Synthesis method of strong base agarose microspheres
The preparation of strong alkali agarose microspheres through multi-step reactions solved the problem of unstable stability and exchange amount in protein and peptide extraction and separation of agarose beads, achieving efficient extraction and separation effects and excellent product performance.
Patent Information
- Application Number
- CN202411818023.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-18
AI Technical Summary
The existing agarose beads have problems of stability and unstable exchange amount in the extraction and isolation and purification of proteins, polypeptides and other substances.
Strong alkali agarose microspheres were prepared by mixing agarose beads with acetone alcohol solution and a catalyst, followed by multi-step reaction with allyl glycidyl ether, sodium acetate solution, water bromine and sodium formate solutions, and finally insulated with trimethylamine.
The prepared strong alkali agarose microspheres have good chemical stability and are not easily dissolved in various solvents. They are suitable for the extraction, separation and purification of proteins, peptides and other substances. The product has excellent performance and meets the needs of different application fields.
Smart Images
Figure CN119638870A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of chemical engineering, in particular to a method for synthesizing strong-base agarose microspheres. Background Art
[0002] In the fields of chemical engineering, materials science and biotechnology, the extraction, separation and purification of proteins, peptides and other substances are important research directions. Agarose beads have a wide range of uses in the fields of biochemistry and bioengineering, mainly due to its two major characteristics: (1) Agarose itself is a macromolecule, containing many hydroxyl groups (-OH) in the molecule, which are very active groups, making it water-soluble and capable of undergoing a variety of chemical reactions; (2) It belongs to the carbohydrate class, is hydrophilic, and has good compatibility with biological macromolecules, making them less likely to be inactivated. Agarose ion exchangers containing strong base exchange functional groups can be used in ion exchange chromatography technology to separate and purify proteins, enzymes, nucleic acids, peptides, oligonucleotides, viruses, bacteriophages and polysaccharides; they can also be used as carriers for affinity chromatography technology to separate and purify enzyme substrates, inhibitors, coenzymes, antibodies, antigens, viruses, cells, polysaccharides, glycoproteins, cell surface receptors, cell nucleic acids, histones, nucleic acid polymerases, binding proteins, hormones and vitamins, carrier proteins, cell surface specific proteins, etc. Summary of the invention
[0003] In order to solve the above problems, the present invention provides a method for synthesizing strong base agarose microspheres. The synthesis method provided by the present invention can prepare an example exchange chromatography medium of a high conductivity working solution, and the exchange capacity of the medium is stable at 0.18-0.20 mmol / ml.
[0004] In order to achieve the above object, the present invention provides the following technical solutions:
[0005] The present invention provides a method for synthesizing strong-base agarose microspheres, comprising the following steps:
[0006] 1) mixing agarose beads, acetol solution and a catalyst and stabilizing for 30 minutes to obtain a stabilizer;
[0007] 2) mixing the stabilizer obtained in step 1) with allyl glycidyl ether and heat-insulating to obtain activated beads;
[0008] 3) mixing the activated beads obtained in step 2) with a sodium acetate solution and 3% by mass bromine water, stabilizing for 1 hour, and then mixing with a sodium formate solution and stabilizing for 30 minutes to obtain brominated beads;
[0009] 4) The brominated beads obtained in step 3) are mixed with trimethylamine and stabilized for 30 minutes, and then incubated for reaction to obtain strong base agarose microspheres.
[0010] Preferably, in step 1), the volume ratio of the mass of agarose beads to the acetol solution is 100 g:250 mL;
[0011] The acetone alcohol solution is acetone and ethanol, and the volume ratio of the acetone to the ethanol is 1:1.
[0012] Preferably, the catalyst in step 1) is sodium carbonate, and the mass ratio of the agarose beads to the catalyst is 100:1.
[0013] Preferably, in step 2), the volume ratio of allyl glycidyl ether to the acetone alcohol solution is 6 to 8:25.
[0014] Preferably, in step 2), allyl glycidyl ether is added dropwise to the stabilizer, and the temperature is controlled at 43-45° C.;
[0015] The temperature of the insulation is 43-45° C., and the time is 30 minutes.
[0016] Preferably, the volume ratio of the activated beads in step 3) to the sodium acetate solution is 100 g:500 mL;
[0017] The mass percentage of the sodium acetate solution is 4%.
[0018] Preferably, the volume ratio of bromine water to sodium acetate solution in step 3) is 6:5;
[0019] The bromine water is added dropwise to the mixture of the activated beads and the sodium acetate solution.
[0020] Preferably, in step 3), the volume ratio of the sodium formate solution to the bromine water is 1:12;
[0021] The mass percentage of the sodium formate solution is 50%.
[0022] Preferably, the volume ratio of the mass of the brominated beads in step 4) to trimethylamine is 100 g:250-400 mL.
[0023] Preferably, the conditions for the heat preservation reaction in step 4) include: temperature of 38° C. and time of 10 h.
[0024] Beneficial effects of the present invention:
[0025] The strong base agarose microspheres synthesized by the present invention have good chemical stability, are basically insoluble in various solvents, and are suitable for the extraction, separation and purification of substances such as proteins and polypeptides. At the same time, the product performance of the present invention is excellent and can meet the needs of different application fields. In summary, the present invention has the advantages of simple operation, mild reaction conditions, excellent product performance, etc. relative to the prior art, and is an ideal preparation method. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0027] Figure 1 It is the synthetic reaction equation of the strong base agarose microsphere of the present invention. DETAILED DESCRIPTION
[0028] The present invention provides a method for synthesizing strong-base agarose microspheres, comprising the following steps:
[0029] 1) mixing agarose beads, acetol solution and a catalyst and stabilizing for 30 minutes to obtain a stabilizer;
[0030] 2) mixing the stabilizer obtained in step 1) with allyl glycidyl ether and heat-insulating to obtain activated beads;
[0031] 3) mixing the activated beads obtained in step 2) with a sodium acetate solution and 3% by mass bromine water, stabilizing for 1 hour, and then mixing with a sodium formate solution and stabilizing for 30 minutes to obtain brominated beads;
[0032] 4) The brominated beads obtained in step 3) are mixed with trimethylamine and stabilized for 30 minutes, and then incubated for reaction to obtain strong base agarose microspheres.
[0033] The present invention mixes agarose beads with an acetol solution and a catalyst and stabilizes them for 30 minutes to obtain a stabilizer. In the present invention, the agarose beads are preferably derived from 6FF cross-linked beads (particle size: 45-165 μm) sold by Ningbo Zhengguang Resin Co., Ltd. In the present invention, the mass of the agarose beads and the volume ratio of the acetol solution are preferably 100g:250mL. In the present invention, the acetol solution is preferably acetone and ethanol, and the volume ratio of acetone and ethanol is preferably 1:1, in order to make the agarose beads and allyl glycidyl ether better fused and reacted. In the present invention, the catalyst is preferably sodium carbonate, and the mass ratio of the agarose beads to the catalyst is preferably 100:1.
[0034] The present invention mixes the obtained stabilizer with allyl glycidyl ether and incubates to obtain activated beads. In the present invention, the volume ratio of allyl glycidyl ether to acetone alcohol solution is preferably 6 to 8:25. In the present invention, the allyl glycidyl ether is preferably added dropwise to the stabilizer, and the temperature is controlled to be 43 to 45° C. In the present invention, the incubation temperature is preferably 43 to 45° C., and the time is preferably 30 minutes. In the present invention, the activated beads are allyl activated and bromine groups are connected.
[0035] The present invention mixes the obtained activated beads with sodium acetate solution and bromine water with a mass percentage of 3% and stabilizes them for 1 hour, then mixes them with sodium formate solution and stabilizes them for 30 minutes to obtain brominated beads. In the present invention, the volume ratio of the mass of the activated beads to the sodium acetate solution is preferably 100g:500mL. In the present invention, the mass percentage of the sodium acetate solution is preferably 4%. In the present invention, the volume ratio of the bromine water to the sodium acetate solution is preferably 6:5. In the present invention, the bromine water is preferably added dropwise to the mixture of the activated beads and the sodium acetate solution. In the present invention, the volume ratio of the sodium formate solution to the bromine water is preferably 1:12. In the present invention, the mass percentage of the sodium formate solution is preferably 50%.
[0036] The present invention mixes the obtained brominated beads with trimethylamine and stabilizes them for 30 minutes, and then performs a heat preservation reaction to obtain strong base agarose microspheres. In the present invention, the mass ratio of the brominated beads to the volume of trimethylamine is preferably 100g:250-400mL. In the present invention, the conditions of the heat preservation reaction preferably include: a temperature of 38°C and a time of 10h.
[0037] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0038] Example 1
[0039] A method for synthesizing strong base agarose microspheres, the specific steps are as follows:
[0040] Step 1: Activation of beads:
[0041] First, 100 g of 6FF cross-linked beads (from Ningbo Zhengguang Resin Co., Ltd., particle size: 45-165 μm) were added to 250 ml of acetone and ethanol solution, wherein the volume ratio of acetone to ethanol was 1: 1. Then, 1 g of catalyst sodium carbonate was added and stabilized for 30 minutes.
[0042] Next, 60 ml of allyl glycidyl ether was added dropwise at a rate of 6 ml / min, and the temperature was controlled at 43° C. After the addition was completed, the mixture was kept warm for 30 minutes and sampled for analysis. The results showed that the double bond content was 0.32 mmol / ml, which met the requirements.
[0043] Finally, the activated beads were washed with water, soaked in anhydrous ethanol for 3 times, each time for 10 minutes, washed with pure water until odorless, and placed in a glass container for the next reaction.
[0044] Step 2: Bromination of beads:
[0045] Add 100g activated beads and 500ml sodium acetate solution (4% by mass) into a flask and keep it at room temperature. Start to add 600ml bromine water dropwise until the solution turns orange. After stabilizing for 1 hour, add 50ml sodium formate solution (50% by mass) dropwise to make the solution colorless. After stabilizing for half an hour, discharge the material. Then, replace the beads with anhydrous ethanol until there is no water, soak in anhydrous ethanol for 30 minutes to react, and obtain brominated beads.
[0046] Step 3: Amination of beads:
[0047] First, 100 g of brominated beads were mixed with 300 ml of trimethylamine, and after stabilization for half an hour, the temperature was raised to 38° C. and kept warm for 10 hours. After the reaction was completed, the material was discharged and the synthesis of agarose microspheres with strong base groups was completed to obtain strong base agarose microspheres.
[0048] 1 Test Principle
[0049] 1.1 Add excess NaOH solution to the agarose strong alkaline medium to completely exchange the strong alkaline medium:
[0050] Bead-Cl+NaOH=Bead-OH+HCl
[0051] 1.2 Use NaCl to remove OH in the medium - Swap down:
[0052] Bead-OH+NaCl=Bead-Cl+NaOH
[0053] 1.3 Titrate the generated iodine OH with HCl standard solution - :
[0054] NaOH + HCl = H 2 O+NaCl.
[0055] Table 1 Test results
[0056]
[0057] Example 2
[0058] A method for synthesizing strong base agarose microspheres, the specific steps are as follows:
[0059] Step 1: Activation of beads:
[0060] First, 100 g of 6FF cross-linked beads were added to 250 ml of acetone and ethanol solution, wherein the volume ratio of acetone to ethanol was 1: 1. Then, 1 g of catalyst sodium carbonate was added and stabilized for 30 minutes.
[0061] Then, 80 ml of allyl glycidyl ether was slowly added dropwise, and the temperature was controlled at 45° C. After the addition was completed, the mixture was kept warm for 30 minutes and sampled for analysis. The results showed that the double bond content was 0.36 mmol / ml, which met the requirements.
[0062] Finally, the activated beads were washed with water, soaked in anhydrous ethanol for 3 times, each time for 10 minutes, washed with pure water until odorless, and placed in a glass container for the next reaction.
[0063] Step 2: Bromination of beads:
[0064] Add 100g activated beads and 500ml sodium acetate solution (4% by mass) into a flask and keep it at room temperature. Start to drop 600ml bromine water until the solution turns orange, then stop dropping. After stabilizing for 1 hour, drop 50ml sodium formate solution (50%) to make the solution colorless. After stabilizing for half an hour, discharge the material. Then, replace the beads with anhydrous ethanol until there is no water, soak in anhydrous ethanol for 30 minutes to react, and obtain brominated beads.
[0065] Step 3: Amination of beads:
[0066] First, 100 g of bromide beads were mixed with 400 ml of trimethylamine, and after stabilization for half an hour, the temperature was raised to 38°C and kept warm for 10 hours, during which sampling and analysis were required. After the reaction was completed, the material was discharged and the synthesis of agarose microspheres with strong base groups was completed to obtain strong base agarose microspheres.
[0067] Table 2 Test results
[0068]
[0069] Example 3
[0070] A method for synthesizing strong base agarose microspheres, the specific steps are as follows:
[0071] Step 1: Activation of beads:
[0072] First, 100 g of 6FF cross-linked beads were added to 250 ml of acetone and ethanol solution, wherein the volume ratio of acetone to ethanol was 1: 1. Then, 1 g of catalyst sodium carbonate was added and stabilized for 30 minutes.
[0073] Then, 70 ml of allyl glycidyl ether was slowly added dropwise, and the temperature was controlled at 45° C. After the addition was completed, the mixture was kept warm for 30 minutes and sampled for analysis. The results showed that the double bond content was 0.30 mmol / ml, which met the requirements.
[0074] Finally, the activated beads were washed with water, soaked in anhydrous ethanol for 3 times, each time for 10 minutes, washed with pure water until odorless, and placed in a glass container for the next reaction.
[0075] Step 2: Bromination of beads:
[0076] Add 100g activated beads and 500ml sodium acetate solution (4% by mass) into a flask and keep it at room temperature. Start to drop 600ml bromine water until the solution turns orange, then stop dropping. After stabilizing for 1 hour, drop 50ml sodium formate solution (50% by mass) to make the solution colorless. After stabilizing for half an hour, discharge the material. Then, replace the beads with anhydrous ethanol until there is no water, soak in anhydrous ethanol for 30 minutes to react, and obtain brominated beads.
[0077] Step 3: Amination of beads:
[0078] First, 100 g of brominated beads were mixed with 250 ml of dimethylethanolamine, and after stabilization for half an hour, the temperature was raised to 38°C and kept warm for 10 hours, during which sampling and analysis were required. After the reaction was completed, the material was discharged and the synthesis of agarose microspheres with strong base groups was completed, strong base agarose microspheres.
[0079] Table 3 Test results
[0080]
[0081] Comparative Example 1
[0082] The comparison with similar foreign products is shown in Table 4.
[0083] Table 4 Comparison results
[0084]
[0085] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for synthesizing strong base agarose microspheres, characterized in that: The following steps are involved: 1) mixing agarose beads, acetol solution and a catalyst and stabilizing for 30 minutes to obtain a stabilizer; 2) mixing the stabilizer obtained in step 1) with allyl glycidyl ether and heat-insulating to obtain activated beads; 3) mixing the activated beads obtained in step 2) with a sodium acetate solution and 3% by mass bromine water, stabilizing for 1 hour, and then mixing with a sodium formate solution and stabilizing for 30 minutes to obtain brominated beads; 4) The brominated beads obtained in step 3) are mixed with trimethylamine and stabilized for 30 minutes, and then incubated for reaction to obtain strong base agarose microspheres.
2. The synthesis method according to claim 1, characterized in that In the step 1), the volume ratio of the mass of the agarose beads to the acetol solution is 100 g:250 mL; The acetone alcohol solution is acetone and ethanol, and the volume ratio of the acetone to the ethanol is 1:
1.
3. The synthesis method according to claim 1, characterized in that The catalyst in step 1) is sodium carbonate, and the mass ratio of the agarose beads to the catalyst is 100:
1.
4. The synthesis method according to claim 1, characterized in that In the step 2), the volume ratio of allyl glycidyl ether to the acetone alcohol solution is 6 to 8:
25.
5. The synthesis method according to claim 1, characterized in that In the step 2), allyl glycidyl ether is added dropwise to the stabilizer, and the temperature is controlled at 43-45° C.; The temperature of the insulation is 43-45° C., and the time is 30 minutes.
6. The synthesis method according to claim 1, characterized in that In step 3), the volume ratio of the activated beads to the sodium acetate solution is 100 g:500 mL; The mass percentage of the sodium acetate solution is 4%.
7. The synthesis method according to claim 1, characterized in that In step 3), the volume ratio of bromine water to sodium acetate solution is 6:5; The bromine water is added dropwise to the mixture of the activated beads and the sodium acetate solution.
8. The synthesis method according to claim 1, characterized in that In step 3), the volume ratio of the sodium formate solution to the bromine water is 1:12; The mass percentage of the sodium formate solution is 50%.
9. The synthesis method according to claim 1, characterized in that In the step 4), the volume ratio of the mass of the brominated beads to trimethylamine is 100 g:250-400 mL.
10. The synthesis method according to claim 1, characterized in that The conditions for the heat preservation reaction in step 4) include: temperature of 38° C. and time of 10 h.