High mechanical strength agarose / sodium alginate composite chromatographic microspheres and a preparation method thereof

Abstract

The present application provides a kind of high mechanical strength agarose / sodium alginate composite chromatography microspheres and its preparation method, wherein, preparation method is added in agarose aqueous solution Sodium alginate, after being stirred sufficiently, form uniform polysaccharide (agarose and sodium alginate) solution, then using emulsifier dispersion, again through agarose cooling gelation polysaccharide composite microspheres are prepared.Calcium chloride aqueous solution is soaked in after being washed polysaccharide composite microspheres, and sodium alginate is gelled by calcium ion, and the mechanical strength of composite microspheres is improved.Back to composite microspheres dropwise epoxy chloropropane and sodium hydroxide aqueous solution are chemically crosslinked, and hydrogen bond between polysaccharide chain segment is converted into covalent bond;In addition, the introduction of sodium alginate makes the hydroxyl group density in the microsphere higher, so that the crosslinking reaction is faster and more sufficient, and the mechanical strength of the microsphere is further improved.The microspheres prepared by the method have high linear flow rate and mechanical strength, and are suitable for separation and purification of biological macromolecules under medium-high pressure.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemistry, and particularly relates to a high-mechanical-strength agarose / sodium alginate composite chromatographic microsphere and a preparation method thereof. BACKGROUND

[0002] The biopharmaceutical industry continuously raises the purity requirements for functional proteins, which continuously promotes the development and research of protein chromatography purification processes. As the stationary phase of chromatography technology, the performance of the medium is one of the most critical factors determining the success of the separation process. Agarose is widely used in the preparation of chromatographic microspheres due to its rich raw materials, low price, low non-specific adsorption, and easy chemical modification. Agarose gel microspheres are constructed by non-covalent bonds to form a three-dimensional network hydrogel. When subjected to a large pressure, plastic deformation often occurs, resulting in microsphere breakage. At this time, with the increase of flow rate, the back pressure rises sharply, and plugging occurs in the chromatography column, which seriously limits the application of agarose gel microspheres in medium-high pressure chromatography environment.

[0003] To improve the mechanical strength of polysaccharide gel microspheres, researchers have developed a number of methods, of which the widely used methods mainly include physical crosslinking and chemical crosslinking. Physical crosslinking usually involves the addition of inorganic nanoparticles or other polymers to natural polysaccharide polymers, which are combined through weak interactions such as van der Waals force, hydrogen bond, and coordination bond, significantly improving the mechanical properties and thermal stability of polysaccharide gel microspheres. Chemical crosslinking converts the hydrogen bonds between polysaccharide chains into covalent bonds by reacting the active functional groups at both ends of the crosslinking agent with the hydroxyl groups on the polysaccharide, constructing a more robust polysaccharide gel network.

[0004] The agarose chromatography microspheres prepared at present have problems such as low mechanical strength and low linear flow rate, which leads to microsphere fragmentation in subsequent high-pressure environments such as protein separation, rapid pressure rise, affects the equipment, and greatly reduces the separation efficiency. Zhao et al. proposed a pre-crosslinking method, that is, adding a bifunctional crosslinking agent, allyl glycidyl ether, to the polysaccharide solution. At this time, the polysaccharide segments are uniformly distributed in the solution, and the crosslinking agent can immediately contact with the hydroxyl group, greatly improving the reaction efficiency. However, due to the limitation of the amount added, the mechanical strength cannot be significantly improved (Zhao X, Huang L, Wu J, et al. Fabrication of rigid and macroporous agarose microspheres by pre-cross-linking and surfactant micelles swelling method [J]. Colloids and Surfaces B: Biointerfaces, 2019, 182: 110377.). Xiao et al. developed a new crosslinking agent and prepared agarose microspheres by one-step pre-crosslinking with cyclic anhydride. Pre-crosslinking gives agarose stronger mechanical strength, allowing it to separate model proteins at a higher linear flow rate under low pressure, but it is still difficult to adapt to industrial situations under medium and high pressure (Xiao Q, Ma M, Chen J, et al. Preparation of macroporous rigid agarose microspheres by pre-crosslinking with cyclic anhydride [J]. International Journal of Biological Macromolecules, 2022, 222: 41-54.). Patent document CN102989400A reports a crosslinking method for agarose microspheres, using pentaerythritol glycidyl ether and epichlorohydrin as crosslinking agents for two-step crosslinking. However, the maximum pressure resistance of the microspheres obtained is only 0.1 MPa, and the maximum linear flow rate is only 400 cm / h. Patent document CN115739050A reports a method of using short-chain alkane dihalides, short-chain diacids, and cyclic anhydride substances as crosslinking agents to crosslink first and then form spheres. The maximum linear flow rate of the agarose microspheres prepared is only 900 cm / h. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a high-mechanical-strength agarose / sodium alginate composite chromatography microsphere and a preparation method thereof. The preparation method prepares composite microspheres by doping sodium alginate in an agarose solution, and combines the crosslinking of sodium alginate with Ca 2+The ion cross-linking, the physical cross-linking between polysaccharide molecules and the chemical cross-linking of epichlorohydrin together build the high-stable polysaccharide gel network, and realize the preparation of the high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0006] A preparation method of high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres, comprising the following steps:

[0007] (1) Dissolve agarose in water, heat to 80-100℃, stir until clear, then stir to cool to 40-90℃, add sodium alginate and stir, to obtain an agarose / sodium alginate solution;

[0008] (2) Disperse sorbitan monooleate and sorbitan monooleate polyoxyethylene ether in toluene to prepare an oil phase;

[0009] (3) Heat the oil phase and inject into the agarose / sodium alginate solution, stir to emulsify, then cool to room temperature, wash the reactants with distilled water, sieve, soak in calcium chloride aqueous solution, then take out and drain to obtain the composite microspheres;

[0010] (4) Mix the composite microspheres, anhydrous sodium sulfate and acetone aqueous solution, and drop epichlorohydrin and sodium hydroxide aqueous solution under heating and stirring, after the drop is completed, carry out heat preservation reaction, after the reaction is completed, wash the product with anhydrous ethanol and distilled water, soak in calcium chloride aqueous solution, to obtain the high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0011] In the above step (1):

[0012] As preferred, the heating temperature of agarose dissolved in water is 90-100℃.

[0013] As preferred, the mass percentage of agarose in water is 1-8%. As further preferred, the mass percentage of agarose in water is 2-6%. More further preferred is 3-4%.

[0014] As preferred, the mass ratio of sodium alginate to agarose is 1:(0.1-20). As further preferred, the mass ratio of sodium alginate to agarose is 1:(0.5-15). More further preferred is 1:(1-2).

[0015] As preferred, the stirring time after adding sodium alginate is 2-18h. As further preferred, the stirring time after adding sodium alginate is 2-14h. More further preferred is 3-12h. As more further preferred, the stirring time after adding sodium alginate is 4-8h.

[0016] As preferred, the time for stirring cooling is 30min.

[0017] As a preference, the temperature is reduced to 70-90°C while stirring. Further preferably, the temperature is 80°C.

[0018] In the above step (2):

[0019] As a preference, the mass of sorbitan monooleate (Span-80) is 0.5-10% of the mass of toluene. Further preferably, the mass of sorbitan monooleate is 1-8% of the mass of toluene. More preferably, the mass of sorbitan monooleate is 3-4% of the mass of toluene.

[0020] As a preference, the mass of sorbitan monooleate polyoxyethylene ether (Tween-80) is 0.2-5% of the mass of toluene. Further preferably, the mass of sorbitan monooleate polyoxyethylene ether is 0.5-4% of the mass of toluene. More preferably, the mass of sorbitan monooleate polyoxyethylene ether is 0.8-1.2% of the mass of toluene.

[0021] In the above step (3):

[0022] As a preference, the heating temperature of the oil phase is 60-70°C. Further preferably, the heating temperature is 65°C.

[0023] As a preference, the stirring and emulsifying time is 10-120 min. Further preferably, the stirring and emulsifying time is 20-40 min. More preferably, the stirring and emulsifying time is 30 min.

[0024] As a preference, the cooling time after emulsification is 10-20 min. Further preferably, the cooling time is 15 min.

[0025] The reaction product is washed with distilled water to remove the upper layer of toluene and emulsifier, and is sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres are soaked in a calcium chloride aqueous solution, and are taken out and drained to obtain the composite microspheres.

[0026] As a preference, the mass concentration of the calcium chloride aqueous solution is 1-15%. Further preferably, the mass concentration is 2-12%. More preferably, the mass concentration is 2-5%.

[0027] As a preference, the soaking time of the microspheres in the calcium chloride aqueous solution is 15-40 h. Further preferably, the soaking time is 20-30 h. More preferably, the soaking time is 24 h.

[0028] As a preference, the mass ratio of the agarose / sodium alginate solution to the oil phase is 1:(0.5-2). Further preferably, the mass ratio is 1:1.

[0029] In the above step (4):

[0030] As a preference, the volume ratio of acetone to water in the aqueous acetone solution is 1 : (0.1 to 10). As a further preference, the volume ratio of acetone to water in the aqueous acetone solution is 1 : (0.5 to 8). Still further preference is 1 : (0.5 to 2).

[0031] As a preference, the concentration of the aqueous sodium hydroxide solution is 50%.

[0032] As a preference, the mass-to-volume ratio of the composite microspheres to the aqueous acetone solution is 1 : (0.1 to 3) g / mL. As a further preference, the mass-to-volume ratio of the composite microspheres to the aqueous acetone solution is 1 : (0.2 to 1) g / mL. Still further preference is 1 : (0.5 to 0.6) g / mL.

[0033] As a preference, the mass-to-volume ratio of the anhydrous sodium sulfate to the aqueous acetone solution is 1 : (0.5 to 8) g / mL. As a further preference, the mass-to-volume ratio of the anhydrous sodium sulfate to the aqueous acetone solution is 1 : (1 to 5) g / mL. Still further preference is 1 : (3 to 4) g / mL.

[0034] As a preference, the temperature of the system is raised to 40 to 60°C before the dropwise addition of the epichlorohydrin and the aqueous sodium hydroxide solution. Further preference is 50°C.

[0035] As a preference, the mass-to-volume ratio of the composite microspheres to the epichlorohydrin is 1 : (0.05 to 0.5) g / mL. As a further preference, the mass-to-volume ratio of the composite microspheres to the epichlorohydrin is 1 : (0.05 to 0.2) g / mL. Still further preference is 1 : (0.08 to 0.12) g / mL.

[0036] As a preference, the mass-to-volume ratio of the composite microspheres to the aqueous sodium hydroxide solution is 1 : (0.05 to 0.5) g / mL. As a further preference, the mass-to-volume ratio of the composite microspheres to the aqueous sodium hydroxide solution is 1 : (0.05 to 0.2) g / mL. Still further preference is 1 : (0.08 to 0.12) g / mL.

[0037] As a preference, the dropwise addition time of the epichlorohydrin and the aqueous sodium hydroxide solution is 0.5 to 40 h. As a further preference, the dropwise addition time of the epichlorohydrin and the aqueous sodium hydroxide solution is 0.5 to 20 h. Still further preference is 1 to 15 h. As still further preference, the dropwise addition time of the epichlorohydrin and the aqueous sodium hydroxide solution is 5 to 7 h.

[0038] As a preference, the reaction time for the incubation reaction is 15 to 20 h. Further preference is 18 h.

[0039] As a preference, the mass concentration of the aqueous calcium chloride solution is 1 to 15%. Further preference is 2 to 12%. Still further preference is 4 to 6%.

[0040] As preferred, the microspheres are soaked in the calcium chloride aqueous solution for 15-40 h. Further preferred is 20-30 h. Still further preferred is 24 h.

[0041] The application discloses a preparation method of high-mechanical-strength agarose / sodium alginate composite chromatography microspheres. The preparation method comprises the following steps: adding sodium alginate into an agarose aqueous solution, fully stirring to form a uniform polysaccharide (agarose and sodium alginate) solution, then dispersing by using an emulsifier, and then preparing polysaccharide composite microspheres by agarose cooling gelation. The washed polysaccharide composite microspheres are soaked in a calcium chloride aqueous solution, and calcium ions are used to make sodium alginate gelate, so that the mechanical strength of the composite microspheres is improved. The epoxy chloropropane and sodium hydroxide aqueous solution are added dropwise into the composite microspheres, chemical cross-linking is realized, hydrogen bonds between polysaccharide chains are converted into covalent bonds, and the strength of the microspheres is further improved. The microspheres prepared by the method have high linear flow rate and mechanical strength, and are suitable for separation and purification of biological macromolecules under medium-high pressure.

[0042] The high-mechanical-strength agarose / sodium alginate composite chromatography microspheres are prepared by the preparation method of the high-mechanical-strength agarose / sodium alginate composite chromatography microspheres.

[0043] Compared with the prior art, the application has the following beneficial effects:

[0044] The preparation method of the high-mechanical-strength agarose / sodium alginate composite chromatography microspheres disclosed by the application dopes sodium alginate into agarose microspheres, and the introduced sodium alginate is ion-crosslinked with calcium ions; flexible alginate coiled chains are integrated into the grid of the agarose network, and serve as reinforcing fillers to form a more stable and elastic gel network, so that the mechanical strength and linear flow rate of the microspheres are improved.

[0045] Meanwhile, the preparation method of the application combines chemical cross-linking of epoxy chloropropane, reacts active functional groups at two ends of the cross-linking agent with hydroxyl groups on polysaccharides (agarose and sodium alginate), converts hydrogen bonds between polysaccharides into covalent bonds, and further improves the mechanical strength of the microspheres.

[0046] The preparation method of the application is simple in operation and easy to scale up, and the prepared composite chromatography microspheres are stable in structure and suitable for separation and purification of various biological macromolecules. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 It is an optical microscope photo of the high-mechanical-strength agarose / sodium alginate composite chromatography microspheres prepared in Example 2.

[0048] Figure 2Back pressure-flow rate plot of the high mechanical strength agarose / sodium alginate composite chromatographic microspheres prepared in Example 2. DETAILED DESCRIPTION

[0049] In order to make the purposes, technical solutions and advantages of the present application more clear and understandable, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are used to explain the present application and are not used to limit the present application. Those skilled in the art can make modifications or equivalent replacements on the basis of understanding the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the protection scope of the present application. The raw materials used in the following specific embodiments are all purchased from the market.

[0050] In the following examples, room temperature refers to 25℃ unless otherwise specified.

[0051] In the following examples, the measurement method of the linear flow rate and mechanical strength (maximum pressure resistance) of the composite chromatographic microspheres: the microspheres are packed in a chromatographic column (XK16 / 20, Cytiva, USA) by homogenization and connected to a protein chromatography system (Autopre100D, Inscinstech, China), and the packing height is 10±0.2 cm. The linear flow rate is increased at certain intervals, and the back pressure value after each stable is recorded. When the pressure (back pressure) changes rapidly, it is the maximum back pressure (maximum pressure resistance) of the medium, and at this time the flow rate is the maximum linear flow rate.

[0052] Example 1

[0053] (1) 1.8 g of agarose was dissolved in 60 mL of water and heated to 95℃ and stirred, and after the solution was clear and transparent, it was stirred for another 30 min to cool to 80℃, 1.8 g of sodium alginate was added, and the stirring was continued for 4 h to form a uniform agarose / sodium alginate solution.

[0054] 1.8 g of sorbitan monooleate (Span-80) and 0.6 g of sorbitan monooleate polyoxyethylene ether (Tween-80) were dispersed in 60 g of toluene to prepare an oil phase, and heated to 65℃ for preheating; the above agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and after stirring for 30 min, it was cooled to room temperature within 15 min. Then the reaction was washed with a large amount of distilled water for several times to remove the upper toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm; finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain the composite microspheres.

[0055] (2) After the water-drained composite microspheres 50 g and 8 g of anhydrous sodium sulfate, 26 mL of an acetone aqueous solution (wherein the volume ratio of acetone to water is 1:1) are thoroughly mixed to obtain a mixed solution; the mixed solution is warmed to 50°C and gently stirred, while 5 mL of epichlorohydrin and 5 mL of a 50% concentration sodium hydroxide aqueous solution are dropped into the mixed solution, the dropping time is controlled to be 6 h, after the dropping is completed, the reaction is preserved for 18 h; after the reaction is completed, the product is washed with anhydrous ethanol and distilled water in sequence, the washed product is soaked in a 5% calcium chloride aqueous solution for 24 h, and is taken out and drained to obtain high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0056] The above agarose / sodium alginate composite chromatographic microspheres are tested, and it is measured in a chromatographic column that the maximum linear flow rate of the agarose / sodium alginate composite chromatographic microspheres is 1020 cm / h, and the maximum pressure resistance is 0.331 MPa.

[0057] Example 2

[0058] (1) 2.4 g of agarose is dissolved in 60 mL of water and heated to 95°C and stirred, after the solution is clear and transparent, the stirring is continued for 30 min to cool to 80°C, 1.8 g of sodium alginate is added, and the stirring is continued for 6 h to form a uniform agarose / sodium alginate solution.

[0059] 1.8 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether are dispersed in 60 g of toluene to prepare an oil phase, and heated to 65°C for preheating; the above agarose / sodium alginate solution is injected into the preheated oil phase for emulsification, after the stirring is continued for 30 min, it is cooled to room temperature within 15 min. Then the reaction is washed with a large amount of distilled water for multiple times to remove the upper toluene and emulsifier, and is sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm; finally, the microspheres are soaked in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain the composite microspheres.

[0060] (2) After the water-drained composite microspheres 50 g and 8 g of anhydrous sodium sulfate, 26 mL of an acetone aqueous solution (wherein the volume ratio of acetone to water is 1:1) are thoroughly mixed to obtain a mixed solution; the mixed solution is warmed to 50°C and gently stirred, while 5 mL of epichlorohydrin and 5 mL of a 50% concentration sodium hydroxide aqueous solution are dropped into the mixed solution, the dropping time is controlled to be 6 h, after the dropping is completed, the reaction is preserved for 18 h; after the reaction is completed, the product is washed with anhydrous ethanol and distilled water in sequence, the washed product is soaked in a 5% calcium chloride aqueous solution for 24 h, and is taken out and drained to obtain high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0061] The optical microscope photograph of the high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres prepared in this example is as shown inFigure 1 The agarose / sodium alginate composite chromatography microspheres were prepared as shown in the following flow chart: Figure 1 It can be seen that the above prepared composite chromatography microspheres are smooth and transparent spherical particles.

[0062] The agarose / sodium alginate composite chromatography microspheres were tested, and the test results are shown in the following table: Figure 2 The agarose / sodium alginate composite chromatography microspheres were prepared as shown in the following flow chart: Figure 2 It can be seen that the maximum linear flow rate of the agarose / sodium alginate composite chromatography microspheres measured in the chromatography column is 1200 cm / h, and the maximum pressure resistance is 0.370 MPa.

[0063] Example 3

[0064] (1) 2.4 g of agarose was dissolved in 60 mL of water and heated to 95°C while stirring, and after the solution became clear and transparent, it was stirred for another 30 min to cool to 80°C. Then, 1.2 g of sodium alginate was added, and the stirring was continued for 4 h to form a uniform agarose / sodium alginate solution.

[0065] An oil phase was prepared by dispersing 1.8 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether in 60 g of toluene and heating to 65°C for preheating. The above agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and after stirring for 30 min, it was cooled to room temperature within 15 min. Then, the reaction product was washed with a large amount of distilled water for several times to remove the upper toluene and emulsifier, and a standard sieve was used for sieving to obtain polysaccharide composite microspheres with a size of 100-300 μm. Finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain the composite microspheres.

[0066] (2) 50 g of the composite microspheres after draining water and 8 g of anhydrous sodium sulfate, 26 mL of an aqueous acetone solution (wherein the volume ratio of acetone to water is 1:1) were thoroughly mixed to obtain a mixed solution. The mixed solution was warmed to 50°C and stirred gently, and at the same time, 5 mL of epichlorohydrin and 5 mL of a 50% sodium hydroxide aqueous solution were added dropwise into the mixed solution, and the dropping time was controlled for 6 h. After the dropping was completed, the reaction was maintained for 18 h. After the reaction was completed, the microsphere product was washed with anhydrous ethanol and distilled water in sequence, and then placed in a 5% calcium chloride aqueous solution for 24 h of soaking. The product was taken out and drained to obtain agarose / sodium alginate composite chromatography microspheres with high mechanical strength.

[0067] The agarose / sodium alginate composite chromatography microspheres were tested, and the test results are shown in the following table:

[0068] Example 4

[0069] (1) 2.4 g of agarose was dissolved in 60 mL of water and heated to 95 °C while stirring, until the solution was clear and transparent, and then stirred for 30 min to cool to 80 °C, 1.8 g of sodium alginate was added, and stirring was continued for 8 h to form a uniform agarose / sodium alginate solution.

[0070] An oil phase was prepared by dispersing 1.8 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether in 60 g of toluene and heating to 65 °C for preheating; the agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and stirring was continued for 30 min, and then cooled to room temperature within 15 min. The reaction product was washed with a large amount of distilled water several times to remove the upper layer of toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain the composite microspheres.

[0071] (2) 50 g of the composite microspheres after draining water and 8 g of anhydrous sodium sulfate, 26 mL of an aqueous acetone solution (acetone to water in a volume ratio of 1:1) were thoroughly mixed to obtain a mixed solution; the mixed solution was warmed to 50 °C and stirred gently, while 5 mL of epichlorohydrin and 5 mL of a 50% sodium hydroxide aqueous solution were added dropwise to the mixed solution, the dropwise addition was controlled for 6 h, and the reaction was maintained for 18 h after the dropwise addition was completed; after the reaction was completed, the product was washed with anhydrous ethanol and distilled water in sequence, and then placed in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain agarose / sodium alginate composite chromatographic microspheres with high mechanical strength.

[0072] The agarose / sodium alginate composite chromatographic microspheres were tested, and the maximum linear flow rate of the agarose / sodium alginate composite chromatographic microspheres was 1150 cm / h, and the maximum pressure resistance was 0.362 MPa.

[0073] Example 5

[0074] (1) 2.4 g of agarose was dissolved in 60 mL of water and heated to 95 °C while stirring, until the solution was clear and transparent, and then stirred for 30 min to cool to 80 °C, 1.8 g of sodium alginate was added, and stirring was continued for 4 h to form a uniform agarose / sodium alginate solution.

[0075] An oil phase was prepared by dispersing 2.4 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether in 60 g of toluene and heating to 65°C for preheating; the above agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and after stirring for 30 min, it was cooled to room temperature within 15 min. The reaction was then washed with a large amount of distilled water several times to remove the upper layer of toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain the composite microspheres.

[0076] (2) The composite microspheres after draining water were mixed with 50 g of anhydrous sodium sulfate, 26 mL of an aqueous solution of acetone (1:1), and 8 g of anhydrous sodium sulfate to obtain a mixed solution; the mixed solution was warmed to 50°C and stirred gently, and 5 mL of epichlorohydrin and 5 mL of a 50% sodium hydroxide aqueous solution were added dropwise to the mixed solution, the dropwise addition time was controlled for 6 h, and the reaction was maintained for 18 h after the dropwise addition was completed; after the reaction was completed, the product was washed with anhydrous ethanol and distilled water in turn, and then soaked in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0077] The above agarose / sodium alginate composite chromatographic microspheres were tested, and the maximum linear flow rate of the agarose / sodium alginate composite chromatographic microspheres was 1000 cm / h and the maximum pressure resistance was 0.342 MPa in a chromatographic column.

[0078] Example 6

[0079] (1) 2.4 g of agarose was dissolved in 60 mL of water and heated to 95°C with stirring, and after the solution became clear and transparent, it was cooled to 80°C with stirring for 30 min, 1.8 g of sodium alginate was added, and stirring was continued for 4 h to form a uniform agarose / sodium alginate solution.

[0080] An oil phase was prepared by dispersing 2.4 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether in 60 g of toluene and heating to 65°C for preheating; the above agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and after stirring for 30 min, it was cooled to room temperature within 15 min. The reaction was then washed with a large amount of distilled water several times to remove the upper layer of toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain the composite microspheres.

[0081] (2) After draining water, the composite microspheres 50 g and 8 g of anhydrous sodium sulfate, 26 mL of acetone aqueous solution (wherein the volume ratio of acetone to water is 1:1) are mixed thoroughly to obtain a mixed solution; the mixed solution is warmed to 50°C and stirred gently, while 5 mL of epichlorohydrin and 5 mL of 50% sodium hydroxide aqueous solution are dropped into the mixed solution, the dropping time is controlled for 6 h, and after the dropping is completed, the reaction is preserved for 18 h; after the reaction is completed, the product is washed with anhydrous ethanol and distilled water in sequence, and the washed product is placed in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0082] The agarose / sodium alginate composite chromatographic microspheres are tested, and it is measured in a chromatographic column that the maximum linear flow rate of the agarose / sodium alginate composite chromatographic microspheres is 1100 cm / h, and the maximum pressure resistance is 0.358 MPa.

[0083] Example 7

[0084] (1) 2.4 g of agarose is dissolved in 60 mL of water and heated to 95°C and stirred, after the solution is clear and transparent, the temperature is lowered to 80°C after stirring for 30 min, 1.8 g of sodium alginate is added, and the stirring is continued for 4 h to form a uniform agarose / sodium alginate solution.

[0085] 1.8 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether are dispersed in 60 g of toluene to prepare an oil phase, and heated to 65°C for preheating; the agarose / sodium alginate solution is injected into the preheated oil phase for emulsification, and after stirring for 30 min, it is cooled to room temperature within 15 min. Then the reaction product is washed with a large amount of distilled water for multiple times to remove the upper toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres are soaked in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain the composite microspheres.

[0086] (2) After draining water, the composite microspheres 50 g and 8 g of anhydrous sodium sulfate, 26 mL of acetone aqueous solution (wherein the volume ratio of acetone to water is 1:5) are mixed thoroughly to obtain a mixed solution; the mixed solution is warmed to 50°C and stirred gently, while 5 mL of epichlorohydrin and 5 mL of 50% sodium hydroxide aqueous solution are dropped into the mixed solution, the dropping time is controlled for 6 h, and after the dropping is completed, the reaction is preserved for 18 h; after the reaction is completed, the product is washed with anhydrous ethanol and distilled water in sequence, and the washed product is placed in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0087] The above agarose / sodium alginate composite chromatography microspheres were tested, and the maximum linear flow rate of the agarose / sodium alginate composite chromatography microspheres was 950 cm / h, and the maximum pressure resistance was 0.312 MPa.

[0088] Example 8

[0089] (1) 2.4 g of agarose was dissolved in 60 mL of water and heated to 95°C and stirred until the solution was clear and transparent, then stirred for another 30 min to cool to 80°C, 1.8 g of sodium alginate was added, and the stirring was continued for 4 h to form a uniform agarose / sodium alginate solution.

[0090] An oil phase was prepared by dispersing 1.8 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether in 60 g of toluene and heating to 65°C for preheating; the above agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and the stirring was continued for 30 min, then cooled to room temperature within 15 min. Then the reaction product was washed with a large amount of distilled water for several times to remove the upper layer of toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain the composite microspheres.

[0091] (2) 50 g of the composite microspheres after draining water and 8 g of anhydrous sodium sulfate, 26 mL of an aqueous solution of acetone (the volume ratio of acetone to water is 1:1) were thoroughly mixed to obtain a mixed solution; the mixed solution was warmed to 50°C and stirred gently, while 5 mL of epichlorohydrin and 5 mL of 50% sodium hydroxide aqueous solution were added dropwise into the mixed solution, the dropping time was controlled for 1 h, and the reaction was kept for 18 h after the dropping was completed; after the reaction was completed, the product was washed with anhydrous ethanol and distilled water in turn, and then soaked in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain agarose / sodium alginate composite chromatography microspheres with high mechanical strength.

[0092] The above agarose / sodium alginate composite chromatography microspheres were tested, and the maximum linear flow rate of the agarose / sodium alginate composite chromatography microspheres was 900 cm / h, and the maximum pressure resistance was 0.319 MPa.

[0093] Example 9

[0094] (1) 2.4 g of agarose was dissolved in 60 mL of water and heated to 95°C and stirred until the solution was clear and transparent, then stirred for another 30 min to cool to 80°C, 1.8 g of sodium alginate was added, and the stirring was continued for 4 h to form a uniform agarose / sodium alginate solution.

[0095] An oil phase was prepared by dispersing 1.8 g of sorbitan monooleate and 0.6 g of sorbitan monooleate polyoxyethylene ether in 60 g of toluene and heating to 65 °C for preheating; the above agarose / sodium alginate solution was injected into the preheated oil phase for emulsification, and after stirring for 30 min, it was cooled to room temperature within 15 min. The reaction was then washed with a large amount of distilled water several times to remove the upper layer of toluene and emulsifier, and then sieved with a standard sieve to obtain polysaccharide composite microspheres of 100-300 μm. Finally, the microspheres were soaked in a 5% calcium chloride aqueous solution for 24 h, and then taken out and drained to obtain the composite microspheres.

[0096] (2) The composite microspheres 50 g after draining water and 8 g of anhydrous sodium sulfate, 10 mL of acetone aqueous solution (wherein the volume ratio of acetone to water is 1:1) were thoroughly mixed to obtain a mixed solution; the mixed solution was warmed to 50 °C and stirred gently, while 5 mL of epichlorohydrin and 5 mL of 50% sodium hydroxide aqueous solution were added dropwise into the mixed solution, the dropwise time was controlled for 6 h, and after the dropwise addition was completed, the reaction was kept for 18 h; after the reaction was completed, the product was washed with anhydrous ethanol and distilled water in turn, and then the washed product was soaked in a 5% calcium chloride aqueous solution for 24 h, taken out and drained to obtain high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres.

[0097] The above agarose / sodium alginate composite chromatographic microspheres were tested, and the maximum linear flow rate of the agarose / sodium alginate composite chromatographic microspheres was 1050 cm / h and the maximum pressure resistance was 0.289 MPa in the chromatographic column.

Claims

1. A method for preparing a high mechanical strength agarose / sodium alginate composite chromatographic microsphere, characterized in that, The method comprises the following steps: (1) Dissolve agarose in water and heat to 80-100 ℃, stir until clear, then stir to cool to 40-90 ℃, add sodium alginate and stir to obtain an agarose / sodium alginate solution; (2) Disperse sorbitan monooleate and sorbitan monooleate polyoxyethylene ether in toluene to prepare an oil phase; (3) Heat the oil phase and inject it into the agarose / sodium alginate solution, stir to emulsify, then cool to room temperature, wash the reactants with distilled water, sieve, soak in a calcium chloride aqueous solution, then take out and drain to obtain the composite microspheres; (4) Mix the composite microspheres, anhydrous sodium sulfate and an acetone aqueous solution, and drop in epoxy chloropropane and a sodium hydroxide aqueous solution under heating and stirring, after the drop is completed, carry out a heat preservation reaction, after the reaction is completed, wash the product with anhydrous ethanol and distilled water, and soak in a calcium chloride aqueous solution to obtain the high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres. In step (1), the mass percentage of agarose in water is 1-8 %; The mass ratio of sodium alginate to agarose is 1:(1-2); In step (4), the mass-volume ratio of the composite microspheres to epoxy chloropropane is 1:(0.08-0.5) g / mL.

2. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In step (1), the stirring time after adding sodium alginate is 2-18 h.

3. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In step (2), the mass percentage of sorbitan monooleate in toluene is 0.5-10 %; The mass percentage of sorbitan monooleate polyoxyethylene ether in toluene is 0.2-5 %.

4. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1, wherein, In step (3), the mass ratio of the agarose / sodium alginate solution to the oil phase is 1:(0.5-2); The stirring and emulsifying time is 10-120 min.

5. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In step (4), the volume ratio of acetone to water in the acetone aqueous solution is 1:(0.1-10); The concentration of the sodium hydroxide aqueous solution is 50 %.

6. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In step (4), the mass-volume ratio of the composite microspheres to the acetone aqueous solution is 1:(0.1-3) g / mL; The mass-volume ratio of anhydrous sodium sulfate to the acetone aqueous solution is 1:(0.5-8) g / mL.

7. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In step (4), the mass-volume ratio of the composite microspheres to the sodium hydroxide aqueous solution is 1:(0.05-0.5) g / mL.

8. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In step (4), the drop time of epoxy chloropropane and the sodium hydroxide aqueous solution is 0.5-40 h; The reaction time of the heat preservation reaction is 15-20 h.

9. The process for the preparation of high mechanically strong agarose / sodium alginate composite chromatographic microspheres as claimed in claim 1 wherein, In steps (3) and (4), the mass concentration of the calcium chloride aqueous solution is independently selected from 1-15 %.

10. A high mechanical strength agarose / sodium alginate composite chromatographic microsphere, characterized in that, The high-mechanical-strength agarose / sodium alginate composite chromatographic microspheres are prepared by the method of any one of claims 1-9.

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