Separation agent

Abstract

Provided is a separation agent having a carrier and a ligand that is supported on the surface of the carrier by means of physical adsorption or chemical bonding, wherein the carrier is core-shell particles composed of an inorganic non-porous core and a porous shell, the shell contains silica gel, the ligand is at least one selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids, and the maximum pore diameter is 15 nm or more. The separation agent achieves high resolution.

Description

Separating agent

[0001] The present disclosure relates to a separating agent.

[0002] In industrial fields such as pharmaceuticals, agricultural chemicals, and biochemistry, separating and purifying target substances to be produced is an extremely important issue, and methods using separating agents have long been used as such separation techniques. The principles by which such separating agents separate target substances include those that utilize the affinity between the separating agent and the target substance, and those that utilize the optical activity of the separating agent and the target substance. Conventionally, separating agents have been known in which fully porous silica gel is used as a carrier and various ligands are supported on the carrier depending on the target substance to be separated. Known separating agents obtained using fully porous silica gel as a carrier include, for example, those supported with optically active polymers. When such separating agents with optically active polymers supported on a carrier are used, optical resolution becomes possible.

[0003] Although fully porous materials are mainly used as supports for supporting ligands, core-shell particles having a non-porous core and a porous shell on the outer surface thereof are also known. For example, Patent Documents 1 and 2 disclose core-shell particles that can be used for chromatographic separation.

[0004] International Publication No. WO 2013 / 176215 International Publication No. WO 2014 / 087937

[0005] As described above, although the technology of using core-shell particles as a carrier is generally known, there have been insufficient reports examining the influence of the configuration conditions of the porous shell. In particular, the configuration conditions of the porous shell have not been examined much from the viewpoint of separation degree, and there is still room for improvement. Therefore, an object of the present disclosure is to provide a separating agent that has high separation degree while using core-shell particles consisting of a non-porous core and a porous shell as a carrier.

[0006] As a result of extensive research, the present inventors have found that the above-mentioned problems can be solved by setting the maximum pore diameter within a specific range in a configuration in which a specific ligand is supported on a core-shell particle consisting of a non-porous core and a porous shell, and have arrived at the invention of the present disclosure.

[0007] That is, the invention according to the present disclosure has the following features: [1] A separating agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding, wherein the carrier is a core-shell particle consisting of an inorganic non-porous core and a porous shell, the shell containing silica gel, the ligand is at least one selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids, and the maximum pore diameter is 15 nm or more. [2] A separating agent having a specific surface area of ​​40 to 109 m ２ [3] The separating agent according to [1], wherein the pore volume of the separating agent is 0.19 to 0.31 cm 3 / g. ３ / g. [4] The separating agent according to any one of [1] to [3], wherein the mass ratio of the ligand to the entire separating agent is 0.6 to 6.2 mass%. [5] The separating agent according to any one of [1] to [4], wherein the optically active polymer is at least one selected from the group consisting of polysaccharides, polysaccharide derivatives, optically active poly(meth)acrylic acid amides, optically active polyamino acids, and optically active polyamides. [6] The separating agent according to any one of [1] to [5], wherein the protein is at least one selected from the group consisting of glycoproteins, protein A, protein G, and proteins, and functional variants thereof. [7] The separating agent according to any one of [1] to [6], wherein the optically inactive polyester is at least one selected from the group consisting of polybutylene terephthalate, polyethylene terephthalate, and polylactic acid. [8] The separating agent according to any one of [1] to [7], wherein the nucleic acid is at least one selected from the group consisting of DNA, a DNA derivative, RNA, and an RNA derivative, and the number of bases of the nucleic acid is 5 to 10,000. [9] The separating agent according to any one of [1] to [8], which is a separating agent for chromatography.

[0008] According to the present disclosure, it is possible to provide a separating agent that has a high degree of separation while using core-shell particles consisting of a non-porous core and a porous shell as a carrier.

[0009] FIG. 10 is a schematic diagram of peaks for explaining elements related to calculation of resolution.

[0010] The embodiments of the present disclosure are described in detail below. However, each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications of the configurations are possible as appropriate within the scope of the present disclosure. The present disclosure is not limited by the embodiments, but only by the scope of the claims. In this disclosure, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits, and "A to B" means A or greater and B or less. Furthermore, when a numerical range expressed as "A to B" or "A or greater and B or less" is stated in stages (e.g., in order of preference), the upper and lower limits of each numerical range can be arbitrarily combined. Furthermore, although multiple embodiments are described in this disclosure, various conditions in each embodiment may be applied to each other to the extent applicable. Furthermore, the expression "A or B" in this disclosure can be interpreted as "at least one selected from the group consisting of A and B." Furthermore, in this disclosure, "multiple" means "two or more." In addition, in the present disclosure, the term "core-shell particles" can be appropriately read as "separating agent."

[0011] <Separating agent> A separating agent (hereinafter also simply referred to as "separating agent") according to one embodiment of the present disclosure is a separating agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding, wherein the carrier is a core-shell particle consisting of an inorganic non-porous core and a porous shell, and the shell contains silica gel, the ligand is at least one selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids, and the maximum pore diameter is 15 nm or greater.

[0012] The above separating agent uses core-shell particles consisting of an inorganic non-porous core and a porous shell as a carrier for the ligand, and has a large maximum pore diameter of 15 nm or more, which allows the ligand substance to penetrate into the inside of the shell, which the inventors speculate is why separation efficiency is improved.

[0013] The maximum pore diameter of the separating agent may be 15 nm or more, preferably 15 nm to 200 nm, more preferably 23 nm to 200 nm, even more preferably 23 nm to 100 nm, and particularly preferably 23 nm to 44 nm. If the maximum pore diameter is above the lower limit of the above range, the degree of separation is likely to be improved. Furthermore, if the maximum pore diameter is above the lower limit of the above range, the large pore diameter allows the ligand substance to sufficiently penetrate into the pores of the shell, ensuring a sufficient loading amount, thereby strengthening the interaction between the particle surface and the target compound, making it easier to achieve good separation. Furthermore, the maximum pore diameter can be controlled, for example, by adjusting the pH of the aqueous solution used when stacking the shells and performing polycondensation. Specifically, increasing the pH can be considered to increase the maximum pore diameter. The maximum pore diameter of the separating agent can be measured by mercury intrusion porosimetry. Mercury intrusion porosimetry is a method in which pressure is applied to cause mercury to penetrate the openings in the separating agent (effectively the openings in the shell portion after ligand loading), and the diameter of the pores, assumed to be cylindrical, is calculated from the pressure value and the corresponding volume of invaded mercury using the Washburn equation. JIS R 1655:2003 (Test method for pore size distribution of fine ceramics molded bodies by mercury intrusion porosimetry, established on May 20, 2003), which is specified for ceramic molded bodies, can be applied mutatis mutandis.

[0014] [Carrier] The separating agent uses core-shell particles consisting of an inorganic non-porous core and a porous shell as a carrier for carrying a ligand. Compared to fully porous carriers, core-shell particles have the advantages of being easier to narrow the particle size distribution and therefore more easily achieve dense packing, and of being easier to suppress solute diffusion because the core hinders solute diffusion, and of being able to shorten the diffusion distance of solutes within the porous portion because the porous portion is thin.

[0015] The term "non-porous" as used herein refers to a core particle having a specific surface area (m ２ / g) is defined as A, and the surface area calculated from the core particle diameter (calculated from the particle radius r, 4πr ２ ) can be calculated from the surface area per unit weight (m ２ When (A−B) / B×100 is B, (A−B) / B×100 is less than 20. On the other hand, the term “porous” in the present disclosure refers to a material having a specific surface area of ​​10 mm2 or less as measured by the BET method. ２ / g or more.

[0016] (Core) The core is inorganic and non-porous, that is, there is no particular limitation as long as it is a non-porous inorganic material. Examples of the shape of the core include spherical, granular, powdery, or any irregular shape. Among these, spherical is preferred from the viewpoint that the uniform particle shape makes it easier to ensure packing density. Note that "spherical" includes not only a perfect sphere but also an approximately spherical shape that can be generally recognized as a sphere.

[0017] The average particle diameter of the core is not particularly limited, but is preferably 0.1 μm to 200 μm, more preferably 0.1 μm to 100 μm, even more preferably 0.5 μm to 50 μm, and particularly preferably 1 μm to 50 μm. The particle diameter of the core can be measured, for example, by observing the separating agent or core-shell particles with an electron microscope such as a transmission electron microscope (TEM). Specifically, a thin sample of an arbitrarily selected separating agent or core-shell particles is prepared, and the thin sample is irradiated with an electron beam accelerated at high voltage. The maximum length of the non-porous portion on the photograph obtained by analyzing the transmitted electron beam is taken as the particle diameter of the core.

[0018] The core material constituting the core-shell particles is an inorganic substance, and specific examples thereof include non-porous particles selected from the group consisting of glass, metals such as titanium and zirconium and their metal oxides; and clay minerals such as bentonite and mica.

[0019] (Shell) The shell is not particularly limited as long as it is porous and contains silica gel, and may be composed of only silica gel. In addition, the silica gel is preferably a hydrolyzate of polyalkoxysiloxane. The external shape of the shell (substantially the external shape of the core-shell particles) may be, for example, spherical, granular, powdery, or any irregular shape. Among these, spherical is preferred from the viewpoint that the uniform particle shape makes it easier to ensure packing density.

[0020] The average shell thickness is not particularly limited, but is preferably 0.1 μm to 100 μm, more preferably 0.1 μm to 50 μm, even more preferably 0.1 μm to 10 μm, and particularly preferably 0.1 μm to 1 μm. When the average thickness is above the lower limit of the above range, the porous portion can be sufficiently loaded with the ligand substance, thereby allowing the sample to be sufficiently retained. Furthermore, when the average thickness is below the upper limit of the above range, the proportion of the shell in the entire core-shell particle is reduced, making it easier to demonstrate the advantages of the core-shell particle in terms of suppressing solute diffusion in the porous portion. The shell thickness can be measured, for example, by observing the core-shell particle with an electron microscope such as a transmission electron microscope (TEM). Specifically, a thin slice sample of an arbitrarily selected core-shell particle is prepared, and the thin slice sample is irradiated with an electron beam accelerated by a high voltage. The electron beam that passes through the sample is analyzed. The maximum length of the entire particle on the photograph obtained is subtracted from the maximum length of the non-porous portion, and the resulting value is divided by 2 to obtain the shell thickness.

[0021] The ratio of the average particle diameter of the core to the average thickness of the shell is not particularly limited, but is preferably 0.1 to 100, more preferably 1.0 to 10, and even more preferably 2.0 to 4.0. If this ratio is above the lower limit of the above range, the proportion of the shell in the entire core-shell particle is small, making it easier to demonstrate the advantage of core-shell particles in terms of suppressing solute diffusion in the porous portion. On the other hand, if this ratio is below the upper limit of the above range, the proportion of the porous portion in the core-shell particle is large, which makes it possible to sufficiently support the ligand substance in the porous portion, thereby allowing the sample to be sufficiently retained.

[0022] The shell contains silica gel. An embodiment containing silica gel (particularly an embodiment consisting of silica gel only) is preferred from the viewpoint of easy production of core-shell particles. When the silica gel is a hydrolyzed product of polyalkoxysiloxane, the form of the polyalkoxysiloxane is not particularly limited, and may be, for example, a product obtained by further hydrolyzing a polyalkoxysiloxane obtained by partial hydrolysis of an alkoxysilane. The alkoxysilane is preferably a tetraalkoxysilane, and among these, tetramethoxysilane, tetraethoxysilane, tetrapropoxysilane, or tetrabutoxysilane is preferred, and tetraethoxysilane is more preferred. For the production of core-shell particles, reference can be made to JP 49-36396 A. Specifically, first, an alkoxysilane is partially hydrolyzed to produce a polyalkoxysiloxane. Then, the polyalkoxysiloxane obtained is dissolved in a solvent such as ether, acetone, or dichloromethane to prepare a polyalkoxysiloxane solution. This solution is applied to the core particles or the core particles are immersed in this solution, and then the solvent is removed to deposit polyalkoxysiloxane as a shell on the surface of the core particles.The deposited polyalkoxysiloxane is then subjected to polycondensation (hydrolysis) in the presence of water, thereby obtaining core-shell particles.

[0023] As the core-shell type particles, commercially available products may be used.

[0024] The core-shell particles may be subjected to a surface treatment, for example, a method using a silane coupling agent having an amino group, such as 3-aminopropyltriethoxysilane.

[0025] [Ligand] In the present disclosure, the term "ligand" refers to a substance supported on a core-shell particle carrier, which exhibits physical affinity or is capable of chiral recognition for a target substance to be separated. The ligand is at least one selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids, from the viewpoints of affinity and specificity for a specific target molecule to be analyzed or separated, stability, reproducibility, etc. These components are described in detail below.

[0026] The average ligand loading rate SR (hereinafter also referred to simply as "loading rate") relative to the entire separating agent is expressed by the following formula (1). The loading rate SR is not particularly limited, but is preferably 0.6% by mass or more and 25% by mass or less, more preferably 0.6% by mass or more and 6.2% by mass or less, and even more preferably 2.7% by mass or more and 6.2% by mass or less. When the loading rate is equal to or greater than the lower limit of the above range, sufficient loading of the ligand substance facilitates separation through the interaction between the particle surface and the target compound. Furthermore, when the loading rate is equal to or less than the upper limit of the above range, the partition coefficient between the ligand on the core-shell particle surface and the target compound is reduced, thereby realizing rapid mass transfer and improving the number of theoretical plates.

[0027]

[0028] In the above formula (1), % C ＣＳＰ is the carbon content of the entire separating agent (mass%), %C Ｓｕｐｐｏｒｔ is the carbon content (mass%) of the entire support, %C Ｌｉｇａｎｄrepresents the carbon content (mass%) of the ligand. The carbon content of each of the above-mentioned substances (separating agent, carrier, ligand) can be measured using an elemental analysis (CHN analysis) device (for example, "Flash Smart CHNS MAS Plus" manufactured by Yamato Scientific Co., Ltd.).

[0029] (Optically Active Polymer) In the present disclosure, an optically active polymer refers to a polymer that has optical rotation, i.e., chirality, that rotates the plane of polarization when plane-polarized light is transmitted through a solution in which the polymer is dissolved. More specifically, examples of the optically active polymer include a polymer in which a monomer for constituting the optically active polymer has optical activity, and a polymer in which an optically inactive monomer is polymerized using an optically active polymerization catalyst.

[0030] The weight average molecular weight of the optically active polymer is not particularly limited, but is preferably 1,000 or more and 1,000,000 or less.

[0031] Polysaccharides or polysaccharide derivatives can be used as optically active polymers. Examples of polysaccharides include β-1,4-glucan (cellulose), α-1,4-glucan (amylose or amylopectin), α-1,6-glucan (dextran), β-1,6-glucan (pustulan), β-1,3-glucan (curdlan, schizophyllan), α-1,3-glucan, and β-1,2-glucan (Crown Gall polysaccharide), β-1,4-galactan, β-1,4-mannan, α-1,6-mannan, β-1,2-fructan (inulin), β-2,6-fructan (levan), β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan, β-1,4-N-acetylchitosan (chitin), pullulan, agarose, alginic acid, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or nigeran, or starch containing amylose.

[0032] Among these, from the viewpoint of easily obtaining high-purity polysaccharides, cellulose, amylose, β-1,4-chitosan, chitin, β-1,4-mannan, β-1,4-xylan, inulin, curdlan, pullulan, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, nigeran, and the like are preferred, and cellulose, amylose, pullulan, and nigeran are more preferred.

[0033] The number-average degree of polymerization of the polysaccharide (the average number of pyranose rings or furanose rings contained in one molecule) is not particularly limited, but from the viewpoint of ease of handling such as solubility and viscosity, it is preferably from 5 to 1,000, more preferably from 10 to 1,000, and even more preferably from 10 to 500.

[0034] For these polysaccharides, for example, ester derivatives or carbamate derivatives obtained by chemically modifying cellulose or amylose can be used as ligands. Such polysaccharide derivatives are known to have high optical resolution ability as chiral stationary phases. Specific examples of ester derivatives or carbamate derivatives include, for example, a cellulose derivative disclosed in Japanese Patent Publication No. 4-42371 in which the hydroxyl groups of cellulose are modified with a substituent in which some of the hydrogen atoms on the aromatic ring of phenyl carbamate are replaced with halogen (fluorine or chlorine), and a cellulose derivative or amylose derivative disclosed in Japanese Patent Publication No. 2005-315668 in which the hydroxyl groups of cellulose or amylose are modified with a substituent in which some of the hydrogen atoms on the aromatic ring of phenyl carbamate are replaced with fluorine, an alkyl group, or an alkoxy group. In such phenyl carbamate derivatives, the substituents substituting the hydrogen atoms on the aromatic rings include halogen groups only, alkyl groups only, or both halogen groups and alkyl groups. In this case, the halogen group is preferably a chlorine group, and the alkyl group is preferably an alkyl group having 1 to 3 carbon atoms, with a methyl group being particularly preferred. Of the above polysaccharides or polysaccharide derivatives, it is particularly preferred to use one selected from the polysaccharide derivatives described above, from the viewpoint of the separation performance of the optical isomers to be separated and the ease of loading onto core-shell particles. The polysaccharide derivative is not limited to those described above, and can be used as appropriate.

[0035] When a polysaccharide or a polysaccharide derivative is used as the ligand, the mass ratio of the polysaccharide or its derivative to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.

[0036] To support the polysaccharide or polysaccharide derivative on the core-shell particles by physical adsorption, the core-shell particles are immersed in a solution containing the polysaccharide or polysaccharide derivative and a solvent, and then the solvent is distilled off to allow physical adsorption onto the core-shell particles. The support rate of the polysaccharide or polysaccharide derivative on the core-shell particles is preferably 1.0 to 25% by mass.

[0037] Methods for chemically bonding a polysaccharide or polysaccharide derivative to a core-shell particle include a method of forming a chemical bond between the core-shell particle and the polysaccharide or polysaccharide derivative; a method of chemically bonding the core-shell particle and the polysaccharide or polysaccharide derivative via a third component (spacer); and a method of physically adsorbing the polysaccharide or polysaccharide derivative onto a carrier and crosslinking the polysaccharide or polysaccharide derivative on the carrier.

[0038] A method for forming a chemical bond between a core-shell particle and a polysaccharide or a polysaccharide derivative (hereinafter abbreviated as "reductive amination method") can be, for example, the method described in JP-A-07-138301. Specifically, this method involves forming a Schiff base between the reducing end of the polysaccharide and a surface-treated support, for example by reacting an aldehyde group with an amino group, and then reducing this to a secondary amine in the presence of a reducing agent (see ELISABETH KALLIN et al., Glycoconjugate J (1986) 3, 311-319), thereby supporting the polysaccharide on the support through a chemical bond, and then derivatizing the polysaccharide as needed.

[0039] A method for chemically bonding a core-shell particle and a polysaccharide or polysaccharide derivative via a third component (spacer) can be, for example, the method described in the examples of JP-A No. 2002-148247. Specifically, this method involves copolymerizing a polymerizable polysaccharide derivative having a polymerizable group such as a vinyl group introduced therein with a core-shell particle having a polymerizable group such as a vinyl group introduced therein in the presence of a third component (polymerizable monomer) having a polymerizable group such as a vinyl group.

[0040] A method for physically adsorbing a polysaccharide or polysaccharide derivative onto a support and crosslinking the polysaccharide or polysaccharide derivative on the support can be, for example, the method described in the Examples of JP-A-11-510193. Specifically, this method involves supporting a polysaccharide derivative on core-shell particles by coating, and then irradiating the polysaccharide derivative on the core-shell particles with light from an immersion mercury lamp to photochemically crosslink the polysaccharide derivative. The method for crosslinking the polysaccharide or polysaccharide derivative is not limited to the above method, but may also be used, for example, a method for crosslinking by a reaction induced by irradiation with radiation such as gamma rays or electromagnetic waves such as microwaves; or a method for crosslinking by a radical reaction using a radical initiator, etc.

[0041] As a method for crosslinking a polysaccharide or polysaccharide derivative coated on core-shell particles by a reaction induced by irradiation with radiation such as gamma rays or electromagnetic waves such as microwaves, for example, the method described in JP-A-2004-167343 can be used. This method involves coating a polysaccharide or polysaccharide derivative on surface-treated core-shell particles, and then irradiating the polysaccharide or polysaccharide derivative on the core-shell particles with gamma rays, thereby crosslinking the polysaccharide or polysaccharide derivative.

[0042] Other methods for chemically bonding core-shell particles to polysaccharides or polysaccharide derivatives include, for example, coating the core-shell particles with a polysaccharide derivative obtained by introducing alkoxysilyl groups into some of the hydroxyl groups or amino groups of the polysaccharide or polysaccharide derivative, and then carrying out a reaction such as polycondensation of the alkoxysilyl groups in an arbitrary solvent.

[0043] Poly(meth)acrylic acid amides can be used as optically active polymers, for example, poly(meth)acrylic acid amides. Poly(meth)acrylic acid amides obtained by polymerizing optically active (meth)acrylic acid amides represented by the following formula (I) are preferably used. Examples of such polymerization reactions include radical polymerization using a radical polymerization initiator such as AIBN (azobisisobutyronitrile) in the presence of a Lewis acid catalyst. The Lewis acid used here is preferably a metal Lewis acid, which is a metal salt (MX), such as scandium triflate, yttrium triflate, magnesium bromide, hafnium chloride, ytterbium triflate, or lutetium triflate. In the polymerization reaction, if the (meth)acrylic acid amide is a liquid at room temperature and normal pressure, the polymerization reaction can be carried out even in the absence of a solvent. However, if the (meth)acrylic acid amide is a solid, any conventional organic solvent without radical scavenging effect can be used as the reaction solvent. Tetrahydrofuran, chloroform, or methanol is more preferred. Other polymerization conditions can be appropriately adjusted by referring to WO 02 / 088204.

[0044]

[0045] In formula (I), R １ , R ２ and R ３ are different from each other and represent a hydrogen atom, a monovalent hydrocarbon group having 1 to 30 carbon atoms, or a monovalent atomic group containing a heteroatom; R ４ represents a hydrogen atom or a monovalent hydrocarbon group having 1 to 30 carbon atoms; R ５ represents a hydrogen atom or a methyl group.

[0046] The above R １ , R ２ and R ３ are preferably different from one another and are a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group, an aralkyl group, a carboalkoxy group, a carbamoyl group, a substituted alkyl group having 1 to 6 carbon atoms and substituted with an amino group, an amino group, an alkyl group having 1 to 6 carbon atoms and substituted with an alkoxy group, an alkoxy group, or a silyl group. ４is preferably a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group, or an aralkyl group, and particularly preferably a hydrogen atom.

[0047] When poly(meth)acrylic acid amide is used as the ligand, the mass ratio of poly(meth)acrylic acid amide to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.

[0048] Poly(meth)acrylic acid amide can be supported on core-shell particles by physical adsorption by immersing the core-shell particles in a solution in which the polyamino acid is dissolved (for example, using chloroform or dichloromethane as the solvent), and then distilling off the solvent.

[0049] The method of making poly(meth)acrylic acid amide supported on core-shell type particles by chemical bonding can be, for example, the method of introducing reactive functional groups into core-shell type particles, and making this reactive functional group react with the amide group that poly(meth)acrylic acid amide has; and the method of introducing reactive functional groups into core-shell type particles, and making the functional group that can react with this reactive functional group react with poly(meth)acrylic acid amide, and making both reactive functional groups react.The former method is carried out by, for example, carrying out surface treatment using a silane coupling agent or the like that has epoxy group, introducing epoxy group into core-shell type particles, and making this epoxy group react with the amide group that poly(meth)acrylic acid amide has.

[0050] Other methods for supporting poly(meth)acrylic acid amide on core-shell type particles by chemical bonding include, for example, a method in which a polymerizable functional group is introduced into the core-shell type particles by surface treatment using a silane coupling agent or the like having a polymerizable functional group such as a vinyl group and a (meth)acryloyl group, and a polymerizable functional group is also introduced into the poly(meth)acrylic acid amide by reaction with an isocyanate ester or the like (see JP 2006-177795 A), and the polymerizable functional groups of both are copolymerized.

[0051] Polyamino acids: For example, polyamino acids can be used as optically active polymers. Polyamino acids do not include proteins, which will be described later. Examples of such polyamino acids include those represented by the following formula (II). Such polyamino acids can be synthesized, for example, by the method described in JP-A-60-193538.

[0052]

[0053] In the above formula (II), n １ is 5 or more; R ６ represents an alkyl group having 1 to 5 carbon atoms, a phenyl group, an aralkyl group having 7 to 12 carbon atoms, or a heterocyclic group, and these groups may have a substituent such as a hydroxyl group, a carboxyl group, a mercapto group, an amino group, or a methylthio group; R ７ represents an alkyl group having 1 to 5 carbon atoms, and is preferably a methyl group or an ethyl group. Examples of the heterocyclic ring constituting the heterocyclic group include 5-pyrazolone, pyrazole, triazole, oxazolone, isoxazolone, barbituric acid, pyridone, pyridine, rhodanine, pyrazolidinedione, pyrazolopyridone, or Meldrum's acid, or heterocyclic rings thereof; or fused heterocyclic rings in which a hydrocarbon aromatic ring and a heterocyclic ring are fused together.

[0054] Examples of α-aminocarboxylic acids for constituting the above polyamino acids include alanine, valine, leucine, phenylalanine, proline, glutamic acid, aspartic acid, etc. In addition, amino acid derivatives such as benzyl aspartate, methyl glutamate, benzyl glutamate, carbobenzoxylysine, carbobenzoxyornithine, acetyltyrosine, and benzylserine can also be used as constituent materials of polyamino acids.

[0055] In the above formula (II), n １ is preferably 100 or less, and more preferably 10 to 40.

[0056] When poly(meth)acrylic acid amide is used as the ligand, the mass ratio of poly(meth)acrylic acid amide to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.

[0057] To support the polyamino acid on the core-shell particles by physical adsorption, the core-shell particles can be immersed in a solution in which the polyamino acid is dissolved (using, for example, dimethylformamide or dioxane as the solvent), and then the solvent can be evaporated.

[0058] The method of supporting polyamino acid on core-shell type particles by chemical bonding includes the method of introducing reactive functional group into core-shell type particles, and reacting this reactive functional group with the amino group of polyamino acid; and the method of introducing reactive functional group into core-shell type particles, and introducing functional group that can react with this reactive functional group into polyamino acid, and reacting both reactive functional groups;.The reactive functional group that is bonded to core-shell type particles can be, for example, epoxy group that reacts with the amino group of polyamino acid.The method of introducing reactive functional group into core-shell type particles can be, for example, surface-treating core-shell type particles with silane coupling agent having epoxy group.

[0059] Other methods for supporting polyamino acids on core-shell particles by chemical bonding include, for example, a method in which a surface treatment is performed using a silane coupling agent having a polymerizable functional group such as a vinyl group and a (meth)acryloyl group to introduce a polymerizable functional group into the core-shell particles, and a method in which a compound having a group reactive with the amino group of the polyamino acid and a polymerizable functional group, such as acrylic acid chloride, glycidyl methacrylate, and chloromethylstyrene, is reacted with the polyamino acid to introduce a polymerizable functional group into the polyamino acid, and the polymerizable functional groups of both are copolymerized.

[0060] Polyamides: For example, polyamides can be used as optically active polymers. Examples of such polyamides include those having one optically active amino acid residue in the main chain of the repeating unit. Examples of combinations of monomer components used in the synthesis of such optically active polyamides include combinations of N-substituted amino acids, which are optically active dicarboxylic acids, with diamines. Examples of N-substituted amino acids that can be used include N-substituted glutamic acid or N-substituted aspartic acid, and examples of diamines that can be used include aromatic diamines such as 4,4'-diaminodiphenylmethane or 1,3-phenylenediamine.

[0061] An example of a method for synthesizing polyamide will be described. As described above, polyamide can be synthesized by polymerizing an N-substituted amino acid, which is an optically active dicarboxylic acid, with a diamine. Specifically, a solution in which N-methylpyrrolidone (NMP) and pyridine (Py) are mixed in a volume ratio of, for example, 4:1, and lithium chloride (LiCl), for example, 4% by weight (hereinafter also referred to as "NMP-Py mixed solution") is added, for example, to 7.5 cm ３ A predetermined amount, for example, 3 mmol of benzoyl-L-glutamic acid (an N-substituted amino acid, which is an optically active dicarboxylic acid), an equimolar amount, for example, 3 mmol of 4,4'-diaminodiphenylmethane (a diamine), and twice the molar amount, for example, 6 mmol of triphenyl phosphite, are heated with stirring at a predetermined temperature, for example, 80°C, for a predetermined time, for example, 3 hours. After completion of the reaction, the product is added dropwise to methanol, which is then filtered to obtain a polymer, which is then dried under reduced pressure. Since the above polyamide is synthesized using an N-substituted amino acid, which is an optically active dicarboxylic acid, the polymer contains a D- or L-optical recognition site, and optical resolution can be performed using this optical recognition site.

[0062] In addition to the above polyamides, polyamides that can be used include those represented by the following general formula (III) or (IV).

[0063]

[0064]

[0065] In the above formulas (III) and (IV), R ８ and R ９ represents an alkylene group having 2 to 20 carbon atoms which may have a branched structure, a divalent group having 6 to 10 carbon atoms and one or more aromatic ring structures, or a divalent group having 3 to 10 carbon atoms and one or more alicyclic structures. ２ and n ３ is an integer from 50 to 100,000.

[0066] The polyamides represented by the formulae (III) and (IV) above may be obtained by the method described in Japanese Patent Publication No. 4-77737. The starting material is easily obtained by reacting (+) or (-)-trans-stilbenediamine with the corresponding dicarboxylic acid or its derivative. The dicarboxylic acid may be HOOC-R. １０ -COOH can be used. １０ is R ８ or R ９ and may be an alkylene group having 4, 6, 8, or 10 carbon atoms, a phenylene group, an oxydiphenylene group, or a cycloalkylene group having a cycloalkane structure such as cyclohexane or cyclobutane.

[0067] The synthesis method of polyamide is not limited to the above method, and synthesis may be performed by methods other than those described above. Furthermore, the appropriate reaction temperature and reaction time vary depending on the reagents used in the reaction and their amounts. The reaction time, reaction temperature, and amounts of reagents in the synthesis examples described above are examples of conditions under which an optically active polymer can be obtained, and can be changed as appropriate.

[0068] When polyamide is used as the ligand, the mass ratio of polyamide to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.

[0069] Polyamide can be supported on core-shell particles by physical adsorption by immersing the core-shell particles in a solution in which polyamide is dissolved (using, for example, hexafluoroisopropanol, dimethylformamide, or dichloromethane as the solvent) and then distilling off the solvent.

[0070] The method of making polyamide supported on core-shell particle by chemical bonding can be, for example, the method of introducing reactive functional group into core-shell particle, and making this reactive functional group react with the amide group that polyamide has; and the method of introducing reactive functional group into core-shell particle, and making the functional group that can react with this reactive functional group react with polyamide, and making both reactive functional groups react.The former method is carried out by, for example, carrying out surface treatment using a silane coupling agent etc. that has epoxy group, introducing epoxy group into core-shell particle, and making this epoxy group react with the amide group that polyamide has.

[0071] Other methods for supporting polyamide on core-shell particles by chemical bonding include, for example, a method in which a surface treatment is performed using a silane coupling agent or the like having a polymerizable functional group such as a vinyl group or a (meth)acryloyl group to introduce a polymerizable functional group into the core-shell particles, and a method in which a polymerizable functional group is also introduced into the polyamide by a reaction with an isocyanate ester or the like (see JP 2006-177795 A), and the polymerizable functional groups of both are copolymerized.

[0072] (Optically Inactive Polyester) Specific examples of optically inactive polyesters include polyethylene terephthalate, polybutylene terephthalate, polyethylene-polybutylene terephthalate, polytrimethylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, polylactic acid, polyglycolic acid, polyε-caprolactone, poly(oxycarbonyloxy-1,4-phenylene-2,2-isopropylidene-1,4-phenylene) (polycarbonate of bisphenol A), etc. Among these, at least one selected from the group consisting of polybutylene terephthalate, polyethylene terephthalate, and polylactic acid is preferred from the viewpoints of affinity and specificity for specific target molecules to be analyzed or separated, stability, reproducibility, etc.

[0073] The weight average molecular weight (Mw) of the optically inactive polyester is preferably 10,000 or more and 1,000,000 or less, more preferably 20,000 or more and 200,000 or less, from the viewpoints of strength of physical adsorption to the carrier and ease of handling due to increased viscosity of the solvent in which the polymer is dissolved.

[0074] When an optically inactive polyester is used as the ligand, the mass ratio of the optically inactive polyester to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.

[0075] When the optically inactive polyester is supported on the core-shell particles by physical adsorption, this can be achieved by immersing the core-shell particles in a solution in which the optically inactive polyester is dissolved, and then distilling off the solvent.

[0076] As a method for supporting an optically inactive polyester on a core-shell particle by chemical bonding, for example, a method of introducing a reactive functional group into the core-shell particle, introducing a functional group capable of reacting with this reactive functional group into the optically inactive polyester, and reacting both reactive functional groups can be mentioned. Such a method is carried out, for example, by carrying out a surface treatment using a silane coupling agent having an epoxy group to introduce an epoxy group into the core-shell particle, and carrying out a chemical treatment using a polyamine to introduce an amino group into the optically inactive polyester, and then reacting the epoxy group introduced into the core-shell particle with the amino group introduced into the polyester in vacuum or air.

[0077] Examples of polyamines for introducing amino groups into optically inactive polyesters include (poly)alkylene polyamines having 2 to 8 carbon atoms. Specific examples of (poly)alkylene polyamines having 2 to 8 carbon atoms include ethylene diamine, propylene diamine, butylene diamine, diethylene triamine, triethylene tetramine, and tetraethylene pentamine. Preferably, the polyamine is one or more selected from the group consisting of ethylene diamine, diethylene triamine, triethylene tetramine, and tetraethylene pentamine, with diethylene triamine being particularly preferred. In particular, the use of a polyamine containing at least three amino groups improves the introduction of amino groups into the polyester. Furthermore, low molecular weight amines are preferred because they are easily removed after chemical treatment.

[0078] An example of a chemical treatment method using a polyamine is a method in which an optically inactive polyester is heat-treated in a solution in which a polyamine is dissolved in an organic solvent or a mixed solvent of water and an organic solvent.

[0079] Separation materials using optically inactive polyesters as ligands are expected to be useful for separating high molecular weight molecules such as proteins.

[0080] (Protein) A protein can be used as the ligand. The protein has a molecular weight of 3 to 300 kDa, preferably 30 to 150 kDa, and can be a substance that has affinity for the protein to be separated, such as an antibody. Among these, from the viewpoint of high selectivity when used to separate antibodies, at least one selected from the group consisting of glycoprotein, protein A, protein G, protein L, albumin, and functional variants thereof is preferred, and at least one selected from the group consisting of glycoprotein, protein A, protein G, protein L, and functional variants thereof is more preferred. When the main purpose is to separate antibodies, the ligand is preferably one that can specifically bind to a portion of immunoglobulin.

[0081] The glycoprotein that can be used is α1-acid glycoprotein. Origins of α1-acid glycoprotein include mammals such as humans, cows, and rabbits, and birds such as chickens, swans, and turkeys. Of these, specific examples of preferred α1-acid glycoproteins include human α1-acid glycoprotein (hereinafter also referred to as h-AGP) and chicken AGP (hereinafter also referred to as c-AGP).

[0082] Human α1-acid glycoprotein (h-AGP) can be a commercially available product (for example, manufactured by Merck Co., Ltd.) If necessary, the commercially available product may be purified by high performance liquid chromatography before immobilization.

[0083] Chicken α1-acid glycoprotein (c-AGP) can be obtained by separating ovomucoid and chicken α1-acid glycoprotein (c-AGP) from crude chicken ovomucoid by liquid chromatography using a cation exchange carrier (e.g., "SP Sepharose" manufactured by Merck) using a stepwise elution method with ammonium acetate buffer (pH 4.6). At this time, a fraction containing c-AGP may be collected and further purified using an ion exchange chromatography carrier such as SP Sepharose.

[0084] Human α1-acid glycoprotein is a glycoprotein consisting of 183 amino acid residues and five sugar chains with a molecular weight of approximately 41,000 to 43,000. Chicken α1-acid glycoprotein has the same amino acid residues and sugar chains as human α1-acid glycoprotein, but its molecular weight is approximately 30,000.

[0085] Examples of albumin include ovalbumin and human serum albumin (molecular weight approximately 66 kDa).

[0086] The functional variant refers to a protein that has at least one alteration in its native amino acid sequence and has at least one function associated with the native sequence. The native sequence includes an amino acid sequence that originally occurs in nature. Alterations of the native amino acid sequence can include, for example, substitution of one or more amino acids with other amino acids; deletion of one or more amino acids; addition of one or more amino acids; and combinations thereof (e.g., a combination of substitution of one or more amino acids with other amino acids, addition of one or more amino acids, and deletion of one or more amino acids from the native sequence).

[0087] Functional variants can also include fragments or domains of a protein. The amino acid sequence of a functional variant can be 70% or more identical, 75% or more identical, 80% or more identical, 85% or more identical, 90% or more identical, 95% or more identical, or 98% or more identical to the native amino acid sequence.

[0088] Alternatively, cellobiohydrolases (CBHs) known as cellobiohydrolases I and II shown below can also be used as proteins. Cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II) are known to be major enzymes that account for the majority of cellulases (80% or more together, with small amounts of endoglucanases, β-glucosidases, and the like also being included). Methods for producing these proteins include expressing large amounts of the desired cellobiohydrolase in host cells using genetic recombination techniques.

[0089] From the viewpoint of obtaining better separation performance, the protein loading rate in the separating agent is preferably 1.0 mass% or more and 25.0 mass% or less, more preferably 2.0 mass% or more and 20.0 mass% or less, even more preferably 3.0 mass% or more and 15.0 mass% or less, still more preferably 4.0 mass% or more and 10.0 mass% or less, and particularly preferably 5.0 mass% or more and 8.0 mass% or less.

[0090] When a protein is used as the ligand, the mass ratio of the protein to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.

[0091] To make core-shell particles carry proteins by physical adsorption, the core-shell particles may be immersed in a solution in which the protein is dissolved in an appropriate solvent, such as hexane or chloroform, and then the solvent is removed by evaporation, for example, by drying under reduced pressure.

[0092] Methods for chemically bonding a protein to a core-shell particle typically include, but are not limited to, a method in which reactive functional groups are introduced into the core-shell particle and then this reactive functional group is reacted directly with a functional group possessed by the protein; or a method in which reactive functional groups are introduced into the core-shell particle and then the core-shell particle and the protein are chemically bonded via a compound having one or more functional groups capable of reacting with the reactive functional groups bonded to the core-shell particle and one or more functional groups capable of reacting with a functional group possessed by the protein (hereinafter, such compounds are collectively referred to as a "spacer").

[0093] An example of the former method is a method in which, when a ligand having an amino group (e.g., Protein A) is carried out, the core-shell particles are surface-treated with a silane coupling agent or the like having a functional group such as an epoxy group that can react with an amino group, thereby introducing a functional group that reacts with an amino group into the core-shell particles, and then this functional group is allowed to react directly with Protein A.

[0094] Examples of the latter method include a method in which an amino acid (amine carboxylic acid) is used as a spacer, and the amino group of the amino acid is reacted with an epoxy group introduced into the core-shell particle, and then the carboxyl group of the amino acid is reacted with an amino group of Protein A; and a method in which a diamine or diol and a diglycidyl compound such as (poly)ethylene glycol diglycidyl ether are successively used as spacers, and the epoxy group introduced into the core-shell particle is reacted with one end group of the diamine or diol, and then the other end group of the diamine or diol is reacted with one epoxy group of the diglycidyl compound, thereby introducing an epoxy group into the core-shell particle and reacting this epoxy group with Protein A.

[0095] Examples of the diamine spacer include aliphatic diamines such as tetramethylenediamine and hexamethylenediamine. Examples of the diol spacer include aliphatic diols such as propylene glycol, butanediol, diethylene glycol, and triethylene glycol, and polyalkylene glycols such as polyethylene glycol.

[0096] The spacer preferably has a linear structure, taking into consideration the reactivity with the ligand and the steric hindrance with the core-shell particles when supporting by chemical bonding. A separating agent in which the core-shell particles and the ligand are bonded via a spacer with a linear structure is less likely to suffer from problems caused by steric hindrance, such as the prevention of the formation of affinity bonds between the protein ligand and the target to be separated (e.g., antibody), and therefore is more likely to ensure good separation performance.

[0097] Alternatively, the protein can be reacted with carbonic acid disuccinimide (N,N'-disuccinimidyl carbonate: DSC) and then reacted with core-shell particles to which amino groups have been introduced using 3-aminopropyltriethoxysilane or the like, thereby allowing the protein to be supported on the core-shell particles by chemical bonding.

[0098] (Nucleic Acid) Nucleic acids can be used as ligands. The nucleic acids are not particularly limited, but examples include DNA, RNA, oligonucleotides, and modified oligonucleotides. DNA or RNA derivatives can also be used. The DNA or RNA may be natural or artificial, but considering its stability as a separation agent, it is preferable to use structurally stable artificial forms. Artificial forms can form sequences that do not exist in natural forms. Among these, at least one selected from the group consisting of DNA, DNA derivatives, RNA, and RNA derivatives is preferred from the viewpoints of affinity, specificity, stability, reproducibility, etc., for the specific target molecule to be analyzed or separated. The number of bases in the nucleic acid is not particularly limited, but is preferably 5 to 10,000. Artificial nucleic acids with 50 to 200 bases are particularly preferred, and those with approximately 100 bases are preferred from the viewpoint of enabling efficient synthesis. In artificial nucleic acids, it is preferable that thymines are not adjacent to each other to prevent thymine dimerization.

[0099] Furthermore, in consideration of durability as a separation agent, the nucleic acid may be derivatized with a protecting group, specifically, the hydroxyl groups at either or both of the 5'- and 3'-positions may be derivatized with a phosphate ester group, an acyl group, an alkoxycarbonyl group, a benzyl group, a substituted benzyl group, an allyl group, or the like.

[0100] When nucleic acids are used as ligands, the mass ratio of the nucleic acids to the entire separating agent is preferably 0.5% by mass or more and 25% by mass or less. If the mass ratio is less than the lower limit of the above range, the nucleic acids cannot be stably present in the separating agent, and sufficient separation performance cannot be obtained. On the other hand, if the mass ratio is more than the upper limit of the above range, the nucleic acids cannot be fully supported on the core-shell particles, resulting in the generation of free nucleic acids, which may adversely affect separation performance.

[0101] To make the core-shell particles carry nucleic acids by physical adsorption, for example, the core-shell particles are dispersed in distilled water to form a suspension, and the nucleic acid is added to the suspension either as is or in the form of an aqueous solution of nucleic acid in distilled water, followed by drying. In this case, a portion of the nucleic acid may be added as is without being converted into an aqueous solution, and the remaining portion may be added in the form of an aqueous solution.

[0102] To make the core-shell particles carry the nucleic acid by chemical bonding, for example, there is mentioned a method of immobilizing the nucleic acid on the carrier via chitosan and the amino group of the chitosan, as described in JP-A-2010-259405.

[0103] In this method, core-shell particles are first subjected to a vapor deposition reaction with an aminosilane such as 3-aminopropyltriethoxysilane, followed by a heat treatment. The aminosilane-treated core-shell particles are then immersed in a glutaraldehyde solution, washed, and air-dried. The glutaraldehyde-treated core-shell particles are then immersed in a chitosan solution and washed with ultrapure water. This allows the aldehyde groups of the glutaraldehyde to react with the amino groups of the chitosan, introducing numerous amino groups onto the surface of the core-shell particles and increasing the surface area for binding nucleic acids. The chitosan-treated core-shell particles are then immersed in a glutaraldehyde solution, washed, and air-dried. This introduces aldehyde groups into the core-shell particles. An avidin solution is dropped onto the core-shell particles and allowed to stand, whereby the aldehyde groups introduced into the core-shell particles react with the amino groups of the avidin, immobilizing the avidin to the core-shell particles via chitosan. A biotin-labeled nucleic acid solution is then dropped onto the avidin-immobilized core-shell particles for reaction, whereby the nucleic acid is immobilized to the core-shell particles via chemical bonds.

[0104] Among the above-mentioned ligands, polymeric ligands have stacked interaction sites compared to low molecular weight ligands, and these interact in a complex manner with the target compound, enabling the resolution of a wide variety of compounds. From this viewpoint, the ligand is preferably at least one selected from the group consisting of polysaccharides, polysaccharide derivatives, optically active poly(meth)acrylic acid amides, optically active polyamino acids, and optically active polyamides.

[0105] <Method for Evaluating Separating Agents> In the present disclosure, the separation performance of a separating agent is evaluated using the separation factor (α), the number of theoretical plates (N1), and the resolution (Rs) when the separating agent is used as a chromatographic separating agent. In the present disclosure, a separating agent is evaluated as having good separation performance if it has high values ​​for one or more, preferably two, and more preferably all, of the separation factor (α), the number of theoretical plates (N1), and the resolution (Rs). Each index is defined as follows:

[0106] Separation factor (α) α = k2 / k1 Where, k1: Retention factor of the component that is more weakly retained, and is calculated by the following formula: k1 = (t1 - t0) / t0 k2: Retention factor of the component that is more strongly retained, and is calculated by the following formula: k2 = (t2 - t0) / t0 t0: Dead time (The time from when a substance that does not interact with the separating agent is introduced into the column until it is eluted. For convenience, the elution time of tri-tert-butylbenzene is taken as the dead time.) t1: Elution time of the component that is more weakly retained t2: Elution time of the component that is more strongly retained

[0107] Number of theoretical plates (N1) N1 = 16 × (tr / W) ２ tr: retention time W: peak width

[0108] ・Separation degree (Rs)

[0109]

[0110] t Ｒ１ , t Ｒ２ : Retention time (t Ｒ１ ≦t Ｒ２ ) (See Figure 1) W １ , W２ : Peak width (see Figure 1) W ０．５ｈ１ , W ０．５ｈ２ : Peak width at half the peak height (half width; see Figure 1)

[0111] A resolution Rs of 1.5 or more generally indicates complete separation (baseline separation). According to a standard resolution curve for two peaks with a peak height ratio of 1 / 1, an Rs of 1.25 or more indicates that the valley depth between the two peaks is 99.4%, indicating nearly complete baseline separation. Therefore, an Rs of 1.25 or more is preferred, and an Rs of 1.5 or more is more preferred.

[0112] The specific surface area of ​​the separating agent is not particularly limited, but is preferably 20 m ２ / g or more 200m ２ / g or less, and ２ / g or more 109m ２ / g or less is more preferable, and 40m ２ / g or more 73m ２ / g or less is even more preferable. If the specific surface area is equal to or greater than the lower limit of the above range, the pore volume can be secured, allowing sufficient loading of the ligand substance, and facilitating separation through the interaction between the particle surface and the target compound. Furthermore, if the specific surface area is equal to or less than the upper limit of the above range, the pore diameter is large, allowing the ligand substance to penetrate sufficiently deep into the shell pores, ensuring a sufficient loading amount, strengthening the interaction between the particle surface and the target compound, and facilitating successful separation. The specific surface area can be controlled, for example, by adjusting the pH of the aqueous solution used when stacking the shells and polycondensing the particles. Specifically, decreasing the pH can be considered to increase the specific surface area. The specific surface area can be measured by mercury intrusion porosimetry, similar to the measurement of the maximum pore diameter described above.

[0113] The pore volume of the separating agent is not particularly limited, but is preferably 0.1 cm ３ / g or more 0.5cm ３ / g or less, and ３ / g or more 0.31cm ３ / g or less, and more preferably 0.19 cm ３ / g or more 0.29cm ３ / g or less is even more preferable. When the pore volume is equal to or greater than the lower limit of the above range, the pore volume can be secured, allowing sufficient support of the ligand substance, and facilitating separation through the interaction between the particle surface and the target compound. Furthermore, when the pore volume is equal to or less than the upper limit of the above range, the pore diameter is large, allowing the ligand substance to penetrate sufficiently into the pores of the shell, ensuring a sufficient support amount, thereby strengthening the interaction between the particle surface and the target compound, and facilitating good separation. The pore volume can be controlled, for example, by adjusting the pH of the aqueous solution used when laminating the shells and carrying out polycondensation. Specifically, decreasing the pH can be considered to increase the pore volume. The pore volume can be measured by mercury intrusion porosimetry, similar to the measurement of the maximum pore diameter described above.

[0114] The bulk density of the separating agent is not particularly limited, but is preferably 0.45 g / cm ３ 1.0g / cm or more ３ It is preferable that the density is 0.56 g / cm or less. ３ 0.62g / cm or more ３ It is more preferable that the core volume is less than 0.05. If the core volume of the core-shell particle is too large, the shell cannot sufficiently support the ligand, while if it is too small, the diffusion of the target compound in the porous portion increases, making it difficult to realize the advantages of the core-shell particle. Furthermore, if the bulk density is high, the packed layer approaches a close-packed structure, and the voids become small, making it easier to suppress the diffusion of the sample. The above bulk density can be measured by the tapping method.

[0115] The d90 / d10 of the core-shell particles is usually 1.00 or more and 1.38 or less, preferably 1.05 or more and 1.36 or less, more preferably 1.10 or more and 1.17 or less, even more preferably 1.15 or more and 1.17 or less, and still more preferably 1.17.

[0116] By setting the d90 / d10 of the core-shell particles within the above range, the separation performance of the separating agent can be improved.

[0117] In the present disclosure, "d10" and "d90" refer to particle sizes at which the cumulative distribution is 10% and 90%, respectively, in a number-based particle size distribution determined by observing the core-shell particles using a scanning electron microscope (SEM) (for example, "SU-5000" manufactured by Hitachi High-Technologies Corporation), randomly selecting approximately 300 particles from the obtained image, and analyzing the particles for circularity using image analysis software (for example, "Image J" manufactured by Wyne Rasband). The d10 and d90 of core-shell particles can be determined not only by SEM observation of the core-shell particles but also by SEM observation of the separating agent.

[0118] The average particle size of the core-shell particles is not particularly limited, but is preferably 0.5 μm or more and 1000 μm or less, more preferably 1.0 μm or more and 200 μm or less, even more preferably 1.5 μm or more and 50 μm or less, still more preferably 2.0 μm or more and 10 μm or less, and particularly preferably 2.5 μm or more and 5.0 μm or less.

[0119] In the present disclosure, the term "average particle size" refers to the number-average particle size. The number-average particle size is determined as the arithmetic mean value of the particle sizes of particles measured by observing particles with a scanning electron microscope (SEM) (for example, "SU-5000" manufactured by Hitachi High-Technologies Corporation), randomly selecting about 300 particles from the obtained image, and analyzing the circular particle size using image analysis software (for example, "Image J" manufactured by Wyne Rasband).

[0120] <Method for producing separating agent> The method for producing the separating agent is not particularly limited, and the separating agent can be produced by a known method or a combination of known methods while referring to the above-mentioned explanation of the separating agent.

[0121] <Uses of the Separating Agent> The separating agent can be preferably used for chromatography. In particular, when an optically active ligand is used, the separating agent can be used as a separating agent for optical isomers, and when an optically inactive ligand is used, the separating agent can be used as a separating agent for affinity chromatography. These separating agents can be used as packing materials for liquid chromatography, as well as packing materials for capillary columns for supercritical fluid chromatography, gas chromatography, electrophoresis, or capillary electrochromatography (CEC); or for CZE (capillary zone electrophoresis) or MEKC (micellar electrokinetic chromatography), etc.

[0122] The present disclosure will be described in more detail below with reference to examples, but the present disclosure should not be construed as being limited to the following examples.

[0123] <Evaluation of Properties> The following describes the evaluation methods for properties measured using the separating agent and column prepared by the methods described below, and the evaluation results are shown in Tables 1 and 2.

[0124] [Separation Test] The separating agent obtained in the examples was pressurized and packed into a stainless steel column of φ0.21 cm×L25 cm by a slurry packing method to prepare a chromatography column.

[0125] The prepared chromatography column was connected to a liquid chromatograph ("X-LC" manufactured by JASCO Corporation). Optical resolution of the racemic mixture of trans-stilbene oxide (TSO) was carried out using this liquid chromatograph under the analytical conditions described above, and the separation performance (k1, k2, N1, Ps1, α, Rs) of the separating agent was evaluated.

[0126] (Separation conditions) Mobile phase: n-Hexane / 2-Propanol=9 / 1 (v / v) Flow rate: 0.21 ml / min Temperature: 25°C Detection: 254 nm

[0127] [Ligand Loading Rate] The ligand loading rate was calculated from the following formula (1): % C ＣＳＰ is the carbon content of the entire separating agent (mass%), %C Ｓｕｐｐｏｒｔ is the carbon content (mass%) of the entire support, %CＬｉｇａｎｄ represents the carbon content (mass%) of the ligand, and the carbon content of each object (separating agent, carrier, ligand) was measured using an elemental analysis (CHN analysis) device ("Flash Smart CHNS MAS Plus" manufactured by Yamato Scientific Co., Ltd.).

[0128]

[0129] [Bulk density] The bulk density of the separating agent was measured by the tapping method. Specifically, a predetermined amount of filler was weighed into a 5 mL measuring cylinder and dropped vertically from a height of 2 to 3 cm. This operation was repeated until the following termination conditions were met to determine the bulk density. (Termination conditions) (1) The above operation was performed at least 300 times. (2) The volume change after performing the above operation 50 times consecutively was 0.1 cm. ３ It must be less than or equal to:

[0130] [Maximum pore diameter, specific surface area, and pore volume] The maximum pore diameter of the separating agent was measured by mercury intrusion porosimetry. Specifically, JIS R 1655 was applied, and mercury was forced into the pores by applying pressure. The diameter of the pores, assumed to be cylindrical, was calculated using the pressure value and the corresponding volume of invaded mercury according to the Washburn equation. The specific surface area and pore volume of the separating agent were also measured by mercury intrusion porosimetry.

[0131] <Experiment A> [Preparation of Separation Column] (Example 1) (1) Synthesis of Ligand Component 3-chloro-4-methylphenyl isocyanate and cellulose were reacted in pyridine solvent under the conditions described in B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39 to obtain a white solid (1) (cellulose tris(3-chloro-4-methylphenylcarbamate)). (2) Preparation of Core-Shell Particles 0.6 g of the white solid (1) was dissolved in 4.8 mL of acetone. The solution was uniformly applied to 5.4 g of core-shell silica gel (average particle size of core-shell particles: 2.7 μm, shell pore diameter: 100 nm (catalog value; measured by gas adsorption method), average core particle size: 1.7 μm, core material: glass, average shell thickness: 0.5 μm, ratio of average core particle size / average shell thickness: 3.4, shell material: silica gel (hydrolyzed polyalkoxysiloxane)) so that the mass ratio of cellulose tris(3-chloro-4-methylphenylcarbamate) to the total particles was 10 mass %, and the solvent was distilled off under reduced pressure to produce particles on which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported by physical adsorption. Next, 3.3 g of the resulting particles were suspended in 500 mL of acetonitrile / water = 60 / 40 (vol. / vol.) and stirred. The resulting suspension was then irradiated with an immersion mercury lamp (Philips, HPK-125 watts, quartz-sheathed) for 10 minutes. The precipitate was collected by filtration, washed with tetrahydrofuran, and dried to obtain core-shell particles (separating agent) in which cellulose tris(3-chloro-4-methylphenylcarbamate) was immobilized (chemically bonded) to silica. The yield was 3.1 g. (3) Filling of separating agent into column The core-shell particles prepared in (2) were pressurized and packed using a slurry packing method, and a stainless steel column measuring φ0.21 cm x L25 cm was packed to prepare a column.

[0132] Example 2 The core-shell silica gel was changed to a core-shell silica gel having the following conditions: average particle diameter of the core-shell particles: 2.7 μm, shell pore diameter: 50 nm (catalog value; measured by gas adsorption method), average core particle diameter: 1.7 μm, core material: glass, average shell thickness: 0.5 μm, average core particle diameter / average shell thickness ratio: 3.4, shell material: silica gel (hydrolyzed polyalkoxysiloxane). Under the same conditions as in Example 1, except that the core-shell silica gel was changed to a core-shell silica gel having the following conditions, particles on which cellulose tris(3-chloro-4-methylphenylcarbamate) was supported by physical adsorption were prepared. Next, 3.0 g of the obtained particles were irradiated with light under the same conditions as in Example 1, and core-shell particles (separating agent) in which cellulose tris(3-chloro-4-methylphenylcarbamate) was immobilized (chemically bonded) on silica were obtained. The yield was 2.9 g. Finally, the separating agent was packed into a stainless steel column in the same manner as in Example 1 to prepare a column.

[0133] (Example 3) (1) Synthesis of Ligand Component 3-Chloro-4-methylphenyl isocyanate and amylose were reacted in pyridine solvent under the conditions described in B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39 to obtain a white solid (2) (amylose tris(3-chloro-4-methylphenylcarbamate)). (2) Preparation of Core-Shell Particles 0.6 g of the white solid (2) was dissolved in 4.8 mL of tetrahydrofuran. The solution was uniformly applied to 5.4 g of core-shell silica gel (average particle size of core-shell particles: 2.7 μm, shell pore diameter: 100 nm (catalog value; measured by gas adsorption), average core particle size: 1.7 μm, core material: glass, average shell thickness: 0.5 μm, shell material: silica gel (hydrolyzed polyalkoxysiloxane)) so that the mass ratio of amylose tris(3-chloro-4-methylphenylcarbamate) to the total particles was 10% by mass. The solvent was then distilled off under reduced pressure to produce particles carrying amylose tris(3-chloro-4-methylphenylcarbamate) supported by physical adsorption. Next, 3.0 g of the resulting particles were suspended in 500 mL of acetonitrile / water = 60 / 40 (vol. / vol.) and stirred. The resulting suspension was then irradiated with an immersion mercury lamp (Philips, HPK-125 watt, quartz-sealed) for 18 minutes. The precipitate was collected by filtration, washed with tetrahydrofuran, and dried to obtain core-shell particles (separating agent) in which amylose tris(3-chloro-4-methylphenylcarbamate) was immobilized (chemically bonded) to silica. The yield was 2.6 g. (3) Filling of separating agent into column The core-shell particles prepared in (2) were pressurized and packed by a slurry packing method, and packed into a stainless steel column measuring φ0.21 cm x L25 cm to prepare a column.

[0134] Comparative Example 1 The core-shell silica gel was changed to a core-shell silica gel having the following conditions: average particle diameter of the core-shell particles: 2.7 μm, shell pore diameter: 16 nm (catalog value; measured by gas adsorption method), average core particle diameter: 1.7 μm, core material: glass, average shell thickness: 0.5 μm, average core particle diameter / average shell thickness ratio: 3.4, shell material: silica gel (hydrolyzed polyalkoxysiloxane). Particles carrying cellulose tris(3-chloro-4-methylphenylcarbamate) by physical adsorption were prepared under the same conditions as in Example 1. Next, 3.2 g of the obtained particles were irradiated with light under the same conditions as in Example 1 to obtain core-shell particles (separating agent) in which cellulose tris(3-chloro-4-methylphenylcarbamate) was immobilized (chemically bonded) on silica. The yield was 2.9 g. Finally, the separating agent was packed into a stainless steel column in the same manner as in Example 1 to prepare a column.

[0135] In Table 1 below, "-" is entered in the Ps1 column for Example 2 and Comparative Example 1, which means that measurement was not possible.

[0136]

[0137] Table 1 shows that in a form in which the ligand is chemically bonded to the surface of the carrier, the resolution Rs is improved by setting the maximum pore diameter of the separating agent to 15 nm or more. In particular, in Examples 1 and 3, complete separation (baseline separation) is achieved because Rs is 1.5 or more, and in Example 2, almost baseline separation is achieved because Rs is 1.25 or more.

[0138] <Experiment B> [Production of Separation Column] (Example 4) A column was produced in the same manner as in Example 1, except that light irradiation was not performed.

[0139] <Experiment B> (Example 5) A column was prepared in the same manner as in Example 2, except that light irradiation was not performed.

[0140] Example 6 A column was prepared in the same manner as in Example 3, except that light irradiation was not performed.

[0141] Comparative Example 2 A column was prepared in the same manner as in Comparative Example 1, except that light irradiation was not performed.

[0142] In Table 2 below, "-" in the bulk density column means that the density was not measured.

[0143]

[0144] From Table 2, it can be seen that even in an embodiment in which the ligand is physically adsorbed on the surface of the carrier, an excellent resolution Rs can be ensured in an embodiment in which the maximum pore diameter is 15 nm or more.

[0145] From the above, it was found that by making the maximum pore diameter of the core-shell particles carrying the ligand 15 nm or more, it is possible to provide a separating agent with high separation efficiency while using the core-shell particles consisting of a non-porous core and a porous shell as a carrier.

Claims

1. A separating agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding, wherein the carrier is a core-shell particle consisting of an inorganic non-porous core and a porous shell, the shell containing silica gel, the ligand is at least one selected from the group consisting of optically active polymers, optically inactive polyesters, proteins, and nucleic acids, and the maximum pore diameter is 15 nm or greater.

2. The specific surface area of ​​the separating agent is 40 to 109 m ２ The separating agent according to claim 1, wherein the molecular weight of the separating agent is 1 / g.

3. The pore volume of the separating agent is 0.19 to 0.31 cm ３ The separating agent according to claim 1 or 2, wherein the molecular weight of the separating agent is 1 / g.

4. The separating agent according to any one of claims 1 to 3, wherein the mass ratio of the ligand to the entire separating agent is 0.6 to 6.2 mass%.

5. The separating agent according to any one of claims 1 to 4, wherein the optically active polymer is at least one selected from the group consisting of polysaccharides, polysaccharide derivatives, optically active poly(meth)acrylic acid amides, optically active polyamino acids, and optically active polyamides.

6. The separating agent according to any one of claims 1 to 5, wherein the protein is at least one selected from the group consisting of glycoprotein, protein A, protein G, and protein, and functional variants thereof.

7. The separating agent according to any one of claims 1 to 6, wherein the optically inactive polyester is at least one selected from the group consisting of polybutylene terephthalate, polyethylene terephthalate, and polylactic acid.

8. The separating agent according to any one of claims 1 to 7, wherein the nucleic acid is at least one selected from the group consisting of DNA, DNA derivatives, RNA, and RNA derivatives, and the number of bases of the nucleic acid is 5 or more and 10,000 or less.

9. The separation material according to any one of claims 1 to 8, which is a chromatographic separation material.

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