JP2811789B2 - Carrier for separating catabolic hemoglobin - Google Patents

Description

The present invention relates to a carrier for separating catabolic hemoglobin.

More specifically, the present invention relates to a carrier for separating catabolic hemoglobin having a specific structure, a small standard deviation of the particle size distribution, a small particle size, and a high degree of cross-linking, so that the mechanical strength and the number of separation stages are high. .

[Conventional technology]

It is conventionally known that the pathology of various diseases can be estimated by separating and quantifying hemoglobin, particularly catabolic hemoglobin, in blood. As a chromatography carrier for this purpose, a separation carrier based on a synthetic polymer or silica gel is used.

As the polymer carrier, for example, Japanese Patent Publication No. 1-15024
In the gazette, there is a description of an acrylic carrier having a carboxyl group, the catabolic hemoglobin in a short time,
A method capable of separating and quantifying with high accuracy is disclosed. However, the carrier is manufactured by a suspension polymerization method, and has a small particle size,
Moreover, it is difficult to produce a carrier having a narrow particle size distribution, and as a result, there is a disadvantage that the number of separation stages is inferior.

On the other hand, the silica gel carrier has the advantage of high mechanical strength and a high number of separation stages, but has the disadvantage that the short-time separation of various catabolic hemoglobins is not satisfactory in performance.

[Problems to be solved by the invention]

The present invention improves the above-mentioned disadvantages, and provides catabolic hemoglobin,
It is an object of the present invention to provide a chromatographic carrier that can be separated and quantified accurately in a short time.

[Means for solving the problem]

That is, the present invention provides: (1) a unit derived from glycidyl methacrylate represented by the following formula having a weight fraction of at least 3%; (R ¹ and R ^{2 each} represent a hydrogen atom and / or an organic group having a cation exchange group.) Includes a polyester of a polyhydric alcohol and a carboxylic acid having at least one polymerizable double bond in one molecule. A monomer having a polymerizable double bond in one molecule other than glycidyl methacrylate (hereinafter, referred to as monomer B) in which the weight fraction of a unit derived from a crosslinkable monomer (hereinafter, referred to as monomer A) is 80 to 97%. The weight fraction of the unit derived from is 0-5% and the exchange capacity is 0.1-0.5 per dry weight.
A carrier for separating catabolic hemoglobin characterized by comprising a cation exchange resin having a milliequivalent amount, and (2) the carrier for separating catabolic hemoglobin produced by the following steps are summarized.

1st step The weight fraction of glycidyl methacrylate is 85 to 100%,
When the weight fraction of the monomer A is 0 to 1% and the monomer B is
Is produced by dispersion polymerization of a monomer mixture having a weight fraction of 0 to 15% in the presence of a medium that dissolves these monomer mixtures but does not dissolve the resulting polymer.

Second step After adding 2 to 20 times by weight of the polymerizable monomer of the seed polymer to the seed polymer produced in the first step,
A seed epoxy polymer is produced by seed polymerization.

However, the polymerizable monomer, in the polymerizable monomer,
It contains 95 to 100% by weight of the monomer A and 0 to 5% by weight of glycidyl methacrylate and / or the monomer B.

Third Step In the second step, 0.1 to 0.5 milliequivalents of cation exchange groups are introduced per 1 g of dry weight using the prepared raw epoxy polymer.

 Hereinafter, the present invention will be described in detail.

The unit derived from glycidyl methacrylate of the carrier of the present invention has a weight fraction of at least 3%, preferably in the range of 3% to 20%, wherein R ¹ and R ² are each a hydrogen atom. Other examples include an organic group having a cation exchange group such as a carboxyl group or a sulfonyl group.

Such carboxymethyl group Specific examples of the organic groups, carboxyalkyl groups such as carboxymethyl ethyl group; sulfonyl group such as sulfonyl propyl; -CH ₂ C
_{H 2 OCH 2 CH 2 m OCH} 2 n -COOH, -CH 2 CH 2 OCH
₂ CH _{2 m} OCH _{2 n} -SO ₃ H (m is 0 or more, preferably 0
To 5 and n represents 1 or more, preferably 1 to 4).

Incidentally, those as the units derived from glycidyl methacrylate, depending on manufacturing conditions, indicates the R ¹ and R ² are simultaneously hydrogen atoms, at least one of R ¹ and R ² represent an organic group having a cation exchange group May be included at the same time.

The monomer A used in the present invention is a crosslinkable monomer containing a polyester of a polyhydric alcohol and a carboxylic acid having at least one polymerizable double bond in one molecule. Examples of the polyhydric alcohol include tetramethylolmethane, tetramethylolethane, trimethylolpropane, dipentaerythritol, ethylene glycol, polyethylene glycol, propylene glycol, and polypropylene glycol. Examples of monomers that are polyesters of carboxylic acids having at least one polymerizable double bond include ethylene glycol diacrylate (or dimethacrylate), glycerin diacrylate (or dimethacrylate), glycerin triacrylate (or trimethacrylate). Acrylic cross-linkable resins such as trimethylolpropane triacrylate (or trimethacrylate) and diethylene glycol diacrylate (or dimethacrylate). It can be mentioned mer. These may be used alone or as a mixture. It is preferable that at least 50% of the monomer which is a polyester of the polyhydric alcohol and the carboxylic acid is contained in the monomer A component. Here, the porosity properties (pore volume, pore radius, surface area, etc.) of the epoxy polymer obtained by seed polymerization based on the type and amount of the monomer A and the combination with the diluent, etc.
Can be changed. Further, as a monomer A other than the above, a polyvinyl compound such as divinylbenzene is added so as to be 30% by weight or less of the monomer A component,
It is also possible to control the hydrophobicity of the raw material epoxy polymer obtained by using it in combination to adjust the holding power of catabolic hemoglobin.

By using the monomer B component in the present invention,
By slightly changing the porosity and hydrophobicity of the raw material epoxy polymer, the retention behavior of catabolic hemoglobin can be adjusted. The monomer B is a monomer having one polymerizable double bond in one molecule other than glycidyl methacrylate, and can be variously used from a hydrophilic monomer to a hydrophobic monomer. Examples of this include glycidyl acrylate, 2-hydroxyethyl acrylate (or methacrylate), glycerin monoacrylate (or methacrylate), methyl acrylate (or methacrylate), 2-ethylhexyl acrylate (or methacrylate), and the like. Further, monomers having a cation exchange group, for example, acrylic acid, methacrylic acid, crotonic acid, maleic acid and the like are also used. Further, as the monomer B, these exemplified monomers may be used alone or as a mixture.

Next, a method for producing the carrier for separating catabolic hemoglobin of the present invention will be described.

The carrier for catabolic hemoglobin separation is, in the first step,
A seed polymer is produced by dispersion polymerization, and in the second step, the seed polymer produced in the first step is impregnated with a polymerizable monomer containing a large amount of a crosslinkable monomer and then polymerized (seed polymerization) to enlarge the seed polymer. In the third step, the polymer is produced in each step of introducing a cation exchange group into the enlarged polymer (raw material epoxy polymer) produced in the second step.

In the first step, a seed polymer is produced by dispersion-polymerizing glycidyl methacrylate. In order to obtain polymer particles having a desired particle size, it is necessary to select a medium to be used.

As the medium to be used, a medium that dissolves the monomer but does not dissolve the produced polymer (a poor solvent for the produced polymer) is used.

Specifically, alcohols such as methanol, ethanol, 2-propanol and 2-methyl-2-propanol,
Further, there may be mentioned ethers / alcohols such as these alcohols and 2-methoxy-ethanol, ethers such as tetrahydrofuran and dioxane, and mixtures of these various solvents with water.

Further, among these solvents, the stronger the property as a poor solvent for the produced polymer, the smaller the particle size of the polymer tends to be.

Further, by adding a small amount of a good solvent to a medium mainly composed of a poor solvent as described above, the particle size of the polymer tends to increase.

By utilizing various properties of such a solvent, a solvent is appropriately selected so as to obtain a desired particle size, and these are used alone or as a mixture as a medium.

On the other hand, another factor affecting the particle size of the produced polymer includes the concentration of the charged monomer. When the concentration is high, the particle size tends to increase, and when the concentration is low, the particle size tends to decrease.

At the time of dispersion polymerization, aggregation, deformation,
It is preferable to use a dispersion stabilizer in order to prevent fusion and increase the dispersion stability. As the dispersion stabilizer, various kinds of synthetic polymer compounds such as homopolymers, copolymers, graft polymers, and block polymers, and natural polymer compounds and derivatives thereof can be used. Specific examples include polyvinyl pyrrolidone, polyvinyl methyl ether, polyethylene imine, polyacrylic acid, vinyl alcohol-vinyl acetate copolymer, ethyl cellulose, hydroxypropyl cellulose and the like. Particularly, those which dissolve in the medium used are preferable.

Further, at the time of dispersion polymerization, an auxiliary stabilizer can be used in addition to the above-mentioned dispersion stabilizer in order to promote the polymerization more stably and increase the dispersion stability. Examples of such a co-stabilizer include anionic surfactants, nonionic surfactants, quaternary ammonium salts, and long-chain alcohols. Specific examples include sodium di (2-ethylhexyl) sulfosuccinate, nonylphenoxypolyethoxyethanol, methyltricaprylylammonium chloride, and cetyl alcohol.

Further, as polymerization initiators that can be preferably used in dispersion polymerization, 2,2′-azobisisobutyronitrile, 4,4′-azobis- (4-cyanopentanoic acid),
2,2'-azobis- (2-methylbutyronitrile), 2,
Examples include azo-based polymerization initiators such as 2'-azobis- (2,4-dimethylvaleronitrile) and peroxides such as benzoyl peroxide.

In addition, as the polymerization temperature during the dispersion polymerization in the present invention,
40 ° C to 90 ° C, preferably 60 ° C to 80 ° C.

The particle diameter of the seed polymer is preferably in the range of not less than 1/3 and not more than 2/3 of the particle diameter of the desired enlarged polymer, specifically from 0.5 μm to 5 μm, more preferably from 1 μm to
4 μm.

Examples of the polymerization initiator that can be used in the second step of the present invention include 2,2′-azobisisobutyronitrile,
4,4'-azobis- (4-cyanopentanoic acid), 2,2'-
Azo-based polymerization initiators such as azobis- (2-methylbutyronitrile) and 2,2'-azobis- (2,4-dimethylvaleronitrile), or benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, 3 And peroxides such as 3,3 ', 5-trimethylhexanoyl peroxide.

Further, porous polymer particles can be obtained by adding an appropriate diluent to the monomer phase during seed polymerization. By making it porous, the amount of functional groups that can be introduced increases, the performance as a carrier for separating catabolic hemoglobin improves, and because the surface area increases, the speed at which the resin is contaminated is reduced, and the durability is improved. . Halogenated hydrocarbons are preferably used as the diluent, and aliphatic or aromatic hydrocarbons, and esters, ketones and ethers thereof can also be used. In the preparation of the monomer dispersion,
It is preferable to use a dispersion stabilizer to enhance dispersibility. As such a dispersion stabilizer, known ones can be used, but those which finely stabilize the monomer are preferable, and specific examples thereof include anions such as sodium dodecylbenzenesulfonate, sodium lauryl sulfate and sodium dialkylsulfosuccinate. Nonionic surfactants such as polyoxyethylene nonylphenyl ether, polyethylene glycol monostearate, sorbitan monostearate, polyvinylpyrrolidone, polyethyleneimine, vinyl alcohol-vinyl acetate copolymer, etc. Can be used in combination.

The amount of the polymerizable monomer to be impregnated in the second step is 2 to 20 times by weight of the seed polymer, preferably 4 to 10 times.
Weight times. When the impregnation amount of the monomer A, which is a main component of the polymerizable monomer, is too low, the crosslink density of the raw material epoxy polymer obtained by seed polymerization becomes low,
Since the mechanical strength is reduced, it is difficult to pass the solution at a high flow rate, and rapid separation of catabolic hemoglobin is not possible. Therefore, the method is not suitable for clinical analysis. Further, the rate of extraction and removal of the polymer containing glycidyl methacrylate as a main component generated in the first step increases, which is not preferable. On the other hand, when the amount of the monomer A impregnated is too high, the weight fraction of the glycidyl methacrylate component in the raw material epoxy polymer becomes low, so that the amount of the introduced cation exchange groups becomes too low, which is not preferable.

In the present invention, in the case of seed polymerization, a dispersion stabilizer is used to absorb the polymerization initiator and the polymerizable monomer, prevent the aggregated seed polymer from agglomerating, deforming and fusing, and increase its dispersion stability. It is preferable to use As such a dispersion stabilizer, known anionic or nonionic surfactants and synthetic polymers such as polyvinylpyrrolidone, polyethyleneimine, and vinyl alcohol-vinyl acetate copolymer can be used.

The polymerization temperature in the seed polymerization of the present invention is usually 40
C. to 90C, preferably 50C to 80C.

The preferred particle size range of the enlarged polymer (raw material epoxy polymer) obtained in the second step is 1.5 μm to 7 μm, more preferably 2 μm to 5 μm.

Further, it is preferable that 90% or more of the particles are in the range of the average particle size ± 1 μm.

Next, a cation exchange group is introduced in the third step.
As a method for introducing the cation-exchange group, a cation exchange group is bonded to a hydroxyl group formed by ring-opening modification of an epoxy group derived from glycidyl methacrylate with water, glycerin, ethylene glycol or the like (for example, monohalogenoalkyl carboxylic acid, monohalogenoalkyl sulfone). A method of reacting an acid or the like to bond a carboxyalkyl group or a sulfonylalkyl group, specifically, a method of reacting monochloroacetic acid to bond a carboxymethyl group), or positively reacting the epoxy group directly or via a spacer. A method of bonding an ion exchange group (for example, a hydroxyalkyl carboxylic acid, specifically, glycolic acid is reacted, and a carboxymethyl group is directly bonded to a ring-opened epoxy group, or ethylene glycol or poly (2-6 Ethylene glycol) Reacting Le, mono halogenoalkyl acid after forming the spacers, and the like is reacted with mono-halogenoalkyl acid) and the like.

Examples of the cation exchange group include strongly acidic and weakly acidic exchange groups, and among them, a carboxyl group is preferable. Here, an organic group is introduced into at least one of two hydroxyl groups generated by ring-opening modification of an epoxy group derived from glycidyl methacrylate. The amount of the cation exchange group introduced, that is, the exchange capacity, can be determined by titration of the resin, but is preferably 0.1 to 0.5 meq / g of dry weight in order to obtain good separation. In order for the exchange capacity to be within the above range, it is necessary that the weight fraction of units derived from glycidyl methacrylate in the raw epoxy polymer is at least 3%.

In the carrier for catabolic hemoglobin separation of the present invention produced by the above steps, the unit derived from glycidyl methacrylate, the unit derived from monomer A, the weight fraction of each of the units derived from monomer B, the pyrolysis gas It can be estimated by analytical means such as chromatography. In particular, at the time of seed polymerization, the polymerizable monomer is impregnated at least 4 times the weight of the seed polymer, and the polymerization conversion is reduced.
When it is 70% or more, it is recognized that the weight fraction of the unit derived from each monomer of the produced polymer is close to the charging ratio of the raw material monomer.

[Action and effect]

The carrier for separating catabolic hemoglobin of the present invention is a carrier suitable for separating catabolic hemoglobin. It is a carrier in which the properties of the seed polymer are reflected in the enlarged polymer by being produced by a specific production method. That is, since the particle diameter of the seed polymer is uniform, the uniformity of the particle diameter of the obtained enlarged polymer is high. For this reason, the eluent can be flowed at a higher speed than in the case of using a carrier having the same average particle size, and the separation pattern is sharp and the number of theoretical plates is high. In addition, a simple copolymerization is performed to absorb and polymerize a polymerizable monomer mainly composed of a low-polarity crosslinkable monomer (monomer A) in a seed polymer containing a large amount of a highly polar monomer (glycidyl methacrylate) unit. In contrast, a seed polymer component composed of a highly polar monomer unit tends to collect on the surface layer of the enlarged polymer particles. As a result, a carrier for the dissimilar hemoglobin separation having a high cation exchange group introduction rate despite a high degree of crosslinking is obtained. And exhibit excellent separation performance.

In addition, since the catabolic hemoglobin separation carrier has a high degree of cross-linking, it has excellent mechanical strength, allows the eluent to flow at high speed, and enables rapid analysis. Furthermore, even when the concentration of the eluent is changed, the swelling and shrinkage are small, and the method is suitable for elution in a gradient mode. In order to make the weight fraction derived from the monomer A as high as 80 to 97%, when the carrier of the present invention is used for separating catabolic hemoglobin, not only the ion exchange effect but also the hydrophobic interaction is combined. Analysis can be performed well. Such features of the carrier for separating catabolic hemoglobin of the present invention are not heretofore available. Further, since the carrier of the present invention is produced by a specific method, its particle size can be reduced, and as a result, it provides a low measurement pressure and excellent separation performance, and can separate catabolic hemoglobin very quickly and efficiently. Can do it.

〔Example〕

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Production of Raw Epoxy Polymer 7.2 g of polyvinylpyrrolidone (trade name K-30: manufactured by Tokyo Chemical Industry Co., Ltd., average molecular weight 40,000), 50 g of glycidyl methacrylate and 0.5 g of 2,2'-azobisisobutyronitrile
Was dissolved in 342 g of ethanol, and the dissolved enzyme was replaced with nitrogen gas, followed by dispersion polymerization at 70 ° C. for 8 hours. After cooling, the polymer particles were isolated and washed with water. The polymerization conversion in this polymerization was 90%, and it was confirmed by observation with a scanning electron microscope that the particles were spherical particles having an extremely narrow particle size distribution with a particle size of 1.4 to 1.5 μm. The following operation was performed using this as a seed polymer.

First, 2,2'-azobisisobutyronitrile 0.7 g, ethylene glycol dimethacrylate 70 g, 1,2-dichloromethane 70 g, sodium dodecyl sulfate 1.31 g and water 560 g
Was finely dispersed such that the particle diameter of most of the oil droplets was in the range of 0.1 to 3 μm, to prepare an emulsion. Next, this emulsion was added dropwise to an aqueous dispersion composed of 10 g of the seed polymer obtained in the above dispersion polymerization, 0.1 g of sodium dodecyl sulfate, and 100 g of water at room temperature over 2 hours.
The oil droplets were absorbed by the seed polymer with slow stirring for 13 hours. Next, 263 g of a 2% aqueous polyvinyl alcohol solution was added thereto, and seed polymerization was performed at 70 ° C. for 8 hours.
After standing to cool, it was isolated by centrifugation and washed with water and methanol. The yield after drying was 80%. Observation with a scanning electron microscope of the polymer particles obtained by this polymerization revealed that the average particle size was 3.0 μm, and 90% or more of the particles were within the average particle size ± 1 μm. When the composition was analyzed by pyrolysis gas chromatography,
The weight fraction of glycidyl methacrylate unit is 5.9%, and the weight fraction of ethylene glycol dimethacrylate unit is 9
It was estimated at 4.1%. Further, when the specific surface area was estimated by the BET method, it was found to be 10.5 m ² / g of porous particles.

(2) Introduction of cation exchange groups 50 g of the raw material epoxy polymer obtained in (1) was added to 10% H ₂ S
Hydrolysis was carried out by heating at 50 ° C. for 5 hours in 535 g of an O ₄ aqueous solution to carry out a ring opening reaction of the epoxy ring. After completion of the reaction, the polymer was separated by filtration, sufficiently washed with demineralized water, and dried. To 50 g of the polymer having hydroxyl groups thus obtained, 190 ml of a 24% aqueous NaOH solution was added, and the mixture was added at room temperature for 30 minutes.
Stirred for minutes. Next, 200 ml of a 2-propanol solution of 80 g of monochloroacetic acid was added dropwise at 50 ° C. with stirring, and then reacted at 90 ° C. for 5.5 hours. After completion of the reaction, the polymer was filtered off, washed with 2500 g of demineralized water and 250 ml of 1N-HCl, and further washed with demineralized water until the washing solution became neutral to obtain a resin having a carboxymethyl group, and further washed with hot water and purified. did. The cation exchange capacity of the obtained resin was 0.26 meq / g of dry weight.

(3) Evaluation of separation of catabolic hemoglobin Resin having carboxymethyl group obtained in (2) 2.
1 g (wet state) was packed into a stainless steel column having an inner diameter of 6 mm and a length of 40 mm by a constant pressure pump, and the obtained packed column was connected to a high performance liquid chromatograph LC-6A system manufactured by Shimadzu Corporation. Hemoglobin Alc standard sample (US B
IO-RAD) was prepared by mixing with a predetermined amount of distilled water, and 10 μm of the sample was introduced into a column to evaluate the separation of catabolic hemoglobin. Elution was performed in an isocratic mode using a 70 mM phosphate buffer (pH 6.2) as an eluent. The flow rate of the eluent was set at 1.5 ml per minute. Detection was performed by absorption of visible light at 415 nm. The obtained chromatogram is shown in FIG. Alc component is 0.66 minutes, Ao component is 2.7
It eluted in 1 minute and was able to be separated very quickly.

Example 2 3.6 g of polyvinylpyrrolidone K-30 (trade name), 60 g of glycidyl methacrylate and 0.3 g of 2,2'-azobisisobutyronitrile were dissolved in 145.9 g of ethanol, and the dissolved oxygen was replaced with nitrogen gas. For 12 hours.
After cooling, the polymer particles were isolated and washed with water. The polymerization conversion in this polymerization was 88%, and it was confirmed by observation with a scanning electron microscope that the particles were spherical particles having an extremely narrow particle size distribution with a particle size of 2.5 to 2.7 μm. next,
Using this as a seed polymer, seed polymerization was carried out in the same manner as in Example 1. First, 2,2'-azobisisobutyronitrile 0.7 g, diethylene glycol dimethacrylate
70 g, 28 g of 1,2-dichloroethane, 28 g of toluene, 1.31 g of sodium dodecyl sulfate and 560 g of water were finely dispersed such that the particle diameter of most oil droplets was in the range of 0.1 to 3 μm, to prepare an emulsion. . Next, this emulsion was mixed with 10 g of the seed polymer obtained by the dispersion polymerization, and sodium dodecyl sulfate.
To the aqueous dispersion composed of 0.1 g and 100 g of water was added dropwise over 2 hours at room temperature, and the oil droplets were absorbed by the seed polymer while slowly stirring at the same temperature for 13 hours.

Next, 263 g of 2% aqueous polyvinyl alcohol solution
Was added and seed polymerization was performed at 70 ° C. for 8 hours. After cooling down,
Isolated by centrifugation and washed with water and methanol. The yield after drying was 85%. The polymer particles obtained by this polymerization had an average particle size of 5.0 μm when observed by a scanning electron microscope, and 90% or more of the particles had an average particle size of ± 1 μm.
m. Further, when the composition was analyzed by pyrolysis gas chromatography, the weight fraction of glycidyl methacrylate units was 8.6%, and the weight fraction of diethylene glycol dimethacrylate units was 91.4%.
%Met.

Then, the obtained raw material epoxy polymer was treated in the same manner as in Example 1 to obtain a resin having a carboxymethyl group, and further washed with hot water and purified. The cation exchange capacity of the obtained resin was 0.34 meq / g of dry weight.

2.2 g of the obtained resin having a carboxymethyl group (wet state) was packed into a stainless steel column having an inner diameter of 4.6 mm and a length of 70 mm by a constant pressure pump. The obtained packed column was subjected to separation evaluation of catabolic hemoglobin in exactly the same manner as in Example 1. The flow rate of the eluent was set at 1.5 ml per minute. Alc
The component eluted at 1.71 minutes and the Ao component eluted at 5.90 minutes, enabling rapid separation.

Example 3 5.2 g of polyvinylpyrrolidone K-30 (trade name), 45 g of glycidyl methacrylate, 5 g of 2-hydroxyethyl methacrylate and 0.5 of 2,2'-azobisisobutyronitrile
g was dissolved in 230 g of ethanol, the dissolved oxygen was replaced with nitrogen gas, and dispersion polymerization was carried out at 70 ° C. for 4 hours.
230 g of an aqueous solution of sodium dodecyl sulfate was added, and about 280 ml of ethanol containing water was distilled off under reduced pressure. A part of the polymer particles was taken out from the obtained aqueous dispersion, washed, and observed with a scanning electron microscope. As a result, it was confirmed that the particles were 1.7 to 1.8 μm in particle diameter and extremely narrow in particle size distribution. Next, seed polymerization was carried out using the aqueous dispersion of the polymer particles as they were. First, 2,2'-azobisisobutyronitrile 0.7 g, ethylene glycol dimethacrylate 70
g, 28 g of 1,2-dichloroethane, 28 g of toluene, 1.31 g of sodium dotesyl sulfate and 560 g of water were finely dispersed such that the particle diameter of most of the oil droplets was in the range of 0.1 to 3 μm to prepare an emulsion. . Next, this emulsion was added at room temperature to 110 g of the aqueous dispersion of the seed polymer obtained by the dispersion polymerization at room temperature.
The oil was dropped over a period of time, and the oil droplets were absorbed by the seed polymer while slowly stirring at the same temperature for 13 hours. Next, 263 g of a 2% aqueous polyvinyl alcohol solution was added thereto, and seed polymerization was performed at 70 ° C. for 8 hours. After standing to cool, it was isolated by centrifugation and washed with water, acetone and methanol. The yield after drying was 82%.

The polymer particles obtained by this polymerization had an average particle size of 3.5 μm when observed with a scanning electron microscope, and 90%
It was confirmed that the above particles were within an average particle size of ± 1 μm. Composition analysis by pyrolysis gas chromatography revealed that the weight fraction of glycidyl methacrylate unit was 10.6%, the weight fraction of ethylene glycol dimethacrylate unit was 88.4%, and the weight fraction of 2-hydroxyethyl methacrylate was Was estimated to be 1.0%.

Then, the obtained raw material epoxy polymer was treated in the same manner as in Example 1 to obtain a resin having a carboxymethyl group, and further washed with hot water and purified. The cation exchange capacity of the obtained resin was 0.30 meq / g of dry weight.

2.1 g of the obtained resin having a carboxymethyl group (wet state) was packed in a stainless steel column having an inner diameter of 6 mm and a length of 40 mm by a constant pressure pump. The packed column thus obtained was used in Example 1
The evaluation of separation of catabolic hemoglobin was performed in exactly the same manner as described above. The flow rate of the eluent was set at 1.5 ml per minute. The Alc component eluted at 1.02 minutes and the Ao component eluted at 3.83 minutes, enabling rapid separation.

Example 4 4.5 g of polyvinylpyrrolidone K-30 (trade name), 42.5 g of glycidyl methacrylate, 5.0 glycidyl acrylate
g, 2.5 g of stearyl methacrylate and 0.5 g of 2,2'-azobisisobutyronitrile were dissolved in 195 g of ethanol, and the dissolved oxygen was replaced with nitrogen gas, followed by dispersion polymerization at 70 ° C. for 6 hours. Post-processing was performed similarly. Observation of the obtained polymer particles with a scanning electron microscope confirmed that they were spherical particles having a particle size of 1.9 to 2.1 μm and having a very narrow particle size distribution. Next, seed polymerization was performed in the same manner as in Example 1 using this as a seed polymer. First
2,2'-azobisisobutyronitrile 0.5 g, ethylene glycol dimethacrylate 50 g, 1,2-dichloroethane 50
g, 0.94 g of sodium dodecyl sulfate and 400 g of water were finely dispersed such that the particle diameter of most of the oil droplets was in the range of 0.1 to 3 μm to prepare an emulsion. Next, this emulsion was added dropwise to an aqueous dispersion composed of 10 g of the seed polymer obtained in the previous dispersion polymerization, 0.1 g of sodium dodecyl sulfate, and 100 g of water over 2 hours at room temperature, and left at that temperature for 13 hours. The oil droplets were absorbed by the seed polymer with slow stirring.
Next, 188 g of a 2% aqueous polyvinyl alcohol solution was added thereto, and seed polymerization was performed at 70 ° C. for 8 hours. After cooling, it was isolated by centrifugation and washed with water and methanol. The yield after drying was 88%. Observation with a scanning electron microscope showed that the polymer particles obtained by this polymerization had an average particle size of 3.6 μm, and 90% or more of the particles had an average particle size of ± 1 μm.
It was confirmed that it was within. Composition analysis by pyrolysis gas chromatography revealed that the weight fraction of glycidyl methacrylate units was 13.2% and the weight fraction of ethylene glycol dimethacrylate units was 84.4%. Was estimated to be 2.4%. Then, the obtained raw material epoxy polymer was treated in the same manner as in Example 1 to obtain a resin having a carboxymethyl group, and further washed with hot water and purified. The cation exchange capacity of the obtained resin was 0.45 meq / g of dry weight.

2.1 g of the obtained resin having a carboxymethyl group (wet state) was packed in a stainless steel column having an inner diameter of 6 mm and a length of 40 mm by a constant pressure pump. The packed column thus obtained was used in Example 1
The evaluation of separation of catabolic hemoglobin was performed in exactly the same manner as described above. The flow rate of the eluent was set at 1.5 ml per minute. The Alc component eluted at 1.14 minutes and the Ao component eluted at 10.51 minutes, enabling rapid separation.

Example 5 7.2 g of polyvinylpyrrolidone K-30 (trade name), 49.85 g of glycidyl methacrylate, 0.15 g of ethylene glycol dimethacrylate and 0.5 g of 2,2'-azobisisobutyronitrile were dissolved in 342 g of ethanol, and nitrogen gas was used. After replacing the dissolved oxygen, dispersion polymerization was carried out at 70 ° C. for 6 hours, and post-treatment was carried out in the same manner as in Example 1. Observation of the obtained polymer particles with a scanning electron microscope confirmed that the particles were spherical particles having an extremely narrow particle size distribution with a particle size of 1.4 to 1.5 μm.
Next, seed polymerization was performed in exactly the same manner as in Example 1 using this as a seed polymer. First, 0.5 g of 2,2'-azobisisobutyronitrile, 50 g of ethylene glycol dimethacrylate, 60 g of 1,2-dichloroethane, 0.94 g of sodium dodecyl sulfate, and 400 g of water were used. It was finely dispersed so as to have a range of 3 μm to prepare an emulsion. Next, this emulsion was mixed with 12.5 g of the seed polymer obtained by the dispersion polymerization, and sodium dodecyl sulfate.
To the aqueous dispersion composed of 0.125 g and 125 g of water was added dropwise over 2 hours at room temperature, and oil droplets were absorbed by the seed polymer while slowly stirring at the same temperature for 13 hours. Then
Add 200 g of a 2% aqueous solution of polyvinyl alcohol to this, and add
Seed polymerization was performed at 8 ° C. for 8 hours. After standing to cool, it was isolated by centrifugation and washed with water, acetone and methanol. The yield after drying was 94%. The polymer particles obtained by this polymerization had an average particle diameter of 2.3 μm when observed by a scanning electron microscope, and 90% or more of the particles had an average particle diameter of ± 1 μm.
m. The composition was analyzed by pyrolysis gas chromatography. The weight fraction of glycidyl methacrylate units was 18.8%, and the weight fraction of ethylene glycol dimethacrylate units was 81.2%.
Met. Next, a resin having a carboxymethyl group was obtained from the obtained raw material epoxy polymer in the same manner as in Example 1, and further washed with hot water and purified. The cation exchange capacity of the obtained resin was 0.43 meq / g of dry weight.

2.1 g of the obtained resin having a carboxymethyl group (wet state) was packed in a stainless steel column having an inner diameter of 6 mm and a length of 40 mm by a constant pressure pump. The packed column thus obtained was used in Example 1
The evaluation of separation of catabolic hemoglobin was performed in exactly the same manner as described above. The flow rate of the eluent was set at 1.5 ml per minute. The Alc component eluted at 0.74 minutes and the Ao component eluted at 2.92 minutes, enabling rapid separation.

Example 6 Dispersion polymerization was carried out in exactly the same manner as in Example 1 to obtain polymer particles. Next, seed polymerization was performed in the same manner as in Example 1 using the obtained polymer particles as a seed polymer. First, 0.7 g of 2,2'-azobisisobutyronitrile,
66.5 g of ethylene glycol dimethacrylate, 3.5 g of glycidyl methacrylate, 70 g of 1,2-dichloroethane, 1.31 g of sodium dodecyl sulfate and 560 g of water are finely dispersed so that the particle diameter of most oil droplets is in the range of 0.1 to 3 μm. And
An emulsion was prepared. Next, this emulsion was added at room temperature to an aqueous dispersion comprising 10 g of the seed polymer obtained in the previous dispersion polymerization, 0.1 g of sodium dodecyl sulfate and 100 g of water.
The oil was dropped over a period of time, and the oil droplets were absorbed by the seed polymer while slowly stirring at the same temperature for 13 hours. Next, 263 g of a 2% aqueous polyvinyl alcohol solution was added thereto, and seed polymerization was performed at 70 ° C. for 8 hours. After standing to cool, it was isolated by centrifugation and washed with water, acetone and methanol. The yield after drying was 81%. Observation with a scanning electron microscope of the polymer particles obtained by this polymerization revealed that the average particle size was 3.1 μm, and that 90% or more of the particles were within the average particle size ± 1 μm. Further, when the composition was analyzed by pyrolysis gas chromatography, the weight fraction of glycidyl methacrylate units was 10.3.
%, The weight fraction of ethylene glycol dimethacrylate units was estimated to be 89.7%. Next, the obtained raw material epoxy polymer was treated in the same manner as in Example 1 to obtain a resin having a carboxymethyl group. The cation exchange capacity of the obtained resin was 0.35 meq / g of dry weight.
2.1 g of the obtained resin having a carboxymethyl group (wet state) was packed in a stainless steel column having an inner diameter of 6 mm and a length of 40 mm by a constant pressure pump. The obtained packed column was subjected to separation evaluation of catabolic hemoglobin in exactly the same manner as in Example 1. The flow rate of the eluent was set at 1.5 ml per minute. The Alc component eluted at 1.22 minutes and the Ao component eluted at 10.74 minutes, enabling rapid separation.

[Brief description of the drawings]

FIG. 1 is a view showing a chromatogram when catabolic hemoglobin is separated using the separation carrier of the present invention. 1 ... Alo, 2 ... Ao

Continuation of front page (72) Inventor Masahiko Annaka 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Yokohama, Japan Mitsubishi Chemical Research Institute (56) References JP-A-58-760 (JP, A) (58) Field (Int.Cl. ⁶ , DB name) G01N 33/72 G01N 30/88

Claims (2)

(57) [Claims] (1) a unit derived from glycidyl methacrylate represented by the following formula having a weight fraction of at least 3%: (R ¹ and R ² represent a hydrogen atom and / or an organic group having a cation exchange group.) A polyhydric alcohol and at least one polymerizable 2 in one molecule
A weight fraction of a unit derived from a crosslinkable monomer including a polyester with a carboxylic acid having a heavy bond of 80 to 97%,
Cation having a weight fraction of 0 to 5% of a unit derived from a monomer having one polymerizable double bond in one molecule other than glycidyl methacrylate and having an exchange capacity of 0.1 to 0.5 milliequivalent per dry weight. A carrier for separating catabolic hemoglobin, characterized by comprising an exchange resin. 2. The method according to claim 1, wherein the composition is manufactured by the following steps.
2. The carrier for separating catabolic hemoglobin according to item 1. First Step The weight fraction of glycidyl methacrylate is 85 to 100%, and the weight fraction of a crosslinkable monomer containing a polyester of a polyhydric alcohol and a carboxylic acid having at least one polymerizable double bond in one molecule is zero. -1%, a monomer mixture in which the weight fraction of a monomer having one polymerizable double bond in one molecule other than glycidyl methacrylate is 0 to 15%. The seed polymer is produced by dispersion polymerization in the presence of a non-existent medium. Second Step After adding a polymerizable monomer in an amount of 2 to 20 times the weight of the seed polymer to the seed polymer produced in the first step, seed polymerization is performed to produce a raw material epoxy polymer. However, the polymerizable monomer in the polymerizable monomer, 95
Other than glycidyl methacrylate and / or glycidyl methacrylate from 0 to 100% by weight of a crosslinkable monomer containing a polyester of a polyhydric alcohol and a carboxylic acid having at least one polymerizable double bond in one molecule; Contains a monomer having one polymerizable double bond in one molecule. Third Step In the second step, 0.1 to 0.5 milliequivalents of cation exchange groups are introduced per 1 g of dry weight using the prepared raw epoxy polymer.