An ordered macroporous polysaccharide microsphere chromatography medium and a method for preparing the same
By using regular calcium carbonate whiskers as pore-forming agents and combining them with sieving methods, ordered macroporous polysaccharide microsphere chromatographic media were prepared, solving the problem of discontinuous pore structure and achieving high-efficiency mass transfer performance and separation of biomacromolecules.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA UNIV OF SCI & TECH
- Filing Date
- 2026-04-24
- Publication Date
- 2026-07-28
AI Technical Summary
The pore structure of existing polysaccharide chromatography media is discontinuous, which easily leads to the formation of isolated pores, resulting in limited improvement in mass transfer performance.
Regular calcium carbonate whiskers are used as pore-forming templates, and ordered macroporous polysaccharide microsphere chromatographic media are formed by filtration and sieving, ensuring that the pores are interconnected, uniform, and free of narrow pore throats.
It significantly improved mass transfer efficiency, reduced mass transfer resistance, and enhanced the separation efficiency and production capacity of biomacromolecules.
Smart Images
Figure CN122076411B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chromatographic media technology, and in particular to an ordered macroporous polysaccharide microsphere chromatographic media and its preparation method. Background Technology
[0002] The separation and purification of biomacromolecules is a critical bottleneck in their production process, typically accounting for 60-90% of the total cost, with product yields generally only 5-40%. Especially with the development of high-expression cell lines, the application of large bioreactors and new culture technologies, the expression levels of upstream biomacromolecules have significantly increased, making downstream separation and purification the main technical bottleneck in the preparation of biomacromolecule products. Unlike traditional small molecule chemical products, the characteristics of biomacromolecule separation and purification processes are mainly reflected in the special nature and complexity of the biological products: (1) biomacromolecules have low stability and are easily denatured and inactivated; (2) they have large molecular weights, resulting in low separation, purification, diffusion, and mass transfer rates; (3) the target product has a similar structure to its coexisting impurities, making separation difficult. Therefore, the quality and efficiency of the separation process directly affect the value of the bioproducts.
[0003] Chromatography, due to its high separation precision, simple equipment, and convenient operation, is widely used in downstream biological separation and purification processes. Statistics show that over 95% of protein drugs worldwide are produced using chromatographic separation techniques. Against this backdrop, achieving high efficiency in chromatographic technology—namely, high adsorption capacity, high activity yield, and high mass transfer rate—can minimize the denaturation and inactivation of biomolecules, increase production capacity, and reduce costs, making it a key focus for researchers in the field of bioseparation engineering.
[0004] Polysaccharide chromatographic media, such as cellulose, agarose, and dextran, were among the earliest types of chromatographic media used for bioseparation. This is mainly because polysaccharide media contain a large number of hydroxyl groups, making them easy to modify with functional ligands, and exhibit weak non-specific adsorption, making them particularly suitable for the separation and purification of unstable biomolecules. To improve the dynamic separation capacity and column efficiency of chromatographic columns, researchers have developed perfusion chromatographic media, also known as flow-through chromatographic media or perfusion chromatographic media. These media possess two different pore sizes: macropores that permeate the entire chromatographic medium, called convection pores, and micropores that intersect and connect the macropores, called diffusion pores. During chromatography, convection pores promote the flow of the mobile phase, reduce mass transfer resistance, achieve convective mass transfer, and significantly improve the mass transfer rate of biomolecules within the medium; while diffusion pores provide a large number of adsorption sites, ensuring a relatively high adsorption capacity of the medium. Therefore, perfusion separation media can maintain a high dynamic adsorption capacity even at high mobile phase flow rates, making them the preferred choice for the separation of biomolecules.
[0005] Currently, commonly used pore-forming methods mainly include solid template methods, dual emulsification methods, and phase separation methods. However, these methods still have their own limitations. For example, solid template methods tend to form isolated macropores that are not connected to micro / mesopores, resulting in limited improvement in separation performance. High concentrations of solid templates are often required to avoid forming isolated pore structures, but this can lead to an overly porous pore structure and weak mechanical properties. For instance, CN101036876A discloses a method for preparing a macroporous cellulose microsphere protein adsorption medium. It uses biocompatible solid calcium carbonate microparticles as a pore-forming agent, combined with sodium cellulose xanthate and transformer oil, and prepares a cellulose microsphere protein adsorption medium with ultra-large pore size and high porosity through suspension preparation, dispersion emulsification, gel solidification, and cross-linking processes. However, the above scheme uses calcium carbonate microparticles as a pore-forming agent. Due to the small particle size of calcium carbonate, it is easy to form isolated pores, and the improvement in mass transfer is not significant. Furthermore, the dual-emulsion strategy utilizes the thermodynamic instability of dual emulsions to allow the dispersed phase within the primary emulsion and the continuous phase outside the secondary emulsion to come into contact and fuse, thereby forming a pervasive macroporous structure within the primary emulsion continuous phase. However, excessively large emulsion droplets make it difficult to control the macroporous structure; the pore structure formed by phase separation method has a smaller pore size and is greatly affected by the environment, resulting in high diffusion resistance of biomolecules within the pores and slow separation rate.
[0006] Therefore, the above traditional methods generally suffer from the problem of discontinuous macroporous structures, which easily lead to island-like pore structures or a large number of narrow pore throats, resulting in limited improvement in mass transfer performance. Summary of the Invention
[0007] The purpose of this invention is to overcome at least one of the defects of the prior art, such as discontinuous pore structure, easy formation of isolated pores, and limited improvement in mass transfer performance, and to provide an ordered macroporous polysaccharide microsphere chromatographic medium and its preparation method.
[0008] The objective of this invention can be achieved through the following technical solutions: This invention first provides a method for preparing ordered macroporous polysaccharide microsphere chromatographic media, the preparation method comprising the following steps: S1: Dissolve the polysaccharide powder in an alkaline urea solution to obtain a polysaccharide solution; S2: Using regular calcium carbonate whiskers as a pore-forming template, it is added to a polysaccharide solution and stirred to obtain a mixed solution; the mixed solution is added to the oil phase as an aqueous phase to form a water-in-oil emulsion under the action of a surfactant; S3: After emulsification for a period of time, the microspheres are solidified and calcium carbonate whiskers are removed using an acid regeneration method; S4: Using the length of calcium carbonate whiskers as the sieving standard, a filter screen with a pore size equal to the length of the calcium carbonate whiskers is used for sieving. Microspheres with a size smaller than the length of the calcium carbonate whiskers are the ordered macroporous polysaccharide microsphere chromatographic medium. When there is a length distribution of calcium carbonate whiskers, the minimum length is used as the sieving standard.
[0009] Further, in step S1, the polysaccharide is any one of cellulose, agarose, chitosan, chitin, and dextran.
[0010] Further, in step S1, the concentration of the polysaccharide solution is 2~10 wt%.
[0011] Furthermore, in step S1, the alkaline urea solutions are all alkaline aqueous solutions composed of alkaline compounds and urea compounds.
[0012] Furthermore, the concentration of the alkaline compound in the alkaline urea solution is 5-20 wt%.
[0013] Furthermore, the alkaline compound is any one or more of NaOH, KOH, and LiOH.
[0014] Furthermore, the concentration of the urea compound is 2-10 wt%.
[0015] Furthermore, the urea compound is any one or more of urea and thiourea.
[0016] Furthermore, in step S2, the diameter of the calcium carbonate whiskers ranges from 0.1 to 10 μm, the length ranges from 10 to 500 μm, and the aspect ratio is greater than 10.
[0017] Further, in step S2, the mass ratio of the polysaccharide to calcium carbonate whiskers is 10:(1~50).
[0018] Furthermore, in step S2, the ratio of the aqueous phase to the oil phase in the water-in-oil emulsion is less than 3:7.
[0019] Further, in step S2, the oil phase is any one of liquid paraffin, isooctane, cyclohexane, white oil, silicone oil, petroleum ether, and vegetable oil.
[0020] Further, in step S2, the surfactant is any one or more of Span20, Span40, Span60, Span80, and Span85.
[0021] Furthermore, in step S3, the emulsification is performed using any one of suspension emulsification, microfluidics, or membrane emulsification.
[0022] Furthermore, in step S3, the emulsification time is 0.5~2 h.
[0023] Furthermore, in step S4, the acid used for acid regeneration is an inorganic acid or an organic acid.
[0024] Furthermore, the inorganic acid is any one or more of sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid.
[0025] Furthermore, the organic acid is any one or more of formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, adipic acid, maleic acid, lactic acid, and citric acid.
[0026] The present invention further provides an ordered macroporous polysaccharide microsphere chromatographic medium, which is prepared by any of the above preparation methods.
[0027] Furthermore, the diameter of the ordered macroporous polysaccharide microsphere chromatographic medium is 1~500 μm.
[0028] Furthermore, the macropore size of the ordered macroporous polysaccharide microsphere chromatographic medium is 0.1-10 μm.
[0029] Compared with the prior art, the present invention has the following technical advantages: (1) This invention innovatively utilizes regular calcium carbonate whiskers as pore-forming agents to construct a series of ordered macroporous polysaccharide microsphere chromatographic media. Since the pore-forming template is a regular whisker, the length of the calcium carbonate whisker is used as the sieving standard. Through filtration and sieving, microspheres with a diameter smaller than the length of the calcium carbonate whisker have an ordered, interconnected, uniform, and regular cylindrical through-pore structure without narrow pore throats. The resulting ordered macroporous polysaccharide microsphere chromatographic media has lower mass transfer resistance and significantly improved mass transfer efficiency of target molecules.
[0030] (2) This invention innovatively utilizes regular calcium carbonate whiskers as a pore-forming agent. Since the length of the calcium carbonate whiskers is greater than the particle size of the microspheres, the interconnection of the macroporous structure within the microspheres can be guaranteed, thereby significantly improving the mass transfer effect. In addition, this invention can control the number of interconnected pores by adjusting the amount or ratio of the calcium carbonate whisker pore-forming agent, and control the size of the interconnected pores by adjusting the diameter of the calcium carbonate whiskers.
[0031] (3) The preparation process of the ordered macroporous polysaccharide microsphere chromatographic medium of the present invention is simple, the pore structure is interconnected and uniform, and it is applicable to the preparation of various polysaccharide microspheres. It can effectively promote the mass transfer of target molecules in the microsphere medium and is expected to serve as a new type of high-performance chromatographic medium platform microsphere. Attached Figure Description
[0032] Figure 1 This is a scanning electron microscope image of the macroporous cellulose microspheres in Example 1 of the present invention.
[0033] Figure 2This is a scanning electron microscope image of the macroporous chitin microspheres in Example 2 of the present invention.
[0034] Figure 3 This is a scanning electron microscope image of the macroporous agarose microspheres in Example 3 of the present invention.
[0035] Figure 4 This is a scanning electron microscope image of the cellulose microspheres in Comparative Example 1 of the present invention.
[0036] Figure 5 This is a scanning electron microscope image of the cellulose microspheres in Comparative Example 2 of the present invention.
[0037] Figure 6 These are the back pressure curves of the cellulose microspheres prepared in Example 1, Comparative Example 1, and Comparative Example 2 of this invention. Detailed Implementation
[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.
[0039] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
[0040] To address the challenges in preparing macroporous polysaccharide microsphere chromatographic media, this invention innovatively proposes a novel method for preparing ordered macroporous polysaccharide microsphere chromatographic media. The key to this invention lies in utilizing regular calcium carbonate whiskers as a solid template pore-forming agent. Calcium carbonate whiskers exhibit a micron-sized fibrous structure. Using uniform calcium carbonate whiskers ensures the absence of isolated pore structures. Furthermore, this invention uses the length of the calcium carbonate whiskers as a sieving standard. Through sieving, microspheres with a diameter smaller than the length of the calcium carbonate whiskers can form a continuous, uniform macroporous structure without narrow throats, extending from the surface of the microsphere inwards. Compared to previously reported methods, this method not only simplifies the preparation process and produces a continuous, uniform pore structure without narrow throats, but also allows for control of the pore size by adjusting the amount or ratio of the calcium carbonate pore-forming agent.
[0041] This invention uses calcium carbonate whiskers as a pore-forming template. Calcium carbonate whiskers have a regular straight-line shape, and the through-holes generated by using them as templates are ordered regular cylindrical shapes. However, when conventional calcium carbonate microparticles are used as templates, the construction of through-holes depends on the contact between microparticles. This contact is generated randomly, resulting in non-uniform pores and a tendency to form island-like pore structures. That is, calcium carbonate microparticles are isolated in microspheres and do not contact other particles, resulting in the formation of closed pores.
[0042] Furthermore, this invention does not simply obtain ordered macroporous microspheres by using calcium carbonate whiskers; it also requires a specific sieving method. By using regular calcium carbonate whiskers in conjunction with a sieving method, microspheres smaller than the sieve mesh size have a through-pore structure, while microspheres larger than the sieve mesh size do not.
[0043] The first aspect of this invention provides a method for preparing ordered macroporous polysaccharide microsphere chromatographic media, the preparation method comprising the following steps: S1: Dissolve the polysaccharide powder in an alkaline urea solution to obtain a polysaccharide solution; S2: Using regular calcium carbonate whiskers as a pore-forming template, it is added to a polysaccharide solution and stirred to obtain a mixed solution; the mixed solution is added to the oil phase as an aqueous phase to form a water-in-oil emulsion under the action of a surfactant; S3: After emulsification for a period of time, the microspheres are solidified and calcium carbonate whiskers are removed using an acid regeneration method; S4: Using the length of calcium carbonate whiskers as the sieving standard, a filter screen with a pore size equal to the length of the calcium carbonate whiskers is used for sieving. Microspheres with a size smaller than the length of the calcium carbonate whiskers are the ordered macroporous polysaccharide microsphere chromatographic medium. When there is a length distribution of calcium carbonate whiskers, the minimum length is used as the sieving standard.
[0044] In some specific embodiments, in step S1, the polysaccharide is any one of cellulose, agarose, chitosan, chitin, and dextran.
[0045] In some specific embodiments, in step S1, the concentration of the polysaccharide solution is 2-10 wt%, preferably 4-8 wt%.
[0046] In some specific embodiments, in step S1, the dissolution temperature is -20 to -4°C.
[0047] In some specific embodiments, in step S1, the alkaline urea solution is an alkaline aqueous solution composed of alkaline compounds and urea compounds.
[0048] In some more specific embodiments, the concentration of the alkaline compound in the alkaline urea solution is 5-20 wt%.
[0049] In some more specific embodiments, the alkaline compound is any one or more of NaOH, KOH, and LiOH, preferably NaOH.
[0050] In some more specific embodiments, the concentration of the urea compound is 2 to 10 wt%.
[0051] In some more specific embodiments, the urea compound is any one or more of urea and thiourea, preferably urea.
[0052] In some specific embodiments, in step S2, the diameter of the calcium carbonate whiskers ranges from 0.1 to 10 μm, the length ranges from 10 to 500 μm, and the aspect ratio is greater than 10.
[0053] In some specific embodiments, in step S2, the mass ratio of the polysaccharide to calcium carbonate whiskers is 10:(1~50).
[0054] In some specific embodiments, in step S2, the ratio of the aqueous phase to the oil phase in the water-in-oil emulsion is less than 3:7.
[0055] In some specific embodiments, in step S2, the oil phase is any one of liquid paraffin, isooctane, cyclohexane, white oil, silicone oil, petroleum ether, and vegetable oil.
[0056] In some specific embodiments, in step S2, the surfactant is any one or more of Span20, Span40, Span60, Span80, and Span85.
[0057] In some specific implementations, in step S3, the emulsification is carried out using any one of suspension method, microfluidics, or membrane emulsification.
[0058] In some specific implementations, the emulsification time in step S3 is 0.5 to 2 hours.
[0059] In some specific embodiments, in step S4, the acid used for acid regeneration is an inorganic acid or an organic acid.
[0060] In some more specific embodiments, the inorganic acid is any one or more of sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid, preferably sulfuric acid or hydrochloric acid.
[0061] In some more specific embodiments, the organic acid is any one or more of formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, adipic acid, maleic acid, lactic acid, and citric acid.
[0062] The present invention further provides an ordered macroporous polysaccharide microsphere chromatographic medium, which is prepared by any of the above preparation methods.
[0063] In some specific embodiments, the diameter of the ordered macroporous polysaccharide microsphere chromatographic medium is 1~500μm, corresponding to the length of the calcium carbonate whiskers used in the preparation.
[0064] In some specific embodiments, the macropore diameter of the ordered macroporous polysaccharide microsphere chromatographic medium is 0.1-10 μm, corresponding to the diameter of the calcium carbonate whiskers used in the preparation.
[0065] Each of the above embodiments can be implemented individually, or in any combination of two or more. The following detailed description of specific examples will further illustrate these embodiments.
[0066] Example 1: This embodiment provides an ordered macroporous cellulose microsphere, the preparation method of which is as follows: (1) Dissolve 5 g of cellulose in 95 g of alkaline urea solution (11 wt% NaOH / 4 wt% urea) at -12 ℃ to form a 5 wt% transparent cellulose solution; (2) Add 3 g of calcium carbonate whiskers (1 μm in diameter and 100 μm in length) to 97 g of alkaline urea solution (11 wt% NaOH / 4 wt% urea) and mix at -20 ℃ for 10 min to form a calcium carbonate whisker suspension.
[0067] (3) The obtained cellulose solution and calcium carbonate whisker solution were mixed in an ice bath at a volume ratio of 8:2 for 20 min to form a cellulose / calcium carbonate whisker mixture. 10 mL of the mixture was added to 100 mL of liquid paraffin, and then 2.5 g of Span80 was added. The mixture was emulsified for 1 h.
[0068] (4) Pour 70 mL of 5 wt% hydrochloric acid into the emulsion system and stir for 15 min to solidify the cellulose and remove the calcium carbonate whiskers. Use a filter screen with a pore size of 100 μm to sieve the microspheres, and the filtered microspheres are the macroporous cellulose microspheres prepared in this example.
[0069] Figure 1 The microstructure of the cellulose microspheres using calcium carbonate whiskers as a porogen in Example 1 is shown. As can be seen from the figure, the overall size of the microspheres is approximately 30-90 μm, and the macropore diameter is approximately 1 μm.
[0070] Example 2: This embodiment provides an ordered macroporous chitin microsphere, the preparation method of which is as follows: (1) Add 4 g of chitosan to 96 g of alkaline urea solution (11 wt% NaOH / 4 wt% urea), freeze the solution in a -25 ℃ freezer for 5 h, and then thaw it by mechanical stirring. After thawing, place the solution in a -25 ℃ freezer again and repeat the above operation 3 times to form a 4 wt% transparent chitosan solution.
[0071] (2) Add 20 g of Span85 to the chitin solution from the previous step and stir the mixture thoroughly. Add 10 g of calcium carbonate whiskers (1 μm in diameter and 100 μm in length) to the above solution and stir at 500 rpm. Add 10 mL of the mixture to 100 mL of liquid paraffin, then add 2.5 g of Span80 and emulsify for 2 h.
[0072] (3) Add 100 mL of 5 wt% sulfuric acid solution to the above emulsion and stir for 5 min to obtain chitin microspheres; use a 100 μm filter screen to screen the microspheres, and the microspheres with a size smaller than 100 μm are macroporous chitin microspheres.
[0073] pass Figure 2 As can be seen, the chitin microspheres prepared in this embodiment have an overall size of about 10~100 μm and the surface also has a micron-scale macroporous structure with a pore diameter of about 1 μm.
[0074] Example 3: This embodiment provides an ordered macroporous agarose microsphere, the preparation method of which is as follows: (1) Mix 4 g of agarose powder with 96 g of alkali urea solution (4.5 wt% LiOH / 7 wt% KOH / 8 wt% urea) for 5 min. Freeze the above solution in a -25 ℃ freezer for 5 h, and then thaw it by mechanical stirring. After thawing, place the solution in a -25 ℃ freezer again and repeat the above operation 3 times to form an agarose solution with a concentration of 4 wt%.
[0075] (2) Calcium carbonate whiskers (1 μm in diameter and 60 μm in length) were mixed with agarose solution at a mass ratio of 15:85 for 2 h to obtain a homogeneous agarose / calcium carbonate whisker mixed solution. 10 mL of this solution was taken as the dispersed phase, 100 mL of isooctane was used as the oil phase, and 3 g of surfactant Span80 was added. The mixture was emulsified for 2 h to obtain an emulsion.
[0076] (3) Add 100 mL of 5 wt% sulfuric acid solution to the above emulsion and stir for 5 min to obtain agarose microspheres. Use a 60-micron filter screen to sieve the microspheres. Microspheres smaller than 60 microns are macroporous agarose microspheres.
[0077] pass Figure 3 As can be seen, the overall size of the agarose microspheres prepared in this embodiment is about 10~60 μm, and the surface also has a micron-scale macroporous structure with a pore diameter of about 1 μm.
[0078] Comparative Example 1: This comparative example provides microporous cellulose microspheres prepared without using a pore-forming template. The difference from Example 1 is that this comparative example does not use a pore-forming template; instead, it obtains the microspheres directly through emulsification and regeneration of a cellulose solution. Other steps are the same as in Example 1.
[0079] Depend on Figure 4 The scanning electron microscope images show that the microporous cellulose microspheres prepared in this comparative example do not have obvious macroporous structures on their surface.
[0080] Comparative Example 2: This comparative example provides macroporous cellulose microspheres. The difference from Example 1 is that this comparative example uses conventional calcium carbonate particles (particle size 0.1-10 micrometers) as the porogen. Other steps are the same as in Example 1.
[0081] Depend on Figure 5 The scanning electron microscope images show that the macropores on the surface of the macroporous cellulose microspheres prepared in this comparative example are island-like, making it difficult to form a through-pore structure suitable for mass transfer.
[0082] Based on the successful preparation of the above embodiments and comparative examples, the present invention further conducts the following flow kinetic tests on the above polysaccharide microsphere chromatographic medium.
[0083] Flow kinetics experiments were performed using an AKTA Explorer 100 system (Amersham Biosciences). Microsphere media were loaded into an HR 5 / 10 column, and the back pressure of the sample column was measured at different flow rates by varying the mobile phase flow rate.
[0084] like Figure 6 As shown, at the same flow rate, both types of calcium carbonate porous microspheres exhibited lower back pressure compared to unporous cellulose microspheres, indicating that the macroporous structure improved the mass transfer performance of the microspheres. More importantly, compared to calcium carbonate particle porous microspheres, calcium carbonate whisker porous microspheres showed a maximum back pressure reduction of 40% at the same flow rate and pore-forming agent dosage, demonstrating that using calcium carbonate whisker templates can form a through-pore structure, significantly reducing mass transfer resistance.
[0085] In summary, this invention innovatively utilizes regular calcium carbonate whiskers as a pore-forming agent and combines it with a specific sieving method to construct ordered macroporous polysaccharide microsphere chromatographic media. The polysaccharide chromatographic microspheres prepared using the method described in this invention simultaneously possess macroporous, mesoporous, and microporous pore structures. More importantly, because the pore-forming template is a regular whisker, the resulting pores are uniform, interconnected, ordered, and without narrow throats, significantly improving the mass transfer performance of the microsphere media.
[0086] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
Claims
1. A method for preparing an ordered macroporous polysaccharide microsphere chromatographic medium, characterized in that, The preparation method includes the following steps: S1: Dissolve the polysaccharide powder in an alkaline urea solution to obtain a polysaccharide solution; S2: Using regular calcium carbonate whiskers as a pore-forming template, it is added to a polysaccharide solution and stirred to obtain a mixed solution; the mixed solution is added to the oil phase as an aqueous phase to form a water-in-oil emulsion under the action of a surfactant; The diameter of the calcium carbonate whiskers ranges from 0.1 to 10 μm, the length ranges from 10 to 500 μm, and the aspect ratio is greater than 10; the mass ratio of the polysaccharide to the calcium carbonate whiskers is 10:(1~50). S3: After emulsification for a period of time, the microspheres are solidified and calcium carbonate whiskers are removed using an acid regeneration method; S4: Using the length of calcium carbonate whiskers as the sieving standard, a filter screen with a pore size equal to the length of the calcium carbonate whiskers is used for sieving. Microspheres with a size smaller than the length of the calcium carbonate whiskers are the ordered macroporous polysaccharide microsphere chromatographic medium. When there is a length distribution of calcium carbonate whiskers, the minimum length is used as the sieving standard.
2. The method for preparing the ordered macroporous polysaccharide microsphere chromatographic medium according to claim 1, characterized in that, In step S1, the polysaccharide is any one of cellulose, agarose, chitosan, chitin, and dextran.
3. The method for preparing the ordered macroporous polysaccharide microsphere chromatographic medium according to claim 1, characterized in that, In step S1, the concentration of the polysaccharide solution is 2-10 wt%.
4. The method for preparing the ordered macroporous polysaccharide microsphere chromatographic medium according to claim 1, characterized in that, In step S1, the alkaline urea solutions are all alkaline aqueous solutions composed of alkaline compounds and urea compounds; The alkaline urea solution contains alkaline compounds at a concentration of 5-20 wt% and urea compounds at a concentration of 2-10 wt%. The alkaline compound is any one or more of NaOH, KOH, and LiOH, and the urea compound is any one or more of urea and thiourea.
5. The method for preparing the ordered macroporous polysaccharide microsphere chromatographic medium according to claim 1, characterized in that, In step S2, the ratio of the aqueous phase to the oil phase in the water-in-oil emulsion is less than 3:7; The oil phase is any one of liquid paraffin, isooctane, cyclohexane, white oil, silicone oil, petroleum ether, and vegetable oil; The surfactant is any one or more of Span20, Span40, Span60, Span80, and Span85.
6. The method for preparing the ordered macroporous polysaccharide microsphere chromatographic medium according to claim 1, characterized in that, In step S3, the emulsification is performed using any one of suspension emulsification, microfluidics, or membrane emulsification. The emulsification time is 0.5 to 2 hours.
7. The method for preparing the ordered macroporous polysaccharide microsphere chromatographic medium according to claim 1, characterized in that, In step S3, the acid used for acid regeneration is an inorganic acid or an organic acid; The inorganic acid is any one or more of sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid, and the organic acid is any one or more of formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, adipic acid, maleic acid, lactic acid, and citric acid.
8. A chromatographic medium of ordered macroporous polysaccharide microspheres, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.
9. The ordered macroporous polysaccharide microsphere chromatographic medium according to claim 8, characterized in that, The ordered macroporous polysaccharide microsphere chromatographic medium has a sphere diameter of 1~500 μm and a macropore diameter of 0.1-10 μm.