JP2002323483A - Novel column for separating optical isomers, method for producing the same, and method for separation using the same - Google Patents

Abstract

(57) [Problem] To provide an optical isomer separation column capable of separating an extremely small amount of sample, reducing the amount of solvent, and having a long column life. SOLUTION: A capillary column for optical isomer separation in which a copolymer of a polysaccharide derivative having a polymerizable functional group and a monomer copolymerizable therewith is chemically bonded to a capillary, a method for producing the same, and an optical method using the same Method for separating isomers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel capillary column for separating optical isomers, a method for producing the same, and a method for separating optical isomers using the same. The present invention relates to an excellent capillary column for separation of optical isomers, which enables rapid and minute analysis.

[0002]

2. Description of the Related Art Many organic compounds have optical isomers, but the physical and chemical properties of these optical isomers, such as the boiling point, melting point, and solubility, are quite low. Are identical. However, it is widely known that optical isomers often differ in terms of physiological activity. For this reason, in the field of pharmaceuticals, remarkable differences are often observed in terms of the efficacy and toxicity between optical isomers, and in the case of fragrances and food additives, there are differences in terms of smell and taste. There is.

[0003] For this reason, the Ministry of Health and Welfare states in its drug production guidelines that "if the drug is racemic, it is desirable to study the absorption, distribution, metabolism and excretion kinetics of each isomer." In the pharmaceutical field, it has stated that optical isomers should be strictly distinguished.

[0004] Under such social demands, means for producing only one of the optical isomers has been widely studied.
At the same time, techniques for separating and analyzing optical isomers have been studied and advanced. As mentioned above, the physical and chemical properties of the optical isomers are exactly the same, so the usual boiling point and solubility,
Analysis cannot be performed by means of separation due to differences in physical properties such as the distribution coefficient and the magnitude of charge.

[0005] Therefore, as a means for separating and analyzing optical isomers capable of discriminating the stereoscopic configuration between optical isomers under conditions close to room temperature (asymmetry identification), optical isomers by high performance liquid chromatography (HPLC) are used. Body separation methods have evolved. As the HPLC column used here, an asymmetric discriminating agent itself or a chiral stationary phase in which the asymmetric discriminating agent is supported on a suitable carrier is used. For example, optically active poly (triphenylmethyl methacrylate) (JP-A-57-150432)
No.), cellulose, amylose derivatives (Y. Okamoto, M. Ka
washima and K. Hatada, J. Am. Chem. Soc., 106, 5337, 198
4), ovomucoid (JP-A-63-307829) and the like have been developed. Among these chiral stationary phases for HPLC, an optical resolution column in which a cellulose derivative or an amylose derivative is supported on silica gel is known to have high asymmetric discrimination ability for a very wide range of compounds.

However, in these HPLC methods, a certain amount (about 0.1 μg or more) of a sample is required for separation, but the absolute amount of the sample is required for use in the above-mentioned fields of biological components and metabolism. Because it is extremely small, for example, 10-
There is a need for a method that can analyze 0.1 pg samples with high accuracy. In addition, since a large amount of mobile phase solvent is discharged in the HPLC method, it has been pointed out that the reduction of the amount of these solvents is an environmental problem.

Therefore, Chromatographia Vol. 42, No. 9/1
In 0,521,1996, cellulose derivatives are used in columns for Open-Tubular liquid chromatography. Among them, it is stated that the problem is that the column life is short and the reproductivity is poor. One of the causes is a step of filling the column. Furthermore, it has been suggested that this problem is resolved by immobilization of the stationary phase.

An object of the present invention is to provide a column for separating optical isomers which can separate even a very small amount of sample, can reduce the amount of solvent, and has a long column life.

[0009]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have completed the present invention.

That is, the present invention is characterized in that a copolymer of a polysaccharide derivative having a polymerizable functional group and a monomer copolymerizable therewith (hereinafter referred to as a polymerizable monomer) is chemically bonded to the capillary. A capillary column for separating optical isomers, a method for producing the same, and a method for separating optical isomers using the same.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The capillary column of the present invention
A polymerization reaction between a polysaccharide derivative having a polymerizable functional group and a polymerizable monomer, which is obtained by polymerizing a polysaccharide derivative having a polymerizable functional group and a polymerizable monomer in a capillary having a polymerizable functional group introduced on the inner surface thereof, And chemical bonding with the capillary are simultaneously performed in the capillary.

That is, a polysaccharide derivative having a polymerizable functional group,
By performing a polymerization reaction of the polymerizable monomer and the polymerizable functional group introduced into the inner surface of the capillary, a chemical bond is formed between the polysaccharide derivative, the polymer composed of the polymerizable monomer, and the capillary.

The polysaccharide constituting the polysaccharide derivative used in the present invention may be any of optically active substances, regardless of any of synthetic polysaccharide, natural polysaccharide and modified natural polysaccharide. Those with high regularity are desirable. For example, β-1,4-glucan (cellulose),
α-1,4-glucan (amylose, amylopectin), α-1,6-glucan (dextran), β-
1,6-glucan (Bustulan), β-1,3-glucan (eg curdlan, schizophyllan, etc.), α-1,3
-Glucan, β-1,2-glucan (Crown Gall polysaccharide), β-1,4-galactan, β-1,4-mannan, α-1,6-mannan, β-1,2-fructan (inulin) , Β-2,6-fructan (levan), β
-1,4-xylan, β-1,3-xylan, β-1,
4-chitosan, α-1,4-N-acetylchitosan (chitin), pullulan, agarose, alginic acid and the like;
Also included are starches containing amylose. Among these, cellulose, amylose, β-1,4-xylan, β-1,4-chitosan, chitin, β-1,4-mannan, inulin, curdlan, etc., from which high-purity polysaccharides can be easily obtained. Cellulose and amylose are particularly preferable.

The number average polymerization degree of these polysaccharides (average number of pyranose or furanose rings contained in one molecule) is preferably 5 or more, more preferably 10 or more, and there is no particular upper limit, but it is 500 or less. Is desirable in terms of ease of handling.

The polysaccharide derivatives used in the present invention include:
Examples of the compound obtained by derivatizing a compound having a functional group capable of reacting with the hydroxyl group in a part of the hydroxyl group of the polysaccharide as described above by forming an ester bond, a urethane bond, or an ether bond by a conventionally known method. Can be Here, the compound having a functional group capable of reacting with a hydroxyl group may be an isocyanic acid derivative, carboxylic acid, ester, acid halide, acid amide, halide, epoxy compound, aldehyde, alcohol, or other compound having a leaving group. Any of these may be used, and aliphatic, alicyclic, aromatic and heteroaromatic compounds, and inorganic acids such as nitric acid can be used. Particularly, an ester derivative or a carbamate derivative is preferable.

The polymerizable functional group to be introduced into the polysaccharide derivative may be any functional group capable of participating in a polymerization reaction such as radical polymerization, anionic polymerization, cationic polymerization, or polycondensation. More desirable. As a method for introducing a polymerizable functional group into the polysaccharide derivative, for example, a compound having a functional group capable of reacting with the hydroxyl group of the polysaccharide and a polymerizable functional group is reacted with the polysaccharide derivative, and an ester bond, a urethane bond, or an ether bond is formed. A method of forming a bond or the like can be given. Examples of the compound having a functional group capable of reacting with a hydroxyl group of the polysaccharide and a polymerizable functional group include a compound having a functional group capable of reacting with the above-mentioned hydroxyl group, and a compound further having a polymerizable functional group. And phenyl isocyanate.
The ratio of the polymerizable functional group introduced into the polysaccharide derivative is preferably 0.01 to 2.9 as a degree of substitution of the three hydroxyl groups of the pyranose or furanose ring, and is preferably 0.1 to 1.5.
Is more preferred.

The capillary column is not particularly limited as long as it is used for ordinary liquid chromatography. The inner diameter of the capillary is preferably 1 to 1000 μm,
~ 200 µm is more preferred. The capillary may have any length, but preferably has a length of 20 to 100 cm.

The material of the capillary is not particularly limited, but is preferably a material containing glass or silica.
Most preferred are fused silica capillaries. The polymerizable functional group introduced into the inner surface of the capillary may be any functional group that can participate in a polymerization reaction such as radical polymerization, anionic polymerization, cationic polymerization, or polycondensation, but a vinyl group is more preferable. Examples of a method for introducing a polymerizable functional group into the inner surface of the capillary include a method in which a compound having a polymerizable functional group typified by an acrylic acid derivative, a methacrylic acid derivative, styrene, or the like is placed in the capillary and heated.

As the polymerizable monomer, any monomer can be used as long as it can participate in a polymerization reaction such as radical polymerization, anionic polymerization, cationic polymerization, polycondensation, etc., and more preferably an acrylic acid derivative, a methacrylic acid derivative, It is a vinyl monomer represented by styrene and the like.

In the capillary column of the present invention, a polysaccharide derivative having a polymerizable functional group, a polymerizable monomer and a polymerization initiator are dissolved in a solvent and added to a capillary in which a polymerizable functional group has been introduced into the inner wall in advance, and a polymerization reaction is carried out. It can be obtained by: The solvent used here is not particularly limited as long as it can dissolve the polysaccharide derivative having a polymerizable functional group and the polymerizable monomer. Further, as the polymerization initiator, those used in a conventionally known method can be used.

The capillary column of the present invention is useful as a column for liquid chromatography, in particular, a column for high performance liquid chromatography. The mobile phase used in this liquid chromatography method may be any of an aqueous type and an organic solvent type. .

The capillary column of the present invention can be used not only for liquid chromatography, but also for gas chromatography, electrophoresis, particularly for capillary electrochromatography (for CEC), CZE (capillary zone electrophoresis), MEKC (micellar dynamics). It can also be used as a capillary column in the (electrochromatography) method.

[0023]

EXAMPLES The present invention will be described in more detail below, but the present invention is not limited to these examples.

Example 1 Introduction of Polymerizable Functional Group into Inner Surface of Capillary Alkaline pretreatment of a fused silica capillary having an inner diameter of 75 μm, dilute hydrochloric acid and ion-exchanged water were passed, washed well, acetone was passed, and dried. Dry with nitrogen bubbling.

The fused silica capillary was added in an amount of 10% by weight.
A solution of 0.37 mg of 1,1'-diphenyl-2-picryl hydroxide containing benzene, 0.9 ml of 3-trimethoxysilylpropyl methacrylate and 2.1 ml of dry N, N-dimethylformamide (DMF) Fill, 120 ℃
For 6 hours. Thereafter, the well was washed well with DMF, further substituted with methanol, and then dried with nitrogen to produce a fused silica capillary into which the desired polymerizable functional group was introduced.

Synthesis of Cellulose Derivative Having Polymerizable Functional Group at Position 6 Under nitrogen atmosphere, 200 ml of dry pyridine was added to 10 g of cellulose, and triphenylmethyl chloride (trityl chloride) 2 was added thereto.
After adding 0.6 g and heating and stirring at 115 ° C. for 24 hours,
The mixture was poured into 3 L of methanol to obtain a cellulose derivative having a trityl group at position 6.

After dissolving 10 g of 6-tritylcellulose in 150 ml of dry pyridine, adding 18.6 g of 3,5-dichlorophenylisocyanate, heating and stirring at 115 ° C. for 48 hours, poured into 3 L of methanol, and Tritylized
A cellulose derivative having 2,3-position 3,5-dichlorophenylcarbamate was obtained. The obtained 6-trityl-2,3-
Bis (3,5-dichlorophenylcarbamate) cellulose, 10 g, was added to a mixed solution of 1 L of methanol and 0.25 g of concentrated hydrochloric acid, stirred for 12 hours at room temperature, and then filtered to remove 6-position trityl group. -2,3-Bis (3,5-dichlorophenylcarbamate) cellulose was obtained.

5 g of this was dissolved in 70 ml of dry pyridine, and 405 mg of 4-vinylphenylisocyanate was added to add 1 g.
After heating and stirring at 15 ° C for 24 hours, 1.05 g of 3,5-dichlorophenylisocyanate was added, and further at 115 ° C.
Heating and stirring were performed for 24 hours. The reaction mixture was poured into 2 L of methanol to obtain a cellulose 3,5-dichlorophenylcarbamate derivative in which a polymerizable vinylphenylcarbamate was introduced at a part of the desired 6-position. The degree of substitution of the polymerizable vinyl phenyl carbamate is 0.25.

[0029] 64 mg of a cellulose derivative having a polymerizable functional group and 6.4 mg of styrene obtained by a polymerization reaction in a capillary are dissolved in 0.9 ml of tetrahydrofuran (THF), and 2.78 mg of 2,2'-azobisisobutyi is added thereto. A solution of lonitrile (AIBN) / THF (1.0 ml) was added. This mixed solution is
The polymer was filled with a fused silica capillary having a polymerizable functional group introduced into the inner surface prepared in the above, and both ends were sealed to carry out a polymerization reaction at 60 ° C. for 20 hours. After the polymerization reaction, the THF is thoroughly passed through, the unreacted substances are removed, and dry nitrogen is passed through, and the copolymer of the cellulose derivative having a polymerizable functional group and styrene is chemically bonded to the capillary. A capillary column for isomer separation was obtained. The capillary column for optical isomer separation is presumed to have a structure represented by the following formula.

[0030]

Embedded image

Example 2 Introduction of Polymerizable Functional Group into Inner Surface of Capillary In the same manner as in Example 1, a fused silica capillary having a polymerizable functional group introduced was prepared.

Synthesis of Cellulose Derivative Having Polymerizable Functional Group at Position 6 Under a nitrogen atmosphere, 200 ml of dry pyridine was added to 10 g of cellulose, and triphenylmethyl chloride (trityl chloride) 2 was added thereto.
After adding 0.6 g and heating and stirring at 115 ° C. for 24 hours,
The mixture was poured into 3 L of methanol to obtain a cellulose derivative having a trityl group at position 6.

After dissolving 10 g of 6-tritylcellulose in 150 ml of dry pyridine, 18.6 g of 3,5-dimethylphenylisocyanate was added, and the mixture was heated and stirred at 115 ° C. for 48 hours. Is tritylated
A cellulose derivative in which 2,3-position was 3,5-dimethylphenylcarbamate was obtained. The obtained 6-trityl-2,3-
Bis (3,5-dimethylphenylcarbamate) cellulose, 10 g, was added to a mixed solution of 1 L of methanol and 0.25 g of concentrated hydrochloric acid, stirred for 12 hours at room temperature, and filtered to remove the 6-position trityl group. Hydroxy-2,3-bis (3,5-dimethylphenylcarbamate) cellulose was obtained.

5 g of this was dissolved in 70 ml of dry pyridine, and 405 mg of 4-vinylphenyl isocyanate was added to add 1 g.
After heating and stirring at 15 ° C for 24 hours, 1.05 g of 3,5-dimethylphenylisocyanate was added, and further at 115 ° C.
Heating and stirring were performed for 24 hours. The reaction mixture was poured into 2 L of methanol to obtain a cellulose 3,5-dimethylphenylcarbamate derivative in which a polymerizable vinylphenylcarbamate was introduced at a part of the desired 6-position. The degree of substitution of the polymerizable vinyl phenyl carbamate is 0.25.

Using 64 mg of the cellulose derivative having a polymerizable functional group obtained by the polymerization reaction in the capillary, a copolymer of the cellulose derivative having a polymerizable functional group and styrene was obtained in the same manner as in Example 1. A capillary column for optical isomer separation chemically bonded to the capillary was obtained. The capillary column for optical isomer separation is presumed to have a structure represented by the following formula.

[0036]

Embedded image

Example 3 Using the capillary column obtained in Example 1, Prince
capillary electrophoresis instrument (Lauerlabs,
Emmen, The Metherlands) system, Jasco as detector
Using CE 917 UV Intelligent (manufactured by JASCO Corporation), the following four racemates (a) (trans stilbene oxide), (b) (laudanosine), (c) (etzoline), (d)
(Piprozoline) was subjected to optical resolution under the following conditions. The result is shown in FIG.

[0038]

Embedded image

Mobile phase: methanol Detection: UV 214 nm Effective capillary length: 50 cm Pumping solution (constant pressure method): racemic (a) 8 mbar, racemic (b) 100 mbar, racemic (c) and (d) 50 mbar Using the capillary column obtained in Example 2, Prince
capillary electrophoresis instrument (Lauerlabs,
Emmen, The Metherlands) system, Jasco as detector
Using CE 917 UV Intelligent (manufactured by JASCO Corporation), the following six racemates (e) to (j) were subjected to optical resolution under the following conditions. Table 1 shows the results. In Table 1, the separation coefficient (α) is defined by the following equation.

Α = k ₂ ′ / k ₁ ′ Here, k ₁ ′ represents the retention coefficient of the optical isomer that is retained more weakly, and k ₂ ′ represents the retention coefficient of the optical isomer that is retained more strongly.

[0041]

Embedded image

Mobile phase: hexane / isopropanol = 9
/ 1 (v / v) Detection: UV 214 nm Effective capillary length: 63.5 cm Pumping solution (constant pressure method): 80 mbar

[0043]

[Table 1]

Example 5 Using the capillary column obtained in Example 1, Prince
capillary electrophoresis instrument (Lauerlabs,
Emmen, The Metherlands) system, Jasco as detector
Using CE 917 UV Intelligent (manufactured by JASCO Corporation), the following three racemates (k) to (m) were subjected to optical resolution under the following conditions. Table 2 shows the results.

[0045]

Embedded image

Mobile phase: hexane / isopropanol = 9
5/5 (v / v) Detection: UV 214 nm Effective capillary length: 50 cm Pumping solution (constant pressure method): 200 mbar

[0047]

[Table 2]

[0048]

According to the present invention, a very small amount of sample can be efficiently separated, and the amount of solvent can be significantly reduced as compared with the conventional HPLC method. Can be provided.

[Brief description of the drawings]

FIG. 1 shows the results of optical resolution performed in Example 3, where (a) is a racemic form (a), (b) is a racemic form (b), and (c)
Is the result of racemic (c) and (d) is the result of racemic (d).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/48 G01N 30/48 W (72) Inventor Tomoyo Yamamoto 654-265 Kamiyacho, Kasugai-shi, Aichi Prefecture

Claims (9)

[Claims] An optical isomer characterized in that a copolymer of a polysaccharide derivative having a polymerizable functional group and a monomer copolymerizable therewith (hereinafter referred to as a polymerizable monomer) is chemically bonded to a capillary. Capillary column for separation. 2. The optical isomer according to claim 1, wherein the polymerization reaction between the polysaccharide derivative having a polymerizable functional group and the polymerizable monomer and the chemical bond with the capillary are simultaneously performed in the capillary. Capillary column for separation. 3. A polymerization reaction between a polysaccharide derivative having a polymerizable functional group, a polymerizable monomer, and a polymerizable functional group introduced into the inner surface of the capillary, whereby a polymer comprising the polysaccharide derivative, the polymerizable monomer, and the capillary are formed. The capillary column for separating optical isomers according to claim 1 or 2, wherein a chemical bond is formed in the capillary column. 4. The capillary column for separating optical isomers according to claim 1, wherein the polysaccharide derivative having a polymerizable functional group is an ester derivative or a carbamate derivative of cellulose or amylose. 5. The polymerizable functional group of the polysaccharide derivative is a vinyl group, the polymerizable monomer is a monomer having a vinyl group, and the polymerizable functional group introduced into the inner surface of the capillary is a vinyl group. The capillary column for separating optical isomers according to claim 3. 6. The capillary column for separating optical isomers according to claim 1, wherein the capillary is a capillary made of fused silica. 7. The capillary column for separating optical isomers according to claim 1, wherein the column is a column for high performance liquid chromatography. 8. The polysaccharide derivative having a polymerizable functional group and a polymerizable monomer are polymerized in a capillary having a polymerizable functional group introduced into an inner surface thereof. Method for producing a capillary column for optical isomer separation. 9. A method for separating optical isomers, comprising using the capillary column according to any one of claims 1 to 7 to separate optical isomers.