JPH11183460A - Packing material for liquid chromatography and its production and use - Google Patents

Abstract

(57) [Summary] (Modifications) [Problem] To provide a packing material for liquid chromatography which is easy to synthesize, has excellent synthesis reproducibility, and has synthesized particles having excellent mechanical strength and chemical stability. To provide. SOLUTION: The porous particles coated with polyvinyl alcohol have a cation exchange group in a hydroxyl group of the coating layer.
Packing material for liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a packing material for liquid chromatography of a cation exchanger, a method for producing the same, and a method for using the same.

[0002]

2. Description of the Related Art In recent years, various polymer particles have been used in the fields of adsorbents, cosmetics, paints, building materials, pharmaceuticals, analytical chemistry, and the like. In the field of high-performance liquid chromatography in analytical chemistry, ion-exchange groups were introduced on the surface of organic polymer substrates and silica substrates that had been surface-modified using silane treatment agents, etc. Fillers are used. As a method for preparing an ion exchanger of an organic polymer base material, a base material obtained by radically copolymerizing a monomer having a functional group for introducing an ion exchange group later and a polyfunctional monomer for maintaining the strength of the base material is used. ,
Ion exchange groups are introduced on the surface.

[0003] As a method of synthesizing a sulfonic acid type cation exchanger, a method of sulfonating styrene-divinylbenzene copolymer fine particles or a method of preparing copolymer fine particles of glycidyl methacrylate and a crosslinkable monomer to directly sulfonate glycidyl groups are used. Or a method of introducing an allyl alcohol into a glycidyl group and sulfonating the glycidyl group. In addition, as a method for preparing a carboxylic acid type cation exchanger, a method of preparing copolymer fine particles of glycidyl methacrylate and a crosslinkable monomer, generating a hydroxyl group on the particle surface, and then introducing α-chloroacetic acid, etc., butadiene-maleic acid on silica gel A method of crosslinking and coating a copolymer resin, a method of coating and curing a styrene-based polymer with a polyfunctional epoxy compound and a polyfunctional carboxylic acid, and the like have been implemented.

[0004] These cation exchangers include proteins,
It is used as a column packing for analysis of amino acids, inorganic cations and amines.

[0005]

The cation exchanger using fine particles of an organic polymer has a higher resistance when used in liquid chromatography than a silica-based fine particle even when an aqueous eluent is used. Widely used. However, when these fine particles are used as a filler, hydrophilic fine particles must be used because it is necessary to reduce the hydrophobic interaction between the filler and the measurement sample. Therefore, it is necessary to use a hydrophilic monomer when preparing the fine particles, which makes it difficult to maintain the mechanical strength as a filler for liquid chromatography. Even when the column is filled with the packing material, there are restrictions on the mechanical strength of the packing material, so that the length of the column that can be packed and the particle size of the packing material are limited, and the separation performance is also limited.

In terms of the synthesis method, in the case of hydrophilic acrylic copolymer fine particles, a polymerization step for preparing a base material, a hydrophilization step for generating a hydroxyl group in order to introduce an ion exchange group into the surface of the base material, In addition, there is a problem that multiple steps such as a step of introducing an ion exchange group are required. In particular, the hydrophilization step
It is not a step in which the reaction proceeds quantitatively, and it is known that reproducibility is poor. Further, there is a problem that the exchange capacity of the obtained cation exchanger is not sufficient.

In the case of sulfonic acid type styrene fine particles, styrene and divinylbenzene copolymer fine particles are first prepared,
Since this is sulfonated using fuming sulfuric acid or the like, it is very difficult to control the amount of ion-exchange group introduced, and it is difficult to introduce a functional group other than sulfonic acid due to stability restrictions in the sulfonation process. There is also a problem.

[0008]

Means for Solving the Problems As a result of studying the above problems, the inventors of the present invention have found that, if porous particles crosslinked and coated with polyvinyl alcohol have a cation exchange group in the hydroxyl group of the coating layer, the synthesis method It is possible to produce fine particles with excellent mechanical reproducibility and excellent mechanical strength and chemical stability as physical properties of the obtained fine particles, and this is used as a filler for liquid chromatography. Thus, the present invention was found to have excellent performance, and the present invention was completed. Further, the method for introducing an ion exchange group of the present invention can be used for various substrates, and has been found to be an excellent method for introducing a cation exchange group. Hereinafter, the packing material for liquid chromatography of the present invention and its production method and use method will be described.

[0009] The packing material for liquid chromatography of the present invention is a fine particle having a cation exchange group in the coating layer of porous particles crosslinked and coated with polyvinyl alcohol. The degree of polymerization of the polyvinyl alcohol coated with the crosslinked porous particles is 2400 or less, particularly preferably 1700 or less, in order to reduce the influence of the physical properties before crosslinking on the pore characteristics of the porous particles. The ratio of the crosslinked polyvinyl alcohol to the porous particles is preferably 5% by weight or more from the viewpoint of maintaining the hydrophilicity of the particles, and is preferably 40% by weight or less from the viewpoint of performance when used in liquid chromatography. As a crosslinked structure of polyvinyl alcohol, a crosslinked structure via an ether bond is preferable when used as a filler for liquid chromatography, because the water resistance of the filler is good. As the structure to which the cation exchange group is bonded, a structure in which the residual hydroxyl group of the crosslinked and coated polyvinyl alcohol is bonded via an ether bond or an ester bond is desirable.

The porous particles do not need to have a functional group capable of forming a chemical bond with the polyvinyl alcohol resin to be cross-linked, and examples thereof include a styrene-divinylbenzene copolymer and a styrene-bismaleimide. Copolymer, divinylbenzene polymer, glycidyl methacrylate / ethylene glycol dimethacrylate copolymer, 2
-Hydroxyethyl methacrylate / ethylene glycol dimethacrylate copolymer, ethylene glycol dimethacrylate polymer, glycidyl methacrylate / divinylbenzene copolymer, 2-hydroxyethyl methacrylate / divinylbenzene copolymer, (anhydrous) maleic acid / divinylbenzene copolymer Examples thereof include organic polymer porous particles such as polymers, itaconic acid / divinylbenzene copolymer, maleimide / divinylbenzene copolymer, and crosslinked sugar, and inorganic porous particles such as silica gel, titanium oxide, zirconium oxide, and alumina. . For packing materials for liquid chromatography, in terms of mechanical strength and solvent resistance,
It is preferable to use styrene-based porous particles.

The process for producing the packing material for liquid chromatography of the present invention comprises the steps of cross-linking and coating porous particles with polyvinyl alcohol and introducing a cation exchange group into the hydroxyl group of the coating layer. A method of cross-linking the porous particles with the polyvinyl alcohol after introducing the alcohol cation exchange group can also be used.

In the step of cross-linking and coating the porous particles with polyvinyl alcohol, first, the porous particles are dispersed in an aqueous solution of polyvinyl alcohol, a cross-linking agent and, if necessary, a catalyst are added, and the polyvinyl alcohol is formed into porous particles. Cross-link coating. When the ratio of polyvinyl alcohol to the porous particles is 40% by weight or more, the porous particles are aggregated. Examples of the crosslinking agent used at this time include dialdehydes such as glutaraldehyde and terephthalaldehyde and epichlorohydrin.

When dialdehyde is used as a cross-linking agent, the cross-linking reaction can be promptly advanced by adding an inorganic acid such as hydrochloric acid or sulfuric acid and using it as a catalyst. The reaction temperature is preferably from room temperature to 100 degrees.
When epichlorohydrin is used as a crosslinking agent, the crosslinking reaction may be advanced by dispersing porous particles in a mixed aqueous solution of polyvinyl alcohol and epichlorohydrin and dropping an aqueous solution of an inorganic base such as caustic soda. The amount of the inorganic base is preferably in the range of the equivalent of epichlorohydrin to 1.5 equivalents. The reaction temperature can be exemplified by dropping an inorganic base at room temperature and heating to about 70 ° C. after the dropping to terminate the crosslinking reaction. After the completion of the reaction, the porous particles coated with polyvinyl alcohol by crosslinking can be washed with pure water to remove the reaction catalyst and salts.

The step of introducing a cation exchange group into the hydroxyl group of the polyvinyl alcohol coating layer cross-linked to the porous particles may be a method of introducing an aldehyde compound having a cation exchange group into the coating layer hydroxyl group by an acetalization reaction or a polyfunctional compound. This is a step of introducing a carboxylic acid compound into the coating resin layer by an esterification reaction.

The method of introducing an aldehyde compound having a cation exchange group by an acetalization reaction is a method of acetalization reaction by dehydration of aldehyde groups of an aldehyde compound having a cation exchange group and hydroxyl groups of polyvinyl alcohol crosslinked and coated on porous particles. This is a method of introducing a cation exchange group. Examples of the aldehyde compound having a cation exchange group used herein include glyoxylic acid, carboxybenzaldehydes, benzaldehyde sulfonic acids, benzaldehyde disulfonic acids, and naphthyl aldehyde sulfonic acids. The acetalization reaction can proceed quickly using an acid catalyst. Examples of the acid catalyst include hydrochloric acid, sulfuric acid, benzenesulfonic acid, and toluenesulfonic acid. As a reaction mode, polyvinyl alcohol cross-linked porous particles are dispersed in a solvent, and an aldehyde compound having a cation exchange group and an acid catalyst are dissolved to allow an acetalization reaction to proceed, or the porous particles and the aldehyde A reaction form in which a compound and an acid catalyst are dispersed and dissolved in a volatile solvent, and the volatile solvent is distilled off, followed by heating to progress the acetalization reaction can be exemplified.

The following is an example of a method for introducing an aldehyde compound having a cation exchange group by an acetalization reaction according to a synthesis method.

A: Porous particles obtained by dissolving an aldehyde compound having a cation exchange group in a solvent which can be azeotroped with water such as benzene or toluene and which is not reactive with the aldehyde compound, and which is cross-linked and coated with polyvinyl alcohol. Disperse. After adding a trace amount of an acid catalyst such as sulfuric acid, benzenesulfonic acid or toluenesulfonic acid, the mixture is heated to remove water generated by the acetalization reaction by azeotropic distillation with the solvent. Thereafter, the particles are collected by filtration, washed with a good solvent of an aldehyde compound having a cation exchange group, further washed with a good solvent of an acid catalyst such as alcohol, and finally replaced with water.

B: Porous particles obtained by dissolving in an aldehyde compound having a cation exchange group and a low boiling point solvent capable of dissolving a small amount of an acid catalyst such as sulfuric acid, benzenesulfonic acid and toluenesulfonic acid and crosslinking-coating polyvinyl alcohol. Spread. Then the solvent is distilled off. The mixture is then
By heating at a temperature of at least 100 degrees, preferably at least 100 degrees and at most 130 degrees, water generated by the acetalization reaction is removed. After cooling, the particles are dispersed in a good solvent of an aldehyde compound having a cation exchange group, the particles are collected by filtration, washed with the solvent, further washed with a good solvent of an acid catalyst such as alcohol, and finally washed. Replace with water.

C: The porous particles coated with an aldehyde compound having a cation exchange group and polyvinyl alcohol by cross-linking are dispersed and dissolved in an aqueous solution of 2N or more hydrochloric acid, sulfuric acid or the like, and stirred at room temperature. Thereafter, the particles are collected by filtration, and washed with a good solvent for an aldehyde compound having a cation exchange group,
Further washing is performed using a good solvent of an acid catalyst such as alcohol, and finally the water is replaced.

D: The porous particles coated with an aldehyde compound having a cation exchange group and polyvinyl alcohol by crosslinking are dispersed and dissolved in water. The pH of the solution is adjusted to about 2 using an acid catalyst such as hydrochloric acid or sulfuric acid. After that, over 40 degrees,
Preferably, heating is performed at a temperature of 60 degrees or more and 100 degrees or less. Thereafter, the particles are collected by filtration, washed with a good solvent of an aldehyde compound having a cation exchange group, further washed with a good solvent of an acid catalyst such as alcohol, and finally replaced with water.

The method of introducing a polyfunctional carboxylic acid compound into the coating resin layer by an esterification reaction is a method of esterifying the hydroxyl groups of polyvinyl alcohol cross-linked to the porous particles and the carboxylic acid groups of the polyfunctional carboxylic acid compound by dehydration. In this method, a cation exchange group is introduced by a reaction. Examples of the polyfunctional carboxylic acids used herein include tricarbanilic acid and butanetetracarboxylic acid as examples of aliphatic polyfunctional carboxylic acids, and trimellitic acid and pyromellitic acid as examples of aromatic polyfunctional carboxylic acids. And tetrahydrofurantetracarboxylic acid as an example of the heterocyclic polyfunctional carboxylic acid. The esterification reaction is
The polyfunctional carboxylic acid can be easily prepared by dispersing the polyfunctional carboxylic acid on the polyvinyl alcohol cross-linked porous particles and heating.

The following is an example of a method for introducing a polyfunctional carboxylic acid compound into the coating resin layer by an esterification reaction.
The polyfunctional carboxylic acid is dissolved in a low-boiling solvent in which the polyfunctional carboxylic acid can be dissolved, and the porous particles crosslinked and coated with polyvinyl alcohol are dispersed in the solution. Then the solvent is distilled off. Subsequently, the mixture is heated at 140 ° C. or more, preferably 160 ° C. or more and 180 ° or less to remove water generated by the esterification reaction. After cooling, the particles are dispersed in a good solvent for the polyfunctional carboxylic acid compound, and then the particles are collected by filtration, washed with the solvent, and finally replaced with water.

The packing material having a cation exchange group obtained by the above method can be packed in a column for liquid chromatography and used for liquid chromatography analysis. By using the filler of the present invention, it is possible to produce a filler having high physical durability, it was difficult in the conventional filler for hydrophilic cation exchange liquid chromatography due to restrictions on the physical durability of the filler, It is also possible to increase the length of the column and to reduce the particle size of the packing material, thereby improving the separation performance when used in liquid chromatography. In addition, since the method of introducing a cation exchange group into the cross-linked coated polyvinyl alcohol is a method that does not select the fine particles of the base material, it is useful as an ion exchange group introduction reaction for fine particles such as silica, cross-linked sugar, and acrylic polymer. It is. The column in which the packing material of the present invention is packed into a column for liquid chromatography is used for analysis of proteins, peptides, and other biological macromolecules by ion exchange, analysis of organic acids by ion exclusion, and ion chromatography of inorganic cations and amines. It can be used for analysis by photography.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1 2 L equipped with a stirrer, a reflux condenser and a nitrogen gas inlet tube
Set the three-neck separable flask in the oil bath,
Add 400 g of pure water and add polyvinyl alcohol (PVA2
24, manufactured by Kuraray Co., Ltd.). Separately, as a monomer solution, 100 g of high-purity divinylbenzene monomer, 20 g of toluene, and 1 g of isoamyl alcohol
30 g and 5 g of benzoyl peroxide were mixed and charged into the three-necked flask. Nitrogen was introduced while stirring at 500 rpm, and stirring was continued at room temperature for 30 minutes. Then, the temperature of the oil bath was increased to 80 ° C, and polymerization was performed for 16 hours. After the polymerization was completed, the gel was washed with 70 ° C. warm water, and further washed with acetone. The obtained gel was classified by a conventional method to obtain a gel having a central particle size of 5 μm. The obtained gel was packed in a column for liquid chromatography, and the elution position of standard polystyrene having a known molecular weight was measured to examine the pore distribution characteristics under the following conditions. The results are shown in FIG.

(Conditions) Column: 7.8 mm D. × 30 cmL. Eluent: THF Flow rate: 0.8 L / min (using CCPD (Tosoh Corp. pump)) Temperature: 40 degrees (CO-8020 (Tosoh Corp. column oven)) : Change in absorbance at UV 254 nm (UV-8020
Example 2 A three-necked flask connected to a stirrer was set in an oil bath, 100 mL of pure water was added, and polyvinyl alcohol (P) was used.
2.0 g of VA 217C (manufactured by Kuraray Co., Ltd.). 10 g (dry) of the gel prepared in Example 1, which had been replaced with water in advance, was placed in a flask and stirred at room temperature with a 50% solution of glutaraldehyde (reagent) in 0.88.
g, and then about 2 mL of a 1N aqueous hydrochloric acid solution was added to adjust the pH to 2.0 or less. Set the bath temperature to 60 degrees,
Stir for 4 hours. After the reaction was completed, the gel was washed with warm water at 70 degrees to obtain a polyvinyl alcohol-coated gel. The pore characteristics of the obtained gel were measured in the same manner as in Example 1. The measurement results are shown in FIG. It was found that even after the polyvinyl alcohol was cross-linked, the polyvinyl alcohol was uniformly cross-linked without greatly affecting the pore characteristics of the original porous particles.

The three-necked flask connected with the stirrer was set in an oil bath, and the polyvinyl alcohol-coated gel was mixed with pure water 1.
The dispersion dispersed in 00 mL was added, and while stirring, 6.33 g of sodium benzaldehyde-2,4-disulfonate (reagent) was added. After adding 5 mL of 1N-HCl, the oil bath was set at 60 degrees and heated with stirring for 4 hours. After cooling to room temperature and filtering with a glass filter, methanol, 1N-HCl aqueous solution, bridge 35
Washing was performed in the order of a 1% aqueous solution and pure water. When the ion exchange capacity was measured with a 0.1N-NaOH aqueous solution, 60 μe
q / gel mL. 4.6 mm of the obtained gel
I. D. A × 10 cmL column was packed and standard protein separation was attempted. FIG. 2 shows the measurement results. The measurement conditions are shown below. (Measurement conditions) Eluent (Solution A): 20 mM acetate buffer (adjusted to pH 5.0 with NaOH) (Solution B): 20 mM acetate buffer (pH 5.0) +0.5 M Na ₂ SO ₄ Flow rate: 1.0 mL / Min Elution: solvent gradient (linear gradient from 100% of solution A to 100% of solution B for 10 minutes) Column temperature: room temperature Sample: trypsinogen (Sigma reagent) 1 mg / mL concentration 5 μL ribonuclease (Sigma reagent) 1 mg / mL concentration 5 μL cytochrome Measurement of C (Sigma reagent) 1 mg / mL, concentration 5 μL As a result of the measurement, it was found that the sample was separated in a short time with a sharp peak with good resolution.

Example 3 A polyvinyl alcohol-coated gel was prepared in the same manner as in Example 2. A polyvinyl alcohol-coated gel was placed in an eggplant type flask, dispersed in methanol, and then benzaldehyde-2,
After 3.5 g of sodium 4-disulfonate and 0.5 g of toluene sulfonic acid (reagent) were added and stirred well,
Methanol was distilled off with an evaporator. 1 flask
It was placed in an oven set at 10 degrees and heated for 3 hours. After cooling, methanol was put into the flask, and the gel was well dispersed, and then filtered through a glass filter. Then methanol,
It wash | cleaned in order of 1N-HCl aqueous solution, bridge 35 1% aqueous solution, and pure water. The ion exchange capacity of the obtained gel was set to 0.1.
When measured using a 1N-NaOH aqueous solution, 197 μm
eq / mL mL.

Example 4 A polyvinyl alcohol-coated gel was prepared in the same manner as in Example 1. Pour polyvinyl alcohol-coated gel into eggplant-shaped flask, disperse in methanol, and then add 50 wt% glyoxylic acid
After 1.5 g of a 1.5% aqueous solution and 0.5 g of toluenesulfonic acid (reagent) were added and thoroughly stirred, methanol was distilled off with an evaporator. The flask was placed in an oven set at 110 degrees and heated for 3 hours. After cooling, methanol was put into the flask, and the gel was well dispersed, and then filtered through a glass filter. Next, washing was carried out in the order of methanol, 1N-HCl aqueous solution, 1% aqueous solution of bridge 35 and pure water.
When the ion exchange capacity of the obtained gel was measured using a 0.1N-NaOH aqueous solution, it was 230 μeq / mL of gel. The obtained gel was 4.6 mm I.D. D. × 10c
mL columns were packed and standard protein separations were attempted. FIG. 3 shows the measurement results. The measurement conditions are shown below.

(Measurement Conditions) Eluent (Solution A): 20 mM acetate buffer (adjusted to pH 5.0 with NaOH) (Solution B): 20 mM acetate buffer (pH 5.0) +0.5 M Na ₂ SO ₄ Flow rate: 1 0.0 mL / min Elution: Solvent gradient (linear gradient from 100% of solution A to 100% of solution B for 10 minutes) Column temperature: room temperature Sample: 1 mg / mL trypsinogen (Sigma reagent) 5 μL Ribonuclease (Sigma reagent) 1 mg / mL Concentration 5 μL Cytochrome C (Sigma reagent) 1 mg / mL Concentration 5 μL α-Trypsinogen (Sigma reagent) 1 mg / mL Concentration 5 μL Lysozyme (Sigma reagent) 1 mg / mL Concentration 5 μL As a result of the measurement, the sample was separated with a sharp peak with good resolution. I understood that. In addition, standard inorganic cations were analyzed using this column. FIG. 4 shows the measurement results. The measurement conditions are shown below.

(Measurement conditions) Eluent: 2 mM HNO ₃ aqueous solution Flow rate: 0.8 mL / min Elution condition: isocratic Column temperature: 40 ° C Sample: Li (1 ppm), Na (5 ppm), NH
₄ (5 ppm), K (10 ppm), Mg (5 pp
m), 20 μL of an aqueous solution of Ca (10 ppm) showed that this column was also capable of simultaneous analysis of mono- and divalent cations.

Example 5 A polyvinyl alcohol-coated gel was prepared in the same manner as in Example 2. Pour polyvinyl alcohol-coated gel into eggplant type flask, disperse in benzene, and then add 50 wt% of glyoxylic acid
1.5 g of the aqueous solution and 0.5 g of toluenesulfonic acid (reagent) were added and stirred well. A Soxhlet extractor containing molecular sieves 4A was connected to the flask, heated in an oil bath, and benzene was refluxed to remove water from the flask. After cooling, methanol was put into the flask, and the gel was well dispersed, and then filtered through a glass filter. Then, methanol, 1N-HCl aqueous solution, bridge 35
Washing was performed in the order of a 1% aqueous solution and pure water. When the ion exchange capacity of the obtained gel was measured using a 0.1N-NaOH aqueous solution, it was 130 μeq / mL of gel.

Example 6 A three-necked flask connected to a stirrer was set in an oil bath, and 100 mL of pure water was added.
1.0 g of VA217C (manufactured by Kuraray Co., Ltd.) was dissolved.
10 g of silica gel having a maximum pore diameter of 150 Å and an average particle diameter of 5.0 μm previously dried with water (dry)
Into a flask and stirring at room temperature while adding 0.44 g of 50% glutaraldehyde solution (reagent).
Approximately 2m of 1N-hydrochloric acid aqueous solution
L was put. The bath temperature was set to 60 degrees and the mixture was stirred for 4 hours.
After the reaction was completed, the gel was washed with warm water at 70 degrees to obtain a polyvinyl alcohol-coated gel. Thereafter, glyoxylic acid was introduced in the same manner as in Example 4. When the ion exchange capacity of the obtained gel was measured using a 0.1N-NaOH aqueous solution,
It was 24 μeq / mL gel. The obtained gel was 4.6
mm I. D. Packed in a x10 cmL column and analyzed for standard inorganic cations. FIG. 5 shows the measurement results.
The measurement conditions are shown below.

(Measurement conditions) Eluent: 2 mM HNO ₃ aqueous solution Flow rate: 0.6 mL / min Elution condition: isocratic Column temperature: 40 ° C Sample: Li (1 ppm), Na (5 ppm), NH
₄ (5 ppm), K (10 ppm), Mg (5 pp
m), 20 μL of an aqueous solution of Ca (10 ppm) showed that the sample was separated with a sharp peak with good resolution. It was found that the method for introducing ion exchange groups can be applied to various porous particles.

Example 7 A polyvinyl alcohol-coated gel was produced in the same manner as in Example 2. A polyvinyl alcohol-coated gel was placed in an eggplant-shaped flask, dispersed in methanol, and then mixed with meso-butane-1,2,2.
4.52 g of 3,4-tetracarboxylic acid (reagent) was dissolved. The methanol was distilled off using an evaporator, and the flask was heated in an oven set at 160 degrees for 1 hour. After cooling, the mixture was dispersed in methanol and filtered with a glass filter. Then methanol, 1N-HCl aqueous solution,
The bridge 35 was washed with a 1% aqueous solution and pure water in this order. When the ion exchange capacity of the obtained gel was measured using a 0.1N-NaOH aqueous solution, it was 880 μeq / mL of gel. The obtained gel was 4.6 mm I.D. D. × 10cmL
The column was packed and standard protein analysis was performed. FIG. 6 shows the measurement results. The measurement conditions were the same as in Example 4. In addition, analysis of inorganic cations was also performed. FIG. 7 shows the measurement results. The measurement conditions are shown below.

(Measurement conditions) Eluent: 2 mM HNO ₃ aqueous solution Flow rate: 0.8 mL / min Elution condition: isocratic Column temperature: 40 ° C Sample: Li (1 ppm), Na (5 ppm), NH
₄ (5 ppm), K (10 ppm), Mg (5 pp
m), Ca (10 ppm), Rb (20 ppm), Ce
(30 ppm), Sr (20 ppm), Ba (30 pp)
20 μL of the aqueous solution of m) It was found that all samples were separated with good separation ability.

Comparative Example A standard protein was analyzed using an ordinary sulfonic acid type ion exchange column. FIG. 8 shows the results. The measurement conditions are shown below.

Column: SP-5PW (manufactured by Toso Corporation, 7.5 mm ID × 7.5 cm L) (Measurement conditions) Eluent (solution A): 20 mM acetate buffer (pH 5.0 with NaOH) (Solution B): 20 mM acetate buffer (pH 5.0) + 0.5 M Na ₂ SO ₄ Flow rate: 1.0 mL / min Elution: Solvent gradient (from 100% solution A to 100% solution B for 60 minutes) (Gradient) Column temperature: room temperature Sample: Trypsinogen (Sigma reagent) 1 mg / mL Concentration 5 μL Ribonuclease (Sigma reagent) 1 mg / mL Concentration 5 μL Cytochrome C (Sigma reagent) 1 mg / mL Concentration 5 μL α-Trypsinogen (Sigma reagent) 1 mg / mL Concentration 5μL

[0039]

According to the present invention, the packing material for liquid chromatography having a cation exchange group in the hydroxyl group of the coating layer of the porous particles obtained by cross-linking the polyvinyl alcohol of the present invention has high mechanical strength and high hydrophilicity. By using the packing material in a column for liquid chromatography analysis, the analysis time can be shortened and the separation ability can be improved. In addition, it is possible to prepare a filler having good durability against the eluent, and it is expected that the column durability will be improved. In addition, the reaction of crosslinking the porous particles of the present invention with polyvinyl alcohol and introducing a cation exchange group into the hydroxyl group of the coating layer can be performed using any porous particles regardless of the type of the porous particles. This method is easy in introducing an exchange group and has excellent reproducibility, and is useful as a method for producing a packing material for liquid chromatography.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the pore characteristics of porous particles of divinylbenzene polymer prepared in Example 1 and particles obtained by cross-linking the porous particles prepared in Example 2 with polyvinyl alcohol using standard polystyrene having a known molecular weight. FIG.

FIG. 2 is a chromatogram of a standard protein mixture in Example 2.

FIG. 3 is a chromatogram of a standard protein mixture in Example 4.

FIG. 4 is a chromatogram of an inorganic cation mixture in Example 4.

FIG. 5 is a chromatogram of an inorganic cation mixture in Example 6.

FIG. 6 is a chromatogram of a standard protein mixture in Example 7.

FIG. 7 is a chromatogram of an inorganic cation mixture in Example 7.

FIG. 8 is a chromatogram of a standard protein mixture in a comparative example.

Claims (10)

[Claims] (1) a porous particle formed by coating polyvinyl alcohol having a cation exchange group in a hydroxyl group of the coating layer;
Packing material for liquid chromatography. 2. The packing material for liquid chromatography according to claim 1, wherein the coating amount of the polyvinyl alcohol is 5 to 40% by weight based on the porous particles. 3. The packing material for liquid chromatography according to claim 1, wherein the cation exchange group is a sulfonic acid group or a carboxylic acid group. 4. The method for liquid chromatography according to claim 1, comprising a step of cross-linking and coating the porous particles with polyvinyl alcohol and a step of introducing an aldehyde compound having a cation exchange group into a hydroxyl group of the coating layer by an acetalization reaction. A method for producing a filler. 5. The method for producing a packing material for liquid chromatography according to claim 4, wherein the cation exchange group is a sulfonic acid group or a carboxylic acid group. 6. A step of cross-linking and coating a porous particle with polyvinyl alcohol and a step of introducing a polyfunctional carboxylic acid compound into a hydroxyl group of the coating layer by an esterification reaction.
A method for producing a packing material for liquid chromatography according to claim 1. 7. The method for producing a packing material for liquid chromatography according to claim 4, wherein the coating amount of the polyvinyl alcohol is 5 to 40% by weight based on the porous particles. 8. A column for liquid chromatography, wherein the packing material for liquid chromatography according to claim 1 is packed in a column for liquid chromatography. 9. An analysis method using the column for liquid chromatography according to claim 8 for biopolymer analysis. 10. An analytical method using the column for liquid chromatography according to claim 8 for ion chromatography.