WO2025249499A1 - Separation agent - Google Patents

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

This disclosure relates to a separating agent.

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.
As a separating agent, there have been known a separating agent in which a carrier made of fully porous silica gel is supported on the carrier with various ligands depending on the target substance to be separated.
As a separating agent obtained by using a fully porous silica gel as a carrier, for example, one carrying an optically active polymer is known. When such a separating agent carrying an optically active polymer is used, optical resolution becomes possible.

While fully porous materials are primarily used as supports for carrying ligands, core-shell particles with a non-porous core and a porous shell on the outer surface are also known. For example, Patent Documents 1 and 2 disclose core-shell particles that can be used for chromatographic separations.

International Publication No. 2013/176215 International Publication No. 2014/087937

As mentioned above, the technology of using core-shell particles as a carrier is generally known, but there have been few reports examining the influence of the porous shell configuration conditions. In particular, there has been little investigation into the porous shell configuration conditions from the viewpoint of separation efficiency, and there is still room for improvement.
Therefore, an object of the present disclosure is to provide a separating agent that uses core-shell particles consisting of a non-porous core and a porous shell as a carrier and has a high degree of separation.

After extensive research, the inventors discovered 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 core-shell particles consisting of a non-porous core and a porous shell, and thus arrived at the invention disclosed herein.

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,
the carrier is a core-shell particle having 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 an optically active polymer, an optically inactive polyester, a protein, and a nucleic acid;
The maximum pore diameter is 15 nm or more.
Separating agent.
[2] The separating agent according to [1], wherein the specific surface area of the separating agent is 40 to 109 m ² /g.
[3] The separating agent according to [1] or [2], wherein the separating agent has a pore volume of 0.19 to 0.31 cm 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 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. [1] The separating agent according to any one of [4] to [5].
[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 glycoprotein, protein A, protein G, and protein, 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 nucleic acid is at least one selected from the group consisting of DNA, a DNA derivative, RNA, and an RNA derivative;
The separating agent according to any one of [1] to [7], wherein the number of bases of the nucleic acid is 5 or more and 10,000 or less.
[9] The separating agent according to any one of [1] to [8], which is a separating agent for chromatography.

This disclosure makes it possible to provide a separating agent with high separation performance using core-shell particles consisting of a non-porous core and a porous shell as a carrier.

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

Although the embodiments of the present disclosure will be described in detail below, the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited by the embodiments, but is limited only by the scope of the claims.
In the present disclosure, a numerical range expressed using "to" means a range that includes the numerical values written before and after "to" as the lower and upper limits, and "A to B" means that the range is 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 written in stages (for example, in order of preference), the upper and lower limits of each numerical range can be combined in any way.
Furthermore, although multiple embodiments will be described in this disclosure, various conditions in each embodiment may be applied to each other to the extent that they are applicable.
Furthermore, the expression "A or B" in the present disclosure can be read as "at least one selected from the group consisting of A and B."
Also, in this disclosure, "plurality" means "two or more."
In addition, in the present disclosure, the term "core-shell particles" can be appropriately read as "separating agent."

<Separating agent>
The separating agent according to one embodiment of the present disclosure (hereinafter also simply referred to as "separating agent") comprises:
A separation agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding,
the carrier is a core-shell particle having 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 an optically active polymer, an optically inactive polyester, a protein, and a nucleic acid;
The maximum pore diameter is 15 nm or more.
It is a separating agent.

The above-mentioned separating agent uses core-shell particles consisting of an inorganic non-porous core and a porous shell as a ligand carrier, and has a large maximum pore diameter of 15 nm or more. The inventors speculate that this allows the ligand substance to penetrate into the interior of the shell, thereby improving separation efficiency.

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. When the maximum pore diameter is equal to or greater than the lower limit of the above range, the degree of separation is likely to be improved. Furthermore, when the maximum pore diameter is equal to or greater than 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, and 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.

[Carrier]
The separating agent uses core-shell particles consisting of an inorganic nonporous 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 able to narrow the particle size distribution and therefore achieve dense packing, of being able to easily suppress solute diffusion because the core obstructs it, and of being able to shorten the diffusion distance of solutes within the porous portion because the porous portion is thin.

In the present disclosure, non-porous refers to a material in which (A-B)/B x 100 is less than 20, where A is the specific surface area (m ² /g) of the surface of the core particle measured by the BET method, and B is the surface area per unit weight (m ² /g) that can be calculated from the surface area obtained from the core particle diameter (4πr ² calculated from the particle radius r).
On the other hand, the term "porous" as used herein refers to a material having a specific surface area of 10 mm ² /g or more as measured by the BET method.

(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. The shape of the core can be, for example, spherical, granular, powdery, or any irregular shape. Among these, spherical is preferred from the viewpoint that 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 spherical.

The average particle size of the core is not particularly limited, but is preferably 0.1 μm or more and 200 μm or less, more preferably 0.1 μm or more and 100 μm or less, even more preferably 0.5 μm or more and 50 μm or less, and particularly preferably 1 μm or more and 50 μm or less.
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 specimen of an arbitrarily selected separating agent or core-shell particles is prepared, and the thin specimen is irradiated with an electron beam accelerated at a high voltage, and the transmitted electron beam is analyzed, and the maximum length of the non-porous part on the obtained photograph is taken as the particle diameter of the core.

The core material that makes up 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.

(shell)
The shell is porous and is not particularly limited as long as it contains silica gel, and may be composed only of silica gel. The silica gel is preferably a hydrolyzate of polyalkoxysiloxane. The shell's external shape (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, since uniform particle shape makes it easier to ensure packing density.

The average thickness of the shell 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. If the average thickness is equal to or greater than 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, if the average thickness is equal to or less than 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 core-shell particles in terms of suppressing solute diffusion in the porous portion.
The shell thickness can be measured, for example, by observing the core-shell particles with an electron microscope such as a transmission electron microscope (TEM). Specifically, a thin sample of an arbitrarily selected core-shell particle is prepared, and the thin sample is irradiated with an electron beam accelerated by a high voltage. The transmitted electron beam is analyzed, and the obtained photograph is obtained by subtracting the maximum length of the non-porous portion from the maximum length of the entire particle, and dividing the result by 2 to obtain the shell thickness.

There are no particular restrictions on the ratio of the average particle diameter of the core to the average thickness of the shell, but it 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 will be small, making it easier to demonstrate the benefits of core-shell particles in terms of suppressing solute diffusion in the porous portion. Furthermore, if this ratio is below the upper limit of the above range, the proportion of the porous portion in the core-shell particle will be large, making it possible to sufficiently support the ligand substance in the porous portion, thereby allowing the sample to be sufficiently retained.

The shell contains silica gel. An embodiment containing silica gel (particularly an embodiment consisting of silica gel only) is preferred from the viewpoint of facilitating the production of core-shell particles.
When the silica gel is a hydrolyzate of a polyalkoxysiloxane, the form of the polyalkoxysiloxane is not particularly limited, and it may be, for example, a polyalkoxysiloxane obtained by partial hydrolysis of an alkoxysilane, which is further hydrolyzed.
The alkoxysilane is preferably a tetraalkoxysilane, and among these, it is preferable to use tetramethoxysilane, tetraethoxysilane, tetrapropoxysilane, tetrabutoxysilane, or the like, and it is more preferable to use tetraethoxysilane.
For the preparation of core-shell particles, reference can be made to Japanese Patent Laid-Open Publication No. 49-36396. Specifically, first, alkoxysilane is partially hydrolyzed to produce polyalkoxysiloxane. The polyalkoxysiloxane thus obtained is then 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 the solvent is then removed, thereby depositing the 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. This allows core-shell particles to be obtained.

Commercially available core-shell particles may be used.

The core-shell particles may be surface-treated. One example of a surface treatment method is to use a silane coupling agent having an amino group, such as 3-aminopropyltriethoxysilane.

[Ligand]
In the present disclosure, the term "ligand" refers to a substance that is supported on a core-shell particle that serves as a carrier, and that 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 will be described in detail below.

The average ligand loading rate SR (hereinafter simply referred to as "loading rate") relative to the entire separating agent is expressed by the following formula (1). There are no particular restrictions on the loading rate SR, but it 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. If the loading rate is above the lower limit of the above range, sufficient ligand substances are loaded, making it easier to achieve separation through interactions between the particle surface and the target compounds. Furthermore, if the loading rate is below the upper limit of the above range, the partition coefficient between the ligands on the core-shell particle surface and the target compounds is reduced, allowing for rapid mass transfer and improving the number of theoretical plates.

In the above formula (1), %C ^CSP represents the carbon content (mass%) of the entire separating agent, %C ^Support represents the carbon content (mass%) of the entire support, and %C ^Ligand represents 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.).

(Optical 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 include an embodiment in which a monomer for constituting an optically active polymer has optical activity, or an embodiment in which an optically inactive monomer is polymerized using an optically active polymerization catalyst.

There are no particular restrictions on the weight-average molecular weight of the optically active polymer, but it is preferably 1,000 or more and 1,000,000 or less.

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.

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, etc. are preferred, with cellulose, amylose, pullulan, and nigeran being more preferred.

There are no particular restrictions on the number-average degree of polymerization of the polysaccharide (the average number of pyranose rings or furanose rings contained in one molecule), but from the standpoint of ease of handling, such as solubility and viscosity, it is preferably 5 to 1,000, more preferably 10 to 1,000, and even more preferably 10 to 500.

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 as chiral stationary phases.
Specific examples of ester derivatives or carbamate derivatives that can be used include, for example, JP-B-4-42371 discloses a cellulose derivative 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 substituted with halogen (fluorine or chlorine); and JP-A-2005-315668 discloses a cellulose derivative or amylose derivative 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 substituted with fluorine, an alkyl group, or an alkoxy group.
In such phenyl carbamate derivatives, the substituent with which hydrogen on the aromatic ring is substituted may be only a halogen group, only an alkyl group, or only a halogen group and an alkyl group. 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 preferable 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 the core-shell particles.
The polysaccharide derivative is not limited to those mentioned above, and any other suitable polysaccharide derivative can be used.

When a polysaccharide or 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.

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 loading rate of the polysaccharide or polysaccharide derivative relative to the core-shell particles is preferably 1.0 to 25% by mass.

Methods for chemically supporting polysaccharides or polysaccharide derivatives on core-shell particles include: forming a chemical bond between the core-shell particles and the polysaccharide or polysaccharide derivative; chemically bonding the core-shell particles and the polysaccharide or polysaccharide derivative via a third component (spacer); and physically adsorbing the polysaccharide or polysaccharide derivative onto a carrier and crosslinking the polysaccharide or polysaccharide derivative on the carrier.

As a method for forming a chemical bond between a core-shell particle and a polysaccharide or polysaccharide derivative (hereinafter abbreviated as the "reductive amination method"), for example, the method described in JP 07-138301 A can be used. 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 Glycoconjugate J (1986) 3, 311-319 ELISABETH KALLIN et al.), thereby supporting the polysaccharide on the support through a chemical bond, and then derivatizing the polysaccharide as needed.

As a method for chemically bonding core-shell particles and polysaccharides or polysaccharide derivatives via a third component (spacer), for example, the method described in the examples of JP 2002-148247 A can be used. Specifically, this method involves copolymerizing a polymerizable polysaccharide derivative into which a polymerizable group such as a vinyl group has been introduced with core-shell particles into which a polymerizable group such as a vinyl group has been introduced in the presence of a third component (polymerizable monomer) having a polymerizable group such as a vinyl group.

A method for physically adsorbing a polysaccharide or polysaccharide derivative onto a carrier and crosslinking the polysaccharide or polysaccharide derivative on the carrier can be, for example, the method described in the examples of JP-A-11-510193. Specifically, this method involves supporting the 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 other methods may also be used, such as crosslinking by a reaction induced by irradiation with radiation such as gamma rays or electromagnetic waves such as microwaves; and crosslinking by a radical reaction using a radical initiator.

As a method for crosslinking polysaccharides or polysaccharide derivatives 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 2004-167343 A 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.

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 in which alkoxysilyl groups have been introduced into some of the hydroxyl or amino groups of the polysaccharide or polysaccharide derivative, and then carrying out a reaction such as polycondensation of the alkoxysilyl groups in a solvent of your choice.

Poly(meth)acrylic acid amide: For example, poly(meth)acrylic acid amide can be used as the optically active polymer. The poly(meth)acrylic acid amide is preferably obtained by polymerizing an optically active (meth)acrylic acid amide represented by the following formula (I):
Such a polymerization reaction may be, for example, 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 pressure, the polymerization reaction can be carried out without solvent, but if it is a solid, any ordinary organic solvent that does not have a radical scavenging effect can be used as the reaction solvent, more preferably tetrahydrofuran, chloroform, or methanol.
Other polymerization conditions can be appropriately adjusted by referring to WO 02/088204.

In formula (I), R ¹ , R ² and R ³ are each different from one another 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; and R ⁵ represents a hydrogen atom or a methyl group.

It is preferable that R ¹ , R ² and R ³ are each different from one another and represent 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.
^R4 is 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.

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.

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 above-mentioned polyamino acid is dissolved (using, for example, chloroform or dichloromethane as the solvent), and then evaporating the solvent.

Examples of methods for supporting poly(meth)acrylic acid amide on core-shell particles by chemical bonding include a method in which a reactive functional group is introduced into the core-shell particles and this reactive functional group is reacted with an amide group possessed by the poly(meth)acrylic acid amide; and a method in which a reactive functional group is introduced into the core-shell particles, a functional group capable of reacting with this reactive functional group is introduced into the poly(meth)acrylic acid amide, and both reactive functional groups are reacted.
The former method is carried out by, for example, introducing epoxy groups into the core-shell particles by surface treatment using a silane coupling agent having an epoxy group, and then reacting the epoxy groups with amide groups of the poly(meth)acrylic acid amide.

Other methods for chemically bonding poly(meth)acrylic acid amide to core-shell particles include, for example, introducing polymerizable functional groups into the core-shell particles by surface treatment using a silane coupling agent or the like having polymerizable functional groups such as vinyl groups and (meth)acryloyl groups, and also introducing polymerizable functional groups into the poly(meth)acrylic acid amide by reaction with an isocyanate ester or the like (see JP 2006-177795 A), and then copolymerizing the polymerizable functional groups of both.

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.

In the above formula (II), ⁿ¹ is 5 or more; ^R6 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, which may have a substituent such as a hydroxyl group, a carboxyl group, a mercapto group, an amino group, or a methylthio group; and ^R7 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.

Examples of α-aminocarboxylic acids that can be used to form the polyamino acids described above include alanine, valine, leucine, phenylalanine, proline, glutamic acid, and aspartic acid. Additionally, amino acid derivatives such as benzyl aspartate, methyl glutamate, benzyl glutamate, carbobenzoxylysine, carbobenzoxyornithine, acetyltyrosine, and benzylserine can also be used as constituent materials for polyamino acids.

In the above formula (II), ⁿ¹ is preferably 100 or less, and more preferably 10 to 40.

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.

Polyamino acids can be supported on core-shell particles by physical adsorption by immersing the core-shell particles in a solution in which the polyamino acids have been dissolved (using, for example, dimethylformamide or dioxane as the solvent), and then evaporating the solvent.

Methods for chemically bonding polyamino acids to core-shell particles include introducing reactive functional groups into the core-shell particles and reacting these reactive functional groups with amino groups in the polyamino acids; and introducing reactive functional groups into the core-shell particles, introducing functional groups reactive with these reactive functional groups into the polyamino acids, and reacting the two reactive functional groups. Examples of reactive functional groups bonded to core-shell particles include epoxy groups, which react with amino groups in polyamino acids. Examples of methods for introducing reactive functional groups into core-shell particles include surface-treating the core-shell particles with a silane coupling agent having an epoxy group.

Other methods for chemically bonding polyamino acids to core-shell particles include, for example, surface treatment with a silane coupling agent having polymerizable functional groups such as vinyl groups and (meth)acryloyl groups to introduce polymerizable functional groups into the core-shell particles, and then reacting the polyamino acid with 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, or chloromethylstyrene, to introduce the polymerizable functional group into the polyamino acid and copolymerize the polymerizable functional groups of both.

Polyamides Examples of optically active polymers that can be used include polyamides having one optically active amino acid residue in the main chain of the repeating unit.
The combination of monomer components for synthesizing this optically active polyamide can be, for example, a combination of an N-substituted amino acid, which is an optically active dicarboxylic acid, and a diamine. The N-substituted amino acid can be, for example, N-substituted glutamic acid or N-substituted aspartic acid, and the diamine can be, for example, 4,4'-diaminodiphenylmethane or an aromatic diamine such as 1,3-phenylenediamine.

An example of a method for synthesizing polyamides will be described. As described above, polyamides can be synthesized by polymerizing an N-substituted amino acid, an optically active dicarboxylic acid, with a diamine. Specifically, a solution (hereinafter also referred to as an "NMP-Py mixed solution") is prepared by adding, for example, 4% by weight of lithium chloride (LiCl) to a mixture of N-methylpyrrolidone (NMP) and pyridine (Py) at a volume ratio of, for example, 4:1. A predetermined amount, for example, ³ mmol of benzoyl-L-glutamic acid (an N-substituted amino acid, an optically active dicarboxylic acid), an equimolar amount, for example, 3 mmol of 4,4'-diaminodiphenylmethane (diamine), and twice the molar amount, for example, 6 mmol of triphenyl phosphite, are added to, for example, 7.5 cm3 of the solution (hereinafter also referred to as an "NMP-Py mixed solution"), and the mixture is heated with stirring at a predetermined temperature, for example, 80°C, for a predetermined time, for example, 3 hours. After the reaction is complete, the product is added dropwise to methanol, filtered to obtain a polymer, and then dried under reduced pressure.
The polyamides described above are synthesized using N-substituted amino acids, which are optically active dicarboxylic acids, and therefore have D- or L-optical recognition sites within the polymer, and optical resolution can be carried out by utilizing these optically active recognition sites.

In addition to the polyamides listed above, polyamides that can be used include those represented by the following general formula (III) or (IV):

In the above formulas (III) and (IV), ^R8 and ^R9 represent 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.
ⁿ² and ⁿ³ are integers from 50 to 100,000.

The polyamides represented by the above formulas (III) and (IV) may be obtained by the method described in Japanese Patent Publication No. 4-77737. As a starting material, they can be easily obtained by reacting (+)- or (-)-trans-stilbenediamine with the corresponding dicarboxylic acid or its derivative.
The dicarboxylic acid may be one represented by the formula HOOC-R ¹⁰ -COOH, where R ¹⁰ has the same meaning as 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.

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

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.

Polyamide can be supported on core-shell particles by physical adsorption by immersing the core-shell particles in a solution containing dissolved polyamide (using, for example, hexafluoroisopropanol, dimethylformamide, or dichloromethane as the solvent) and then evaporating the solvent.

Examples of methods for supporting polyamide on core-shell particles by chemical bonding include a method of introducing a reactive functional group into the core-shell particles and reacting this reactive functional group with an amide group possessed by the polyamide; and a method of introducing a reactive functional group into the core-shell particles, introducing a functional group capable of reacting with this reactive functional group into the polyamide, and reacting the two reactive functional groups.
The former method is carried out by, for example, introducing epoxy groups into the core-shell particles by surface treatment using a silane coupling agent having an epoxy group, and then reacting these epoxy groups with amide groups possessed by the polyamide.

Other methods for chemically bonding polyamide to core-shell particles include, for example, surface treatment with a silane coupling agent having polymerizable functional groups such as vinyl groups and (meth)acryloyl groups to introduce polymerizable functional groups into the core-shell particles, and then introducing polymerizable functional groups into the polyamide by reaction with an isocyanate ester or the like (see JP 2006-177795 A), and then copolymerizing the polymerizable functional groups of both.

(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, and poly(oxycarbonyloxy-1,4-phenylene-2,2-isopropylidene-1,4-phenylene) (polycarbonate of bisphenol A). 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, and the like.

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.

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.

When optically inactive polyester is supported on 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 has been dissolved, and then evaporating off the solvent.

Examples of methods for supporting an optically inactive polyester on a core-shell particle by chemical bonding include a method in which a reactive functional group is introduced into the core-shell particle, a functional group capable of reacting with the reactive functional group is introduced into the optically inactive polyester, and the two reactive functional groups are reacted with each other.
Such a method is carried out, for example, by introducing epoxy groups into the core-shell particles by surface treatment using a silane coupling agent having an epoxy group, and by introducing amino groups into an optically inactive polyester by chemical treatment using a polyamine, and then reacting the epoxy groups introduced into the core-shell particles with the amino groups introduced into the polyester in vacuum or air.

Polyamines for introducing amino groups into optically inactive polyesters include (poly)alkylenepolyamines having 2 to 8 carbon atoms. Specific examples of (poly)alkylenepolyamines having 2 to 8 carbon atoms include ethylenediamine, propylenediamine, butylenediamine, diethylenetriamine, triethylenetetramine, and tetraethylenepentamine. Preferably, they are one or more selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, and tetraethylenepentamine, with diethylenetriamine being particularly preferred. In particular, using a polyamine containing at least three amino groups results in better introduction of amino groups into the polyester. Furthermore, low molecular weight amines are preferred because they are easier to remove after chemical treatment.

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

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

(protein)
The ligand may be a protein, which has a molecular weight of 3 to 300 kDa, preferably 30 to 150 kDa, and has affinity for the protein to be separated, such as an antibody.
Among these, from the viewpoint of high selectivity when used for antibody separation, 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.

α1-acid glycoprotein can be used as the glycoprotein. Sources of α1-acid glycoprotein include mammals such as humans, cows, and rabbits, and birds such as chickens, swans, and turkeys. Specific examples of preferred α1-acid glycoproteins include human α1-acid glycoprotein (hereinafter simply referred to as h-AGP) and chicken AGP (hereinafter simply referred to as c-AGP).

Human α1-acid glycoprotein (h-AGP) can be commercially available (e.g., manufactured by Merck). Commercially available products may be purified by high-performance liquid chromatography before loading, if necessary.

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

Human α1-acid glycoprotein is a glycoprotein consisting of 183 amino acid residues and five sugar chains, with a molecular weight of approximately 41,000-43,000. 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.

Examples of albumin include egg albumin and human serum albumin (molecular weight approximately 66 kDa).

The above-mentioned functional variant refers to a protein that has at least one modification in its native amino acid sequence and that has at least one function associated with the native sequence. A native sequence includes an amino acid sequence that originally occurs in nature. Modifications of a native amino acid sequence can include, for example, substituting one or more amino acids with other amino acids; deleting one or more amino acids; adding one or more amino acids; and combinations thereof (for example, a combination of substituting one or more amino acids with other amino acids, adding one or more amino acids, and deleting one or more amino acids from the native sequence).

Functional variants may also include fragments or domains of a protein. The amino acid sequence of a functional variant may 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 naturally occurring amino acid sequence.

Furthermore, cellobiohydrolases (CBH), 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 the main enzymes that account for the majority of cellulases (over 80% together, with small amounts of endoglucanases and β-glucosidases also included). Methods for producing these proteins include using genetic recombination technology to express large amounts of the desired cellobiohydrolase in host cells.

In order to obtain better separation performance, the protein loading rate in the separating agent is preferably 1.0% by mass or more and 25.0% by mass or less, more preferably 2.0% by mass or more and 20.0% by mass or less, even more preferably 3.0% by mass or more and 15.0% by mass or less, even more preferably 4.0% by mass or more and 10.0% by mass or less, and particularly preferably 5.0% by mass or more and 8.0% by mass or less.

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

One method for physically adsorbing proteins onto core-shell particles is to immerse the core-shell particles in a solution of the protein dissolved in an appropriate solvent, such as hexane or chloroform, and then evaporate the solvent by drying under reduced pressure, etc.

Methods for chemically bonding proteins to core-shell particles typically include, but are not limited to, a method in which reactive functional groups are introduced into the core-shell particles and then these reactive functional groups are reacted directly with functional groups on the protein; or a method in which reactive functional groups are introduced into the core-shell particles and then the core-shell particles and the protein are chemically bonded via a compound having one or more functional groups reactive with the reactive functional groups bonded to the core-shell particles and one or more functional groups reactive with functional groups on the protein (hereinafter such compounds are collectively referred to as "spacers").

An example of the former method is to carry out a surface treatment using a silane coupling agent or the like that has a functional group, such as an epoxy group, that can react with amino groups, to introduce functional groups that react with amino groups into the core-shell particles, and then directly react these functional groups with Protein A.

Examples of the latter method include a method using an amino acid (amine carboxylic acid) as a spacer, reacting the amino group of the amino acid with an epoxy group introduced into the core-shell particle, and then reacting the carboxyl group of the amino acid with the amino group of Protein A; and a method using a diamine or diol and a diglycidyl compound such as (poly)ethylene glycol diglycidyl ether as a spacer in succession, reacting the epoxy group introduced into the core-shell particle with one end group of the diamine or diol, and then reacting the other end group of the diamine or diol 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.

Examples of diamines that serve as spacers include aliphatic diamines such as tetramethylenediamine and hexamethylenediamine. Examples of diols that serve as spacers include aliphatic diols such as propylene glycol, butanediol, diethylene glycol, and triethylene glycol; and polyalkylene glycols such as polyethylene glycol.

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

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 chemically bonding the protein to the core-shell particles.

(nucleic acid)
Nucleic acids can be used as the ligand. The nucleic acid is not particularly limited, and examples include DNA, RNA, oligonucleotides, and modified oligonucleotides. Derivatives of DNA or RNA 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. The 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.
In particular, artificial nucleic acids having 50 to 200 bases are preferred, and from the viewpoint of enabling efficient synthesis, it is preferable to use those having about 100 bases. In the artificial nucleic acid, from the viewpoint of preventing thymine dimerization, it is preferable that thymines are not adjacent to each other.

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 can be derivatized with a phosphate ester group, an acyl group, an alkoxycarbonyl group, a benzyl group, a substituted benzyl group, or an allyl group, etc.

When a nucleic acid is used as the ligand, the mass ratio of the nucleic acid to the entire separating agent is preferably 0.5 mass % or more and 25 mass % or less.
If the mass ratio is below the lower limit of the above range, the nucleic acid cannot be stably present in the separation agent, and sufficient separation performance cannot be obtained, while if the mass ratio exceeds the upper limit of the above range, the nucleic acid cannot be fully supported on the core-shell particles, resulting in the generation of free nucleic acid, which may have an adverse effect on separation performance.

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

One way to chemically bond nucleic acids to core-shell particles is to immobilize nucleic acids on a carrier via chitosan and the amino groups of the chitosan, as described in JP 2010-259405 A, for example.

In this method, core-shell particles are first vapor-deposited with an aminosilane such as 3-aminopropyltriethoxysilane, followed by a heat treatment. Next, the aminosilane-treated core-shell particles are immersed in a glutaraldehyde solution, then washed and air-dried. Next, the glutaraldehyde-treated core-shell particles are immersed in a chitosan solution, then washed with ultrapure water. This causes 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. Next, the chitosan-treated core-shell particles are immersed in a glutaraldehyde solution, then washed and air-dried. This introduces aldehyde groups into the core-shell particles. An avidin solution is dropped onto these core-shell particles and allowed to stand, causing the aldehyde groups introduced into the core-shell particles to react with the amino groups of the avidin, immobilizing the avidin to the core-shell particles via the chitosan. A biotin-labeled nucleic acid solution is then dropped onto the core-shell particles with the avidin immobilized thereon and allowed to react, immobilizing the nucleic acid to the core-shell particles via a chemical bond.

Among the above ligands, polymeric ligands have stacked interaction points 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 perspective, it is preferable to use 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.

<Method for evaluating separating agents>
In this disclosure, the indices used to evaluate the separation performance of a separating agent are 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 this disclosure, a high value 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) is considered to indicate good separation performance. Each indices is defined as follows:

Separation factor (α)
α = k2/k1
During the ceremony,
k1: retention coefficient of the more weakly retained component, calculated by the following formula:
k1= (t1-t0)/t0
k2: retention coefficient of the more strongly retained component, 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 when it is eluted. For convenience, the elution time of tri-tert-butylbenzene is taken as the dead time.)
t1: elution time of the weaker retained component t2: elution time of the stronger retained component

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

・Separation degree (Rs)

t _R1 , t _R2 : Retention time (t _R1 ≦t _R2 ) (see Figure 1)
W ₁ , W ₂ : Peak width (see Figure 1)
W _0.5h1 , W _0.5h2 : Peak width at the position where the peak height is 1/2 (half-value width; see FIG. 1)

A resolution Rs of 1.5 or higher 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 higher indicates that the valley depth between the two peaks is 99.4%, indicating near-baseline separation. Therefore, an Rs of 1.25 or higher is preferred, and an Rs of 1.5 or higher is even more preferred.

The specific surface area of the separating agent is not particularly limited, but is preferably 20 m ² /g or more and 200 m ² /g or less, more preferably 40 m ² /g or more and 109 m ² /g or less, and even more preferably 40 m ² /g or more and 73 m ² /g or less. 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 the ligand substance to be sufficiently supported, making it easier to achieve 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 large pore diameter allows the ligand substance to sufficiently penetrate into the pores of the shell, ensuring the supported amount, thereby strengthening the interaction between the particle surface and the target compound, making it easier to achieve good separation. The specific surface area can be controlled, for example, by adjusting the pH of the aqueous solution used when stacking the shells and performing polycondensation. Specifically, reducing the pH can be considered to increase the specific surface area.
The specific surface area can be measured by mercury intrusion porosimetry in the same manner as in the measurement of the maximum pore diameter.

The pore volume of the separating agent is not particularly limited, but is preferably 0.1 cm ³ /g or more and 0.5 cm ³ /g or less, more preferably 0.19 ^{cm 3 /} g or more and 0.31 cm ³ /g or less, and even more preferably 0.19 cm ^{3 /g or more and 0.29 cm 3} /g or less. If the pore volume is equal to or greater than the lower limit of the above range, the pore volume can be secured, allowing the ligand substance to be sufficiently supported, and making it easier to achieve separation through the interaction between the particle surface and the target compound. Furthermore, if the pore volume is equal to or less than the upper limit of the above range, the large pore diameter allows the ligand substance to sufficiently penetrate into the pores of the shell, and making it possible to secure the supported amount, thereby strengthening the interaction between the particle surface and the target compound, making it easier to achieve good separation. The pore volume can be controlled, for example, by adjusting the pH of the aqueous solution used when stacking the shells and performing polycondensation. Specifically, reducing the pH can be considered to increase the pore volume.
The pore volume can be measured by mercury intrusion porosimetry in the same manner as in the measurement of the maximum pore diameter.

The bulk density of the separating agent is not particularly limited, but is preferably 0.45 g/ ^cm3 or more and 1.0 g/ ^cm3 or less, and more preferably 0.56 g/ ^cm3 or more and 0.62 g/ ^cm3 or less. If the volume of the core in the core-shell particle is too large, the shell cannot sufficiently support the ligand, and if it is too small, the diffusion of the target compound in the porous portion increases, making it difficult to demonstrate the advantages of the core-shell particle. Furthermore, if the bulk density is high, the packed layer becomes close to a close-packed structure, and the voids become small, making it easier to suppress the diffusion of the sample.
The bulk density can be measured by a tapping method.

The d90/d10 of the core-shell particles is typically 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 even more preferably 1.17.

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

In the present disclosure, "d10" and "d90" mean particle sizes at which the cumulative distribution is 10% and 90%, respectively, in a number-based particle size distribution obtained by observing 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 particles using image analysis software (for example, "Image J" manufactured by Wyne Rasband).
The d10 and d90 of the 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.

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, even 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.

In this disclosure, "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 size of each particle measured by observing with a scanning electron microscope (SEM) (e.g., "SU-5000" manufactured by Hitachi High-Technologies Corporation), randomly selecting approximately 300 particles from the resulting image, and analyzing the circular particle size using image analysis software (e.g., "Image J" manufactured by Wyne Rasband).

<Method of manufacturing the 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.

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

The following examples further illustrate the present disclosure. However, the present disclosure should not be construed as being limited to the following examples.

<Evaluation of characteristics>
The methods for evaluating the properties measured using the separating agent and column prepared by the methods described below are described below, and the evaluation results are shown in Tables 1 and 2.

[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.

The prepared chromatography column was connected to a liquid chromatograph ("X-LC" manufactured by JASCO Corporation). Using the analytical conditions described above, optical separation of the racemic form of trans-stilbene oxide (TSO) was performed with this liquid chromatograph, and the separation performance (k1, k2, N1, Ps1, α, Rs) of the separation agent was evaluated.

(separation conditions)
Mobile phase; n-Hexane/2-Propanol=9/1 (v/v)
Flow rate: 0.21ml/min
Temperature: 25℃
Detection: 254 nm

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

[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, and the operation was repeated until the following end condition was met to determine the bulk density.
(Ending conditions)
(1) Perform the above operation at least 300 times.
(2) When the above operation is performed 50 times in succession, the volume change is 0.1 ^cm3 or less.

[Maximum pore diameter, specific surface area, pore volume]
The maximum pore diameter of the separating agent was measured by mercury intrusion porosimetry, in accordance with JIS R 1655. Specifically, pressure was applied to cause mercury to penetrate the pores, and the diameter of the assumed cylindrical pores was calculated using the Washburn equation based on the pressure value and the corresponding volume of penetrated mercury.
The specific surface area and pore volume of the separating agent were also measured by mercury intrusion porosimetry.

<Experiment A>
[Manufacture 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), average core particle size: 1.7 μm, core material: glass, average shell thickness: 0.5 μm, ratio of average core particle size to 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 prepare 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 watt, quartz-encased) 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) Packing of Separating Agent into Column The core-shell particles prepared in (2) were packed into a stainless steel column of φ0.21 cm×L25 cm by pressurizing and packing using a slurry packing method to prepare a column.

Example 2
Particles carrying cellulose tris(3-chloro-4-methylphenylcarbamate) by physical adsorption were prepared under the same conditions as in Example 1, except that the core-shell silica gel was changed to one 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). Next, 3.0 g of the resulting 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) to the 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.

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 mass%. The solvent was then distilled off under reduced pressure to prepare particles carrying amylose tris(3-chloro-4-methylphenylcarbamate) supported by physical adsorption. Next, 3.0 g of the resulting particles was suspended in 500 mL of acetonitrile/water = 60/40 (vol./vol.) and stirred. The resulting suspension was then irradiated with a mercury immersion lamp (Philips, HPK-125 watts, quartz-encased) 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) Packing of Separating Agent into Column The core-shell particles prepared in (2) were packed into a stainless steel column of φ0.21 cm×L25 cm by pressurizing and packing using a slurry packing method to prepare a column.

(Comparative Example 1)
Particles carrying cellulose tris(3-chloro-4-methylphenylcarbamate) by physical adsorption were prepared under the same conditions as in Example 1, except that the core-shell silica gel was changed to one 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). Next, 3.2 g of the resulting particles were irradiated with light under the same conditions as in Example 1, yielding core-shell particles (separating agent) in which cellulose tris(3-chloro-4-methylphenylcarbamate) was immobilized (chemically bonded) to the 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.

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

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, near baseline separation is achieved because Rs is 1.25 or more.

<Experiment B>
[Manufacture of separation column]
Example 4
A column was prepared in the same manner as in Example 1, except that light irradiation was not performed.

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

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

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

In Table 2 below, a "-" is listed in the bulk density column, which means that it was not measured.

Table 2 shows that even in cases where the ligand is physically adsorbed to the surface of the carrier, excellent resolution Rs can be ensured when the maximum pore diameter is 15 nm or greater.

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

Claims (9)

A separation agent having a carrier and a ligand supported on the surface of the carrier by physical adsorption or chemical bonding,
the carrier is a core-shell particle having 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 an optically active polymer, an optically inactive polyester, a protein, and a nucleic acid;
The maximum pore diameter is 15 nm or more.
Separating agent. 2. The separating agent according to claim 1, wherein the specific surface area of the separating agent is 40 to 109 m ² /g. 3. The separating agent according to claim 1, wherein the separating agent has a pore volume of 0.19 to 0.31 cm ³ /g. The separation agent according to any one of claims 1 to 3, wherein the mass ratio of the ligand to the entire separation agent is 0.6 to 6.2 mass%. 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. The separation 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. 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. the nucleic acid is at least one selected from the group consisting of DNA, a DNA derivative, RNA, and an RNA derivative;
The separating agent according to any one of claims 1 to 7, wherein the number of bases of the nucleic acid is 5 or more and 10,000 or less. The separation material according to any one of claims 1 to 8, which is a separation material for chromatography.