CN101531734A - Preparation and application of composite TiO*copolymer microspheres - Google Patents
Preparation and application of composite TiO*copolymer microspheres Download PDFInfo
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- CN101531734A CN101531734A CN200810101947A CN200810101947A CN101531734A CN 101531734 A CN101531734 A CN 101531734A CN 200810101947 A CN200810101947 A CN 200810101947A CN 200810101947 A CN200810101947 A CN 200810101947A CN 101531734 A CN101531734 A CN 101531734A
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Abstract
The invention relates to a method for preparing composite TiO2 copolymer microspheres and application of the composite TiO2 copolymer microspheres in natural product separation and purification. According to the method, an oil phase containing surface-modified TiO2, benzoyl peroxide serving as an initiator, GMA serving as a monomer, DVB serving as a crosslinking agent and the oil phase of a pore forming agent is added into a composite aqueous phase containing gelatin and sodium chloride, and the mixture is heated to cure to form microspheres; and beta-CD is directly immobilized on the microspheres serving as a immobilization matrix in the presence of sodium hydride to form the composite TiO2 copolymer microspheres. The copolymer microspheres can be used as an expanded bed adsorption medium to separate isoflavones from soybean waste molasses and purify the isoflavones. The method has the advantages of simple preparation process and easy control and amplification. The prepared TiO2 copolymer microspheres have the advantages of good medium sphericity, mild elution conditions, high biocompatibility, excellent hydrophilicity and wide application prospect in the natural product separation and purification.
Description
Technical field
The present invention is specifically related to a kind of composite Ti O
2The preparation of copolymer microsphere and the application in natural product (soybean isoflavones) separation and purification thereof.
Background technology
Expanded Bed Adsorption (Expanded Bed Adsorpt ion, EBA) technology is a kind of novel protein separating and purifying technology that last century, the nineties grew up, can directly from fermented liquid, cell culture fluid or homogenate, catch target product, collect solid-liquid separation, concentrate and preliminary purification among an operating unit, reduced operation steps, improve product yield, reduced purifying expense and capital contribution, be described as first the new unit operation that occurred in the last few years.The performance characteristic of expanding bed is between fluidized-bed and fixed bed, and sorbent material relies on the liquid countercurrent motion to suspend in bed, and the density and the particle diameter of sorbent material have certain distribution simultaneously, therefore can form stable grade bed in expanding bed.
The Expanded Bed Adsorption matrix that exploitation is suitable for is one of expanding bed technology key in application.The adsorbent matrix that is applied to the expanding bed technology mainly contains two kinds: a kind of is at the hydrophilic natural macromolecular material of inert high-density nuclear outsourcing last layer, as agarose-quartz sand, agarose-stainless steel, dextran-silica gel, Mierocrystalline cellulose-titanium dioxide etc.; Another kind is directly to use highdensity material, as sintered glass, zirconium dioxide-hydrogel, (per) fluoropolymer etc.Expansion bed substrate has been successfully used to intestinal bacteria homogenate, inclusion body at present, the intestinal bacteria nutrient solution, yeast cell homogenate, yeast culture liquid, the extraction of Hybridoma Cell Culture liquid and animal tissues's product, also expanding bed can be used as bio-reactor, but the expansion bed substrate that do not appear in the newspapers as yet is in the Separation of Natural Products Application in Purification.Synthetic copolymer has obtained widely using in the fixed bed chromatography as matrix but as the also rarely seen report of Expanded Bed Adsorption agent matrix.
Summary of the invention
The object of the present invention is to provide a kind of composite Ti O
2The preparation method of copolymer microsphere Expanded Bed Adsorption matrix and the application in soy molasses separation and purification soybean isoflavones thereof.
A kind of composite Ti O provided by the present invention
2The preparation method of copolymer microsphere may further comprise the steps:
1) gelatin and sodium-chlor are added in the deionized water, heated and stirred is warming up to 60 ℃, obtains containing the aqueous solution of gelatin and sodium-chlor, and the massfraction of gelatin and sodium-chlor is respectively 0.5～2.5% and 4～12% in the aqueous solution;
2) with the TiO of surface modification
2Join and stir the organic phase solution that obtains stable dispersion in the mixing solutions of initiator, monomer, linking agent and pore-creating agent, wherein said initiator is a benzoyl peroxide, described monomer glycidyl methacrylate (GMA), described linking agent is divinylbenzene (DVB), the mass ratio of GMA and DVB is 4～1:1, described pore-creating agent is the miscellany of polyoxyethylene glycol and butylacetate, and the add-on of initiator is 0.5～2% of a monomer mass, TiO
2Add-on be 5～80% of monomer mass, the add-on of pore-creating agent is 50～130% of a monomer mass;
3) aqueous solution and step 2 that step 1) is made) Zhi Bei organic phase solution by volume 2～7:1 mix, stir and to be warming up to 90 ℃ of reaction 9h after fixing, filtration, washing, dryings and to obtain copolymer microsphere;
4) with the copolymer microsphere of step 3) preparation, join exsiccant N, swelling in the dinethylformamide (DMF);
5) (β-CD) is dissolved among the DMF and adds sodium hydride NaH rapidly, and add-on is 0.1～0.5% of β-CD quality, till stirring reaction to no hydrogen is emitted in shaking table under the normal temperature, obtains β-CD sodium salt solution with beta-cyclodextrin;
6) swelling in the step 4) is good copolymer microsphere is added in the β-CD sodium salt solution of clear in the step 5), the consumption of β-CD sodium salt solution is 1～5 times of copolymer microsphere quality, stirring reaction 15～20h in 60～80 ℃ shaking bath, reaction finishes back suction filtration, washing, and oven dry makes composite Ti O
2Copolymer microsphere.
Described composite Ti O
2The particle diameter of copolymer microsphere is that 40～400 μ m, wet true density are 1.1～1.5g/cm
3
A kind of composite Ti O provided by the present invention
2Copolymer microsphere is as Expanded Bed Adsorption matrix separation and purification soybean isoflavones from soy molasses, and concrete steps are as follows:
1) with composite Ti O
2After the copolymer microsphere dress is opened column, with 0.01～0.05mol/L, the phosphate buffer soln equilibrium adsorption chromatography column of pH5～8, and Expanded Bed Adsorption post on the waste molasses after will diluting;
2) after absorption finishes, there is not absorption peak at ultraviolet spectrometer 254nm with deionized water wash cylinder to washings;
3) be that 50%～80% ethanolic soln carries out gradient elution with concentration, elution volume is 6～12 column volumes, isolates soybean isoflavones.
The present invention has following beneficial effect:
1) the prepared composite Ti O of the present invention
2The copolymer microsphere regular shape helps the stable of expanding bed operation;
2) the prepared composite Ti O of the present invention
2Copolymer microsphere matrix has suitable density and size distribution, is applicable to expanding bed;
3) the prepared composite Ti O of the present invention
2The density of copolymer microsphere and size can be regulated, and can satisfy the needs of different application environment;
4) preparation technology of the present invention is simple, is easy to control and amplification;
5) the prepared composite Ti O of the present invention
2Copolymer microsphere matrix elution requirement gentleness, good biocompatibility, good hydrophilic property is being with a wide range of applications aspect the Separation of Natural Products purifying.
The invention will be further described below in conjunction with embodiment.
Embodiment
Embodiment 1
1) in the 1L there-necked flask that agitator, prolong and thermometer are housed, adds 4g gelatin, 21g sodium-chlor and 500g deionized water, be warming up to 60 ℃ and stir half an hour;
2) with the TiO of 30g surface modification
2Join in the mixing solutions of 100g glycidyl methacrylate, 64g divinylbenzene, 1.4g benzoyl peroxide, 44.2g poly(oxyethylene glycol) 400 and 18g butylacetate, stir the organic phase solution that obtains stable dispersion;
3) with step 2) organic phase solution that obtains slowly joins the aqueous phase that step 1) obtains, regulate mixing speed to 350rpm, it is constant to keep rotating speed, and 60 ℃ of low suspensions disperse 45min, be warming up to 90 ℃, insulation 9h, curing obtains white microballoon, filters, hot wash 5 times, pore-creating agent is removed in the acetone extracting, and vacuum-drying obtains copolymer microsphere to constant;
4) take by weighing the copolymer microsphere that the 100g step 3) prepares, join exsiccant N, swelling is spent the night in the dinethylformamide (DMF);
5) with the 100g beta-cyclodextrin (β-CD) is dissolved among the DMF and adds 0.2g sodium hydride (NaH) rapidly, under the normal temperature in common shaking table stirring reaction when no hydrogen is emitted till;
6) copolymer microsphere that swelling in the step 4) is good is added in the β-CD sodium salt solution of the clear that step 5) obtains, stirring reaction 20h in 70 ℃ shaking bath, reaction finishes final vacuum pump suction filtration, solid product is earlier with twice of DMF drip washing, again with till a large amount of hot washes are not when having absorption with ultraviolet spectrometer detection washings, obtaining size distribution by screening is 58～320 μ m, and density is the composite Ti O of 1.21g/mL
2Copolymer microsphere.
With the composite Ti O that makes
2Copolymer microsphere dress post, loading height is 12cm, use 0.01mol/L, the phosphate buffered saline buffer of pH=6.8 is at linear rate of flow 220cm/h, under ratio of expansion 2.1 conditions behind the equilibrium adsorption chromatography column, with sample on the soy molasses after diluting.After absorption finishes, with 3 column volumes of deionized water wash, not having absorption peak at ultraviolet spectrometer 254nm to washings, is that 55%～70% ethanolic soln carries out gradient elution according to fixed bed pattern concentration then, and elution volume is 8 column volumes.It is 63.51% that the soybean isoflavones that obtains detects purity through HPLC.
Embodiment 2
1) in the 1L there-necked flask that agitator, prolong and thermometer are housed, adds 4g gelatin, 21g sodium-chlor and 500g deionized water, be warming up to 60 ℃ and stir half an hour;
2) with the TiO of 50g surface modification
2Join the mixing solutions of 100g glycidyl methacrylate, 55g divinylbenzene, 1.4g benzoyl peroxide, 90.2g poly(oxyethylene glycol) 400 and 36.5g butylacetate, stir the organic phase solution that obtains stable dispersion;
3) step 2) organic phase solution that obtains slowly joins the aqueous phase that step 1) obtains, regulate mixing speed to 480rpm, it is constant to keep rotating speed, and 60 ℃ of low suspensions disperseed 45 minutes, be warming up to 90 ℃, insulation 9h, curing obtains white microballoon, filters, hot wash 5 times, pore-creating agent is removed in the acetone extracting, and vacuum-drying obtains copolymer microsphere to constant;
4) take by weighing the 80g copolymer microsphere, add exsiccant N, swelling is spent the night in the dinethylformamide (DMF);
5) with the 120g beta-cyclodextrin (β-CD) is dissolved among the DMF and adds rapidly under 0.45g sodium hydride (NaH) normal temperature, in common shaking table stirring reaction when no hydrogen is emitted till;
6) copolymer microsphere that swelling in the step 4) is good is added in the β-CD sodium salt solution of the clear that step 5) obtains, stirring reaction 18h in 80 ℃ shaking bath, reaction finishes final vacuum pump suction filtration, solid product is earlier with twice of DMF drip washing, again with till a large amount of hot washes are not when having absorption with ultraviolet spectrometer detection washings, obtaining size distribution by screening is 40～380 μ m, the composite Ti O of density 1.33g/mL
2Copolymer microsphere.
With the composite Ti O that makes
2Copolymer microsphere dress post, loading height is 17cm, use 0.01mol/L, the phosphate buffered saline buffer of pH=6.8 is at linear rate of flow 250cm/h, under ratio of expansion 2.5 conditions behind the equilibrium adsorption chromatography column, with sample on the soy molasses after diluting.After absorption finishes, with 3 column volumes of deionized water wash, not having absorption peak at ultraviolet spectrometer 254nm to washings, is that 55%～70% ethanolic soln carries out gradient elution according to fixed bed pattern concentration then, and elution volume is 6 column volumes.It is 66.83% that the soybean isoflavones that obtains detects purity through HPLC.
Claims (3)
1, a kind of composite Ti O
2The preparation method of copolymer microsphere is characterized in that, may further comprise the steps:
1) gelatin and sodium-chlor are added in the deionized water, heated and stirred is warming up to 60 ℃, obtains containing the aqueous solution of gelatin and sodium-chlor, and the massfraction of gelatin and sodium-chlor is respectively 0.5～2.5% and 4～12% in the aqueous solution;
2) with the TiO of surface modification
2Join and stir the organic phase solution that obtains stable dispersion in the mixing solutions of initiator, monomer, linking agent and pore-creating agent, wherein said initiator is a benzoyl peroxide, described monomer is glycidyl methacrylate GMA, described linking agent is divinylbenzene DVB, the mass ratio of GMA and DVB is 4～1:1, described pore-creating agent is the miscellany of polyoxyethylene glycol and butylacetate, and the add-on of initiator is 0.5～2% of a monomer mass, TiO
2Add-on be 5～80% of monomer mass, the add-on of pore-creating agent is 50～130% of a monomer mass;
3) aqueous solution and step 2 that step 1) is made) Zhi Bei organic phase solution by volume 2～7:1 mix, stir and to be warming up to 90 ℃ of reaction 9h after fixing, filtration, washing, dryings and to obtain copolymer microsphere;
4) with the copolymer microsphere of step 3) preparation, join exsiccant N, swelling among the dinethylformamide DMF;
5) beta-cyclodextrin β-CD is dissolved among the DMF and the rapid sodium hydride NaH that adds, add-on is 0.1～0.5% of β-CD quality, till stirring reaction to no hydrogen is emitted in shaking table under the normal temperature, obtains β-CD sodium salt solution;
6) swelling in the step 4) is good copolymer microsphere is added in the β-CD sodium salt solution of clear in the step 5), the consumption of β-CD sodium salt solution is 1～5 times of copolymer microsphere quality, stirring reaction 15～20h in 60～80 ℃ shaking bath, reaction finishes back suction filtration, washing, and oven dry makes composite Ti O
2Copolymer microsphere.
2, preparation method according to claim 1 is characterized in that, described composite Ti O
2The particle diameter of copolymer microsphere is that 40～400 μ m, wet true density are 1.1～1.5g/cm
3
3, a kind of composite Ti O
2The application of copolymer microsphere is characterized in that, described composite Ti O
2Copolymer microsphere is as Expanded Bed Adsorption matrix separation and purification soybean isoflavones from soy molasses, and concrete steps are as follows:
1) uses composite Ti O
2After the copolymer microsphere dress is opened column, with 0.01～0.05mol/L, the phosphate buffer soln equilibrium adsorption chromatography column of pH5～8, and Expanded Bed Adsorption post on the waste molasses after will diluting;
2) after absorption finishes, there is not absorption peak at ultraviolet spectrometer 254nm with deionized water wash cylinder to washings;
3) be that 50%～80% ethanolic soln carries out gradient elution with concentration, elution volume is 6～12 column volumes, isolates soybean isoflavones.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810101947A CN101531734A (en) | 2008-03-14 | 2008-03-14 | Preparation and application of composite TiO*copolymer microspheres |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810101947A CN101531734A (en) | 2008-03-14 | 2008-03-14 | Preparation and application of composite TiO*copolymer microspheres |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103623798A (en) * | 2013-10-29 | 2014-03-12 | 西南交通大学 | Single-layer mono-dispersed hollow TiO2 microsphere array fixed on Ti substrate |
| CN105821025A (en) * | 2016-04-06 | 2016-08-03 | 浙江丰安生物制药有限公司 | Extraction method of thrombin |
| CN106589222A (en) * | 2016-12-05 | 2017-04-26 | 黄晖 | Monodisperse high-molecular microspheres and preparation method thereof |
| CN108250256A (en) * | 2018-03-05 | 2018-07-06 | 北京化工大学 | A kind of method for preparing high-purity punicalagins crystalline powder |
-
2008
- 2008-03-14 CN CN200810101947A patent/CN101531734A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103623798A (en) * | 2013-10-29 | 2014-03-12 | 西南交通大学 | Single-layer mono-dispersed hollow TiO2 microsphere array fixed on Ti substrate |
| CN103623798B (en) * | 2013-10-29 | 2015-05-20 | 西南交通大学 | Single-layer mono-dispersed hollow TiO2 microsphere array fixed on Ti substrate |
| CN105821025A (en) * | 2016-04-06 | 2016-08-03 | 浙江丰安生物制药有限公司 | Extraction method of thrombin |
| CN106589222A (en) * | 2016-12-05 | 2017-04-26 | 黄晖 | Monodisperse high-molecular microspheres and preparation method thereof |
| CN108250256A (en) * | 2018-03-05 | 2018-07-06 | 北京化工大学 | A kind of method for preparing high-purity punicalagins crystalline powder |
| CN108250256B (en) * | 2018-03-05 | 2020-10-27 | 北京化工大学 | Method for preparing punicalagin crystal powder |
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Open date: 20090916 |