JPH0339047B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a packing material for liquid chromatography that enables separation of optical isomers. More specifically, it relates to a packing material for liquid chromatography 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, etc. It is.

Conventionally, ligand exchange chromatography has been used as a method for separating optical isomers by liquid chromatography, and ion exchange resins, octadecylsilane for reversed-phase chromatography, chemically bonded silica gel, etc. are used as packing materials. has been done.

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

Therefore, it may be possible to chemically bond the optically active substance to the packing material itself for easier separation, but if the packing material is non-polar like styrene gel, the adsorption properties other than ligand exchange will be lower than that of the aqueous solution. A new separation method is desired, as undesirable phenomena occur especially with aromatic samples, such as broadening of peaks (reduction in the number of theoretical plates) and prolongation of analysis time.

As a result of intensive research in view of the above problems, the present inventor has found that
We have completed the present invention by immobilizing optically active amino acids on silica gel, which has relatively low adsorption properties, and found a packing material suitable for separating optical isomers such as amino acids.

That is, the present invention is a liquid chromatography system suitable for separating optical isomers such as amino acids by aminopropylating the silica gel pore surface using a conventional method and immobilizing amino acids by applying a reagent capable of introducing activated N-acyl. The present invention provides fillers for use in

The present invention will be explained in detail below.

The present invention involves reacting aminopropylated silica gel, which has been aminopropylated using a silane coupling agent having an amino group, with a reagent capable of introducing active N-acyl and an optically active amino acid having a protecting group in an organic solvent. By adding amino acids to the silica gel pore surface, 0.3
~0.5m mol/g immobilized porous sphere,
Average particle size 1-50μ, preferably 5-40μ, average pore size 10-500A, preferably 50-300A, surface area
The filler has an area of 100 to 500 m ² /g, preferably 200 to 350 m ² /g, and a mechanical strength of 300 Kg/cm ² or more.

When the average particle size is less than 1μ, a column with excellent separation efficiency can be obtained, but it is difficult to fill the column.On the other hand, when the particle size exceeds 50μ, it is easy to fill the column and the sample load is large, but separation is difficult. This is not preferable because it reduces efficiency. If the average pore diameter is less than 10 Å, the effective surface area becomes small, and if it exceeds 500 Å, the mechanical strength becomes weak, which is not preferable.

In the present invention, an ordinary aminopropylation reaction can be used to obtain the aminopropylated silica gel. That is, it is porous and spherical, with an average particle diameter of 1 to 50 μ, an average pore diameter of 10 to 500 Å, and a surface area of 100
An aminopropylated silica gel is obtained by reacting ~500 cm ² /g of silica gel with a silane coupling agent having an amino 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.

Examples of the silane coupling agent having an amino group include 3-amino-propyltriethoxysilane,
N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane, N-(2-aminoethyl)
Examples include -γ-aminopropyltrimethoxysilane, and 3-amino-propyltriethoxysilane is particularly 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.

Optically active amino acids having a protecting group include histidine, phenylalanine, phenylalanine, and butyloxycarbonyl (hereinafter referred to as Boc.) group, tosyl group, benzyl group, and carbobendoxy group, in order to prevent deactivation of the optically active amino acid. Examples include tryptophan, but Boc. histidine tosylate is particularly preferred.

Reagents that can introduce activated N-acyl include:
Examples include 1,1'-carbonyldiimidazole, N-acetylimidazole, and succinic anhydride, with 1,1'-carbonyldiimidazole being particularly preferred.

Optically active amino acids with protecting groups and activated N-
The reaction with a reagent capable of introducing an acyl can take any form in which an optically active amino acid having a protecting group and a reagent capable of introducing an activated N-acyl are mixed at 0°C or lower in the presence of an organic solvent, but in particular, , by dissolving an optically active amino acid with a protecting group in an organic solvent, adding a reagent capable of introducing activated N-acyl with stirring, cooling the reaction vessel with ice, and continuing to stir until carbon dioxide generation stops, resulting in a uniform solution. Preferably, a reaction solution is obtained.

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

Add the above aminopropylated silica gel to the homogeneous reaction solution obtained by the above operation, and
C, preferably 20 to 25 C, for 15 hours or more, preferably 20 to 30 hours, to immobilize the amino acid having a protecting group on the surface of the silica gel pores.

If the temperature is lower than 10°C, immobilization will proceed rapidly and the pore surfaces of the silica gel may be covered, which is not preferable. Further, if the stirring time is less than 15 hours, sufficient immobilization cannot be achieved, which is not preferable. The amount of immobilized amino acid having a protecting group can be determined by Kjeldahl nitrogen analysis and weight change.

Finally, removal of the protecting group, e.g. removal of the Boc.
This is carried out by treatment with trifluoroacetic acid. That is, silica gel immobilized with Boc. amino acids is added to a 30 wt% solution of trifluoroacetic acid in dichloromethane that has been distilled and dried, and the Boc. group of the Boc. amino acids is removed by reacting at room temperature for 1 hour. . Removal of each protecting group can be easily confirmed by the change in absorption band of the infrared absorption spectrum before and after the reaction.

The filler of the present invention obtained by the above method can immobilize multiple amino acids on silica gel compared to conventional fillers obtained by immobilizing optically active amino acids on silica gel. Various amino acid optical isomers can be separated well.

Hereinafter, the present invention will be explained by examples.

Examples 1 to 6 5 g of silica gel with a particle size of 10 μ, a pore size of 100 Å, and a surface area of 350 m ² /g was mixed with 3-amino-propyltriethoxysilane 5 in 70 ml of anhydrous toluene at 90° C. for 24 hours.
Aminopropylated silica gel was synthesized by reacting with g.

Next, dry dimethylformamide (hereinafter referred to as
Dissolve 10 g of Boc. histidine tosylate in 70 ml of DMF, and add 3.2 g of 1,1'-carbonyldiimidazole (hereinafter referred to as CDI) while stirring. 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 amino acid propylation of the aminopropylated silica gel is 2 mmole/
It was hot at g.

The gel synthesized in this way was mixed with DMF, methanol,
Wash thoroughly sequentially with dichloromethane. The amount of Boc. histidine tosylate immobilized on amino silica gel was determined to be 0.36 m by Kjeldahl nitrogen analysis.
It was calculated as mole/g. This value completely matched the value determined by weight change.

The Boc. group was removed by trifluoroacetic acid treatment ^* , and the tosylerto group was removed by hydroxybenzotriazole treatment ^** . The infrared absorption spectra before and after the removal of each protecting group are as follows: Boc. group at 1700 cm ^-1 , Tosyl group at 1420-1330 cm
This was confirmed by the absorption change in ^{the -1} absorption band.

The immobilized silica gel was filled into a 2 mmφ, 100 mm stainless steel column as an equilibrium slurry consisting of tetrabromoethane, methanol, and carbon tetrachloride. After filling, Cu ²⁺ ions were loaded.

As Examples 1 to 6, tryptophan, phenylalanine,
Tyrosine, valine, dopamine, mandelic acid L
The divided chromatograms of D and D are shown.

Equipment High performance liquid chromatograph (manufactured by Toyo Soda Kogyo Co., Ltd., product name,
HLC803D) Column Stainless steel 2mmφ, 100mm Eluent 1/15M phosphoric acid buffer (PH4.6) +
10 ^-4 MCu ²⁺ Flow rate 1.0ml/min Detector Ultraviolet, 210nm *Trifluoroacetic acid treatment Add 30% trifluoroacetic acid to 100ml of distilled and dried dichloromethane and react at room temperature for 1 hour to obtain Boc. Take the base.

**Hydroxybenzotriazole treatment Hydroxybenzotriazole in 100ml THF
Add a slight excess of 0.5 g and react at room temperature for 2 hours.
After 2 hours, the generation of isobutylene has ceased and the reaction is complete.

Comparative Example 7.5 g of L-proline is added to 17.5 ml of 3-triethoxysilylpropylamine in 175 ml of pyridine. The mixture was stirred at room temperature for 15 hours and refluxed for 5 hours under dry conditions. Then, keep it at 0°C for 15 hours. Unreacted L-proline was removed with a filter, and the solution was evaporated at 70°C under 17 mmHg. Dry benzene is added to remove any L-proline still in solution by recrystallization.
By subsequently vacuum drying the solution at 60°C
15 g of N-[3-(triethoxysilyl-propyl)]-L-prolinamide were obtained.

Add 5 g of silica gel to 70 ml of dry toluene and remove the adsorbed water by redistillation. When redistillation temperature reached 109℃, 2.5mg N in 15ml toluene
-[3-(triethoxysilyl-propyl)]-L-
Add the dissolved solution of prolinamide. The mixed solution was refluxed under anhydrous conditions for 8 hours. The chemically bonded silica gel thus obtained is washed with toluene, ethanol, water, ethanol, toluene in this order, and finally with diethyl ether. The chemically bonded silica gel is dried in vacuum at 60°C for 8 hours.

The L-proline-bonded stationary phase obtained in this way was mixed with a slurry using carbon tetrachloride as a solvent, and
Pack a 4.8mm ID stainless steel column using ethanol in the usual way. After packing, 0.02M copper sulfate solution is applied to the column.

Equilibrium was reached with 0.7 mole of Cu() ions fixed per 1 mole of L-proline.

The column thus obtained was connected to an HPLC device, and the D-form, L-form and L-form of tryptophan, phenylalanine and tyrosine were measured under the following measurement conditions.
The chromatograms of the -body are shown in Figures 7-9.

Equipment High performance liquid chromatograph (trade name manufactured by Spectra Physics, Inc.,
SP8000) Column Stainless steel 4.8mm ID, 250mm Eluent H ₂ O−CH ₃ CN (47:53) −0.15M
NH ₄ Cl flow rate 1.0ml/min Detector Ultraviolet, 210nm

[Brief explanation of the drawing]

Figures 1 to 6 are chromatograms in which the L-form and D-form of tryptophan, phenylalanine, tyrosine, valine, dopamine, and mandelic acid obtained using the packing material of the present invention are separated. . Figures 7 to 9 are chromatograms in which the L-form and D-form of tryptophan, phenylalanine, and tyrosine were separated using a conventional packing material in which optically active amino acids were immobilized on silica gel. be. 1 L-form of various amino acids and mandelic acid 2 D-form of various amino acids and mandelic acid

Claims (1)

[Claims] 1. A packing material for separating optical isomers, which is obtained by immobilizing histidine on the surface of silica gel pores.