JP4151901B2 - Separation method by liquid chromatography. - Google Patents

Description

  The present invention relates to a method for separating an organic mixture by liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient.

  Conventionally, a polysaccharide derivative or a separating agent carried on a carrier such as silica gel exhibits excellent characteristics as a stationary phase for liquid chromatography, and therefore various structurally similar compound groups including optical isomers. (Hereinafter sometimes referred to as “organic mixture”).

  In liquid chromatography using this separating agent, depending on the type of compound to be separated, the peak shape of the elution curve becomes abnormally wide and the separation may be insufficient. Several methods have been proposed to improve the segregation.

For example, Patent Document 1 discloses that when liquid chromatography is performed using a separating agent containing a polysaccharide derivative as an active ingredient, a perchlorine mixture of 100: 0 to 0: 100 of water and a water-soluble organic solvent as a mobile phase. There has been proposed a separation method by liquid chromatography, which is characterized by using a solution to which a highly cationic salt such as sodium acid is added.
Patent Document 2 proposes a separation method by liquid chromatography characterized by adding various salts to a normal phase mobile phase when performing liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient. Has been.
Patent Document 3 discloses a method for separating optical isomers using a basic mobile phase under reversed-phase conditions in the separation of optical isomers by liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient. ing.

  However, even when the techniques described in the above Patent Documents 1 to 3 are applied, when a basic compound such as an adenine derivative is separated by a normal phase mobile phase, separation is impossible or insufficient. There were many problems.

JP-A-5-85947 Japanese Patent Laid-Open No. 5-180818 JP 11-189551 A

  The present invention has been made in view of the above-mentioned problems, and in a method for separating an organic mixture by liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient, a basic compound such as an adenine derivative is particularly sure. It is an object of the present invention to provide a method for separating an organic mixture by liquid chromatography that can be separated into two.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor, when separating by liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient, when adding a little water to the normal phase mobile phase, It was found that the separation of the optical isomer mixture of the adenine derivative that could not be separated could be improved, and a sharper peak of the elution curve could be obtained, and the present invention was completed.

Thus, according to the present invention, a separation method by liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient, wherein water having a concentration of 500 to 5000 ppm is added to a normal phase mobile phase comprising an organic solvent. A method for separating an organic mixture by liquid chromatography is provided.
In the separation method of the present invention, the organic mixture is preferably a mixture of optical isomers of a basic organic compound.

  According to the present invention, in a separation method by liquid chromatography using a separating agent containing a polysaccharide derivative as an active ingredient, it is difficult or impossible to separate conventionally by a simple method of adding a small amount of water to a normal phase mobile phase. The organic mixture that was present can be reliably separated.

  Although the detailed mechanism of action of the present invention is unknown, (i) the polarity of the surface of the filler is increased, and the adsorption of the adenine derivative is less likely to occur, (ii) the trace amount in the silica gel that is the filler carrier of the optically active column When an adenine derivative is coordinated to a metal and tailing, adding water to the mobile phase covers the silica gel surface with water molecules, preventing coordination and improving the peak shape. Conceivable.

  In conventional high performance liquid chromatography (HPLC) analysis, the change in the amount of water in the mobile phase causes the retention time to fluctuate. For this reason, a silica gel tower is installed on the mobile phase bottle, etc. It prevented moisture absorption. Similarly, in the separation using a normal phase chiral column, it is known that water in the mobile phase adversely affects the degree of separation. The amount of water that has an influence is about 100 ppm to 700 ppm, and when it exceeds 500 ppm, the degree of separation suddenly decreases and separation may not be possible. It was common to have to control.

  On the other hand, in the separation method of the present invention, by adding water to the normal phase mobile phase so as to have a concentration of 500 ppm to 5000 ppm, the peak shape of the elution curve is sharpened, and organic compounds that have conventionally been difficult to separate Separation is possible.

  The method for separating an organic mixture of the present invention by liquid chromatography uses a separating agent containing a polysaccharide derivative as an active ingredient, and water having a concentration of 500 to 5000 ppm is added to a normal phase mobile phase comprising an organic solvent. It is characterized by adding.

The separating agent used in the present invention contains a polysaccharide derivative as an active ingredient.
The polysaccharide derivative to be used is one in which a part or all of the hydrogen atoms on the hydroxyl group of the polysaccharide, preferably 85% or more, are substituted with a substituent.
The polysaccharide used as a raw material for the polysaccharide derivative may be any optically active material, regardless of whether it is a synthetic polysaccharide, a natural polysaccharide, or a naturally-modified polysaccharide. Preferably, a high-purity polysaccharide can be easily obtained. Cellulose, amylose, β-1,4-chitosan, chitin, β-1,4-mannan, β-1,4-xylan, inulin, α-1,3-glucan, β-1,3-glucan . The number average degree of polymerization (average number of pyranose rings or furanose rings contained in one molecule) of these polysaccharides is 5 or more, and there is no particular upper limit, but from the viewpoint of handleability, it is preferably 500 or less.

Examples of the polysaccharide derivative used in the present invention include ester derivatives, carbamate derivatives, ether derivatives and the like of the above polysaccharides. Of these, carbamate derivatives or ester derivatives are preferable, and aromatic carbamate derivatives or aromatic ester derivatives are particularly preferable.
Preferable specific examples of the polysaccharide derivative used in the present invention include those shown below.

(In the formula, n represents an arbitrary natural number, R ′ represents an alkyl group such as a methyl group, an alkoxy group such as a methoxy group, a substituent such as a halogen atom such as chlorine, and m represents an integer of 0 to 5. In addition, when m is 2 or more, R ′ may be the same or different.)

  Among these, in the present invention, use of amylose tris (3,5-dimethylphenylcarbamate), cellulose tris (3,5-dimethylphenylcarbamate), cellulose tris (benzoate), or cellulose tris (4-methylbenzoate) Is particularly preferred.

  In order to use the above-mentioned polysaccharide derivative as a separating agent, it is made into particles or supported on a carrier such as silica gel. Usually, many supported on a carrier are used. Moreover, what is marketed as an optical isomer resolving agent can also be used as it is.

  In the present invention, a normal phase mobile phase (normal phase mobile phase) is used as a developing solvent for liquid chromatography. The normal phase system means that a mobile phase having a polarity lower than that of a stationary phase (a separating agent containing a polysaccharide derivative as an active ingredient) is used. As the organic solvent component of the normal phase mobile phase, for example, a nonpolar solvent such as n-hexane and a polar solvent compatible with the nonpolar solvent, preferably a mixture of alcohols as one component or a multicomponent mixture can be used.

The separation method of the present invention is characterized by using a mixed solvent in which water is added to the organic solvent component described above as a normal phase mobile phase so as to have a concentration of 500 to 5000 ppm, preferably 1000 to 5000 ppm.
The water added to the organic solvent component is not particularly limited, but it is preferable to use distilled water or deionized water with few impurities.

  According to the present invention, the peak shape of the elution curve can be improved by a simple method of adding a predetermined amount of water to the mobile phase, and an organic mixture that has been impossible or difficult to separate can be reliably ensured. It becomes possible to separate.

  According to the method of the present invention, there is no concern about the memory effect. The memory effect means that when an acidic compound is separated by a chiral column using a normal phase mobile phase, an organic acid (TFA or the like) is added to the mobile phase and analyzed for the purpose of improving the peak shape of the elution curve. When analysis of a neutral compound is performed using a mobile phase containing no TFA, the peak shape and resolution of the elution curve are deteriorated, and reproducibility cannot be obtained. In normal-phase chiral columns, the “memory effect” is often a problem, but according to the method of the present invention, such a memory effect does not occur. Therefore, even if a normal phase mobile phase containing no water is used after the method of the present invention is carried out, the resolution does not decrease.

  Further, according to the method of the present invention, even a compound having UV absorption only on the low wavelength side can be sufficiently detected, and high detection sensitivity can be obtained. That is, when an organic acid or organic base is added to the mobile phase for the purpose of improving the peak shape of the elution curve and analysis is performed using a UV detector, there is a problem of UV cutoff due to the addition of the organic acid or organic base. . That is, by adding an organic acid / organic base to the mobile phase, the background increases and the measurable UV wavelength is limited to the high wavelength side. For example, when n-hexane / ethanol based mobile phase is used, if 0.1% of DEA is added to the mobile phase, it should be possible to analyze at UV 230 nm originally, but it does not exceed 250 nm due to the addition of DEA. The background becomes high and analysis becomes difficult. There is no problem if all the compounds to be analyzed have UV absorption on the high wavelength side, but in reality, compounds having absorption only on the low wavelength side are not uncommon.

On the other hand, the method of the present invention is to add a small amount of water to the normal phase mobile phase. Although water absorbs UV having a wavelength of 185 nm or less, it has substantially no UV absorption, so even a compound having UV absorption only on the low wavelength side can be sufficiently detected, and the detection sensitivity can be increased. .
Further, since a very small amount of water is added to the mobile phase, post-treatment at the time of fractionation is also advantageous.

  Regarding the influence on the analytical column due to the addition of water to the mobile phase, the water-added mobile phase was continuously passed for 150 hours, then switched to a mobile phase to which no water was added, the adenine derivative was analyzed, the retention time and Since the peak shape of the tailed elution curve was the same as that before the addition of water, it is considered that there is no such influence.

  The organic mixture targeted by the separation method of the present invention is a mixture of organic compounds having similar chemical structures, physical properties, and chemical properties. Examples thereof include optical isomer mixtures, stereoisomer mixtures such as diastereomers, positional isomer mixtures, geometric isomer mixtures, compounds having different degrees of unsaturation, and compounds belonging to homologues.

  Among these, the separation method of the present invention is preferably used in the case where a mixture of optical isomers of a basic organic compound is separated into each optical isomer, and more preferably, a mixture isomer mixture of adenine derivatives is separated from each optical isomer. It can be applied when separating into isomers.

  In order to carry out the present invention, a normal liquid chromatography method may be used as it is. That is, a separation agent containing a polysaccharide derivative as an active ingredient is packed in a column, the organic mixture to be separated is charged on the top of the column, and the organic mixture is completely adsorbed on the packing, and then the normal phase is fed from the top at a constant rate. This can be done by letting the mobile phase flow down.

  The separation method of the present invention is to use a mixed solvent containing 500 to 5000 ppm of water as the normal phase mobile phase, thereby sharpening the peak shape of the elution curve of the organic matter and improving the resolution. In some cases, the separation ability can be further increased by changing the composition of the mobile phase and the flow velocity thereof.

  EXAMPLES Next, although an Example and a comparative example demonstrate this invention further in detail, this invention is not limited to a following example.

(1) Compound to be separated (R) -9- (2′-hydroxypropyl) adenine (compound (1R)) and (S) -9- (2′-hydroxypropyl) adenine (compound (1S)) shown below The mixed sample solution was analyzed. As for the sample, about 0.5 mg of each of the compound (1R) and the compound (1S) is weighed, dissolved in 5 ml of ethanol, filtered through a 0.2 μm Millipore filter, about 1 ml of each is weighed and mixed. Prepared.

（２）キラルカラム
キラルカラムについては、次のものを使用した。
・キラルカラム（Ａ）：セルローストリス（３，５−ジメチルフェニルカーバメート）（商品名：Ｏｐｔｉ−Ｐａｋ ＸＣ、ダイセル化学（株）製）
・キラルカラム（Ｂ）：セルローストリス（４−メチルベンゾエート）（商品名：ＣＨＲＡＬＣＥＬＯＪ−Ｈ、ダイセル化学（株）製）

(2) Chiral column The following were used about the chiral column.
Chiral column (A): cellulose tris (3,5-dimethylphenyl carbamate) (trade name: Opti-Pak XC, manufactured by Daicel Chemical Industries, Ltd.)
Chiral column (B): Cellulose tris (4-methylbenzoate) (trade name: CHLARECELOJ-H, manufactured by Daicel Chemical Industries, Ltd.)

(3) Separation conditions
(i) Analytical column: the above chiral column (A) or (B)
Average particle size: 10 μm
Column size: 3.9 mmφ × 300 mm (S / NC8420371)
(ii) Flow rate: 0.4 ml / min
(iii) Column oven: 40 ° C
(iv) Detection wavelength: UV254nm
(v) Detection sensitivity: 0.032 AUFS (detector integrator output: 1 V / AU)

Example 1
Using the chiral column (A), the mobile phase composition was n-hexane / 2-propanol = 800/200 (volume ratio), and water in an amount of 1000 ppm was added to the mobile phase. By adding 2-propanol as a compatible solvent, n-hexane and water were made uniform. An elution curve when 1000 ppm of water is added to the mobile phase is shown in FIG. In FIG. 1, the horizontal axis represents retention time (minutes), and the vertical axis represents peak intensity (the same applies to the following figures). From FIG. 1, it was possible to separate the compound (1R) and the compound (1S) by adding water to a normal normal phase mobile phase.

Comparative Example 1
In Example 1, it operated similarly to Example 1 except having made the mobile phase composition n-hexane / ethanol = 1000/100 (volume ratio). The elution curve is shown in FIG.
As is clear from FIG. 2, the compound (1R) and the compound (1S) could not be separated in a normal normal phase mobile phase to which water was not added.

Comparative Example 2
In Comparative Example 1, 0.1 vol% of normal phase mobile phase hediethylamine was added and the change in peak shape was observed. The elution curves are shown in FIGS. 3 (a) and 3 (b). As is clear from FIGS. 3A and 3B, the peak shape of the elution curve did not become sharp even when 0.1% by volume of diethylamine was added to the normal phase mobile phase of Comparative Example 1 (peak shape). Was not improved).

  Further, although not shown, in Comparative Example 1, a change in peak shape was observed by adding 0.5% by volume of normal phase mobile phase to diethylamine, but the peak shape of the elution curve was not sharp. .

Example 2
In Example 1, a chiral column (B) was used, and the mobile phase composition was n-hexane / 2-propanol / water = 900/100/2 (volume ratio) to perform separation by liquid chromatography. The elution curve is shown in FIG.
From FIG. 4, it was possible to separate the compound (1R) and the compound (1S) by adding water to a normal normal phase mobile phase.

Comparative Example 3
In Example 2, the same operation as in Example 2 was performed except that the mobile phase composition was n-hexane / 2-propanol = 900/100 (volume ratio). The elution curve is shown in FIG.
As is clear from FIG. 5, the compound (1R) and the compound (1S) could not be separated in a normal normal phase mobile phase to which water was not added.

Confirmation of damage to chiral column In Examples 1 and 2, it was found that the peak shape of the elution curve could be improved by adding water to the mobile phase, and the compound (1R) and the compound (1S) could be separated. However, water in the mobile phase of a normal-phase analytical column may deteriorate the separation state and damage the analytical column. Therefore, a mobile phase of n-hexane / 2-propanol = 800/200 (volume ratio) was sufficiently passed through the chiral columns (A) and (B) in which water was added to the mobile phase, and water on the surface of the filler was removed. After removing, the mobile phase was switched to n-hexane / 2-propanol = 900/150 (volume ratio), the adenine derivative was analyzed, and the damage state of the chiral column was confirmed.

  As a result of the examination, as before the addition of water to the mobile phase, both the R and S isomers had strong peak tailing and were not separated at all, and the state when the examination was started was reproduced. That is, it was confirmed that there was no damage to the chiral columns (A) and (B). In addition, the effect of adding water became clear.

  The present invention relates to a method for separating a compound and a compound by liquid chromatography, and can be used for the purpose of purification by analysis or fractionation in research and production management.

2 is a diagram illustrating an elution curve of a sample in Example 1. FIG. 6 is a diagram illustrating an elution curve of a sample in Comparative Example 1. FIG. 6 is a diagram illustrating an elution curve of a sample in Comparative Example 2. FIG. 6 is a diagram illustrating an elution curve of a sample in Example 2. FIG. It is a figure showing the elution curve of the sample in the comparative example 3.

Claims (2)

  A liquid chromatography separation method using a separation agent comprising a polysaccharide derivative as an active ingredient, wherein water having a concentration of 500 to 5000 ppm is added to a normal phase mobile phase comprising an organic solvent. Separation of organic mixtures by chromatography. The separation method according to claim 1, wherein the organic mixture is a mixture of optical isomers of a basic organic compound.

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