JPH05215736A - Chromato separation method - Google Patents

Abstract

PURPOSE:To enable using a separation agent including multisugar carbonic acid ester and spread the range of separation object by using a mixture of shock absorber liquid and water soluble organic solvent with a specific ratio for a moving phase. CONSTITUTION:As for a water soluble organic solvent mingling with water, acetonitryl, methanol or ethanol are favourable. Buffer liquid, for example, acetic acid buffer liquid or phosphoric acid buffer liquid, etc., is favourable in the pH range of 2 to 8. The mixing ratio of the shock absorber and the water soluble organic solvent is in volume ratio of 99:1 to 1:99 and is favourable in the range of 90:10 to 10:90. A separation agent to be used has polysaccharide carbonic acid ester for an effective component and is favourable to be esterized by carbonic acid for over 85% of the hydrogen atoms on hydroxil group which the polysaccharide. The compound separated by this method is not limited and capable of separating steroisomer, position isomer, geometric metamer, compounds with different unsaturation and compounds belonging to homologue.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating compounds from each other by liquid chromatography, which can be used for the purpose of analysis in research / manufacturing control or purification by fractionation.

[0002]

2. Description of the Related Art Polycarboxylic acid esters of polysaccharides, or separating agents which carry these on a carrier such as silica gel, have various structures including optical isomers as stationary phases for liquid chromatography. It has been revealed that it shows excellent properties in the separation of chemically similar compounds (stereoisomers such as diastereomers, regioisomers, geometric isomers, compounds with different degrees of unsaturation, compounds belonging to homologues). (The 28th HPLC Research Symposium Abstracts, p25, 26). When using this type of stationary phase, conventionally, a substantially normal phase mobile phase (specifically,
Mixture of non-polar organic solvent such as hexane and alcohols)
I have used only. However, it is very difficult to bring a highly polar compound as an object to be separated in the normal phase mobile phase. It is an object of the present invention to broaden the range of objects to be separated so that a separating agent using a carboxylic acid ester of a polysaccharide having excellent separating performance can be somehow used in a reverse phase system.

In response to this problem, the present inventors have recently proposed that a system using a mixture of a water-soluble organic solvent and water or an aqueous solution containing various salts as a mobile phase of a reverse phase system can separate various compounds. It was found to be practically effective (Japanese Patent Application No. 3-24).
No. 5681). However, there is a problem that the pH cannot be adjusted only by adding a salt and the pH is unstable.

[0004]

[Means for Solving the Problems] The present inventors have conducted extensive studies to solve the above-mentioned problems of the conventional chromatography system, and as a result, 99% of the buffer solution and the water-soluble organic solvent were used. It has been found that when a mixed liquid of 1: 1 to 1: 99 (volume ratio) is used as a mobile phase, better separation can be given, and further there is no concern about hydrolysis of the carboxylic acid ester of the polysaccharide. It came to completion. That is, the present invention uses a separating agent containing a carboxylic acid ester of a polysaccharide as an active ingredient, and when performing liquid chromatography, 99: 1 to 1:99 (volume ratio of a buffer solution and a water-soluble organic solvent as a mobile phase). The present invention provides a method for separating various compounds by liquid chromatography, which is characterized in that a mixed solution of (1) is used.

The water-soluble organic solvent used in the present invention may be any one as long as it is miscible with water. Preferred are acetonitrile, methanol and ethanol. Other known water-soluble organic solvents are preferred. Can also be used. Any buffer may be used in the present invention. Examples include acetate buffer, phosphate buffer, perchlorate buffer and the like. The preferred pH range of the buffer used in the present invention is 2-8. The method for preparing the mobile phase of the present invention may be any method,
The easiest method is to mix the above-mentioned arbitrarily prepared buffer solution and the water-soluble organic solvent in a volume ratio of 99: 1 to 1:99, preferably 90:10 to 10:90.

The separating agent used in the present invention contains a carboxylic acid ester of a polysaccharide as an active ingredient, and specifically, a carboxylic acid ester of a synthetic polysaccharide, a natural polysaccharide or a natural product-modified polysaccharide is used, preferably Cellulose, amylose, β-1,4-
Some hydrogen atoms on the hydroxyl groups of polysaccharides such as chitosan, chitin, β-1,4-mannan, β-1,4-xylan, inulin, α-1,3-glucan, β-1,3-glucan Alternatively, all, preferably 85% or more, are esterified with a carboxylic acid. Examples of the most effective separating agent among these include cellulose tris (4-methyl) benzoate and the like.

The compound separated by the method of the present invention is not particularly limited, and includes optical isomers, stereoisomers such as diastereomers, positional isomers, geometric isomers, compounds having different degrees of unsaturation. , Various structurally similar compounds such as compounds belonging to the homologue can be separated.

[0008]

EXAMPLES The present invention will be described below in detail with reference to Examples and Comparative Examples, but it is clear from the above-mentioned reasons that the present invention is not limited to these Examples. The parameters k ′ and α used in Examples and Comparative Examples are defined as follows.

[0009]

[Equation 1]

Example 1 As a mobile phase, pH ₃ , 0.1M HClO ₄ -NaClO ₄ aqueous solution / CH ₃ C
The optical isomers of mianserin were separated using a mixed solution of N = 80/20 (volume ratio). As the column, a commercially available CHIRALCEL OJ (manufactured by Daicel Chemical Industries, Ltd.) having a length of 25 cm and an inner diameter of 0.46 cm packed with a stationary phase in which cellulose tris (4-methyl) benzoate was supported on silica gel was used. The mobile phase flow rate was 1.0 ml / min, and the column temperature was 40 ° C. An ultraviolet detector was used to detect the eluting optical isomer, and the wavelength was set to 254 nm. As a separation result, a chromatogram is shown in FIG. 1, and retention times, volume ratios and separation coefficients of both enantiomers are shown in Table 1.

Comparative Example 1 As a mobile phase, a mixed solution of H ₂ O / CH ₃ CN = 80/20 (volume ratio) was used to separate optical isomers of mianserin. The experimental conditions were the same as in Example 1. Both enantiomers did not elute within 1 hour.

[0012]

[Table 1]

Example 2 As a mobile phase, pH ₃ , 0.1 M HClO ₄ -NaClO ₄ aqueous solution / CH ₃ C
The enantiomers of trimebutine were separated using a mixture of N 2 = 80/20 (volume ratio). As the column, a commercially available CHIRALCEL OJ (manufactured by Daicel Chemical Industries, Ltd.) having a length of 25 cm and an inner diameter of 0.46 cm packed with a stationary phase in which cellulose tris (4-methyl) benzoate was supported on silica gel was used. The mobile phase flow rate was 1.0 ml / min, and the column temperature was 40 ° C. An ultraviolet detector was used to detect the eluting optical isomer, and the wavelength was set to 254 nm. As a result of separation, a chromatogram is shown in FIG. 2, and retention times, volume ratios and separation coefficients of both enantiomers are shown in Table 2.

Comparative Example 2 As a mobile phase, 0.1 M NaClO ₄ aqueous solution / CH ₃ CN = 80/20
The optical isomers of trimebutine were separated using the mixed solution (volume ratio). The experimental conditions were the same as in Example 2. Both enantiomers did not elute within 1 hour.

Comparative Example 3 As a mobile phase, a mixed solution of H ₂ O / CH ₃ CN = 80/20 (volume ratio) was used to separate optical isomers of trimebutine. The experimental conditions were the same as in Example 2. Both enantiomers did not elute within 1 hour.

[0016]

[Table 2]

[0017]

Industrial Applicability By the method of using the specific mobile phase of the present invention, various compounds can be favorably separated, and the range of compounds to be separated in the separating agent using the carboxylic acid ester of polysaccharide is expanded. There was no concern about damage such as hydrolysis of the carboxylic acid ester of the polysaccharide used as a separating agent, and conversely, by making the pH more acidic, the life of the silica gel carrier became longer. In addition, the retention time was shorter than the addition of only salt, which enabled analysis in a short time. Further, in this mobile phase, the solubility of a basic substance such as an amine as an object to be separated is also increased. Therefore, the method of the present invention facilitates the general analysis of various compounds, particularly the substances contained in sample liquids such as plasma, serum, and body fluids, which are mainly water.

[Brief description of drawings]

FIG. 1 is a chromatogram showing the separation result of Example 1.

FIG. 2 is a chromatogram showing the separation result of Example 2.

Claims (1)

[Claims] 1. When performing liquid chromatography using a separating agent containing a carboxylic acid ester of a polysaccharide as an active ingredient, 99: 1 to 1: 1 of a buffer solution and a water-soluble organic solvent as a mobile phase.
A method for separating various compounds by liquid chromatography, which comprises using a mixed solution of 1:99 (volume ratio).