CN1233438C - Method for preparing microspheric PGDT separating medium with two kinds of pore forms - Google Patents
Method for preparing microspheric PGDT separating medium with two kinds of pore forms Download PDFInfo
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- CN1233438C CN1233438C CN 01134656 CN01134656A CN1233438C CN 1233438 C CN1233438 C CN 1233438C CN 01134656 CN01134656 CN 01134656 CN 01134656 A CN01134656 A CN 01134656A CN 1233438 C CN1233438 C CN 1233438C
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- separating medium
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- foaming agent
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Abstract
The present invention discloses a method for preparing a micro sphere PGDT separating medium with two pore forms, which comprises the following steps: a monomer, crosslinking agents, a liquid pore-forming agent and an initiating agent are uniformly mixed; a solid pore-forming agent is added, and a uniformly mixed process is carried out; a micro sphere is formed by suspension polymerization; the extracting process by anhydrous alcohol, the acid washing process and the drying process of the micro sphere are carried out, and then the separating medium with two pore forms can be obtained. The method for preparing a micro sphere PGDT separating medium with two pore forms is characterized in that the solid pore-forming agent is calcium carbonate, and the solid pore-forming agent accounts for 10 to 30% of volume content of a reaction mixture; the volume ratio of the solid pore-forming agent to the liquid pore-forming agent is from 0.5 to 1; the use level of the pore-forming agents accounts for 20 to 60% of volume content of the reaction mixture; the substance amount ratio of one crosslinking agent to the other crosslinking agent is from 0.5 to 2, and the ratio of the use level of the crosslinking agents to the substance amount of the monomer is from 0.3 to 0.6; under ordinary pressure, the temperature of polymerization is from 65 to 85 DEG C by control. The decorated separating medium manufactured by the method for preparing a micro sphere PGDT separating medium with two pore forms can be used for separating biological macromolecules, such as proteins, etc., and the separating medium has the advantages of large absorption capacity, mild elution requirement and good application prospect in the preparation type separation of large-scale biological macromolecules.
Description
Technical field
The present invention relates to have two class pass PGDT a kind of the preparation with suspension polymerization, promptly the method for poly-(GMA-divinylbenzene-trimerization triallyl isocyanurate) microspheric separating medium belongs to the chromatography media technology of preparing that is used for the separation and purification of biological macromolecule process.
Background technology
In the separation and purification process of large biological molecule, LC is acknowledged as a kind of highly effective means.Along with continuous growth, also more and more higher to the requirement of LC dielectric behavior to the biological product demand.Therefore, the chromatography media of processability excellence becomes the important topic of bioseparation technology research.
In the past four during the decade, people have developed and manyly have been used to filter or as the film and the macroporous polymer particle of catalyst carrier, the aperture reaches the 100-1000 nanometer.Noticeable is when the aperture of film reaches 500 nanometers, as long as there is small pressure differential on both sides, just can cause the convective mass transfer in the hole.People such as Van Kreveld point out in the paper of " Journal of Chromatography " the 397th curly hair table in 1987, as long as this especially big convective mass transfer hole of crossing particle is arranged on the chromatographic stuffing particle, then separated solute just can be with the bore area that flows mutually together rapidly near medium, rather than carry out diffusion mass transfer, thereby can accelerate mass transport process by concentration gradient.
The U.S. Pat Pat5019270 that people such as Afeyan and Regnier applied for perfusion chromatography in 1989.The key of perfusion chromatography is two class pass separating mediums with the POROS name.This is that the diapsid separating medium of skeleton contains two kinds of ducts that vary in size with styrene-divinylbenzene: big bore dia is 600-800nm, and fluid is to pass through streamed; Hole diameter is 50-100nm, and fluid passes through with the diffusion form.This type of medium itself has enough big cross section, hole, makes the relative current that flows by absorbent particles, greatly reduces the resistance to mass tranfer of stagnant flow phase in the hole, has increased substantially the post effect.Its preparation process is at first synthetic microballoon, by emulsification it is further assembled forming aggregation then, and aggregation reassociates and synthesizes the separating medium of required particle diameter.And, because of the hydrophobicity of medium skeleton structure strong, non-specific adsorption to biological substances such as protein is strong, in it separates, except being directly used in the rp mode, in other pattern, when using in ion-exchange, the affine and hydrophobic interaction pattern, generally need dielectric surface is carried out modification, adopt coating usually.As seen, its preparation process is very complicated, the cost height.
The paper that people such as Yihua Yu deliver in 1999 " Journal of Chromatophy A " the 855th volume shows with the GMA to be monomer, divinylbenzene and trimerization triallyl isocyanurate are crosslinking agent, liquid organic solvent toluene and normal heptane are pore-foaming agent, have prepared poly (glycidyl methacrylate) type ball-type separating medium by suspension polymerization.Experimental result shows that the non-specific adsorption of this class ball-type separating medium is lower, has higher mechanical strength.
The paper that people such as Minlian Zhang deliver in rolling up in calendar year 2001 " Journal of Chromatography A " the 922nd shows with the GMA to be monomer, divinylbenzene and trimerization triallyl isocyanurate are crosslinking agent, liquid organic solvent cyclohexanol and dodecanol are pore-foaming agent, have prepared by a step home position polymerization reaction to have two class pass poly (glycidyl methacrylate) separating mediums.Experiment shows that fluid mass transfer behavior in two class pass separating mediums improves greatly.But two class pass separating mediums of in-situ polymerization preparation make its application be subjected to strict restriction because fragmentation takes place under high flow rate its out-of-shape easily.
Summary of the invention
Purpose of the present invention is exactly to go up the defective that exists at above-mentioned separating medium and preparation method thereof, provides a kind of particle diameter controlled, the preparation method of the microballoon PGDT separating medium of the two class passes that mechanical performance is high.
Technical scheme of the present invention is: earlier with the monomer GMA, crosslinking agent divinylbenzene and trimerization triallyl isocyanurate, liquid porogen toluene and normal heptane, and the initator azodiisobutyronitrile mixes, adding the solid pore-foaming agent again mixes, then, by suspension polymerization synthetic polymer microballoon.Microballoon with the absolute ethyl alcohol extracting removing liquid porogen, with comprising that chlorohydric acid pickling removes solid pore-foaming agent, drying again, can obtain two class pass PGDT separating mediums, it is characterized in that the solid pore-foaming agent is a calcium carbonate, consumption accounts for 10～30% of reactant mixture volume content; Solid pore-foaming agent and liquid porogen volume ratio are 0.5～1; The pore-foaming agent consumption accounts for 20～60% of reactant mixture volume content; The crosslinking agent divinylbenzene is 0.5～2 with the amount of substance ratio of trimerization triallyl isocyanurate, and dosage of crosslinking agent is 0.3～0.6 with the ratio of the amount of substance of monomer GMA; Under normal pressure, polymerization temperature is controlled between 65～85 ℃.
The particle diameter of above-mentioned calcium carbonate granule is 0.5～3 μ m, and density is 2.71g/ml.
Below the present invention is described in detail.
Key technology of the present invention has 4 points: the one, and the selection of solid pore-foaming agent.The purpose that adds the solid pore-foaming agent be in separating medium, generate more than the 100nm to discharge orifice, the solid particle that is used for pore should have suitable particle diameter and density, adopting the calcium carbonate superfine powder particle among the present invention is the solid pore-foaming agent, its density is 2.71g/ml, particle diameter is 0.5～3 μ m, can be used for preparing two class pass separating mediums of structure homogeneous.Two of key technology is selections of organic pore-foaming agent.According to the type of monomer and crosslinking agent, select suitable organic pore-foaming agent, so that synthetic two class pass separating mediums with big specific area.It is liquid porogen that the present invention adopts toluene and normal heptane, can make synthetic separating medium specific area big, and adsorption capacity is big, has mechanical strength preferably simultaneously.Three of key technology is the proportionings of regulating solid pore-foaming agent and liquid porogen, can realize the control to the mass transfer and the absorption property of prepared separating medium.Four of key technology is selections of polymerization.The suspension polymerization that the present invention selects present most separating medium preparation to be adopted.
Two class pass PGDT separating mediums of the present invention's preparation are compared with existing two class pass separating mediums, and its tangible advantage is: preparation process is simple, easy to operate, cost is low; Can realize that the aperture to discharge orifice and diffusion hole is simultaneously controlled in the two class pass separating mediums, change the proportioning and the consumption of crosslinking agent, the proportioning of organic solvent and consumption can be regulated the aperture of diffusion hole, and the particle diameter of change solid particle can be regulated the aperture to discharge orifice; The skeleton structure of this separating medium is a poly (glycidyl methacrylate) type polymer, and its good hydrophilic property is a little less than the non-specific adsorption; Contain a large amount of epoxy radicals in the skeleton structure, be easy to modify aglucon, for example, ion-exchange group, affinity ligands etc. are used in the separation of protein and other, and adsorption capacity is big, the elution requirement gentleness; Preparing type large-scale large biological molecule has a good application prospect in separating.
Description of drawings
Accompanying drawing is to take phasor with the Electronic Speculum of two prepared class pass PGDT separating mediums of the inventive method.Among the figure, 1 for penetrating porose area, and 2 are the diffusion porose area.
The specific embodiment
Following example will give further instruction to method provided by the invention.
Embodiment 1
Take by weighing 5.00 gram GMAs (GMA), 1.70 gram divinylbenzene (DVB), 1.46 gram trimerization triallyl isocyanurate (TAIC), 1.84 gram toluene, 1.33 the gram normal heptane, 0.12 gram azobisisobutyronitrile joins in the 50ml conical flask, mixes, add 12.9 gram calcium carbonate, mix.Pre-polymerization 24 hours in thermostatic water-circulator bath under 40 ℃.In the there-necked flask that agitator, reflux condensing tube and thermometer are housed, add 1% poly-vinyl alcohol solution after, reactant mixture is added.Under nitrogen protection, regulate mixing speed, treat that drop is dispersed into suitable granularity after, slowly be warming up to 65 ℃ with 1 ℃/3 minutes speed, reacted 3 hours, be warming up to 75 ℃ with same speed again, reacted 1 hour, be warming up to 85 ℃ at last, reacted 2 hours.Then polymer microballoon is changed in the nylon sandbag, ethanol extracting 24 hours removes organic pore-foaming agent.Again the microballoon after the extracting is immersed in the 0.2M hydrochloric acid solution, removes the solid pore-foaming agent, after the vacuum drying, obtain above-mentioned two class pass PGDT separating mediums, 6.0 grams.Utilize diethylamine to modify separating medium, after getting 5.0 gram separating mediums and 25ml dioxane and 25ml diethylamine mixing, descend to react 6.5 hours in 60 ℃.After question response finished, distilled water fully washed, and (＜1Torr 1Torr=133.332Pa), obtains can be used for two class pass PGDT separating mediums of biological substance such as isolated protein under the anion exchange pattern in vacuum drying.The volume average particle size of this separating medium is 45.6 μ m, and specific area is 52.1m
2/ g, static adsorption capacity is the wet separating medium of 73.3mgBSA/g.Settling methods dress post, under the flow velocity of 180.0cm/h, dynamic adsorption capacity is up to 33.2mgBSA/ml column volume (51.8mgBSA/g wet separating medium).
Embodiment 2
Take by weighing 5.00 gram GMA, 1.01 gram DVB, 1.05 gram TAIC, 1.19 gram toluene, 0.87 gram normal heptane, 0.12 gram azobisisobutyronitrile joins in the 50ml conical flask, mixes, and adds 14 gram calcium carbonate, mixes.By synthetic PGDT two class hole separating mediums 6.0 grams that can be used for adhesion protein under the anion exchange pattern of the method among the embodiment 1.Volume average particle size is 49.1 μ m, and its specific area is 28.7m
2/ g, static adsorption capacity is the wet separating medium of 34.9mgBSA/g.Settling methods dress post.Under the flow velocity of 180.0cm/h, dynamic adsorption capacity reaches 13.4mgBSA/ml column volume (21.3mgBSA/g wet separating medium).
Claims (2)
1. the preparation method of the microballoon PGDT separating medium of a class pass, earlier with the monomer GMA, crosslinking agent divinylbenzene and trimerization triallyl isocyanurate, liquid porogen toluene and normal heptane, and the initator azodiisobutyronitrile mixes, adding the solid pore-foaming agent again mixes, then, by suspension polymerization synthetic polymer microballoon, microballoon with the absolute ethyl alcohol extracting to remove liquid porogen, comprise with chlorohydric acid pickling to remove the solid pore-foaming agent, drying can obtain two class pass PGDT separating mediums again, it is characterized in that, the solid pore-foaming agent is a calcium carbonate, and consumption accounts for 10～30% of reactant mixture volume content; Solid pore-foaming agent and liquid porogen volume ratio are 0.5～1; The pore-foaming agent consumption accounts for 20～60% of reactant mixture volume content; The crosslinking agent divinylbenzene is 0.5～2 with the amount of substance ratio of trimerization triallyl isocyanurate, and dosage of crosslinking agent is 0.3～0.6 with the ratio of the amount of substance of monomer GMA; Under normal pressure, polymerization temperature is controlled between 65～85 ℃.
2. press the preparation method of the microballoon PGDT separating medium of the described two class passes of claim 1, the particle diameter that it is characterized in that calcium carbonate granule is 0.5～3 μ m, and density is 2.71g/ml.
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| CN 01134656 CN1233438C (en) | 2001-11-09 | 2001-11-09 | Method for preparing microspheric PGDT separating medium with two kinds of pore forms |
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| CN 01134656 CN1233438C (en) | 2001-11-09 | 2001-11-09 | Method for preparing microspheric PGDT separating medium with two kinds of pore forms |
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| CN1233438C true CN1233438C (en) | 2005-12-28 |
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| CN101869778B (en) * | 2009-04-24 | 2012-06-27 | 西安瑞仁生物技术有限公司 | Composite GMA (Glycidyl Methacrylate)/ZrO2 copolymer microsphere as well as preparation and application thereof |
| CN101625347B (en) * | 2009-08-07 | 2012-05-30 | 清华大学 | Method for synthesizing integral bed for plasmid analysis |
| CN102432744A (en) * | 2011-09-07 | 2012-05-02 | 天津大学 | Method for preparing monodispersed functional polymer microspheres |
| CN103360542A (en) * | 2012-03-31 | 2013-10-23 | 重庆赛英思医疗器械股份有限公司 | Synthesis of LDL (low-density lipoprotein) specific adsorbent in serum |
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