Strong acid cation exchange chromatography material and preparation method thereof

Abstract

The invention discloses a strongly acidic cation exchange chromatography material and a preparation method thereof. The chromatography material takes microspheres as a matrix material, and takes halogenated alkyl sodium benzenesulfonate and hydroxyl-containing halogenated alkyl sodium sulfonate as functional groups. The preparation method comprises the following steps: epoxy activation: dispersing microspheres in a continuous phase, adding a reducing agent, an alkaline solution and a grafting reagent, and reacting to obtain epoxy activated microspheres; preliminary sulfonation: enabling the epoxy activated microspheres to react with hydroxyl-containing halogenated alkyl sodium sulfonate serving as a first sulfonating agent under an alkaline condition to obtain preliminarily sulfonated microspheres; deep sulfonation: enabling the preliminarily sulfonated microspheres to react with halogenated sodium alkyl benzene sulfonate serving as a second sulfonating agent to obtain a target product. The strongly acidic cation exchange chromatography material provided by the invention can achieve the effects of high protein adsorption capacity and high ion exchange site density.

Description

Technical Field

[0001] The present invention relates to a liquid chromatography separation medium, in particular to a strongly acidic cation exchange chromatography material and a preparation method thereof. Background Art

[0002] Ion exchange chromatography is a method for separation and purification by the ion exchange process between charged solute molecules and exchangeable ions in the ion exchange chromatography medium. Therefore, it has also become an important method for the separation and purification of proteins, nucleic acids, polypeptides and most fermentation products. The ion exchange process includes the following steps: diffusion of ions in the solution on the surface of the chromatography material, diffusion of ions in the solution to the charged part of the pore, ion exchange process, diffusion of the exchanged ions through the pores to the surface of the chromatography medium, and diffusion of the exchanged ions from the surface to the solution. Among them, the pore diffusion rate is limited by the ions and the pore size. Therefore, the three-dimensional network skeleton of the chromatography medium and the number of exchange sites play a crucial role in the chromatography effect.

[0003] According to the nature of the charge carried by the ion exchange functional group, the chromatography medium can be divided into two categories: cation exchange chromatography materials and anion exchange chromatography materials. Further, according to the ionization strength of the functional group, the cation exchange chromatography materials can be divided into strong acid type and weak acid type. Among them, the strong acid type materials usually contain sulfonic groups, while the weak acid type materials often contain phosphoric groups or carboxylic groups, etc. The preparation of cation exchange chromatography materials is achieved by introducing negatively charged functional groups (such as sulfonic groups, carboxyl groups or phosphoric groups) on a matrix (such as agarose, cellulose, silica gel or polymer microspheres), so as to realize the separation of positively charged substances.

[0004] In the traditional preparation process of strongly acidic cation exchange chromatography materials, the sulfonation process usually uses inorganic compounds such as concentrated sulfuric acid, sulfur trioxide or chlorosulfonic acid to prepare strongly acidic ion exchange resins through one-step sulfonation or sulfonation-hydrolysis reactions. However, this method has problems such as harsh reaction conditions, high operation risks, and a large amount of waste acid generated during the production process, posing a greater hidden danger to environmental protection and production safety. Taking strongly acidic ion exchange materials based on styrene or styrene / divinylbenzene as an example, their sulfonation process needs to be carried out under strong acidic conditions, which is not applicable to carriers with a three-dimensional network skeleton such as biological polysaccharides. For carriers such as polyglycidyl methacrylate and polysaccharide skeletons, common sulfonating agents include inorganic salts such as sodium bisulfite and sodium thiosulfate. However, the use of such sulfonating agents may disrupt the redox balance of the reaction system, resulting in the generation of by-products, thereby affecting the performance of the materials. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a strongly acidic cation exchange chromatography material with high protein adsorption capacity and high ion exchange site density.

[0006] Another object of the present invention is to provide a preparation method of a strongly acidic cation exchange chromatography material with simple operation process, reduced environmental pollution, wide application range and easy industrialization.

[0007] Technical solution: The strongly acidic cation exchange chromatography material of the present invention uses a hydroxyl-containing microsphere as a matrix, grafts an epoxide spacer arm at the hydroxyl end, grafts a hydroxyl-containing sodium haloalkylsulfonate at the spacer arm end, and grafts sodium haloalkylbenzenesulfonate at one end of the spacer arm and / or sodium haloalkylsulfonate.

[0008] Preferably, the chemical formula of the chromatography material is as follows:

[0009]

[0010] Wherein, R1, R2, R3, R4, and R5 represent alkyl groups.

[0011] Preferably, the microsphere is a porous microsphere or a non-porous microsphere; more preferably, it is one or more of an agarose microsphere, a chitosan microsphere, a polystyrene-divinylbenzene microsphere with a hydroxyl group on the hydrophilic modified surface, and a poly(methyl methacrylate glycidyl ether) microsphere modified by epoxy hydrolysis.

[0012] The preparation method of the chromatography material of the present invention includes the following steps:

[0013] (1) Epoxy activation: Dispersing the microspheres in the continuous phase, adding a reducing agent, an alkaline solution, and an epoxy grafting reagent, and reacting to obtain epoxy-activated microspheres;

[0014] (2) Preliminary sulfonation: Reacting the epoxy-activated microspheres with a hydroxyl-containing sodium haloalkylsulfonate as the first sulfonating agent under alkaline conditions to obtain preliminarily sulfonated microspheres;

[0015] (3) Deep sulfonation: Reacting the preliminarily sulfonated microspheres with sodium haloalkylbenzenesulfonate as the second sulfonating agent under alkaline conditions to obtain the target product.

[0016] Preferably, the continuous phase in step (1) is an N,N-dimethylformamide aqueous solution.

[0017] Preferably, the particle size of the microspheres in step (1) is 2-200 μm, and the pore size is

[0018] Preferably, the microspheres in step (1) are porous microspheres or non-porous microspheres; more preferably, they are one or more of an agarose microsphere, a chitosan microsphere, a polystyrene-divinylbenzene microsphere with a hydroxyl group on the hydrophilic modified surface, and a poly(methyl methacrylate glycidyl ether) microsphere modified by epoxy hydrolysis; even more preferably, it is an agarose microsphere.

[0019] Preferably, the reducing agent in step (1) is sodium borohydride; the alkaline solution is an aqueous solution of 35wt% - 50wt% NaOH or KOH.

[0020] Preferably, the epoxy grafting reagent in step (1) is a diepoxy substance or a monoepoxy substance; more preferably, it is one or more of 1,4-butanediol diglycidyl ether, dipropylene glycol diglycidyl ether, and epichlorohydrin.

[0021] Preferably, the reaction time in step (1) is 8 - 14 hours, and the reaction temperature is 45 - 60 °C;

[0022] Preferably, the general structural formula of the hydroxy-containing haloalkylsulfonate sodium in step (2) is

[0023]

[0024] wherein R1 and R2 represent alkyl groups, and X represents a halogen; more preferably, it is one or more of sodium 3-chloro-2-hydroxypropane sulfonate and sodium 4-chloro-1-hydroxybutane sulfonate.

[0025] Preferably, the reaction time in step (2) is 8 - 14 hours, and the reaction temperature is 45 - 60 °C;

[0026] Preferably, the general structural formula of the haloalkylbenzenesulfonate sodium in step (3) is

[0027] Y-R3-PhSO3Na,

[0028] R3 represents an alkyl group, and Y represents a halogen; more preferably, it is one or more of sodium 4-(bromomethyl)benzenesulfonate and sodium 4-(2-bromoethyl)benzenesulfonate.

[0029] Principle of the invention: By increasing the length of the connecting chain on the microsphere carrier, the protein adsorption capacity of the ion exchange chromatography material can be improved to a certain extent. The microsphere surface is distributed with abundant functional groups. By introducing a long-chain connecting ion exchange group on the active functional group, the steric hindrance between the active groups can be effectively reduced, thereby enhancing the adsorption ability of the material to proteins. The phenyl group in the haloalkylbenzenesulfonate sodium has hydrophobicity, and BSA (bovine serum albumin) is a non-glycosylated single-chain polypeptide and also has a certain degree of hydrophobicity. Under the combined action of ions and hydrophobicity, the binding ability to BSA can be increased.

[0030] By using sodium haloalkylsulfonate containing hydroxyl group as the first sulfonating agent, this method can efficiently form ion exchange groups on the surface of microspheres; subsequently, sodium haloalkylbenzenesulfonate is added as the second sulfonating agent to fully utilize the active hydroxyl groups on the spacer arms of the microspheres and the branched hydroxyl groups of the first sulfonating agent to graft sulfonic acid groups, thereby increasing the ion exchange site density of the chromatography material. For example, by using sodium 3-chloro-2-hydroxypropane sulfonate as the first sulfonating agent, the epoxy groups on the spacer arms of the microspheres can undergo affinity substitution with sodium 3-chloro-2-hydroxypropane sulfonate; the sodium haloalkylbenzenesulfonate added in step (3) reacts with the hydroxyl groups on the sodium 3-chloro-2-hydroxypropane sulfonate grafted in step (2) and the hydroxyl groups on the spacer arms under specific conditions, increasing the density of sulfonic acid groups, thereby enhancing the adsorption capacity for target ions and further increasing the ion exchange capacity and dynamic loading capacity.

[0031] Advantages: Compared with the prior art, the present invention has the following remarkable advantages: (1) By using sodium haloalkylsulfonate containing hydroxyl group as the first sulfonating agent, this method can efficiently form ion exchange groups on the surface of microspheres, enabling the dynamic loading capacity of the chromatography material to reach as high as 79.53 mg / mL; (2) In the third step of this method, sodium haloalkylbenzenesulfonate is added as the second sulfonating agent to fully utilize the active hydroxyl groups on the branched chains of the microspheres to graft sulfonic acid groups, increasing the ion exchange site density of the chromatography material; (3) This method uses two different sulfonating agents to sulfonate the raw materials twice, increasing the number of sulfonic acid groups as functional groups and providing more active sites; (4) No strong acid substances are used throughout the synthesis process of this method, enhancing the applicability to the polysaccharide microsphere carrier and reducing environmental pollution at the same time; (5) By grafting epoxy groups onto the microspheres, not only the reaction activity on the surface of the microspheres is improved, but also the protein adsorption capacity of the ion exchange chromatography material is significantly enhanced due to the presence of the intermediate long chain; (6) Compared with the weakly acidic cation exchange chromatography material, due to the strong ionization ability of the sulfonic acid group, the strong acidic material prepared by this method has a faster ion exchange rate, can exchange more types of ions, and has a wider application range. Brief Description of the Drawings

[0032] Figure 1 is one of the structural general formulas of the chromatography material prepared by the present invention;

[0033] Figure 2 is the dynamic loading capacity spectrum of the chromatography material prepared in Example 1;

[0034] Figure 3 is the dynamic loading capacity spectrum of the chromatography material prepared in Comparative Example 1. Detailed Embodiments

[0035] The technical solutions of the present invention will be further described below in conjunction with the embodiments.

[0036] Example 1

[0037] As shown in the figure, the strongly acidic cation exchange chromatography material of the present invention uses agarose microspheres as the matrix material and sodium halide alkyl benzene sulfonate and hydroxyl-containing halide alkyl sulfonate as the functional groups, and its chemical formula is as follows:

[0038]

[0039] The preparation method of the chromatography material of the present invention includes the following steps:

[0040] (1) Add 60 ml of N,N-dimethylformamide and 60 ml of pure water into a 500-ml four-necked flask, then add 120 ml of agarose microspheres (particle size D50 = 75 μm, pore size ), stir for 5 - 10 min to fully disperse, then add 0.11 g of sodium borohydride, and raise the temperature in the reaction vessel to 50 °C; weigh 5.02 g of 1,4-butanediol diglycidyl ether and 8.36 ml of 45% potassium hydroxide solution, and simultaneously drop them into the flask using a syringe pump, and control the dropping to be completed within 4 h; after the dropping is completed, continue to stir and keep warm for 14 h, and obtain epoxy-activated microspheres after washing with ethanol and pure water.

[0041] (2) Add the epoxy-activated microspheres into 120 ml of pure water, then add 1.97 g of 3-chloro-2-hydroxypropane sulfonate as the first sulfonating agent, control the temperature in the reaction vessel at 25 °C, and stir for 2 h; then add 5.2 ml of 45% potassium hydroxide solution and 0.17 g of sodium hypophosphite, and continue to raise the temperature to 50 °C and stir for 12 h; after the reaction is completed, obtain the preliminary sulfonated microspheres after washing with ethanol and pure water.

[0042] (3) Add 80 ml of pure water and 40 ml of N,N-dimethylformamide into the reaction vessel, then add 1.27 g of anhydrous sodium carbonate and 0.17 g of sodium bicarbonate, mix and stir for 15 min, add 8.25 g of 4-(bromomethyl) benzene sulfonate and stir for 15 min, and finally add the preliminary sulfonated microspheres obtained in step (2), and react at 25 °C for 14 h. Obtain the strongly acidic cation exchange chromatography material, that is, the target product, after washing with ethanol and pure water.

[0043] The reaction formula is as follows:

[0044]

[0045] Example 2

[0046] The same parts as in Example 1 in this example will not be described in detail, and the differences are as follows:

[0047] Replace 5.02 g of 1,4-butanediol diglycidyl ether in step (1) with 3.70 ml of epichlorohydrin.

[0048] Example 3

[0049] The same parts as in Example 1 in this example will not be described in detail. The differences are as follows:

[0050] Replace 120 ml of agarose microspheres in step (1) with 95 ml of epoxy hydrolyzed modified poly(methyl methacrylate glycidyl ether) microspheres (particle size D50 = 60 μm, pore size ).

[0051] Comparative Example 1

[0052] (1) Add 60 ml of N,N-dimethylformamide and 60 ml of pure water into a 500 ml four-necked flask, then add 120 ml of agarose microspheres (particle size D50 = 75 μm, pore size ), stir for 5 - 10 min for sufficient dispersion, then add 0.11 g of sodium borohydride, and raise the temperature to 50 °C in the reaction vessel; weigh 5.02 g of 1,4-butanediol diglycidyl ether and 8.36 ml of 45% potassium hydroxide solution, and simultaneously add them dropwise into the flask using a syringe pump, and control to complete the dropwise addition in 4 h; after the dropwise addition is completed, continue to stir and keep warm for 14 h, and obtain epoxy-activated microspheres after washing with ethanol and pure water.

[0053] (2) Add the epoxy-activated microspheres into 120 ml of pure water, then add 1.97 g of 3-chloro-2-hydroxypropanesulfonic acid sodium salt, control the temperature in the reaction vessel at 30 °C, and stir for 2 h; then add 5.2 ml of 45% potassium hydroxide solution and 0.17 g of sodium hypophosphite, and continue to raise the temperature to 50 °C and stir for reaction for 12 h; after the reaction is completed, obtain a strongly acidic cation exchange chromatography material after washing with ethanol and pure water.

[0054] Comparative Example 2

[0055] (1) Add 60 ml of N,N-dimethylformamide and 60 ml of pure water into a 500 ml four-necked flask, then add 120 ml of agarose microspheres (particle size D50 = 75 μm, pore size ), stir for 5 - 10 min for sufficient dispersion, then add 0.11 g of sodium borohydride, and raise the temperature to 50 °C in the reaction vessel; weigh 5.02 g of 1,4-butanediol diglycidyl ether and 8.36 ml of 45% potassium hydroxide solution, and simultaneously add them dropwise into the flask using a syringe pump, and control to complete the dropwise addition simultaneously in 4 h; after the dropwise addition is completed, continue to stir and keep warm for 14 h, and obtain epoxy-activated microspheres after washing with ethanol and pure water.

[0056] (2) Add 80 ml of pure water and 40 ml of N,N-dimethylformamide into the reaction vessel, then add 1.27 g of anhydrous sodium carbonate and 0.17 g of sodium bicarbonate, mix and stir for 15 min, add 8.25 g of sodium 4-(bromomethyl)benzenesulfonate and stir for 15 min. Finally, add the microspheres activated by epoxy in step (1), and react at 25 °C for 14 h. After cleaning with ethanol and pure water, a strongly acidic cation exchange chromatography material is obtained.

[0057] Comparative Example 3

[0058] (1) Take 120 ml of agarose microspheres (particle size D50 = 75 μm, pore size ), add them to 120 ml of pure water, stir for 5 - 10 min to disperse evenly, then add 1.97 g of 3-chloro-2-hydroxypropanesulfonic acid sodium salt, control the temperature in the reaction vessel at 30 °C, and stir for 2 h; then add 5.2 ml of 45% potassium hydroxide solution and 0.17 g of sodium hypophosphite, continue to raise the temperature to 50 °C and stir for 12 h; after the reaction is completed, after cleaning with ethanol and pure water, the preliminarily sulfonated microspheres are obtained.

[0059] (2) Add 80 ml of pure water and 40 ml of N,N-dimethylformamide into the reaction vessel, then add 1.27 g of anhydrous sodium carbonate and 0.17 g of sodium bicarbonate, mix and stir for 15 min, add 8.25 g of sodium 4-(bromomethyl)benzenesulfonate and stir for 15 min. Finally, add the preliminarily sulfonated microspheres in step (1), and react at 25 °C for 14 h. After cleaning with ethanol and pure water, a strongly acidic cation exchange chromatography material is obtained.

[0060] Comparative Example 4

[0061] The same parts as in Example 1 in this example will not be elaborated, and the differences are as follows:

[0062] Replace sodium 4-(bromomethyl)benzenesulfonate in step (3) with 3-chloro-2-hydroxypropanesulfonic acid sodium salt.

[0063] Comparative Example 5

[0064] The same parts as in Example 1 in this example will not be elaborated, and the differences are as follows:

[0065] Change "continue to raise the temperature to 50 °C and stir for 12 h" in step (2) to "control the temperature in the reaction vessel at 25 °C and stir for 6 h".

[0066] Perform performance tests on the prepared strongly acidic cation exchange chromatography materials, including ion exchange capacity and dynamic binding capacity tests.

[0067] 1. Determination of ion exchange capacity

[0068] Take 5 mL of the target product and add it to 100 mL of a sulfuric acid solution with a concentration of 0.5 M. Mix overnight, and then wash and filter it with pure water and ethanol respectively. Transfer it to a conical flask after filtering thoroughly.

[0069] Add 80 mL of sodium hydroxide with a concentration of C OH = 0.05 mol / L to the conical flask, heat it in a water bath at 35 °C for 1 h. Weigh 80 mL of 0.5 mol / L sodium chloride solution and add it to the sintered glass funnel to wash the packing four times. Filter it dry and retain the filtrate.

[0070] Mix the above collected filtrates evenly. Take 20 mL of the filtrate and add 2 - 3 drops of phenolphthalein indicator. Titrate it with a standard hydrochloric acid solution with a concentration of C H = 0.05 mol / L. When it changes from colorless to purplish red and does not change color within 30 s, it reaches the end point.

[0071] Record the consumed volume V H of the standard hydrochloric acid solution, and calculate the ion exchange capacity (unit: eq / L):

[0072] IEC = (C OH *V OH - 8 * C H *V H ) / V

[0073] where V is the sample volume, unit: L; V OH is the volume of the weighed sodium hydroxide solution, unit: L; V H is the volume of the hydrochloric acid solution consumed in the titration, unit: L.

[0074] 2. Determination of dynamic binding capacity

[0075] The instrument model used in this test is the Sepax SCG protein chromatography system. Load the prepared target product into the chromatographic column, use BSA as the test sample, and take the amount of BSA sample corresponding to 10% of the breakthrough volume as the dynamic binding capacity of the chromatographic column. The specific experimental parameters are as follows:

[0076] Wavelength: 280 nm; Chromatographic column: 4.6 × 50 mm;

[0077] Sample loading flow rate: 0.5 mL / min;

[0078] Equilibration buffer: 50 mM NaAC; pH 4.50;

[0079] Elution buffer: 50 mM NaAC + 1 M NaCl; pH 4.50;

[0080] Sample loading solution: 50 mM NaAC + 2.0 mg / mL BSA;

[0081] Test the loading capacity of the chromatographic column. First, elute the chromatographic column with the eluent at a flow rate of 1 ml / min for 20 min. Then, balance the chromatographic column with the equilibration solution at a flow rate of 1 ml / min for 20 min, and then change the flow rate to 0.5 ml / min and balance for 5 min. Next, load the sample solution at a flow rate of 0.5 ml / min for 150 min. After loading, rinse the chromatographic column with 0.5 M NaOH solution at a flow rate of 1 ml / min for 30 min. Finally, elute the chromatographic column with the eluent for 20 min.

[0082] The ion exchange capacity and dynamic binding capacity of the chromatography materials prepared in Examples 1-3 and Comparative Examples 1-5 were tested respectively, and the test results are shown in Table 1.

[0083] Table 1 Performance characterization of the chromatography materials prepared in Examples 1-3 and Comparative Examples 1-5

[0084] Sample Ion exchange capacity (eq / L) Dynamic loading (mg / mL) Example 1 0.140 79.53 Example 2 0.125 71.46 Example 3 0.132 74.22 Comparative Example 1 0.095 60.81 Comparative Example 2 0.111 62.39 Comparative Example 3 0.047 26.70 Comparative Example 4 0.121 68.57 Comparative Example 5 0.084 55.46

[0085] According to the data of Example 1 and Example 2, it can be seen that the performance of the chromatography material synthesized using the diepoxy substance as the grafting reagent is better than that synthesized using the monoepoxy substance. On the one hand, it may be because the spacer arm of the chromatography material obtained in Example 1 is longer than that in Example 2, weakening the steric hindrance effect of the sulfonation reaction; on the other hand, the hydroxyl groups formed during the reaction of the diepoxy grafting reagent can serve as subsequent sulfonation reaction sites, improving the group density. The dynamic loading capacity of the chromatography material prepared in Example 1 is 79.53 mg / ml, as Figure 2 shown.

[0086] According to Table 1, compared with Example 1, removing the deep sulfonation process in step (3) in Comparative Example 1 resulted in a decrease in the number of sulfonic acid groups on the surface of the chromatography material, and the dynamic loading capacity of its finished product is 60.81 mg / ml, as Figure 3As shown, it is lower than that of Example 1, indicating that the deep sulfonation process can further improve the material properties. Compared with Example 1, in Comparative Example 2, the preliminary sulfonation process in step (2) is removed. The epoxy-activated microspheres can undergo a sulfonation reaction with sodium 4-(bromomethyl)benzenesulfonate, and the dynamic loading capacity of the obtained chromatography material is 62.39 mg / ml. However, the lack of the reaction process of sodium 3-chloro-2-hydroxypropanesulfonate results in a reduction in the sulfonic acid groups on the surface of the chromatography material, and its dynamic loading capacity is 21.56% lower than that of Example 1. Compared with Example 1, Comparative Example 3 lacks the process of increasing the spacer arm epoxy activation of the hydroxyl groups on the microsphere surface in step (1). The hydroxyl groups on the microsphere surface are less active than the epoxy groups, resulting in a dynamic loading capacity of only 26.70 mg / ml. Ideally, in Example 1, if the hydroxyl groups on the spacer arm of the preliminary sulfonated microspheres obtained in step (2) and the hydroxyl groups after grafting sodium 3-chloro-2-hydroxypropanesulfonate completely react with sodium 4-(bromomethyl)benzenesulfonate, the sulfonic acid groups on the microsphere surface can be greatly increased. In fact, under specific reaction conditions, due to the influence of the activity and steric hindrance of the hydroxyl groups on the side chain, the conversion efficiency of this reaction is not very high, but the dynamic loading capacity of Example 1 is overall increased by about 31% compared with Comparative Example 1. The difference in the dynamic loading capacity between Example 1 and Comparative Example 3 also reflects the necessity of epoxy activation before sulfonation on the microsphere surface.

[0087] Compared with Example 1, in Comparative Example 4, the sulfonating agents used in the two sulfonation reactions are both halogenated alkyl sulfonates containing hydroxyl groups, while the second sulfonating agent in Example 1 is a phenyl-containing sulfonate, and the performance of the obtained chromatography material is better. The reason is that phenyl has hydrophobicity, and BSA (bovine serum albumin) is a non-glycosylated single-chain polypeptide and also has a certain degree of hydrophobicity. Under the combined action of ions and hydrophobicity, the binding ability to BSA can be increased, so the dynamic loading capacity is higher than that of Comparative Example 4.

[0088] According to the data of Example 1 and Comparative Example 5, shortening the preliminary sulfonation time and lowering the temperature are not conducive to the progress of the reaction.

Claims

1. A strongly acidic cation exchange chromatography material, characterized in that, The chromatographic material described above uses hydroxy-containing microspheres as the matrix, grafts an epoxide spacer arm at the hydroxy terminus, grafts a hydroxy-containing haloalkylsulfonate at the spacer arm terminus, and grafts a haloalkylbenzenesulfonate at one end of the spacer arm and / or the haloalkylsulfonate.

2. The strongly acidic cation exchange chromatography material according to claim 1, wherein The chemical formula of the chromatographic material described above is as follows: Wherein, R1, R2, R3, R4, and R5 represent alkyl groups.

3. A method for preparing the chromatographic material according to claim 1, characterized in that, It includes the following steps: (1) Epoxy activation: Disperse the microspheres in the continuous phase, add a reducing agent, an alkaline solution, and an epoxy grafting reagent, and obtain epoxy-activated microspheres after reaction; (2) Preliminary sulfonation: React the epoxy-activated microspheres with a hydroxy-containing haloalkylsulfonate as the first sulfonating agent under alkaline conditions to obtain preliminarily sulfonated microspheres; (3) Deep sulfonation: React the preliminarily sulfonated microspheres with a haloalkylbenzenesulfonate as the second sulfonating agent under alkaline conditions to obtain the target product.

4. The preparation method according to claim 3, characterized in that, The microspheres described in step (1) are one or more of agarose microspheres, chitosan microspheres, polystyrene-divinylbenzene microspheres with hydroxy groups on the hydrophilic modified surface, and poly(methyl methacrylate glycidyl ether) microspheres modified by epoxy hydrolysis.

5. The preparation method according to claim 3, characterized in that, The reducing agent described in step (1) is sodium borohydride; the alkaline solution is an aqueous solution of 35wt% - 50wt% NaOH or KOH.

6. The preparation method according to claim 3, characterized in that, The grafting reagent described in step (1) is one or more of 1,4-butanediol diglycidyl ether, epichlorohydrin, and dipropylene glycol diglycidyl ether.

7. The preparation method according to claim 3, characterized in that, The general structural formula of the hydroxy-containing haloalkylsulfonate described in step (2) is Wherein R1 and R2 represent alkyl groups, and X represents a halogen.

8. The preparation method according to claim 3, characterized in that, The hydroxy-containing haloalkylsulfonate described in step (2) is one or more of 3-chloro-2-hydroxypropanesulfonate and 4-chloro-1-hydroxybutanesulfonate.

9. The preparation method according to claim 3, characterized in that The general structural formula of the haloalkylbenzenesulfonate described in step (3) is Y-R3-PhSO3Na, where R3 represents an alkyl group and Y represents a halogen.

10. The preparation method according to claim 3, characterized in that, The haloalkylbenzenesulfonate described in step (3) is one or more of 4-(bromomethyl)benzenesulfonate and 4-(2-bromoethyl)benzenesulfonate.

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