JP2559525B2 - Method for producing packing material for liquid chromatography - Google Patents

Description

The present invention relates to a packing material suitable for liquid chromatography,
Particularly, it relates to a packing material suitable for weak cation exchange chromatography.

(Prior Art) Liquid chromatography is used for separation or detection of various substances, and water-based gel permeation chromatography, ion exchange chromatography, etc. are used for separation of proteins from biological samples, especially for separation of hydrophilic substances. It's being used. The ion-exchange chromatography method is a method in which a packing material having an ion-exchange group is used and the separation is performed according to the difference in the ion-exchange adsorptivity of the component ions to be separated. Those having a carboxyl group as an ion exchange group are useful for analysis of proteins and peptides as weak ion exchange chromatography.

The most commonly used filler for weak cation exchange chromatography is a gel in which a carboxyl group is introduced on the surface of styrene-divinylbenzene crosslinked polymer particles,
Alternatively, there are crosslinked copolymer gels of styrene, divinylbenzene and a monomer having a carboxyl group. Such a synthetic polymer-based filler is usually prepared by the method disclosed in JP-A-58-221164, that is, suspension polymerization by adding a polymerization initiator to a crosslinkable monomer and a monomer having a carboxyl group. It is prepared by Alternatively, styrene, divinylbenzene, and a monomer having a functional group capable of forming a carboxyl group by a hydrolysis reaction (hereinafter referred to as a hydrolyzable group) are copolymerized and then the functional group is hydrolyzed. A gel having a carboxyl group as a group can also be used. To improve the pressure resistance of such a filler, it is necessary to increase the degree of cross-linking, but since the cross-linking portion is hydrophobic, increasing the degree of cross-linking increases the hydrophobicity of the gel and causes non-specific adsorption of proteins.
Therefore, the amount of the crosslinking agent is limited, and it is difficult to obtain sufficient pressure resistance. Further, the filler obtained by the above method, because the carboxyl groups are dispersed throughout the polymer particles,
It easily swells and shrinks in an aqueous solvent, and for this reason also the pressure resistance is insufficient.

A silica-based filler in which a carboxyl group is chemically bonded to the surface of porous silica gel is known as a filler having excellent pressure resistance, capable of relatively high-speed treatment, and excellent in separation ability. However, this filler has a property of adsorbing a substance having a basic group such as a protein due to the influence of residual silanol groups on the surface. Furthermore, since silica gel dissolves in acids and alkalis,
The pH of the eluent is limited to 3-8.

Furthermore, as a packing material that can be used in the packing material for the weak cation exchange chromatography and has relatively excellent pressure resistance,
JP-A-59-18705, JP-A-62-63856 and JP-A-63-
There is a filler obtained by the so-called seed polymerization method disclosed in 79064. In this polymerization method, a cross-linked polymer particle is impregnated with a polymerization initiator and a monomer, and these are subjected to suspension polymerization to obtain particles having a two-layer structure. In this method, if a monomer having a carboxyl group is used as the monomer to be impregnated into the crosslinked polymer particles,
A packing material for weak cation exchange chromatography is obtained. Also, a similar packing material for weak cation exchange chromatography can be obtained by impregnating a monomer having a hydrolyzable group, polymerizing, and then hydrolyzing. However, since a carboxyl group is present inside the obtained filler particles, it tends to swell and shrink in the aqueous solvent for the same reason as above, and therefore the pressure resistance is still insufficient.

(Problems to be Solved by the Invention) The present invention is to solve the above-mentioned conventional drawbacks, and an object thereof is a packing material for chromatography suitable for separation of hydrophilic substances such as proteins, It is an object of the present invention to provide a method for producing a filler having high pressure resistance, low swelling / contraction, and low nonspecific adsorption of proteins and the like.
Another object of the present invention is to provide a method for producing a packing material having the above-mentioned excellent properties and particularly suitable for weak cation exchange chromatography.

(Means for Solving Problems) The method for producing a packing material for liquid chromatography of the present invention comprises a step of preparing hydrophobic cross-linked polymer particles impregnated with a polymerization initiator; In the dispersed liquid dispersion, a monomer having a functional group capable of generating a carboxyl group by a hydrolysis reaction is added and dissolved, and the monomer is polymerized at the surface portion of the hydrophobic crosslinked polymer particles, A layer of a polymer having a functional group capable of generating a carboxyl group by a hydrolysis reaction is formed on the surface of the hydrophobic crosslinked polymer particles.
A step of forming to a thickness of 10 to 300Å; and a step of hydrolyzing the functional group and coating the surface of the hydrophobic crosslinked polymer particles with a polymer having a carboxyl group to a thickness of 10 to 300Å, Thereby, the above object is achieved.

The material of the hydrophobic crosslinked polymer particles used in the present invention is a (co) polymer obtained by (co) polymerizing a hydrophobic crosslinkable monomer or a hydrophobic crosslinkable monomer and a hydrophobic non-crosslinkable monomer. Examples thereof include a copolymer with a crosslinkable monomer. Since these (co) polymers are the skeleton portion of the filler of the present invention as described above, they should form a polymer that does not chemically react (for example, hydrolyze) under the hydrolysis reaction conditions described later. is necessary. Therefore, the above-mentioned monomer serving as a raw material is a monomer having no functional group capable of chemically reacting under the hydrolysis reaction condition. The above 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-crosslinking 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. Also, it can be used as a material if necessary,
Examples of the hydrophobic non-crosslinkable monomer include styrene-based monomers such as styrene and methylstyrene. When the crosslinkable and non-crosslinkable monomers are mixed and used,
The crosslinking 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 all the monomers.

In the present invention, the material of the polymer coating the hydrophobic crosslinked polymer particles is obtained by polymerizing a monomer having a hydrolyzable group. Examples of such a monomer include alkyl esters of acrylic acid or methacrylic acid (hereinafter referred to as (meth) acrylic acid) such as methyl acrylate and methyl methacrylate; (meth) acrylamide; (meth) acrylonitrile, etc. Is mentioned. Two or more kinds of these monomers having a hydrolyzable group may be mixed and used, if necessary. The amount of the monomer used varies depending on the kind of the monomer, but is 5 to 50 parts by weight with respect to 100 parts by weight of the hydrophobic crosslinked polymer.

The above-mentioned monomer having a hydrolyzable group is polymerized on the surface of the hydrophobic crosslinked polymer to coat the polymer, and the average thickness of the coating layer is 10 to 300Å. The coating layer is hydrolyzed after polymerization to generate a carboxyl group,
It has weak cation exchange ability and hydrophilicity. Therefore, if the average thickness exceeds 300Å,
The swelling / shrinking effect of the coating layer portion becomes so large that it cannot be ignored, and the resolution and pressure increase easily occur. On the other hand, if the average thickness exceeds 300 Å, it takes time to equilibrate with the eluent, which lowers the resolution and prolongs the analysis time, which is not preferable. If the average thickness is less than 10Å, the coating is incomplete and the exposed surface of the hydrophobic crosslinked polymer particles is likely to occur. With such exposure,
The substance to be separated (eg, protein) may be specifically adsorbed on the packing material.

Observation of the coating layer and measurement of its thickness in the present invention are carried out as follows. From the filler particles used as the sample, make a section of 1000 Å or less with a microtome. The section is treated with a labeling agent or staining reagent specific to the carboxyl group, and observed and photographed with a transmission electron microscope. For example, by treating with a silver nitrate solution, the carboxyl group reacts with silver to become -COOAg, so the distribution state of the carboxyl group in the filler particles, the state of the coating layer and the average thickness are measured by a transmission electron microscope. I can do things.

Next, typical production examples 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.

To prepare the packing material for weak ion exchange chromatography of the present invention, hydrophobic crosslinked polymer particles are first prepared. The hydrophobic crosslinked polymer particles can be prepared by any known aqueous suspension polymerization method. First, the above-mentioned hydrophobic monomer (hydrophobic crosslinkable monomer and, if necessary, hydrophobic non-crosslinkable monomer) and a polymerization initiator are dissolved in a diluent. The polymerization initiator and the polymerization initiator to be impregnated in the obtained hydrophobic cross-linked polymer particles (described later) are catalysts that generate radicals and are not particularly limited as long as they are hydrophobic. For example, organic peroxides such as benzoyl peroxide, acetyl peroxide, cumene peroxide; azo compounds such as azobisisobutyronitrile and azobisisobutyroamide;
Any of the known radical generating catalysts can be used.

The above diluent is added as a pore-forming agent, and any organic solvent that dissolves the above monomer and does not dissolve the polymer thereof can be used. For example, aromatic hydrocarbons such as toluene, xylene, diethylbenzene and dodecylbenzene; hexane, heptane, octane,
Saturated hydrocarbons such as decane; alcohols such as isoamyl alcohol, hexyl alcohol and octyl alcohol. The amount used is not particularly limited, but it is preferably 200 parts by weight or less relative to 100 parts by weight of the monomer mixture.

The above monomer mixture (monomer, initiator, diluent) is added to an aqueous phase in which a suspension stabilizer such as polyvinyl alcohol or calcium phosphate is dissolved, and the mixture is replaced with nitrogen and stirred for 40 to 10 minutes.
Suspension polymerization is performed by heating to 0 ° C. Since an organic solvent as a diluent is dispersed and present in the obtained polymer particles, porous spherical particles can be obtained by removing the organic solvent after the polymerization is completed. By using various organic solvents having different compatibility with the monomer mixture as a diluent,
It is possible to arbitrarily change the size of the pores of the porous polymer.

Next, the obtained hydrophobic crosslinked polymer particles are impregnated with a polymerization initiator. 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 solvent. This causes the polymerization initiator to penetrate into the particles. If necessary, the solvent is distilled off by heating at a temperature not higher than the decomposition point of the polymerization initiator to obtain hydrophobic crosslinked polymer particles containing the polymerization initiator inside. The polymerization initiator-containing particles are dispersed in a dispersion medium in which the monomer having a hydrolyzable group is dissolved, or the monomer having a hydrolyzable group is dispersed in a dispersion medium in which the particles are dispersed. Add, dissolve and replace with nitrogen, then heat with stirring to carry out polymerization. By this polymerization, a monomer having a hydrolyzable group is polymerized on the surface of the hydrophobic crosslinked polymer particles to coat the particles. As the dispersion medium, water or an organic solvent that dissolves a monomer having a hydrolyzable group, or a mixture of both may be used. To stabilize the dispersibility of the hydrophobic cross-linked polymer, a dispersion stabilizer such as carboxymethyl cellulose or polyvinyl alcohol may be added to the dispersion medium. The temperature and time of polymerization vary depending on the type of the monomer having a hydrolyzable group to be reacted and the type of the polymerization initiator, but are 0.5 to 40 ° C. at 100 ° C.
~ 20 hours. Above method (hereinafter referred to as impregnation method)
Thus, the above-mentioned two-layer structure polymer particles are prepared.

In addition to the method of subjecting the hydrophobic crosslinked polymer particles impregnated with the above-mentioned polymerization initiator to the polymerization reaction of the monomer having a hydrolyzable group, the hydrolyzable group is continuously added to the preparation of the hydrophobic crosslinked polymer particles. The above-mentioned two-layer structure polymer particles can also be prepared by a method of reacting a monomer having a (hereinafter referred to as a continuous method). In this method, first, a polymerization reaction for preparing the hydrophobic crosslinked polymer particles is started. When the polymerization proceeds to some extent and the unreacted polymerization initiator remains, the above-mentioned monomer having a hydrolyzable group is added to the reaction system. In such a state, since the polymerization initiator is present in the oil phase in the system and inside the produced hydrophobic crosslinked polymer particles, the polymerization of the monomer having a hydrolyzable group occurs subsequently, and A layer of the polymer having a hydrolyzable group is formed so as to cover the surface portion of the hydrophobic crosslinked polymer particles. Therefore, also by this continuous method, the same bilayer structure particles as in the above impregnation method can be obtained.

The polymer particles obtained by each of the above methods are thoroughly washed with hot water, an organic solvent or the like to remove the suspension stabilizer, solvent, residual monomer and the like contained or attached to the particles.

By hydrolyzing the obtained polymer particles with an acid catalyst or an alkali catalyst, the hydrolyzable functional group present in the coating layer on the particle surface is hydrolyzed to a carboxyl group. For example, when methyl acrylate is used as the monomer having a hydrolyzable group, the polymer particles are reacted in a 15-25 wt% sodium hydroxide solution in methanol at a temperature of 60-80 ° C. for 4-10 hours. -COOCH _{3 on the} particle surface
The group becomes a carboxyl group.

After the hydrolysis reaction, the polymer particles are collected by filtration, washed several times and dried, and if necessary, the particles are classified to obtain a packing material for weak cation exchange chromatography. The filler of the present invention is a polymer particle having a two-layer structure having a hydrophobic cross-linked polymer as a skeleton and the surface portion of the hydrophobic cross-linked polymer is covered with a polymer having a carboxyl group. By using a polymer having a high degree of cross-linking as the skeleton, it is possible to obtain a packing material for liquid chromatography which has extremely high mechanical strength and excellent pressure resistance. 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 carboxyl group, there is no nonspecific adsorption of proteins and the like, and it is suitable as a packing material for weak cation exchange chromatography. This filler can be used in a wide pH range. Furthermore, since the pressure resistance is large and the degree of swelling / shrinkage is extremely low as described above, the particle size can be reduced, and as a result, separation can be performed with high accuracy. Since 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 coating the coated polymer particles used as the filler in an epoxy resin, a slice having a thickness of about 900 Å is obtained using a Reichert-Jung Microtome ULTRACUTE. This section was labeled with a silver nitrate solution (for volumetric analysis, manufactured by Wako Pure Chemical Industries, Ltd.), observed and photographed with a transmission electron microscope JEM100S manufactured by JEOL Ltd., and the distribution state of carboxyl groups and The average thickness of the coating layer was measured.

"Evaluation Method of Filler" The obtained filler was filled in a stainless steel column having an inner diameter of 6 mm and a thickness of 75 mm, and the pressure resistance and the swelling property in 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 obtained from the change in column pressure when liquids with different ionic strengths were passed.

Carboxyl groups on the surface of the filler were quantified by an automatic potentiometric titrator AT-310 manufactured by Kyoto Electronics Manufacturing Co., Ltd. In addition, human blood was analyzed using Hi-AUTO A _1C manufactured by Kyoto Daiichi Kagaku Co., Ltd., and the separability was compared with that of the conventional product. The measuring method is as follows. As a human blood sample, a blood sample of the same person (healthy person) to which heparin was added immediately after being collected was used. The blood sample is automatically collected by the dedicated hemolyzed blood 21L (phosphate buffer solution containing nonionic surfactant) attached to the device.
It is diluted twice and hemolyzed. As the eluents, A solution (pH 5.9 phosphate buffer solution), B solution (pH 7.2 phosphate buffer solution) and C solution (pH 5.9 phosphate buffer solution), which are dedicated reagents attached to this device, are used as eluents. used. Separately, a protein standard substance was separated using a liquid chromatograph system SSLC-20 manufactured by Sekisui Chemical Co., Ltd.

Example 1 200 g of polystyrene gel HSG-50 manufactured by Sekisui Chemical Co., Ltd. was used as the hydrophobic cross-linked polymer particles, and this was immersed in acetone 1 in which 0.5 g of acetyl peroxide (polymerization initiator) was dissolved. The polymerization initiator was impregnated. next,
Acetone was distilled off under reduced pressure at 20 ° C. Disperse the above-mentioned impregnated hydrophobic cross-linked polymer in 50% methanol aqueous solution 2, add 50 g of methyl acrylate (monomer having hydrolyzable group) with stirring, and replace with nitrogen to 70 ° C.
The polymerization reaction was carried out for 5 hours. The product was washed successively with hot water and acetone and dried. 150 g of the obtained fine polymer particles was added to a 20 wt% methanol solution of sodium hydroxide.
It was added to 500 ml and heated at 75 ° C. for 5 hours to hydrolyze the ester portion of methyl polyacrylate. After cooling the reaction mixture to room temperature, the polymer particles were filtered off, washed several times and dried. The obtained polymer particles were used as an air classifier Turbo Classifier TC-15 manufactured by Nisshin Engineering Co., Ltd.
Particles having a particle size of 8 to 10 μm were collected by classification with N to obtain a filler. This was packed in a stainless steel column having an inner diameter of 6 mm and a length of 75 mm. The filling was performed by adding 2 g of the filler to 35 ml of purified water, stirring for 5 minutes, and then filling 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 pressure resistance evaluation, the pressure loss was proportional to the flow velocity up to 150 kg / cm ² . When the swelling test was performed, the eluent was 40 mM
No increase in column pressure was observed when the phosphate buffer solution was changed to a 200 mM phosphate buffer solution. The ion exchange capacity of the packing material surface was measured by titration and found to be 0.7 meq / g. The filler was treated with the silver nitrate solution by the above method, and the average thickness of the coating layer was measured to be about 100Å
Met. Human blood was analyzed by Hi-AUTO A _1C manufactured by Kyoto Daiichi Kagaku Co., Ltd. The chromatogram obtained as a result is shown in FIG. In FIG. 1 and FIGS. 3 and 5 described later, 1 is hemoglobin (hereinafter referred to as Hb) A _1a and
A _1b , 2 are fetal Hb (F), 3 are unstable HbA _1c , 4 are stable HbA _1c , and 5 is a peak due to normal Hb (A ₀ ).

Furthermore, a mixture of several proteins (manufactured by Sigma) was separated using a liquid chromatography system SSLC-20 manufactured by Sekisui Chemical Co., Ltd. Elute with 20 mM phosphate buffer (pH
7.0, hereinafter referred to as solution A); and an equal mixture of solution A and 0.5 M NaCl (hereinafter referred to as solution B), from solution A 100% to B
It was performed by a linear gradient method to a liquid of 100%. The chromatogram obtained as a result is shown in FIG. In FIG. 2 and FIGS. 4 and 6 described later, peak 6 is due to myoglobin, 7 is trypsinogen, 8 is ribonuclease A, 9 is cytochrome C, and 10 is lysozyme.

Example 2 100 g of styrene (hydrophobic non-crosslinkable monomer), 200 g of divinylbenzene (hydrophobic crosslinkable monomer), 1 g of benzoyl peroxide (polymerization initiator) and 200 g of toluene (diluent)
Dissolved in. This is a 4% aqueous solution of polyvinyl alcohol
After adding to 2.5 and adjusting the particle size with stirring, the suspension was polymerized by heating to 80 ° C. under nitrogen substitution. After polymerizing at 80 ° C. for 8 hours, the product was washed successively with hot water and acetone and dried to obtain fine hydrophobic crosslinked polymer particles.

200 g of the hydrophobic crosslinked polymer particles were immersed in acetone 1 in which 0.5 g of acetyl peroxide (polymerization initiator) was dissolved to impregnate the polymerization initiator. Next, the acetone was distilled off under reduced pressure at 20 ° C. Suspend the above hydrophobic cross-linked polymer impregnated in 50% aqueous methanol solution 2, add 50 g of acrylonitrile (monomer having hydrolyzable group) with stirring, replace with nitrogen, and at 70 ° C for 10 hours. A polymerization reaction was carried out. 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, regarding pressure resistance, the pressure loss was proportional to the flow velocity up to 150 kg / cm ² . When the swelling test was conducted, no increase in column pressure was observed when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer. The ion exchange capacity of the packing material surface was measured by titration to be 0.4 meq
It was / g. The filler was treated with a silver nitrate solution, and the average thickness of the coating layer was measured according to the above method, and it was about 70Å. Human blood and various proteins were separated in the same manner as in Example 1. The resulting chromatograms are shown in FIGS. 3 and 4.

Example 3 In this example, a continuous process was employed in which the preparation of the hydrophobic crosslinked polymer particles was followed by the reaction of the monomers having hydrolyzable groups.

100g of styrene, 200g of divinylbenzene and 1g of benzoyl peroxide were dissolved in 200g of toluene, added to 2.5% of 4% polyvinyl alcohol aqueous solution, sized with stirring, and then heated to 80 ° C under nitrogen substitution for suspension polymerization went. After polymerizing at 80 ° C for 2 hours, 50 g of acrylamide (monomer having a hydrolyzable group) was added to the above system and further polymerized at 80 ° C for 2 hours, and the product was washed successively with hot water and acetone. Dried. Next, 200 g of the dried gel is placed in 500 ml of a 20% (by weight) solution of sodium hydroxide in 500 ml for 10 hours 75
Heated to ° C. The reaction mixture was cooled to room temperature, washed with water several times, and dried. The obtained minute polymer gel was classified and filled in the same manner as in Example 1 and evaluated. As a result, the pressure loss was proportional to the pressure loss and the flow velocity up to 150 kg / cm ² . When the swelling test was performed, the eluent was 40 mM
No increase in column pressure was observed when the phosphate buffer solution was changed to a 200 mM phosphate buffer solution. The ion exchange capacity of the packing material surface was measured by titration and found to be 0.5 meq / g. Further, the filler was treated with a silver nitrate solution, and the average thickness of the coating layer was measured according to the above method,
It was about 80Å. Human blood and various proteins were separated in the same manner as in Example 1. The resulting chromatogram was similar to that shown in FIGS. 1 and 2.

Example 4 Also in this example, the preparation of the hydrophobic cross-linked polymer particles is followed by the reaction of the monomer having a hydrolyzable group,
The continuous method was adopted.

Styrene 100 g, divinylbenzene 200 g and benzoyl peroxide 1 g were dissolved in toluene 200 g, added to 4% polyvinyl alcohol aqueous solution 2 and sized with stirring, then heated to 80 ° C. under nitrogen substitution to carry out suspension polymerization. went. After polymerizing at 80 ° C for 4 hours, 50 g of 2-hydroxyethylmethacrylate (monomer having a hydrolyzable group) was added to the above system and further polymerized at 80 ° C for 2 hours. It was washed successively with acetone and dried. The obtained fine polymer particles are hydrolyzed in the same manner as in Example 1,
Evaluation was performed by classifying and filling. As a result, the pressure loss was proportional to the pressure loss and the flow velocity up to 150 kg / cm ² . A swelling test was performed and the eluent was changed from 40 mM phosphate buffer.
No increase in column pressure was observed when the buffer was changed to 200 mM phosphate buffer. The ion exchange capacity of the packing material surface was measured by titration and found to be 0.6 meq / g. The filler was treated with a silver nitrate solution, and the average thickness of the coating layer was measured according to the above method, and it was about 100Å. Human blood and various proteins were separated in the same manner as in Example 1. The resulting chromatogram is the first
It was similar to the figure and FIG.

Comparative Example 1 100 g of styrene, 200 g of divinylbenzene, 70 g of methyl acrylate (monomer having a hydrolyzable group) and 1 g of acetyl peroxide were dissolved in 200 g of toluene, added to 2.5% of 4% polyvinyl alcohol aqueous solution, and stirred. While granulating, the mixture was heated to 70 ° C. to carry out suspension polymerization. After polymerizing at 70 ° C. for 8 hours, the product was hydrolyzed, classified and filled in the same manner as in Example 1 and evaluated.

As a result of performing the same evaluation as in Example 1, the pressure resistance was proportional to the flow velocity up to 80 kg / cm ² . A swelling test showed that the eluent was 200m from 40mM phosphate buffer.
When changing to M phosphate buffer, the column pressure is 20 kg / cm ²
Rose. Thus, it is clear that the pressure resistance and swelling resistance are inferior to those of the filler of Example 1. Further, when the filler was treated with a silver nitrate solution and the average thickness of the coating layer was measured according to the method described above, the carboxyl groups were evenly present in the filler particles. Further, in the same manner as in Example 1, human blood and various proteins were separated. The results are shown in FIGS. 5 and 6. 1 and 2
It is clear that the resolution is inferior when compared to the figure.

Comparative Example 2 As Example 1 using 200 g of polystyrene gel HSG-50 manufactured by Sekisui Chemical Co., Ltd. as the hydrophobic cross-linked polymer particles and 300 g of methyl acrylate as the monomer having a hydrolyzable group. The filler was prepared in the same manner as above and evaluated.

As a result, the pressure loss was proportional to the pressure loss and the flow velocity up to 150 kg / cm ² . When the swelling test was conducted, when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer, the column pressure increased by 25 kg / cm ² . This is considered to be because the swelling resistance was deteriorated because the coating layer was thick, as described later. The ion exchange capacity of the filler surface was measured by titration and found to be 1.2 meq / g. The filler was treated with a silver nitrate solution, and the average thickness of the coating layer was measured according to the above method, and it was about 400Å. However, the coating layer was thick and the pores were almost blocked.

The protein was separated in the same manner as in Example 1. The resulting chromatogram is shown in FIG. The holding force is weaker than in FIGS. 2 and 4, but this is because the surface area is reduced because the pores are blocked as described above.

Comparative Example 3 As Example 1 using 200 g of polystyrene gel HSG-50 manufactured by Sekisui Chemical Co., Ltd. as the hydrophobic crosslinked polymer particles and 10 g of methyl acrylate as the monomer having a hydrolyzable group. The filler was prepared in the same manner as above and evaluated.

As a result, the pressure loss was proportional to the pressure loss and the flow velocity up to 150 kg / cm ² . When the swelling test was conducted, no increase in column pressure was observed when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer. The ion exchange capacity of the packing material surface measured by titration was 0.1 m
It was eq / g. Also, treating the filler with a silver nitrate solution,
When the average thickness of the coating layer was measured according to the above method, it was about 8Å. In addition, there was an uncoated portion on a part of the surface of the filler.

The protein was separated in the same manner as in Example 1. The resulting chromatogram is shown in FIG. Although the elution order is different from that in FIGS. 2 and 4, this is probably because the coating is insufficient and the separation is performed by the hydrophobic interaction.

(Effect of the Invention) According to the present invention, as described above, a water-based packing material for liquid chromatography having excellent pressure resistance, less swelling and shrinkage, and non-specific adsorption of proteins can be obtained.
Such a packing material can be widely used as a packing material for weak cation exchange chromatography in the isolation or analysis of various hydrophilic substances.

[Brief description of drawings]

FIGS. 1, 3 and 5 are chromatograms obtained when blood analysis was performed using the columns packed with the packing materials obtained in Example 1, Example 2 and Comparative Example 1, respectively. Indicates. 2, FIG. 4 and FIGS. 6 to 8 show that various proteins were separated using the columns packed with the packing materials obtained in Example 1, Example 2 and Comparative Examples 1 to 3, respectively. The chromatogram obtained at the time is shown.

Claims (1)

(57) [Claims] 1. A method for producing a packing material for liquid chromatography, comprising the step of preparing hydrophobic crosslinked polymer particles impregnated with a polymerization initiator; a dispersion liquid in which the hydrophobic crosslinked polymer particles are dispersed. , A monomer having a functional group capable of generating a carboxyl group by a hydrolysis reaction is added and dissolved, and the monomer is polymerized on the surface portion of the hydrophobic crosslinked polymer particles to obtain the hydrophobic crosslinked polymer. Forming a layer of a polymer having a functional group capable of generating a carboxyl group on the surface of the particle by a hydrolysis reaction to a thickness of 10 to 300Å; and hydrolyzing the functional group to form the hydrophobic cross-linked polymer. A step of coating the surface of the coalesced particles with a polymer having a carboxyl group to a thickness of 10 to 300Å;