Quaternary ammonium salt-modified strong anion-exchange monolithic column

Abstract

Disclosed in the present invention is a monolithic column material. Quaternary ammonium groups are grafted onto the inner surfaces of pores and channels of the monolithic column material. The quaternary ammonium group is present in the form of EP-OCH2CH(OH)CH-N+R1R2R3. The monolithic column prepared in the present invention is rich in hydroxyl, has excellent compatibility with an eluted analyte containing biomacromolecules, has excellent specific adsorption capacity for nucleic acid substances, and is suitable for enrichment and separation of DNA and / or RNA substances.

Description

A Quaternary Ammonium Salt Modified Strong Anion Monolithic Column

[0001] This application claims priority to Chinese patent application 2024105777496, filed on May 10, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a monolithic column material. More specifically, it relates to a monolithic column material for nucleic acid enrichment and separation. Background Technology

[0003] Currently, the separation and purification of biomolecules primarily utilizes chromatographic microspheres for surface ligand modification to achieve separation and purification with different functions, yielding relatively pure molecules for use as pharmaceutical and diagnostic reagents, raw materials in biochemical processes, and additives in food and cosmetics. However, microsphere media as a stationary phase have the following drawbacks: 1) The pore distribution is relatively small, resulting in poor separation ability for larger biomolecules; 2) The diffusion effect of chromatographic microspheres is significant, requiring a long retention time to fully diffuse to the theoretical specific surface area of ​​the microspheres, which takes even longer for biomolecules; 3) The operating pressure of microsphere-packed chromatographic columns is relatively high, which is not conducive to the efficient operation of the system.

[0004] Monolithic column technology is a new generation of chromatographic stationary phases developed in recent years. Currently, it offers significant advantages over traditional porous microsphere-packed media for the rapid separation and purification of biomolecules. A monolithic column consists of a single, highly interconnected channel within which fluid flows. The monolithic column matrix is ​​made of polymer, and the average diameter of its internal channels is 2 μm. Inside the monolithic column, each channel is connected to dozens of other channels. This high connectivity facilitates uniform fluid distribution, reduces operating pressure, and enables high flow rates and low column pressures.

[0005] Currently, monolithic column materials are polymethyl methacrylate (PMMA) polymers or polystyrene polymers. These have many commercially successful products on the market and represent relatively successful monolithic column solutions. However, PMMA and polystyrene polymers also have the following problems: 1) High molding temperatures lead to complex process control due to thermal effects when preparing larger blocks, resulting in poor uniformity of pore distribution and potential dual-pore size distribution. Furthermore, curing typically takes 12-24 hours at 60-80°C; 2) After curing, they exhibit strong hydrophobicity, leading to significant non-specific adsorption during the purification of biomolecules. Hydrophilic modification (e.g., using molecules rich in hydroxyl groups) is generally required to reduce non-specific adsorption; 3) When curing to larger pore sizes, their poor toughness makes them prone to damage, affecting performance. These are the technical problems encountered by monolithic columns in the enrichment and separation of biomolecules, and these are the technical problems that this invention aims to solve.

[0006] CN202211600949.6 discloses an epoxy resin-based chromatography material, which features a uniform internal structure, good reproducibility in preparation, high toughness, modifiability for functional ligands, and high hydrophilicity. CN113145088A discloses a hydrophilic monolithic material and its preparation and application, in which triglycidyl isocyanurate (TGIC) and 1,4,7,10-tetraazacyclododecane (cyclen) are ultrasonically dissolved in an organic solvent, followed by an epoxy-amine ring-opening polymerization reaction under heating conditions to form the monolithic material. Because the reaction between epoxy and amine generates a large number of hydroxyl groups, the resulting monolithic material exhibits good hydrophilicity. EP2485836A1 discloses a monolithic column with a fixed monomeric avidin for enriching and identifying biotinylated species, comprising a stationary phase made of a monolithic material containing reactive groups, to which avidin binds, monomerizes, and refolds.

[0007] Currently, when dealing with the separation of biomolecules, especially the enrichment and separation of nucleic acid substances from mixtures including proteins and nucleic acids, monolithic columns need to further enhance their specific adsorption of biomolecules, in addition to their general excellent adsorption performance, uniform results, low operating pressure, and high flow rate. Summary of the Invention

[0008] The technical problem this invention aims to solve is to overcome the shortcomings of existing monolithic columns, which lack optimization for biomolecules, especially nucleic acids, resulting in low yields (generally 60%-80%), poor stability, inconsistent linear scale-up reproducibility, and complex purification process optimization and control. Furthermore, the complex temperature control and long curing cycle during the curing process lead to high costs. This invention proposes an epoxy resin-based monolithic column material with specific adsorption capacity for nucleic acids. It retains the excellent adsorption performance, uniform results, low operating pressure, and high flow rate characteristics of epoxy resin monolithic columns, while also possessing excellent specific enrichment capacity for nucleic acids.

[0009] To achieve the above objectives, the present invention proposes the following technical solution.

[0010] A first aspect of the present invention is to provide an integral column.

[0011] A monolithic column in which quaternary ammonium salts are grafted onto the inner surfaces of its pores and channels.

[0012] In some embodiments, the quaternary ammonium group is EP-OCH2CH(OH)CH-N + R 1 R 2 R 3 It exists in the form of.

[0013] in:

[0014] EP is epoxy resin;

[0015] R 1 R 2 R 3 The groups are alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine)yl, polyvinyl alcohol, and polyvinylamine; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine), polyvinyl alcohol, and polyvinylamine groups are optionally surrounded by amino, hydroxyl, or -C groups. 1-6 Alkyl, -C 1-6 Hydroxyalkyl, -C 1-6 It is aminoalkyl substituted or hybridized with O, N, Si, P, or S.

[0016] The alkyl group is preferably methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 1-methylpropyl, 1,1-dimethylethyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

[0017] The alkenyl group is preferably vinyl, propenyl, propenyl, 2-propenyl, 2-methylpropenyl, butenyl, butenyl, butenyl, 3-butenyl, butane-1,3-dienyl, 2-methylbutenyl, hexenyl, hexenyl, 2-dienyl, hexenyl, 4-dienyl, and hexenyl-1,3-dienyl groups.

[0018] The alkynyl group is preferably ethynyl, 1-propynyl, 2-propynyl (propynyl), 1-butynyl, 2-butynyl, and 3-butynyl groups.

[0019] The cycloalkyl group is preferably cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, or cyclooctyl group.

[0020] The heterocyclic alkyl group is preferably pyrrolidinyl, imidazolidinone, pyrazolyl, piperidinyl, 2,5-piperazinyl, pyranyl, or morpholinyl.

[0021] The poly(alkylene oxide) group is preferably polyethylene glycol, polypropylene glycol, or polybutylene glycol, with a preferred end group being hydroxyl or amino, and a preferred molecular weight of 500-5000, 1000-5000, 2000-4000, 2500-3500, or 3000.

[0022] The poly(alkyleneamine) group is preferably polyethylenediamine, polypropylenediamine, or polybutylenediamine, and the terminal group is preferably hydroxyl, amino, or C-terminated. 1-6 The substituted amino group preferably has a molecular weight of 500-5000, more preferably 1000-5000, more preferably 2000-4000, more preferably 2500-3500, and more preferably 3000.

[0023] The polyvinyl alcohol group is preferably the end or middle part of polyvinyl alcohol with EP-OCH2CH(OH)CH-N +Connected, preferably with a molecular weight of 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, preferably 3000.

[0024] The polyvinylamine group is preferably the end or middle part of polyvinyl alcohol with EP-OCH2CH(OH)CH-N + Connected, preferably with a molecular weight of 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, preferably 3000.

[0025] In some embodiments, the EP-OCH2CH(OH)CH-NR 1 R 2 R 3 The anion exchange capacity of the epoxy resin in the form of the epoxy resin is 20 μmol / mL-200 μmol / mL, preferably 20 μmol / mL, 30 μmol / mL, 40 μmol / mL, 50 μmol / mL, 60 μmol / mL, 70 μmol / mL, 80 μmol / mL, 90 μmol / mL, 100 μmol / mL, 110 μmol / mL, 120 μmol / mL, 130 μmol / mL, 140 μmol / mL, 150 μmol / mL, 160 μmol / mL, 170 μmol / mL, 180 μmol / mL, 190 μmol / mL, and 200 μmol / mL.

[0026] In some embodiments, the pore size of the integral column is 2μm-6μm, preferably 2.0μm, 2.1μm, 2.2μm, 2.3μm, 2.4μm, 2.5μm, 2.6μm, 2.7μm, 2.8μm, 2.9μm, 3.0μm, 3.1μm, 3.2μm, 3.3μm, 3.4μm, 3.5μm, 3.6μm, 3.7μm, 3 .8μm, 3.9μm, 4.0μm, 4.1μm, 4.2μm, 4.3μm, 4.4μm, 4.5μm, 4.6μm, 4.7μm, 4.8μm, 4.9 μm, 5.0μm, 5.1μm, 5.2μm, 5.3μm, 5.4μm, 5.5μm, 5.6μm, 5.7μm, 5.8μm, 5.9μm, 6.0μm.

[0027] In some embodiments, the pore volume of the monolithic column is 1.1 mL / g-1.8 mL / g, preferably 1.10 mL / g, 1.15 mL / g, 1.20 mL / g, 1.25 mL / g, 1.30 mL / g, 1.35 mL / g, 1.40 mL / g, 1.45 mL / g, 1.50 mL / g, 1.55 mL / g, 1.60 mL / g, 1.65 mL / g, 1.70 mL / g, 1.75 mL / g, or 1.80 mL / g.

[0028] In some embodiments, the porosity of the integral column is 55%-68%, preferably 55.0%, 55.5%, 56.0%, 56.5%, 57.0%, 57.5%, 58.0%, 58.5%, 59.0%, 59.5%, 60.0%, 60.5%, 61.0%, 61.5%, 62.0%, 62.5%, 63.0%, 63.5%, 64.0%, 64.5%, 65.0%, 65.5%, 66.0%, 66.5%, 67.0%, 67.5%, or 68.0%.

[0029] In some embodiments, the raw material for the monolithic column includes substance A containing polyepoxy groups.

[0030] The term "substance containing polyepoxy groups" refers to a substance whose structure contains two or more epoxy groups.

[0031] In some embodiments, the substance containing a polyepoxy group is preferably a polyepoxy group glycidyl ether or a polyepoxy group glycidyl ester.

[0032] In some embodiments, the glycidyl ether may be one or more monomers satisfying the following structural formula I.

[0033] In structural formula I, R 1 Selected from hydrogen atoms, substituted or unsubstituted C1-C atoms 10 Alkyl groups, or epoxy groups;

[0034] n takes the form of a positive integer between 0 and 10;

[0035] L 1 Selected from oxygen or nitrogen atoms;

[0036] L 2 Selected from oxygen or nitrogen atoms;

[0037] X is selected from substituted or unsubstituted C1-C. 10 Alkylene, substituted or unsubstituted C3-C 10 cycloalkylene, Or, a benzene ring.

[0038] Preferably, the glycidyl ether is one or more of the following monomers (a)-(m);

[0039] The structure (a) above is the structure of glycerol triglycidyl ether.

[0040] The structure (b) above is the structure of pentaerythritol tetraglycidyl ether.

[0041] The structure (j) described above is the structure of 1,4-butanediol diglycidyl ether.

[0042] The structure (k) described above is the structure of trihydroxymethylethane triglycidyl ether.

[0043] The structure (l) described above is the structure of bisphenol A diglycidyl ether.

[0044] The structure (m) described above is the structure of bisphenol F diglycidyl ether.

[0045] In some embodiments, the glycidyl ether may be one or more polymers that satisfy the following structural formula II.

[0046] In structural formula II, R 2 It is selected from hydrogen atoms, substituted or unsubstituted C1-C 10 Alkyl groups, or,

[0047] m takes the form of a positive integer between 2 and 40.

[0048] Preferably, the glycidyl ether is one or more of the following polymers (1)-(4);

[0049] In some embodiments, the glycidyl ester may be one or more monomers that satisfy the following structural formula III;

[0050] In structural formula III, Y is selected from substituted or unsubstituted C1-C. 10 Alkylene, substituted or unsubstituted C3-C 10 Cycloalkylene, or benzene ring.

[0051] Preferably, the glycidyl ester is a monomer (A) and / or (B) thereof;

[0052] In some embodiments, the substance containing a polyepoxy group is preferably one or more of the following monomers (I)-(IV);

[0053] In some embodiments, the substance containing polyepoxy groups is preferably a polyepoxy group glycidyl ether monomer and / or a polyepoxy group glycidyl ether polymer.

[0054] The polyepoxy group glycidyl ether monomers may be glycerol triglycidyl ether and / or pentaerythritol tetraglycidyl ether.

[0055] The polyepoxy group glycidyl ether polymer may be polyglycerol glycidyl ether and / or polypentaerythritol tetraglycidyl ether.

[0056] In some embodiments, when the substance containing polyepoxy groups is a mixture of the polyepoxy group glycidyl ether monomer and the polyepoxy group glycidyl ether polymer, the mixing volume ratio of the polyepoxy group glycidyl ether monomer and the polyepoxy group glycidyl ether polymer can be (0.1-9):1, for example 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1. For example, a mixture of polyglycerol triglycidyl ether and pentaerythritol tetraglycidyl ether = 1:1, or polypentaerythritol tetraglycidyl ether and glycerol triglycidyl ether = 1:1.

[0057] In some embodiments, the molecular weight of the substance containing the polyepoxy group can be 300-2000, for example, 550, 700, 1000, 1200, 1500, or 1800.

[0058] In some embodiments, raw material A may further include a substance containing a monoepoxy group. The substance containing a monoepoxy group refers to a substance whose structure contains one epoxy group.

[0059] The substance containing a monoepoxy group is preferably a monoepoxy group glycidyl ether or a monoepoxy group glycidyl ester.

[0060] The monoepoxy group glycidyl ether can be a conventional material containing one epoxy group in its structure, preferably phenyl glycidyl ether and / or butyl glycidyl ether.

[0061] The monoepoxy group glycidyl ester may be one or more of phenyl glycidyl ether, butyl glycidyl ether, pentyl glycidyl ether, octyl glycidyl ether, octadecyl glycidyl ether and naphthyl glycidyl ether, preferably phenyl glycidyl ether and / or butyl glycidyl ether.

[0062] In some embodiments, the raw material for the monolithic column includes pore-forming agent B.

[0063] In some embodiments, the porogen B is a solvent that is miscible with the substance A containing polyepoxy groups and does not undergo condensation polymerization. Preferably, it is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether; more preferably, it is one or more of toluene, dioxane, and methyl tert-butyl ether, such as toluene and dioxane, toluene and methyl tert-butyl ether, or dioxane and methyl tert-butyl ether.

[0064] In some embodiments, when the porogen B is a mixture of two different substances, the volume ratio of the two different substances may be (0.1-9):1, for example 0.5:1, 1:1, 2:1, 3:1, 5:1 or 7:1.

[0065] In some embodiments, the weight percentage of raw material A to the total weight of raw material A and porogen B is preferably 22%-70%, for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 58%, 65%, or 68%. The weight percentage of porogen B to the total weight of raw material A and porogen B is preferably 35%-80%, for example 40%, 45%, 50%, 60%, 65%, 70%, 75%, or 78%.

[0066] In some embodiments, raw material A is a polyepoxy group glycidyl ether monomer and / or a polyepoxy group glycidyl ether polymer; pore-forming agent B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether.

[0067] In some embodiments, raw material A is a polyepoxy group glycidyl ether polymer and a monoepoxy group glycidyl ether monomer; pore-forming agent B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether.

[0068] In some embodiments, raw material A is polyglycerol triglycidyl ether and monoepoxy group glycidyl ether monomers; pore-forming agent B is one or more of toluene, xylene, dichloromethane, dichloroethane, dioxane, formamide (DMF), dimethyl sulfoxide (DMSO), and methyl tert-butyl ether; the monoepoxy group glycidyl ether monomers are preferably phenyl glycidyl ether and / or butyl glycidyl ether.

[0069] In some embodiments, raw material A is polyglycerol triglycidyl ether and monoepoxy group glycidyl ether monomers; pore-forming agent B is one or more of toluene, dioxane, and methyl tert-butyl glycerol ether; the monoepoxy group glycidyl ether monomers are preferably phenyl glycidyl ether and / or butyl glycidyl ether.

[0070] In some embodiments, the raw material for the monolithic column includes catalyst C.

[0071] In some embodiments, the catalyst C may be a Lewis acid and / or a complex of a Lewis acid.

[0072] The Lewis acid may be one or more of aluminum trichloride, boron trifluoride, ferric bromide, ferric chloride, zinc chloride, niobium trichloride, and sulfur trioxide, such as boron trifluoride.

[0073] The Lewis acid complex may be one or more of boron trifluoride diethyl ether, boron trifluoride acetonitrile, boron fluoride dimethyl carbonate, and boron trifluoride ethylamine.

[0074] In some embodiments, the weight percentage of the catalyst C relative to the total weight of the raw material A and the porogen B is preferably 0.3‰-1‰, for example 0.4‰, 0.5‰, 0.6‰, 0.7‰, 0.8‰ or 0.9‰.

[0075] In some embodiments, the raw material for the monolithic column includes quaternary ammonium salt modifier D.

[0076] In some embodiments, the quaternary ammonium salt modifier D has NR 1 R 2 R 3 It exists in the form of.

[0077] Among them, R 1 R 2 R 3 The groups are alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine)yl, polyvinyl alcohol, and polyvinylamine; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine), polyvinyl alcohol, and polyvinylamine groups are optionally surrounded by amino, hydroxyl, or -C groups. 1-6 Alkyl, -C 1-6 Hydroxyalkyl, -C 1-6 It is aminoalkyl substituted or hybridized with O, N, Si, P, or S.

[0078] The alkyl group is preferably methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 1-methylpropyl, 1,1-dimethylethyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

[0079] The alkenyl group is preferably vinyl, propenyl, propenyl, 2-propenyl, 2-methylpropenyl, butenyl, butenyl, butenyl, 3-butenyl, butane-1,3-dienyl, 2-methylbutenyl, hexenyl, hexenyl, 2-dienyl, hexenyl, 4-dienyl, and hexenyl-1,3-dienyl groups.

[0080] The alkynyl group is preferably ethynyl, 1-propynyl, 2-propynyl (propynyl), 1-butynyl, 2-butynyl, and 3-butynyl groups.

[0081] The cycloalkyl group is preferably cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, or cyclooctyl group.

[0082] The heterocyclic alkyl group is preferably pyrrolidinyl, imidazolidinone, pyrazolyl, piperidinyl, 2,5-piperazinyl, pyranyl, or morpholinyl.

[0083] The poly(alkylene oxide) group is preferably polyethylene glycol, polypropylene glycol, or polybutylene glycol, with a preferred end group being hydroxyl or amino, and a preferred molecular weight of 500-5000, 1000-5000, 2000-4000, 2500-3500, or 3000.

[0084] The poly(alkyleneamine) group is preferably polyethylenediamine, polypropylenediamine, or polybutylenediamine, and the terminal group is preferably hydroxyl, amino, or C-terminated. 1-6 The substituted amino group preferably has a molecular weight of 500-5000, more preferably 1000-5000, more preferably 2000-4000, more preferably 2500-3500, and more preferably 3000.

[0085] The polyvinyl alcohol group is preferably the end or middle portion of polyvinyl alcohol with N. +Connected, preferably with a molecular weight of 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, preferably 3000.

[0086] The polyvinylamine group is preferably the end or middle portion of polyvinyl alcohol with N. + Connected, preferably with a molecular weight of 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, preferably 3000.

[0087] A second aspect of the present invention is to provide a method for preparing a monolithic column.

[0088] The preparation method includes the following steps:

[0089] S1. Hydroxylate the entire column;

[0090] S2. The hydroxylated monolithic column is quaternized with ammonium salt.

[0091] In step S1, hydroxylation includes the following steps:

[0092] S11. Clean the entire column;

[0093] S12. Drying monolithic column;

[0094] S13. Hydroxylated monolithic column;

[0095] S14. Post-processing.

[0096] In step S11, a monolithic column is taken and rinsed with 10-20 times its volume of deionized water to remove the protective solution.

[0097] In step S12, the entire column is dried using a vacuum pump. The drying time is 10-120 min, preferably 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, or 110 min. The drying temperature is 5-50℃, preferably 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, or 45℃.

[0098] In step S13, an acidic aqueous solution is added to the dried monolithic column, and the temperature is raised to react, so that the residual epoxy groups in the monolithic column are converted into hydroxyl groups.

[0099] The acid in the acidic aqueous solution is sulfuric acid, hydrochloric acid, nitric acid, formic acid, or acetic acid, and the concentration of the acid in the acidic aqueous solution is 0.1-0.5M, preferably 0.2M, 0.3M, or 0.4M.

[0100] The volume ratio of the acidic aqueous solution to the monolithic column is (3-30):1, preferably 5:1, 7:1, 10:1, 15:1, 20:1, 25:1, or 30:1.

[0101] The heating reaction involves heating the system to 40-80℃ and reacting for 1-6 hours. The preferred temperatures are 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃. The preferred reaction times are 1.0h, 1.5h, 2.0h, 2.5h, 3.0h, 3.5h, 4.0h, 4.5h, 5.0h, 5.5h, and 6.0h.

[0102] In step S14, the hydroxylated monolithic column is rinsed with deionized water until the pH is neutral.

[0103] In some embodiments, after step S1, the overall column is determined by epoxy group density measurement to determine that the density of hydroxylated epoxy groups is 0 or undetectable.

[0104] In step S2, quaternization includes the following steps:

[0105] S21. Clean the entire column;

[0106] S22. Drying monolithic column;

[0107] S23. Quaternized monolithic column;

[0108] S24. Neutralization and Post-processing.

[0109] In step S21, a monolithic column is taken and rinsed with 10-20 times its volume of deionized water to remove the protective solution.

[0110] In step S22, the entire column is dried using a vacuum pump. The drying time is 10-120 min, preferably 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, or 110 min. The drying temperature is 5-50℃, preferably 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, or 45℃.

[0111] In step S23, a quaternary ammonium salt modifier D solution is added to the monolithic column. After thorough wetting, a catalyst is added, and the temperature is raised to react, so that the hydroxyl groups in the monolithic column are converted into quaternary ammonium salt groups.

[0112] In step S23, the quaternary ammonium salt modifier D solution can be mixed with the catalyst and then added to the monolithic column. The temperature is then raised to react and convert the hydroxyl groups in the monolithic column into quaternary ammonium salt groups.

[0113] The quaternary ammonium salt modifier D solution is an aqueous solution of quaternary ammonium salt modifier D with a concentration of 40%-70% (mass-volume ratio g / mL), preferably 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0114] The catalyst is cerium ammonium nitrate, which is either alkaline or acidic.

[0115] The alkali is one or more of potassium hydroxide and sodium hydroxide, with a concentration of 3%-10%, preferably 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0116] The acidic cerium ammonium nitrate is a mixture of cerium ammonium nitrate and an acid, wherein the acid is one or more of sulfuric acid, hydrochloric acid, nitric acid, formic acid, and acetic acid.

[0117] The amount of cerium ammonium nitrate added is 1-10% (mass-volume ratio g / mL (solvent volume)), preferably 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The amount of acid added is 0.1%-1% (mass-volume ratio g / mL (solvent volume)), preferably 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.

[0118] The amount of the quaternary ammonium salt modifier D solution added is 5mL-20mL / block, preferably 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL, 14mL, 15mL, 16mL, 17mL, 18mL, 19mL, or 20mL.

[0119] The heating reaction involves heating the system to 40-80℃ and reacting for 10-48 hours. The preferred temperatures are 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃. The preferred reaction times are 10h, 12h, 15h, 18h, 21h, 24h, 27h, 30h, 33h, 36h, 39h, 42h, 45h, and 48h.

[0120] In step S24, the monolithic column after quaternization is cleaned with a pH adjuster and then washed with deionized water until the pH is neutral.

[0121] The pH adjuster is an acid or a base. The acid is one or more of sulfuric acid, hydrochloric acid, nitric acid, formic acid, and acetic acid. The base is one or more of sodium hydroxide, potassium hydroxide, and ammonia water.

[0122] After step S24 is completed, in order to protect the entire column, it can be stored in a 20%-70% ethanol solution for later use.

[0123] A third aspect of the present invention is to provide an application of a monolithic column for the enrichment and separation of DNA or RNA.

[0124] The monolithic column has the function of specifically adsorbing DNA or RNA. Specifically, it can specifically adsorb DNA and / or RNA in a mixed solution containing biological macromolecules, enabling them to be enriched in the monolithic column and then eluted for separation.

[0125] A fourth aspect of the present invention is to provide a chromatographic column comprising the aforementioned monolithic column.

[0126] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0127] The reagents and raw materials used in this invention are all commercially available.

[0128] The positive and progressive effects of this invention are as follows:

[0129] 1. The monolithic column prepared by this invention is rich in hydroxyl groups and has excellent compatibility with elution targets containing biomacromolecules, making it particularly suitable for the field of biomacromolecule chromatography.

[0130] 2. The monolithic column prepared by this invention retains the excellent adsorption performance, uniform results, low operating pressure, and high flow rate characteristics of epoxy resin monolithic column materials. The pores are adjustable, ranging from submicron to micron in size, with a relatively uniform distribution exhibiting a single-peak normal distribution and high ligand utilization. The pores are through-holes, eliminating diffusion effects, eliminating the need for increased retention time, shortening the process cycle, exhibiting no significant eddy current phenomenon, low shear force, and no impact on sample loading at high flow rates, demonstrating high toughness.

[0131] 3. The monolithic column prepared by this invention has excellent specific adsorption capacity for nucleic acid substances, and is suitable for the enrichment and separation of DNA and / or RNA substances. Attached Figure Description

[0132] Figure 1 shows the aperture distribution of the integral column block prepared in Example 3.

[0133] Figure 2 shows the pore size distribution of the integral column block obtained in Example 7.

[0134] Figure 3 is a scanning electron microscope image of the monolithic column obtained in Example 1.

[0135] Figure 4 is a scanning electron microscope image of the monolithic column obtained in Comparative Example 12.

[0136] Figure 5 is a chromatographic diagram of the monolithic substrate prepared in Example 1.

[0137] Figure 6 is a chromatographic diagram of the monolithic block prepared in Example 7.

[0138] Figure 7 is a chromatographic diagram of the monolithic substrate prepared in Example 13. Detailed Implementation

[0139] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0140] Unless otherwise specified, the reagents used in the following examples and comparative examples are all commercially available products in the art.

[0141] Preparation Example

[0142] S1 hydroxylation: Take 1 mL of the entire column block and rinse it with 20 times its volume of deionized water through a sintered glass funnel to remove the protective solution. Dry the column using a vacuum pump through the sintered glass funnel for 60 min at an ambient temperature of 25°C.

[0143] Transfer the 1 mL monolithic column block after desiccation to a glass reaction flask, add 0.2 M deionized sulfuric acid aqueous solution, and add 10 mL of reaction solution per block (1 mL); sonicate for 30 min to accelerate the rapid immersion of the reaction solution into the monolithic column block; after sonication, transfer the glass reaction flask to a water bath, heat to 60 °C, and keep warm for 4 h; place the hydroxylated monolithic column block on a sintered sand funnel and rinse with deionized water until the pH is neutral (6.5-7).

[0144] The density of the bulk epoxy group after hydroxylation was determined to be 0 by epoxy group density measurement;

[0145] Table 1. Performance of examples of hydroxylated monolithic columns N / A: Hydroxylation-free treatment

[0146] The determination of epoxy group density indicates that all residual epoxy groups in Preparation Examples 1-4 have been converted into hydroxyl groups, which can be used for further coupling treatment. Preparation Comparative Examples 1-4 demonstrate that the residual epoxy group density is negatively correlated with pore size.

[0147] Example

[0148] Method 1:

[0149] 1. First, prepare a 40-70% aqueous solution of solvent A (mass / volume ratio g / mL) in a glass reaction flask (10mL / ml).

[0150] 2. Add the hydroxylation chromatography block to the above reaction system, shake quickly, and sonicate / evacuate for 15 minutes.

[0151] 3. Weigh 5% NaOH (mass-volume ratio g / mL (solvent volume)) of the reaction solvent and add it to the above system. After adding, shake quickly and sonicate for 15 min.

[0152] 4. After the ultrasound is completed, place the device in a water bath shaking table at 50-70℃ and 100 rpm for 24 hours.

[0153] 5. First, clean with 1M hydrochloric acid, then clean with deionized water until the pH value is neutral (6.5-7), and store in 20% ethanol for later use.

[0154] Method 2:

[0155] 1. Prepare a 40-70% aqueous solution of solvent A (mass-volume ratio g / mL) and a 5% aqueous solution of NaOH (mass-volume ratio g / mL (solvent volume)) of the reaction solvent in a glass reaction flask (10mL / piece).

[0156] 2. Add the hydroxylation chromatography block to the above reaction system, shake quickly, and sonicate / evacuate for 15 minutes.

[0157] 3. After the ultrasound is completed, place the device in a water bath shaking table at 50-70℃ and 100 rpm for 24 hours.

[0158] 4. First, clean with 1M hydrochloric acid, then clean with deionized water until the pH value is neutral (6.5-7), and store in 20% ethanol for later use.

[0159] Method 3:

[0160] 1. Prepare a 5% cerium ammonium nitrate and 0.5% sulfuric acid aqueous solution (mass-volume ratio g / mL (solvent volume)), and mix thoroughly in a glass reaction flask (10mL / ml).

[0161] 2. Add the hydroxylated chromatographic block to the above reaction system, shake quickly, and sonicate / evacuate for 15 minutes;

[0162] 3. The above system is pre-reacted for 30 min at 30°C; 20-50% of solvent A is added to the above system, mixed well, and sonicated / vacuumed for 15 min.

[0163] 4. Place in a water bath and react at 30-50℃ for 6 hours;

[0164] 5. First, clean with 1M sodium hydroxide, then clean with deionized water until the pH value is neutral (6.5-7), and store in 20% ethanol for later use.

[0165] Method 4:

[0166] 1. Prepare an aqueous solution of 5% cerium ammonium nitrate, 0.5% sulfuric acid, and 20-50% solvent A (mass-volume ratio g / mL (solvent volume)), and mix thoroughly in a glass reaction flask (10mL / ml).

[0167] 2. Add the hydroxylated chromatographic block to the above reaction system, shake quickly, and sonicate / evacuate for 15 minutes;

[0168] 3. Place in a water bath and react at 30-50℃ for 6 hours;

[0169] 4. First, clean with 1M sodium hydroxide, then clean with deionized water until the pH value is neutral (6.5-7), and store in 20% ethanol for later use.

[0170] Table 2. Processes of Examples and Comparative Examples

[0171] Effect test

[0172] Pore ​​size distribution, pore volume, and porosity determination

[0173] Test method: Mercury porosimetry

[0174] Test equipment: USA - McMurray-Autopore V 9620

[0175] Test results:

[0176] After hydroxylation and stacking modification, the pore size, porosity, and pore volume remained essentially stable. This indicates that the pore structure was not damaged during the modification process.

[0177] Figure 1 shows the pore size distribution of the hydroxylated block prepared in Example 3, and Figure 2 shows the pore size distribution of the quaternized ammonium salt prepared in Example 7 after column finishing. The pore size distribution results are consistent.

[0178] The test results for each embodiment and comparative example are shown in Table 3.

[0179] Table 3. Test results of pore size distribution, pore volume, and porosity.

[0180] Field emission electron microscopy scanning test

[0181] Test subjects: Example 1, Comparative Example 12

[0182] Testing equipment: Zeiss Sigma 300 (Germany), Oxford Spectroscopy.

[0183] Test results:

[0184] Electron microscopy results clearly show that the pore structure remains consistent after coupling modification. Even under higher concentrations and temperatures, Comparative Example 5 showed no impact on the pore structure. This is consistent with mercury intrusion porosimetry results, indicating that this method can functionalize the monolithic column while maintaining its basic properties such as pore size.

[0185] Epoxy ligand density determination

[0186] Test subjects: Examples 1-4 and Comparative Examples 1-4 in Table 1

[0187] Test method: Sodium thiosulfate-hydrochloric acid titration method.

[0188] After being prepared using epoxy-modified materials, the chromatographic materials of Examples 1-4 and Comparative Examples 1-4 were thoroughly cleaned with deionized water and then vacuum-dried in a sintered glass funnel for 10 minutes. One piece (approximately 0.5 g) of each material was then weighed and placed in a ground-glass conical flask. 3 mL of 1.3 mol / L sodium thiosulfate and 1-2 drops of phenolphthalein indicator were added. The flasks were sealed and allowed to react at room temperature for 1 hour. The supernatant was titrated with 0.1 mol / L hydrochloric acid standard solution until the red solution turned colorless. The epoxy group density was calculated by substituting the volume of hydrochloric acid standard solution consumed into the following formula:

[0189] S = 1000 * [M HCl [(V0-V1)*ρ / W]

[0190] S: Epoxy ligand density, μmol / mL;

[0191] M HCl Hydrochloric acid concentration, mmol / mL;

[0192] V0, V1: Volumes of HCl before and after titration, in mL;

[0193] ρ: Medium density (1.2 g / mL);

[0194] W: The mass (g) of the whole block weighed.

[0195] Test equipment: acid-base titration apparatus.

[0196] Test results: See Table 1.

[0197] Table 1 shows that the epoxy groups were almost undetectable in the entire column after hydroxylation treatment, while residual epoxy groups remained in the unhydroxylated column. Furthermore, the epoxy group density was negatively correlated with pore size; the larger the pore, the lower the epoxy group density. This indicates that hydroxylation treatment is a necessary step for subsequent coupling with quaternary ammonium compounds.

[0198] Anion exchange capacity determination

[0199] Test subjects: Quaternary ammonium salt anion exchange monolithic columns prepared in Examples 1-18 and Comparative Examples 1-11 of Table 2 above.

[0200] Test method: GB 5760-86.

[0201] Test equipment: acid-base titration apparatus.

[0202] Test results are shown in Table 4 below.

[0203] Table 4. Anion exchange capacity results for each example and comparative example.

[0204] In Examples 1-18, the anion exchange capacity of quaternary ammonium salts gradually decreased with increasing pore size. Furthermore, the ion exchange capacity was generally in the range of 70-180 μmol / mL, suitable for the ligand density requirements of chromatography. Alkene quaternary ammonium salt compounds showed relatively ideal coupling efficiency, with their anion exchange capacity generally exceeding 120 μmol / mL in a monolithic column with a relatively small pore size (average pore size 2.7 μm). Except for Example 6, this may be because bromide ions have a larger atomic radius than chloride ions, resulting in more active electrons, stronger ion interactions, and greater exchange difficulty. Other alkene quaternary ammonium salt compounds, after coupling, still have carbonyl, amide, methyl, and hydroxyl groups, which can be used to adjust the ion elution intensity of biomolecules through hydrogen bonding, dipole moment, and polar / nonpolar differences. Considering industrial preparation cost and efficiency, 2,3-epoxypropyltrimethylammonium chloride is the preferred choice, followed by allyltrimethylammonium chloride and others.

[0205] The anion exchange capacity results of Example 7 and Comparative Example 11 were 138.2 μmol / mL and 90.8 μmol / mL, respectively, indicating that the hydroxylation process significantly improved the anion exchange density.

[0206] The anion exchange capacity results of Example 7 and Comparative Example 1 were 138.2 μmol / mL and 98.2 μmol / mL, respectively. This indicates that the coupling efficiency of adding sodium hydroxide after premixing the 2,3-epoxypropyltrimethylammonium chloride solution with the monolithic column block and sonicating for 15 min in Method 1 is high. In Method 2, the one-pot method of premixing all reagents may be because the 2,3-epoxypropyltrimethylammonium chloride is partially destroyed by strong sodium oxide and does not have time to contact the surface of the pores inside the monolithic column.

[0207] The anion exchange capacities of Comparative Examples 2, 3, 4, and 5 were 112.8 μmol / mL, 120.8 μmol / mL, 122.5 μmol / mL, and 124.8 μmol / mL, respectively. Compared with Example 7, the anion exchange densities were all lower, indicating that both excessively high and low temperatures, as well as excessively low concentrations of the 2,3-epoxypropyltrimethylammonium chloride solution, can affect the coupling efficiency. It is speculated that excessively high solvent concentrations affect the diffusion effect of the solid-liquid reaction, while excessively low concentrations reduce the contact opportunities on the local pore surfaces. Excessively high temperatures may cause the reaction rate of 2,3-epoxypropyltrimethylammonium chloride with the overall column to be slower than the rate of epoxy degradation.

[0208] The anion exchange capacities of Comparative Examples 6-10 were 101.2 μmol / mL, 98.2 μmol / mL, 91.2 μmol / mL, 98.6 μmol / mL, and 102.0 μmol / mL, respectively. Compared with Example 8 (121.6 μmol / mL), Comparative Example 6 used the one-pot method (Method 4), indicating that the coupling efficiency was significantly lower than that of Method 3, which involved a pre-reaction followed by the addition of solvent A. The lower anion exchange densities of Comparative Examples 7-10 indicate that excessively high or low amounts of solvent A, or excessively high temperatures, can affect the coupling efficiency.

[0209] Bacterial lysate enrichment of plasmid DNA test

[0210] Test subjects: Examples 1-18 and Comparative Examples 1-12

[0211] Test method: as follows

[0212] 1. Preparation of bacterial lysate:

[0213] 1.1. Bacterial strain amplification and culture, strain (Escherichia coli TOP10, pET-30a-rspa, approximately 6500bp). (Bacterial strain amplification is a routine operation in the field of bioengineering and will not be described in detail here);

[0214] 1.2. Alkaline lysis of bacterial cells and amplification: After centrifugation, the bacterial cells are collected as solids and the crude plasmid is extracted using the classic three-step alkaline lysis method (this is a routine operation in the field of bioengineering and will not be described in detail here).

[0215] 1.3. Ammonium sulfate / calcium chloride precipitation method for RNA removal, generally with a salt concentration of 0.3-2M calcium chloride or ammonium sulfate (this is a routine operation in the field of bioengineering and will not be described in detail here);

[0216] 1.4. Collect the supernatant by centrifugation, which is the crude plasmid before column loading;

[0217] 2. Column chromatography test on lysis buffer:

[0218] 2.1 Place the reacted quaternary ammonium salt anion exchange chromatography block into a 1 mL radial flow chromatography apparatus (Yuji biological chromatography apparatus, specifically the device described in Chinese Utility Model Patent Publication No. CN219743990U, the reference of which is incorporated herein by reference in its entirety);

[0219] 2.2. Connect the chromatography fixture containing the quaternary ammonium salt anion exchange block to the liquid chromatography equipment, and use equilibration buffer W: 50mM Tris-HCl + 10mM EDTA, pH 7.2-7.4, equilibrate the column by 5-10 times its volume until the conductivity, pH, and UV baselines are stable.

[0220] 2.3. Take the crude plasmid (pET-30a-rspa, 6500bp), dilute it to a conductivity ≤30ms / cm, and inject 150mL through the chromatography system at a flow rate of 5mL / min; use (5%-25% elution buffer E + 75%-95% equilibration buffer W) to wash away impurities.

[0221] 2.4 Use elution buffer E (50mM Tris-HCl + 10mM EDTA + 2M NaCl, pH 7.2-7.4) for gradient elution and collect plasmid DNA at a flow rate of 2-3 mL / min; collect the elution samples separately for micro-ultraviolet spectrophotometry and agarose gel electrophoresis.

[0222] 2.5. Regeneration: First, regenerate the chromatographic column with 100% eluent at a flow rate of 2-3 mL / min; then wash the column with 1M sodium hydroxide, and immediately wash the column with deionized water until neutral.

[0223] Chromatographic test results: See Table 5 below:

[0224] Table 5 Chromatographic test results

[0225] As shown in Table 5, the monolithic column modified with quaternary ammonium salts possesses the ability to enrich plasmid DNA from bacterial lysis and remove some impurities through gradient elution. Figures 5, 6, and 7 are the chromatograms for Examples 1, 7, and 13, respectively. FT represents the flow-through portion, i.e., non-negatively charged or weakly negatively charged molecules in the lysis buffer; W represents the eluted portion of negatively charged molecules bound to the column (such as RNA, HCP, and other negatively charged small molecules); E represents the eluted, relatively pure plasmid DNA; and C represents the sodium hydroxide regeneration peak. In Examples 1, 7, and 13, the pore size increases sequentially, the specific surface area decreases sequentially, and the corresponding ligand density gradually decreases. Therefore, the strong retention decreases sequentially. Smaller pore sizes result in excessively high local ligand densities, leading to some plasmid DNA not effectively dissociating under certain ionic strength elution conditions, as shown in Figures 5-6.

[0226] The recoverable plasmid loadings shown in Table 5 are basically consistent with the anion exchange capacity results in Table 4, showing a positive correlation. For example, the loadings of Example 7 and Comparative Example 1 are 3.02 mg (unsaturated loading) and 1.24 mg, respectively. Due to the different coupling methods, the amount of plasmid loaded on the monolithic column with 4.2 μm pores is significantly different. The loadings of Comparative Examples 2-5 are 2.12 mg, 2.77 mg, 2.83 mg, and 2.76 mg, respectively, which are consistent with the above-mentioned ion exchange capacity results. Similarly, Examples 8 and Comparative Examples 6-10 are shown in Table 5.

[0227] The loading of Comparative Example 11 was 1.43 mg, which was significantly lower than that of Example 7, indicating that the hydroxylation process had a significant effect on increasing the loading.

[0228] The loading capacities in Example 1 and Comparative Example 12 were 3.01 mg (unsaturated loading, as some plasmid DNA was eluted by sodium hydroxide due to the small pore size) and 2.71 mg, respectively. Excessive concentration or temperature actually reduced the loading capacity, consistent with the anion exchange capacity results. The decrease in loading capacity was mainly due to coupling efficiency rather than the effect of concentration or temperature on the pore size, as shown in Figures 3-4.

[0229] Examples 1-6 show elution salt concentrations ranging from 65% to 85%; Examples 7-12 show elution salt concentrations ranging from 55% to 78%; Examples 13-18 show elution salt concentrations ranging from 50% to 72%. As the pore size increases, the ligand density gradually decreases, and the elution ion strength gradually decreases, resulting in a gradually decreasing elution salt ratio. Compared to conventional weak anion exchange DEAE, strong anion exchange requires a higher salt concentration because the strength of quaternary ammonium salts and hydroxide anions is greater than that of chloride anions, thus requiring a higher proportion of ion strength for exchange. Examples 5, 11, and 17 involve N,N,N-trimethyl-3-[1-oxo-2-propenylamino]propylammonium chloride ligand coupling. As shown in Table 5, their elution salt concentration ratios are relatively low, possibly because they contain amide groups, which have certain hydrogen bonding interactions, allowing for relatively gentle elution. The elution strength of different ligands can be comprehensively considered based on the binding strength of the purified substance, such as plasmid DNA, the difference in binding strength of impurities, and the selection of pore size. The chromatography process can be optimized and balanced in terms of purity, yield, and efficiency to achieve better results.

Claims

1. A monolithic column, characterized in that, Quaternary ammonium salts are grafted onto the inner surfaces of the holes and channels in the integral column.

2. The integral column as described in claim 1, characterized in that, The quaternary ammonium salt group is EP-OCH2CH(OH)CH-N + R 1 R 2 R 3 It exists in the form of; in: EP is epoxy resin; R 1 R 2 R 3 The groups are alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine)yl, polyvinyl alcohol, and polyvinylamine; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine), polyvinyl alcohol, and polyvinylamine groups are optionally surrounded by amino, hydroxyl, or -C groups. 1-6 Alkyl, -C 1-6 Hydroxyalkyl, -C 1-6 It is aminoalkyl substituted or hybridized with O, N, Si, P, or S.

3. The integral column as described in claim 2, characterized in that, The alkyl group is preferably methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 1-methylpropyl, 1,1-dimethylethyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl. The alkenyl group is preferably vinyl, propenyl, propenyl, 2-propenyl, 2-methylpropenyl, butenyl, butenyl, butenyl, 3-butenyl, butane-1,3-dienyl, 2-methylbutenyl, hexenyl, hexenyl, hexenyl, hexenyl, hexenyl, hexenyl, and hexenyl-1,3-dienyl groups. The alkynyl group is preferably ethynyl, 1-propynyl, 2-propynyl (propynyl), 1-butynyl, 2-butynyl, and 3-butynyl groups; The cycloalkyl group is preferably cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, or cyclooctyl group. The heterocyclic alkyl group is preferably pyrrolidinyl, imidazolidinone, pyrazolyl, piperidinyl, 2,5-piperazinyl, pyranyl, or morpholinyl group; The poly(alkylene oxide) group is preferably polyethylene glycol, polypropylene glycol, or polybutylene glycol, with the terminal group preferably being hydroxyl or amino, and the molecular weight preferably being 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, and preferably 3000. The poly(alkyleneamine) group is preferably polyethylenediamine, polypropylenediamine, or polybutylenediamine, and the terminal group is preferably hydroxyl, amino, or C-terminated. 1-6 The substituted amino group preferably has a molecular weight of 500-5000, more preferably 1000-5000, more preferably 2000-4000, more preferably 2500-3500, and more preferably 3000; The polyvinyl alcohol group is preferably the end or middle part of polyvinyl alcohol with EP-OCH2CH(OH)CH-N + Connected, preferably with a molecular weight of 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, preferably 3000; The polyvinylamine group is preferably the end or middle part of polyvinyl alcohol with EP-OCH2CH(OH)CH-N + Connected, preferably with a molecular weight of 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, preferably 3000.

4. The integral column as described in any one of claims 1-3, characterized in that, The EP-OCH2CH(OH)CH-NR 1 R 2 R 3 The anion exchange capacity of the epoxy resin in the form of the epoxy resin is 20 μmol / mL-200 μmol / mL, preferably 20 μmol / mL, 30 μmol / mL, 40 μmol / mL, 50 μmol / mL, 60 μmol / mL, 70 μmol / mL, 80 μmol / mL, 90 μmol / mL, 100 μmol / mL, 110 μmol / mL, 120 μmol / mL, 130 μmol / mL, 140 μmol / mL, 150 μmol / mL, 160 μmol / mL, 170 μmol / mL, 180 μmol / mL, 190 μmol / mL, and 200 μmol / mL. The pore size of the integral column is 2μm-6μm, preferably 2.0μm, 2.1μm, 2.2μm, 2.3μm, 2.4μm, 2.5μm, 2.6μm, 2.7μm, 2.8μm, 2.9μm, 3.0μm, 3.1μm, 3.2μm, 3.3μm, 3.4μm, 3.5μm, 3.6μm, 3.7μm, or 3.8μm. , 3.9μm, 4.0μm, 4.1μm, 4.2μm, 4.3μm, 4.4μm, 4.5μm, 4.6μm, 4.7μm, 4.8μm, 4.9μm , 5.0μm, 5.1μm, 5.2μm, 5.3μm, 5.4μm, 5.5μm, 5.6μm, 5.7μm, 5.8μm, 5.9μm, 6.0μm; The pore volume of the monolithic column is 1.1 mL / g-1.8 mL / g, preferably 1.10 mL / g, 1.15 mL / g, 1.20 mL / g, 1.25 mL / g, 1.30 mL / g, 1.35 mL / g, 1.40 mL / g, 1.45 mL / g, 1.50 mL / g, 1.55 mL / g, 1.60 mL / g, 1.65 mL / g, 1.70 mL / g, 1.75 mL / g, or 1.80 mL / g. The porosity of the integral column is 55%-68%, preferably 55.0%, 55.5%, 56.0%, 56.5%, 57.0%, 57.5%, 58.0%, 58.5%, 59.0%, 59.5%, 60.0%, 60.5%, 61.0%, 61.5%, 62.0%, 62.5%, 63.0%, 63.5%, 64.0%, 64.5%, 65.0%, 65.5%, 66.0%, 66.5%, 67.0%, 67.5%, and 68.0%.

5. The integral column as described in any one of claims 1-4, characterized in that, The raw materials for the monolithic column include substance A containing polyepoxy groups, pore-forming agent B, and quaternary ammonium salt modifier D; The quaternary ammonium salt modifier D possesses NR 1 R 2 R 3 It exists in the form of; Among them, R 1 R 2 R 3 The groups are alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine)yl, polyvinyl alcohol, and polyvinylamine; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, poly(alkyleneoxy)yl, poly(alkyleneamine), polyvinyl alcohol, and polyvinylamine groups are optionally surrounded by amino, hydroxyl, or -C groups. 1-6 Alkyl, -C 1-6 Hydroxyalkyl, -C 1-6 It is aminoalkyl substituted or hybridized with O, N, Si, P, or S.

6. A method for preparing a monolithic column as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Hydroxylate the entire column; S2. The hydroxylated monolithic column is quaternized with ammonium salt.

7. The preparation method according to claim 6, characterized in that, Step S1 includes: S13. Add an acidic aqueous solution to the dried monolithic column and heat it to react, so that the residual epoxy groups in the monolithic column are converted into hydroxyl groups.

8. The preparation method according to any one of claims 6-7, characterized in that, Step S2 includes: S23. Add quaternary ammonium salt modifier D solution to the monolithic column, thoroughly wet it, add the catalyst, and heat the reaction to convert the hydroxyl groups in the monolithic column into quaternary ammonium salt groups; or S23. After mixing the quaternary ammonium salt modifier D solution with the catalyst, add the monolithic column and heat the reaction to convert the hydroxyl groups in the monolithic column into quaternary ammonium salt groups.

9. The preparation method according to claim 8, characterized in that, The catalyst is a base or acidic cerium ammonium nitrate; the base is one or more of potassium hydroxide and sodium hydroxide; the acidic cerium ammonium nitrate is a mixture of cerium ammonium nitrate and an acid, and the acid is one or more of sulfuric acid, hydrochloric acid, nitric acid, formic acid, and acetic acid.

10. The monolithic column as described in any one of claims 1-5 or the monolithic column prepared by the preparation method as described in any one of claims 6-9 is used for DNA or RNA enrichment and separation.

11. A chromatographic column, characterized in that, It includes the integral column as described in any one of claims 1-5 or the integral column prepared by the preparation method as described in any one of claims 6-9.

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