JPH05133948A - Packing agent of ligand exchange chromatography method of resolving optical isomer using the same - Google Patents

Abstract

PURPOSE:To obtain a means of resolving an optical isomer by a ligand exchange chromatography which performs a detection at higher sensitivity with a lower background and allows the minimization of the amount of a chiral reagent to be used. CONSTITUTION:This method of resolving an optical isomer uses a ligand exchange chromatography packing agent which has a carbon based particles coated with amino acid containing N-aryl sulfonyl-optically active aromatic ring.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a ligand exchange chromatography packing material which can be used for resolution of optical isomers such as amino acids, and a method for resolving optical isomers using the packing material.

[Prior Art]

Conventionally, as a method for resolving optical isomers,
A method called ligand exchange chromatography is known. For example, in Japanese Examined Patent Publication No. 60-11023, a silica is used as a packing material, and a chiral reagent, N- (p-toluenesulfonyl) -L-phenylalanine, and copper sulfate are used as a mobile phase to measure the amino acid content by reverse phase chromatography. Methods for resolving optical isomers are described.
Similarly, in JP-A-61-93145, silica is used as a filler and N which is a chiral reagent is used as a mobile phase.
A method for resolving optical isomers of amino acids by reverse phase chromatography using-(p-toluenesulfonyl) -D-phenylglycine and copper sulfate is described. However, when attempting to resolve the optical isomers of amino acids by a method that includes an amino acid-based chiral reagent in the mobile phase, the background for detection will be high, so if the amino acid concentration in the sample is not high, the UV However, there was a problem in that the sensitivity of the detection was lowered because it could not be detected by measuring the absorbance. In addition, since the amount of chiral reagent required is large, the cost is high.

[0003]

Therefore, an object of the present invention is to provide a reversed-phase ligand exchange chromatography which has a low background of detection, can be performed with good sensitivity, and requires a small amount of chiral reagent. The purpose is to provide a means for the resolution of the optical isomers by chromatography.

[0004]

As a result of earnest research, the inventors of the present invention have used carbon-based particles coated with an amino acid containing an N-arylsulfonyl-optically active aromatic ring as a packing material for ligand exchange chromatography. It is found that the above-mentioned purpose can be achieved by using
The present invention has been completed.

That is, the present invention provides a ligand-exchange chromatography packing material in which carbon-based particles are coated with an amino acid containing an N-arylsulfonyl-optically active aromatic ring. Furthermore, the present invention comprises conducting a ligand exchange chromatography by passing a sample containing optical isomers in the presence of a salt of a divalent or higher valent transition metal through a column containing the packing material of the present invention. A method for resolving isomers is provided.

As described above, in the filler of the present invention, carbon-based particles are coated with N-arylsulfonyl-optically active aromatic ring-containing amino acid as a chiral reagent. The N-arylsulfonyl-optically active aromatic ring-containing amino acid is not particularly limited, but N- (1-naphthalene) sulfonyl-D or L-phenylglycine and N
-(P-Toluene) sulfonyl-D- or L-phenylglycine is preferred. The N-arylsulfonyl-optically active aromatic ring-containing amino acid may be, as described in detail later,
The carbon-based particles can be coated by a so-called dynamic coating method, which comprises passing a solution of N-arylsulfonyl-amino acid containing an optically active aromatic ring through a column packed with carbon-based particles. When silica particles are coated with such a chiral reagent, the bond between the silica particles and the chiral reagent is weak due to the hydrophobic bond, and thus the chiral reagent is not sufficiently retained on the silica particles. Therefore, in the prior art using silica-based particles, it was necessary to include the chiral reagent in the mobile phase as described above. In the present invention, since the carbon-based particles are used, the N-arylsulfonyl-optically active aromatic ring amino acid is firmly bound to the particles, and the filler is repeatedly used without including the chiral reagent in the mobile phase. be able to. The bond between the N-arylsulfonyl-optically active aromatic ring-containing amino acid and the carbon-based particle is probably π-.
This is due to the π-electron bond, which is stronger than the hydrophobic bond.

The carbonaceous particles used in the present invention are volatile organic substances, melt-soluble organic substances, meltable polymers, thermosetting polymers, meltable coal, non-meltable coal, infusible fibrous organic substances,
Carbon black, pyrolytic carbon, made from wood, etc.
Carbon whiskers, activated carbon, glassy carbon, coke, artificial graphite, isotropic graphite, molecular sieve carbon, mesophase carbon, carbon fibers and the like are molded into a preferable particle size as a filler, and any one can be prepared if the particle size is adjusted. It may be carbon-based particles. When used for analysis, the particle size is 1 to 100 μm, preferably 1 to 20 μm
5 to 500μ particle size when used for preparative separation
m, preferably about 10 to 200 μm. Examples of particularly preferable carbon-based particles include those described in Japanese Patent Application No. 2-128633.

The filler of the present invention can be manufactured by the so-called dynamic coating method as follows. The coating solution used for dynamic coating is prepared as follows. Arylsulfonyl optically active aromatic ring-containing amino acid is dissolved in an organic solvent such as acetonitrile or methanol, and then this is mixed with an aqueous solution of a sodium salt of a weak acid such as sodium carbonate, and finally a transition metal salt having a valence of 2 or more (for example, Salts of copper, zinc, nickel, cadmium, cobalt and the like can be mentioned, but a metal salt of copper is preferable) and dissolved to prepare a coating solution. The filler of the present invention can be produced by passing this coating solution through a column filled with carbon-based particles. The above-mentioned method is based on the dynamic coating method, but it is also possible to fill the column with what is coated by bringing the carbon-based particles into contact with the solution in the container.

The substance capable of resolving optical isomers using the column packed with the packing material of the present invention is a substance having two or more atoms capable of coordinative bonding, such as amino acids and oxyacids. , Preferably amino acids.

Reversed phase ligand exchange chromatography according to the method of the present invention using a column packed with the packing material of the present invention can be carried out basically in the same manner as the conventional method. However, in the method of the present invention, it is not necessary to include a chiral reagent in the mobile phase, and therefore, the mobile phase does not contain the transition metal salt coated on the column in water and / or acetonitrile or methanol having an affinity for water. Only the metal salt contained in such a solvent may be used. For example,
A 0.5 mM copper sulfate aqueous solution or a solution obtained by adding acetonitrile thereto can be preferably used. The flow rate of the mobile phase may be about 1 ml / min. Also, chromatography can be performed at room temperature. Fraction detection
Preferably, it can be carried out by measuring the absorbance at the maximum absorption wavelength of the sample substance. For example, when the sample is an amino acid, the absorbance at 230 nm based on the complex formation between the amino acid and copper (II) ion is measured. It can be done by

[0011]

INDUSTRIAL APPLICABILITY In the ligand exchange chromatography using the packing material of the present invention, since the chiral reagent is stably retained by the carbon-based particles, it is not necessary to include the chiral reagent in the mobile phase. Therefore, when separating amino acids using an amino acid-based chiral reagent, the background of detection is low, and therefore it is possible to detect in the ultraviolet region, which is the maximum absorption wavelength range of amino acids, and the sensitivity of chromatography is high. Get higher In addition, the amount of chiral reagent used can be reduced. Furthermore, since the chiral reagent is stably retained on the carbon-based particles, chromatography using the packing material of the present invention is highly reproducible. Furthermore, the filler of the present invention can be repeatedly regenerated and used for a long period of time.

[0012]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 5% by volume of vacuum distillation residual oil having an average molecular weight of 600, 5% by volume of divinylbenzene, 1% by weight of polyvinyl alcohol, 0.25% by weight of azobisisobutyronitrile, 5% by volume of toluene and ion-exchanged water (remainder) The mixture consisting of 1) was stirred at a high speed using a laboratory diperser at a temperature of 20 ° C. or lower. The mixture was then heated to 80 ° C. for 6 hours with stirring. The resulting beads are then collected by filtration,
It was dried at 100 ° C. The beads were then infusibilized by heating in air at 350 ° C. for 3 hours. This was fired at 2500 ° C. in a nitrogen gas atmosphere. After firing, ultrasonic treatment in benzene, washing with methanol / ether, drying at 100 ° C. and classification were carried out to obtain carbon-based particles having a particle size of 7 μm.

The results of elemental analysis of the obtained particles are carbon 10
It was 0%, and hydrogen, oxygen, nitrogen and sulfur were not detected. The total pore volume is 0.4442 ml / g, and the volume of pores with a radius of 1 to 10 nm is 0.0768 ml / g, 10 to 10 nm.
The volume of pores of 50 nm is 0.3569 ml / g and the volume of pores of 50 nm or more is 0.0105 ml / g. Therefore, the pore volume index (1
0-50) / (1-50) was 82.3%.

The carbon particles thus obtained were packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 100 mm, and the coating solution prepared as described below was used at a flow rate of 1 ml / min.
The solution was allowed to flow in ml, and carbon-based particles were dynamically coated with N- (1-naphthalene) sulfonyl-D-phenylglycine. The coating amount of N- (1-naphthalene) sulfonyl-D-phenylglycine is about 200 μmol /
It was g. The coating solution used for dynamic coating was prepared as follows. 400 mg of N- (1
-Naphthalene) sulfonyl-D-phenylglycine 4
A coating solution was prepared by dissolving in 0 ml of acetonitrile, then mixing this in a 100 mg / l aqueous solution of sodium carbonate, and finally adding and dissolving 125 mg of copper sulfate.

Using the column obtained, a concentration of 1 mg / ml
5 μl of the D, L-alanine aqueous solution was injected and the chromatography was performed. A 0.5 mM aqueous copper sulfate solution was used as the mobile phase. The flow rate was 1 ml / min and the operation was performed at room temperature. The detection of amino acids was performed by measuring the absorbance at 230 nm based on the complex formation between amino acids and copper (II) ions.

The obtained chromatogram is shown in FIG. As shown in FIG. 1, the D-alanine peak and the L-alanine peak are clearly separated, and it can be seen that the optical isomers of amino acids can be resolved using the packing material of the present invention.

Example 2 A sample amino acid was D, L-leucine, and the mobile phase was 0.
The same operation as in Example 1 was carried out except that it was composed of a 5 mM copper sulfate aqueous solution 80%, acetonitrile 16% and water 4%. The obtained chromatogram is shown in FIG. As shown in FIG. 2, the D-leucine peak and the L-leucine peak were clearly separated.

Example 3 The same operation as in Example 2 was performed except that the sample amino acid was D, L-valine. The obtained chromatogram is shown in FIG. As shown in FIG. 3, the D-leucine peak and the L-leucine peak were clearly separated.

[Brief description of drawings]

FIG. 1 is a diagram showing a chromatogram obtained by subjecting D, L-alanine to ligand exchange chromatography using the packing material of the present invention.

FIG. 2 is a diagram showing a chromatogram obtained by subjecting D, L-leucine to ligand exchange chromatography using the packing material of the present invention.

FIG. 3 is a diagram showing a chromatogram obtained by subjecting D, L-valine to ligand exchange chromatography using the packing material of the present invention.

Claims (3)

[Claims] 1. Carbon-based particles containing N-arylsulfonyl-
A ligand exchange chromatography packing material which is formed by coating an amino acid containing an optically active aromatic ring. 2. The N-arylsulfonyl-optically active aromatic ring-containing amino acid is N- (1-naphthalene) sulfonyl-D-phenylglycine, N- (1-naphthalene).
Sulfonyl-L-phenylglycine, N- (p-toluene) sulfonyl-D-phenylglycine and N- (p-
The filler according to claim 1, which is selected from the group consisting of toluene) sulfonyl-D-phenylglycine. 3. Ligand exchange chromatography is carried out by passing a sample containing optical isomers through a column containing the packing according to claim 1 or 2 in the presence of a salt of a divalent or higher valent transition metal. Method for resolving optical isomers consisting of.