JP6056230B2 - Cation exchanger for liquid chromatography - Google Patents

Description

The present invention relates to a cation exchanger characterized in that a polyvinyl sulfonic acid copolymer is fixed on the surface of substantially non-porous particles.

  Cation exchangers for liquid chromatography are (1) highly resistant to target substances such as proteins (hereinafter sometimes simply referred to as “targets”) in order to be less susceptible to the influence of the salt concentration contained in the sample. (2) have sufficient target adsorption capacity to maintain the separation performance of trace components, (3) have high resolution to improve separation and analysis accuracy, (4) separation・ Can be used at high operating flow rates to reduce analysis time (has mechanical strength to withstand high pressures associated with high operating flow rates), (5) Does not cause a decrease in operating flow rates (low operating pressure) And (6) It is required that the interaction between the exchanger and the subject is small except for cation exchange.

The cation exchanger for liquid chromatography is obtained by introducing a cation exchange group (ligand) into particles constituting the exchanger. For the purpose of improving the above (3), it has been proposed to use non-porous particles having no intra-particle diffusion (Non-Patent Document 1) or to use particles having a particle size of about 10 μm. Yes. In recent years, it has been proposed to use particles having a particle diameter of 5 μm or less as the particle diameter is further reduced. Further, it is necessary to apply a higher operating pressure in order to prevent the separation / analysis time from being extended due to the use of such fine particles, and the mechanical strength (4) required for the particles is about 100 MPa. On the other hand, as the cation exchange group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, and the like have been conventionally known. Among them, the sulfonic acid group has a small pKa, and when the pH of the eluent is lowered for the purpose of ion-dissociating the target, the target can be stably maintained even if the pH fluctuates. As a method of introducing sulfonic acid groups into particles, a method of allowing sulfur trioxide, chlorosulfuric acid or concentrated sulfuric acid to act on styrene copolymer particles, and introducing epoxy groups or allyl groups into particles Further, a method in which sulfurous acid is allowed to act and a method in which propane sultone, butane sultone or sulfur trisulfide is allowed to act on the particles having a hydroxyl group are used.

Non-porous particles (synthetic polymer particles) having a synthetic polymer as a skeleton can achieve (4) together with (3) above. Therefore, it is conceivable to apply a conventional method for introducing a cation exchange group to a non-porous polymer particle, but a synthetic polymer particle generally has a functional group for introducing a cation exchange group to the particle surface later. In order to increase the mechanical strength of the particles, the polyfunctional unsaturated crosslinkable monomer is used in order to increase the mechanical strength of the particles. When a large amount is used, the amount of functional groups on the particle surface decreases, and a sufficient amount of cation exchange groups cannot be introduced on the particle surface, resulting in a decrease in the cation exchange group density on the particle surface. As a result, the holding power of the target becomes insufficient (the above (1) cannot be achieved), and the adsorption capacity for the target, particularly biopolymers such as proteins, is increased due to the small surface area due to the non-porous property. The above (2) is impaired, and the target elution peak is broadened.
Therefore, for the purpose of improving the above (1) and (2), a method of grafting and fixing a vinyl polymer chain having a cation exchange capacity on the particle surface (Patent Documents 1 to 4), a water-soluble polymer in the pores There has been proposed a method (Patent Document 5) for immobilizing the above. However, when the polymer chain is fixed to the particle surface by grafting, the operating pressure becomes high, and the operating flow rate is lowered against the above (5). Also, the method of fixing the water-soluble polymer in the pores cannot be applied to non-porous particles.

  For the purpose of improving the above (2) and (5), a copolymer of a monomer having a sulfonic acid group and a monomer having a carboxylic acid group, and a polymer having a functional group capable of reacting with the carboxylic acid group, A method of fixing to the surface by partial cross-linking has also been proposed (Patent Document 6). However, in this method, when applied to fine particles having a particle diameter of about 5 μm, the operation flow rate is lowered against the requirement (5). In order to improve (1) and (6) by this method, the polymer formed by partial crosslinking has a small ratio of main chains and side chains other than sulfonic acid groups, and has a high cation exchange group density, and In addition, it is also required that an interaction other than cation exchange (for example, a hydrophobic interaction) is difficult to occur with the object.

  In the above method, use of a vinyl sulfonic acid polymer (Patent Documents 6 to 10) is considered as a copolymer of a monomer having a sulfonic acid group and a monomer having a carboxylic acid group that can improve (1) and (6). However, in the case of a homopolymer of vinyl sulfonic acid, the method for fixing it to the particle surface is limited. For example, Patent Document 11 describes that vinyl sulfonic acid is introduced by reacting with a hydroxyl group on the particle surface, but only one vinyl sulfonic acid is introduced per one hydroxyl group on the particle surface, Said (2) will be impaired. Moreover, it is difficult to produce a vinyl sulfonic acid having a high molecular weight (Non-patent Documents 2 and 12), and as a molecular weight of 20,000 or more, Patent Document 13 is slightly disclosed as a polyvinyl sulfonic acid polymer (homopolymer). However, since it is a homopolymer, it cannot be applied to the method of fixing to the surface of the particle as described above by partial crosslinking.

US Pat. No. 5,453,186 U.S. Pat. No. 3,723,306 Japanese Patent Publication No.57-23694 Japanese Patent Publication No.57-58373 JP 2008-232764 A JP 2002-306974 A Special table 2003-512171 gazette Japanese Patent No. 3474567 JP 2006-519273 A Japanese Patent Laid-Open No. 10-60142 Special table 2009-506340 JP-A-7-173226 JP 2009-227965 A

High-performance ion-exchange chromatography of proteins on non-porous ionexchangers, Journal of Chromatography A Volume 398, 1987, Page 327-334 J. et al. Am. Chem. Soc., 76, 6399-6401 (1954)

The object of the present invention is to meet the demands of (1) to (6), have a high holding power for the target, have a sufficient target adsorption capacity, have a high resolution, and have a sufficient mechanical strength. An object of the present invention is to provide a cation exchanger for liquid chromatography having a low operating pressure and small interaction other than cation exchange.

  The cation exchanger for liquid chromatography of the present invention made to solve the above-mentioned problems is characterized in that a polyvinyl sulfonic acid copolymer is fixed on the surface of non-porous particles. Hereinafter, the present invention will be described in detail.

  The cation exchanger of the present invention is based on non-porous particles for improving the separation ability of (3) above. The term “non-porous” in the present invention includes not only particles having no micropores but also particles having pores of a size into which an object to be separated using a cation exchanger cannot enter. It is. A method for controlling the pore size of the particles is conventionally known, for example, US Pat. No. 4,382,124.

  For the purpose of improving (3), the non-porous particles preferably have a particle size of 5 μm or less. For example, inorganic substrates (for example, silica, zirconia, alumina, etc.) and organic substrates (for example, crosslinked polysaccharides) Or a crosslinked vinyl monomer such as acrylamide, acrylic steal, or styrene). These particles include monofunctional vinyl monomers (glycidyl methacrylate, vinyl benzyl glycidyl ether, etc.) and polyfunctional vinyl monomers (ethylene glycol diester) as disclosed in, for example, JP-B-58-58026 and JP-A-53-90991. (Methacrylate, polyethylene glycol dimethacrylate, glycene polymethacrylate, divinylbenzene, etc.) produced by suspension polymerization of a mixed solution, for example, by seed polymerization as described in JP-A-2001-2716 Can be manufactured. When non-porous particles of an organic substrate are used, a copolymer of a monofunctional vinyl monomer and a polyfunctional vinyl monomer, particularly a structural unit derived from a monofunctional vinyl monomer is represented by the following formula (1). (In the formula, R1 represents a hydrogen atom or CH3. R2 represents a polyvinylsulfonic acid copolymer introduced via a polyoxyalkylene bond or directly). For the purpose of improving (4), the polyfunctional vinyl monomer content is preferably 5% by weight or more. For the purposes of improving (1) to (3), a sufficient amount of polyvinyl sulfonic acid copolymer In order to secure a functional group for fixing, the ratio of the polyfunctional vinyl monomer is preferably 50% by weight or less. For example, in the case of a copolymer of a monofunctional vinyl monomer having an epoxy group, such as glycidyl methacrylate, and a polyfunctional vinyl monomer, the epoxy group is directly used, the epoxy group is hydrolyzed and the ring is opened to form a water-soluble polyvalent monomer. Hydroxylation can be performed by hydrophilizing with alcohol or the like, and if it is a particle having a hydroxyl group, for example, a polyfunctional epoxy compound such as epichlorohydrin, ethylene glycol diglycidyl ether or the like can be obtained by a known method. Can be introduced.

本発明のカチオン交換体は、カチオン交換基としてのスルホン酸基を有するポリビニルスルホン酸共重合体を固定したものであるが、ポリビニルスルホン酸共重合体としては、特に上記（１）〜（３）及び（５）を改善するために、その分子量が５０００から４００００の範囲のものが好ましい。分子量が小さいと（１）から（３）の改善効果が小さく、分子量が大すぎると操作圧が上昇して（５）の改善効果が小さくなるからである。

The cation exchanger of the present invention is obtained by fixing a polyvinyl sulfonic acid copolymer having a sulfonic acid group as a cation exchange group. As the polyvinyl sulfonic acid copolymer, the above-mentioned (1) to (3) are particularly preferable. And in order to improve (5), the thing whose molecular weight is the range of 5000 to 40000 is preferable. This is because when the molecular weight is small, the improvement effects (1) to (3) are small, and when the molecular weight is too large, the operating pressure increases and the improvement effect (5) becomes small.

  The polyvinyl sulfonic acid copolymer of the present invention can be obtained by copolymerizing vinyl sulfonic acid and a vinyl monomer having a functional group capable of binding to a functional group on the particle surface for immobilization on the particle. For example, when an epoxy group or a carboxyl group is present on the particle surface, a vinyl monomer having a hydroxyl group, an amino group, a thiol group or the like that can be bonded to these may be copolymerized with vinyl sulfonic acid. Among them, vinyl monomers having a hydroxyl group that can be easily copolymerized with vinyl sulfonic acid are preferable. Specifically, 2-hydroxyethyl (meth) acrylic acid ester, polyethylene glycol (meth) acrylic acid ester, and glycerol (meth) acrylic are used. Examples include (meth) acrylic acid esters of esters and other polyol compounds, N-hydroxymethyl (meth) acrylamide, N-hydroxyethyl (meth) acrylamide, N- (tris (hydroxymethyl) methyl) (meth) acrylamide, and the like. Can do.

  The structural unit of vinyl sulfonic acid constituting the polyvinyl sulfonic acid copolymer is preferably in the range of 99.5 to 50 mol% for the purpose of improving (1) to (3) and (6). The amount of the polyvinyl sulfonic acid copolymer immobilized on the particle surface is preferably adjusted in the range where the cation exchange capacity of the cation exchanger is 5 to 30 meq / L for the purpose of improving (5). By controlling the amount of such immobilization, a cation exchanger having low operating pressure can be realized.

  As a method for fixing the polyvinyl sulfonic acid copolymer to the particle surface, for example, when an epoxy group is present on the particle surface, a vinyl sulfonic acid and a vinyl monomer having a hydroxyl group are copolymerized to form a polyvinyl sulfonic acid copolymer. For example, the particles can be dispersed in an aqueous solution in which the copolymer is dissolved to change the solution from neutral to alkaline. In this case, the concentration of 5 to 50% is selected as the usage amount of the polyvinyl sulfonic acid copolymer, and 0.1 to 0.5 molar sodium hydroxide is added to the solution at 30 to 60 degrees for 1 to 24 hours. A specific example is heating. Further, for example, when an epoxy group or a carboxyl group is present on the particle surface, a vinyl sulfonic acid and a vinyl monomer having a hydroxyl group are copolymerized to produce a polyvinyl sulfonic acid copolymer, and the copolymer is used as a volatile solvent. For example, the particles having an epoxy group or a carboxyl group are dispersed in a dissolved state and the solvent is distilled off, followed by drying and heating at 90 to 150 degrees for 1 to 5 hours. In this case, the amount of the polyvinyl sulfonic acid copolymer used can be specifically exemplified as 5% by weight or more of the particles by dry weight.

  The cation exchanger of the present invention can be used for separation and purification of biological samples such as various proteins containing glycohemoglobin by packing in a column for liquid chromatography, for example.

  Since the cation exchanger of the present invention is obtained by fixing cation exchange groups to non-porous particles, (3) can be improved, particularly when fine particles of 5 μm or less are used. it can. In the present invention, when a vinyl monomer copolymer (crosslinked product) is used, mechanical strength that can withstand 100 MPa can be realized, and (4) can be improved. In addition, the cation exchanger of the present invention uses (1) to (3) and (6) because a sulfonic acid group is used as the cation exchange group and a sufficient amount of the cation exchange group can be fixed to the particles. In addition, (5) can be improved by controlling the fixed amount.

  As described above, the cation exchanger of the present invention (1) has a high holding power with respect to the target in order to make it less susceptible to the influence of the salt concentration contained in the sample, and (2) has the ability to separate trace components. Sufficient target adsorption capacity to maintain, (3) High resolution to improve separation / analysis accuracy, (4) High operating flow rate for fast separation / analysis time (fast (It has mechanical strength to withstand the high pressure associated with the operation flow rate), (5) Does not cause a decrease in the operation flow rate (is a low operation pressure), and (6) Cations exchange between the exchanger and the object It is a cation exchanger that can meet the conventional requirement that the interaction other than is small (interaction other than small cation exchange).

It is a figure which shows the result of Example 1, the comparative example 1, and the comparative example 2. FIG. 1 is a diagram showing a chromatogram of a protein (glycohemoglobin) in Example 1. FIG.

Hereinafter, the cation exchanger of the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
[Creation of non-porous substrate]
Non-porous particles were produced by the method (seed polymerization method) described in Japanese Patent Publication No. 2001-2716. 20 g of benzyl methacrylate and 0.95 g of 2-ethylhexyl mercaptoacetate were mixed in a 500 mL three-necked flask, and 200 g of ion-exchanged water was added. A magnetic stirrer was put in, attached to an oil bath set at 85 degrees, a nitrogen inlet tube was installed, and stirring was performed at 150 rpm. Separately, 0.6 g of potassium persulfate and 20 g of ion-exchanged water were measured and dissolved in a 50 mL container. After 30 minutes, an aqueous potassium persulfate solution was charged with a syringe from a rubber stopper installed in a three-necked flask. Soap-free emulsion polymerization was carried out at a rotation speed of 300 rpm. After continuing the polymerization for 2 hours, the agglomerate was removed and the seed solution was recovered. The solid content of the seed solution was 6.98%, and the particle size was 0.39 μm as measured by an electron microscope.
2,3 Epoxypropyl methacrylate 64 g, Ethylene dimethacrylate 16 g, 2,2′-azobis (2,4-dimethylvaleronitrile) (trade name V-65, manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 g and dodecyl 0.2 g of sodium sulfate was weighed into a 300 mL flask, a stirring bar was added, and the mixture was mixed with a magnetic stirrer. 100 mL of ion-exchanged water was added and emulsified with an ultrasonic homogenizer while stirring with a magnetic stirrer. 10.90 g of seed solution prepared as described above (solid content 0.760 g) and 50 mL of 4% aqueous polyvinyl alcohol solution were added, and the mixture was stirred well for 1 minute and allowed to stand. After standing at room temperature for 30 minutes, the mixture was allowed to stand in a water bath set at 60 degrees and polymerized for 2 hours. The obtained polymerization solution was filtered through a glass filter, and washed with warm water, acetone, and warm water in this order to obtain non-porous particles.

  The obtained non-porous particles were put into a 500 mL separable flask, 300 mL of ion-exchanged water was added, placed in an oil bath set at 90 degrees, and heated for 24 hours with stirring to hydrolyze the epoxy group. . The non-porous particles after hydrolyzing the epoxy groups were particles having a uniform particle diameter of 1.8 μm as observed with an electron microscope.

100 g of the obtained non-porous particles (water suction dry), 103 g of epichlorohydrin and 100 g of ion-exchanged water were placed in a 500 mL separable flask, and placed in a water bath set at 45 degrees and stirred. Separately, 88 g of 48% aqueous sodium hydroxide solution was weighed, placed in a disposable syringe, placed in a syringe pump, and charged into a separable flask with stirring at a rate of 0.5 mL / min. After the addition, the reaction was continued for 2 hours, and the reaction product was filtered through a glass filter, washed with water and acetone in this order, and dried by ventilation to obtain epoxidized non-porous particles.
Example 1
Sodium vinyl sulfonate (hereinafter abbreviated as “VSNa”) 25% aqueous solution (manufactured by Tokyo Chemical Industry) 10.0 g, 2-hydroxyethyl methacrylate (hereinafter abbreviated as “HEMA”) 0.216 g (ratio with VSNa) 10 mol%) and 0.026 g of potassium persulfate were placed in a 10 mL glass container and polymerized in a water bath set at 65 degrees for 5 hours. A small amount of the polymerization solution was sampled, and the molecular weight of the copolymer was measured by gel permeation chromatography under the following conditions. As a result, the weight average molecular weight was 11700, and the polymerization rate of vinyl sulfonic acid was 94%.

Conditions for gel permeation chromatography Column: TSKgel (registered trademark) G4000PW _XL and G2500PW _XL
Eluent: 0.1 mol / L Sodium nitrate Flow rate: 1.0 mL / min Detection: Refractometer Sample: 10-fold dilution, 5 uL injection The above polymerization solution is purified by reprecipitation with 200 mL of methanol, and dried by ventilation under a nitrogen stream. A white powder of vinyl sulfonic acid copolymer was obtained.
To 5 g (dry weight) of epoxidized non-porous particles, 1.0 g of vinyl sulfonic acid copolymer (20 wt% per dry weight of epoxidized non-porous particles) and 20 mL of pure water are placed in a 100 mL eggplant-shaped flask. It was well dispersed in an ultrasonic bath. An eggplant-shaped flask was attached to the evaporator, and water was distilled off under reduced pressure. The eggplant-shaped flask was placed in a warm air superheater set at 150 degrees and heated for 1 hour to fix the vinyl sulfonic acid copolymer on the surface of the epoxidized non-porous particles. After cooling the eggplant-shaped flask, water was added, well dispersed, and washed with water, 0.1N hydrochloric acid, 0.1N sodium hydroxide, and water in this order using a glass filter to obtain a cation exchanger. The obtained cation exchanger was loaded into a liquid chromatography column having a diameter of 4.6 mm and a length of 50 mm as a dispersion with 10% ion-exchanged water as a dispersion. The obtained column was subjected to liquid chromatography under the following conditions to measure protein separation performance. The operating pressure of the cation exchanger was the operating pressure when the eluent A was fed at a flow rate of 0.5 mL / min. The cation exchange capacity of the cation exchanger is such that a 25 mmol / L citric acid solution is passed through the column for 10 minutes at a flow rate of 0.4 mL / min, followed by elution of ion-exchanged water at a flow rate of 0.4 mL / min. The cation exchanger extracted from the column was dispersed with a 0.5 mol / L sodium chloride aqueous solution in a 200 mL beaker, and then with a 0.01 mol / L sodium hydroxide aqueous solution. Measurement was performed by automatic titration with pH 7 as the end point.

Liquid chromatography conditions Eluent A: 10 mmol / L sodium phosphate buffer (pH 6.5)
Eluent B: 10 mmol / L sodium phosphate buffer (pH 6.5) containing 0.5 mol / L NaCl
Gradient: 5-100% eluent B linear gradient, 9.5 minutes Flow rate: 0.4 mL / min Target: α-chymotrypsinogen A (1 g / L)
Ribonuclease A (1 g / L)
Lysotium (1 g / L)
Injection volume: 3, 5, 7, 10, 20, 30, and 50 μL
The chromatogram when 10 μL of the target is injected is as shown in FIG. 1. The three types of target are well separated, and the cation exchanger of the present invention has sufficient holding power for all the targets. You can see that The operating pressure of the cation exchanger was as low as 26.8 MPa, and the cation exchange capacity was 16.6 meq per liter of cation exchanger.
Moreover, the chromatogram at the time of analyzing glycohemoglobin under the following conditions is shown in FIG. It can be seen that the cation exchanger of the present invention shows good separation even when glycated hemoglobin is targeted.
Analysis conditions for glycohemoglobin Eluent A: 20 mmol / L sodium phosphate buffer (pH 6.3)
Eluent B: 20 mmol / L sodium phosphate buffer (pH 6.3) containing 0.2 mol / L NaClO4
Gradient: 0 to 100% eluent B linear gradient, 10 minutes Flow rate: 0.5 mL / min Sample: HbA1c control (1.0 g / L) (manufactured by Tosoh Corporation)
Injection volume: 5 μL
For the cation exchanger produced in Example 1, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected It is shown in 1.
Example 2
In producing a vinyl sulfonic acid copolymer, instead of HEMA, 1 mol% of N-hydroxyethylacrylamide (hereinafter abbreviated as “HEAA”) (manufactured by Sigma) in a ratio with VSNa was used and obtained. In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same operation as in Example 1 was performed except that the amount used was 5% by weight per dry weight of the epoxidized non-porous particles. A cation exchanger was obtained. Regarding the cation exchanger produced in Example 2, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.
Example 3
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 2 was carried out except that the amount used was 10% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 3, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 4
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 2 was performed except that the amount used was 20% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 4, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.
Example 5
When the amount of HEAA used is 5 mol% in proportion to VSNa, and the obtained vinyl sulfonic acid copolymer is fixed to the epoxidized nonporous particles, the amount used is determined by drying the epoxidized nonporous particles. A cation exchanger was obtained in the same manner as in Example 1 except that the amount was 5% by weight. Regarding the cation exchanger produced in Example 5, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 6
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 5 was carried out except that the amount used was 10 wt% per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 6, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected It is shown in 1.
Example 7
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 5 was performed except that the amount used was 20% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 6, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected It is shown in 1.
Example 8
When the amount of HEAA used is 10 mol% in proportion to VSNa, and the obtained vinyl sulfonic acid copolymer is fixed to the epoxidized nonporous particles, the amount used is determined by drying the epoxidized nonporous particles. A cation exchanger was obtained in the same manner as in Example 1 except that the amount was 5% by weight. Regarding the cation exchanger produced in Example 8, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A is injected are shown. It is shown in 1.
Example 9
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 8 was carried out except that the amount used was 10% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 9, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 10
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 8 was performed except that the amount used was 20% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. For the cation exchanger produced in Example 10, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 11
When the amount of HEAA used is 25 mol% in proportion to VSNa, and the obtained vinyl sulfonic acid copolymer is fixed to the epoxidized nonporous particles, the amount used is determined by drying the epoxidized nonporous particles. A cation exchanger was obtained in the same manner as in Example 1 except that the amount was 3% by weight per weight. Regarding the cation exchanger produced in Example 11, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.

Example 12
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 11 was carried out except that the amount used was 5 wt% per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 12, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.

Example 13
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 11 was performed except that the amount used was 10% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 13, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected It is shown in 1.

Example 14
In producing a vinyl sulfonic acid copolymer, 0.5 mol% of HEMA was used in a ratio with VSNa, and in fixing the obtained vinyl sulfonic acid copolymer to epoxidized non-porous particles, A cation exchanger was obtained in the same manner as in Example 1 except that the amount used was 5 wt% per dry weight of the epoxidized non-porous particles. Regarding the cation exchanger produced in Example 14, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 15
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 14 was performed except that the amount used was 10% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 15, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.

Example 16
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 14 was carried out except that the amount used was 20% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 16, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.

Example 17
In producing a vinyl sulfonic acid copolymer, 5 mol% of HEMA was used in a ratio with VSNa, and the obtained vinyl sulfonic acid copolymer was used for fixing to epoxidized non-porous particles. A cation exchanger was obtained in the same manner as in Example 1 except that the amount was 5% by weight per dry weight of the epoxidized non-porous particles. Regarding the cation exchanger produced in Example 17, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.

Example 18
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 17 was carried out except that the amount used was 10% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 18, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.

Example 19
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 17 was carried out except that the amount used was 20% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 19, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.

Example 20
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the amount used was 20% by weight per dry weight of the epoxidized non-porous particles, and the heating time was 5 hours. The same operation was performed to obtain a cation exchanger. Regarding the cation exchanger produced in Example 20, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 21
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 1 was carried out except that the amount used was 5 wt% per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 21, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Example 22
In fixing the vinyl sulfonic acid copolymer to the epoxidized non-porous particles, the same procedure as in Example 1 was carried out except that the amount used was 10% by weight per dry weight of the epoxidized non-porous particles. An exchange was obtained. Regarding the cation exchanger produced in Example 22, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown. It is shown in 1.
Example 23
In Example 1, 20.0 g of 25% aqueous solution of VSNa, 0.5 g of N- [Tris (hydroxymethyl) methyl] acrylamide (Sigma, Tris-AA) (7.5 mol% with respect to VSNa) in a 30 mL glass container A cation exchanger was obtained using a vinyl sulfonic acid copolymer obtained by performing the same operation except that it was placed in direct sunlight for 3 days. Regarding the cation exchanger produced in Example 23, the molecular weight of the immobilized vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-value width when 10 μL of α-chymotrypsinogen A was injected are shown in the table. It is shown in 1.
Comparative Example 1
Particles were taken out from a commercially available column (TSKgel (registered trademark) SP-NPR), packed into the same 4.6 mm inner diameter × 50 mm length column used in Example 1, and under the same conditions as in Example 1. Protein separation was measured. The operating pressure was a low operating pressure of 6.2 MPa, but the holding power was weak and the width of the elution peak was broad. The results are shown in FIG. 1, and the peak half-value width when 10 μL of operating pressure and α-chymotrypsinogen A are injected are shown in Table 1.
Comparative Example 2
Particles were taken out from a commercially available column (TSKgel (registered trademark) SP-STAT), packed into the same 4.6 mm inner diameter × 50 mm length column used in Example 1, and under the same conditions as in Example 1. Protein separation was measured. The operating pressure was as low as 1.6 MPa, but the holding power was weak and the width of the elution peak was broad. The results are shown in FIG. 1, and the peak half-value width when 10 μL of operating pressure and α-chymotrypsinogen A are injected are shown in Table 1.

Comparative Example 3
In producing the vinyl sulfonic acid copolymer, 30 mol% of HEAA in proportion to VSNa is used instead of HEMA, and the obtained vinyl sulfonic acid copolymer is fixed to the epoxidized non-porous particles. At that time, a cation exchanger was obtained in the same manner as in Example 1 except that the amount used was 20% by weight per dry weight of the epoxidized non-porous particles. Regarding the cation exchanger produced in Comparative Example 3, the molecular weight of the fixed vinyl sulfonic acid copolymer, the polymerization rate of VSNa, the cation exchange capacity, the operating pressure, and the peak half-width when 10 μL of α-chymotrypsinogen A was injected are shown. As shown in Fig. 1, the operation pressure was high and measurement was difficult.

Comparative Example 4
4 g of the epoxidized non-porous particles used in Example 1, 3 g of dextran (molecular weight 60000), 7 g of ion-exchanged water and 60 μL of sodium hydroxide were placed in a 40 mL capacity centrifuge tube and shaken in a water bath set at 50 degrees for 16 hours. did. The resulting reaction product was centrifuged to remove the supernatant, and 10 mL of ion exchange water was added and dispersed, followed by further centrifugation. The operation consisting of removal of the supernatant, addition of ion exchange water and centrifugation was repeated three times in total. After removing the supernatant, 4.3 g of ion exchange water, 1.2 g of 1,3 propane sultone and 48% hydroxylation were added to the centrifuge tube. A 1.5 g sodium aqueous solution was added and stirred. The reaction solution was filtered through a glass filter and washed with water, 0.1N hydrochloric acid, 0.1N sodium hydroxide and water in this order to obtain a cation exchanger.

  The obtained cation exchanger was packed in the same 4.6 mm inner diameter × 50 mm length column used in Example 1, and protein separation was attempted under the same conditions as in Example 1. It was 60 MPa or more, and it was difficult to carry out the separation operation.

Claims (4)

It is composed of a copolymer of a monofunctional vinyl monomer and a polyfunctional vinyl monomer, and a polyvinyl sulfonic acid copolymer is fixed on the surface of non-porous particles having a particle size of 5 μm or less. Cation exchanger for liquid chromatography.   The cation exchanger for liquid chromatography according to claim 1, wherein the molecular weight of the polyvinyl sulfonic acid copolymer is from 5,000 to 40,000. The cation exchanger for liquid chromatography according to claim 1 or 2 , wherein the introduced amount of the polyvinyl sulfonic acid copolymer is 30% by weight or less of the non-porous particles. A column for liquid chromatography comprising the cation exchanger for liquid chromatography according to any one of claims 1 to 3 .

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