. It does not include homopolymers of group-containing monomers.

In the seventh step, the obtained polymer is treated with water and / or
Or a step of washing with an organic solvent. This step is performed in order to wash the produced polymer with water and / or an organic solvent to remove excess additives, unnecessary products, and the like. It is not always necessary to wash with both water and an organic solvent.
When both water and an organic solvent are used, the order of washing is not particularly limited.

The substance removed by the above washing includes a dispersant, a residual monomer, a non-crosslinked polymer and the like. Particularly, since the cation exchange group-containing monomer is added in a large excess in the fourth step, it is necessary to wash the monomer and its polymer in order to remove it.

The organic solvent used in the case of washing with an organic solvent is selected in consideration of the fact that the obtained polymer particles do not dissolve and the solubility of the substance to be removed. For example, acetone, methanol, ethanol , Isopropanol and the like.

In the above washing method, water or an organic solvent, or a mixture thereof may be added to the polymerization product and stirred, or the polymerization product is immersed in the organic solvent, or the organic solvent is added to the polymerization product. It can be carried out by circulating extraction or by other known methods. In the washing step, heating may be performed to such an extent that the polymer particles are not damaged. Water and organic solvent used for washing are removed by a method such as decantation or centrifugation.

(Application to Packing Material for Liquid Chromatography) The polymer particles obtained by the above-mentioned process are used as packing material for liquid chromatography by satisfying the following particle size and particle size distribution. The average particle size is 0.1 to 50
μm is preferable, and 0.5 to 20 μm is more preferable. In the particle size distribution, CV is preferably 20% or less, more preferably 15% or less. The above conditions can be satisfied by classifying the polymer particles obtained in the above step as necessary. A known method such as a dry method or a wet method can be used for the classification.

When used as a packing material for liquid chromatography, a column for liquid chromatography can be constituted by packing the packing material into a column made of stainless steel or resin. At the time of packing, an appropriate method can be used. A wet method (slurry method) in which a predetermined amount of a filler is dispersed in a dispersion medium such as a solvent used as an eluent, and the mixture is pressed into a column via a packer or the like is used. Particularly preferred.

The substances to be measured in the separation and measurement using the packing material for liquid chromatography obtained in the present invention are all substances conventionally separated by cation exchange liquid chromatography or ion chromatography. In particular, biologically-related substances such as catecholamine derivatives, nucleotides, peptides, and proteins are suitable.

The liquid chromatograph to which the filler obtained in the present invention can be applied may be a known liquid chromatograph, and includes, for example, a liquid sending pump, a sample introduction device, a column, a detector and the like. In addition, other accessories (such as a thermostat and an eluent deaerator) may be appropriately attached to these.

For liquid chromatography analysis using the packing material obtained in the present invention, a known eluent is used.
For example, various buffers containing the following substances and the like can be mentioned. Inorganic acids such as phosphoric acid, nitric acid, hydrochloric acid and perchloric acid and salts thereof; organic acids such as acetic acid, malic acid, tartaric acid, succinic acid and citric acid and salts or halides thereof; sodium hydroxide and potassium hydroxide Basic substances; other inorganic or organic salts. Good's buffer can also be used. Further, for example, an organic solvent such as acetone, acetonitrile, dioxane, methanol, and ethanol can be used, and water or a mixture of the above-mentioned buffer and an organic solvent can also be used.

[0056]

In the present invention, first, hydrophobic cross-linked polymer particles having a high degree of cross-linking and having a small number of hydrophilic groups such as ion-exchange groups inside the particle skeleton are prepared, and then cation exchange is performed near the surface of the particles. The group-containing monomer is polymerized. Therefore, (1)
Since the obtained filler has little swelling and shrinkage, it quickly equilibrates to changes in the eluent, so that the measurement time can be shortened especially in various gradient elution methods using a plurality of eluents; Since the filled filler has high pressure resistance, high flow rate analysis can be performed, which is advantageous for shortening the measurement time. Similarly, the column can be packed under a high pressure, so that the life of the column can be extended. In addition, in the method of the present invention, when adding the cation exchange group-containing monomer, the temperature is once cooled to around room temperature (10 to 40 ° C.), and then the mixture is added. As a result, the production stability is dramatically improved. In addition, since the cation exchange group-containing monomer is added in a large excess amount and is stopped at a stage of a low polymerization rate, it is possible to perform polymerization with good reproducibility even for a monomer that is extremely easily aggregated. it can.

[0057]

EXAMPLES Examples of the method of the present invention will be described below. Example 1 Benzoyl peroxide (polymerization initiator: Wako Pure Chemical Industries, Ltd.) was added to 400 g of triethylene glycol dimethacrylate (hydrophobic crosslinkable monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.).
1.5 g were mixed and dissolved. This was dispersed in 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol (Gohsenol GH-20, manufactured by Nippon Synthetic Chemical Industry Co., Ltd.), and the temperature was increased while stirring under a nitrogen atmosphere, followed by polymerization at 75 ° C. for 1.2 hours. After 1.2 hours, the reaction system was cooled to 35 ° C. in about 30 minutes, and 200 g of methacrylic acid (a carboxyl group-containing monomer: manufactured by Wako Pure Chemical Industries, Ltd.) was added at once. Then, after stirring at 25 ° C. for 60 minutes, the temperature was raised to 80 ° C. and polymerization was performed for 1 hour. After the polymerization, the product was washed with water and acetone, and classified to obtain a filler having an average particle size of 5 μm.

Example 2 250 g of divinylbenzene (hydrophobic crosslinkable monomer: manufactured by Kishida Chemical Co.) and 50 g of styrene (non-crosslinkable monomer: manufactured by Wako Pure Chemical Industries) were mixed, and 100 g of toluene was added thereto. And benzoyl peroxide 1.5
g was mixed and dissolved. This was dispersed in 2.5 liters of a 5% by weight aqueous solution of polyvinyl alcohol, and the temperature was increased while stirring under a nitrogen atmosphere, and polymerization was performed at 80 ° C. for 2.5 hours. After 2.5 hours, the reaction system was cooled to 30 ° C. in about 40 minutes, and 150 g of acrylic acid (carboxyl group-containing monomer: manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto all at once. Then at 25 ° C 6
After stirring for 0 minutes, the temperature was raised to 80 ° C. again, and polymerization was performed for 1 hour. After the polymerization, the product was washed with water and acetone and classified to obtain a filler having an average particle size of 5 μm.

Example 3 350 g of tetraethylene glycol dimethacrylate (hydrophobic crosslinking monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.) and tetramethylolmethane tetraacrylate (hydrophobic crosslinking monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.) 1.5 g of benzoyl peroxide was mixed and dissolved in 50 g. This was dispersed in 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol, and the temperature was increased while stirring under a nitrogen atmosphere, and polymerization was performed at 80 ° C. for 1.3 hours. After 1.3 hours, the reaction system was cooled to 35 ° C. in about 30 minutes, and 80 g of methacrylic acid (carboxyl group-containing monomer: manufactured by Wako Pure Chemical Industries) was used.
And 80 g of itaconic acid (carboxyl group-containing monomer: manufactured by Tokyo Chemical Industry Co., Ltd.) were added all at once. Then, after stirring at 25 ° C. for 60 minutes, the temperature was raised to 80 ° C. and polymerization was performed for 1 hour. After the polymerization, the product was washed with water and acetone, and classified to obtain a filler having an average particle size of 5 μm.

Example 4 350 g of triethylene glycol dimethacrylate and tetramethylol methane triacrylate (hydrophobic crosslinkable monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.)
1.0 g of benzoyl peroxide was mixed and dissolved in 50 g of the mixture. This was dispersed in 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol, and the temperature was increased while stirring under a nitrogen atmosphere, followed by polymerization at 80 ° C. for 1.5 hours.
After 1.5 hours, the reaction system was cooled to room temperature in about 20 minutes, and when the temperature in the reaction system reached 30 ° C., 2-acrylamido-2-methylpropanesulfonic acid (sulfonic acid group-containing monomer : Tokyo Chemical Industry Co., Ltd.) 50% aqueous solution 40
0 ml was added dropwise over 10 minutes. Then at 25 ° C 6
After stirring for 0 minutes, the temperature was raised to 80 ° C. again, and polymerization was performed for 1 hour. After the polymerization, the product was washed with water and acetone and classified to obtain a filler having an average particle size of 5 μm.

Example 5 200 g of methyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1200 g of ion-exchanged water. After the temperature was raised to 70 ° C. under a nitrogen atmosphere with stirring, 10 ml of an aqueous solution of 0.5% by weight of potassium persulfate (manufactured by E. Merk) and an aqueous solution of 2% by weight of sodium styrenesulfonate (manufactured by Wako Pure Chemical Industries) were used. 10 ml was added to the reaction. After polymerization at 70 ° C. for 24 hours, the reaction product was filtered to obtain a CV3.5 having an average particle size of 0.7 μm and a particle size of 3.5.
% Monodispersed particles were obtained.

A 0.5% by weight aqueous solution of sodium lauryl sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) in which 1 g of the above monodispersed particles is dispersed
20 ml of 3% by weight polyvinyl alcohol aqueous solution
0 ml was added and stirred for 1 hour at room temperature (monodisperse particle dispersion). Meanwhile, 1.0 g of benzoyl peroxide was dissolved in 50 g of triethylene glycol dimethacrylate. 900 ml of a 0.5% by weight aqueous solution of sodium lauryl sulfate was added thereto, and the mixture was homogenized (IKA Labotechni).
(manufactured by K Company) at 24,000 rpm for 15 minutes. The obtained emulsion was added to the monodispersed particle dispersion and stirred at room temperature for 24 hours to allow the monomer and the polymerization initiator to be absorbed by the monodispersed particles. Thereafter, the temperature was increased while stirring under a nitrogen atmosphere, and polymerization was carried out at 80 ° C. for 1.5 hours. After a lapse of 1.5 hours, the reaction system was cooled to 35 ° C. in about 30 minutes, and 30 g of methacrylic acid was added all at once. Then 60 at 25 ° C
After stirring for minutes, the temperature was raised again to 80 ° C., and polymerization was performed for 1 hour. After the polymerization, the product was washed with water and acetone and classified to obtain a filler having an average particle size of 3.5 μm.

Example 6 Example 5 was repeated except that 25 g of 2-acrylamido-2-methylpropanesulfonic acid was used instead of 30 g of methacrylic acid.
By the same operation as described above, a filler having an average particle size of 3.5 μm was obtained.

Comparative Example 1 Benzoyl peroxide (polymerization initiator) was added to a mixture of 400 g of diethylene glycol dimethacrylate (hydrophobic crosslinking monomer) and 200 g of methacrylic acid (monomer having a carboxyl group).
5 g were mixed and dissolved. This was dispersed in 2.5 liters of a 4% by weight aqueous solution of polyvinyl alcohol, the temperature was increased while stirring under a nitrogen atmosphere, and polymerization was carried out at 80 ° C. for 8 hours.
After the polymerization, the product was washed and classified to obtain a filler having an average particle size of 5 μm.

Comparative Example 2 250 g of divinylbenzene (hydrophobic crosslinkable monomer: manufactured by Kishida Chemical Co., Ltd.), 50 g of styrene (non-crosslinkable monomer: manufactured by Wako Pure Chemical Industries, Ltd.), 100 parts of toluene
g, 150 g of acrylic acid and 1.5 g of benzoyl peroxide were mixed and dissolved. This was dispersed in 2.5 liters of a 5% by weight aqueous solution of polyvinyl alcohol, the temperature was increased while stirring under a nitrogen atmosphere, and polymerization was performed at 80 ° C. for 8 hours. After the polymerization, the product was washed with water and acetone and classified to obtain a filler having an average particle size of 5 μm.

(Comparative Example 3) Example of introduction of carboxyl group by post-treatment: glycidyl methacrylate (manufactured by NOF Corporation)
350 g, ethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) 50 g and azobisisobutyronitrile 1
0 g, mixed in a 4% by weight aqueous solution of polyvinyl alcohol, and heated with stirring under a nitrogen atmosphere.
For 10 hours. After washing the obtained polymer, it was classified to obtain a polymer having an average particle size of 5 μm. The polymer 100
g was added to 300 g of xylene. The polymer impregnated with xylene is added to 1 liter of a 4% by weight aqueous solution of polyvinyl alcohol containing 15 g of concentrated sulfuric acid, and stirred while stirring.
Heated at 0 ° C. for 1 hour. Then, it was washed with water and acetone and dried. 100 g of the obtained polymer is diluted with 50 dioxane.
0 ml of boron trifluoride etherate (5
5%) and heated at 50 ° C. for 8 hours. After washing with acetone, it was dried.

Further, 20 g of the polymer was dispersed in 1000 ml of water, 35 g of sodium monochloroacetate, 20 g of potassium iodide and 60 g of 50% by weight sodium hydroxide were added, and the mixture was reacted at 60 ° C. for 3 hours with stirring. The obtained polymer was washed and dried to obtain a filler having an average particle size of 5 μm.

(Comparative Example 4) Example of introduction of sulfonic acid group by post-treatment: 2-hydroxyethyl methacrylate 400
g, 50 g of diethylene glyzyl dimethacrylate, 50 g of methyl methacrylate and 1.5 g of benzoyl peroxide were mixed and dispersed in 2.5 L of a 4% by weight aqueous solution of polyvinyl alcohol. The temperature was raised while stirring under a nitrogen atmosphere, and polymerization was carried out at 80 ° C. for 8 hours. After washing the obtained polymer, it was classified to obtain a polymer having an average particle size of 5 μm. 100 g of the polymer was dispersed in 100 ml of a 20% by weight aqueous sodium hydroxide solution. To this, 40 g of epichlorohydrin was added and reacted for 5 hours. After dispersing 100 g of the obtained epoxy group-containing polymer in 100 ml of a 20% by weight aqueous sodium sulfate solution,
The reaction was performed for 5 hours. The obtained polymer was washed and dried to obtain a filler having an average particle size of 5 μm.

Comparative Example 5 Reference of Y. Ohtsuka et al
[J. Applied Polym. Sci., 27 (1982) 3279-3288]: 100 g of ethylene glycol dimethacrylate (hydrophobic crosslinking monomer: manufactured by Shin-Nakamura Chemical Co., Ltd.) and 1.0 g of benzoyl peroxide are mixed. Dissolved. This was dispersed in 2.5 liters of a 1% by weight aqueous solution of polyvinyl alcohol, and heated to 80 ° C. while stirring under a nitrogen atmosphere. After 1 hour, 50 g of methacrylic acid was added to the reaction system, and the mixture was further polymerized at 80 ° C. for 24 hours. After the polymerization, the product was washed and classified to obtain a filler having an average particle size of 5 μm.

(Comparative Example 6) Document of Y. Ohtsuka et al
Method according to [J. Applied Polym. Sci., 27 (1982) 3279-3288]: Instead of 50 g of methacrylic acid in Comparative Example 5, sodium polystyrene sulfonate (monomer containing a sulfonic acid group: manufactured by Wako Pure Chemical Industries, Ltd.) ) Except that 50g was used
The same operation as in Comparative Example 5 was performed to obtain a filler.

Comparative Example 7 Two-Step Polymerization Functional Group (COO
H) (Method division method of Japanese Patent Publication No. 8-7197): 1.5 g of benzoyl peroxide was dissolved in 400 g of triethylene glycol dimethacrylate and dispersed in 2.5 liter of a 5% by weight aqueous solution of polyvinyl alcohol. The temperature was raised with stirring, and polymerization was carried out at 80 ° C. for 8 hours. The obtained product was washed with water and acetone to obtain crosslinked polymer particles. 300 g of the particles were added to 1 liter of an acetone solution containing 1.0 g of benzoyl peroxide (polymerization initiator) to impregnate the particles with the polymerization initiator. Next, acetone was distilled off under reduced pressure at 20 ° C. 1% by weight
The obtained polymerization initiator-containing particles were dispersed in 2.5 liters of an aqueous polyvinyl alcohol solution, and 200 g of methacrylic acid was added with stirring, followed by purging with nitrogen, followed by polymerization at 80 ° C. for 1 hour. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 5 μm.

Comparative Example 8 Two-Step Polymerization Functional Group (COO
H) (Continuous method of Japanese Patent Publication No. 8-7197): 1.5 g of benzoyl peroxide was dissolved in 400 g of triethylene glycol dimethacrylate and dispersed in 2.5 liter of a 5% by weight aqueous solution of polyvinyl alcohol. The temperature was raised while stirring, and polymerization was carried out at 80 ° C. for 1 hour. One hour later, 200 g of methacrylic acid was added to the reaction system, and polymerization was further performed at 80 ° C for one hour. After the polymerization, the polymer was washed and classified to obtain a filler having an average particle size of 5 μm.

(Evaluation of Performance) Examples 1 to 6 and Comparative Examples 1 to
The performance of the filler No. 8 was evaluated as follows. (1) Production of a column for liquid chromatography: 0.7 g of a filler was collected, and a 50 mM phosphate buffer (pH 6.
0) Dispersed in 30 ml, sonicated for 5 minutes, and then stirred well. The whole amount is made of stainless steel empty column (4.6φ × 3
(5 mm) was injected into a connected packer (Umeya Seiki Co., Ltd.). A liquid pump (manufactured by Sanuki Kogyo KK) was connected to the packer, and the solution was packed at a constant pressure of 200 kg / cm ² to produce a column for liquid chromatography.

(2) Swelling resistance test: An eluent having a different pH is passed through a column packed with a carboxyl group-containing filler as follows, and the column pressure fluctuates at that time:
The degree of swelling was measured and compared based on the time required for the column pressure to stabilize (time required for equilibration). That is, a 200 mM phosphate buffer solution (pH 5.7: solution A) was passed through the column filled with the filler obtained in each of Examples 1 to 3 and Comparative Examples 1 to 30 for 30 minutes. After the column pressure became constant, 300 mM phosphate buffer (pH 8.
5: Solution B), and the change in column pressure was observed. Table 1 shows the obtained results.

[0075]

[Table 1]

The fillers of Examples 1 to 3 and 5 were the same as Comparative Example 1
As compared with the fillers of Nos. 1 to 3, the increase in the pressure value was smaller, and the time required for stabilizing the pressure value (time until equilibration) was found to be extremely short.

Next, 100 mM phosphate buffer (pH 3.0: C solution) was added to the columns each filled with the filler having a sulfonic acid group obtained in Examples 4 and 6 and Comparative Example 4.
The solution was passed for 0 minutes. After the column pressure becomes constant, 300
The mixture was passed through a phosphate buffer (pH 8.5: D solution), and the change in column pressure and the time required for the column pressure to stabilize (time until equilibration) were observed. The results are shown in Table 2.

[0078]

[Table 2]

The fillers of Examples 4 and 6 have a smaller rise in pressure value and a very short time required for stabilization of the pressure value (time until equilibration) as compared with the filler of Comparative Example 4. I understood.

As described above, the fillers of Examples 1 to 6 obtained by the method of the present invention were obtained by a conventional one-stage polymerization method (Comparative Examples 1 and 2) or a cation exchange group introduction method by post-treatment. It was found that, as compared with the filler (Comparative Examples 3 and 4), the equilibration with respect to the change of the eluent was earlier, and the swelling was difficult.

(3) Measurement of hemoglobins: Glycohemoglobin (Hb) in human blood, which is an index for diagnosing diabetes, is contained using columns filled with the fillers obtained in Examples and Comparative Examples. Hb was measured. (Measurement conditions) System: Liquid pump: LC-9A (manufactured by Shimadzu Corporation) Autosampler: ASU-420 (manufactured by Sekisui Chemical Co., Ltd.) Detector: SPD-6AV (manufactured by Shimadzu Corporation) Eluent: phosphate and Elution was performed by a step gradient method using two kinds of buffers containing perchlorate. Eluent 1: Elution from the beginning to before hemoglobin A0 (HbA0). Stable hemoglobin A1c (stable HbA
The concentration was adjusted between 10 and 200 mM and the pH between 5.0 and 5.8 so that 1c) was eluted at the most appropriate retention time. Eluent 2: elute HbA0. Concentration is 300 mM, pH
7.2. Flow rate: 1.5 ml / min Detection wavelength: 415 nm Sample injection volume: 10 μl

(Measurement Sample) Blood of a healthy person was sampled from sodium fluoride to prepare the following samples. Sample a) Glucose-loaded blood: An aqueous glucose solution was added to healthy human blood at a concentration of 500 mg / dl, reacted at 37 ° C. for 5 hours, and then diluted with hemolysis (0.1% by weight polyethylene glycol mono-4-).
Hemolyzed with octyl phenyl ether (Triton X-100) (manufactured by Tokyo Chemical Industry Co., Ltd., phosphate buffer solution (pH 7.0)) and diluted 150-fold to obtain sample a. Sample b) Sample containing carbamylated Hb (CHb): 10 ml of healthy human blood
Was added with 1 ml of a physiological saline solution of 0.3% by weight of sodium cyanate, and reacted at 37 ° C. for 3 hours.
Hemolysis dilution (0.1% by weight polyethylene glycol mono-4-octyl phenyl ether (Triton X-10)
0) (manufactured by Tokyo Chemical Industry Co., Ltd.) in a phosphate buffer solution (pH 7.0).
The sample was hemolyzed in 0)) and diluted 150-fold to obtain a sample b. Sample c) Sample containing acetylated Hb (AHb): healthy human blood 1
To 0 ml, 1 ml of a 0.3% by weight aqueous solution of acetaldehyde in physiological saline was added, and reacted at room temperature for 3 hours. Then, a hemolysis diluent (0.1% by weight of polyethylene glycol mono-4-octylphenyl ether (Triton X- 1
00) (manufactured by Tokyo Chemical Industry Co., Ltd.) in a phosphate buffer solution (pH 7.0).
The sample was hemolyzed by 0)) and diluted 150 times to obtain a sample c.

(Measurement Results) (A) Fillers of Examples 1 to 6: (Sample a) A chromatogram obtained by measuring sample a using the filler of Example 1 is shown in FIG. Shown in FIG.
In (a), peak 1 shows hemoglobin A1a and b
(HbA1a and b); peak 2 is hemoglobin F (H
bF); peak 3 shows unstable HbA1c; peak 4 shows stable HbA1c; peak 5 shows HbA0. HbF
(Peak 2) and stable HbA as an indicator of diabetes diagnosis
In order to maintain good quantification of the 1c peak (peak 4), HbF, unstable HbA1c, stable HbA1
It must be eluted in the order of c and HbA0. Especially HbF
Since HbA1c usually has a smaller peak than HbA1c and HbA0, if eluted between, for example, stable HbA1c and HbA0, the quantitativeness of HbF is extremely reduced. In FIG. 1 (a), each peak is eluted in the above order, and H
bF and stable HbA1c are well separated from peaks of other components.

(Sample b) The chromatogram obtained by measuring the sample b using the filler of Example 1 is shown in FIG. In FIG. 1B, peak 6 indicates CHb.
CHb is well separated from stable HbA1c. (Sample c) FIG. 1C shows a chromatogram obtained by measuring the sample c using the filler of Example 1. FIG.
In (c), peak 7 indicates AHb. AHb is well separated from stable HbA1c.

Similar results were obtained when the fillers of Examples 2 to 6 were used.

(B) Fillers of Comparative Examples 1 to 3 (Sample a) A chromatogram obtained by measuring Sample a using the filler of Comparative Example 1 is shown in FIG. Hb
F, the unstable HbA1c and the stable HbA1c are eluted in this order, but the separation between the unstable HbA1c and the stable HbA1c is poor despite the long measurement time. (Sample b) The chromatogram obtained by measuring the sample b using the filler of Comparative Example 1 is shown in FIG. CHb
And stable HbA1c are poorly separated. (Sample c) FIG. 2C shows a chromatogram obtained by measuring Sample c using the filler of Comparative Example 1. AHb
And stable HbA1c are poorly separated.

Similar results were obtained when the fillers of Comparative Examples 2 and 3 were used.

(C) Filler of Comparative Example 4: (Sample a) A chromatogram obtained by measuring Sample a using the filler of Comparative Example 4 is shown in FIG. Although unstable HbA1c and stable HbA1c are separated,
HbF could not be measured. In addition, the measurement time is long. (Sample b) A chromatogram obtained by measuring the sample b using the filler of Comparative Example 4 is shown in FIG. CHb
And stable HbA1c are poorly separated. (Sample c) The chromatogram obtained by measuring the sample c using the filler of Comparative Example 4 is shown in FIG. AHb
And stable HbA1c are poorly separated.

(D) Fillers of Comparative Examples 5 and 6: (Sample a) A chromatogram obtained by measuring sample a using the filler of Comparative Example 5 is shown in FIG. (Sample b) A chromatogram obtained by measuring Sample b using the filler of Comparative Example 5 is shown in FIG. (Sample c) The chromatogram obtained by measuring the sample c using the filler of Comparative Example 5 is shown in FIG. In any case, despite the long measurement time, the separation of HbF, unstable HbA1c, and stable HbA1c is poor.

Similar results were obtained when the filler of Comparative Example 6 was used.

(E) Fillers of Comparative Examples 7 and 8: (Sample a) A chromatogram obtained by measuring sample a using the filler of Comparative Example 7 is shown in FIG. (Sample b) FIG. 5B shows a chromatogram obtained by measuring the sample b using the filler of Comparative Example 7. Unstable HbA1c and CHb are stable HbA
1c was well separated. (Sample c) FIG. 5C shows a chromatogram obtained by measuring Sample c using the filler of Comparative Example 7. AHb
Could not be separated from stable HbA1c.

Similar results were obtained when the filler of Comparative Example 8 was used.

As described above, when the filler according to the embodiment of the method of the present invention is used, stable HbA1c and HbF which are not affected by the prior art can be determined without being affected by unstable HbA1c, CHb and AHb. We can see that we can do it.

(F) Column durability: The column durability of the packing materials of Example 4 and Comparative Example 4 was compared. The sample a was repeatedly measured, and the change in the stable HbA1c value calculated by the following equation from the obtained chromatogram was examined. Stable HbA1c (%) = (peak area of stable HbA1c) ÷ (total peak area) × 100

FIG. 6 shows the obtained results. It is shown as a relative value when the initial value of the measurement is 100. 6, the filler of Example 4 is more stable than the filler of Comparative Example 4.
It can be seen that the bA1c value is stable for a long time and the column durability is excellent.

(G) Polymerization reproducibility: The fillers of Examples 1 and 4 and Comparative Examples 3 to 8 were each prepared 30 times under the same conditions.
The difference between each lot was examined. (Measurement method) In the above measurement method, the stable HbA1c
Eluent 1 was prepared such that the peak retention time was about 10 minutes. Using the filler of each lot, the sample a was measured, and the retention time of the stable HbA1c peak was examined. 30
Table 3 shows the average value, the standard deviation, and the coefficient of variation of the retention time of the stable HbA1c peak when the polymerization state of the lot was good. Table 3 also shows the number of lots in which agglomeration occurred during preparation and evaluation as a filler was impossible.

[0097]

[Table 3]

As is clear from Table 3, in the examples according to the method of the present invention, the reproducibility of the performance as a filler is extremely good without causing aggregation during the polymerization. On the other hand, a manufacturing method by a post-treatment which is a conventional technique (Comparative Examples 3 and 4)
In this case, the performance of the filler varies greatly. Further, a method of adding a cation exchange group-containing monomer during polymerization,
In the method of polymerizing the cation-exchange group-containing monomer by impregnating the crosslinked particles with a polymerization initiator (Comparative Examples 5 to 8), it can be seen that in addition to the variation in performance, aggregation during the polymerization is likely to occur.

(4) Analysis example of protein mixture: Carboxyl group-containing filler (Examples 1-3, Comparative Examples 1-3,
The mixture of protein standards was analyzed using 5, 7, 8). (Measurement conditions) System: Same as the above-mentioned system for measuring hemoglobins (3) Eluent: Elution was performed by a linear gradient method using two kinds of buffers containing sodium phosphate. Eluent 3: 30 mM (pH 7.1) Eluent 4: Linear gradient from 100% of eluent 3 + 300 mM Na ₂ SO ₄ eluent 3 to 100% of eluent 4 Flow rate: 1.5 ml / min Detection wavelength: 280 nm Sample Injection volume: 10 μl

(Measurement sample) Myoglobin, α-chymotrypsinogen, ribonuclease A, lysozyme (Si
gma) mixture

(Measurement Results) The obtained chromatogram is shown in FIG. FIG. 7A is a chromatogram when the fillers of Examples 1 to 3 are used, and FIG.
The chromatogram at the time of using 3, 5, 7, 8 fillers is shown. In FIG. 7, peak 8 indicates myoglobin, peak 9 indicates α-chymotrypsinogen, peak 10 indicates ribonuclease A, and peak 11 indicates lysozyme. From the figure, when the fillers of Examples 1 to 3 are used, the respective peaks are well separated in a short time, but when the fillers of Comparative Examples 1 to 3, 5, 7, and 8 are used. It can be seen that, despite the long measurement time, each peak lacks sharpness.

(5) Example of Analysis of Peptide Mixture: Using a sulfonic acid group-containing filler (Examples 4, 6 and Comparative Examples 4, 6), a mixture of peptide standard substances was analyzed. (Measurement conditions) System: Same as the above-mentioned system for measuring hemoglobins (3) Eluent: Elution was performed by a linear gradient method using two kinds of buffers containing acetic acid. Eluent 5: 30 mM (pH 2.8) Eluent 6: Linear gradient from 100% of eluent 5 + 300 mM Na ₂ SO ₄ eluent 5 to 100% of eluent 6 Flow rate: 1.5 ml / min Detection wavelength: 215 nm Sample Injection volume: 10 μl

(Measurement sample) Mixture of γ-endorphin, calcitonin, substance P, insulin, β-endorphin (manufactured by Sigma)

(Results of Measurement) The obtained chromatogram is shown in FIG. 8A is a chromatogram when the fillers of Examples 4 and 6 are used, FIG. 8B is a chromatogram when the filler of Comparative Example 4 is used, and FIG. ,
9 shows a chromatogram when the filler of Comparative Example 6 was used. 8, peak 12 indicates γ-endorphin, peak 13 indicates calcitonin, peak 14 indicates substance P, peak 15 indicates insulin, and peak 16 indicates β-endorphin. As shown in the figure, when the fillers of Examples 4 and 6 were used, each peak was well separated in a short time.
When the filler of Comparative Example 4 was used, each peak was found to lack sharpness despite the long measurement time, and when the filler of Comparative Example 6 was used, each peak was not separated. Was.

(Reproducibility of Polymerization) Examples 4 and 6: The polymerization methods of Comparative Examples 4 and 6 were each prepared 30 times under the same conditions.
The difference between each lot was examined. (Measurement method) In the above measurement method, eluent 5 was prepared so that the retention time of the β-endorphin (peak 16) peak was about 20 minutes. Using the filler of each lot, the above sample was measured, and the retention time of the β-endorphin peak was examined. Table 4 shows the average value, the standard deviation, and the coefficient of variation of the retention time of the β-endorphin peak when the polymerization state of the 30 lots was measured for those having a good polymerization state. Table 4 also shows the number of lots in which agglomeration occurred during preparation and evaluation as a filler was impossible.

[0106]

[Table 4]

As described above, the sulfonic acid group-containing filler obtained by the method of the present invention can separate peptides in a shorter time and with higher precision than the filler produced by the conventional production method, and can reduce the amount of polymerization lot. It can be seen that the variation is very small.

[0108]

According to the packing material for liquid chromatography obtained by the method of the present invention, the backbone is composed of hydrophobic crosslinked particles, and the functional groups having ion exchange ability are unevenly distributed near the surface. Equilibrium is fast to the external environment due to composition change, etc., and it has excellent swelling resistance. Therefore, conventionally, a filler obtained by mixing and polymerizing a crosslinkable monomer and a cation exchange group-containing monomer, a hydrophilic monomer and a crosslinkable monomer are copolymerized. After that, it has a high resolution within a shorter measurement time than a filler having many hydrophilic functional groups inside the particle, such as a filler obtained by introducing a cation exchange group by post-treatment.

Further, the method of the present invention is characterized in that, when the monomer containing a cation exchange group is added, the polymerization system is cooled to a temperature near room temperature to terminate the polymerization reaction or to extremely slow the polymerization rate; After the addition of the monomer, the mixture is stirred at a temperature near room temperature for a certain period of time to stabilize the added cation exchange group-containing monomer in the reaction system and then polymerize the monomer. This makes it possible to add a cation exchange group-containing monomer during polymerization or to impregnate a crosslinkable polymer with a polymerization initiator and then polymerize the cation exchange group-containing monomer. As a result, the polymerization stability becomes extremely high.

Further, in the conventional method, polymerization was carried out for about 24 hours after the addition of the cation exchange group-containing monomer to increase the degree of polymerization. The monomers are also restricted. In addition, the conditions for introducing a sufficient cation exchange group for the ion exchange reaction and the conditions for preventing aggregation are inconsistent, and setting the conditions is difficult. In the present invention,
By adding a large excess of the cation exchange group-containing monomer and terminating at a low polymerization rate stage, even if the monomer is easily aggregated, sufficient ions for the ion exchange reaction can be obtained without reducing the polymerization reproducibility. Exchange capacity can be imparted to the filler.

Further, since the polymerization is completed by a series of polymerization operations, the operation is simpler and simpler than the method of introducing a cation exchange group by post-treatment, and the reproducibility is also good. In addition, since all the cation exchange groups are derived from monomers, the reproducibility of performance when applied to liquid chromatography fillers is good, which is extremely advantageous in production.

[Brief description of the drawings]

FIG. 1 shows that Hb was obtained using the filler obtained in Example 1.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 2 shows that Hb was obtained using the filler obtained in Comparative Example 1.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 3 shows that Hb was obtained using the filler obtained in Comparative Example 4.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 4 shows that Hb was obtained using the filler obtained in Comparative Example 5.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 5 shows that Hb was obtained using the filler obtained in Comparative Example 7.
The figure which shows the chromatogram obtained when performing the measurement of the class.

FIG. 6 shows the results of a column durability test.

FIG. 7 is a view showing a chromatogram obtained when a measurement of a protein mixture is performed.

FIG. 8 is a view showing a chromatogram obtained when measuring a peptide mixture.

[Explanation of symbols]

 1 HbA1a and b 2 HbF 3 unstable HbA1c 4 stable HbA1c 5 HbA06 carbamylated Hb 7 acetylated Hb 8 myoglobin 9 α-chymotrypsinogen 10 ribonuclease A 11 lysozyme 12 γ-endorphinin 13 subsylcinthine β-subrinthin β-subsulfinine 13 subsylcinto-subrinthin 13 subsylcinto-subsulfinin 13 sub-calcin Endorphin

Claims (1)

[Claims] 1. A method for producing a packing material for liquid chromatography, comprising the following steps: (I) a first step of dispersing a polymerizable mixture containing a hydrophobic crosslinkable monomer and a polymerization initiator in an aqueous dispersion medium; (ii) raising the dispersion obtained in the first step to 50 to 100 ° C. A second step of heating and conducting a polymerization reaction for 20 to 240 minutes, (iii) a third step of cooling the reaction system to 10 to 40 ° C.
(Iv) a fourth step of adding a cation exchange group-containing monomer to the reaction system, (v) after completion of the addition, the reaction system is heated at 10 to 40 ° C. for 0.5 to 0.5 ° C.
A fifth step of stirring for 24 hours, (vi) raising the temperature of the reaction system to 50 to 100 ° C. to carry out a polymerization reaction, 20 to 240 minutes after the initiation of polymerization, and the addition amount of the cation exchange group-containing monomer. A sixth step of terminating the polymerization reaction at a stage of a polymerization rate of 0.1 to 20%, (vii) a seventh step of washing the obtained polymer with water and / or an organic solvent.