JP2896589B2 - Optical isomer separating agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a separating agent for liquid chromatography suitable for separating optical isomers.

[Conventional technology]

Optical isomers are the same compounds chemically, but have different effects on living organisms, so it is an important issue to obtain optically pure products in the fields of medicine, pesticides, biochemistry, etc. . For example, in pharmaceuticals and agricultural chemicals, the stereochemistry of a compound not only has a great effect on the medicinal effect, but also plays an extremely important role in terms of absorption, metabolism, side effects, and the like. There is a strong demand for a drug having only a specific pharmacological action without side effects or the like, and it is expected that the development of a drug having a specific tertiary structure will become more and more important in the future. In order to selectively produce a compound having a specific steric structure, stereoselective synthesis methods have been studied, but this method is industrially applicable only in a limited range, and separation of a mixture is indispensable. It is. As a method for separating optical isomers, a preferred crystallization method and a diastereomer method are well known,
The range of application was limited, and it was inefficient to purify to high purity. For this reason, in recent years, the development of a chromatographic apparatus and the development of a filler have led to active separation of isomers by the chromatographic method. Above all, liquid chromatography has attracted attention in terms of milder separation conditions and higher processing capacity than gas chromatography.

Liquid chromatographic separating agents used for the separation of optical isomers include those in which a low-molecular compound having asymmetric discrimination ability is bound to a suitable support (mostly silica gel), and those that have an asymmetric discrimination ability. Are used as such or in combination with a support. Examples of the use of amino acids (W.
Santi, et al, J. Chromatogr., Vol. 203, 377 (1981)),
Those using crown ethers (M. Sugiura, et al, J.
Chromatogr., Vol. 405, 145 (1987)), for example, those using derivatives of polysaccharides (K. Htada, et a.
l, J. Am. Chem. Soc., vol. 106, 5357 (1984)), those using proteins (J. Hermasson, J. Chromtogr., vol. 269, 71).
(1983)). In order to be used as a separating agent for liquid chromatography, requirements such as high mechanical strength, porosity, uniform distribution of the active site in the particles, and chemical stability are required. In order to satisfy these requirements, it has been widely practiced to bond or carry a substance having a separating ability to porous silica gel particles, preferably spherical particles. However, in this case, the silica gel particles themselves are expensive and require an operation of binding or supporting, and as a result, the separating agent becomes very expensive, and the silica gel is vulnerable to alkali and the use conditions are limited. There are drawbacks such as.

In order to remedy these drawbacks, attention was paid to the fact that cellulose, which is an example of a macromolecular compound produced by living organisms, and certain cellulose derivatives have a function of discriminating the three-dimensional structure. Examples of use for body separation have been reported. J. Chromatogr., Vol. 387, 56
2 (1987) and J. High Resolut. Chromatogr. Commun., Vo
l.3, 31 (1980), as an example of using a cellulose derivative, microcrystalline cellulose triacetate (hereinafter referred to as MCT) obtained by acetylating crystalline cellulose under heterogeneous conditions.
Abbreviation) is used in J. Chromatogr., Vol. 405, 155
(1987). However, these methods use fine amorphous particles as they are, and it is difficult to use them at a high flow rate as a separating agent used under column packing. For this reason, a method of supporting a cellulose derivative, for example, MCT on silica gel is known (J. Liq. Ch.
romatogr., vol. 9, 313 (1986)). However, this method involves problems such as the need for carrying operation as described above and the high cost.

[Problems to be solved by the invention]

An object of the present invention is to overcome the disadvantages of the prior art described above,
An object of the present invention is to provide an optical isomer separating agent for liquid chromatography which can be prepared without requiring a supporting operation and can be used at a high flow rate.

[Means and actions for solving the problem]

The present inventors have studied a method for separating optical isomers using cellulose and a cellulose derivative, and have maintained the three-dimensional structure discriminating ability of cellulose and made only itself into spherical particles without using a carrier and crosslinked cellulose and cellulose derivatives. However, the present inventors have found that the above-mentioned disadvantages of the prior art can be eliminated, and that the agent can be used as a suitable optical isomer separating agent for liquid chromatography. That is, the present invention mainly has an optical isomer separating agent for liquid chromatography obtained by forming cellulose or a cellulose derivative into spherical particles and cross-linking them. With such a configuration, the separating agent of the present invention is packed in a column for liquid chromatography, a mixture of optical isomers is added thereto, and the adsorbate is eluted with a suitable eluent to elute the adsorbate. Isomers can be separated under high purity.

The present invention and the separating agent related thereto can be produced by any known method. A method in which cellulose is dissolved in a copper ammonia solution, cadoxene, thiocyanate solution, or the like, and the resulting solution is formed into droplets in a liquid or gaseous phase, followed by coagulation and regeneration (JP-A-55-44312)
No.), b. A method in which a solution in which a cellulose organic acid ester is dissolved in an organic solvent is suspended in an aqueous dispersion medium, and then the organic solvent is removed, and the thus obtained particles are saponified and regenerated with an alkaline substance (Japanese Patent Application Laid-Open No. 56-24430). , C. The a. And b. A method of crosslinking the spherical cellulose particles obtained by the above method with a crosslinking agent (JP-A-55-129156).
The a. ~ C. A method of introducing a substituent into cellulose particles obtained by the method described in (Chemical Society of Japan, vol. 1981, p. 1989); B. And a method of partially saponifying the cellulose organic acid ester spherical particles obtained by the above method.

Among them, c. And in connection with this. The cross-linking and separating agents typically obtained in the above are included in the present invention, but these are particularly preferable because the mechanical strength is further improved and the optical isomer can be separated under high purity. The particle size of the separating agent of the present invention is suitably from 1 to 1000 μm, preferably from 3 to 500 μm. For example, a column for liquid chromatography, which is generally used, may be filled with the separating agent of the present invention, and the operation may be performed in accordance with a general liquid chromatography method. In the above-mentioned operation, for example, a. 2 to 10 times the apparent gel volume of the eluent is applied to the column to wash and equilibrate the gel (prior to this, if necessary, washing with an acid or alkaline solution, an organic solvent, etc.). B. Inject a solution of the desired optical isomer mixture (hereinafter referred to as a sample solution), c. Flow an eluent, and d.
This can be performed by monitoring the elution state of each component with a detector connected to the column outlet, dividing the mixture into appropriate fractions, and obtaining an eluate.

The eluate is a solvent shown below depending on the target substance, that is, water or a buffer solution, a solution in which a salt is dissolved therein, a mixed solution of any of the above solutions and a water-soluble organic solvent, and an organic solvent. You can choose from From the solution containing the target substance thus obtained, optical isomers of high optical purity can be easily obtained by performing operations such as concentration, desalting, and drying as necessary.

〔The invention's effect〕

The separating agent of the present invention is obtained by forming cellulose or a cellulose derivative having high optical isomer resolving power and high mechanical strength into spherical particles and cross-linking without using a carrier. Easy and economical,
When this is packed in a column and applied to liquid chromatography, effects such as separation of optical isomers at a high flow rate can be obtained.

 Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 (Reference Example) 320 g of cellulose triacetate (degree of oxidation 61%) was methylene chloride.
Dissolved in 4000 ml, and the resulting solution
Add dropwise to 00 ml. Then, methylene chloride was distilled off at 35 ° C. with stirring to obtain spherical particles of cellulose triacetate. This was washed with water and saponified in an aqueous solution of NaOH-ethanol to obtain spherical cellulose particles.

Example 2 Dry product 50 of the spherical cellulose particles obtained in Example 1
g, a surfactant 0.5 g, and ligroin 350 ml are placed in a reaction vessel equipped with a stirrer and dispersed by stirring. Then, 57 g of a 20% by weight aqueous sodium hydroxide solution was gradually added to the dispersion, and 30 to 3
After stirring at 5 ° C for 3 hours, 16 g of monochloroacetic acid was added, and
The reaction was carried out at a temperature of 5 ° C. for 5 hours, followed by cooling to room temperature, neutralization with acetic acid and filtration. After dispersing the obtained filtrate in warm water, ligroin was removed by decantation, and then further washed with water to obtain carboxymethylated cellulose particles. 50 g of the carboxymethylated cellulose particles thus obtained, 0.1 g of a surfactant and 400 ml of ligroin are placed in a vessel equipped with a stirrer and dispersed by stirring.
Next, a solution of sodium chloride dissolved in 150 g of a 5% by weight aqueous solution of sodium hydroxide until the dispersion was saturated was added to the dispersion, and the mixture was stirred at room temperature for 2 hours. The mixture was heated and reacted for 2 hours, and then cooled to room temperature, neutralized with acetic acid, and filtered. After the filtrate was dispersed in warm water, ligroin was removed by decantation, and then washed with water to obtain a crosslinked product of carboxymethylated cellulose particles. The thus-obtained crosslinked product particles had a true spherical shape, and the ion exchange capacity was 1.1 meq per 1 g of the dry particles.

Example 3 50 g of the cellulose particles of Example 1 and 5 g of a surfactant were dispersed in 10 l of heptane with stirring. To this dispersion was added 2.3 kg of a 20% by weight aqueous sodium hydroxide solution, and the mixture was stirred at room temperature for 2 hours. Thereafter, 500 g of epichlorohydrin was further added, and the mixture was stirred at 45 ° C. for 8 hours.
The reaction was allowed to proceed for a period of time, followed by cooling to room temperature, neutralization with acetic acid, and filtration. After the filtrate was dispersed in warm water, heptane was removed by decantation and then washed sequentially with methanol and water to obtain crosslinked cellulose particles.

Example 4 50 g of cellulose powder (CF-1 type manufactured by Whatman) was added to
The dispersion was stirred and dispersed in 800 g of an aqueous solution containing 40% by weight of calcium thiocyanate and 20% by weight of calcium chloride, and the dispersion was kept at 110 ° C. under reduced pressure to dissolve the contained cellulose powder. The solution thus obtained was added dropwise to dichlorobenzene (containing 2% nonionic surfactant) at 110 ° C., stirred and dispersed, and the resulting dispersion was added to a large amount of cold methanol with stirring. . After filtration, the product was washed with methanol and then with water to obtain spherical cellulose particles. The cellulose particles were subjected to the same treatment as in Example 3 to obtain crosslinked cellulose particles. 50 g of this crosslinked cellulose, 0.8 g of a surfactant and 0.8 g of sodium borohydride were dispersed in 700 ml of heptane with stirring, 430 g of a 7% by weight aqueous sodium hydroxide solution was added, and the mixture was stirred at 35 ° C. for 1 hour, and then diethylaminoethyl chloride was added. Add 70g of hydrochloride and add 50 ℃
At room temperature overnight, followed by cooling to room temperature, neutralization with acetic acid and filtration. The obtained filtrate was dispersed in warm water, heptane was removed by decantation, and then further washed with water to obtain a crosslinked product of diethylaminoethylated cellulose particles. The crosslinked product particles thus obtained are spherical, and the ion exchange capacity is 1 g of dry particles.
It was 0.8meq per hit.

Application Example The spherical particles of cellulose and cellulose derivatives obtained in Examples 1 to 4 were each subjected to wet classification by 40 to 100 μm.
The particles thus obtained were packed into a column having an inner diameter of 15 mm and a length of 1500 mm by a slurry method. Then, under this column, using a 0.3 M saline solution as an eluent, a separation process by a liquid chromatography method was performed on an optical isomer mixture of a cobalt (III) complex represented by the following formula (I).

In the above separation treatment, the eluate from the column was 10 ml
Of each fraction, and the UV absorbance of each fraction was measured. As is clear from the results shown in FIG. 1, when any of the particles of Examples 1 to 4 (the numbers in the figure correspond to those of the example), the particles are divided into two peaks. 2 optical isomers
It was confirmed that it was separated into components. Furthermore, for each of the two peaks in each Example, the results were as shown in Table 1 when the optical rotation was measured by mixing three fractions at the center, and the two peaks were compounds each having an optical isomer relationship with each other. It is understood that this corresponds to.

Comparative Example 1 The crystalline cellulosic Avicel TG- which is said to be capable of separating optical isomers in place of the classified particles described in the application example
An attempt was made to separate optical isomers by liquid chromatography in the same manner as in the application example except that 101 (manufactured by Asahi Kasei Kogyo Co., Ltd.) or MCT obtained by heterogeneously acetylating the same in benzene was used. There was almost no liquid flow, and the treatment was practically impossible.

Comparative Example 2 A liquid chromatography method was performed in the same manner as in the application example except that the classified particles described in the application example were replaced with a liquid chromatography filler SP-Sephadex (manufactured by Pharmacia) based on the dextran polysaccharide. An attempt was made to separate optical isomers, but only one peak was shown, and optical isomers could not be separated.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between fraction No. and ultraviolet absorbance for explaining the effect of the embodiment of the present invention. 1 ... Example 1, 2 ... Example 2, 3 ... Example 3, 4
...

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 30/48 G01N 30/48 T // C07M 7:00

Claims (2)

(57) [Claims] 1. An optical isomer separating agent for liquid chromatography, characterized in that cellulose or a cellulose derivative is formed into spherical particles, and the spherical particles thus formed are crosslinked. 2. The separating agent according to claim 1, wherein the spherical particles have a particle size of 1 to 1,000 μm.