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Crosslinking method of agarose 4B microspheres

A cross-linking method and agarose technology, applied in the cross-linking field of agarose 4B microspheres, can solve the problems of inability to meet industrial pressure and flow rate, poor mechanical strength, and high water content, and achieve easy control and amplification, and less environmental pollution. , the effect of high degree of cross-linking

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Due to the high water content and large pore size of agarose 4B microspheres (agarose content is 4%), they can be used for the purification and separation of macromolecules such as proteins, but their mechanical strength is relatively poor, which greatly limits their application
Although many literatures have reported the cross-linking method of agarose 4B microspheres, it still cannot meet the needs of industrial pressure and flow rate.

Method used

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  • Crosslinking method of agarose 4B microspheres
  • Crosslinking method of agarose 4B microspheres
  • Crosslinking method of agarose 4B microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Take 40g of Sepharose 4B microspheres in a 500mL Erlenmeyer flask with a stopper, add 60mL of 1.0mol / L NaOH solution, let it stand at 30°C for 10min, then add 32mL of dimethyl sulfoxide, 6mL of absolute ethanol and 10mL of pentaerythritol glycidol in sequence Ether was reacted in a water-bath shaker at 30° C. and 170 rpm for 6 h, and washed with 20% ethanol, acetone, and deionized water in sequence.

[0020] Take 40g of cross-linked microspheres in a 500mL Erlenmeyer flask with a stopper, add 60mL, 3.0mol / L NaOH solution, let stand at 30°C for 10min, then add 32mL dimethyl sulfoxide and 10mL epichlorohydrin in turn, put in In a water-bath shaker at 170 rpm, wash with 20% ethanol, acetone, and deionized water at 30° C., 35° C., and 40° C. for 2 hours respectively. After high temperature sterilization, the loss rate is about 1.5%.

[0021] The agarose 4B microspheres after the secondary cross-linking were packed into a column (column inner diameter 1cm, column height 3cm...

Embodiment 2

[0023] Take 40g of Sepharose 4B microspheres in a 500mL Erlenmeyer flask with a stopper, add 60mL of 1.0mol / L NaOH solution, let it stand at 30°C for 10min, then add 34mL of dimethyl sulfoxide, 6mL of absolute ethanol and 8mL of pentaerythritol glycidol in sequence Ether was reacted at 30° C. in a water-bath shaker at 170 rpm for 6 h, and washed with 20% ethanol, acetone, and deionized water in sequence.

[0024] Take 40 g of cross-linked microspheres into a 500 mL Erlenmeyer flask with a stopper, add 60 mL of 3.0 mol / L NaOH solution, let it stand at 30°C for 10 min, then add 34 mL of dimethyl sulfoxide and 8 mL of epichlorohydrin in sequence, and place in In a water-bath shaker at 170 rpm, wash with 20% ethanol, acetone, and deionized water at 30° C., 35° C., and 40° C. for 2 hours respectively. After high temperature sterilization, the loss rate is about 1.6%. The agarose 4B microspheres after the secondary cross-linking were packed into a column (column inner diameter 1cm,...

Embodiment 3

[0026] Take 40g of Sepharose 4B microspheres in a 500mL Erlenmeyer flask with a stopper, add 50mL of 1.0mol / L NaOH solution, let it stand at 30°C for 10min, then add 32mL of dimethyl sulfoxide, 6mL of absolute ethanol and 15mL of pentaerythritol glycidol Ether was reacted at 30° C. in a water-bath shaker at 170 rpm for 5 h, and washed with 20% ethanol, acetone, and deionized water in sequence.

[0027] Take 40 g of once cross-linked microspheres into a 500 mL Erlenmeyer flask with a stopper, add 50 mL of 3.0 mol / L NaOH solution, let it stand at 30°C for 10 min, then add 32 mL of dimethyl sulfoxide and 15 mL of epichlorohydrin in sequence, and place in In a water-bath shaker at 170 rpm, wash with 20% ethanol, acetone, and deionized water at 30° C., 35° C., and 40° C. for 2 hours respectively. After high temperature sterilization, the loss rate is about 1.8%.

[0028] The agarose 4B microspheres after the secondary cross-linking were packed into a column (column inner diameter ...

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Abstract

The invention discloses a crosslinking method of agarose 4B microspheres. The agarose 4B microspheres as materials are subjected to crosslinking by two-steps by using pentaerythritol glycidyl ether and epoxy chloropropane as a crosslinking agent. The method comprises the following steps: 1), the primary crosslinking of the agarose 4B microspheres: arranging agarose 4B microspheres, sodium hydroxide and dimethylsulfoxide, absolute ethyl alcohol and pentaerythritol glycidyl ether in a three-corner flask with a plug, shaking in a water bath shaker, and mechanically stirring for a certain time, so as to obtain primarily cross-linked agarose 4B microspheres; and 2), the secondary cross linking of agarose 4B microspheres: mixing the agarose 4B microspheres after the primary cross linking with sodium hydroxide solution uniformly, standing, sequentially adding dimethylsulfoxide and epoxy chloropropane, shaking in the water bath shaker, so as to obtain the secondarily cross-linked agarose 4B microspheres. The agarose 4B microspheres have excellent sphericity, high temperature and high pressure resistance, excellent hydrodynamics characteristics, excellent chemical stability and the like, and can be used as a substrate for biological macromolecules chromatography separation.

Description

technical field [0001] The invention relates to a cross-linking method of agarose 4B microspheres Background technique [0002] Chromatography is a widely used technique for separating and purifying macromolecular substances, and the chromatographic medium is in the most critical position in the chromatographic system. The chromatographic separation medium based on natural polysaccharide agarose is a classic biological molecular separation material. It is widely used due to its incomparable advantages such as porosity, hydrophilicity, electrical neutrality, and easy derivatization. For the separation and purification of various bioactive macromolecules. However, the skeleton structure of uncrosslinked agarose gel is fixed by hydrogen bonds, and it is a soft matrix, which is easy to compress during application, and the back pressure of the column bed is high, which cannot meet the requirements of high flow rate chromatography. [0003] In recent years, through the chemical ...

Claims

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Application Information

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IPC IPC(8): B01J13/14C08J3/24C08L5/12
Inventor 夏海锋郑梦杰吴璞强
Owner JIANGNAN UNIV
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