CN1087641C - Method for synthesizing metal chelated affinity membrane chromatographic medium for radial chromatographic column - Google Patents
Method for synthesizing metal chelated affinity membrane chromatographic medium for radial chromatographic column Download PDFInfo
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- CN1087641C CN1087641C CN97105052A CN97105052A CN1087641C CN 1087641 C CN1087641 C CN 1087641C CN 97105052 A CN97105052 A CN 97105052A CN 97105052 A CN97105052 A CN 97105052A CN 1087641 C CN1087641 C CN 1087641C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N2030/386—Radial chromatography, i.e. with mobile phase traversing radially the stationary phase
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Abstract
The present invention relates to a method for synthesizing a metal chelated affinity membrane chromatographic medium for a radial chromatographic column. The method is characterized in that after epoxy propyl methylacrylate carries out automatic polymerization reaction to prepare a polymer, the polymer and a cotton fiber carry out grafting reaction; then, the reaction product and iminoacetic acid carry out ring cleavage reaction; and finally, metal ions are of transition to carry out chelation reaction. The method has the advantages of low price and easy obtainment of raw materials, simple reaction conditions, easy control and large binding capacity of the prepared chelated medium. The radial chromatographic column which is filled with the medium is used for separating and purifying biological macromolecules comprising albumin human, catalase, urokinase, etc. The method has the advantages of large treating capacity, high flow speed, high purification multiple, convenient linear amplification, etc.
Description
The invention provides a kind of is matrix to lack cotton fiber, behind certain living polymer polymkeric substance and fibre grafting, but introduce the part---imido-acetic acid of chelating transition metal ion again, make the method that is suitable for the metal-chelating film chromatography medium that separation and purification of biological macromolecule uses.
Bioengineering and engineered developing rapidly to society provides increasing biologics, however have only the biologics that passes through separation and purification and reach certain purity to be utilized by people, especially for clinical biochemical drug.The separation and purification of biological macromolecule technology of classics that with soft gel is matrix can not adapt to the needs of current development far away because flow velocity is slow, pressure drop is big, disengaging time is long, shortcoming such as difficult amplification when handling on a large scale.The mid-80 has been released radially membrane chromatography technology, it is that membrane chromatography technology and chromatographic resolution principle are combined, adopt the Radial Flow technology, overcome the shortcoming of traditional colour spectral technology, can be when less bed height, obtain bigger treatment capacity, can also reach big flow velocity, in addition, it can carry out linear amplification, is promptly keeping the post footpath constant and when only increasing column length, can increase the treatment capacity of sample in proportion, therefore, radially the membrane chromatography technology is the effective tool that a kind of comparatively ideal bioengineering separates purification on a large scale.
The affinity chromatography technology is the important means of separation and purification biomacromolecule, is characterized in that selectivity is good, purifying multiple height.The metal chelate affinity chromatography that with the transition metal ion is the versatility part is with its low cost of manufacture, good, the higher separation and purification that is widely used in biologics of purifying multiple of versatility.Affinity chromatography technology and above-mentioned radially membrane chromatography technology combined to form a kind of new separation technology, we are referred to as radially affinity film chromatography technology, and it has concurrently, and membrane chromatography technology flow velocity radially is fast, treatment capacity big, the easy advantage of linear amplification and affinity chromatography technology high selectivity.It is a kind of very potential isolation technics.Cellulosic non-specific adsorption is less, suitable aperture structure is arranged, it can be by methods such as esterification, etherificate, oxidations, making the cellulose affinity media on reactive group or the affinity ligand introducing cellulose skeleton, but this several method can only obtain very limited binding capacity under the situation of not destroying cellulosic structure, make that the column capacity of corresponding pillar is very little, bring difficulty for this class medium application in radial chromatographic column.
The purpose of this invention is to provide the synthetic method of the radial chromatography column packing one metal chelating mould assembly affinity media that a kind of Selective Separation purifying that is suitable for biomacromolecule adopts, the raw material of this synthetic method utilization cheaply is easy to get, reaction conditions is simple, easy to control.
The synthetic method of radial chromato bar immobilized metal ion afinity chromatography medium of the present invention, be to adopt to carry out self-polymeric reaction with glytidyl methacrylate or acrylic acid epoxy propyl ester and obtain polymkeric substance, carry out graft reaction with cellulose again, add imido-acetic acid then and carry out ring-opening reaction as the chelating aglucon, add the process that metal ion solution reacts at last, autohemagglutination and graft reaction carry out in aqueous medium, and temperature of reaction is 50～90 ℃.And in above-mentioned reaction, add initiating agent, and azoisobutyronitrile for example, n-BuLi or ammonium persulfate and sodium thiosulfate, the initiating agent addition is 1～5% of a polymerization single polymerization monomer weight.In addition, to be controlled at pH be 8～13 to chelating aglucon imido-acetic acid acid ring-opening reaction in the above-mentioned reaction, temperature of reaction is 40～80 ℃, and can be by adding inorganic salts, for example sodium chloride or sodium sulphate improve the conversion ratio of ring-opening reaction as ring-opening reaction promoter, to improve the aglucon concentration on the chelating media that is synthesized.The mother liquor of ring-opening reaction is capable of circulation to be applied mechanically.Other process in the above-mentioned course of reaction, even can carrying out with reference to technique known.Specifically, synthetic method of the present invention is pressed step and is carried out:
1. glytidyl methacrylate or acrylic acid epoxy propyl ester carry out the self-polymeric reaction synthetic polymer;
2. cellulose and polymkeric substance carry out graft reaction;
3. add chelating reagent and carry out ring-opening reaction;
4. add transition metal ion (as Cu
2+, Zn
2+, Fe
3+, Ni
2+Deng) solution carries out chelatropic reaction.
In above-mentioned 1,2 reaction, the control temperature of reaction is 50～90 ℃, and 0.5～2 hour self-polymeric reaction time, the graft reaction time is 0.5～2.5 hour.Be reflected in the deionized water and carry out.In course of reaction, for improving the epoxy content on polymerizate and the cellulosic graft reaction product, the consumption of (methyl) acrylic acid epoxy propyl ester should be excessive, and reactant is pressed (weight) cellulose: (methyl) acrylic acid epoxy propyl ester=1: 2～10 add.Be accelerated reaction simultaneously, can add initiating agent to bring out reaction, as adopting initiating agent azo isobutyronitrile, n-BuLi or sodium thiosulfate and the ammonium persulfate of anionic polymerisation, the addition of initiating agent is 1～5% of a polymerization single polymerization monomer.The ring-opening reaction temperature is controlled at 40～80 ℃, and the reaction time can be controlled at according to the aglucon concentration of required acquisition 0.5～8 hour.Ring-opening reaction is carried out in weak alkaline medium, and the pH value is controlled at 8～13.Ring-opening product is the chelating aglucon, should be as the addition of imido-acetic acid (IDA) above the amount of the epoxide group on the graft polymerization post-modification cellulose, and with 1～5: 1 is advisable.For further improving the concentration that is connected to the chelating aglucon on the modified cellulose, an amount of inorganic salts can be added, in reaction mixture as sodium chloride, sodium sulphate etc., the adding weight of inorganic salts is 1～10% of cellulose amount, and reacted mother liquor is capable of circulation after filtering to be applied mechanically, to reduce cost.Give further instruction below by example to synthetic reaction of the present invention.
The preparation of example 1. imido-acetic acid type (IDA) metal-chelating media
The building-up process of imido-acetic acid type metal-chelating medium is undertaken by following chemical reaction:
In above-mentioned ring-opening reaction 3, the ring-opening reaction thing is imido-acetic acid (IDA), control pH=8～13 of reaction mixture in the reaction with the sodium carbonate liquor of 2mol/l, temperature of reaction is controlled at 40～80 ℃, and the addition of inorganic salts is 1～5% of a reactant liquor cumulative volume.Experimentation is, stirrer is being housed, temperature is taken into account and is added the short cotton fibers of 65 grams in the 5 liter there-necked flasks of condenser, 3000 ml deionized water, be warming up to 80 ℃ under stirring, stir, the glytidyl methacrylate that adds 300 milliliters subsequently stirred after 10 minutes, added the mixed solution that contains ammonium persulfate and each 10 gram of sodium thiosulfate again and made initiating agent, insulation reaction 2 hours, be cooled to 70 ℃ then, add imido-acetic acid solution and carry out ring-opening reaction, and with pH=8～13 of sodium carbonate liquor conditioned reaction potpourri, insulation reaction 1～8 hour, after stopping reaction, be washed till neutrality, soak with acetone or alcohol then with a large amount of deionized waters, the residue on flush away modified cellulose surface is again with a large amount of deionization washings.Add the copper-bath of 2% (weight/volume), stirred～2 hours, filter, be washed till no copper ion with a large amount of deionized waters.Vacuum drying, the percent grafting of gained modified cellulose are 140% (weight), are the dried media of 150 μ mol/g with the aglucon concentration of nitrogen elemental microanalysis method or aas determination metal-chelating medium.Chelatropic reaction 4 also can be done film forming in cellulose media and carry out later on again.
The application of example 2 metal-chelating media
IDA type copper ion chelating media prepared in the example 1 is made paper shape film with paper technology, be filled into radially in (or axially) chromatography column, with 0.05mol/l phosphate buffer (pH=7.0) the balance pillar that contains 1mol/l sodium chloride, beef liver catalase crude product solution (1000U/mg albumen) is gone up sample separates, with the 0.01mol/l imidazoles wash-out that contains 1mol/l sodium chloride, the purity of the beef liver catalase behind the purifying has improved 7 times, and the recovery reaches 70%.Chelating media can repeatedly use after 0.005mol/l EDTA-NaCl regeneration repeatedly, and it is stable that separating property keeps.
By above-mentioned example, it is big to the binding capacity of protein or enzyme to adopt synthetic method provided by the invention can prepare radially (or axially) look synthetic chelating media easily, be easy to regeneration, but repeated multiple times is used.Radially (or axially) chromatographic column of using this chelating media filling can be rich in the protein or the enzyme of residues such as histidine, cysteine, tryptophane to many surfaces, for example, seralbumin, immunoglobulin (Ig), urokinase, peroxidase etc. carry out the Selective Separation purifying, not only treatment capacity is big, flow velocity fast, it is active good to keep, and selectivity is better, and purifying is doubly higher.The present invention is for the radial chromatography medium has increased new kind, and the synthetic method of the radially metal chelated affinity membrane chromatographic medium that is provided is a kind of biochemical isolation technics that prospects for commercial application is arranged very much.
Claims (7)
1. the synthetic method of a radial chromato bar immobilized metal ion afinity chromatography medium, it is characterized in that this synthetic method is to adopt to carry out self-polymeric reaction with glytidyl methacrylate or acrylic acid epoxy propyl ester and obtain polymkeric substance, carry out graft reaction with cellulose again, add imido-acetic acid then and carry out ring-opening reaction, add the process that metal ion solution carries out chelatropic reaction at last as the chelating aglucon.Autohemagglutination and graft reaction carry out in aqueous medium, and autohemagglutination and graft reaction temperature are 50～90 ℃.
2. by the described synthetic method of claim 1, the same amount that it is characterized in that reactant glytidyl methacrylate or acrylic acid epoxy propyl ester should be excessive, and reactant weight is in glytidyl methacrylate or acrylic acid epoxy propyl ester: cellulose=2～10: 1 ratio.
3. according to the described synthetic method of claim 1, it is characterized in that adding initiating agent azoisobutyronitrile, n-BuLi or ammonium persulfate and sodium thiosulfate in autohemagglutination and the graft reaction, the initiating agent addition is 1～5% of a polymerization single polymerization monomer weight.
4. according to the described synthetic method of claim 1, it is characterized in that it is 8～13 that chelating aglucon imido-acetic acid ring-opening reaction is controlled at pH, temperature of reaction is 40～80 ℃, and can improve the conversion ratio of ring-opening reaction as ring-opening reaction promoter by adding sodium chloride, to improve the aglucon concentration on the chelating media that is synthesized, the mother liquor of ring-opening reaction is capable of circulation to be applied mechanically.
5. according to the described synthetic method of claim 4, it is characterized in that the addition of chelating aglucon imido=acetate should surpass the amount of the epoxy radicals on the graft polymerization post-modification cellulose, its ratio is 1～5: 1.
6. according to the described synthetic method of claim 1, it is characterized in that ring-opening reaction adds inorganic salts sodium chloride or sodium sulphate is done ring-opening reaction promoter, it adds weight is 1～10% of cellulose amount.
7. according to the described synthetic method of claim 1, it is characterized in that adding transition metal ion Cu
2+, Zn
2+, Fe
3+Or Ni
2+Solution carries out chelatropic reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97105052A CN1087641C (en) | 1997-01-24 | 1997-01-24 | Method for synthesizing metal chelated affinity membrane chromatographic medium for radial chromatographic column |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97105052A CN1087641C (en) | 1997-01-24 | 1997-01-24 | Method for synthesizing metal chelated affinity membrane chromatographic medium for radial chromatographic column |
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| CN1188681A CN1188681A (en) | 1998-07-29 |
| CN1087641C true CN1087641C (en) | 2002-07-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11325104B2 (en) | 2017-12-07 | 2022-05-10 | Emp Biotech Gmbh | System and method of applied radial technology chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103108693A (en) * | 2010-07-29 | 2013-05-15 | Emd密理博公司 | Grafting method to improve chromatography media performance |
| CN103012228B (en) * | 2012-11-30 | 2014-07-02 | 西北农林科技大学 | Method for separating and purifying astaxanthin by silver ion chelating chromatographic column |
| CN104630169A (en) * | 2014-12-31 | 2015-05-20 | 唯美度科技（北京）有限公司 | CAT (catalase) extracted from earthworms |
| CN112892211B (en) * | 2021-01-28 | 2023-06-09 | 中国工程物理研究院核物理与化学研究所 | Column type hydrogen helium separation and concentration coupling device and method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4627918A (en) * | 1985-11-04 | 1986-12-09 | Sepragen Corporation | Chromatography column using horizontal flow |
| CN1072421A (en) * | 1991-11-20 | 1993-05-26 | 中国科学院大连化学物理研究所 | Synthesizing of weak positive ion medium for radial cbromato bar |
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- 1997-01-24 CN CN97105052A patent/CN1087641C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4627918A (en) * | 1985-11-04 | 1986-12-09 | Sepragen Corporation | Chromatography column using horizontal flow |
| CN1072421A (en) * | 1991-11-20 | 1993-05-26 | 中国科学院大连化学物理研究所 | Synthesizing of weak positive ion medium for radial cbromato bar |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11325104B2 (en) | 2017-12-07 | 2022-05-10 | Emp Biotech Gmbh | System and method of applied radial technology chromatography |
| US11731107B2 (en) | 2017-12-07 | 2023-08-22 | Emp Biotech Gmbh | System and method of applied radial technology chromatography |
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| CN1188681A (en) | 1998-07-29 |
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