Alkali-resistant chromatographic stationary phase material based on covalent organic framework (COFs) as well as preparation and application of alkali-resistant chromatographic stationary phase material

Abstract

The invention relates to a method for constructing a novel chiral chromatographic stationary phase by using a covalent organic framework (COFs) material to carry out one-pot in-situ wrapping on alkali-resistant biological chiral resolution molecules in a base catalysis formation process under a green and mild condition, and the method is used for chiral separation research. The COFs of various bonding types involved in the invention are formed by connecting a series of organic monomers through covalent bonds, and have excellent stability and good crystallinity. The covalent organic framework material is creatively designed and developed as a novel carrier material with wide applicability aiming at the possible problems of poor stability, low loading rate of a chiral resolving agent, easiness in loss of the chiral resolving agent, limited applicability and the like existing in a carrier material of a traditional chiral chromatographic column stationary phase. An alkali-resistant chiral resolving agent (such as protein, polysaccharide, cyclodextrin and the like) is efficiently loaded through a water-phase base catalysis in-situ wrapping means, so that various efficient and durable chiral stationary phases are prepared, and the chiral stationary phases are used as novel high-performance chromatographic column packing for chromatography chiral separation (such as high performance liquid chromatography, capillary chromatography and the like). Various chiral stationary phases prepared by the technology have good separation efficiency, high stability and durability, and are successfully applied to efficient separation of various chiral substances such as chiral amino acids and chiral drugs, and the separation degree is gt; and 1.0. According to the technology, the application range and the service life of chiral chromatographic column separation are greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of chromatographic chiral separation, specifically relating to the preparation of novel composite chiral stationary phases that use covalent organic frameworks as carriers and biomolecules with natural chiral units as chiral resolving agents, suitable for various chromatographic separation methods such as high performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE). Background Technology

[0002] Chirality is ubiquitous in nature. Biomolecules that make up living organisms, including proteins, nucleic acids, and enzymes, as well as many drugs and foods, all possess a single chiral structure. Chiral compounds generally have two corresponding isomers that are almost identical in physical and chemical properties. However, enantiomers with different optical activities exhibit significant differences in biological activity, metabolic processes, and toxicity. Therefore, obtaining a single-configuration isomer with high biological activity is currently a key research focus in chromatographic chiral separation.

[0003] Currently, chromatography is the most commonly used method for enantiomer separation, including gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). For various chromatographic methods, the chromatographic column is the heart of the chromatograph, and the separating material (stationary phase) packed within the column is crucial for achieving separation. Therefore, high-performance chromatographic column packing materials are one of the richest and most creative aspects of chromatographic research. Chiral stationary phases (CSPs) packed within chiral columns are typically prepared by loading substances with chiral recognition and separation capabilities (chiral resolving agents) onto a support through methods such as chemical bonding or physical coating. These mainly include: polysaccharide derivative CSPs, cyclodextrin CSPs, protein CSPs, macrocyclic antibiotic CSPs, ligand exchange CSPs, and crown ether CSPs. The first three types of chiral stationary phases are the most commonly used in various chromatographic separation methods. Appropriate chiral stationary phases can be selected based on the type and properties of the analyte to achieve different separation effects. The performance of the support used to load chiral resolving agents has a significant impact on the separation effect of CSP. An ideal support should have high loading capacity, be resistant to leakage of the loaded chiral resolving agent, have high stability, and high applicability (it can be used to load different types of chiral resolving agents). However, the mainstream commercial CSP supports are currently silicon-based substrates, which have poor stability, especially in alkaline environments, severely limiting the expansion and application of chiral resolving agents.

[0004] As a newly emerging functional porous material in recent years, porous framework materials (such as metal-organic frameworks, MOFs; covalent organic frameworks, COFs; and hydrogen-bonded organic frameworks, HOFs) have seen rapid development in applications such as gas adsorption and separation, sensors, and catalysis due to their advantages including high specific surface area, tunable pore size, diverse structures, ease of modification, and excellent thermal and chemical stability. For example, patent CN 109569026 A (Preparation of a chromatographic stationary phase based on a porous framework material for chiral separation) has successfully immobilized chiral resolving agents for protein or macrocyclic antibiotic biomolecules in porous frameworks, which can then be used for chiral separation in chromatography. However, the porous framework material involved in this patent fails to solve the problem of poor alkali stability, and the chiral recognition molecules used are also not alkali-resistant, thus limiting its application in chiral separation. This application addresses the problems of poor alkali stability, inability of chiral resolving agents to withstand alkali, low loading rate, and easy leakage of traditional chiral stationary phase support materials. It creatively screens and designs covalent organic frameworks as chiral stationary phase supports, utilizing their excellent properties such as high stability, high specific surface area, and porosity. Alkali-resistant chiral resolving agents (such as proteins, polysaccharides, cyclodextrins, etc.) are loaded through in-situ encapsulation under aqueous alkaline catalytic conditions to prepare highly efficient chiral stationary phases (CSPs), thereby overcoming the application limitations of traditional materials in chiral chromatography columns.

[0005] On the other hand, we explored the synthesis of COFs under alkaline conditions in an aqueous phase. By leveraging the inherent properties of alkali-resistant biomolecules, we achieved in-situ encapsulation of biomolecules by COFs. This effectively improved the biomolecules' tolerance to harsh conditions during separation while maintaining their separation activity. Patent 202211322784.0 discloses an aqueous synthesis method for covalent organic framework materials, but the synthesized COFs used acid catalysis, which cannot meet the requirements for immobilizing alkali-resistant biomolecules. Therefore, we further developed an alkaline-catalyzed synthesis method for these COFs. Accordingly, the alkaline-catalyzed COFs achieve in-situ encapsulation of alkali-resistant biomolecules, overcoming size limitations, improving the loading capacity and efficiency of chiral resolving agents in porous framework materials, effectively reducing and preventing leakage of chiral resolving agents, thereby expanding the separation capability of chiral columns and improving their separation effect and reusability. On the other hand, unlike traditional support materials (such as silica and agarose), porous framework materials possess excellent crystallinity, a defined structure, and a well-defined pore environment. This facilitates the study and analysis of the interactions and related mechanisms between these materials and the chiral resolving agents they support, enabling continuous improvement and optimization of the performance of such CSPs. Furthermore, porous framework materials offer flexibility in composition and structure, allowing for the convenient introduction of specific functional groups or structures based on the structure and properties of the chosen chiral resolving agent, thus achieving highly efficient loading of certain chiral resolving agents. Therefore, this method can be used to design and prepare different types of chiral stationary phases (CSPs) as high-performance chiral chromatographic column packing materials to meet diverse separation needs, thus possessing broad application value. Summary of the Invention

[0006] This application addresses the problems existing in traditional chiral stationary phase support materials by creatively utilizing covalent organic framework materials as carriers and loading various alkali-resistant chiral resolving agents (such as proteins, polysaccharides, cyclodextrins, etc.) through in-situ coating and immobilization methods to prepare novel, efficient, and durable chiral chromatographic stationary phases as high-performance chiral chromatographic column packing materials.

[0007] On the one hand, the present invention provides a covalent organic framework material that can be synthesized under aqueous alkaline catalytic conditions, characterized in that it is obtained by a Schiff base condensation reaction of a monomer containing an aldehyde functional group and a monomer containing an amino or hydrazide functional group, and the synthesis conditions are mild and environmentally friendly.

[0008] Specifically, this invention provides a method for synthesizing covalent organic frameworks (COFs), characterized in that the covalent organic framework is a covalent organic framework linked by vinyl groups, imide groups, borate ester groups, boron-oxygen six-membered ring groups, imine bonds, azazine groups, keto-enol groups, hydrazone bonds, β-keto-amine bonds, or triazine bonds, and is synthesized through the following steps: Organic monomer 1 and organic monomer 2 are dissolved or dispersed in the reaction solvent, mixed and then subjected to a condensation reaction under catalytic conditions. The final product is obtained after separation and purification.

[0009] Preferably, the structure of the organic monomer is as follows: Figure 1 As shown, the monomer type is di-linked, tri-linked, or tetra-linked, and X, Y, and M in the figure are any one of aldehyde, amino, and hydrazide groups.

[0010] Preferably, organic monomer 1 is a monomer containing an aldehyde functional group. Further preferred monomers containing aldehyde functional groups include terephthalaldehyde, 4,4'-biphenyldiphenylaldehyde, 1,4-bis(4-aldehydephenyl)benzene, 4,4''-dialdehyde tetraphenyl, 4,4''-dialdehyde pentaphenyl, 4,4'-((2,2'-bipyridine)-5,5'-diphenyl)diphenylaldehyde, 5',5'''-(1,4-phenyl)bis(2,2'-bipyridine-5-aldehyde), 1,10-phenanthroline-3,8-dialdehyde, 4,4'-(1,10-phenanthroline)-3,8-diphenylaldehyde, 1,3,5-triphenylaldehyde (TFp), 2-hydroxy-1,3,5-benzenetrialdehyde, 2,6-dihydroxy-1,3,5-benzenetrialdehyde, trialdehyde-phloroglucinol (Tp), and 1,3,5-tris(p-formylbenzene). 2,4,6-Tris(4-aldehydephenyl)-1,3,5-triazine (TFPT), 4,4',4''-[benzyl-1,3,5-triyltris(ethynyl-2,1-diyl)]tribenzaldehyde (TFEPB), 4,4',4''-(benzyl-1,3,5-tris(ethynyl-2,1-diyl))-tris(salicylaldehyde) (TFEPB-OH) 4',4''',4'''''-(1,3,5-triazine-2,4,6-triyl)tris(([1,1'-biphenyl]-4-carboxaldehyde)) (TFBPB), 2,4,6-tris-(4-formyl-biphenyl-4-yl)-1,3,5-triazine (TFBPT), tetra(4-formylphenyl)methane, 4,4',4'' The monomer is selected from 4”'-silanetetraethyltetrabenzaldehyde, tetra-(4-formylphenyl)ethylene, 1,3,5,7-tetraformyl-adamantane, and 1,3,5,7-tetra(4-benzaldehyde)-adamantane; the monomer containing the aldehyde functional group may be optionally substituted with a substituent selected from one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I. More preferably, the organic monomer 1 is 1,3,5-triformaldehyde (TFp).

[0011] Preferably, the organic monomer 2 is a monomer containing an amino or hydrazide functional group. More preferably, the monomer containing the amino functional group is hydrazine hydrate, p-phenylenediamine, benzidine, 5,5'-diamino-2,2'-bipyridine, or 4,4'-diaminoterphenyl, 4,4''-diaminotetraphenyl, 4,4''-diaminopentaphenyl, 4,4'-((2,2'-bipyridine)-5,5'-diphenyl)diphenylamine, 5',5'''-(1,4-phenyl)bis(2,2'-bipyridine-5-amine), 1,10-phenanthroline-3,8-diamine, 4,4'-(1,10-phenanthroline)-3,8-diphenylamine, 1,3,5- Triphenylamine, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT), 4,4',4''-[benzyl-1,3,5-triyltris(ethynyl-2,1-diyl)]triphenylamine (TAEPB), 4',4''',4'''''-(1,3,5-triazine-2,4,6-triyl)tri(([1,1'-biphenyl]-4-amine)) (TABPB), 2,4,6-tris-(4-aminobiphenyl-4-yl)-1,3,5-triazine (TABPT), tetra(4-aminophenyl)methane, 4,4',4'' 4”'-Silanetetraethyltetraphenylamine, tetra-(4-aminophenyl)ethylene, 1,3,5,7-tetraaminoadamantane, 1,3,5,7-tetra(4-anilino)adamantane; the monomer containing the amino functional group may optionally be substituted with a substituent selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, -I; the monomer containing the hydrazide functional group is optionally R-substituted terephthalohydrazide; where R is... , , , or When multiple substitutions are used, each R is independently selected from the structures described above. More preferably, the organic monomer 2 is hydrazine hydrate or 2,5-bis(2-methoxyethoxy)terephthalohydrazide.

[0012] Preferably, the catalyst includes, but is not limited to, catalysts containing OH. - CH3COO - NH 3+ CO3 2- S 2- SO3 2- PO4 3- ClO - CN -The catalyst is a basic inorganic compound, and a basic organic compound containing primary, secondary, tertiary, or quaternary amino groups. More preferably, the catalyst is any one or more of sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, sodium acetate, potassium carbonate, potassium bicarbonate, potassium acetate, sodium sulfide, potassium sulfide, sodium sulfite, potassium sulfite, sodium phosphate, potassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hypochlorite, potassium hypochlorite, sodium cyanide, potassium cyanide, aniline, methylamine, dimethylamine, ethylamine, diethylamine, triethylamine, diisopropylamine, pyridine, and pyrimidine. The catalyst may optionally be substituted with substituents selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I.

[0013] Preferably, the reaction solvent is water or a mixture of water and a small amount of miscible organic solvent. More preferably, the reaction solvent is water, or a mixed solvent of organic solvent and water in a volume ratio of 0.01-1:1. Even more preferably, the volume ratio of organic solvent to water is 0.05-0.3:1.

[0014] Preferably, the organic solvent has a certain solubility in water. More preferably, the organic solvent includes, but is not limited to, one or more of tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, 1,4-dioxane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.

[0015] Preferably, the reaction temperature is 4℃-100℃, more preferably 25℃-80℃, and even more preferably 37℃-60℃.

[0016] Preferably, the molar ratio of aldehyde functional group monomer to amino or hydrazide functional group monomer in the reaction system is 1:(1-4), and more preferably 1:1.5.

[0017] Preferably, the initial concentration of each monomer in the reaction system is 0.001-0.05 mol / L, more preferably 0.002-0.01 mol / L.

[0018] Preferably, the concentration of the alkaline catalyst in the reaction system is 0.001-10 mol / L, more preferably 0.01-1 mol / L.

[0019] Preferably, the synthesis temperature of the covalent organic framework material is 0℃-100℃, more preferably 37℃-60℃.

[0020] Preferably, the synthesis reaction time of the covalent organic framework material is 1 min-72 h, more preferably 60 min-6 h.

[0021] Preferably, the covalent organic framework (COFs) comprises hydrazone bonds, imine bonds, azazine bonds, and β-ketoamines, and has good thermal and solvent stability.

[0022] Preferably, the covalent organic framework materials include, but are not limited to, NKCOF-98, NKCOF-99, NKCOF-98-butene, NKCOF-98-butyne, NKCOF-98-dimethylethylamine, NKCOF-98-3, NKCOF-98-5, and NKCOF-98-7, the specific structures of which are described in [reference needed]. Figure 2 .

[0023] More specifically, the present invention provides a method for synthesizing covalent organic framework materials, characterized in that the materials are obtained from monomers via Schiff base condensation under aqueous-phase alkaline catalysis, comprising the following steps: 1) The aldehyde functional group monomer and the amino or hydrazide functional group monomer are dissolved or dispersed in a reaction solvent, and an inorganic base or organic base is used as a catalyst to carry out the reaction. 2) After the reaction is complete, centrifuge to remove unreacted monomers.

[0024] Preferably, in step 2), unreacted monomers are removed by ethanol exchange and tetrahydrofuran extraction.

[0025] Preferably, after step 2), the purified product is further processed under vacuum conditions to obtain the final product.

[0026] On the other hand, this invention provides a method for in-situ encapsulating alkali-resistant chiral resolving agents of biomolecules with COFs under aqueous alkaline catalysis to form a chiral stationary phase. Compared with the existing methods for synthesizing chiral stationary phases, such as covalent and coating methods, its advantages are: 1) The in-situ synthesized composite material has a high loading capacity of biomolecules; 2) The in-situ encapsulation method can encapsulate biomolecules of different sizes because it does not rely on the pores of the COFs themselves, and is therefore not limited by the COFs carrier structure and its pores (such as hydrophilicity / hydrophobicity, size); 3) It is firmly fixed, almost leak-free, and has strong protective force; 4) It has a wide range of applicable pH and solvents (buffer solutions, ethanol, methanol, isopropanol, acetone, dichloromethane, n-hexane, etc.) and is applicable to various chromatographic separation modes; 5) It is simple, fast, and easy to implement. Its key feature is that, during the COFs formation process, a one-pot method can be used to in-situ encapsulate biomolecules in the system to prepare biomolecule@COFs composite chiral stationary phase materials, under green and mild conditions, and with a simple synthesis method.

[0027] Specifically, it is a method for in-situ synthesis of a bio-composite chiral stationary phase material based on a covalent organic framework material, characterized in that the bio-composite chiral stationary phase material based on the covalent organic framework material includes a covalent organic framework material and biomolecules, wherein the covalent organic framework material in-situ encapsulates the biomolecules.

[0028] Preferably, the covalent organic framework material is a covalent organic framework material linked by vinyl groups, imide groups, borate ester groups, boron-oxygen six-membered ring groups, imine bonds, azazine groups, keto-enol groups, hydrazone bonds, β-keto-amine bonds, or triazine bonds; the biomolecule is selected from proteins, polysaccharides, and cyclodextrins; specifically, it is synthesized through the following steps: Organic monomer 1 and organic monomer 2 are dissolved or dispersed in the reaction solvent, mixed, and then biomolecules are added. A condensation reaction is carried out under alkaline catalyst conditions, and the final product is obtained after separation and purification.

[0029] Preferably, organic monomer 1 is a monomer containing an aldehyde functional group. More preferably, the monomer containing an aldehyde functional group is (a) 1,3,5-tricarboxaldehyde (TFp), (b) 2-hydroxy-1,3,5-benzenetricarboxaldehyde, (c) 2,6-dihydroxy-1,3,5-benzenetricarboxaldehyde, (d) trialdehyde-resorcinol (Tp), (e) 1,3,5-tris(p-formylphenyl)benzene (TFPB), (f) 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine (TFPT), (g) 4,4',4''-[benzene-1,3,5-triyltris(acetylene-2, The monomer is 1,3,5-triphenylformaldehyde (TFEPB) or (h)4,4',4''-(benzene-1,3,5-tris(acetylene-2,1-diyl))-tris(salicylaldehyde) (TFEPB-OH); the monomer containing the aldehyde functional group may be optionally substituted with a substituent selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I. More preferably, the organic monomer 1 is 1,3,5-triformaldehyde (TFp).

[0030] Preferably, the organic monomer 2 is a monomer containing an amino or hydrazide functional group. More preferably, the monomer containing an amino functional group is any one of (a) hydrazine hydrate, (b) p-phenylenediamine, (c) benzidine, (d) 5,5'-diamino-2,2'-bipyridine, or (e) substituted 4,4'-diaminoterphenyl; the monomer containing an amino functional group may optionally be substituted with a substituent selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I; the monomer containing a hydrazide functional group is optionally R-substituted terephthalohydrazide; where R is... , , , , or When multiple substitutions are used, each R is independently selected from the structures described above. More preferably, the organic monomer 2 is hydrazine hydrate or 2,5-bis(2-methoxyethoxy)terephthalohydrazide.

[0031] Preferably, the biomolecule is selected from proteins, polysaccharides, and cyclodextrins. More preferably, the biomolecule is selected from one or more of lipases, alkaline proteases, human serum albumin (HSA), α-acid glycoprotein (AGP), ovomucoid (OVM), cellulase, glucosidase, amylose, amylopectin, cellulose, chitosan, dextran, and modified or unmodified α, β, and γ-cyclodextrins.

[0032] Preferably, the catalyst includes, but is not limited to, catalysts containing OH. - CH3COO - NH 3+ CO3 2- S 2- SO3 2- PO4 3- ClO - CN -The catalyst is a basic inorganic compound, and a basic organic compound containing primary, secondary, tertiary, or quaternary amino groups. More preferably, the catalyst is any one or more of sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, sodium acetate, potassium carbonate, potassium bicarbonate, potassium acetate, sodium sulfide, potassium sulfide, sodium sulfite, potassium sulfite, sodium phosphate, potassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hypochlorite, potassium hypochlorite, sodium cyanide, potassium cyanide, aniline, methylamine, dimethylamine, ethylamine, diethylamine, triethylamine, diisopropylamine, pyridine, and pyrimidine. The catalyst may optionally be substituted with substituents selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I.

[0033] Preferably, the reaction solvent is water or a mixture of water and a small amount of miscible organic solvent. More preferably, the reaction solvent is water, or a mixed solvent of organic solvent and water in a volume ratio of 0.01-1:1. Even more preferably, the volume ratio of organic solvent to water is 0.05-0.3:1.

[0034] Preferably, the organic solvent has a certain solubility in water. More preferably, the organic solvent includes, but is not limited to, one or more of tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, 1,4-dioxane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.

[0035] Preferably, the reaction temperature is 4℃-100℃, more preferably 25℃-80℃, and even more preferably 37℃-60℃.

[0036] Preferably, the molar ratio of aldehyde functional group monomer to amino or hydrazide functional group monomer in the reaction system is 1:(1-4), and more preferably 1:1.5.

[0037] Preferably, the initial concentration of each monomer in the reaction system is 0.001-0.05 mol / L, more preferably 0.002-0.01 mol / L.

[0038] Preferably, the concentration of the alkaline catalyst in the reaction system is 0.001-10 mol / L, more preferably 0.01-1 mol / L.

[0039] Preferably, the covalent organic framework (COFs) comprises hydrazone bonds, imine bonds, azazine bonds, and β-ketoamines, and has good thermal and solvent stability.

[0040] Preferably, the covalent organic framework materials include, but are not limited to, NKCOF-98, NKCOF-99, NKCOF-98-butene, NKCOF-98-butyne, NKCOF-98-dimethylethylamine, NKCOF-98-3, NKCOF-98-5, and NKCOF-98-7, the specific structures of which are described in [reference needed]. Figure 2 .

[0041] Preferably, the biomolecule @COFs composite chiral stationary phase material includes, but is not limited to, alkaline protease @NKCOF-98, alkaline protease @NKCOF-99, alkaline protease @NKCOF-98-butene, alkaline protease @NKCOF-98-butyne, alkaline protease @NKCOF-98-dimethylethylamine, alkaline protease @NKCOF-98-3, alkaline protease @NKCOF-98-5, alkaline protease @NKCOF-98-7, amylose @NKCOF-98, cellulose @NKCOF-98, chitosan @NKCOF-98, and β-cyclodextrin @NKCOF-98.

[0042] Preferably, the concentration of the biomolecule solution is 0.01-100 g / L, more preferably 5-20 g / L.

[0043] Preferably, the synthesis temperature of the biomolecule@COFs composite chiral stationary phase material is 0℃-100℃, more preferably 37℃-60℃.

[0044] Preferably, the synthesis reaction time of the biomolecule@COFs composite chiral stationary phase material is 1 min-72 h, more preferably 60 min-6 h.

[0045] The biomolecules @COFs are synthesized through in-situ coating, and have a higher loading capacity and lower leakage rate compared with covalent and coated chiral stationary phase materials.

[0046] The novel method for preparing composite chiral stationary phases provided by this invention broadens the scope of methods for immobilizing chiral resolving agents of biomolecules and their industrial applications.

[0047] On the other hand, the present invention provides an application of the aforementioned composite chiral stationary phase material, characterized in that the biomolecule@COFs composite chiral stationary phase material obtained by in-situ encapsulation and immobilization of alkali-resistant biomolecules using covalent organic framework materials under aqueous alkaline catalysis conditions maintains the chiral recognition ability of biomolecules and can be used as a novel, highly efficient, and durable chiral chromatographic stationary phase as a high-performance chiral chromatographic column packing material, thereby significantly improving the stability and applicability of chiral resolving agents, reducing and avoiding leakage of chiral resolving agents, and thus improving the durability of chiral chromatographic columns.

[0048] Preferably, due to the low density and uniform particle size of covalent organic framework materials, the chiral columns obtained by filling them have the characteristics of good permeability, uniform and stable column pressure, good column efficiency repeatability, and high batch-to-batch stability.

[0049] Preferably, the high-performance chiral chromatographic column includes, but is not limited to, monolithic or packed columns for high-performance liquid chromatography, gas chromatography, and capillary chromatography prepared by in-situ growth or direct packing of composite chiral stationary phase materials.

[0050] Further preferred, the novel chiral stationary phase can be applied to the separation of amino acids, drugs, and small organic molecules under reversed-phase chromatography, polar organic phase chromatography, and normal-phase chromatography conditions.

[0051] Preferably, protein, polysaccharide, and cyclodextrin molecules, in addition to their hydrophobic sites, typically possess hydrophilic groups and ionizable groups, thus being amphiphilic molecules. Therefore, biomolecular CSPs are suitable for both aqueous phase (i.e., reversed-phase chromatography) and organic phase (i.e., normal-phase chromatography), and are applicable to both acidic and alkaline separation environments. Furthermore, they all exhibit high selectivity for enantiomers, demonstrating broad chiral recognition capabilities. Taking alkaline protease CSP, amylose CSP, and β-cyclodextrin CSP as examples, they all exhibit high selectivity factors α and resolution Rs for various enantiomers, indicating that they can completely separate various chiral substances.

[0052] More preferably, the chiral separation capability based on alkaline protease CSP includes, but is not limited to, the chiral separation of DL-phenylalanine, DL-leucine, RS-flurbiprofen, RS-sulindac, RS-zatopibuprofen, and RS-tenofovir.

[0053] More preferably, the chiral separation capability based on amylose CSP includes, but is not limited to, the chiral separation of RS-promethazine, RS-atenolol, RS-propranolol, and RS-chlorpheniramine.

[0054] More preferably, the chiral separation capability based on β-cyclodextrin CSP includes, but is not limited to, the chiral separation of RS-sulprofen, RS-flurbiprofen, RS-sulinic acid, RS-zatoibuprofen, and RS-ibuprofen.

[0055] More preferably, the chiral separation based on the novel CSP has a resolution Rs≥1.0, and more preferably, the resolution Rs≥1.5.

[0056] The chiral stationary phase prepared by this invention, due to the amphiphilic and biionic nature of its chiral resolving agent and the designability of the covalent organic framework material, is widely applicable to various chromatographic conditions. Furthermore, the chiral resolving agent is firmly fixed, maintaining its separation effect even with repeated mobile phase changes. In addition, commercially available chiral column solid matrices are generally silicon-based, and because silicon-based substrates are sensitive to strong acids and bases, their pH range is limited to 4-8. The chiral stationary phase prepared by the method of this application, due to the stability of the covalent organic framework material, is applicable to conditions in the pH range of 1-13. Attached Figure Description

[0057] Figure 1 : The monomer structure used to synthesize COFs.

[0058] Figure 2 Schematic diagrams of several COF structures.

[0059] Figure 3 Various characterizations of NKCOF-98 and alkaline protease@NKCOF-98.

[0060] Figure 4 PXRD results for alkaline protease @NKCOF-98-butene, alkaline protease @NKCOF-98-butyne, alkaline protease @NKCOF-98-dimethylethylamine, alkaline protease @NKCOF-98-3, alkaline protease @NKCOF-98-5, and alkaline protease @NKCOF-98-7.

[0061] Figure 5 FT-IR results for alkaline protease @NKCOF-98-butene, alkaline protease @NKCOF-98-butyne, alkaline protease @NKCOF-98-dimethylethylamine, alkaline protease @NKCOF-98-3, alkaline protease @NKCOF-98-5, and alkaline protease @NKCOF-98-7.

[0062] Figure 6 The loading results of NKCOF-98 on the chiral resolving agents alkaline protease, amylose, and β-cyclodextrin.

[0063] Figure 7 Stability of free alkaline protease and alkaline protease@NKCOF-98 in citrate buffer, 100°C, organic solvents DMF, SDS, and urea.

[0064] Figure 8 : Cyclic catalytic performance of alkaline protease@NKCOF-98.

[0065] Figure 9 Separation diagrams of chiral columns prepared using alkaline protease@NKCOF-98 as the chiral stationary phase for DL-phenylalanine, DL-leucine, RS-flurbiprofen, RS-sulindac, RS-zatopibuprofen, and RS-tenofovir.

[0066] Figure 10 Separation diagrams of chiral columns prepared using commercially available cyclodextrin as the chiral stationary phase for DL-phenylalanine, DL-leucine, RS-flurbiprofen, RS-sulindac, RS-zatopibuprofen, and RS-tenofovir.

[0067] Figure 11 Separation diagrams of RS-promethazine, RS-atenolol, RS-propranolol, and RS-chlorpheniramine using a chiral column prepared with amylose@NKCOF-98 as the chiral stationary phase.

[0068] Figure 12 Separation diagrams of RS-sulprofen, RS-flurbiprofen, RS-sulinic acid, RS-zatobuprofen, and RS-ibuprofen prepared using a chiral column with β-cyclodextrin@NKCOF-98 as the chiral stationary phase.

[0069] Figure 13 Stability of a chiral column prepared with alkaline protease@NKCOF-98 as the chiral stationary phase for repeated injection of DL-phenylalanine.

[0070] Figure 14 Flowchart for the preparation of alkali-resistant chromatographic stationary phase materials based on covalent organic frameworks (COFs). Detailed Implementation

[0071] Unless otherwise stated in the context of this application, the technical terms and abbreviations used herein have their conventional meanings as known to those skilled in the art; unless otherwise stated, the raw material compounds used in the following examples were all commercially available. The alkaline protease was derived from Bacillus subtilis and branded as XINSE; the monomers used to synthesize COFs, such as trimesaldehyde, and the chiral recognition molecules amylose and β-cyclodextrin, were purchased from Shanghai BIDE Pharmaceutical Technology Co., Ltd.; the organic reagents, such as acetonitrile and hydrazine hydrate, were purchased from Tianjin Bohua Chemical Reagent Co., Ltd.

[0072] The synthesis of covalent organic framework materials, characterization and testing of various properties, loading methods for chiral resolving agents, and chiral separation performance as described in this invention are specifically implemented as follows. The following examples are for further explanation and illustration of this invention only and should not be considered as limiting the scope of the invention, which is to be limited only by the claims.

[0073] Examples 1-7 illustrate the loading of covalent organic framework materials onto chirally separated biomolecules.

[0074] Example 1:

[0075] The synthesis of NKCOF-98 was carried out as follows: 3.3 mg of trimesin and 10.3 mg of 2,5-bis(2-methoxyethoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. The reaction was carried out at 60 °C for 2 hours. After the reaction was completed, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 10.2 mg of NKCOF-98 as a white solid powder, with a yield of 75%. Figure 3 As shown in (a, b, c), the results demonstrate the successful synthesis of NKCOF-98, and X-ray powder diffraction (PXRD), infrared (FT-IR), and gas adsorption characterization tests were performed.

[0076] The basic protease @NKCOF-98 was synthesized as follows: 3.3 mg of trimesin and 10.3 mg of 2,5-bis(2-methoxyethoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. The basic protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of the enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was completed, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 15.2 mg of basic protease @NKCOF-98 as a white solid powder, with a yield of 72%.

[0077] Example 2:

[0078] Characterization of alkaline protease @NKCOF-98: NKCOF-98 and alkaline protease @NKCOF-98 were characterized by X-ray powder diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR), gas adsorption, and confocal laser imaging. The results of these multiple characterizations corroborate each other, demonstrating the successful synthesis of NKCOF-98 and the successful construction of alkaline protease @NKCOF98. The characterization results are shown below. Figure 3 a) PXRD results for both NKCOF-98 and alkaline protease@NKCOF-98 show characteristic peaks at 3.1° and 6.5°, consistent with the simulated peak positions, indicating good crystallinity and successful material synthesis; b) and c) Compared to NKCOF-98, the specific surface area of ​​alkaline protease@NKCOF-98 is significantly reduced, but the pore size distribution remains almost unchanged, indicating that NKCOF-98 successfully encapsulates and loads alkaline protease using defect sites; d) In the infrared data, the aldehyde groups of monomers TB and BMTH in alkaline protease@NKCOF-98 (~3320 cm⁻¹) show a characteristic peak at 3.1° and 6.5°, consistent with the simulated peak positions, indicating good crystallinity and successful material synthesis; -1) and acylhydrazine amino (~1680 cm -1 The characteristic peak disappears, and a new imine bond absorption characteristic peak (~1640 cm⁻¹) appears simultaneously. -1 In addition, alkaline protease @NKCOF-98 also showed a distinct characteristic peak of alkaline protease (~1530 cm⁻¹). -1 This indicates that NKCOF-98 was successfully synthesized and loaded with alkaline protease. Furthermore, observation under a laser confocal microscope showed that the fluorescently labeled FITC-alkaline protease@NKCOF-98 exhibited a significant fluorescent signal, proving that the encapsulation was successfully achieved.

[0079] Example 3:

[0080] Synthesis and characterization of alkaline proteases @NKCOF-98-butene, @NKCOF-98-butyne, @NKCOF-98-dimethylethylamine, @NKCOF-98-3, @NKCOF-98-5, and @NKCOF-98-7.

[0081] Synthesis of alkaline protease @NKCOF-98-butene: 3.3 mg of trimesin and 10.0 mg of 2,5-bis(3-buten-1-oxo)terephthalohydrazide were dispersed in a 9 mL aqueous solution (50 mM disodium hydrogen phosphate, pH=10.5) and a 1 mL tetrahydrofuran mixture. The alkaline protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of this enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was complete, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 14.6 mg of alkaline protease @NKCOF-98-butene as a white solid powder, with a yield of 69%.

[0082] Synthesis of alkaline protease @NKCOF-98-butyne: 3.3 mg of trimesin and 10.0 mg of 2,5-bis(3-butyn-1-oxo)terephthalohydrazide were dispersed in a 9 mL aqueous solution (50 mM disodium hydrogen phosphate, pH=10.5) and a 1 mL tetrahydrofuran mixture. The alkaline protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of this enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was complete, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 18.2 mg of alkaline protease @NKCOF-98-butyne as a white solid powder, with a yield of 75%.

[0083] Synthesis of alkaline protease @NKCOF-98-dimethylethylamine: 3.3 mg of trimesin and 10.0 mg of 2,5-bis(2-(dimethylamine)ethoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. The alkaline protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of this enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was complete, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 13.1 mg of alkaline protease @NKCOF-98-dimethylethylamine as a yellow solid powder, with a yield of 65%.

[0084] The alkaline protease @NKCOF-98-3 was synthesized using the following steps: 3.3 mg of trimesin and 9.2 mg of 2,5-bis(2-methoxymethoxy)terephthalohydrazide were dispersed in a 10 mL aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. The alkaline protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of this enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was complete, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 14.2 mg of alkaline protease @NKCOF-98-3 as a yellow solid powder, with a yield of 63%.

[0085] The basic protease @NKCOF-98-5 was synthesized using the following steps: 3.3 mg of trimesin and 10.8 mg of 2,5-bis(2-methoxypropoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. The basic protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of this enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was complete, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 17.2 mg of basic protease @NKCOF-98-5 as a white solid powder, with a yield of 71%.

[0086] The basic protease @NKCOF-98-7 was synthesized using the following steps: 3.3 mg of trimesin and 12.6 mg of 2,5-bis(2-(2-methoxyethoxy)ethoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. The basic protease was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of this enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was complete, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 19.2 mg of basic protease @NKCOF-98-5 as a white solid powder, with a yield of 82%.

[0087] Example 4:

[0088] The alkaline proteases @NKCOF-98-butene, @NKCOF-98-butyne, @NKCOF-98-dimethylethylamine, @NKCOF-98-3, @NKCOF-98-5, and @NKCOF-98-7 were characterized by X-ray powder diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR). Multiple characterization results corroborated each other, jointly demonstrating the successful synthesis of alkaline proteases @COFs. Characterization results are shown below. Figure 4-5 PXRD data showed that alkaline protease @NKCOF-98-butene, alkaline protease @NKCOF-98-butyne, alkaline protease @NKCOF-98-dimethylethylamine, alkaline protease @NKCOF-98-3, alkaline protease @NKCOF-98-5, and alkaline protease @NKCOF-98-7 all exhibited characteristic peaks at 3.1° and 6.5°, consistent with the simulated peak positions, indicating good crystallinity. The infrared spectra of each material showed peaks at ~1640 cm⁻¹. -1 A characteristic absorption peak for imine bonds appears at ~1530 cm⁻¹, and also at ~1530 cm⁻¹. -1 The presence of characteristic absorption peaks belonging to alkaline proteases further confirms the successful synthesis of enzyme@COFs materials.

[0089] Example 5:

[0090] The synthesis of amylose@NKCOF-98 was carried out as follows: 3.3 mg of trimesin and 10.3 mg of 2,5-bis(2-methoxyethoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. Amylose was dissolved in distilled water to prepare a 10 mg / mL enzyme solution. 1 mL of the enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was completed, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 15.1 mg of amylose@NKCOF-98 as a white solid powder, with a yield of 71%.

[0091] The synthesis of β-cyclodextrin @NKCOF-98 was carried out as follows: 3.3 mg of trimesin and 10.3 mg of 2,5-bis(2-methoxyethoxy)terephthalohydrazide were dispersed in 10 mL of an aqueous solution containing 50 mM disodium hydrogen phosphate at pH 10.5. β-cyclodextrin was dissolved in distilled water to prepare an enzyme solution of 10 mg / mL. 1 mL of the enzyme solution was added to the above system and mixed thoroughly. The reaction was carried out at 60 °C for 2 hours. After the reaction was completed, the solid was separated by centrifugation, washed three times with distilled water, and then dried in a vacuum freeze dryer to obtain 15.6 mg of β-cyclodextrin @NKCOF-98 as a white solid powder, with a yield of 76%.

[0092] After synthesis, the UV absorbance of the supernatant was measured, and the amount of immobilized biomolecules was calculated by comparing it with the solution before synthesis. The results are as follows: Figure 6 The results showed that, compared to alkaline protease, NKCOF-98 had essentially the same loading capacity for amylose and β-cyclodextrin.

[0093] Example 6:

[0094] The stability of alkaline protease and alkaline protease@NKCOF-98 in acidic (citric acid) solution, high temperature (100℃), surfactant (SDS, sodium dodecyl sulfate), organic solvent DMF, and denaturant urea was studied. The specific implementation steps are as follows: Alkaline protease activity assay: The enzymatic activity of free and immobilized alkaline protease was assessed spectrophotometrically by recording the increase in absorbance at 405 nm after enzymatic hydrolysis of Boc-L-alanine 4-nitrophenyl ester (NPA). The reaction mixture consisted of 1.98 mL of 50 mM sodium phosphate buffer (containing 20% ​​ethanol) at pH 7, with 20 μL of 100 mM NPA dissolved in acetonitrile added. The mixture was preheated at 25 °C, and then 20 μg of alkaline protease (or alkaline protease@COF containing 20 μg of the enzyme) was added. After incubation for 5 minutes, the sample was centrifuged, and the clear supernatant was used for testing.

[0095] The stability test in citric acid solution was conducted as follows: 1 mg of free alkaline protease or alkaline protease@NKCOF-98 containing 1 mg of alkaline protease was immersed in 3 mL of citric acid buffer (50 mM, pH=4) for one hour. The activity of the free alkaline protease or the composite material was then tested. The results showed that after treatment, the activity of the free alkaline protease was almost completely lost, while the alkaline protease@NKCOF-98 retained approximately 90% of its activity. Figure 7 As shown.

[0096] The stability test in sodium dodecyl sulfate was conducted as follows: 1 mg of free alkaline protease or alkaline protease@NKCOF-98 containing 1 mg of alkaline protease was immersed in a 20 mg / mL sodium dodecyl sulfate solution for 1 hour. The activity of the free alkaline protease or the composite material was then tested. The results showed that after treatment, the activity of the free alkaline protease was almost completely lost, while the alkaline protease@NKCOF-98 retained approximately 90% of its activity. Figure 7 As shown.

[0097] The high-temperature stability test was conducted as follows: 1 mg of free alkaline protease or alkaline protease@NKCOF-98 containing 1 mg of alkaline protease was treated at 100°C for 20 minutes, after which the activity of the free alkaline protease or the composite material was detected. The results showed that after treatment, the activity of the free alkaline protease was almost completely lost, while the alkaline protease@NKCOF-98 retained approximately 90% of its activity. Figure 7 As shown.

[0098] The organic solvent stability test was conducted as follows: 1 mg of free alkaline protease or alkaline protease@NKCOF-98 containing 1 mg of alkaline protease was immersed in 5 mL of DMF organic solvent for one hour. The activity of the free alkaline protease or the composite material was then tested. The results showed that after treatment, the activity of the free alkaline protease was almost completely lost, while the alkaline protease@NKCOF-98 retained approximately 90% of its activity. Figure 7 As shown.

[0099] The stability test of the denaturing agent urea was conducted as follows: 1 mg of free alkaline protease or alkaline protease@NKCOF-98 containing 1 mg of alkaline protease was immersed in 3 mL of urea solution (8 M) for one hour, after which the activity of the free alkaline protease or the composite material was tested. The results showed that after treatment, the activity of the free alkaline protease was almost completely lost, while the alkaline protease@NKCOF-98 retained approximately 90% of its activity. Figure 7As shown.

[0100] Example 7:

[0101] The cyclic catalytic performance of alkaline protease @NKCOF-98 was investigated. The specific steps were as follows: 19.8 mL of phosphate buffer (50 mM, pH 7.0, containing 20% ​​ethanol) was added to a 50 mL centrifuge tube. Then, alkaline protease @NKCOF-98 containing 200 μg of alkaline protease was added, followed by 200 μL of 100 mM Boc-L-alanine 4-nitrophenyl acetonitrile solution. The mixture was reacted at room temperature for 5 min in a shaker, centrifuged, and the supernatant was collected. The absorbance was measured at 405 nm. The material was recovered, washed with distilled water, and the catalytic reaction was repeated. The results showed that after 10 cycles of catalytic catalysis, alkaline protease @NKCOF-98 retained approximately 80% of its initial catalytic activity, while the free alkaline protease completely dissolved in the catalytic system and could not achieve cyclic catalysis. Figure 8 The cyclic catalytic performance of alkaline protease@NKCOF-98.

[0102] Examples 8-14 are tests of the separation capability of the novel chiral stationary phase.

[0103] Example 8:

[0104] Preparation of the HPLC chiral stationary phase using biomolecule @NKCOF-98: 1 g of alkaline protease @NKCOF-98, amylose @NKCOF-98, or β-cyclodextrin @NKCOF-98 was prepared according to the method in Example 1 and air-dried. The prepared materials were ground and added to 50 mL of water, ultrasonically dispersed for 10 min, and the resulting homogeneous chiral stationary phase was packed into a homogenizing tube. Using water as the displacement solvent, the packing was pressurized into a ferrule-type empty chromatographic column using a pneumatic pump. The column specifications were 100 × 4.6 mm (I.D.). The packing pressure was 5000 psi.

[0105] Example 9:

[0106] Mobile phase and sample preparation: Acidic, basic, and neutral samples were selected for testing. Samples were first prepared with water or methanol to a concentration of 1 mg / mL, then diluted to 20 μg / mL with the mobile phase before testing. The injection volume was 20 μL. Reversed-phase mobile phase: A pH 10, 50 mM phosphate buffer solution was prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate, with 10% methanol added as an organic adjuster. The solution was filtered before use. Normal-phase mobile phase: Hexane and acetonitrile were sonicated for 30 min before use. The flow rate was 0.5 mL / min.

[0107] Example 10:

[0108] The separation effect of alkaline protease@NKCOF-98 stationary phase was investigated using a reversed-phase mobile phase. The results are as follows: Figure 9 Table 1: Table 1: Separation results of chiral samples under reverse-phase conditions: Substrate Detection wavelength (nm) <![CDATA[Retention time RT1 (min)]]> <![CDATA[Retention time RT2 (min)]]> α <![CDATA[R s ]]> DL-phenylalanine 260 3.12 5.20 2.75 1.57 DL-Leucine 220 3.20 5.11 2.75 1.50 RS-Flurbiprofen 257 3.12 5.11 2.75 1.51 RS-Sullinacid 260 3.12 5.28 2.80 1.53 RS-Zatopirofen 260 3.12 4.94 2.67 1.55 RS-Tylenolfovir 260 4.30 15.32 5.79 1.50 Conclusion: As can be seen from the separation results in Table 1 above, this chiral stationary column has a better separation effect on the reversed phase of the samples in Table 1, and its overall performance is stronger than that of commercial chiral columns.

[0109] Example 11:

[0110] A commercially available cyclodextrin stationary phase was used to separate the above chiral samples as a control. A reversed-phase mobile phase (water + acetonitrile) was used for separation, and the results are as follows: Figure 10 Table 2: Table 2: Separation results of chiral samples using commercial cyclodextrin columns: Substrate Detection wavelength (nm) <![CDATA[Retention time RT1 (min)]]> <![CDATA[Retention time RT2 (min)]]> α <![CDATA[R s ]]> DL-phenylalanine 260 / / / 0 DL-Leucine 220 / / / 0 RS-Flurbiprofen 257 41.32 71.06 1.78 3.53 RS-Sullinacid 260 15.41 25.48 1.81 2.52 RS-Zatopirofen 260 5.58 6.92 1.52 1.81 RS-Tylenolfovir 260 / / / 0 Conclusion: As can be seen from the separation results in Table 2 above, the commercial cyclodextrin chiral stationary phase can only separate some chiral samples, and its adaptability is significantly insufficient. Furthermore, when using an alkaline reversed-phase mobile phase, the chiral column is unstable and cannot be used for testing.

[0111] Example 12:

[0112] The separation effect of amylose@NKCOF-98 stationary phase was investigated using a normal-phase mobile phase. The results are as follows: Figure 11 Table 3: Table 3: Separation results of chiral samples under normal phase conditions: Substrate Detection wavelength (nm) <![CDATA[Retention time RT1 (min)]]> <![CDATA[Retention time RT2 (min)]]> α <![CDATA[R s ]]> RS-Promethazine 260 2.86 5.55 2.44 1.02 RS-Propranolol 260 1.82 3.73 3.70 1.07 RS-Atenol 260 2.95 6.93 3.08 1.05 RS-chlorpheniramine 260 1.91 5.03 4.30 1.06 Conclusion: As can be seen from the separation results in Table 3 above, this chiral stationary column has a better separation effect on the normal phase of the samples in Table 3, and its overall performance is stronger than that of commercial chiral columns.

[0113] Example 13:

[0114] The separation effect of β-cyclodextrin@NKCOF-98 stationary phase was demonstrated by using a reversed-phase mobile phase, and the results are as follows: Figure 12 Table 4: Table 4: Separation results of chiral samples under reverse-phase conditions: Substrate Detection wavelength (nm) <![CDATA[Retention time RT1 (min)]]> <![CDATA[Retention time RT2 (min)]]> α <![CDATA[R s ]]> RS-Sulprofen 260 1.30 6.41 7.11 1.05 RS-Flurbiprofen 260 1.13 5.72 7.72 1.03 RS-Sullinacid 260 1.22 5.55 7.14 1.15 RS-Zatopirofen 260 1.22 5.64 7.43 1.13 RS-Ibuprofen 260 1.22 5.64 7.29 1.07 Conclusion: As can be seen from the separation results in Table 4 above, this chiral stationary column has a better separation effect on the reversed phase of the samples in Table 4, and its overall performance is stronger than that of commercial chiral columns.

[0115] Example 14:

[0116] The stability of the alkaline protease@NKCOF-98 stationary phase was determined by repeatedly injecting DL-phenylalanine into the reversed-phase mobile phase and observing the trend of resolution changes. The stationary phase maintained a resolution >1.0 for DL-phenylalanine even after 2400 injections, demonstrating its high chiral separation stability. Results are as follows... Figure 13 .

Claims

1. A method for synthesizing covalent organic framework materials (COFs), characterized in that, The covalent organic framework material is a covalent organic framework material linked by vinyl, imide, borate ester, boron oxide six-membered ring, imine, azazine, keto-enol, hydrazone, β-ketoamine, or triazine bonds, and is synthesized through the following steps: Organic monomer 1 and organic monomer 2 are dissolved or dispersed separately in a reaction solvent, mixed, and then subjected to a condensation reaction under alkaline catalysis. After separation and purification, the final product is obtained. Among them, organic monomer 1 is a monomer containing an aldehyde functional group; The organic monomer 2 is a monomer containing an amino or hydrazide functional group; The catalyst is an OH-containing catalyst. - CH3COO - NH 3+ CO3 2- S 2- SO3 2- PO4 3- ClO - CN - The catalyst is any one or more of basic inorganic compounds, such as primary amine groups, secondary amine groups, tertiary amine groups, and quaternary amine groups. The catalyst may be optionally substituted with substituents selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I. The reaction solvent is water, or a mixture of organic solvent and water in a volume ratio of 0.01-1:1; The reaction temperature is 4℃-100℃.

2. The synthesis method according to claim 1, characterized in that, The monomer containing the aldehyde functional group is any one of (a) 1,3,5-tricarboxaldehyde (TFp), (b) 2-hydroxy-1,3,5-benzenetricarboxaldehyde, (c) 2,6-dihydroxy-1,3,5-benzenetricarboxaldehyde, (d) trialdehyde-resorcinol (Tp), (e) 1,3,5-tris(p-formylphenyl)benzene (TFPB), (f) 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine (TFPT), (g) 4,4',4''-[benzene-1,3,5-triyltris(acetylene-2,1-diyl)]tribenzaldehyde (TFEPB), or (h) 4,4',4''-(benzene-1,3,5-tris(acetylene-2,1-diyl))-tris(salicylaldehyde) (TFEPB-OH); The monomer containing an amino functional group is any one of (a) hydrazine hydrate, (b) p-phenylenediamine, (c) benzidine, (d) 5,5'-diamino-2,2'-bipyridine, or (e) substituted 4,4'-diaminoterphenyl. The monomers containing aldehyde functional groups and amino functional groups may be optionally substituted with substituents selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I. The monomer containing the hydrazide functional group is optionally R-substituted terephthalohydrazide; wherein R is , , , or When multiple substitutions are used, each R is independently selected from the above structures. Preferably, the organic monomer 1 is 1,3,5-tricarboxaldehyde (TFp); the organic monomer 2 is hydrazine hydrate, 2,5-bis(2-methoxyethoxy)terephthaloylhydrazine, 2,5-bis(3-butene-1-oxo)terephthaloylhydrazine, 2,5-bis(3-butyn-1-oxo)terephthaloylhydrazine, 2,5-bis(2-(dimethylamine)ethoxy)terephthaloylhydrazine, 2,5-bis(2-methoxymethoxy)terephthaloylhydrazine, 2,5-bis(2-methoxypropoxy)terephthaloylhydrazine, or 2,5-bis(2-(2-methoxyethoxy)ethoxy)terephthaloylhydrazine.

3. The synthesis method as described in claim 1, characterized in that, The synthesis method specifically includes the following steps: 1) Dissolve or disperse aldehyde functional group monomers and amino or hydrazide functional group monomers in a reaction solvent, and add an alkaline catalyst to carry out the reaction; 2) After the reaction is complete, separate and remove unreacted monomers. The reaction solvent is water or a mixture of water and a small amount of miscible organic solvent, wherein the organic solvent is selected from any one or more of tetrahydrofuran, methanol, ethanol, acetonitrile, acetone, 1,4-dioxane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.

4. An in-situ synthesis method for biocomposite chiral stationary phase materials based on covalent organic framework materials, characterized in that, The biocomposite chiral stationary phase material based on covalent organic framework material includes a covalent organic framework material and an alkali-resistant biomolecule chiral resolving agent, wherein the covalent organic framework material encapsulates the biomolecules in situ. The covalent organic framework material is a covalent organic framework material linked by vinyl, imide, borate ester, boron-oxygen six-membered ring, imine, azazine, keto-enol, hydrazone, β-ketoamine, or triazine bonds; the biomolecule is selected from proteins, polysaccharides, and cyclodextrins, and is synthesized through the following steps: Organic monomer 1 and organic monomer 2 are dissolved or dispersed separately in a reaction solvent, mixed, and then added to a biomolecule solution. A condensation reaction is carried out under alkaline catalytic conditions, followed by separation and purification to obtain the final product. Among them, organic monomer 1 is a monomer containing an aldehyde functional group; The organic monomer 2 is a monomer containing an amino or hydrazide functional group; The catalyst is an OH-containing catalyst. - CH3COO - NH 3+ CO3 2- S 2- SO3 2- PO4 3- ClO - CN - Basic inorganic compounds such as primary amine groups, secondary amine groups, tertiary amine groups, quaternary amine groups, etc.; The reaction solvent is water, or a mixture of organic solvent and water in a volume ratio of 0.01-1:

1.

5. The synthesis method as described in claim 4, characterized in that, The monomer containing the aldehyde functional group is any one of (a) 1,3,5-tricarboxaldehyde (TFp), (b) 2-hydroxy-1,3,5-benzenetricarboxaldehyde, (c) 2,6-dihydroxy-1,3,5-benzenetricarboxaldehyde, (d) trialdehyde-resorcinol (Tp), (e) 1,3,5-tris(p-formylphenyl)benzene (TFPB), (f) 2,4,6-tris(4-aldehydephenyl)-1,3,5-triazine (TFPT), (g) 4,4',4''-[benzene-1,3,5-triyltris(acetylene-2,1-diyl)]tribenzaldehyde (TFEPB), or (h) 4,4',4''-(benzene-1,3,5-tris(acetylene-2,1-diyl))-tris(salicylaldehyde) (TFEPB-OH); The monomer containing an amino functional group is any one of (a) hydrazine hydrate, (b) p-phenylenediamine, (c) benzidine, (d) 5,5'-diamino-2,2'-bipyridine, or (e) substituted 4,4'-diaminoterphenyl. The monomers containing aldehyde functional groups and amino functional groups may be optionally substituted with substituents selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I. The monomer containing the hydrazide functional group is optionally R-substituted terephthalohydrazide; wherein R is , , , or When multiple substitutions are used, each R is selected independently from the above structures; Preferably, the organic monomer 1 is 1,3,5-triformaldehyde (TFp); the organic monomer 2 is hydrazine hydrate, 2,5-bis(2-methoxyethoxy)terephthaloylhydrazine, 2,5-bis(3-butene-1-oxy)terephthaloylhydrazine, 2,5-bis(3-butyn-1-oxy)terephthaloylhydrazine, 2,5-bis(2-(dimethylamine)ethoxy)terephthaloylhydrazine, 2,5-bis(2-methoxymethoxy)terephthaloylhydrazine, 2,5-bis(2-methoxypropoxy)terephthaloylhydrazine or 2,5-bis(2-(2-methoxyethoxy)ethoxy)terephthaloylhydrazine.

6. The synthesis method according to claim 4, characterized in that, The biomolecules are selected from one or more of the following: lipase, alkaline protease, human serum albumin (HSA), α-acid glycoprotein (AGP), ovomucoid (OVM), cellulase, glucosidase, amylose, amylopectin, cellulose, chitosan, dextran, and modified or unmodified α, β, γ-cyclodextrins.

7. The synthesis method according to claim 4, characterized in that, The catalyst is an OH-containing catalyst. - CH3COO - NH 3+ CO3 2- S 2- SO3 2- PO4 3- ClO - CN - The catalyst is any one or more of basic inorganic compounds, such as primary amine, secondary amine, tertiary amine, and quaternary amine groups, and may be optionally substituted with substituents selected from any one or more of -H, -OH, -NH2, -CN, -NO2, -OCH3, -SO3H, -CH3, -COOH, -COOCH3, -F, -Cl, -Br, and -I.

8. The synthesis method according to claim 4, characterized in that, Includes the following steps: 1) Dissolve or disperse aldehyde functional group monomers and amino or hydrazide functional group monomers in a reaction solvent, add enzyme solution, and react with an alkaline catalyst at a reaction temperature of 4℃-100℃. 2) After the reaction is complete, separate and remove unreacted monomers and biomolecules; 3) The purified product is dried to obtain the final product.

9. The novel chiral stationary phase prepared according to claims 4-8, characterized in that, It can prepare monolithic or packed columns for high performance liquid chromatography, gas chromatography, and capillary chromatography using in-situ growth or direct packing methods.

10. The use of the biocomposite chiral stationary phase material based on covalent organic framework materials prepared according to claims 4-8, characterized in that, Novel chiral stationary phases can be applied to the separation of chiral amino acids, drugs, and small organic molecules under reversed-phase chromatography, polar organic phase chromatography, and normal-phase chromatography conditions.

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