Chromatography support, method for producing chromatography support, and polymer having amine-based side chain

Abstract

A stationary phase for chromatography, which is obtained by supporting a polymer having an amine-based side chain on a support, the polymer having one or more repeating units selected from the group consisting of a repeating unit represented by formula (1) and a repeating unit represented by formula (2) (in the formulae, R 11 and R 21 represent a hydrogen atom or a methyl group; R 12 , R 13 , R 22 , and R 23 represent a saturated aliphatic hydrocarbon group; Y 1 and Y 2 represent an oxygen atom, NH, or NCH3; and n1 and n2 represent an integer of 1 or more and 8 or less).

Description

Technical Field

[0001] This disclosure relates to stationary phases for chromatography, methods for manufacturing stationary phases for chromatography, and polymers having amine side chains. Background Technology

[0002] Chromatography is the most widely used method for the separation and analysis of compounds, especially organic compounds. This method separates different substances by utilizing the inherent distribution ratio of substances relative to the solid (stationary phase) spatially fixed within a tube called a column or capillary, and the fluid (mobile phase) moving within its gaps.

[0003] In the life sciences, chromatography is indispensable for the separation and analysis of carbohydrates, nucleic acids, nucleosides, peptides, and metabolites. These substances are mostly hydrophilic and highly polar, and are typically separated and analyzed using chromatography with hydrophilic or highly polar stationary phases.

[0004] For example, Patent Document 1 discloses a method for separating and analyzing sugars by using a filler having a primary or secondary amino group as a hydrophilic functional group and by hydrophilic interaction chromatography.

[0005] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2017-116421 Summary of the Invention

[0006] The problem that the invention aims to solve However, even with conventional hydrophilic stationary phases, separation can be difficult depending on the substance. Therefore, to increase the freedom of stationary phase selection, it is necessary to develop novel hydrophilic stationary phases.

[0007] The subject of this disclosure is a novel hydrophilic stationary phase for chromatography.

[0008] Methods for solving problems To address the aforementioned issues, the inventors of this disclosure conducted in-depth research. They discovered that by supporting polymers with amine side chains on a support, novel stationary phases for chromatography exhibiting excellent hydrophilicity can be obtained. In other words, the main points of this disclosure are as follows.

[0009] [1] Chromatographic methods use a stationary phase, which is prepared by supporting a polymer with amine side chains on a support. The aforementioned polymers having amine side chains have one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2).

[0010] [Chemical Formula 1] (In equations (1) and (2), R) 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8. [2] The chromatographic method described in [1] uses a stationary phase in which the aforementioned polymer having amine side chains is supported on the aforementioned support by chemical bonds.

[0011] [3] The chromatographic method described in [1] or [2] uses a stationary phase for hydrophilic interaction chromatography.

[0012] [4] The stationary phase for chromatography as described in any one of [1] to [3], wherein the aforementioned support is a porous inorganic support or a non-porous inorganic support.

[0013] [5] Separation methods, including: In the separation process, the chromatographic stationary phase described in any one of [1] to [4] is used to separate substances by hydrophilic interaction chromatography.

[0014] [6] A method for manufacturing a stationary phase for chromatography, comprising any one of the steps (a) to (h) below, The monomers with amine side chains in (a) to (g) below are selected from one or more of the group consisting of monomers represented by formula (3) and monomers represented by formula (4). The polymers with amine side chains in (h) below have one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2).

[0015] (a) A process of free radical polymerization of monomers with amine side chains in the presence of a support with polymerizable functional groups. (b) A step of chain transfer polymerization of a monomer having an amine side chain in the presence of a compound having a crosslinkable silyl group and a chain transfer functional group to obtain a polymer having the aforementioned crosslinkable silyl group and amine side chain; and a step of combining the aforementioned crosslinkable silyl group of the polymer with functional groups on the surface of a support. (c) The process of polymerizing a monomer having amine side chains with a silane coupling agent having polymerizable functional groups, and the process of attaching the resulting polymer to a support via a silane coupling reaction. (d) The process of polymerizing a monomer with amine side chains with a silane coupling agent with polymerizable functional groups in the presence of a support. (e) A process of chain transfer polymerization of monomers with amine side chains in the presence of a support incorporating chain transfer functional groups. (f) A process of free radical polymerization of monomers with amine side chains in the presence of a support incorporating atom transfer radical polymerization initiator groups. (g) A step of subjecting a monomer having an amine side chain to atom transfer radical polymerization in the presence of a compound having a crosslinkable silyl group and an atom transfer radical polymerization initiator group to obtain a polymer having the aforementioned crosslinkable silyl group and amine side chain, and a step of combining the aforementioned crosslinkable silyl group of the polymer with functional groups on the surface of a support. (h) The process of coating a polymer with amine side chains onto the surface of a support. [Chemical Formula 2] (In equations (1) to (4), R) 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8. [7] The method for manufacturing a stationary phase for chromatography as described in [6], wherein the aforementioned support is a porous inorganic support or a non-porous inorganic support.

[0016] [8] A polymer having an amine side chain, having one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2).

[0017] [Chemical Formula 3] (In equations (1) and (2), R) 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8. [9] Polymers with amine side chains, as described in [8], are used as stationary phases in chromatography.

[0018]

[10] A method for manufacturing polymers having amine side chains, comprising a polymerization step of polymerizing monomers having amine side chains. The aforementioned monomers having amine side chains are selected from one or more of the group consisting of monomers represented by formula (3) and monomers represented by formula (4).

[0019] [Chemical Formula 4] (In equations (3) and (4), R) 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8.

[11] The method for manufacturing a polymer having amine side chains as described in

[10] , wherein the aforementioned polymerization is atom transfer radical polymerization, free radical polymerization, or chain transfer polymerization.

[0020] Invention Effects According to this disclosure, it is possible to obtain the effect of providing a novel stationary phase for chromatography that is hydrophilic.

[0021] Furthermore, the subject matter and effects of this disclosure are not specifically limited to the content described above, but also include the content that a person skilled in the art would understand based on the entire specification. Attached Figure Description

[0022] [ Figure 1 The graph is a plot obtained by drawing a graph with the stationary phase of the chromatographic method used in the embodiment, with the vertical axis being α (Tb / Tp) and the horizontal axis being α (U / 2dU).

[0023] [ Figure 2 The graph is a plot obtained by drawing a stationary phase for the chromatographic method of the embodiment, with the vertical axis being α (U / 5FU) and the horizontal axis being α (2'dU / 2'd5FU).

[0024] [ Figure 3 The graph is a plot obtained by drawing a stationary phase for the chromatographic method of the embodiment, with the vertical axis being α (5FU / 2'd5FU) and the horizontal axis being α (U / 2'dU).

[0025] [ Figure 4 The graph is a plot obtained by drawing a stationary phase for the chromatographic method of the embodiment, with the vertical axis being α (U / 2'dU) and the horizontal axis being α (I / isol). Detailed Implementation

[0026] The present disclosure is described below with specific examples, but each embodiment and its combination is merely an example, and appropriate additions, omissions, substitutions and other changes to the structure may be made without departing from the spirit of the present disclosure. The present disclosure is not limited to the embodiments.

[0027] Furthermore, the various methods disclosed in this specification can also be combined with any other features disclosed in this specification.

[0028] In this disclosure, "X~Y" indicating a range means "X or more, Y or less". Furthermore, when the numerical ranges represented by "X~Y" or "X or more, Y or less" are described in stages (e.g., in a preferred order), the upper and lower limits of each numerical range can be arbitrarily combined.

[0029] In this published text, the phrase “choose one or more groups consisting of X, Y, and Z” means any one of X, Y, Z, combinations of X and Y, combinations of X and Z, combinations of Y and Z, or combinations of X, Y, and Z.

[0030] In this public text, references such as "x1, x2, and x3, etc. X" refer to x1, x2, and x3 as examples of X, and do not imply that X is limited to x1, x2, and x3, etc.

[0031] 1. Stationary phase used in chromatography The first embodiment of this disclosure is a chromatographic stationary phase (hereinafter sometimes simply referred to as "stationary phase") formed by supporting a polymer having amine side chains on a support.

[0032] The stationary phase described in this embodiment exhibits high hydrophilicity due to its specific amine side chains. Therefore, the stationary phase described in this embodiment interacts strongly with both hydrophilic and polar substances, effectively retaining these substances and thus demonstrating high separation performance for both types of substances.

[0033] 1-1. Carrier The support for polymers with amine side chains can be a non-porous support or a porous support, preferably a porous support.

[0034] In this disclosure, porous support means a support in which fine pores are formed throughout the entire support, and also includes surface porous support (core-shell support) with a structure in which a non-porous core is covered by a porous layer.

[0035] When the support is a porous support, the average pore size of the support is not particularly limited, but is preferably 1 nm or more and 1,000 nm or less, more preferably 5 nm or more and 500 nm or less, and even more preferably 10 nm or more and 100 nm or less.

[0036] The average pore size of the support was determined based on JIS Z 8831-2:2010 (Powder (solid) pore size distribution and pore characteristics - Part 2: Methods for determination of mesopores and macropores based on gas adsorption; formulated on April 20, 2010).

[0037] When the support is a porous support, the specific surface area of ​​the support is not particularly limited, but is preferably 5 m². 2 / g or more 1,000m 2 / g or less, preferably 10m 2 / g or more 500m 2 / g or less.

[0038] When the support is a non-porous support, the specific surface area of ​​the support is not particularly limited, but is preferably 0.005 m². 2 / g or more and less than 5m 2 / g, more preferably 0.01m 2 / g or more 4m 2 / g or less.

[0039] The specific surface area of ​​the support was determined using nitrogen via the BET multipoint method, based on JIS Z 8830:2013 (Determination of specific surface area of ​​powders (solids) based on gas adsorption; established on July 1, 1990; revised on January 21, 2013).

[0040] The shape of the support is not particularly limited, and examples include particles, membranes, and porous cylinders (monoliths) that are liquid-tightly contained in a column tube. From the viewpoint of ensuring uniform separation performance, the shape of the support is preferably a particle, and more preferably a spherical particle.

[0041] In this disclosure, "spherical" includes not only the shape of a perfect sphere, but also shapes with a circular, approximately circular, elliptical, or approximately elliptical cross-section, such as elongated spheres and oblate spheroids. More specifically, if the aspect ratio of the support is 2.0 or less, its shape is considered spherical. The aspect ratio of a spherical support is preferably 1.5 or less.

[0042] It should be noted that the aspect ratio of the support is determined using the following method. The support is randomly scattered on the observation stage and observed from directly above using an electron microscope or optical microscope. In any frame observing more than 10 independent primary particles (not in contact with or overlapping any other particles), the major axis and minor axis (the length of the longest portion perpendicular to the major axis) of each independent primary particle are calculated, and the ratio of these two axes is taken as the aspect ratio of each particle. The arithmetic mean of the aspect ratios of all independent primary particles in the frame is taken as the aspect ratio of the support. Here, a primary particle refers to a particle whose interface between particles can be clearly observed. Typically, primary particles are moderately dispersed to avoid overlap on the stage, but accidental overlap is difficult to avoid. Additionally, there are clumps of primary particles that aggregate; these are removed from the observation.

[0043] When the carrier is a particle, the particle size is not particularly limited, but is preferably 0.1 μm or more and 50.0 μm or less, more preferably 1.0 μm or more and 40.0 μm or less, even more preferably 1.0 μm or more and 30.0 μm or less, and particularly preferably 1.0 μm or more and 10.0 μm or less.

[0044] In the case of spherical particles, the particle size of the carrier refers to the median particle size, measured by a laser diffraction / scattering particle size distribution measuring device, that is, the 50% cumulative value of the cumulative volume distribution curve, i.e., the 50% cumulative diameter of the volume reference (D). 50 ).

[0045] When the particles are amorphous, the particle size of the support is expressed as the diameter of a sphere with the same volume as the particle. In this case, the particle size of the support is determined using a device that measures microscopic images, such as the Malvern Mastersizer 2000E.

[0046] When the support is a particle, the stationary phase also has a particle shape. Generally, when a polymer is supported on a support, the BET specific surface area does not change by more than one error before and after support. Therefore, when the support is a particle, the particle size of the stationary phase can be considered to be approximately the same as the particle size of the support. That is, the range of the stationary phase particle size, including its preferred range, is the same as the range described above for the particle size of the support.

[0047] Materials that can be used as supports include organic materials, inorganic materials, and organic-inorganic hybrid materials. Hereinafter, supports composed of organic materials, inorganic materials, and organic-inorganic hybrid materials will be referred to as organic supports, inorganic supports, and organic-inorganic hybrid supports, respectively.

[0048] Examples of organic materials that constitute organic carriers include polystyrene, poly(meth)acrylamide, and poly(meth)acrylate.

[0049] Inorganic materials constituting an inorganic support include silica gel, alumina, zirconium oxide, titanium oxide, magnesium oxide, glass, kaolin, silicates, and hydroxyapatite, with silica gel, alumina, or glass being preferred, and silica gel being more preferred.

[0050] As an example of an organic-inorganic hybrid support, an organic-inorganic hybrid support formed by the sol-gel reaction of alkoxysilanes with alkyl-substituted or alkylene-substituted alkoxysilane compounds can be cited.

[0051] The support is preferably a porous inorganic support or a porous inorganic support, more preferably a porous inorganic particle or a porous inorganic particle, even more preferably a porous inorganic particle, and particularly preferably a porous silica gel particle.

[0052] 1-2. Polymers with amine side chains In this embodiment, the polymer supported on the support, having amine side chains, has one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2). That is, the amine side chains in the polymer having amine side chains refer to side chains containing tertiary amine-N-oxide groups (hereinafter sometimes simply referred to as "N-oxide groups") in formula (1) and side chains containing tertiary amino groups in formula (2). The polymer having amine side chains preferably has at least one repeating unit represented by formula (1).

[0053] [Chemical Formula 5] In equations (1) and (2), R 11 and R 21 Each represents a hydrogen atom or a methyl group, preferably a hydrogen atom.

[0054] In equations (1) and (2), R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group. It should be noted that, in this disclosure, a saturated aliphatic hydrocarbon group can be any of the following: linear, branched, or cyclic.

[0055] The number of carbon atoms in the aliphatic hydrocarbon group is not particularly limited, but is preferably 1 to 8, more preferably 1 to 4, even more preferably 1 to 3, and particularly preferably 1.

[0056] Specific examples of aliphatic hydrocarbon groups include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, and 2-ethylhexyl; and cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 3,5-dimethylhexyl, preferably alkyl groups.

[0057] R 12 and R 13 Preferably, they are the same group. Additionally, R 22 and R 23 Preferably, they are the same group.

[0058] In equations (1) and (2), Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3, preferably an oxygen atom or NH.

[0059] In equations (1) and (2), n1 and n2 each represent an integer of 1 to 8, preferably an integer of 1 to 6, more preferably an integer of 1 to 4, and even more preferably an integer of 2 to 3.

[0060] As specific examples of repeating units represented by equation (1) and equation (2), the following repeating units can be cited.

[0061] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] Polymers having amine side chains may include repeating units other than those represented by formula (1) and formula (2) without prejudice to the effects of this disclosure. There are no particular limitations on other repeating units; for example, repeating units derived from a support with polymerizable functional groups used in step (a) described later; repeating units derived from silane coupling agents with polymerizable functional groups used in steps (c) and (d) described later; and repeating units derived from any polymerizable compound such as styrene compounds and olefin compounds; etc.

[0062] In polymers having amine side chains, the total content of one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2) is not particularly limited as long as the effect of this disclosure can be obtained. From the viewpoint of more effectively utilizing the separation characteristics brought about by polymers having amine side chains, it is preferably 80 mol% or more and 100 mol% or less, more preferably 90 mol% or more and 100 mol% or less, and even more preferably 95 mol% or more and 100 mol% or less.

[0063] There are no particular limitations on the method of supporting polymers with amine side chains onto the support. For example, preferred methods include: supporting the polymers with amine side chains onto the support via chemical bonds; and physically adsorbing (coating) the polymers with amine side chains onto the support; etc. More preferably, the polymers with amine side chains are supported on the support via chemical bonds. This further improves the separation performance of the stationary phase. Furthermore, when performing separation based on chromatography, even if the mobile phase contains a good solvent for the polymers with amine side chains, it is possible to prevent the polymers with amine side chains from dissolving in the mobile phase solvent and being removed from the stationary phase, thus suppressing the degradation of the stationary phase's functionality.

[0064] The method for synthesizing (manufacturing) polymers having amine side chains is selected according to the aforementioned loading method. In the case where the stationary phase involved in this embodiment is a stationary phase obtained by loading a polymer having amine side chains onto a support via chemical bonds, the stationary phase involved in this embodiment can be manufactured by a method including any of the following steps (a) to (h). When the stationary phase is manufactured by a method including steps (a), (d), (e), or (f), the synthesis of the polymer having amine side chains and the loading of the polymer having amine side chains onto the support occur in parallel. On the other hand, when the stationary phase is manufactured by a method including steps (b), (c), or (g), the polymer having amine side chains is temporarily synthesized and then loaded onto the support via chemical bonds. On the other hand, when the stationary phase involved in this embodiment is a stationary phase obtained by physically adsorbing a polymer having amine side chains onto a support, the stationary phase involved in this embodiment can be manufactured by a method including the following step (h).

[0065] (a) A process of free radical polymerization of monomers with amine side chains in the presence of a support with polymerizable functional groups. (b) A step of chain transfer polymerization of a monomer having an amine side chain in the presence of a compound having a crosslinkable silyl group and a chain transfer functional group to obtain a polymer having the aforementioned crosslinkable silyl group and amine side chain; and a step of combining the aforementioned crosslinkable silyl group of the polymer with functional groups on the surface of a support. (c) The process of polymerizing a monomer having amine side chains with a silane coupling agent having polymerizable functional groups, and the process of attaching the resulting polymer to a support via a silane coupling reaction. (d) The process of polymerizing a monomer with amine side chains with a silane coupling agent with polymerizable functional groups in the presence of a support. (e) A process of chain transfer polymerization of monomers with amine side chains in the presence of a support incorporating chain transfer functional groups. (f) A process of free radical polymerization of monomers with amine side chains in the presence of a support incorporating atom transfer radical polymerization initiator groups. (g) A step of subjecting a monomer having an amine side chain to atom transfer radical polymerization in the presence of a compound having a crosslinkable silyl group and an atom transfer radical polymerization initiator group to obtain a polymer having the aforementioned crosslinkable silyl group and amine side chain, and a step of combining the aforementioned crosslinkable silyl group of the polymer with functional groups on the surface of a support. (h) The process of coating a polymer with amine side chains onto the surface of a support. It should be noted that, in any of these methods, the stereoregularity of polymers with amine side chains can be controlled by selecting the polymerization temperature, polymerization solvent, and additives.

[0066] The following describes process (a).

[0067] Examples of polymerizable functional groups used as the support for polymerizable functional groups in step (a) include vinyl, allyl, isopropenyl, and alkenyl groups with 4 to 12 carbon atoms having a double bond at the ω position, which have alkene-type unsaturated bonds. Vinyl, allyl, or isopropenyl are preferred.

[0068] When porous or non-porous silica gel (hereinafter, the concept encompassing both is sometimes referred to simply as "silica gel") is used as a support, the polymerizable functional groups of silica gel are chemically bonded to the support through the silanol groups.

[0069] When using a support other than silica gel, surface treatment of the support can inhibit excessive adsorption of the target substance to the support itself, and enable chemical bonding with polymerizable functional groups through groups introduced during surface treatment. Examples of surface treatment agents include silane coupling agents such as 3-aminopropyltrimethoxysilane; titanium coupling agents; and aluminate coupling agents; etc.

[0070] Supports with polymerizable functional groups can be obtained by, for example, a silane coupling reaction of a compound represented by formula (I) with a support, preferably silica gel.

[0071] WXY-SiR 3-n Z n (I) In formula (I), W represents a polymerizable functional group (free radical polymerizable functional group). As a polymerizable functional group, a group having an olefinic unsaturated bond is preferred, more preferably vinyl, allyl, isopropenyl, or an alkenyl group having a double bond at the ω position with 4 to 12 carbon atoms, and even more preferably vinyl, allyl, or isopropenyl.

[0072] In formula (I), X represents an amide group, an ester group, an N-alkylamide group with 1 to 3 carbon atoms, an ether group, a sulfoxide group, a sulfonyl group, a thioether group, or a phosphate ester group. X is preferably an amide group, an N-alkylamide group with 1 to 3 carbon atoms, or an ester group.

[0073] In formula (I), Y represents an alkylene group having 1 to 30 carbon atoms. Y is preferably an alkylene group having 1 to 10 carbon atoms, more preferably an alkylene group having 1 to 5 carbon atoms, and even more preferably methylene, ethylene, or trimethylene.

[0074] In formula (I), each R independently represents an alkyl group having 1 to 5 carbon atoms. R is preferably an alkyl group having 1 to 3 carbon atoms, more preferably methyl, ethyl, or n-propyl, and even more preferably methyl or ethyl.

[0075] In formula (I), Z independently represents an alkoxy group with 1 to 5 carbon atoms, a halogen atom, an alkyl thio group with 1 to 20 carbon atoms, an amino group, or an allyl group with 1 to 5 carbon atoms. Examples of alkoxy groups with 1 to 5 carbon atoms include methoxy and ethoxy. Examples of halogen atoms include chlorine, bromine, and iodine. Examples of alkyl thio groups with 1 to 20 carbon atoms include methyl thio and ethyl thio. Examples of amino groups include dimethylamino, diethylamino, pyrrolidinyl, and imidazolyl. Examples of allyl groups with 1 to 5 carbon atoms include alkyl-substituted or unsubstituted allyl groups, such as allyl and 2-methyl-2-propenyl. From the perspective of a good balance between ease of processing and reactivity, Z is preferably an alkoxy group with 1 to 5 carbon atoms, and more preferably a methoxy or ethoxy group.

[0076] In equation (I), n represents an integer between 1 and 3. n is preferably 3.

[0077] The compound represented by formula (I) can be a commercially available compound, or it can be obtained by reacting a compound having W with a compound having -Y-SiR 3-n Z nThe compounds represented by the indicated groups are reacted to produce the product. It should be noted that the reaction between these compounds generates the "-X-" in formula (I).

[0078] Examples of compounds containing W include acrylic acid, whose hydrogen atom on the carbon atom bonded to the vinyl group can be replaced by an alkyl group having 1 to 12 carbon atoms; its halides; and so on.

[0079] As a Y-SiR 3-n Z n Compounds that represent the group, for example, include silane coupling agents that have an alkoxy group having 1 to 5 carbon atoms as Z.

[0080] The support with polymerizable functional groups is preferably a surface-modified silica gel obtained by the silane coupling reaction of a compound represented by formula (I) with silica gel.

[0081] In step (a), when a polymer with amine side chains is supported on a support, the polymer with amine side chains is manufactured by free radical polymerization of the polymerizable functional groups of the monomer with amine side chains and the polymerizable functional groups bound to the support. The reaction conditions for free radical polymerization can be any conditions known in the field of polymer synthesis or conditions based thereon.

[0082] The monomer having an amine side chain is selected from one or more monomers in the group consisting of the monomers represented by formula (3) and the monomers represented by formula (4). The monomers represented by formula (3) and the monomers represented by formula (4) respectively generate the repeating unit represented by formula (1) and the repeating unit represented by formula (2).

[0083] [Chemical Formula 10] In equation (3), R 11 R 12 R 13 Y 1 , and n1 are respectively related to R in equation (1) 11 R 12 R 13 Y 1 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0084] In equation (4), R 21 R 22 R 23 Y 2 , and n2 are respectively related to R in equation (2) 21 R 22 R 23 Y 2The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0085] In free radical polymerization, it is preferable to carry out the polymerization in the presence of a catalytic amount of a free radical initiator. Any free radical initiator, including known free radical initiators and those based thereon, can be used. Examples of known free radical initiators include azo compounds and peroxides.

[0086] Formula (II) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support in step (a). The polymer in Formula (II) can be a block copolymer or a random copolymer.

[0087] [Chemical Formula 11] In formula (II), W' and W” each represent groups formed by the polymerization of W, which is a polymerizable functional group in formula (I). Specific examples of W' include single bonds and alkylene groups with 1 or more carbon atoms, preferably single bonds or methylene groups. Specific examples of W” include hydrogen atoms or methyl groups, etc.

[0088] In equation (II), X and Y have the same meaning as X and Y in equation (I), and their preferred methods are also the same.

[0089] In formula (II), Z' represents a linking group or single bond formed between the silicon atom and the support in formula (I) via a silane coupling reaction. For example, if Z in formula (I) is an alkoxy group with 1 to 5 carbon atoms and the support is silica gel, then Z' is -O-.

[0090] In formula (II), V is a group formed by the coupling of Z, R, Z' with a support, or by coupling between compounds represented by formula (I). Z and R have the same meaning as Z and R in formula (I), and their preferred methods are also the same. It should be noted that when V is a group formed by coupling between compounds represented by formula (I), n in formula (I) is 2 or 3.

[0091] In formula (II), when n=1, V=R; when n=2, the total proportion of unreacted Z and R relative to the total number of V is more than 5% and less than 100%; when n=3, the proportion of unreacted Z relative to the total number of V is more than 0% and less than 100%.

[0092] In equation (II), R 11 R 12 R 13 Y 1 , and n1 are respectively related to R in equation (1)11 R 12 R 13 Y 1 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0093] In equation (II), R 21 R 22 R 23 Y 2 , and n2 are respectively related to R in equation (2) 21 R 22 R 23 Y 2 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0094] In formula (II), p represents 1 to 10. Preferably, p is 1 to 5.

[0095] In formula (II), q1, q2, and q1+q2 each represent 10 or more and 3,000 or less. q1, q2, and q1+q2 are preferably 15 or more and 2,500 or less, more preferably 20 or more and 2,000 or less. Either q1 or q2 can be 0.

[0096] The following describes process (b).

[0097] Compounds with crosslinkable silyl groups and chain transfer functional groups used in step (b) can be exemplified by compounds represented by formula (III). Crosslinkable silyl groups are groups that, although dependent on reaction conditions, can induce homocoupling reactions and reactions with functional groups on the surface of the support. In formula (III), the crosslinkable silyl group is -SiR. 3-n Z n The term "silyl group" indicates a methyl silyl group.

[0098] TY-SiR 3-n Z n (III) In formula (III), T represents a chain transfer functional group. A chain transfer functional group refers to a functional group that actively participates in chain transfer reactions accompanying the transfer of growth active species and the re-initiation reaction during free radical polymerization. By possessing a chain transfer functional group, the molecular weight and terminal structure of polymers with amine side chains can be controlled to some extent. Examples of chain transfer functional groups preferably include alkyl halogens with 1 to 12 carbon atoms, alkyl halogens with 1 to 12 carbon atoms having a thiol group at the terminal, and alkyl halogens with 1 to 12 carbon atoms having a disulfide group within the group. Examples of halogens with 1 to 12 carbon atoms include chlorine, bromine, and iodine. Preferably, alkyl halogens with 1 to 3 carbon atoms are used as alkyl halogens.

[0099] In equation (III), Y, R, Z, and n have the same meaning as Y, R, Z, and n in equation (I), and their preferred methods are also the same.

[0100] The monomer with amine side chain used in step (b) is the same as the monomer with amine side chain used in step (a), that is, it is one or more of the monomers represented by formula (3) and the monomers represented by formula (4).

[0101] Chain transfer polymerization is preferably carried out in the presence of a catalytic amount of a radical initiator. Any radical initiator, including known radical initiators and those based thereon, can be used. Examples of known radical initiators include azo compounds and peroxides. In this case, the molecular weight can be controlled to some extent by adjusting the molar ratio of the chain transfer agent to the monomer.

[0102] By chain transfer polymerization of monomers with amine side chains in the presence of compounds having crosslinkable silyl groups and chain transfer functional groups, polymers represented by formula (IV), i.e., polymers having crosslinkable silyl groups and amine side chains, can be obtained. This polymer, as shown in formula (IV), has crosslinkable silyl groups at its ends.

[0103] [Chemical Formula 12] In formula (IV), T' represents a group derived from T in formula (III). In other words, T' is a residue of T generated by chain transfer polymerization. For example, in the case of T being 12-mercaptododecyl, -T'-Y-SiR 3-n Z n -SC 12 H 24 -Y-SiR 3-n Z n .

[0104] In equation (IV), Y, R, Z, and n have the same meaning as Y, R, Z, and n in equation (III), and their preferred methods are also the same.

[0105] In equation (IV), R 11 R 12 R 13 Y 1 , and n1 are respectively related to R in equation (1) 11 R 12 R 13 Y 1 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0106] In equation (IV), R 21 R 22 R 23 Y 2 , and n2 are respectively related to R in equation (2) 21 R 22 R 23 Y 2 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0107] In formula (IV), q1, q2, and q1+q2 each represent 10 or more and 3,000 or less. q1, q2, and q1+q2 are preferably 15 or more and 2,500 or less, more preferably 20 or more and 2,000 or less. Either q1 or q2 can be 0.

[0108] As a method for binding the crosslinked silyl group of a polymer having crosslinked silyl and amine side chains to functional groups on the surface of a support, a silane coupling reaction can be employed. As the silane coupling reaction, known reactions or any reaction based thereon can be used.

[0109] Formula (V) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support in step (b).

[0110] [Chemical Formula 13] In equation (V), Z' and V have the same meaning as Z' and V in equation (II).

[0111] In equation (V), Y, T', and R 11 R 12 R 13 Y 1 n1, R 21 R 22 R 23 Y 2 n2, q1, q2, and q1+q2 each correspond to Y, T', and R in equation (IV). 11 R 12 R 13 Y 1 n1, R 21 R 22 R 23 Y 2 The meanings of n2, q1, q2, and q1+q2 are the same, and their optimization methods are also the same.

[0112] The following describes process (c).

[0113] As a silane coupling agent with polymerizable functional groups used in step (c), a compound represented by formula (I) is preferably exemplified, for example. When the compound represented by formula (I) is used in step (c), its preferred manner is the same as when it is used in step (a).

[0114] The monomer with amine side chain used in step (c) is the same as the monomer with amine side chain used in step (a), that is, it is one or more of the monomers represented by formula (3) and the monomers represented by formula (4).

[0115] The method for polymerizing monomers with amine side chains with silane coupling agents having polymerizable functional groups is not particularly limited, and any polymerization method can be used, preferably free radical polymerization, and more preferably living free radical polymerization. In free radical polymerization, it is preferable to carry out the polymerization in the presence of a catalytic amount of a free radical initiator. As the free radical initiator, any free radical initiator, such as known free radical initiators and free radical initiators based thereon, can be used. Examples of known free radical initiators include azo compounds and peroxides. Furthermore, from the perspective of facilitating the control of the molecular weight of polymers with amine side chains, polymerization is preferably carried out in the presence of a chain transfer agent.

[0116] As a method for binding the obtained polymer (i.e., a polymer with amine side chains) to a support via a silane coupling reaction, any method, such as known silane coupling reactions and reactions based thereon, can be used.

[0117] Formula (II) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support through step (c).

[0118] The following describes process (d).

[0119] As a silane coupling agent with polymerizable functional groups used in step (d), compounds represented by formula (I) are preferably used. When the compound represented by formula (I) is used in step (d), its preferred manner is the same as when it is used in step (a).

[0120] The monomer with amine side chain used in step (d) is the same as the monomer with amine side chain used in step (a), that is, it is one or more of the monomers represented by formula (3) and the monomers represented by formula (4).

[0121] When a monomer with amine side chains is polymerized with a silane coupling agent having polymerizable functional groups in the presence of a support, the polymerization method is not particularly limited, and any polymerization method can be used, but free radical polymerization is preferred. In free radical polymerization, it is preferably carried out in the presence of a catalytic amount of a free radical initiator. As the free radical initiator, any free radical initiator, such as known free radical initiators or free radical initiators based thereon, can be used. Examples of known free radical initiators include azo compounds and peroxides.

[0122] Formula (II) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support through step (d).

[0123] The following describes process (e).

[0124] The chain transfer functional group on the surface of the support used in step (e) that is introduced (chemically bonded) can be the same group as the chain transfer functional group of the compound used in step (b) that has a crosslinkable silane and a chain transfer functional group. Therefore, as a support obtained by introducing a chain transfer functional group onto the surface, for example, a support obtained by bonding the compound represented by formula (III) to the support via a silane coupling reaction can be preferably described.

[0125] The monomer with amine side chain used in step (e) is the same as the monomer with amine side chain used in step (a), that is, it is one or more of the monomers represented by formula (3) and the monomers represented by formula (4).

[0126] Chain transfer polymerization is preferably carried out in the presence of a catalytic amount of a radical initiator. Any radical initiator, including known radical initiators and those based thereon, can be used. Examples of known radical initiators include azo compounds and peroxides. In this case, the molecular weight can be controlled to some extent by adjusting the molar ratio of the chain transfer agent to the monomer.

[0127] Formula (V) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support in step (e).

[0128] The following describes process (f).

[0129] The atom transfer radical polymerization initiator group used in step (f) that incorporates the atom transfer radical polymerization initiator group is preferably an α-haloalkyl carbonyl group. Examples of α-haloalkyl carbonyl groups include α-bromoisobutyryl, α-bromopropionyl, bromoacetyl, and chloroacetyl, with α-bromoisobutyryl being the most preferred.

[0130] When silica gel is used as a support, the silanol groups and atom transfer radical polymerization initiator groups of silica gel are chemically bonded to the support.

[0131] When using a support other than silica gel, surface treatment of the support can inhibit excessive adsorption of the target substance to the support itself, and enable chemical bonding with the atom transfer radical polymerization initiator groups through the groups introduced during the surface treatment. Examples of surface treatment agents include silane coupling agents such as 3-aminopropyltrimethoxysilane; titanium coupling agents; and aluminate coupling agents; etc.

[0132] Supports incorporating atom transfer radical polymerization initiator groups can be obtained by, for example, a silane coupling reaction of a compound represented by formula (VI) with a support, preferably silica gel.

[0133] AQY-SiR 3-n Z n (VI) In formula (VI), A represents an atom transfer radical polymerization initiator group. Preferably, an α-haloalkyl carbonyl group is preferred as the atom transfer radical polymerization initiator group; more preferably, an α-bromoisobutyryl group, an α-bromopropionyl group, a bromoacetyl group, or a chloroacetyl group is preferred; and even more preferably, an α-bromoisobutyryl group is preferred.

[0134] In formula (VI), Q represents an oxygen atom, NH, or NCH3, preferably NH.

[0135] In equation (VI), Y, R, Z, and n have the same meaning as Y, R, Z, and n in equation (I), and their preferred methods are also the same.

[0136] The compound represented by formula (VI) can be a commercially available compound, or it can be produced by reacting a compound having A with a compound having -Y-SiR. 3-n Z n The compounds represented by the indicated groups are reacted to produce the product. It should be noted that the reaction between these compounds generates the "-X-" in formula (VI).

[0137] Compounds having the structure represented by A can be exemplified by compounds obtained by combining halogen atoms such as chlorine, bromine, and iodine atoms with an atom transfer radical polymerization initiator group. Examples of such compounds include α-bromoisobutyryl bromide, α-bromoisobutyryl chloride, α-bromopropionyl bromide, α-bromopropionyl chloride, bromoacetyl bromide, bromoacetyl chloride, chloroacetyl bromide, and chloroacetyl bromide chloride, with α-bromoisobutyryl bromide being preferred.

[0138] As a Y-SiR 3-n Z nCompounds that represent the group, for example, include silane coupling agents that have an alkoxy group having 1 to 5 carbon atoms as Z.

[0139] The support incorporating an atom transfer radical polymerization initiator group is preferably a surface-modified silica gel obtained by the silane coupling reaction of a compound represented by formula (VI) with silica gel.

[0140] When a polymer with amine side chains is supported on a support in step (f), the polymer with amine side chains is manufactured by atom transfer radical polymerization of the monomer with amine side chains using an atom transfer radical polymerization initiator group bonded to the support as an initiator. The reaction conditions for atom transfer radical polymerization can be any conditions known in the field of polymer synthesis or conditions based thereon.

[0141] The monomer with amine side chain used in step (f) is the same as the monomer with amine side chain used in step (a), that is, it is one or more of the monomers represented by formula (3) and the monomers represented by formula (4).

[0142] Formula (VII) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support through step (f).

[0143] [Chemical Formula 14] In formula (VII), A' represents a group derived from A in formula (VI). In other words, A' represents a residue of A generated by atom transfer radical polymerization. For example, if A is α-bromoisobutyryl, then -A'-QY-SiR 3-n Z n -(CH3)2CCO-QY-SiR 3-n Z n .

[0144] In equation (VII), Z' and V have the same meaning as Z' and V in equation (II).

[0145] In equation (VII), Y and Q have the same meaning as Y and Q in equation (VI), and their preferred methods are also the same.

[0146] In equation (VII), R 11 R 12 R 13 Y 1 , and n1 are respectively related to R in equation (1) 11 R 12 R 13 Y 1The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0147] In equation (VII), R 21 R 22 R 23 Y 2 , and n2 are respectively related to R in equation (2) 21 R 22 R 23 Y 2 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0148] In formula (VII), q1, q2, and q1+q2 each represent 10 or more and 3,000 or less. q1, q2, and q1+q2 are preferably 15 or more and 2,500 or less, more preferably 20 or more and 2,000 or less. Either q1 or q2 can be 0.

[0149] The following describes process (g).

[0150] Compounds with crosslinking silyl groups and atom transfer radical polymerization initiator groups used in step (g) can be exemplified by compounds represented by formula (VI).

[0151] The monomer with an amine side chain used in step (g) is the same as the monomer with an amine side chain used in step (a), that is, it is one or more of the monomers represented by formula (3) and the monomers represented by formula (4).

[0152] By subjecting monomers with amine side chains to atom transfer radical polymerization in the presence of a compound having crosslinkable silyl groups and an atom transfer radical polymerization initiator group, a polymer represented by formula (VIII) can be obtained, i.e., a polymer having crosslinkable silyl groups and amine side chains. This polymer, as shown in formula (VIII), has crosslinkable silyl groups at its ends.

[0153] [Chemical Formula 15] In equation (VIII), Q, Y, R, Z, and n have the same meaning as Q, Y, R, Z, and n in equation (VI), and their preferred methods are also the same.

[0154] In equation (VIII), A' has the same meaning as A' in equation (VII).

[0155] In equation (VIII), R 11 R 12 R 13 Y 1 , and n1 are respectively related to R in equation (1) 11R 12 R 13 Y 1 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0156] In equation (VIII), R 21 R 22 R 23 Y 2 , and n2 are respectively related to R in equation (2) 21 R 22 R 23 Y 2 The meanings of n1 and n2 are the same, and their preferred methods are also the same.

[0157] In formula (VIII), q1, q2, and q1+q2 each represent 10 or more and 3,000 or less. q1, q2, and q1+q2 are preferably 15 or more and 2,500 or less, more preferably 20 or more and 2,000 or less. Either q1 or q2 can be 0.

[0158] As a method for binding the crosslinked silyl group of a polymer having crosslinked silyl and amine side chains to functional groups on the surface of a support, a silane coupling reaction can be employed. As the silane coupling reaction, known reactions or any reaction based thereon can be used.

[0159] Formula (IX) shows a structure that is presumably one of the preferred methods for obtaining a stationary phase by loading a polymer having amine side chains onto a support through step (g).

[0160] [Chemical Formula 16] In equation (IX), Z' and V have the same meaning as Z' and V in equation (II).

[0161] In formula (IX), Y, Q, A', R 11 R 12 R 13 Y 1 n1, R 21 R 22 R 23 Y 2 n2, q1, q2, and q1+q2 each correspond to Y, Q, A', and R in equation (VIII). 11 R 12 R 13 Y 1 n1, R 21 R 22 R 23 Y 2The meanings of n2, q1, q2, and q1+q2 are the same, and their optimization methods are also the same.

[0162] The following is a description of process (h).

[0163] As a method for coating a polymer having amine side chains onto the surface of a support, any method, including known methods and methods based thereon, can be employed. As a known method, a preferred approach is to coat (physically adsorb) a coating solution containing a polymer having amine side chains and a solvent onto the surface of the support, and then remove the solvent. The solvent for the coating solution is not particularly limited as long as it can dissolve the polymer having amine side chains; examples include water; aprotic polar solvents such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), and N-methyl-2-pyrrolidone (NMP); cyclic amines such as pyridine; cyclic ethers such as THF and 1,4-dioxane; alcohols such as methanol and ethanol; and mixtures of two or more solvents selected from these; etc. Among these, considering the higher solubility of polymers with amine side chains, the solvent of the coating liquid is preferably water, an aprotic polar solvent, a cyclic amine, a cyclic ether, or a mixture of two or more solvents selected therefrom, more preferably water, an aprotic polar solvent, or a mixture of two or more solvents selected therefrom, and even more preferably water, DMF, or a mixture therefrom.

[0164] Polymers having amine side chains can be manufactured by performing a polymerization process in which one or more monomers having amine side chains selected from the group consisting of monomers represented by formula (3) and monomers represented by formula (4) are polymerized. If the polymer having amine side chains contains other repeating units, it can be further polymerized together with monomers having amine side chains to generate other repeating units. Examples of monomers generating other repeating units include any polymerizable compounds such as styrene compounds and olefin compounds.

[0165] There are no particular limitations on the polymerization method for monomers with amine side chains; any polymerization method such as atom transfer radical polymerization, free radical polymerization, and chain transfer polymerization can be used. As for the polymerization reaction conditions, any conditions known in the field of polymer synthesis or conditions based thereon can be used.

[0166] In any of the steps (a) to (h), if the polymer with amine side chains has repeating units represented by formula (1), the monomer represented by formula (4) may be polymerized first to synthesize a polymer with repeating units represented by formula (2), and then an oxidation step may be performed to oxidize the obtained polymer, converting part or all of the tertiary amines in the side chains into N-oxide groups, thereby producing a polymer with amine side chains. Any oxidizing agent such as mCPBA may be used in the oxidation step.

[0167] Furthermore, polymers with amine side chains can have cross-linked structures. Specifically, when manufacturing the stationary phase using a method including step (h), it is preferable to perform a cross-linking step after step (h) to introduce a cross-linked structure into the polymer. This renders the polymer with amine side chains insoluble and firmly fixed onto the support. Therefore, during chromatographic separation, the dissolution of the polymer with amine side chains in the mobile phase is suppressed, thereby preventing a decrease in the functionality of the stationary phase.

[0168] Examples of methods for crosslinking polymers with amine side chains include radiation crosslinking based on irradiation with gamma rays, X-rays, and electron beams; ultraviolet crosslinking based on ultraviolet radiation; thermal crosslinking based on heating; crosslinking using compounds with crosslinking groups; and combinations thereof. As a crosslinking method, it is preferable to select one or more of radiation crosslinking and thermal crosslinking methods, considering minimal impact on the composition of the polymer with amine side chains and minimal residual reagent problems. The radiation dose in radiation crosslinking and ultraviolet crosslinking, and the heating temperature and heating time in thermal crosslinking, can be selected according to the degree of crosslinking and the degree of degradation of the polymer with amine side chains.

[0169] The number average molecular weight (Mn) of the polymer having amine side chains is not particularly limited, but is preferably 2,000 or more and 600,000 or less, more preferably 2,500 or more and 500,000 or less, and even more preferably 3,000 or more and 400,000 or less.

[0170] The weight-average molecular weight (Mw) of the polymer having amine side chains is not particularly limited, but from the perspective of ensuring solubility in solvents and preventing particle aggregation when the polymer having amine side chains is supported on a carrier, it is preferably 2,000 or more and 600,000 or less, more preferably 2,500 or more and 500,000 or less, and even more preferably 3,000 or more and 400,000 or less.

[0171] The polydispersity index (PDI) of polymers having amine side chains is not particularly limited, but is preferably 1.0 or more and 5.0 or less, more preferably 1.0 or more and 3.0 or less, and even more preferably 1.0 or more and 2.0 or less.

[0172] The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of polymers having amine side chains can be calculated based on polystyrene conversion using size exclusion chromatography (SEC). Size exclusion chromatography (SEC) is performed under the conditions described in the examples.

[0173] However, when the stationary phase is manufactured by a method including steps (a), (d), (e), or (f), the synthesis of the polymer with amine side chains and the loading of the polymer with amine side chains onto the support occur in parallel, so the weight-average molecular weight is calculated using the supernatant of the polymerization solution.

[0174] On the other hand, when the stationary phase is manufactured by a method including steps (b), (c), or (g), its weight-average molecular weight is determined before the polymer having amine side chains is combined with the support.

[0175] The polydispersity index (PDI) of polymers with amine side chains is calculated by dividing the weight-average molecular weight (Mw) calculated according to the above method by the number-average molecular weight (Mn).

[0176] The average thickness (loading per 1g of support / specific surface area of ​​support) of the polymer with amine side chains supported on the support is not particularly limited, but is preferably 5 nm to 25 nm, more preferably 10 nm to 20 nm. If the average thickness of the polymer with amine side chains is within the above range, there is a tendency to obtain sharp peaks, which is preferred.

[0177] The content of the polymer with amine side chains in 100 parts by weight of the stationary phase obtained by supporting the polymer with amine side chains on a support is not particularly limited, but is preferably 10% by mass or more and 45% by mass or less, more preferably 10% by mass or more and 30% by mass or less, and even more preferably 20% by mass or more and 25% by mass or less. By keeping the content of the polymer with amine side chains within the above range, it is possible to appropriately exert the adsorption capacity of the polymer with amine side chains while suppressing peak broadening. The content of the polymer with amine side chains in the stationary phase is determined by elemental analysis.

[0178] The content of polymers with amine side chains in 100 parts by mass of the stationary phase was determined by elemental analysis. Based on the carbon content determination results of the support without amine side chains and the stationary phase, all carbon other than that contained in the support without amine side chains was taken as carbon from the polymers with amine side chains, and the content of polymers with amine side chains in the stationary phase was calculated.

[0179] 1-3. Chromatography There are no particular limitations on the chromatographic method used as the stationary phase in the first embodiment, but liquid chromatography and supercritical fluid chromatography are preferred examples.

[0180] Examples of liquid chromatography methods include, but are not limited to, normal-phase chromatography, reversed-phase chromatography, size exclusion chromatography, and ion-exchange chromatography. The stationary phase involved in this embodiment, as shown in the examples described later, exhibits good retention based on hydrophilic partitioning, and is therefore suitable for normal-phase chromatography, particularly hydrophilic interaction chromatography (HILIC).

[0181] 2. Methods for preparing stationary phases for chromatography.

[0182] The second embodiment of this disclosure is a method for manufacturing a stationary phase for chromatography, and includes any one of steps (a) to (h). The monomer having an amine side chain in steps (a) to (g) is one or more monomers selected from the group consisting of monomers represented by formula (3) and monomers represented by formula (4). Furthermore, the polymer supported on the support in steps (a) to (h) is a polymer having an amine side chain, and has one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2).

[0183] In this embodiment, each of steps (a) to (h) has the same meaning as steps (a) to (h) described in the description of the first embodiment, and their preferred methods are also the same. In addition, the repeating unit represented by formula (1), the repeating unit represented by formula (2), the monomer represented by formula (3), the monomer represented by formula (4), and the carrier each have the same meaning as the repeating unit represented by formula (1), the repeating unit represented by formula (2), the monomer represented by formula (3), the monomer represented by formula (4), and the carrier in the first embodiment, and their preferred methods are also the same.

[0184] The manufacturing method described in this embodiment enables the manufacture of the stationary phase for chromatography described in the first embodiment.

[0185] The manufacturing method described in this embodiment may include any steps in addition to those described above. Examples of such arbitrary steps include, for instance, the oxidation step and crosslinking step described in the description of the first embodiment.

[0186] 3. Separation Method The third embodiment of this disclosure is a separation method including a separation step in which substances are separated by chromatography using the stationary phase described in the first embodiment. As the chromatography method, liquid chromatography is preferred, and normal-phase chromatography is more preferred. The stationary phase described in the first embodiment, as shown in the examples described later, exhibits excellent retention due to its hydrophilic partitioning; therefore, hydrophilic interaction chromatography (HILIC) is particularly preferred as the chromatography method.

[0187] Chromatography can be performed using commercially available chromatographs, such as liquid chromatographs and supercritical fluid chromatographs. Column equilibration conditions and flow rates, among other factors, can be selected based on column size, sample capacity, and the type of mobile phase.

[0188] The substances separated by the separation method according to this embodiment are not particularly limited, but are preferably one or more selected from the group consisting of hydrophilic substances and polar substances. As shown in the examples described later, the stationary phase according to the first embodiment exhibits excellent retention of hydrophilic partitions and OH group selectivity; therefore, the separation method according to this embodiment can be considered suitable for the separation of these substances. Examples of hydrophilic and polar substances include, for example, sugars, glycosides, nucleic acids, and nucleosides. Furthermore, the stationary phase according to the first embodiment also exhibits good configuration selectivity and positional isomer selectivity; therefore, the separation method according to this embodiment can also effectively separate diastereomers, enantiomers, and positional isomers.

[0189] When performing separation based on liquid chromatography, the solvent used as the mobile phase is not particularly limited, but is preferably an organic solvent capable of dissolving the substance to be separated. Examples include organic solvents, water, and mixtures of water and organic solvents.

[0190] There are no particular limitations on the organic solvent used; examples include acetonitrile, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, 2-methyltetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMAc), acetone, methyl ethyl ketone, diethyl methyl ketone, methyl tert-butyl ether, chloroform, and dichloromethane. Additionally, organic solvents can also be mixtures of hydrocarbons with 5 to 8 carbon atoms and alcohols commonly used in chromatographic methods.

[0191] Organic solvents can be used alone, or in any combination and ratio of two or more.

[0192] Of the above, from the perspective of high chemical stability, the organic solvent is preferably selected from one or more of acetonitrile, methanol, ethanol, n-propanol, 2-propanol, and tetrahydrofuran, more preferably selected from one or more of methanol and acetonitrile, and even more preferably acetonitrile.

[0193] When separation is performed using supercritical fluid chromatography, examples of solvents used as the mobile phase include supercritical carbon dioxide, mixtures of supercritical carbon dioxide and organic solvents, and mixtures of supercritical carbon dioxide, organic solvents, and water. As the organic solvent contained in the aforementioned mixtures, solvents described above that are used as organic solvents in the mobile phase of liquid chromatography can be appropriately used.

[0194] Example The present disclosure is illustrated in more detail below with reference to the embodiments, but the present disclosure is not limited to the embodiments described below, as long as they do not depart from its spirit.

[0195] [Synthetic Example 1: Synthesis of 3-dimethylaminopropylacrylamide N-oxide] 3-Dimethylaminopropylacrylamide was dissolved in approximately twice its volume of methanol to obtain a 3-dimethylaminopropylacrylamide solution. While cooling the resulting solution in an ice bath, hydrogen peroxide in an equivalent molar amount to the dimethylamino group was added to the solution using a 35% aqueous hydrogen peroxide solution, and the mixture was stirred at room temperature for several hours. The reaction was monitored by TLC until the 3-dimethylaminopropylacrylamide spot disappeared from the TLC. Then, a further 35% aqueous hydrogen peroxide solution was added to the reaction mixture, and the reaction was continued at room temperature. After confirming the disappearance of the 3-dimethylaminopropylacrylamide spot on the TLC, the methanol was removed using an evaporator, thus yielding 3-dimethylaminopropylacrylamide N-oxide.

[0196] [Synthetic Example 2: Synthesis of silica incorporating atom transfer radical polymerization initiator groups] 3.53 g of 3-aminopropyltriethoxysilane and triethylamine were dissolved in 20 mL of tetrahydrofuran while stirring in an ice bath. A solution of 4.23 g of α-bromoisobutyryl bromide in tetrahydrofuran (30 mL) was added dropwise over 20 minutes. The tetrahydrofuran was then removed under reduced pressure to give a colorless solid. 80 mL of ethyl acetate was added to the solid, and after removing insoluble matter, the ethyl acetate was removed by distillation to give 6.31 g of 3-(α-bromoisobutyramide)propyltriethoxysilane (ATRPini).

[0197] Porous silica gel (ChromatoRex SPS100-5, manufactured by Fuji Silysia Chemical Ltd., with a particle size of 5 μm and a specific surface area of ​​300 m²) was used.2 3.78 g of silica (with an average pore size of 10 nm), 3.94 g of ATRPini, 0.87 g of pyridine, and 80 mL of toluene were mixed and degassed under reduced pressure for 30 minutes under ultrasonic irradiation. The resulting reaction solution was heated under reflux for 3 hours, then filtered, and the solid was recovered. The solid was washed with acetone and dried to obtain 4.24 g of silica (ATRPini-silica) incorporating atom transfer radical polymerization initiator groups. ATRPini-silica is presumed to have the following structure.

[0198] [Chemical Formula 17] [Example 1: Preparation of stationary phase for chromatography] 0.75 g of 3-dimethylaminopropylacrylamide N-oxide (DMAPAAm N-Oxide), 0.75 g of ATRPini-silica, 0.10 g of tris[2-(dimethylamino)ethyl]amine (Me6TREN), 0.075 g of tetraethylammonium bromide (TEAB), 6 mL of water, and 3 mL of acetonitrile were added to a flask and degassed under reduced pressure for 15 minutes under ultrasonic irradiation. The resulting reaction solution was heated to 40°C in a water bath while stirring and bubbling with argon gas for 25 minutes. 48 mg of copper bromide (I) and 3 mL of acetonitrile were then added, and the reaction solution was heated in a 40°C water bath for 3 hours while stirring.

[0199] The reaction mixture was then transferred to a centrifuge tube and centrifuged for 10 minutes. The supernatant was removed, and the mixture was then mixed with 0.10 g of disodium ethylenediaminetetraacetate dihydrate and 30 mL of ammonium acetate buffer (100 mM, pH 4.6) and stirred overnight at room temperature. The resulting mixture was centrifuged for 10 minutes, and the supernatant was removed, yielding a precipitate. The precipitate was dispersed in water, filtered, and the solid was recovered. The solid was washed with methanol and acetone, respectively, and dried at 100 °C for 1 hour to obtain 0.841 g of the chromatographic stationary phase (PDMAPAAm N-oxide silica). PDMAPAAm N-oxide silica is presumed to have the following structure.

[0200] [Chemical Formula 18] [Example 2: Preparation of stationary phase for chromatography] Except for replacing 3-dimethylaminopropylacrylamide N-oxide (DMAPAAm N-Oxide) with 3-dimethylaminopropylacrylamide (DMAPAAm), the procedure was the same as in Example 1 to obtain the stationary phase (PDMAPAAm silica) for chromatography. PDMAPAAm silica is presumed to have the following structure.

[0201] [Chemical Formula 19] [Comparative Example 1: Stationary Phase in Chromatography] Porous silica gel ("CHROMATOREX SPS100-5" manufactured by Fuji Silysia Chemical Ltd.) was used as the stationary phase for chromatography in Comparative Example 1.

[0202] [Comparative Example 2: Preparation of Stationary Phase for Chromatography] Except for replacing 3-dimethylaminopropylacrylamide N-oxide (DMAPAAm N-Oxide) with 3-trimethylaminopropylacrylamide chloride (TMAPPAm), the procedure was the same as in Example 1 to obtain the stationary phase (PTMAPPAm silica) for chromatography. PTMAPPAm silica is presumed to have the following structure.

[0203] [Chemical Formula 20] [Reference Example 1: Stationary Phase in Chromatography] The ATRPini-silica obtained in Synthesis Example 2 was used as the stationary phase for the chromatography of Reference Example 1.

[0204] [Column Test] The stationary phase was dispersed in 10 mL of a mixed solvent (50 / 50 (v / v)) formed by water and a slurry solvent (Chemco Plus Co., Ltd. "Chemco Slurry Mix SB") and packed into a stainless steel column (3 mm inner diameter), thereby preparing a column for hydrophilic interaction chromatography (HILIC).

[0205] Using the prepared HILIC column, the evaluation of the items shown in Table 1 was carried out according to the column test method, based on the following test conditions. The results are shown in Tables 2 and 3.

[0206] Measurement conditions • Mobile phase (Examples, Comparative Examples, Reference Examples): Acetonitrile (ACN) / AcONH4 buffer (100 mM, pH 4.76) (ACN / AcONH4 buffer = 90 / 10 (v / v)) • Mobile phase (other samples): α(AX), α(CX)…acetonitrile (ACN) / AcONH4 buffer (100mM, pH 4.76) (ACN / AcONH4 buffer = 90 / 10 (v / v)) Other than the items mentioned above… Acetonitrile (ACN) / AcONH4 buffer (20mM, pH 4.76) Column temperature: 30℃ • Linear velocity: 1.0 mm / sec • Flow rate: 0.3 mL / min • Detector: UV detector (254nm) Evaluation Project The retention coefficient k(X) of substance X = (t) R -t0) / t0 t R Retention time of substance X t0: Elution time of non-retaining substances • The selectivity of substance Y relative to substance Z is α(Y / Z) = k(Y) / k(Z) k(Y): Retention coefficient of substance Y k(Z): Retention coefficient of substance Z [Table 1] 1: N,N,N-4-nitrophenyl α-D-glucopyranoside 2: N,N,N-4-nitrophenyl β-D-glucopyranoside [Table 2] [Table 3] The k(U) values ​​of the stationary phases in Comparative Example 1, Comparative Example 2, and Reference Example 1 were 0.78, 3.48, and 0.06, respectively. In contrast, the k(U) values ​​of the stationary phases in Example 1 and Example 2 were 13.75 and 12.75, respectively, which are significantly higher than the k(U) values ​​of the stationary phases in Comparative Example 1, Comparative Example 2, and Reference Example 1. Furthermore, the k(U) values ​​of the stationary phases in Example 1 and Example 2 significantly exceeded the k(U) value (4.58) of the commercially available HILIC column "TSKgel Amide-80" manufactured by TOSOH CORPORATION, which has the highest retention rate. Therefore, it can be seen that the stationary phases in Example 1 and Example 2 have very strong interactions with hydrophilic and polar substances, and exhibit excellent retention of hydrophilic substances.

[0207] The stationary phases of Examples 1 and 2 have α(U / 2'dU) values ​​of 2.71 and 2.65, respectively. On the other hand, the unmodified porous silica gel of Comparative Example 1 has a smaller (U / 2'dU) value of 1.14 compared to the aforementioned values. This indicates that the stationary phases of Examples 1 and 2 exhibit high selectivity for OH groups.

[0208] The stationary phases of Examples 1 and 2 showed α(U / 5MU), α(V / A), α(α / β), and α(2d / 3d) equal or greater than those of Comparative Examples 1 and 2. Therefore, the stationary phases of Examples 1 and 2 exhibited high hydrophobicity selectivity, configuration selectivity, and positional isomer selectivity.

[0209] The stationary phases of Examples 1 and 2 have an α(Tb / Tp) of 1 or less, and can therefore be considered as stationary phases with an alkaline surface. On the other hand, the unmodified porous silica gel of Comparative Example 1 has an α(U / 5MU) α(Tb / Tp) greater than 1, and can therefore be considered as a stationary phase with an acidic surface.

[0210] The α(I / isoI) of the unmodified porous silica gel in Comparative Example 1 was 3.42. On the other hand, the α(I / isoI) of the stationary phases in Examples 1 and 2 were 34.91 and 39.52, respectively, which were larger values ​​than those of the unmodified porous silica gel in Comparative Example 1. Therefore, the stationary phases in Examples 1 and 2 exhibited high selectivity for diols.

[0211] [Graphing of the separation characteristics of the stationary phase used in chromatography, Part I] The vertical axis is plotted as α (Tb / Tp), representing the surface pH of the stationary phase, and the horizontal axis is plotted as α (U / 2dU), representing hydrophilicity. A graph is created for each stationary phase shown in Table 4. The results are presented below. Figure 1 .

[0212] Figure 1 In this context, a surface α(Tb / Tp) of around 1 indicates neutrality, a value greater than 1 indicates weak acidity (cation exchange), and a value less than 1 indicates basicity (anion exchange). Furthermore, a higher α(U / 2'dU) indicates higher OH group selectivity. Figure 1 In the diagram, the lower left region of the straight line, which is shown as an auxiliary feature, represents the separation characteristics of monomolecular modified stationary phases, which are typically obtained by modifying a surface with a monomolecular length of several nm. The upper right region of the straight line represents the separation characteristics of polymer-modified stationary phases, which are typically obtained by modifying a polymer with a thickness of about 15 nm.

[0213] [Table 4] Depend on Figure 1It is evident that the stationary phases of Examples 1 and 2 exhibit the same separation characteristics as polymer-modified stationary phases, rather than the same separation characteristics as unimolecular-modified stationary phases. Furthermore, it is evident that the stationary phases of Examples 1 and 2 exhibit novel separation characteristics that cannot be categorized into any of the following types: amide-type, zwitterionic, unmodified silica, neutral molecular type, and amine-type. In particular, the N-oxide-type stationary phase of Example 1 shows that, unlike amine-type stationary phases, it exhibits basicity similar to alkoxides.

[0214] [Graphing of the separation characteristics of the stationary phase in chromatography II] The vertical axis represents α (U / 5FU), an indicator of amino selectivity, and the horizontal axis represents α (2'dU / 2'd5FU), another indicator of amino selectivity. A graph was plotted for each stationary phase shown in Table 5, and the results are presented below. Figure 2 .

[0215] [Table 5] Table 5 Figure 2 In the diagram, the distribution is roughly linear. It can be argued that the separation is achieved through partitioning rather than adsorption.

[0216] Depend on Figure 2 It is evident that, with a mobile phase pH of 4.7, the stationary phase of Example 1 exhibits separation characteristics where both α(U / 5FU) and α(2'd / 2'd5FU) are above 1, showing a difference from amine-type stationary phases where these values ​​are mostly distributed below 1. On the other hand, it is evident that, with a mobile phase pH of 7.0, the stationary phase of Example 1 exhibits different behavior depending on the buffer solution.

[0217] Furthermore, it is known that the stationary phase of Example 2 exhibits the same separation characteristics as the amine-type stationary phase under any conditions where the pH of the mobile phase is 4.7 or 7.0.

[0218] [Graphing of the separation characteristics of the stationary phase in chromatography III] The vertical axis represents α (5FU / 2'd5FU), an indicator of amino selectivity, and the horizontal axis represents α (U / 2'dU), an indicator of hydrophilicity. A graph was plotted for each stationary phase shown in Table 5. The results are presented below. Figure 3 .

[0219] Figure 3In the examples, stationary phases 1 to 29 were broadly categorized into two groups: those distributed along the auxiliary lines shown in solid lines and those distributed along the auxiliary lines shown in dashed lines. More specifically, amine-type stationary phases were distributed along the solid lines with steeper slopes, while non-modified silica, neutral molecular-type stationary phases, amide-type stationary phases, zwitterionic stationary phases, acidic stationary phases, and quaternary ammonium-type stationary phases (Comparative Example 2) were distributed along the dashed lines with gentler slopes.

[0220] On the other hand, by Figure 3 It can be seen that the stationary phase of Example 1 deviates from both the solid and dashed lines. Furthermore, the stationary phase of Example 2 is plotted close to the solid line when the mobile phase pH is 4.7, but deviates from both the solid and dashed lines when the mobile phase pH is 7.0. Therefore, it can also be seen that the stationary phases of Examples 1 and 2 exhibit separation characteristics different from conventional stationary phases.

[0221] [Graphing of the separation characteristics of stationary phases in chromatography, IV] The vertical axis represents α (U / 2'dU) as an indicator of hydrophilicity, and the horizontal axis represents α (I / isol) as an indicator of the selectivity of inosine relative to acetal-protected inosine. Graphs were plotted for each stationary phase shown in Table 5. The results are presented in… Figure 4 .

[0222] Figure 4 In the diagram, the stationary phases of Examples 1 and 2 are shown in the upper right corner, revealing very high selectivity for OH groups. Therefore, it can be said that the stationary phases of Examples 1 and 2 are suitable for separating polar substances with more than one OH group.

Claims

1. The stationary phase used in chromatography is prepared by supporting a polymer with amine side chains on a support. The polymer having amine side chains has one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2). [Chemical Formula 1] In equations (1) and (2), R 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8.

2. The stationary phase for chromatography as described in claim 1, wherein, The polymer with amine side chains is supported on the support by chemical bonds.

3. The stationary phase for chromatography as described in claim 1, which is used in hydrophilic interaction chromatography.

4. The stationary phase for chromatography as described in claim 1, wherein, The support is a porous inorganic support or a non-porous inorganic support.

5. Separation methods, including: The separation process uses the chromatographic stationary phase as described in any one of claims 1 to 4 to separate substances by hydrophilic interaction chromatography.

6. A method for manufacturing a stationary phase for chromatography, comprising any one of the following steps (a) to (h), The monomers with amine side chains in (a) to (g) below are selected from one or more of the group consisting of monomers represented by formula (3) and monomers represented by formula (4). The polymers with amine side chains in (h) below have one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2). (a) A process of free radical polymerization of monomers with amine side chains in the presence of a support with polymerizable functional groups; (b) A step of chain-transfer polymerization of a monomer having an amine side chain in the presence of a compound having a crosslinkable silyl group and a chain-transfer functional group to obtain a polymer having the crosslinkable silyl group and an amine side chain; and a step of combining the crosslinkable silyl group of the polymer with functional groups on the surface of a support. (c) The process of polymerizing a monomer having an amine side chain with a silane coupling agent having a polymerizable functional group, and the process of attaching the resulting polymer to a support via a silane coupling reaction. (d) A process of polymerizing a monomer with amine side chains with a silane coupling agent with polymerizable functional groups in the presence of a support; (e) A process of chain transfer polymerization of monomers having amine side chains in the presence of a support with chain transfer functional groups; (f) A process of free radical polymerization of monomers with amine side chains in the presence of a support incorporating an atom transfer radical polymerization initiator group; (g) A step of performing atom transfer radical polymerization on a monomer having an amine side chain in the presence of a compound having a crosslinked silyl group and an atom transfer radical polymerization initiator group to obtain a polymer having the crosslinked silyl group and an amine side chain, and a step of combining the crosslinked silyl group of the polymer with functional groups on the surface of a support. (h) The process of coating a polymer with amine side chains onto the surface of a support. [Chemical Formula 2] In equations (1) to (4), R 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8.

7. The method for manufacturing a stationary phase for chromatography as described in claim 6, wherein, The support is a porous inorganic support or a non-porous inorganic support.

8. A polymer having an amine side chain, having one or more repeating units selected from the group consisting of repeating units represented by formula (1) and repeating units represented by formula (2), [Chemical Formula 3] In equations (1) and (2), R 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8.

9. The polymer having amine side chains as described in claim 8, used as a stationary phase for chromatography.

10. A method for manufacturing a polymer having amine side chains, comprising a polymerization step of polymerizing monomers having amine side chains. The monomer having an amine side chain is selected from one or more monomers represented by formula (3) and formula (4). [Chemical Formula 4] In equations (3) and (4), R 11 and R 21 Each represents a hydrogen atom or a methyl group; R 12 R 13 R 22 and R 23 Each represents a saturated aliphatic hydrocarbon group; Y 1 and Y 2 Each represents an oxygen atom, NH, or NCH3; n1 and n2 each represent an integer between 1 and 8.

11. The method for manufacturing a polymer having amine side chains as described in claim 10, wherein, The polymerization is atom transfer radical polymerization, free radical polymerization, or chain transfer polymerization.

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