JP2590184B2 - Separation method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for efficiently separating a mixture of compounds having a carboxyl group such as an amino acid by liquid chromatography.

[Conventional technology and problems]

Various studies have been made on a method for efficiently separating a mixture of compounds containing a carboxyl group such as an amino acid, for example, a mixture of optical isomers, by liquid chromatography.

However, none of the conventionally proposed methods has been satisfactory in terms of separation performance.

[Means for solving the problem]

The present inventors have made intensive studies on such problems, and as a result, completed the present invention.

That is, the present invention provides a separation method comprising, when separating a mixture of compounds containing a carboxyl group in a normal phase system in liquid chromatography, adding 0.01 to 20% by weight of an acid to a developing solvent. About.

In the present invention, the normal phase system refers to a case where the mobile phase has lower polarity than the stationary phase, and refers to a normal phase system, for example, a case where silica gel is used as the stationary phase and hexane or the like is used as the mobile phase.

In the present invention, the compound containing a carboxyl group is a compound containing at least one carboxyl group in the molecule of the organic compound. The mixture includes a mixture composed of optical isomers. Examples include optically active amino acids.

Also, there is a mixture of other isomers such as geometric isomers and positional isomers. By way of example, geometric isomers include cis-trans isomers, ortho, para, meta isomers, and the like. Regioisomers include various cyclic polysaccharides. In addition, there is a mixture of various organic compounds containing a carboxyl group.

In the present invention, if the organic compound containing a carboxyl group has a functional group other than the carboxyl group, it may be chemically modified to increase the separation efficiency.

As described above, the separation method of the present invention is most suitable for separation of various isomers, particularly optical isomers.

As a filler used in the separation method of the present invention, when various solvents or a mixture thereof are used as a developing solvent for liquid chromatography, it is possible to select a filler that shows a normal phase system in at least one kind of solvent system. is important.

Such fillers include inorganic fillers and organic fillers. Examples of the inorganic filler include silica gel, alumina, and glass, and examples of the organic filler include homopolymers such as polystyrene, polyacrylamide, and polyacrylate, and copolymers composed of various reactive monomers.

In particular, as a filler for separating isomers, for example, optical isomers, a compound having an asymmetric center, an asymmetric center, or a molecule having an asymmetric center, that is, a compound having an asymmetric carbon is used as an active ingredient. Examples of the filler include those using an optically active amino acid. As a concrete example, see
1350 and JP-A-60-66162.
Examples of the polymer type include polyamino acids, polysaccharides such as cellulose and agarose, and derivatives thereof.

When the polysaccharide or a derivative thereof is specifically shown, the polysaccharide here refers to any one of a synthetic polysaccharide, a natural polysaccharide, and a modified natural polysaccharide, as long as it is optically active,
Preferably, it is a highly regular homoglycan and the binding mode is constant. More preferably cellulose, amylose, β-1,4-chitosan, chitin, β-1,4-xylan, which can easily obtain a high-purity polysaccharide,
Inulin, α-1,3-glucan, β-1,3-glucan and the like. The polysaccharide derivative means that some or all of the hydrogen atoms on the hydroxyl group or amino group of the polysaccharide, that is, 30% or more, preferably 50% or more, and more preferably 85% or more are substituted with another atomic group. Things. The atomic group here is R ″ is an aliphatic group having 1 to 3 oxygen atoms, 3 to 8
A cyclic aliphatic group consisting of an aromatic group having 6 to 20 carbon atoms or a heteroaromatic group having 4 to 20 carbon atoms,
As the atomic group, one kind of these may be used, or two or more kinds may be used. This is preferably selected in terms of separation performance.

Further, any of them may have a substituent. Examples of the substituent herein include an alkyl group which may have a C _{1 to} C ₅ branch such as a methyl group and a t-butyl group, and a halogen such as chlorine. Various substituents can be used in a range in which is improved. In addition, the number of substituents can be selected to be 1 or more as long as the performance of separation is improved. Further, for example, when an aromatic group such as a phenyl group is used, when one kind of substituent is used, the position of the substituent can be selected within the range of improving the separation performance such as ortho, meta and para positions. Further, when there are two kinds of substituents, various arrangements can be selected in the same manner as in the 3,4 and 3,5 positions.

These derivatives can be easily obtained using various known chemical reactions. For example, when a cellulose derivative containing an aromatic group is shown as an example of synthesis, a part or all of hydrogen of a hydroxyl group of cellulose is substituted with an aromatic group. Examples of the type of bond in this substitution include an ester bond, an ether bond, and a urethane bond.

As an example of the polysaccharide or a derivative thereof as described above, examples of the polysaccharide include microcrystalline cellulose, and examples of the polysaccharide derivative include cellulose triacetate, cellulose tribenzoate, cellulose tris (p-methylbenzoate), cellulose trisphenyl carbamate, Cellulose tris (p-methylphenyl carbamate), cellulose tris (3,5-dimethylphenyl carbamate), cellulose tris (p-chlorophenyl carbamate), cellulose tris (pt-butylphenyl carbamate), cellulose tricinnamate, etc. .

In addition, derivatives of polysaccharides other than cellulose, such as amylose, chitosan, xylan, and dextran, as described above can also be used.

Examples of the compound having molecular asymmetry include, for example, an optically active polymer mainly containing a polymerizable monomer represented by the following formula.

Wherein R ₁ , R ₂ and R ₃ are the same or different Y is an alkyl group, halogen or amino group which may have a C _{1 to} C ₄ branch, and l and m indicate the number of substituents and are integers from 1 to 5.

Also, JP-A-61-160059, JP-A-61-162750-1 and JP-A-51-160059.
81891 and the like.

Filler used in the present invention, both inorganic and organic, as it is, 1 μm to 10 mm, preferably 1 μm to 300 μm
It can be used as a carrier having a particle size of m. Preferably, it is porous and has an average pore size of 10 to 100 μm.
m, preferably 30 ° to 1000 °.

In addition, for organic materials, it is preferable that the carrier retain 1 to 100% by weight, preferably 5 to 50% by weight.

As the carrier, an organic carrier may be used, but an inorganic carrier such as silica gel is preferable, and the properties thereof are as described above. Further, as a method for retaining, there are a physical method, a method of chemically bonding and the like, and it is preferable that the carrier is subjected to a surface treatment such as a silane treatment using an organic silane compound.

The normal phase solvent used in the present invention includes a compound having low polarity, and is preferably a hydrocarbon which may have a C _{1 to} C ₁₀ branch. However in some cases polar solvents in a range that does not break the normal phase, for example, C ₁ -C may be such as the addition of ₁₀ alcohol and the better the improved separation efficiency.

Examples of the acid used in the present invention include organic acids and inorganic acids. Examples of the organic acid include formic acid, acetic acid, propionic acid, dichloroacid, trichloroacetic acid, and trifluoroacetic acid. , Phosphoric acid and the like.

It is particularly preferable to use an acid stronger than the acid strength of the compound containing a carboxyl group to be separated.

More preferably, the acid strength pKA should be 5 or less, preferably 4 or less. Examples thereof include formic acid (3.7) and trifluoroacetic acid (0.23).

In the separation method of the present invention, an acid is added to a normal phase developing solvent, but the amount of the acid used is based on the entire normal phase developing solvent.
The content is preferably 0.01 to 20% by weight, more preferably 0.1 to 10% by weight.

〔Example〕

Hereinafter, the present invention will be described in detail by Synthesis Examples and Examples,
The present invention is not limited to these examples. In addition, the definition of the term represented in an Example is as follows.

Synthesis Example 1 (filler) Synthesis of cellulose tris (3,5-dimethylphenylcarbamate) 1 g of microcrystalline cellulose (manufactured by Merck, degree of polymerization 100), pyridine 50 ml, 3,5-dimethylphenyl isocyanate 6.5
After heating the ml at 100 ° C. for 17 hours, the reaction mixture was poured into 500 ml of methanol. The resulting precipitate was separated by filtration and dried to obtain a 3,5-dimethylphenylcarbamate derivative. Substitution degree is almost
100%.

Yield 3.26g Yield 88% One part of the obtained cellulose tris (3,5-dimethylphenylcarbamate) was dissolved in 8 parts of acetone, and treated with diphenylsilane-treated silica gel (manufactured by Merck, Inc., Lichrosph).
er Si-1000), and the acetone was distilled off under reduced pressure to obtain a filler. The filler was packed into a column having an inner diameter of 0.46 cm and a length of 25 cm by a slurry method using methanol.

Example 1 Table 1 shows the results of optically resolving various optically active carboxylic acids at a developing solvent temperature of 25 ° C. and a flow rate of 0.5 ml / min using the filler obtained in the above Synthesis Example.

Example 2 A similar filler was prepared using Amylose instead of Cellulose in Synthesis Example 1,
Table 2 shows the results of various separations using the 5-dimethylphenylcarbamate derivative.

Example 3 Various fillers were synthesized in the same manner as in Synthesis Example 1, and the results of separation were shown in Table-3.

Example 4 Tables 4 and 5 show the results of the examination of the developing solvent using the filler of Synthesis Example 1.

[Effects of the Invention] According to the present invention, by using an acid in a normal phase system, the efficiency of separation of a carboxyl group-containing compound by liquid chromatography can be significantly improved.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location C07C 59/125 9450-4H C07C 59/125 B 59/50 59/50 61/04 61/04 61 / 09 61/09 61/28 61/28 61/39 61/39 271/22 271/22 C07D 209/42 C07D 209/42

Claims (1)

(57) [Claims] 1. A method for separating a mixture of compounds having a carboxyl group in a normal phase system by adding 0.01 to 20% by weight of an acid to a developing solvent in liquid chromatography. .