JP3176423B2 - Chromatography carrier and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a carrier for chromatography.

[0002]

2. Description of the Related Art Conventionally, silica gel and crosslinked polymers have been generally used as chromatography carriers. However, in recent years, hydroxyapatite has been used as a chromatographic carrier for separating biological macromolecules such as proteins, nucleic acids, sugars, and glycosides by utilizing its biocompatibility (Japanese Patent Publication No. Hei. No. 37706).
For use as a carrier for chromatography, it is preferable that the shape is spherical. However, since hydroxyapatite is generally difficult to mold into a sphere,
Improvements have been attempted. Recently, a method for obtaining particulate hydroxyapatite by spraying and drying a suspension of hydroxyapatite fine crystal particles has been developed,
It has also been used as a carrier for chromatography (JP-A-62-91410, JP-A-1-23).
0413, JP-A-2-47551 and JP-A-64-42311).

[0003] Hydroxyapatite particle carriers are generally low in mechanical strength and irregular in particle size as compared with commonly used chromatography carriers, for example, silica-based carriers or polymer-based carriers. Shortcomings have been noted. The particle size of the carrier is generally 1 to 10 μm for analysis and 50 to 1000 μm for preparative,
Particle size uniformity has a significant effect on separation. Furthermore, in order to shorten the separation time, it must have a pressure resistance that can withstand a high flow rate. In other words, the particle size is smaller and uniform,
In addition, it is necessary that the particles have high mechanical strength, but the hydroxyapatite particle carrier is still insufficient for these requirements. Furthermore, since a baking step is usually included in the production process, it is difficult to control the properties of the particles, for example, the pore volume and the specific surface area, and it has not been possible to obtain hydroxyapatite particles with good reproducibility.

[0004] In order to compensate for the above-mentioned drawback, a chromatography carrier comprising core particles having high pressure resistance and coated with hydroxyapatite has been developed (JP-A-63-15797).
No. 3, JP-A-63-182563, JP-A-63-182563
JP-A-3-221844 and JP-A-2-147857
No.). In any of the above-mentioned methods for producing the carrier, a suspension of hydroxyapatite particles is applied to the core particles by spraying, and the core particles are coated with the hydroxyapatite particles by drying. Furthermore, there is a method in which the hydroxyapatite particles are firmly fixed by baking at a high temperature following the above step. As the core particles of the carrier, inorganic substances, for example, spherical glass beads (JP-A-63-157977 and JP-A-63-182563) and silica-based solids (JP-A-2-147857) are mainly used. Gazette). On the other hand, JP-A-63-157973, JP-A-63-1825
Japanese Patent Application Laid-Open No. 63-221844 and Japanese Patent Application Laid-Open No. 63-221844 disclose that polymer particles can be used as core particles, but there is no description of Examples thereof. In practice, when a method of spraying a suspension of hydroxyapatite particles onto polymer core particles to coat the core particles is employed, high-temperature treatment cannot be performed to firmly fix the hydroxyapatite particles. Therefore, its immobilization strength is insufficient.

[0005] On the other hand, as a method for producing hydroxyapatite particles, the Tizerius method (A. Tiselius et al . , Arch. Biochem.
Biophys. , 65, 132-155 (1956)). In this method, a precipitate of hydroxyapatite particles is formed by reacting a water-soluble calcium salt and a phosphate in an aqueous solution. Recently, it has been reported that, in the above-mentioned wet mixing method, the formation of hydroxyapatite is promoted or inhibited depending on predetermined conditions by adding a polymer containing a phosphoric acid group during the production of hydroxyapatite ( S.Shimabayashi and Y.Tanizawa, Chem.Pharm.Bu
ll. , 38, 1810-1814 (1990)). In the production of the hydroxyapatite, it is considered that calcium ions present in the reaction solution bind to the phosphate group of the polymer, and this portion serves as a nucleus to produce amorphous calcium phosphate, which grows to the hydroxyapatite. ing.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional disadvantages, and has as its object the following.
An object of the present invention is to provide a chromatography support coated with hydroxyapatite, which has excellent pressure resistance and swelling resistance, has good separation accuracy, and a method for producing the same.

[0007]

In view of the above demand for a hydroxyapatite carrier in the above-mentioned chromatographic analysis, the present inventors have conducted intensive studies on polymers containing a phosphate group and found that phosphorylation during the formation of hydroxyapatite was carried out. By utilizing the effect of the polymer, it has been found that it is possible to provide a chromatography support coated with hydroxyapatite that can achieve the above object,
The present invention has been completed.

[0008] The first chromatography carrier of the present invention has homogeneous core particles composed of a crosslinked polymer having a phosphate group, and a coating layer, and the coating layer is formed on the surface of the core particles chemically. Formed with bound hydroxyapatite particles.

The first method for producing a chromatographic carrier of the present invention comprises a step of bringing a solution containing a calcium salt and a phosphate into contact with homogeneous core particles made of crosslinked polymer particles having a phosphate group. Include.

[0010] The second chromatography carrier of the present invention has a core particle composed of a crosslinked polymer having a phosphate group only on the surface and a coating layer, and the coating layer is formed on the surface of the core particle by a chemical reaction. Formed by hydroxyapatite particles bound to

The second method for producing a chromatography carrier according to the present invention comprises a step of bringing a solution containing a calcium salt and a phosphate into contact with core particles composed of crosslinked polymer particles having a phosphate group only on the surface. Is included.

The material of the crosslinked polymer particles (core particles) used in the preparation of the first and second chromatography carriers of the present invention is obtained by (co) polymerizing a hydrophobic crosslinkable monomer. A (co) polymer or a copolymer of a hydrophobic crosslinkable monomer and a hydrophobic non-crosslinkable monomer is used. The hydrophobic crosslinkable monomer and the hydrophobic non-crosslinkable monomer,
Each can be used alone or in combination of two or more. Examples of the hydrophobic crosslinkable monomer include di (meth) acrylates such as ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate, and polypropylene glycol di (meth) acrylate. Acrylic acid esters; poly (meth) acrylic acid esters of polyhydric alcohols such as tetramethylolmethane tri (meth) acrylate and tetramethylolmethanetetra (meth) acrylate; divinyltoluene, divinylxylene,
Aromatic hydrocarbons having two or more vinyl groups such as divinylnaphthalene are used. Examples of the hydrophobic non-crosslinkable monomer include carboxylic acid vinyl esters such as vinyl acetate, vinyl propionate and vinyl butyrate; and aromatic hydrocarbons having a vinyl group such as styrene, vinyl toluene and vinyl naphthalene. ; Methyl (meth) acrylate, propyl (meth) acrylate, (meth)
(Meth) acrylic acid derivatives such as butyl acrylate, 2-hydroxyethyl methacrylate, glycidyl methacrylate, and (meth) acrylonitrile; allyl compounds such as triallyl isocyanate; vinylpyridine; The crosslinked polymer particles prepared by the method described below using the above monomers are not particularly limited, but are preferably spherical, and can be synthesized by suspension polymerization. The particle size is usually 0.001 to 10 mm, preferably 0.00
2-1 mm.

To prepare the first carrier of the present invention, first, homogeneous core particles made of a crosslinked polymer having a phosphate group are prepared. The core particles are prepared by mixing a monomer having a phosphate group (hereinafter simply referred to as a phosphate monomer) with the above-mentioned crosslinkable monomer and, if necessary, a non-crosslinkable monomer. It is obtained by turbid polymerization. As the phosphoric acid monomer, mono (2-acryloyloxyethyl) acid phosphate, mono (2-methacryloyloxyethyl) acid phosphate, mono (acryloyloxypolypropylene glycol) acid phosphate,
Mono (methacryloyloxy polypropylene glycol)
Acid phosphate is used. Two or more phosphoric acid monomers may be used as a mixture. The use amount of the phosphoric acid monomer is not particularly limited, but is preferably 10 to 200 parts by weight based on 100 parts by weight of the monomer other than the phosphoric acid monomer. A compound having a functional group which can be easily converted to a phosphate group by a chemical reaction, for example, a compound having a phosphate group, can also be used as the phosphate monomer. When polymerization is performed using such a phosphate monomer, the functional group is converted into a phosphate group by a chemical reaction after the polymerization is completed.
However, the chemical reaction must be performed under conditions that do not adversely affect the crosslinked polymer itself.

A monomer mixture containing such a phosphoric acid monomer (a phosphoric acid monomer, a crosslinkable monomer and, if necessary, a non-crosslinkable monomer, and a polymerization initiator) and, if necessary, To carry out suspension polymerization using a diluent. As the polymerization initiator,
Any of the radical generating catalysts can be used.
For example, benzoyl peroxide, acetyl peroxy
Organic peroxides such as azobisisobutyronitrile
And azo compounds such as azobisisobutyroamide
Used. In addition, as the diluent, the above monomer
Organic solvents that dissolve and do not dissolve the polymer
A shift can also be used. For example, toluene, xylene
Fragrances such as benzene, diethylbenzene, dodecylbenzene
Aromatic hydrocarbons; hexane, heptane, octane, decane
Such saturated hydrocarbons; isoamyl alcohol, hexyl
Alcohol, octyl alcohol
And the like. Add such diluent as needed
Thereby, porous crosslinked polymer particles are obtained,
The surface area of the particles can be increased. Use of diluent
Although the amount is not limited, the above monomer mixture (monomer and
Polymerization initiator) not more than 300 parts by weight per 100 parts by weight
It is preferably a ratio.

The above suspension polymerization is carried out, for example, by mixing the above monomer
(Monomer and polymerization initiator) and dilution if necessary
Agents such as polyvinyl alcohol and calcium phosphate
Add the suspension stabilizer to the dissolved aqueous phase, replace with nitrogen, and stir.
By heating to 40-100 ° C while heating
Can be. After the polymerization is completed, the obtained crosslinked polymer particles are
Wash thoroughly with organic solvent and contained in particles
Suspension stabilizers, diluents and residues
Spherical particles can be obtained by removing existing monomers
You. When a diluent is added, porous particles are obtained.
Different compatibility with the above hydrophobic monomer mixture as diluent
By using various organic solvents, porous polymer particles
It is possible to arbitrarily change the size of the pore
You. Further, by classifying the particles as necessary, the crosslinked polymer particles having a phosphate group used in the present invention are obtained and used as core particles.

In order to form a coating layer composed of hydroxyapatite particles on the surface of the core particles, a chemical bonding method can be used. That is, the crosslinked polymer particles may be added to a solution capable of producing hydroxyapatite, that is, a solution containing a calcium salt and a phosphate, or the medium in which the polymer particles are suspended may produce hydroxyapatite. A solution containing substances, for example, calcium chloride and potassium phosphate, or a method of adding calcium chloride and potassium phosphate is used. The phosphate groups of the polymer particles are bonded with calcium ions, so that the phosphate groups of the polymer become nuclei for hydroxyapatite formation, and consequently the crosslinked polymer particles are coated with the hydroxyapatite particles. A first carrier of the invention is obtained.

In an analysis system using a hydroxyapatite carrier, it is hydroxyapatite that interacts with the sample, and the core particles used at that time are preferably inert having mechanical strength. Therefore, the phosphate group serving as a nucleus for hydroxyapatite formation in the present invention is preferably present only on the surface portion of the core particle. If the phosphate groups are uniformly distributed in the core particles, the remaining phosphate groups after the hydroxyapatite generation reaction may cause ionic interaction during analysis. The presence of phosphate groups, i.e. hydrophilic groups, in the particles
This may cause a decrease in mechanical strength and a delay in equilibration when switching the eluent in the chromatographic analysis. In view of such a point, the present inventor further attempted to prepare the following second carrier.

The core particle used in the second carrier of the present invention is a hydrophilic polymer obtained by polymerizing a phosphoric acid monomer having a crosslinked polymer particle as a skeleton, and only a surface portion of the crosslinked polymer particle. Is a polymer having a two-layer structure coated with. To prepare the core particles, first, crosslinked polymer particles having a phosphate group only on the surface are prepared. It first of all, of the above-mentioned
Mixing with crosslinkable monomers or, if necessary, non-crosslinkable monomers
Polymerization initiator (but do not use phosphoric acid monomer)
And polymerizing by the same method as described above.
Ri was prepared having no crosslinked polymer particle ion exchange groups,
Next, the obtained crosslinked polymer particles are impregnated with a polymerization initiator. In order to impregnate the polymerization initiator, the initiator is dissolved in a solvent having a low boiling point and a good affinity for the crosslinked polymer particles, and the above crosslinked polymer particles are immersed therein. This allows the polymerization initiator to penetrate into the particles. This is heated at a temperature lower than the decomposition point of the polymerization initiator, if necessary, to remove the solvent, thereby obtaining crosslinked polymer particles containing the polymerization initiator inside, mainly near the surface. The polymerization initiator-containing particles are dispersed in an aqueous dispersion medium in which the phosphoric acid monomer is dissolved, or the phosphoric acid monomer is added to an aqueous dispersion medium in which the particles are dispersed, and dissolved. Then, after purging with nitrogen, the mixture is heated with stirring to carry out polymerization. By this polymerization, the phosphoric acid monomer is polymerized on the surface of the crosslinked polymer particle, and coats the particle surface. Further, when the crosslinked polymer particles are porous, a polymer layer having a phosphate group is also formed on the inner surface of the pores. In order to stabilize the dispersibility of the crosslinked polymer particles in the aqueous dispersion medium, carboxymethyl cellulose,
A dispersion stabilizer such as polyvinyl alcohol can also be added. The temperature and time of the polymerization vary depending on the type of the hydrophilic monomer to be reacted and the type of the polymerization initiator,
It is about 0.5 to 40 hours at 40 to 100 ° C. By the above method, crosslinked polymer particles having a phosphate group only on the surface are obtained and used as core particles. Here, "only on the surface" refers to the surface or a portion of the region extending from the surface to 1% or less of the particle size.

In addition to the method of subjecting the crosslinked polymer particles impregnated with the polymerization initiator to a polymerization reaction with a phosphoric acid monomer,
A crosslinked polymer particle having a phosphate group only on the surface can also be prepared by a continuous method of reacting a phosphoric acid monomer following the preparation of the porous crosslinked polymer particle. In this method, a polymerization reaction for preparing the porous crosslinked polymer particles is first started. When the polymerization has progressed to some extent and the unreacted polymerization initiator remains, the phosphoric acid monomer is added to the reaction system and dissolved in the dispersion medium. In such a state, since the polymerization initiator remains on the organic phase in the system and on the surface of the generated hydrophobic polymer particles, polymerization of the phosphoric acid monomer subsequently occurs, and only the surface of the crosslinked polymer is coated. A phosphoric acid polymer layer is formed. When the phosphoric acid monomer is added, the form of addition is not particularly limited, and may be any of a method of adding all at once, a method of adding several times or a method of adding dropwise. Although it is not necessary to lower the temperature at the time of the addition, it is better to cool once, add the phosphoric acid monomer and dissolve well in the aqueous phase, and then raise the temperature again to improve polymerization reproducibility. Further, the temperature may be changed before and after the addition of the phosphoric acid monomer. The polymerization conditions after the addition of the phosphoric acid monomer vary depending on the monomer used, but are from 40 to 100 ° C. for 0.5 to 10 hours.
The polymerization is stopped at the stage where aggregation does not occur due to the homopolymer of the phosphoric acid monomer. After completion of the polymerization, the polymer particles are washed, and the particles are further classified, if necessary, to obtain crosslinked polymer particles having a phosphate group only on the surface used in the present invention and used as core particles. . The formation of a coating layer composed of hydroxyapatite particles on the surface of the core particles is performed by the same chemical bonding method as the method for producing the first carrier of the present invention.

According to each of the above-mentioned methods, a chromatography carrier in which the coating layer on the core particle surface is formed of hydroxyapatite is prepared.

The chromatographic support of the present invention has high pressure resistance and little swelling / shrinkage because the surface of the crosslinked polymer is coated with hydroxyapatite. Therefore, analysis can be performed at a high flow rate, and the analysis time can be reduced.
Further, since the carrier itself is spherical and the particle diameter is uniform, the separation accuracy is good. The chromatography carrier of the present invention can be used as a chromatography carrier for separating biopolymers by utilizing the above-mentioned performance.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to embodiments.

The packing of the carrier for chromatography and the evaluation of the column were carried out by the following methods.

"Method of filling carrier" 2.3 g of the carrier was weighed, dispersed in 30 ml of purified water, and stirred for 5 minutes. A packer manufactured by Umetani Seiki Co., Ltd. was connected to a stainless steel empty column (6 mmφ × 75 mm), and the dispersion was poured into the packer, and a liquid pump LC manufactured by Shimadzu Corporation was used.
It was connected to -6AD and filled at a constant pressure of 100 kg / cm ² .

"Evaluation of pressure resistance of carrier" Purified water was passed through a column packed by the above method, and the relationship between flow rate and pressure loss was examined. The liquid was sent by a liquid sending pump LC-6AD manufactured by Shimadzu Corporation, and the pressure of a pressure gauge of the pump was monitored by a recorder.

"Evaluation of Swelling Resistance of Carrier" The particle size in a dry state and a state immersed in purified water was measured with a Coulter Multisizer (manufactured by Coulter KK), and the change in the particle size in a dry state and a state immersed in purified water was measured. Was performed by examining

[0027] using a column packed with "Evaluation method of carrier" above method was subjected to liquid chromatography using a Sekisui Chemical Co. liquid chromatography graphs system SSLC-20. The analysis conditions were set as follows.

Eluent: Elution by concentration gradient method using phosphate buffer (pH 6.8) Flow rate: 1 ml / min Detection: Absorbance at 280 nm Sample: Standard solution of the following proteins (each 1 mM aqueous solution) Bovine serum albumin Lysozyme cytochrome c Injection volume: 50 μl

Example 1 In this example, a carrier was prepared in which crosslinked polymer particles having a phosphate group uniformly in the particles were used as core particles, and the coating layer was formed of hydroxyapatite.

100 g of tetraethylene glycol dimethacrylate (crosslinkable monomer), 100 g of tetramethylol methane triacrylate (crosslinkable monomer),
1 g of benzoyl peroxide (polymerization initiator) is dissolved in a mixed solution of 50 g of (methacryloyloxyethyl) acid phosphate (phosphate monomer), added to 2.5 liters of a 4% aqueous solution of polyvinyl alcohol, and stirred. After sizing, the suspension was heated to 80 ° C. under nitrogen substitution to perform suspension polymerization. After polymerization at 80 ° C. for 5 hours, the product was washed with hot water and acetone sequentially and dried, and the obtained crosslinked polymer particles were obtained.
Nippon Steel Mining Co., Ltd. air classifier ELBOW-JET L
Classification was performed using ABO EJ-L-3 .

The resulting fine crosslinked polymer particles containing phosphate groups were coated with hydroxyapatite by the following chemical method. First, 100 g of the obtained particles was added to 2.5 mM
Immersed in 1 liter of KH ₂ PO ₄ solution.
One liter of a mM CaCl ₂ solution was added and stirred.
After reacting at room temperature for 100 minutes, the carrier was separated by filtration and dried.

The obtained particles were subjected to scanning electron microscopy (
Observation by Hitachi, Ltd.) revealed that the particle surfaces were covered with hydroxyapatite. The pressure resistance and the swelling resistance were evaluated by the above methods. In the evaluation of the pressure resistance, the pressure loss was proportional to at least up to 350 kg / cm ² . In the evaluation of the swelling resistance, no change in the particle size was observed, and the evaluation was good. Further, chromatography was performed by the above method. The resulting chromatogram is
It is shown in FIG. In FIG. 1 and FIG. 2 and FIG.
Peak due to bovine serum albumin, 2 is lysozyme
And peak 3 due to cytochrome c.
It is.

Comparative Example 1 In this comparative example, a separation example using crushed hydroxyapatite will be described.

When hydroxyapatite (produced by Daiichi Kagaku Kagaku Kogyo Co., Ltd.) was suspended in purified water and tried to be filled by the above method, the filling liquid did not flow. Fill pressure 1
The pressure was reduced from 00 kg / cm ² to 10 kg / cm ² to perform constant pressure filling.

The pressure resistance and the swelling resistance were evaluated by the above methods. In the evaluation of pressure resistance, 20 kg / c
Only up to m ² was proportional to the flow rate. In the evaluation of the swelling resistance, one having an average particle size of 8.5 μm in the dry state swelled to 10.6 μm in the immersion state. Further, chromatography was performed by the above method. The resulting chromatogram is shown in Figure 2. As compared with Example 1 , the separation was poor and the analysis time was long.

Example 2 In this example, a carrier was prepared in which a crosslinked polymer particle having a phosphate group only on the surface was used as a core particle, and the coating layer was formed of hydroxyapatite.

Diethylene glycol dimethacrylate
(Crosslinkable monomer) 100 g, tetramethylolmethanete
100 g of triacrylate (crosslinkable monomer) and ben
1 g of zoyl peroxide (polymerization initiator) was mixed to obtain
2.5% 4% aqueous solution of polyvinyl alcohol
After adding to the turtle and granulating with stirring, replace with nitrogen
The suspension polymerization was carried out by heating to 80 ° C under the following conditions. 8 hours at 80 ° C
After polymerization, wash the product sequentially with hot water and acetone
And dried. Nippon Steel Mining Co., Ltd.
ELBOW-JET LABOE Air Classifier
Classification was performed using a JL-3 model. 100 g of this polymer particle
Was immersed in 1 liter of acetone in which 0.5 g of benzoyl peroxide (polymerization initiator) was dissolved to impregnate the polymerization initiator. Then the acetone was distilled off at 20 ° C. under reduced pressure. The obtained initiator-containing polymer was dispersed in 1 liter of a 50% aqueous methanol solution, and the mixture was stirred with mono (2-methacryloyloxy) acid phosphate 3
0 g was added and dissolved. This solution is replaced with nitrogen and then 80 ℃
For 5 hours. The product was washed with hot water and acetone and dried. The obtained crosslinked polymer particles were coated with hydroxyapatite in the same manner as in Example 1 .

When the obtained particles were observed with a scanning electron microscope, the surfaces of the particles were covered with hydroxyapatite. The pressure resistance and the swelling resistance were evaluated by the above methods. In the evaluation of pressure resistance, at least 35
The flow rate and the pressure loss were proportional to 0 kg / cm ² . In the evaluation of the swelling resistance, no change in the particle size was observed, and the evaluation was good. Further, chromatography was performed by the above method. The resulting chromatogram is shown in Figure 3. In addition to the comparative examples described above, a better separation was obtained than in each example, and the analysis could be performed in a short time.

Example 3 In this example, the continuous method of preparing a hydrophobic crosslinked polymer particle and reacting a phosphoric acid monomer as described in the text was employed.

1 g of benzoyl peroxide was dissolved in 200 g of triethylene glycol dimethacrylate (crosslinkable monomer), and 2.5 g of a 4% aqueous solution of polyvinyl alcohol was added.
Added to liters. After the solution was replaced with nitrogen while stirring, the solution was heated to 80 ° C. to perform polymerization.

After polymerization for 2 hours, the mixture was cooled to 30 ° C.
60 g of mono (2-methacryloyloxy) acid phosphate was added and dissolved in the reaction solvent. It was heated again and polymerization was carried out at 80 ° C. for 5 hours. The obtained product was sequentially washed with hot water and acetone and dried. Using the obtained polymer, a hydroxyapatite-coated support was obtained in the same manner as in Example 1 .

When the obtained particles were observed with a scanning electron microscope, the surface of the particles was covered with hydroxyapatite. The pressure resistance and the swelling resistance were evaluated by the above methods. In the evaluation of pressure resistance, at least 35
The flow rate and pressure loss were proportional to 0 kg / cm ² . In the evaluation of the swelling resistance, no change in the particle size was observed, and the evaluation was good. Further, chromatography was performed by the above method. The resulting chromatogram was as in FIG.

[0043]

According to the present invention, a hydroxyapatite-coated carrier for chromatography having excellent pressure resistance and swelling resistance and high resolution can be obtained. This is due to the use of highly crosslinked polymer particles as the core particles. Furthermore, according to the method of the present invention, these carriers can be produced with good reproducibility.

[Brief description of the drawings]

FIG. 1 shows a chromatogram obtained as a result of performing chromatography using the carrier obtained in Example 1 .

FIG. 2 shows a chromatogram obtained as a result of performing chromatography using the carrier used in Comparative Example 1.

FIG. 3 shows a chromatogram obtained as a result of performing chromatography using the carriers obtained in Examples 2 and 3 .

Claims (4)

(57) [Claims] 1. A homogeneous chromatographic carrier comprising a crosslinked polymer having a phosphate group, and a chromatographic carrier having a coating layer, wherein the coating layer is a hydroxyapatite chemically bonded to the surface of the core particle. A chromatography carrier formed of particles. To 2. A homogeneous core particles made of a crosslinked polymer having a phosphoric acid group, comprising contacting a solution containing the calcium salt and phosphate, the chromatography matrix of claim 1 Production method. 3. Core particles comprising a crosslinked polymer having a phosphoric acid group only on the surface thereof, and a chromatographic carrier having a coating layer, wherein the coating layer comprises a hydroxyl group chemically bonded to the surface of the core particles. A chromatographic carrier formed of apatite particles. 4. The chromatography carrier according to claim 3 , further comprising a step of bringing a solution containing a calcium salt and a phosphate into contact with core particles made of a crosslinked polymer having a phosphate group only on the surface. Manufacturing method.