JPH09119923A - Chromatographic separating/purifying method using urea - Google Patents

Abstract

PROBLEM TO BE SOLVED: To facilitate the separation/purification of substances which have been difficult to be separated by employing granular urea as fixed phase and isopropylalcohol as moving phase. SOLUTION: Spherical urea having grain size of 3mm is filled in a column having diameter of 2cm until column length of 2cm is obtained. A solution produced by introducing 2g of a fatty acid ester into isopropylalcohol is then introduced into the column and developed at a flow rate of 3ml/min. The flow- out liquid is then demarcated up to 300mI with 50mI as one demarcation. Subsequently, isopropylalcohol is evaporated and urea is extracted from the residue using hexane before evaporating hexane to obtain a sample. Finally, the quantity of ERA is determined based on the ratio of carbon-carbon double bond to the ethyl group of the sample by nuclear magnetic resonance method. 1.54g of EPA mixture having purity of 97% or higher is obtained from 2g of EPA mixture having purity of 85% using second and third demarcation samples which exhibits the separation/purification effect clearly.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a separation technique applying a chromatograph.

[0002]

2. Description of the Related Art The use of chromatographic techniques for separating organic substances is widely practiced. Especially in the case of liquid phase system, it is possible to handle those with relatively large molecular weight,
Further, since it can be easily scaled up, it is applied to industrial separation and purification of substances and put to practical use.

These chromatographic techniques make use of the difference in binding property or affinity between a substance and a stationary phase, and silica gel, aluminum oxide, cellulose, diatomaceous earth, activated carbon, etc. have been used as the stationary phase. Also,
These stationary phases that have been conventionally used have specific characteristics, that is, the stationary phase with various chemical modifications is used, or various resins having a characteristic functional group are immobilized. It is also being used as a phase.

However, even in these conventional techniques, it is difficult to separate and purify a large amount of organic substances, and it is necessary to perform the purification by another technique such as precision distillation under reduced pressure. However, precision distillation requires an extremely large-scale and expensive device, and the higher the precision required, the lower the yield, and the large amount of control know-how, etc., which makes it difficult to carry out. It was

Examples of such substances which are difficult to separate and purify include various esters of linear unsaturated acids such as eicosapentaenoic acid and docosahexaenoic acid. These are often used as pharmaceuticals, and in that case, the purification thereof is required to be particularly accurate.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above problems of the prior art and to provide a method capable of easily separating and purifying a substance which has been difficult to separate in the past.

[0007]

The present invention is, as described in claim 1, a chromatographic separation and purification method using urea as a stationary phase.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The urea used as the stationary phase is preferably granular. Particle size is 0.3mm-1
0 mm is suitable. If it is less than 0.3 mm, problems such as moisture absorption during the handling, dissolution in the mobile phase, or clogging may occur, and the pressure loss also increases. However, by defining conditions (adjustment of handling environment, selection of mobile phase liquid, combined use of spacers, use of pump, etc.) that will not cause these problems,
A particle size of less than 3 mm can also be used.

On the other hand, when the particles having a particle size of more than 10 mm are used, the surface area is decreased and the number of theoretical plates is decreased. In addition, such large granular urea is liable to be cracked and is apt to be clogged. However, when separating and refining a large amount of raw materials, countermeasures that take these into consideration (spacer combination,
This does not apply if multiple columns are used in series).

As the shape of the stationary phase, spherical, tablet-shaped, disk-shaped, hemispherical, needle-shaped, polyhedral, or angulated peculiar shapes such as konpeito can be selected. Such a material can be obtained by applying a known technique such as a drum granulation method, a pan granulation method, a fluidized bed granulation method, or a spouted bed granulation method. When choosing a stationary phase, one must be careful to choose one with as high a breaking strength as possible. It should be noted that it is also possible to mold and use a material that does not elute in the mobile phase liquid together with urea as a binder,
Tablet molding may be applied by applying a tablet molding technique.

The particle size distribution is preferably as large as possible in order to prevent an increase in pressure loss and to easily control the number of theoretical plates. However, in order to facilitate the separation and purification of specific substances,
It is also possible to use stationary phases with a unique particle size distribution.

Urea as the granular stationary phase can be used in combination with a commonly used stationary phase. Examples thereof include silica gel, celite, diatomaceous earth, alumina, activated carbon, cellulose, various polymers, their derivatives, and those chemically modified. In use, urea and these commonly used stationary phases may be mixed and granulated, or the separately granulated particles may be mixed and used. In addition, these may be stacked in a column and used.

Further, it is possible to use, as a stationary phase, a substance having a surface coated with urea as a core. Such a thing can be produced by, for example, precipitating urea on the surface of the substance which becomes the core. In addition,
It is also possible to improve the separation efficiency by reducing the pressure loss by combining a spacer and a filler. As the shape of the column, it is possible to apply all the shapes used in the known chromatographic method. It is also possible to combine several columns having urea as a stationary phase, or to combine with a column having another stationary phase.

As the mobile phase, it is necessary to select one having a low urea solubility. As long as it is such a solvent, a relatively hydrophilic solvent such as alcohol, ester and ketone, a non-hydrophilic solvent such as methylene chloride, hexane and benzene, or a mixture thereof can be appropriately used. When alcohol, especially isopropyl alcohol, tert-butyl alcohol or the like having 3 to 5 carbon atoms is used as a mobile phase, good results can be obtained when separating and purifying a mixture containing a fatty acid ester or the like. Thus, the separation and purification ability can be improved by appropriately selecting the solvent according to the target substance.

When the mixture of organic substances is liquid, it can be directly introduced into the column for separation and purification without being dissolved in these solvents. In addition, when these mobile phase liquids dissolve urea even a little, if the saturated solution of urea is separately used before use,
It is desirable because there is no fear that urea in the column will be consumed.

When isopropyl alcohol or ethyl acetate is used among the above-mentioned solvents, urea in the column is not consumed, or even if it is consumed, fine-grained urea can be used. Good separation and purification ability can be obtained. If there is no consumption of urea,
After the treatment, urea is practically not mixed in the mobile phase containing the target substance, so that the target substance can be obtained without the urea removal step. Furthermore, when a mobile phase liquid in which isopropyl alcohol and ethyl acetate are mixed is used, it is possible to reduce the dissolution of urea in the transfer phase liquid while keeping the resolution optimum by the mixing ratio.

It is desirable that a mobile phase liquid is previously introduced into the column before it is used for separation and purification. This eliminates turbulence in the flow of mobile phase in the column,
It is possible to prevent obstacles such as a decrease in the number of theoretical plates. However, this does not apply if the mobile phase liquid is introduced from the lower part of the column and is allowed to flow out from the upper part.

The temperature at the time of separation and purification can be appropriately set according to the intended product, but it is desirable to keep it constant during the separation and purification.
In addition to obtaining stable results, changes in the solubility of the mobile phase liquid with urea due to temperature changes may cause obstacles due to a decrease in the stationary phase, or precipitation of urea when urea is saturated and dissolved in the mobile phase liquid. It is possible to prevent clogging due to. However, this is not the case when a temperature change is required for separating the target substance.

As a raw material to be separated and purified by the chromatographic separation and purification method using urea of the present invention as a stationary phase, naturally, a compound having no affinity with urea has an affinity with urea. A mixture of compounds, also
It is a mixture of compounds that differ in the strength of their affinity.

There are a large number of such compounds, and some of them are exemplified by pentane, hexane or higher linear hydrocarbons, 1-octene or higher linear α-olefins, 1-phenyloctadecane, 3- 2 carbon atoms such as methyl eicosane, 1-cyclohexyl eicosane, 1-cyclohexyl eicosane, 1-cyclopentyl hen eicosane
Zero or more hydrocarbons and their various derivatives (acid amides,
Acid ester, etc.), oils and fats derived from organisms such as animals and plants, fish, and mineral oil, etc., and can be purified and separated.

In the above, various esters such as octadecatetraenoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid (also known as icosapentaenoic acid), docosapentaenoic acid and docosahexaenoic acid (methyl, ethyl, various Esters with various alcohols such as propyl, various butyls and various pentyls) are also included.

In the chromatographic separation and purification method according to the present invention, among the raw materials introduced into the column, the saturated compound has a large interaction with urea, so that the unsaturated compound flows out first. Therefore, it is particularly advantageous in terms of purity to carry out separation and purification using an unsaturated compound as a target substance.

The target substance is recovered from the mobile phase liquid by removing the mobile phase liquid by means such as evaporation. When urea is dissolved in the mobile phase liquid, after removing the mobile phase liquid by evaporation as described above, the residue is extracted with a solvent (hexane or the like) that does not dissolve urea, The target substance can be easily recovered by removing the solvent by evaporation. Further, when the target substance is not water-soluble, it can be recovered by dissolving urea in the residue and removing it.

The column used for separation and purification can be washed to some extent by continuously flowing the mobile phase liquid. Here, when the mobile phase liquid used is a hydrophilic solvent, it is retained in the column by washing with a non-hydrophilic solvent, and when a non-hydrophilic solvent is used as the mobile phase liquid, washing with a hydrophilic solvent. It is easy to remove and collect the components that are present. Further, a plurality of solvents having different properties may be mixed and used. As one of the applied techniques of the present invention, it is possible to use it as a kind of chromatographic analysis device by providing various detection means used in a liquid chromatograph device such as ultraviolet spectroscopic analysis at the column outlet. Is.

[0025]

【Example】

Example 1 A spherical urea having a particle diameter of 3 mm was packed in a column having a diameter of 2 cm so that the column length would be 20 cm. In this column, a fatty acid ester liquid derived from fish oil (85% by weight of ethyl eicosapentaenoate (hereinafter referred to as "EPA"), 10% by weight of ethyl palmitate, and 5% by weight of ethyl ester of another organic acid) 2. A solution in which 0 g was dissolved in isopropyl alcohol was introduced, and the flow rate of isopropyl alcohol was 3 ml / min for development.

50 ml of the effluent is used as one fraction and 300
Fractionation (first to sixth fractionation) was performed up to ml. For each of these, isopropyl alcohol was removed by evaporation. Since this residue contains urea, it was extracted with hexane (extracted 3 times with 10 ml of hexane).
Samples (first to sixth fractions) obtained by removing these hexanes by evaporation
And

The weight of these samples was measured, and carbon-to-ethyl groups of these samples were measured by nuclear magnetic resonance.
The amount of EPA was calculated from the ratio of carbon double bonds. Table 1 shows the results. In addition, ethyl palmitate was not detected in the samples of the second and third fractions.

[0028]

[Table 1]

Focusing on the second and third fraction samples in Table 1, it was found that 2.0 g of a mixture of 85% EPA purity yielded 1.54 g of a EPA mixture of 97% or higher purity. The effect of the separation and purification method of the present invention is clear. The properties of ethyl palmitate are very close to those of EPA and are therefore very difficult to separate and purify. Usually, fine distillation under reduced pressure is carried out for separation. Although mixing was said to be unavoidable, it could be completely removed in this example.

Example 2 200 g of granular urea having a diameter of 1 to 2 mm packed in a column having a diameter of 3 cm was used as a stationary phase, isopropyl alcohol was used as a mobile phase liquid, and 203 mg of methyl palmitate and retinoic acid were used in this column. A mixture of 198 mg of methyl dissolved in 3 ml of isopropyl alcohol was introduced and
Flow the mobile phase liquid at a flow rate of 1 / min and 30m at the column outlet.
Chromatographic separation was performed by sampling 1 by 1. Isopropyl alcohol in the sample of each of these fractions was removed by evaporation, then dissolved in methylene chloride, and then washed with water using a separating funnel to remove urea, and the sample was analyzed by nuclear magnetic resonance. The three-fraction sample (36 mg) consisted of methyl retinoic acid only,
The fourth fraction (134 mg) was found to consist of 97% by weight methyl retinoate and 3% by weight methyl palmitate.

[Example 3] 1.5 cm in diameter filled with 13 g of granular urea crushed to a diameter of 0.5 mm or less
Column as the stationary phase and ethyl acetate as the mobile phase liquid, 123 mg of methyl palmitate and 122 mg of methyl retinoic acid were dissolved in 3 ml of ethyl acetate and introduced into this column, and the mobile phase liquid was supplied at a flow rate of 1 ml / min. Then, 20 ml of each sample was sampled at the column outlet for chromatographic separation. When ethyl acetate was removed from this first fraction (20 ml), all the remaining 118 mg was methyl retinoate.

[Embodiment 4] Similar to Embodiment 3, except that
125 mg of methyl palmitate and 130 mg of methyl retinoate were used as a mobile phase liquid using an ethyl acetate-isopropyl alcohol mixed solvent prepared by mixing ethyl acetate and isopropyl alcohol in a volume ratio of 5: 1.
And were dissolved in 3 ml of this mixed solvent and introduced into the column,
The mobile phase liquid was flushed. 20 ml of each sample was sampled at the column outlet, washed with water using a separating funnel, and then the solvent was evaporated. At this time, it was found that the sample of the second fraction consisted only of retinoic acid methyl.

Example 5 A column having a diameter of 2 cm was packed with 60 g of urea ground in the same manner as in Example 3, ethyl acetate was used as a mobile phase liquid, and 102 m of n-decane was added to this column.
g and trans-5-decene (113 mg) were dissolved in 2 ml of ethyl acetate and introduced. The stationary phase liquid was caused to flow at 2 ml / min, and 30 ml was sampled at the column outlet. When the solvent was removed from each fraction and the non-evaporated components were analyzed by gas chromatography, 113 mg of the non-evaporated components contained in the solution of the second fraction were n-.
It was found to contain 8 mg of decane and 103 mg of trans-5-decene.

[0034]

INDUSTRIAL APPLICABILITY The separation and purification method of the present invention applies the difference in affinity of various organic substances with urea as a separation principle of the chromatographic method, and can be used for separation and purification of various organic substances. Further, the separation and purification method of the present invention is excellent in that it does not require special equipment such as a precision distillation column and can be easily carried out. In addition, urea used as a stationary phase is a substance that is produced in large amounts by fertilizers, etc., and is an organic solvent that is also frequently used for various liquids used as a mobile phase, and since it can be recovered, the separation and purification method of the present invention is It can be implemented at a very low cost.

Incidentally, as a conventional separation method using urea,
Urea treatment method is known, but this is to separate only by the presence or absence of affinity with urea, it was not possible to utilize the strength of the affinity for separation and purification, the separation and purification method of the present invention Then, by maximally utilizing the difference in the strength of affinity, it is possible to easily separate and purify the material, which was conventionally difficult to separate.

In the urea chromatographic separation / purification method of the present invention, when isopropyl alcohol, ethyl acetate and a mixed solvent thereof are used as the mobile phase liquid, fine particles of urea can be used as the stationary phase urea. Good resolution is obtained. It is just surprising that the ester ethyl acetate or a mixed solvent thereof as the ester can be used as the mobile phase liquid as clarified in the above Examples, and in this case, very good separation and purification was performed. It is possible to eliminate the need for the urea removal step.

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Claims (10)

[Claims] 1. A chromatographic separation / purification method, wherein urea is used as a stationary phase. 2. The chromatographic separation and purification method according to claim 1, wherein the urea is granular. 3. The chromatographic separation and purification method according to claim 1 or 2, wherein isopropyl alcohol is used as a mobile phase. 4. The chromatographic separation and purification method according to claim 1 or 2, wherein ethyl acetate is used as a mobile phase. 5. The chromatographic separation / purification method according to claim 1, wherein a mixed solvent of ethyl acetate and isopropyl alcohol is used as a mobile phase. 6. A method for separating and purifying an organic substance, which comprises using a chromatograph using urea as a stationary phase. 7. A method for separating and purifying a saturated compound and an unsaturated compound, which comprises using a chromatograph with urea as a stationary phase. 8. A method for separating and purifying a fatty acid ester, which comprises using a chromatograph having urea as a stationary phase. 9. A method for separating and purifying ethyl eicosapentaenoate, which comprises using a chromatograph with urea as a stationary phase. 10. A chromatographic analyzer characterized in that urea is used as a stationary phase.