JPH06308108A - Adsorption carrier for chromatography and its manufacture - Google Patents

Abstract

(57) [Summary] [Purpose] An adsorption carrier for chromatography, which has excellent asymmetric discrimination ability, is easy to produce, and has excellent reproducibility and durability, and a method for producing the same. An adsorption carrier for chromatography which comprises reacting a vinyl group-containing cyclodextrin or a derivative thereof with a vinyl group on a water-insoluble support, and a method for producing the same.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel adsorption carrier for chromatography and a method for producing the same, and more particularly to an adsorption carrier for chromatography having an asymmetric discrimination ability and a method for producing the same.

[0002]

2. Description of the Related Art Cyclodextrin (hereinafter referred to as CD) is a cylindrical cyclic compound in which D-glucopyranoside is bonded to α-1,4-glucoside, and the outside of the cylinder of the cyclic compound has many hydroxyl groups and is hydrophilic. However, the inside of the cylinder has hydrophobicity. Since the inside of the cylinder is hydrophobic, this CD is known as a host compound in which various organic compounds having a hydrophobic group such as a phenyl group and a naphthyl group are included. Further, since CD has 5 optically active carbons per D-glucopyranoside molecule, it is also known as a compound having an asymmetric discrimination ability.

In recent years, the importance of separation / purification of optically active compounds has increased, and there has been an increasing tendency to utilize the asymmetric discrimination ability of CD for this purpose. For example, US Pat.
According to JP-A-9,399, as an adsorption carrier for chromatography, silica gel having an epoxy group or an adsorption carrier obtained by reacting CD with a synthetic polymer is used.
A method for separating and purifying an optically active compound is known.
Further, JP-A-61-237057 exemplifies a method for producing an adsorption carrier for chromatography with a CD derivative having the same content as described above.

However, the above-mentioned conventional methods have problems that the production of the carrier is complicated, the reproducibility and durability are poor, and the separation ability is insufficient.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide an adsorption carrier for chromatography which is excellent in asymmetric discrimination ability, is easy to produce the carrier, and is excellent in reproducibility and durability, and a method for producing the same. It is in.

[0006]

The present invention has been made in order to achieve the above object, and comprises reacting a water-insoluble support with a vinyl group-containing CD or its vinyl group derivative. The present invention relates to an adsorption carrier for chromatography and a method for producing the same.

The adsorption carrier for chromatography of the present invention and the method for producing the same will be described below. The water-insoluble support in the present invention, a functional group capable of binding to a vinyl group by performing graft polymerization, for example, an alcoholic hydroxyl group, an alkenyl group, an alkynyl group, a mercapto group,
Amino group, formyl group, acetyl group, carboxyl group,
There is no particular limitation as long as it has a functional group such as a halogen group or can be introduced, and agarose, dextran, polysaccharides such as cellulose and so-called soft beads such as polyacrylamide, polystyrene, polymethacrylate, polyvinyl alcohol. And hard synthetic polymer beads such as copolymers thereof, or inorganic hard beads such as silica gel and alumina. The bead size, shape, presence / absence of pores and the pore size are not particularly limited, but are usually 1 to 1000 μm.
m, preferably 3 to 120 μm, and the shape of the beads is preferably porous sphere.

The CD used in the present invention includes:
Examples include α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and C
As the D derivative, for example, a hydroxyalkylated cyclodextrin, a glycosylated cyclodextrin modified with a substituent having a hydroxyl group, or a methylated cyclodextrin or an acylated cyclodextrin having at least one hydroxyl group can be used. Any CD may be used, and a mixture thereof or a derivative thereof may be used.

The vinyl group-containing CD or its derivative used in the present invention is, for example, an acrylic acid ester or a methacrylic acid ester having a CD or a derivative residue thereof, or an acryloyloxy hydroxy group having a CD or a derivative residue thereof. Examples thereof include propyl ether or methacryloyloxy hydroxypropyl ether, and methacryloyloxyethyl carbamate having a CD or a derivative residue thereof.

The adsorption carrier for chromatography of the present invention can be produced by the method described below. That is,
To obtain a CD having a vinyl group, the CD is dissolved or dispersed in an appropriate solvent, and then a vinyl derivative having both a vinyl group and a group capable of reacting with the hydroxyl group of CD is added.

As the solvent, any solvent can be used without particular limitation as long as it can dissolve or disperse CD without inhibiting the reaction between CD and the vinyl derivative. Examples of such a solvent include water, and organic solvents include alkyl amides such as N, N-dimethylformamide and N, N-dimethylacetamide, isopropyl ether, 1,4-
Dioxane, ether such as tetrahydrofuran, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, ester such as isoamyl acetate, chloroform,
Examples thereof include halogenated hydrocarbons such as dichloromethane and carbon tetrachloride, aromatic hydrocarbons such as benzene and toluene, and amines such as pyridine, quinoline and triethylamine. These solvents may be used alone or in combination of two or more kinds.

The vinyl derivative is not particularly limited as long as it has both a vinyl group and a group capable of reacting with the hydroxyl group of CD, and examples thereof include acrylic acid chloride and methacrylic acid chloride which directly react with the hydroxyl group of CD. Compounds having various halide groups, glycidyl acrylate,
In addition to compounds having an epoxy group such as glycidyl methacrylate, or compounds having an isocyanate group such as 2-methacryloyloxyethyl isocyanate, acrylic acid and methacrylic acid that react by adding a hydroxyl group of CD and a condensing agent. Examples thereof include compounds having a carboxyl group.

When a vinyl derivative which directly reacts with a hydroxyl group of CD is used, the vinyl derivative is dissolved or dispersed in the above-mentioned solvent and then the vinyl derivative is added in an amount of 0.5 to 5 per equivalent of CD.
Add 0 equivalent, preferably 1 to 7 equivalent, and add vinyl derivative to C
Introduce to D. In this reaction, a reactor equipped with a dryer containing a dehydrating agent is used for 0.5 to 48 hours, preferably 1
Allow to react for ~ 24 hours. The reaction temperature at this time is 0 ° C to 80 ° C.
It is desirable to keep the temperature at 0 ° C, preferably 0 ° C to 30 ° C. Further, a poor solvent is added to the reaction solution until a precipitate is precipitated, and the precipitate is collected by filtration, washed well with the poor solvent and dried to obtain the vinyl group-containing CD.
Examples of the poor solvent include ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclohexanone.

When a vinyl derivative which reacts with the hydroxyl group of CD by adding a condensing agent is used, for example, Nobuo Izumiya et al. "Basics and Experiments of Peptide Synthesis" 41-88
This can be done as described on the page (Maruzen). Specifically, CD and vinyl derivatives are suitable solvents such as water, and organic solvents include alkyl amides such as N, N-dimethylformamide and N, N-dimethylacetamide, isopropyl ether, 1,4-dioxane, and tetrahydrofuran. Ether system such as methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, isoamyl acetate, etc., halogenated hydrocarbon system such as chloroform, dichloromethane, carbon tetrachloride, aromatic hydrocarbon system such as benzene, toluene, etc. After being dissolved or dispersed in an amine system such as pyridine, quinoline, or triethylamine, a condensing agent such as N, N'-dicyclohexylcarbodiimide, N, N'-disuccinimidyl carbonate, N, N'-carbonyldiimidazole, N-ethyl-N'-dimethylami React using nopropylcarbodiimide, thionyl chloride, etc. The amount of the vinyl derivative used in this reaction is 0.5 to 50 equivalents, preferably 1 to 7 equivalents per CD equivalent, and the amount of the condensing agent used is 0.6 per CD equivalent.
-60 equivalents, preferably 1-7 equivalents.

This reaction is carried out under the same reaction conditions as in the case of using the vinyl derivative which directly reacts with the hydroxyl group of CD. When obtaining a CD derivative having a vinyl group, a CD derivative is used in place of CD and the same operation is performed. Furthermore, when performing these reactions, it is desirable to dry the CD or its derivatives before use. The CD derivative can be obtained by a known method (Tetrahedron, 1983, 3).
9, P.I. 1417), or a commercially available product such as heptakis (2,6-di-O-methyl) -β-cyclodextrin (manufactured by Aldrich). Specifically, for example, when obtaining hydroxypropyl-β-cyclodextrin, it can be obtained by heating β-cyclodextrin and monochloropropyl alcohol in the presence of an alkali.

When the water-insoluble support having a functional group capable of reacting with the vinyl group is reacted with CD having a vinyl group or a derivative thereof, the support is dispersed in a suitable solvent, A CD having a vinyl group obtained by the above-mentioned method by generating radicals on the support.
Alternatively, a derivative thereof is reacted. As a method of generating radicals, for example, a method of irradiating light, radiation or the like,
Examples include a method of adding a radical initiator. As a method of irradiating light, radiation, etc., for example, Shu Kanbara et al.
It can be carried out as described in "Polymer Reaction", pages 171-189 (Kyoritsu Shuppan).

As a method for adding a radical initiator, the support is dispersed in an appropriate solvent, the radical initiator is added, and the vinyl group-containing CD or its derivative obtained by the above-mentioned method is further added. React. As the radical initiator, for example, a peroxide type, an azobis type, a redox type or the like can be selected according to the solubility in the polymerization solvent. Examples of the polymerization initiator include peroxides such as hydrogen peroxide, benzoyl peroxide, t-butylperoxymaleic acid, succinic acid peroxide, benzoyl peroxide, lauroyl peroxide, and acetyl peroxide. , 2'-azobisisobutyronitrile, 2,2'-azobis (2-aminodipropane) dihydrochloride, 2,2'-azobis [2-methyl-N- (hydroxymethyl) propionamide], etc. Examples thereof include azobis type, cerium ammonium nitrate, and redox type such as potassium persulfate.

The amount of the radical initiator used in this reaction is 0. 1 with respect to the vinyl group-containing CD or its derivative.
It is 001 to 5% by weight, preferably 0.005 to 3% by weight. The solvent used for the polymerization can be used without particular limitation as long as it can dissolve the vinyl group-containing CD or its derivative without inhibiting radical polymerization. As such a solvent, water, as an organic solvent, N,
Alkyl amides such as N-dimethylformamide, N, N-dimethylacetamide, isopropyl ether,
Ether-based, such as dioxane, tetrahydrofuran,
Methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, ester series such as isoamyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone, ketone series such as cyclohexanone, aromatic hydrocarbon series such as benzene, xylene, toluene, nitrotoluene, chloroform, Examples thereof include halogenated hydrocarbons such as dichloromethane and carbon tetrachloride. These solvents may be used alone or in combination of two or more kinds.

In this reaction, if necessary, in a reactor equipped with a drying device containing a dehydrating agent for 0.5 to 48 hours,
The reaction is preferably carried out for 1 to 24 hours. The reaction temperature at this time is desirably maintained at 10 ° C to 80 ° C, preferably 20 ° C to 60 ° C. The reaction product is collected by filtration and, if necessary, further washed with the solvent to obtain the adsorption carrier for chromatography of the present invention. At this time, in order to further improve the resolution, if the immobilization amount is further increased, the above operation is repeated several times to obtain an adsorption carrier for chromatography having a high immobilization amount of CD or its derivative.

According to the present invention, it is possible to obtain an adsorption carrier for chromatography which is characterized by reacting a water-insoluble support with the vinyl group of a cyclodextrin having a vinyl group or a derivative thereof. , Such an adsorption carrier for chromatography can be used as an adsorption carrier for high performance liquid chromatography from an adsorption carrier for simple liquid chromatography, and not only optical isomers but also positional isomers and geometric isomers can be obtained by packing in a column. Initially, various isomers can be separated.

[0021]

The present invention will be described in more detail with reference to examples. However, it goes without saying that these are examples of the adsorption carrier for chromatography of the present invention, and the present invention is not limited to these.

Example 1 Preparation of mono (6-O- (2-methacryloyloxyethylcarbamoyl))-β-cyclodextrin-immobilized carrier In an epoxy group-containing gel-like copolymer obtained from glycidyl methacrylate and glycerin dimethacrylate The epoxy group of was subjected to ring opening modification with water and dried under vacuum for 5 hours. 10 g of this carrier was mixed with 10 g of 1,4-butanediol diglycidyl ether (hereinafter abbreviated as 1,4-BGE).
g, and 20m of 1M sodium hydroxide aqueous solution
After adding 1 liter and shaking at 30 ° C. for 5 hours, the carrier was collected by filtration and water was added to
Wash with M sodium chloride and acetone. This 1,4-BGE-immobilized carrier (epoxy group; dry carrier 1 g
(250 μmol per 10 μg), 50 ml of 0.1 M sulfuric acid aqueous solution was added, the mixture was shaken at 70 ° C. for 1 hour, and the carrier was collected by filtration.
Wash with water and acetone. Thus, a support having a group capable of binding to a CD having a vinyl group and a derivative thereof was produced.

10 g of β-cyclodextrin was added to N, N-
Dissolve in 200 ml of dimethylformamide, and under ice cooling, 2-methacryloyloxyethyl isocyanate 1.5
4 g is added dropwise with stirring for 10 minutes. Then, 3 ml of triethylamine was added, and after stirring at 20 ° C. for 12 hours, 300 ml of acetone was added to the reaction solution. The precipitate thus formed was collected by filtration, and the residue was washed with acetone to give a white solid of mono (6-O- (2-methacryloyloxyethylcarbamoyl))-β-cyclodextrin 5.7.
9 g was obtained.

The physical properties of this compound are shown below. ¹ H nuclear magnetic resonance spectrum (solvent; heavy water, internal standard; trimethylsilylpropane sulfonate) ppm 1.79 (3H, singlet) 3.40 to 4.00 (42
H, multiplet) 4.92 (7H, doublet) 5.59 (1H, singlet) 6.01 (1H, singlet) Infrared spectrum (KBr) cm ^-1 1635, 1720 (main) Only absorption)

Support 1 obtained by the method as described above
0 g to 250 ml water and cerium ammonium nitrate 0.0
8 g was dispersed and the mono- (6
Add 2.5 g of -O- (2-methacryloyloxyethylcarbamoyl))-β-cyclodextrin at 25 ° C.
After stirring for 10 hours, the carrier was collected by filtration and washed well with water and methanol. The thus obtained carrier was analyzed by elemental analysis for N and contained β-cyclodextrin in an amount of about 0.
It was confirmed that 025 mmol was immobilized.

Example 2 Preparation of Mono (6-O-methacryloyl) -β-cyclodextrin-Immobilized Carrier Instead of mono (6-O- (2-methacryloyloxyethylcarbamoyl))-β-cyclodextrin in Example 1. In addition, mono (6-O-methacryloyl) -β-
Mono (6-O-methacryloyl) was obtained by the same procedure as in Example 1 except that cyclodextrin was used.
A -β-cyclodextrin-immobilized carrier was obtained. It was confirmed that β-cyclodextrin was immobilized on the thus obtained carrier in an amount of 0.05 mmol per 1 g of the dried carrier, based on the weight increase rate.

Example 3 Preparation of Mono (6-O-acryloyl) -β-cyclodextrin-Immobilized Carrier Instead of mono (6-O- (2-methacryloyloxyethylcarbamoyl))-β-cyclodextrin in Example 1. In the same manner as in Example 1, except that mono (6-O-acryloyl) -β-cyclodextrin was used, mono (6-O-acryloyl) -β was obtained.
-A cyclodextrin-immobilized carrier was obtained. It was confirmed that β-cyclodextrin was immobilized on the thus obtained carrier in an amount of 0.05 mmol per 1 g of the dried carrier, based on the weight increase rate.

Comparative Example Preparation of β-Cyclodextrin-Immobilized Carrier β was added to 10 g of the 1,4-BGE-immobilized carrier used in Example 1.
-Added 20 g of cyclodextrin and added 0.067
Add 100 ml of N sodium hydroxide solution and add 4 at 30 ° C.
After stirring for 8 hours, the carrier was filtered and washed with water and acetone. The carrier thus obtained contained β-cyclodextrin in an amount of 0.025 mm per 1 g of the dry carrier depending on the weight increase rate.
It was confirmed that ol was immobilized.

Application Example The mono (6-O- (2-methacryloyloxyethylcarbamoyl))-β-cyclodextrin-immobilized carrier obtained in Example 1 and the β-cyclodextrin-immobilized carrier obtained in Comparative Example were used. , Each inner diameter 4.6mm, length 150mm
The stainless steel chromatograph tube of No. 1 was filled with the slurry method, and (+,-)-aminoglutethiimide was analyzed using a high performance liquid chromatograph. FIG. 1 shows a chromatogram analyzed using the adsorption carrier for chromatography obtained in Example 1, and FIG. 2 shows a chromatogram analyzed using the β-cyclodextrin-immobilized carrier obtained in Comparative Example. .

The analysis conditions are as follows. Eluent: 2% acetic acid-triethylamine buffer (pH 6.
5): Acetonitrile (9: 1) Flow rate: 0.25 ml / min Column temperature: 10 ° C. Detector: Ultraviolet spectrophotometer (trade name: Shodex UV-41; Showa Denko KK) Detection wavelength: 254 nm Further, it is clear that the adsorption carrier for chromatography obtained by the present invention has a higher chiral discrimination ability than the adsorption carrier for chromatography obtained by the conventional method.

[0031]

As is apparent from the application examples, the adsorption carrier for chromatography of the present invention is obtained by graft-polymerizing the vinyl group of a cyclodextrin having a vinyl group or its derivative on a water-insoluble support. It has a higher asymmetric discrimination ability than a conventional adsorption carrier for chromatography which is obtained by reacting CD with a water-insoluble support having an epoxy group. Therefore, the adsorption carrier for chromatography according to the present invention has the possibility of separating various optical isomers, and is a powerful means for measuring the application to a wide range of industrial separation and purification equipment in addition to simple analysis and preparative means. It is clear that

[Brief description of drawings]

FIG. 1 is a chromatogram using the carrier of Example 1.

FIG. 2 is a chromatogram using a carrier of a comparative example.

Claims (2)

[Claims] 1. An adsorption carrier for chromatography, which comprises reacting a water-insoluble support with the vinyl group of a cyclodextrin having a vinyl group or a derivative thereof. 2. A method for producing an adsorption carrier for chromatography, which comprises graft-polymerizing cyclodextrin or a derivative thereof having a vinyl group on a water-insoluble support.