CN1233438C - Method for preparing microspheric PGDT separating medium with two kinds of pore forms - Google Patents

Abstract

The invention discloses a preparation method of a microsphere PGDT separation medium with two types of pores. The method is to mix the monomer, cross-linking agent, liquid porogen, and initiator first, then add the solid porogen and mix evenly, and form microspheres through suspension polymerization, extract the microspheres with absolute ethanol, and pickle , and dry, two types of pore-type separation media can be obtained. It is characterized in that: the solid porogen is calcium carbonate, and the dosage accounts for 10-3% of the volume content of the reaction mixture; the volume ratio of the solid porogen to the liquid porogen is 0.5-1; the dosage of the porogen occupies 10% of the volume content of the reaction mixture. 20-60%; the ratio of the amount of the two cross-linking agents is 0.5-2, and the ratio of the amount of cross-linking agent to the amount of the monomer is 0.3-0.6; under normal pressure, the polymerization temperature is controlled at 65-85 between ℃. The separation medium prepared by the method can be used for the separation of biomacromolecules such as proteins after modification, has large adsorption capacity and mild elution conditions, and has good application prospects in large-scale preparative separation of biomacromolecules.

Description

Preparation method of microsphere PGDT separation medium with two types of pores

Technical field

The present invention relates to a method for preparing PGDT with two types of holes by means of suspension polymerization, i.e. poly(glycidyl methacrylate-divinylbenzene-triallyl isocyanurate) microsphere separation medium, It belongs to the chromatography medium preparation technology used in the separation and purification process of biological macromolecules.

Background technique

In the separation and purification process of biological macromolecules, liquid chromatography is recognized as a very effective means. With the increasing demand for biological products, the performance requirements of liquid chromatography media are also getting higher and higher. Therefore, the preparation of chromatographic media with excellent performance has become an important topic in the research of bioseparation technology.

In the past forty years, many membranes and macroporous polymer particles with pore sizes of 100-1000 nm have been developed for filtration or as catalyst supports. Remarkably, when the pore diameter of the membrane reaches 500 nanometers, as long as there is a slight pressure difference between the two sides, convective mass transfer in the pores can be induced. Van Kreveld et al. pointed out in a paper published in Volume 397 of "Journal of Chromatography" in 1987 that as long as there are such large convective mass transfer pores that cross the particles on the chromatography packing particles, the separated solutes will follow the flow. The phase quickly approaches the surface of the inner hole of the medium instead of relying on the concentration gradient for diffusion and mass transfer, thus speeding up the mass transfer process.

Afeyan and Regnier et al. applied for the US Patent US Pat5019270 of perfusion chromatography in 1989. The key to perfusion chromatography is the two types of pore-type separation media named by POROS. This dual-pore separation medium with styrene-divinylbenzene as the skeleton contains two kinds of pores with different sizes: the diameter of the large pores is 600-800nm, and the fluid passes through in the form of convection; the diameter of the small pores is 50-100nm, and the fluid passes through Diffusion forms pass. This type of medium itself has a large enough pore cross-section to allow the flow to flow through the adsorbent particles, which greatly reduces the mass transfer resistance of the stagnant mobile phase in the pores and greatly improves the column efficiency. The preparation process is to first synthesize microspheres, and then further aggregate them to form aggregates through emulsification, and then aggregate to form a separation medium with a required particle size. Moreover, due to the strong hydrophobicity of the framework structure of the medium, the non-specific adsorption of biological substances such as proteins is strong. In its separation, in addition to being directly used in reversed-phase mode, it can also be used in other modes, such as ion exchange, affinity and hydrophobic interaction. When used in the action mode, it is generally necessary to modify the surface of the medium, usually by coating. It can be seen that the preparation process is very complicated and the cost is high.

The paper published by Yihua Yu et al. in Volume 855 of "Journal of Chromatophy A" in 1999 showed that glycidyl methacrylate was used as a monomer, and divinylbenzene and triallyl trimeric isocyanurate were used as crosslinking agents. , the liquid organic solvents toluene and n-heptane were used as porogens, and polyglycidyl methacrylate spherical separation media were prepared by suspension polymerization. Experimental results show that this type of spherical separation media has low non-specific adsorption and high mechanical strength.

The paper published by Minlian Zhang et al. in Volume 922 of "Journal of Chromatography A" in 2001 showed that glycidyl methacrylate was used as a monomer, divinylbenzene and triallyl trimeric isocyanurate were used as crosslinking agents , the liquid organic solvents cyclohexanol and dodecanol were used as porogens, and polyglycidyl methacrylate separation media with two types of pores were prepared by one-step in-situ polymerization. Experiments show that the mass transfer behavior of fluid in the two types of pore-type separation media is greatly improved. However, the two types of pore-type separation media prepared by in-situ polymerization are prone to breakage at high flow rates due to their irregular shapes, which severely limits their application.

Contents of Invention

The purpose of the present invention is to provide a method for preparing a microsphere PGDT separation medium with controllable particle size and two types of pores with high mechanical properties for the defects in the above-mentioned separation medium and its preparation method.

The technical scheme of the present invention is: first make monomer glycidyl methacrylate, cross-linking agent divinylbenzene and triallyl isocyanurate, liquid porogen toluene and n-heptane, and initiator azo The diisobutyronitrile is mixed evenly, and then the solid porogen is added to mix evenly, and then polymer microspheres are synthesized by suspension polymerization. The microspheres are extracted with absolute ethanol to remove the liquid porogen, washed with hydrochloric acid to remove the solid porogen, and then dried to obtain two types of pore-type PGDT separation media, characterized in that the solid porogen is Calcium carbonate, the amount accounts for 10-30% of the volume content of the reaction mixture; the volume ratio of the solid porogen to the liquid porogen is 0.5-1; the amount of the porogen accounts for 20-60% of the volume content of the reaction mixture; The mass ratio of vinylbenzene to triallyl isocyanurate is 0.5 to 2, and the mass ratio of the amount of crosslinking agent to monomer glycidyl methacrylate is 0.3 to 0.6; at normal pressure Under this condition, the polymerization temperature is controlled between 65 and 85°C.

The above-mentioned calcium carbonate particles have a particle diameter of 0.5-3 μm and a density of 2.71 g/ml.

The present invention will be described in detail below.

The key technology of the present invention has four points: the one, the selection of solid porogen. The purpose of adding solid porogen is to generate convective holes above 100nm in the separation medium, and the solid particles used for pore formation should have suitable particle size and density. Among the present invention, ultrafine calcium carbonate particles are adopted as solid porogen. It has a density of 2.71g/ml and a particle size of 0.5-3μm, and can be used to prepare two types of pore-type separation media with uniform structures. The second key technology is the selection of organic porogens. According to the types of monomers and cross-linking agents, suitable organic porogens are selected in order to synthesize two types of pore-type separation media with large specific surface areas. The invention adopts toluene and n-heptane as liquid porogens, which can make the synthesized separation medium have large specific surface area, large adsorption capacity and good mechanical strength. The third key technology is to adjust the proportion of solid porogen and liquid porogen, which can realize the control of mass transfer and adsorption performance of the prepared separation medium. The fourth key technology is the choice of aggregation method. The present invention selects the suspension polymerization method currently used in the preparation of most separation media.

Compared with the existing two types of pore type separation media, the two types of pore type PGDT separation media prepared by the present invention have obvious advantages: simple preparation process, convenient operation, and low cost; it can realize convection in the two types of pore type separation media The pore size of the hole and the diffusion hole can be controlled at the same time, changing the ratio and dosage of the crosslinking agent, the ratio and dosage of the organic solvent can adjust the pore size of the diffusion hole, changing the particle size of the solid particle can adjust the pore size of the convection hole; this separation medium The skeleton structure is a polyglycidyl methacrylate polymer, which has good hydrophilicity and weak non-specific adsorption; the skeleton structure contains a large number of epoxy groups, which are easy to modify ligands, such as ion exchange groups, affinity Ligands, etc., are used in the separation of biological macromolecules such as proteins, with large adsorption capacity and mild elution conditions; they have good application prospects in large-scale preparative separation of biomacromolecules.

Description of drawings

The accompanying drawing is an electron microscope phase diagram of two types of pore-type PGDT separation media prepared by the method of the present invention. In the figure, 1 is the penetrating hole area, and 2 is the diffusion hole area.

Detailed ways

The following examples will further illustrate the method provided by the invention.

Example 1

Weigh 5.00 grams of glycidyl methacrylate (GMA), 1.70 grams of divinylbenzene (DVB), 1.46 grams of triallyl isocyanurate (TAIC), 1.84 grams of toluene, 1.33 grams of n-heptane, 0.12 Add 12.9 grams of calcium carbonate to a 50ml Erlenmeyer flask and mix evenly. Prepolymerize for 24 hours in a super constant temperature water bath at 40°C. In a three-necked flask equipped with a stirrer, a reflux condenser and a thermometer, after adding 1% polyvinyl alcohol solution, the reaction mixture was added. Under the protection of nitrogen, adjust the stirring speed. After the droplets are dispersed into an appropriate particle size, slowly raise the temperature to 65°C at a speed of 1°C/3 minutes, react for 3 hours, then raise the temperature to 75°C at the same speed, and react for 1 hour. Finally, the temperature was raised to 85° C., and the reaction was carried out for 2 hours. Then the polymer microspheres were transferred into nylon sandbags and extracted with ethanol for 24 hours to remove the organic porogen. Then soak the extracted microspheres in 0.2M hydrochloric acid solution, remove the solid porogen, and vacuum dry to obtain 6.0 g of the above-mentioned two types of pore-type PGDT separation medium. The separation medium was modified with diethylamine, and 5.0 g of the separation medium was mixed with 25 ml of dioxane and 25 ml of diethylamine, and reacted at 60° C. for 6.5 hours. After the reaction is finished, it is fully washed with distilled water and dried in vacuum (<1Torr, 1Torr=133.332Pa) to obtain two types of pore-type PGDT separation media that can be used to separate biological substances such as proteins in anion exchange mode. The volume average particle diameter of this separation medium is 45.6 μm, the specific surface area is 52.1 m ² /g, and the static adsorption capacity is 73.3 mgBSA/g wet separation medium. The column is packed by gravity settling method. At the flow rate of 180.0cm/h, the dynamic adsorption capacity is as high as 33.2mgBSA/ml column volume (51.8mgBSA/g wet separation medium).

Example 2

Weigh 5.00 grams of GMA, 1.01 grams of DVB, 1.05 grams of TAIC, 1.19 grams of toluene, 0.87 grams of n-heptane, and 0.12 grams of azobisisobutyronitrile into a 50ml Erlenmeyer flask, mix well, add 14 grams of calcium carbonate, mix uniform. According to the method in Example 1, 6.0 grams of PGDT two-type pore separation medium that can be used for protein adsorption in anion exchange mode was synthesized. The volume average particle size is 49.1μm, the specific surface area is 28.7m ² /g, and the static adsorption capacity is 34.9mgBSA/g wet separation medium. Column packing by gravity settling method. At a flow rate of 180.0 cm/h, the dynamic adsorption capacity reached 13.4 mgBSA/ml column volume (21.3 mgBSA/g wet separation medium).

Claims (2)

1. A preparation method for a microsphere PGDT separation medium of two types of pore types. First, the monomer glycidyl methacrylate, the cross-linking agent divinylbenzene and triallyl isocyanurate, and the liquid pore-forming The agent toluene and n-heptane, and the initiator azobisisobutyronitrile are mixed evenly, and then the solid porogen is added and mixed evenly, then, the polymer microspheres are synthesized by suspension polymerization, and the microspheres are extracted with absolute ethanol to remove the liquid The porogen includes pickling with hydrochloric acid to remove the solid porogen, and then drying to obtain two types of pore-type PGDT separation media. It is characterized in that the solid porogen is calcium carbonate, and the amount accounts for 50% of the volume content of the reaction mixture. 10-30%; the volume ratio of solid porogen to liquid porogen is 0.5-1; the amount of porogen accounts for 20-60% of the volume content of the reaction mixture; the cross-linking agent divinylbenzene and trimeric isocyanurate The material ratio of allyl ester is 0.5-2, and the ratio of the amount of crosslinking agent to the monomer glycidyl methacrylate is 0.3-0.6; under normal pressure, the polymerization temperature is controlled at 65-85°C between. 2. by the preparation method of the microsphere PGDT separation medium of two kinds of pore types claimed in claim 1, it is characterized in that the particle diameter of calcium carbonate particle is 0.5～3 μm, and density is 2.71g/ml.