JP3124350B2 - Liquid chromatography separation method - Google Patents

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 a compound from a compound by liquid chromatography, and can be used for the purpose of purification by analysis or fractionation in research and production control.

[0002]

2. Description of the Related Art A polysaccharide derivative or a separating agent in which the polysaccharide derivative is supported on a carrier such as silica gel is used as a stationary phase for liquid chromatography as a variety of structurally similar compounds including optical isomers. (Stereoisomers such as diastereomers, positional isomers, geometric isomers, compounds with different degrees of unsaturation, compounds belonging to homologues, etc.) have been shown to exhibit excellent properties in separation (1991) January 7, 28th HPLC Research Symposium Abstracts, pp. 25-26). Conventionally, this type of stationary phase is mainly composed of a normal phase mobile phase, that is, when the polarities of a stationary phase and a mobile phase are compared, a stationary phase having a relatively high polarity and a mobile phase having a low polarity (typically, Hexane and alcohols). However, when the compound to be separated is a carboxylic acid, an amine, or the like, the peak shape may be unusually wide. For example, an acid (eg, trifluoroacetic acid, acetic acid, formic acid, etc.) may be used to separate the carboxylic acid. In the case of amines, it has been found that adding an amine (eg, diethylamine, triethylamine, etc.) to the mobile phase has the effect of narrowing the peak width and improving the separation.

However, compounds to be separated include, for example, salts of strong acids such as sulfonic acid, salts of strong bases such as guanidinium, so-called onium salts (eg, quaternary ammonium, tertiary oxonium, phosphonium, tertiary sulfonium, etc.). In addition, in the case of zwitterionic substances (for example, compounds having an amino group and a carboxyl group in the molecule), the use of the above-mentioned additives has no effect, and separation is impossible or peaks are significantly deformed and separation is difficult. There were many bad things.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems of the conventional liquid chromatography system, and as a result, have found that various types of liquids are contained in the normal-phase mobile phase. By adding a salt, it was possible to improve the separation of a compound that was difficult to separate or a compound that could not be separated, and found that a sharper peak could be obtained, and completed the present invention. . That is, the present invention provides a method for performing liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient, by performing liquid chromatography of various compounds characterized by adding various salts to a normal-phase mobile phase. It provides a separation method.

[0005] The separating agent used in the present invention contains a polysaccharide derivative as an active ingredient.
No. 944, p 363, namely, synthetic polysaccharides, natural polysaccharides and derivatives of modified natural polysaccharides are used, and preferably, cellulose, amylose, β-1, 4--chitosan, chitin, β-
1,4-mannan, β-1,4-xylan, inulin, α-
Part or all of the hydrogen atoms on the hydroxyl groups of polysaccharides such as 1,3-glucan and β-1,3-glucan, preferably 85
% Or more is replaced with another atomic group. An example of the most effective separating agent among these is cellulose tris (4
-Methyl) benzoate, cellulose trisphenyl carbamate and the like.

The various salts referred to in the present invention are those obtained by removing water from a mixture of an acidic substance and a basic substance or, in the case of a combination of a Bronsted acid and a Bronsted base, from water. Acidic substances include perchloric acid, hydroiodic acid, carboxylic acids (including perfluoroalkanoic acids),
Sulfonic acid, phosphonic acid, alkyl sulfate, tetraphenylboric acid (HBPh ₄ ), tetrafluoroboric acid (HBF ₄ ), boron trifluoride (BF ₃ ), hydrosilicofluoric acid (H ₂ SiF ₆ or HSiF
₅ ), BR ₃ , phenols, cyanoform and the like,
Preferred are carboxylic acids, especially trifluoroacetic acid. Examples of the basic substance include all primary, secondary and tertiary amines (including guanidine and imidine), quaternary ammonium hydroxide, tertiary sulfonium salt hydroxide, tertiary oxonium hydroxide, and quaternary phosphonium. Hydroxides, secondary iodonium hydroxides and the like can be mentioned, and preferred is diethylamine.

In the present invention, the normal phase mobile phase to which the above-mentioned various salts are added may be any mobile phase as long as the salt can be dissolved. Preferably, a non-polar solvent such as hexane and a polar solvent compatible therewith are used. It is a liquid obtained by mixing a solvent, preferably an alcohol, with one or more components. The mobile phase may further contain other components for adjusting the strength of retention and the good separation. Among the other components mentioned here, any of the above-mentioned acidic substances and the above-mentioned basic substances may be in excess. The method for preparing the salt of the present invention is not limited to mixing the above-mentioned acidic substance and the above-mentioned basic substance, but may be any substance such as a double decomposition product. For example, to prepare an amine carboxylate, for example, a volatile solvent such as methylene chloride, in which both components are easily dissolved, may be dissolved in a slightly excessive amount, either amine or carboxylic acid, and then mixed. After evaporating the volatile solvent, a salt can be obtained. In particular, when it is not necessary to maintain the liquidity of the mobile phase at around neutral, it is not necessary to isolate all the salts, and appropriate amounts of the acidic substance and the basic substance are separately added to the eluate. It may be added (however, it goes without saying that water is by-produced in the case of a combination of a Bronsted acid and a Bronsted base). In practice, when using the mobile phase of the present invention, one or more of the above salts are sent to the column as a solution in which the concentration is from 0.001% (v / v) to saturation.

[0008]

The method of the present invention has made it possible to separate compounds which could not be separated by the conventional method, but the effect is that the ionic compound existing in the salt state can be separated from the mobile phase to which the salt described in the present invention is added. Among them, ion-exchanged and behaves as an ion pair most suitable for chromatographic separation as shown in the following formulas (1) and (2), or when the added salt is a conjugate acid (C
⁺⁾ And conjugate base (D ^- to include both), ion pair A
^- · B ⁺ or A ^- · C each anion component of ^+, it is considered that mainly due to play a role to prevent even excessive adsorption to any cationic component not clear.

[0009]

Embedded image

[0010]

EXAMPLES The present invention will be described below in detail with reference to Examples and Comparative Examples. However, it is clear that the present invention is not limited to these Examples for the reasons already described. The parameters k ′ and α used in the examples and comparative examples are defined as follows.

[0011]

(Equation 1)

Example 1 The optical isomer of trimebutine maleate (maleate of trimebutine) was used as a mobile phase in hexane / 2-propanol = 95/5 (v / v) at 1000 ppm of diethylamine.
Separation was performed using a mixed solution to which trifluoroacetate (hereinafter referred to as DEA / TFA salt) was added. The chromatogram is shown in FIG. 1, and the retention time, volume ratio and separation coefficient of both enantiomers are shown in Table 1. The column is CHIRALCEL using cellulose tris (4-methyl) benzoate as the separating agent.
OJ (manufactured by Daicel Chemical Industries, Ltd.) was used.

Comparative Example 1- (1) The same trimebutine maleate as in Example 1 was subjected to optical isomer separation using a mixed solution of hexane / 2-propanol = 95/5 (v / v) as a mobile phase. The chromatogram is shown in FIG. 2, and the retention time, volume ratio and separation coefficient of both enantiomers are shown in Table 1. Note that the stationary phase was prepared in Example 1
The same column was used.

Comparative Example 1- (2) Trimebutine isolated from trimebutine maleate of Example 1 was used as a mobile phase in the same hexane / 2-propanol = 95/5 (v / v) as in Comparative Example 1- (1). Was used to separate optical isomers. The chromatogram is shown in FIG.
Table 1 shows the retention time, volume ratio and separation coefficient of both enantiomers. The same column as in Example 1 was used for the stationary phase.

[0015]

[Table 1]

Example 2 An optical isomer of mianserin hydrochloride (mianserin hydrochloride) was used as a mobile phase in hexane / 2-propanol = 95 /
Separation was carried out using a mixture of 5 (v / v) and 1000 ppm of DEA / TFA salt. The chromatogram is shown in FIG. 4, and the retention time, volume ratio and separation coefficient of both enantiomers are shown in Table 2. The column used was CHIRALCEL OJ (manufactured by Daicel Chemical Industries, Ltd.) using cellulose tris (4-methyl) benzoate as a separating agent.

Comparative Example 2- (1) The same mianserin hydrochloride as in Example 2 was subjected to optical isomer separation using a mixed solution of hexane / 2-propanol = 95/5 (v / v) as a mobile phase. The chromatogram is shown in FIG. 5, and the retention time, volume ratio and separation coefficient of both enantiomers are shown in Table 2. The same column as in Example 2 was used for the stationary phase.

Comparative Example 2- (2) Mianserin isolated from the same mianserin hydrochloride as in Example 2 was used as a mobile phase in the same hexane / methanol as in Comparative Example 2- (1).
Optical isomers were separated using a mixture of 2-propanol = 95/5 (v / v). The chromatogram is shown in FIG. 6, and the retention time, volume ratio and separation coefficient of both enantiomers are shown in Table 2. In addition, the stationary phase includes Example 2 and Comparative Example 2.
-The same column as (1) was used.

[0019]

[Table 2]

[0020]

The method using the mobile phase of the present invention has been
Chromatographic separation of a mixture of strongly basic substances or salts thereof and zwitterionic compounds, for which it has been difficult to obtain good separation, has been facilitated. In the case where a simple weakly basic or weakly acidic substance is present in the form of a salt, it was conventionally necessary to isolate the free base or free acid. Can be provided. These results have allowed mixtures of various similar compounds, especially those which have been previously impossible or have simplified the analysis and separation of optical isomers.

[Brief description of the drawings]

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

FIG. 2 is a chromatogram showing a separation result of Comparative Example 1- (1).

FIG. 3 is a chromatogram showing a separation result of Comparative Example 1- (2).

FIG. 4 is a chromatogram showing a separation result of Example 2.

FIG. 5 is a chromatogram showing the separation results of Comparative Example 2- (1).

FIG. 6 is a chromatogram showing the results of separation of Comparative Example 2- (2).

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI G01N 30/88 G01N 30/88 W (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 30/26 B01D 15 / 08 C07B 57/00 340 C07B 63/00 G01N 30/48 G01N 30/88 CA (STN) JICST file (JOIS)

Claims (1)

(57) [Claims] When a liquid chromatography is carried out using a separating agent containing a polysaccharide derivative as an active ingredient, various salts are added to a normal-phase mobile phase, and the separation of various compounds is performed by the liquid chromatography. Law.