Weak anion-modified monolithic column

Abstract

Disclosed is a monolithic column material, primary amine groups or secondary amine groups or tertiary amine groups being grafted on the internal surfaces of pores and channels of the monolithic column material. The primary amine groups, secondary amine groups or tertiary amine groups exist in the form of EP-OCH2CH(OH)CH-NR1R2. A monolithic column prepared by the present invention is rich in hydroxyl groups, has excellent compatibility with elution objects containing biological macromolecules, and also has excellent specific adsorption capacity for nucleic acid substances, making it suitable for enrichment and separation of DNA and / or RNA substances.

Description

A weakly anion-modified monolithic column

[0001] This application claims priority to Chinese patent application 2024105777528, 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] An integral column having primary, secondary, or tertiary amine groups grafted onto the inner surfaces of its pores and channels.

[0012] In some embodiments, the primary, secondary, or tertiary amine group is represented by EP-OCH2CH(OH)CH-NR. 1 R 2 It exists in the form of.

[0013] in:

[0014] EP is epoxy resin;

[0015] R 1 R 2 The groups are hydrogen, 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 connected to the end or middle part of the polyvinyl alcohol with EP-OCH2CH(OH)CH-N, and the molecular weight is preferably 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, and preferably 3000.

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

[0025] In some embodiments, the EP-OCH2CH(OH)CH-NR 1 R 2 The anion exchange capacity of the epoxy resin in the form of [the resin] is 30 μmol / mL to 300 μmol / mL, preferably 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, 200 μmol / mL, 210 μmol / mL, 220 μmol / mL, 230 μmol / mL, 240 μmol / mL, 250 μmol / mL, 260 μmol / mL, 270 μmol / mL, 280 μmol / mL, and 290 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 a weak anionic modifier D.

[0076] In some embodiments, the weak anionic modifier D possesses NHR 1 R 2 It exists in the form of.

[0077] Among them, R 1 R 2 The groups are hydrogen, 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 part of the polyvinyl alcohol connected to NH, and the molecular weight is preferably 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, and preferably 3000.

[0086] The polyvinylamine group is preferably the end or middle part of polyvinyl alcohol connected to NH, and the molecular weight is preferably 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, and 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. Oxidize the hydroxylated monolithic ring;

[0091] S3. The epoxidized monolithic column is weakly anionic modified.

[0092] In step S1, hydroxylation includes the following steps:

[0093] S11. Clean the entire column;

[0094] S12. Drying monolithic column;

[0095] S13. Hydroxylated monolithic column;

[0096] S14. Post-processing.

[0097] In step S11, a monolithic column is taken and rinsed with 10-20 times its volume of deionized water to remove the protective solution.

[0098] 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℃.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] In step S14, the hydroxylated monolithic column is rinsed with deionized water until the pH is neutral.

[0104] 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.

[0105] In step S2, epoxidation includes the following steps:

[0106] S21. Clean the entire column;

[0107] S22. Drying monolithic column;

[0108] S23. Epoxidized monolithic column;

[0109] S24. Post-processing.

[0110] In step S21, a monolithic column is taken and rinsed with 10-20 times its volume of deionized water to remove the protective solution.

[0111] 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℃.

[0112] In step S23, a solvent is added to the dried monolithic column, and after it is fully wetted, epichlorohydrin is added, followed by the addition of an alkali, and the temperature is raised to react, so that the hydroxyl groups in the monolithic column are converted into epoxy groups.

[0113] The solvent is a DMSO:H2O mixed solvent in a ratio of 3:7-7:3, preferably 4:6, 5:5, or 6:4. The volume ratio of the solvent to the overall column is (3-30):1, preferably 5:1, 7:1, 10:1, 15:1, 20:1, 25:1, or 30:1.

[0114] The amount of epichlorohydrin added is 3-30% of the solvent mass, preferably 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29%.

[0115] The alkali is one or more of sodium hydroxide, potassium hydroxide, and ammonia water. The amount added is 0.5-5% of the solvent mass, preferably 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, or 4.5%.

[0116] The heating reaction involves heating the system to 40-80℃ and reacting for 1-8 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, 6.0h, 6.5h, 7.0h, and 7.5h.

[0117] In step S24, the epoxidized monolithic column is rinsed with ethanol and deionized water until the pH is neutral.

[0118] In step S3, the weak anion modification includes the following steps:

[0119] S31. Mixed solvent and weak anionic modifier D;

[0120] S32. Wet the entire column with the mixed solution described in S31;

[0121] S33 weakly anion-modified monolithic column;

[0122] S34 post-processing.

[0123] The solvent in step S31 is one or more of DMF, DMSO, and DMAc. The amount of solvent added is 3-30% of the total column volume, preferably 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29%. The amount of the weak anionic modifier D added is 10-50% (v / v) of the solvent volume, preferably 15%, 20%, 25%, 30%, 35%, 40%, or 45%. Vibration, ultrasound, or other methods can also be used to assist mixing in step S31.

[0124] In step S32, the epoxidized monolithic column prepared in step S2 is added to the mixed solution described in S31 and thoroughly impregnated. In step S32, vibration, ultrasound, vacuuming, or other methods can also be used to assist in impregnation.

[0125] In step S33, the temperature is increased to allow the epoxy groups in the overall column to fully react with the weak anionic modifier D.

[0126] The heating reaction involves heating the system to 60-100℃ and reacting for 2-15 hours. The preferred temperatures are 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, and 100℃. The preferred reaction times are 2.0h, 2.5h, 3.0h, 3.5h, 4.0h, 4.5h, 5.0h, 5.5h, 6.0h, 6.5h, 7.0h, 7.5h, 8.0h, 8.5h, 9.0h, 9.5h, 10.0h, 10.5h, 11.0h, 11.5h, 12.0h, 12.5h, 13.0h, 13.5h, 14.0h, 14.5h, and 15.0h.

[0127] In step S34, the entire column is cooled and removed, and then rinsed with ethanol and deionized water until the pH is neutral.

[0128] After step S34 is completed, in order to protect the entire column, it can be stored in a 20%-70% ethanol solution for later use.

[0129] 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.

[0130] 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.

[0131] A fourth aspect of the present invention is to provide a chromatographic column comprising the aforementioned monolithic column.

[0132] 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.

[0133] The reagents and raw materials used in this invention are all commercially available.

[0134] The positive and progressive effects of this invention are as follows:

[0135] 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.

[0136] 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.

[0137] 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

[0138] Figure 1 shows the pore size distribution of the integral column block obtained in Example 8.

[0139] Figure 2 shows the pore size distribution of the integral column block prepared in Comparative Example 3.

[0140] Figure 3 shows the pore size distribution of the integral column block prepared in Comparative Example 4.

[0141] Figure 4 is a scanning electron microscope image of the monolithic column obtained in Preparation Example 3.

[0142] Figure 5 is a scanning electron microscope image of the monolithic column obtained in Example 8.

[0143] Figure 6 is a scanning electron microscope image of the monolithic column obtained in Comparative Example 3.

[0144] Figure 7 is a scanning electron microscope image of the monolithic column obtained in Comparative Example 4.

[0145] Figure 8 is a chromatographic diagram of the monolithic column block prepared in Example 7. Detailed Implementation

[0146] 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.

[0147] Unless otherwise specified, the reagents used in the following examples and comparative examples are all commercially available products in the art.

[0148] Preparation Example

[0149] 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.

[0150] Transfer the 1ml monolithic column block after desiccation to a glass reaction flask, add 0.2M deionized sulfuric acid aqueous solution, and add 10ml of reaction solution per block (1mL); sonicate for 30min 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℃, and keep warm for 4h; place the hydroxylated monolithic column block on a sintered glass funnel and rinse with deionized water until the pH is neutral (6.5-7).

[0151] The density of the bulk epoxy group after hydroxylation was determined to be 0 by epoxy group density measurement;

[0152] S2. Epoxidation: Take 1 mL of hydroxylated monolithic column, rinse with 20 times its volume of deionized water through a sand core funnel, and then use a vacuum pump to dry it through the sand core funnel for 60 min at an ambient temperature of 25℃.

[0153] Transfer the 1 mL monolithic column block after vacuum drying to a glass reaction flask. First, add solvent (50% DMSO deionized water), 10 mL per block (1 mL). Sonicate for 20 min to fully wet the monolithic column block. Then add epichlorohydrin, 10% of the solvent mass, and continue sonicating for 30 min. Finally, add 2% sodium hydroxide and continue sonicating for 10 min, controlling the temperature at 20℃, and mix thoroughly. Transfer to a constant temperature water bath at 60℃ and react for 6 hours. Rinse with ethanol and water alternately, each time with 20 times the volume of the block. After washing, vacuum dry and store at low temperature.

[0154] Table 1. Performance of Epoxidized Monolithic Column Preparation Examples

[0155] By measuring the epoxy group density in Examples 1-4, the average pore size gradually increased. After hydroxylation followed by epoxidation, the epoxy group density decreased with increasing pore size.

[0156] Comparative Examples 1 and 2 are examples 1 and 4, respectively, where epoxidation was performed directly without hydroxylation. The ligand density in these examples was significantly lower than the values ​​obtained after hydroxylation followed by epoxidation. This indicates that a small amount of epoxy groups remained after the monolithic column was formed. Hydroxylation of these epoxy groups converted them into hydroxyl groups, which, along with the hydroxyl groups already present in the original column, acted as reactants. In the subsequent epoxidation step, all hydroxyl groups were converted back to epoxy groups, increasing the epoxy group density in the monolithic column. Furthermore, hydroxylation and epoxidation increased the flexibility of the epoxy groups, and the presence of C3 linking groups between the epoxy groups and the monolithic column substrate allowed specifically adsorbed groups to extend from the substrate surface, enhancing the trapping ability.

[0157] Example

[0158] Add DMF solvent to the reaction flask at a volume of 10 mL / block (1 mL). Then add solution A as shown in the table below, at a volume of 30% of the solvent mass. Mix the two thoroughly and sonicate for 10 min.

[0159] Add the prepared monolithic epoxidized column to the above reaction solution, and sonicate or vacuum for 10 minutes to ensure thorough wetting. Transfer the reaction flask to a 60°C oil bath and react for 2 hours. Then raise the temperature to 80°C and react for 10 hours. After cooling, remove the reacted monolithic column and wash it twice with alternating ethanol and water, with a washing volume of 20 times the column volume, until the pH of the washing solution reaches 6.5-7. Finally, store the monolithic column in 20%-70% ethanol for later use.

[0160] Table 2. Processes of Examples and Comparative Examples

[0161] Effect test

[0162] Pore ​​size distribution, pore volume, and porosity determination

[0163] Test method: Mercury porosimetry

[0164] Test equipment: USA - McMurray-Autopore V 9620

[0165] Test results:

[0166] Examples 1-6 show that after the monolithic column with an average pore size of 2.7 μm was modified with amine epoxy, the pore size, pore volume and porosity parameters did not change significantly.

[0167] Examples 7-12 show that after the monolithic column with an average pore size of 4.2 μm was modified with amine epoxy, the pore size, pore volume and porosity parameters did not change significantly.

[0168] Examples 13-18 show that after the monolithic column with an average pore size of 5.2 μm was modified with amine epoxy, the pore size, pore volume and porosity parameters did not change significantly.

[0169] Comparative Examples 1-4 show a significant decrease in pore size, indicating that increasing the reaction temperature or the amount of polyethyleneimine significantly affects the pore size. By controlling the temperature and dosage, the modification reaction can be prevented from affecting the overall pore structure of the column. In Comparative Examples 1-3, the pore size decreased from 4.2 μm to 1.73 μm, 0.56 μm, and 0.15 μm, respectively. Comparative Example 4 exhibits an abnormal pore structure, with most pores potentially completely destroyed or blocked.

[0170] Table 3. Test results of pore size distribution, pore volume, and porosity.

[0171] Figure 1 shows the pore size distribution of Example 8, Figure 2 shows the pore size distribution of Comparative Example 3, and Figure 3 shows the pore size distribution of Comparative Example 4. The figures clearly show that after the excessive high-temperature reaction of polyethyleneimine 5000, the pore size distribution becomes wider and the pore size significantly smaller. In Comparative Example 4, the pore size distribution is abnormal, with no normal distribution curve, suggesting that the pore structure is essentially destroyed and blocked. Only residual pores of varying sizes remain.

[0172] Field emission electron microscopy scanning test

[0173] Test subjects: Preparation Example 3, Example 8, Comparative Example 3, Comparative Example 4

[0174] Testing equipment: Zeiss Sigma 300 (Germany), Oxford Spectroscopy.

[0175] Test results:

[0176] Figure 4 is an electron microscope image of Preparation Example 3; Figure 5 is an electron microscope image of Example 8; Figure 6 is an electron microscope image of Comparative Example 3; and Figure 7 is an electron microscope image of Comparative Example 4. Based on the electron microscope results, it can be clearly seen that the pore size of Comparative Examples 3 and 4 is significantly smaller, and many pore gaps are completely blocked. The results are consistent with the pore size distribution, pore volume, and porosity test results obtained using the aforementioned mercury intrusion porosimetry method.

[0177] Epoxy ligand density determination

[0178] Test subjects: Examples 1-4 and Comparative Examples 1-2 in Table 1

[0179] Test method: Sodium thiosulfate-hydrochloric acid titration method.

[0180] After being prepared using epoxy-modified materials, the chromatographic materials of Examples 1-4 and Comparative Examples 1-2 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:

[0181] S = 1000 * [M HCl [(V0-V1)*ρ / W]

[0182] S: Epoxy ligand density, μmol / mL;

[0183] M HCl Hydrochloric acid concentration, mmol / mL;

[0184] V0, V1: Volumes of HCl before and after titration, in mL;

[0185] ρ: Medium density (1.2 g / mL);

[0186] W: The mass (g) of the whole block weighed.

[0187] Test equipment: acid-base titration apparatus.

[0188] Test results: See Table 1.

[0189] Anion exchange capacity determination

[0190] Test subjects: the monolithic anion exchange columns prepared in Examples 1-18 and Comparative Examples 1-4 as shown in Table 2 above.

[0191] Test method: GB 5760-86.

[0192] Test equipment: acid-base titration apparatus.

[0193] Test results are shown in Table 4 below.

[0194] Table 4. Anion exchange capacity results for each example and comparative example.

[0195] Bacterial lysate enrichment of plasmid DNA test

[0196] Test subjects: Examples 1-18 and Comparative Examples 1-3

[0197] Test method: as follows

[0198] 1. Preparation of bacterial lysate:

[0199] 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);

[0200] 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).

[0201] 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);

[0202] 1.4. Collect the supernatant by centrifugation, which is the crude plasmid before column loading;

[0203] 2. Column chromatography test on lysis buffer:

[0204] 2.1 Place the well-reacted weak anion exchange chromatography block (DEAE-Plus) into a 1 mL radial flow chromatography apparatus (Yuji Biochromatographic Apparatus);

[0205] 2.2. Connect the chromatography fixture containing the DEAE-Plus block to the liquid chromatography equipment, and use equilibration buffer W: 50mM Tris-HCl + 10mM EDTA, pH 7.2-7.4, to equilibrate the column by 5-10 times its volume until the conductivity, pH, and UV baselines are stable.

[0206] 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.

[0207] 2.4 Gradient elution was performed using elution buffer E (50mM Tris-HCl + 10mM EDTA + 2M NaCl, pH 7.2-7.4) to collect plasmid DNA at a flow rate of 2-3 mL / min. The elution samples were then analyzed using a micro-volume UV spectrophotometer and agarose gel electrophoresis.

[0208] 2.5. Regeneration: First, regenerate the DEAE-Plus column with 100% eluent at a flow rate of 2-3 mL / min; then wash the column with 1M sodium hydroxide, and immediately rinse the column with deionized water until neutral.

[0209] Chromatographic test results: See Table 5 below:

[0210] Table 5 Chromatographic test results

[0211] As can be clearly seen from Table 5, the purity of the crude plasmid increased significantly after whole-column enrichment (when OD260 / 280 < 1.8, it indicates contamination by impurities such as proteins; when OD260 / 280 > 2, it indicates contamination by small RNA in the plasmid sample; when 1.8 ≤ OD260 / 280 ≤ 2, the plasmid purity is relatively high and can be used for subsequent experiments); and the concentration of plasmid recovered by elution buffer E was significantly higher than that of the original sample; the OD260 / 280 ratio of the plasmid purity after elution E was between 1.8 and 1.96.

[0212] The elution buffer E ratio is proportional to the salt concentration in the elution buffer. The results show that 3-diethylaminopropylamine, diethylamine, and hexamethylenediamine can recover plasmids at lower salt concentrations, while polyethyleneimine 5000 requires a higher salt concentration for elution. This may be due to the steric hindrance of its polymer ligands and its complex amine composition, requiring a higher ionic strength to elute the plasmids. Furthermore, larger pore sizes result in easier elution, smaller elution volumes, and higher elution concentrations. Overall, in Examples 1-18, plasmid DNA of 6500 bp was generally recovered well, with yields typically around 1.5 mg.

[0213] The pore size of Comparative Examples 1-3 was smaller, but Comparative Example 1 could still recover about 1.5 mg of plasmid from 6500 bp plasmid DNA. In contrast, Comparative Examples 2-3 recovered about 1 mg and 0.8 mg of plasmid, respectively. Furthermore, the elution volume and the proportion of elution buffer E gradually increased in Comparative Examples 1-3, resulting in a decrease in yield. This was related to the smaller pore size and the resulting damage and blockage of the pore channels.

[0214] Figure 8 shows the chromatography diagram of Example 7. FT represents the injection flow-through peak, mainly consisting of impurities such as amino acids, proteins, pigments, fatty acids, and other small molecule metabolites in the E. coli lysate. W mainly represents residual RNA and some protein impurities eluted. E represents the plasmid portion eluted and enriched by the elution buffer. The chromatography diagram clearly shows good peak shape, a standard normal distribution of the elution peaks, and no obvious tailing. The maximum value of the FT flow-through peak is relatively stable at 750 mAU, indicating that the plasmid DNA did not elute.

[0215] 15000bp large plasmid loading and yield test

[0216] Test subjects: Examples 4, 10, and 16

[0217] Test method: as follows

[0218] 3.1. The anion exchange chromatography stack (DEAE-Plus) was placed in 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] 3.2. Connect the chromatography fixture containing the DEAE-Plus block to the liquid chromatography equipment, and use the equilibration solution: 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] 3.3. Take the plasmid sample (pHelper-CA plasmid DNA (approximately 15000bp, provided by Kaituo Biotechnology). This sample is pure and only the binding and elution efficiency of the ultra-large plasmid is being investigated), dilute it to 1 mg / mL, and inject 5 mg through the chromatography equipment injection system at a flow rate of 5 mL / min.

[0221] 3.4 Use eluent E (50mM Tris-HCl + 10mM EDTA + 2M NaCl, pH 7.2-7.4) for gradient elution, and elute the target substance with 75% eluent + 25% equilibration buffer at a flow rate of 2-3 mL / min.

[0222] 3.5. Regeneration: First, regenerate the DEAE-Plus column with 100% eluent at a flow rate of 2-3 mL / min; then wash the column with 1M sodium hydroxide, and immediately rinse the column with deionized water until neutral.

[0223] Test results: See Table 6

[0224] Table 6. Results of 15000bp plasmid loading and yield tests

[0225] The results in Table 6 clearly show that for ultra-large plasmid DNA (15000bp), the larger the pore size of the monolithic column, the higher the yield. The 5.2μm weak anion exchange chromatography monolithic column has the advantage of specific enrichment and separation in the chromatographic purification of ultra-large plasmid DNA.

Claims

1. A monolithic column, characterized in that, The integral column has primary, secondary, or tertiary amine groups grafted onto the inner surfaces of the holes and channels.

2. The integral column as described in claim 1, characterized in that, The primary, secondary, or tertiary amine group exists in the form of EP-OCH2CH(OH)CH-NR1R2; in: EP is epoxy resin; R 1 R 2 The groups are hydrogen, 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 connected to the end or middle part of the polyvinyl alcohol with EP-OCH2CH(OH)CH-N, and the molecular weight is preferably 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, and preferably 3000. The polyvinylamine group is preferably connected to the end or middle of polyvinyl alcohol with EP-OCH2CH(OH)CH-N, and the molecular weight is preferably 500-5000, preferably 1000-5000, preferably 2000-4000, preferably 2500-3500, and 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 The anion exchange capacity of the epoxy resin in the form of [the resin] is 30 μmol / mL to 300 μmol / mL, preferably 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, 200 μmol / mL, 210 μmol / mL, 220 μmol / mL, 230 μmol / mL, 240 μmol / mL, 250 μmol / mL, 260 μmol / mL, 270 μmol / mL, 280 μmol / mL, and 290 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, porogen B, and weak anionic modifier D; The weak anionic modifier D possesses NHR. 1 R 2 It exists in the form of; Among them, R 1 R 2 The groups are hydrogen, 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. Oxidize the hydroxylated monolithic ring; S3. The epoxidized monolithic column is weakly anionic modified.

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 solvent to the monolithic column, fully wet it, then add epichlorohydrin, followed by alkali, and heat the column to react, so that the hydroxyl groups in the monolithic column are converted into epoxy groups.

9. The preparation method according to any one of claims 6-8, characterized in that, Step S3 includes: S32. Add the epoxidized monolithic column prepared in step S2 to the mixed solution described in S31 and allow it to fully impregnate; S33. Heating reaction to allow the epoxy groups in the overall column to fully react with the weak anionic modifier D.

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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