Mercaptopyridine modified monolithic column

Abstract

Disclosed in the present invention is a monolithic column material, which has pyridine groups grafted onto the internal surfaces of pores and channels thereof. The pyridine groups are present in the form of EP-OCH2CH(OH)CH-SPy. The monolithic column prepared in the present invention is rich in hydroxyl, has excellent compatibility with elution objects containing biomacromolecules, and also has excellent specific adsorption capacity for nucleic acid substances, thereby being suitable for the enrichment and separation of DNA and / or RNA substances.

Description

A monolithic column modified with mercaptopyridine

[0001] This application claims priority to Chinese patent application 2024105777513, 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 having pyridine groups grafted onto the inner surfaces of its pores and channels.

[0012] In some embodiments, the pyridine group is present in the form of EP-OCH2CH(OH)CH-SPy.

[0013] in:

[0014] EP stands for epoxy resin, and Py is a substituted or unsubstituted pyridine ring.

[0015] The unsubstituted pyridine ring, which serves as the basic framework or functional group bond of the object, consists only of hydrogen atoms or their deuterium atoms.

[0016] The substituents in the substituted pyridine ring are alkyl, cycloalkyl, heterocyclic, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, hydroxyl, thiol, alkoxy, alkylthio, aryl ether, aryl thioether, halogen, cyano, aldehyde, acyl, carboxyl, ester, amide, sulfonyl, sulfonate, sulfonamide, amino, nitro, and silylalkyl groups.

[0017] The saturated aliphatic hydrocarbon groups such as alkylmethyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl can be substituted or unsubstituted. There are no particular limitations on the additional substituents added when substituted; examples include alkyl, halogen, aryl, and heteroaryl groups, and this will be consistent throughout the following description. Furthermore, there is no particular limitation on the number of carbon atoms in the alkyl group; however, for ease of acquisition or cost considerations, a range of 1 to 20, more preferably 1 to 8, is preferred.

[0018] The term cycloalkyl refers to saturated alicyclic hydrocarbon groups such as cyclopropyl, cyclohexyl, norbornyl, and adamantyl, which may be substituted or unsubstituted. The number of carbon atoms in the cycloalkyl group is not particularly limited, but is preferably in the range of 3 to 20.

[0019] The term "heterocyclic group" refers to an aliphatic ring, such as a pyran ring, piperidine ring, or cyclic amide, which has atoms other than carbon atoms within the ring. This group may be substituted or unsubstituted. The number of carbon atoms in the heterocyclic group is not particularly limited, but is preferably in the range of 2 or more and 20 or less.

[0020] The term "alkenyl" refers to unsaturated aliphatic hydrocarbon groups containing double bonds, such as vinyl, allyl, and butadienyl, which may be substituted or unsubstituted. The number of carbon atoms in the alkenyl group is not particularly limited, but is preferably in the range of 2 to 20.

[0021] The term "cycloalkenyl" refers to unsaturated alicyclic hydrocarbon groups containing a double bond, such as cyclopentenyl, cyclopentadienyl, and cyclohexenyl. These groups may be substituted or unsubstituted. The number of carbon atoms in the cycloalkenyl group is not particularly limited, but is preferably in the range of 3 to 20.

[0022] The term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group containing a triple bond, such as ethynyl, which may be substituted or unsubstituted. The number of carbon atoms in the alkynyl group is not particularly limited, but is preferably in the range of 2 to 20.

[0023] The term "aryl" can be any of the monocyclic or fused ring types, such as phenyl, naphthyl, fluorenyl, benzo[a]fluorenyl, dibenzo[a]fluorenyl, phenanthryl, anthracene, benzo[a]phenanthryl, benzo[a]anthryl, pyrene, fluoranthyl, triphenylenyl group, benzo[a]fluoranthyl, dibenzo[a]anthryl, peryl, helicenyl group, and other aromatic hydrocarbon groups. Phenyl, naphthyl, fluorenyl, phenanthryl, and anthracene are preferred. The aryl group may be substituted or unsubstituted. The number of carbon atoms in the aryl group is not particularly limited, but is preferably in the range of 6 to 40, more preferably 6 to 30. Furthermore, in the case of a phenyl group, if each of the two adjacent carbon atoms has a substituent, these substituents may form a ring structure with each other. As a result, the resulting group, depending on its structure, can be equivalent to one or more of the following: "substituted phenyl", "aryl group with a structure of two or more cyclic condensation", and "heteroaryl group with a structure of two or more cyclic condensation".

[0024] The term "heteroaryl" can refer to any of the monocyclic or fused ring types, such as pyridinyl, furanyl, thiopheneyl, quinolinyl, isoquinolinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, naphridinyl, cinnamyl, phthalazinyl, quinoxalinyl, quinazolinyl, benzofuranyl, benzothiopheneyl, indolyl, dibenzofuranyl, dibenzothiopheneyl, carbazoyl, benzocarbazoyl, carbolinyl group, indolocarbazoyl, benzofuranocarbazoyl, benzothiophenocarbazoyl, dihydroindocarbazoyl, benzoquinolinyl, acridineyl, dibenzoacridyl, benzoimidazoyl, imidazopyridyl, benzooxazolyl, benzothiazoyl, phenanthrolinel, and other cyclic aromatic groups having atoms other than carbon and hydrogen in one or more rings, i.e., heteroatoms. The heteroatom is preferably a nitrogen atom, an oxygen atom, or a sulfur atom. The heteroaryl group may be substituted or unsubstituted. The number of carbon atoms in the heteroaryl group is not particularly limited, but is preferably in the range of 2 or more and 40 or less, more preferably in the range of 2 or more and 30 or less.

[0025] The term alkoxy refers to a functional group such as methoxy, ethoxy, or propoxy that has an aliphatic hydrocarbon group bonded via an ether bond. It may be substituted or unsubstituted. The number of carbon atoms in the alkoxy group is not particularly limited, but is preferably in the range of 1 to 20.

[0026] An alkathio group refers to an alkoxy group in which the oxygen atom in the ether bond is replaced by a sulfur atom. This substitution may or may not be performed. The number of carbon atoms in the alkathio group is not particularly limited, but is preferably in the range of 1 to 20.

[0027] The term "aryl ether group" refers to a functional group, such as a phenoxy group, to which an aromatic hydrocarbon group is bonded via an ether bond. This group may be substituted or unsubstituted. The number of carbon atoms in the aryl ether group is not particularly limited, but is preferably in the range of 6 to 40.

[0028] The term "aryl thioether" refers to an aryl ether group in which the oxygen atom of the ether bond is replaced by a sulfur atom. Further substitution is possible. The number of carbon atoms in the aryl thioether group is not particularly limited, but is preferably in the range of 6 to 40.

[0029] The term "halogen" refers to atoms selected from fluorine, chlorine, bromine, and iodine.

[0030] The term "cyano" refers to a functional group represented by the structure -C≡N. Here, the carbon atom is bonded to the other functional groups.

[0031] An aldehyde group refers to a functional group represented by the structure -C(=O)H. Here, the carbon atom is bonded to other functional groups.

[0032] The term "acyl group" refers to functional groups such as acetyl, propionyl, benzoyl, and acryloyl, which are bonded to alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl groups via a carbonyl group, and these groups may be further substituted. The number of carbon atoms in the acyl group is not particularly limited, but is preferably 2 or more and 40 or less, more preferably 2 or more and 30 or less.

[0033] The term "ester group" refers to a functional group formed by ester bonds, such as alkyl, cycloalkyl, aryl, and heteroaryl groups, which may be further substituted. The number of carbon atoms in the ester group is not particularly limited, but is preferably in the range of 1 to 20. More specifically, examples include: methyl ester groups such as methoxycarbonyl, ethyl ester groups such as ethoxycarbonyl, propyl ester groups such as propoxycarbonyl, butyl ester groups such as butoxycarbonyl, isopropyl ester groups such as isopropoxymethoxycarbonyl, hexyl ester groups such as hexoxycarbonyl, and phenyl ester groups such as phenoxycarbonyl.

[0034] The term "amide group" refers to a functional group formed by alkyl, cycloalkyl, aryl, heteroaryl, etc., bonded via an amide bond, which may be further substituted. The number of carbon atoms in the amide group is not particularly limited, but is preferably in the range of 1 to 20. More specifically, examples include: methylamide, ethylamide, propylamide, butylamide, isopropylamide, hexylamide, phenylamide, etc.

[0035] The term sulfonyl group refers to a functional group formed by alkyl, cycloalkyl, aryl, heteroaryl, etc., bonded via a -S(=O)2- bond, which may be further substituted. The number of carbon atoms in the sulfonyl group is not particularly limited, but is preferably in the range of 1 to 20.

[0036] The term "sulfonate group" refers to a functional group formed by alkyl, cycloalkyl, aryl, or heteroaryl groups bonded via a sulfonate bond. Here, the sulfonate bond refers to a bond in which the carbonyl group (i.e., -C(=O)-) of an ester bond is substituted with a sulfonyl group (i.e., -S(=O)2-). Further substitution is possible. The number of carbon atoms in the sulfonate group is not particularly limited, but is preferably in the range of 1 to 20.

[0037] The term "sulfonamide group" refers to a functional group formed by alkyl, cycloalkyl, aryl, or heteroaryl groups bonded via a sulfonamide bond. Here, the sulfonamide bond refers to a bond in which the carbonyl group (i.e., -C(=O)-) of the amide bond is substituted with a sulfonyl group (i.e., -S(=O)2-). Further substitution is possible. The number of carbon atoms in the sulfonamide group is not particularly limited, but is preferably in the range of 1 to 20.

[0038] The term "amino" refers to an amino group, whether substituted or unsubstituted. Examples of substituents that are substituted include aryl, heteroaryl, straight-chain alkyl, and branched alkyl groups. Here, phenyl, naphthyl, pyridyl, and quinolinyl are preferred as aryl and heteroaryl groups. Further substitution is also possible. The number of carbon atoms is not particularly limited, but is preferably 2 or more and 50 or less, more preferably 6 or more and 40 or less, and particularly preferably 6 or more and 30 or less.

[0039] The term silyl group refers to a functional group bonded with substituted or unsubstituted silicon atoms, such as alkylsilyl groups like trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, propyldimethylsilyl, and vinyldimethylsilyl; or arylsilyl groups like phenyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, and trinaphthylsilyl. Further substitution is also possible. The number of carbon atoms in the silyl group is not particularly limited, but is preferably in the range of 1 to 30.

[0040] In some embodiments, the substituents in the substituted pyridine ring are methyl, sulfonamide, carboxyl, or nitro.

[0041] In some embodiments, the -Spy is derived from one or more of 2-mercaptopyridine, 4-mercaptopyridine, 2-mercapto-5-nitropyridine, 6-methylpyridine-2-mercapto, 6-mercaptopyridine-3-carboxylic acid, 4-mercaptopyridine-3-sulfonamide, and 6-mercaptopyridine-3-sulfonamide.

[0042] 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.

[0043] 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.

[0044] 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%.

[0045] In some embodiments, the raw material for the monolithic column includes substance A containing polyepoxy groups.

[0046] The term "substance containing polyepoxy groups" refers to a substance whose structure contains two or more epoxy groups.

[0047] In some embodiments, the substance containing a polyepoxy group is preferably a polyepoxy group glycidyl ether or a polyepoxy group glycidyl ester.

[0048] In some embodiments, the glycidyl ether may be one or more monomers satisfying the following structural formula I.

[0049] In structural formula I, R 1 Selected from hydrogen atoms, substituted or unsubstituted C1-C atoms 10 Alkyl groups, or epoxy groups;

[0050] n takes the form of a positive integer between 0 and 10;

[0051] L 1 Selected from oxygen or nitrogen atoms;

[0052] L 2 Selected from oxygen or nitrogen atoms;

[0053] X is selected from substituted or unsubstituted C1-C. 10 Alkylene, substituted or unsubstituted C3-C 10 cycloalkylene, Or, a benzene ring.

[0054] Preferably, the glycidyl ether is one or more of the following monomers (a)-(m);

[0055] The structure (a) above is the structure of glycerol triglycidyl ether.

[0056] The structure (b) above is the structure of pentaerythritol tetraglycidyl ether.

[0057] The structure (j) described above is the structure of 1,4-butanediol diglycidyl ether.

[0058] The structure (k) described above is the structure of trihydroxymethylethane triglycidyl ether.

[0059] The structure (l) described above is the structure of bisphenol A diglycidyl ether.

[0060] The structure (m) described above is the structure of bisphenol F diglycidyl ether.

[0061] In some embodiments, the glycidyl ether may be one or more polymers that satisfy the following structural formula II.

[0062] In structural formula II, R 2 It is selected from hydrogen atoms, substituted or unsubstituted C1-C 10 Alkyl groups, or,

[0063] m takes the form of a positive integer between 2 and 40.

[0064] Preferably, the glycidyl ether is one or more of the following polymers (1)-(4);

[0065] In some embodiments, the glycidyl ester may be one or more monomers that satisfy the following structural formula III;

[0066] 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.

[0067] Preferably, the glycidyl ester is a monomer (A) and / or (B) thereof;

[0068] In some embodiments, the substance containing a polyepoxy group is preferably one or more of the following monomers (I)-(IV);

[0069] In some embodiments, the substance containing polyepoxy groups is preferably a polyepoxy group glycidyl ether monomer and / or a polyepoxy group glycidyl ether polymer.

[0070] The polyepoxy group glycidyl ether monomers may be glycerol triglycidyl ether and / or pentaerythritol tetraglycidyl ether.

[0071] The polyepoxy group glycidyl ether polymer may be polyglycerol glycidyl ether and / or polypentaerythritol tetraglycidyl ether.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] The substance containing a monoepoxy group is preferably a monoepoxy group glycidyl ether or a monoepoxy group glycidyl ester.

[0076] 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.

[0077] 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.

[0078] In some embodiments, the raw material for the monolithic column includes pore-forming agent B.

[0079] 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.

[0080] 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.

[0081] 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%.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] In some embodiments, the raw material for the monolithic column includes catalyst C.

[0087] In some embodiments, the catalyst C may be a Lewis acid and / or a complex of a Lewis acid.

[0088] 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.

[0089] 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.

[0090] 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‰.

[0091] In some embodiments, the raw material for the monolithic column includes mercaptopyridine modifier D.

[0092] In some embodiments, the mercaptopyridine modifier D exists in the form of HS-Py.

[0093] Wherein, Py is a substituted or unsubstituted pyridine ring; the substitution positions of HS- on the pyridine ring are 2-, 3-, or 4-.

[0094] The unsubstituted pyridine ring, which serves as the basic framework or functional group bond of the object, consists only of hydrogen atoms or their deuterium atoms.

[0095] The substituents in the substituted pyridine ring are alkyl, cycloalkyl, heterocyclic, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, hydroxyl, thiol, alkoxy, alkylthio, aryl ether, aryl thioether, halogen, cyano, aldehyde, acyl, carboxyl, ester, amide, sulfonyl, sulfonate, sulfonamide, amino, nitro, and silylalkyl groups. The definitions of each group are the same as before.

[0096] A second aspect of the present invention is to provide a method for modifying a monolithic column.

[0097] The preparation method includes the following steps:

[0098] S1. Hydroxylate the entire column;

[0099] S2. Oxidize the hydroxylated monolithic ring;

[0100] S3. Modify the epoxidized monolithic column with mercaptopyridine.

[0101] In step S1, hydroxylation includes the following steps:

[0102] S11. Clean the entire column;

[0103] S12. Drying monolithic column;

[0104] S13. Hydroxylated monolithic column;

[0105] S14. Post-processing.

[0106] In step S11, a monolithic column is taken and rinsed with 10-20 times its volume of deionized water to remove the protective solution.

[0107] 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℃.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] In step S14, the hydroxylated monolithic column is rinsed with deionized water until the pH is neutral.

[0113] 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.

[0114] In step S2, epoxidation includes the following steps:

[0115] S21. Clean the entire column;

[0116] S22. Drying monolithic column;

[0117] S23. Epoxidized monolithic column;

[0118] S24. Post-processing.

[0119] In step S21, a monolithic column is taken and rinsed with 10-20 times its volume of deionized water to remove the protective solution.

[0120] 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℃.

[0121] 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.

[0122] 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.

[0123] The amount of epichlorohydrin 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%.

[0124] The alkali is one or more of sodium hydroxide, potassium hydroxide, and ammonia water. The amount added is 0.5-5% of the total column weight, preferably 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, or 4.5%.

[0125] 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.

[0126] In step S24, the epoxidized monolithic column is rinsed with ethanol and deionized water until the pH is neutral.

[0127] In step S3, the modification of mercaptopyridine includes the following steps:

[0128] S31. Mixed solvent, catalyst and mercaptopyridine modifier D;

[0129] S32. Wet the entire column with the mixed solution described in S31;

[0130] S33 mercaptopyridine modified monolithic column;

[0131] S34 post-processing.

[0132] The solvent in step S31 is an alcohol solvent, preferably one or more of methanol and ethanol. The amount of solvent added is 5-30 mL / mL, preferably 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, or 30 mL.

[0133] The catalyst is sodium alkoxide, preferably one or more of sodium methoxide and sodium ethoxide. The concentration of the catalyst is 3-30 mol% of the solution, preferably 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, or 30 mol%.

[0134] The concentration of the mercaptopyridine modifier D in the solution is 1-10% (w / v), preferably 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0135] Step S31 can also use vibration, ultrasound or other methods to assist mixing.

[0136] 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.

[0137] In step S33, the temperature is increased to allow the epoxy groups in the overall column to fully react with the mercaptopyridine modifier D.

[0138] The heating reaction involves heating the system to 40℃-80℃ and reacting for 10-36 hours. The preferred temperatures are 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃. The preferred reaction times are 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours, and 36 hours.

[0139] In step S34, the entire column is cooled and removed, and then rinsed with ethanol and deionized water until the pH is neutral.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] A fourth aspect of the present invention is to provide a chromatographic column comprising the aforementioned monolithic column.

[0144] 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.

[0145] The reagents and raw materials used in this invention are all commercially available.

[0146] The positive and progressive effects of this invention are as follows:

[0147] 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.

[0148] 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.

[0149] 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

[0150] Figure 1 shows the pore size distribution of the integral column block prepared in Example 1.

[0151] Figure 2 shows the pore size distribution of the integral column block obtained in Example 6.

[0152] Figure 3 is a scanning electron microscope image of the monolithic column obtained in Example 3.

[0153] Figure 4 is a scanning electron microscope image of the monolithic column obtained in Example 7.

[0154] Figure 5 is a chromatographic diagram of the monolithic column block prepared in Example 1. Detailed Implementation

[0155] 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.

[0156] Unless otherwise specified, the reagents used in the following examples and comparative examples are all commercially available products in the art.

[0157] Preparation Example

[0158] 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.

[0159] 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).

[0160] The density of the bulk epoxy group after hydroxylation was determined to be 0 by epoxy group density measurement;

[0161] 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℃.

[0162] 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 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.

[0163] Table 1. Performance of Epoxidized Monolithic Column Preparation Examples N: Indicates that this step was not performed.

[0164] 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.

[0165] 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.

[0166] Example

[0167] Weigh 20 mol% of reagent B as described in Table 2 and add it to the solution of solvent C as described in Table 2 (addition amount is 10 mL / mL block), and mix thoroughly in a glass reactor. Separately weigh 5% (w / v) of reagent A as described in Table 2 and add it to the aforementioned solution, and mix thoroughly.

[0168] Add the epoxidized chromatographic block to the aforementioned mixed solution and sonicate for 15 min.

[0169] The reaction mixture was then placed in a water bath shaker, heated to 60°C, and shaken at a frequency of 100 rpm for 24 hours.

[0170] After the reaction is complete, remove the monolithic column and wash it alternately with ethanol / deionized water until the pH of the droplet is neutral. Store the monolithic column in 20% ethanol for later use.

[0171] Table 2. Processes of Examples and Comparative Examples

[0172] Effect test

[0173] Pore ​​size distribution, pore volume, and porosity determination

[0174] Test method: Mercury porosimetry

[0175] Test equipment: USA - McMurray-Autopore V 9620

[0176] Test results:

[0177] The results from the examples show that the pore parameters remained largely normal after the click chemical coupling of thiol and epoxy. No pore collapse, blockage, or other abnormal phenomena were observed. Therefore, this method can successfully prepare the thiol-pyridine modified monolithic column without affecting its basic properties such as pore size distribution, pore volume, and porosity.

[0178] Table 3. Test results of pore size distribution, pore volume, and porosity.

[0179] Figure 1 shows the pore size distribution of Example 1, and Figure 2 shows the pore size distribution of Example 6. As can be seen from the figures, the pore size distribution after modification is concentrated and there is no significant change in the pore size distribution. It can be seen that the modification step has no effect on the basic properties such as pore size.

[0180] Field emission electron microscopy scanning test

[0181] Test subjects: Preparation Example 3, Example 7

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

[0183] Test results:

[0184] Figure 3 is an electron microscope image of Preparation Example 3; Figure 4 is an electron microscope image of Example 7. Preparation Example 3 and Example 7 are monolithic columns modified with epoxidation and quaternary ammonium salt, respectively, with pore sizes of approximately 4.2 μm. By comparing the electron microscope images, the pore structure after mercaptopyridine modification is normal and consistent with the porous structure of the unmodified monolithic column.

[0185] Epoxy ligand density determination

[0186] Test subjects: Examples 1-4 and Comparative Examples 1-2 in Table 1

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

[0188] After epoxidation modification, 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 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 reacted 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 / L;

[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] 15000bp large plasmid loading and yield test

[0198] Test subjects: Examples 1-22

[0199] Test method: as follows

[0200] 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);

[0201] 3.2. Connect the chromatography fixture containing the PYSH block to the liquid chromatography equipment and use the equilibration solution: 50mM Tris-HCl + 10mM EDTA + 2.5-3M (NH4)2SO4, pH 7.2-7.4. Equilibrate the column by 5-10 times its volume until the conductivity, pH, and UV baselines are stable.

[0202] 3.3. Take the plasmid sample (pHelper-CA plasmid DNA (approximately 15000bp, provided by Kaituo Biotechnology), supercoiled plasmid purity 90%), dilute it to 1 mg / mL, and inject 5 mL through the chromatography system at a flow rate of 3-5 mL / min.

[0203] 3.4 Elution was performed using eluent E (50mM Tris-HCl + 10mM EDTA + 1.7M (NH4)2SO4 + 0.3M NaCl) at a flow rate of 2-3 mL / min.

[0204] 3.5. Regeneration: First, regenerate the PYSH column with deionized water 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.

[0205] Test results: See Table 4

[0206] Table 4. Results of 15000bp plasmid loading and yield tests

[0207] For Examples 1-6, after modifying the 2.7 μm monolithic column, the yield was 50%-60%; the loading was ≥4.5 mg. As determined in Example 1, the injection was not supersaturated, and the flow-through FT portion consisted of open-ring plasmids (meaning that during injection, the mobile phase could be adjusted to allow impurities to flow through, indirectly improving the overall column utilization, as shown in Figure 5). The conventional microsphere-based thiopyridine method is the classic three-step chromatography method using agarose microspheres, including molecular sieves, 2-thiopyridine modified packing material, and anion exchange microspheres. When purifying ultra-large plasmid DNA with 2-thiopyridine packing material, the yield and loading are relatively low due to the diffusion effect required by the microsphere packing material. The yield is generally 30-40%, and the loading is 1-2 mg / mL. This type of packing column is generally suitable for separating plasmid molecules below 6000 bp.

[0208] Other similar monolithic column materials, such as high-density C4 polymethacrylate monolithic columns, separate supercoiled structures through hydrophobic interactions. Sample loading requires a high salt concentration to increase these hydrophobic interactions. Thiolpyridine purification plasmids, also known as thiophilic plasmids, contain a pyridine ring and carbon-sulfur bonds. The pyridine ring provides some hydrophobic interaction, while nitrogen provides ionic and hydrogen bonding, and sulfur is a strong electrophilic group. Compared to open-ring plasmids, supercoiled plasmids, due to their stronger topologically compressed helical configuration, have a relatively higher density of hydrogen bonds, hydrophobicity, and charge on the phosphate backbone, thus enabling separation through this difference.

[0209] Examples 7-12 show that after modifying a 4.2 μm monolithic chromatographic column with mercaptopyridine, the yield was 88%-90% and the loading was 3-4 mg. Examples 13-18 show that after modifying a 5.2 μm monolithic chromatographic column with mercaptopyridine, the yield was 92%-97% and the loading was 1-2 mg.

[0210] As shown in Table 4, the loading capacity gradually decreases while the yield gradually increases with increasing pore size. The purity of the supercoiled plasmids improved from the original 90%, indicating that the open-ring plasmids exhibited certain enrichment and separation effects on several coupled ligands. Regarding separation efficiency, the purity of 2-mercaptopyridine (Examples 1, 7, 13, 19-22), 4-mercaptopyridine (Examples 2, 8, 14), and 2-mercapto-6-methylpyridine (Examples 4, 10, 16) was generally above 95%. The purity of the other pyridine-extended compounds showed a corresponding increase, albeit a smaller one of about 2%. This may be because the nitro, carboxyl, and sulfonamide groups on the pyridine ring altered the overall hydrophobicity, polarity, hydrogen bonding, and ionic bonding of the ligands. Although the separation degree can be optimized through chromatographic process conditions, the relationship between purity, loading capacity or efficiency, and yield is a near-impossible triangle, requiring a balance that is quite challenging. 2-Mercaptopyridine, 4-mercaptopyridine, and 2-mercapto-6-methylpyridine can meet the purification requirements.

Claims

1. A monolithic column, characterized in that, The pores and channels of the integral column are grafted with pyridine groups.

2. The integral column as described in claim 1, characterized in that, The pyridyl group exists in the form of EP-OCH2CH(OH)CH-SPy; in: EP stands for epoxy resin, and Py is a substituted or unsubstituted pyridine ring. The substituents in the substituted pyridine ring are alkyl, cycloalkyl, heterocyclic, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, hydroxyl, thiol, alkoxy, alkylthio, aryl ether, aryl thioether, halogen, cyano, aldehyde, acyl, carboxyl, ester, amide, sulfonyl, sulfonate, sulfonamide, amino, nitro, and silylalkyl groups.

3. The integral column as described in claim 2, characterized in that, In some embodiments, the substituents in the substituted pyridine ring are methyl, sulfonamide, carboxyl, or nitro.

4. The integral column as described in any one of claims 1-3, characterized in that, 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 mercaptopyridine modifier D; The mercaptopyridine modifier D exists in the form of HS-Py; Wherein, Py is a substituted or unsubstituted pyridine ring; the substitution positions of HS- on the pyridine ring are 2-, 3-, or 4-.

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. Modify the epoxidized monolithic column with mercaptopyridine.

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 dried monolithic column, fully impregnate it, then add epichlorohydrin, followed by alkali, and heat the reaction to convert the hydroxyl groups in the monolithic column into epoxy groups.

9. The preparation method according to any one of claims 6-8, characterized in that, Step S3 includes: S31. Mixed solvent, catalyst and mercaptopyridine modifier D; S32. Wet the entire column with the mixed solution described in S31; S33 mercaptopyridine modified monolithic column; The solvent in step S31 is an alcohol solvent, preferably one or more of methanol and ethanol; The catalyst is sodium alkoxide, preferably one or more of sodium methoxide and sodium ethoxide.

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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