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Method for synthesizing agarose gel hydrogen bond adsorbing chromatography medium by using quercetin as genin
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A technology of agarose gel and hydrogen bond adsorption, which is applied in the preparation method of peptides, chemical instruments and methods, oxytocin/vasopressin, etc., can solve the problems of complex process, low yield, low purity, etc. Achieve the effect of simple and easy steps
Active Publication Date: 2011-01-26
浙江华军药业有限公司 +1
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[0007] The purpose of the present invention is to overcome the problems of low purity, low yield and complicated process of purifying oxytocin from the synthetic solution by using traditional methods, and prepare a hydrogen bonded oxytocin with agarose gel as the matrix and quercetin as the ligand. Adsorption medium, providing a high-selectivity, high-purity, high-yield chromatographic separation medium for one-step purification of oxytocin
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Embodiment 1
[0020] Example 1. Preparation of agarose gel hydrogen bond adsorption chromatographic media for purification of oxytocin by using short-chain epoxidation reagentepichlorohydrin to link the ligand quercetin
[0021] 1) 1 liter of highly cross-linked agarose gel microspheres with a concentration of 12% and an average particle size of 30 μm, washed with water and transferred to a reaction flask or reactor;
[0022] 2) Add 200ml of water, add 10g of sodiumsulfate, and stir for 2h;
[0023] 3) Add 200ml of water and 50ml of 30% sodiumhydroxide and stir at 60rpm;
[0024] 4) Add 40ml epichlorohydrin, stir at 25°C, 60rpm, and react for 6h;
[0026] 6) Add 6mg quercetin and hesperetin, 30% sodium hydroxide, 25°C, 60rpm stirring, and react for 12h;
[0027] 7) Adjust pH to 6 with hydrochloric acid and wash with 10 times volume of water.
Embodiment 2
[0028] Example 2. Using long-chain epoxidation reagent 1,4-bis-(2,3-epoxypropoxy)-butane to connect quercetin to prepare agarose gel hydrogen bond adsorption for oxytocin purification Chromatographic medium
[0029] 1) 1 liter of highly cross-linked agarose gel microspheres with a concentration of 12% and an average particle size of 30 μm, washed with water and transferred to a reaction flask or reactor;
[0030] 2) Add 200ml water, add 15g sodium sulfate, and stir for 3h;
[0031] 3) Add 200ml water and 50ml 50% sodium hydroxide, stir at 60rpm;
[0032] 4) Add 60ml of 1,4-bis-(2,3-epoxypropoxy)-butane, stir at 25°C, 60rpm, and react for 8h;
[0034] 6) Add 9mg of quercetin and hesperetin, 30% sodium hydroxide, 25℃, 60rpm stirring, and react for 15h;
[0035] 7) Adjust pH to 6 with hydrochloric acid and wash with 10 times volume of water.
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Abstract
The invention relates to a method for synthesizing a hydrogen bond adsorbing medium by taking agarose gel as a matrix and taking quercetin as genin. The surface of an agarose gel medium is provided with a great amount of hydroxyl for reactions and a great amount of polar groups such as ether bonds, semiacetal, primary alcohol, secondary alcohol and the like which can react with peptide bonds (amido bonds) in peptides molecules, and alcoholic hydroxyl groups, phenolichydroxyl, sulfhydryl groups and the like in a side chain to form hydrogen bonds. The agarose gel hydrogen bond adsorbing chromatography medium for oxytocin is prepared by connecting short link epoxidereagentepichlorohydrin and long link epoxidereagent 1,4-dual-(2,3-epoxy propyl group)-butane activated agarose gel with the quercetin serving as the genin. The medium is used for the oxytocin by liquid-phase synthesis and solid-phase synthesis.
Description
Technical field [0001] The invention relates to a method for synthesizing a chromatographic medium, in particular to a method for synthesizing a quercetin agarose gel hydrogen bond adsorption chromatographic medium. [0002] The invention belongs to the field of biochemical separation of bioengineering. Background technique [0003] Agarose gel is a natural polysaccharide chromatographic medium. It was invented in the 1960s. It has many characteristics of an ideal medium: high pore size (90-96%), which can maintain high pore size even at high concentrations; The open structure of the connecting fibers; the high connectivity effectively ensures the intramolecular material transfer; the large specific surface area provides a high adsorption capacity, and it is easy to prepare spherical particles with different agarose concentrations and different sizes to meet the needs of different fields; It is stable in sodiumhydroxide and can meet the harsh in-place cleaning steps; low non-spec...
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