Dual-basic amino acid separation filler as well as preparation method and application thereof

Abstract

The invention relates to the field of synthesis methods of amino acid separation fillers. The invention provides a preparation method of a dual-basic amino acid separation filler. The preparation method comprises the following steps: (1) preparing chloromethylated microspheres; (2) preparing a reaction system: adding an alkaline reagent solution at room temperature, and uniformly stirring; (3) adding chloromethylated microspheres and reacting; and (4) post-treatment: filtering out waste liquid from the reacted resin, washing with a solvent, and washing with ultrapure water until the resin is neutral, thereby obtaining the dual-basic amino acid separation filler. The invention also provides the double-basic amino acid separation filler. The invention also provides application of the dual-basic amino acid separation filler in separation of 17 amino acids. The double-basic amino acid separation filler prepared by the invention has both a quaternary ammonium strong-basic anion exchange group and a tertiary amine weak-basic anion exchange group which jointly act on carboxyl and amino in amino acid, so that 17 common proteolysis amino acids are separated.

Description

Technical Field

[0001] The invention relates to the field of synthetic methods for amino acid separation fillers, in particular to a double basic amino acid separation filler and a preparation method and application thereof. Background Art

[0002] Amino acids refer to a large class of substances in the organic compound system that contain both amino and carboxyl groups. According to the position of the amino group relative to the carboxyl group on the main chain, they can be divided into α, β and γ amino acids. The amino acids that make up proteins in natural products in nature are all α-amino acids, also known as standard amino acids. Typical acidic amino acids and basic amino acids in standard amino acids are shown in formula (1).

[0003]

[0004] Formula (1) Since the amino acid molecular formula contains the basic group amino group and the acidic group carboxyl group, it is an amphoteric substance. At present, in the analytical science community, low cross-linking cation exchange resins (cross-linking degree <10%) and a series of acidic buffers are used as regeneration fluids for the separation, detection and purification of amino acids. By adjusting the acidity of different buffers, the ionization of amino acids is controlled, thereby achieving the separation process of various amino acids on the cation exchange resin. At present, this separation method and separation system are widely used in ion exchange chromatography amino acid separation analysis and high-purity separation and purification processes.

[0005] However, low cross-linked cation exchange resins have low mechanical strength, and the cross-linking degree of the resin is generally controlled below 10%, which is easy to break, especially under high pressure or high flow rate conditions, which may lead to decreased column efficiency and poor separation effect; the selectivity for certain amino acids such as threonine / serine, glycine / alanine may not be high enough, resulting in poor separation effect; the separation speed of low cross-linked resins is relatively slow, especially when high resolution is required, exceeding 60 minutes, and the separation time may be long.

[0006] Therefore, the prior art needs to be improved urgently. Summary of the invention

[0007] In view of the technical problems in the prior art, the present invention proposes a dibasic amino acid separation filler and a preparation method and application thereof. The preparation method of a dibasic amino acid separation filler provided by the present invention solves the problem of a single separation scheme. According to the amphoteric properties of amino acids, a dibasic amino acid separation filler is developed and applied to the separation and analysis process of amino acids. The surface of the dibasic amino acid separation filler and the inner surface of the pore diameter have both quaternary ammonium strong alkaline anion exchange groups and tertiary amine weak alkaline anion exchange groups. Under different pH concentration eluent environments, the two groups show different dissociation forms and act together on the carboxyl and amino groups in the amino acids to separate 17 common proteolytic amino acids. The dibasic amino acid separation filler involved in the present invention is limited to being used to separate standard amino acids in protein hydrolysis.

[0008] The technical solution provided by the present invention is: A method for preparing a dibasic amino acid separation filler comprises the following steps: (1) Preparation of chloromethylated microspheres: polystyrene polymer microspheres are prepared by swelling polymerization method, appropriate particle size is selected for washing and drying, and surface chloromethylated microspheres are prepared by Blanc reaction; (2) Prepare the reaction system: Add the alkaline reagent solution at room temperature to a three-necked flask equipped with a water bath heater, an electric stirrer, a reflux condenser, and a thermometer, and stir evenly; (3) Adding chloromethylated microspheres and reacting: adding the surface chloromethylated microspheres in step (1) to the solution obtained in step (2) under stirring, stirring at room temperature for 20 to 60 minutes, and then heating to raise the temperature; (4) Post-treatment: After the reaction, the resin is filtered to remove the waste liquid, washed with a solvent, and then washed with ultrapure water until it becomes neutral to obtain the acid-base amino acid separation filler.

[0009] In the method for preparing a double basic amino acid separation filler as described above, the crosslinking degree of the polystyrene polymer microspheres in step (1) is 40-60%, and the pore size is 50-150nm. Preferably, the crosslinking degree of the polystyrene polymer microspheres in step (1) is 50-60%, and the pore size is 80-120nm. Under this condition, the polystyrene polymer microspheres will have sufficient rigidity to withstand sufficient pressure and organic solvent tolerance; preferably, the pore size has a sufficient specific surface area and a moderate pore size so that the amino acid molecules enter the inner surface of the pore size and increase the effective exchange area. Most preferably, the crosslinking degree of the polystyrene polymer microspheres in step (1) is 55%, and the pore size is 80-120nm.

[0010] In the method for preparing a dibasic amino acid separation filler as described above, the polystyrene polymer microspheres with a suitable particle size selected in step (1) are polystyrene polymer microspheres with a particle size of 2-10 μm. Preferably, the polystyrene polymer microspheres with a suitable particle size selected in step (1) are polystyrene polymer microspheres with a particle size of 5-7 μm. Under this condition, the preferred particle size has both sufficient column efficiency per unit length and moderate working pressure to obtain the best separation efficiency.

[0011] In the method for preparing a dibasic amino acid separation filler as described above, the alkaline reagent in step (2) is selected from any one of tetramethylethylenediamine, tetramethylpropylenediamine, tetramethylbutylenediamine, tetramethylpentanediamine, and tetramethylhexanediamine, or a mixture thereof. Preferably, the alkaline reagent in step (2) is selected from tetramethylethylenediamine. Under this condition, the functional group has a moderate main chain length so that the amino acid functional group can act on the dibasic group at the same time.

[0012] In the method for preparing a dibasic amino acid separation filler as described above, the alkaline reagent solution in step (2) is a di-tertiary amino alkaline reagent aqueous solution, and the concentration of the di-tertiary amino alkaline reagent aqueous solution is 0.05M-0.2M. Preferably, the concentration of the di-tertiary amino alkaline reagent aqueous solution is 0.05M-0.1M. Under this condition, the reaction has an optimal reaction speed to avoid the base ball from being broken due to too fast reaction during the reaction.

[0013] In the method for preparing a double basic amino acid separation filler as described above, the molar to mass ratio of the alkaline reagent to the chloromethylated microspheres in step (3) is 1:500-1:2000. Preferably, the molar to mass ratio of the alkaline reagent to the chloromethylated microspheres in step (3) is 1:500-1:1000. Under this condition, the reaction can be ensured to be thorough without excessive waste of raw materials.

[0014] In the method for preparing a dibasic amino acid separation filler as described above, the post-heating in step (3) is specifically as follows: heating to 30-50°C for reaction for 1-3h, and then heating to at least 50-90°C for 2-20h. Preferably, the post-heating in step (3) is specifically as follows: heating to 35-50°C for reaction for 1-2h, and then heating to at least 60-80°C for 3-15h. Under these conditions, the reaction speed is moderate, and the reaction process can be ensured to be thorough without breaking the polystyrene polymer microspheres.

[0015] In the method for preparing a dibasic amino acid separation filler as described above, the solvent in step (4) is a solution of methanol and water in a volume ratio of 1:1.

[0016] Based on the same inventive concept, the present invention provides a dibasic amino acid separation filler, which is prepared according to the preparation method described above.

[0017] Based on the same inventive concept, the present invention provides a double basic amino acid separation filler prepared according to the preparation method described above or the use of the double basic amino acid separation filler described above in separating 17 kinds of amino acids. The 17 kinds of proteolytic amino acids are arginine (Arg), lysine (Lys), alanine (Ala), threonine (Thr), glycine (Gly), valine (Val), serine (Ser), proline (Pro), isoleucine (Ile), leucine (Leu), methionine (Met), histidine (His), phenylalanine (Phe), glutamic acid (Glu), aspartic acid (Asp), cystine (Cys), and tyrosine (Tyr). The double basic amino acid separation filler described above is used to separate 17 kinds of amino acids in about 35 minutes, with good separation effect and short separation time.

[0018] The beneficial effects of the technical solution provided by the present invention are: 1. The present invention provides a method for preparing a dibasic amino acid separation filler. Based on the amphoteric properties of amino acids, a dibasic amino acid separation filler is developed and applied to the separation and analysis process of amino acids, providing a new filler for amino acid separation.

[0019] 2. The present invention provides a dual basic amino acid separation filler, which has both a quaternary ammonium strong basic anion exchange group and a tertiary amine weak basic anion exchange group, which act together on the carboxyl group and the amino group in the amino acid to separate 17 common proteolytic amino acids in about 35 minutes, with good separation effect and short separation time.

[0020] 3. The present invention provides a new chromatographic separation mode for protein hydrolyzed amino acids. This separation mode can be completed by gradient elution with sodium hydroxide, and the preparation of sodium hydroxide or online electrolysis is a simple operation, which saves time and effort compared with the traditional complex buffer preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a whole SEM image of the chloromethylated microspheres prepared in step (1) of Example 1 of the present invention; Figure 2 This is an enlarged SEM image of the surface of the chloromethylated microspheres prepared in step (1) of Example 1 of the present invention; Figure 3 A separation diagram of 17 hydrolyzed standard amino acids separated by a chromatographic column prepared from the double basic amino acid separation filler prepared in Example 1 of the present invention; Figure 4A separation diagram of 17 hydrolyzed standard amino acids separated by a chromatographic column prepared by the amino acid separation filler prepared in Comparative Example 1 of the present invention; Figure 5 The separation diagram of 17 hydrolyzed standard amino acids separated by a chromatographic column prepared by the amino acid separation filler prepared in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0022] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more related listed items. It should be understood that, unless otherwise specified, various raw materials in the present invention can be obtained commercially.

[0024] Embodiment 1: This embodiment provides a method for preparing a dibasic amino acid separation filler, comprising the following steps: (1) Polystyrene polymer microspheres with a crosslinking degree of 55% and a pore size of 80-120 nm were prepared by the swelling polymerization method (reference: Li Lu, Wen Xiufang, Pi Pihui, et al. Preparation of styrene-divinylbenzene porous crosslinked microspheres by seed swelling polymerization [J]. Fine Chemicals, 2006, 23 (6)). The 5-7 μm particle size fraction was screened and fully washed, extracted and dried for use. In a 250 mL three-necked flask equipped with a water bath heating, air flow duct, electric stirrer, reflux condenser (with a waterproof device at the outlet) and a thermometer, 9 g of trioxymethylene, 5 g of polystyrene polymer microspheres that had been extracted and thoroughly dried, 50 mL of concentrated hydrochloric acid, and 35 g of anhydrous zinc chloride were added. The entire reaction system was quickly sealed, and the stirrer was turned on to stir the reaction base for 10 min. The temperature was slowly raised to 35°C and stirred for 30 min. Dry HCl gas was introduced at a rate of 1.5 mL / min, and the temperature was slowly raised to 50°C. The reaction was kept warm for 4 hours. After the reaction is finished, the entire reaction system is poured into a large amount of deionized water in a fume hood to terminate the reaction, and the surface chloromethylated polystyrene microspheres are obtained after sufficient washing and drying; (2) In a 250 mL three-necked flask equipped with a water bath heater, an electric stirrer, a reflux condenser, and a thermometer, add 100 mL of a 0.1 M tetramethylethylenediamine aqueous solution at room temperature, start the electric stirrer, and control the stirring speed of the electric stirrer to be 120 r / min; (3) Stirring at a speed of 120 r / min, add 5 g of the surface chloromethylated polystyrene microspheres obtained in step (1) under continuous stirring, stir at room temperature for 30 min, turn on the heating, react at 35 °C for 2 h; raise the temperature to 60 °C and keep the temperature for 10 h; (4) The product after the reaction in step (3) is filtered to remove the waste liquid, washed three times with a solution of methanol and water in a volume ratio of 1:1, and washed with ultrapure water until neutral, thereby obtaining a dibasic amino acid separation filler.

[0025] Figure 1 This is the overall SEM image of the chloromethylated microspheres prepared in step (1) of Example 1 of the present invention. Figure 2 This is an enlarged SEM image of the chloromethylated microspheres prepared in step (1) of Example 1 of the present invention. Figure 1 and Figure 2 It can be seen that the chloromethylated microspheres are regular spherical, with smooth surface, uniform dispersion, no obvious aggregation, and uniform particle size.

[0026] The bibasic amino acid separation filler prepared in this example was loaded into a 4.0×250 mm stainless steel column tube by homogenization method at a pressure of 30 MPa. The obtained chromatographic column was used for qualitative and quantitative analysis of protein hydrolysis standard amino acids. The test results are shown in FIG. Figure 3 As shown, Figure 3 The separation diagram of 17 hydrolyzed standard amino acids separated by a chromatographic column prepared with the double basic amino acid separation filler prepared in Example 1 of the present invention.

[0027] The analytical conditions for protein hydrolysis of standard amino acids are as follows: Detector: Three-electrode integrating pulse amperometric detector, where the working electrode is Au electrode; the reference electrode is Ag / AgCl (saturated KCl); the counter electrode is a graphite electrode; the pulse scanning cycle potential and time are set as follows: E1=-0.2V, T1=10ms; E2=0.3V, T2=30ms; E3=-2.0V, T3=10ms; E4=0.8V, T4=10ms Mobile phase: Gradient elution, the mobile phase, ratio and time are set as follows: A: ultrapure water; B: 0.25 mol / L NaOH; C: 1.0 mol / L NaAc; flow rate: 0.22 mL / min

[0028] like Figure 3 As shown, the injection volume is 20 μL, and the standard solution concentration is 100 nmol / mL. From the analysis results, it can be seen that the double basic amino acid separation filler prepared by the method of the parameters of the present invention can well separate 17 kinds of proteolytic amino acids (arginine, lysine, alanine, threonine, glycine, valine, serine, proline, isoleucine, leucine, methionine, histidine, phenylalanine, glutamic acid, aspartic acid, cystine, tyrosine), and its separation order conforms to the characteristics of amino acid amphoteric substances under alkaline solution conditions, and avoids the problem of lysine / ammonium group and threonine / serine, glycine / alanine, glutamic acid / proline being difficult to separate and interfering with each other in the commonly used acidic separation system.

[0029] Embodiment 2: This embodiment provides a method for preparing a dibasic amino acid separation filler, comprising the following steps: (1) preparing surface chloromethylated polystyrene microspheres according to the method in step (1) of Example 1; (2) In a 250 ml three-necked flask equipped with a water bath heater, an electric stirrer, a reflux condenser and a thermometer, add 100 ml of a 0.1 M tetramethylethylenediamine aqueous solution at room temperature, start the stirrer and control the stirring speed to 120 r / min; (3) Stirring at a speed of 120 r / min, add 5 g of the surface chloromethylated polystyrene microspheres obtained in step (1) under continuous stirring, stir at room temperature for 30 min, turn on the heating, react at 50 °C for 1 h; raise the temperature to 70 °C and keep the temperature for 3 h; (4) The product after the reaction in step (3) is filtered to remove the waste liquid, washed three times with a solution of methanol and water in a volume ratio of 1:1, and washed with supernatant water until neutral, thereby obtaining a dibasic amino acid separation filler.

[0030] The filler was loaded into a 4.0×250 mm stainless steel column tube by homogenization method for protein hydrolysis standard amino acid test. This example does not provide a spectrum.

[0031] Embodiment 3: This embodiment provides a method for preparing a dibasic amino acid separation filler, comprising the following steps: (1) preparing surface chloromethylated polystyrene microspheres according to the method in step (1) of Example 1; (2) In a 250 mL three-necked flask equipped with a water bath heater, an electric stirrer, a reflux condenser, and a thermometer, add 170 mL of a 0.05 M tetramethylethylenediamine aqueous solution at room temperature, start the stirrer, and control the stirring speed to 120 r / min; (3) Stirring at a speed of 120 r / min, add 5 g of the surface chloromethylated polystyrene microspheres obtained in step (1) under continuous stirring, stir at room temperature for 30 min, turn on the heating, react at 35 °C for 2 h; raise the temperature to 80 °C and keep the temperature for 15 h; (4) The product after the reaction in step (3) is filtered to remove the waste liquid, washed three times with a solution of methanol and water in a volume ratio of 1:1, and washed with supernatant water until neutral, thereby obtaining a dibasic amino acid separation filler.

[0032] The filler was loaded into a 4.0×250 mm stainless steel column tube by homogenization method for protein hydrolysis standard amino acid test. This example does not provide a spectrum.

[0033] Comparative Example 1: This comparative example provides a method for preparing an amino acid separation filler, comprising the following steps: (1) preparing surface chloromethylated polystyrene microspheres according to the method in step (1) of Example 1; (2) In a 250 mL three-necked flask equipped with a water bath heater, an electric stirrer, a reflux condenser, and a thermometer, add 100 mL of a 0.1 M trimethylamine aqueous solution at room temperature, start the stirrer, and control the stirring speed to 120 r / min; (3) Stirring at a speed of 120 r / min, add 5 g of the surface chloromethylated polystyrene microspheres obtained in step (1) under continuous stirring, stir at room temperature for 30 min, turn on the heating, react at 35 °C for 2 h; raise the temperature to 60 °C and keep the temperature for 10 h; (4) The product after the reaction in step (3) is filtered to remove the waste liquid, washed three times with a solution of methanol and water in a volume ratio of 1:1, and washed with supernatant water until neutral, thereby obtaining an amino acid separation filler.

[0034] The filler was loaded into a 4.0×250 mm stainless steel column tube by homogenization method. The obtained chromatographic column was used for qualitative and quantitative analysis of protein hydrolysis standard amino acids. The analysis conditions were carried out according to the conditions of Example 1. The test results are shown in FIG. Figure 4 shown.

[0035] from Figure 4It can be seen that the same resin-based ball, under the same process conditions, uses a filler prepared by trimethylamine with a common monoquaternary ammonium salt group (universal strong basic anion resin, a single strong basic group, and no tertiary amino weak basic group) and has no separation ability for 17 protein hydrolyzed amino acid standard solutions under the same test conditions. The reason is that, firstly, the quaternary ammonium salt group carries a positive charge, which has nothing to do with the pH value of the surrounding environment. The pH of the mobile phase has little effect on the charge density of the monoquaternary ammonium salt matrix, that is, the adsorption capacity of the monoquaternary ammonium salt group for anions changes little with the pH of the solution; the weakly basic tertiary amino group is just the opposite, and its adsorption capacity changes greatly with the pH of the mobile phase; secondly, the monoquaternary ammonium salt group lacks a carbon chain between nitrogen atoms, and conjugated adsorption cannot occur, and it is not easy to occur, and it is impossible to use other types of interactions such as hydrogen bonds and hydrophobic interactions to enhance adsorption capacity and selectivity.

[0036] Comparative Example 2: This comparative example provides a method for preparing an amino acid separation filler, comprising the following steps: (1) preparing surface chloromethylated polystyrene microspheres according to the method in step (1) of Example 1; (2) In a 250 ml three-necked flask equipped with a water bath heater, electric stirrer, reflux condenser and thermometer, add 100 ml of 0.1 M dimethylethanolamine aqueous solution at room temperature, start the stirrer and control the stirring speed to 120 r / min; (3) Stirring at a speed of 120 r / min, add 5 g of the surface chloromethylated polystyrene microspheres obtained in step (1) under continuous stirring, stir at room temperature for 30 min, turn on the heating, react at 35 °C for 2 h; raise the temperature to 60 °C and keep the temperature for 10 h; (4) The product after the reaction in step (3) is filtered to remove the waste liquid, washed three times with a solution of methanol and water in a volume ratio of 1:1, and washed with supernatant water until neutral, thereby obtaining an amino acid separation filler.

[0037] The filler was loaded into a 4.0×250 mm stainless steel column tube using the homogenization method for protein hydrolysis standard amino acid test.

[0038] Comparative Example 3: This comparative example provides a method for preparing an amino acid separation filler, comprising the following steps: (1) preparing surface chloromethylated polystyrene microspheres according to the method in step (1) of Example 1; (2) In a 250 mL three-necked flask equipped with a water bath heater, an electric stirrer, a reflux condenser, and a thermometer, add 100 mL of a 0.1 M tetramethylethylenediamine aqueous solution at room temperature and start stirring at a speed of 120 r / min.

[0039] (3) Add 5 g of the surface chloromethylated polystyrene microspheres obtained in step (1) under continuous stirring at a stirring speed of 120 r / min, stir at room temperature for 10 min, turn on the heating, and keep the reaction at 35°C for 10 h; (4) The product after the reaction in step (3) is filtered to remove the waste liquid, washed three times with methanol and water solution in a volume ratio of 1:1, and washed with supernatant water until neutral, thereby obtaining an amino acid separation filler.

[0040] The filler was loaded into a 4.0×250 mm stainless steel column tube by homogenization method. The prepared chromatographic column was used for qualitative and quantitative analysis of protein hydrolysis standard amino acids. The test results are shown in Figure 5 shown.

[0041] from Figure 5 It can be seen that, using the standard ratio raw materials of this comparative example 3, under different process conditions, the heating and temperature preservation step is not carried out, that is, the reaction does not go through a "ripening" process at a certain temperature, the reaction is not fully carried out, and the obtained filler cannot effectively separate the 17 kinds of protein hydrolysis standard amino acids. The reason is that in organic chemical reactions, the molecular spatial structure of the reaction substrate is large and different, and temperature is the primary factor affecting the progress of the reaction. Under certain conditions, temperature is the primary condition for promoting the thoroughness of the reaction; for microsphere reaction substrates with a physical scale of micrometers, di-tertiary amino molecules smaller than one nanometer must react and be completely in the base ball, and temperature is the first factor. Therefore, the heating and temperature preservation step (i.e., the "ripening" process) of the present invention ensures that the reaction is fully carried out. Under this condition, an efficient amino acid separation filler is obtained.

[0042] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0043] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a dibasic amino acid separation filler, characterized in that: The following steps are involved: (1) Preparation of chloromethylated microspheres: polystyrene polymer microspheres are prepared by swelling polymerization method, and appropriate particle sizes are selected for washing and drying to prepare surface chloromethylated microspheres; (2) Prepare the reaction system: Add alkaline reagent solution to a three-necked flask at room temperature and stir evenly; (3) Adding chloromethylated microspheres and reacting: adding the surface chloromethylated microspheres in step (1) to the solution obtained in step (2) under stirring, stirring at room temperature for 20 to 60 minutes, and then heating to raise the temperature; (4) Post-treatment: After the reaction, the resin is filtered to remove the waste liquid, washed with a solvent, and then washed with ultrapure water until it becomes neutral to obtain a dibasic amino acid separation filler.

2. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: In the step (1), the cross-linking degree of the polystyrene polymer microspheres is 40-60%, and the pore size is 50-150 nm.

3. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: The polystyrene polymer microspheres with a suitable particle size screened in the step (1) are polystyrene polymer microspheres with a particle size of 2-10 μm.

4. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: The alkaline reagent in step (2) is selected from any one of tetramethylethylenediamine, tetramethylpropylenediamine, tetramethylbutylenediamine, tetramethylpentanediamine, and tetramethylhexanediamine, or a mixture of several thereof.

5. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: The alkaline reagent solution in step (2) is a di-tertiary amino alkaline reagent aqueous solution, and the concentration of the di-tertiary amino alkaline reagent aqueous solution is 0.05M-0.2M.

6. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: The molar to mass ratio of the alkaline reagent to the chloromethylated microspheres in step (3) is 1:500-1:2000.

7. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: The post-heating in step (3) is specifically as follows: heating to 30-50°C for reaction for 1-3h, and then heating to at least 50-90°C for 2-20h.

8. The method for preparing a double basic amino acid separation filler according to claim 1, characterized in that: The solvent in step (4) is a solution of methanol and water in a volume ratio of 1:

1.

9. A double basic amino acid separation filler, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the doubly basic amino acid separation filler prepared according to the preparation method according to any one of claims 1 to 8 or the doubly basic amino acid separation filler according to claim 9 in separating 17 kinds of amino acids.

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