JPH05293372A - Carrier for chromatography and its production - Google Patents

Abstract

PURPOSE:To produce a carrier coated with hydroxyapatite, excellent in pressure resistance and swelling resistance and having satisfactory separation accuracy. CONSTITUTION:This carrier for chromatography has core granules made of cross-linked polymer having no ion exchange group and coating layers formed with hydroxyapatite particles physically fused in the surface of the core granules.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a carrier for chromatography.

[0002]

2. Description of the Related Art Conventionally, silica gel or a cross-linked polymer has been generally used as a carrier for chromatography. However, in recent years, hydroxyapatite has come to be used as a chromatographic carrier for separating biopolymers such as proteins, nucleic acids, sugars and glycosides by utilizing its biocompatibility (Japanese Patent Publication No. 37706).
When used as a carrier for chromatography, it is preferably spherical in shape. However, in general, hydroxyapatite is difficult to mold into a spherical shape,
The improvement is being tried. Recently, a method for obtaining particulate hydroxyapatite by spraying and drying a suspension of hydroxyapatite fine crystal particles has been developed,
It has also come to be used as a carrier for chromatography (JP-A-62-91410, JP-A-1-23).
0413, JP-A-2-47551 and JP-A-64-42311).

Hydroxyapatite particle carriers are generally low in mechanical strength and uneven in particle size, as compared with commonly used carriers for chromatography, for example, silica-based carriers or polymer-based carriers. Disadvantages are pointed out. The particle size of the carrier is generally 1 to 10 μm for analysis and 50 to 1000 μm for preparative separation,
Uniformity of particle size greatly affects separation. Furthermore, in order to shorten the separation time, it must have pressure resistance to withstand a high flow rate. In other words, the particle size is smaller and uniform,
And, it is necessary that the particles have high mechanical strength, but the hydroxyapatite particle carrier is still insufficient in these requirements. Furthermore, since a calcination step is usually included in the manufacturing process, it becomes difficult to control the properties of the particles, for example, the pore volume and the specific surface area, and hydroxyapatite particles cannot be obtained with good reproducibility.

In order to make up for the above drawbacks, a chromatographic carrier comprising a core particle having a high pressure resistance and hydroxyapatite coated thereon has been developed (Japanese Patent Laid-Open No. 63-15797).
No. 3, JP-A-63-182563, JP-A-Sho 6
JP-A-3-221844 and JP-A-2-147857.
Publication). Each of the above-mentioned methods for producing the carrier is a method in which a suspension of hydroxyapatite particles is sprayed onto the core particles to be adhered thereto, and the core particles are coated with the hydroxyapatite particles by drying. Further, there is also a method of firmly fixing the hydroxyapatite particles by firing at a high temperature subsequent to the above steps. The core particles of the carrier are mainly inorganic substances such as spherical glass beads (JP-A-63-157973 and JP-A-63-182563) and silica-based solids (JP-A-2-147857). Gazette) is used. On the other hand, JP-A-63-157973 and JP-A-63-1825.
No. 63 and JP-A No. 63-221844 describe that it is possible to use polymer particles as the core particles, but the examples thereof are not described. When a method of actually spraying a suspension of hydroxyapatite particles onto the polymer core particles to coat the core particles, high temperature treatment cannot be performed in order to firmly fix the hydroxyapatite particles. Therefore, its immobilization strength is insufficient.

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)) and so-called wet mixing methods. This method is a method of producing a precipitate of hydroxyapatite particles by reacting a water-soluble calcium salt and a phosphate in an aqueous solution. Recently, in the above wet mixing method, it has been reported that the formation of hydroxyapatite is promoted or inhibited under certain conditions by adding a polymer containing a phosphate group when hydroxyapatite is produced ( S. Shimabayashi and Y. Tanizawa, Chem.Pharm.Bu
ll. , 38, 1810-1814 (1990)). In the production of the above hydroxyapatite, calcium ions present in the reaction solution are bound to the phosphate group of the polymer, and this portion becomes the nucleus to produce amorphous calcium phosphate, which is considered to grow to hydroxyapatite. ing.

[0006]

SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned conventional drawbacks, and its object is to:
It is an object of the present invention to provide a hydroxyapatite-coated chromatographic carrier having excellent pressure resistance and swelling resistance and good separation accuracy, and a method for producing the same.

[0007]

Means for Solving the Problems In view of the demand for the hydroxyapatite carrier in the above-mentioned chromatographic analysis, the present inventor diligently studied a polymer containing a phosphate group, and as a result, phosphorylation during the formation of hydroxyapatite. It was found that by utilizing the influence of the polymer, it is possible to provide a chromatographic carrier coated with hydroxyapatite that can achieve the above object,
The present invention has been completed.

The first chromatographic carrier of the present invention has core particles made of a crosslinked polymer having no ion exchange group and a coating layer, and the coating layer is physically fused to the surface of the core particles. Formed by the hydroxyapatite particles.

The second chromatographic carrier of the present invention has a homogeneous core particle composed of a cross-linked polymer having a phosphoric acid group, and a coating layer, and the coating layer chemically reacts on the surface of the core particle. It is formed of bound hydroxyapatite particles.

A second method for producing a carrier for chromatography of the present invention comprises a step of bringing a solution containing a calcium salt and a phosphate into contact with a homogeneous core particle composed of a crosslinked polymer particle having a phosphate group. Include.

The third chromatographic carrier of the present invention has core particles made of a cross-linked polymer having a phosphoric acid group only on the surface, and a coating layer, and the coating layer chemically reacts on the surface of the core particles. Formed of hydroxyapatite particles bound to.

The third method for producing a carrier for chromatography of the present invention is a step of bringing a solution containing a calcium salt and a phosphate salt into contact with a core particle composed of a crosslinked polymer particle having a phosphoric acid group only on the surface. Includes.

The material for the crosslinked polymer particles (core particles) used for preparing the first, second and third chromatography carriers of the present invention is (co) polymerized with a hydrophobic crosslinkable monomer. A (co) polymer obtained by the above 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 may be used alone or in combination of two or more kinds. Examples of the hydrophobic crosslinkable monomer include ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate,
Di (meth) acrylic acid esters such as propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate; polyhydric alcohol poly (tetramethylolmethane tri (meth) acrylate, tetramethylolmethane tetra (meth) acrylate) (Meth) acrylic acid ester; aromatic hydrocarbons having two or more vinyl groups such as divinyltoluene, divinylxylene and divinylnaphthalene are used.
Examples of the hydrophobic non-crosslinkable monomer include vinyl acetate, vinyl propionate, vinyl butyrate, and other carboxylic acid vinyl esters; styrene, vinyltoluene, vinylnaphthalene, and other aromatic hydrocarbons having a vinyl group. (Meth) acrylic acid derivatives such as methyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, 2-hydroxyethyl methacrylate, glycidyl methacrylate, (meth) acrylonitrile; triallyl isocyanate, etc. Allyl compound; vinyl pyridine, etc. are used. The cross-linked polymer particles prepared by the method described below using the above-mentioned 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 0.002 to 1 mm.

To prepare the first carrier of the present invention, first, crosslinked polymer particles having no ion exchange group are prepared as core particles. The crosslinked polymer particles are obtained by adding a polymerization initiator to the above-mentioned crosslinkable monomer or, if necessary, a mixture with a non-crosslinkable monomer, and polymerizing the mixture. As the polymerization initiator used in this case, any radical-generating catalyst can be used. Examples thereof include organic peroxides such as benzoyl peroxide and acetyl peroxide; and azo compounds such as azobisisobutyronitrile and azobisisobutyroamide. Further, by adding a diluent as needed, porous crosslinked polymer particles can be obtained, and the surface area of the particles can be increased. As the diluent, any organic solvent that dissolves the monomer and does not dissolve the polymer can be used. Examples thereof include aromatic hydrocarbons such as toluene, xylene, diethylbenzene and dodecylbenzene; saturated hydrocarbons such as hexane, heptane, octane and decane; alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol. The amount of the diluent used is not limited, but is preferably 300 parts by weight or less with respect to 100 parts by weight of the monomer mixture (monomer and polymerization initiator).

For example, the above-mentioned monomer mixture (monomer and polymerization initiator) and, if necessary, a diluent are added to an aqueous phase in which a suspension stabilizer such as polyvinyl alcohol or calcium phosphate is dissolved, and after substitution with nitrogen. , 40 to 1 with stirring
Suspension polymerization is carried out by heating to 00 ° C. After completion of the polymerization, the resulting crosslinked polymer particles are thoroughly washed with hot water and an organic solvent to remove the suspension stabilizer, diluent and residual monomer contained in or attached to the particles. Thereby, spherical particles are obtained. Porous particles are obtained when a diluent is added. By using various organic solvents having different compatibility with the above-mentioned hydrophobic monomer mixture as a diluent, it is possible to arbitrarily change the size of the pores of the porous polymer particles. Further, by classifying the particles as necessary, the crosslinked polymer particles having no ion exchange group used in the present invention can be obtained and used as the core particles.

To form a coating layer of hydroxyapatite particles on the surface of the core particles, the following physical method is used. That is, the first carrier of the present invention can be obtained by adding hydroxyapatite powder to the crosslinked polymer particles obtained above and stirring and mixing at a suitable temperature. To perform this operation, a commercially available surface fusion device, for example, a mechanofusion system manufactured by Hosokawa Micron Co., Ltd. is used. The device is an application of a mechanochemical reaction, in which strong mechanical energy is applied to the surface of powder particles of a plurality of different materials to cause a strong bond between particles (this is called surface fusion). By this,
The surface is modified and particles with new physical properties are formed (Yokoyama, Urayama, Naito and Kato, Grinding, 32, 78 (1988)).
Hydroxyapatite particles are used in an amount of 5 to 100 parts by weight, preferably 10 to 50 parts by weight, based on 100 parts by weight of the crosslinked polymer particles. The crosslinked polymer particles and the hydroxyapatite particles are set in the apparatus, and the temperature is about 30 ° C.
Rotor speed of about 1000 rp at a temperature of ~ 100 ° C
By mixing at m to 3000 rpm, the first carrier of the present invention is obtained. Other surface fusion devices such as Nara Machinery Co., Ltd. Hybridization System; Fuji Paudal Co., Ltd. New / Marumerizer; Kawasaki Heavy Industries, Ltd. Kryptron System KOSMOS-C are commercially available. Can be used in the invention.

To prepare the second carrier of the present invention, first, homogeneous core particles made of a cross-linked polymer having a phosphate group are prepared. The core particles are prepared by mixing a monomer having a phosphoric acid group (hereinafter, simply referred to as a phosphoric acid monomer) together with the above-mentioned crosslinkable monomer and, if necessary, a non-crosslinkable monomer. Obtained by turbid polymerization. Examples of the phosphoric acid monomer include mono (2-acryloyloxyethyl) acid phosphate, mono (2-methacryloyloxyethyl) acid phosphate, mono (acryloyloxypolypropylene glycol) acid phosphate,
Mono (methacryloyloxy polypropylene glycol)
Acid phosphate or the like is used. Two or more kinds of the above phosphoric acid monomers may be mixed and used. The amount of the phosphoric acid monomer used is not particularly limited, but is preferably 10 to 200 parts by weight with respect to 100 parts by weight of the monomer other than the phosphoric acid monomer. A compound having a functional group that can be easily converted into a phosphoric acid group by a chemical reaction, for example, a phosphoric acid ester group can also be used as the phosphoric acid monomer. When polymerization is performed using such a phosphoric acid monomer, the functional group is converted into a phosphoric acid group by a chemical reaction after the completion of the polymerization.
However, the chemical reaction must be carried out under conditions that do not adversely affect the crosslinked polymer itself. A monomer mixture containing such a phosphoric acid monomer (phosphoric acid monomer, crosslinkable monomer and optionally non-crosslinkable monomer, and polymerization initiator) and, if necessary, diluent Is subjected to suspension polymerization by the same method as described above. After the completion of the polymerization, the polymer particles are washed and, if necessary, classified, whereby the phosphoric acid group-containing crosslinked polymer particles used in the present invention are obtained and used as core particles.

A chemical bonding method may be used to form a coating layer of hydroxyapatite particles on the surface of the core particles. That is, the crosslinked polymer particles can be added to a solution capable of producing hydroxyapatite, that is, a solution containing a calcium salt and a phosphate salt, or hydroxyapatite can be produced in a medium in which the polymer particles are suspended. A solution containing a substance, for example, calcium chloride and potassium phosphate, or a method of adding calcium chloride and potassium phosphate is used. By binding the phosphate groups and calcium ions on the surface of the polymer particles, the phosphate groups of the polymer serve as nuclei for forming hydroxyapatite, and as a result, the crosslinked polymer particles are coated with the hydroxyapatite particles. A second carrier of the invention is obtained.

In an analytical system using a hydroxyapatite carrier, it is hydroxyapatite that interacts with the sample, and the core particles used at that time are preferably inert particles having mechanical strength. Therefore, it is preferable that the phosphoric acid group serving as the nucleus of hydroxyapatite formation in the present invention is present only on the surface portion of the core particle. If the phosphoric acid groups are uniformly distributed in the core particles, it is considered that the residual phosphoric acid groups after the hydroxyapatite formation reaction have an ionic interaction during the analysis. The presence of a phosphate group in the particle, that is, a hydrophilic group,
It can also be a cause of a decrease in mechanical strength and a delay in equilibration when switching the eluent in a chromatographic analysis. In consideration of such a point, the present inventor further tried to prepare the third carrier shown below.

The core particle used in the third carrier of the present invention is a hydrophilic polymer obtained by polymerizing a phosphoric acid monomer with a cross-linked polymer particle as a skeleton, and only the surface portion of the cross-linked polymer particle. Is a polymer having a two-layer structure. To prepare the core particles, first, crosslinked polymer particles having a phosphoric acid group only on the surface are prepared. First, a crosslinked polymer particle having no ion exchange group, which is used as the first carrier of the present invention, is prepared by the same method as described above, and then the obtained crosslinked polymer particle is added with a polymerization initiator. Impregnate. 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 in the solvent. This causes the polymerization initiator to penetrate into the particles. If necessary, this is heated at a temperature not higher than the decomposition point of the polymerization initiator to remove the solvent, to obtain crosslinked polymer particles containing the polymerization initiator in the inside thereof, 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 above phosphoric acid monomer is added to an aqueous dispersion medium in which the particles are dispersed, and dissolved. Then, after substituting with nitrogen, polymerization is carried out by heating with stirring. By this polymerization, the phosphoric acid monomer is polymerized on the surface of the crosslinked polymer particles to coat the particle surface. Further, when the crosslinked polymer particles are porous, a polymer layer having a phosphoric acid group is also formed on the inner surface of the pores. In order to stabilize the dispersibility of the crosslinked polymer particles in the above aqueous dispersion medium, carboxymethyl cellulose,
A dispersion stabilizer such as polyvinyl alcohol can also be added. The temperature and time of polymerization vary depending on the type of hydrophilic monomer to be reacted and the type of polymerization initiator,
It is about 0.5 to 40 hours at 40 to 100 ° C. By the above method, crosslinked polymer particles having a phosphoric acid group only on the surface are obtained and used as core particles. Here, “only on the surface” refers to a surface or a portion of a region extending from the surface to 1% or less of the particle diameter.

In addition to the method of subjecting the crosslinked polymer particles impregnated with the above polymerization initiator to the polymerization reaction with the phosphoric acid monomer,
Crosslinked polymer particles having a phosphoric acid group only on the surface can also be prepared by a continuous method in which a phosphoric acid monomer is reacted subsequently to the preparation of porous crosslinked polymer particles. In this method, first, a polymerization reaction for preparing the above-mentioned porous crosslinked polymer particles is started. When the polymerization proceeds 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, the phosphoric acid monomer is subsequently polymerized to cover only the surface of the crosslinked polymer. In this way, 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 a method of adding all at once, a method of adding in several times or a method of adding dropwise. Further, it is not necessary to lower the temperature at the time of addition, but it is preferable that the temperature is once cooled, the phosphoric acid monomer is added and well dissolved in the aqueous phase, and then the temperature is raised again to improve the reproducibility of the polymerization. Furthermore, the temperature may be changed before and after the addition of the phosphoric acid monomer. The polymerization conditions after addition of the phosphoric acid monomer vary depending on the monomer used, but are 40 to 100 ° C. and 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 the completion of the polymerization, the polymer particles are washed, and further, if necessary, the particles are classified to obtain a crosslinked polymer particle having a phosphoric acid group only on the surface, which is used in the present invention and is used as a core particle. .. The coating layer made of hydroxyapatite particles is formed on the surface of the core particles by the same chemical bonding method as the method for producing the second carrier of the present invention.

A chromatographic carrier in which the coating layer on the surface of the core particle is formed of hydroxyapatite is prepared by each of the above methods.

In the chromatography carrier of the present invention, the surface portion of the crosslinked polymer is coated with hydroxyapatite, so that it has high pressure resistance and little swelling / shrinkage. Therefore, the analysis can be performed at a high flow rate, and the analysis time can be shortened.
Furthermore, since the carrier itself is spherical and has a uniform particle size, the separation accuracy is good. The chromatographic carrier of the present invention can be used as a chromatographic carrier for separating biopolymers by utilizing the above properties.

[0024]

EXAMPLES The present invention will be described below with reference to examples.

The packing and evaluation of the column for the carrier for chromatography was carried out by the following method.

"Carrier filling method" 2.3 g of the carrier was weighed, dispersed in 30 ml of purified water, and stirred for 5 minutes. Umeya Seiki Co., Ltd. packer with stainless steel empty column (6 mmφ
X 75 mm), pour the above dispersion into a packer,
It was connected to a liquid-sending pump LC-6AD manufactured by Shimadzu Corporation, and constant pressure filling was performed at 100 kg / cm ² .

"Evaluation of pressure resistance of carrier" Purified water was passed through the column packed by the above method, and the relationship between flow velocity and pressure loss was investigated. Liquid delivery LC-6A manufactured by Shimadzu Corporation
The pressure of the pressure gauge of the pump was monitored by a recorder.

"Evaluation of carrier swelling resistance" The particle size in a dry state and in a state of being immersed in purified water was measured by a Coulter Multisizer (manufactured by Coulter Co.), and the change of the particle size in a state of being dried and immersed in purified water was measured. Was done by investigating.

"Method for evaluating carrier" Using the column packed by the above method, liquid chromatography was carried out using a liquid chromatography fluff system SSLC-20 manufactured by Sekisui Chemical Co., Ltd. The analysis conditions were set as follows.

Eluent: Elution by concentration gradient method with phosphate buffer (pH 6.8) Flow rate: 1 ml / min Detection: Absorbance at 280 nm Sample: Standard solution of the following proteins (1 mM aqueous solution) Bovine serum albumin Lysozyme cytochrome c Injection amount: 50 μl (Example 1) In the present Example and Example 2, crosslinked polymer particles having no ion-exchange group were used as core particles, which were coated with hydroxyapatite to obtain a carrier.

100 g of diethylene glycol dimethacrylate (crosslinkable monomer), 100 g of tetramethylolmethane tetraacrylate (crosslinkable monomer) and 1 g of benzoyl peroxide (polymerization initiator) were mixed, and the resulting solution was mixed with 4% polyvinyl. The mixture was added to 2.5 liters of an aqueous alcohol solution, and the particles were sized with stirring, and then heated to 80 ° C. under nitrogen substitution to carry out suspension polymerization. After polymerizing at 80 ° C. for 8 hours, the product was washed successively with hot water and acetone and dried. The obtained crosslinked polymer particles were air classifier ELBOW-JET LABO E manufactured by Nittetsu Mining Co., Ltd.
Particles having a particle size of 10 to 20 μm were collected by classification with a JL-3 type. Then, the obtained particles (core particles) 50
and 10 g of hydroxyapatite (manufactured by Daiichi Rare Element Chemical Industry Co., Ltd .: crushed) are set in Mechanofusion System AM-15F (manufactured by Hosokawa Micron Corp.), and the rotor rotation speed is changed from 2000 rpm to 2 in 10 minutes.
At 600 rpm, the temperature was raised from 35 ° C to 60 ° C.

The obtained carriers were collected and observed with a scanning electron microscope (manufactured by Hitachi, Ltd.).
The surface of the carrier was coated with crushed hydroxyapatite. Using this carrier, the pressure resistance and swelling resistance were evaluated by the methods described above. In the evaluation of pressure resistance, the column pressure and flow rate are 350 k, which is the limit of the pump used.
Proportional to g / cm ² . In the evaluation of swelling resistance,
No change in particle size was observed, which was good. Further, chromatography was performed by the above method. The resulting chromatogram is shown in FIG. In FIG. 1 and FIGS. 2, 3 and 4 described later, 1 is a peak due to bovine serum albumin, 2 is a peak due to lysozyme,
3 is a peak attributed to cytochrome c.

(Example 2) Styrene (non-crosslinkable monomer)
150 g and divinylbenzene (crosslinkable monomer; p-
Body, m-body mixed product, product containing 55% ethyl vinylbenzene) and 1 g of benzoyl peroxide (polymerization initiator) were mixed and dissolved. This was added to 2.5 liters of a 4% polyvinyl alcohol aqueous solution, and the particles were sized with stirring, and then heated at 80 ° C. under nitrogen substitution to carry out suspension polymerization. After polymerizing at 80 ° C. for 8 hours, the product was washed successively with hot water and acetone and dried to obtain fine crosslinked polymer particles. A carrier was prepared according to Example 1 using the hydrophobic crosslinked polymer particles.

Using this carrier, the pressure resistance and swelling resistance were evaluated by the methods described above. In the evaluation of pressure resistance, it was proportional to the flow velocity up to the maximum pressure as in Example 1. In the evaluation of swelling resistance, no change in particle size was observed and it was good. Further, chromatography was performed by the above method. The resulting chromatogram was similar to that shown in FIG.

(Example 3) In this example, a crosslinked polymer particle having a phosphoric acid group uniformly in the particle was used as a core particle, and a carrier having a coating layer formed of hydroxyapatite was prepared.

100 g of tetraethylene glycol dimethyl acrylate (crosslinkable monomer), 100 g of tetramethylolmethane triacrylate (crosslinkable monomer), mono (2
1 g of benzoyl peroxide (polymerization initiator) was dissolved in a mixed solution of 50 g of (methacryloyloxyethyl) acid phosphate (phosphoric acid monomer), added to 2.5 liter of 4% polyvinyl alcohol aqueous solution, and stirred. After particle size regulation, suspension polymerization was carried out by heating to 80 ° C. under nitrogen substitution. After polymerizing at 80 ° C. for 5 hours, the product was washed with hot water and acetone in that order, dried, and classified in the same manner as in Example 1.

The obtained fine crosslinked polymer particles containing a phosphoric acid group were coated with hydroxyapatite by the following chemical method. First, 100 g of the obtained particles is 2.5 mM
Dip into 1 liter of KH ₂ PO ₄ solution of
1 liter of mM CaCl ₂ solution was added and stirred.
After reacting for 100 minutes at room temperature, the carrier was filtered off and dried.

Observation of the obtained particles with a scanning electron microscope revealed that 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, the pressure loss was proportional to at least 350 kg / cm ² . In the evaluation of swelling resistance, no change in particle size was observed and it was good. Further, chromatography was performed by the above method. The resulting chromatogram is shown in FIG.

Comparative Example 1 In this comparative example, an example of separation using crushed hydroxyapatite is shown.

When hydroxyapatite (manufactured by Daiichi Rare Element Chemical Industry Co., Ltd.) was suspended in purified water and an attempt was made to fill by the above method, the filling liquid stopped flowing. Filling pressure is 1
Constant pressure filling was performed by lowering the pressure from 00 kg / cm ² to 10 kg / cm ² .

The pressure resistance and swelling resistance were evaluated by the above methods. In the evaluation of pressure resistance, 20 kg / c
It was proportional to flow rate only up to m ² . In the evaluation of the swelling resistance, the average particle size in the dry state was 8.5 μm, but it was swollen to 10.6 μm in the immersed state. Further, chromatography was performed by the above method. The resulting chromatogram is shown in FIG. Separation was poor and analysis time was long as compared with Examples 1 to 3 above.

Example 4 In this example, a carrier was prepared in which a crosslinked polymer particle having a phosphoric acid group only on the surface was used as a core particle and a coating layer of which was formed of hydroxyapatite.

First, hydrophobic crosslinked polymer particles having no ion exchange group were prepared in the same manner as in Example 1, and 100 g of the obtained polymer particles were dissolved with 0.5 g of benzoyl peroxide (polymerization initiator). It was immersed in 1 liter of acetone to impregnate the polymerization initiator. Then add 2 parts of acetone
Evaporate under reduced pressure at 0 ° C. The obtained initiator-containing polymer is dispersed in 1 liter of a 50% aqueous methanol solution,
While stirring, 30 g of mono (2-methacryloyloxy) acid phosphate was added and dissolved. After this solution was replaced with nitrogen, a polymerization reaction was performed at 80 ° C. for 5 hours. The product was washed with hot water and acetone and dried. The crosslinked polymer particles obtained were coated with hydroxyapatite in the same manner as in Example 3.

Observation of the obtained particles with a scanning electron microscope revealed that 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 swelling resistance, no change in particle size was observed and it was good. Further, chromatography was performed by the above method. The chromatogram obtained as a result is shown in FIG. Not only the above-mentioned comparative examples but also better separations than the respective examples were obtained, and further, the analysis was possible in a short time.

(Example 5) In this example, the continuous method of reacting a phosphoric acid monomer subsequent to the preparation of the hydrophobic cross-linked polymer particles described above was adopted.

1 g of benzoyl peroxide was dissolved in 200 g of triethylene glycol dimethacrylate (crosslinking monomer) to prepare a 4% polyvinyl alcohol aqueous solution 2.5.
Added to liters. This solution was replaced with nitrogen while stirring and then heated to 80 ° C. to carry out polymerization.

After polymerizing for 2 hours, it 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 polymerized at 80 ° C. for 5 hours. The obtained product was washed successively with hot water and acetone and dried. Using the obtained polymer, the same procedure as in Example 3 was carried out to obtain a hydroxyapatite-coated carrier.

Observation of the obtained particles with a scanning electron microscope revealed that 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 pressure loss were proportional to 0 kg / cm ² . In the evaluation of swelling resistance, no change in particle size was observed and it was good. Further, chromatography was performed by the above method. The resulting chromatogram was similar to that shown in FIG.

[0049]

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

[Brief description of drawings]

FIG. 1 shows a chromatogram obtained as a result of performing chromatography using the carriers obtained in Examples 1 and 2.

FIG. 2 shows a chromatogram obtained as a result of performing chromatography using the carrier obtained in Example 3.

FIG. 3 shows a chromatogram obtained as a result of performing chromatography using the carrier used in Comparative Example 1.

FIG. 4 shows a chromatogram obtained as a result of chromatography using the carriers obtained in Examples 4 and 5.

Claims (5)

[Claims] 1. A chromatographic carrier having a core particle made of a cross-linked polymer having no ion-exchange group and a coating layer, wherein the coating layer is physically fused to the surface of the core particle. A chromatographic carrier formed by. 2. A homogenous core particle made of a crosslinked polymer having a phosphate group, and a chromatographic carrier having a coating layer, wherein the coating layer is hydroxyapatite chemically bonded to the surface of the core particle. A chromatographic carrier formed of particles. 3. The chromatography carrier according to claim 2, which comprises a step of bringing a homogeneous core particle made of a crosslinked polymer having a phosphate group into contact with a solution containing a calcium salt and a phosphate salt. Production method. 4. A chromatographic carrier having a core particle made of a cross-linked polymer having a phosphoric acid group only on the surface thereof, and a coating layer, wherein the coating layer is a hydroxy compound chemically bonded to the surface of the core particle. A chromatographic carrier formed of apatite particles. 5. The carrier for chromatography according to claim 4, which includes 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 phosphoric acid group only on the surface. Manufacturing method.