JPH0437818B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a separation method for optically resolving enantiomers such as amino acids and their derivatives by liquid chromatography.

[Conventional technology]

Up to now, methods for determining the optical purity of amino acids using liquid chromatography include methods of converting amino acids into diastereomers and then separating them, methods using a chiral stationary phase, and methods of adding a chiral compound to the mobile phase. It was hot. The method of converting into diastereomers is problematic because it requires racemization during derivatization and complicated operations. In addition, in the method of adding a chiral compound to the mobile phase, the optical purity of the added chiral compound becomes a problem.
A post-label method must be used as a detection method, making the device complex. In addition, as a method using a chiral stationary phase, there is a method using a packing material in which a chiral amino acid is supported on silica gel via a spacer, and the packing material is described in J.
Chromatogr., 203 , 377 (1981), K. Sugden et al., J. Chromatogr., 192 , 228 (1980), or
Angew.Chem.Int.Ed. by VADavankov et al.
Engl., 21 , 930 (1982). All of these are immobilized amino acids that are coordinately or ionicly bonded to divalent copper ions, and the difference in free energy of interaction with each enantiomer of racemic amino acids that can coordinate this is determined by This is a so-called ligand exchange type filler that utilizes

[Problem that the invention seeks to solve]

However, the method described in the above literature uses a KH ₂ PO ₄ aqueous solution, a cupric acetate aqueous solution, or a cupric acetate aqueous solution mixed with acetonitrile as a mobile phase (solvent), and these are all negative. Because the ionic moiety has a relative affinity for copper ions, it either detaches the fixed copper ions or exists in the vicinity of the copper ions and has an interfering effect that makes it difficult for ligand exchange with racemic free amino acids to occur. As a result, a chromatograph with good quantitative performance cannot be obtained, and the column life is shortened, among other disadvantages.

[Means for Solving the Problems] In order to further improve the performance of a packing material for optical resolution in which a chiral amino acid is supported on silica gel, the present inventors conducted intensive studies on the mobile phase thereof, and found that divalent copper The present invention was completed by discovering that the above problems could be solved by using a strong acid salt.

That is, the present invention provides a method for separating enantiomers by liquid chromatography using a packing material in which a metal salt of an optically active amino acid is bonded to silica gel via a silanizing agent, in which a strong acid salt of divalent copper is used as a mobile phase. The present invention relates to a method for separating enantiomers, which is characterized in that the enantiomers are added to the enantiomers.

The present invention provides a means for dramatically improving the separation ability of a method for separating enantiomers using a chiral stationary phase and obtaining good chromatography.

Examples of strong acid salts of divalent copper used in the present invention include cupric chloride, cupric nitrate, copper sulfate, copper perchlorate, and hydrates thereof, which are used as aqueous solutions.

In the present invention, any known silane treatment agent may be used to form the spacer portion of the filler formed by bonding an optically active amino acid metal salt to silica gel via a silane treatment agent. Specifically, 3-chloropropyltrimethoxysilane, 3-chloropropyldimethoxymethylsilane, 3-chloropropylmethyldichlorosilane, 3-chloropropyltrichlorosilane, 3-
Bromopropyldimethylchlorosilane, 3-bromopropyltrichlorosilane, 3-bromopropyltrimethoxysilane, 2-(3,4-epoxycyclohexylethyl)trimethoxysilane, 3
Examples include -glycidoxypropyltrimethoxysilane, diethoxy-3-glycidoxypropylmethylsilane, 3-glycidoxypropyldimethylethoxysilane, and 8-bromooctyltrichlorosilane. Optically active metal salts of amino acids include natural or unnatural α-amino acids and β-amino acids.
-Metal salts of amino acids are exemplified. More specifically, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, ornithine, methionine, phenylalanine, serine,
Examples include metal salts of threonine, tryptophan, tyrosine, valine, phenylglycine, proline, hydroxyproline, and allohydroxyproline. Examples of metals that form salts include divalent copper, nickel, and trivalent cobalt.
If these are expressed as a general formula, it will be as shown in the following formula ().

[However, at least one of Y ¹ , Y ² , and Y ³ in the formula
One represents silica gel and the siloxane bonding part with silica gel, and the remaining represent hydrogen and carbon number 1, respectively.
-6 alkyl group, C6-14 aryl group,
C7-20 arylalkyl group, halogen,
It represents 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 an optically active metal salt of an amino acid.] These are used as a packing material for liquid chromatography, and a strong acid salt of divalent copper is added to the mobile phase to separate enantiomers. The amount of the strong divalent copper salt added is preferably 0.05mM to 5mM, more preferably 0.1mM to 1mM.

In the present invention, the cylindrical column used for liquid chromatography may be made of either glass or stainless steel, but is usually selected based on the working pressure and pressure resistance. Although any column size is possible in principle, an appropriate column size is selected based on the sample injection amount, analysis time, solvent flow rate, etc.

The above-mentioned column is filled with the packing material represented by the above general formula (), and either dry or wet packing methods are possible, but for small columns, it is usually a wet method, that is, a slurry, which allows a high number of theoretical plates to be obtained. It is better to fill it according to the method. The packed column is connected to a pump to allow the solvent to flow through it, and a syringe is provided between the pump and the column, through which the enantiomers to be separated are injected into the solvent, and separation is performed in the column. The separated enantiomers are detected by a detector provided at the column outlet, and any possible detection method may be used.

Compounds that can be optically resolved by the separation method of the present invention include, for example, amino acids, but amino acids usually require only one enantiomer, and the separation method using such an optical separation filler is not suitable for optical resolution. It is an important analytical tool for determining the purity of active amino acids in the production of them.

〔Effect of the invention〕

The separation method provided by the present invention not only makes it possible to obtain particularly good chromatography when enantiomers of amino acids and their derivatives are optically resolved by liquid chromatography, but also slows down the deterioration of the packing material. becomes possible.

〔Example〕

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

Comparative Example 1 Using a silane treatment agent having a glycidoxypropylsilyl group, a filler in which a copper salt of L-proline was bonded to silica gel was formed into a length of 25 mm by a slurry method.
The amino acid enantiomers were separated by liquid chromatography using 0.05M KH ₂ PO ₄ as a solvent. Amino acid chromatography using an ultraviolet detector showed broad peaks and deformation.

Example 1 In a method similar to Comparative Example 1, 0.5 m of solvent
When a cupric nitrate aqueous solution of M was used, an extremely sharp peak was obtained. Also length 25cm, inner diameter
When a 0.46 cm stainless steel cylindrical column was used and 2 ml of solvent was flowed per minute at 50°C, the initial resolution of phenylalanine was 2.1 and remained unchanged even after 120 hours.

Comparative Example 2 In the same method as Comparative Example 1, the solvent was further added.
When 0.5 mM cupric acetate was added, the resolution of phenylalanine after 30 hours was significantly lowered to 1.5 (initial value 2.1).

Comparative Example 3 In the same method as in Example 1, when an aqueous magnesium sulfate solution was used as the solvent, optical resolution was possible, but the peak became significantly broader.

Comparative Example 4 In the same method as in Example 1, when an aqueous potassium hydrogen sulfate solution was used as the solvent, the copper ions in the stationary phase were desorbed in a short time, resulting in loss of optical resolution ability.

Comparative Example 5 In the same method as in Example 1, when an aqueous potassium nitrate solution was used as the solvent, optical resolution was possible, but the peak became significantly broad and a concentration of 1 to 10 mM was required to obtain a sharp peak. It was hot.

Synthesis example 1 Silica gel Lichrosorb SI 100, particle size 10μm
(manufactured by E. Merck) in a vacuum for 5 hours at 120-150°C.
Heat and dry. 20 g of dried silica gel is suspended in 120 ml of anhydrous benzene, 8 ml of glycidoxypropyltrimethoxysilane is added thereto, and the suspension is heated to reflux under a stream of dry nitrogen. The methanol produced at this time is removed from the system and the reaction is allowed to proceed for 3 hours. After the reaction is completed, it is cooled to room temperature and filtered through a glass filter. The obtained modified silica gel is washed with anhydrous benzene and then dried in vacuo at 40°C. Dissolve 1.24 g of sodium salt of L-tryptophan in 30 ml of anhydrous methanol, and add the above modified silica gel to the solution.
Add 3.5 g, suspend, and shake at room temperature for 4 days. The modified silica gel was filtered and washed with methanol, and then transferred to an aqueous solution in which 2 g of copper sulfate was dissolved in 50 ml of pure water to obtain a copper salt. This was filtered again and washed with pure water to obtain silica gel to which the copper salt of L-tryptophan was chemically bonded.

Example 2 The filler obtained in Synthesis Example 1 was prepared with an inner diameter of 0.46 cm and a length of
Packed into a 25cm stainless steel cylindrical column, 0.25m
When an aqueous copper sulfate solution of M was used, an extremely sharp peak was obtained, making optical resolution conventionally difficult. α-alanine, leucine, aspartic acid,
Optical resolution of enantiomers such as glutamic acid and glutamine has become possible.

Claims (1)

[Claims] 1. In a method for separating enantiomers by liquid chromatography using a packing material made of silica gel bound to an optically active metal salt of an amino acid via a silanizing agent, a strong acid salt of divalent copper is added to the mobile phase. A method for separating enantiomers characterized by: