JP2559526C - - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a packing material for liquid chromatography, particularly a packing material for liquid chromatography suitable for gel permeation chromatography, and a method for producing the packing material. (Prior art) Liquid chromatography is used for separation or detection of various substances, and aqueous gel permeation chromatography (hereinafter referred to as GPC) for separation of hydrophilic substances such as separation of proteins from biological samples. Is used. The GPC method is based on the principle that when molecules in a sample are diffused into the pores inside the packing material, small molecules enter the pores, causing a delay in the elution time, and eluting the larger molecules in order. How to do. As a filler often used for aqueous GPC for separating a hydrophilic substance, a natural polymer gel such as dextran gel and agarose gel has been conventionally used. These gels are excellent fillers with little non-specific adsorption of proteins and the like, but are inferior in pressure resistance due to the softness of the gels, making high-speed processing impossible. Examples of fillers that can be processed at a relatively high speed as compared with these natural polymers include synthetic polymer fillers composed of a crosslinkable polymer gel. Examples of the crosslinkable polymer gel include a copolymer of divinylbenzene and vinyl acetate and a copolymer of polyethylene glycol dimethacrylate and hydroxyethyl methacrylate. Synthetic polymer-based fillers made of the above materials and used in aqueous systems are usually
It is prepared by the method disclosed in No. 58, that is, suspension polymerization by adding a polymerization initiator to a crosslinkable monomer and a hydrophilic monomer. Alternatively, according to the method disclosed in JP-A-52-138077, a monomer having a functional group capable of forming a hydroxyl group by a hydrolysis reaction (hereinafter referred to as a hydrolyzable group) such as vinyl acetate Is polymerized, the obtained polymer is hydrolyzed, and further post-crosslinked with epichlorohydrin to prepare a filler. In these fillers, it is necessary to increase the degree of cross-linking to improve pressure resistance, but since the cross-linking portion is hydrophobic, increasing the degree of cross-linking increases the hydrophobicity of the gel,
Non-specific adsorption of proteins occurs. For this reason, the amount of the crosslinking agent is limited, and it is difficult to obtain sufficient pressure resistance. Furthermore, the filler obtained by the above method is easily swelled and shrunk in an aqueous solvent because the hydrophilic monomer is dispersed throughout the polymer particles, and for this reason the pressure resistance is also high. Not enough. As a filler excellent in pressure resistance, capable of relatively high-speed processing, and excellent in separation ability, there is a silica-based filler obtained by chemically treating the surface of porous silica gel. However, this filler has a property of adsorbing a substance having a basic group such as a protein due to the effect of residual silanol groups on the surface. Furthermore, silica gel dissolves in acids and alkalis,
The pH of the eluent is limited to 3-8. Further, as a filler which can be used in the above-mentioned gel for aqueous GPC and has relatively excellent pressure resistance, disclosed in JP-A-59-18705, JP-A-62-63856 and JP-A-63-79064. ,
There are fillers obtained by a so-called seed polymerization method. This polymerization method is a method in which a crosslinked polymer particle is impregnated with a polymerization initiator and a monomer, and is subjected to suspension polymerization to obtain particles having a two-layer structure. In this method, when a monomer having a hydroxyl group is used as a monomer to be impregnated in the crosslinked polymer particles, an aqueous GPC filler is obtained. Further, the same filler for GPC can be obtained by impregnating with a monomer having a hydrolyzable group, polymerizing, and then hydrolyzing. However, since a hydroxyl group is present inside the obtained filler particles, the filler particles are easily swelled and shrunk in an aqueous solvent for the same reason as described above, and thus the pressure resistance is still insufficient. (Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional disadvantages, and an object of the present invention is to provide a packing material for chromatography suitable for separating hydrophilic substances such as proteins.
An object of the present invention is to provide a method for producing a filler having high pressure resistance, little swelling / shrinkage, and little nonspecific adsorption of proteins and the like. Another object of the present invention is to provide a method for producing a filler having the above excellent properties, and particularly suitable for GPC. (Means for Solving the Problems) The method for producing a packing material for liquid chromatography of the present invention comprises a step of impregnating a previously isolated hydrophobic crosslinked polymer particle with a polymerization initiator; In the dispersion in which is dispersed, a monomer having a functional group capable of generating a hydroxyl group by a hydrolysis reaction is added and dissolved, and the monomer is polymerized on the surface portion of the hydrophobic cross-linked polymer particles, Forming a layer of a polymer having a functional group capable of generating a hydroxyl group by a hydrolysis reaction on the surface portion of the hydrophobic crosslinked polymer particles to a thickness of 10 to 300 °; and hydrolyzing the functional group; A step of coating the surface of the hydrophobic crosslinked polymer particles with a polymer having a hydroxyl group in a thickness of 10 to 300 °, thereby achieving the above object. The filler obtained by the production method of the present invention has a hydrophobic crosslinked polymer particle as a skeleton, and a surface portion of the hydrophobic crosslinked polymer particle is coated with a polymer having a hydrolyzable group.
The surface of the polymer particles having a two-layer structure is hydrolyzed. As a material of the hydrophobic crosslinked polymer particles, a (co) polymer obtained by (co) polymerizing a hydrophobic crosslinkable monomer or a hydrophobic crosslinkable monomer and a hydrophobic non-crosslinkable monomer And copolymers thereof. Since these (co) polymers are the skeleton portion of the filler of the present invention as described above,
It is necessary that the polymer does not undergo a chemical reaction (for example, hydrolysis) in a hydrolysis reaction described later. Therefore, the monomer as a material, under the hydrolysis reaction conditions,
It is a monomer without a functional group that can react chemically. The above-mentioned hydrophobic crosslinked polymer is a homopolymer of a hydrophobic crosslinkable monomer or a copolymer composed of two or more kinds of crosslinkable monomers. If necessary, one or more non-crosslinkable monomers can be added. Examples of the hydrophobic crosslinkable monomer include aromatic compounds having two or more vinyl groups such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene. Examples of the non-crosslinkable hydrophobic monomer include styrene monomers such as styrene and methylstyrene. When the crosslinkable and non-crosslinkable monomers are used as a mixture, the crosslinkable monomer is used in an amount of 10 parts by weight or more, preferably 20 parts by weight or more based on 100 parts by weight of the total monomers. Is done. In the present invention, the polymer material covering the hydrophobic crosslinked polymer particles is obtained by polymerizing a monomer having a hydrolyzable group. Examples of such monomers include vinyl carboxylate esters such as vinyl acetate, vinyl propionate, vinyl butyrate, and vinyl valerate; and glycidyl compounds such as glycidyl methacrylate. The monomers having a hydrolyzable group may be used as a mixture of two or more as necessary. The amount of the monomer used depends on the type of the monomer, but the hydrophobic cross-linked polymer
The ratio is 5 to 50 parts by weight based on 100 parts by weight. The monomer having a hydrolyzable group is polymerized on the surface of the hydrophobic cross-linked polymer to coat the polymer, and the thickness of the coating layer is 10 to 300 mm. Since the coating layer is hydrolyzed to generate a hydroxyl group, it becomes hydrophilic. Therefore, when the average thickness of the coating layer is 300 mm or more, the swelling / shrinking effect of the coating layer portion becomes so large that it cannot be ignored, and the effect appears in the form of a decrease in separation ability and an increase in pressure. Absent. In addition, it takes a long time to equilibrate with the eluent, and disadvantages such as a decrease in resolution and an increase in analysis time are not preferable. Further, the coating layer needs to completely cover the surface of the hydrophobic cross-linked polymer. This is because if there is a portion where the hydrophobic cross-linked polymer is exposed, non-specific adsorption may occur when the portion comes into contact with a substance to be separated (such as a protein) in aqueous chromatography. The observation of the coating layer and the measurement of the average thickness of the coating layer are performed as follows. From the filler particles used as a sample, make a section of 1000 mm or less with a microtome. The section is treated with a hydroxyl group-specific labeling agent or staining reagent, and observed and photographed with a transmission electron microscope. For example, if a section is treated with osmic acid, the distribution of hydroxyl groups, the state of the coating layer, and the average thickness of the coating layer can be measured by transmission electron microscopy. Next, a typical production example for preparing the filler of the present invention will be described. However, the filler of the present invention is not limited to the one obtained by the following method. In preparing the filler for GPC of the present invention, first, hydrophobic crosslinked polymer particles are prepared. The hydrophobic crosslinked polymer particles can be prepared by any known aqueous suspension polymerization method. First, the above-mentioned hydrophobic monomer (hydrophobic crosslinking monomer and, if necessary, hydrophobic non-crosslinking monomer) and a polymerization initiator are dissolved in a diluent. The polymerization initiator and the polymerization initiator to be impregnated into the obtained hydrophobic crosslinked polymer particles (described later) are catalysts that generate radicals, and are not particularly limited as long as they are hydrophobic. Any of known radical generating catalysts such as organic peroxides such as benzoyl peroxide, acetyl peroxide and cumene peroxide; azo compounds such as azobisisobutyronitrile and azobisisobutyramide can be used. . The diluent is added as a pore-forming agent, and any organic solvent that dissolves the monomer and does not dissolve the polymer can be used. For example, aromatic hydrocarbons such as toluene, xylene, diethylbenzene and dodecylbenzene; saturated hydrocarbons such as hexane, heptane, octane and decane; and alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol. The amount used is not particularly limited, but is preferably 15 to 200 parts by weight based on 100 parts by weight of the monomer mixture. The above monomer mixture is added to an aqueous phase in which a suspension stabilizer such as polyvinyl alcohol, calcium phosphate or the like is dissolved. After the replacement with nitrogen, the mixture is heated to 40 to 100 ° C. with stirring to carry out suspension polymerization. Since an organic solvent as a diluent is dispersed in the obtained polymer particles, porous spherical particles are obtained by removing the organic solvent after the polymerization. By using various organic solvents having different compatibility with the monomer mixture as a diluent, it is possible to arbitrarily change the pore size of the porous polymer. Next, the obtained hydrophobic crosslinked polymer particles are isolated, and the isolated hydrophobic crosslinked polymer particles are impregnated with a polymerization initiator. In order to impregnate the polymerization initiator, the polymerization initiator is dissolved in a solvent having a low boiling point and a good affinity for the hydrophobic crosslinked polymer, and the hydrophobic crosslinked polymer particles are immersed in the solution. This allows the polymerization initiator to penetrate into the particles. If necessary, this is heated at a temperature below the decomposition point of the polymerization initiator to distill off the solvent, whereby hydrophobic crosslinked polymer particles containing the polymerization initiator therein can be obtained. The polymerization initiator-containing particles are dispersed in a dispersion medium in which the monomer having a hydrolyzable group is dissolved, or a monomer having a hydrolyzable group is added to a dispersion medium in which the particles are dispersed. Then, the mixture is dissolved, replaced with nitrogen, and heated under stirring to carry out polymerization. By this polymerization,
A monomer having a hydrolyzable group is polymerized on the surface of the hydrophobic cross-linked polymer particle, and coats the particle. As the dispersion medium, water or an organic solvent that dissolves a monomer having a hydrolyzable group, or a mixture of both can be used. In order to stabilize the dispersibility of the hydrophobic crosslinked polymer, a dispersion stabilizer such as carboxymethylcellulose and polyvinyl alcohol can be added to the dispersion medium. The polymerization temperature and time vary depending on the type of the monomer having a hydrolyzable group to be reacted and the type of the polymerization initiator, but it is about 0.5 to 20 hours at 40 to 100 ° C. By the above method, the polymer particles having the above two-layer structure are prepared. The polymer particles obtained by the above method are sufficiently washed with hot water, an organic solvent, or the like to remove the suspension stabilizer, solvent, residual monomer, and the like contained in or attached to the particles. By hydrolyzing the obtained polymer particles with an acid catalyst or an alkali catalyst, the hydrolyzable functional groups present in the coating layer on the particle surface are hydrolyzed to hydroxyl groups.
For example, when vinyl acetate is used as the monomer having a hydrolyzable group, the polymer particles are prepared in a 15 to 25% by weight methanol solution of sodium hydroxide at a temperature of 60 to 80 ° C. for 4 to 4 hours.
By reacting for 10 hours, the —O—COCH ₃ group on the particle surface becomes a hydroxyl group. After the hydrolysis reaction, the polymer particles are collected by filtration, washed several times, dried and, if necessary, classified to obtain a filler for GPC. The filler of the present invention is a two-layer polymer particle having a hydrophobic cross-linked polymer as a skeleton and a surface group of the hydrophobic cross-linked polymer coated with a polymer having a hydroxyl group. By using a polymer having a high degree of cross-linking as the skeleton portion, a packing material for liquid chromatography having extremely high mechanical strength and excellent pressure resistance can be obtained. Since there is no hydrophilic group in the skeleton portion of this filler, the degree of swelling and shrinking is extremely small. Since the surface is coated with a hydrophilic polymer having a hydroxyl group, it has no non-specific adsorption of proteins and the like, and is suitable as a filler for GPC. This filler can be used in a wide pH range. Further, as described above, since the pressure resistance is large and the degree of swelling / shrinking is extremely low, the particle size can be reduced, and as a result, separation with high accuracy is possible. Because it can be used under high pressure conditions, rapid analysis can be performed. (Example) Hereinafter, the present invention will be described with reference to examples. The methods for measuring the physical properties and evaluating the performance of the fillers obtained in the following Examples and Comparative Examples are as follows. "Measurement method of average thickness of coating layer" After embedding coated polymer particles used for filler in epoxy resin, Reichert-Jung
Using a microtome ULTRACUTE made by the company, make a section about 900 mm thick. The obtained section was stained with an osmic acid solution (for electron microscope, manufactured by Wako Pure Chemical Industries, Ltd.), observed and photographed with a transmission electron microscope JEM100S manufactured by JEOL Ltd., and the distribution of hydroxyl groups was determined. The condition and the average thickness of the coating layer were measured. "Evaluation Method of Filler" The filler was packed in a stainless steel column having an inner diameter of 7.5 mm and a length of 500 mm, and pressure resistance and swelling property with water were examined. The pressure resistance was measured by flowing purified water through the column, changing the flow rate, and measuring the relationship between the flow rate and the pressure loss. The swelling property was evaluated by measuring the difference in particle size between a dry state and a state immersed in purified water using a centrifugal sedimentation type particle size distribution analyzer SA-CP3 (manufactured by Shimadzu Corporation). Furthermore, a calibration curve was prepared using a standard sample having a known molecular weight, and the exclusion limit molecular weight (
Hereinafter, it is referred to as MLim). The calibration curve in GPC is a curve showing the relationship between the molecular weight of the substance to be separated and the elution volume in the chromatogram. As shown in FIG. 1, the vertical axis indicates the molecular weight (M) of the substance to be separated (polymer or oligomer). Logarithm,
It is a curve obtained by plotting the elution volume (Ve) on a horizontal axis on a graph. In FIG. 1, the value on the vertical axis at the point where the extension of the inclined straight line and the extension of the line parallel to the horizontal axis intersect is the exclusion limit molecular weight MLim. The analysis conditions for preparing the calibration curve are shown below. Column Stainless steel inner diameter 7.5 mm Length 500 mm Liquid chromatograph Liquid chromatograph system manufactured by Sekisui Chemical Co., Ltd. SSLC-20 sample Dextran 0.5% aqueous solution manufactured by Sigma 0.5% aqueous solution of polyethylene glycol manufactured by Wako Pure Chemical Industries, Ltd. Flow rate 1.0 ml / Min Eluent Purified water Detector MLim was determined for the GPC packing material of each example by differential refractometer SE-51 manufactured by Showa Denko KK Further, GPC analysis of various proteins was performed using 50 mM phosphate buffer as an eluent, and a calibration curve was drawn. Example 1 Polystyrene-based gel HSG-50 2 manufactured by Sekisui Chemical Co., Ltd. was used as hydrophobic cross-linked polymer particles.
This was immersed in 1 liter of acetone in which 0.5 g of decanoyl peroxide (polymerization initiator) was dissolved to impregnate the polymerization initiator. Then the acetone was distilled off at 20 ° C. under reduced pressure. Disperse the impregnated hydrophobic cross-linked polymer in 2 liters of a 50% aqueous methanol solution, add 50 g of vinyl acetate (monomer having a hydrolyzable group) with stirring, and replace with nitrogen for 10 hours at 65 ° C. A polymerization reaction was performed. The product was washed successively with hot water and acetone and dried. 150 g of the obtained fine polymer particles were added to 500 ml of a 20 wt% methanol solution of sodium hydroxide, and heated at 75 ° C. for 5 hours to hydrolyze the ester portion of polyvinyl acetate. After cooling the reaction mixture to room temperature, the polymer particles were collected by filtration, washed several times and dried. The obtained polymer particles were classified using an air classifier Turbo Classifier TC-15N manufactured by Nisshin Engineering Co., Ltd. to collect particles having a particle size of 8 to 10 μm to obtain a filler. This was packed in a stainless steel column having an inner diameter of 7.5 mm and a length of 500 mm. Filling was performed by taking 15 g of the filler in 120 ml of purified water, stirring the mixture for 5 minutes, and filling the mixture at a constant flow rate of 2.0 ml / min. The pressure resistance and the swelling property were evaluated by the above methods. In the evaluation of pressure resistance,
The pressure loss was proportional to the flow rate up to 150 kg / cm ² . When a swelling test was performed, it was found that there was no difference in particle size between the dried state and the state immersed in purified water, and it was found that the particles did not swell in an aqueous solvent. After staining with osmic acid, the film was observed and photographed with a transmission electron microscope, and the average thickness of the coating layer was measured to be about 50 °. Further, a calibration curve was prepared according to the above evaluation method. The obtained calibration curve is
Shown in the figure. MLim was 5.0 × 10 ⁵ . Here, FIG. 2 and FIGS.
The plots 1 to 6 in the calibration curve of FIG. 7 are the results obtained using the dextran sample, and the molecular weight of each dextran is 2,000,000 for 1 and 500,00 for 2
0, 3 is 100,000, 4 is 70,000, 5 is 40,000, and 6 is 10,000. Plot 7-1
0 is the result obtained using the polyethylene glycol sample, and the molecular weights are 7,000 for 7, 7 for 6,000, 9 for 2,000, and 10 for 600. As a result of analyzing various proteins (manufactured by Sigma), the obtained calibration curve
Shown in the figure. Here, plots 11 to 20 in FIG. 3 and FIG. 5 to be described later are respectively thyroglobulin (molecular weight: 660,000; hereinafter, the molecular weight is shown in parentheses), and 12 is γ-.
Globulin (156,000), 13 is bovine serum albumin (67,000), 14 is ovalbumin (4
3,000), 15 is peroxidase (40,200), 16 is β-lactoglobulin (35,000),
17 is the result obtained using myoglobin (16,900), 18 is the result obtained using ribonuclease A (13,700), 19 is the result obtained using cytochrome C (12,400), and 20 is the result obtained using glycine tetramer (246). Example 2 100 g of styrene (hydrophobic non-crosslinkable monomer), 200 g of divinylbenzene (hydrophobic crosslinkable monomer) and 1 g of benzoyl peroxide (polymerization initiator) were dissolved in 200 g of toluene (diluent). This was added to 2.5 liters of a 4% aqueous solution of polyvinyl alcohol, and the resulting mixture was sized with stirring, and then heated to 80 ° C. under nitrogen substitution to carry out suspension polymerization. 80
After polymerization at 8 ° C. for 8 hours, the product was sequentially washed with hot water and acetone, and dried to obtain fine hydrophobic crosslinked polymer particles. 200 g of the hydrophobic cross-linked polymer particles were treated with benzoyl peroxide (polymerization initiator) 0.
The polymerization initiator was impregnated by immersion in 1 liter of acetone in which 5 g was dissolved. Then the acetone was distilled off at 20 ° C. under reduced pressure. The impregnated hydrophobic crosslinked polymer was suspended in 2 liters of a 50% aqueous methanol solution, and 50 g of glycidyl methacrylate (monomer having a hydrolyzable group) was added thereto with stirring. The polymerization reaction was performed for an hour. The product was washed successively with hot water and acetone and dried. The polymer particles were hydrolyzed, classified and filled in the same manner as in Example 1 and evaluated. As a result, as for the pressure resistance, the pressure loss was proportional to the flow velocity up to 150 kg / cm ² . When a swelling test was performed, it was found that there was no difference in particle size between the dried state and the state immersed in purified water, and it was found that the particles did not swell in an aqueous solvent. Microscopic observation and photography and measurement of the average thickness of the coating layer revealed that the average thickness was about 100 mm. MLim was 1.0 × 10 ⁵ . The obtained calibration curve is
The figure and the results of the protein analysis are shown in FIG. Comparative Example 1 100 g of styrene, 200 g of divinylbenzene, 70 g of vinyl acetate (a monomer having a hydrolyzable group) and 1 g of decanoyl peroxide were dissolved in 200 g of toluene, and added to 2.5 l of a 4% aqueous solution of polyvinyl alcohol. After granulating with stirring, heat to 65 ° C,
Suspension polymerization was performed. After polymerization at 65 ° C. for 10 hours, the product was hydrolyzed, classified, filled and evaluated in the same manner as in Example 1. As a result of performing the same evaluation as in Example 1, the pressure loss was proportional to the flow velocity up to 80 kg / cm ² for the pressure resistance. When the swelling test was performed, the average particle size was 9.2 μm to 12.5 μm.
m and the degree of swelling of the filler was found to be high. When the average thickness of the coating layer was measured by microscopic observation and photographing after staining, the hydroxyl groups were uniformly distributed inside the particles. An attempt to obtain MLim by drawing a calibration curve showed that polyethylene glycol having a higher molecular weight was eluted later. This is thought to be due to the hydrophobic interaction between the polyethylene glycol and the filler caused by the hydrophobicity of the filler surface. It is considered that the higher the molecular weight of polyethylene glycol, the higher the hydrophobicity, and therefore the stronger the hydrophobic interaction with the filler. The resulting calibration curve is shown in FIG. Comparative Example 2 Polystyrene gel HSG-50 2 manufactured by Sekisui Chemical Co., Ltd. as hydrophobic crosslinked polymer particles
A filler was obtained and evaluated in the same manner as in Example 1 except that 100 g of vinyl acetate was used as a monomer having a hydrolyzable group. As a result, as for the pressure resistance, the pressure loss and the flow velocity were proportional to 350 kg / cm ² . When the swelling test was performed, there was no difference in particle size between the dried state and the state immersed in purified water.
It was found that it did not swell in aqueous solvents. Further, the average thickness of the coating layer was measured by microscopic observation and photographing after dyeing and found to be about 400 mm. In addition, when trying to obtain MLim by drawing a calibration curve, it was not possible to separate samples of each molecular weight. This is because the surface coating layer is too thick,
This is probably because the pores of the filler were almost completely blocked. The calibration curve obtained is shown in FIG. Comparative Example 3 Polystyrene gel HSG-50 2 manufactured by Sekisui Chemical Co., Ltd. as hydrophobic crosslinked polymer particles
A filler was obtained in the same manner as in Example 1 except that 100 g of vinyl acetate was used as the monomer having a hydrolyzable group, and 10 g of vinyl acetate was used. As a result, as for the pressure resistance, the pressure loss and the flow velocity were proportional to 350 kg / cm ² . When the swelling test was performed, there was no difference in particle size between the dried state and the state immersed in purified water.
It was found that it did not swell in the aqueous solvent. When the average thickness of the coating layer was measured by microscopic observation and photographing after staining, it was about 8 mm, and some uncoated portions were observed. When a calibration curve was drawn to obtain MLim, it was found that polyethylene glycol having a higher molecular weight was eluted later. This may be due to the hydrophobic interaction between the polyethylene glycol and the filler, which occurred due to the hydrophobicity of the filler surface. The resulting calibration curve was similar to FIG. (Effects of the Invention) According to the present invention, an aqueous liquid chromatography packing material having excellent pressure resistance, little swelling / shrinking, and no nonspecific adsorption of proteins can be obtained.
Such a filler can be widely used as a filler for GPC in the isolation or analysis of various hydrophilic substances.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a calibration curve showing the relationship between the molecular weight of a substance to be separated and the elution volume of an eluent in gel permeation chromatography. FIGS. 2, 4, 6, and 7 show the packings obtained in Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively, packed in a column, and GPC analysis of a standard sample. The calibration curve when performed is shown. FIG. 3 and FIG. 5 show calibration curves when the packings obtained in Examples 1 and 2 were packed in columns and proteins were separated.

Claims (1)

[Claims] 1. A method for producing a packing material for liquid chromatography, comprising: impregnating a polymerization initiator into hydrophobic cross-linked polymer particles isolated in advance; hydrolyzing a dispersion in which the hydrophobic cross-linked polymer particles are dispersed; A monomer having a functional group capable of forming a hydroxyl group by a reaction is added and dissolved, and the monomer is polymerized on the surface portion of the hydrophobic cross-linked polymer particle. A polymer layer having a functional group capable of generating a hydroxyl group by a hydrolysis reaction,
A step of hydrolyzing the functional group and coating the surface of the hydrophobic crosslinked polymer particles with a polymer having a hydroxyl group in a thickness of 10 to 300 °. Manufacturing method of filler.