JPH03261729A - Separating material for optical isomer - Google Patents

Abstract

PURPOSE:To obtain the subject separating material used for liquid chromatography suitable for separation of optical isomers and capable of usage under a high flow rate by granulating cellulose or a cellulose derivative into a crosslinked spherical particle having a specified particle size. CONSTITUTION:The subject separating material having 1-1000mum (preferably 3-500mum) particle size is obtained, e.g. by dissolving cellulose in a cuprammonium solution, cadoxen, thiocyanate solution, etc., forming droplets of the resultant solution in a liquid phase or a gas phase, subsequently coagulating the droplets, regenerating cellulose for formation of spherical cellulose particles and crosslinking the resultant particles using a crosslinking agent. The obtained separating material is recommendably used by packing the above-mentioned separating material in a column for liquid chromatography and carrying out operations according to the conventional liquid chromatography method. The above-mentioned separating material can be produced readily and economically and has a high mechanical strength.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a separating agent for liquid chromatography suitable for separating optical isomers.

[Prior art] Although optical isomers are chemically the same compounds, they have different effects on living organisms, so it is difficult to separate and obtain optically pure substances in fields such as medicine, agricultural chemicals, and biochemistry. This has become an important issue. For example, in pharmaceuticals and agricultural chemicals, the stereochemistry of compounds not only has a great effect on their efficacy, but also on absorption and
It also plays an extremely important role in terms of metabolism, side effects, etc.

There is a strong demand for drugs that have only specific pharmacological effects without side effects, and it is expected that the need to develop drugs with specific three-dimensional structures will increase in the future. Stereoselective synthetic methods are being investigated to selectively produce compounds with specific steric structures, but they are only applicable in a limited range industrially, and separation of mixtures is essential. It is.

The preferential crystallization method and the diastereomer method are well known as methods for separating optical isomers, but their scope of application is limited and they are inefficient when attempting to purify them to a high degree of purity. Therefore, in recent years, with the development of chromatography equipment and packing materials, separation of isomers by cyclomography has become popular. Among these, liquid chromatography is attracting attention because of its milder separation conditions and greater throughput compared to gas chromatography (7F).

Separating agents for liquid chromatography used to separate optical isomers include: (1) a low-molecular compound with asymmetric discrimination ability bonded to an appropriate support (mostly silica gel), and (2) It is known that a polymer compound having an asymmetric discrimination ability is used in combination with the polymer compound 11 or a support. Examples of ■ include those using amino acids (W,
8anti.

et al., J.' Chromatogr.
vol. 20: L 377 (1981)), those using crown ether (M, Sugiura 嘗e
tale J-Chromatogr-+Mao1.4
05, 145 (1987)). An example of ■ is one using a polysaccharide derivative (K.

Htada, et ale J-Am-Chem-S
oc, , vol.

106.5357 (1984)), those using proteins (J-Hermagson, J, Chromt
ogr-t vol.

269.71 (1983)), four-liquid atomography separation agents must have high mechanical strength, porosity, uniform distribution of active sites on particles, and chemical stability. Requirements such as: In order to meet these requirements, conventionally, it has been widely practiced to bind or support substances having separation ability on porous silica gel particles, preferably casings, or spherical particles. However, in this case, the silica gel particles themselves are expensive, require bonding or supporting operations, and as a result, the separating agent becomes very expensive, and silica gel is weak against alkalis and its usage conditions are limited. There were drawbacks such as.

In order to improve these drawbacks, we focused on the fact that cellulose and certain cellulose derivatives, which are examples of macromolecular compounds produced by living organisms, have the ability to identify three-dimensional structures.
Examples of its use in separating optical isomers by liquid chromatography have been reported. Examples of using cellulose itself as a total separation agent include J-Chromatogr,
, vol, 387.

562 (1987) and J-High Re5olu
t.

Chromatogr-Commun, vol.
3, 31 (1980), and as an example of using cellulose derivatives, mlcrocrystallina e, which acetylated crystalline cellulose under heterogeneous conditions,
@1luiose triacetate (hereinafter referred to as M
The method using J, Chromato
gr-vol, 405.155 (1987).

However, these methods produce fine amorphous particles! However, it is difficult to use it at high flow rates as a separation agent used in column packing.

For this reason, a method is known in which cellulose derivatives such as MCTk are supported on silica gel (J.

Liq-Chromatogr-, vol, 9, 3
13 (1986)), this method requires a carrying operation as described above and is expensive.

[Problem to be solved by the invention]

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

[Means and actions for solving section Mt]

The present inventors have researched methods for separating optical isomers using cellulose and cellulose derivatives, and have developed the cellulose and cellulose derivatives described above that retain the three-dimensional structure discrimination ability of cellulose and are made into spherical particles without using a carrier. The inventors have discovered that the present invention can be achieved by eliminating the disadvantages of the prior art, and can be used as a suitable optical isomer separating agent for liquid chromatography. That is, the present invention
The main component is an optical isomer separating agent for liquid chromatography made of spherical particles of cellulose or cellulose derivatives. With such a configuration, the separation agent of the present invention is packed into a full liquid chromatography column, a mixture of optical isomers is added thereto, and the adsorbed substance is eluted with an appropriate eluent. Separation of optical isomers can be performed.

The separating agent of the present invention can be produced by any known method, but for example, (a) it can be dissolved in a cellulose gold steel ammonia solution, cadoxene, thiocyanate solution, etc., and in the resulting solution gold liquid or in the gas phase. A method of solidifying and regenerating the droplets after forming them into droplets (l#Kai No. 55-44312), (b) suspending a solution of cellulose organic acid ester t-W in a solvent in an aqueous dispersion medium, and then adding an organic solvent to the organic solvent. and a method for saponifying and regenerating the thus obtained particles with an alkaline substance (Japanese Unexamined Patent Publication No. 56-24430), c.

2. A method of crosslinking spherical cellulose particles obtained using a crosslinking agent using a crosslinking agent (JP-A-55-129156); 2. A method of introducing a substituent into the cellulose particles obtained in the above-mentioned a. to c. (Journal of the Chemical Society of Japan, vol. 1981,
(p. 1980) and Ho.

A method for partially saponifying cellulose organic acid ester spherical particles obtained by Jingjikou can be shown.

Among these, the crosslinking separation agent typically obtained by C.
The particle size of the separation agent of the present invention is particularly preferable because the mechanical strength is further improved.
0 pm is suitable. To use it, for example, a commonly used liquid chromatography column may be filled with the separating agent of the present invention, and the following operation may be performed according to the method of in-shell liquid chromatography. The operation may include, for example,
a. Wash and equilibrate the gel by flowing an eluent with an apparent volume of 2 to 10 times the gel volume through the column (prior to this).

If necessary, washing with an acid or alkaline solution, an organic solvent, etc. can be performed), b. Injecting a solution of the desired optical isomer mixture (hereinafter referred to as sample solution),
This can be done by flowing the C0 eluent, monitoring the elution form E of each component with a detector connected to the outlet of the force 2, and dividing the eluate into appropriate 7 fractions to obtain the eluate.

Among these, the eluate contains the target substance and the following solvents: water or a buffer solution with a salt 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 among them. From the solution containing the target substance obtained in this way, it is absolutely necessary! Concentrate, desalt, and
Optical isomers with high optical purity can be easily obtained by performing operations such as drying.

[Effects of the Invention] The separating agent of the present invention is made by forming cellulose or a cellulose derivative, which has the ability to separate optical isomers and has high mechanical strength, into spherical particles without using a carrier.
The method for producing it is easy and economical, and when liquid chromatography is applied by packing this product into a column, effects such as separation of optical isomers at a high flow rate can be obtained.

Hereinafter, embodiments of the present invention will be explained in more detail.

Example 1 Cellulose triacetate (degree of acetylation 61%) 320 f was mixed with 4000 m7 of methylene chloride. The resulting solution was added to a 4% gelatin aqueous solution 70G0IdKfi4. Then, methylene chloride was distilled off at 35° C. while stirring to obtain spherical particles of cellulose triacetate. After washing this product with water, it was saponified in an OH-ethanol aqueous solution to obtain spherical cellulose particle tubes.

Example 2 50f of dried spherical cellulose particles obtained in Example 1
, surfactant (15F, LiGloy 2350s7?) [Pour into a reaction vessel equipped with a stirrer and stir and disperse.

Next, 57 ft of a 20% by weight aqueous sodium hydroxide solution was gradually added to this dispersion, and after stirring at 30 to 35°C for 3 hours, 16 ft of monochrome acetic acid was added and reacted at 70°C for 5 hours. Cooling at 1, neutralization with acetic acid and filtration were performed. The obtained filtrate was dispersed in hot water, decantation was performed to remove silibroy, and the mixture was further washed with water to obtain carboxymethylated cellulose particles.

Carboxymethylated cellulose particles 501 thus obtained, surfactants o, ip and ligroin 400
g/? Place in a container with a stirrer and stir to disperse. Next, to this dispersion, 150 % of aqueous sodium water solution was added.
Add sodium chloride to the solution tube in a saturated case and stir at room temperature for 2 hours, then add shrimp chlorohydrin 1
01f was added thereto, the temperature was raised to 50°C, and the reaction was allowed to proceed for 2 hours. After that, the mixture was sequentially cooled to room temperature, neutralized with acetic acid, and filtered. After dispersing in hot water, siligroin was removed by decantation, and then further washed with water to obtain crosslinked particles of carboxymethylated cellulose particles. The particles were perfectly spherical, and the ion exchange capacity was 1.1 meq based on dry particles.

Example 3 Cellulose particles 50F of Example 1, surfactant 5ff, Hegetan 101! Stir and disperse. Add 20x to this dispersion.
After adding 2.3 kg of % sodium hydroxide aqueous solution and stirring at room temperature for 2 hours, shrimp chlorohydrin 500
f was added and reacted at 45°C for 8 hours, followed by cooling to room temperature 1, neutralization with acetic acid, and filtration. After dispersing the filtrate in hot water, hepta:/f was removed by decantation, and then sequentially washed with methanol and water to obtain crosslinked cellulose particles.

Example 4 Cellulose powder (Whatman IICF-1 type) 50f'i, 40% by weight calcium thiocyanate and 2
The dispersion was stirred and dispersed in an aqueous solution containing 01% calcium chloride at 80°F, and then the dispersion was maintained at 110°C under reduced pressure to dissolve the cellulose powder contained therein. The solution thus obtained was added dropwise to 110"C of dichlorobenzene (containing 2% nonionic surfactant), stirred and dispersed, and then the resulting dispersion was added to a large amount of O-cooled methanol under stirring. did.

After the product was completely filtered, it was washed with methanol and then water to obtain spherical cellulose particles. This cellulose particle tube was subjected to the same treatment as in Example 3 to obtain crosslinked cellulose particles. Profit &. 50f of this crosslinked cellulose, 0.8f of surfactant and 0.8f't heptane 7001# of sodium boronohydride were dispersed therein under stirring, and 430ft of a 7% by weight aqueous sodium hydroxide solution was added thereto at 35°C. Stir for 1 hour, then add 70ft of diethylaminoethyl chloride hydrochloride for 5 hours.
The reaction was allowed to proceed overnight at 0° C., followed by cooling in a room temperature cabinet, neutralization with acetic acid, and filtration. The obtained filtrate was dispersed in warm water, and then heptane was removed by decantation, and then further washed with water to obtain a crosslinked product of diethylaminoethylated cellulose particles.

The crosslinked particles thus obtained were perfectly spherical, and the ion exchange capacity was 0.8 m@q per dry particle 1f.Cellulose and cellulose derivatives obtained in Examples 1 to 4 Each spherical particle tube is subjected to wet classification.
The particles thus obtained were packed into a column with an inner diameter of 15 mg and a length of 150 Qsar by a slurry method. Subsequently, under the same column, the optical isomer mixture of the cobal)ID complex represented by the following formula (I) was separated by liquid chromatography using 0.3M saline as an eluent.

Even when misaligned particles were used, the peaks were divided into two, confirming that the optical isomers were separated into two components. Furthermore, for each of the two peaks in each example, the three central fractions were mixed and the optical rotation was measured, and the results shown in Table 1 were obtained. It is understood that this corresponds to

Table 1 Note that in the above separation process, the eluate Fi1 from the column
The sample was divided into 0- fractions, and the ultraviolet llI light intensity of each fraction was measured. It is clear from the results shown in FIG. Avicel TG, a crystalline cellulose material that is said to be possible.
101 (manufactured by Asahi Kasei Kogyo ■) or MCT' obtained by heterogeneously acetylating it in benzene. The flow of liquid is almost good,
It was virtually impossible to treat.

Comparative Example 2 A liquid chromatography method was carried out in the same manner as in the application example, except that SP-Sephadex (manufactured by Pharmacia), a packing material for liquid chromatography based on the polysaccharide dextran, was used instead of the classified particles described in the application example. An attempt was made to separate the optical isomers, but only one peak was observed and the optical isomers could not be separated.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between fraction and ultraviolet absorbance for explaining the effects of the embodiment of the present invention. l...Example 1.2...Example 2.3...Example 3.4...Example 4° Fraction No.

Claims (3)

[Claims] (1) An optical isomer separating agent for liquid chromatography made of spherical particles of cellulose or a cellulose derivative. (2) The separating agent according to claim (1), wherein the spherical particles are crosslinked. (3) The separating agent according to claim (1) or claim (2), wherein the spherical particles have a particle size of 1 to 1,000 μm.