JPH0627093B2 - Separation method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a separation method for optically resolving the enantiomer of α-hydroxycarboxylic acid or a salt thereof by liquid chromatography.

[Conventional technology]

Up to now, the optical resolution of α-hydroxycarboxylic acid using liquid chromatography has been described by Tanimura et al., JS Chromatogr., 291 (1984) by The Analytical Society of Japan, 30th Lecture Summary, P15 (1981). ),
155. These are methods in which a reversed-phase partitioning packing is used and divalent copper and an optically active amino acid or an optically active N, N-dialkylamino acid are used as a mobile phase, and a trivalent iron salt solution is used as a coloring reagent for detection. The post label detection method is adopted.

[Problems to be solved by the invention]

At the same time, however, the method described in the above-mentioned document has a problem that an overwhelmingly large amount of the optically active substance is consumed as a mobile phase due to the optical resolution of a sample which is a very small amount of the optically active substance. .

In addition, the mobile phase described in the literature exhibits a strong absorbance with respect to ultraviolet rays and visible rays, so the ultraviolet / visible absorptiometer (UV-V1S detector) which is most commonly used as a detector for liquid chromatography. However, there is a drawback in that the device must use a complicated post label detection method.

[Means for solving problems]

The present inventors have diligently studied a separation base material (a packing material for liquid chromatography) as a method for separating an enantiomer of α-hydroxycarboxylic acid or a salt thereof without using an optically active substance as a mobile phase. As a result, they have found that the above problems can be solved by using a filler in which a metal salt of L-proline is bound to silica gel, and completed the present invention.

That is, the present invention is a method for separating an enantiomer of an α-hydroxycarboxylic acid or / and a salt thereof by liquid chromatography using a filler obtained by binding a metal salt of L-proline to silica gel.

Examples of the carrier used in the present invention include silica gel, a porous synthetic polymer, and a polysaccharide. More specifically, examples of the porous synthetic polymer include styrene-divinylbenzene copolymer and acrylic acid ester of polyhydric alcohol. Polymer of
Dextran and cellulose are exemplified as the polysaccharide, and silica gel is most preferable.

The filler used in the present invention using silica gel as a carrier is represented by the following general formula (I).

[However, in the formula, at least one of Y ¹ , Y ² , and Y ³ represents silica gel and a siloxane bond portion with silica gel, and the rest are hydrogen, an alkyl group having 1 to 6 carbon atoms, and a carbon atom having 6 to 14 carbon atoms, respectively. Represents an aryl group, an arylalkyl group having 7 to 20 carbon atoms, a halogen, a hydroxy group, an alkoxy group having 1 to 6 carbon atoms, or any combination thereof. X represents a spacer having 1 to 20 carbon atoms.
R represents a metal salt of L-proline] Any known silane-treating agent for forming the spacer portion of the filler represented by the general formula (I) can be used, and specifically, 3-chloropropyl is used. Trimethoxysilane, 3-chloropropyldimethoxymethylsilane, 3-chloropropylmethyldichlorosilane, 3-chloropropyltrichlorosilane, 3-bromopropyldimethylchlorosilane, 3-promopropyltrichlorosilane, 3-bromopropyltrimethoxysilane, 2 −
(3,4-epoxycyclohexylethyl) trimethoxysilane, 3-glycidoxypropyltrimethoxysilane, diethoxy-3-glycidoxypropylmethylsilane, 3-glycidoxypropyldimethylethoxysilane, 8-bromooctyltrichlorosilane Are exemplified. Examples of the metal salt of L-proline include salts of divalent copper, nickel and trivalent cobalt.

In the separation method of the present invention, the substance represented by the general formula (I) is used as a packing material for liquid chromatography, and an aqueous solution of a divalent copper strong acid salt or a mixture thereof with acetonitrile and alcohol is used as a mobile phase. Is preferred. Examples of the strong acid salt of divalent copper include cupric chloride, cupric nitrate, copper sulfate, copper perchlorate and hydrates thereof, which are used as an aqueous solution thereof.

The cylindrical column used in the liquid chromatography for separating the enantiomers of the α-hydroxycarboxylic acid and its salt in the present invention may be either glass or stainless steel, but it is usually selected from the working pressure and pressure resistance. It Although any column size is possible in principle, an appropriate column size is selected depending on the injection amount of sample, analysis time, solvent flow rate and the like.

The packing represented by the general formula (I) is packed in the column, and either a dry method or a wet method can be used as a packing method, but in a small column, a wet method which usually provides a high theoretical plate number. It is better to fill by the method, that is, the slurry method. The packed column is connected to a pump to allow the solvent to flow, but an injector is provided between the pump and the column, from which the enantiomers to be separated are injected, and the column is divided. The separated enantiomers are detected by a detector provided at the column outlet, but the method of detection may be any possible method, and a commonly used UV detector is often adequate for the purpose.

The target compounds that can be optically resolved according to the present invention are racemic mixtures of α-hydroxycarboxylic acids and their salts. Specific examples include lactic acid, mandelic acid, malic acid, glyceric acid and their sodium salts, potassium salts, calcium salts and the like.

〔The invention's effect〕

α-Hydroxycarboxylic acid is an important substance as an intermediate in synthesizing an optically active substance, especially a physiologically active substance, and in many cases, only one of the antipodes is required. The analysis of the body and its isolation is important. The separation method provided by the present invention shows extremely good optical resolution with respect to the enantiomers of α-hydroxycarboxylic acid and its salt, and the D-form and the L-form which are enantiomers show a sufficient difference in the elution time. Since it elutes and the peaks of the respective components are separated into sharps, it can be advantageously used for qualitative / quantitative analysis of enantiomers and fractionation thereof.

〔Example〕

The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Synthesis Example 1 Silica gel Lichrosorb SI 100, particle size 10 μm (E.Me
(manufactured by rck) in vacuum for 5 hours at 120 to 150 ° C. and dried. 20 g of dried silica gel is suspended in 120 ml of anhydrous benzene, 8 ml of glycidoxypropyltrimethoxysilane is added thereto, and the mixture is heated to reflux under a stream of dry nitrogen. The methanol produced at this time is allowed to react for 3 hours so as to be removed from the system. After completion of the reaction, the mixture is cooled to room temperature and passed through a glass filter. The obtained modified silica gel is washed with anhydrous benzene and then dried in vacuum at 40 ° C. L-
1.24 g of sodium salt of proline in anhydrous methanol 30
It is dissolved in ml, 3.5 g of the above-mentioned modified silica gel is added and suspended, and the mixture is shaken at room temperature for 4 days. After passing through the modified silica gel and washing with methanol, 2 g of copper sulfate was added to 50 ml of pure water.
To obtain a copper salt. This was filtered again and washed with pure water to obtain silica gel in which the copper salt of L-proline was chemically bound.

Example 1 The filler obtained in Synthesis Example 1 was used to perform optical resolution of racemates of various α-hydroxycarboxylic acids and salts thereof. That is, the average particle size is 10 μm and the average pore size is 100 μm.
2.5 × 10 ^{-4 A} high-performance liquid chromatographic stainless steel column (25 cm × 0.46 cm) was filled with a packing material consisting of a copper salt of L-proline bound to the all-porous silica gel of Å.
Optical resolution of the racemic α-hydroxycarboxylic acid or its salt shown below was carried out at room temperature at a flow rate of 1 ml / min using M copper sulfate aqueous solution as the solvent, and the results shown in Table 1 below were obtained.

In Table-1, k ', α, and R _S are defined as follows.

That is, as shown by the values of α and R _S in Table-1, in each compound, a sufficient difference was observed in the elution time of the enantiomers D-form and L-form, and the two were completely separated as peaks. Therefore, it is understood that the method of the present invention is an excellent separation method that can be used for qualitative / quantitative analysis of enantiomers and preparative separation thereof.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C07C 59/50 8930-4H G01N 30/48 W 8310-2J // C07B 57/00 346 7419-4H

Claims (1)

[Claims] 1. A separation method, characterized in that the enantiomers of α-hydroxycarboxylic acid or / and a salt thereof are separated by liquid chromatography using a packing obtained by binding a metal salt of L-proline to silica gel.