JPH05249093A - Column for liquid chromatography - Google Patents

Abstract

PURPOSE:To obtain a column, for liquid chromatography use, which is extremely useful for measuring the purity of, and for separating, the mixture of an optically active substance or the mixture of a structural isomer. CONSTITUTION:The title column is a column, for liquid chromatography use, which has been prepared from a filler in which a carrier has been coated with a polysaccharide derivative. When 5mum of (+ or -)-alpha-phenylethyl alcohol at a concentration of 1mg/ml is injected under the following conditions, the theoretical step height of a first peak which has been obtained is 0.025mm or lower: mobile- phase n-hexane/2-propanol = 9/1; temperature: 25 deg.C; and flow rate: 0.5ml/min.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a column for liquid chromatography, more specifically, a packing material prepared by coating a carrier with a polysaccharide derivative, and a packing material prepared from the packing material.
For example, it relates to a column for liquid chromatography which is extremely useful for optical resolution.

[0002]

Okamoto et al. Have already reported an optical resolving agent using a filler obtained by coating a silica gel with a polysaccharide derivative (Japanese Patent Publication No. 63-12850). However, the number of column stages was low. Therefore, the object of the present invention is to
An object of the present invention is to provide a column for liquid chromatography that has a high number of column stages and is useful for measuring the purity and separating a mixture of an optically active substance or a mixture of structural isomers.

[0003]

Means for Solving the Problems The inventors of the present invention have made earnest studies on a method of coating a carrier and a method of packing to solve the above problems, and as a result, have found an excellent packing material and a column, and completed the present invention. .. That is, the present invention uses the mobile phase n-
Hexane / 2-propanol = 9/1, temperature 25 ℃, flow rate
Under the condition of 0.5 ml / min, a concentration of 1 mg / ml (±) -α-
A column for liquid chromatography prepared from a packing material coated with a polysaccharide derivative, characterized in that the theoretical plate height of the first peak obtained when 5 μl of phenylethyl alcohol was injected was 0.025 mm or less. Is provided.

Here, the theoretical height of the first peak is a value obtained by the following equation. First peak theoretical plate height (mm) = column length (mm) / number of first peak plates

[0005]

[Equation 1]

The carrier in the present invention is 500 to 5000.
Those having a pore size of Å are preferable, and more preferably 10
It has a pore size of 00 to 4000Å. For example, silica, alumina, zirconia, magnesia, glass, kaolin, titanium oxide, silicate, etc. are preferable, and silica gel is preferable. The surface of these carriers may be treated in order to improve the affinity of the polysaccharide derivative and to modify the surface of the carrier itself. The particle size of the carrier in the present invention is preferably a porous carrier having an average particle size of a value between 3 μm and 10 μm, and most preferably an average particle size of a value between 3 μm and 5 μm.

The polysaccharide in the present invention may be any one of optically active ones, whether synthetic polysaccharides, natural polysaccharides or natural product-modified polysaccharides, but preferably those having a high regularity of binding mode. .. For example, β-1,4-glucan (cellulose), α-1,4-glucan (amylose, amylopectin), α-1,6-glucan (dextran), β-1,6-glucan (pustulan), β-1,3
-Glucans (eg curdlan, schizophyllan, etc.),
α-1,3-glucan, β-1,2-glucan (CrownGall polysaccharide), β-1,4-galactan, β-1,4-mannan, α
-1,6-mannan, β-1,2-fructan (inulin)
, Β-2,6-fructan (levan), β-1,4-xylan, β-1,3-xylan, β-1,4-chitosan, β-
1,4-N-acetylchitosan (chitin), pullulan, agarose, alginic acid, α-cyclodextrin, β-
Examples thereof include cyclodextrin and γ-cyclodextrin, and also include starch containing amylose. Particularly preferred are cellulose, amylose, β-1,4-chitosan, chitin, which can easily obtain high-purity polysaccharides,
β-1,4-mannan, β-1,4-xylan, inulin,
For example, a curd run. The number average degree of polymerization of these polysaccharides (the average number of pyranose or furanose rings contained in one molecule) is 5 or more, preferably 10 or more, but there is no particular upper limit, but 500 or less is easy to handle. preferable.

As the polysaccharide derivative in the present invention, 50% to 100% of the hydroxyl group and amino group of the polysaccharide are an aromatic or aromatic alkyl or aromatic alkynyl ester group or carbamoyl group which may have a substituent. Substituted derivatives and the like are mentioned, and specifically, a hydroxyl group and /
Alternatively, the hydrogen atom of the amino group is substituted with a group represented by the following general formula (1), (2), (3) or (4).

[0009]

[Chemical 1]

In the formula, Q is an aromatic group which may contain a hetero atom, which may be unsubstituted or has 1 to 1 carbon atoms.
12 alkyls, 1 to 12 carbons alkoxy, 1 to 1 carbons
From 12 alkylthio, cyano, halogen, acyl having 1 to 8 carbons, acyloxy having 1 to 8 carbons, hydroxy, alkoxycarbonyl having 1 to 12 carbons, nitro, and di (alkyl having 1 to 8 carbons) amino It may be substituted by one or more groups of the group Illustrative aromatic groups include phenyl, naphthyl, phenanthryl, anthracyl, indenyl, indanyl, furyl, thionyl, pyryl, benzofuryl, benzthionyl, indyl, pyridyl, pyrimidyl, quinolyl and isoquinolyl. Particularly preferred are phenyl, naphthyl and pyridyl.

X is a divalent hydrocarbon having 1 to 4 carbon atoms and may contain a double bond or a triple bond. Two
Examples of valent hydrocarbon groups are methylene, ethylene, ethylidene, ethenylene, ethynylene, 1,2- or 1,3-propylene, 1,1- or 2,2-propylidene and the like.

In the present invention, the amount of the polysaccharide derivative when the carrier is coated with the polysaccharide derivative is 1 to the carrier.
100% by weight is preferable, and 5 to 50% by weight is more preferable.
Examples of the method for coating the carrier with the polysaccharide derivative according to the present invention include a method in which a solution in which the polysaccharide derivative is dissolved is dropped while stirring the porous carrier, and the carrier is dried by reducing pressure or injecting gas. The solvent for dissolving the polysaccharide derivative, which is used when the polysaccharide derivative is coated on the carrier in the present invention, is a polysaccharide derivative-soluble solvent having a boiling point of 100 ° C or lower that may contain 30% or less of a solvent having a boiling point of 100 ° C or higher ..

The packing of the packing material obtained as described above in a column is carried out by alkane having 5 to 10 carbon atoms, alcohol having 1 to 10 carbon atoms, or alkane having 5 to 10 carbon atoms and 1 to 1 carbon atoms. A method of dispersing a filler in a mixed solution of 10 alcohols and pressurizing with the same solvent can be mentioned. To exemplify an alkane having 5 to 10 carbon atoms, n-pentane,
Examples thereof include n-hexane, cyclohexane, n-heptane, isoheptane, n-octane, isooctane and n-nonane, and n-hexane is preferable. To exemplify an alcohol having 1 to 10 carbon atoms, methanol, ethanol, n-propanol, 2-propanol, n-
Examples thereof include butanol, 2-butanol, t-butanol and the like, but preferably methanol, ethanol and 2-
It is propanol.

The column obtained by the present invention can be used for measuring the purity of a mixture of optically active substances or a mixture of structural isomers, and for separation.

[0015]

EXAMPLES The present invention will be described below with reference to examples.
The invention is not limited to these examples.

Example 1 A 500 ml three-necked flask was charged with an average particle size of 5 μm and a pore size of 1000.
Add 9.6 g of Å silica gel and stir at a rotation speed of about 200 rpm with a stirring blade using a mechanical stirrer. Cellulose tris (3,5-dimethylphenyl carbamate)
2.4 g of acetone / tetramethylurea = 9/1 mixed solution 1
The solution dissolved in 8 ml is gradually added dropwise with a dropping funnel. After completion of dropping, nitrogen is flowed at a flow rate of 2 liters per minute while continuing stirring to distill off the organic solvent. Disperse the obtained particles in methanol, and use a slurry method in a stainless steel column with an inner diameter of 0.46 cm and a length of 25 cm for 300 kg / methanol.
A column for liquid chromatography was obtained by pressurizing and filling cm ² .

Example 2 The column obtained in Example 1 was loaded with mobile phase n-hexane / 2.
-Propanol = 9/1, temperature 25 ° C, flow rate 0.5 ml / min
Under the conditions described above, 5 μl of (±) -α-phenylethyl alcohol having a concentration of 1 mg / ml was injected, and the theoretical plate height of the first peak was measured by the following formula. The results are shown in Table 1. First peak theoretical plate height (mm) = column length (mm) /
Number of steps in the first peak

[0018]

[Equation 2]

Example 3 A 500 ml three-necked flask was charged with an average particle size of 7 μm and a pore size of 1000.
Add 9.6 g of Å silica gel and stir at a rotation speed of about 200 rpm with a stirring blade using a mechanical stirrer. A solution prepared by dissolving 2.4 g of cellulose tri (p-methylbenzoate) in 15 ml of a mixed solution of methylene chloride / methyl benzoate = 9/1 is gradually added dropwise with a dropping funnel. After completion of the dropping, nitrogen is flown at a flow rate of 2 liters per minute while continuing stirring to distill off the organic solvent. The obtained particles were dispersed in methanol, and a stainless steel column having an inner diameter of 0.46 cm and a length of 25 cm was charged with 300 kg / cm ² of methanol by a slurry method and packed to obtain a column for liquid chromatography.

Example 4 The column obtained in Example 3 was loaded with mobile phase n-hexane / 2.
-Propanol = 9/1, temperature 25 ° C, flow rate 0.5 ml / min
Under the above conditions, 5 μl of (±) -α-phenylethyl alcohol having a concentration of 1 mg / ml was injected, and the theoretical plate height of the first peak was measured in the same manner as in Example 1. The results are shown in Table 1.

Example 5 A 500 ml three-necked flask was charged with an average particle size of 5 μm and a pore size of 1000.
Add 9.6 g of Å silica gel and stir at a rotation speed of about 200 rpm with a stirring blade using a mechanical stirrer. Add 2.4 g of cellulose tribenzoate to bromochloromethane /
Nitrobenzene = 86/14 A solution dissolved in 15 ml of mixed solution is gradually added dropwise with a dropping funnel. After completion of the dropping, nitrogen is flown at a flow rate of 2 liters per minute while continuing stirring to distill off the organic solvent. The obtained particles were dispersed in methanol, and a stainless steel column having an inner diameter of 0.46 cm and a length of 25 cm was charged with 300 kg / cm ² of methanol by a slurry method and packed to obtain a column for liquid chromatography.

Example 6 The column obtained in Example 5 was charged with mobile phase n-hexane / 2.
-Propanol = 9/1, temperature 25 ° C, flow rate 0.5 ml / min
Under the above conditions, 5 μl of (±) -α-phenylethyl alcohol having a concentration of 1 mg / ml was injected, and the theoretical plate height of the first peak was measured in the same manner as in Example 1. The results are shown in Table 1.

[0023]

[Table 1]

Measurement conditions Mobile phase n-hexane / 2-propanol = 9/1 Flow rate 0.5 ml / min Temperature 25 ° C. Detection UV (254 nm) In addition, liquid chromatography equipment manufactured by JASCO Corporation ( 880-PU pump, 875-UV detector) was used.

Claims (8)

[Claims] 1. A (±) -α-phenylethyl alcohol 5 having a concentration of 1 mg / ml under the conditions of mobile phase n-hexane / 2-propanol = 9/1, temperature 25 ° C. and flow rate 0.5 ml / min.
A column for liquid chromatography prepared from a packing material in which a carrier is coated with a polysaccharide derivative, characterized in that the theoretical plate height of the first peak obtained when μl is injected is 0.025 mm or less. 2. The column according to claim 1, which is prepared from a packing material in which a polysaccharide derivative is coated on a porous carrier having a pore size of 500 to 5000Å. 3. The column according to claim 1, which is prepared from a packing in which a polysaccharide derivative is coated on a porous carrier having an average particle diameter of a value between 3 μm and 10 μm. 4. A polysaccharide derivative in which 50% or more and 100% or less of a hydroxyl group or an amino group of a polysaccharide is substituted with an ester group of an aromatic, aromatic alkyl or aromatic alkynyl which may have a substituent or a carbamoyl group. The column according to claim 1, which is prepared from a packing material in which the carrier is coated. 5. The column according to claim 1, 2, 3 or 4, which is used for measuring the purity or separating a mixture of an optically active substance or a mixture of structural isomers. 6. The polysaccharide derivative is treated with a solvent having a boiling point of 100 ° C. or higher.
The method for producing a filler according to claim 1, wherein the porous carrier is coated with a solution dissolved in a soluble solvent having a boiling point of 100 ° C or less, which may contain 30% or less, and dried. 7. The method for producing a filler according to claim 1, wherein the solution in which the polysaccharide derivative is dissolved is added dropwise while stirring the porous carrier and dried by reducing the pressure or injecting a gas. 8. Particles obtained by coating a porous carrier with a polysaccharide derivative are alkanes having 5 to 10 carbon atoms, alcohols having 1 to 10 carbon atoms, or alkanes having 5 to 10 carbon atoms and 1 carbon atom.
The method for manufacturing a column according to claim 1, wherein the column is dispersed in a mixed solution of alcohols of 10 to 10 and the column is packed by a slurry method using pressure with the same solvent.