JP2010133733A - Cation exchanger, method for manufacturing the same and application thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cation exchanger capable of efficiently introducing a carboxylic acid group or sulfonic acid group into the surface of a carrier for chromatography under a condition more moderate than before using a small amount of a reagent. <P>SOLUTION: The cation exchanger is manufactured by the first step (1) for introducing an epoxy group into the carrier for chromatography and the second step (2) for reacting a compound having a thiol group or carboxylic acid group in its molecule or a compound having the thiol group or sulfonic acid group in its molecule with the epoxy group. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cation exchanger, a production method thereof, and an application thereof. The cation exchanger of the present invention is particularly useful as a chromatographic separation agent.

  Carriers having ion exchange groups are widely used in the field of chromatographic separation as separation materials. For example, a cation exchanger into which a carboxylic acid group or a sulfonic acid group is introduced as an ion exchange group is used for the purpose of analysis and fractionation of amino acids, peptides, proteins, etc. in the field of liquid chromatography.

  The carrier having a carboxylic acid is a method in which a halogenated acetate is reacted with a carrier having a hydroxyl group in the presence of a high concentration alkali (for example, see Patent Document 1), and a method in which a carrier having an amino group is reacted with a dicarboxylic acid anhydride ( For example, see Patent Document 2), a method of reacting a carrier having an epoxy group with an amino compound having a carboxylic acid group such as aminocaproic acid, a method of oxidizing a carrier having an allyl group with permanganates (for example, Patent Document 3) Etc.).

  In addition, the carrier having a sulfonic acid may be a method of reacting a halogenated ethanesulfonate salt or a sultone with a carrier having a hydroxyl group in the presence of a high concentration alkali (for example, see Patent Document 1), or a carrier having an allyl group with oxygen. It is prepared by a method of reacting bisulfites in the presence (see, for example, Patent Document 3).

  However, in the method of reacting a halogenated acetic acid, a halogenated ethanesulfonic acid, or a halogenated propanesulfonic acid with a carrier having a hydroxyl group in the presence of a high concentration of alkali, a reaction in which the halogen group is replaced with a hydroxyl group also proceeds with an alkali. Therefore, it is necessary to use a large amount of medicine. Further, it cannot be used for silica that dissolves in alkali or a carrier that undergoes hydrolysis.

  In the method of reacting a dicarboxylic acid anhydride with a carrier having an amino group, an unreacted amino group remains, so that the remaining amino group must be blocked with acetic anhydride or the like.

  On the other hand, in a chromatographic support, a spacer is generally introduced between the support and the ion exchange group in order to reduce non-specific interaction between the target compound to be separated and the support. As the compound that forms such a spacer, for example, epichlorohydrin, a polyfunctional epoxy compound, or a compound having an allyl group is used. Specifically, a spacer is introduced into the chromatographic support, and an ion exchange group is introduced into the introduced spacer end.

  In order to introduce an ion exchange group to the introduced spacer end, a method of reacting an epoxy group at the end of the spacer with an amino compound having a carboxylic acid group such as aminocaproic acid is generally used. However, since the reactivity of amino groups is low, the amount of carboxylic acid groups introduced is extremely small. Moreover, in the coupling | bonding of an epoxy group and an amino group, the couple | bonded amino group also has ion exchange property, and becomes an amphoteric ion exchanger.

  On the other hand, when a compound having an allyl group is used as a spacer, an ion exchange group can be introduced into the end of the spacer by oxidizing the allyl group into a carboxylic acid or by adding sodium bisulfite to the allyl group to form a sulfone. However, each method has a problem that an unreacted allyl group having high hydrophobicity remains.

JP-A-1-262468 US Pat. No. 4,560,704 U.S. Pat. No. 4,029,583

  The present invention has been made in view of the above-described background art, and its object is to efficiently use a small amount of a reagent on a surface of a chromatography carrier or a sulfone group on a surface of a chromatographic support under mild conditions as compared with conventional methods. An object of the present invention is to provide a cation exchanger capable of introducing an acid group, a production method thereof, and a use thereof.

  As a result of intensive studies to achieve the above object, the present inventors have found the cation exchanger of the present invention and have completed the present invention. That is, this invention is a cation exchanger as shown below, its manufacturing method, and its use.

  [1] A cation exchanger having a carboxylic acid group containing a thiol group or a sulfonic acid group containing a thiol group as an ion exchange group on the surface of a chromatography carrier.

  [2] The amount of the carboxylic acid group containing a thiol group or the sulfonic acid group containing a thiol group is 0.005 to 0.5 meq per 1 mL (wet capacity) of the cation exchanger. The cation exchanger according to [1] above.

  [3] The carboxylic acid group containing the thiol group or the sulfonic acid group containing a thiol group is represented by the following formula (1).

（上記式中、Ｒは炭素数１〜３のアルキレン基を表し、Ａはカルボン酸基、スルホン酸基又はそれらの塩を表す。）
で示されるイオン交換基であることを特徴とする上記［１］又は［２］に記載のカチオン交換体。

(In the above formula, R represents an alkylene group having 1 to 3 carbon atoms, and A represents a carboxylic acid group, a sulfonic acid group, or a salt thereof.)
The cation exchanger according to [1] or [2] above, which is an ion exchange group represented by

  [4] The cation exchange according to any one of [1] to [3], wherein the chromatography carrier is a natural polymer carrier, a synthetic polymer carrier, or an inorganic carrier. body.

  [5] The cation according to any one of the above [1] to [4], wherein the chromatographic support is in the form of spherical particles, non-spherical particles, a membrane, or a monolith (continuum). Exchanger.

[6] The method for producing a cation exchanger according to any one of [1] to [5], including the following steps.
(1) a first step of introducing an epoxy group into a chromatographic support; and
(2) A second step of reacting the epoxy group with a compound having a thiol group and a carboxylic acid group in the molecule, or a compound having a thiol group and a sulfonic acid group in the molecule.

  [7] In the above [6], in the first step described above, an epoxy group is introduced into a chromatographic carrier using epichlorohydrin, a polyfunctional epoxy compound or an epoxy group-containing silane treating agent. A method for producing a cation exchanger.

  [8] The above-mentioned polyfunctional epoxy compound is ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, glycerol. The method for producing a cation exchanger according to [6] above, which is one or more compounds selected from the group consisting of diglycidyl ether, glycerol triglycidyl ether, and sorbitol polyglycidyl ether.

  [9] The epoxy group-containing silane treating agent is selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, and 3-glycidoxypropyltriethoxysilane. It is a 1 type, or 2 or more types of compound, The manufacturing method of the cation exchanger as described in said [7] characterized by the above-mentioned.

  [10] In the second step, a compound having a thiol group and a carboxylic acid group in the molecule, or a compound having a thiol group and a sulfonic acid group in the molecule is represented by the following formula (2):

（上記式中、Ｒは炭素数１〜３のアルキレン基を表し、Ａはカルボン酸基、スルホン酸基又はそれらの塩を表す。）
からなる群より選ばれる１種又は２種以上の化合物であることを特徴とする上記［６］乃至［９］のいずれかに記載のカチオン交換体の製造方法。

(In the above formula, R represents an alkylene group having 1 to 3 carbon atoms, and A represents a carboxylic acid group, a sulfonic acid group, or a salt thereof.)
The method for producing a cation exchanger according to any one of the above [6] to [9], which is one or more compounds selected from the group consisting of:

  [11] The compound represented by the formula (2) is one or more compounds selected from the group consisting of mercaptoacetic acid, mercaptopropionic acid, mercaptoethanesulfonic acid, mercaptopropanesulfonic acid, and salts thereof. The method for producing a cation exchanger according to [10] above, wherein

  [12] A chromatographic separation agent comprising the cation exchanger according to any one of [1] to [4].

  [13] A packed column for chromatography formed by packing the cation exchanger according to any one of [1] to [4] in a chromatography column.

  The cation exchanger of the present invention can efficiently introduce a carboxylic acid group or a sulfonic acid group onto the surface of a chromatographic support under a mild condition as compared with the conventional method, with a small amount of reagent, and chromatography. Since it can be suitably used as a filler for the industrial use, it is extremely useful industrially.

  The cation exchanger of the present invention has a carboxylic acid group containing a thiol group or a sulfonic acid group containing a thiol group as an ion exchange group on the surface of the chromatography carrier.

  As such a carboxylic acid group containing a thiol group or a sulfonic acid group containing a thiol group, for example, the following formula (1)

（上記式中、Ｒは炭素数１〜３のアルキレン基を表し、Ａはカルボン酸基、スルホン酸基又はそれらの塩を表す。）
で示されるイオン交換基が挙げられる。

(In the above formula, R represents an alkylene group having 1 to 3 carbon atoms, and A represents a carboxylic acid group, a sulfonic acid group, or a salt thereof.)
The ion exchange group shown by these is mentioned.

  As a material for the chromatographic carrier constituting the cation exchanger of the present invention, any material can be used without particular limitation as long as it can introduce an epoxy group, which will be described later, and examples thereof include natural polymer carriers and synthetic polymers. System carriers, inorganic carriers and the like can be used.

  In the present invention, examples of the natural polymer carrier include polysaccharides such as cellulose, agarose, and dextran. Examples of the synthetic polymer carrier include hydroxyl group-containing monomers such as 2-hydroxyethyl (meth) acrylate, 2-hydroxymethyl (meth) acrylate, hydroxypropyl (meth) acrylate, ethylene glycol di (meth) acrylate, Examples thereof include those prepared by mixing with a crosslinkable monomer such as divinylbenzene and polymerizing in the presence of a polymerization initiator. Examples of the inorganic carrier include silica and zeolite. Examples of the form of the carrier include, but are not particularly limited to, spherical particles, non-spherical particles, membranes, monoliths (continuous bodies), and the like.

Although it does not specifically limit as a manufacturing method of the cation exchanger of this invention, For example, manufacturing by the following processes is preferable.
(1) a first step of introducing an epoxy group into a chromatographic support; and
(2) A second step of reacting the epoxy group with a compound having a thiol group and a carboxylic acid group in the molecule or a compound having a thiol group and a sulfonic acid group in the molecule.

  In the step (1), the epoxy group can be introduced into the chromatography carrier by a known method. For example, in the case of a natural polymer carrier or a synthetic polymer carrier, a method of reacting epichlorohydrin or a polyfunctional epoxy compound in the presence of an alkali such as NaOH or KOH can be mentioned. Specific examples of the polyfunctional epoxy compound include ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, Examples thereof include glycerol diglycidyl ether, glycerol triglycidyl ether, sorbitol polyglycidyl ether, and the like.

  In the case of an inorganic carrier, for example, a method of reacting with an epoxy group-containing silane treating agent can be mentioned. Specific examples of the epoxy group-containing silane treating agent include 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, and 3-glycidoxypropyltriethoxysilane.

  In the step (2), the cation exchanger of the present invention has a chromatographic carrier (hereinafter sometimes referred to as an epoxidized carrier) into which an epoxy group has been introduced in the step (1). It is prepared by reacting a compound having a thiol group and a carboxylic acid group, or a compound having a thiol group and a sulfonic acid group in the molecule. Specifically, these are preferably reacted in a solution (in the range of pH 7 to 9) at a reaction temperature of 25 to 40 ° C. for 5 to 24 hours.

  The amount of the epoxy group introduced into the epoxidized carrier is not particularly limited, but is usually in the range of 0.02 to 2 mol per 1 g (dry weight) of the epoxidized carrier.

  In the step (2), examples of the compound having a thiol group and a carboxylic acid group in the molecule or the compound having a thiol group and a sulfonic acid group in the molecule include the following formula (2):

（上記式中、Ｒは炭素数１〜３のアルキレン基を表し、Ａはカルボン酸基、スルホン酸基又はそれらの塩を表す。）
で示される化合物が挙げられる。分子内にチオール基とカルボン酸基を有する化合物としては、具体的には、メルカプト酢酸（チオグリコール酸）、メルカプトプロピオン酸、メルカプトブタン酸、又はそれらの塩類等が例示され、これらのうち、メルカプト酢酸、メルカプトプロピオン酸又はそれらの塩類がより好ましい。また、分子内にチオール基とスルホン酸基を有する化合物としては、具体的には、メルカプトエタンスルホン酸、メルカプトプロパンスルホン酸又はそれらの塩類が例示される。

(In the above formula, R represents an alkylene group having 1 to 3 carbon atoms, and A represents a carboxylic acid group, a sulfonic acid group, or a salt thereof.)
The compound shown by these is mentioned. Specific examples of the compound having a thiol group and a carboxylic acid group in the molecule include mercaptoacetic acid (thioglycolic acid), mercaptopropionic acid, mercaptobutanoic acid, and salts thereof. Among these, mercapto Acetic acid, mercaptopropionic acid or their salts are more preferred. Specific examples of the compound having a thiol group and a sulfonic acid group in the molecule include mercaptoethanesulfonic acid, mercaptopropanesulfonic acid, and salts thereof.

  The amount of the carboxylic acid group containing a thiol group or the sulfonic acid group containing a thiol group introduced into the cation exchanger of the present invention (that is, the ion exchange capacity) is not particularly limited, A range of 0.005 to 0.5 meq per 1 mL (wet volume) of cation exchanger is preferred.

  The cation exchanger of the present invention is particularly useful as a packing material for chromatography.

  If the cation exchanger of the present invention is packed in a chromatography column made of, for example, glass or metal, it can be used as a chromatography column for separation, analysis or fractionation of anionic proteins and the like. .

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is limited to these Examples and is not interpreted. In the following examples, the amount of epoxy groups and the ion exchange capacity were measured as follows.

Epoxy group quantification method.
100 mL of concentrated sulfuric acid was placed in a 500 mL separable flask, and 30 mL of concentrated hydrochloric acid was added dropwise using a dropping funnel. The generated hydrogen chloride was blown into a 2 L separable flask to which 1500 mL of dioxane was added with a glass tube to prepare a 0.2 N hydrochloric acid-dioxane solution. Epoxidized carrier Wg (dry weight) was placed in a 200 mL Erlenmeyer flask, 25 mL of 0.2 N hydrochloric acid-dioxane solution was added, and the mixture was stirred with a magnetic stirrer at 25 ° C. for 3 hours. After stirring, 50 mL of ethyl alcohol and 1 mL of phenolphthalein solution were added, and excess hydrochloric acid was titrated with a 0.1 N NaOH aqueous solution (titrated amount AmL). Similarly, 25 mL of 0.2N hydrochloric acid-dioxane solution was titrated with 0.1N-NaOH aqueous solution without adding an epoxidized support (titer amount TmL). The amount of the epoxy group was calculated from the following formula.

イオン交換容量の定量方法．
５０ｍＬビーカーに１０ｍＬ（湿潤容量）のカチオン交換体、０．５Ｎ−ＨＣＬ溶液２０ｍＬを加え分散させた。このスラリーをグラスフィルターでろ過し、カチオン交換体をろ液が中性になるまで純水で洗浄した。このカチオン交換体を２００ｍＬのビーカーに加え、０．５ｍｏｌ／Ｌ塩化ナトリウム水溶液１００ｍＬに分散させた。スターラーチップを入れ、攪拌させながら０．５Ｎ−ＮａＯＨ水溶液を用いて自動滴定装置（平沼産業社製、ＣＯＭ−４５０）で終点をｐＨ７として滴定した（滴定量ＢｍＬ）。カチオン交換体のイオン交換容量は以下の式より算出した。

Quantification method of ion exchange capacity.
In a 50 mL beaker, 10 mL (wet capacity) cation exchanger and 20 mL of 0.5 N HCl solution were added and dispersed. This slurry was filtered with a glass filter, and the cation exchanger was washed with pure water until the filtrate became neutral. This cation exchanger was added to a 200 mL beaker and dispersed in 100 mL of a 0.5 mol / L aqueous sodium chloride solution. A stirrer chip was added, and the mixture was titrated with an automatic titrator (COM-450, manufactured by Hiranuma Sangyo Co., Ltd.) using 0.5N-NaOH aqueous solution while stirring, with the end point set to pH 7 (titration BmL). The ion exchange capacity of the cation exchanger was calculated from the following equation.

実施例１．
クロマトグラフィー用担体として、デキストランをベースとしたＳｅｐｈａｄｅｘ（登録商標） Ｇ−２５ Ｆｉｎｅ（ＧＥ社製）を純水で洗浄した後、その湿潤重量６０ｇを滴下ロート付の三口フラスコに入れ、純水６６ｇ、エピクロルヒドリン６０ｇ添加し、４０℃で分散させた。水酸化ナトリウム２１ｇを純水３６ｇに溶解させた後、この溶液を滴下ロートより２時間で滴下した。滴下後更に２時間反応させた。反応終了後、ガラスフィルターを用いて担体を分離し、純水で洗浄し、エポキシ化担体を得た。導入されたエポキシ基の量はエポキシ化担体１ｇ（乾燥重量）当たり０．８３ｍｍｏｌであった。

Example 1.
Sephadex (registered trademark) G-25 Fine (manufactured by GE) based on dextran as a chromatographic carrier was washed with pure water, and its wet weight of 60 g was put into a three-necked flask with a dropping funnel, and 66 g of pure water. 60 g of epichlorohydrin was added and dispersed at 40 ° C. After 21 g of sodium hydroxide was dissolved in 36 g of pure water, this solution was dropped from the dropping funnel in 2 hours. It was made to react for 2 hours after dripping. After completion of the reaction, the carrier was separated using a glass filter and washed with pure water to obtain an epoxidized carrier. The amount of the epoxy group introduced was 0.83 mmol per 1 g (dry weight) of the epoxidized support.

  The epoxidized carrier (50 g) obtained as described above was weighed into a 300 mL three-necked flask, and then added with 100 g of 0.5 mol / L sodium thioglycolate aqueous solution (pH 8.5) and reacted at 40 ° C. for 16 hours. I let you. After completion of the reaction, the product cation exchanger was separated with a glass filter and washed with pure water. The ion exchange capacity of the cation exchanger thus produced was 0.23 meq per 1 mL (wet capacity) of the cation exchanger.

Example 2
Epoxidation was carried out in the same steps as in Example 1 except that the carrier for chromatography was changed to TOYOPEARL (registered trademark) HW-65C (manufactured by Tosoh Corporation), which is a hydrophilic vinyl polymer having a hydroxyl group. The amount of the epoxy group introduced was 0.40 mmol per 1 g (dry weight) of the epoxidized support.

  The epoxidized carrier (50 g) obtained as described above was weighed into a 300 mL three-necked flask, and then added with 100 g of 0.5 mol / L sodium thioglycolate aqueous solution (pH 8.5) and reacted at 40 ° C. for 16 hours. I let you. After completion of the reaction, the product cation exchanger was separated with a glass filter and washed with pure water. The ion exchange capacity of the cation exchanger thus produced was 0.067 meq per 1 mL (wet capacity) of the cation exchanger.

  The cation exchanger thus obtained was 7.5 mm I.V. D. The protein mixture was separated by packing in a × 75 mmL column.

Measuring device: AKTAprime (manufactured by GE),
Eluent A: 20 mmol / L phosphate buffer (pH 6.8),
Eluent B: Eluent A + 0.5 mol / L sodium chloride (pH 6.8),
Gradient: linear gradient from 100% eluent A to 100% eluent B in 60 minutes.
Flow rate: 1.0 mL / min,
Sample: Ribonuclease A (10 mg / mL),
Cytochrom C (5 mg / mL),
Lysozyme (2.5 mg / mL),
Sample injection volume: 100 microliters,
Measurement wavelength: UV 280 nm.

  FIG. 1 shows the result of the above measurement. 1 that the cation exchanger obtained in Example 2 can be used for protein separation.

Example 3 FIG.
The reaction was carried out in the same steps as in Example 2 except that sodium mercaptopropane sulfonate was used as the sulfonic acid having a thiol group. The ion exchange capacity of the cation exchanger thus produced was 0.055 meq per 1 mL (wet capacity) of the cation exchanger.

  The cation exchanger thus obtained was 7.5 mm I.V. D. The protein mixture was separated by packing in a × 75 mmL column. The measurement apparatus and measurement conditions are the same as those in Example 2.

  FIG. 2 shows the result of the above measurement. 2 that the cation exchanger of Example 3 can be used for protein separation.

Example 4
As a carrier for chromatography, TOYOPEARL (registered trademark) HW-65C (manufactured by Tosoh Corporation), which is a hydrophilic vinyl polymer having a hydroxyl group, was washed with pure water, and its wet weight was 60 g and a 0.6 mol / L NaOH aqueous solution ( 60 g), 1,4-butanediol diglycidyl ether (60 g) was added and reacted at 25 ° C. for 16 hours. After completion of the reaction, the carrier was separated using a glass filter and washed with pure water to obtain an epoxidized carrier. The amount of the epoxy group introduced was 0.22 mmol per 1 g (dry weight) of the epoxidized support.

  Carboxylic acid groups were introduced into the epoxidized carrier (50 g) obtained as described above under the same conditions as in Example 2. The ion exchange capacity of the produced cation exchanger was 0.040 meq per 1 mL (wet volume) of the cation exchanger.

Embodiment 5 FIG.
As a carrier for chromatography, 10 g of Wakogel TMC-300 (manufactured by Wako Pure Chemical Industries, Ltd.), which is silica gel, was washed with pure water and then added to a 300 mL three-necked flask. 95 g of pure water and 5 g of 3-glycidoxypropyltrimethoxysilane were added to adjust the pH to 6.0, followed by reaction at 90 ° C. for 30 minutes. After completion of the reaction, the carrier was separated using a glass filter and washed with pure water to obtain an epoxidized carrier. The amount of the epoxy group introduced was 0.40 mmol per 1 g (dry weight) of the epoxidized carrier.

  The epoxidized carrier (10 g) obtained as described above was weighed into a 100 mL three-necked flask, and then 1.0 g / L sodium thioglycolate aqueous solution (pH 8.0) and 50 g were added, and the mixture was added at 25 ° C. for 16 hours. Reacted. After completion of the reaction, the product cation exchanger was separated with a glass filter and washed with pure water. The ion exchange capacity of the cation exchanger thus produced was 0.19 meq per 1 mL (wet capacity) of the cation exchanger.

FIG. 3 is a diagram showing a liquid chromatography chart of protein separation performed in Example 2. FIG. 4 is a diagram showing a liquid chromatography chart of protein separation performed in Example 3.

Claims (13)

A cation exchanger having a thiol group-containing carboxylic acid group or a thiol group-containing sulfonic acid group as an ion exchange group on the surface of a chromatography carrier. The amount of a carboxylic acid group containing a thiol group or a sulfonic acid group containing a thiol group is in the range of 0.005 to 0.5 meq per 1 mL (wet capacity) of the cation exchanger. The cation exchanger according to 1. A carboxylic acid group containing a thiol group or a sulfonic acid group containing a thiol group is represented by the following formula (1):

(In the above formula, R represents an alkylene group having 1 to 3 carbon atoms, and A represents a carboxylic acid group, a sulfonic acid group, or a salt thereof.)
The cation exchanger according to claim 1 or 2, which is an ion exchange group represented by the following formula. The cation exchanger according to any one of claims 1 to 3, wherein the chromatography carrier is a natural polymer carrier, a synthetic polymer carrier, or an inorganic carrier. The cation exchanger according to any one of claims 1 to 4, wherein the chromatographic support is in the form of spherical particles, non-spherical particles, a membrane, or a monolith (continuum). The method for producing a cation exchanger according to any one of claims 1 to 5, comprising the following steps.
(1) a first step of introducing an epoxy group into a chromatographic support; and
(2) A second step of reacting the epoxy group with a compound having a thiol group and a carboxylic acid group in the molecule, or a compound having a thiol group and a sulfonic acid group in the molecule. The cation exchanger according to claim 6, wherein in the first step, an epoxy group is introduced into the chromatographic support using epichlorohydrin, a polyfunctional epoxy compound or an epoxy group-containing silane treating agent. Method. Polyfunctional epoxy compound is ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, glycerol diglycidyl ether, glycerol The method for producing a cation exchanger according to claim 7, which is one or more compounds selected from the group consisting of triglycidyl ether and sorbitol polyglycidyl ether. The epoxy group-containing silane treating agent is one or two selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, and 3-glycidoxypropyltriethoxysilane The method for producing a cation exchanger according to claim 7, which is the above compound. In the second step, a compound having a thiol group and a carboxylic acid group in the molecule, or a compound having a thiol group and a sulfonic acid group in the molecule is represented by the following formula (2):

(In the above formula, R represents an alkylene group having 1 to 3 carbon atoms, and A represents a carboxylic acid group, a sulfonic acid group, or a salt thereof.)
The method for producing a cation exchanger according to any one of claims 6 to 9, wherein the compound is one or more compounds selected from the group consisting of: The compound represented by formula (2) is one or more compounds selected from the group consisting of mercaptoacetic acid, mercaptopropionic acid, mercaptoethanesulfonic acid, mercaptopropanesulfonic acid, and salts thereof. The method for producing a cation exchanger according to claim 10. A chromatographic separation agent comprising the cation exchanger according to any one of claims 1 to 4. A packed column for chromatography comprising the cation exchanger according to any one of claims 1 to 4 packed in a chromatography column.

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