CN101301593A - A kind of preparation method of temperature-sensitive paper base film - Google Patents

Abstract

A method for preparing a temperature-sensitive paper base film belongs to the field of separation membranes. The steps of the present invention: soak the filter paper base membrane with deionized water, dry it after cleaning, cut it and put it into a reactor in a nitrogen atmosphere; use the mixed solution of ketone and deionized water replaced by nitrogen as a solvent, and N-isopropyl Acrylamide or the copolymer of N-isopropylacrylamide and acrylate is the solute, and a photosensitizer with a mass of 0.5% to 3% of the solute is added; the monomer solution is added to the filter paper to completely wet the filter paper, and irradiated with ultraviolet light 10-15 minutes to initiate graft polymerization; after removing unpolymerized monomers and residual photosensitizers, dry the filter paper of grafted polymers. The separation membrane realizes protein adsorption and elution desorption only by changing the temperature, so as to achieve the purpose of separation and purification. The membrane prepared by the invention has the advantages of small operating pressure drop, good biocompatibility (anti-pollution), readily available materials, low cost, no environmental pollution, and simple equipment operation.

Description

A kind of preparation method of temperature-sensitive paper base film

technical field

The invention relates to a method for preparing a temperature-sensitive separation membrane using paper as a base membrane.

Background technique

As a novel separation and purification technology, membrane chromatography has many advantages over traditional column chromatography, such as low pressure drop, large adsorption capacity, fast mass transfer, and easy scale-up. The working principle of membrane chromatography is to use a membrane with a certain pore size as a medium, connect ligands, and use the interaction between membrane ligands and target molecules such as proteins to separate and purify. Therefore, the membrane material is the key factor to determine the separation effect of membrane chromatography.

Commonly used chromatographic membrane materials include natural-based polymer materials such as modified cellulose, chitosan and their modified products, and artificially synthesized polymer materials such as polysulfone and polyamide. Since the artificially synthesized polymer materials are mostly single homopolymers, and most polymer membranes are hydrophobic, it is easy to cause biofouling (membrane fouling).

Cellulose is a resource-rich natural polymer compound with good hydrophilicity and biocompatibility. Using it as a membrane substrate is expected to reduce membrane fouling. Filter paper is made of fiber, has a porous structure, is widely used as a filter medium, and has a certain mechanical strength, and can be used as a membrane medium. Eli Ruckenstein et al. (Wei Guo, Eli Ruckenstein.Separation and purification of horseradish peroxidase by membrane affinity chromatography.Journal of Membrane Science211(2003)101～111) used filter paper as substrate to prepare affinity membrane chromatography for horseradish peroxidation Separation and purification of biological macromolecules such as enzymes. In addition, Deqiang Yu et al. (Deqiang Yu, Xiaonong Chen, Robert Pelton, Raja Ghos.Paper-PEG-Based Membranes for Hydrophobic Interaction Chromatography: Purification of Monoclonal Antibody. Biotechnology and Bioengineering, 2008, 99(6): 1434～1442) will Grafted onto filter paper, a hydrophobic chromatographic membrane was prepared, and the adsorption and elution separation and purification of hIgG1-CD4 were realized by using the salting-out effect. These studies indicate that filter paper can be used as a substrate for separation membranes.

Contents of the invention

The invention aims to prepare a novel paper-based separation membrane, which can achieve protein adsorption, elution and desorption only by changing the temperature, so as to achieve the purpose of separation and purification. The membrane works by adsorbing proteins from aqueous media at relatively high temperatures and desorbing them by rinsing at lower temperatures. Therefore, grafting temperature-sensitive polymers on filter paper base membranes is the key to obtain such membranes. The graft polymer of the present invention is poly-N-isopropylacrylamide and its copolymers, and the temperature-sensitive transition temperature of these polymers is between 5-32°C. Since the porous structure of filter paper is retained after grafting, this membrane has the advantages of small operating pressure drop, good biocompatibility (anti-pollution), easy-to-obtain materials, low cost, no pollution to the environment, and simple equipment operation.

The preparation method of the above-mentioned paper-based protein chromatographic separation membrane comprises the following process steps:

1. Treatment of matrix membrane

Soak the filter paper base membrane with deionized water, wash it, dry it, cut it into a suitable size, and put it into a reactor with a nitrogen atmosphere.

2. Preparation of monomer solution

Preparation of monomer solution: The monomer solution uses a mixture of nitrogen-substituted ketone and deionized water as the solvent, the mass ratio of the two is 0.5-2:1, N-isopropylacrylamide or N-isopropyl The copolymer of acrylamide and acrylate is a solute, and the total monomer mass concentration is 3% to 30%. Add a photosensitizer (such as benzophenone, but not limited to Benzophenone).

3. Graft polymerization

Add the prepared monomer solution to the filter paper to completely wet the filter paper. Irradiate with UV light to initiate graft polymerization.

4. Post-treatment of separation membrane

After the reaction, soak and wash the obtained polymer-grafted filter paper membrane with a mixed solution of acetone and distilled water to remove unpolymerized monomers and residual photosensitizers. Finally, the filter paper of the grafted polymer was dried.

The separation process of the protein by the paper-based chromatographic separation membrane provided by the invention: when the temperature is higher than the lower critical solution temperature (LCST) of poly-N-isopropylacrylamide (PNIPAM) or its copolymer, the grafted chain is in a hydrophobic state , the hydrophobic interaction with the hydrophobic groups of the protein occurs, and the protein is adsorbed; when the temperature is lower than the LCST of the grafted chain, the grafted chain becomes hydrophilic, the hydrophobic interaction with the protein disappears, and the protein desorbs. The difference in the hydrophobicity of different proteins, that is, the difference in the hydrophobic interaction force of the grafted chains, is used to achieve protein separation.

The present invention has the following effects:

1. The filter paper used in the paper-based chromatographic separation membrane prepared by the present invention is a hydrophilic natural material, has good biocompatibility, and reduces the risk of protein denaturation and membrane fouling.

2. The graft polymerization induced by ultraviolet light can control the grafting rate and maintain the porous structure of the membrane, so that the membrane has low operating pressure drop and high flux.

3. The paper-based separation membrane prepared by the present invention can realize protein adsorption at a lower temperature by controlling the graft chain composition and its temperature-sensitive transition temperature, thereby saving energy consumption and reducing the risk of protein deformation.

4. No high saline medium is required for protein adsorption, and the separation process is more environmentally friendly.

5. The base material of filter paper is easy to obtain and low in cost.

Description of drawings

Fig. 1 is example 1 electron micrograph;

Fig. 2 is example 2 electron micrographs;

Fig. 3 is the electron micrograph of example 6.

Detailed ways

Implementation example 1:

In this example, analysis filter paper (qualitative analysis filter paper of Hangzhou Special Paper Co., Ltd.) was used as the matrix membrane, and graft polymerization was initiated by ultraviolet light, and poly N-isopropylacrylamide chains were grafted on the surface of the matrix membrane.

Specific steps are as follows:

1. Cut 10×10 cm filter paper and place it in a light-transmitting and nitrogen-filled reactor;

2. Prepare the monomer solution, the monomer solution is a mixture of acetone and deionized water replaced by nitrogen (acetone 1.6g, deionized water 1.3g) as a solvent, and N-isopropylacrylamide (0.25g) as a solvent The solute, the monomer concentration is 7.9%, and the photosensitizer is benzophenone (0.005g).

3. Inject the prepared solution onto the filter paper with a syringe to completely wet the filter paper to carry out the polymerization reaction. The irradiation time was 10 minutes.

4. After the reaction, soak and wash the filter paper with a mixed solution of acetone and deionized water, and then soak it repeatedly with deionized water to remove unpolymerized monomers and residual photosensitizers.

5. Finally, dry the filter paper in a drying oven at 60° C. after natural drying, and weigh it after placing it at room temperature. The grafting rate is calculated to be 4.2% (wt). The temperature-sensitive transition temperature (LCST) of the grafted polymer is 32°C.

The separation membrane surface porous structure that this example makes is as shown in Fig. 1 electron microscope photo:

Implementation example 2:

In this example, the filter paper is the same as that in Example 1, and the graft polymerization is initiated by ultraviolet light, and poly-N-isopropylacrylamide chains are grafted on the surface of the matrix membrane. Specific steps are as follows:

1. Cut 10×10 cm filter paper and place it in a light-transmitting and nitrogen-filled reactor;

2. Prepare the monomer solution, the monomer solution is a mixture of acetone and deionized water replaced by nitrogen (acetone 1.2g, deionized water 1.0g) as a solvent, and N-isopropylacrylamide (0.5g) as a solvent Solute, monomer concentration is 18.5%, photosensitizer is benzophenone (0.01g).

3. Inject the prepared solution onto the filter paper with a syringe to completely wet the filter paper to carry out the polymerization reaction. The irradiation time was 15 minutes.

4. After the reaction, soak and wash the filter paper with a mixed solution of acetone and deionized water, and then soak it repeatedly with deionized water to remove unpolymerized monomers and residual photosensitizers.

5. Finally, dry the filter paper in a drying oven at 60° C. after natural drying, and weigh it after placing it at room temperature. The grafting rate is 38% (wt). Our transition temperature (LCST) of the grafted polymer was 32°C.

The separation membrane surface porous structure that this example makes is as shown in Fig. 2 electron microscope photo:

Implementation example 3:

In this example, the filter paper is the same as that in Example 1, and the graft polymerization is initiated by ultraviolet light, and poly-N-isopropylacrylamide chains are grafted on the surface of the matrix membrane. Specific steps are as follows:

1. Cut 10×10 cm filter paper and place it in a light-transmitting and nitrogen-filled reactor;

2. Prepare a monomer solution, the monomer solution is a mixture of acetone and deionized water replaced by nitrogen (3g of acetone, 1.5g of deionized water) as a solvent, and N-isopropylacrylamide (0.5g) as a solute , the monomer concentration is 10%, and the photosensitizer is benzophenone (0.01 g).

3. Inject the prepared solution onto the filter paper with a syringe to completely wet the filter paper to carry out the polymerization reaction. The irradiation time was 15 minutes.

4. After the reaction, soak and wash the filter paper with a mixed solution of acetone and deionized water, and then soak it repeatedly with deionized water to remove unpolymerized monomers and residual photosensitizers.

5. Finally, dry the filter paper in a drying oven at 60° C. after natural drying, and weigh it after placing it at room temperature. The grafting rate is 12.2% (wt). Our transition temperature (LCST) of the grafted polymer was 32°C.

Implementation example 4:

In this example, the filter paper is the same as that in Example 1, and the graft polymerization is initiated by ultraviolet light, and poly-N-isopropylacrylamide chains are grafted on the surface of the matrix membrane. Specific steps are as follows:

1. Cut 10×10 cm filter paper and place it in a light-transmitting and nitrogen-filled reactor;

2. prepare the monomer solution, the monomer solution is the mixture of acetone and deionized water (acetone 2.2g, deionized water 4.4g) through nitrogen replacement as solvent, and N-isopropylacrylamide (1.8g) as solvent The solute, the monomer concentration is 21.4%, and the photosensitizer is xanthone (0.009g).

3. Inject the prepared solution onto the filter paper with a syringe to completely wet the filter paper to carry out the polymerization reaction. The irradiation time was 15 minutes.

4. After the reaction, soak and wash the filter paper with a mixed solution of acetone and deionized water, and then soak it repeatedly with deionized water to remove unpolymerized monomers and residual photosensitizers.

5. Finally, the filter paper was dried naturally in a drying oven at 60° C., and weighed after placing it at room temperature. The grafting rate was 41.0% (wt). Our transition temperature (LCST) of the grafted polymer was 32°C.

Implementation example 5:

In this example, the filter paper is the same as that in Example 1, and the graft polymerization is initiated by ultraviolet light, and poly-N-isopropylacrylamide chains are grafted on the surface of the matrix membrane. Specific steps are as follows:

1. Cut 10×10 cm filter paper and place it in a light-transmitting and nitrogen-filled reactor;

2. prepare monomer solution, monomer solution is solvent with the mixture (butanone 2.2g, deionized water 1.8g) of butanone and deionized water replaced by nitrogen, with N-isopropylacrylamide (0.17g ) is the solute, the monomer concentration is 4.0%, and the photosensitizer is benzophenone (0.0051g).

3. Inject the prepared solution onto the filter paper with a syringe to completely wet the filter paper to carry out the polymerization reaction. The irradiation time was 15 minutes.

4. After the reaction, soak and wash the filter paper with a mixed solution of butanone and deionized water, and then soak it repeatedly with deionized water to remove unpolymerized monomers and residual photosensitizers.

5. Finally, dry the filter paper in a drying oven at 60° C. after natural drying, and weigh it after placing it at room temperature. The grafting rate is 3.0% (wt). Our transition temperature (LCST) of the grafted polymer was 32°C.

Implementation example 6:

In this example, the filter paper is the same as that in Example 1, and the graft polymerization is initiated by ultraviolet light, and poly-N-isopropylacrylamide chains are grafted on the surface of the matrix membrane. Specific steps are as follows:

1. Cut 10×10 cm filter paper and place it in a light-transmitting and nitrogen-filled reactor;

2. prepare monomer solution, monomer solution is solvent with the mixture (acetone 2.6g, deionized water 2.1g) of acetone and deionized water replaced by nitrogen, with N-isopropylacrylamide (0.6g) and Ethyl acrylate (0.11 g) was the solute, the monomer concentration was 11.3%, and the photosensitizer was benzophenone (0.012 g).

3. Inject the prepared solution onto the filter paper with a syringe to completely wet the filter paper to carry out the polymerization reaction. The irradiation time was 15 minutes.

4. After the reaction, soak and wash the filter paper with a mixed solution of acetone and deionized water, and then soak it repeatedly with deionized water to remove unpolymerized monomers and residual photosensitizers.

5. Finally, dry the filter paper in a drying oven at 60° C. after natural drying, and weigh it after placing it at room temperature. The grafting rate is 25% (wt). The temperature-sensitive transition temperature (LCST) of the grafted polymer is 20°C.

The separation membrane surface porous structure that this example makes is as shown in Figure 3 electron microscope photo:

The separation membrane with a grafting rate of 12.2% in Example 3 was used to carry out protein adsorption experiments. The protein used in the experiment was bovine serum albumin (BSA). Changes in protein concentration were characterized by measuring changes in light transmittance of protein solutions with an ultraviolet spectrophotometer. The specific process includes:

1. Prepare a BSA aqueous solution (pH=6.0) with a concentration of 500 μg/ml.

2. Adsorption: Cut the membrane into a size of 3cm×3cm, put it into a bottle with a volume of 50ml, add 20ml of protein solution (the membrane is completely immersed in the solution), seal it and put it in a constant temperature oscillator, and absorb it at 37°C for 2 Hour.

3. Measure the light transmittance of the original protein solution in step 1 and the protein solution after adsorption in step 2 by an ultraviolet spectrophotometer, calculate the corresponding protein concentration, and then calculate the adsorption amount.

Measurement results: the light transmittance of unadsorbed BSA solution is 26.1% (BSA concentration is 500 μg/ml), the light transmittance of BSA solution after filter membrane adsorption is 44.2%, and the corresponding protein concentration is 225 μg/ml. According to the decrease of BSA concentration (275μg/ml), the total volume of the protein solution in the adsorption experiment (20ml) and the area of the paper-based separation membrane (18cm ² ), the adsorption capacity of the separation membrane to BSA was calculated to be 611.1μg/cm ² ) .

Claims (1)

1. the preparation method of a thermosensitive paper-based membrane is characterized in that, may further comprise the steps:

(1), the processing of matrix membrane

The filter paper basement membrane is soaked, cleans the back drying with deionized water, put into the reactor of nitrogen atmosphere after the cutting;

(2), the preparation of monomer solution

The preparation of monomer solution: with the ketone crossed through nitrogen replacement and the mixed solution of deionized water is solvent, the mass ratio of ketone and deionized water is 0.5～2: 1, the copolymer of N-N-isopropylacrylamide or N-N-isopropylacrylamide and acrylate is a solute, total monomer mass concentration is 3%～30%, adds the sensitising agent of solute quality 0.5%-3% in this solution;

(3), glycerol polymerization

The above-mentioned monomer solution for preparing is added on the filter paper, makes the filter paper complete wetting, with UV-irradiation 10-15 minute with the initiation grafting polymerization;

(4), the post processing of diffusion barrier

Reaction finishes the back and soaks, washs resulting polymers grafting filter paper film with above-mentioned ketone and deionized water mixed solution, to remove unconverted monomer, remaining sensitising agent; At last, with the filter paper drying of graft polymers.

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