CN115160603A - 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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- 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 1
- 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 1
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Images
Classifications
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- 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 imidazole-based ionic liquid to obtain a cellulose solution; dissolving agarose in deionized water to obtain an agarose aqueous solution; 2) Slowly adding the agarose aqueous solution into the cellulose solution under the condition of stirring, and uniformly stirring to obtain a water phase; 3) Adding the water phase into the oil phase, stirring, and then cooling 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 a polysaccharide microsphere-based sphere; 5) Mixing a crosslinking activator with the polysaccharide microsphere base spheres, then adding an organic solvent, mixing, heating a mixed system, adding sodium hydroxide for reaction to obtain the epoxy group activated composite polysaccharide microspheres, and then cleaning to be neutral. The polysaccharide microsphere prepared by the invention still has stronger mechanical strength when having larger aperture.
Description
Technical Field
The invention belongs to the technical field of preparation of polysaccharide microspheres, 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 that a mobile phase of a mixed sample carrying different components flows through a plurality of stationary phases, a series of physical or chemical interactions exist between the sample and the stationary phases, after multiple distribution, part of the sample is adsorbed in the stationary phases due to different interactions between different components of the sample and the stationary phases, and then the interaction force between the sample and the stationary phases is changed through elution solutions with different conditions to elute, so that the effect of mutually separating different components of the sample is achieved.
The stationary phase of chromatography (also known as chromatography media) is typically in the form of micron-sized spherical particles, which can be prepared from polymers or natural polysaccharides. The polymer matrix chromatographic medium has stronger mechanical strength and is beneficial to use, but has poor biocompatibility and may have nonspecific adsorption with a separated sample; media of natural polysaccharide matrices (e.g., agarose) are more biocompatible, but generally have lower mechanical strength, which is detrimental to scale-up and cost reduction.
A method for improving the rigidity of gel beads using monofunctional crosslinkers containing masking functionality is described in EP 203049. In another example, a manufacturing process for cross-linking polysaccharide gels to obtain large pores 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, generally requiring more solvent and multiple crosslinks. In CN 111989155A, it is described that the mechanical strength of the natural polysaccharide microspheres can be enhanced by embedding the fibers, but this method requires microfibrillation of the embedded fibers in advance (to embed the fibers in the microspheres without affecting the spherical shape), and has complicated process and high cost. In CN 112619612A, a preparation method of a high-strength cellulose/agarose composite microsphere is disclosed, in the method, alkaline thiourea is used as a solvent to dissolve cellulose, the prepared microsphere has a small pore passage, and if the method is applied to the field of chromatographic separation, the problems of large elution volume, low elution concentration, low loading capacity and the like caused by low mass transfer speed can be solved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a high-rigidity macroporous polysaccharide microsphere and a preparation method thereof.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a preparation method of high-rigidity macroporous polysaccharide microspheres comprises the following steps:
1) Dissolving microcrystalline cellulose in imidazole-based ionic liquid to obtain a cellulose solution; dissolving agarose in deionized water to obtain an agarose aqueous solution;
2) Slowly adding the agarose aqueous solution into the cellulose solution under the condition of stirring, and uniformly stirring to obtain a water phase;
3) According to the water phase: oil phase mass ratio 1: (1.2-1.5) adding the water phase into the oil phase, stirring at 100-300rpm for 20-80min, and then cooling 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 a polysaccharide microsphere-based sphere;
5) Mixing the polysaccharide microsphere base spheres with a crosslinking activator according to a volume ratio of 5 (1-4), then adding an organic solvent according to a volume ratio of 1 (1-1.5) of the polysaccharide microsphere base spheres to the organic solvent, mixing, heating the mixed system to 30-50 ℃ (optimally 35-40 ℃), adding 50% sodium hydroxide according to a volume ratio of 5 (1-3) of the polysaccharide microsphere base spheres to 50% sodium hydroxide, reacting for 3-24h (optimally 5-8 h) to obtain epoxy group activated composite polysaccharide microspheres, and cleaning to neutrality.
Preferably, in the step 1), the microcrystalline cellulose and the imidazole-based ionic liquid are mixed according to the mass ratio of 1 (30-100), and are heated to 100-170 ℃ (optimally 100-120 ℃) for dissolution.
More preferably, the imidazolyl ionic liquid includes, but is not limited to, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride or 1-butyl-3-methylimidazolium bromide.
Preferably, in the step 1), agarose and deionized water are mixed according to the mass ratio of 1 (5-15), and are 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 deg.C (preferably 60-70 deg.C) under stirring, adding span 80, span 60 and tween 20 at a mass ratio of 10 (1-2) to (0-1), and stirring to completely dissolve.
Preferably, in the step 4), the polysaccharide microspheres are washed by using ethanol and deionized water, and the organic phase is removed to obtain the microsphere-based spheres.
Preferably, in step 5), deionized water is used to clean the epoxy group activated composite polysaccharide microspheres, and organic reagents and sodium hydroxide are removed to make the microspheres neutral.
Preferably, the crosslinking activator includes, but is not limited to, one or two 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 (chromatography medium), which comprises the following steps: 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, and can be used as a skeleton of the microsphere; the agarose has the characteristics of looseness and porosity, so that the microspheres retain the characteristic of high specific surface area of common agarose microspheres. The present invention does not require a pre-treated embedded fiber. The polysaccharide microsphere prepared by the method can reach the same rigidity of a product sold in the market after a small amount of cross-linking agent is used, and can obtain rigidity far exceeding that of the product sold in the market after secondary cross-linking, and has a good pore structure.
Drawings
FIG. 1 shows the results of the pressure and flow rate measurements of various samples in the test examples of the present invention.
FIG. 2 is the SEM topography of example 2 sample 2 of the present invention.
FIG. 3 is an SEM topography of 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 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 is not to be taken as limiting the scope of the invention.
Example 1:
preparing an aqueous phase: 9g of microcrystalline cellulose was dissolved in 600ml of 1-ethyl-3-methylimidazolium acetate solution, heated to 100 ℃ and dissolved. 31g of low electroosmosis 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: weighing 1.2kg of liquid paraffin and 0.2kg of petroleum ether, adding into a reaction kettle, heating to 60 ℃, then adding 8g of span 80 and 1g of span 60, and stirring for dissolving.
Slowly adding the water phase into the oil phase, obtaining polysaccharide microspheres with proper particle size by adjusting the rotating speed (50-300 rpm) (the particle size of the microspheres can be observed by a microscope in a sampling manner, the rotating speed is proper when the particle size of the microspheres is 150 mu m), emulsifying for about 30min, and cooling the reaction system to below 25 ℃ by ice water.
And adding the emulsification system into 4L of absolute ethyl alcohol, stirring uniformly, standing, decanting, and washing off an organic phase in the system by using the absolute ethyl alcohol and deionized water to obtain the polysaccharide microsphere-based sphere.
And (3) crosslinking: weighing 500ml of the polysaccharide microsphere-based spheres obtained in the steps, adding 200ml of epoxy chloropropane, mixing, then adding 500ml of dimethyl sulfoxide, mixing, heating to 35 ℃, then adding 100g of 50% sodium hydroxide, and continuing to react for 6 hours. After the reaction was completed, the polysaccharide microspheres were washed to neutrality with deionized water, which was "sample 1".
Example 2:
based on example 1, the "cross-linking" step is repeated once to obtain "sample 2", the SEM topography is shown in FIG. 2, and the polysaccharide microspheres prepared in this example have 200-1000nm through holes.
Example 3:
preparing a water phase: 18g of microcrystalline cellulose are dissolved in 600ml of 1-ethyl-3-methylimidazolium acetate solution, heated to 100 ℃ and dissolved. 22g of low electroosmosis 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: weighing 1.2kg of liquid paraffin and 0.2kg of petroleum ether, adding into a reaction kettle, heating to 60 ℃, then adding 8g of span 80 and 1g of span 60, and stirring for dissolving.
Slowly adding the water phase into the oil phase, adjusting the rotation speed to obtain polysaccharide microspheres with proper particle size (the particle size of the microspheres can be observed by a microscope for sampling), emulsifying for about 30min, and cooling the reaction system to below 25 ℃ by ice water.
And adding the emulsification system into 4L of absolute ethyl alcohol, stirring uniformly, standing, decanting, and washing off an organic phase in the system by using the absolute ethyl alcohol and deionized water to obtain the polysaccharide microsphere-based sphere.
And (3) crosslinking: weighing 500ml of the polysaccharide microsphere-based spheres obtained in the steps, adding 200ml of epoxy chloropropane, mixing, adding 500ml of dimethyl sulfoxide, mixing, heating to 35 ℃, adding 100g of 50% sodium hydroxide, and continuing to react for 6 hours. After the reaction is finished, deionized water is used for cleaning the polysaccharide microspheres to be neutral, the polysaccharide microspheres are sample 3, the SEM topography is shown in figure 3, after the use amount of cellulose is increased, the pore diameter of the polysaccharide microspheres prepared in the embodiment is obviously increased, the maximum pore diameter is about 5 microns, but the mechanical strength is weakened due to the overlarge pore diameter, and the polysaccharide microspheres are easy to crack.
Comparative example:
this example is a comparative example without the addition of cellulose.
Preparing a water phase: 40g of low electroosmosis agarose was dissolved in 100ml of deionized water, heated to 95 ℃ and dissolved.
Preparing an oil phase: weighing 1.2kg of liquid paraffin and 0.2kg of petroleum ether, adding into a reaction kettle, heating to 60 ℃, then adding 8g of span 80 and 1g of span 60, and stirring for dissolving.
Slowly adding the water phase into the oil phase, regulating the rotating speed to obtain polysaccharide microspheres with proper particle size (the particle size of the microspheres can be observed by a microscope as a sample), emulsifying for about 30min, and cooling the reaction system to below 25 ℃ by ice water.
And adding the emulsification system into 4L of absolute ethyl alcohol, stirring uniformly, standing, decanting, and washing off an organic phase in the system by using the absolute ethyl alcohol and deionized water to obtain the polysaccharide microsphere-based sphere.
And (3) crosslinking: weighing 500ml of the polysaccharide microsphere-based spheres obtained in the steps, adding 200ml of epoxy chloropropane, mixing, adding 500ml of dimethyl sulfoxide, mixing, heating to 35 ℃, adding 100g of 50% sodium hydroxide, and continuing to react for 6 hours. After the reaction is finished, the polysaccharide microspheres are washed to be neutral by using deionized water.
The above "cross-linking" step was repeated once to obtain "sample 4", the SEM topography is shown in FIG. 4, and the polysaccharide microspheres in this comparative example had a small pore size of only 10-30nm.
Test example:
the sample 1, the sample 2, the sample 3 and the sample 4 are respectively loaded into a chromatographic column with the inner diameter of 26mm and the column height of 15cm, deionized water is used as a test fluid, and the pressure flow rate test is carried out under the condition of room temperature, and the test result is shown in figure 1.
Comparing the sample 1 and the sample 4, the polysaccharide microsphere prepared by the invention can obtain a flow rate which is higher than that of the common microsphere after being crosslinked for two times only once; comparing the sample 2 with the sample 4, the polysaccharide microsphere prepared by the invention can obtain a flow rate which is 3-7 times higher than that of the common microsphere after twice crosslinking; comparing sample 3 and sample 1, the polysaccharide microsphere prepared by the invention can obtain higher flow rate after increasing the dosage of microcrystalline cellulose.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications can be made on the basis of the above description, and all the implementation methods cannot be exhaustive, and the obvious variations or modifications introduced in the technical scheme of the present invention are within the protection scope of the present invention.
Claims (10)
1. A preparation method of high-rigidity macroporous polysaccharide microspheres comprises the following steps:
1) Dissolving microcrystalline cellulose in imidazole-based ionic liquid to obtain a cellulose solution; dissolving agarose in deionized water to obtain an agarose aqueous solution;
2) Slowly adding the agarose aqueous solution into the cellulose solution under the condition of stirring, and uniformly stirring to obtain a water phase;
3) According to the water phase: oil phase mass ratio 1:1.2-1.5 adding the water phase into the oil phase, stirring at 100-300rpm for 20-80min, and cooling 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 a mass ratio of 1;
5) Mixing the polysaccharide microsphere base sphere with a crosslinking activator according to a volume ratio of 5.
2. The preparation method of the high-rigidity macroporous polysaccharide microsphere according to claim 1, wherein in the step 1), microcrystalline cellulose and the imidazolyl ionic liquid are mixed according to a mass ratio of 1 to 30-100, and are heated to 100-170 ℃ for dissolution.
3. The method for preparing high-rigidity macroporous polysaccharide microspheres according to claim 1, wherein the imidazolyl ionic liquid comprises 1-ethyl-3-methylimidazole acetate, 1-butyl-3-methylimidazole chloride salt or 1-butyl-3-methylimidazole bromide salt.
4. The method for preparing highly rigid macroporous polysaccharide microspheres according to claim 1, wherein in step 1), agarose and deionized water are mixed according to a mass ratio of 1.
5. The method for preparing high rigidity macroporous polysaccharide microspheres 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, then adding span 80, span 60 and tween 20 which are mixed according to the mass ratio of 10.
6. The method for preparing high-rigidity macroporous polysaccharide microspheres according to claim 1, wherein in the step 4), the polysaccharide microspheres are washed by ethanol and deionized water, and an organic phase is removed to obtain microsphere-based spheres.
7. The method for preparing highly rigid macroporous polysaccharide microspheres according to claim 1, wherein in step 5), the epoxy-activated composite polysaccharide microspheres are washed with ethanol and deionized water to remove organic reagents and sodium hydroxide, thereby achieving neutrality.
8. The method for preparing high rigidity macroporous polysaccharide microspheres according to claim 1, wherein the crosslinking activator comprises one or two of epichlorohydrin and 1,4 butanediol diglycidyl ether.
9. The method for preparing highly rigid macroporous polysaccharide microspheres according to claim 1, wherein the organic solvent comprises one or more of acetone, 1, 4-dioxane, and dimethyl sulfoxide.
10. A high-rigidity macroporous polysaccharide microsphere prepared by the method for preparing a high-rigidity macroporous polysaccharide microsphere as claimed in any one of claims 1 to 9.
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