JPS59173756A - Packing material for separating optical isomer - Google Patents

Abstract

PURPOSE:To prevent attraction of optical isomers except the optical isomer intended to be separated and to enable the sepn. of the optical isomer with easy sepn. efficiency in a short time by using the particles formed by immobilizing an optically active amino acid on the pore surface of silica gel as a packing material for a liquid chromatography. CONSTITUTION:A silane coupling agent such as 3-amino-propyl triethoxy silane or the like is brought into reaction with the pore surface of silica gel, thereby forming porous spherical particles of aminopropylated silica gel. An optically active amino acid having a butyl oxycarbonyl group, etc. as a protective group and a reagent (1,1'-carbonyl di-imidazole, etc.) into which an activated N-acyl group can be introduced are brought into reaction with such particles, by which the particles immobilized with the optically active amino acid are obtd. Chromatography is performed by using the silica gel particles as a packing agent, by which the sepn. of an optical isomer of various amino acids, mandelic acid, etc. can be efficiently performed. The packing material has high strength, is easy to handle and contributes to an increase in the immobilizing capacity of the amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a liquid chromatography agent that enables the separation of optical isomers. More specifically, it relates to a liquid chromatography Idemitsu agent that immobilizes optically active amino acids on a hydrophilic gel used in gel permeation chromatography in an aqueous solution system and is capable of separating optical isomers of amino acids and the like. .

Traditionally, ligand exchange chromatography has been used as a method for separating optical isomers by liquid chromatography.
Ion conversion resins, octadecylsilane for reverse phase chromatography, chemically bonded silica gel, etc. are used as packing materials.

However, in the above method, it is necessary to add the optically active amino acid to the bath separation liquid, and the separation method is complicated and expensive.

Therefore, a method of chemically bonding the optically active substance to the packing material itself for easier separation can be considered, but if the packing material is non-polar like styrene gel, the adsorption properties other than ligand exchange will be reduced by the water bath. The new fraction 1ξ:L method is observed in un-eluted liquids, and causes undesirable phenomena such as peak width (decreased number of theoretical plates), prolongation of analysis time, etc., especially with aromatic samples. But ¥;! It's rare.

In view of the above-mentioned problems, the present inventors conducted intensive research and found a suitable photonic agent and completed the present invention.

That is, in the present invention, the pore surface of silica gel is aminopropylated by a conventional method to provide a filler fZA for activated N-acyl chromatography.

The Honto Meikin will be explained in detail below.

In the present invention, the aminopropylated silica gel is subjected to aminozolopylation using a silano coupling agent having an amine 'Al-', and the aminopropylated silica gel is protected with a reagent capable of introducing active N-acyl in a bath medium and has optical activity. By adding amine 1β to the reaction solution, amine r9 was added to the silica gel pore table 1ii in an amount of 0.6 to [L 5 to mol
/f Fixed porous spherical shape, average particle size 1-50μ
, preferably 5 to 40μ, average pore diameter IUA to 50 [J
A, good -1<+f150~600 large, surface 1100
~50' Om2/V, dexterously'-1,1200~
3 b Om27f/, Ka-ko, hQlrfi,
Target 1iJNovember is a filling process in which the amount is 300 kg/Furi" or more.

If the average particle size is less than 1μ, a column with excellent fractionation plasticity can be obtained, but it is difficult to fill the column.On the other hand, if the particle size is larger than 50μ, it is difficult to fill the column. Although the sample load is large, this is not preferable because the separation efficiency deteriorates. If the average pore diameter is less than 10A, the effective surface area will be small, and if it exceeds 500A, the mechanical strength will deteriorate, which is not preferable.

In the present invention, the method for obtaining aminopropylated silica gel uses a conventional aminopropylated gel I5f with a particle size of 1 to 5.
0μ, average pore diameter 10-500A, surface area 100-50.
Aminopropylated silica gel is obtained by reacting 0 cm2/q of silica gel with a silane coupling agent having an amine group in an aromatic organic solvent at a temperature of 80 to 150°C for a reaction time of 15 to 48 hours.

Examples of the aromatic organic solvent include benzene, toluene, xylene, methylbenzene, and cyclohexane, with toluene being particularly preferred.

As a silane coupling agent having an amine group, 6-
Amitupropyltriethoxysilane.

Examples include N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane, N-(2-aminoethyl)-γ-amino, propyltrimethoxysilane, and especially 3-amino-propyltriethoxysilane. 27
Ran is preferred.

Next, the optically active amino acid having a protecting group is reacted with a reagent capable of introducing activated N-acyl, and the above aminopropylated silica gel is added thereto to immobilize the optically active amino acid.

As an optically active amino acid having a protecting group, butyloxycarbonyl (
(hereinafter referred to as Boa) group, tosyl group.

histidine having a benzyl group or a carbobendoxy group,
Examples include phenylalanine, tryptophan, etc., especially Boc. Histidine tosylate is preferred.

As a reagent that can introduce activated N-acyl, 1.1'
-carbonyldiimidazole, N-acetylimidazole, and succinic anhydride, but in particular 1.
1'-carbonyldiimidazole is preferred.

The reaction between an optically active amino acid having a protecting group and a reagent capable of introducing activated N-acyl is carried out by mixing the optically active amino acid having a protecting group and a reagent capable of introducing activated N-acyl at 0°C or lower in the presence of an organic solvent. In particular, an optically active amino acid having a protecting group is dissolved in an organic solvent, a reagent capable of introducing activated N-acyl is added with stirring, the reaction vessel is ice-cooled, and the reaction vessel is cooled with ice. It is preferable to obtain a homogeneous reaction bath liquid by continuing stirring until the generation of carbon stops.

The amount of reagent capable of introducing activated N-acyl used in this reaction is 1 molar ratio to aminopropylated silica gel.
~4 times the amount, preferably 2 to 3 times the amount. If the molar ratio is less than 1, less amino acids will be immobilized,
If the amount exceeds 4 times, the amount of immobilized amino acids remains unchanged, which is uneconomical and unfavorable.

The above aminopropylated silica gel was added to the homogeneous reaction solution obtained by the above operation, and the mixture was heated at 10 to 40°C, preferably 20 to 25°C, for 15 hours or more.
Noic acid is immobilized on the surface of the silica gel pores by stirring for 60 hours using a barrier.

When the temperature is 10℃, it takes a long time to make one room, and the temperature is 40℃.
If the temperature exceeds .degree. C., immobilization will proceed rapidly and the pore surfaces of the silica gel may be bridged, which is not preferable. Unfortunately, if the stirring time is less than 15 hours, sufficient immobilization cannot be achieved, which is not preferable. The amount of immobilized amino acid with a protecting group is
Performed with Kjeldahl nitrogen. That is, 7J, distilled, trifluorovinegar in dry dichloromethane, 11! Silica gel immobilized with Boc and amino 1m was placed in a 0wt% bath solution, reacted for 1 hour at room temperature, and Boc
, which removes the Boc group of Aminohare. '6 The packing material of the present invention obtained by the above-described method has a higher concentration of amino acid than the conventional filler obtained by immobilizing an optically active amino acid on silica gel. Since it can be immobilized on silica gel, various amino acid optical isomers can be separated favorably.

The present invention will be explained below using examples.

Examples 1 to 6 Grain i10μ, pore size 100X, surface) lA350m2/l
of silica gel 52 in 70 ql of anhydrous toluene at 90°C.
24 hours, 6-amitubrovyltriethoxosilane 5
1 to synthesize aminopropylated silica gel.

Next, 70 mlt/CBoc of dried dimethylformamide (hereinafter referred to as DMF) and histidine tosylate 101 were dissolved and, without stirring, 1,1'-carbonyldiimidazole (hereinafter referred to as CDI)5. Add zr.

Cool the reaction vessel with ice and continue stirring until the evolution of carbon dioxide stops. Thereafter, the previously synthesized aminopropylated silica gel was added to the reaction mixture, and the mixture was stirred at room temperature for 24 hours. The amount of CDI added at this time is twice the number of moles as the amount of amino groups in the aminopropylated silica gel. Here, the degree of aminopropylation of the aminopropylated silica gel is 2 m mo
It was le/P.

Thus synthesized/gel fD MF, methanol.

Wash thoroughly sequentially with dichloromethane. The amount of Boc and histidine tosylate immobilized on the amine silica gel was determined to be 0.56 nn mole by Kjeldahl Deposit Laboratory. This value completely matched the value of 1st shift determined by weight change.

Removal of the Boc group was carried out by a trifluorochemical treatment, and removal of the tosyl group was confirmed by a change in the absorption of hydroxyl group.

The immobilized silica gel is 2yymφ iQO+, +m
A stainless steel column was filled with an equilibrium slurry consisting of tetracybromoethane, methanol, and carbon tetrachloride.

A friend who goes to Cu2 + iono page r = f' after filling.

As Examples 1 to 6, tryptophan and phenylalanine are shown in FIGS. 1 to 6 under the following measurement conditions.

Chiro7no, Balin, Dopamine, Mandel KeinoL together,
D shows an integrated segmented chromatogram.

Equipment High performance liquid chromatograph (manufactured by Toyo Soda Kogyo Co., Ltd., product name, HLC803) Column Stainless steel 2mmφ, 10100i brood liquid
1/15M phosphorus H buffer (pH 4,6) +1o =+
Flow rate 1.0 mg/min Detector Ultraviolet, 210 nm *Trifluoroacetic acid treatment Add 50% trifluoroacetic acid to 7100 ml of distilled and dried dichloromethane and react at room temperature for 1 hour to detect BOc.
, takes the amino acid BOc group.

**Hydroxybenzotriazole treated THF100+
A slight excess of hydroxybenzotriazole a5f' is added in ++J, and the mixture is allowed to react at room temperature for 2 hours. After 2 hours, the generation of inbutylene ceases and the reaction is complete.

Comparative Example In 175 ml of pyridine, 7.52 ml of L-proline are added to 17.5 rnl of 5-triethoxysilylpropylamine. The mixture was stirred at room temperature for 15 hours + N) and refluxed for 5 hours under dry conditions. Then, it is kept at 0°C for 15 hours. Unreacted proline was removed with a filter, and the solution was evaporated at 70° C. and 17 mmHg. 711] of dry benzene and remove the L-proline still remaining in solution by recrystallization. Subsequently, the solution was vacuum-dried at 60°C to obtain 159cr)N-,[
5-()Liethoxosilylubrovir)1-L-zololinamide was obtained.

Add 52 silica gel to 70 d of dry toluene,
Remove adsorbed water by redistillation. When the redistillation temperature reached 109°C, 2.5Il19 of N- to 15- of toluene
(,5-()ethoxysilyluprovir)]-]L-
Add the dissolved solution of glolin.

The mixed solution was refluxed under anhydrous conditions for 8 hours.

The chemically bonded silica gel thus obtained is mixed with toluene.

Wash with ethanol, water, ethanol, toluene, and finally diethyl ether. The chemically bonded silica gel is dried in vacuum at 60° C. for 8 hours.

A slurry of the L-proline-bonded stationary phase obtained in this manner was used as a tetranomized carbon ge solvent, and the length was 25 cm.

A 4,8 Mu stainless steel column is backed with ethanol in the usual manner. . After backing,
002M copper sulfate bath solution is passed through the column.

L-Zolorif 1 mole p (17 mole of Cu
(Engineering I) Io/ is fixed and equilibrium is reached.

The column thus obtained was connected to an HP L,0 apparatus, and the chromatograms of D and L of tryptophan, phenylalanine, and tyrosine, respectively, under the following measurement conditions are shown in FIGS. 7 to 9.

Equipment High performance liquid chromatograph (trade name manufactured by Spectra-Physics, 5paooo) Column Stainless steel 4.8 mm, 250 m
+x eluent H2O-CH,ON (47: 53)-
0,15M NH4Cl flow rate 1. , Ome/min Detector Ultraviolet 210 nm

[Brief explanation of drawings]

Figures 1 to 6 are chromatograms in which tryptophan, phenylalanine, telo-7, valine, and do-no (mine and Mandel's salts) are separated, obtained using the photostimulant of the present invention. Figures 7 to 9 are chromatograms in which the L-unit and D-unit of tryptophan, phenylalanine, and tyrosine were separated using a conventional photochromic agent in which optically active amino acids were immobilized on silica gel. 1. Each jiii amino acid and mandel's L unit 2. Various amino acids and mandelic acid D unit patent applicant Toyo Soda Kogyo Co., Ltd. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5
Figure 6 Figure 7 Figure 8 331-9th nj

Claims (1)

[Claims] Optical isomer separation agent obtained by immobilizing optically active amino acids on the surface of silica gel pores