CN115160603B - High-rigidity macroporous polysaccharide microsphere and preparation method thereof - Google Patents
High-rigidity macroporous polysaccharide microsphere and preparation method thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 69
- 150000004676 glycans Chemical class 0.000 title claims abstract description 60
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 60
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 30
- 238000003756 stirring Methods 0.000 claims abstract description 24
- 239000012071 phase Substances 0.000 claims abstract description 23
- 229920000936 Agarose Polymers 0.000 claims abstract description 20
- 239000008367 deionised water Substances 0.000 claims abstract description 19
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims abstract description 17
- 229920002678 cellulose Polymers 0.000 claims abstract description 15
- 239000001913 cellulose Substances 0.000 claims abstract description 15
- 235000010980 cellulose Nutrition 0.000 claims abstract description 15
- 238000004132 cross linking Methods 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 125000002883 imidazolyl group Chemical group 0.000 claims abstract description 8
- 239000002608 ionic liquid Substances 0.000 claims abstract description 8
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims abstract description 7
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims abstract description 7
- 239000008108 microcrystalline cellulose Substances 0.000 claims abstract description 7
- 229940016286 microcrystalline cellulose Drugs 0.000 claims abstract description 7
- 239000012074 organic phase Substances 0.000 claims abstract description 7
- 239000003960 organic solvent Substances 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 239000002131 composite material Substances 0.000 claims abstract description 6
- 239000004593 Epoxy Substances 0.000 claims abstract description 5
- 239000012190 activator Substances 0.000 claims abstract description 5
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 5
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 5
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 claims description 5
- 239000008346 aqueous phase Substances 0.000 claims description 5
- 229940057995 liquid paraffin Drugs 0.000 claims description 5
- 239000003208 petroleum Substances 0.000 claims description 5
- ZXLOSLWIGFGPIU-UHFFFAOYSA-N 1-ethyl-3-methyl-1,2-dihydroimidazol-1-ium;acetate Chemical compound CC(O)=O.CCN1CN(C)C=C1 ZXLOSLWIGFGPIU-UHFFFAOYSA-N 0.000 claims description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical group CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 235000019441 ethanol Nutrition 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- IAZSXUOKBPGUMV-UHFFFAOYSA-N 1-butyl-3-methyl-1,2-dihydroimidazol-1-ium;chloride Chemical compound [Cl-].CCCC[NH+]1CN(C)C=C1 IAZSXUOKBPGUMV-UHFFFAOYSA-N 0.000 claims description 2
- OIWSIWZBQPTDKI-UHFFFAOYSA-N 1-butyl-3-methyl-2h-imidazole;hydrobromide Chemical compound [Br-].CCCC[NH+]1CN(C)C=C1 OIWSIWZBQPTDKI-UHFFFAOYSA-N 0.000 claims description 2
- SHKUUQIDMUMQQK-UHFFFAOYSA-N 2-[4-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COCCCCOCC1CO1 SHKUUQIDMUMQQK-UHFFFAOYSA-N 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 229940078552 o-xylene Drugs 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 abstract description 4
- 239000011148 porous material Substances 0.000 abstract description 4
- 239000002245 particle Substances 0.000 description 9
- 230000005526 G1 to G0 transition Effects 0.000 description 6
- 230000001804 emulsifying effect Effects 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 239000003431 cross linking reagent Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000012501 chromatography medium Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012876 topography Methods 0.000 description 3
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- 239000013065 commercial product Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/12—Powdering or granulating
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/12—Agar-agar; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2401/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives
- C08J2401/02—Cellulose; Modified cellulose
- C08J2401/04—Oxycellulose; Hydrocellulose
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Processes Of Treating Macromolecular Substances (AREA)
Abstract
The invention provides a high-rigidity macroporous polysaccharide microsphere and a preparation method thereof, wherein the preparation method comprises the following steps: 1) Dissolving microcrystalline cellulose in an imidazolyl ionic liquid to obtain a cellulose solution; dissolving agarose in deionized water to obtain agarose aqueous solution; 2) Slowly adding the agarose water solution into the cellulose solution under the stirring condition, and uniformly stirring to obtain a water phase; 3) Adding the water phase into the oil phase, stirring, and then reducing the system temperature to below 25 ℃ within 1 h; 4) Mixing the mixed system obtained in the step 3) with absolute ethyl alcohol, uniformly stirring, standing, then cleaning, and removing an organic phase to obtain polysaccharide microsphere base spheres; 5) Mixing a crosslinking activator with the polysaccharide microsphere base spheres, then adding an organic solvent, mixing, heating a mixed system, adding sodium hydroxide into the mixed system to react, obtaining epoxy-activated composite polysaccharide microspheres, and then cleaning to be neutral. The polysaccharide microsphere prepared by the invention can still have stronger mechanical strength when having larger pore diameter.
Description
Technical Field
The invention belongs to the technical field of polysaccharide microsphere preparation, and relates to a high-rigidity macroporous polysaccharide microsphere and a preparation method thereof.
Background
In the field of biotechnology, chromatography is a very common separation method. Chromatography generally refers to flowing a mobile phase carrying a mixed sample of different components through stationary phases, wherein the sample and the stationary phases have a series of physical or chemical interactions, after multiple partitioning, due to different interactions between the different components of the sample and the stationary phases, part of the sample is adsorbed in the stationary phases, and then the interaction force between the sample and the stationary phases is changed by eluting solutions under different conditions to perform elution, so as to achieve the effect of mutual separation of the different components of the sample.
The stationary phase of chromatography (also known as the chromatography medium) is typically a micron-sized sphere-like particle, which can be prepared from polymers or natural polysaccharides. The polymer matrix chromatographic medium has stronger mechanical strength, is favorable for use, but has poorer biocompatibility, and can have nonspecific adsorption with a separation sample; the media of natural polysaccharide matrices (e.g., agarose) have better biocompatibility but generally have lower mechanical strength, which is detrimental to scale up and cost reduction.
In EP 203049 a method is described for improving the rigidity of gel beads using monofunctional crosslinkers comprising masking functional groups. In another example, a manufacturing process for cross-linking polysaccharide gels to obtain macropores and high rigidity is described in WO 97/38018, which comprises the step of introducing a cross-linking agent into the polysaccharide solution prior to gel formation. In these methods, the mechanical strength is increased by bridging the polysaccharide molecular chains with a crosslinking agent, which generally requires more solvent and multiple crosslinks. In CN 111989155A, the mechanical strength of natural polysaccharide microspheres can be enhanced by embedding fibers, but this method requires a microfibrillation treatment (treatment to be able to be embedded in microspheres without affecting the sphericity) of the embedded fibers in advance, and is complicated in process and high in cost. In CN 112619612A, a preparation method of high-strength cellulose/agarose composite microspheres is disclosed, wherein alkaline thiourea is used as a solvent for cellulose dissolution, and prepared microspheres have smaller pore channels, and if the method is applied to the field of chromatographic separation, the problems of larger elution volume, low elution concentration, lower loading capacity and the like are caused by slower mass transfer speed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the high-rigidity macroporous polysaccharide microsphere and the preparation method thereof, and the prepared polysaccharide microsphere still can have stronger mechanical strength when having larger aperture (used for separating samples with larger particle size), and has good biocompatibility and low nonspecific adsorption.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a preparation method of high-rigidity macroporous polysaccharide microspheres comprises the following steps:
1) Dissolving microcrystalline cellulose in an imidazolyl ionic liquid to obtain a cellulose solution; dissolving agarose in deionized water to obtain agarose aqueous solution;
2) Slowly adding the agarose water solution into the cellulose solution under the stirring condition, and uniformly stirring to obtain a water phase;
3) According to the aqueous phase: oil phase mass ratio 1: (1.2-1.5) adding the water phase into the oil phase, stirring for 20-80min at 100-300rpm, and then reducing the system temperature to below 25 ℃ within 1 h;
4) Mixing the mixed system obtained in the step 3) with absolute ethyl alcohol according to the mass ratio of 1 (3-5), uniformly stirring, standing, then cleaning, and removing an organic phase to obtain polysaccharide microsphere base spheres;
5) Mixing the polysaccharide microsphere base sphere with a crosslinking activator according to the volume ratio of 5 (1-4), adding an organic solvent according to the volume ratio of the polysaccharide microsphere base sphere to the organic solvent of 1 (1-1.5), mixing, heating the mixed system to 30-50 ℃ (optimal 35-40 ℃), adding 50% sodium hydroxide into the mixed system according to the volume ratio of the polysaccharide microsphere base sphere to 50% sodium hydroxide of 5 (1-3), reacting for 3-24 hours (optimal 5-8 hours), and obtaining the epoxy-activated composite polysaccharide microsphere, and cleaning to be neutral.
Preferably, in step 1), microcrystalline cellulose and imidazolyl ionic liquid are mixed according to a mass ratio of 1 (30-100), and heated to 100 ℃ -170 ℃ (optimally 100 ℃ -120 ℃) for dissolution.
More preferably, the imidazolyl ionic liquid includes, but is not limited to, 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole chloride or 1-butyl-3-methylimidazole bromide.
Preferably, in the step 1), agarose and deionized water are mixed according to the mass ratio of 1 (5-15), and heated to 80-130 ℃ for dissolution.
Preferably, the oil phase is prepared by the following method: heating one or more of liquid paraffin, petroleum ether, toluene and o-xylene to 50-90 ℃ under stirring (optimally 60-70 ℃), adding span 80, span 60 and tween 20 mixed according to the mass ratio of 10 (1-2) (0-1), and stirring to completely dissolve for standby.
Preferably, in step 4), the polysaccharide microsphere is washed with ethanol and deionized water to remove the organic phase and obtain a microsphere base sphere.
Preferably, in step 5), deionized water is used to clean the epoxy-activated composite polysaccharide microspheres, and the organic reagent and sodium hydroxide are removed to neutrality.
Preferably, the crosslinking activator includes, but is not limited to, one or both of epichlorohydrin, 1,4 butanediol diglycidyl ether (or other crosslinking agent having multiple functional groups).
Preferably, the organic solvent includes, but is not limited to, one or more of acetone, 1, 4-dioxane, dimethyl sulfoxide.
The invention also provides a high-rigidity macroporous polysaccharide microsphere prepared by the preparation method.
The invention has the beneficial effects that:
the invention provides a preparation method of polysaccharide microspheres (chromatographic media): microsphere preparation was performed using a mixed solution of cellulose and agarose. After balling, cellulose molecules have stronger hydrogen bond action and stronger mechanical strength, so that the cellulose molecules can be used as a skeleton of the microsphere; agarose has the characteristics of loose and porous structure, so that the microspheres retain the characteristic of high specific surface area of common agarose microspheres. The present invention does not require pre-treated embedded fibers. The polysaccharide microsphere prepared by the method can reach the same rigidity of a commercial product after less crosslinking agent and less crosslinking, and can obtain the rigidity far exceeding the rigidity of the commercial product after secondary crosslinking and has a good pore channel structure.
Drawings
FIG. 1. Results of pressure flow rate tests of different samples in the test examples of the present invention.
Fig. 2 is a SEM topography of example 2 of the present invention.
Fig. 3 is an SEM topography of example 3, example 3 of the present invention.
Fig. 4 is an SEM topography of comparative example 4 of the present invention.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and with reference to the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Example 1:
preparing an aqueous phase: 9g of microcrystalline cellulose was dissolved in 600ml of 1-ethyl-3-methylimidazole acetate solution, heated to 100℃and dissolved. 31g of low electroosmotic agarose was dissolved in 400ml of deionized water, heated to 95℃and dissolved. The aqueous agarose solution was then slowly added to the cellulose solution with stirring.
Preparing an oil phase: 1.2kg of liquid paraffin and 0.2kg of petroleum ether are weighed and added into a reaction kettle, the temperature is heated to 60 ℃, then 8g of span 80 and 1g of span 60 are added, and stirring and dissolution are carried out.
Slowly adding the water phase into the oil phase, regulating the rotation speed (50-300 rpm) to obtain polysaccharide microsphere with proper particle size (the particle size of the microsphere can be observed by a microscope, the maximum microsphere particle size is proper rotation speed when 150 μm), emulsifying for about 30min, and cooling the reaction system to below 25 ℃ by ice water.
Adding the emulsifying system into 4L absolute ethyl alcohol, stirring uniformly, standing and decanting, and then washing an organic phase in the system by using absolute ethyl alcohol and deionized water to obtain the polysaccharide microsphere base sphere.
Crosslinking: the polysaccharide microsphere base spheres obtained in the above steps are measured by 500ml, 200ml of epichlorohydrin is added, mixed, then 500ml of dimethyl sulfoxide is added, mixed, heated to 35 ℃, then 100g of 50% sodium hydroxide is added, and the reaction is continued for 6 hours. After the reaction was completed, the polysaccharide microsphere was washed to neutrality using deionized water to give "sample 1".
Example 2:
based on example 1, the step of "crosslinking" was repeated once to obtain "sample 2", the SEM morphology of which is shown in fig. 2, and the polysaccharide microsphere prepared in this example had through holes of 200-1000 nm.
Example 3:
preparing an aqueous phase: 18g of microcrystalline cellulose was dissolved in 600ml of 1-ethyl-3-methylimidazole acetate solution, heated to 100℃and dissolved. 22g of low electroosmotic agarose was dissolved in 400ml of deionized water, heated to 95℃and dissolved. The aqueous agarose solution was then slowly added to the cellulose solution with stirring.
Preparing an oil phase: 1.2kg of liquid paraffin and 0.2kg of petroleum ether are weighed and added into a reaction kettle, the temperature is heated to 60 ℃, then 8g of span 80 and 1g of span 60 are added, and stirring and dissolution are carried out.
Slowly adding the water phase into the oil phase, regulating the rotation speed to obtain polysaccharide microsphere with proper particle size (the particle size of the microsphere can be observed by a microscope), emulsifying for about 30min, and cooling the reaction system to below 25 ℃ by ice water.
Adding the emulsifying system into 4L absolute ethyl alcohol, stirring uniformly, standing and decanting, and then washing an organic phase in the system by using absolute ethyl alcohol and deionized water to obtain the polysaccharide microsphere base sphere.
Crosslinking: the polysaccharide microsphere base spheres obtained in the above steps are measured by 500ml, 200ml of epichlorohydrin is added, mixed, 500ml of dimethyl sulfoxide is added, mixed, heated to 35 ℃,100 g of 50% sodium hydroxide is added, and the reaction is continued for 6 hours. After the reaction, the polysaccharide microsphere is washed to be neutral by deionized water, and is a sample 3, the SEM morphology chart is shown in figure 3, after the dosage of cellulose is increased, the aperture of the polysaccharide microsphere prepared by the embodiment is obviously increased, and the maximum aperture is about 5 mu m, but the mechanical strength is weakened due to overlarge aperture, and the polysaccharide microsphere is easy to crack.
Comparative example:
this example is a comparative example where no cellulose was added.
Preparing an aqueous phase: 40g of low electroosmotic agarose was dissolved in 100ml of deionized water, heated to 95℃and dissolved.
Preparing an oil phase: 1.2kg of liquid paraffin and 0.2kg of petroleum ether are weighed and added into a reaction kettle, the temperature is heated to 60 ℃, then 8g of span 80 and 1g of span 60 are added, and stirring and dissolution are carried out.
Slowly adding the water phase into the oil phase, regulating the rotation speed to obtain polysaccharide microsphere with proper particle size (the particle size of the microsphere can be observed by a microscope), emulsifying for about 30min, and cooling the reaction system to below 25 ℃ by ice water.
Adding the emulsifying system into 4L absolute ethyl alcohol, stirring uniformly, standing and decanting, and then washing an organic phase in the system by using absolute ethyl alcohol and deionized water to obtain the polysaccharide microsphere base sphere.
Crosslinking: the polysaccharide microsphere base spheres obtained in the above steps are measured by 500ml, 200ml of epichlorohydrin is added, mixed, 500ml of dimethyl sulfoxide is added, mixed, heated to 35 ℃,100 g of 50% sodium hydroxide is added, and the reaction is continued for 6 hours. After the reaction is completed, the polysaccharide microsphere is washed to be neutral by deionized water.
The cross-linking step was repeated once to obtain a sample 4, the SEM morphology is as shown in FIG. 4, and the polysaccharide microsphere in this comparative example has a smaller pore size of only 10-30nm.
Test example:
sample 1, sample 2, sample 3 and sample 4 were respectively packed into a chromatographic column having an inner diameter of 26mm and a column height of 15cm, and were subjected to pressure flow rate test at room temperature with deionized water as a test fluid, and the test results are shown in FIG. 1.
As can be seen from the comparison of the samples 1 and 4, the polysaccharide microsphere prepared by the method can obtain the flow velocity which is higher than that of the common microsphere and is twice crosslinked after only one time of crosslinking; as can be seen from the comparison sample 2 and the comparison sample 4, the polysaccharide microsphere prepared by the method can obtain 3-7 times higher flow rate than that of the common microsphere after being crosslinked twice; as can be seen from the comparison of the sample 3 and the sample 1, the polysaccharide microsphere prepared by the method can obtain higher flow velocity after the dosage of microcrystalline cellulose is increased.
It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and not limiting of the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments of the present invention, and all obvious changes and modifications that come within the scope of the invention are defined by the following claims.
Claims (6)
1. A preparation method of high-rigidity macroporous polysaccharide microspheres comprises the following steps:
1) 9g of microcrystalline cellulose and 600mL of imidazolyl ionic liquid are mixed, heated to 100-170 ℃ and dissolved in the imidazolyl ionic liquid to obtain cellulose solution; 31g of agarose and 400mL of deionized water are mixed, heated to 80-130 ℃ for dissolution, and an agarose aqueous solution is obtained, wherein the volume ratio of the imidazolyl ionic liquid to the deionized water is 3:2;
2) Slowly adding the agarose water solution into the cellulose solution under the stirring condition, and uniformly stirring to obtain a water phase;
3) According to the aqueous phase: oil phase mass ratio 1:1.2-1.5 adding the water phase into the oil phase, stirring for 20-80min at 100-300rpm, and then cooling the system to below 25 ℃ within 1 h;
4) Mixing the mixed system obtained in the step 3) with absolute ethyl alcohol according to the mass ratio of 1:3-5, uniformly stirring, standing, and then cleaning the polysaccharide microsphere by using ethyl alcohol and deionized water to remove an organic phase to obtain a polysaccharide microsphere base sphere;
5) Mixing the polysaccharide microsphere base spheres with a crosslinking activator according to the volume ratio of 5:1-4, adding an organic solvent according to the volume ratio of the polysaccharide microsphere base spheres to the organic solvent of 1:1-1.5, mixing, heating the mixed system to 30-50 ℃, adding 50% sodium hydroxide into the mixed system according to the volume ratio of the polysaccharide microsphere base spheres to the 50% sodium hydroxide of 5:1-3, reacting for 3-24 hours to obtain epoxy-activated composite polysaccharide microspheres, and cleaning the epoxy-activated composite polysaccharide microspheres by using ethanol and deionized water to remove the organic reagent and the sodium hydroxide so as to achieve neutrality.
2. The method for preparing the high-rigidity macroporous polysaccharide microsphere according to claim 1, wherein the imidazolyl ionic liquid comprises 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole chloride or 1-butyl-3-methylimidazole bromide.
3. The method for preparing the high-rigidity macroporous polysaccharide microsphere according to claim 1, wherein the oil phase is prepared by the following method: heating one or more of liquid paraffin, petroleum ether, toluene and o-xylene to 50-90 ℃ under stirring, adding span 80, span 60 and tween 20 mixed according to the mass ratio of 10:1-2:0-1, and stirring to completely dissolve for standby.
4. The method for preparing high-rigidity macroporous polysaccharide microsphere according to claim 1, wherein the crosslinking activator comprises one or two of epichlorohydrin and 1, 4-butanediol diglycidyl ether.
5. The method for preparing the high-rigidity macroporous polysaccharide microsphere according to claim 1, wherein the organic solvent comprises one or more of acetone, 1, 4-dioxane and dimethyl sulfoxide.
6. A high-rigidity macroporous polysaccharide microsphere prepared by the method of any one of claims 1-5.
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