CN1472002A - Macropore high-capacity agarose gel media preparing method - Google Patents
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Abstract
A process for preparing high-capacity microreticular agarose gel medium includes such steps as dispersing the granular calcium carbonate as hole-forming agent in the aqueous solution of agarose to obtain suspension, adding it to salad oil, adding emulsifier Span80, emulsifying while stirring, quick cooling for sphericizing, adding epoxy chloropropane, cross linking, reducing with sodium borohydride, and using diluted hydrochloric acid to remove granular calcium carbonate.
Description
Technical field
The present invention relates to a kind of preparation method of high power capacity macroporous Ago-Gel medium, belong to the technology of preparing of Ago-Gel medium.
Background technology
In the process of biological downstream, chromatography has the irreplaceable status of other separation means at present.The development of chromatographic technique is to be prerequisite with the exploitation of separating medium and procedural theory perfect, and wherein the exploitation of novel chromatography media is one of the research focus in chromatography field always.Desirable chromatography media not only should have higher adsorption capacity, and should satisfy fast, the requirement of high-resolution and low non-specific adsorption.But the aperture of present most chromatography media is all in nanometer scale.The mass tranfer coefficient of protein in above-mentioned medium seriously influenced the mass transfer of large biological molecule in chromatography media than low two to three orders of magnitude of small-molecule substance.On the other hand, be that the mechanical stability of this class homogeneous pattern chromatography media of representative is all not high with the Ago-Gel medium, can't realize the requirement that separates fast.So the quick separation that strengthen the mass transfer of solute in medium, realizes large biological molecule is one of ultimate challenge of facing of chromatography technology.
The thinking that addresses the above problem mainly contains two kinds:
The first, reduce the particle diameter of medium.It mainly is conceived to reduce the chromatography media particle diameter, mass-transfer efficiency is improved in shortening mass transfer path.Owing to adopted the medium of small particle diameter, so the back pressure of chromatographic column increases in the operating process, and the mechanical strength of medium is had higher requirement.This method is mainly used in the preparation of analytical chromatography medium at present.
The second, the exploitation macroporous matrix.In the chromatography media preparation process, add certain pore-foaming agent (as cyclohexane, hexane etc.), when generating the nanoscale diffusion hole, form a certain amount of micron-sized opening.Solution flows with the form of convection current in opening, in diffusion hole then with the diffusion way transmission.Though lost the theoretical adsorption capacity of part in the preparation process medium, the generation of opening realizes for strengthening mass transfer in the medium, increasing effective adsorbance that the quick separation of big molecule solute has important function.At present, this method is the prefered method that realizes that large biological molecule separates fast.
1991, people such as Afeyan proposed the notion of macroporous matrix and have announced the patent (US5,019,270) of utilizing styrene and divinylbenzene to prepare macroporous matrix.Utilize the operations flows scooter 1500cm/h of the macroporous matrix of this technology preparation.After this, people such as Afeyan has strengthened protection to this patented technology by a series of patent.But above-mentioned diplopore medium is the organic polymer synthetic medium, and non-specific adsorption that this class medium is higher and complicated preparation technology have restricted its application to a certain extent.Gustavsson and Larsson have reported that with oil phase (cyclohexane) be liquid porogen in " Journal of Chromatography A " the 734th volume, adopt the profit two phase process to prepare method (the P.E.Gustavsson and P.O.Larsson.Superporous agarose of super big hole Ago-Gel medium, a new material for chromatography, Journal of Chromatography A, 1996,743,231-240).This class super big hole medium had both kept the original network structure of Ago-Gel medium, possessed the characteristic of opening simultaneously.On this basis, Larsson discloses and has adopted the profit two phase process to obtain the patented technology (US5,723,601) of super big hole medium.Diffusion hole and the aperture opening more than 0.5 micron that the medium that utilizes this method to prepare has aperture 2～50 nanometers.Owing to adopted liquid porogen, the aperture of opening is difficult to control (as U.S. Pat 5,723, the pore-size distribution of the Ago-Gel medium opening of being reported in 601 is at 0.5～1000 μ m) in the above-mentioned macroporous matrix, and the preparation process more complicated; In addition, residual organic pore-foaming agent also can produce certain influence to the performance and the use of medium in the chromatography media.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of high power capacity macroporous Ago-Gel medium.With the separating medium of this method preparation have mechanical strength good, than the chromatography medium of high-adsorption-capacity and mass transfer rate, controllable bore diameter and porosity and particle diameter.
The present invention is realized by following technical proposals.Adopting the solia particle of biocompatibility, is water with the agarose solution, is oil phase with edible salad oil, the method for the separating medium of preparation macroporous type.This method mainly comprises processes such as the crosslinked and solia particle removal of preparation, dispersion and emulsion, gel solidification and the gel of suspension.It is characterized in that, under 85-90 ℃, with the particle diameter 0.86 μ m-4.58 μ m (10%-90%) that distributes, density is that to be scattered in concentration be 2-8% to the calcium carbonate microparticle of 2.3-2.6g/mL, volume is in the agarose solution of 20～40 times of calcium carbonate microparticle volumes, fully stir and make finely dispersed suspension, then this suspension being added volume is in the water volume 4-10 salad oil doubly, and add emulsifying agent Span80 by 10-50g/L and carry out stirring reaction, reactant mixture is cooled to 10～30 ℃ rapidly, solidify to form microballoon, adding volume ratio in the microballoon after Xiang Jingshui cleans is the epoxychloropropane of 1-5%, and under 0.3-0.7mol/L NaOH condition, carry out crosslinked, under 0.8-1.4mol/L NaOH condition, use the 4-6g/L sodium borohydride reduction afterwards, microballoon after the reduction cleaning adds watery hydrochloric acid and removes the calcium carbonate microparticle that wraps up in the microballoon, makes the macroporous Ago-Gel medium that particle diameter is 70～200 μ m.
Above-mentioned optimal conditions: the concentration of agarose solution is 4～6%, and the aqueous phase agarose solution is 20-25 with the ratio of calcium carbonate granule volume, and the salad oil volume is 6-7 a times of water volume, and the addition of emulsifying agent Span80 is 25-35g/L; Solidification temperature is 10～20 ℃; The employing epoxychloropropane is a crosslinking agent, under 0.4-0.7mol/L sodium hydroxide solution condition, the concentration of epoxychloropropane is 2-4% (percent by volume), is reducing agent with the sodium borohydride, and its consumption is 4.5～5.5g/L under 0.8-1.4mol/L NaOH condition.
The macroporous Ago-Gel medium of the present invention preparation is compared with existing chromatography medium, and its tangible advantage is, raw material is relatively cheap, the easy economy of preparation process, and equipment is simple; Solid carbonic acid calcium particulate is nontoxic; Medium has the characteristics of organic polymer macroporous matrix concurrently when preserving original Ago-Gel class medium advantage; The mass transfer rate height, adsorption capacity is big, and the opening aperture is controlled; Having of macropore helps realize quick separation in the medium, chromatography stream scooter 1200cm/h; Active group is many on the Ago-Gel, by modifying different aglucons, and for example pigment aglucon, ion-exchange aglucon etc., the relative homogeneous medium, adsorption capacity can improve 2～3 times, can satisfy multiple adsorbing separation requirement, and the elution requirement gentleness; Good biocompatibility, good hydrophilic property is with a wide range of applications at the aspects such as immobilization, cell separation, DNA and protein purification of enzyme.
Below the present invention is described in detail.
Key technology of the present invention has 7 points: the one, and the selection of calcium carbonate microparticle.Density is 2.3-2.6g/mL, and particle diameter is 2.67 μ m, and nontoxic calcium carbonate microparticle can be used as the solid pore-foaming agent.Two of key technology is preliminary treatment of calcium carbonate microparticle.Because the compatibility of calcium carbonate microparticle and water is very poor, with calcium carbonate microparticle in containing the water of surfactant, stirs, ultrasonic, fully after the mixing, discard cleaning solution.Three of key technology is dispersion and the content of calcium carbonate microparticle in agarose solution.Calcium carbonate microparticle and the agarose solution handled well are fully mixed, and the content of calcium carbonate is directly connected to porosity, influences mass transfer rate.Four of key technology adopts the profit two phase process to prepare macroporous Ago-Gel medium.In the profit two-phase, react, the microballoon that contains calcium carbonate microparticle of acquisition, calcium carbonate microparticle is evenly distributed in medium.Five of key technology is the control of mixing speed in preparation process.In course of reaction, to keep the stable of rotating speed; Simultaneously, the regulation and control rotating speed also is to obtain one of different-grain diameter method.Six of key technology is that the suitable crosslinked and reduction step that adopts is handled, and has both helped improving the stability and the mechanical strength of macroporous matrix; Do not influence simultaneously the distinctive network structure of Ago-Gel self again.Removing of seven calcium carbonate microparticles of key technology.Medium is soaked to remove the wherein calcium carbonate microparticle of parcel with watery hydrochloric acid in crosslinked back.
Description of drawings
Fig. 1 is the photo (multiplication factor is 160) of the macroporous Ago-Gel medium that contains calcium carbonate microparticle of the present invention's preparation under the light microscope
Fig. 2 is the photo (calcium carbonate has been removed, and multiplication factor is 160) of the high power capacity macroporous Ago-Gel medium of the present invention's preparation under the light microscope
Fig. 3 has described the relation of macropore and homogeneous pattern Ago-Gel medium flow velocity and back pressure, and wherein (△) is corresponding to the homogeneous pattern Ago-Gel medium, () corresponding to macroporous Ago-Gel medium.
Fig. 4 has described macroporous matrix of the present invention, homogeneous medium and the Sepharose FF static adsorption isotherm to bovine serum albumin(BSA) in 0.01mol/L Tris-HCl buffer solution (pH7.6), wherein (△) is corresponding to macroporous Ago-Gel medium (vehicle economy AE-CA), (zero) corresponding homogeneous pattern medium (vehicle economy AE-A, do not add calcium carbonate, other conditions are identical with macroporous Ago-Gel), () corresponding to DEAE-Sepharose FF (vehicle economy AE-FF).
Fig. 5 has described standard chromatographic column (HR5/5, Amersham Biosciences) macroporous matrix, homogeneous medium and Sepharose FF are to the dynamic adsorption curve of bovine serum albumin(BSA) in, wherein flowing is 0.01mol/L Tris-HCl buffer solution (pH7.6) mutually, flow velocity 612cm/h, bovine serum albumin(BSA) feed concentration are 2mg/mL.1 corresponding to DEAE-FF among the figure, and 2 corresponding to DEAE-A, and 3 corresponding to DEAE-CA.
The specific embodiment
Following example will give further instruction to method provided by the invention.
Under 90 ℃, the particle diameter 2.67 μ m of the 4.5g that handles well, 0.86～4.58m calcium carbonate microparticle that distributes are evenly dispersed in the 50mL 60g/L agarose solution by quick stirring.Then this suspension is poured into fast in the 300mL organic facies that contains 30g/L Span80, under 90 ℃, control mixing speed 1200rpm; Behind the reaction 30min, be cooled to 20 ℃ rapidly, keeping under the constant condition of mixing speed, continue curing reaction 40min; In above-mentioned reaction system, add the water that doubles its volume, obtain the Ago-Gel medium of parcel calcium carbonate microparticle by natural subsidence.The microballoon of collecting is successively removed remaining organic facies with acetone and distilled water cyclic washing.After getting the clean microballoon of 50mL and isopyknic 1mol/L sodium hydroxide solution mixing, add the 500mg sodium borohydride, the concentration that makes sodium borohydride in the suspension is 5g/L.Said mixture under the 170rpm condition, reacts 30min in 25 ℃ in shaking bath; The epoxychloropropane that adds 2mL then under these conditions, continues reaction 5h.Reaction finishes, and arrives neutral with the distilled water cyclic washing.In cleaning back microballoon (50mL), add the isopyknic sodium hydroxide solution of 2mol/L, make suspension again; Add the 500mg sodium borohydride then, the concentration that makes sodium borohydride in the solution is 5g/L.In 30 ℃, under the 170rpm, in shaking bath, react 6h.Reaction finishes, and adds the distilled water cyclic washing to neutral.Add a certain amount of water (be about medium volume about 8 times) in the microballoon after cleaning, add 0.1mol/L watery hydrochloric acid again, and put into shaking table and react with volume; After a period of time, repeat the step of above-mentioned removal calcium carbonate again.Use 0.05mol/L salt acid soak 30 minutes at last, further remove remaining calcium carbonate microparticle, so just be prepared into macroporous matrix (as shown in Figure 1).(HR5/5, macroporous matrix, homogeneous pattern medium and the Sepharose FF medium that loads 1mL in AmershamBiosciences) respectively investigate under different in flow rate the chromatographic column back pressure with the Changing Pattern of flow velocity at the standard chromatographic column.Macroporous matrix can produce convection current owing to there are many macropores in medium, reduced pressure drop relatively, can (1250cm/h) operate under higher flow velocity, and (612cm/h) of the upper limit relative homogeneous medium of flow velocity is doubled many (as shown in Figure 2).
Under 85 ℃, the particle diameter 2.67 μ m of the 6g that handles well, the calcium carbonate microparticle of 0.86～4.58 μ m that distributes are evenly dispersed in the 50mL 60g/L agarose solution by quick stirring.Then this suspension is poured into fast in the 300mL organic facies that contains 30g/L Span80, under 85 ℃ of temperature of parcel, mixing speed 1300rpm, reaction 15min; Be cooled to 15 ℃ rapidly, keeping under the constant condition of mixing speed curing reaction 30min; In above-mentioned system, add the water that doubles its volume, obtain the Ago-Gel medium of parcel calcium carbonate microparticle by natural subsidence.The microballoon of collecting is successively used acetone and distilled water cyclic washing, removes fully until organic facies.After getting the clean microballoon of 50mL and equal-volume 1mol/L sodium hydroxide solution mixing, add the 400mg sodium borohydride, the concentration that makes sodium borohydride in the suspension is 4g/L.Said mixture under the 200rpm condition, reacts 30min in 25 ℃ in shaking bath; The epoxychloropropane that adds 2mL then under these conditions, continues reaction 5h.Reaction finishes, and arrives neutral with the distilled water cyclic washing.In cleaning back microballoon (50mL), add the isopyknic sodium hydroxide solution of 2mol/L, make suspension again; Add the 600mg sodium borohydride then, the concentration that makes sodium borohydride in the solution is 6g/L.In 30 ℃, under the 200rpm, in shaking bath, react 6h.Reaction finishes, and adds the distilled water cyclic washing to neutral.After adding a certain amount of water (be about medium volume about 8 times) in the microballoon that obtains, add 0.1mol/L watery hydrochloric acid again, and put into shaking table and react with volume; Repeat the step of above-mentioned removing calcium carbonate microparticle again.Use 0.05mol/L salt acid soak 20 minutes at last,, so just be prepared into macroporous matrix to remove remaining calcium carbonate microparticle.Get the above-mentioned macroporous Ago-Gel medium of 10g (having removed calcium carbonate), move among the Erlenmeyer flask I of 250mL, add 20mL 3.0mol/L DEAE-Cl and mix, the Erlenmeyer flask II that other gets a 250mL adds 20mL 3.5mol/LNaOH, puts into the preheating of 60 ℃ of constant temperature shaking tables after the sealing.Behind the 10min NaOH among the Erlenmeyer flask II is added Erlenmeyer flask I, seal.Behind the reaction 1h, take out Erlenmeyer flask I water cooling.The macroporous matrix of having modified is extremely neutral with deionized water rinsing in sand core funnel.It is the ion exchange capacity of 1.9mmol/mL that this macropore is modified density.Under 0.01mol/L Tris-HCl buffer solution (pH7.6), the static adsorbance of macroporous Ago-Gel medium (vehicle economy AE-CA) is the wet microballoon of 276.0mg bovine serum albumin(BSA)/mL; The static adsorbance of homogeneous pattern medium (vehicle economy AE-A) is the wet microballoon of 193.0mg bovine serum albumin(BSA)/mL; The static adsorbance of DEAE-Sepharose FF (vehicle economy AE-FF) is the wet microballoon (as shown in Figure 3) of 128.0mg bovine serum albumin(BSA)/mL.Using standard chromatographic column (HR5/5, Amersham Biosciences), flow velocity is under the 612cm/h, and the dynamic adsorbance of DEAE-CA is the wet microballoon of 43.4 bovine serum albumin(BSA)s/mL; The dynamic adsorbance of DEAE-A is the wet microballoon of 28.7mg bovine serum albumin(BSA)/mL; The dynamic adsorbance of DEAE-FF is that the wet microballoon of 6.9mg bovine serum albumin(BSA)/mL (all refers to the dynamic adsorbance calculated, as shown in Figure 4) under 5% breakthrough point.
Claims (2)
1. the preparation method of a high power capacity macroporous Ago-Gel medium, this method mainly comprises the preliminary treatment that solia particle is used, the preparation of suspension, emulsion reaction, solidify and cross-linking process, the process that solia particle is removed, it is characterized in that: under 85-90 ℃, it is 4-8% that particle diameter distribution 0.86～4.58 μ m calcium carbonate microparticle of handling well is scattered in concentration equably, volume is in the agarose solution of 15～30 times of calcium carbonate microparticle volumes, fully stir and make finely dispersed suspension, then this suspension being added volume is in the water volume 4-10 salad oil doubly, and press 10-50g/L and add emulsifying agent Span80, after emulsion reaction is carried out in stirring, be cooled to 10～30 ℃ rapidly, solidify to form microballoon, add the epoxychloropropane that volume ratio is 1-5% again, and under 0.3-0.7mol/L NaOH condition, carry out crosslinked, under 0.8-1.4mol/L NaOH condition, use the 4-6g/L sodium borohydride reduction afterwards, remove calcium carbonate microparticle with hydrochloric acid at last, just can be prepared into macroporous matrix.
2. the preparation method of high power capacity macroporous Ago-Gel medium according to claim 1, it is characterized in that: the concentration of agarose solution is 4～6%, the aqueous phase agarose solution is 15-25 with the ratio of calcium carbonate microparticle, the salad oil volume is 6-7 a times of water volume, and the addition of emulsifying agent Span80 is 25-35g/L; Adopting concentration 2-4% (percent by volume) epoxychloropropane is crosslinking agent, carries out crosslinkedly under the 0.4-0.7mol/LNaOH condition, is reducing agent with the sodium borohydride, and its consumption is 4.5～5.5g/L under 0.8-1.4mol/L NaOH condition.
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| CN100368075C (en) * | 2005-04-05 | 2008-02-13 | 天津大学 | Method for preparing ultra macropore type rigid macromolecule medium by using suspension liquid of fine particles of calcium carbonate |
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| CN103182199B (en) * | 2013-03-22 | 2014-12-24 | 西北大学 | Method for purifying polyphenols |
| CN103182199A (en) * | 2013-03-22 | 2013-07-03 | 西北大学 | Method for purifying polyphenols |
| CN105944686A (en) * | 2016-05-18 | 2016-09-21 | 绿麒（厦门）海洋生物科技有限公司 | Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres |
| CN105944686B (en) * | 2016-05-18 | 2018-04-20 | 绿麒（厦门）海洋生物科技有限公司 | A kind of agarose gel microsphere of the glycosyl containing aminoglucose and preparation method thereof |
| CN108531563A (en) * | 2018-02-05 | 2018-09-14 | 深圳市尚维高科有限公司 | The purposes and lysate of porous microsphere and the application method of lysate |
| CN115197479A (en) * | 2022-06-17 | 2022-10-18 | 苏州百奥吉生物科技有限公司 | Polysaccharide microsphere with uniform particle size and preparation method thereof |
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