Method for producing highly unsaturated fatty acid ester composition

Abstract

Provided is a method for efficiently producing the desired highly unsaturated fatty acid ester composition in high purity. Provided is a method for producing a highly unsaturated fatty acid ester composition from an oil / fat composition, the method comprising: the removal, by subjecting the oil / fat composition to a first column chromatography, of some or all of at least one component having a relative value for the peak top retention time of 1.8-3.5 in the first column chromatography when the peak top retention time of the desired substance is 1.0; and the removal, by subjecting an eluate comprising the desired substance obtained by the first column chromatography to a second column chromatography, of some or all of at least one component having a relative value for the peak top retention time of 0.90-1.1 in the second column chromatography when the peak top retention time of the desired substance is 1.0.

Description

Method for producing highly unsaturated fatty acid ester composition

[0001] The present invention relates to a method for producing a composition of polyunsaturated fatty acid (PUFA) esters using column chromatography.

[0002] Polyunsaturated fatty acids are used in pharmaceuticals and health foods. Polyunsaturated fatty acids are produced from natural raw materials, such as vegetable oils and marine oils, which are rich in polyunsaturated fatty acids. Natural raw materials also contain other components, such as fatty acids with different numbers of double bonds, e.g., saturated fatty acids, monounsaturated fatty acids, and fatty acids with different carbon chain lengths. The content of polyunsaturated fatty acids in natural raw materials is not high. To use polyunsaturated fatty acids in pharmaceuticals and health foods, it is necessary to selectively separate the desired polyunsaturated fatty acids (Patent Document 1).

[0003] Column chromatography is known as a method for separating highly unsaturated fatty acids (Patent Document 2), while simulated moving bed chromatography is used as a type of chromatography that allows continuous operation (Patent Documents 3 to 7).

[0004] International Publication No. 2014 / 054435 Special Publication No. 2014-511406 Publication Special Publication No. Hei 8-512336 Publication Japanese Patent Application Publication No. Hei 8-218091 Publication Special Publication No. 2016-508156 Publication Publication No. 2019-207234 Publication Special Publication No. 2013-516398

[0005] Simulated moving bed chromatography requires a complex system and equipment and is more expensive than chromatography using a single column. Therefore, there remains a need for a method for efficiently producing a target highly unsaturated fatty acid ester composition with high purity without requiring a complex device such as simulated moving bed chromatography.

[0006] The present inventors have unexpectedly found that a multistage separation method using a first column chromatography and a second column chromatography can efficiently produce the desired polyunsaturated fatty acid, thereby addressing the above-mentioned problems, and have completed the present invention. The gist of the present invention is a method for producing a polyunsaturated fatty acid ester composition from the following oil and fat composition.

[0007] [1-1] A method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition, comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column to remove all or part of at least one component having a peak top retention time relative to 1.8 to 3.5, where the peak top retention time of the target highly unsaturated fatty acid ester in the first column chromatography is taken as 1.0, thereby obtaining an eluate containing the target product by the first column chromatography; (b) subjecting the eluate containing the target product obtained by the first column chromatography to a second column chromatography using a second column to remove all or part of at least one component having a peak top retention time relative to 0.90 to 1.1, where the peak top retention time of the target product in the second column chromatography is taken as 1.0, thereby obtaining an eluate by the second column chromatography; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing the target product, wherein the obtained composition contains 95% by weight or more of the target product.

[0008] [1-2] The method according to [1-1], wherein the loading interval until the next column chromatography can be started after the start of one column chromatography run is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns. [1-3] The method according to [1-2], wherein the loading interval until the next column chromatography run can be started after the start of one column chromatography run is shorter than that in the case of column chromatography using only the second column.

[0009] [1-4] The method according to any one of [1-1] to [1-3], wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the target substance in the first column chromatography is at least one component other than a highly unsaturated fatty acid ester in the oil or fat composition. [1-5] The method according to any one of [1-1] to [1-4], wherein the component that is completely or partially removed by subjecting the eluate containing the target substance obtained in the first column chromatography to a second column chromatography using a second column is at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of the target substance in the second column chromatography. [1-6] The method according to any one of [1-1] to [1-5], wherein the first column can be connected to the second column so that the eluate from the first column can be injected into the second column, and the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of at least one component contained in the eluate is in the range of 1.1 or less when the retention time of the peak top of the target substance in second column chromatography is set to 1.0. [1-7] The method according to [1-6], wherein the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.90 to 1.1 when the retention time of the peak top of the target substance in second column chromatography is set to 1.0.

[0010] [1-8] The method according to [1-6], wherein the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.94 to 1.1 when the retention time of the peak top of the target compound in the second column chromatography is taken as 1.0. [1-9] The method according to [1-6], wherein the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.96 to 1.1 when the retention time of the peak top of the target compound in the second column chromatography is taken as 1.0. [1-10] The method according to [1-6], wherein the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.93 to 1.09 when the retention time of the peak top of the target compound in the second column chromatography is taken as 1.0. [1-11] The method according to [1-6], wherein the eluate from the first column having a retention time relative to 3.5 or more when the retention time of the peak top of the target compound in the first column is taken as 1.0 is discharged, and a new mobile phase is injected into the second column. [1-12] The method according to any one of [1-1] to [1-11], wherein the first column chromatography and the second column chromatography use reverse-phase column chromatography.

[0011] [1-13] The method according to any one of [1-1] to [1-12], wherein the first column chromatography and the second column chromatography are fixed-bed column chromatography. [1-14] The method according to any one of [1-1] to [1-13], wherein the first column chromatography and the second column chromatography use the same mobile phase. [1-15] The method according to any one of [1-1] to [1-14], wherein the second column is packed with a larger amount of stationary phase than the first column.

[0012] [1-16] The method according to any one of [1-1] to [1-15], wherein the second column has the same inner diameter as the first column but is longer than the first column. [1-17] The method according to any one of [1-1] to [1-16], wherein the oil or fat composition is derived from any one of fish oil, vegetable oil, algae, and microorganisms. [1-18] The method according to any one of [1-1] to [1-17], wherein the target substance is an ester of any one of eicosapentaenoic acid (EPA), dihomo-γ-linolenic acid (DGLA), and arachidonic acid (ARA). [1-19] The method according to any one of [1-1] to [1-18], wherein at least one component removed in whole or in part by the first column chromatography is an ester of any one of C22:0, C20:0, C18:0, and C20:1. [1-20] The method according to [1-18] or [1-19], wherein the ester is an ethyl ester.

[0013] [1-21] The method according to any one of [1-1] to [1-20], wherein the content of the at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.3 wt % or less, based on the total fatty acid esters contained in the eluate subjected to the second column chromatography. [1-22] The method according to [1-21], wherein the content of the at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.1 wt % or less or 0.05 wt % or less, based on the total fatty acid esters contained in the eluate subjected to the second column chromatography.

[0014] [1-23] The method according to any one of [1-1] to [1-22], wherein the composition obtained in (c) contains 96% by weight or more of the target substance. [1-24] The method according to any one of [1-1] to [1-23], further comprising preparing the oil or fat composition by distillation from the raw material composition. [1-25] The method according to any one of [1-1] to [1-24], wherein (a), (b), and (c) are repeated two or more times. [1-26] The method according to any one of [1-1] to [1-25], further comprising, before (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target substance. [1-27] The method according to any one of [1-1] to [1-26], wherein the eluate containing the target substance obtained by the first column chromatography is the eluate obtained up to the end of elution of the target substance from the first column.

[0015] [2-1] A method for producing an eicosapentaenoic acid ester composition from an oil or fat composition, the method comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column to obtain an eluate containing an eicosapentaenoic acid ester, the eicosapentaenoic acid ester containing 0.3 wt% or less of a C20:0 ester relative to all fatty acid esters contained in the eluate; (b) subjecting the eluate containing the eicosapentaenoic acid ester obtained by the first column chromatography to a second column chromatography using a second column to obtain an eluate; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing an eicosapentaenoic acid ester, wherein the obtained composition contains 96.5 wt% or more of an eicosapentaenoic acid ester and at least one component other than an eicosapentaenoic acid ester, and the at least one component other than an eicosapentaenoic acid ester contains 0.2 wt% or less each of stearidonic acid, C19:4 ester, and C21:5 ester. [2-2] The method according to [2-1], wherein the obtained composition contains 0.01 wt % to 0.2 wt % of stearidonic acid, C19:4 ester, and C21:5 ester.

[0016] [2-3] The method according to [2-1] or [2-2], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns. [2-4] The method according to [2-3], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using only the second column.

[0017] [2-5] The method according to any one of [2-1] to [2-4], wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the eicosapentaenoic acid ester in the first column chromatography, where the retention time of the peak top of the eicosapentaenoic acid ester in the first column chromatography is taken as 1.0, is at least one component other than the eicosapentaenoic acid ester in the oil or fat composition. [2-6] The method according to any one of [2-1] to [2-5], wherein the component that is completely or partially removed by subjecting the eluate containing the eicosapentaenoic acid ester obtained in the first column chromatography to a second column chromatography using a second column is at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of the eicosapentaenoic acid ester in the second column chromatography, where the retention time of the peak top of the eicosapentaenoic acid ester in the second column chromatography is taken as 1.0. [2-7] The method according to any one of [2-1] to [2-6], wherein the first column can be connected to the second column so that the eluate from the first column can be injected into the second column, and the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of at least one component other than the eicosapentaenoic acid ester contained in the eluate is in the range of 1.1 or less, when the retention time of the peak top of the eicosapentaenoic acid ester in second column chromatography is set to 1.0. [2-8] The method according to [2-7], wherein the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of the eicosapentaenoic acid ester contained in the eluate is in the range of 0.90 to 1.1, when the retention time of the peak top of the eicosapentaenoic acid ester in second column chromatography is set to 1.0.

[0018] [2-9] The method according to [2-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than the eicosapentaenoic acid ester contained in the eluate, when the retention time of the peak top of the eicosapentaenoic acid ester in second column chromatography is taken as 1.0, is in the range of 0.94 to 1.1. [2-10] The method according to [2-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of the eicosapentaenoic acid ester contained in the eluate, when the retention time of the peak top of the eicosapentaenoic acid ester in second column chromatography is taken as 1.0, is in the range of 0.96 to 1.1. [2-11] The method according to [2-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than the eicosapentaenoic acid ester contained in the eluate, when the retention time of the peak top of the eicosapentaenoic acid ester in second column chromatography is taken as 1.0, is in the range of 0.93 to 1.09. [2-12] The method according to [2-7], wherein the eluate from the first column, when the relative value of the retention time of the peak top of the target substance in the first column is taken as 1.0, is 3.5 or more, is discharged, and a new mobile phase is injected into the second column.

[0019] [2-13] The method according to any one of [2-1] to [2-12], wherein the first column chromatography and the second column chromatography use reversed-phase column chromatography. [2-14] The method according to any one of [2-1] to [2-13], wherein the first column chromatography and the second column chromatography use fixed-bed column chromatography. [2-15] The method according to any one of [2-1] to [2-14], wherein the first column chromatography and the second column chromatography use the same mobile phase.

[0020] [2-16] The method according to any one of [2-1] to [2-15], wherein the second column is packed with a larger amount of stationary phase than the first column. [2-17] The method according to any one of [2-1] to [2-16], wherein the second column has the same inner diameter as the first column but is longer than the first column. [2-18] The method according to any one of [2-1] to [2-17], wherein at least one component removed in whole or in part by the first column chromatography is an ester of any one of C20:0, C18:0, and C20:1. [2-19] The method according to any one of [2-1] to [2-18], wherein the oil or fat composition is derived from any one of fish oil, vegetable oil, algae, and microorganisms. [2-20] The method according to any one of [2-1] to [2-19], wherein the ester is an ethyl ester.

[0021] [2-21] The method according to any one of [2-1] to [2-20], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing an eicosapentaenoic acid ester and subjected to the second column chromatography is 0.3 wt % or less, based on the total amount of fatty acid esters contained in the eluate subjected to the second column chromatography. [2-22] The method according to [2-21], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing an eicosapentaenoic acid ester and subjected to the second column chromatography is 0.1 wt % or less or 0.05 wt % or less, based on the total amount of fatty acid esters contained in the eluate subjected to the second column chromatography.

[0022] [2-23] The method according to any one of [2-1] to [2-22], wherein the obtained composition contains 96% by weight or more of eicosapentaenoic acid esters. [2-24] The method according to any one of [2-1] to [2-22], further comprising preparing the oil or fat composition by distillation from a raw material composition. [2-25] The method according to any one of [2-1] to [2-24], wherein (a), (b), and (c) are repeated two or more times.

[0023] [2-26] The method according to any one of [2-1] to [2-25], wherein in (b), all or part of at least one component having a peak top retention time of 0.90 to 1.1 relative to the retention time of the peak top of the eicosapentaenoic acid ester in the second column chromatography, where the retention time of the peak top of the eicosapentaenoic acid ester in the second column chromatography is taken as 1.0, is removed. [2-27] The method according to any one of [2-1] to [2-26], further comprising, before (a), subjecting the oil or fat composition to column chromatography using the first column to confirm the retention time of the peak top of the eicosapentaenoic acid ester. [2-28] The method according to any one of [2-1] to [2-27], wherein the eluate containing the eicosapentaenoic acid ester obtained in the first column chromatography is the eluate obtained up to the completion of elution of the eicosapentaenoic acid ester from the first column.

[0024] [3-1] A method for producing a dihomo-γ-linolenic acid ester composition from an oil or fat composition, the method comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column to obtain an eluate containing dihomo-γ-linolenic acid esters, the eluate containing C22:0 esters in an amount of 0.5% by weight or less relative to all fatty acid esters contained in the eluate; (b) subjecting the eluate containing dihomo-γ-linolenic acid esters obtained by the first column chromatography to a second column chromatography using a second column to obtain an eluate; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing eicosapentaenoic acid esters, wherein the obtained composition contains 95.0% by weight or more of dihomo-γ-linolenic acid esters and at least one component other than dihomo-γ-linolenic acid esters, and the at least one component other than dihomo-γ-linolenic acid esters contains 0.60% by weight or less of C20:4n-6 esters. [3-2] The method according to [3-1], wherein the obtained composition contains 0.01% by weight to 0.2% by weight of C20:4n-6 ester.

[0025] [3-3] The method according to [3-1] or [3-2], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns. [3-4] The method according to [3-3], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using only the second column.

[0026] [3-5] The method according to any one of [3-1] to [3-4], wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of a dihomo-γ-linolenic acid ester in the first column chromatography is at least one component other than a dihomo-γ-linolenic acid ester in the oil or fat composition. [3-6] The method according to any one of [3-1] to [3-5], wherein the component that is completely or partially removed by subjecting the eluate containing the target substance obtained by the first column chromatography to a second column chromatography using a second column is at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of a dihomo-γ-linolenic acid ester in the second column chromatography. [3-7] The method according to any one of [3-1] to [3-6], wherein the first column can be connected to the second column so that an eluate from the first column can be injected into the second column, and the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of at least one component other than dihomo-γ-linolenic acid ester contained in the eluate, when the retention time of the peak top of dihomo-γ-linolenic acid ester in second column chromatography is taken as 1.0, is 1.1 or less.

[0027] [3-8] The method according to [3-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than dihomo-γ-linolenic acid ester contained in the eluate, when the retention time of the peak top of the dihomo-γ-linolenic acid ester in second column chromatography is taken as 1.0, is in the range of 0.90 to 1.1. [3-9] The method according to [3-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than dihomo-γ-linolenic acid ester contained in the eluate, when the retention time of the peak top of the dihomo-γ-linolenic acid ester in second column chromatography is taken as 1.0, is in the range of 0.94 to 1.1. [3-10] The method according to [3-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than dihomo-γ-linolenic acid ester contained in the eluate, when the retention time of the peak top of the dihomo-γ-linolenic acid ester in second column chromatography is taken as 1.0, is in the range of 0.96 to 1.1. [3-11] The method according to [3-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than dihomo-γ-linolenic acid ester contained in the eluate, when the retention time of the peak top of the dihomo-γ-linolenic acid ester in second column chromatography is taken as 1.0, is in the range of 0.93 to 1.09. [3-12] The method according to [3-7], wherein the eluate from the first column having a relative retention time of 3.5 or more, where the retention time of the peak top of the dihomo-γ-linolenic acid ester in the first column is set to 1.0, is discharged, and a new mobile phase is injected into the second column. [3-13] The method according to any one of [3-1] to [3-12], wherein the first column chromatography and the second column chromatography use reversed-phase column chromatography.

[0028] [3-14] The method according to any one of [3-1] to [3-13], wherein the first column chromatography and the second column chromatography are fixed-bed column chromatography. [3-15] The method according to any one of [3-1] to [3-14], wherein the first column chromatography and the second column chromatography use the same mobile phase. [3-16] The method according to any one of [3-1] to [3-15], wherein the second column is packed with a larger amount of stationary phase than the first column.

[0029] [3-17] The method according to any one of [3-1] to [3-16], wherein the second column has the same inner diameter as the first column and is longer than the first column. [3-18] The method according to any one of [3-1] to [3-17], wherein at least one component removed in whole or in part by the first column chromatography is an ester of any one of C22:0, C20:0, C18:0, and C20:1. [3-19] The method according to any one of [3-1] to [3-18], wherein the oil or fat composition is derived from any one of fish oil, vegetable oil, algae, and microorganisms. [3-20] The method according to any one of [3-1] to [3-19], wherein the ester is an ethyl ester.

[0030] [3-21] The method according to any one of [3-1] to [3-20], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing dihomo-γ-linolenic acid esters and subjected to the second column chromatography is 0.3 wt % or less, based on the total amount of fatty acid esters contained in the eluate subjected to the second column chromatography. [3-22] The method according to [3-21], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing dihomo-γ-linolenic acid esters and subjected to the second column chromatography is 0.1 wt % or less or 0.05 wt % or less, based on the total amount of fatty acid esters contained in the eluate subjected to the second column chromatography. [3-23] The method according to any one of [3-1] to [3-22], wherein the obtained composition contains 96 wt % or more of dihomo-γ-linolenic acid esters.

[0031] [3-24] The method according to any one of [3-1] to [3-23], wherein in (b), all or part of at least one component having a peak top retention time of 0.90 to 1.1 relative to the retention time of the peak top of a dihomo-γ-linolenic acid ester in the second column chromatography, where the retention time of the peak top of the dihomo-γ-linolenic acid ester in the second column chromatography is taken as 1.0, is removed. [3-25] The method according to any one of [3-1] to [3-24], further comprising, before (a), subjecting the oil or fat composition to column chromatography using the first column to confirm the retention time of the peak top of the dihomo-γ-linolenic acid ester. [3-26] The method according to any one of [3-1] to [3-25], wherein the eluate containing the dihomo-γ-linolenic acid ester obtained in the first column chromatography is the eluate obtained up to the end of elution of the dihomo-γ-linolenic acid ester from the first column.

[0032] [4-1] A method for producing an arachidonic acid ester composition from an oil or fat composition, comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column to obtain an eluate containing arachidonic acid esters, the eluate containing C22:0 esters being 0.5 wt% or less relative to all fatty acid esters contained in the eluate; (b) subjecting the eluate containing arachidonic acid esters obtained by the first column chromatography to a second column chromatography using a second column to obtain an eluate; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing arachidonic acid esters, wherein the obtained composition contains 95.0 wt% or more of arachidonic acid esters and at least one component other than arachidonic acid esters, and the at least one component other than arachidonic acid esters contains 0.25 wt% or less of C18:3n-6 esters. [4-2] The method according to [4-1], wherein the obtained composition contains 0.01% by weight to 0.2% by weight of C18:3n-6 ester.

[0033] [4-3] The method according to [4-1] or [4-2], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns. [4-4] The method according to [4-3], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using only the second column.

[0034] [4-5] The method according to any one of [4-1] to [4-4], wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the arachidonic acid ester in the first column chromatography, where the retention time of the peak top of the arachidonic acid ester in the first column chromatography is taken as 1.0, is at least one component other than the arachidonic acid ester in the oil or fat composition. [4-6] The method according to any one of [4-1] to [4-5], wherein the component that is completely or partially removed by subjecting the arachidonic acid ester-containing eluate obtained by the first column chromatography to a second column chromatography using a second column is at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of the arachidonic acid ester in the second column chromatography, where the retention time of the peak top of the arachidonic acid ester in the second column chromatography is taken as 1.0. [4-7] The method according to any one of [4-1] to [4-6], wherein the first column can be connected to the second column so that the eluate from the first column can be injected into the second column, and the relative value of the retention time of the peak top of at least one component other than arachidonic acid esters contained in the eluate, when the retention time of the peak top of the arachidonic acid esters in second column chromatography is taken as 1.0, is 1.1 or less. [4-8] The method according to any one of [4-7], wherein the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of at least one component other than arachidonic acid esters contained in the eluate, when the retention time of the peak top of the arachidonic acid esters in second column chromatography is taken as 1.0, is 0.90 to 1.1.

[0035] [4-9] The method according to [4-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than arachidonic acid ester contained in the eluate is in the range of 0.94 to 1.1 when the retention time of the peak top of the arachidonic acid ester in the second column chromatography is set to 1.0. [4-10] The method according to [4-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than arachidonic acid ester contained in the eluate is in the range of 0.96 to 1.1 when the retention time of the peak top of the arachidonic acid ester in the second column chromatography is set to 1.0. [4-11] The method according to [4-7], wherein the eluate from the first column is injected into a second column, and the relative value of the retention time of the peak top of at least one component other than arachidonic acid ester contained in the eluate, when the retention time of the peak top of the arachidonic acid ester in the second column chromatography is taken as 1.0, is in the range of 0.93 to 1.09. [4-12] The method according to [4-7], wherein the eluate from the first column, when the relative value of the retention time of the peak top of the arachidonic acid ester in the first column is taken as 1.0, is 3.5 or more, is discharged, and a new mobile phase is injected into the second column.

[0036] [4-13] The method according to any one of [4-1] to [4-12], wherein the first column chromatography and the second column chromatography use reversed-phase column chromatography. [4-14] The method according to any one of [4-1] to [4-13], wherein the first column chromatography and the second column chromatography use fixed-bed column chromatography. [4-15] The method according to any one of [4-1] to [4-14], wherein the first column chromatography and the second column chromatography use the same mobile phase. [4-16] The method according to any one of [4-1] to [4-15], wherein the second column is packed with a larger amount of stationary phase than the first column.

[0037] [4-17] The method according to any one of [4-1] to [4-16], wherein the second column has the same inner diameter as the first column and is longer than the first column. [4-18] The method according to any one of [4-1] to [4-17], wherein at least one component removed in whole or in part by the first column chromatography is an ester of any one of C22:0, C20:0, C18:0, and C20:1. [4-19] The method according to any one of [4-1] to [4-18], wherein the oil or fat composition is derived from any one of fish oil, vegetable oil, algae, and microorganisms. [4-20] The method according to any one of [4-1] to [4-19], wherein the ester is an ethyl ester.

[0038] [4-21] The method according to any one of [4-1] to [4-20], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing arachidonic acid esters and subjected to the second column chromatography is 0.3 wt% or less, based on the total amount of fatty acid esters contained in the eluate subjected to the second column chromatography. [4-22] The method according to [4-21], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing arachidonic acid esters and subjected to the second column chromatography is 0.1 wt% or less or 0.05 wt% or less, based on the total amount of fatty acid esters contained in the eluate subjected to the second column chromatography. [4-23] The method according to any one of [4-1] to [4-22], wherein the obtained composition contains 96 wt% or more of arachidonic acid esters.

[0039] [4-24] The method according to any one of [4-1] to [4-23], further comprising preparing the oil or fat composition from a raw material composition by distillation. [4-25] The method according to any one of [4-1] to [4-24], further comprising repeating (a), (b), and (c) two or more times.

[0040] [4-26] The method according to any one of [4-1] to [4-25], wherein in (b), all or part of at least one component having a peak top retention time of 0.90 to 1.1 relative to the retention time of the peak top of the arachidonic acid ester in the second column chromatography, where the retention time of the peak top of the arachidonic acid ester in the second column chromatography is taken as 1.0, is removed. [4-27] The method according to any one of [4-1] to [4-26], further comprising, before (a), subjecting the oil or fat composition to column chromatography using the first column to confirm the retention time of the peak top of the arachidonic acid ester. [4-28] The method according to any one of [4-1] to [4-27], wherein the eluate containing the arachidonic acid ester obtained in the first column chromatography is the eluate obtained up to the end of elution of the arachidonic acid ester from the first column.

[0041] [5-1] A method for shortening the time required for column chromatography in producing a highly unsaturated fatty acid ester composition from an oil or fat composition, comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column, thereby removing all or part of at least one component having a peak top retention time relative to the peak top retention time of the target highly unsaturated fatty acid ester in the first column chromatography, where the retention time of the peak top of the target product in the first column chromatography is taken as 1.0, thereby obtaining an eluate containing the target product by the first column chromatography; (b) subjecting the eluate containing the target product obtained by the first column chromatography to a second column chromatography using a second column, thereby removing all or part of at least one component having a peak top retention time relative to the peak top retention time of the target product in the second column chromatography, where the retention time of the peak top of the target product in the second column chromatography is taken as 1.0, thereby obtaining an eluate by the second column chromatography; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing the target product, wherein the obtained composition contains 95% by weight or more of the target product, A method in which, after the start of one column chromatography run, the loading interval until another column chromatography run can be started is shorter than in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns.

[0042] [5-2] The method according to [5-1], wherein the loading interval after the start of one column chromatography run until the next column chromatography run can be started is shorter than that in the case of column chromatography using only the second column. [5-3] The method according to [5-1] or [5-2], wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the target substance in the first column chromatography run, which is set to 1.0, is at least one component other than a highly unsaturated fatty acid ester in the oil or fat composition.

[0043] [5-4] The method according to any one of [5-1] to [5-3], wherein the eluate containing the target product obtained by the first column chromatography is subjected to second column chromatography using a second column, and at least one component having a peak top retention time of 0.93 to 1.09 relative to the peak top retention time of the target product in the second column chromatography, is removed in whole or in part. [5-5] The method according to any one of [5-1] to [5-4], wherein the first column can be connected to the second column so that the eluate from the first column is injected into the second column, and the eluate from the first column is injected into the second column, and the relative peak top retention time of a component contained in the eluate is 1.1 or less, relative to the peak top retention time of the target product in the second column chromatography, is 1.0. [5-6] The method according to [5-5], wherein the eluate from the first column is injected into a second column, and the relative values ​​of the retention times of the peak tops of the components contained in the eluate, when the retention time of the peak top of the target substance in second column chromatography is taken as 1.0, are in the range of 0.90 to 1.1. [5-7] The method according to [5-5], wherein the eluate from the first column is injected into a second column, and the relative values ​​of the retention times of the peak tops of the components contained in the eluate, when the retention time of the peak top of the target substance in second column chromatography is taken as 1.0, are in the range of 0.94 to 1.1.

[0044] [5-8] The method according to [5-5], wherein the eluate from the first column is injected into the second column, and the relative value of the peak top retention time of the components contained in the eluate is in the range of 0.96 to 1.1 when the retention time of the peak top of the target substance in the second column chromatography is set to 1.0. [5-9] The method according to [5-5], wherein the eluate from the first column is injected into the second column, and the relative value of the peak top retention time of the components contained in the eluate is in the range of 0.93 to 1.09 when the retention time of the peak top of the target substance in the first column is set to 1.0. [5-10] The method according to [5-5], wherein the eluate from the first column having a retention time of 3.5 or more relative to the retention time of the peak top of the target substance in the first column is discharged, and a new mobile phase is injected into the second column. [5-11] The method according to any of [5-1] to [5-10], wherein the first column chromatography and the second column chromatography use reversed-phase column chromatography.

[0045] [5-12] The method according to any one of [5-1] to [5-11], wherein the first column chromatography and the second column chromatography are fixed-bed column chromatography. [5-13] The method according to any one of [5-1] to [5-12], wherein the first column chromatography and the second column chromatography use the same mobile phase. [5-14] The method according to any one of [5-1] to [5-13], wherein the second column is packed with a larger amount of stationary phase than the first column.

[0046] [5-15] The method according to any one of [5-1] to [5-14], wherein the second column has the same inner diameter as the first column but is longer than the first column. [5-16] The method according to any one of [5-1] to [5-15], wherein the target product is an ester of any one of eicosapentaenoic acid (EPA), dihomo-γ-linolenic acid (DGLA), and arachidonic acid (ARA). [5-17] The method according to any one of [5-1] to [5-16], wherein at least one component removed in whole or in part by the first column chromatography is an ester of any one of C22:0, C20:0, C18:0, and C20:1. [5-18] The method according to any one of [5-1] to [5-17], wherein the oil or fat composition is derived from any one of fish oil, vegetable oil, algae, and microorganisms. [5-19] The method according to [5-17] or [5-18], wherein the ester is an ethyl ester.

[0047] [5-20] The method according to any one of [5-1] to [5-19], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.3 wt % or less, based on the total fatty acid esters contained in the eluate subjected to the second column chromatography. [5-21] The method according to [5-20], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.1 wt % or less or 0.05 wt % or less, based on the total fatty acid esters contained in the eluate subjected to the second column chromatography. [5-22] The method according to any one of [5-1] to [5-21], wherein the highly unsaturated fatty acid ester composition contains 96 wt % or more of the target substance.

[0048] [5-23] The method according to any one of [5-1] to [5-22], further comprising preparing the oil or fat composition by distillation from a raw material composition. [5-24] The method according to any one of [5-1] to [5-23], wherein (a), (b), and (c) are repeated two or more times. [5-25] The method according to any one of [5-1] to [5-24], further comprising, before (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target substance. [5-26] The method according to any one of [5-1] to [5-25], wherein the eluate containing the target substance obtained by the first column chromatography is the eluate obtained up to the end of elution of the target substance from the first column.

[0049] [6-1] A method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition, comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column, thereby removing all or part of at least one component having a peak top retention time relative to 1.8 to 3.5, where the retention time of the peak top of the target highly unsaturated fatty acid ester in the first column chromatography is taken as 1.0, to obtain an eluate containing the target product by the first column chromatography; and (b) subjecting the eluate containing the target product obtained by the first column chromatography to a second column chromatography using a second column, thereby removing all or part of at least one component having a peak top retention time relative to 0.90 to 1.1, where the retention time of the peak top of the target product in the second column chromatography is taken as 1.0, to obtain an eluate by the second column chromatography.

[0050] [6-2] The method according to [6-1], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns. [6-3] The method according to [6-2], wherein the loading interval after the start of one column chromatography run until the start of another column chromatography run is shorter than that in the case of column chromatography using only the second column.

[0051] [6-4] The method according to any one of [6-1] to [6-3], wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the target substance in the first column chromatography, where the retention time is 1.0, is at least one component other than a highly unsaturated fatty acid ester in the oil or fat composition. [6-5] The method according to any one of [6-1] to [6-4], wherein the component that is completely or partially removed by subjecting the eluate containing the target substance obtained in the first column chromatography to a second column chromatography using a second column is at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of the target substance in the second column chromatography, where the retention time is 1.0. [6-6] The method according to any one of [6-1] to [6-5], wherein the first column can be connected to the second column so that the eluate from the first column can be injected into the second column, and the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of at least one component contained in the eluate is in the range of 1.1 or less, when the retention time of the peak top of the target substance in second column chromatography is set to 1.0. [6-7] The method according to [6-6], wherein the eluate from the first column is injected into the second column, and the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.90 to 1.1, when the retention time of the peak top of the target substance in second column chromatography is set to 1.0.

[0052] [6-8] The method according to [6-6], wherein the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.94 to 1.1 when the retention time of the peak top of the target compound in the second column chromatography is taken as 1.0. [6-9] The method according to [6-6], wherein the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.96 to 1.1 when the retention time of the peak top of the target compound in the second column chromatography is taken as 1.0. [6-10] The method according to [6-6], wherein the relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.93 to 1.09 when the retention time of the peak top of the target compound in the second column chromatography is taken as 1.0. [6-11] The method according to [6-6], wherein the eluate from the first column having a retention time relative to 3.5 or more when the retention time of the peak top of the target compound in the first column is taken as 1.0 is discharged, and a new mobile phase is injected into the second column. [6-12] The method according to any one of [6-1] to [6-11], wherein the first column chromatography and the second column chromatography use reverse-phase column chromatography.

[0053] [6-13] The method according to any one of [6-1] to [6-12], wherein the first column chromatography and the second column chromatography are fixed-bed column chromatography. [6-14] The method according to any one of [6-1] to [6-13], wherein the first column chromatography and the second column chromatography use the same mobile phase. [6-15] The method according to any one of [6-1] to [6-14], wherein the second column is packed with a larger amount of stationary phase than the first column.

[0054] [6-16] The method according to any one of [6-1] to [6-15], wherein the second column has the same inner diameter as the first column but is longer than the first column. [6-17] The method according to any one of [6-1] to [6-16], wherein the oil or fat composition is derived from fish oil, vegetable oil, algae, or a microorganism. [6-18] The method according to any one of [6-1] to [6-17], wherein the target substance is an ester of any one of eicosapentaenoic acid (EPA), dihomo-γ-linolenic acid (DGLA), and arachidonic acid (ARA). [6-19] The method according to any one of [6-1] to [6-18], wherein at least one component removed in whole or in part by the first column chromatography is an ester of any one of C22:0, C20:0, C18:0, and C20:1. [6-20] The method according to [6-18] or [6-19], wherein the ester is an ethyl ester.

[0055] [6-21] The method according to any one of [6-1] to [6-20], wherein the content of the at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.3 wt % or less, based on the total fatty acid esters contained in the eluate subjected to the second column chromatography. [6-22] The method according to [6-21], wherein the content of the at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.1 wt % or less or 0.05 wt % or less, based on the total fatty acid esters contained in the eluate subjected to the second column chromatography.

[0056] [6-23] The method according to any one of [6-1] to [6-22], wherein the obtained composition contains 96% by weight or more of the target substance. [6-24] The method according to any one of [6-1] to [6-23], further comprising preparing the oil or fat composition by distillation from a raw material composition. [6-25] The method according to any one of [6-1] to [6-24], wherein (a), (b), and (c) are repeated two or more times. [6-26] The method according to any one of [6-1] to [6-25], further comprising, before (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target substance. [6-27] The method according to any one of [6-1] to [6-26], wherein the eluate containing the target substance obtained by the first column chromatography is the eluate obtained up to the end of elution of the target substance from the first column.

[0057] In the present invention, it has been found that a composition containing highly unsaturated fatty acid ethyl esters such as EPA ethyl ester, DGLA ethyl ester, and ARA ethyl ester at high concentrations can be effectively produced in a shorter time and with less solvent usage than when a single column is used. In the present invention, fatty acids such as C20:0 ethyl ester can be removed.

[0058] In one aspect of the present invention, the "composition" can contain one or more PUFAs or ester derivatives thereof. Compositions containing PUFAs or ester derivatives thereof can be obtained from raw materials containing highly unsaturated fatty acids as constituent fatty acids. For example, suitable oil and fat compositions can be obtained from raw material compositions obtained by esterifying natural oils and fats, including plant and animal oils and fats, as well as from raw material compositions obtained by esterifying oils obtained from genetically modified plants, animals, and microorganisms, including yeast and filamentous fungi. For example, a composition obtained by esterifying oils obtained from microorganisms of the genus Mortierella, such as filamentous fungi, can be used. Examples include compositions obtained by ethyl esterifying fish oils, algae oils, and microalgae oils, as well as compositions obtained by ethyl esterifying vegetable oils, such as borage oil, scutellaria oil, and evening primrose oil. In one embodiment, the raw material composition is a composition obtained by ethyl esterifying fish oil. In another embodiment, the raw material composition is a composition obtained by ethyl esterifying algal oil.

[0059] In one embodiment of the present invention, the oil and fat composition is obtained by subjecting a raw material composition to distillation. In one embodiment of the present invention, the highly unsaturated fatty acid ester composition is a composition obtained by the method of the present invention, and is a composition containing the target highly unsaturated fatty acid ester. The target highly unsaturated fatty acid ester can be any ester, for example, an ethyl ester, of eicosapentaenoic acid (EPA), dihomo-γ-linolenic acid (DGLA), or arachidonic acid (ARA).

[0060] As used herein, the term "fatty acid" refers to long-chain aliphatic carboxylic acids (alkanoic acids) of various chain lengths, ranging from approximately C12 to C22 (where the number refers to the total number of carbon atoms in the chain). The predominant chain length is C16 to C22. Fatty acid structures can be represented using a simple "X:Y" notation, where X is the total number of carbon atoms in the particular fatty acid and Y is the number of double bonds. For example, a saturated fatty acid with 20 carbon atoms may be represented as "C20:0," a monounsaturated fatty acid with 18 carbon atoms may be represented as "C18:1," etc., and arachidonic acid may be represented as "C20:4,n-6," etc. The "n-" indicates the position of the double bond beginning from the methyl end of the fatty acid; for example, "n-6" indicates that the double bond begins at the sixth position counting from the methyl end of the fatty acid. This method is well known to those skilled in the art, and fatty acids represented according to this method can be readily identified by those skilled in the art.

[0061] Fatty acids are carboxylic acids with long aliphatic chains, which can be either saturated or unsaturated. Fatty acids are usually produced industrially by hydrolysis of triglycerides or phospholipids from natural sources. Some are also produced synthetically. Regardless of the method of production, purification methods are required to obtain pure products for food, cosmetic, or industrial use.

[0062] As used herein, the term "polyunsaturated fatty acid (PUFA)" refers to a fatty acid with more than one double bond. The polyunsaturated fatty acid herein is in the form of an ester. The ester is typically an alkyl ester, e.g., C １ -C ６ Alkyl ester, or C １ -C ４ It is an alkyl ester. An example of an ester is an ethyl ester.

[0063] In one embodiment of the present invention, the highly unsaturated fatty acids include eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, stearidonic acid, C18:3, C19:4, C20:4, and C21:5. In one embodiment of the present invention, the highly unsaturated fatty acids include eicosapentaenoic acid, dihomo-γ-linolenic acid, and arachidonic acid. In one embodiment of the present invention, the at least one component removed in whole or in part by the second column chromatography includes C18:3, C19:4, C20:4, and C21:5. The at least one component removed in whole or in part by the second column chromatography may be an analog of the highly unsaturated fatty acid of interest.

[0064] As used herein, the term "eicosapentaenoic acid (EPA)" is the common name for cis-5,8,11,14,17-eicosapentaenoic acid; this fatty acid is a C20:5n-3 fatty acid. "EPA-EE" may specifically refer to EPA ethyl ester. As used herein, "dihomo-gamma-linolenic acid (DGLA)" is the common name for cis-8,11,14-eicosatrienoic acid; this fatty acid is a C20:3n-6 fatty acid. As used herein, "arachidonic acid (ARA)" is the common name for cis-5,8,11,14-eicosatetraenoic acid; this fatty acid is a C20:4n-6 fatty acid.

[0065] In one embodiment of the present invention, the fat and oil composition typically comprises a highly unsaturated fatty acid and at least one fatty acid. Therefore, more typically, the fat and oil composition comprises a highly unsaturated fatty acid and at least one fatty acid and / or its ester. Typical fatty acid esters are the same as those defined above for highly unsaturated fatty acids. Preferably, the fat and oil composition comprises at least one fatty acid having 20 carbon atoms and a fatty acid ethyl ester. More preferably, the fat and oil composition comprises a highly unsaturated fatty acid and at least one fatty acid selected from C18:0 (stearic acid), C20:0 (arachidic acid), C20:1 (eicosenoic acid), C22:0 (behenic acid), and esters thereof. In one embodiment of the present invention, the at least one component removed in whole or in part by the first column chromatography is at least one fatty acid ester described above.

[0066] In one embodiment of the present invention, at least one component other than the target substance (for example, highly unsaturated fatty acid ester) contained in the oil or fat composition has a lower polarity than the target substance.

[0067] In one embodiment of the present invention, the oil and fat composition contained in the eluate subjected to the second column chromatography contains at least one component selected from C18:0, C20:0, C20:1, and C22:0 esters in an amount of 0.5 wt % or less, 0.3 wt % or less, 0.1 wt % or less, 0.05 wt % or less, 0.03 wt % or less, or 0.01 wt % or less, based on the total oil and fat composition contained in the eluate subjected to the second column chromatography.

[0068] "Column chromatography," as used herein, refers to the process of selectively retaining or retarding one or more components of a fluid solution as the fluid flows through a column containing a stationary phase(s) composed of finely divided substances and / or materials with capillary channels. Retention results from the relative partitioning of the components of the mixture between the stationary phase and a bulk fluid phase (e.g., a mobile phase), the latter of which migrates through the stationary phase. Column chromatography is used for the analysis and separation of mixtures of two or more substances. Column chromatography includes, for example, preparative chromatography, analytical chromatography, high-performance liquid chromatography, simulated moving bed chromatography, real moving bed chromatography, and supercritical fluid chromatography (SFC).

[0069] The terms "non-polar" and "polar" to describe a mobile phase or a highly unsaturated fatty acid ester can be used relative to one another. For example, "non-polar" can refer to the least polar solvent in the mobile phase, while "polar" can refer to a solvent in the mobile phase that is more polar than the "non-polar" solvent.

[0070] In one embodiment of the present invention, the column chromatography comprises passing the oil or fat composition through one or more columns. Accordingly, the first column chromatography comprises passing the oil or fat composition through the first column and removing all or part of at least one component that elutes later than the target highly unsaturated fatty acid ester (i.e., the relative value of the peak top retention time is greater than 1.0, when the peak top retention time of the target compound in the first column is set to 1.0). The first column may comprise a combination of multiple columns in tandem and / or in parallel. The second column chromatography comprises passing the eluate containing the target compound obtained from the first column chromatography through the second column and removing all or part of related substance components to obtain a highly unsaturated fatty acid ester composition containing a predetermined amount of the target compound. The second column may comprise a combination of multiple columns in tandem and / or in parallel. Any known column may be used in the claimed method. In one embodiment of the present invention, the eluate containing the target compound obtained in the first column chromatography is the eluate obtained up to the end of elution of the target compound from the first column. In one embodiment of the present invention, the removal of all or part of at least one component other than the target substance in the first column chromatography is carried out by collecting the eluate up to the end of elution of the target substance from the first column.

[0071] The first column chromatography is performed using a first column. In one aspect of the present invention, the first column chromatography is performed for the purpose of removing all or part of components having a retention time longer than that of the target component. In one embodiment of the present invention, the retention time of the component removed all or part by the first column chromatography is 1.5 or more, 1.7 or more, or 1.8 or more, or 10 or less, 8 or less, 5 or less, 3.5 or less, 3.0 or less, or 1.5 to 10, 1.7 to 8, 1.8 to 5, 1.8 to 3.5, 1.8 to 3.0, or 1.8 to 2.5, where the retention time of the target component in the first column chromatography is taken as 1. In one embodiment of the present invention, when at least one component is partially removed by the first column chromatography, 99% or more, 95% or more, or 90% or more of the component is removed.

[0072] The second column chromatography is performed using a second column. In one aspect of the present invention, the second column chromatography is performed for the purpose of removing all or part of components having retention times similar to those of the target substance. Components having retention times similar to those of the target substance may be referred to as "related substances" herein. In one embodiment of the present invention, the retention times of the components removed by the second column chromatography are 0.8 or more, 0.9 or more, or 1.5 or less, 1.3 or less, 1.2 or less, or 1.1 or less, or 0.8 to 1.5, 0.9 to 1.3, 0.9 to 1.2, or 0.9 to 1.1, where the retention time of the target substance in the second column chromatography is taken as 1. In one embodiment of the present invention, when components having retention times similar to those of the target substance are partially removed by the second column chromatography, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more of the components are removed.

[0073] In one embodiment of the present invention, a fraction of the eluate from the first chromatography that was not subjected to the second chromatography and a fraction of the eluate from the second chromatography that contains the fatty acid esters removed from the highly unsaturated fatty acid ester composition may be recovered and subjected to a third column chromatography. The third column chromatography is carried out using a third column.

[0074] In one aspect of the present invention, the term "first column" refers to a column used in the first column chromatography, the term "second column" refers to a column used in the second column chromatography, and the term "third column" refers to a column used in the third column chromatography. The dimensions of the columns used are not particularly limited and will depend to some extent on the volume of the oil or fat composition to be refined. The diameter of each column is 1 mm or more, 2 mm or more, 4 mm or more, 8 mm or more, 16 mm or more, 32 mm or more, 64 mm or more, 128 mm or more, 256 mm or more, or 500 mm or more, or 4000 mm or less, 2000 mm or less, 1000 mm or less, 500 mm or less, or 1 to 4000 mm, 2 to 2000 mm, 4 to 1000 mm, 8 to 1000 mm, 16 to 1000 mm, 32 to 500 mm, 30 to 800 mm, or 400 to 800 mm. In one embodiment of the present invention, the first column and the second column can have the same or different diameters. The ratio of the diameters of the first and second columns is 1 for the first column, and the ratio of the diameters of the second column is 0.1 or more, 0.3 or more, 0.5 or more, or 10 or less, 3 or less, 2 or less, or 1.5 or less, or 0.1 to 10, 0.3 to 3, 0.5 to 2, or 0.5 to 1.5. In one embodiment of the present invention, columns with the same diameter are used as the first and second columns. In one embodiment of the present invention, when third-column chromatography is performed, the first to third columns can have the same or different diameters. The third column can have the same or different diameters as either the first column or the second column. In one embodiment of the present invention, the second column has the same diameter as the first column and is longer than the first column. As used herein, the first column and the second column having the same or identical diameter means that the ratio of the diameters of the first column and the second column is 0.8 to 1.2, for example, 0.9 to 1.1 or 1.

[0075] In one embodiment of the present invention, the first and second columns have approximately circular cross sections, and the ratio of the cross-sectional areas of the first and second columns, where the cross-sectional area of ​​the first column is 1, is 0.1 or more, 0.2 or more, or 10 or less, 4 or less, 2 or less, or 0.1 to 10, 0.2 to 4, or 0.2 to 2. In one embodiment of the present invention, columns having the same cross-sectional area are used as the first and second columns. In one embodiment of the present invention, when third column chromatography is performed, the first to third columns can have the same or different cross-sectional areas. The third column can have the same or different cross-sectional area as the first column or the second column, respectively.

[0076] In one embodiment of the present invention, the length of each column is 5 cm or more, 10 cm or more, 20 cm or more, or 800 cm or less, 400 cm or less, 200 cm or less, 150 cm, 120 cm or less, or 5 to 800 cm, 10 to 400 cm, 20 to 200 cm, 20 to 150 cm, or 20 to 120 cm. In one embodiment of the present invention, the first column is shorter than the second column. The ratio of the lengths of the first and second columns, relative to the length of the first column being 1, is 0.5 or more, 1 or more, 1.1 or more, 1.5 or more, or 10 or less, 7 or less, 5 or less, or 0.5 to 10, 1 to 7, 1.1 to 5, or 1.5 to 5. In one embodiment of the present invention, when third-column chromatography is performed, the third column can have the same or different length from the first column or the second column. In one embodiment of the present invention, the second column has the same inner diameter as the first column and a longer length than the first column. The second column has the same inner diameter as the first column, and has a length that is, for example, 1.1 times or more, 1.5 times or more, or 5 times or less, 7 times or less, or 10 times or less than the length of the first column. As used herein, "the first column and the second column have the same or identical inner diameter" refers to a ratio of the inner diameters of the first column to the second column of 0.8 to 1.2, for example, 0.9 to 1.1, or 1.

[0077] The column is cylindrical, having an outer diameter, an inner diameter, and a length. In certain embodiments, the column is a preparative chromatography column. Preparative chromatography columns can have an inner diameter of 5 mm or more, 10 mm or more, 20 mm or more, 50 mm or more, or 4 m or less, 2 m or less, 1 m or less, 80 cm or less, or from about 5 mm to about 4 m, from about 10 mm to about 2 m, from about 20 mm to about 1 m, or from about 50 mm to 80 cm, and a length of from about 100 mm to about 5 m, from about 2 cm to about 2 m, or from about 10 cm to about 1.5 m. In certain embodiments, the column is an analytical chromatography column. Analytical columns can have an inner diameter of from about 1 mm to about 10 cm and a length of from about 10 mm to about 500 mm. The dimensions can be selected such that the inner diameter is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 220%, about 240%, about 260%, about 280%, about 300%, about 320%, about 340%, about 360%, about 380%, about 400%, about 450%, or about 500% of the length. The outer diameter can be about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5%, about 7.5%, about 10%, about 15%, about 20%, about 25%, or about 30% larger than the inner diameter.

[0078] The term "peak" as used herein refers to a peak in a chromatogram. A chromatogram is a graph showing detected values ​​over time. When an eluent is detected, the detected value increases and the chromatogram shows a "peak." Each peak in a chromatogram indicates the presence of a component in the sample. Each peak is labeled with a retention time, and time in the chromatogram increases from left to right. "Peak top" refers to the point in the chromatogram where the detected value of each peak is maximum.

[0079] As used herein, "separation" refers to a process characterized by the spatial separation of components of a composition containing highly unsaturated fatty acid esters based on their differential distribution between phases (e.g., a mobile phase and a stationary phase) in relative motion. Separation results from loading a sample onto a column and subsequent elution from the column. "Fractionation" is a separation process in which a volume of a mixture is separated into several smaller volumes during a phase transition, where the composition varies according to a gradient. Different fractions are collected at different times based on differences in the specific properties of the individual components (highly unsaturated fatty acid esters within the mixture or sample), such as their affinity for the stationary phase and / or the mobile phase.

[0080] As used herein, "subjecting to column chromatography" means loading a sample containing highly unsaturated fatty acid esters onto a column until the entire sample is accommodated within the column. In this specification, the sample is an oil or fat composition. The sample is typically loaded onto the top of the stationary phase of a packed column. Here, "top" refers to the end of the stationary phase that first receives the mobile phase as it elutes through the column. The oil or fat composition can be subjected to column chromatography by a syringe, a pump, or by directly applying the sample to the top of the stationary phase. The sample can be mixed with a minimal amount of mobile phase or other solvent for loading.

[0081] As used herein, "elution" means that a component loaded onto a column passes through the column and flows out as a solution in the mobile phase. As used herein, "eluent" means the mobile phase eluted from the column, which may contain components contained in the oil or fat composition loaded onto the column. In one embodiment, it is a solution containing components contained in the oil or fat composition in the mobile phase eluted from the column. In one embodiment of the present invention, "eluate containing the target product obtained by the first column chromatography" means an eluate that has passed through the first column and contains the target product.

[0082] "Eluate," as used herein, refers to a component discharged from a column in column chromatography. In one embodiment of the present invention, the "eluate to be subjected to the second column chromatography" is the eluate from the first column chromatography.

[0083] The "components" refer to components contained in the oil and fat composition subjected to column chromatography, including highly unsaturated fatty acid esters and fatty acid esters. In one aspect of the present invention, the "at least one component removed in whole or in part by the first column chromatography" can be a fatty acid ester having a longer retention time than the target highly unsaturated fatty acid ester, for example, any one of C22:0, C20:0, C18:0, and C20:1 esters. The "at least one component in the second column chromatography" refers to a component other than the target highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained from the oil and fat composition, and can be stearidonic acid or any one of C19:4, C21:5, C20:4, and C18:3 esters.

[0084] In one embodiment of the present invention, the "at least one component removed in whole or in part by the first column chromatography" and the "at least one component in the second column chromatography" may be different. In one embodiment of the present invention, the "at least one component in the second column chromatography" may contain the same component as the "at least one component removed in whole or in part by the first column chromatography."

[0085] "Biphenyl," "C30," "C22," "C18," "C8," "C5," and "C4," as used herein, refer to functional groups present on the column packing material (stationary phase). For example, a biphenyl column exposes materials flowing through the column to unsubstituted biphenyl groups, while a C18 column exposes materials flowing through the column (e.g., mobile phase and components) to unsubstituted straight- or branched-chain 18-carbon alkyl groups.

[0086] "Chromatographic conditions" refers to the parameters under which column chromatography is operated, including, by way of example, packing pressure, mobile and stationary phase composition, slurry concentration, pressure at which the column is operated, column temperature, mobile phase temperature, mobile phase gradient, mobile phase flow rate, column type used, detection instrumentation and parameters used, sample preparation protocol employed, sedimentation time and pressure at which sedimentation is performed, and stand time and pressure at which stand is performed.

[0087] "Gradient" refers to a change in mobile phase composition over time while column chromatography is performed. The composition of the mobile phase can change as the solvent is eluted through the column. Different mobile phases can be added at increasing percentages over time during elution.

[0088] "Purity" refers to a ratio indicating the content of a major component of a composition, and can be calculated, for example, from the results of gas chromatography (GC) using an internal standard. In one embodiment of the present specification, the major component is a highly unsaturated fatty acid ester, such as an EPA ester, a DGLA ester, or an ARA ester.

[0089] "Target product" refers to the target highly unsaturated fatty acid ester, such as EPA ester, DGLA ester, or ARA ester, obtained from a single fraction or a combination of multiple fractions obtained by elution and fractionation.

[0090] In one embodiment of the present invention, the concentration includes reducing the pressure and / or heating the eluate containing the target highly unsaturated fatty acid ester, and recovering the solvent by distillation. In one embodiment of the present invention, the concentration is performed using an evaporator. Examples of the evaporator include an eluate evaporator and / or a thin-film evaporator. Any type of evaporator can be used for the concentration, including, for example, a thin-film evaporator, a single evaporator, a multiple-effect evaporator, a multi-chamber evaporator, a natural circulation evaporator, a forced circulation evaporator, a falling thin-film evaporator, an ascending thin-film evaporator, and combinations thereof. In one aspect of the present invention, examples of the natural circulation evaporator include an external heating type and a calandria type.

[0091] Certain ranges are provided herein by numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically stated number, an unstated number that is close to or approaching the stated number can be a number that provides a substantial equivalent of the specifically stated number in the context in which it is given. In some embodiments, about can refer to ±5%, ±2.5%, or ±1% of the number to which it refers.

[0092] In the English specification corresponding to this specification, the terms "a," "an," and "the" and similar designations in the context of describing components are to be construed as including both the singular and the plural, unless otherwise limited or negated by context, particularly in the context of the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, and representative exemplary methods and materials are described herein.

[0093] Where a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the scope of the invention, and where one or both limits are included, subject to any specifically excluded limits, ranges excluding either or both of those included limits are also included in the invention.

[0094] The present invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0095] In one embodiment of the present invention, a method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition includes, prior to performing the first column chromatography, subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target compound. In one embodiment of the present invention, the first column chromatography removes all or part of at least one component whose peak top retention time is later than that of the target highly unsaturated fatty acid. "Retention time" refers to the time a component spends in the column after being injected into the column. Typically, retention time refers to the time at which the peak top appears in a chromatogram.

[0096] As used herein, "retention time of the peak top of the target highly unsaturated fatty acid" refers to the retention time of the peak top of the target substance in the chromatograms of the first and second chromatography. In one aspect of the present invention, "relative retention time (RRT)" (which can be used interchangeably with "relative value" and "relative value of retention time") can be calculated based on the retention time measurement results. "Relative retention time" indicates the relationship between the retention time of a component and the retention time of the target substance. As used herein, "relative value of peak top retention time" refers to the value obtained by dividing the retention time of the peak top of a component other than the target substance by the retention time of the peak top of the target substance in the chromatograms of the first and second chromatography, where the retention time of the peak top of the target substance is set to 1.

[0097] An eluate having a "relative retention time" equal to or greater than a predetermined value means an eluate collected after the time when the "relative retention time" reaches that value. An eluate having a "relative retention time" equal to or less than a predetermined value means an eluate collected before the time when the "relative retention time" reaches that value.

[0098] In one embodiment of the present invention, the retention time of the peak top can be determined by measuring with a UV detector.

[0099] In one embodiment of the present invention, the retention time of the peak top can be determined by measuring each fraction by fatty acid analysis using gas chromatography.

[0100] As used herein, the term "single column chromatography" refers to a process in which a batch of an oil or fat composition is passed through one or more columns to obtain a highly unsaturated fatty acid ester composition containing a predetermined concentration of a target substance. In a single column chromatography, the batch of an oil or fat composition can be passed through one or two columns. When a batch of an oil or fat composition is passed through two columns in a single column chromatography, the two columns are referred to as the first column and the second column and are connected in series. When two columns are used in a single column chromatography, a portion of the eluate from the first column is discharged without being injected into the second column. In one aspect of the present invention, the first column and the second column can be connected to each other so that the eluate from the first column is injected into the second column. In one aspect of the present invention, the phrase "the eluate from the first column is injected into the second column" means that the eluate from the first column can be injected into the second column. In one aspect, the eluate can be directly applied to the second column without treatment such as concentration. In one embodiment of the present invention, being communicable means that the first column and the second column can be switched from a communicated state to a non-communicated state, or vice versa.

[0101] In one embodiment of the present invention, "discharging the eluate from the first column" means discharging the eluate from the first column as waste without injecting it into the second column. In one embodiment of the present invention, when the relative retention time is 3.5 or greater, the first column is not connected to the second column, and the eluate from the first column is discharged. In one embodiment of the present invention, the eluate discharged from the first column can be injected into a third column.

[0102] In one aspect of the present invention, the "loading interval after the start of one column chromatography run until a second column chromatography run can be started" refers to the time from the time when an oil or fat composition is loaded onto a first or second column once until a second loading (e.g., a second loading) of the oil or fat composition can be started onto the same column in column chromatography using a single column or two or more columns. In this case, the second loading of the oil or fat composition onto the same column is started with a time interval such that elution of the target substance in the reloaded oil or fat composition starts after the elution of the last component in the first column chromatography run is completed. In one aspect of the present invention, the "loading interval after the start of one column chromatography run until a second column chromatography run can be started" is calculated or measured as the time from the time when elution of the target substance starts on the first or second column until the elution of the last component is completed. In one embodiment of the present invention, the "loading interval after the start of one column chromatography run until a second column chromatography run can be started" is calculated as the sum of the time from the time the oil or fat composition is subjected to a first column chromatography run until the elution of the target substance from the first column is completed, and the time from the time elution of the target substance in the second column chromatography run until the last component is discharged from the second column. In one aspect of the present invention, a first column and a second column are used, and the last component is eluted from either the first column or the second column. In one embodiment of the present invention, the "loading interval after the start of one column chromatography run until a second column chromatography run can be started" is the same as the time from the time elution of the target substance in the first column chromatography run until the last component is discharged from the first column.In another aspect of the present invention, the "loading interval after the start of one column chromatography until another column chromatography can be started" is the sum of the time from the time the oil or fat composition is subjected to first column chromatography to the time when the elution of the target substance from the first column is completed and the time from the time when elution of the target substance in the second column chromatography starts to the time when the last component is discharged from the second column, or the time from the time when elution of the target substance in the second column chromatography starts to the time when the last component is discharged from the second column. In one aspect of the present invention, the time from the time when elution of the target substance in the first column chromatography starts to the time when the last component is discharged from the first column is shorter than the time from the time when elution of the target substance in the first column chromatography starts to the time when the last component is discharged from the first column. In one embodiment of the present invention, the sum of the time from when the oil or fat composition is subjected to first column chromatography to when the elution of the target substance from the first column is completed and the time from when elution of the target substance in the first column chromatography starts to when the last component is discharged from the first column, or the time from when elution of the target substance in the second column chromatography starts to when the last component is discharged from the second column, is shorter than the time from when elution of the target substance in the first column starts to when the last component is discharged from the second column in column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns. Also, in another embodiment of the present invention, the time from when elution of the target substance in the first column starts to when elution of the last component is completed is shorter than the time from when elution of the target substance in the second column starts to when elution of the last component is completed in column chromatography using only the second column.In another aspect of the present invention, the total time from the time when the oil or fat composition is subjected to first column chromatography to the time when the elution of the target substance from the first column is completed, plus the total time from the time when elution of the target substance in the second column chromatography starts to the time when the last component is discharged from the second column, is shorter than the time from the time when elution of the target substance in the second column starts to the time when elution of the last component is completed in column chromatography using only the second column.

[0103] As used herein, a "single column having a length equal to the sum of the lengths of the first and second columns" refers to, for example, a single column having the same inner diameter and stationary phase as the first and / or second columns. In one embodiment, a single column having a length equal to the sum of the lengths of the first and second columns refers to a single column having the same inner diameter and stationary phase as the second column, and having a length in the flow direction equal to the sum of the lengths of the first and second columns.

[0104] In one embodiment of the present invention, reversed-phase column chromatography is suitable as the column chromatography. Any adsorbent of a reversed-phase distribution system can be used as the stationary phase, without any particular specification. For example, polymer beads such as polystyrene reticulated with DVB (divinylbenzene), or silica gel bonded with C4, C8, or C18 alkyl groups, particularly an ODS column using octadecylsilyl (ODS), can be used. Each chromatography column can contain the same or different adsorbents. Typically, the first column and the second column can contain the same or different adsorbents. In one embodiment of the present invention, the first column and the second column contain the same adsorbent. In one embodiment of the present invention, when a third column chromatography is performed, the third column can use the same or different adsorbent as the first column or the second column.

[0105] The stationary phase can be selected from C30, C22, C18, C8, C4, biphenyl, fluorophenyl, hydrophilic interaction liquid chromatography (HILIC) stationary phase, acrylamide, silica, phenylhexyl stationary phase, polar embedded alkyl, fluorophenylpropyl, or any stationary phase known in the art of chromatography. In some embodiments, a chiral stationary phase is used. The choice of stationary phase will be apparent to those skilled in the art and may depend on the components to be purified by chromatography. Non-polar components, such as highly unsaturated fatty acid esters, may require the use of a reversed-phase stationary phase, such as C18 (ODS). Various different types of octadecyl silica (ODS), including fully end-capped, partially end-capped, and base-deactivated, can be used. More polar components may require a normal-phase stationary phase, such as unbonded silica, amino phase, or cyano phase.

[0106] In certain aspects, the stationary phase has a diameter of 1 μm or more, 20 μm or more, 40 μm or more, 60 μm or more, 80 μm or more, 100 μm or more, 1000 μm or more, 2000 μm or more, 3000 μm or more, 4000 μm or more, or 20 μm or less, 40 μm or less, 60 μm or less, 80 μm or less, 100 μm or less, 1000 μm or less, 2000 μm or less, 3000 μm or less, 40 The particles may have a median particle size of 00 μm or less, 5000 μm or less, or from about 1 μm to about 20 μm, from about 20 μm to about 40 μm, from about 40 μm to about 60 μm, from about 60 μm to about 80 μm, from about 80 μm to about 1000 μm, from about 1000 μm to about 2000 μm, from about 2000 μm to about 3000 μm, from about 3000 μm to about 4000 μm, or from about 4000 μm to about 5000 μm. In certain embodiments, the stationary phase comprises particles having a median particle size greater than 50 μm, greater than 45 μm, greater than 40 μm, greater than 35 μm, greater than 30 μm, greater than 25 μm, greater than 20 μm, greater than 15 μm, or greater than 10 μm, and / or less than 5000 μm, less than 4000 μm, less than 3000 μm, less than 2000 μm, less than 1000 μm, less than 500 μm, less than 100 μm, less than 80 μm, less than 60 μm, or between 10 μm and 5000 μm, 15 μm and 4000 μm, 20 μm and 3000 μm, 25 μm and 2000 μm, 30 μm and 1000 μm, 35 μm and 500 μm, 40 μm and 100 μm, 45 μm and 80 μm, or 50 μm and 60 μm. The median particle size can be measured by laser diffraction scattering methods, as will be understood by those skilled in the art.

[0107] In one embodiment of the present invention, the first column and the second column may use the same or different stationary phases. When different stationary phases are used, the first column may use, for example, polystyrene reticulated with DVB (divinylbenzene), silica gel bonded with a C8 alkyl group, or octadecylsilyl (ODS), while the second column may use a stationary phase different from that of the first column, selected from polystyrene reticulated with DVB (divinylbenzene), alkyl group, or silica gel bonded with octadecylsilyl (ODS). In one embodiment of the present invention, when a third column chromatography is performed, the third column may use the same or different stationary phase from that of the first column or the second column.

[0108] Furthermore, the first column and the second column may use stationary phases containing particles with the same or different median particle sizes. When stationary phases containing particles with different median particle sizes are used, the first column may have a particle size of, for example, 1 μm or more, 20 μm or more, 40 μm or more, 60 μm or more, 80 μm or more, 1000 μm or more, 2000 μm or more, 3000 μm or more, or 4000 μm or more; or 20 μm or less, 40 μm or less, 60 μm or less, 80 μm or less, 1000 μm or less, 2000 μm or less, 3000 μm or less; A stationary phase comprising particles having a median particle size of 4000 μm or less, 5000 μm or less, or from about 1 μm to about 20 μm, from about 20 μm to about 40 μm, from about 40 μm to about 60 μm, from about 60 μm to about 80 μm, from about 80 μm to about 1000 μm, from about 1000 μm to about 2000 μm, from about 2000 μm to about 3000 μm, from about 3000 μm to about 4000 μm, or from about 4000 μm to about 5000 μm may be used. and in the second column, the diameter is 1 μm or more, 20 μm or more, 40 μm or more, 60 μm or more, 80 μm or more, 1000 μm or more, 2000 μm or more, 3000 μm or more, 4000 μm or more, or 20 μm or less, 40 μm or less, 60 μm or less, 80 μm or less, 1000 μm or less, 2000 μm or less, 3000 μm or less, 4000 μm or less, 5000 μm or less, or about 1 μm to about The stationary phase may be different from that of the first column, and may contain particles having a median particle size selected from the group consisting of 20 μm, about 20 μm to about 40 μm, about 40 μm to about 60 μm, about 60 μm to about 80 μm, about 80 μm to about 1000 μm, about 1000 μm to about 2000 μm, about 2000 μm to about 3000 μm, about 3000 μm to about 4000 μm, and about 4000 μm to about 5000 μm. In one aspect of the present invention, when third column chromatography is performed, the third column may use a stationary phase containing particles having the same or different median particle size as the first column or the second column.

[0109] In one embodiment of the present invention, the amount of the packed stationary phase is 1 to 1000 kg, 10 to 900 kg, 20 to 800 kg, 30 to 700 kg, 40 to 600 kg, or 50 to 500 kg by weight. In one embodiment of the present invention, the amounts of the packed stationary phase in the first column and the second column may be the same or different. When the amounts of the packed stationary phases are different, the amount of the packed stationary phase in the first column is, for example, 1 to 100 kg, 10 to 100 kg, or 30 to 100 kg. The amount of the packed stationary phase in the second column is, for example, 1 to 1000 kg, 10 to 800 g, or 50 to 800 g. The ratio of the amount of the packed stationary phase in the first column to the amount of the packed stationary phase in the second column is 0.5 to 10, 1 to 7, 1.1 to 5, or 2 to 4.5, relative to the amount of the packed stationary phase in the first column. In one embodiment of the present invention, when third column chromatography is performed, the third column may be packed with the same or a different amount of stationary phase as the first or second column. In one embodiment of the present invention, the amount of stationary phase packed in the second column is greater than the amount of stationary phase packed in the first column. The amount of stationary phase packed in the second column is 1.1 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, or 4 times or less, 4.5 times or less, 5 times or less, 6 times or less, 7 times or less, or 10 times or less than the amount of stationary phase packed in the first column.

[0110] In one embodiment of the present invention, the column chromatography comprises elution with a mobile phase comprising one or more of water, an organic solvent, or supercritical carbon dioxide. In some embodiments of these methods, the mobile phase comprises methanol. In some embodiments of these methods, the chromatography comprises a mobile phase comprising an additive selected from one or more of formic acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide.

[0111] In one embodiment of the present invention, the solvent used as the mobile phase is an organic solvent selected from alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane. Examples of alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. Methanol and ethanol are preferred. Methanol is more preferred. Examples of ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE). Examples of esters include methyl acetate and ethyl acetate. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone (MIBK). An example of a nitrile is acetonitrile.

[0112] In one embodiment of the present invention, the mobile phases used in the first and second column chromatography may be the same or different, but are preferably the same. When different mobile phases are used, for example, methanol, ethanol, diethyl ether, diisopropyl ether, methyl t-butyl ether, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone (MIBK), acetonitrile, hexanes, and dichloromethane can be used in the first column chromatography, and a mobile phase different from that used in the first column can be used in the second column, and is selected from methanol, ethanol, diethyl ether, diisopropyl ether, methyl t-butyl ether, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone (MIBK), acetonitrile, hexanes, and dichloromethane. In one embodiment of the present invention, when a third column chromatography is performed, the third column can use the same or different mobile phase as the first or second column.

[0113] The mobile phase may further contain additives, including buffers and pH adjusters. The choice of additive may be determined based on the mobile phase used, the stationary phase used, and the component to be purified. In some embodiments, the mobile phase contains an additive selected from one or more of formic acid, trifluoroacetic acid, heptafluorobutyric acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide. In some embodiments, the mobile phase may be additive-free.

[0114] In one aspect of the invention, the column chromatography comprises a mobile phase gradient. In one aspect of the invention, the column chromatography comprises the purification of one or more highly unsaturated fatty acid esters. In one embodiment of the present invention, the highly unsaturated fatty acid ester is selected from the group consisting of docosahexaenoic acid, crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosapentaenoic acid, and herring acid. acid), propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceric acid, silicic acid, geddic acid and polyunsaturated fatty acids selected from one or more of: hexatriacontylic acid, celloplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid. In one aspect of the invention, the ester is a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl ester.In one embodiment of the invention, column chromatography comprises purification of eicosapentaenoic acid ethyl ester.

[0115] In some embodiments, the highly unsaturated fatty acid ester composition contains the target highly unsaturated fatty acid ester with a purity of about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 86% or more, about 90% or more, about 95% or more, about 96.5% or more, about 98% or more, about 99% or more, about 99.5% or more, about 99.8% or more, or about 99.9% or more, and / or less than about 97%, less than about 98%, less than about 99.0%, less than about 99.95%, or less than about 99.99%. In some embodiments, the yield is 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, and / or less than 75%, less than 80%, 85%, less than 90%, or less than 95%. In some embodiments, the recovery is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.7% or more, and / or less than 95%, less than 97%, less than 98%, less than 99%, less than 99.5%, less than 99.7%, or less than 99.9%.

[0116] The mobile phase can include one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other solvent known in the art. The selection of the mobile phase can require consideration of the highly unsaturated fatty acid ester to be purified and the stationary phase used. For a non-polar highly unsaturated fatty acid ester, a reversed-phase stationary phase should be selected in which the polarity of the mobile phase is sufficient to elute the highly unsaturated fatty acid ester of interest, but not so rapid that elution approaches the solvent front.

[0117] In some embodiments, a solvent gradient can be used as the mobile phase during elution. The primary purpose of gradient elution is to elute strongly retained analytes faster from the column while weakly retained analytes elute more slowly, so that the eluted analytes produce well-resolved peaks upon detection. For example, in reversed-phase chromatography, starting with a low content of nonpolar solvent in the eluent allows weakly retained analytes to be separated. Strongly retained analytes will remain on the adsorbent surface at the top of the column or will migrate very slowly. Increasing the amount of nonpolar components (e.g., acetonitrile) in the eluent allows strongly retained components to migrate faster due to steadily increasing competition for adsorption sites by the nonpolar solvent.

[0118] Thus, in reversed-phase chromatography used for non-polar highly unsaturated fatty acid esters, the solvent at the start of the chromatography elution may contain a high percentage of polar solvent A, e.g., water, selected from about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 0%. Solvent B can be a solvent less polar than solvent A, e.g., methanol (when solvent A is water). Solvent B will constitute the remaining percentage of the mobile phase. As the column is run and the solvent elutes through the stationary phase and the column, a gradient will result, resulting in a gradual increase in the concentration of solvent B over time. In some embodiments, a single solvent can be used as the mobile phase. In some embodiments, the single solvent can be one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other solvent known in the art, used alone or in combination.

[0119] In some embodiments, the rate of increase of solvent B over time can be constant. In some embodiments, there is no gradient and the mobile phase is isocratic during elution. In some embodiments, different rates of increase in the percentage of solvent B at different time ranges within a chromatographic method can be utilized. In some embodiments, the mobile phase can be isocratic for certain time ranges of a chromatographic method and include a gradient at other time ranges.

[0120] In some embodiments, the first and second column chromatography may both be isocratic, may both include a gradient, or may both be isocratic over a certain time range and include a gradient over another time range. In some embodiments, the first column chromatography may be isocratic and the second column chromatography may include a gradient, or the first column chromatography may include a gradient and the first column chromatography may be isocratic. In some embodiments, if a third column chromatography is included, the third column chromatography may be isocratic or may include a gradient.

[0121] The multiple solvents used in the mobile phase can be stored separately in a mobile phase supply and mixed using a pump before elution through the column. The mobile phase supply includes a mobile phase source and a solvent delivery system. Such a solvent delivery system is a pumping device, such as a commercially available column chromatography pump, that provides the solvent or mobile phase to the column. Such pumps generally provide pulse-free flow, flow rates ranging from 0.1 to 100 L / min, precise control of flow rate, high pressure (up to 6000 psi), and corrosion- and solvent-resistant components. Reciprocating pumps consist of a small chamber into which the solvent is pumped by the back-and-forth motion of a motor-driven piston. Two check valves, which alternately open and close, control the direction and flow of the solvent into and out of the cylinder. Single-piston pumps use specially designed cams to allow very rapid refill times and produce a more continuous flow. The disadvantage of pulsed flow with reciprocating pumps is often overcome by using a pulse damper. The use of a dual-piston pump, operated with pistons moving out of phase with each other, provides a rational solution for pulse-free fluid delivery. Linear velocity in a column represents the speed at which a fluid passes through the cross section of the column. Linear velocity (m / time) = Flow rate (m ３ / time) / column cross-sectional area (m ２ )) can be about 0.2 to 20.0 m / hr, about 1.0 to 15.0 m / hr, about 1.0 to 10.0 m / hr, about 1.5 to 10.0 m / hr, or about 2.0 to 9.0 m / hr. In one embodiment, the linear velocity is about 4.0 to 9.0 m / hr.

[0122] In one embodiment of the present invention, the linear velocity in the first column and the second column may be the same or different. When the linear velocities are different, the linear velocity in the first column may be faster or slower than the linear velocity in the second column. The ratio of the linear velocities in the first and second columns is 0.5 to 2.0, 0.5 to 1.5, or 1 to 1.5, relative to the linear velocity in the first column, in the second column. In one embodiment of the present invention, the linear velocities in the first column and the second column are the same. In one embodiment of the present invention, when third column chromatography is performed, the third column may be performed at a linear velocity that is the same as or different from that of the first column or the second column.

[0123] In one embodiment of the invention, the column chromatography is carried out at room temperature or above room temperature. Preferably, the method is carried out at a temperature above room temperature. The first and second column chromatography steps can be carried out at the same temperature or at different temperatures, but are preferably at the same temperature.

[0124] In one embodiment of the present invention, the temperature higher than room temperature is 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, or 60°C or lower, 55°C or lower, 50°C or lower, 45°C or lower, or 20 to 60°C, 25 to 55°C, 30 to 50°C, or 35 to 45°C.

[0125] In one embodiment of the present invention, the column chromatography may use two or more columns. The column chromatography may be performed using a known fixed-bed chromatography device. Such column chromatography is called fixed-bed chromatography. In one embodiment of the present invention, the first and second column chromatography are fixed-bed chromatography.

[0126] In some embodiments, a detector is used to monitor the mobile phase eluting from the column for the presence of a component or components. Detection methods known in the art, such as mass spectrometry (MS), UV / Vis absorbance, fluorescence, refractive index, or conductivity, can be used.

[0127] In other embodiments, any of a variety of standard column chromatography detectors can be used to detect the eluate immediately after elution from the column. In other embodiments, each fraction can be individually monitored for the presence of components by analysis. In some embodiments, the analysis is fatty acid analysis using gas chromatography.

[0128] In some embodiments, the eluate from the column is detected as a peak in a chromatogram. The retention time of the peak is used to identify the compound, and the peak height (or area) is proportional to the amount of eluate in the oil / fat composition. "Retention time" is the time required for the eluate to pass through the column and is measured from the time of injection (or loading) of the oil / fat composition to the time of elution. Ideally, each eluate of interest will have a characteristic retention time. However, the retention of the eluate will vary depending on the eluent, stationary phase, temperature, and column chromatography settings. Therefore, the retention time of the eluate is compared with the retention time of one or more standard compounds under the same conditions. Suitable detectors exhibit good sensitivity, good stability, reproducibility, linear response over several orders of magnitude when used for quantitative purposes, short response time, and ease of operation. Such detectors include, but are not limited to, UV / Vis absorbance detectors, photodiode array detectors, fluorescence detectors, refractive index detectors, and conductivity detectors.

[0129] A UV / Vis absorbance detector consisting of a scanning spectrophotometer with grating optics can be used. The use of a deuterium source (ultraviolet range, 190-360 nm) either independently or in combination with a tungsten source (visible range, 360-800 nm) provides a simple means of detecting absorbing species as they emerge from the column.

[0130] Photodiode array (PDA)-based instruments are ultraviolet / visible absorbance detectors that allow for very rapid collection of data over a selected spectral range. Absorbance spectral data for each chromatographic peak can be collected and stored. The stored data can be compared to the spectra of pure standards from a library. PDA detectors are useful in identifying components that are difficult to separate (overlapping peaks) because the characteristic spectra for each of the unresolved components are likely to be different.

[0131] Fluorescence detectors are useful in detecting analytes that exhibit chemiluminescent properties such as fluorescence or phosphorescence. They are at least an order of magnitude more sensitive than UV absorbance detectors. Fluorescence is observed by detecting grating-separated emission radiation, typically at a 90-degree angle to the excitation beam. The number of fluorescent species can be enhanced by post-column derivatization (PCD) of the eluted compounds (or pre-column derivatization of the sample itself) with special reagents.

[0132] Refractive index (RI) detectors respond to almost all solutes. Differences in the refractive index of the reference mobile phase relative to the column effluent result in the detection of separated components as peaks on the chromatogram. Due to its extremely high sensitivity to the mobile phase, this detector cannot be used without sufficient pulse attenuation in the LC pump, and it is also not suitable for gradient applications due to the varying mobile phase composition. The detection limit is usually lower than that observed with absorbance detectors.

[0133] Conductivity detectors offer sensitive detection of all charged species. They can be used with LC systems for simple and reliable detection of anions, cations, metals, organic acids, and surfactants down to ppb levels. The addition of a chemical suppressant between the column and the conductivity detector serves to reduce the conductivity of the eluate, allowing the use of gradient elution and ppb-level determination with minimal baseline drift. For typical determination of low levels of anions, the eluate is diluted with a weakly ionized, low-conductivity acid (e.g., Na ２ CO ３ Concurrently, analyte anions are converted to their corresponding high conductivity acids (e.g., NaCl to HCl), increasing the relative analyte signal.

[0134] In one embodiment of the present invention, the purity of the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained by the second column chromatography is 93% by weight or more, 95% by weight or more, 96% by weight or more, 96.5% by weight or more, 97% by weight or more, 97.5% by weight or more, 98% by weight or more, 98.5% by weight or more, 99% by weight or more, or 99.5% by weight or more, based on the total highly unsaturated fatty acid ester composition.

[0135] The retention time of the target substance is about 0.5 minutes to about 2 minutes, about 2 minutes to about 4 minutes, about 4 minutes to about 6 minutes, about 6 minutes to about 8 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 12 minutes, about 12 minutes to about 14 minutes, about 14 minutes to about 16 minutes, about 16 minutes to about 18 minutes, about 18 minutes to about 20 minutes, about 20 minutes to about 22 minutes, about 22 minutes to about 24 minutes, about 24 minutes to about 26 minutes, about 26 minutes to about 28 minutes, and about 28 minutes to about 30 minutes. , about 30 minutes to about 32 minutes, about 32 minutes to about 34 minutes, about 34 minutes to about 36 minutes, about 36 minutes to about 38 minutes, or about 38 minutes to about 40 minutes, or greater than about 0.5 minutes, greater than about 1 minute, greater than about 1.5 minutes, greater than about 2 minutes, or greater than about 5 minutes, and / or less than 50 minutes, less than 40 minutes, less than 30 minutes, or less than 20 minutes.

[0136] In one embodiment of the present invention, the retention time of the peak top of the target compound in the first column chromatography is shorter than the retention time of the peak top of the target compound in the second column chromatography. The retention time of the peak top of the target compound in the first column chromatography may be about 0.5 minutes to about 2 minutes, about 2 minutes to about 4 minutes, about 4 minutes to about 6 minutes, about 6 minutes to about 8 minutes, or about 8 minutes to about 10 minutes, or less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. The retention time of the peak top of the target compound in the second column chromatography was about 6 minutes to about 8 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 12 minutes, about 12 minutes to about 14 minutes, about 14 minutes to about 16 minutes, about 16 minutes to about 18 minutes, about 18 minutes to about 20 minutes, about 20 minutes to about 22 minutes, about 22 minutes to about 24 minutes, about 24 minutes to about 26 minutes, about 26 minutes to about 28 minutes, about 28 minutes to about 30 minutes, and about 30 minutes to about It can be 32 minutes, about 32 minutes to about 34 minutes, about 34 minutes to about 36 minutes, about 36 minutes to about 38 minutes, or about 38 minutes to about 40 minutes, or less than 40 minutes, less than 38 minutes, less than 36 minutes, less than 34 minutes, less than 32 minutes, less than 30 minutes, less than 28 minutes, less than 26 minutes, less than 24 minutes, less than 22 minutes, less than 20 minutes, less than 18 minutes, less than 16 minutes, less than 14 minutes, less than 12 minutes, less than 10 minutes, or less than 8 minutes.

[0137] In one aspect of the present invention, an internal standard can be used during analysis by gas chromatography (GC).The internal standard can be added to a sample as a reference marker to determine the relative retention time of an analyte relative to the internal standard or to aid in the quantification of the analyte.The internal standard can be appropriately selected by those skilled in the art to be a compound that is very similar to, but not identical to, the target analyte, for example, a deuterated derivative of the target analyte.When used for quantification purposes, the internal standard can then be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the standard analyte concentration, where the standard is a sample of known concentration prepared by those skilled in the art to be used as a reference for the unknown analyte sample to be quantified.

[0138] In one embodiment, the highly unsaturated fatty acid ester composition may comprise any of docosahexaenoic acid, crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, osbondo acid, sardine acid, tetracosapentaenoic acid, herring acid, propionic acid, butyric acid, valeric acid, and caproic acid. , enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboseric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, russellic acid, silicic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, tetracontylic acid, or an ester thereof.

[0139] In some embodiments, the highly unsaturated fatty acid ester composition may have a purity of greater than about 70%, greater than about 80%, greater than about 85%, greater than about 86%, greater than about 90%, greater than about 95%, greater than about 96.5%, greater than about 98%, greater than about 99%, greater than about 99.5%, greater than about 99.8%, or greater than about 99.9%. In some embodiments, the highly unsaturated fatty acid ester composition may have a purity of less than about 100%, less than about 99.9999%, less than about 99.5%, less than 99%, less than 98.5%, or less than about 98%. In some embodiments, the highly unsaturated fatty acid ester composition may have a purity of 70 to 98%, 80 to 98%, 85 to 98.5%, 86 to 98.5%, 90 to 98.5%, 95 to 99%, 96.5 to 99%, 98 to 99.5%, 99 to 99.5%, 99.5 to 99.9999%, 99.8 to 99.9999%, or 99.8 to 100%. In some embodiments, the highly unsaturated fatty acid ester composition may be obtained with a yield of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%. In some embodiments, the highly unsaturated fatty acid ester composition may have a yield of less than 100%, less than 95%, less than 90%, or less than 80%. In some embodiments, the highly unsaturated fatty acid ester composition may have a yield of 50-80%, 55-90%, 60-90%, 65-95%, 70-95%, 75-100%, or 80-100%. In some embodiments, the highly unsaturated fatty acid ester composition may have a recovery rate of greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, or greater than 99.7%. In some embodiments, the highly unsaturated fatty acid ester composition may have a recovery rate of less than 100%, or less than 99.9999%.In certain embodiments, the highly unsaturated fatty acid ester composition may have a purity of 55 to 99.9999%, 60 to 99.9999%, 65 to 99.9999%, 70 to 99.9999%, 75 to 99.9999%, 80 to 99.9999%, 85 to 99.9999%, 90 to 99.9999%, 95 to 99.9999%, 98 to 99.9999%, 99 to 100%, or 99.7 to 100%.

[0140] In one embodiment of the present invention, the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained in the second column chromatography is an EPA ester, for example, EPA ethyl ester, and its purity is 93% by weight or more, 95% by weight or more, 96% by weight or more, 96.5% by weight or more, 97% by weight or more, 97.5% by weight or more, 98% by weight or more, 98.5% by weight or more, 99% by weight or more, or 99.5% by weight or more, based on the total highly unsaturated fatty acid ester composition.

[0141] In one embodiment of the present invention, the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained in the second column chromatography is a DGLA ester, for example, DGLA ethyl ester, and its purity is 93% by weight or more, 95% by weight or more, 96% by weight or more, 96.5% by weight or more, 97% by weight or more, 97.5% by weight or more, 98% by weight or more, 98.5% by weight or more, 99% by weight or more, or 99.5% by weight or more, based on the total highly unsaturated fatty acid ester composition.

[0142] In one embodiment of the present invention, the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained by the second column chromatography is an ARA ester, for example, ARA ethyl ester, and has a high purity, such as 93% by weight or more, 95% by weight or more, 96% by weight or more, 96.5% by weight or more, 97% by weight or more, 97.5% by weight or more, 98% by weight or more, 98.5% by weight or more, 99% by weight or more, or 99.5% by weight or more, based on the total highly unsaturated fatty acid ester composition.

[0143] In one embodiment of the present invention, the highly unsaturated fatty acid ester composition obtained in the second column chromatography contains at least one fatty acid ester selected from C18:0, C20:0, C20:1, and C22:0 esters in an amount of 0.6 wt % or less, 0.3 wt % or less, 0.2 wt % or less, 0.1 wt % or less, 0.05 wt % or less, 0.03 wt % or less, or 0.01 wt % or less, based on the total highly unsaturated fatty acid ester composition.

[0144] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0145] Example 1 In this test, a reduction in the loading interval was verified using a column model by comparing a single serial column with a dual serial column. Samples containing EPA-EE, C20:0 ethyl ester, and C20:1 ethyl ester were prepared from crudely refined sardine oil (the preparation method was the same as in Example 2 below). The single serial column consisted of three serially connected columns with an internal diameter of 20 mm and lengths of 300 mm, 500 mm, and 500 mm. The dual serial column consisted of a first column with an internal diameter of 20 mm and a length of 300 mm, and a second column consisting of two serially connected columns with an internal diameter of 20 mm and a length of 500 mm. The column temperature was 40°C. The flow rate of the single serial column was 37 mL / min. In the dual serial column setup, the flow rate of the first column was 32 mL / min, and the flow rate of the second column was 37 mL / min. The stationary phase used was Daisopaq SP-120-50-ODS-B (Osaka Soda Co., Ltd.). The mobile phase was HPLC-grade methanol (Kanto Chemical Co., Ltd.). Detection was by UV at 230 nm. With a single tandem column, EPA-EE began eluting 13 minutes after the start of column chromatography, and the elution of C20:0 ethyl ester ended 24 minutes after the start of EPA-EE elution. With a twin-column tandem system, EPA-EE elution from the first column ended 3.5 minutes after the start of column chromatography. With the second column, EPA-EE elution began 10.5 minutes after the start of column chromatography, and the elution of C20:1 ethyl ester ended 12 minutes after the start of EPA-EE elution.

[0146] In the case of a single tandem column, the fatty acid furthest from EPA-EE is C20:0 ethyl ester. In the case of a double tandem column system, if the eluate up to 3.5 minutes, the time when EPA-EE elution ends in the first column, is collected and loaded onto the second column, the fatty acid furthest from EPA-EE in the second column is C20:1 ethyl ester. This is because C20:0 ethyl ester is removed in the first column. In the case of a single tandem column, elution of C20:0 ethyl ester ends 24 minutes after the start of EPA-EE elution. Therefore, by setting the loading interval to 24 minutes, the C20:0 ethyl ester eluted last in the previous load will not be mixed into the EPA-EE fraction detected in the next load. On the other hand, in the case of a dual-column system, the elution of C20:1 ethyl ester ends 12 minutes after the start of EPA-EE elution in the second column, so if the loading interval is 12 minutes, the C20:1 ethyl ester eluted last in the previous loading will not be mixed into the EPA-EE fraction eluted in the next loading. In other words, the introduction of a dual-column system shortened the loading interval in half from 24 minutes to 12 minutes, which was the case with a single column, demonstrating that this is an effective method for reducing the amount of solvent used.

[0147] [Example 2] The conditions for column chromatography were as follows. The HPLC equipment used was as follows: the pump was NP-KX500 (Nihon Seimitsu Kagaku Co., Ltd.), the detector was S-3702 (Soma Optical Co., Ltd.), and the column oven was CO705 (GL Sciences Inc.). The ODS packing material used was Daisopaq SP-120-50-ODS-B (Osaka Soda Co., Ltd.). The first column had an inner diameter of 20 mm and a length of 300 mm, and the second column had an inner diameter of 20 mm and a length of 1000 mm. The flow rate of the first column was 32 mL / min, and the flow rate of the second column was 37 mL / min. The solvent was HPLC-grade methanol (Kanto Chemical Co., Ltd.). Detection was by UV at 230 nm. 2.4 g of EPA-EE (80%) was loaded.

[0148] EPA-EE (80%) was prepared as follows: crude sardine oil was subjected to short path distillation (SPD). The SPD-treated oil was subjected to ethanolysis with ethyl alcohol in the presence of an alkali catalyst to form EPA-EE, a fish oil ethyl ester. 80% EPA-EE was prepared from the fish oil ethyl ester by precision distillation.

[0149] First, a fractionation test was conducted on the first column. 2.4 g of EPA-EE (80%) was loaded onto the first column, and fraction (Fr. 0) was fractionated from immediately after injection until the UV increased. Fractions 1 to 20 were then fractionated every 10 seconds, followed by Fraction 29 for 30 minutes. Methanol was removed from the collected fractions using a vacuum evaporator, and a 1 mg / mL hexane solution of C23:0 methyl ester was added as an internal standard. The fractions were then analyzed by gas chromatography (GC). The amount of eluted compounds in each fraction was calculated from the GC peak area and the area ratio between the internal standard. The elution start and end times for EPA-EE (93% content) were determined at 3 minutes 30 seconds and 4 minutes 20 seconds, respectively, and the composition of related substances at those times was determined. Therefore, it was decided that the elution range from 3 minutes 30 seconds to 4 minutes 20 seconds after the start of column chromatography would be transferred from the first column to the second column.

[0150] Next, 2.4 g of the sample was loaded onto the first column, and the elution range of 93% or more EPA-EE (from 3 minutes 30 seconds to 4 minutes 20 seconds) was transferred to the second column. The time up to the rise in UV in the second column was defined as Fr. 0, and fractions 1 to 38 were collected every 10 seconds, followed by fraction 39, which was fractionated for 35 minutes. Similar to the fractionation test for the first column, GC analysis was performed to determine the composition of related substances when EPA-EE was 97% or more.

[0151] The conditions used for GC analysis were as follows: The instrument was a 7890A Network GC System (Agilent) with a DB-WAX 30 m x 0.25 mm x 0.25 μm column. The column temperature was 210°C. The injection temperature was 250°C, the split rate was 1:50, and the injection volume was 1 μL. An FID detector at 250°C was used. Helium carrier gas with a linear velocity of 31 cm / min was used.

[0152]

[0153] [Example 3] The conditions for column chromatography performed to concentrate DGLA were as follows. The columns used were axial compression columns LC-600 type (Kurita Water Industries Ltd.) with an inner diameter of 600 mm, packed with 50 μm particle size ODS packing material (SP-120-50-ODS-B, Osaka Soda Co., Ltd.) at a packing pressure of 3.5 MPa, with 75 kg in the first column and 115 kg in the second column. The column length of the first column was 50 cm, and the column length of the second column was 80 cm. Methanol was used as the mobile phase, and the flow rate was 28 L / min for both the first and second columns.

[0154] Oil composition 1 (DGLA content, 87.7%) was prepared as follows. Microbial oil 1 derived from a Mortierella microorganism containing 37.2 wt% DGLA in its fatty acid composition was ethyl-esterified with an alkali catalyst according to a conventional method, and the C20 fraction was concentrated by precision distillation to obtain oil composition 1. First, fractionation of the first column was performed. 8.6 kg (4.5% of the weight of the adsorbent in the first and second columns) of oil composition 1 (DGLA content, 87.7%) was loaded onto the first column, and fraction (Fr. 1) was fractionated immediately after the end of injection. Fraction 1 was fractionated for 180 seconds, Fractions 2 to 19 for 30 seconds each, and Fractions 20 to 33 for 120 seconds each. 1 mL of each fraction was dispensed into a Falcon tube, and 1 mL of a 1 mg / mL hexane solution of methyl tricosanoate was added using a volumetric pipette. 5 mL of a 5% aqueous NaCl solution was then added and vigorously stirred to separate the two layers. The upper layer was then collected in a GC vial and subjected to GC-FID analysis as a GC sample. The GC conditions were as follows: The amount of compound eluted in each fraction was calculated from the area ratio between the GC peak area and the internal standard.

[0155] <GC analysis conditions> Apparatus: Capillary gas chromatograph GC-2025, Shimadzu Corporation Column: DB-WAX (30 m x 0.25 mm ID, film thickness: 0.25 μm) Carrier gas: Hydrogen, 1 mL / min Split ratio: 1:30 Column temperature: 230°C (30 min) Injection port temperature: 250°C Detector type, temperature: FID, 250°C Injection volume: 1 μL

[0156] As a result, the elution start time of the target substance, DGLA, in the first column was 4.5 minutes and the elution end time was 30 minutes. However, by deliberately setting the elution start time of the target fraction (DGLA content, 92.7%) to be sent to the second column to 4.5 minutes and the elution end time to 9 minutes, the elution of C22:0, which takes a long time to elute, was suppressed to 0.0%.

[0157] Next, after loading 8.6 kg of oil / fat composition 1 onto the first column, the time for transferring the oil / fat composition from the first column to the second column was within the range (4.5 to 9 minutes) specified for the fractionation of the first column. Fractionation of the eluate from the second column began immediately after the completion of transferring oil / fat composition 1 from the first column to the second column, with Fraction 1 being fractionated for 360 seconds, Fractions 2 to 24 for 30 seconds each, and Fractions 25 to 34 for 120 seconds each. GC analysis was performed in the same manner as in the fractionation test for the first column, and the recovered fractions were identified so that DGLA was 96.5% or more in the fatty acid composition of the recovered fractions, and the fatty acid composition of the recovered fractions at that time was determined.

[0158]

[0159] [Example 4] The conditions for column chromatography carried out to concentrate ARA were as follows: The same equipment and conditions as in Example 2 were used for the HPLC column and mobile phase conditions.

[0160] Oil composition 2 (ARA content, 79.4%) was prepared as follows. Microbial oil 2 derived from a Mortierella microorganism containing 43.6 wt% ARA in its fatty acid composition was ethyl-esterified with an alkali catalyst according to a conventional method, and the C20 fraction was concentrated by precision distillation to obtain oil composition 2. First, fractionation of the first column was performed. 1.0 kg (0.55% of the total adsorbent weight of the first and second columns) of oil composition 2 (ARA content, 79.4%) was loaded onto the first column, and fraction (Fr. 1) was fractionated immediately after the end of injection. Fraction 1 was fractionated for 361 seconds, Fractions 2 to 13 for 30 seconds, Fractions 14 to 16 for 300 seconds, and Fractions 17 to 29 for 60 seconds. Each fraction was subjected to GC analysis in the same manner as in Example 2 to determine the amount of compound eluted in each fraction. As a result, the elution start time of ARA, the target component after the first column, was 6 minutes and the elution end time was 40 minutes, but the elution start time of the target fraction (ARA content, 78.9%) to be sent to the second column was deliberately set to 6.5 minutes and 12 minutes, respectively, thereby suppressing C22:0, which takes a long time to elute, to 0.0%.

[0161] Next, after loading 1.0 kg of oil / fat composition 2 onto the first column, the time for transferring the eluate from the first column to the second column was within the range specified for the fractionation of the first column (6.5 to 12 minutes). Fractionation of the eluate from the second column began after loading of oil / fat composition 2 onto the first column, with Fraction 1 being fractionated for 1051 seconds, Fractions 2 to 31 being fractionated for 10 seconds each, and Fractions 32 to 33 being fractionated for 600 seconds each. GC samples prepared by pretreatment in the same manner as in the fractionation test for the first column were analyzed under the GC conditions below. The recovered fractions were specified so that ARA was 96.5% or more in the fatty acid composition of the recovered fractions, and the fatty acid composition of the recovered fractions was determined.

[0162] <GC analysis conditions> Apparatus: Capillary gas chromatograph GC-2025, Shimadzu Corporation Column: DB-WAX (30 m x 0.25 mm ID, film thickness: 0.25 μm) Carrier gas: Hydrogen, 31.8 cm / sec constant linear velocity control Split ratio: 1:30 Column temperature: 210°C (30 min) Injection port temperature: 250°C Detector type, temperature: FID, 250°C Injection volume: 1 μL

[0163]

Claims

1. A method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition, comprising: (a) subjecting the oil or fat composition to a first column chromatography using a first column, thereby removing all or a part of at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the target highly unsaturated fatty acid ester in the first column chromatography, which is taken as 1.0; (b) subjecting the eluate containing the target product obtained from the first column chromatography to a second column chromatography using a second column, thereby removing all or a part of at least one component having a peak top retention time of 0.90 or more and 1.1 or less relative to the retention time of the peak top of the target product in the second column chromatography, which is taken as 1.0; and (c) concentrating the eluate obtained from the second column chromatography to obtain a composition containing the target product, wherein the obtained composition contains 95% by weight or more of the target product.

2. The method according to claim 1, wherein the loading interval after the start of one column chromatography run until another column chromatography run can be started is shorter than that in the case of column chromatography using a single column having a length equal to the combined length of the first and second columns.

3. The method according to claim 1 or 2, wherein the at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the target substance in the first column chromatography, which is taken as 1.0, is at least one component other than highly unsaturated fatty acid esters in the oil and fat composition.

4. The method according to any one of claims 1 to 3, wherein the at least one component which is removed in whole or in part in (b) is at least one component having a peak top retention time which is 0.93 or more and 1.09 or less relative to the retention time of the peak top of the target substance in the second column chromatography, where the retention time is 1.

0.

5. The method according to any one of claims 1 to 4, wherein the first column is connectable to the second column such that eluate from the first column is injected into the second column.

6. The method according to any one of claims 1 to 5, wherein the first column chromatography and the second column chromatography use reverse phase column chromatography.

7. The method according to any one of claims 1 to 6, wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.

8. The method according to any one of claims 1 to 7, wherein the first column chromatography and the second column chromatography use the same mobile phase.

9. The method of any one of claims 1 to 8, wherein the second column has a larger amount of packed stationary phase than the first column, or has the same inner diameter as the first column and a longer length than the first column.

10. The method according to any one of claims 1 to 9, wherein the target substance is an ester of any one of eicosapentaenoic acid (EPA), dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (ARA).

11. The method according to any one of claims 1 to 10, wherein the at least one component that is completely or partially removed in the first column chromatography is any one of C22:0, C20:0, C18:0 and C20:1 esters.

12. The method of claim 10 or 11, wherein the ester is an ethyl ester.

13. The method according to any one of claims 1 to 12, wherein the highly unsaturated fatty acid ester composition contains 96% by weight or more of the target substance.

14. The method of any one of claims 1 to 13, wherein (a), (b) and (c) are repeated two or more times.

15. The method according to any one of claims 1 to 14, wherein the target product is an eicosapentaenoic acid ester and at least one component removed in whole or in part by the second column chromatography is an ester of stearidonic acid, C19:4, or C21:

5.

16. The method according to any one of claims 1 to 14, wherein the target product is a dihomo-gamma-linolenic acid ester and at least one component that is completely or partially removed by the second column chromatography is a C20:4n-6 ester.

17. The method according to any one of claims 1 to 14, wherein the target product is an arachidonic acid ester, and at least one component that is completely or partially removed by the second column chromatography is a C18:3n-6 ester.

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