JPS60208940A - Separation and purification of long-chain saturated acid - Google Patents

Abstract

PURPOSE:Long-chain unsaturated fatty acids are separated from a mixture thereof with long chain fatty acids by reverse phase partition chromatography using a specific organic solvent as an eluent to collect long-chain unsaturated fatty acids efficiently through simple operations. CONSTITUTION:Long-chain fatty acid mixture is subjected to reverse phase partition chromatography using, as an eluent, an organis solvent with a solubility parameter of 11-20 to separate long-chain unsaturated fatty acids such as eicosapentaenoic acid or docosahexaenoic acid, which are used as a starting materials of prostaglandin. As an eluent, is cited acetonitrile, methanol, ethanol, N-methylformamide or formamide. Methanol is most preferred, because it has good separation activity and easiness in solvent removal. The solvent is previously refluxed to eliminate oxygen whereby the formation of peroxides can be avoided.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying long chain unsaturated fatty acid compounds from a mixture of long chain fatty acid oxides of various carbon numbers.

Unsaturated fats such as eicosapentaenoic acid C (hereinafter referred to as EPA) and docosahegisaenoic acid (hereinafter referred to as DHA) are mainly found in the bodies of fish such as sardines, salmon, and cod. It is contained in the form of glyceride along with long fKJ% fatty acids, and is known as a substance that has the effect of preventing thrombotic diseases such as cerebral infarction and myocardial infarction in vivo. In recent years, EPA has also attracted attention as a raw material for prostaglandins, along with arachidonic acid and dihomo r-salunic acid, and research on its pharmacological effects has become active. Under these circumstances, the development of an industrial method for producing highly purified EPA and DHA has been awaited recently.

Conventionally, as a method for separating and refining EPA and DHA present in fish oil, etc., glyceride was first processed in a methanol tank.
A method is known in which a lower alcohol such as Tan-# is converted into a varnish, distilled, and further concentrated by urea inclusion treatment (for example, JP-A-58-8037). However, the purity of the esters of EPA and DHA obtained by this method is about 20 to 60% at most, and it is difficult to achieve higher purity because they are a mixture of substances with similar boiling points.

In addition, a method using reversed phase chromatography is being considered as a method for obtaining highly pure DPA JpDHA. Kaisho 5
8-88339), water, lower alcohols, and aliphatic ketones using a gel-based or synthetic polymer-based reversed-phase partition chromatography column.

A method has been proposed in which the solution is a mixture of at least one component selected from the group consisting of ethyl acetate or acetonitrile (Japanese Patent Laid-Open No. 109444/1983). However, since these methods use an eluent containing water, the solubility of the sample is low, and it is necessary to prepare a dilute sample solution over time or to prepare an organic solution. The drawback is that the sample, which is prepared as a concentrated solution and deposited on the column, must be gradually dissolved by manually pouring solvent over the column.

In addition, in order to recover the target component from the fraction I):J, after volatilizing the water-soluble organic solvent, add a water-insoluble organic solvent to extract the target component, and then oil-water separation. It is necessary to either evaporate the organic solvent by evaporating the organic solvent, or to evaporate all the solvent by directly subjecting the fractionated fraction to vacuum drying. However, the former is a complicated process, and the latter requires treatment at low temperatures because EP and DMA are sensitive to heat, and both are industrially uneconomical methods.

Therefore, the present inventors conducted intensive research to improve this drawback of the method applying reversed phase chromatography, and as a result, they remembered that it is effective to use a specific eluent, and completed the present invention. reached. -Thus, according to the present invention, the desired length can be obtained by performing reverse phase partition chromatography from a mixture of long chain fatty acid compounds including long chain unsaturated fatty acid compounds using a column packed with a carrier for reverse phase partition chromatography. A method for separating and purifying a long-chain unsaturated fatty acid compound is provided, which is characterized in that an organic solvent having a solubility parameter of 11 to 20 is used as an eluent.

The raw material used in the present invention is a mixture of long chain fatty acid compounds including the desired long chain unsaturated fatty acid compound. Here, the long-chain unsaturated fatty acid compound means a fatty acid having 18 or more carbon atoms, preferably 20 to 22 carbon atoms, and 3 or more double bonds, or a derivative such as an ester or amide of the fatty acid, and particularly Lower alkyl esters are awarded.

Such mixtures can be obtained according to conventional methods.

For example, a mixture containing the target octopus unsaturated fatty acids can be obtained by saponifying lipids extracted from fish such as sardines, salmon, and cod with an alkali and then neutralizing them. A mixture containing the desired long-chain unsaturated fatty acid ester can be obtained by esterifying with. Further, if necessary, the target component can be concentrated and used by distilling the obtained mixture or subjecting it to urea inclusion treatment.

The column packing material used in the present invention may be any commonly used column packing material, and specific examples thereof include silica gel-based and polymer-based ones.

The former is usually one in which an alkyl group having 6 to 24 carbon atoms is chemically bonded to silica, and preferably an alkyl group having 8 to 18 carbon atoms is chemically bonded to silica. The alkyl group is on the surface of the silica particle and has the function of distributing the long chain fatty acid compound of the sample to the solvent, and as the number of carbon atoms decreases, the ability to hold the sample decreases.

The particle size of such silica filler is usually 1 to 100 μm.

The diameter is preferably 5 to 50 μm, and the diameter of the pores on the particle surface is usually 20 to 2 ood, preferably 50 to 150 Å.

On the other hand, the latter is a porous synthetic polymer that has long-chain alkyl groups or fatty acid residues in its side chains, and its configurations include polyvinyl stearate, polyoctadecyl acrylate,
Examples include polyoctadecyl methacrylate.

In the present invention, the solubility parameter δ is used as the eluent.
It is essential to use an organic solvent having a value of 11 to 20, preferably 12.5 to 18.5. Here dissolve L
The parameter is defined by the following formula.

ΔE=evaporation energy, U=molecular volume [cIna] Specific examples of organic solvents used include acetonitrile (11
,9), ethanol (12J9), ethanol (14
, 5), N-methylformamide (1° true 641), and formamide (18,3), among which methanol is particularly prized for its separation properties and ease of solvent removal. Note that the numerical value in parentheses represents the δ value.

In addition, these solvents Vi can be appropriately mixed and used, and even if the δ value is outside the above range, the δ value of the mixed solvent (the δ value of each solvent multiplied by the volume fraction and added) If the value (value) is within the above range, it is included in the scope of the present invention.

It is preferable to heat and reflux such an eluent in advance to remove dissolved oxygen in order to prevent the formation of peroxides.

Other operating conditions can be appropriately selected according to conventional methods. For example, the amount of eluent used is usually 50
~10100O, preferably about ~700ml, and the elution rate is not necessarily constant depending on the type of filler, particle size distribution, pore size, etc. J speed from 0.05 to 2゜h, -1, preferably 0
．． Surkotka is suitable for 15-10 hr-'.

Thus, according to the present invention, the desired long-chain unsaturated fatty acid compound can be efficiently purified with simple operations.

Examples of the present invention are shown below, but the present invention is not limited thereto. The compositional analysis of the raw materials and separated and purified products was conducted using gas chromatography under the following conditions.

Gas chromatography conditions Column: 2mX3Mφ, 10% DEGS, Cel port
545.

6G~80mesh Thirst: Inlet 250℃, column 200℃Detector:
FID Example 1 Sardine oil was subjected to methyl esterification 4 by a conventional method and urea inclusion treatment in a methanol solution to obtain fatty acid methyl ester with a high degree of unsaturation from liquid P. The i) content of 1iPA methyl ester was 27.0%. This sample 41 was
, using a column (4,1crnφX40crn) packed with silylated silica (average particle size 38 μm) 300F, and expanded with methanol at 10 m4/min.

The eluate from the column was differentially 183 #? When analyzed using #i, the chart shown in FIG. 1 was obtained. 130 ml of both portions indicated as A in Figure 1 were collected, and the composition was measured using gas chromatography.
99.1% of the stores on the 1st floor contained 0EPA ester.

Example 2 Urea inclusion treatment sample 4 f of sardine oil methyl ester used in Example 1 (hPA methyl ester content 27.0%
, a large rat preparative high-speed liquid chromatograph equipped with a column (5,76nφ x 30crn) packed with 31 tons of C1° silylated silica (particle size 35-105μ) containing 14.5% of DIIA methyl ester. Manufactured by Waters.

Prep500A) with acetonitrile/methanol (15/85, V/V) mixed solvent 1 o o n
It was developed with 1t15k Ic.

When the eluate from the column was analyzed in the same manner as in Example 1, the chart shown in FIG. 2 was obtained.

Fractions A and B shown in Figure 2 were separated and subjected to rotary evaporation to remove the solvent, resulting in a colorless oil with 606 m? , 507 mf was obtained. The composition of both parts A is 97.5% BPA methyl ester,
DHA methyl ester θ%, and the composition of fraction B is DH
The A methyl ester content was 99.0%, and the EPA methyl ester content was θ%.

Example 3 A column containing 0.3F (EPA ethyl ester content 12.0%) prepared by ethyl esterification of cod liver oil and C, silylated silica (average particle size 5 μm) 309 as a packing material (
Formamide 0 using
, 8 m and 47 minutes. Differential bending D of column eluate
A chart based on the r meter is shown in Figure 3. i+,!
After adding 500 mt of water to 10.5 mt of 7i min A and extracting with 100 m4 of petroleum needle, the mixture was applied to a rotary 9 evaporator to obtain 24.2 mW of colorless oil. The composition of EPA ethyl ester determined by gas chromatography was 99.3%.

Comparative Example 1 Example 1 except that dioxane (solubility parameter 10.2) was used instead of methanol as the developing solvent.
An experiment was conducted in the same manner. As a result, the state of separation detected by the differential refractometer was as shown in FIG. 4, and the separation was insufficient.

Comparative Example 2 The experiment was carried out according to Example 1, except that a mixture of methanol/acetone/water == 52/30/1B (Solubility parameter 14.7) was used as the developing solvent instead of methanol. went. Since this solution had poor sample solubility, it required 800 mL to dissolve 4 tons of sample. This was just equal to the column packing bed volume. Therefore, as shown in Figure 5, the column eluate is overloaded with multiple components.
However, the separation was insufficient.

[Brief explanation of drawings]

Figures 1 to 3 show the elution curves of Examples 1 to 3, and Figure 4 shows the elution curves of Examples 1 to 3.
Figures 5 to 5 show the elution I lines of Comparative Examples 1 and 2. Figure 1 Development time (hours) Figure 2 Figure 3 0 1 2 3 4 Development time (hours)

Claims (1)

[Claims] 1. The desired long-chain unsaturated fatty acid compound is obtained from a mixture of long-chain fatty acid compounds containing the long-chain unsaturated fatty acid compound by performing reverse-phase partition chromatography using a column needle packed with a carrier for reverse-phase partition chromatography. 1. A method for separating and purifying long-chain unsaturated fatty acid compounds, characterized in that the method uses an organic solvent having a solute M degree parameter of 11 to 20 as an eluent.