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Method of preparing zinc contained chelating affinity super-macro-porosity crystal gel medium for column-chromatography

A technology of crystal gel medium and column chromatography, applied in chemical instruments and methods, and other chemical processes, can solve the problem of low adsorption capacity, achieve high separation efficiency, easy large-scale production, easy elution and regeneration

Inactive Publication Date: 2009-07-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chelated Cu was successfully developed in Europe in 2002 2+ Acrylamide-based ultra-macroporous crystal-linked gel medium, but its adsorption capacity is low (the adsorption capacity for bovine serum albumin BSA or lysozyme is 0.1-0.2mg / mL bed layer)
However, there is currently no column chromatography with Zn 2+ Chelating-affinity super-large pore continuous bed gel media

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Dissolve 1.45g of AAm monomer, 0.06g of cross-linking agent MBAAm and 0.1g of AGE in 15mL of deionized water. After stirring evenly, quickly add 9mg of TEMED and 23mg of APS. In the column, after being sealed, in a programmable temperature-controlled constant temperature cooling system, the temperature is lowered from 0°C to -40°C, and then the temperature is kept constant to carry out cooling crystallization and porogenous polymerization. After 39 hours, the temperature was raised to room temperature to obtain a super-large-pore continuous bed crystal gel medium matrix.

[0033] At 20°C, use 0.2 matrix volume of 0.06M Na 2 CO 3 solution, 0.01M IDA solution with 5 times the volume of the matrix, respectively activated the obtained matrix for 24 hours, and then used 0.5M ZnSO with 2 times the volume of the matrix 4 The solution undergoes a chelation reaction to obtain Zn 2+ Chelating affinity gel medium. Its porosity is 56%, and SEM shows that its pore diameter range...

Embodiment 2

[0035] Dissolve 1.95g of DMAAm monomer, 0.5g of crosslinking agent N,N'-diacryloylethylenediamine and 0.6g of AGE and ethylene glycol diglycidyl ether (mass ratio 1:1) mixture ligand material in 50ml After stirring evenly in deionized water, quickly add 29mg TEMED and 53mg APS, put the resulting mixture into a chromatographic column with an inner diameter of 26mm and a length of 200mm, seal it, and carry out cooling and crystallization in a programmable temperature-controlled constant temperature cooling system Hole-causing. The temperature change process is: (A) cooling: from 0°C to -65°C; (B) heating: heating to -7°C; (C) constant temperature: constant temperature for 5 hours; (D) cooling: cooling from -7°C again to -20°C; (E) constant temperature: constant temperature for 16 hours; (F) temperature rise: heat up to room temperature to obtain a super-large-pore continuous bed crystal gel medium matrix.

[0036] The obtained matrix was activated with 12 times the matrix volum...

Embodiment 3

[0038] Dissolve 0.8g of DMAEMA monomer, 0.41g of cross-linking agent MBAAm and 0.4g of ligand material ethylene glycol diglycidyl ether in 11ml of deionized water. After stirring evenly, quickly add 49mg of TEMED and 45mg of APS, and put the resulting mixture into In a glass chromatography column with an inner diameter of 10mm and a length of 200mm, after sealing, the temperature is lowered to -19°C in a programmable temperature-controlled constant temperature cooling system for cooling and crystallization to form pores. Then, the crystals were melted at room temperature to obtain a super-macroporous continuous bed crystal gel medium matrix.

[0039] The obtained medium matrix was reacted with 1M NaOH solution 4 times the volume of the matrix at 50°C, then reacted with 0.8M EDT solution 3 times the volume of the matrix at 50°C for 10 h, and finally reacted with 5M ZnCl 10 times the volume of the matrix 2 Solution chelation reaction to get Zn 2+ Chelating affinity gel medium. ...

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Abstract

The invention relates to a metal ion chelating affinity type ultra-large pore continuous bed crystal gel medium for column chromatography and a preparation method thereof. The crystal gel medium is columnar or disc-shaped, the inner surface is solidly loaded with Zn2+ affinity ligand, the porosity is 50-98%, the pore diameter range is 5-500 μm, and the diameter or equivalent diameter of the crystal gel medium is greater than or equal to 10mm . The crystal gel medium can be directly placed in the chromatography column for chromatographic operation. It has good connectivity and can be operated in a high flow rate range; the crystal gel medium has good adsorption and separation performance, large adsorption capacity, high separation efficiency, and easy elution and regeneration. easy. It is suitable for large-scale separation and purification of conventional fermentation products, biochemical drugs, downstream targets of genetic engineering, etc., and has a wide range of applications. The preparation of the crystal gel medium is easy to obtain monomer materials, the process is simple and fast, the cost is low, and the large-scale production is very easy.

Description

(1) Technical field [0001] The invention relates to the technical fields of bioseparation and medicine, and relates to a metal ion chelation affinity type columnar or disc-shaped ultra-large-pore continuous bed crystal glue bioseparation medium for column chromatography and a preparation method thereof. (2) Background technology [0002] The ultra-large pore continuous bed chromatography separation method is a new type of biological chromatography separation technology. The medium of the ultra-large pore continuous bed is called crystal gel medium, and its pore size reaches tens to hundreds of microns, which can directly pass the microbial cells or cell fragments in the fermentation broth. Therefore, for complex feed fluids such as fermentation broth, culture fluid, and lysate, pretreatments such as centrifugation, sedimentation, and filtration can be used, and this technology can be used to directly extract and separate biomacromolecular target substances from these raw flu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/282B01J20/30
Inventor 姚克俭沈绍传贠军贤
Owner ZHEJIANG UNIV OF TECH