JP4371900B2 - Chromatographic packing material - Google Patents

Description

  The present invention relates to a chromatographic filler. More specifically, a phosphorylcholine group and a specific ion-exchange group, or a phosphorylcholine group and a hydrophobic group, or a phosphorylcholine group and an aromatic group are directly covalently bonded to the substrate surface, and exchange with a GFC mode. The present invention relates to a packing material for chromatography, which realizes separation in which modes and / or reversed-phase modes are mixed, and extremely reduces non-specific irreversible adsorption of proteins and peptides.

  Polymers having a phosphorylcholine group have been studied as biocompatible polymers. Biocompatible materials in which this polymer is coated on various bases have been developed.

  For example, Patent Document 1 describes a medical material coated with a polymer having a phosphorylcholine group. Patent Document 2 discloses a separating agent coated with a polymer having a phosphorylcholine group.

  The above materials have a phosphorylcholine structure by reacting mainly an acrylic monomer having a hydroxyl group with 2-chloro-1,3,2-dioxaphosphorane-2-oxide and further converting to quaternary ammonium with trimethylamine. The surface is coated with a polymer obtained by synthesizing a monomer and polymerizing the monomer (see Patent Documents 3 and 4 for the method for producing the polymer).

  Patent Document 3 manufactures a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid ester, and Patent Document 4 manufactures a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.

  On the other hand, there are many commercially available GFC fillers for separating biological samples such as proteins and polypeptides having a molecular weight smaller than that of proteins by size exclusion. The filler for GFC includes a filler based on a crosslinked hydrophilic polymer and a filler based on silica gel.

  A filler based on a cross-linked hydrophilic polymer has a wide mobile phase pH range and high versatility. However, the filler based on the polymer is more difficult to control the pore size than the filler based on the silica gel (1) because it is difficult to obtain a high theoretical plate number from the viewpoint of size exclusion. (2) In many cases, reproducible chromatograms cannot be obtained due to poor strength against high pressure conditions when used in high performance liquid chromatography (HPLC) and swelling of particles by mobile phase solvent. .

The filler based on silica gel has a problem of adsorption of protein or polypeptide onto the surface of the silica gel substrate. Therefore, for the purpose of suppressing adsorption of proteins and polypeptides in the analysis sample to the silica gel, a filler using a silica gel whose surface is modified with a non-dissociable hydrophilic group is commercially available.
For example, Shodex PROTEIN KW-803 (product name) is commercially available from Showa Denko KK as a silica gel-based GFC column. This silica gel column is described in the catalog as being a silica gel GFC mode column suitable for the analysis of proteins having a molecular weight of about several thousand to one million.
YMC-Pack Diol (product name) is commercially available from YMC Co., Ltd. This is also a silica gel-based column for GFC, which is obtained by covalently bonding a functional group having a diol structure to a silica gel substrate, and is described as being applicable to separation of several thousand to several hundred thousand proteins.

  Non-Patent Document 1 describes that protein adsorption is reduced by phosphorylcholine groups chemically grafted onto a substrate.

  However, it is difficult to effectively coat the entire surface by a method of coating and modifying the surface of an object with a polymer having a phosphorylcholine group. Further, since the coated polymer is peeled off from the object, there is a problem in durability. Furthermore, since the surface of the object is coated with a polymer, the basic properties required for the object itself are lost, deviating from the purpose of imparting the intended function such as biocompatibility by the phosphorylcholine group. There is also.

  Even when a phosphorylcholine derivative is introduced into an object as a low molecule, a method in which a functional group capable of reacting with a phosphorylcholine derivative is first introduced into the object and then the phosphorylcholine derivative is reacted is not reacted. Since the functional group remains on the surface of the object, the biocompatibility is lowered.

  For example, when an amino group is first introduced on the surface of the object and then an aldehyde derivative of phosphorylcholine is reacted with the amino group on the object surface, many unreacted amino groups remain. When many amino groups remain on the surface of the substance, the amino groups have strong basicity, so that acidic proteins mainly exhibit extremely strong electrostatic interactions, and most of them are adsorbed. When taken as a packing material for chromatography, it causes deterioration of the recovery rate of the protein and significant tailing of the peak. Furthermore, protein adsorption brings about its denaturation, and when taken as a biocompatible material, it causes inflammation and the like, which is not preferable.

  On the other hand, apart from the GFC column, an ion exchange column and a reverse phase column already exist independently and are widely used for separation and purification of proteins.

An ion exchange column is a column packed with a filler having a functional group having ion exchange properties in a modified chain. For example, the following columns are commercially available.
(1) Strong anion exchange combined with -N (CH ₃ ) ³⁺ (Product name: Shodex IEC QA-825, manufactured by Showa Denko KK)
(2) Weak anion exchange with -N (C ₂ H ₅ ) ³⁺ (Product name: Shodex IEC DEAE-825, manufactured by Showa Denko KK)
(3) -COO ^- weak cation exchange was bound to (Showa Denko Co., Ltd. Product name: Shodex IEC CM-825)
(4) Strong cation exchange combined with -SO ^3- (Product name: Shodex IEC SP-825, manufactured by Showa Denko KK)

  Both ion exchange columns achieve separation by adsorbing proteins once and eluting the adsorbed proteins with a salt concentration gradient or pH gradient. Moreover, as a filler base material, both hydrophilic polymer and silica gel are commercialized.

  The ion exchange column is particularly suitable for trace protein analysis due to (1) the protein adsorbed to the ion exchange group does not normally desorb, (2) undesirable non-specific irreversible adsorption to other than the ion exchange group. In this case, low protein recovery is a problem. When an inorganic matrix typified by silica gel is used as a base material, the phenomenon (2) occurs due to an undesired group present on the surface of the matrix. Moreover, it is thought that the phenomenon of (2) is more dominant than (1) as a cause in the low recovery rate of protein. When silica gel is used as the inorganic matrix, a typical group that causes an undesirable interaction is a silanol group.

  Patent Document 5 describes a method of inactivating a silanol group by adhering a monomer to a silica gel surface and polymerizing the monomer as it is. According to this document, a monomer having an ion exchange group or a functional group capable of ionization and a monomer having a nonionic polar substituent are mixed and brought into close contact with the silica gel surface, and then on the matrix surface. By polymerizing the monomer mixture in close contact, it is said that a filler having an ion exchange group on the silica gel surface and having a silanol group covered with a hydrophilic polymer and inactivated can be produced. .

  Patent Document 6 describes an ion exchanger in which the surface of the ion exchanger is coated with a nonionic polar substance. According to this document, it is explained that undesirable non-specific irreversible adsorption of proteins and cells can be suppressed by coating the surface of an ion exchanger with polysaccharides, polyols, polyamides and the like.

  In the method of coating a silica gel surface with a polymer as disclosed in Patent Document 5 and Patent Document 6, it is difficult to form a uniform monomolecular layer on the silica gel surface. That is, since uneven irregularities may be formed during polymerization of the monomer or the entire pores may be blocked, the uniformity of pore diameter, which is a great advantage when silica gel is used as a substrate, cannot be utilized. In addition, since there is no covalent bond between the polymer covering the substrate surface and the substrate surface, there is a problem in terms of durability.

On the other hand, a method of imparting ion exchange properties to the GFC column is also used. The GFC mode is a mode in which the interaction with the analysis target substance is substantially eliminated and the separation is performed only by the difference in the diffusion coefficient of the analysis target substance. When there is no interaction with the filler surface, only the molecular size is mainly reflected in the diffusion coefficient. Therefore, the approximate molecular weight can be measured from the elution time by using a GFC column. In such a GFC column substantially free of interaction with the analyte, it is generally known that the protein recovery rate is good.
Asahipak GS-320 is commercially available from Showa Denko KK for an attempt to achieve both good separation and high recovery by deliberately presenting a group that causes weak interaction on a GFC substrate that provides high recovery. Yes. This column is based on a polymer, resulting in weak hydrophobicity by polyvinyl alcohol, the main chain of the polymer, and weak cation exchange by the carboxyl groups remaining on the surface, resulting in a unique separation. ing. However, since the polymer is used as a base material, swelling and shrinkage due to the solvent cannot be avoided, and the solvent used is limited for the above-mentioned column. Specifically, the upper limit of acetonitrile concentration is 50%. In addition, since the remaining ion exchange groups and the properties of the polymer main chain are utilized, a wide range of ion exchange properties and hydrophobicity cannot be imparted to the filler.
On the other hand, when silica gel is used as the base material, there is no limitation on the solvent, and the uniformity of pore diameter, which is a very important factor in the GFC base material, is based on silica gel rather than polymer base material. The material is better.

JP 2000-279512 A JP 2002-98676 A JP-A-9-3132 JP-A-10-298240 JP 7-509173 A Jian R. Lu et al., Langmuir 2001, 17, 3382-3389.

  In order to solve the above-mentioned problems, the present invention achieves both a high recovery rate of proteins and peptides and a high resolution by ion exchange interaction or hydrophobic interaction, and the pore characteristics of the filler base material. The purpose is to develop a filler based on an inorganic matrix that is excellent in durability without losing quality, and provides a new filler with high recovery rate and high resolution without denaturing proteins. Is.

Ｒは炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、式（１）は炭素骨格の炭素原子数が１〜３０であって、その修飾鎖に上記ホスホリルコリン基を一つ以上有するホスホリルコリン基含有化合物であれば良い。
（２）

Ｒ’は炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、式（２）は炭素骨格の炭素原子数が１〜３０であって、その修飾鎖上にカルボキシル基を一つ以上有する化合物であれば良い。 That is, the present invention provides a chromatographic packing characterized in that a compound represented by the following formula (1) and a compound represented by the following formula (2) are directly covalently bonded to the substrate surface. An agent is provided.
(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, Formula (1) may be any phosphorylcholine group-containing compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more phosphorylcholine groups in the modified chain.
(2)

R ′ represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, Formula (2) may be a compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more carboxyl groups on the modified chain.

Ｒは炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、式（１）は炭素骨格の炭素原子数が１〜３０であって、その修飾鎖に上記ホスホリルコリン基を一つ以上有するホスホリルコリン基含有化合物であれば良い。
（３）

Ｒ’は炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、式（３）は炭素骨格の炭素原子数が１〜３０であって、その修飾鎖にスルホン酸基を一つ以上有する化合物であれば良い。 Further, the present invention provides a chromatographic packing, wherein the compound represented by the following formula (1) and the compound represented by the following formula (3) are directly covalently bonded to the substrate surface. An agent is provided.



(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, Formula (1) may be any phosphorylcholine group-containing compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more phosphorylcholine groups in the modified chain.
(3)

R ′ represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, Formula (3) may be a compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more sulfonic acid groups in the modified chain.

Ｒは炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、式（１）は炭素骨格の炭素原子数が１〜３０であって、その修飾鎖に上記ホスホリルコリン基を一つ以上有するホスホリルコリン基含有化合物であれば良い。


（４）

Ｒ’は炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、炭素骨格の炭素原子数が１〜３０であって、その修飾鎖上に４級アンモニウム基を一つ以上有する化合物であれば良い。
また、Ｒ²、Ｒ³、Ｒ⁴は、それぞれ独立に、メチル基、エチル基、プロピル基、イソプロピル基、ｎ−ブチル基、ｔ−ブチル基である。 Furthermore, the present invention provides the packing for chromatography, wherein the compound represented by the following formula (1) and the compound represented by the following formula (4) are directly covalently bonded to the substrate surface. An agent is provided.
(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, Formula (1) may be any phosphorylcholine group-containing compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more phosphorylcholine groups in the modified chain.


(4)

R ′ represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, it may be a compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more quaternary ammonium groups on the modified chain.
R ² , R ³ and R ⁴ are each independently a methyl group, ethyl group, propyl group, isopropyl group, n-butyl group or t-butyl group.

Ｒは炭素数１〜３０の任意の修飾鎖を表す。修飾鎖の任意の位置に、枝分かれ構造、不飽和結合、ベンゼン核、窒素原子、酸素原子、硫黄原子、リン原子、ケイ素原子、塩素原子、フッ素原子、臭素原子を一つ以上有しても良く、また、これらの組み合わせを有して良い。具体的には、炭素−炭素二重結合または三重結合、ベンゼン核、二級または三級アミン、カルボニル、エーテル結合、エステル結合、アミド結合、ウレタン結合、尿素結合、リン酸エステル、シロキサン結合のうち一種以上含んでも良く、そのＣ−Ｈ結合のうち一つまたはそれ以上が塩素原子、臭素原子、フッ素原子によって置換されていても良い。
すなわち、式（１）は炭素骨格の炭素原子数が１〜３０であって、その修飾鎖に上記ホスホリルコリン基を一つ以上有するホスホリルコリン基含有化合物であれば良い。 Furthermore, the present invention provides a chromatographic method characterized in that a compound represented by the following formula (1) and a saturated alkyl group, an unsaturated alkyl group or an aromatic compound are directly covalently bonded to the surface of a substrate. The present invention provides a lithographic filler.
The saturated alkyl chain and the unsaturated alkyl chain are those having 1 to 30 carbon atoms, and the aromatic compound is, for example, a benzene nucleus such as a phenylpropyl group or a naphthyl group, or a heterocyclic ring such as a pyrrole ring or a pyridine ring. is there.
(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. Specifically, among carbon-carbon double bond or triple bond, benzene nucleus, secondary or tertiary amine, carbonyl, ether bond, ester bond, amide bond, urethane bond, urea bond, phosphate ester, siloxane bond One or more of them may be contained, and one or more of the C—H bonds may be substituted with a chlorine atom, a bromine atom, or a fluorine atom.
That is, Formula (1) may be any phosphorylcholine group-containing compound having 1 to 30 carbon atoms in the carbon skeleton and having one or more phosphorylcholine groups in the modified chain.

  The present invention also provides the above-mentioned chromatography filler, wherein the base material is spherical porous silica gel.

  Furthermore, the present invention provides the above-mentioned chromatographic filler, wherein the spherical porous silica gel has an average particle diameter of 1 to 200 μm.

  The present invention also provides the above-mentioned chromatographic packing material, wherein the spherical porous silica gel has an average pore diameter of 10 to 1000 angstroms.

When the chromatographic packing material of the present invention is used in the GFC mode (a mode using a mobile phase not containing an organic solvent and containing a salt of about 300 mmol / L), the adsorption of proteins and polypeptides is extremely small and a high separation ability is achieved. Demonstrate.
Inherently, groups that interact with biological samples such as ion exchange groups and alkyl chains cause non-specific irreversible adsorption of proteins. However, the chromatographic packing material of the present invention does not interact with biological samples. These interactions and a high protein recovery rate can be achieved by mixing the effecting group and the highly biocompatible phosphorylcholine group at the molecular level.

  The chromatographic packing material of the present invention has ion exchange property and / or hydrophobicity in addition to the protein adsorption inhibiting effect of the phosphorylcholine group. Therefore, separation according to the interaction is possible, and the strength of the interaction can be adjusted by the salt concentration and pH of the mobile phase, and also by the mixing ratio of both functional groups on the filler surface. It is possible to adjust to.

  Since the GFC mode uses only water as a mobile phase solvent, it is an extremely important mode as a separation / analysis method while maintaining protein activity. When only a group that interacts with a protein / peptide, such as an ion exchange group or a hydrophobic group, is present on the substrate surface, the protein causes nonspecific irreversible adsorption in the GFC mode using a mobile phase that does not contain an organic solvent. On the other hand, the presence of phosphorylcholine groups on the surface significantly improves the protein recovery rate, and a protein recovery rate equal to or higher than that of a general GFC column can be obtained.

  In addition, the surface modification method of the present invention is different from the method of covering the substrate surface with a polymer, in order to introduce a phosphorylcholine group and a group that interacts with a biological sample such as a protein or peptide as a low molecule. In addition, the surface of the base material can be modified with a monolayer thickness, and the pore shape unique to the base material can be maintained. In isocratic conditions where a mobile phase having a constant composition is kept flowing without performing elution by gradient, uniformity of the pore diameter of the substrate is important in order to obtain a high theoretical plate number. Then, a very good peak shape can be obtained without losing it.

  Furthermore, since the surface modification method of the present invention has a covalent bond between the substrate surface and the modified chain, the durability is very good as compared with the method of covering the substrate surface with a polymer.

  The GFC mode does not use an organic solvent, and the analyte can be recovered without flowing an eluent with strong acidity or ionic strength. Therefore, since these can be separated and purified without inactivating the protein, it is expected that the high resolution of the column packing of the present invention is useful also in terms of isolation of unknown biological samples and medical applications.

  In the packing material for chromatography of the present invention, a compound having a phosphorylcholine group of formula (1), a compound having an ion-exchangeable group of formulas (2) to (4), and a saturated alkyl group or unsaturated alkyl group or That the phenyl group and benzene nucleus are directly covalently bonded to the surface of the substrate means that they are introduced to the surface of the substrate in a chemically bonded state, and are coated with a polymer having both. This means that the base material into which both are introduced is not included. The chemical bond is not particularly limited.

（６）

式中、ｍは２〜６、ｎは１〜４である。ＯＭｅは、ＯＥｔ、Ｃｌであってもよい。またＳｉと結合するＯＭｅまたはＯＥｔまたはＣｌの内、２つまではメチル基、エチル基、プロピル基、イソプロピル基、ブチル基、イソブチル基でも良い。 In order to introduce the functional group represented by the formula (1), a method of introducing a phosphorylcholine group into silica gel with a phosphorylcholine group-containing compound represented by the following formula (5) or (6) is preferable. In the introduction, only the compound (5) may be used, and (6) may be mixed. The production method of the following formula (5) or (6) will be described below.
(5)

(6)

In the formula, m is 2 to 6, and n is 1 to 4. OMe may be OEt or Cl. Further, up to two of OMe, OEt, or Cl bonded to Si may be a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, or an isobutyl group.

“Method for Producing Phosphorylcholine Group-Containing Compound of Formula (5) or (6)”
A phosphorylcholine derivative represented by the following formula (7) is dissolved in distilled water. The phosphorylcholine derivative of the following formula (3) is a known compound, and a commercially available product can be obtained.
(7)

（９）

An aqueous solution of the compound of the formula (7) was cooled in an ice-water bath, and 2 equivalents of sodium periodate was added to the compound of the formula (7) and stirred for 5 hours. The reaction solution is concentrated under reduced pressure and dried under reduced pressure, and a phosphorylcholine derivative having an aldehyde group represented by the following formula (8) is extracted with methanol. The structural formula and 1H-NMR spectrum are shown in FIG. Since the compound of formula (8) is in equilibrium with the compound of formula (9) in water, the actual spectrum reflects both formulas (8) and (9).
(8)

(9)

  Next, 0.5 equivalent of 3-aminopropyltrimethoxysilane is added to the methanol solution of the formula (8). The mixed solution is stirred at room temperature for a predetermined time, and then ice-cooled, and 2 equivalents of sodium cyanohydroborate are added, and the mixture is returned to room temperature and stirred for 16 hours. During this time, dry nitrogen is continuously supplied to the reaction vessel. After filtering the precipitate, a methanol solution of formula (5) and / or formula (6) is obtained.

The above procedure can be carried out in the same manner even if m and n in the compound represented by formula (5) or (6) are changed. The procedure shown here is for m = 3 and n = 2. The reaction solvent is not particularly limited. In addition to the above-described methanol, aprotic such as water, alcohols such as ethanol, propanol, and butanol, N, N-dimethylformamide (abbreviation: DMF), dimethyl sulfoxide (abbreviation: DMSO), and the like. A solvent can be used. However, in order to prevent polymerization during the reaction, a dehydrated solvent is preferable, and dehydrated methanol is particularly preferable.
Further, when the methoxy group (OMe) in (5) or (6) is an ethoxy group (OEt), the reaction is carried out by changing methanol to ethanol, and in the case of Cl, it is only necessary to change to dimethylformamide or dimethyl sulfoxide. .
Further, the above method is also applicable when two or one of OMe, OEt, or Cl bonded to Si is substituted with any of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, and an isobutyl group. Can be produced in exactly the same way.

“Production of fillers in which phosphorylcholine groups and carboxyl groups are directly covalently bonded to the substrate surface”
By mixing a methanol solution of the compound of formula (5) and / or (6) with a silane coupling agent having a group that interacts with proteins, peptides, etc., and introducing it into silica gel, the purpose is extremely simple. A filler having the following functions can be produced.
Specifically, a phosphorylcholine group and a group having an interaction by a dehydration reaction between a hydroxyl group present on the substrate surface and the compounds of formulas (5) and (6) or a methoxy group or an ethoxy group of a commercially available silane coupling agent. Are simultaneously introduced into the substrate surface. Here, the group having an interaction refers to a functional group having ion exchangeability or ionization, or a saturated alkyl group, an unsaturated alkyl group, a phenyl group or the like having a hydrophobic interaction.

0.73 g of triethoxysilylpropyl succinic anhydride (Amax Co., Ltd.) is dissolved in 10 mL of N, N-dimethylformamide (Wako Pure Chemical Industries, Ltd.), 4 mL of distilled water and 0.35 g of 4-dimethylaminopyridine are added, The mixture is stirred at room temperature for 16 hours to obtain a silane coupling agent having a carboxyl group represented by the following formula (9). This reaction is a hydrolysis reaction of succinic anhydride with 4-dimethylaminopyridine and is a known reaction.
(10)

A solution containing the compound represented by the formula (10) is added to a methanol solution containing 4.8 mmol of the compounds of the formulas (5) and (6). Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) with an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 100 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Then, it is filtered and washed with 50 mL of a 50% aqueous solution of N, N-dimethylformamide and 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain the desired powder. As the reaction solvent, in addition to the water-methanol mixed solution, protic solvents such as water, ethanol, 2-propanol, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, toluene, diethyl ether can be used. They can be used singly or in combination.

  When there is no hydroxyl group on the substrate surface, it is effective to dissolve the compounds of formulas (5) and (6) in a volatile solvent, apply the solution to the material surface, and then dry the solvent. Specifically, an appropriate amount of a methanol solution (0.3 mmol / mL) of the compounds of formulas (5) and (6) is directly applied to the substance according to the specific surface area of the substance. Next, methanol is vaporized by natural drying, heat drying or the like. At this time, Si—OMe of the compounds of the formulas (5) and (6) cause a dehydration reaction to generate a Si—O—Si bond, thereby covering the surface of the substance. This dehydration reaction is known. The film formed in this way during the volatilization of methanol can be subjected to surface modification relatively strongly because it forms bonds with hydroxyl groups present in a trace amount on most material surfaces. This method is an extremely effective surface modification method not only for a substance having no hydroxyl group but also for a substance having a hydroxyl group.

  Already disclosed techniques, such as a method of inactivating silanol groups by adhering a monomer to the silica gel surface and polymerizing the monomer as it is, or ion exchange with the surface of the ion exchanger coated with a nonionic polar substance In the method of obtaining a body, it is difficult to form a uniform monomolecular layer on the surface of silica gel, and it is impossible to utilize the uniformity of pore diameter, which is an advantage when silica gel is used as a substrate. That is, since uneven irregularities are formed during polymerization of the monomer, the effective pore diameter may vary, or the pores themselves may be filled with the polymer. In addition, since there is no covalent bond between the polymer covering the substrate surface and the substrate surface, there is a problem in terms of durability.

On the other hand, in the method of the present invention, since the surface treatment is performed with a silane coupling agent, the surface modification can be performed on the surface of the base material with a thickness almost equal to that of the monomolecular layer. Furthermore, the durability is extremely good because a strong covalent bond is formed between the silane coupling agent and the substrate surface. When the substrate is silica gel, a strong Si—O—Si bond is formed. By using the compounds of the formulas (5) and (6), it is possible to introduce a phosphorylcholine group excellent in the effect of suppressing protein adsorption, which has been difficult until now, at a single molecule level, and to provide a general interaction Mixing with silane coupling agent became easy. Furthermore, the mixing ratio of groups that can interact with the compounds of the formulas (5) and (6) can also be set freely.
The mixing ratio of the compounds of the formulas (5) and (6) in the present invention to the group that interacts with the biological sample is 5% to 95% as the molar ratio of the compounds of (5) and (6). .

The powder in which the carboxyl group is mixed with the phosphorylcholine group can be obtained by the following method in addition to using the compound represented by the formula (10). Specific examples (A to D) are shown below.
A: 3-Cyanopropyltrimethoxysilane (Amax Co., Ltd.) is mixed with a methanol solution containing the compounds of formulas (5) and (6) at a predetermined ratio, and silica gel is added and refluxed at 80 ° C. for 5 hours. Next, it is filtered and washed with methanol to obtain a powder in which cyanopropyl groups and phosphorylcholine groups are mixed. Next, the obtained powder is dispersed in a 5% hydrochloric acid aqueous solution (Wako Pure Chemical Industries, Ltd.), stirred for 5 hours at 80 ° C., and converted from a cyano group to a carboxyl group, thereby mixing phosphorylcholine groups and carboxyl groups. Powder can be obtained. The conversion of a cyano group into a carboxyl group with a hydrochloric acid aqueous solution is a known reaction.
B: 3-aminopropyltrimethoxysilane (Amax Co., Ltd.) is mixed with a methanol solution containing the compounds of formulas (5) and (6) at a predetermined ratio, and silica gel is added and refluxed at 80 ° C. for 5 hours. Next, it is filtered and washed with methanol to obtain a powder in which aminopropyl groups and phosphorylcholine groups are mixed. Next, the obtained powder was dispersed in N, N-dimethylformamide (Wako Pure Chemical Industries, Ltd.), an amino acid having a carboxyl group protected with a protective group, carbonyldiimidazole (Wako Pure Chemical Industries, Ltd.), triethylamine. (Wako Pure Chemical Industries, Ltd.) Each 1 equivalent amount is added and stirred at room temperature for 2 hours to obtain a powder in which phosphorylcholine groups and carboxyl groups are mixed. In addition, the urea bond formation reaction by carbonyldiimidazole of amino groups is a well-known reaction.
C: 3-aminopropyltrimethoxysilane (Amax Co., Ltd.) is mixed with a methanol solution containing the compounds of formulas (5) and (6) at a predetermined ratio, and silica gel is added and refluxed at 80 ° C. for 5 hours. Next, it is filtered and washed with methanol to obtain a powder in which aminopropyl groups and phosphorylcholine groups are mixed. Next, the obtained powder is dispersed in N, N-dimethylformamide (Wako Pure Chemical Industries, Ltd.) and succinic anhydride (Wako Pure Chemical Industries, Ltd.) or maleic anhydride (Wako Pure Chemical Industries, Ltd.). 1 equivalent, HOBt (N-hydroxybenzotriazole) (Wako Pure Chemical Industries, Ltd.) 1.2 equivalent, WSC · HCl (1-ethyl-3- (3′-dimethylaminopropyl) carbodiimide) (Wako Pure Chemical) Kogyo Co., Ltd.) By adding 1.2 equivalents at 0 ° C., a powder in which phosphorylcholine groups and carboxyl groups are mixed can be obtained.
D: N- (trimethoxysilylpropyl) -O-polyethylene oxide urethane (Amax Co., Ltd.) was mixed with a methanol solution containing the compounds of formulas (5) and (6) at a predetermined ratio, and silica gel was added to add 80. Reflux at 5 ° C. for 5 hours. Next, it is filtered and washed with methanol to obtain a powder in which hydroxyl groups and phosphorylcholine groups are mixed. Disperse the resulting powder in a 1% aqueous sodium hydroxide solution, add 1.2 equivalents of 2-bromoacetic acid, and reflux at 80 ° C. for 5 hours to obtain a powder in which phosphorylcholine groups and carboxyl groups are mixed. Can do.

The base material used in the present invention includes inorganic porous bodies such as silica, silica gel, activated carbon, zeolite, alumina, clay mineral, and porous organic polymer resins. The substrate is preferably a powder. Spherical or crushed porous silica gel is preferred. The average particle diameter of the spherical porous silica gel is 1 to 200 μm, preferably 1 to 10 μm, the average diameter of the pores of the spherical porous silica gel is 10 to 500 Å, preferably 80 to 300 Å, and the specific surface area is 50 to 800 m ^2. / G, preferably 100 to 600 m ² / g.

  When the chromatographic packing material of the present invention is used as a column for GFC, the adsorption of proteins and polypeptides is extremely small, and a high separation ability by ion exchange interaction and / or hydrophobic interaction is exhibited. Since there is no production restriction on the blending of groups that exert an interaction, various characteristics such as anion exchangeability, cation exchangeability, hydrophobicity, and π-electron interaction can be introduced at an arbitrary ratio.

  The chromatographic packing material of the present invention is a column packing material having a higher separation ability based on the weak charge and hydrophobicity of the sample. Thus, a packing material in which a phosphorylcholine group interacts with a molecular level at a molecular level has never been seen before, and can be said to be a completely new type of multi-mode column packing material. By changing the pH and salt concentration of the mobile phase, it is possible to arbitrarily control the strength of the interaction between the filler surface and the protein and polypeptide. Furthermore, in a wide range of conditions, the protein is equivalent to or better than the GFC column. It has a recovery rate. The target protein or polypeptide can be freely retained by optimizing the pH and salt concentration of the mobile phase. Since the GFC mode that can be used in the packing material for chromatography of the present invention can separate and purify proteins and enzymes without inactivating them, the column packing of the present invention can also be used for isolation of unknown biological samples and medical applications. It is expected that high resolution and high recovery of the agent will be useful.

The chromatographic packing material of the present invention is specifically obtained as a high-resolution column packing material with very little protein and polypeptide adsorption, for example, separation of proteins in human serum or trypsin digestion of proteins. It is excellent for separation of polypeptides contained in the obtained sample or separation / sorting based on activity evaluation of unknown proteins contained in the living body.
Moreover, it can be applied not only to separation of proteins and peptides but also low molecular weight compounds having strong hydrophilicity such as amino acids and biological hormones. Due to the extremely strong hydrophilicity of the phosphorylcholine group, separation by hydrophilic interaction with these analytes can be achieved under mobile phase conditions including an organic solvent. At this time, more excellent separation can be achieved by ion exchange properties, hydrophobicity, and π-electron interaction due to groups other than phosphorylcholine groups existing on the surface.

Next, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.

（９）

"Synthesis Example 1 Aldehyde Compound Containing Phosphorylcholine Group"
L-α-glycerophosphorylcholine (450 mg) was dissolved in 15 ml of distilled water and cooled in an ice-water bath. Sodium periodate (750 mg) was added and stirred for 5 hours. The reaction solution was concentrated under reduced pressure and dried under reduced pressure, and the target product was extracted with methanol. The following compound (8) has a structure. A 1H-NMR spectrum of the compound of formula (8) is shown in FIG. Since the compound of formula (8) is in equilibrium with the compound of formula (9) in water, the actual spectrum reflects both formulas (8) and (9).
(8)

(9)

（１２）

"Synthesis Example 2: Production of compounds of formulas (5) and (6)"
7.5 g of the compound of Synthesis Example 1 is dissolved in 30 mL of dehydrated methanol, and the inside of the container is replaced with dry nitrogen. Next, 3.6 g of 3-aminopropyltrimethoxysilane was added to the methanol solution of Compound 1. The mixed solution was stirred at room temperature for 5 hours and then ice-cooled, 2.5 g of sodium cyanohydroborate was added, and the mixture was returned to room temperature and stirred for 16 hours. During this time, dry nitrogen was kept flowing in the reaction vessel. After filtration of the precipitate, a methanol solution of the compounds of the following formulas (11) and (12), which are target substances, was obtained.
(11)

(12)

“Synthesis Example 3: Production of Compound of Formula (9)”
As a compound of the formula (2), 0.73 g of triethoxysilylpropyl succinic anhydride (Amax Co., Ltd.) is dissolved in 10 mL of N, N-dimethylformamide (Wako Pure Chemical Industries, Ltd.), 4 mL of distilled water and 4-dimethylaminopyridine are dissolved. 0.35 g was added and stirred at room temperature for 16 hours to obtain a silane coupling agent having a carboxyl group represented by the following formula (9). This reaction is a hydrolysis reaction using 4-dimethylaminopyridine, which is succinic anhydride, as a catalyst, and is a known reaction.
(10)

"Example 1: Production of packing material for chromatography"
A solution containing the compound represented by the formula (10) was added to a methanol solution containing 4.8 mmol of the compounds of the formulas (11) and (12). Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) having an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 140 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Then, it was filtered and washed with 50 mL of a 50% aqueous solution of N, N-dimethylformamide and 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain the intended packing material for chromatography. As the reaction solvent, in addition to the water-methanol mixed solvent, protic solvents such as water, ethanol, 2-propanol, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, toluene, diethyl ether can be used. They can be used singly or in combination.

「表１」より、実施例１のクロマトグラフィー用充填剤では、０．１２ｍｍｏｌ／ｇ_gelのリン元素を含有することが分かる。このことから、クロマトグラフィー用充填剤には、その表面に０．１２ｍｍｏｌ／ｇ_gelのホスホリルコリン基が直接結合して導入されたことが分かる。 "Proof of PC group introduction by quantitative determination of phosphorus"
The phosphorus element of the packing material for chromatography made of the modified powder having phosphorylcholine groups and carboxyl groups on the surface produced in Example 1 was quantified. In the production method of the present invention, the phosphorus element is an element inherent to the phosphorylcholine group, and the phosphorylcholine group actually introduced can be quantified by quantifying the phosphorus element present on the powder surface.
Phosphorus element was quantified by molybdic acid coloring method. The quantitative method will be described below.
1) Weigh a predetermined amount of powder into a thoroughly cleaned test tube.
2) Add 3 mL of 60% aqueous perchloric acid to 1 powder.
3) To avoid liquid dispersion, attach a cooling tube to the top of the two test tubes and heat at 120 ° C. for 1 hour and then at 180 ° C. for 2 hours. In this operation, all the modified chains on the powder surface are oxidized, and in particular phosphorus element is liberated as phosphoric acid.
4) Centrifuge the solution of 3 (3000 rpm, 5 minutes) and transfer the supernatant to a 1 mL sample tube.
5) Add 1 mL of distilled water, 500 μL of 0.5 M sodium molybdate, and 500 μL of 0.5 M ascorbic acid to the solution of 4.
6) Heat the solution of 5 in a 95 ° C. water bath for 5 minutes and then cool with cold water.
7) 200 μL of 6 colored solution is dropped onto a 96-well plate, and the color intensity at 710 nm is measured with a plate reader.
8) The amount of phosphorus element contained in the sample is quantified based on a calibration curve obtained in advance from the color intensity of a phosphoric acid solution having a known concentration.

The calibration curve used in this determination is shown in FIG.
In addition, Table 1 shows the results of phosphorus determination in the chromatographic filler comprising the modified powder having phosphorylcholine groups and carboxyl groups on the surface produced in Example 1.

From "Table 1", it can be seen that the chromatography filler of Example 1 contains 0.12 mmol / g _gel of phosphorus element. This shows that 0.12 mmol / g _gel phosphorylcholine group was directly bonded to the surface of the chromatography filler.

“Proof of introduction of carboxyl group by FT-IR spectrum”
Next, FIG. 3 shows an FT-IR spectrum of the chromatography filler of Example 1. According to FIG. 3 characteristic absorption of carboxyl group near 1725 cm ^-1 and near 1560 cm ^-1 was confirmed, it can be seen that a carboxyl group is introduced.
As described above, the quantitative determination of phosphorus element and the presence of the characteristic absorption of the carboxyl group in the FT-IR spectrum indicate that the chromatography filler of Example 1 has covalently bonded the phosphorylcholine group and the carboxyl group directly to the surface. .

“Column packing and separation of the chromatographic packing material of Example 1”
The chromatographic packing material of Example 1 was packed into a stainless steel empty column having an inner diameter of 4.6 mm and a length of 250 mm by an ordinary slurry method.
Chromatogram acquisition conditions are as follows.
Mobile phase: 50 mmol / L phosphate buffer + NaCl (predetermined concentration) pH 6.9
Flow rate: 200 μL / min
Temperature: 25 ° C
Detection: UV 280nm

Twelve kinds of proteins and peptides shown in “Table 2” were each injected into a column packed with the chromatography packing material of Example 1, and detailed evaluation was performed from their elution time and peak area. Table 2 shows the proteins and peptides used in the study, their isoelectric points (pI), molecular weights, and sample concentrations.
FIG. 4 shows the relationship between the molecular weight and elution time when the column of Example 1 was used at a mobile phase NaCl concentration of 500 mmol / l. According to FIG. 4, it can be seen that a substance having a higher molecular weight elutes earlier under this condition. It can be seen that when the mobile phase salt concentration is 500 mmol / l, there is very little interaction between the protein and the filler surface, and as a result, separation in the GFC mode in which elution in the order of molecular weight occurs.

  Next, FIG. 5 shows the relationship between the molecular weight and elution time under the condition that the NaCl concentration of the mobile phase is lowered to 250 mmol / l. Compared to FIG. 4 where the NaCl concentration of the mobile phase is 500 mmol / l, it can be seen that the elution time of some proteins has increased. FIG. 6 shows the relationship between the rate of increase of elution time and the isoelectric point of the protein in order to clarify from the viewpoint of ion exchange properties what kind of protein is specifically retained due to the decrease in the salt concentration of the mobile phase. Indicated. As is clear from FIG. 6, it can be seen that the column of the present invention having phosphorylcholine groups and carboxyl groups on its surface has the ability to retain basic proteins with high isoelectric points. This retention ability was expressed by reducing the salt concentration. In general, it is known that the ion exchange interaction becomes stronger as the salt concentration of the mobile phase is lower. Therefore, the specific retention behavior for the basic protein by the column of the present invention is due to the ion exchange interaction, and specifically, the result of the ability to exchange cation by the carboxyl group present on the surface of the filler. It is.

Next, a comparison with a packing material for chromatography composed of powder into which only formulas (11) and (12) are introduced will be described. This comparison is intended to clarify the superiority of the present invention over the case where only the phosphorylcholine group is introduced.
First, the production and packing of a chromatographic filler composed of a powder into which only formulas (11) and (12) are introduced will be described.
“Comparative Example 1: Production of a packing material for chromatography consisting of powder into which only formulas (11) and (12) are introduced and column packing”
Distilled water (35 mL) is added to the methanol solution containing the compounds of formulas (11) and (12) produced in Synthesis Example 2, and 14 g of silica gel having an average particle size of 5 μm, an average pore size of 30 nm and a specific surface area of 140 m ² / g is added. did. This powder dispersion was refluxed at 80 ° C. for 5 hours to be coupled. After refluxing, the product was filtered and washed with 100 mL of methanol to obtain the target substance. The elemental analysis values of the modified powder treated with the surface modifier of Example 1 in the above procedure are shown in “Table 3”. C% or N% in the table indicates mass% of carbon element or nitrogen element contained in the powder. From this value, the atomic ratio (C / N) of carbon and nitrogen after the surface treatment is 5.08. C / N after the coupling of the surface modifiers of the formulas (11) and (12) is 5, indicating that the surface modifier was introduced into the powder without being destroyed. The produced modified powder was packed into an empty column having an inner diameter of 4.6 mm and a length of 250 mm by an ordinary slurry method as a chromatography filler.

"Comparative test 1: Effect obtained by mixing groups that interact with each other"
The column packed with the chromatographic packing material of Example 1 and the column packed with the powder introduced only with the formulas (11) and (12) manufactured in Comparative Example 1 were used for evaluation in Example 1. Evaluation and comparison were performed using 12 kinds of proteins.
Increase rate of elution time of each protein when the mobile phase NaCl concentration is reduced from 500 mmol / l to 250 mmol / l in the column packed with the powder introduced only with the formulas (11) and (12) produced in Comparative Example 1 Is shown in FIG. The column of the packing material for chromatography manufactured in Example 1 can be compared with FIG. 6 and FIG. Specifically, (1) the column packed with the powder produced in Comparative Example 1 retains acidic protein having a low isoelectric point (FIG. 7), and (2) the column for the packing material for chromatography in Example 1 In FIG. 6, a basic protein having a high isoelectric point was retained (FIG. 6). (1) is retention by phosphorylcholine group and anion exchange interaction caused by secondary amine contained in the modified chain, and (2) is a carboxyl group introduced in a mixed manner with phosphorylcholine group as “group having interaction” Is a cation exchange interaction.

  Further, in order to clarify the difference between the two, a chromatogram when human serum albumin and lysozyme are injected at each salt concentration is shown in FIG. As is apparent from FIG. 8, the powder produced in Comparative Example 1 has anion exchange ability due to the influence of the secondary amine present in the phosphorylcholine group and the spacer, and negatively charged human serum albumin has a low salt concentration. On the other hand, the chromatographic packing material produced in Example 1 has a cation exchange ability due to the influence of the carboxyl group, and tends to hold positively charged cytochrome C at a low salt concentration. I understand that there is. The column packing material of the present invention is a completely new packing material that imparts a cation exchange ability that was impossible in a powder having only a conventional phosphorylcholine group introduced.

“Comparison test 2: Comparison of elution time and recovery rate with general GFC column”
Proteins in a human serum sample (Concerer N (product name) diluted twice with distilled water) using a commercially available chromatography column (Shodex PROTEIN KW803, Showa Denko KK) as it is commercially available Attempted separation. The column mentioned in the comparative example has the same inner diameter and length as the column described in Example 1.
This filler is described as a filler for size exclusion mode in which a hydrophilic group is covalently bonded to the surface of porous silica gel. The average pore diameter is 300 angstroms and the average particle diameter is 5 μm, which is suitable for comparison with the filler described in Example 1. It is described that it is suitable for separation of proteins having a molecular weight of 10,000 to several hundred thousand.
Since Shodex PROTEIN KW803 uses a non-dissociable hydrophilic group, it does not have a charged functional group unlike a filler prepared using the surface modifier of the present invention. Therefore, it is considered that there is almost no ionic interaction with the protein.
As in Example 1, using a mobile phase in which 500 mmol / l sodium chloride was added to 50 mmol / l phosphate buffer (prepared from Na ₂ HPO ₄ and KH ₂ PO ₄ ), the flow rate was 0.1 ml / min, column Human serum albumin (5 mg / ml) and lysozyme (1 mg / ml) were injected at an oven temperature of 25 ° C. Detection was performed at UV 280 nm. The results are shown in FIG.

In KW803, which is a general GFC column, a change in retention time due to a decrease in salt concentration is not observed for both human serum albumin that is an acidic protein and lysozyme that is a basic protein.
On the other hand, in Example 1 of this invention, it turns out that only the lysozyme which is basic protein is hold | maintained with the fall of salt concentration unlike the column for conventional GFC. As described above, the retention of the basic protein, which is peculiar to the powder of the present invention, is due to the carboxyl group coexisting with the phosphorylcholine group. This means that the target protein can be separated from other components by changing the mobile phase conditions.

「表３」より明らかなように、本願発明のカラムにおいては検討したすべてのタンパク質について一般的なＧＦＣカラムと同等以上の回収率を誇る。γ-グロブリンを初めとする数種類のタンパク質については、既存のＧＦＣカラムよりも１０％以上回収率が高い。
移動相ＮａＣｌ濃度が５００ｍｍｏｌ／ｌの場合では、本願発明のカラム特有であるイオン交換性がほとんど現れないために相互作用が存在せず、回収率は極めて良好である。さらには、上述の通り塩基性タンパク質に保持が生じ始め、カチオン交換性を併発する条件である移動相ＮａＣｌ濃度が２５０ｍｍｏｌ／ｌの場合においても、一般に回収率が良いとされるＧＦＣカラム以上の回収率を誇る。本来タンパク質を吸着させる性質を有するカルボキシル基が存在しながらもこれほどまでに回収率が高いのは、カルボキシル基と分子レベルで共存するホスホリルコリン基の影響である。
このように、本来タンパク質・ペプチドの非特異的不可逆吸着をもたらす「相互作用を及ぼす基」であっても、ホスホリルコリン基と共存させることによって相互作用に基づいた分離と優れた回収率を両立させることができる。 Furthermore, the recovery rate by comparison of peak areas when each protein is injected into a column packed with KW803 and the powder of the present invention will be described. The examination was carried out using the protein and peptide samples shown in Table 2. Table 4 shows the peak areas when the sample was injected into the chromatographic packing material of Example 1 of the present invention when the peak area when the sample was injected into KW803 was 100.

As is apparent from “Table 3”, the column of the present invention has a recovery rate equivalent to or higher than that of a general GFC column for all the proteins studied. For several types of proteins including γ-globulin, the recovery rate is 10% or more higher than that of the existing GFC column.
When the mobile phase NaCl concentration is 500 mmol / l, the ion exchange property peculiar to the column of the present invention hardly appears, so there is no interaction, and the recovery rate is very good. Furthermore, as described above, the basic protein begins to be retained, and even when the mobile phase NaCl concentration is 250 mmol / l, which is a condition that causes cation exchange, the recovery is more than the GFC column, which generally has a good recovery rate. Boasting rate. The reason why the recovery rate is so high even though the carboxyl group originally has the property of adsorbing protein is due to the influence of the phosphorylcholine group coexisting with the carboxyl group at the molecular level.
In this way, even if it is an “interaction group” that inherently causes non-specific irreversible adsorption of proteins and peptides, coexistence with the phosphorylcholine group achieves both separation based on interaction and excellent recovery. Can do.

合成例２で製造した式（１１）および（１２）の化合物４．８ｍｍｏｌを含むメタノール溶液に、式（３）の化合物として上記式（１３）に示す化合物を０．４０ｇ添加する。さらに蒸留水２０ｍＬと平均粒子径５μｍ、平均細孔径３００オングストローム、比表面積１４０ｍ²／ｇの球状高純度シリカゲル（洞海科学製）４．０ｇを添加し、オイルバス中８０℃で５時間還流する。その後、反応溶液をろ過し、次いで５０％メタノール水溶液５０ｍＬでろ過洗浄し、さらにメタノール５０ｍＬでろ過洗浄し、８０℃で３時間減圧乾燥させ、目的とする粉体を得る。反応溶媒は水-メタノール混合溶媒以外にも、水、エタノール、２−プロパノール等のプロトン性溶媒や、ジメチルスルホキシド、ジメチルホルムアミド、トルエン、ジエチルエーテル等の非プロトン性溶媒を使用でき、これらを単一、または組み合わせて用いることができる。なお、本粉体改質法は、式（１３）に示した化合物を用いずとも、スルホン酸および、シリカゲルに存在するシラノール基とシロキサン結合を形成する官能基を分子内に有するあらゆる化合物について全く同様に適用することが可能である。 "Example 2: Production of a packing material for chromatography having phosphorylcholine groups and sulfonic acid groups on the substrate surface"
Although the compound of Formula (3) will not be specifically limited if it is a silane coupling agent which has a sulfonic acid, Manufacture by 3- (trihydroxysilyl) -1-propanesulfonic acid (Amax Co., Ltd.) shown to Formula (13) An example is described.


(13)

0.40 g of the compound represented by the above formula (13) is added as a compound of the formula (3) to a methanol solution containing 4.8 mmol of the compounds of the formula (11) and (12) produced in Synthesis Example 2. Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) having an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 140 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Thereafter, the reaction solution is filtered, then filtered and washed with 50 mL of 50% aqueous methanol solution, further filtered and washed with 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain the desired powder. As the reaction solvent, in addition to the water-methanol mixed solvent, protic solvents such as water, ethanol, 2-propanol, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, toluene, diethyl ether can be used. Or can be used in combination. This powder modification method does not use the compound represented by the formula (13), but uses sulfonic acid and any compound having a functional group that forms a siloxane bond with a silanol group present in silica gel. It is possible to apply similarly.

合成例２で製造した式（１１）および（１２）の化合物４．８ｍｍｏｌを含むメタノール溶液に上述の式（１４）に示す化合物を０．６２ｇ添加する。さらに蒸留水２０ｍＬと平均粒子径５μｍ、平均細孔径３００オングストローム、比表面積１４０ｍ²／ｇの球状高純度シリカゲル（洞海科学製）４．０ｇを添加し、オイルバス中８０℃で５時間還流する。その後、反応溶液をろ過し、次いで５０％メタノール水溶液５０ｍＬでろ過洗浄し、さらにメタノール５０ｍＬでろ過洗浄し、８０℃で３時間減圧乾燥させ、目的とする粉体を得る。反応溶媒は水-メタノール混合溶媒以外にも、水、エタノール、２−プロパノール等のプロトン性溶媒や、ジメチルスルホキシド、ジメチルホルムアミド、トルエン、ジエチルエーテル等の非プロトン性溶媒を使用でき、これらを単一、または組み合わせて用いることができる。なお、本粉体改質法は、式（１４）に示した化合物を用いずとも、４級アンモニウムおよび、シリカゲルに存在するシラノール基とシロキサン結合を形成する官能基を分子内に有するあらゆる化合物について全く同様に適用することが可能である。
また、以下の方法によっても目的とする改質粉体を得ることができる。
合成例２で製造した式（１１）および（１２）の化合物４．８ｍｍｏｌを含むメタノール溶液に３−アミノプロピルトリメトキシシラン（アヅマックス株式会社）を０．７０ｇ添加する。さらに蒸留水２０ｍＬと平均粒子径５μｍ、平均細孔径３００オングストローム、比表面積１４０ｍ²／ｇの球状高純度シリカゲル（洞海科学製）４．０ｇを添加し、オイルバス中８０℃で５時間還流する。その後、反応溶液をろ過し、次いで５０％メタノール水溶液５０ｍＬでろ過洗浄し、さらにメタノール５０ｍＬでろ過洗浄し、８０℃で３時間減圧乾燥させ、アミノプロピル基とホスホリルコリン基の混在する粉体を得る。この粉体をトルエンに分散させ、ヨウ化メチルをアミノ基に対して５当量添加し、１００℃で５時間還流し、目的とする粉体を得ることができる。 "Example 3: Production of a packing material for chromatography having phosphorylcholine groups and quaternary ammonium groups on the substrate surface"
The compound of formula (3) is not particularly limited as long as it is a silane coupling agent having a quaternary ammonium group, but N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride represented by formula (14) (Amax) A manufacturing example by the company) will be described.
(14)

0.62 g of the compound represented by the above formula (14) is added to a methanol solution containing 4.8 mmol of the compounds of the formula (11) and (12) manufactured in Synthesis Example 2. Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) having an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 140 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Thereafter, the reaction solution is filtered, then filtered and washed with 50 mL of 50% aqueous methanol solution, further filtered and washed with 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain the desired powder. As the reaction solvent, in addition to the water-methanol mixed solvent, protic solvents such as water, ethanol, 2-propanol, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, toluene, diethyl ether can be used. Or can be used in combination. This powder modification method is applicable to any compound having quaternary ammonium and a functional group that forms a siloxane bond with a silanol group present in silica gel in the molecule without using the compound represented by formula (14). It is possible to apply exactly the same.
The target modified powder can also be obtained by the following method.
To a methanol solution containing 4.8 mmol of the compounds of formulas (11) and (12) produced in Synthesis Example 2, 0.70 g of 3-aminopropyltrimethoxysilane (Amax Co., Ltd.) is added. Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) having an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 140 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Thereafter, the reaction solution is filtered, then filtered and washed with 50 mL of 50% aqueous methanol solution, further filtered and washed with 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain a powder in which aminopropyl groups and phosphorylcholine groups are mixed. This powder is dispersed in toluene, 5 equivalents of methyl iodide are added to amino groups, and the mixture is refluxed at 100 ° C. for 5 hours to obtain the desired powder.

合成例２で製造した式（１１）および（１２）の化合物４．８ｍｍｏｌを含むメタノール溶液に上述の式（１５）に示す化合物を０．２０ｇ添加する。さらに蒸留水２０ｍＬと平均粒子径５μｍ、平均細孔径３００オングストローム、比表面積１４０ｍ²／ｇの球状高純度シリカゲル（洞海科学製）４．０ｇを添加し、オイルバス中８０℃で５時間還流する。その後、反応溶液をろ過し、次いで５０％メタノール水溶液５０ｍＬでろ過洗浄し、さらにメタノール５０ｍＬでろ過洗浄し、８０℃で３時間減圧乾燥させ、目的とする粉体を得る。反応溶媒は水-メタノール混合溶媒以外にも、水、エタノール、２−プロパノール等のプロトン性溶媒や、ジメチルスルホキシド、ジメチルホルムアミド、トルエン、ジエチルエーテル等の非プロトン性溶媒を使用でき、これらを単一、または組み合わせて用いることができる。なお、本粉体改質法は、式（１５）に示した化合物を用いずとも、炭素数１〜３０までの飽和または不飽和アルキル鎖および、シリカゲルに存在するシラノール基とシロキサン結合を形成する官能基を分子内に有するあらゆる化合物について全く同様に適用することが可能である。具体的にはｎ−オクタデシルトリメトキシシラン（アヅマックス株式会社）やｎ−オクチルトリメトキシシラン（アヅマックス株式会社）を疎水性相互作用をもたらす修飾鎖として全く同じ方法で適用することができる。 "Example 4: Production of a packing material for chromatography having phosphorylcholine groups and butyl groups on the substrate surface"
In the case where the hydrophobic interaction by the hydrophobic group is mixed with the phosphorylcholine group, an alkyl chain having an arbitrary chain length can be introduced. In this example, a production example using n-butyltrimethoxysilane (Amax Co., Ltd.) represented by the formula (15) will be described.
(15)

0.20 g of the compound represented by the above formula (15) is added to a methanol solution containing 4.8 mmol of the compounds of the formula (11) and (12) manufactured in Synthesis Example 2. Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) having an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 140 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Thereafter, the reaction solution is filtered, then filtered and washed with 50 mL of 50% aqueous methanol solution, further filtered and washed with 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain the desired powder. As the reaction solvent, in addition to the water-methanol mixed solvent, protic solvents such as water, ethanol, 2-propanol, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, toluene, diethyl ether can be used. Or can be used in combination. This powder modification method forms a saturated or unsaturated alkyl chain having 1 to 30 carbon atoms and a silanol group present in silica gel and a siloxane bond without using the compound represented by formula (15). The same application can be applied to any compound having a functional group in the molecule. Specifically, n-octadecyltrimethoxysilane (Amax Co., Ltd.) and n-octyltrimethoxysilane (Amax Co., Ltd.) can be applied in exactly the same manner as a modified chain that causes a hydrophobic interaction.

合成例２で製造した式（１１）および（１２）の化合物４．８ｍｍｏｌを含むメタノール溶液に上述の式（１６）に示す化合物を０．１８ｇ添加する。さらに蒸留水２０ｍＬと平均粒子径５μｍ、平均細孔径３００オングストローム、比表面積１４０ｍ²／ｇの球状高純度シリカゲル（洞海科学製）４．０ｇを添加し、オイルバス中８０℃で５時間還流する。その後、反応溶液をろ過し、次いで５０％メタノール水溶液５０ｍＬでろ過洗浄し、さらにメタノール５０ｍＬでろ過洗浄し、８０℃で３時間減圧乾燥させ、目的とする粉体を得る。反応溶媒は水-メタノール混合溶媒以外にも、水、エタノール、２−プロパノール等のプロトン性溶媒や、ジメチルスルホキシド、ジメチルホルムアミド、トルエン、ジエチルエーテル等の非プロトン性溶媒を使用でき、これらを単一、または組み合わせて用いることができる。なお本粉体改質法は、芳香族化合物および、シリカゲルに存在するシラノール基とシロキサン結合を形成する官能基を分子内に有するあらゆる化合物についても全く同様に適用することが可能である。芳香族化合物とは例えばフェニル基、ナフチル基、ピレン、ピリジン環、ピロール環、フラン環などを示し、具体的な化合物としてはフェネチルトリメトキシシラン（アヅマックス株式会社）、トリフェニルエトキシシラン（アヅマックス株式会社）、Ｎ−（３−トリメトキシシリルプロピル）ピロール（アヅマックス株式会社）、ｍ−フェノキシフェニルジメトキシシラン（アヅマックス株式会社）、１−ナフチルトリメトキシシラン（アヅマックス株式会社）などの適用が可能である。 “Example 5: Production of packing material for chromatography having phosphorylcholine group and aromatic compound on substrate surface”
The case of mixing hydrophobicity and π-electron interaction with the phosphorylcholine group can be realized by applying a silane coupling agent having an aromatic group in the molecule. In this example, a production example using phenyltrimethoxysilane (Amax Co., Ltd.) represented by the formula (16) will be described.
(16)

0.18 g of the compound represented by the above formula (16) is added to a methanol solution containing 4.8 mmol of the compounds of the formula (11) and (12) produced in Synthesis Example 2. Further, 20 mL of distilled water, 4.0 g of spherical high-purity silica gel (manufactured by Dokai Kagaku) having an average particle size of 5 μm, an average pore size of 300 Å, and a specific surface area of 140 m ² / g are added and refluxed at 80 ° C. for 5 hours in an oil bath. . Thereafter, the reaction solution is filtered, then filtered and washed with 50 mL of 50% aqueous methanol solution, further filtered and washed with 50 mL of methanol, and dried under reduced pressure at 80 ° C. for 3 hours to obtain the desired powder. As the reaction solvent, in addition to the water-methanol mixed solvent, protic solvents such as water, ethanol, 2-propanol, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, toluene, diethyl ether can be used. Or can be used in combination. This powder modification method can be applied in the same manner to aromatic compounds and any compounds having in the molecule functional groups that form siloxane bonds with silanol groups present in silica gel. Examples of the aromatic compound include a phenyl group, a naphthyl group, a pyrene, a pyridine ring, a pyrrole ring, and a furan ring. Specific examples of the aromatic compound include phenethyltrimethoxysilane (Amax Co., Ltd.), triphenylethoxysilane (Amax Co., Ltd.). ), N- (3-trimethoxysilylpropyl) pyrrole (Amax Co.), m-phenoxyphenyldimethoxysilane (Amax Co.), 1-naphthyltrimethoxysilane (Amax Co., Ltd.) and the like are applicable.

The characteristics of the packing material for chromatography according to the present invention include not only separation due to the difference in molecular weight due to the GFC mode, but also any anion exchange interaction, cation exchange interaction, hydrophobic interaction, and π electron interaction. The strength of the protein / peptide recovery is extremely excellent.
Furthermore, since these interactions can be adjusted according to the salt concentration, pH, and organic solvent concentration of the mobile phase, it is possible to perform a unique separation according to the protein. In addition, since protein does not cause irreversible adsorption on the powder surface, it can be separated, fractionated, and analyzed without protein alteration or deactivation.

2 is a 1H-NMR spectrum of the compound of Synthesis Example 1. 2 is a calibration curve used for the determination of phosphorus element in Example 1. FIG. 2 is an FT-IR spectrum of the packing material for chromatography of Example 1. FIG. 2 is an elution time-molecular weight plot of the chromatographic packing material of Example 1 (mobile phase NaCl concentration 500 mmol / l). FIG. It is an elution time-molecular weight plot of the chromatography packing material of Example 1 (mobile phase NaCl concentration 250 mmol / l). FIG. 3 is a graph showing the relationship between the rate of increase in elution time accompanying the decrease in mobile phase NaCl concentration and the protein isoelectric point in the chromatographic packing material of Example 1. It is the figure which showed the relationship between the elution time increase rate with the fall of the mobile phase NaCl density | concentration in the packing material for chromatography of the comparative example 1, and a protein isoelectric point. 2 is a chromatogram of human serum albumin and lysozyme at each salt concentration using the chromatographic packing column of Example 1 and Comparative Example 1. FIG. 2 is a chromatogram of human serum albumin and lysozyme at each salt concentration using the chromatographic packing material of Example 1 and KW803.

Claims (7)

A filler for chromatography, wherein a compound represented by the following formula (1) and a compound represented by the following formula (2) are directly covalently bonded to the surface of a substrate.
(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included.
(2)

R ′ represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. A filler for chromatography, wherein a compound represented by the following formula (1) and a compound represented by the following formula (3) are directly covalently bonded to the substrate surface.
(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included.
(3)

R ′ represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included. A packing material for chromatography, wherein a compound represented by the following formula (1) and a compound represented by the following formula (4) are directly covalently bonded to the substrate surface.


(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included.
(4)

R ′ represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included.
R ² , R ³ and R ⁴ are each independently a methyl group, ethyl group, propyl group, isopropyl group, n-butyl group or t-butyl group. A packing material for chromatography, wherein a compound represented by the following formula (1) and a saturated alkyl group, an unsaturated alkyl group or an aromatic compound are directly covalently bonded to the surface of a substrate.
(1)

R represents an arbitrary modified chain having 1 to 30 carbon atoms. It may have one or more branched structure, unsaturated bond, benzene nucleus, nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, silicon atom, chlorine atom, fluorine atom, bromine atom at any position of the modified chain Also, a combination of these may be included.   The chromatographic filler according to claim 1, wherein the base material is a spherical porous silica gel.   6. The chromatographic packing material according to claim 5, wherein the spherical porous silica gel has an average particle size of 1 to 200 [mu] m. The packing material for chromatography according to claim 5 or 6, wherein the average diameter of the pores of the spherical porous silica gel is 10 to 1000 angstroms.

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