A bicontinuous macroporous cellulose / polystyrene composite monolithic column and its preparation method and application

Abstract

The present application relates to the preparation field of organic polymer monolithic column, in particular to a kind of double-continuous supermacroporous cellulose / polystyrene composite monolithic column and its preparation method and application.Firstly, O / W Pickering emulsion is prepared using modified cellulose nanocrystal (CNC), and Janus CNC-PS with single side grafted polystyrene (PS) polymer brush is prepared by electron activation regeneration-atom transfer radical polymerization (AGET-ATRP) reaction system, and double-continuous phase emulsion gel (Bijel) is prepared by one-step stirring method using Janus CNC-PS with different HLB values as stabilizer.Then, double-continuous supermacroporous cellulose / polystyrene composite monolithic column is obtained by in-situ active polymerization through AGET-ATRP reaction using Janus CNC-PS as macromolecular initiator and Bijel as emulsion template.The obtained monolithic column has uniform skeleton structure, double-continuous skeleton and pore, high mechanical strength, and the unmodified side of CNC covers the outer surface of skeleton, so that the pore surface has hydrophilicity and is easy to modify.The monolithic column effectively overcomes the defects of current polymer monolithic column, such as poor biocompatibility, low mechanical strength and low specific surface area, and has great application potential in the field of enzyme and cell immobilization and biomacromolecule separation and analysis.

Description

Technical Field

[0001] This invention relates to the field of preparation of monolithic organic polymer columns, specifically to a bicontinuous macroporous cellulose / polystyrene composite monolithic column, its preparation method, and its application. Background Technology

[0002] With the continuous development of the biopharmaceutical industry globally, the market demand for biopharmaceuticals such as antibodies and vaccines is increasing daily. Compared with upstream cell expression and culture technologies, downstream separation and purification technologies have lagged behind. However, separation and purification are crucial for improving product purity, reducing activity loss, and lowering product costs. Chromatography is an indispensable step in the separation and purification of biopharmaceuticals, and the performance of the separation medium plays a key role in separation efficiency. Compared with microsphere packing materials, monolithic columns are simple to prepare, have high column volume utilization, and good permeability, giving them significant advantages in the rapid separation of biomolecules, and are thus known as the fourth-generation chromatographic separation medium.

[0003] Based on the properties of the materials used in the preparation of monolithic columns, they can be classified into inorganic silica monolithic columns, organic polymer monolithic columns, polysaccharide monolithic columns, and organic-inorganic hybrid monolithic columns. While each type of monolithic column has its own advantages and disadvantages, organic polymer monolithic columns, with their good mechanical strength and chemical stability, high operating flow rates, and resistance to acid and alkali cleaning, have attracted widespread attention in the separation and purification of biomolecules such as antibodies and vaccines. Based on the different reactants and cross-linking agents used in the preparation process, organic polymer monolithic columns can be further classified into polyacrylamide monolithic columns, polymethacrylate monolithic columns, and polystyrene monolithic columns. Polyacrylamide monolithic columns have relatively good biocompatibility and can be used for the separation of biomolecules; however, their matrix is ​​relatively soft and their mechanical strength is low, making them unsuitable as high-flow-rate chromatographic separation media. Polymethacrylate monolithic columns have good mechanical strength and moderate biocompatibility; however, their backbone has generally poor resistance to acids and alkalis, which is detrimental to acid and alkali cleaning and regeneration of monolithic columns in industrial applications. Polystyrene monolithic columns are stable under different pH conditions and have high mechanical strength, but they are strongly hydrophobic, have poor biocompatibility, and lack active groups on the surface of styrene molecules, making them difficult to modify. This limits their application in the separation of biomacromolecules.

[0004] To improve the hydrophilicity and biocompatibility of monolithic polystyrene columns, our laboratory previously prepared a dual-channel hydrophilic bicontinuous polystyrene monolithic column (ZL201811578771.3; Macromol. Rapid Commun. 2021, 42, 2100154) using a bicontinuous internal-phase emulsion (BMIPE) template method. This column features a dual-channel distribution of through-pores (150-5000 nm) and mesopores (10-150 nm). The internal framework is cross-linked polystyrene, while the outer surface is composed of hydroxyl-rich amphiphilic sugar polymers (ADG). This allows it to be used directly as a hydrophilic chromatographic medium and easily derivatized into other chromatographic modes, showing great potential in the rapid separation of biomolecules. ADG is key to the preparation of this monolithic column, serving both as a surfactant in the preparation of BMIPE and as an initiator for the electron activation regeneration-atom transfer radical polymerization (AGET-ATRP) of the monolithic column. However, the synthesis process of ADG is somewhat complicated. It not only requires the synthesis of sugar monomers, but also the precise control of the ratio of the two blocks (hydrophobic and hydrophilic blocks) using living polymerization. This results in low yield and difficulties in scale-up.

[0005] Emulsion templates are an effective method for preparing monolithic columns. Compared to surfactant-stabilized emulsions, solid particle-stabilized Pickering emulsions offer enhanced stability, environmental friendliness, low cost, and wide applicability. Nanocellulose (nanocrystals and nanofibers) is an abundant, low-cost, non-polluting, and biodegradable raw material with a surface rich in hydroxyl groups, making it easy to modify and functionalize. Although nanocellulose is hydrophilic, it has been found that it can aggregate and self-assemble at the interface of two phases, forming steric hindrance, making it an excellent stabilizer for Pickering emulsions. Traditional Pickering emulsions are oil-in-water (O / W) or water-in-oil (W / O) type, with only one continuous phase. This results in monolithic columns prepared using these as templates having mostly closed dead pores, poor permeability, and extremely low permeability coefficients, rendering them unusable. Recently developed colloidal particle-stabilized bicontinuous emulsion gels (Bijel) are complex three-dimensional networks composed of continuous fluid channels, offering significant advantages in preparing bicontinuous porous materials (Applied Physics Reviews, 2021, 8, 021323). Summary of the Invention

[0006] To address the aforementioned issues and building upon previous work, this invention employs Janus CNC-PS cellulose nanocrystals grafted with polystyrene (PS) polymer brushes of varying chain lengths on one side as a stabilizer. These nanocrystals are dispersed in both oil and aqueous phases, and Bijel is prepared using a direct mixing method. Then, using Bijel as a template and Janus CNC-PS as a macromolecular initiator, electron-activated regenerated atom transfer radical polymerization (AGET-ATRP) is employed to prepare a monolithic, bicontinuous, macroporous cellulose / polystyrene (BGCP) composite column with a uniform framework structure. The resulting BGCP composite column shows promising applications in enzyme and cell immobilization, cell culture, and the rapid separation and purification of biomolecules.

[0007] In view of this, the present invention provides a BGCP composite monolithic column, its preparation method and application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a BGCP composite monolithic column includes the following steps:

[0010] (1) The cellulose nanocrystal (CNC) solution was freeze-dried. A certain mass of CNC was dispersed in pyridine and reacted with acyl halide compounds at room temperature for 1-24 h. The molar ratio of acyl halide compounds to hydroxyl groups on CNC ranged from 1:3 to 1:30. After the reaction was completed, the reaction solution was removed by centrifugation. The precipitate was thoroughly washed with ethanol, centrifuged, and vacuum dried to obtain halogen-modified CNC.

[0011] (2) The modified CNC, ascorbic acid, inorganic salt and deionized water obtained in step (1) are mixed as the aqueous phase, and styrene, catalyst and ligand are mixed as the oil phase. The aqueous phase and oil phase are stirred and mixed and deoxygenated by inert gas to obtain a modified CNC stable O / W Pickering emulsion. The modified CNC is used as the AGET-ATRP macromolecular initiator and styrene is used as the monomer. PS polymer brush is grafted onto one side of CNC by AGET-ATRP reaction. The reaction solution is precipitated in ethanol, centrifuged, washed thoroughly with ethanol, centrifuged, and vacuum dried to obtain the product Janus CNC-PS. The reaction temperature is 70-100℃, the reaction time is 3-48h, the molar ratio of monomer to halogen on modified CNC is 10:1 to 200:1, the amount of catalyst added is the same as the molar amount of halogen on modified CNC, and the amount of ligand added is 0.5-3 times that of catalyst.

[0012] (3) The Janus CNC-PS containing longer PS segments obtained in step (2) is mixed with the reactive monomer, crosslinking agent, pore-forming agent, catalyst and ligand as the oil phase. The Janus CNC-PS containing shorter PS segments, ascorbic acid and inorganic salt are added to deionized water and mixed as the aqueous phase. The aqueous phase is slowly added dropwise to the oil phase under stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. Janus CNC-PS not only serves as a stabilizer for Bijel, but also as a macromolecular initiator for AGET-ATRP. The prepared Bijel is deoxygenated by passing an inert gas and used as a polymerization template for AGET-ATRP. It is added to a stainless steel column tube with a syringe and heated to obtain a polymer monolithic column.

[0013] (4) The monolithic column obtained in step (3) is washed with ethanol and water respectively, and then dried under vacuum at room temperature to obtain the BGCP composite monolithic column.

[0014] The CNC machine mentioned in step (1) is purchased from the market and has a diameter of 4-10nm and a length of 100-800nm.

[0015] Preferably, the acyl halide compound in step (1) is 2-bromoisobutyryl bromide, 2-bromoisobutyryl chloride, 2-chloroisobutyryl bromide, 2-chloroisobutyryl chloride, bromoacetyl bromide, bromoacetyl chloride, chloroacetyl chloride or chloroacetyl bromide.

[0016] Preferably, the catalyst in step (2) is a transition metal salt such as CuCl2 or CuBr2; the ligand is a common ligand used in the ATRP reaction, such as N,N,N,N',N'-pentamethyldiethylenetriamine (PMDETA), tris(2-pyridylmethyl)amine (TPMA), N,N,N',N'-tetra(2-pyridylmethyl)ethylenediamine (TPEDA), triphenylphosphine or tributylphosphine, etc.

[0017] In step (2), the amount of PS grafting in Janus CNC-PS is the key to this invention, as it directly affects whether Bijel can be successfully prepared. The amount of PS grafting can be controlled by the number of initiation sites, reaction temperature, reaction time, and catalyst content.

[0018] In step (2), the amount of PS grafted onto Janus CNC-PS can be characterized by the hydrophilic-lipophilic balance (HLB) value of the modified CNC. The HLB value of Janus CNC-PS is calculated using Griffin's formula, as shown below:

[0019]

[0020] Where M H : Molecular weight of the hydrophilic portion, ML : Molecular weight of the hydrophobic portion, M: Molecular weight of Janus CNC-PS. Since the molecular weight of cellulose nanocrystals cannot be determined, the mass of each component is used for calculation.

[0021] The inorganic salts mentioned in steps (2) and (3) can be sodium chloride, potassium chloride, sodium sulfate or magnesium sulfate, etc., accounting for 0.01%-10% of the mass of the aqueous phase, preferably 0.1%-2%; the mass ratio of the aqueous phase to the oil phase should be 1:0.1-1:10, preferably 1:0.8-1:1.2.

[0022] The HLB range of Janus CNC-PS used in step (3) is 2-16; the amount of Janus CNC-PS added is 0.01%-20% of the oil phase mass, preferably 5%-20%; by changing the amount of Janus CNC-PS added and the HLB value, the stability and morphology of Bijel can be changed, thereby controlling the pore structure of the BGCP composite monolithic column.

[0023] The degree of crosslinking in step (3) is 10%-90%, preferably 40%-60%; the porogen is a small molecule porogen such as isopropanol, toluene, xylene, acetonitrile or chloroform, and the amount added is 5%-50% of the mass of the polymerizable monomer, preferably 15%-30%.

[0024] In step (3), the reaction temperature is 60-130℃, preferably 95-110℃; the reaction time is 5-48h, preferably 10-30h.

[0025] This invention also discloses a BGCP composite monolithic column prepared by the above method, wherein the monolithic column has a cross-linked polystyrene framework and a hydrophilic cellulose nanocrystal outer surface; the monolithic column has a uniform framework, a bicontinuous framework and channels, macropores with a diameter of 0.05-10 μm, micropores with a diameter of 2-50 nm, a porosity of 50-80%, and a specific surface area of ​​30-200 m². 2 / g.

[0026] The advantages of this invention are:

[0027] This invention utilizes the AGET-ATRP reaction system to prepare Janus CNC-PS with different HLB values. Using Janus CNC-PS as a stabilizer, Bijel was prepared via a one-step stirring method. Using Janus CNC-PS as a macromolecular initiator and Bijel as an emulsion template, in-situ living polymerization was carried out via AGET-ATRP to obtain a BGCP composite monolithic column. The resulting monolithic column has a uniform framework structure, bicontinuous framework and pores, and high mechanical strength. The unmodified side of the CNC covers the outer surface of the framework, making the pore surface hydrophilic and easy to modify. The BGCP composite monolithic column effectively overcomes the shortcomings of current polymer monolithic columns, such as poor biocompatibility, low mechanical strength, and low specific surface area, and has great application potential in the fields of enzyme and cell immobilization and the separation and analysis of biomolecules. Attached Figure Description

[0028] Figure 1 This is a Bijel inverted fluorescence microscope image prepared in Example 1 of the present invention;

[0029] Figure 2 This is a scanning electron microscope image of the BGCP composite monolithic column prepared in Example 1 of the present invention;

[0030] Figure 3 This is a scanning electron microscope image of the cellulose / polystyrene composite monolithic column prepared in the comparative example. Specific implementation methods

[0031] The specific implementation method of the present invention is as follows:

[0032] Example 1

[0033] (1) 1 g of lyophilized CNC was added to an Erlenmeyer flask, followed by 100 mL of pyridine for dispersion. Then, 0.355 g of 2-bromoisobutyryl bromide was added to the dispersion, and the mixture was reacted at room temperature for 24 h. After the reaction was complete, the reaction solution was removed by centrifugation, the precipitate was thoroughly washed with ethanol, centrifuged again, and dried under vacuum to obtain halogen-modified CNC. The Br content of the modified CNC was determined to be 5% by the Volhard method.

[0034] (2) The modified CNC (0.2g) and ascorbic acid (0.06g) obtained in step (1) were added together to 3mL of deionized water containing 0.1% NaCl as the aqueous phase. CuBr2 (0.123g) and N,N,N,N',N'-pentamethyldiethylenetriamine (PMDETA) (0.191g) were added to 3mL of styrene as the oil phase. The aqueous phase and oil phase were stirred and mixed and deoxygenated by argon gas to obtain a modified CNC-stabilized O / W Pickering emulsion. The emulsion was transferred to a 10mL heart-shaped bottle and placed in a metal bath at 98℃ for 30h. After the reaction was completed, the reaction product was diluted with tetrahydrofuran and precipitated in ethanol multiple times until the product had no obvious blue color. After vacuum drying, the product Janus CNC-PS (1.081g) was obtained and the HLB value was calculated to be 3.7.

[0035] Another heart-shaped flask was prepared with the same feed ratio as above. The reaction time was 12 hours. After vacuum drying, the product Janus CNC-PS (0.408 g) was obtained. The HLB value was calculated to be 9.8.

[0036] (3) Take a 10 mL beaker and add Janus CNC-PS (0.1 g) with HLB=3.7, CuBr2 (0.116 g), styrene (1.09 g), divinylbenzene (0.73 g), toluene (0.18 g), and PMDETA (0.173 g) in sequence. Stir and mix to obtain the oil phase. Take another 10 mL beaker and add Janus CNC-PS (0.1 g) with HLB=9.8, NaCl (0.002 g), ascorbic acid (0.08 g), and 2 mL of deionized water in sequence. Stir and mix to obtain the aqueous phase. Slowly add the aqueous phase to the oil phase while stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. The inverted fluorescence microscope image of Bijel is shown below. Figure 1 Bijel was deoxygenated by argon gas for 15 min, and the emulsion was added to the stainless steel column using a syringe. The reaction was carried out in an oil bath at 80°C for 48 h. (4) The monolithic column obtained in step (3) was washed with ethanol and water respectively, and vacuum dried to obtain the BGCP composite monolithic column. The scanning electron microscope image of the BGCP composite monolithic column is shown in the figure. Figure 2 .

[0037] Example 2

[0038] (1) 1 g of lyophilized CNC was added to an Erlenmeyer flask, followed by 100 mL of pyridine for dispersion. Then, 0.355 g of 2-bromoisobutyryl bromide was added to the dispersion, and the mixture was reacted at room temperature for 24 h. After the reaction was complete, the reaction solution was removed by centrifugation, the precipitate was thoroughly washed with ethanol, centrifuged again, and dried under vacuum to obtain halogen-modified CNC. The Br content of the modified CNC was determined to be 5% by the Volhard method.

[0039] (2) The modified CNC (0.2g) and ascorbic acid (0.06g) obtained in step (1) were added together to 3mL of deionized water containing 0.1% NaCl as the aqueous phase. CuBr2 (0.123g) and N,N,N,N',N'-pentamethyldiethylenetriamine (PMDETA) (0.191g) were added to 3mL of styrene as the oil phase. The aqueous phase and oil phase were stirred and mixed and deoxygenated by argon gas to obtain a modified CNC-stabilized O / W Pickering emulsion. The emulsion was transferred to a 10mL heart-shaped bottle and placed in a metal bath at 98℃ for 34h. After the reaction was completed, the reaction product was diluted with tetrahydrofuran and precipitated in ethanol multiple times until the product had no obvious blue color. After vacuum drying, the product Janus CNC-PS (1.290g) was obtained and the HLB value was calculated to be 3.1.

[0040] Another heart-shaped flask was prepared with the same feed ratio as above. The reaction time was 8 hours. After vacuum drying, the product Janus CNC-PS (0.276 g) was obtained. The HLB value was calculated to be 14.5.

[0041] (3) Take a 10 mL beaker and add Janus CNC-PS (0.2 g), CuBr2 (0.116 g), styrene (0.69 g), divinylbenzene (1.13 g), toluene (0.18 g), and PMDETA (0.173 g) with HLB=3.1 in sequence. Stir and mix to obtain the oil phase. Take another 10 mL beaker and add Janus CNC-PS (0.05 g), NaCl (0.004 g), ascorbic acid (0.08 g), and 2 mL of deionized water with HLB=14.5 in sequence. Stir and mix to obtain the aqueous phase. Slowly add the aqueous phase to the oil phase while stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. Deoxygenate by purging with argon gas for 15 min. Add the emulsion to a stainless steel column using a syringe and react in an oil bath at 80 °C for 48 h.

[0042] (4) The monolithic column obtained in step (3) is washed with ethanol and water respectively, and then vacuum dried to obtain the BGCP composite monolithic column.

[0043] Example 3

[0044] (1) 1 g of lyophilized CNC was added to an Erlenmeyer flask, followed by 100 mL of pyridine for dispersion. Then, 1.419 g of 2-bromoisobutyryl bromide was added to the dispersion, and the mixture was reacted at room temperature for 24 h. After the reaction was complete, the reaction solution was removed by centrifugation, the precipitate was thoroughly washed with ethanol, centrifuged again, and dried under vacuum to obtain halogen-modified CNC. The Br content of the modified CNC was determined to be 22% by the Volhard method.

[0045] (2) The modified CNC (0.2g) and ascorbic acid (0.06g) obtained in step (1) were added together to 3mL of deionized water containing 0.1% NaCl as the aqueous phase. CuBr2 (0.123g) and N,N,N,N',N'-pentamethyldiethylenetriamine (PMDETA) (0.191g) were added to 3mL of styrene as the oil phase. The aqueous phase and oil phase were stirred and mixed and deoxygenated by argon gas to obtain a modified CNC-stabilized O / W Pickering emulsion. The emulsion was transferred to a 10mL heart-shaped flask and placed in a metal bath at 98℃ for 32h. After the reaction was completed, the reaction product was diluted with tetrahydrofuran and precipitated in ethanol multiple times until the product had no obvious blue color. After vacuum drying, the product Janus CNC-PS (1.251g) was obtained and the HLB value was calculated to be 3.2.

[0046] Another flask was prepared with the same feed ratio as above. The reaction time was 10 hours. After vacuum drying, the product Janus CNC-PS (0.3539 g) was obtained. The HLB value was calculated to be 11.3.

[0047] (3) Take a 10 mL beaker and add Janus CNC-PS (0.2 g), CuBr2 (0.116 g), styrene (1.09 g), divinylbenzene (0.73 g), toluene (0.18 g), and PMDETA (0.173 g) with HLB=3.2 in sequence. Stir and mix to obtain the oil phase. Take another 10 mL beaker and add Janus CNC-PS (0.1 g), NaCl (0.002 g), ascorbic acid (0.08 g), and 2 mL of deionized water with HLB=11.3 in sequence. Stir and mix to obtain the aqueous phase. Slowly add the aqueous phase to the oil phase while stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. Deoxygenate by purging with argon gas for 15 min. Add the emulsion to a stainless steel column using a syringe and react in an oil bath at 80 °C for 48 h.

[0048] (4) The monolithic column obtained in step (3) is washed with ethanol and water respectively, and then vacuum dried to obtain the BGCP composite monolithic column.

[0049] Example 4

[0050] (1) 1 g of lyophilized CNC was added to an Erlenmeyer flask, followed by 100 mL of pyridine for dispersion. Then, 0.355 g of 2-bromoisobutyryl bromide was added to the dispersion, and the mixture was reacted at room temperature for 20 h. After the reaction was complete, the reaction solution was removed by centrifugation, the precipitate was thoroughly washed with ethanol, centrifuged again, and dried under vacuum to obtain halogen-modified CNC. The Br content of the modified CNC was determined to be 4% by the Volhard method.

[0051] (2) The modified CNC (0.2g) and ascorbic acid (0.06g) obtained in step (1) were added together to 3mL of deionized water containing 0.1% NaCl as the aqueous phase. CuBr2 (0.123g) and N,N,N,N',N'-pentamethyldiethylenetriamine (PMDETA) (0.191g) were added to 3mL of styrene as the oil phase. The aqueous phase and oil phase were stirred and mixed and deoxygenated by argon gas to obtain a modified CNC-stabilized O / W Pickering emulsion. The emulsion was transferred to a 10mL heart-shaped bottle and placed in a metal bath at 98℃ for 24h. After the reaction was completed, the reaction product was diluted with tetrahydrofuran and precipitated in ethanol multiple times until the product had no obvious blue color. After vacuum drying, the product Janus CNC-PS (0.615g) was obtained and the HLB value was calculated to be 6.5.

[0052] Another heart-shaped bottle was prepared with the same feed ratio as above. The reaction time was 8 hours. After vacuum drying, the product Janus CNC-PS (0.282 g) was obtained. The HLB value was calculated to be 14.2.

[0053] (3) Take a 10 mL beaker and add Janus CNC-PS (0.07 g), CuBr2 (0.116 g), styrene (1.09 g), divinylbenzene (0.73 g), toluene (0.18 g), and PMDETA (0.173 g) with HLB=6.5 in sequence. Stir and mix to obtain the oil phase. Take another 10 mL beaker and add Janus CNC-PS (0.07 g), NaCl (0.002 g), ascorbic acid (0.08 g), and 2.5 mL of deionized water with HLB=14.2 in sequence. Stir and mix to obtain the aqueous phase. Slowly add the aqueous phase to the oil phase while stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. Deoxygenate with argon gas for 15 min. Add the emulsion to a stainless steel column through a syringe and react in an oil bath at 80 °C for 48 h.

[0054] (4) The monolithic column obtained in step (3) is washed with ethanol and water respectively, and then vacuum dried to obtain the BGCP composite monolithic column.

[0055] Example 5

[0056] (1) 1 g of lyophilized CNC was added to an Erlenmeyer flask, followed by 100 mL of pyridine for dispersion. Then, 0.355 g of 2-bromoisobutyryl bromide was added to the dispersion, and the mixture was reacted at room temperature for 24 h. After the reaction was complete, the reaction solution was removed by centrifugation, the precipitate was thoroughly washed with ethanol, centrifuged again, and dried under vacuum to obtain halogen-modified CNC. The Br content of the modified CNC was determined to be 5% by the Volhard method.

[0057] (2) The modified CNC (0.2g) and ascorbic acid (0.06g) obtained in step (1) were added together to 3mL of deionized water containing 0.1% NaCl as the aqueous phase. CuBr2 (0.123g) and N,N,N,N',N'-pentamethyldiethylenetriamine (PMDETA) (0.191g) were added to 3mL of styrene as the oil phase. The aqueous phase and oil phase were stirred and mixed and deoxygenated by argon gas to obtain a modified CNC-stabilized O / W Pickering emulsion. The emulsion was transferred to a 10mL heart-shaped bottle and placed in a metal bath at 90℃ for 30h. After the reaction was completed, the reaction product was diluted with tetrahydrofuran and precipitated in ethanol multiple times until the product had no obvious blue color. After vacuum drying, the product Janus CNC-PS (0.953g) was obtained and the HLB value was calculated to be 4.2.

[0058] Another flask was prepared with the same feed ratio as above. The reaction time was 12 hours. After vacuum drying, the product Janus CNC-PS (0.392 g) was obtained. The HLB value was calculated to be 10.2.

[0059] (3) Take a 10 mL beaker and add Janus CNC-PS (0.1 g), CuBr2 (0.116 g), styrene (1.09 g), divinylbenzene (0.73 g), toluene (0.18 g), and PMDETA (0.173 g) with HLB=4.2 in sequence. Stir and mix to obtain the oil phase. Take another 10 mL beaker and add Janus CNC-PS (0.05 g), NaCl (0.002 g), ascorbic acid (0.08 g), and 2 mL of deionized water with HLB=10.2 in sequence. Stir and mix to obtain the aqueous phase. Slowly add the aqueous phase to the oil phase while stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. Deoxygenate by purging with argon gas for 15 min. Add the emulsion to a stainless steel column using a syringe and react in an oil bath at 80 °C for 48 h.

[0060] (4) The monolithic column obtained in step (3) is washed with ethanol and water respectively, and then vacuum dried to obtain a cellulose / polystyrene composite monolithic column.

[0061] Comparative example:

[0062] (1) 1 g of lyophilized CNC was added to an Erlenmeyer flask, followed by 100 mL of pyridine for dispersion. Then, 0.355 g of 2-bromoisobutyryl bromide was added to the dispersion, and the mixture was reacted at room temperature for 24 h. After the reaction was complete, the reaction solution was removed by centrifugation, the precipitate was thoroughly washed with ethanol, centrifuged again, and dried under vacuum to obtain halogen-modified CNC. The Br content of the modified CNC was determined to be 5% by the Volhard method.

[0063] (2) Take a 5mL heart-shaped flask, add a stir bar, add the modified CNC (0.2g) and CuBr (0.079g) obtained in step (1), and purge with argon gas for 20min under sealed conditions. Then, add styrene (3mL) and PMDETA (0.191g) to the heart-shaped flask with a syringe, and continue to purge with argon gas for 20min. After that, seal the heart-shaped flask and place it in a metal bath at 110℃ for 12h. After the reaction is completed, dilute the reaction product with tetrahydrofuran and precipitate it in ethanol multiple times until the product has no obvious blue color. After vacuum drying, the product CNC-PS (0.688g) is obtained. Calculate HLB = 5.8.

[0064] (3) Take a 10 mL beaker and add CNC-PS (0.1 g), CuBr2 (0.116 g), styrene (1.09 g), divinylbenzene (0.73 g), toluene (0.18 g), and PMDETA (0.173 g) with HLB=5.8 in sequence. Stir and mix to obtain the oil phase. Take another 10 mL beaker and add NaCl (0.002 g), ascorbic acid (0.08 g), and 2 mL of deionized water in sequence. Stir and mix to obtain the aqueous phase. Slowly add the aqueous phase to the oil phase while stirring to obtain a W / O Pickering emulsion stabilized by CNC-PS. Deoxygenate by argon gas for 15 min. Add the emulsion to a stainless steel column using a syringe and react at 80 °C in an oil bath for 48 h. (4) Clean the monolithic column obtained in step (3) with ethanol and water respectively, and vacuum dry to obtain the BGCP composite monolithic column. See the scanning electron microscope image below. Figure 3 .

[0065] The pore size, specific surface area, and porosity of the monolithic columns obtained in the above experiments are shown in Table 1.

[0066] Table 1 compares the pore size, porosity, and specific surface area of ​​the BGCP composite monolithic columns in the examples and comparative examples.

[0067]

[0068] Effect Experiment:

[0069] To verify that the BGCP composite monolithic column of the present invention has a dual continuous framework and pore structure, scanning electron microscopy was used to observe Example 1 ( Figure 2 ) and comparative examples ( Figure 3The morphology of the monolithic columns prepared by bulk polymerization shows that the former has high porosity and a through-hole structure. This is mainly due to the self-assembly of the Bijel template obtained by Janus CNC-PS with different HLB values ​​at the interface, which, after further polymerization, yields three-dimensional through-hole channels. In contrast, the monolithic columns prepared using CNC-PS prepared by bulk polymerization as a stabilizer for Pickering emulsions have channels that are mainly connected through throats. Pressure-flow rate curves were measured for the monolithic columns prepared in Example 1 and the comparative example. The maximum flow rates were 3612 cm / h and 722 cm / h, respectively, and the permeability coefficients calculated using Darcy's law were 1.5 × 10⁻⁶, respectively. -12 m 2 and 2.9×10 -13 m 2 This indicates that the former has better permeability and higher mechanical strength, and can be applied to separation operations at high flow rates.

Claims

1. A method for preparing a bicontinuous, ultraporous cellulose / polystyrene BGCP composite monolithic column, characterized in that, Includes the following steps: (1) The cellulose nanocrystal CNC solution was freeze-dried. A certain mass of CNC was dispersed in pyridine and reacted with acyl halide compounds at room temperature for 1-24 h. The molar ratio of acyl halide compounds to hydroxyl groups on CNC ranged from 1:3 to 1:

30. After the reaction was completed, the reaction solution was removed by centrifugation. The precipitate was thoroughly washed with ethanol, centrifuged, and vacuum dried to obtain halogen-modified CNC. (2) The modified CNC, ascorbic acid, inorganic salt and deionized water obtained in step (1) are mixed as the aqueous phase, and styrene, catalyst and ligand are mixed as the oil phase. The aqueous phase and oil phase are stirred and mixed and deoxygenated by inert gas to obtain the modified CNC stable O / W Pickering emulsion. The modified CNC is used as the initiator for electron activation regeneration atom transfer radical polymerization AGET-ATRP macromolecular polymerization. Styrene is used as the monomer. PS polymer brush is grafted onto one side of CNC by AGET-ATRP reaction. The reaction solution is precipitated in ethanol, centrifuged, washed thoroughly with ethanol, centrifuged, and vacuum dried to obtain the product Janus CNC-PS. The reaction temperature is 70-100 ℃, the reaction time is 3-48 h, the molar ratio of monomer to halogen on modified CNC is 10:1~200:1, the amount of catalyst added is the same as the molar amount of halogen on modified CNC, and the amount of ligand added is 0.5-3 times that of catalyst. (3) The Janus CNC-PS with longer PS segments obtained in step (2) is mixed with the reactant monomer, crosslinking agent, pore-forming agent, catalyst and ligand as the oil phase. The Janus CNC-PS with shorter PS segments, ascorbic acid and inorganic salt are added to deionized water and mixed as the aqueous phase. The aqueous phase is slowly added dropwise to the oil phase under stirring to obtain Bijel stabilized by Janus CNC-PS with different chain lengths. Janus CNC-PS not only serves as a stabilizer for Bijel, but also as a macromolecular initiator for AGET-ATRP. The prepared Bijel is deoxygenated by passing an inert gas and used as a polymerization template for AGET-ATRP. It is added to a stainless steel column tube with a syringe and heated to obtain a polymer monolithic column. (4) The monolithic column obtained in step (3) was washed with ethanol and water respectively, and then dried under vacuum at room temperature to obtain the BGCP composite monolithic column; In step (2), the catalyst is a transition metal salt of CuCl2 or CuBr2; the ligand is selected from at least one of N,N,N,N',N'-pentamethyldiethylenetriamine PMDETA, tris(2-pyridinemethyl)amine TPMA, N,N,N',N'-tetra(2-pyridinemethyl)ethylenediamine TPEDA, triphenylphosphine or tributylphosphine; the inorganic salt in steps (2) and (3) is sodium chloride, potassium chloride, sodium sulfate or magnesium sulfate, accounting for 0.01%-10% of the mass of the aqueous phase; the mass ratio of the aqueous phase to the oil phase is 1:0.1-1:10; the reaction monomer in step (3) is styrene and divinylbenzene.

2. The method for preparing the BGCP composite monolithic column according to claim 1, characterized in that, The CNC mentioned in step (1) is a commercially available product with a diameter of 4-10 nm and a length of 100-800 nm.

3. The method for preparing the BGCP composite monolithic column according to claim 1, characterized in that, The acyl halide compound mentioned in step (1) is 2-bromoisobutyryl bromide, 2-bromoisobutyryl chloride, 2-chloroisobutyryl bromide, 2-chloroisobutyryl chloride, bromoacetyl bromide, bromoacetyl chloride, chloroacetyl chloride or chloroacetyl bromide.

4. The method for preparing the BGCP composite monolithic column according to claim 1, characterized in that, The HLB value of Janus CNC-PS used in step (3) ranges from 2 to 16; the amount added is 0.1% to 20% of the oil phase mass.

5. The method for preparing the BGCP composite monolithic column according to claim 1, characterized in that, In step (3), the degree of crosslinking is 10%-90%; the porogen is at least one of isopropanol, toluene, xylene, acetonitrile, and chloroform, and the amount added is 5%-50% of the mass of the reactant monomer.

6. The method for preparing the BGCP composite monolithic column according to claim 1, characterized in that, In step (3), the polymerization reaction temperature is 60-130 ℃ and the reaction time is 5-48 h.

7. A BGCP composite monolithic column prepared by the preparation method according to claim 1, characterized in that... The monolithic column has a cross-linked polystyrene framework, with hydrophilic cellulose nanocrystals forming the outer surface of the framework. The composite monolithic column has a uniform framework, with both the framework and pores being bicontinuous. The macropore diameter is 0.05-10 μm, the micropore diameter is 2-50 nm, the porosity is 50-80%, and the specific surface area is 30-200 m². 2 / g.

Images