Porous composite gel microspheres for chromatography and preparation method thereof

Abstract

The invention provides porous composite gel microspheres for chromatography and a preparation method thereof.The preparation method of the porous composite gel microspheres for chromatography comprises the following steps that agarose is dissolved in water, chitin is added into the water, the materials are mixed to be uniform, and a water phase is obtained; adding the water phase into the oil phase, stirring and emulsifying to obtain a suspension; wherein the oil phase is a mixed solution of methylbenzene, span 80 and cellulose palmitate sodium sulfate; cooling the suspension until the suspension is solidified into balls to obtain composite microspheres; the composite microspheres and epoxy chloropropane are subjected to a cross-linking reaction under the alkaline condition, and the porous composite gel microspheres for chromatography are obtained. According to the preparation method of the porous composite gel microspheres for chromatography, the mechanical property of agarose gel can be improved, and the obtained porous composite gel microspheres are good in mechanical property, high in pressure resistance and capable of meeting the requirement for repeated use for more times.

Description

Technical Field

[0001] The invention relates to the technical field of chromatography separation, in particular to a porous composite gel microsphere for chromatography and a preparation method thereof. Background Art

[0002] Agarose-based porous gel microspheres are widely used as chromatographic media for bioseparation due to their advantages of high biosafety and low extractables. However, when used as chromatographic media, agarose-based porous gel microspheres have the disadvantage of poor mechanical properties, which results in low pressure tolerance after loading the chromatographic column, few reuse times, and short service life.

[0003] In view of this, it is urgent to propose a gel microsphere to solve the problem of poor mechanical properties of agarose-based porous gel microspheres. Summary of the invention

[0004] The purpose of the present invention is to provide porous composite gel microspheres for chromatography, so as to solve the problems of poor mechanical properties and low pressure tolerance of porous gel microspheres based on agarose in the prior art.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] A method for preparing porous composite gel microspheres for chromatography comprises the following steps:

[0007] (1) dissolving agarose in water, adding chitosan thereto, and mixing well to obtain an aqueous phase;

[0008] (2) adding the aqueous phase obtained in step (1) to the oil phase at a temperature of 70-90° C., stirring and emulsifying to obtain a suspension;

[0009] Wherein, the oil phase is a mixture of toluene, Span 80, and sodium cellulose palmitate sulfate;

[0010] (3) cooling the suspension obtained in step (2) until it solidifies into spheres to obtain composite microspheres;

[0011] (4) The composite microspheres obtained in step (3) are cross-linked with epichlorohydrin under alkaline conditions to obtain porous composite gel microspheres for chromatography.

[0012] Preferably, in step (1), the mass concentration of agarose in water is 4-6wt%; the mass concentration of chitosan in water is 1-5wt%.

[0013] Preferably, in step (2), the stirring and emulsification time is 20-40 min.

[0014] Preferably, in step (2), the volume ratio of the oil phase to the water phase is (4-6):1.

[0015] Preferably, in step (2), the oil phase is a mixed solution of toluene, Span 80, and sodium cellulose palmitate sulfate in a weight ratio of 100:(1-2):(0.3-0.8).

[0016] Preferably, in step (3), the temperature is lowered to below 20°C at a rate of 1-2°C / min.

[0017] Preferably, step (3) further comprises the steps of washing and filtering the obtained composite microspheres with deionized water.

[0018] Preferably, step (4) specifically comprises: adding the composite microspheres obtained in step (3) to an ethanol solution, adding sodium hydroxide thereto, stirring for 5-10 hours, then adding epichlorohydrin and sodium borohydride thereto, reacting at 60-70° C. for 4-6 hours, to obtain porous composite gel microspheres for chromatography.

[0019] Preferably, in step (4), the sodium hydroxide is a sodium hydroxide solution with a mass concentration of 40-45wt%; the volume concentration of the ethanol is 50-60%;

[0020] The volume ratio of the composite microspheres, the ethanol solution, the epichlorohydrin, and the sodium hydroxide solution is 1:(2-4):(1.5-1.8):(1.0-1.2);

[0021] The mass ratio of the sodium borohydride to the epichlorohydrin is 1:(50-60).

[0022] The invention also provides a porous composite gel microsphere for chromatography obtained by the preparation method of the porous composite gel microsphere for chromatography.

[0023] The above solution of the present invention includes at least the following beneficial effects:

[0024] The preparation method of porous composite gel microspheres for chromatography of the present invention comprises the following steps: dissolving agarose in water, adding chitosan thereto, mixing evenly, and obtaining an aqueous phase; adding the aqueous phase to an oil phase at a temperature of 70-90°C, stirring and emulsifying, and obtaining a suspension; wherein the oil phase is a mixture of toluene, Span 80, and sodium cellulose palmitate sulfate; cooling the suspension until solidified into balls, and obtaining composite microspheres; cross-linking the composite microspheres with epichlorohydrin under alkaline conditions, and obtaining porous composite gel microspheres for chromatography. The preparation method of porous composite gel microspheres for chromatography of the present invention can improve the mechanical properties of agarose gel, and the obtained porous composite gel microspheres have good mechanical properties and high pressure tolerance, and can meet the requirements of more times of repeated use.

[0025] The sodium cellulose palmitate sulfate not only acts as a surfactant to play a good emulsifying role, making the gel microspheres more uniform and effectively preventing adhesion between microspheres, but also regulates the cross-linking structure formed by agarose during the cross-linking reaction and enhances the mechanical properties of the microspheres. DETAILED DESCRIPTION

[0026] In the embodiments of the present invention, if the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially. Raw materials of different manufacturers and models do not affect the implementation of the technical solution of the present invention and the realization of the technical effects.

[0027] Example 1

[0028] The method for preparing the porous composite gel microspheres for chromatography in this embodiment comprises the following steps:

[0029] (1) dissolving agarose in water, adding chitosan thereto, and mixing well to obtain an aqueous phase;

[0030] Wherein, the mass concentration of agarose in water is 4wt%; the mass concentration of chitosan in water is 3wt%.

[0031] (2) adding the aqueous phase obtained in step (1) to the oil phase at a temperature of 70° C., stirring and emulsifying for 40 minutes to obtain a suspension;

[0032] The oil phase is a mixture of toluene, Span 80, and sodium cellulose palmitate sulfate in a weight ratio of 100:1:0.6. The volume ratio of the oil phase to the water phase is 6:1;

[0033] (3) cooling the suspension obtained in step (2) to below 20° C. at a rate of 1-2° C. / min to solidify it into spheres, and washing and filtering with deionized water to obtain composite microspheres;

[0034] (4) The composite microspheres obtained in step (3) are added to an ethanol solution, sodium hydroxide is added thereto, and the mixture is stirred for 8 hours. Then, epichlorohydrin and sodium borohydride are added thereto, and the mixture is reacted at 60° C. for 5 hours to obtain porous composite gel microspheres for chromatography.

[0035] Among them, the sodium hydroxide is a sodium hydroxide solution with a mass concentration of 40wt%; the volume concentration of the ethanol is 55%; the volume ratio of the composite microspheres, the ethanol solution, the epichlorohydrin, and the sodium hydroxide solution is 1:2:1.5:1.1; the mass ratio of the sodium borohydride to the epichlorohydrin is 1:55.

[0036] Example 2

[0037] The method for preparing the porous composite gel microspheres for chromatography in this embodiment comprises the following steps:

[0038] (1) dissolving agarose in water, adding chitosan thereto, and mixing well to obtain an aqueous phase;

[0039] Wherein, the mass concentration of agarose in water is 5wt%; the mass concentration of chitosan in water is 5wt%.

[0040] (2) adding the aqueous phase obtained in step (1) to the oil phase at a temperature of 90° C., stirring and emulsifying for 30 minutes to obtain a suspension;

[0041] The oil phase is a mixture of toluene, Span 80, and sodium cellulose palmitate sulfate in a weight ratio of 100:2:0.8. The volume ratio of the oil phase to the water phase is 4:1;

[0042] (3) cooling the suspension obtained in step (2) to below 20° C. at a rate of 1-2° C. / min to solidify it into spheres, and washing and filtering with deionized water to obtain composite microspheres;

[0043] (4) The composite microspheres obtained in step (3) are added to an ethanol solution, sodium hydroxide is added thereto, and the mixture is stirred for 10 hours. Then, epichlorohydrin and sodium borohydride are added thereto, and the mixture is reacted at 65° C. for 6 hours to obtain porous composite gel microspheres for chromatography.

[0044] Among them, the sodium hydroxide is a sodium hydroxide solution with a mass concentration of 45wt%; the volume concentration of the ethanol is 60%; the volume ratio of the composite microspheres, the ethanol solution, the epichlorohydrin, and the sodium hydroxide solution is 1:3:1.6:1.0; the mass ratio of the sodium borohydride to the epichlorohydrin is 1:60.

[0045] Example 3

[0046] The method for preparing the porous composite gel microspheres for chromatography in this embodiment comprises the following steps:

[0047] (1) dissolving agarose in water, adding chitosan thereto, and mixing well to obtain an aqueous phase;

[0048] Wherein, the mass concentration of agarose in water is 6wt%; the mass concentration of chitosan in water is 1wt%.

[0049] (2) adding the aqueous phase obtained in step (1) to the oil phase at a temperature of 80° C., stirring and emulsifying for 20 min to obtain a suspension;

[0050] Wherein, the oil phase is toluene, Span 80, and sodium cellulose palmitate sulfate according to 100:

[0051] The weight ratio of the oil phase to the water phase is 5:1;

[0052] (3) cooling the suspension obtained in step (2) to below 20° C. at a rate of 1-2° C. / min to solidify it into spheres, and washing and filtering with deionized water to obtain composite microspheres;

[0053] (4) The composite microspheres obtained in step (3) are added to an ethanol solution, sodium hydroxide is added thereto, and the mixture is stirred for 5 hours. Then, epichlorohydrin and sodium borohydride are added thereto, and the mixture is reacted at 70° C. for 4 hours to obtain porous composite gel microspheres for chromatography.

[0054] Among them, the sodium hydroxide is a sodium hydroxide solution with a mass concentration of 42wt%; the volume concentration of the ethanol is 50%; the volume ratio of the composite microspheres, the ethanol solution, the epichlorohydrin, and the sodium hydroxide solution is 1:4:1.8:1.2; the mass ratio of the sodium borohydride to the epichlorohydrin is 1:50.

[0055] Example 4

[0056] The method for preparing the porous composite gel microspheres for chromatography in this embodiment comprises the following steps:

[0057] (1) dissolving agarose in water, adding chitosan thereto, and mixing well to obtain an aqueous phase;

[0058] Wherein, the mass concentration of agarose in water is 6wt%; the mass concentration of chitosan in water is 5wt%.

[0059] (2) adding the aqueous phase obtained in step (1) to the oil phase at a temperature of 70° C., stirring and emulsifying for 40 minutes to obtain a suspension;

[0060] Wherein, the oil phase is toluene, Span 80, and sodium cellulose palmitate sulfate according to 100:

[0061] The weight ratio of the oil phase to the water phase is 5:1.

[0062] (3) cooling the suspension obtained in step (2) to below 20° C. at a rate of 1-2° C. / min to solidify it into spheres, and washing and filtering with deionized water to obtain composite microspheres;

[0063] (4) The composite microspheres obtained in step (3) are added to an ethanol solution, sodium hydroxide is added thereto, and the mixture is stirred for 6 hours. Then, epichlorohydrin and sodium borohydride are added thereto, and the mixture is reacted at 70° C. for 6 hours to obtain porous composite gel microspheres for chromatography.

[0064] Among them, the sodium hydroxide is a sodium hydroxide solution with a mass concentration of 45wt%; the volume concentration of the ethanol is 60%; the volume ratio of the composite microspheres, the ethanol solution, the epichlorohydrin, and the sodium hydroxide solution is 1:4:1.8:1.0; the mass ratio of the sodium borohydride to the epichlorohydrin is 1:60.

[0065] Comparative Example 1

[0066] The preparation method of the porous composite gel microspheres for chromatography in this comparative example is the same as that in Example 4, except that chitosan is not added in step (1).

[0067] Comparative Example 2

[0068] The preparation method of the porous composite gel microspheres for chromatography in this comparative example is the same as that in Example 4, except that in step (2), the oil phase is a mixture of toluene and Span 80 in a weight ratio of 100:1.5.

[0069] Comparative Example 3

[0070] The preparation method of the porous composite gel microspheres for chromatography in this comparative example is the same as that in Example 4, except that in step (1), chitosan is not added. In step (2), the oil phase is a mixture of toluene and Span 80 in a weight ratio of 100:1.5.

[0071] Effect experiment example

[0072] In order to verify the technical effect of the method for preparing the porous composite gel microspheres for chromatography described in the present invention, the following experiments were conducted:

[0073] The maximum pressure resistance of the porous composite gel microspheres obtained in Examples 1-4 and Comparative Examples 1-3 was tested by the pressure resistance test method disclosed in Chinese patent document CN115850540B.

[0074] After testing, the results are as follows:

[0075]

[0076] According to the above results, the method for preparing the porous composite gel microspheres for chromatography of the present invention can obtain the porous composite gel microspheres for chromatography with good mechanical properties and high pressure tolerance.

[0077] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. A method for preparing porous composite gel microspheres for chromatography, characterized in that: The steps include: (1) dissolving agarose in water, adding chitosan thereto, and mixing well to obtain an aqueous phase; (2) adding the aqueous phase obtained in step (1) to the oil phase at a temperature of 70-90° C., stirring and emulsifying to obtain a suspension; Wherein, the oil phase is a mixture of toluene, Span 80, and sodium cellulose palmitate sulfate; (3) cooling the suspension obtained in step (2) until it solidifies into spheres to obtain composite microspheres; (4) The composite microspheres obtained in step (3) are cross-linked with epichlorohydrin under alkaline conditions to obtain porous composite gel microspheres for chromatography.

2. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: In step (1), the mass concentration of agarose in water is 4-6wt%; the mass concentration of chitosan in water is 1-5wt%.

3. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: In step (2), the stirring and emulsifying time is 20-40 minutes.

4. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: In step (2), the volume ratio of the oil phase to the water phase is (4-6):

1.

5. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: In step (2), the oil phase is a mixed solution of toluene, Span 80, and sodium cellulose palmitate sulfate in a weight ratio of 100:(1-2):(0.3-0.8).

6. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: In step (3), the temperature is lowered to below 20°C at a rate of 1-2°C / min.

7. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: Step (3) also includes the steps of washing and filtering the obtained composite microspheres with deionized water.

8. The method for preparing porous composite gel microspheres for chromatography according to claim 1, characterized in that: Step (4) specifically comprises: adding the composite microspheres obtained in step (3) into an ethanol solution, adding sodium hydroxide thereto, stirring for 5-10 hours, then adding epichlorohydrin and sodium borohydride thereto, reacting at 60-70° C. for 4-6 hours, and obtaining porous composite gel microspheres for chromatography.

9. The method for preparing porous composite gel microspheres for chromatography according to claim 8, characterized in that: In step (4), the sodium hydroxide is a sodium hydroxide solution with a mass concentration of 40-45wt%; the volume concentration of the ethanol is 50-60%; The volume ratio of the composite microspheres, the ethanol solution, the epichlorohydrin, and the sodium hydroxide solution is 1:(2-4):(1.5-1.8):(1.0-1.2); The mass ratio of the sodium borohydride to the epichlorohydrin is 1:(50-60).

10. Porous composite gel microspheres for chromatography obtained by the method for preparing porous composite gel microspheres for chromatography according to any one of claims 1 to 9.

Images