Method for producing highly unsaturated fatty acid ester composition

Abstract

Provided is a method for efficiently producing a target highly unsaturated fatty acid ester composition with high purity. Provided is a method for producing a highly unsaturated fatty acid ester composition from a fat and oil composition, the method comprising: supplying the fat and oil composition to a first column chromatography, and removing all or part of at least one component in which the relative value of the retention time of the peak top of a target object in the first column chromatography is 1.8-3.5 when the retention time of the peak top is 1.0; and supplying an eluate containing the target obtained in the first column chromatography to a second column chromatography, and removing all or part of at least one component in which the relative value of the retention time of the peak top of the target in the second column chromatography is 0.90-1.1 when the retention time of the peak top is 1.0.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for manufacturing a highly unsaturated fatty acid (PUFA) ester composition using column chromatography. BACKGROUND

[0002] Highly unsaturated fatty acids are used as pharmaceuticals, health foods. Highly unsaturated fatty acids are manufactured from natural raw materials such as highly unsaturated fatty acid-rich plant oils and marine oils. The natural raw materials also contain fatty acids having different numbers of double bonds, such as saturated fatty acids, monounsaturated fatty acids, fatty acids having different carbon chain lengths, and other components. The content of highly unsaturated fatty acids in the natural raw materials is not high. When highly unsaturated fatty acids are used as pharmaceuticals, health foods, it is necessary to selectively separate the target highly unsaturated fatty acid (Patent Literature 1).

[0003] As a separation method for highly unsaturated fatty acids, column chromatography is known (Patent Literature 2). On the other hand, as a chromatography method that can be operated continuously, a simulated moving bed chromatography method can be used (Patent Literatures 3 to 7).

[0004] PRIOR ART DOCUMENTS PATENT LITERATURE Patent Literature 1: International Publication No. 2014 / 054435 Patent Literature 2: Japanese Patent Application Laid-Open No. 2014-511406 Patent Literature 3: Japanese Patent Application Laid-Open No. H8-512336 Patent Literature 4: Japanese Patent Application Laid-Open No. H8-218091 Patent Literature 5: Japanese Patent Application Laid-Open No. 2016-508156 Patent Literature 6: Japanese Patent Application Laid-Open No. 2019-207234 Patent Literature 7: Japanese Patent Application Laid-Open No. 2013-516398 SUMMARY PROBLEMS TO BE SOLVED BY THE INVENTION The simulated moving bed chromatography method requires a complex system, device, and is more expensive than a chromatography method using a single column. Therefore, there is still a demand for a method for manufacturing a target highly unsaturated fatty acid ester composition with high purity and high efficiency without the complex device like the simulated moving bed chromatography method.

[0005] TECHNICAL SOLUTION TO THE PROBLEM The present inventors have unexpectedly found that, by using a multistage separation method using a first column chromatography and a second column chromatography, it is possible to efficiently manufacture a target highly unsaturated fatty acid while addressing the above technical problem, thereby completing the present application.

[0006] The present application relates to a method for producing a highly unsaturated fatty acid ester composition from an oil and fat composition.

[0007] [1-1] A method for producing a highly unsaturated fatty acid ester composition from an oil and fat composition, comprising: (a) removing all or a part of at least one component having a relative value of the peak top retention time of 1.8 to 3.5 when the peak top retention time of a target substance, i.e., a highly unsaturated fatty acid ester, in a first column chromatography is set to 1.0, by subjecting an oil and fat composition to the first column chromatography using a first column, to obtain an eluate containing the target substance by the first column chromatography, (b) removing all or a part of at least one component having a relative value of the peak top retention time of 0.90 to 1.1 when the peak top retention time of the target substance in a second column chromatography is set to 1.0, by subjecting the eluate containing the target substance obtained by the first column chromatography to the second column chromatography using a second column, to obtain 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 substance, The obtained composition contains 95% by weight or more of the target substance.

[0008] [1-2] The method according to [1-1], wherein a load interval from the start of the column chromatography to the time when the column chromatography can be started again is shortened as compared with the case where the column chromatography is performed using a single column having a length equal to the total length of the first and second columns.

[0009] [1-3] The method according to [1-2], wherein the load interval from the start of the column chromatography to the time when the column chromatography can be started again is shortened as compared with the case where the column chromatography is performed using only the second column.

[0010] [1-4] The method according to any one of [1-1] to [1-3], wherein the at least one component having a relative value of the peak top retention time of 1.8 to 3.5 when the peak top retention time of the target substance in the first column chromatography is set to 1.0 is at least one component other than the highly unsaturated fatty acid ester in the oil and fat composition.

[0011] [1-5] The method according to any one of [1-1] to [1-4], wherein the all or a part removed by subjecting the eluate containing the target substance obtained by the first column chromatography to the second column chromatography using the second column is at least one component having a relative value of the peak top retention time of 0.93 to 1.09 when the peak top retention time of the target substance in the second column chromatography is set to 1.0.

[0012] [1-6] The method according to any one of [1-1] to [1-5], wherein the eluate from the first column is injected to the second column so that the eluate from the first column is injected to the second column, the eluate from the first column is injected to 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 from the first column when the retention time of the peak top of the target substance in the second column chromatogram is set to 1.0 is in the range of 1.1 or less.

[0013] [1-7] The method according to [1-6], wherein the eluate from the first column is injected to 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 from the first column when the retention time of the peak top of the target substance in the second column chromatogram is set to 1.0 is in the range of 0.90 to 1.1.

[0014] [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 from the first column when the retention time of the peak top of the target substance in the second column chromatogram is set to 1.0 is in the range of 0.94 to 1.1.

[0015] [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 from the first column when the retention time of the peak top of the target substance in the second column chromatogram is set to 1.0 is in the range of 0.96 to 1.1.

[0016] [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 from the first column when the retention time of the peak top of the target substance in the second column chromatogram is set to 1.0 is in the range of 0.93 to 1.09.

[0017] [1-11] The method according to [1-6], wherein the eluate from the first column having a relative value of the retention time of the peak top of the target substance in the first column set to 1.0 of 3.5 or more is discharged, and a new mobile phase is injected in the second column.

[0018] [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 a reversed phase column chromatography.

[0019] [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.

[0020] [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.

[0021] [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 the stationary phase than the first column.

[0022] [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 and is longer than the first column.

[0023] [1-17] The method according to any one of [1-1] to [1-16], wherein the oil composition is derived from any one of fish oil, vegetable oil, algae and microorganism.

[0024] [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). γ

[0025] [1-19] The method according to any one of [1-1] to [1-18], wherein the at least one component removed in whole or in part in the first column chromatography is an ester of any one of C22:0, C20:0, C18:0 and C20:1.

[0026] [1-20] The method according to [1-18] or [1-19], wherein the ester is an ethyl ester.

[0027] [1-21] The method according to any one of [1-1] to [1-20], wherein the content of the at least one component removed in whole or in part in the first column chromatography in the eluate containing the target substance supplied to the second column chromatography is 0.3% by weight or less, relative to the total fatty acid esters contained in the eluate supplied to the second column chromatography.

[0028] [1-22] The method according to [1-21], wherein the content of the at least one component removed in whole or in part in the first column chromatography in the eluate containing the target substance supplied to the second column chromatography is 0.1% by weight or less or 0.05% by weight or less, relative to the total fatty acid esters contained in the eluate supplied to the second column chromatography.

[0029] [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.

[0030] [1-24] The method according to any one of [1-1] to [1-23], further comprising: preparing the oil composition from the raw material composition by distillation.​

[0031] [1-25] The method according to any one of [1-1] to [1-24], wherein (a), (b), and (c) are repeated 2 or more times.

[0032] [1-26] The method according to any one of [1-1] to [1-25], further comprising, before (a), subjecting the oil and fat composition to column chromatography using a first column, and confirming a retention time of a peak top of the target substance.

[0033] [1-27] The method according to any one of [1-1] to [1-26], wherein the eluate containing the target substance obtained in the first column chromatography is an eluate until the elution of the target substance from the first column is completed.

[0034] [2-1] A method for producing an eicosapentaenoic acid ester composition from an oil and fat composition, comprising: (a) obtaining an eluate containing an eicosapentaenoic acid ester by subjecting an oil and fat composition to first column chromatography using a first column, the eluate containing 0.3% by weight 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 in the first column chromatography to second column chromatography using a second column to obtain an eluate, and (c) concentrating the eluate obtained in the second column chromatography to obtain a composition containing an eicosapentaenoic acid ester, the obtained composition containing 96.5% by weight or more of the eicosapentaenoic acid ester and at least one component other than the eicosapentaenoic acid ester, the at least one component other than the eicosapentaenoic acid ester containing 0.2% by weight or less of each of stearidonic acid, a C19:4 ester, and a C21:5 ester.

[0035] [2-2] The method according to [2-1], wherein the obtained composition contains 0.01% by weight to 0.2% by weight of each of stearidonic acid, a C19:4 ester, and a C21:5 ester.

[0036] [2-3] The method according to [2-1] or [2-2], wherein a load interval from the start of the column chromatography to the time when the next column chromatography can be started is shortened as compared with the case where column chromatography using a single column having a length equal to the total length of the first and second columns is performed.

[0037] [2-4] The method according to [2-3], wherein the load interval from the start of the column chromatography to the time when the next column chromatography can be started is shortened as compared with the case where column chromatography using only the second column is performed.

[0038] [2-5] According to any one of [2-1] to [2-4], wherein at least one component whose relative value of the retention time of the peak of eicosapentaenoic acid ester in the first column chromatography is 1.0 is at least one component other than eicosapentaenoic acid ester in the oil composition.

[0039] [2-6] The method according to any one of [2-1] to [2-5], wherein at least one component, which is a relative value of the retention time of the peak of the eicosapentaenoic acid ester obtained in the first column chromatography is removed by supplying the eluent containing the eicosapentaenoic acid ester obtained in the first column chromatography to the second column chromatography using the second column chromatography, is removed wholly or partially by the eluent containing the eicosapentaenoic acid ester obtained in the first column chromatography, wherein the retention time of the peak of the eicosapentaenoic acid ester obtained in the second column chromatography is set to 1.0.

[0040] [2-7] The method according to any one of [2-1] to [2-6], wherein the first column is in communication with the second column, such that the eluent from the first column is injected into the second column. The eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than eicosapentaenoic acid contained in the eluent is in the range of 1.1 or less when the retention time of the peak of eicosapentaenoic acid in the second column chromatography is set to 1.0.

[0041] [2-8] According to the method of [2-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than eicosapentaenoic acid contained in the eluent when the retention time of the peak of eicosapentaenoic acid in the second column chromatography is set to 1.0 is in the range of 0.90 to 1.1.

[0042] [2-9] According to the method of [2-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than eicosapentaenoic acid contained in the eluent is in the range of 0.94 to 1.1 when the retention time of the peak of eicosapentaenoic acid in the second column chromatography is set to 1.0.

[0043] [2-10] According to the method of [2-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than eicosapentaenoic acid contained in the eluent is in the range of 0.96 to 1.1 when the retention time of the peak of eicosapentaenoic acid in the second column chromatography is set to 1.0.

[0044] [2-11] According to the method of [2-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than eicosapentaenoic acid contained in the eluent when the retention time of the peak of the eicosapentaenoic acid in the second column chromatography is set to 1.0 is in the range of 0.93 to 1.09.

[0045] [2-12] According to the method described in [2-7], wherein the eluent from the first column is discharged when the relative value of the retention time of the peak of the target in the first column is 3.5 or more when the retention time is set to 1.0, and a new mobile phase is injected into the second column.

[0046] [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.

[0047] [2-14] The method according to any one of [2-1] to [2-13], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.

[0048] [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.

[0049] [2-16] The method according to any one of [2-1] to [2-15], wherein the second column is filled with a greater amount of stationary phase than the first column.

[0050] [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 and is longer than the first column.

[0051] [2-18] The method according to any one of [2-1] to [2-17], wherein at least one component that is wholly or partially removed in the first column chromatography is an ester of any one of C20:0, C18:0 and C20:1.

[0052] [2-19] The method according to any one of [2-1] to [2-18], wherein the oil composition is derived from any one of fish oil, vegetable oil, algae and microorganisms.

[0053] [2-20] The method according to any one of [2-1] to [2-19], wherein the ester is an ethyl ester.

[0054] [2-21] According to any one of [2-1] to [2-20], wherein, relative to all fatty acid esters contained in the eluent provided for the second column chromatography, the content of at least one component in the eluent containing eicosapentaenoic acid esters provided for the second column chromatography that has been completely or partially removed in the first column chromatography is 0.3% by weight or less.

[0055] [2-22] According to the method of [2-21], wherein, relative to all fatty acid esters contained in the eluent provided for the second column chromatography, the content of at least one component in the eluent containing eicosapentaenoic acid esters provided for the second column chromatography that has been removed in whole or in part in the first column chromatography is less than 0.1% by weight or less than 0.05% by weight.

[0056] [2-23] The method according to any one of [2-1] to [2-22], wherein the resulting composition contains more than 96% by weight of eicosapentaenoic acid ester.

[0057] [2-24] The method according to any one of [2-1] to [2-22] further comprises: preparing the oil composition from the raw material composition by distillation.

[0058] [2-25] The method according to any one of [2-1] to [2-24], wherein (a), (b) and (c) are repeated more than twice.

[0059] [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 whose relative retention time of the peak when the retention time of the peak of the eicosapentaenoic acid ester in the second column chromatography is set to 1.0 is removed.

[0060] [2-27] The method according to any one of [2-1] to [2-26] further comprises: prior to (a), feeding the oil composition to a column chromatogram using a first column to confirm the retention time of the peak of the eicosapentaenoic acid ester.

[0061] [2-28] The method according to any one of [2-1] to [2-27], wherein the eluent containing eicosapentaenoic acid ester obtained in the first column chromatography is the eluent containing eicosapentaenoic acid ester up to the end of the elution of the first column.

[0062] [3-1] A high-quality, high-efficiency compound made from an oil-oil composition γ A method for assembling a linoleic acid ester composition comprising: (a) By feeding the oil and fat composition into a first column chromatogram using a first column, a high-... γ - Linolenic acid ester eluent, (b) The high-concentration dihydrogen ion obtained from the first column chromatography was used to... γ - The eluent of linolenic acid esters was used for second-column chromatography to obtain the eluent, and (c) The eluent obtained from the second column chromatography was concentrated to give a composition containing eicosapentaenoic acid ester. The resulting composition contains more than 95.0% by weight of di-high- γ -Linolenic acid and high cholesterol- γ -At least one ingredient other than linoleic acid, high-protein- γ - At least one ingredient other than linoleic acid contains less than 0.60% by weight of an ester of C20:4n-6.

[0063] [3-2] According to the method of [3-1], wherein the resulting composition contains 0.01% to 0.2% by weight of an ester of C20:4n-6.

[0064] [3-3] According to the method described in [3-1] or [3-2], the loading interval from the start of one column chromatography to the point where another column chromatography can begin is shortened compared to the case of column chromatography using a single column of length equal to the combined length of the first and second columns.

[0065] [3-4] According to the method described in [3-3], the loading interval from the start of the first column chromatography to the start of the second column chromatography is shortened compared to the case of column chromatography using only the second column.

[0066] [3-5] The method according to any one of [3-1] to [3-4], wherein, in the first column chromatogram, the two high- γ -At least one component of the oil composition having a relative retention time of 1.8 to 3.5 at a retention time of linoleic acid ester peak set to 1.0 is a di-high- γ - At least one ingredient other than linoleic acid esters.

[0067] [3-6] The method according to any one of [3-1] to [3-5], wherein removing all or part of the target analyte from the second column chromatography by supplying the eluent containing the target analyte obtained in the first column chromatography to the second column chromatography using the second column is in the process of removing the target analyte from the second column chromatography. γ - At least one component whose relative retention time at the peak of linolenic acid ester is set to 1.0 is 0.93 to 1.09.

[0068] [3-7] The method according to any one of [3-1] to [3-6], wherein the first column is in communication with the second column, such that the eluent from the first column is injected into the second column. The eluent from the first column is injected into the second column, and the high-density chromatogram in the second column is then... γ The eluent containing high levels of alpha-linolenic acid esters at a retention time of 1.0 times at the peak of the alpha-linolenic acid esters peak contains two high-... γ - The relative retention time of the peak of at least one component other than linoleic acid is in the range of 1.1 or less.

[0069] [3-8] According to the method described in [3-7], wherein the eluent from the first column is injected into the second column, and the high-density chromatogram in the second column is... γ The eluent containing high levels of alpha-linolenic acid esters at a retention time of 1.0 times at the peak of the alpha-linolenic acid esters peak contains two high-... γ The relative retention times of the peaks of at least one component other than linoleic acid esters range from 0.90 to 1.1.

[0070] [3-9] According to the method described in [3-7], wherein the eluent from the first column is injected into the second column, and the high-density chromatogram in the second column is... γ The eluent containing high levels of alpha-linolenic acid esters at a retention time of 1.0 times at the peak of the alpha-linolenic acid esters peak contains two high-... γ The relative retention times of the peaks of at least one component other than linoleic acid esters ranged from 0.94 to 1.1.

[0071] [3-10] According to the method described in [3-7], wherein the eluent from the first column is injected into the second column, and the high-density chromatogram in the second column is... γ The eluent containing high levels of alpha-linolenic acid esters at a retention time of 1.0 times at the peak of the alpha-linolenic acid esters peak contains two high-... γ The relative retention times of the peaks of at least one component other than linoleic acid esters range from 0.96 to 1.1.

[0072] [3-11] According to the method described in [3-7], wherein the eluent from the first column is injected into the second column, and the high-density chromatogram in the second column is... γ The eluent containing high levels of alpha-linolenic acid esters at a retention time of 1.0 times at the peak of the alpha-linolenic acid esters peak contains two high-... γ The relative retention times of the peaks of at least one component other than linoleic acid esters ranged from 0.93 to 1.09.

[0073] [3-12] According to the method described in [3-7], wherein, in the first column, the two high- γ- The relative retention time of the linolenic acid ester peak is set to 1.0. The eluent from the first column is discharged and a new mobile phase is injected into the second column.

[0074] [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.

[0075] [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.

[0076] [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.

[0077] [3-16] The method according to any one of [3-1] to [3-15], wherein the second column is filled with a greater amount of stationary phase than the first column.

[0078] [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.

[0079] [3-18] The method according to any one of [3-1] to [3-17], wherein at least one component that is removed in whole or in part in the first column chromatography is an ester of any one of C22:0, C20:0, C18:0 and C20:1.

[0080] [3-19] The method according to any one of [3-1] to [3-18], wherein the oil composition is derived from any one of fish oil, vegetable oil, algae and microorganisms.

[0081] [3-20] The method according to any one of [3-1] to [3-19], wherein the ester is an ethyl ester.

[0082] [3-21] The method according to any one of [3-1] to [3-20], wherein, relative to all fatty acid esters contained in the eluent supplied to the second column chromatography, the eluent supplied to the second column chromatography contains di-high- γ - The content of at least one component in the eluent of linolenic acid esters that has been completely or partially removed in the first column chromatography is less than 0.3% by weight.

[0083] [3-22] According to the method of [3-21], wherein, relative to all fatty acid esters contained in the eluent supplied to the second column chromatography, the eluent supplied to the second column chromatography contains di-high- γ- The content of at least one component in the eluent of linolenic acid esters that has been completely or partially removed in the first column chromatography is less than 0.1% by weight or less than 0.05% by weight.

[0084] [3-23] The method according to any one of [3-1] to [3-22], wherein the obtained composition contains 96% by weight or more of di-high- γ -Linolenic acid esters.

[0085] [3-24] The method according to any one of [3-1] to [3-23], wherein, in (b), the di-high-carbon dioxide in the second column chromatogram is removed. γ -The relative value of the retention time of the peak of at least one component is 0.90 to 1.1 when the retention time of the peak of -linolenic acid ester is set to 1.0.

[0086] [3-25] The method according to any one of [3-1] to [3-24] further comprises: prior to (a), subjecting the oil composition to column chromatography using a first column to confirm the presence of high-... γ - Retention time of the peak of linolenic acid esters.

[0087] [3-26] The method according to any one of [3-1] to [3-25], wherein the first column chromatography yields a product containing two high-... γ -The eluent for linolenic acid esters is high-quality and high-efficiency.- γ - Linolenic acid esters are the eluent from the end of the first column elution.

[0088] [4-1] A method for manufacturing an arachidonic acid ester composition from an oil composition, comprising: (a) By feeding the oil and fat composition into a first column chromatogram using a first column, an eluent containing arachidonic acid esters is obtained, comprising less than 0.5% by weight of C22:0 esters relative to all fatty acid esters contained in the eluent. (b) The eluent containing arachidonic acid obtained from the first column chromatography is used in a second column chromatography to obtain an eluent, and (c) The eluent obtained from the second column chromatography was concentrated to give a composition containing arachidonic acid esters. The resulting composition contains more than 95.0% by weight of arachidonic acid ester and at least one other component besides arachidonic acid ester, wherein the at least one component besides arachidonic acid ester contains less than 0.25% by weight of a C18:3 n-6 ester.

[0089] [4-2] According to the method of [4-1], wherein the resulting composition contains 0.01% to 0.2% by weight of a C18:3 n-6 ester.

[0090] [4-3] According to the method of [4-1] or [4-2], the loading interval from the start of one column chromatography to the start of another column chromatography is shortened compared to the case of column chromatography using a single column of length equal to the combined length of the first and second columns.

[0091] [4-4] According to the method described in [4-3], the loading interval from the start of the first column chromatography to the start of the second column chromatography is shortened compared to the case of column chromatography using only the second column.

[0092] [4-5] According to any one of [4-1] to [4-4], wherein at least one component whose relative retention time of the peak of arachidonic acid ester in the first column chromatography is 1.0 is 1.8 to 3.5, is at least one component other than arachidonic acid ester in the oil composition.

[0093] [4-6] The method according to any one of [4-1] to [4-5], wherein at least one component, which is removed entirely or partially by supplying the eluent containing arachidonic acid obtained in the first column chromatography to the second column chromatography using the second column, has a relative value of 0.93 to 1.09 for the retention time of the peak of arachidonic acid in the second column chromatography when the retention time of the peak of arachidonic acid in the second column chromatography is set to 1.0.

[0094] [4-7] The method according to any one of [4-1] to [4-6], wherein the first column is in communication with the second column, such that the eluent from the first column is injected into the second column. The eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than arachidonic acid contained in the eluent is in the range of 1.1 or less when the retention time of the peak of arachidonic acid in the second column chromatography is set to 1.0.

[0095] [4-8] The method according to any one of [4-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than arachidonic acid contained in the eluent when the retention time of the peak of arachidonic acid in the second column chromatography is set to 1.0 is in the range of 0.90 to 1.1.

[0096] [4-9] According to the method of [4-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than arachidonic acid contained in the eluent when the retention time of the peak of arachidonic acid in the second column chromatography is set to 1.0 is in the range of 0.94 to 1.1.

[0097] [4-10] According to the method of [4-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than arachidonic acid contained in the eluent when the retention time of the peak of arachidonic acid in the second column chromatography is set to 1.0 is in the range of 0.96 to 1.1.

[0098] [4-11] According to the method of [4-7], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component other than arachidonic acid contained in the eluent when the retention time of the peak of arachidonic acid in the second column chromatography is set to 1.0 is in the range of 0.93 to 1.09.

[0099] [4-12] According to the method of [4-7], wherein the eluent from the first column is discharged when the relative value of the retention time of the peak of the arachidonic acid ester in the first column is 3.5 or more when the retention time is set to 1.0, and a new mobile phase is injected into the second column.

[0100] [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.

[0101] [4-14] The method according to any one of [4-1] to [4-13], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.

[0102] [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.

[0103] [4-16] The method according to any one of [4-1] to [4-15], wherein the second column is filled with a greater amount of stationary phase than the first column.

[0104] [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.

[0105] [4-18] The method according to any one of [4-1] to [4-17], wherein at least one component that is removed in whole or in part in the first column chromatography is an ester of any one of C22:0, C20:0, C18:0 and C20:1.

[0106] [4-19] The method according to any one of [4-1] to [4-18], wherein the oil composition is derived from any one of fish oil, vegetable oil, algae and microorganisms.

[0107] [4-20] The method according to any one of [4-1] to [4-19], wherein the ester is an ethyl ester.

[0108] [4-21] According to any one of [4-1] to [4-20], wherein, relative to all fatty acid esters contained in the eluent provided for the second column chromatography, the content of at least one component in the eluent containing arachidonic acid esters provided for the second column chromatography that has been completely or partially removed in the first column chromatography is 0.3% by weight or less.

[0109] [4-22] According to the method of [4-21], wherein, relative to all fatty acid esters contained in the eluent provided for the second column chromatography, the content of at least one component in the eluent containing arachidonic acid esters provided for the second column chromatography that has been removed in whole or in part in the first column chromatography is less than 0.1% by weight or less than 0.05% by weight.

[0110] [4-23] The method according to any one of [4-1] to [4-22], wherein the resulting composition contains more than 96% by weight of arachidonic acid ester.

[0111] [4-24] The method according to any one of [4-1] to [4-23] further comprises: preparing the oil composition from the raw material composition by distillation.

[0112] [4-25] The method according to any one of [4-1] to [4-24], wherein (a), (b) and (c) are repeated more than twice.

[0113] [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 whose relative retention time at the peak of arachidonic acid ester is 0.90 to 1.1 when the retention time of the peak of arachidonic acid ester in the second column chromatography is set to 1.0 is removed.

[0114] [4-27] The method according to any one of [4-1] to [4-26] further comprises: prior to (a), feeding the oil composition to a column chromatogram using a first column to confirm the retention time of the peak of the arachidonic acid ester.

[0115] [4-28] The method according to any one of [4-1] to [4-27], wherein the eluent containing arachidonic acid ester obtained in the first column chromatography is the eluent containing arachidonic acid ester up to the end of the elution of the first column.

[0116] [5-1] A method for shortening the time required for column chromatography in the preparation of highly unsaturated fatty acid ester compositions from oil compositions. (a) By feeding the oil and fat composition to a first column chromatogram using a first column, removing all or part of at least one component whose relative retention time at the peak of the target compound, i.e., the highly unsaturated fatty acid ester, is 1.0 in the first column chromatogram, an eluent containing the target compound is obtained by passing the first column chromatogram through the first column chromatography. (b) By supplying the eluent containing the target analyte obtained from the first column chromatography to a second column chromatography using a second column, removing all or part of at least one component whose relative retention time at the peak of the target analyte is 0.90 to 1.1 when the retention time of the peak of the target analyte in the second column chromatography is set to 1.0, an eluent is obtained by the second column chromatography, and (c) Concentrate the eluent obtained from the second column chromatography to obtain a composition containing the target analyte. The resulting composition contains more than 95% by weight of the target substance. The loading interval from the start of one column chromatography to the point where another column chromatography can begin is shortened compared to the case of column chromatography using a single column of length equal to the combined length of the first and second columns.

[0117] [5-2] According to the method described in [5-1], the loading interval from the start of one column chromatography to the start of another column chromatography is shortened compared to the case of column chromatography using only a second column.

[0118] [5-3] According to the method of [5-1] or [5-2], wherein at least one component whose relative value of the retention time of the peak of the target analyte in the first column chromatography is 1.0 is 1.8 to 3.5, is at least one component other than highly unsaturated fatty acid esters in the oil and fat composition.

[0119] [5-4] The method according to any one of [5-1] to [5-3], wherein at least one component, which is removed entirely or partially by supplying the eluent containing the target analyte obtained in the first column chromatography to the second column chromatography using the second column, has a relative value of 0.93 to 1.09 for the retention time of the peak of the target analyte in the second column chromatography when the retention time of the peak of the target analyte in the second column chromatography is set to 1.0.

[0120] [5-5] The method according to any one of [5-1] to [5-4], wherein the first column is in communication with the second column, such that the eluent from the first column is injected into the second column. The eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of the component contained in the eluent when the retention time of the peak of the target analyte in the second column chromatography is set to 1.0 is 1.1 or less.

[0121] [5-6] According to the method of [5-5], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of the component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.90 to 1.1.

[0122] [5-7] According to the method of [5-5], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of the component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.94 to 1.1.

[0123] [5-8] According to the method of [5-5], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of the component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.96 to 1.1.

[0124] [5-9] According to the method of [5-5], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of the component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.93 to 1.09.

[0125] [5-10] According to the method described in [5-5], wherein the eluent from the first column is discharged when the relative value of the retention time of the peak of the target in the first column is 3.5 or more when the retention time is set to 1.0, and a new mobile phase is injected into the second column.

[0126] [5-11] The method according to any one of [5-1] to [5-10], wherein the first column chromatography and the second column chromatography use reversed-phase column chromatography.

[0127] [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.

[0128] [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.

[0129] [5-14] The method according to any one of [5-1] to [5-13], wherein the second column is filled with a greater amount of stationary phase than the first column.

[0130] [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 and is longer than the first column.

[0131] [5-16] The method according to any one of [5-1] to [5-15], wherein the target substance is eicosapentaenoic acid (EPA), dihydrogen phosphate (DHP), or EPA. γ - An ester of either linolenic acid (DGLA) or arachidonic acid (ARA).

[0132] [5-17] The method according to any one of [5-1] to [5-16], wherein at least one component that is removed in whole or in part in the first column chromatography is an ester of any one of C22:0, C20:0, C18:0 and C20:1.

[0133] [5-18] The method according to any one of [5-1] to [5-17], wherein the oil composition is derived from any one of fish oil, vegetable oil, algae and microorganisms.

[0134] [5-19] The method according to [5-17] or [5-18], wherein the ester is an ethyl ester.

[0135] [5-20] The method according to any one of [5-1] to [5-19], wherein, relative to all fatty acid esters contained in the eluent containing the target in the second column chromatography, the content of at least one component that has been completely or partially removed in the first column chromatography is 0.3% by weight or less.

[0136] [5-21] According to the method of [5-20], wherein, relative to all fatty acid esters contained in the eluent containing the target in the second column chromatography, the content of at least one component that has been completely or partially removed in the first column chromatography is less than 0.1% by weight or less than 0.05% by weight.

[0137] [5-22] The method according to any one of [5-1] to [5-21], wherein the highly unsaturated fatty acid ester composition contains 96% by weight or more of the target substance.

[0138] [5-23] The method according to any one of [5-1] to [5-22] further comprises: preparing the oil composition from the raw material composition by distillation.

[0139] [5-24] The method according to any one of [5-1] to [5-23], wherein (a), (b) and (c) are repeated more than twice.

[0140] [5-25] The method according to any one of [5-1] to [5-24] further comprises: prior to (a), feeding the oil composition to a column chromatogram using a first column to confirm the retention time of the peak of the target analyte.

[0141] [5-26] The method according to any one of [5-1] to [5-25], wherein the eluent containing the target analyte obtained in the first column chromatography is the eluent containing the target analyte up to the end of elution from the first column.

[0142] [6-1] A method for manufacturing a highly unsaturated fatty acid ester composition from an oil composition. (a) By feeding the oil and fat composition to a first column chromatogram using a first column, removing all or part of at least one component whose relative retention time at the peak of the target compound, i.e., the highly unsaturated fatty acid ester, is 1.0 in the first column chromatogram, an eluent containing the target compound is obtained by the first column chromatogram, and (b) By supplying the eluent containing the target analyte obtained in the first column chromatography to the second column chromatography using the second column, all or part of at least one component whose relative retention time at the peak of the target analyte is 0.90 to 1.1 when the retention time of the peak of the target analyte in the second column chromatography is set to 1.0 is removed by the second column chromatography.

[0143] [6-2] According to the method of [6-1], the loading interval from the start of one column chromatography to the start of another column chromatography is shortened compared to the case of column chromatography using a single column of length equal to the combined length of the first and second columns.

[0144] [6-3] According to the method described in [6-2], the loading interval from the start of the first column chromatography to the start of the second column chromatography is shortened compared to the case of column chromatography using only the second column.

[0145] [6-4] The method according to any one of [6-1] to [6-3], wherein the at least one component whose relative value of the retention time of the peak of the target analyte in the first column chromatography is 1.0 is 1.8 to 3.5, is at least one component other than highly unsaturated fatty acid esters in the oil and fat composition.

[0146] [6-5] The method according to any one of [6-1] to [6-4], wherein at least one component, which is removed entirely or partially by supplying the eluent containing the target analyte obtained in the first column chromatography to the second column chromatography using the second column, has a relative value of 0.93 to 1.09 for the retention time of the peak of the target analyte in the second column chromatography when the retention time of the peak of the target analyte in the second column chromatography is set to 1.0.

[0147] [6-6] The method according to any one of [6-1] to [6-5], wherein the first column is in communication with the second column, such that the eluent from the first column is injected into the second column. The eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of at least one component contained in the eluent when the retention time of the peak of the target analyte in the second column chromatography is set to 1.0 is 1.1 or less.

[0148] [6-7] According to the method of [6-6], wherein the eluent from the first column is injected into the second column, and the relative value of the retention time of the peak of the at least one component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.90 to 1.1.

[0149] [6-8] According to the method of [6-6], wherein the relative value of the retention time of the peak of the at least one component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.94 to 1.1.

[0150] [6-9] According to the method of [6-6], wherein the relative value of the retention time of the peak of the at least one component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.96 to 1.1.

[0151] [6-10] According to the method of [6-6], wherein the relative value of the retention time of the peak of the at least one component contained in the eluent when the retention time of the peak of the target in the second column chromatography is set to 1.0 is in the range of 0.93 to 1.09.

[0152] [6-11] According to the method described in [6-6], wherein the eluent from the first column is discharged when the relative value of the retention time of the peak of the target in the first column is 3.5 or more when the retention time is set to 1.0, and a new mobile phase is injected into the second column.

[0153] [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 reversed-phase column chromatography.

[0154] [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.

[0155] [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.

[0156] [6-15] The method according to any one of [6-1] to [6-14], wherein the second column is filled with a greater amount of stationary phase than the first column.

[0157] [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 and is longer than the first column.

[0158] [6-17] The method according to any one of [6-1] to [6-16], wherein the oil composition is derived from any one of fish oil, vegetable oil, algae and microorganisms.

[0159] [6-18] The method according to any one of [6-1] to [6-17], wherein the target substance is eicosapentaenoic acid (EPA), dihydrogen phosphate (DHP), or EPA. γ - An ester of either linolenic acid (DGLA) or arachidonic acid (ARA).

[0160] [6-19] The method according to any one of [6-1] to [6-18], wherein at least one component that is wholly or partially removed in the first column chromatography is an ester of any one of C22:0, C20:0, C18:0 and C20:1.

[0161] [6-20] The method according to [6-18] or [6-19], wherein the ester is an ethyl ester.

[0162] [6-21] The method according to any one of [6-1] to [6-20], wherein, relative to all fatty acid esters contained in the eluent containing the target in the second column chromatography, the content of the at least one component that was removed in whole or in part in the first column chromatography is 0.3% by weight or less.

[0163] [6-22] According to the method of [6-21], wherein, relative to all fatty acid esters contained in the eluent containing the target in the second column chromatography, the content of the at least one component that was removed in whole or in part in the first column chromatography is less than 0.1% by weight or less than 0.05% by weight.

[0164] [6-23] The method according to any one of [6-1] to [6-22], wherein the resulting composition contains 96% by weight or more of the target substance.

[0165] [6-24] The method according to any one of [6-1] to [6-23] further comprises: preparing the oil composition from the raw material composition by distillation.

[0166] [6-25] The method according to any one of [6-1] to [6-24], wherein (a), (b) and (c) are repeated more than twice.

[0167] [6-26] The method according to any one of [6-1] to [6-25] further comprises: prior to (a), feeding the oil composition to a column chromatogram using a first column to confirm the retention time of the peak of the target analyte.

[0168] [6-27] The method according to any one of [6-1] to [6-26], wherein the eluent containing the target analyte obtained in the first column chromatography is the eluent containing the target analyte up to the end of elution from the first column.

[0169] Invention Effects This invention reveals that, compared to using a single column, it is possible to efficiently manufacture compositions containing high concentrations of highly unsaturated fatty acid ethyl esters such as EPA ethyl ester, DGLA ethyl ester, and ARA ethyl ester in a shorter time and with reduced solvent usage. In this invention, fatty acids such as C20:0 ethyl ester can be removed. Detailed Implementation

[0170] In one aspect of the invention, the "composition" can contain one or more PUFAs or their ester derivatives. The composition containing PUFAs or their ester derivatives can be obtained from raw materials containing highly unsaturated fatty acids as constituent fatty acids. For example, suitable oil compositions can be obtained from raw material compositions derived by esterifying natural oils containing plant, animal, and fat oils, and from raw material compositions derived by esterifying oils obtained from genetically recombinant plants, animals, and microorganisms including yeast and filamentous fungi. For example, as filamentous fungi, compositions derived by esterifying oils obtained from the genus *Mortierella* can be cited. Examples include compositions derived by ethyl esterifying fish oil, oils from algae and microalgae, and plant oils such as borage oil, *Echium plantagineum* oil, and evening primrose oil. In one embodiment, the raw material composition is a composition derived by ethyl esterifying fish oil. In another embodiment, the raw material composition is a composition derived by ethyl esterifying algal oil.

[0171] In one embodiment of the invention, the oil composition is obtained by distilling the raw material composition.

[0172] In one embodiment of the invention, the highly unsaturated fatty acid ester composition refers to a composition obtained by the method of the invention, which is a composition containing the target substance, namely, a highly unsaturated fatty acid ester. The highly unsaturated fatty acid ester of the target substance can be eicosapentaenoic acid (EPA), dihydroxy-2-(2-) ...γ - An ester of either linolenic acid (DGLA) or arachidonic acid (ARA), such as an ethyl ester.

[0173] In this specification, the term "fatty acid" refers to long-chain aliphatic carboxylic acids (alkanoic acids) of various chain lengths, approximately C12 to C22 (the numbers here refer to the total number of carbon atoms in the chain). The main chain length is C16 to C22. The structure of a fatty acid can be represented using the simple expression "X:Y". Here, X is the total number of carbon atoms in a particular fatty acid, and Y is the number of double bonds. For example, a saturated fatty acid with 20 carbon atoms can be expressed as "C20:0", a monovalent unsaturated fatty acid with 18 carbon atoms can be expressed as "C18:1", etc., and arachidonic acid can be expressed as "C20:4, n-6", etc. "n-" indicates the starting position of the double bond counting from the methyl end of the fatty acid; for example, if it is "n-6", it means that the starting position of the double bond is the 6th position counting from the methyl end of the fatty acid. This method is well known to those skilled in the art, and those skilled in the art can easily determine fatty acids expressed according to this method.

[0174] Fatty acids are carboxylic acids with long aliphatic chains, which can be either saturated or unsaturated. Fatty acids are typically manufactured industrially through the hydrolysis of triglycerides or phospholipids from natural sources. Some fatty acids are also produced synthetically. Regardless of the manufacturing method, purification is required to obtain pure products for food, cosmetic, or industrial use.

[0175] In this specification, the term "highly unsaturated fatty acid (PUFA)" refers to a fatty acid having more than one double bond. The highly unsaturated fatty acids in this specification are in the form of esters. Esters are typically alkyl esters, such as C1-C6 alkyl esters or C1-C4 alkyl esters. Ethyl esters are an example of esters.

[0176] In one aspect of the invention, examples of highly unsaturated fatty acids include eicosapentaenoic acid (EPA), docosapentaenoic acid (DFA), and docosahexaenoic acid (DHA). γ -Linolenic acid, arachidonic acid, octadecanoic acid, C18:3, C19:4, C20:4, C21:5. In one aspect of the invention, eicosapentaenoic acid (EPA), dihydroxy-2-(2-) ... γ -Linolenic acid, arachidonic acid. In one aspect of the invention, examples of at least one component that is completely or partially removed in the second column chromatography include C18:3, C19:4, C20:4, and C21:5. The at least one component that is completely or partially removed in the second column chromatography may be an analogue of the target highly unsaturated fatty acid.

[0177] In this specification, the term "eicosapentaenoic acid (EPA)" is the common name for cis-5,8,11,14,17-eicosapentaenoic acid. This fatty acid is a C20:5 n-3 fatty acid. "EPA-EE" may specifically refer to EPA ethyl ester.

[0178] In this instruction manual, "two high- γ "-Linolenic acid (DGLA)" is the common name for cis-8,11,14-eicosatotrienoic acid; this fatty acid is a C20:3 n-6 fatty acid.

[0179] In this specification, "arachidonic acid (ARA)" is the common name for cis-5,8,11,14-eicosatetraenoic acid; this fatty acid is a C20:4 n-6 fatty acid.

[0180] In one aspect of the invention, the oil composition typically comprises a highly unsaturated fatty acid and at least one other fatty acid. More typically, the oil composition comprises a highly unsaturated fatty acid and at least one other fatty acid and / or its ester. Typical fatty acid esters are the same as those specified above for highly unsaturated fatty acids. Preferably, the oil composition comprises at least one of a fatty acid having 20 carbon atoms and an ethyl ester of a fatty acid. More preferably, the 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 (betainic acid), and their esters. In one aspect of the invention, the at least one component removed in whole or in part in the first column chromatography is the at least one fatty acid ester described above.

[0181] In one aspect of the invention, at least one component in the oil composition, other than the target substance (e.g., a highly unsaturated fatty acid ester), has a lower polarity than the target substance.

[0182] In one aspect of the invention, the oil composition contained in the eluent supplied for the second column chromatography contains, relative to the total oil composition contained in the eluent supplied for the second column chromatography, at least one component selected from esters of C18:0, C20:0, C20:1, and C22:0 in an amount of 0.5% by weight or less, 0.3% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.03% by weight or less, or 0.01% by weight.

[0183] "Column chromatography" refers to a process, when used in this specification, of selectively retaining or delaying one or more components of a fluid solution as it flows through a column containing a stationary phase (single or more) composed of finely segmented substances and / or materials with capillary pathways. Retention results in the relative distribution of the components of a mixture between the stationary phase and the host fluid phase (e.g., the mobile phase), the latter moving 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, actual moving bed chromatography, and supercritical fluid chromatography (SFC).

[0184] The terms “nonpolar” and “polar” used to describe mobile phases or highly unsaturated fatty acid esters can be used as relative terms. For example, “nonpolar” can refer to the solvent in the mobile phase with the lowest polarity, while “polar” can refer to the solvent in the mobile phase with a higher polarity than the “nonpolar” solvent.

[0185] In one aspect of the invention, column chromatography includes passing the oil and fat composition through one or more columns. Therefore, the first column chromatography includes: passing the oil and fat composition through a first column; removing all or part of at least one component that elutes more slowly than the target, i.e., a highly unsaturated fatty acid ester (i.e., the relative value of the retention time of the peak when the retention time of the target in the first column is set to 1.0 exceeds 1.0). The first column may be used in combination of multiple columns arranged longitudinally and / or side-by-side. The second column chromatography includes: passing the eluent containing the target obtained from the first column chromatography through a second column, removing all or part of the similar substance components, to obtain a highly unsaturated fatty acid ester composition containing the target in a predetermined amount. The second column may also be used in combination of multiple columns arranged longitudinally and / or side-by-side. Any known column can be used in the method according to the claims.

[0186] In one embodiment of the invention, the eluent containing the target analyte obtained in the first column chromatography is the eluent containing the target analyte up to the end of elution from the first column. In another embodiment of the invention, removal of all or part of at least one component other than the target analyte from the first column chromatography is achieved by recovering the eluent up to the end of elution from the first column.

[0187] The first column chromatography is performed using a first column. In one aspect of the invention, the first column chromatography is performed with the aim of removing all or part of the components whose retention time is longer than that of the target analyte. In one embodiment of the invention, when the retention time of the target analyte in the first column chromatography is set to 1, the retention time for removing all or part of the component by the first column chromatography is 1.5 or more, 1.7 or more, 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. In one embodiment of the invention, when at least one component is partially removed in the first column chromatography, 99% or more, 95% or more, or 90% or more of the component is removed.

[0188] The second column chromatography is performed using a second column. In one aspect of the invention, the second column chromatography is performed with the aim of removing all or part of the components whose retention times are similar to those of the target analyte. Components whose retention times are similar to those of the target analyte can be referred to as similar substances in this specification. In one embodiment of the invention, when the retention time of the target analyte in the second column chromatography is set to 1, the retention time of the component removed by the second column chromatography is 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. In one embodiment of the invention, when a portion of the component whose retention time is similar to that of the target analyte is removed in 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 component is removed.

[0189] In one aspect of the invention, fractions from the eluent of the first chromatography that were not fed into the second chromatography and fractions of fatty acid esters removed from the highly unsaturated fatty acid ester composition from the eluent of the second chromatography can be recovered and fed into a third column chromatography. The third column chromatography is carried out using a third column.

[0190] In one embodiment of the invention, the first column refers to a column used for first-column chromatography, the second column refers to a column used for second-column chromatography, and the third column refers to a column used for third-column chromatography. The size of the columns used is not particularly limited and depends to some extent on the volume of the oil composition to be purified. 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 less than 4000 mm, less than 2000 mm, less than 1000 mm, less than 500 mm, or 1–4000 mm, 2–2000 mm, 4–1000 mm, 8–1000 mm, 16–1000 mm, 32–500 mm, 30–800 mm, or 400–800 mm. In one embodiment of the 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, when the first column is set to 1, 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 invention, columns with the same diameter are used as the first and second columns. In one embodiment of the invention, when performing third column chromatography, the first to third columns can have the same or different diameters. The third column can have the same or different diameters as the first or second column, respectively. In one embodiment of the invention, the second column has the same diameter as the first column and is longer than the first column. In this specification, "the first and second columns have the same or identical diameters" means that the ratio of the diameters of the first and second columns is 0.8 to 1.2, for example, 0.9 to 1.1 or 1.

[0191] In one embodiment of the invention, the cross-sections of the first and second columns are approximately circular. The ratio of the cross-sectional areas of the first and second columns, when the first column is set to 1, is 0.1 or more, 0.2 or more, or 10 or less, 4 or less, or 2 or less, or 0.1 to 10, 0.2 to 4, or 0.2 to 2. In one embodiment of the invention, columns with the same cross-sectional area are used as the first and second columns. In one embodiment of the invention, when performing third-column chromatography, 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 or second column, respectively.

[0192] In one embodiment of the 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 or less, 120 cm or less, or 5–800 cm, 10–400 cm, 20–200 cm, 20–150 cm, or 20–120 cm. In one embodiment of the invention, the first column is shorter than the second column. The ratio of the lengths of the first and second columns, when the first column is set to 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 0.5–10, 1–7, 1.1–5, or 1.5–5. In one embodiment of the invention, when performing third column chromatography, the third column can have the same or different lengths as the first or second column, respectively. In one embodiment of the invention, the second column has the same inner diameter as the first column and is longer than the first column. The second column has the same inner diameter as the first column, for example, a length that is more than 1.1 times, more than 1.5 times, or less than 5 times, less than 7 times, or less than 10 times the length of the first column. In this specification, "the first column and the second column have the same or identical inner diameter" means that the ratio of the inner diameters of the first column to the inner diameter of the second column is 0.8 to 1.2, for example, 0.9 to 1.1 or 1.

[0193] A column is a cylindrical shape having an outer diameter, an inner diameter, and a length. In one embodiment, the column is a preparative chromatographic column. A preparative chromatographic column can have an inner diameter of 5 mm or more, 10 mm or more, 20 mm or more, 50 mm or more, or less than 4 m, less than 2 m, less than 1 m, less than 80 cm, or approximately 5 mm to approximately 4 m, approximately 10 mm to approximately 2 m, approximately 20 mm to approximately 1 m, or approximately 50 mm to 80 cm, and a length of approximately 100 mm to approximately 5 m, approximately 2 cm to approximately 2 m, or approximately 10 cm to approximately 1.5 m. In another embodiment, the column is an analytical chromatographic column. An analytical column can have an inner diameter of approximately 1 mm to approximately 10 cm and a length of approximately 10 mm to approximately 500 mm. The dimensions can be selected such that the inner diameter is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 140%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%, 320%, 340%, 360%, 380%, 400%, 450%, or 500% of the length. The outer diameter can be approximately 0.1%, approximately 0.5%, approximately 1.0%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 3.5%, approximately 4.0%, approximately 4.5%, approximately 5%, approximately 7.5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, or approximately 30% larger than the inner diameter.

[0194] The term "peak," as used in this specification, refers to a peak in a chromatogram. A chromatogram is a graph that represents detected values ​​over time. When eluent is detected, the detected value increases, and the chromatogram displays a "peak." Each peak in the chromatogram represents the presence of a component in the sample. Each peak is labeled with a retention time, which increases from left to right in the chromatogram.

[0195] "Peak" refers to the point in the chromatogram where the detected value of each peak is the largest.

[0196] When used in this specification, "separation" refers to a process characterized by the separation of the constituent elements of a composition containing highly unsaturated fatty acid esters through spatial separation based on the distribution differences between phases in relative motion (e.g., mobile and stationary phases). Separation results in the loading of the sample onto the column and the subsequent elution of the column.

[0197] "Fracturing" is a separation process in which a certain amount of mixture is divided into multiple smaller amounts during a phase transfer process, where the composition varies according to a gradient. Different fractions are collected at different time points based on the specific characteristics of each component (highly unsaturated fatty acid esters in the mixture or sample), for example, differences in their affinity with the stationary phase and / or mobile phase.

[0198] When used in this specification, "for column chromatography" means loading a sample containing highly unsaturated fatty acid esters onto a column until the entire sample is contained within the column. In this specification, the sample is an oil or fat composition. Typically, the sample is loaded onto the top of the stationary phase of a packed column. Here, "top" is the end of the stationary phase that initially receives the mobile phase as it elutes through the column. Oil or fat compositions can be column-chromatographically processed by applying the sample directly to the injector, pump, or top of the stationary phase. The sample can be mixed with a minimal amount of mobile phase or other solvent used for loading.

[0199] When used in this specification, "elution" refers to the elution of a component loaded on a column through a solution that serves as the mobile phase.

[0200] When used in this specification, "eluent" refers to the mobile phase eluted from a column, which may contain components contained in the oil composition loaded on the column. In one embodiment, it is a solution containing components contained in the oil composition in the mobile phase eluted from the column. In one embodiment of the invention, "eluent containing the target analyte obtained in the first column chromatography" means the eluent that has passed through the first column and contains the target analyte.

[0201] When used in this specification, "eluate" refers to the component discharged from the column in column chromatography. In one embodiment of the invention, "eluate for second column chromatography" is the eluate from the first column chromatography.

[0202] "Component" refers to the component contained in the oil and fat composition supplied for column chromatography, and includes highly unsaturated fatty acid esters and fatty acid esters. In one aspect of the invention, "at least one component that is removed in whole or in part in the first column chromatography" can be a fatty acid ester with a longer retention time compared to the target, i.e., the highly unsaturated fatty acid ester, for example, any one of C22:0, C20:0, C18:0, and C20:1. Furthermore, "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 an ester of any one of octadecanoic acid, C19:4, C21:5, C20:4, and C18:3.

[0203] In one embodiment of the invention, "at least one component that has been completely or partially removed in the first column chromatography" and "at least one component in the second column chromatography" may be different. In another embodiment of the invention, "at least one component in the second column chromatography" may include the same component as "at least one component that has been completely or partially removed in the first column chromatography".

[0204] When used in this specification, “biphenyl,” “C30,” “C22,” “C18,” “C8,” “C5,” and “C4” refer to functional groups present on the column packing material (stationary phase). For example, a biphenyl column exposes the material flowing through the column to unsubstituted biphenyl groups, while a C18 column exposes the material flowing through the column (e.g., the mobile phase and components) to unsubstituted straight-chain or branched 18-carbon alkyl groups.

[0205] "Chromatographic conditions" refers to the parameters used in column chromatography. Examples include packing pressure, composition of mobile and stationary phases, slurry concentration, pressure at which the column is run, column temperature, mobile phase temperature, mobile phase gradient, mobile phase flow rate, column type used, detector and parameters used, sample preparation method used, settling time and pressure at which settling is performed, and settling time and pressure at which settling is performed.

[0206] "Gradient" refers to the change in the composition of the mobile phase over time during column chromatography. The composition of the mobile phase can change as the solvent elutes through the column. Different mobile phases can be added during elution, increasing by a percentage over time.

[0207] "Purity" refers to the ratio of the content of the main component in the composition, which can be calculated, for example, by gas chromatography (GC) using an internal standard. In one embodiment of this specification, the main component is a highly unsaturated fatty acid ester, such as an EPA ester, DGLA ester, or ARA ester.

[0208] "Target" refers to a target highly unsaturated fatty acid ester, such as EPA ester, DGLA ester, or ARA ester, obtained by elution and fractionation of a single fraction or a combination of fractions.

[0209] In one aspect of the invention, concentration includes subjecting the eluent containing the target highly unsaturated fatty acid ester to reduced pressure and / or heating, and recovering it as a solvent by distillation. In another aspect of the invention, concentration is performed using an evaporator. Examples of evaporators include eluent evaporators and / or thin-film evaporators. Any type of evaporator can be used in concentration, such as thin-film evaporators, single-effect evaporators, multi-chamber evaporators, natural circulation evaporators, forced circulation evaporators, falling film evaporators, rising film evaporators, and combinations thereof. In one aspect of the invention, examples of natural circulation evaporators include external heating and calandria systems.

[0210] In this specification, specific ranges are given using numerical values ​​prefixed with the term "about". The term "about" is used in this specification to provide the precise number it prefixes and to provide textual support for numbers close to or approximately equal to the number prefixed with the term. In determining whether a number is close to or approximately equal to a specifically stated number, an unstated number close to or close to the stated number may be a quantity substantially equivalent to the specifically stated number given in the context in which it is given. In one manner, about can refer to ±5%, ±2.5%, or ±1% of the number it refers to.

[0211] In the corresponding English specification, the terms “a,” “an,” “the,” and the same indicator words in the context of the description of the ingredients are interpreted as, particularly in the context of the claims, including both single and multiple of these unless otherwise defined or negated by the context.

[0212] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All methods and materials similar to or equivalent to those described in this specification can also be used in the implementation or testing of this invention, and representative illustrative methods and materials are described herein.

[0213] It should be understood that, when a range of values ​​is provided, the individual values ​​between the upper and lower limits of that range (unless the context explicitly indicates otherwise, up to one-tenth of the unit of the lower limit) and any other recorded values ​​within or falling within that recorded range are included in the scope of this invention. The upper and lower limits of their smaller ranges can be independently included in the smaller range, and if they are also included in the scope of this invention as objects of all specifically excluded limits, the range excluding any one or both of the limits they encompass is also included in this invention.

[0214] This invention is not limited to the specific manner described (such manner can, of course, be varied). It should be understood that the scope of this invention is defined only by the appended claims, and therefore the terminology and methods used in this specification are for the purpose of describing particular manners only and are not intended to be limiting.

[0215] In one aspect of the invention, a method for producing a highly unsaturated fatty acid ester composition from an oil composition includes: feeding the oil composition to a column chromatogram using a first column before performing a first column chromatography to confirm the retention time of the peak of the target analyte.

[0216] In one aspect of the invention, at least one component whose retention time at the peak is slower than that of the target analyte, i.e., highly unsaturated fatty acids, is removed in a first column chromatography, either in whole or in part.

[0217] "Retention time" refers to the time a component spends inside the column after being injected. Typically, retention time indicates the time it takes for a peak to appear in the chromatogram.

[0218] In this specification, "retention time of the peak of the target substance, i.e., highly unsaturated fatty acid" refers to the retention time of the peak of the target substance as shown in the chromatograms of the first and second chromatograms.

[0219] In one aspect of the invention, a “relative retention time (RRT)” can be calculated based on the retention time measurement results (which can be used interchangeably with “relative value” and “relative value of retention time”). “Relative retention time” indicates the relationship between the retention time of a component and the retention time of the target analyte.

[0220] In this specification, "relative value of peak retention time" refers to the value obtained by dividing the retention time of the peak of components other than the target analyte by the retention time of the target analyte in the chromatograms of the first and second chromatograms when the retention time of the peak of the target analyte is set to 1.

[0221] [Number 1] Eluent with a "relative retention time" of 1 or higher refers to eluent recovered after the time when the "relative retention time" reaches that value. Eluent with a "relative retention time" of 2 or lower refers to eluent recovered before the time when the "relative retention time" reaches that value.

[0222] In one aspect of the invention, the retention time of the peak can be determined by measuring it using a UV detector.

[0223] In one aspect of the invention, the retention time of the peak can be determined by measuring each fraction using gas chromatography-based fatty acid analysis.

[0224] In this specification, "single-column chromatography" refers to the process of passing a batch of an oil and fat composition through one or more columns to obtain a highly unsaturated fatty acid ester composition containing the target analyte at a specified concentration. In single-column chromatography, a batch of the oil and fat composition can be passed through one or two columns. In the case where a batch of the oil and fat composition is passed through two columns in single-column chromatography, the two columns are referred to as the first column and the second column, and are connected in series. In the case of using two columns in single-column chromatography, a portion of the eluent from the first column is discharged without being injected into the second column. In one aspect of the invention, the first column and the second column can be connected, allowing the eluent from the first column to be injected into the second column. In one aspect of the invention, "eluent from the first column being injected into the second column" means that the eluent from the first column can be injected into the second column. In one aspect, the eluent can be directly supplied to the second column without concentration or other treatment. In one aspect of the invention, "connectivity" means that the first column and the second column can be switched from a connected state to a disconnected state, or vice versa.

[0225] In one embodiment of the invention, "discharging the eluent from the first column" means discharging the eluent from the first column as unwanted material instead of injecting it into the second column. In another embodiment, when the relative retention time is 3.5 or more, the first column is not connected to the second column, thereby discharging the eluent from the first column. In yet another embodiment, the eluent discharged from the first column can be injected into a third column.

[0226] In one aspect of the invention, the "loading interval from the start of one column chromatography to the point where a second column chromatography can begin" refers to the time from the moment the oil composition is loaded once onto the first or second column in column chromatography using one or more columns until the moment the loading of the oil composition onto the same column can begin again (e.g., a second loading). In this case, the reloading of the oil composition onto the same column begins with a time interval allowed, such that the elution of the target substance in the reloaded oil composition begins after the elution of the last component in the first column chromatography has ended. In one aspect of the invention, the "loading interval from the start of one column chromatography to the point where a second column chromatography can begin" is calculated or determined as the time from the moment the elution of the target substance begins in the first or second column to the moment the elution of the last component ends. In one aspect of the invention, the "loading interval from the start of one column chromatography to the point where a second column chromatography can begin" is calculated as the sum of the time from the moment the oil composition is supplied to the first column chromatography to the moment the elution of the target substance ends in the first column chromatography and the time from the moment the elution of the target substance begins in the second column chromatography to the moment the last component is discharged from the second column. In one aspect of the invention, a first column and a second column are used, and the final component is eluted from either the first column or the second column. In one embodiment of the invention, the "loading interval from the start of one column chromatography to the point where a second column chromatography can begin" is equal to the time from the start of elution of the target compound in the first column chromatography to the point where the final component is discharged from the first column. In another embodiment of the invention, the "loading interval from the start of one column chromatography to the point where a second column chromatography can begin" is equal to the sum of the time from the moment the oil composition is fed to the first column chromatography to the point where the target compound is eluted from the first column and the time from the start of elution of the target compound in the second column chromatography to the point where the final component is discharged from the second column; or the time from the start of elution of the target compound in the second column chromatography to the point where the final component is discharged from the second column. In one embodiment of the invention, the time from the start of elution of the target compound in the first column chromatography to the point where the final component is discharged from the first column is shorter than the time from the start of elution of the target compound to the point where the final component is discharged in column chromatography using a single column of length equal to the combined lengths of the first and second columns. In one aspect of the invention, the time from the moment the oil composition is supplied to the first column chromatography to the end of elution of the target compound from the first column, and the total time from the start of elution of the target compound in the first column chromatography to the end of discharge of the last component from the first column, or the time from the start of elution of the target compound in the second column chromatography to the end of discharge of the last component from the second column, is shorter than the time from the start of elution of the target compound to the end of discharge of the last component in column chromatography based on a single column of length equal to the combined length of the first and second columns.Furthermore, in another aspect of the invention, in column chromatography based solely on the second column, the time from the start of elution of the target compound to the end of elution of the final component in the first column is shorter than the time from the start of elution of the target compound to the end of elution of the final component in the second column. Additionally, in another aspect of the invention, the total time from the moment the oil composition is supplied to the first column chromatograph to the end of elution of the target compound from the first column, plus the time from the start of elution of the target compound to the end of elution of the final component from the second column, is shorter than the time from the start of elution of the target compound to the end of elution of the final component in the second column in column chromatography based solely on the second column.

[0227] In this specification, "a single column of length equal to the combined length of the first and second columns" refers, for example, to a single column having the same inner diameter and stationary phase as the first and / or second columns. In one embodiment, a single column of length equal to the combined length of the first and second columns refers to a single column having the same inner diameter and stationary phase as the second column, and whose length in the flow path direction is equal to the combined length of the first and second columns.

[0228] In one aspect of the invention, reversed-phase column chromatography is suitable as column chromatography. As the stationary phase, any reversed-phase partitioning adsorbent can be used without particular specification, such as DVB (divinylbenzene) and polymer beads such as networked polystyrene, or C4, C8, or C18 alkyl-bonded silica gel, particularly ODS columns using octadecylsilane (ODS). Each column can contain the same or different adsorbents. Typically, the first and second columns can contain the same or different adsorbents. In one aspect of the invention, the first and second columns contain the same adsorbent. In one aspect of the invention, when performing third-column chromatography, the third column can use the same or different adsorbents as the first or second columns, respectively.

[0229] 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 field of chromatography.

[0230] In one approach, a chiral stationary phase is used. The choice of stationary phase is obvious to those skilled in the art and can depend on the component to be purified by chromatography. Nonpolar components, such as highly unsaturated fatty acid esters, may require a reversed-phase stationary phase, such as C18 (ODS). Various types of octadecyl-bonded silica (ODS) can be used, including fully capped, partially capped, and base-deactivated substances. More polar components may require a normal-phase stationary phase such as unbonded silica, amino phase, or cyano phase.

[0231] In one manner, the stationary phase comprises having 1 μ m or more, 20 μ m or more, 40 μ m or more, 60 μ m or above, 80 μ m or more, 100 μ m or more, 1000 μ m or more, 2000 μ m or more, 3000 μ m or more, 4000 μ m or more, or 20 μ Below m, 40 μ Below m, 60 μ Below m, 80 μ Below m, 100 μ Below m, 1000 μ Below m, 2000 μ Below m, 3000 μ Below m, 4000 μ Below m, 5000 μ Below m, or about 1 μ m ~ approximately 20 μ m, approximately 20 μ m ~ approximately 40 μ m, approximately 40 μ m ~ Approximately 60 μ m, approximately 60 μ m ~ Approximately 80 μ m, approximately 80 μ m ~ approximately 1000 μ m, approximately 1000 μ m ~ approximately 2000 μ m, approximately 2000 μ m ~ approximately 3000 μ m, approximately 3000 μ m ~ approximately 4000 μ m, approximately 4000 μ m ~ approximately 5000 μ The median value of the particle size m. In one embodiment, the stationary phase comprises particles with a particle size greater than 50.μ m, greater than 45 μ m, greater than 40 μ m, greater than 35 μ m, greater than 30 μ m, greater than 25 μ m, greater than 20 μ m, greater than 15 μ m, or greater than 10 μ m, and / or less than 5000 μ m, less than 4000 μ m, less than 3000 μ m, less than 2000 μ m, less than 1000 μ m, less than 500 μ m, less than 100 μ m, less than 80 μ m, less than 60 μ m, or 10 μ m~5000 μ m, 15 μ m~4000 μ m, 20 μ m~3000 μ m, 25 μ m~2000 μ m, 30 μ m~1000 μ m, 35 μ m~500 μ m, 40 μ m~100 μ m, 45 μ m~80 μ m, 50 μ m~60 μ The particle with a central value of m in diameter. The central value of the particle size, as understood by those skilled in the art, can be determined by laser diffraction scattering.

[0232] In one aspect of the invention, the stationary phases in the first and second columns can be the same or different. When using different stationary phases, for example, polystyrene networked with DVB (divinylbenzene) or silica gel bonded with C8 alkyl or octadecylsilyl (ODS) can be used in the first column, and a stationary phase selected from polystyrene networked with DVB (divinylbenzene) or silica gel bonded with alkyl or octadecylsilyl (ODS), different from the stationary phase of the first column, can be used in the second column. In one aspect of the invention, when performing third column chromatography, the third column can use the same or different stationary phases from the first or second column, respectively.

[0233] Furthermore, a stationary phase containing particles with the same or different center values ​​can be used in both the first and second columns. When using a stationary phase containing particles with different center values, for example, in the first column, a stationary phase containing particles with a center value of 1... μ m or more, 20 μ m or more, 40 μ m or more, 60 μ m or above, 80 μ m or more, 1000 μ m or more, 2000 μ m or more, 3000 μ m or more, 4000 μ m or more, or 20 μ Below m, 40 μ Below m, 60 μ Below m, 80 μ Below m, 1000 μ Below m, 2000 μ Below m, 3000 μ Below m, 4000 μ Below m, 5000 μ Less than m, or about 1 μ m ~ approximately 20 μ m, approximately 20 μ m ~ approximately 40 μ m, approximately 40 μ m ~ Approximately 60 μ m, approximately 60 μ m ~ Approximately 80 μ m, approximately 80 μ m ~ approximately 1000 μ m, approximately 1000 μ m ~ approximately 2000 μ m, approximately 2000 μ m ~ approximately 3000 μ m, approximately 3000 μ m ~ approximately 4000 μ m, approximately 4000 μ m ~ approximately 5000 μ In the second column, a stationary phase containing particles with a central value of a particle size of m can be used, which can include particles with a particle size selected from 1 μ m or more, 20 μ m or more, 40 μ m or more, 60 μ m or above, 80 μ m or more, 1000 μ m or more, 2000 μ m or more, 3000 μ m or more, 4000 μ m or more, or 20μ Below m, 40 μ Below m, 60 μ Below m, 80 μ Below m, 1000 μ Below m, 2000 μ Below m, 3000 μ Below m, 4000 μ Below m, 5000 μ Less than m, or about 1 μ m ~ approximately 20 μ m, approximately 20 μ m ~ approximately 40 μ m, approximately 40 μ m ~ Approximately 60 μ m, approximately 60 μ m ~ Approximately 80 μ m, approximately 80 μ m ~ approximately 1000 μ m, approximately 1000 μ m ~ approximately 2000 μ m, approximately 2000 μ m ~ approximately 3000 μ m, approximately 3000 μ m ~ approximately 4000 μ m, approximately 4000 μ m ~ approximately 5000 μ A stationary phase different from the stationary phase of the first column, consisting of particles with a central value of particle size m. In one embodiment of the invention, when performing third-column chromatography, the third column can use a stationary phase comprising particles having a central value of particle size that is the same as or different from that of the first or second column.

[0234] In one embodiment of the invention, the amount of stationary phase packed is 1–1000 kg, 10–900 kg, 20–800 kg, 30–700 kg, 40–600 kg, or 50–500 kg by weight. In another embodiment, the amounts of stationary phase in the first column and the second column can be the same or different. When the amounts of stationary phase packed are different, the amount of stationary phase in the first column is, for example, 1–100 kg, 10–100 kg, or 30–100 kg. Similarly, the amount of stationary phase in the second column is, for example, 1–1000 kg, 10–800 g, or 50–800 g. The ratio of the amounts of stationary phase in the first and second columns is 0.5–10, 1–7, 1.1–5, or 2–4.5 for the second column when the first column is set to 1. In one embodiment of the invention, when performing third column chromatography, the third column can be packed with the same or different amounts of stationary phase as the first or second column.

[0235] In one embodiment of the invention, the amount of stationary phase filling the second column is greater than the amount of stationary phase filling the first column. The amount of stationary phase filling 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 less than 4 times, less than 4.5 times, less than 5 times, less than 6 times, less than 7 times, or less than 10 times relative to the amount of stationary phase filling the first column.

[0236] In one embodiment of the invention, column chromatography includes elution using a mobile phase comprising one or more of water, an organic solvent, or supercritical carbon dioxide. In a subset of these methods, the mobile phase comprises methanol. In a subset of these methods, chromatography includes a mobile phase comprising one or more additives selected from formic acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide.

[0237] In one embodiment of the 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, isopropanol, n-butanol, isobutanol, sec-butanol, and tert-butanol. Methanol and ethanol are preferred. Methanol is more preferred. Examples of ethers include diethyl ether, diisopropyl ether, and methyl tert-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). Examples of nitriles include acetonitrile.

[0238] In one aspect of the invention, the mobile phase in the first and second column chromatography can be the same or different, but preferably the same. When using different mobile phases, for example, methanol, ethanol, diethyl ether, diisopropyl ether, methyl tert-butyl ether, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone (MIBK), acetonitrile, hexane, and dichloromethane can be used in the first column, while a mobile phase selected from methanol, ethanol, diethyl ether, diisopropyl ether, methyl tert-butyl ether, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone (MIBK), acetonitrile, hexane, and dichloromethane, different from the mobile phase in the first column, can be used in the second column. In one aspect of the invention, when performing third column chromatography, the third column can use a mobile phase that is the same as or different from that used in the first or second column, respectively.

[0239] The mobile phase may further contain additives including buffers and pH adjusters. The selection of additives can 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 one or more additives selected from formic acid, trifluoroacetic acid, heptafluorobutyric acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide. In some embodiments, the mobile phase may not contain additives.

[0240] In one embodiment of the invention, column chromatography includes a mobile phase gradient. In another embodiment of the invention, column chromatography comprises the purification of one or more highly unsaturated fatty acid esters. In another embodiment of the invention, the highly unsaturated fatty acid ester is selected from docosahexaenoic acid, crotonic acid, myristone acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, transoleic acid, cis-11-octadecenoic acid, stigma, eicosaenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, linolenic acid, pinolenic acid, tungsten acid, 5,8,11-eicosatrienoic acid (MEAD acid), and dihydroxy-2- ... μ -Linolenic acid, eicosatrienoic acid, octadecanoic acid, arachidonic acid, eicosatrienoic acid, adrenal acid, bosseopentaenoic acid, eicosapentaenoic acid, cis-4,7,10,13,16-octadecanoic acid, sardine acid, tetracosapentaenoic acid, 4,8,12,15,18,21-octadecanoic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanic acid, stearic acid, nonadecanic acid, arachidic acid, peicosanoic acid, behenic acid, tridecanoic acid, lignoceric acid, pentadecanoic acid, ceric acid, carboceric acid An ester of one or more highly unsaturated fatty acids selected from the following: linaloic acid, nonacosylicacid, beeswax acid, heptanoic acid, laccaeroic acid, psyllic acid, geddic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonatriacontylic acid, and tetracontylic acid. In one embodiment 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 includes the purification of ethyl eicosapentaenoic acid.

[0241] 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, 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%, and 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%, less than 85%, less than 90%, or less than 95%. In some methods, the recovery rate is 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 99.7% or higher, 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%.

[0242] The mobile phase can contain one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexane, dichloromethane, supercritical carbon dioxide, or any other solvent known in this technique. The selection of the mobile phase may require consideration of the highly unsaturated fatty acid esters to be purified and the stationary phase used. For reversed-phase stationary phases targeting nonpolar highly unsaturated fatty acid esters, a polar mobile phase should be selected that is sufficient for eluting the desired highly unsaturated fatty acid esters, but whose elution rate is not excessive as close to the solvent front as possible.

[0243] In one approach, a solvent gradient can be used as the mobile phase during elution. The main purpose of gradient elution is to produce sufficiently separated peaks for the eluted analytes upon detection, eluting strongly retained analytes more quickly and weakly retained analytes more slowly. For example, in reversed-phase chromatography, starting the eluent with a low concentration of a nonpolar solvent allows for the separation of weakly retained analytes. Strongly retained analytes may remain on the adsorbent surface at the top of the column or migrate very slowly. Increasing the amount of a nonpolar component (e.g., acetonitrile) in the eluent steadily increases competition for the adsorption site with the nonpolar solvent, thus allowing the strongly retained components to migrate more quickly.

[0244] Therefore, in reversed-phase chromatography for nonpolar highly unsaturated fatty acid esters, the solvent at the start of elution in the chromatography can contain a high percentage of polar solvent A, such as 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 nonpolar solvent compared to solvent A, such as methanol (if solvent A is water). Solvent B should constitute the remaining percentage of the mobile phase. When the column is run, as the solvent elutes through the stationary phase and the column, the gradient result causes the concentration of solvent B to increase over time. In some embodiments, a single-component solvent can also be used as the mobile phase. In some embodiments, the single-component solvent can be used alone or in combination with one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexane, dichloromethane, supercritical carbon dioxide, or any other solvent known in this art.

[0245] In one mode, the increase rate of solvent B over time can be constant. In another mode, there is no gradient, and the mobile phase has a constant composition during elution. In another mode, it is possible to utilize the different increases in the percentage of solvent B within different time ranges of the chromatography. In yet another mode, the mobile phase can have a constant composition within a specific time range of the chromatography and contain a gradient within other time ranges.

[0246] In one approach, the first and second column chromatograms may both be of constant composition, or both may contain gradients, or both may be of constant composition within a specific time range and contain gradients within other time ranges. In another approach, the first column chromatogram may be of constant composition, and the second column chromatogram may contain gradients; or the first column chromatogram may contain gradients, and the first column chromatogram may be of constant composition. In another approach, when a third column chromatogram is included, the third column chromatogram may be of constant composition or contain gradients.

[0247] Multiple solvents used in the mobile phase can be stored separately in the mobile phase supply section and mixed using a pump before elution through the column. The mobile phase supply section includes a mobile phase source and a solvent delivery system. This solvent delivery system is a pump device, such as a commercially available column chromatography pump, which supplies solvent or mobile phase to the column. Such pumps typically provide pulsation-free flow, flow rates ranging from 0.1 to 100 L / min, accurate flow rate control, high pressure generation (below 6000 psi), and corrosion and solvent resistance. Reciprocating pumps contain a small chamber in which the solvent is pumped in by the back-and-forth movement of a piston driven by a motor. Two check valves, opening and closing alternately, control the direction and flow of the solvent in and out of the cylinder. Single-piston pumps, using specially designed cams, can achieve very rapid replenishment times, producing more continuous flow. The disadvantages of pulsation flow caused by reciprocating pumps are often overcome by using pulse dampers. The use of dual-piston pumps, with pistons moving in different phases, provides a reasonable solution for pulsation-free fluid delivery. The linear velocity in the column represents the velocity of the fluid through the column cross-section. Linear velocity (linear velocity (m / hour) = flow rate (m³ / h) 3 / hour) / Column cross-sectional area (m²) 2 The linear velocity can be approximately 0.2–20.0 m / h, approximately 1.0–15.0 m / h, approximately 1.0–10.0 m / h, approximately 1.5–10.0 m / h, or approximately 2.0–9.0 m / h. In one configuration, the linear velocity is approximately 4.0–9.0 m / h.

[0248] In one embodiment of the invention, the linear velocities of the first and second columns can be the same or different. When the linear velocities are different, the linear velocity in the first column can be faster or slower than that in the second column. When the first column is set to 1, the ratio of the linear velocities in the first and second columns in the second column is 0.5–2.0, 0.5–1.5, or 1–1.5. In one embodiment of the invention, the linear velocities in the first and second columns are the same. In one embodiment of the invention, when performing third column chromatography, the third column can be operated at the same or different linear velocities as the first or second column, respectively.

[0249] In one aspect of the invention, column chromatography is performed at room temperature or above room temperature. Preferably, the method is performed at a temperature above room temperature. The first and second column chromatography can be performed at the same temperature or at different temperatures, preferably at the same temperature.

[0250] In one aspect of the present invention, the temperature above room temperature is 20°C or above, 25°C or above, 30°C or above, 35°C or below, or 60°C or below, 55°C or below, 50°C or below, 45°C or below, or 20-60°C, 25-55°C, 30-50°C or 35-45°C.

[0251] In one aspect of the invention, column chromatography can use two or more columns. Column chromatography can be performed using known fixed-bed chromatography apparatus. Such column chromatography is referred to as fixed-bed chromatography. In one aspect of the invention, the first and second column chromatography are fixed-bed chromatography.

[0252] In some methods, a detector is used to monitor the mobile phase eluted from the column for the presence of one or more components. Detection methods known in this technique (e.g., mass spectrometry (MS), UV / Vis absorbance, fluorescence, refractive index, or conductivity) can be used.

[0253] In other methods, any of the various standard column chromatography detectors can be used to detect eluents that have just been eluted from the column.

[0254] In other methods, the presence of components can be monitored at each fraction separately through analysis. In some methods, the analysis is performed using gas chromatography for fatty acid analysis.

[0255] In some methods, the eluent from the column is detected as peaks 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 eluent in the oil composition. "Retention time" is the time required for the eluent to pass through the column, determined from the moment the oil composition is injected (or loaded) until the moment of elution. Ideally, each eluent of the target compound should have a characteristic retention time. However, the retention of the eluent varies with variations in the eluent, stationary phase, temperature, and column chromatography settings. Therefore, the retention time of the eluent is compared with the retention times of more than one standard compound under the same conditions. A suitable detector exhibits good sensitivity, good stability, reproducibility, linear response across multiple orders of magnitude 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.

[0256] A UV / Vis absorbance detector can be used with a scanning spectrophotometer that includes a grating optics system. The use of a deuterium source (UV range, 190–360 nm) and a tungsten source (visible range, 360–800 nm), either independently or in combination, provides a simple means of detecting absorbant species as they leave the column.

[0257] A photodiode array (PDA) device is a UV / Vis absorbance detector capable of rapidly collecting data within a selected spectral range. It can collect and store absorbance spectral data associated with each chromatographic peak. The stored data can be compared with pure standard spectra from a library. PDA detectors are useful in the identification of difficult-to-separate components (repeating peaks) because the characteristic spectra associated with the unseparated components are highly likely to differ from those of the individual unseparated components.

[0258] Fluorescence detectors are useful in the detection of analytical species 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 typically observed by detecting emission radiation separated by a grating at an angle of 90 degrees relative to the excitation beam. The number of fluorescent species can be enhanced by post-column derivatization (PCD) of eluted compounds using special reagents (or pre-column derivatization of the sample itself).

[0259] The refractive index (RI) detector responds to almost all solutes. The difference in refractive index between the reference mobile phase and the column effluent results in the detection of components that separate as peaks on the chromatogram. Due to its extremely high sensitivity to the mobile phase, this detector cannot be used in an LC pump without sufficient pulse attenuation, and is also unsuitable for gradient applications due to varying mobile phase compositions. The detection limit is typically lower than that observed by an absorbance detector.

[0260] The conductivity detector provides high-sensitivity detection of all charged substances. This detector can easily and reliably detect anions, cations, metals, organic acids, and surfactants down to the ppb level, and therefore can be used with LC systems. The addition of a chemical inhibitor between the column and the conductivity detector reduces the conductivity of the eluent, enabling gradient elution and determination of ppb levels with minimal baseline drift. To typically determine low levels of anions, the eluent is converted to a low-conductivity acid with weak ionization (e.g., from Na₂CO₃ to carbonic acid), thereby reducing background noise. Simultaneously, the anions of the analyte species are converted to their corresponding high-conductivity acids (e.g., from NaCl to HCl), relatively increasing the analyte signal.

[0261] In one aspect of the invention, the purity of the highly unsaturated fatty acid esters contained in the highly unsaturated fatty acid ester composition obtained in 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, relative to the total highly unsaturated fatty acid ester composition.

[0262] The retention time of the target object can be approximately 0.5 minutes to approximately 2 minutes, approximately 2 minutes to approximately 4 minutes, approximately 4 minutes to approximately 6 minutes, approximately 6 minutes to approximately 8 minutes, approximately 8 minutes to approximately 10 minutes, approximately 10 minutes to approximately 12 minutes, approximately 12 minutes to approximately 14 minutes, approximately 14 minutes to approximately 16 minutes, approximately 16 minutes to approximately 18 minutes, approximately 18 minutes to approximately 20 minutes, approximately 20 minutes to approximately 22 minutes, approximately 22 minutes to approximately 24 minutes, and approximately 24 minutes to approximately 26 minutes. Approximately 26 to 28 minutes, approximately 28 to 30 minutes, approximately 30 to 32 minutes, approximately 32 to 34 minutes, approximately 34 to 36 minutes, approximately 36 to 38 minutes or approximately 38 to 40 minutes, or more than approximately 0.5 minutes, more than approximately 1 minute, more than approximately 1.5 minutes, more than approximately 2 minutes, or more than approximately 5 minutes, and / or less than 50 minutes, less than 40 minutes, less than 30 minutes or less than 20 minutes.

[0263] In one embodiment of the invention, the retention time of the peak of the target analyte in the first column chromatogram is shorter than the retention time of the peak of the target analyte in the second column chromatogram. The retention time of the peak of the target analyte in the first column chromatogram can 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, 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 times of the target analyte peak in the second column chromatography can be approximately 6 to 8 minutes, 8 to 10 minutes, 10 to 12 minutes, 12 to 14 minutes, 14 to 16 minutes, 16 to 18 minutes, 18 to 20 minutes, 20 to 22 minutes, 22 to 24 minutes, 24 to 26 minutes, 26 to 28 minutes, 28 to 30 minutes, and 30 to 32 minutes. minutes, approximately 32 to 34 minutes, approximately 34 to 36 minutes, approximately 36 to 38 minutes or approximately 38 to 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, less than 8 minutes.

[0264] In one aspect of the invention, an internal standard can be used during analysis using gas chromatography (GC). To determine the relative retention time of the analyte relative to the internal standard or to aid in the quantification of the analyte, the internal standard can be added to the sample as a reference marker. The internal standard can be suitably selected by those skilled in the art as a compound that is very similar to but not identical to the target analyte, such as a deuterated derivative of the target analyte. For quantification purposes, the internal standard can be calibrated 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 for reference in quantification of unknown analyte samples.

[0265] In one embodiment, the highly unsaturated fatty acid ester composition comprises docosahexaenoic acid, crotonic acid, myristone acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, transoleic acid, cis-11-octadecenoic acid, codenoic acid, eicosaenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, tungsten acid, 5,8,11-eicosatotrienoic acid, and dihydroxy- μ -Linolenic acid, eicosatrienoic acid, octadecanoic acid, arachidonic acid, eicosatrienoic acid, adrenaline, borseopentanoic acid, eicosapentanoic acid, cis-4,7,10,13,16-docosahexaenoic acid, sardine acid, docosapentanoic acid, 4,8,12,15,18,21-docosahexaenoic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid Myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, dodecanoic acid, behenic acid, tridecanoic acid, limonic acid, pentadecanoic acid, ceric acid, heptadecanoic acid, limonic acid, nonadecanoic acid, beeswax acid, tridecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, tetradecanoic acid, or esters thereof, or containing them.

[0266] In one embodiment, the highly unsaturated fatty acid ester composition may have a purity 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 another embodiment, the highly unsaturated fatty acid ester composition may have a purity less than about 100%, less than about 99.9999%, less than about 99.5%, less than about 99%, less than about 98.5%, or less than about 98%. In one embodiment, the highly unsaturated fatty acid ester composition can have a purity of 70–98%, 80–98%, 85–98.5%, 86–98.5%, 90–98.5%, 95–99%, 96.5–99%, 98–99.5%, 99–99.5%, 99.5–99.9999%, 99.8–99.9999%, or 99.8–100%. In another embodiment, the highly unsaturated fatty acid ester composition can be obtained at a yield greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%. In yet another embodiment, the highly unsaturated fatty acid ester composition can have a yield less than 100%, less than 95%, less than 90%, or less than 80%. In one embodiment, 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 another embodiment, the highly unsaturated fatty acid ester composition may have a recovery rate 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 yet another embodiment, the highly unsaturated fatty acid ester composition may have a recovery rate less than 100% or less than 99.9999%. In one manner, the highly unsaturated fatty acid ester composition may have a purity of 55–99.9999%, 60–99.9999%, 65–99.9999%, 70–99.9999%, 75–99.9999%, 80–99.9999%, 85–99.9999%, 90–99.9999%, 95–99.9999%, 98–99.9999%, 99–100%, or 99.7–100%.

[0267] In one embodiment of the invention, the highly unsaturated fatty acid ester composition obtained in the second column chromatography contains EPA esters, such as EPA ethyl ester, with a purity of 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, relative to the total highly unsaturated fatty acid ester composition.

[0268] In one embodiment of the invention, the highly unsaturated fatty acid ester composition obtained in the second column chromatography contains DGLA esters, such as DGLA ethyl ester, with a purity of 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, relative to the total highly unsaturated fatty acid ester composition.

[0269] In one embodiment of the invention, the highly unsaturated fatty acid ester composition obtained in the second column chromatography contains highly unsaturated fatty acid esters such as ARA esters, for example, ARA ethyl ester, which have high purity and are 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, relative to the total highly unsaturated fatty acid ester composition.

[0270] In one aspect of the invention, the highly unsaturated fatty acid ester composition obtained in the second column chromatography contains at least one fatty acid ester selected from esters of C18:0, C20:0, C20:1, and C22:0 in an amount of less than 0.6% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.05% by weight, less than 0.03% by weight, or less than 0.01% by weight relative to the total highly unsaturated fatty acid ester composition.

[0271] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments in any way.

[0272] Example [Example 1] In this experiment, the shortened loading interval was verified using a column model by comparing a single tandem column with a double tandem column. A sample containing EPA-EE, C20:0 ethyl ester, and C20:1 ethyl ester was prepared from crude refined sardine oil (preparation method was the same as in Example 2 below). The single tandem column used a column consisting of columns with inner diameters of 20 mm, lengths of 300 mm, 500 mm, and 500 mm connected in series. The double tandem column used a first column with an inner diameter of 20 mm and a length of 300 mm, and a second column consisting of two columns with an inner diameter of 20 mm and a length of 500 mm connected in series. The column temperature was set to 40°C. The flow rate for the single tandem column was 37 mL / min. In the double tandem column, the flow rate for the first column was 32 mL / min, and the flow rate for the second column was 37 mL / min. The stationary phase used was Daisopak SP-120-50-ODS-B (Osaka Soda Co., Ltd.). The mobile phase was HPLC-grade methanol (Kanto Chemical Co., Ltd.). Detection was performed using UV at 230 nm.

[0273] In a single tandem column, EPA-EE began elution 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.

[0274] In addition, in the two-column tandem column configuration, the EPA-EE elution in the first column ends 3.5 minutes after the start of column chromatography. In the second column, the EPA-EE elution begins 10.5 minutes after the start of column chromatography, and the elution of C20:1 ethyl ester ends 12 minutes after the start of EPA-EE elution.

[0275] In a single tandem column, the fatty acid furthest from EPA-EE is C20:0 ethyl ester. However, in a two-column tandem column, when the eluent from the first column is recovered up to the end of EPA-EE elution (3.5 minutes) 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 a single tandem column, C20:0 ethyl ester elution ends 24 minutes after the start of EPA-EE elution; therefore, by setting the loading interval to 24 minutes, the last C20:0 ethyl ester eluted in the previous loading will not be contaminated in the EPA-EE fraction detected in the next loading.

[0276] On the other hand, in a two-column tandem column, the elution of C20:1 ethyl ester ends 12 minutes after the start of EPA-EE elution in the second column. Therefore, if the loading interval is every 12 minutes, the last C20:1 ethyl ester eluted in the previous loading will not mix into the EPA-EE fraction eluted in the next loading.

[0277] That is, it can be seen that the loading interval in a single column is reduced by half from 24 minutes to 12 minutes by introducing two tandem columns, which is an effective way to reduce the amount of solvent used.

[0278] [Example 2] The column chromatography conditions are as follows. The HPLC equipment used is as follows: The pump was an NP-KX500 (Nippon Seiko Scientific Co., Ltd.), the detector was an S-3702 (Soma Optical Co., Ltd.), and the column oven was a CO705 (GLSciences Co., Ltd.). The ODS packing material used was Daisopak 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 for the first column was 32 mL / min, and the flow rate for the second column was 37 mL / min. The solvent was HPLC-grade methanol (Kanto Chemical Co., Ltd.). Detection was performed using UV at 230 nm. 2.4 g of EPA-EE (80%) was loaded.

[0279] EPA-EE (80%) was prepared as follows: Short-path distillation (SPD) was performed on crude refined sardine oil. For the SPD-treated oil, an alcoholysis reaction with ethanol was carried out in the presence of an alkaline catalyst to form ethyl esters of fish oil, i.e., EPA-EE. The ethyl esters of fish oil were then precisely distilled to prepare 80% EPA-EE.

[0280] First, a fractionation test was performed on the first column. 2.4 g of EPA-EE (80%) was loaded into the first column. Fractionation was initiated shortly after injection of fraction (Fr.0) until the UV index rose. Fractions Fr.1–Fr.20 were then fractionated every 10 seconds, followed by fractionation of Fr.29 for 30 minutes. Methanol was removed from the fractions using a vacuum evaporator. After adding a 1 mg / mL hexane solution of C23:0 methyl ester as an internal standard, analysis was performed using gas chromatography (GC). The elution volume of each fraction was calculated from the ratio of the GC peak area to the internal standard area. The elution of EPA-EE (93%) started at 3 minutes 30 seconds and ended at 4 minutes 20 seconds. The composition of similar substances at this point was determined. Therefore, the elution range from 3 minutes 30 seconds to 4 minutes 20 seconds from the start of column chromatography was determined for transfer from the first column to the second column.

[0281] Next, after loading 2.4 g onto the first column, the elution range (3 min 30 sec to 4 min 20 sec) with EPA-EE ≥ 93% was transferred to the second column. The period until the UV concentration in the second column increased was designated as Fr.0. Fr.1–38 were fractionated every 10 sec, followed by fractionation at Fr.39 for 35 minutes. Similar to the fractionation experiment on the first column, GC analysis was performed to determine the composition of the similar material at EPA-EE ≥ 97%.

[0282] The conditions used for GC analysis are as follows. The equipment is a DB-WAX 30m × 0.25mm × 0.25mm. μ A 7890A networked GC system (Agilent) with m columns. Column temperature: 210°C. Injection temperature: 250°C. Split rate: 1:50. Injection volume: 1... μ L. Use an FID detector at 250°C. Use helium carrier gas with a linear velocity of 31 cm / min.

[0283] [Table 1] [Example 3] The conditions for column chromatography performed for DGLA concentration are as follows.

[0284] The column is used in the LC-600 type axial compression column (Kurita Kogyo Co., Ltd.) with an inner diameter of 600mm and a filling pressure of 3.5MPa. The first column is filled with 50mm particle size. μ A column was obtained by filling 75 kg of ODS packing material (SP-120-50-ODS-B, Osaka Soda Co., Ltd.) with 50 μm particles in a second column. μ The columns were obtained by using 115 kg of ODS packing material (SP-120-50-ODS-B, Osaka Soda Co., Ltd.). The first column was 50 cm long, and the second column was 80 cm long. Methanol was used as the mobile phase, and the flow rate for both the first and second columns was set to 28 L / min.

[0285] Oil composition 1 (DGLA content, 87.7%) was prepared as follows: microbial oil 1 from Mortierella microorganisms containing 37.2% by weight of DGLA in the fatty acid composition was ethylated using a base catalyst according to conventional methods, and the C20 fraction was concentrated by precision distillation to obtain oil composition 1.

[0286] First, fractionation was performed on the first column. 8.6 kg of oil composition 1 (DGLA content, 87.7%) (4.5% relative to the total weight of the adsorbent material in the first and second columns) was loaded into the first column. Shortly after injection, fractionation (Fr.1) was performed: Fr.1 fractionation for 180 seconds, Fr.2 to Fr.19 fractionation for 30 seconds, and Fr.20 to Fr.33 fractionation for 120 seconds. 1 mL of each fraction was dispensed into Falcon tubes, and 1 mL of methyl tridecanoate 1 mg / mL hexane solution was added using a whole pipette, followed by 5 mL of 5% NaCl aqueous solution. After vigorous stirring and two-layer separation, the upper layer was collected in a GC vial for GC-FID analysis. GC conditions were as follows. The elution amount of each compound in each fraction was calculated from the ratio of the GC peak area to the internal standard area.

[0287] GC Analysis Conditions Apparatus: GC-2025 capillary gas chromatograph, Shimadzu Corporation Column: DB-WAX (30m×0.25mmID, film thickness: 0.25 μ m) Carrier gas: hydrogen, 1 mL / min Flow split ratio: 1:30 Column temperature: 230℃ (30 minutes) Inlet temperature: 250℃ Detector type and temperature: FID, 250℃ Injection volume: 1 μ L As a result, the elution time of the target compound DGLA in the first column started at 4.5 minutes and ended at 30 minutes. By specifically setting the elution time of the target fraction (DGLA content, 92.7%) delivered to the second column to start at 4.5 minutes and end at 9 minutes, the time-consuming C22:0 during elution was suppressed to 0.0%.

[0288] Next, after loading 8.6 kg of oil composition 1 into the first column, the eluent transfer time from the first column to the second column was set to the range determined by the fractionation in the first column (4.5 minutes to 9 minutes). Fractionation of the eluent in the second column began shortly after the oil composition 1 was transferred from the first column to the second column: Fr.1 was fractionated for 360 seconds, Fr.2 to Fr.24 for 30 seconds each, and Fr.25 to Fr.34 for 120 seconds each. Similar to the fractionation experiment in the first column, GC analysis was performed to determine the fatty acid composition of the recovered fraction, ensuring that the DGLA content in the fatty acid composition of the recovered fraction was above 96.5%.

[0289] [Table 2] [Example 4] The conditions for column chromatography performed for ARA concentration are as follows.

[0290] The HPLC column and mobile phase conditions were the same as those used in Example 2.

[0291] Oil composition 2 (ARA content, 79.4%) was prepared as follows: Microbial oil 2 from the genus Mortierella, containing 43.6% by weight of ARA in its fatty acid composition, was esterified using a base catalyst according to conventional methods, and the C20 fraction was concentrated by precision distillation to obtain oil composition 2.

[0292] First, fractionation was performed on the first column. 1.0 kg of oil composition 2 (ARA content, 79.4%) (0.55% of the total adsorbent weight of the first and second columns) was loaded into the first column. Shortly after injection, fraction (Fr.1) was fractionated: Fr.1 fractionated for 361 seconds, Fr.2 to Fr.13 fractionated for 30 seconds, Fr.14 to Fr.16 fractionated for 300 seconds, and Fr.17 to Fr.29 fractionated for 60 seconds. For each fraction, GC analysis was performed using the same method as in Example 2 to determine the elution amount of the compound in each fraction. As a result, the elution start time for the target component, ARA, after the first column was 6 minutes, and the elution end time was 40 minutes. By specifically setting the elution start time for the target fraction (ARA content, 78.9%) delivered to the second column to 6.5 minutes, the elution end time was 12 minutes, and the C22:0 elution time was suppressed to 0.0%.

[0293] Next, after loading 1.0 kg of oil composition 2 into the first column, the transport time from the first column to the second column was set to the range determined by the fractionation in the first column (6.5 minutes to 12 minutes). Fractionation of the eluent in the second column began after the loading of oil composition 2 onto the first column, with Fr.1 fractionated for 1051 seconds, Fr.2 to Fr.31 each fractionated for 10 seconds, and Fr.32 to Fr.33 each fractionated for 600 seconds. GC samples prepared under the same pretreatment as the fractionation test in the first column were analyzed under the following GC conditions to determine the fatty acid composition of the recovered fraction, ensuring that the ARA content in the fatty acid composition of the recovered fraction was 96.5% or higher, and the fatty acid composition of the recovered fraction at this point was calculated.

[0294] GC Analysis Conditions Apparatus: GC-2025 capillary gas chromatograph, Shimadzu Corporation Column: DB-WAX (30m×0.25mmID, film thickness: 0.25μ m) Carrier gas: hydrogen, constant linear velocity control at 31.8 cm / sec Flow split ratio: 1:30 Column temperature: 210℃ (30 minutes) Inlet temperature: 250℃ Detector type and temperature: FID, 250℃ Injection volume: 1 μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ γ γ μ μ μ μ μ μ μ μ L [Table 3]

Claims

1. A method for manufacturing a highly unsaturated fatty acid ester composition from an oil and fat composition, characterized in that, The method includes: (a) By feeding the oil and fat composition into a first column chromatogram using a first column, removing all or part of at least one component whose relative retention time at the peak of the target compound, i.e., the highly unsaturated fatty acid ester, is 1.0 when the retention time of the peak in the first column chromatogram is set to 1.

0. (b) By supplying the eluent containing the target analyte obtained from the first column chromatography to a second column chromatography using a second column, all or a portion of at least one component whose relative retention time at the peak of the target analyte, when the retention time at the peak of the target analyte in the second column chromatography is set to 1.0, is removed; and (c) Concentrate the eluent obtained in the second column chromatography to obtain a composition containing the target analyte; The resulting composition contains more than 95% by weight of the target substance.

2. The method according to claim 1, wherein, The loading interval from the start of one column chromatography to the start of another is shorter compared to column chromatography using a single column of length obtained by combining the first and second columns.

3. The method according to claim 1 or 2, wherein, At least one component whose relative retention time at the peak of the target analyte in the first column chromatography is 1.0 and is other than highly unsaturated fatty acid esters in the oil and fat composition is at least one component.

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

0.

5. The method according to any one of claims 1 to 4, wherein, The first column can be connected to the second column, so that the eluent from the first column can be injected into the second column.

6. The method according to any one of claims 1 to 5, wherein, The first and second column chromatograms used reversed-phase column chromatography.

7. The method according to any one of claims 1 to 6, wherein, The first and second column chromatograms were fixed-bed column chromatograms.

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

9. The method according to any one of claims 1 to 8, wherein, The second column contains more stationary phase than the first column, or the second column has the same inner diameter as the first column but is longer than the first column.

10. The method according to any one of claims 1 to 9, wherein, The target substance is eicosapentaenoic acid (EPA) and dihydrogen phosphate. γ - An ester of either linolenic acid (DGLA) or arachidonic acid (ARA).

11. The method according to any one of claims 1 to 10, wherein, At least one component that is removed in whole or in part in the first column chromatography is an ester of any one of C22:0, C20:0, C18:0 and C20:

1.

12. The method according to 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 more than 96% by weight of the target substance.

14. The method according to any one of claims 1 to 13, wherein, Repeat (a), (b) and (c) more than twice.

15. The method according to any one of claims 1 to 14, wherein, The target compound is an eicosapentaenoic acid ester, in which at least one component, octadecanoic acid, C19:4, or C21:5, is removed in whole or in part during the second column chromatography.

16. The method according to any one of claims 1 to 14, wherein, The target is the second high-speed rail. γ - Linolenic acid ester, at least one component of which is removed in whole or in part in the second column chromatography is a C20:4 n-6 ester.

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

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