JP2015030685A - Method for separating liposoluble substance by pseudo moving-bed chromatography using reverse phase column, and device therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly efficient unsaturated fatty acid purification method, capable of solving a problem in an existing unsaturated fatty acid purification method, and a device therefor.SOLUTION: Provided are a separation method and device using SMB chromatography including at least two kinds of columns selected from a group consisting of a C18 column, a C8 column, a C4 column and a C1 column, preferably, at least three C18 columns and at least one of the C8 column and/or the C4 column and/or the C1 column.

Description

  The present invention relates to a separation and purification method by simulated moving bed chromatography using a C18, C8, C4, and / or C1 column, and an apparatus therefor.

  Unsaturated fatty acids are difficult to separate and purify from each other because of their similar molecular weight and chemical properties. Conventionally, as a method for separating a specific fatty acid from a mixture of fatty acids or monoesters, a difference in boiling point, a difference in molecular weight, and a difference in solubility have been mainly used. For example, distillation, molecular sieving, and supercritical fluid extraction are known. Examples of the separation method based on the number of double bonds include wintering, urea addition, silver complex formation, and solvent fractionation.

In addition, there are chromatographic methods using silver ion-treated resins and C18 fillers, but they are simple but insufficiently separated or highly purified, but are not suitable for industrial mass processing. Each has its advantages and disadvantages. The technical problems that have been used to purify unsaturated fatty acids and their derivatives are described below. Representative separation / purification methods include, for example, (1) vacuum precision distillation method, (2) molecular distillation method, (3) urea addition method, (4) silver nitrate treatment method, (5) fixed bed chromatography. And (6) simulated moving bed chromatography. The features and problems of each method are as follows.
(1) Features of vacuum precision distillation method: A method of separation utilizing the difference in boiling point of each component.
Problem: There may be heat denaturation. It takes a long time to purify high purity products.
(2) Features of molecular distillation method: There is little thermal influence during distillation.
Challenge: Low resolution.
(3) Urea addition method features: Utilizes the property of forming hexagonal columnar adduct crystals while taking in linear molecules when dissolved urea crystallizes.
Challenge: Low selectivity. Waste treatment by derivation of urea adducts.
(4) Silver nitrate treatment method characteristics: Utilizes the property that a silver nitrate solution forms a complex with respect to a double bond of a fatty acid.
Challenge: Silver prices are unstable. Embrace foreign objects.
(5) Fixed-layer chromatography characteristics: little thermal influence. High precision separation is possible.
Problem: A large amount of eluent is used. Not suitable for industrial production.
(6) Pseudo moving bed chromatography characteristics: The amount of eluent used is small and suitable for industrialization because of continuous operation.
Problem: C18 fillers often used to separate unsaturated fatty acids are expensive. Since only a single solvent can be used, it cannot be separated by a gradient.

  Pseudo moving bed (hereinafter also referred to as “SMB”) chromatography is often used for industrial chromatographic purification because it uses a small amount of eluent and can be operated continuously unlike conventional fixed bed chromatography. . For example, p-xylene is produced by SMB at 500,000 t / year.

  In general, by using a gradient, it is possible to change elution conditions over time. As a result, separation between components is improved and time until elution is shortened. However, in SMB chromatography, it is not possible to use a gradient that changes the proportion of different solvents over a period of time as is done in single column chromatography, and only a single eluent can be used.

  As a purification technique of eicosapentaenoic acid ethyl ester (hereinafter, EPA ethyl), for example, Patent Documents 1 and 2 mention SMB chromatography as an industrially excellent purification technique for unsaturated fatty acids and derivatives thereof. SMB chromatography continuously removes polyunsaturated fatty acids and their derivatives, which are target components, by keeping the distribution of each fraction formed in the column adsorbent layer in a steady state while continuously supplying raw materials. be able to.

  When separating highly unsaturated fatty acids and derivatives thereof, C18 silica gel is often used as the column filler, but it is expensive for separation performance, and further improvement in performance becomes a problem in industrialization.

  Therefore, there is a need for a highly efficient method for purifying unsaturated fatty acids and an apparatus therefor that solve the problems in existing methods for purifying unsaturated fatty acids.

  In recent years, unsaturated fatty acids are not only attracting attention as supplements as essential fatty acids, but also because EPA ethyl has been approved as a therapeutic agent for obstructive arteriosclerosis and hyperlipidemia as a switch OTC. The ethyl market is expanding.

Special table 2013-516398 JP-A-8-218091

  It is an object of the present invention to provide a highly efficient unsaturated fatty acid purification method and an apparatus therefor by solving the problems in existing unsaturated fatty acid purification methods.

  Also, when separating highly unsaturated fatty acids and their derivatives, C18 silica gel is often used as the column filler, but since it is expensive for the separation performance, a relatively inexpensive method for purifying unsaturated fatty acids and therefore It is an object of the present invention to provide an apparatus and to improve separation performance.

  INDUSTRIAL APPLICABILITY INDUSTRIALLY PRODUCING PURIFICATION TECHNIQUES FROM POLY-UNSATURATED FATTY ACIDS AND DERIVATIVES DERIVED FROM AN ANIMALS AND PLANTS AND MICROBIO FATS AND ITS such as Fish Oil, Algal Extract Oils, Extract Oils From Genetically Modified Plants Provide a way to do it.

  In the present invention, the above-mentioned problems have been solved by using a plurality of types of columns simultaneously in SMB chromatography.

  For example, in the present invention, four or more silica gel columns using C18, C8, C4, and / or C1 columns are used in SMB chromatography. As the eluent, for example, a normal layer system (n-hexane) can be used, but is not limited thereto.

The present invention provides, for example:
(Item 1)
A method for separating a fat-soluble substance using SMB chromatography,
Here, an apparatus for performing separation by the SMB chromatography is as follows:
(1) at least three C18 columns;
(2) at least one column selected from the group consisting of a C8 column, a C4 column, and a C1 column; and
(3) The same number of circulation lines as the number of columns connecting the columns;
Prepared,
Here, the method is as follows:
(A) supplying the eluent to the first circulation line;
(A) supplying the sample to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Where the first to fourth circulation lines are downstream from the upstream, the first circulation line, the second circulation line, the third circulation line, and A method of arranging in order of the fourth circulation line.
(Item 2)
Item 2. The method according to Item 1, wherein the sample is a distilled sample.
(Item 3)
Item 2. The method according to Item 1, wherein the fat-soluble substance is a fatty acid or a fatty acid derivative.
(Item 4)
Item 2. The method according to Item 1, wherein the fat-soluble substance is selected from the group consisting of α-linolenic acid, stearidonic acid, docosapentaenoic acid, eicosapentaenoic acid, docosahexaenoic acid, and lower alcohol esters thereof.
(Item 5)
A method for separating a fat-soluble substance using SMB chromatography,
Here, an apparatus for performing separation by the SMB chromatography is as follows:
(1) at least two columns selected from the group consisting of a C18 column, a C8 column, a C4 column, and a C1 column; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Here, the method is as follows:
(A) supplying the eluent to the first circulation line;
(A) supplying the sample to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Where the first to fourth circulation lines are downstream from the upstream, the first circulation line, the second circulation line, the third circulation line, and A method of arranging in order of the fourth circulation line.
(Item 6)
Item 6. The method according to Item 5, wherein the sample is a distilled sample.
(Item 7)
Item 6. The method according to Item 5, wherein the fat-soluble substance is a fatty acid or a fatty acid derivative.
(Item 8)
Item 6. The method according to Item 5, wherein the fat-soluble substance is selected from the group consisting of α-linolenic acid, stearidonic acid, docosapentaenoic acid, eicosapentaenoic acid, docosahexaenoic acid, and lower alcohol esters thereof.
(Item 9)
Separation apparatus for fat-soluble substances using SMB chromatography, comprising:
(1) at least three C18 columns;
(2) at least one column selected from the group consisting of a C8 column, a C4 column, and a C1 column; and
(3) The same number of circulation lines as the number of columns connecting the columns;
Prepared,
Where the device is:
(A) supplying the eluent to the first circulation line;
(A) supplying the raw material to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Wherein the first circulation line to the fourth circulation line are arranged downstream from the upstream, the first circulation line, the second circulation line, the A device arranged in the order of the third circulation line and the fourth circulation line.
(Item 10)
Separation apparatus for fat-soluble substances using SMB chromatography, comprising:
(1) at least two columns selected from the group consisting of a C18 column, a C8 column, a C4 column, and a C1 column; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Where the device is:
(A) supplying the eluent to the first circulation line;
(A) supplying the raw material to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Wherein the first circulation line to the fourth circulation line are arranged downstream from the upstream, the first circulation line, the second circulation line, the A device arranged in the order of the third circulation line and the fourth circulation line.
(Item 11)
A method for separating a fat-soluble substance using SMB chromatography,
Here, an apparatus for performing separation by the SMB chromatography is as follows:
(1) at least two types of at least four reversed phase columns; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Here, the method is as follows:
(A) supplying the eluent to the first circulation line;
(A) supplying the sample to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Where the first to fourth circulation lines are downstream from the upstream, the first circulation line, the second circulation line, the third circulation line, and A method of arranging in order of the fourth circulation line.
(Item 12)
Separation apparatus for fat-soluble substances using SMB chromatography, comprising:
(1) at least two types of at least four reversed phase columns; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Where the device is:
(A) supplying the eluent to the first circulation line;
(A) supplying the raw material to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Wherein the first circulation line to the fourth circulation line are arranged downstream from the upstream, the first circulation line, the second circulation line, the A device arranged in the order of the third circulation line and the fourth circulation line.

  In accordance with the present invention, a highly efficient unsaturated fatty acid purification process and apparatus therefor are provided.

FIG. 1 is a schematic diagram of SMB chromatography.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, the term “SMB chromatography” is a separation method that utilizes the principle of liquid chromatography, which is a selective adsorption that differs from a specific component in a raw material to another specific component. A plurality of unit packed beds filled with adsorbents with capacity are connected in series, and the unit packed bed at the most downstream part and the unit packed bed at the most upstream part are connected to form an endless circulation system. Refers to the layer.

  The term “raw material supply port” used herein refers to a raw material supply port used for SMB chromatography. As used herein, the term “component extraction position” refers to an outlet from a column of a solution subjected to SMB chromatography.

  As used herein, the term “upstream” refers to the side from which the eluent flows, and the term “downstream” refers to the side from which the eluent flows.

  As used herein, the term “circulation line” refers to a portion connecting adjacent columns. The circulation line connects the upstream component extraction position and the downstream raw material supply port, or connects the most downstream component extraction position and the upstream raw material supply port.

  As used herein, the term “switching the circulation line” refers to the column B, the column B, the column C, and the column D adjacent to each other in the order of column A-column B-column C-column D. The circulation line connecting the inlets is (i) a circulation line connecting the outlet of the column A and the inlet of the column B, or (ii) a circulation line connecting the outlet of the column C and the inlet of the column D. Any one of them.

  The term “reverse phase column” as used herein refers to a column used in a separation system in which the polarity of the mobile phase is higher than the polarity of the stationary phase. Representative reverse phase columns include, but are not limited to, reverse phase silica gel columns.

  As used herein, the term “reverse phase silica gel column” refers to a chromatography column using reverse phase silica gel as a carrier. Representative reverse phase silica gel columns include, but are not limited to, C18 columns, C8 columns, C4 columns, and C1 columns. Reversed-phase silica gel columns can be applied to the separation of a wide range of compounds from fat-soluble to ionic substances, and are currently the most widely used technique.

  As used herein, the term “C18 column” refers to a chromatographic column using chemically bonded porous spherical silica gel whose surface is modified with octadecylsilyl groups as a carrier. Also simply referred to as ODS column.

  The term “C8 column” used herein refers to a chromatographic column using chemically bonded porous spherical silica gel whose surface is modified with an octyl group as a carrier.

  The term “C4 column” used herein refers to a chromatographic column using chemically bonded porous spherical silica gel whose surface is modified with a butyl group as a carrier.

  The term “C1 column” used in the present specification refers to a chromatographic column using chemically bonded porous spherical silica gel whose surface is modified with a trimethyl group as a carrier.

  The SMB chromatography used in the present invention includes four or more columns. The SMB chromatography of the present invention preferably comprises three or more C18 columns and one or more C8 columns and / or C4 columns and / or C1 columns. As a ratio of a preferable combination of the two, C18 column: C8 column and / or C4 column and / or C1 column = 3: 1 to 12 can be mentioned.

  As long as the solvent used for the raw material used for the SMB chromatography of the present invention is a liquid that can be used for column chromatography, a liquid of any purity may be used. For example, for a liquid extracted from a raw material, a liquid obtained by partially concentrating the target fatty acid by a method selected from the group consisting of vacuum precision distillation, filtration, urea addition, silver nitrate treatment, and fixed bed chromatography is used as the raw material. Can do. The raw material used is preferably a free fatty acid or a lower alcohol ester such as ethanol or methanol. When the raw material is a solid, it is dissolved in a solvent such as methanol, ethanol or hexane.

  As used herein, “vacuum precision distillation” means, in the case of EPA, the component having 20 carbon chains including EPA is located at an intermediate boiling point among fish oil fatty acids, In the case of continuous distillation using a single tower distillation apparatus, a two tower apparatus or a four tower apparatus is required. In the case of the two-column system, this means that the component (first fraction) of C19 or less is distilled, the residue is sent to the second column, and the component C20 (main fraction) is fractionated.

  As used herein, “filtration” refers to a method of decolorizing a raw material with a filter and / or an adsorbent and removing a peroxide. Typically, it refers to a method in which a raw material and an adsorbent are mixed and stirred in a nitrogen atmosphere and the adsorbent is removed by a filter press. In the method of the present invention, preferred adsorbents include, but are not limited to, silica gel, activated clay, activated carbon, and zeolite.

  As used herein, “urea addition” refers to a method in which a raw material and a urea methanol solution are mixed and cooled to form a urea adduct incorporating saturated fatty acids and monounsaturated fatty acids, and this is filtered off. . Typically, it refers to a method of obtaining the desired unsaturated fatty acid by extracting n-hexane from a urea adduct, treating with silica gel, and then distilling off n-hexane.

  As used herein, “silver nitrate treatment” means, in the case of highly unsaturated fatty acid ethyl, the raw material and a silver nitrate solution are stirred and the unreacted ester is extracted, for example, by n-hexane, and then the aqueous phase is diluted or heated, A method of concentrating the target highly unsaturated fatty acid ethyl by extracting the liberated ester again with n-hexane.

  As used herein, “fixed bed chromatography” refers to a method in which a column is filled with a packing material, a raw material is passed through an eluent, and a fraction containing a target component is taken out and concentrated. In the method of the present invention, preferred fillers include, but are not limited to, silica gel, reverse phase silica gel and silver nitrate impregnated silica gel.

  As used herein, the term “eluent” is a solution supplied through an eluent supply port D. Depending on the gel to be used and the substance to be separated, those skilled in the art can easily determine the eluent. As an eluent that can be used when a fat-soluble substance is separated using a reverse-phase silica gel column, a highly polar solvent is generally used. Typically, water / methanol, methanol, ethanol, water / ethanol is used. , Water / acetonitrile, acetonitrile, acetone, isopropanol, but are not limited thereto.

  The term “fat-soluble substance” as used herein refers to a compound that is soluble in an organic solvent such as n-hexane or ethanol. Examples of the fat-soluble substance in the present invention include, but are not limited to, fatty acids or derivatives thereof. Examples of the “fat-soluble substance” in the present invention include α-linolenic acid, stearidonic acid, docosapentaenoic acid, eicosapentaenoic acid, ω3 fatty acids such as docosahexaenoic acid, and lower alcohol esters thereof, and γ- Examples thereof include, but are not limited to, ω6 fatty acids such as linolenic acid and arachidonic acid, or their lower alcohol esters of methanol or ethanol.

  As used herein, the term “lower alcohol ester” refers to an ester of a fatty acid or derivative thereof and a lower alcohol. As used herein, the term “lower alcohol” refers to any linear or branched alcohol having 10 or less carbon atoms.

(SMB chromatography)
In SMB chromatography, raw materials and eluent are supplied to an endless circulation system, and the X component (that is, the weak affinity component) that moves at high speed in the column (unit packed bed) and the column within the column at low speed The Y component (that is, the affinity component) that moves is extracted from different positions. The SMB chromatography sequentially moves the raw material supply position, the eluent supply position, the X component extraction position, and the Y component extraction position downstream in the fluid circulation direction while maintaining a fixed positional relationship. A processing operation performed continuously is realized in a pseudo manner. As a result, the distribution state of each component in the layer moves with a substantially constant width, and the extraction position of each component is an operation method that can continue to take a portion with high purity and concentration.

  An exemplary SMB chromatography is shown in FIG. Columns 1 to 4 are connected by a circulation line, and a raw material supply port F, an eluent supply port D, an affinity component outlet A, and a weak affinity component outlet B are provided in the circulation line between the columns to circulate. The valves are sequentially switched in the flow direction at regular intervals (that is, the circulation line is switched).

  For example, the raw material supplied from the raw material supply port F moves from the column 3 toward the column 2 due to the eluent supplied from the eluent supply port D. At that time, the X component, which is a weak affinity component, moves through the column at a higher speed because the affinity for the column is weak. Conversely, the Y component, which is an affinity component, has a strong affinity for the column and therefore moves through the column at a lower speed. As a result, both the X component and the Y component move at different speeds, and as a result, both of them are separated. In order to continuously separate the X component and the Y component, it is necessary to provide the raw material supply port F at a position between them. The Y component moves through the column at a lower speed than the X component, but continues to move for the eluent supplied from the eluent supply port D. Therefore, the valve is sequentially switched every predetermined time in the direction of the circulating flow, It is necessary to maintain the raw material supply port F between the X component and the Y component. That is, after a certain period of time in FIG. 1, the column 4 is moved to the column 3 position, the column 3 is moved to the column 2 position, the column 2 is moved to the column 1 position, and the column 1 is moved to the column 4 position. (In other words, the circulation line connecting column 1 and column 2 is switched to the circulation line connecting column 2 and column 3, and the circulation line connecting column 2 and column 3 is connected to column 3 and column 4. Switch to the circulation line, switch the circulation line that connects column 3 and column 4 to the circulation line that connects column 4 and column 1, and the circulation line that connects column 4 and column 1 to the circulation line that connects column 1 and column 2 Switch to.) As a result, the raw material supply port F moves between the column 4 and the column 3, the weak affinity component outlet B moves between the column 3 and the column 2, and the eluent supply port D connects to the column 2. The affinity component outlet A moves between the column 1 and the column 4.

  In the SMB chromatography of the present invention, four or more columns are used. The number of columns is not particularly limited. Preferably, the number of columns is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13, 15, 15, 16, 17, Although it is 18, 19, and 20, it is not limited to these. When the number of columns is more than four, between the raw material supply port F and the weak affinity component outlet B, between the weak affinity component outlet B and the eluent supply port D, the eluent supply port A plurality of columns may be provided between D and the affinity component outlet A and / or between the affinity component outlet A and the raw material supply port F.

(About C18 silica gel)
C18 silica gel often used for separation of fatty acid esters has a structure in which an octadecylsilyl group is bonded to a hydroxyl group on the surface of the silica gel and a hydrophobic molecule of carbon chain 18 extends from the surface. The hydrophobic interaction generated between the hydrophobic molecule and each component changes the retention force on the filler and separates the components. Regarding the holding strength, there is the following tendency.
(1) The retention is stronger as the number of double bonds in the sample molecule is smaller.
(2) The higher the molecular weight of the sample molecule, the stronger the retention.
For this reason, eicosapentaenoic acid (C20: 5) and docosahexaenoic acid (C22: 6), which are particularly useful among highly unsaturated fatty acids, are eicosatetraenoic acid (C20: 4ω3), which is a naturally derived related substance, The degree of difficulty in separation on C18 silica gel is high among components such as arachidonic acid (C20: 4ω6) and docosapentaenoic acid (C22: 5) due to the relationship between the molecular weight and the number of double bonds. Therefore, when the components are sufficiently separated in order to obtain a high-purity product by the conventional SMB, it is necessary to increase the filler with respect to the sample or reduce the injection amount of the sample.

(About C8, C4, C1 silica gel)
C8 and C4 silica gels have C18 silica gel bonded with hydrophobic molecules with carbon chains 18 whereas hydrophobic molecules with carbon chains 8 and 4 are bonded with each other. For this reason, it has the following characteristics compared with C18 silica gel.
(1) The holding power is weak.
(2) Low stereoselectivity.
This shows a separation pattern that is not found in C18 and gives better separation conditions than C18 silica gel (eg quinine and lidocaine) for some materials.

  Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited thereto.

(Comparative Example 1)
As a comparative example, the result using only C18 silica gel is described.

  As a starting material, an oil containing 75% of EPA ethyl ester was used. The raw material concentration was 200 g / L (methanol solution). The raw material supply amount was 10.5 mL (10.5 mL / LR / h) per liter of filler per hour. As the filler, four C18 silica gels were used. The column size was 10 mm diameter x 500 mm height. Methanol as the eluent was used at 400 mL (0.40 L / LR / h) per liter of filler per hour of eluent volume.

The resulting purified product contained EPA ethyl with a purity of 98.3%. The related substances included the following substances:
Ethyl octadecatetraenoate: 0.31%
Ethyl arachidonate: 0.42%
Ethicosatetraenoic acid ethyl: 0.46%
Other impurities: 0.51%.

  The recovery rate of EPA was 89%, POV (peroxide value) was 1.9, AV (acid value) was 0.03, and AnV (anisidine value) was 0.5. From the above results, it was found that this product can be used in the fields of medicine, cosmetics and food.

Example 1
The results using the C18 column and the C8 column are as follows.

  The starting material was an oil containing 75% EPA ethyl ester. The raw material concentration was 200 g / L (methanol solution), and the raw material supply amount was 21 mL (21 mL / LR / h) per liter of filler per hour. The columns used were three C18 silica gel columns and one C8 silica gel column (column size: diameter 10 mm × height 500 mm). Methanol as eluent was used at 400 mL (0.40 L / L-R / h) of eluent volume per liter of filler per hour.

As a result, EPA ethyl having a purity of 98.3% was obtained. Related substances included the following:
Ethyl octadecatetraenoate: 0.22%
Ethyl arachidonate: 0.32%
Ethicosatetraenoic acid ethyl: 0.55%
Other impurities: 0.61%.

  The recovery rate of EPA was 92%, POV (peroxide value) was 2.2, AV (acid value) was 0.04, and AnV (anisidine value) was 0.6.

  From the above results, compared to Comparative Example 1 using only the C18 column, the raw material supply amount can be processed up to about twice. Therefore, in the present invention, it became clear that pharmaceutical grade EPA ethyl can be produced with a smaller amount of filler than before.

(Example 2)
The results obtained by changing the ratio of the C18 column to the C8 column from Example 1 are as follows.

  As a starting material, an oil containing 75% of EPA ethyl ester was used. The raw material concentration was 200 g / L (methanol solution), and 21 mL (21 mL / LR / h) per 1 L of filler per hour was used as the raw material supply amount. The columns used were two C18 silica gel columns and two C8 silica gel columns (column size: diameter 10 mm × height 500 mm). Methanol as eluent was used at 400 mL (0.40 L / L-R / h) of eluent volume per liter of filler per hour.

As a result, EPA ethyl having a purity of 95.0% was obtained. Related substances included the following:
Ethyl octadecatetraenoate: 0.60%
Ethyl arachidonate: 0.84%
Ethicosatetraenoate: 1.37%
Other impurities: 2.19%
The recovery rate of EPA was 80%, POV (peroxide value) was 2.0, AV (acid value) was 0.02, and AnV (anisidine value) was 0.7.

  From the above results, the purity decreased by 3.3% compared to Comparative Example 1, but the raw material supply amount could be processed up to about twice.

Example 3
The results using the C18 column and the C4 column are as follows.

  As a starting material, an oil containing 75% of EPA ethyl ester was used. The raw material concentration was 200 g / L (methanol solution), and 21 mL (21 mL / LR / h) per 1 L of filler per hour was used as the raw material supply amount. The columns used were three C18 silica gel columns and one C4 silica gel column (column size: diameter 10 mm × height 500 mm). Methanol as eluent was used at 400 mL (0.40 L / L-R / h) of eluent volume per liter of filler per hour.

As a result, EPA ethyl having a purity of 98.2% was obtained. Related substances included the following:
Ethyl octadecatetraenoate: 0.30%
Ethyl arachidonate: 0.29%
Ethicosatetraenoate: 0.44%
Other impurities: 0.77%
The recovery rate of EPA was 87%, POV (peroxide value) was 2.1, AV (acid value) was 0.03, and AnV (anisidine value) was 0.7.

  From the above results, the purity decreased by 0.1% as compared with Comparative Example 1, but the raw material supply amount could be processed up to about twice. It was also found from other data that it could be purified to pharmaceutical grade.

Example 4
The results obtained by changing the ratio of the C18 column and the C4 column from Example 3 are as follows.

  As a starting material, an oil containing 75% of EPA ethyl ester was used. The raw material concentration was 200 g / L (methanol solution), and 21 mL (21 mL / LR / h) per 1 L of filler per hour was used as the raw material supply amount. The columns used were two C18 silica gel columns and two C4 silica gel columns (column size: diameter 10 mm × height 500 mm). Methanol as eluent was used at 400 mL (0.40 L / L-R / h) of eluent volume per liter of filler per hour.

As a result, EPA ethyl having a purity of 98.6% was obtained. Related substances included the following:
Ethyl octadecatetraenoate: 0.24%
Ethyl arachidonate: 0.21%
Ethicosatetraenoic acid ethyl: 0.36%
Other impurities: 0.59%
The recovery rate of EPA was 76%, POV (peroxide value) was 2.5, AV (acid value) was 0.03, and AnV (anisidine value) was 0.7.

  From the above results, the purity increased by 0.3% compared to Comparative Example 1, and the raw material supply amount could be processed up to about twice. It was also found from other data that it could be purified to pharmaceutical grade.

(Combination of C18 silica gel and C8, C4, C1 silica gel)
In the present invention, by using two or more kinds of column packing materials having different separation performances in SMB chromatography, in particular, by using a combination of C18 column and C8 column and / or C4 column and / or C1 column A plurality of substances that are difficult to separate depending on the type of column packing can be easily and easily separated. Moreover, in the present invention, it is possible to perform separation (for example, purification to a pharmaceutical grade) with higher accuracy and in a larger amount than conventional SMB chromatography. In one aspect of the present invention, high-precision separation can be completed in a short time and at a low cost.

  Although not wishing to be bound by theory, C18 silica gel, C8 silica gel, C4 silica gel, and C1 silica gel have low stereoselectivity, so the strength of component retention depends on the difference in polarity rather than molecular weight. . From this, for example, it is considered that the degree of separation is improved for components having the same molecular weights such as eicosapentaenoic acid (C20: 5) and eicosatetraenoic acid (C20: 4ω3) but having a difference in polarity. It is done. In addition, since C8, C4, and C1 silica gels have weak holding power, the time from injection to extraction of components is shortened. Therefore, the processing amount when compared with C18 silica gel increases. As a result, the optimum combination of separation capacity and processing capacity as an SMB column packing material is considered to improve the processing capacity per packing material compared to conventional C18 silica gel and contribute to the reduction of manufacturing costs. .

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  In accordance with the present invention, a highly efficient unsaturated fatty acid purification process and apparatus therefor are provided.

Claims (12)

A method for separating a fat-soluble substance using SMB chromatography,
Here, an apparatus for performing separation by the SMB chromatography is as follows:
(1) at least three C18 columns;
(2) at least one column selected from the group consisting of a C8 column, a C4 column, and a C1 column; and
(3) The same number of circulation lines as the number of columns connecting the columns;
Prepared,
Here, the method is as follows:
(A) supplying the eluent to the first circulation line;
(A) supplying the sample to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Where the first to fourth circulation lines are downstream from the upstream, the first circulation line, the second circulation line, the third circulation line, and A method of arranging in order of the fourth circulation line.   The method of claim 1, wherein the sample is a distilled sample.   The method according to claim 1, wherein the fat-soluble substance is a fatty acid or a fatty acid derivative.   The method according to claim 1, wherein the fat-soluble substance is selected from the group consisting of α-linolenic acid, stearidonic acid, docosapentaenoic acid, eicosapentaenoic acid, docosahexaenoic acid, and lower alcohol esters thereof. . A method for separating a fat-soluble substance using SMB chromatography,
Here, an apparatus for performing separation by the SMB chromatography is as follows:
(1) at least two columns selected from the group consisting of a C18 column, a C8 column, a C4 column, and a C1 column; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Here, the method is as follows:
(A) supplying the eluent to the first circulation line;
(A) supplying the sample to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Where the first to fourth circulation lines are downstream from the upstream, the first circulation line, the second circulation line, the third circulation line, and A method of arranging in order of the fourth circulation line.   The method of claim 5, wherein the sample is a distilled sample.   The method according to claim 5, wherein the fat-soluble substance is a fatty acid or a fatty acid derivative.   The method according to claim 5, wherein the fat-soluble substance is selected from the group consisting of α-linolenic acid, stearidonic acid, docosapentaenoic acid, eicosapentaenoic acid, docosahexaenoic acid, and lower alcohol esters thereof. . Separation apparatus for fat-soluble substances using SMB chromatography, comprising:
(1) at least three C18 columns;
(2) at least one column selected from the group consisting of a C8 column, a C4 column, and a C1 column; and
(3) The same number of circulation lines as the number of columns connecting the columns;
Prepared,
Where the device is:
(A) supplying the eluent to the first circulation line;
(A) supplying the raw material to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Wherein the first circulation line to the fourth circulation line are arranged downstream from the upstream, the first circulation line, the second circulation line, the A device arranged in the order of the third circulation line and the fourth circulation line. Separation apparatus for fat-soluble substances using SMB chromatography, comprising:
(1) at least two columns selected from the group consisting of a C18 column, a C8 column, a C4 column, and a C1 column; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Where the device is:
(A) supplying the eluent to the first circulation line;
(A) supplying the raw material to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Wherein the first circulation line to the fourth circulation line are arranged downstream from the upstream, the first circulation line, the second circulation line, the A device arranged in the order of the third circulation line and the fourth circulation line. A method for separating a fat-soluble substance using SMB chromatography,
Here, an apparatus for performing separation by the SMB chromatography is as follows:
(1) at least two types of at least four reversed phase columns; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Here, the method is as follows:
(A) supplying the eluent to the first circulation line;
(A) supplying the sample to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Where the first to fourth circulation lines are downstream from the upstream, the first circulation line, the second circulation line, the third circulation line, and A method of arranging in order of the fourth circulation line. Separation apparatus for fat-soluble substances using SMB chromatography, comprising:
(1) at least two types of at least four reversed phase columns; and
(2) The same number of circulation lines as the number of columns connecting between columns are provided,
Where the device is:
(A) supplying the eluent to the first circulation line;
(A) supplying the raw material to the third circulation line;
(C) extracting the separation solution from the second circulation line and the fourth circulation line; and
(D) switching the first circulation line, the second circulation line, the third circulation line, and the fourth circulation line;
Wherein the first circulation line to the fourth circulation line are arranged downstream from the upstream, the first circulation line, the second circulation line, the A device arranged in the order of the third circulation line and the fourth circulation line.

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