Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
  • A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
  • A23D9/02—Other edible oils or fats, e.g. shortenings or cooking oils characterised by the production or working-up
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
  • A23L33/12—Fatty acids or derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
  • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/37—Esters of carboxylic acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/32—Bonded phase chromatography
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B3/00—Refining fats or fatty oils
  • C11B3/10—Refining fats or fatty oils by adsorption
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/26—Conditioning of the fluid carrier; Flow patterns
  • G01N30/38—Flow patterns
  • G01N30/46—Flow patterns using more than one column
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/84—Preparation of the fraction to be distributed
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/86—Signal analysis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

• the present invention relates to a method for producing a composition of polyunsaturated fatty acid (PUFA) esters using column chromatography.
• PUFA polyunsaturated fatty acid
• Polyunsaturated fatty acids are used as medicines and health foods. They are produced from natural raw materials such as vegetable oils and marine oils that are rich in polyunsaturated fatty acids. Natural raw materials also contain other components, such as fatty acids with different numbers of double bonds, for example, saturated fatty acids, monounsaturated fatty acids, and fatty acids with different carbon chain lengths. The content of polyunsaturated fatty acids in natural raw materials is not high. To use polyunsaturated fatty acids as medicines or health foods, it is necessary to selectively separate the desired polyunsaturated fatty acids (Patent Document 1).
• Patent Document 2 Column chromatography is known as a method for separating highly unsaturated fatty acids (Patent Document 2).
• Patent Documents 3 to 7 simulated moving bed chromatography is used as a type of chromatography that allows for continuous operation.
• Simulated moving bed chromatography requires complex systems and equipment, and is more expensive than chromatography using a single column. Therefore, there is still a need for a method for efficiently producing the desired highly unsaturated fatty acid ester composition with high purity without requiring complex equipment such as simulated moving bed chromatography.
• the gist of the present invention is a method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition as described below.
• the first column can be connected to the second column so that an eluate from the first column can be injected into the second column;
• [1-13] The method according to any one of [1-1] to [1-12], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.
• [1-14] The method according to any one of [1-1] to [1-13], wherein the first column chromatography and the second column chromatography use the same mobile phase.
• [1-15] The method according to any one of [1-1] to [1-14], wherein the second column is packed with a larger amount of stationary phase than the first column.
• [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.
• [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 microorganisms.
• [1-18] The method according to any one of [1-1] to [1-17], wherein the target substance is an ester of any one of eicosapentaenoic acid (EPA), dihomo-gamma-linolenic acid (DGLA), and arachidonic acid (ARA).
• EPA eicosapentaenoic acid
• DGLA dihomo-gamma-linolenic acid
• ARA arachidonic acid
• [1-23] The method according to any one of [1-1] to [1-22], wherein the composition obtained in (c) contains the target compound in an amount of 96% by weight or more.
• [1-24] The method according to any one of [1-1] to [1-23], further comprising preparing the oil or fat composition by distillation from a raw material composition.
• [1-25] The method according to any one of [1-1] to [1-24], wherein (a), (b) and (c) are repeated two or more times.
• [1-26] The method according to any one of [1-1] to [1-25], further comprising, before (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target substance.
• [1-27] The method according to any of [1-1] to [1-26], wherein the eluate containing the target substance obtained in the first column chromatography is an eluate obtained up to the end of elution of the target substance from the first column.
• a method for producing an eicosapentaenoic acid ester composition from an oil or fat composition comprising the steps of: (a) subjecting the oil and fat composition to a first column chromatography using a first column to obtain an eluate containing eicosapentaenoic acid esters, the eicosapentaenoic acid esters containing 0.3 wt% or less of C20:0 esters relative to the total fatty acid esters contained in the eluate; (b) subjecting the eluate containing the eicosapentaenoic acid ester obtained by the first column chromatography to a second column chromatography using a second column to obtain an eluate; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing the eicosapentaenoic acid ester,
• the first column can be connected to the second column so that an eluate from the first column can be injected into the second column;
• the method according to any one of [2-1] to [2-6], wherein the eluate from the first column is injected into the second column, and the relative value of the peak top retention time of at least one component other than the eicosapentaenoic acid ester contained in the eluate when the peak top retention time of the eicosapentaenoic acid ester in the second column chromatography is set to 1.0 is within the range of 1.1 or less.
• [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 reverse phase column chromatography.
• [2-14] The method according to any one of [2-1] to [2-13], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.
• [2-15] The method according to any one of [2-1] to [2-14], wherein the first column chromatography and the second column chromatography use the same mobile phase.
• [2-16] The method according to any one of [2-1] to [2-15], wherein the second column is packed with a larger amount of stationary phase than the first column.
• [2-17] The method according to any one of [2-1] to [2-16], wherein the second column has the same inner diameter as the first column and is longer than the first column.
• [2-18] The method according to any one of [2-1] to [2-17], wherein the at least one component that is completely or partially removed by the first column chromatography is any one of C20:0, C18:0, and C20:1 esters.
• [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.
• the ester is an ethyl ester.
• a method for producing a dihomo- ⁇ -linolenic acid ester composition from an oil or fat composition comprising the steps of: (a) subjecting an oil and fat composition to a first column chromatography using a first column to obtain an eluate containing dihomo- ⁇ -linolenic acid esters, the eluate containing 0.5% by weight or less of C22:0 esters relative to the total fatty acid esters contained in the eluate; (b) subjecting the eluate containing the dihomo- ⁇ -linolenic acid ester obtained by the first column chromatography to a second column chromatography using a second column to obtain an eluate; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing the eicosapentaenoic acid ester.
• the resulting composition contains 95.0% by weight or more of dihomo-gamma-linolenic acid esters and at least one component other than dihomo-gamma-linolenic acid esters, and the at least one component other than dihomo-gamma-linolenic acid esters contains 0.60% by weight or less of C20:4n-6 esters.
• [3-3] The method according to [3-1] or [3-2], wherein a loading interval until a second column chromatography can be started after a first column chromatography is started is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns.
• [3-4] The method according to [3-3], in which the loading interval until another column chromatography can be started after one column chromatography is started is shorter than that in the case of column chromatography using only the second column.
• the first column can be connected to the second column so that an eluate from the first column can be injected into the second column;
• the method according to any one of [3-1] to [3-6], wherein the eluate from the first column is injected into a second column, and the relative value of the peak top retention time of at least one component other than dihomo- ⁇ -linolenic acid ester contained in the eluate when the peak top retention time of dihomo- ⁇ -linolenic acid ester in the second column chromatography is set to 1.0 is within the range of 1.1 or less.
• [3-14] The method according to any one of [3-1] to [3-13], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.
• [3-15] The method according to any one of [3-1] to [3-14], wherein the first column chromatography and the second column chromatography use the same mobile phase.
• [3-16] The method according to any one of [3-1] to [3-15], wherein the second column is packed with a larger amount of stationary phase than the first column.
• [3-17] The method according to any one of [3-1] to [3-16], wherein the second column has the same inner diameter as the first column and is longer than the first column.
• [3-18] The method according to any one of [3-1] to [3-17], wherein the at least one component that is completely or partially removed by the first column chromatography is any one of C22:0, C20:0, C18:0, and C20:1 esters.
• the oil composition is derived from any one of fish oil, vegetable oil, algae, and microorganisms.
• the ester is an ethyl ester.
• a method for producing an arachidonic acid ester composition from an oil or fat composition comprising the steps of: (a) subjecting the oil and fat composition to a first column chromatography using a first column to obtain an eluate containing arachidonic acid esters, the eluate containing 0.5% by weight or less of C22:0 esters relative to the total fatty acid esters contained in the eluate; (b) subjecting the eluate containing the arachidonic acid ester obtained by the first column chromatography to a second column chromatography using a second column to obtain an eluate; and (c) concentrating the eluate obtained by the second column chromatography to obtain a composition containing the arachidonic acid ester,
• [4-3] The method according to [4-1] or [4-2], wherein a loading interval until a next column chromatography can be started after a first column chromatography is started is shorter than that in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns.
• [4-4] The method according to [4-3], in which the loading interval until another column chromatography can be started after one column chromatography is started is shorter than that in the case of column chromatography using only the second column.
• the first column can be connected to the second column so that an eluate from the first column can be injected into the second column;
• the method according to any one of [4-1] to [4-6], wherein the eluate from the first column is injected into a second column, and the relative value of the peak top retention time of at least one component other than arachidonic acid ester contained in the eluate when the peak top retention time of arachidonic acid ester in the second column chromatography is set to 1.0 is within a range of 1.1 or less.
• [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 reverse phase column chromatography.
• [4-14] The method according to any one of [4-1] to [4-13], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.
• [4-15] The method according to any one of [4-1] to [4-14], wherein the first column chromatography and the second column chromatography use the same mobile phase.
• [4-16] The method according to any one of [4-1] to [4-15], wherein the second column is packed with a larger amount of stationary phase than the first column.
• [4-17] The method according to any one of [4-1] to [4-16], wherein the second column has the same inner diameter as the first column and is longer than the first column.
• [4-18] The method according to any one of [4-1] to [4-17], wherein the at least one component that is completely or partially removed by the first column chromatography is any one of C22:0, C20:0, C18:0, and C20:1 esters.
• [4-20] The method according to any one of [4-1] to [4-19], wherein the ester is an ethyl ester.
• [4-26] The method according to any one of [4-1] to [4-25], wherein in (b), all or a part of at least one component having a peak top retention time of 0.90 to 1.1 relative to the retention time of the peak top of an arachidonic acid ester in the second column chromatography, which is taken as 1.0, is removed.
• [4-27] The method according to any one of [4-1] to [4-26], further comprising, prior to (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of an arachidonic acid ester.
• a method for shortening the time required for column chromatography in the production of a highly unsaturated fatty acid ester composition from an oil or fat composition comprising: (a) subjecting an oil and fat composition to a first column chromatography using a first column to remove all or a part of at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of a highly unsaturated fatty acid ester, which is a target substance, in the first column chromatography, taken as 1.0, thereby obtaining an eluate containing the target substance by the first column chromatography; (b) subjecting the eluate containing the target substance obtained by the first column chromatography to a second column chromatography using a second column to remove all or a part of at least one component having a peak top retention time of 0.90 to 1.1 relative to the retention time of the peak top of the target substance in the second column chromatography, which is taken as 1.0, to obtain an eluate by the second column chromat
• the resulting composition contains the target substance in an amount of 95% by weight or more,
• a method in which, after one column chromatography has been started, the loading interval before another column chromatography can be started is shorter than in the case of column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns.
• [5-4] The method according to any one of [5-1] to [5-3], wherein the eluate containing the target substance obtained in the first column chromatography is subjected to a second column chromatography using a second column, and thereby at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of the target substance in the second column chromatography is removed in whole or in part.
• the first column can be connected to the second column so that an eluate from the first column is injected into the second column;
• the method according to any one of [5-1] to [5-4], wherein an eluate from a first column is injected into a second column, and a relative value of the retention time of the peak top of a component contained in the eluate when the retention time of the peak top of the target substance in the second column chromatography is set to 1.0 is within a range of 1.1 or less.
• [5-12] The method according to any one of [5-1] to [5-11], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.
• [5-13] The method according to any one of [5-1] to [5-12], wherein the first column chromatography and the second column chromatography use the same mobile phase.
• [5-14] The method according to any one of [5-1] to [5-13], wherein the second column is packed with a larger amount of stationary phase than the first column.
• [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.
• [5-16] The method according to any one of [5-1] to [5-15], wherein the target substance is an ester of any one of eicosapentaenoic acid (EPA), dihomo-gamma-linolenic acid (DGLA), and arachidonic acid (ARA).
• EPA eicosapentaenoic acid
• DGLA dihomo-gamma-linolenic acid
• ARA arachidonic acid
• [5-17] The method according to any one of [5-1] to [5-16], wherein the at least one component that is completely or partially removed by the first column chromatography is any one of C22:0, C20:0, C18:0, and C20:1 esters.
• [5-21] The method according to [5-20], wherein the content of at least one component that is completely or partially removed by the first column chromatography in the eluate containing the target substance and subjected to the second column chromatography is 0.1 wt % or less or 0.05 wt % or less based on the total fatty acid esters contained in the eluate subjected to the second column chromatography.
• [5-22] The method according to any one of [5-1] to [5-21], wherein the highly unsaturated fatty acid ester composition contains the target compound in an amount of 96% by weight or more.
• [5-23] The method according to any one of [5-1] to [5-22], further comprising preparing the oil or fat composition from a raw material composition by distillation.
• [5-24] The method according to any one of [5-1] to [5-23], wherein (a), (b) and (c) are repeated two or more times.
• [5-25] The method according to any one of [5-1] to [5-24], further comprising, before (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target substance.
• [5-26] The method according to any one of [5-1] to [5-25], wherein the eluate containing the target substance obtained in the first column chromatography is an eluate obtained up to the end of elution of the target substance from the first column.
• a method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition comprising: The method includes: (a) subjecting an oil or fat composition to a first column chromatography using a first column, thereby removing all or a part of at least one component having a peak top retention time of 1.8 to 3.5 relative to the retention time of the peak top of the target highly unsaturated fatty acid ester in the first column chromatography, which is taken as 1.0, to obtain an eluate containing the target substance by the first column chromatography; and (b) subjecting the eluate containing the target substance obtained by the first column chromatography to a second column chromatography using a second column, thereby removing all or a part of at least one component having a peak top retention time of 0.90 to 1.1 relative to the retention time of the peak top of the target substance in the second column chromatography, which is taken as 1.0, to obtain an eluate by the second column chromatography.
• [6-5] The method according to any one of [6-1] to [6-4], wherein the eluate containing the target substance obtained in the first column chromatography is subjected to a second column chromatography using a second column, whereby at least one component having a peak top retention time of 0.93 to 1.09 relative to the retention time of the peak top of the target substance in the second column chromatography is taken as 1.0 is removed in whole or in part.
• the first column can be connected to the second column so that an eluate from the first column is injected into the second column;
• [6-10] The method according to [6-6], wherein a relative value of the retention time of the peak top of the at least one component contained in the eluate is in the range of 0.93 to 1.09 when the retention time of the peak top of the target substance in the second column chromatography is taken as 1.0.
• [6-11] The method according to [6-6], wherein an eluate from a first column having a retention time of 3.5 or more relative to the retention time of the peak top of the target substance in the first column set to 1.0 is discharged, and a new mobile phase is injected into the second column.
• [6-12] The method according to any one of [6-1] to [6-11], wherein the first column chromatography and the second column chromatography use reverse phase column chromatography.
• [6-13] The method according to any one of [6-1] to [6-12], wherein the first column chromatography and the second column chromatography are fixed bed column chromatography.
• [6-14] The method according to any one of [6-1] to [6-13], wherein the first column chromatography and the second column chromatography use the same mobile phase.
• [6-15] The method according to any one of [6-1] to [6-14], wherein the second column is packed with a larger amount of stationary phase than the first column.
• [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.
• [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.
• [6-18] The method according to any one of [6-1] to [6-17], wherein the target substance is an ester of any one of eicosapentaenoic acid (EPA), dihomo-gamma-linolenic acid (DGLA), and arachidonic acid (ARA).
• EPA eicosapentaenoic acid
• DGLA dihomo-gamma-linolenic acid
• ARA arachidonic acid
• [6-23] The method according to any one of [6-1] to [6-22], wherein the obtained composition contains the target substance in an amount of 96% by weight or more.
• [6-24] The method according to any one of [6-1] to [6-23], further comprising preparing the oil or fat composition by distillation from a raw material composition.
• [6-25] The method according to any one of [6-1] to [6-24], wherein (a), (b) and (c) are repeated two or more times.
• [6-26] The method according to any one of [6-1] to [6-25], further comprising, before (a), subjecting the oil or fat composition to column chromatography using a first column to confirm the retention time of the peak top of the target substance.
• [6-27] The method according to any one of [6-1] to [6-26], wherein the eluate containing the target substance obtained in the first column chromatography is an eluate obtained up to the end of elution of the target substance from the first column.
• a composition containing highly unsaturated fatty acid ethyl esters such as EPA ethyl ester, DGLA ethyl ester, and ARA ethyl ester at high concentrations can be effectively produced in a shorter time and with less solvent usage than when using a single column.
• fatty acids such as C20:0 ethyl ester can be removed.
• the "composition” may include one or more PUFAs or ester derivatives thereof.
• the composition including the PUFAs or ester derivatives thereof may be obtained from a raw material including polyunsaturated fatty acids as constituent fatty acids.
• suitable oil compositions may be obtained from raw material compositions obtained by esterifying natural oils and fats, including vegetable and animal oils and fats, and from raw material compositions obtained by esterifying oils obtained from genetically modified plants, animals, and microorganisms, including yeast and filamentous fungi.
• the filamentous fungi may include compositions obtained by esterifying oils obtained from microorganisms of the genus Mortierella.
• the raw material composition is a composition obtained by ethyl esterifying fish oil.
• the raw material composition is a composition obtained by ethyl esterifying algae oil.
• the fat or oil composition is obtained by subjecting a raw material composition to distillation.
• the highly unsaturated fatty acid ester composition is a composition obtainable by the method of the present invention, and is a composition containing the target highly unsaturated fatty acid ester.
• the target highly unsaturated fatty acid ester can be any ester, for example, an ethyl ester, of eicosapentaenoic acid (EPA), dihomo- ⁇ -linolenic acid (DGLA), or arachidonic acid (ARA).
• fatty acid refers to long-chain aliphatic carboxylic acids (alkanoic acids) of various chain lengths, from about C12 to C22 (where the number refers to the total number of carbon atoms in the chain). The predominant chain length is C16 to C22.
• Fatty acid structures may be represented by a simple notation of "X:Y", where X is the total number of carbon atoms in the particular fatty acid, and Y is the number of double bonds.
• a saturated fatty acid with 20 carbon atoms may be represented as "C20:0”
• a monounsaturated fatty acid with 18 carbon atoms may be represented as “C18:1”
• arachidonic acid may be represented as "C20:4,n-6", etc.
• the “n-” indicates the position at which the double bond begins, counting from the methyl end of the fatty acid, e.g., "n-6" indicates that the double bond begins at the sixth position counting from the methyl end of the fatty acid.
• This method is well known to those skilled in the art, and fatty acids represented according to this method can be easily identified by those skilled in the art.
• Fatty acids are carboxylic acids with long aliphatic chains, which can be either saturated or unsaturated. Fatty acids are usually produced industrially by hydrolysis of triglycerides or phospholipids from natural sources. Some are produced synthetically. Regardless of the method of production, purification methods are required to obtain a pure product for food, cosmetic or industrial use.
• polyunsaturated fatty acid means a fatty acid with more than one double bond.
• the polyunsaturated fatty acid herein is in the form of an ester.
• the ester is typically an alkyl ester, such as a C1 - C6 alkyl ester, or a C1 - C4 alkyl ester. Examples of esters include ethyl esters.
• the highly unsaturated fatty acids include eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, stearidonic acid, C18:3, C19:4, C20:4, and C21:5.
• the highly unsaturated fatty acids include eicosapentaenoic acid, dihomo- ⁇ -linolenic acid, and arachidonic acid.
• the at least one component that is removed in whole or in part by the second column chromatography includes C18:3, C19:4, C20:4, and C21:5.
• the at least one component that is removed in whole or in part by the second column chromatography may be an analog of the highly unsaturated fatty acid of interest.
• EPA eicosapentaenoic acid
• EPA-EE EPA eicosapentaenoic acid
• DGLA dihomo-gamma-linolenic acid
• ARA arachidonic acid
• the fat composition typically comprises a highly unsaturated fatty acid and at least one fatty acid.
• the fat composition comprises a highly unsaturated fatty acid and at least one fatty acid and/or ester thereof.
• Typical fatty acid esters are similar to those defined above for highly unsaturated fatty acids.
• the fat composition comprises at least one fatty acid having 20 carbon atoms and a fatty acid ethyl ester. More preferably, the fat composition comprises a highly unsaturated fatty acid and at least one fatty acid selected from C18:0 (stearic acid), C20:0 (arachidic acid), C20:1 (eicosenoic acid), C22:0 (behenic acid) and esters thereof.
• the at least one component that is completely or partially removed in the first column chromatography is at least one fatty acid ester as described above.
• At least one component other than the target substance (e.g., highly unsaturated fatty acid ester) contained in the oil composition has a lower polarity than the target substance.
• the oil composition contained in the eluate subjected to the second column chromatography contains at least one component selected from C18:0, C20:0, C20:1, and C22:0 esters in an amount of 0.5% 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 or less based on the total oil composition contained in the eluate subjected to the second column chromatography.
• Column chromatography refers to the process of selective retention or retardation of one or more components of a fluid solution as the fluid flows through a column containing a stationary phase or phases composed of finely divided substances and/or materials having capillary channels. Retention results from the relative partitioning of the components of the mixture between the stationary phase and a bulk fluid phase (e.g., a mobile phase), the latter of which migrates through the stationary phase.
• a bulk fluid phase e.g., a mobile phase
• Column chromatography is used for the analysis and separation of mixtures of two or more substances.
• Column chromatography includes, for example, preparative chromatography, analytical chromatography, high performance liquid chromatography, simulated moving bed chromatography, real moving bed chromatography, and supercritical fluid chromatography (SFC).
• non-polar and polar to describe the mobile phase or highly unsaturated fatty acid esters can be used as relative terms to one another.
• non-polar can refer to the solvent in the mobile phase that is the least polar
• polar can refer to the solvent in the mobile phase that is more polar than the “non-polar” solvent.
• the column chromatography includes passing the oil and fat composition through one or more columns.
• the first column chromatography includes passing the oil and fat composition through the first column, and removing all or a part of at least one component that elutes later than the highly unsaturated fatty acid ester of interest (i.e., the relative value of the retention time of the peak top when the retention time of the peak top of the target in the first column is set to 1.0) is greater than 1.0.
• the first column may be a combination of multiple columns in tandem and/or in parallel.
• the second column chromatography includes passing the eluate containing the target obtained from the first column chromatography through the second column, and removing all or a part of the components of related substances to obtain a highly unsaturated fatty acid ester composition containing a predetermined amount of the target.
• the second column may be a combination of multiple columns in tandem and/or in parallel. Any known column may be used in the claimed method.
• the eluate containing the target substance obtained in the first column chromatography is the eluate obtained until the completion of elution of the target substance from the first column.
• the removal of all or a part of at least one component other than the target substance in the first column chromatography is carried out by recovering the eluate obtained until the completion of elution of the target substance from the first column.
• the first column chromatography is performed using a first column.
• the first column chromatography is performed for the purpose of removing all or part of a component having a longer retention time than the target.
• the retention time of the component removed all or part 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, where the retention time of the target in the first column chromatography is 1.
• when at least one type of component is partially removed by the first column chromatography 99% or more, 95% or more, or 90% or more of the component is removed.
• the second column chromatography is performed using a second column.
• the second column chromatography is performed for the purpose of removing all or part of the components whose retention times are close to that of the target. Components whose retention times are close to that of the target can be referred to as related substances in this specification.
• the retention times of the components removed by the second column chromatography are 0.8 or more, 0.9 or more, or 1.5 or less, 1.3 or less, 1.2 or less, or 1.1 or less, or 0.8 to 1.5, 0.9 to 1.3, 0.9 to 1.2, or 0.9 to 1.1, assuming that the retention time of the target in the second column chromatography is 1.
• the components whose retention times are close to that of the target are partially removed by the second column chromatography, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more of the components are removed.
• a fraction of the eluate from the first chromatography that was not subjected to the second chromatography and a fraction of the eluate from the second chromatography that contains the fatty acid esters removed from the highly unsaturated fatty acid ester composition may be recovered and subjected to a third column chromatography.
• the third column chromatography is performed using a third column.
• the first column refers to a column used in the first column chromatography
• the second column refers to a column used in the second column chromatography
• the third column refers to a column used in the third column chromatography.
• the dimensions of the columns used are not particularly limited and will depend to some extent on the volume of the oil composition to be refined.
• each column is 1 mm or more, 2 mm or more, 4 mm or more, 8 mm or more, 16 mm or more, 32 mm or more, 64 mm or more, 128 mm or more, 256 mm or more, or 500 mm or more, or 4000 mm or less, 2000 mm or less, 1000 mm or less, 500 mm or less, or 1 to 4000 mm, 2 to 2000 mm, 4 to 1000 mm, 8 to 1000 mm, 16 to 1000 mm, 32 to 500 mm, 30 to 800 mm, or 400 to 800 mm.
• the first column and the second column can have the same or different diameters.
• the ratio of the diameters of the first column and the second column is 1 for the first column, and the ratio of the diameters of the second column is 0.1 or more, 0.3 or more, 0.5 or more, or 10 or less, 3 or less, 2 or less, or 1.5 or less, or 0.1 to 10, 0.3 to 3, 0.5 to 2, or 0.5 to 1.5.
• columns having the same diameter are used as the first and second columns.
• the first column to the third column can have the same or different diameters.
• the third column can have the same or different diameters as the first column or the second column.
• the second column has the same diameter as the first column and is longer than the first column.
• the first column and the second column have the same or identical diameter, which means that the ratio of the diameters of the first column and the second column is 0.8 to 1.2, for example, 0.9 to 1.1 or 1.
• the cross sections of the first and second columns are approximately circular, and the ratio of the cross-sectional areas of the first and second columns is 0.1 or more, 0.2 or more, or 10 or less, 4 or less, 2 or less, or 0.1 to 10, 0.2 to 4, or 0.2 to 2, when the cross-sectional area of the first column is taken as 1, for the second column.
• columns having the same cross-sectional area are used as the first and second columns.
• 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 each of the first column and the second column.
• 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, and has a length that is, for example, 1.1 times or more, 1.5 times or more, or 5 times or less, 7 times or less, or 10 times or less than the length of the first column.
• the first column and the second column having the same or identical inner diameter refers to the ratio of the inner diameters of the first column and the second column being 0.8 to 1.2, for example, 0.9 to 1.1, or 1.
• the column is cylindrical having an outer diameter, an inner diameter, and a length.
• the column is a preparative chromatography column.
• a preparative chromatography column can include an inner diameter of 5 mm or more, 10 mm or more, 20 mm or more, 50 mm or more, or 4 m or less, 2 m or less, 1 m or less, 80 cm or less, or about 5 mm to about 4 m, about 10 mm to about 2 m, about 20 mm to about 1 m, or about 50 mm to 80 cm, and a length of about 100 mm to about 5 m, about 2 cm to about 2 m, or about 10 cm to about 1.5 m.
• the column is an analytical chromatography column.
• An analytical column can include an inner diameter of about 1 mm to about 10 cm, and a length of about 10 mm to about 500 mm.
• the dimensions can be selected such that the inner diameter is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 220%, about 240%, about 260%, about 280%, about 300%, about 320%, about 340%, about 360%, about 380%, about 400%, about 450% or about 500% of the length.
• the outer diameter can be about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5%, about 7.5%, about 10%, about 15%, about 20%, about 25%, or about 30% larger than the inner diameter.
• peak means a peak in a chromatogram.
• a chromatogram is a plot of detected values over time. When an eluent is detected, the detected value increases and the chromatogram shows a "peak.” Each peak in the chromatogram indicates the presence of a component in the sample. Each peak is labeled with a retention time, and time in the chromatogram increases from left to right. "Peak top” refers to the point in a chromatogram where the detected value of each peak is maximum.
• “Separation,” as used herein, refers to a process characterized by the spatial separation of components of a composition that includes highly unsaturated fatty acid esters based on their differential distribution between phases in relative motion (e.g., a mobile phase and a stationary phase). Separation results from loading of a sample onto a column and subsequent elution of the column.
• “Fractionation” is a separation process in which a volume of a mixture is divided into several smaller volumes during a phase transition, where the composition varies according to a gradient. Different fractions are collected at different times based on differences in the specific properties of the individual components (the highly unsaturated fatty acid esters within the mixture or sample), such as their affinity for the stationary and/or mobile phase.
• Subjecting to column chromatography means loading a sample containing highly unsaturated fatty acid esters onto a column until the entire sample is accommodated within the column.
• the sample is an oil composition.
• the sample is typically loaded onto the top of the stationary phase of a packed column, where "top” is the end of the stationary phase that first receives the mobile phase as it elutes through the column.
• the oil composition may be subjected to column chromatography by a syringe, a pump, or by direct application of the sample to the top of the stationary phase.
• the sample may be mixed with a minimal amount of mobile phase or other solvent for loading.
• Elution means that a component loaded onto a column passes through the column and exits as a solution in the mobile phase.
• the term “eluent” refers to the mobile phase eluted from the column, which may contain components contained in the oil composition loaded onto the column.
• the mobile phase eluted from the column is a solution containing components contained in the oil composition.
• the "eluent containing the target product obtained by the first column chromatography” refers to the eluent that has passed through the first column and contains the target product.
• Eluate refers to the components discharged from a column in column chromatography.
• the "eluate to be subjected to the second column chromatography” is the eluate of the first column chromatography.
• the “component” refers to a component contained in the oil composition subjected to column chromatography, and includes highly unsaturated fatty acid esters and fatty acid esters.
• the "at least one component removed in whole or in part by the first column chromatography” can be a fatty acid ester having a longer retention time than the highly unsaturated fatty acid ester of interest, for example, any of the C22:0, C20:0, C18:0, and C20:1 esters.
• the "at least one component in the second column chromatography” refers to a component contained in the highly unsaturated fatty acid ester composition obtained from the oil composition, other than the highly unsaturated fatty acid ester of interest, and can be any of the esters of stearidonic acid, C19:4, C21:5, C20:4, and C18:3.
• the "at least one component removed in whole or in part by the first column chromatography” and the “at least one component in the second column chromatography” may be different.
• the "at least one component in the second column chromatography” may contain the same component as the "at least one component removed in whole or in part by the first column chromatography.”
• Biphenyl "C30", “C22”, “C18”, “C8”, “C5", and “C4" as used herein refer to functional groups present on the column packing material (stationary phase).
• a biphenyl column exposes materials flowing through the column to unsubstituted biphenyl groups
• a C18 column exposes materials flowing through the column (e.g., mobile phase and components) to unsubstituted straight or branched chain 18-carbon alkyl groups.
• Chromatographic conditions means the parameters under which column chromatography is operated. Examples include packing pressure, mobile and stationary phase composition, slurry concentration, pressure at which the column is operated, column temperature, mobile phase temperature, mobile phase gradient, mobile phase flow rate, column type used, detection instrumentation and parameters used, sample preparation protocol employed, sedimentation time and pressure at which sedimentation is performed, stand time and pressure at which stand is performed.
• “Gradient” refers to the change in mobile phase composition over time while column chromatography is performed.
• the composition of the mobile phase can change as the solvent is eluted through the column.
• Different mobile phases can be added in increasing percentages over time during elution.
• “Purity” is a ratio indicating the content of the main component of the composition, and can be calculated, for example, from the measurement results of gas chromatography (GC) using an internal standard.
• the main component is a highly unsaturated fatty acid ester, for example, an EPA ester, a DGLA ester, or an ARA ester.
• Target product refers to the desired polyunsaturated fatty acid ester, e.g., EPA ester, DGLA ester, or ARA ester, obtained from a single fraction or a combination of fractions obtained by elution and fractionation.
• the concentration includes reducing pressure and/or heating the eluent containing the target highly unsaturated fatty acid ester, and recovering the solvent by distillation.
• the concentration is performed using an evaporator.
• the evaporator may be an eluent evaporator and/or a thin film evaporator. Any type of evaporator may be used for the concentration, such as a thin film evaporator, a single evaporator, a multiple effect evaporator, a multi-chamber evaporator, a natural circulation evaporator, a forced circulation evaporator, a thin film falling evaporator, a thin film ascending evaporator, or a combination thereof.
• the type of natural circulation evaporator may be, for example, an external heating type or a calandria type.
• a method for producing a highly unsaturated fatty acid ester composition from an oil or fat composition includes subjecting the oil or fat composition to column chromatography using a first column and confirming the retention time of the peak top of the target substance prior to carrying out the first column chromatography.
• at least one component having a peak top retention time later than that of the target highly unsaturated fatty acid in the first column chromatography is entirely or partially removed.
• Retention time refers to the time that a component spends in the column after being injected into the column. Typically, retention time refers to the time at which the top of a peak appears in a chromatogram.
• the retention time of the peak top of the target highly unsaturated fatty acid means the retention time of the peak top shown by the target substance in the chromatograms of the first and second chromatography.
• a “relative retention time (RRT)” (which may be used interchangeably with “relative value” and “relative value of retention time”) can be calculated based on the results of retention time measurements.
• Relative retention time indicates the relationship of the retention time of a component to the retention time of a target substance.
• the term "relative value of the retention time of the peak top” refers to a value obtained by dividing the retention time of the peak top of a component other than the target compound by the retention time of the peak top of the target compound in the chromatograms of the first and second chromatography, when the retention time of the peak top of the target compound is set to 1.
• An eluate having a "relative retention time” equal to or greater than a predetermined value means an eluate collected after the time when the "relative retention time” reaches that value. Also, an eluate having a "relative retention time” equal to or less than a predetermined value means an eluate collected before the time when the "relative retention time” reaches that value.
• the retention time of the peak top can be determined by measuring with a UV detector.
• the peak top retention time can be determined by measuring each fraction by fatty acid analysis using gas chromatography.
• one-time column chromatography means a process in which a batch of an oil composition is passed through one or more columns to obtain a highly unsaturated fatty acid ester composition containing a target substance at a predetermined concentration.
• the batch of an oil composition can be passed through one or two columns.
• the two columns are called the first column and the second column and are connected in series.
• two columns are used in one-time column chromatography, a part of the eluate from the first column is discharged without being injected into the second column.
• the first column and the second column can be connected so that the eluate from the first column is injected into the second column.
• the eluate from the first column is injected into the second column means that the eluate from the first column can be injected into the second column.
• the eluate can be directly applied to the second column without being subjected to a process such as concentration.
• being communicable means that the first column and the second column can be switched from a communicated state to a non-communicated state, or vice versa.
• "discharging the eluate from the first column” means discharging the eluate from the first column as waste without injecting it into the second column.
• the first column when the relative retention time is 3.5 or more, the first column is not connected to the second column, and the eluate from the first column is discharged.
• the eluate discharged from the first column can be injected into a third column.
• the "loading interval until re-column chromatography can be started after the start of one column chromatography” means the time from the time when the oil composition is loaded once onto the first or second column until re-loading (e.g., second loading) of the oil composition onto the same column can be started in column chromatography using a single column or two or more columns.
• re-loading of the oil composition onto the same column is started with a time interval such that elution of the target substance in the re-loaded oil composition starts after the elution of the last component in one column chromatography is completed.
• the "loading interval until re-column chromatography can be started after the start of one column chromatography" is calculated or measured as the time from the time when elution of the target substance starts on the first or second column until elution of the last component is completed.
• the "loading interval until re-column chromatography can be started after the start of one column chromatography" is calculated as the total time from the time when the oil or fat composition is subjected to the first column chromatography to the time when the elution of the target substance from the first column is completed, and the time from the time when the elution of the target substance in the second column chromatography is started to the time when the last component is discharged from the second column.
• a first column and a second column are used, and the last component is eluted from the first column or the second column.
• the "loading interval until re-column chromatography can be started after the start of one column chromatography" is a time equal to the time from the time when the elution of the target substance in the first column chromatography is started to the time when the last component is discharged from the first column.
• the "loading interval after the start of one column chromatography until another column chromatography can be started” is equal to the sum of the time from the time when the oil or fat composition is subjected to the first column chromatography to the time when the elution of the target substance is completed from the first column and the time from the time when the elution of the target substance starts in the second column chromatography to the time when the last component is discharged from the second column, or the time from the time when the elution of the target substance starts in the second column chromatography to the time when the last component is discharged from the second column.
• the time from the time when the elution of the target substance starts in the first column chromatography to the time when the last component is discharged from the first column is shorter than the time from the time when the elution of the target substance starts in the first column chromatography to the time when the last component is discharged from the first column in column chromatography using a single column having a length equal to the sum of the lengths of the first and second columns.
• the time from the time the elution of the target substance starts in the first column to the time the elution of the last component is completed is shorter than the time from the time the elution of the target substance starts in the second column to the time the elution of the last component is completed in column chromatography using only the second column.
• the total time from the time when the oil composition is subjected to the first column chromatography to the time when the elution of the target substance from the first column is completed, and the total time from the time when the elution of the target substance in the second column starts to the time when the last component is discharged from the second column is shorter than the time from the time when the elution of the target substance in the second column starts to the time when the elution of the last component is completed in column chromatography using only the second column.
• a "single column having a length equal to the sum of the lengths of the first and second columns” refers to, for example, a single column having the same inner diameter and stationary phase as the first and/or second column.
• a single column having a length equal to the sum of the lengths of the first and second columns refers to a single column having the same inner diameter and stationary phase as the second column, and a length in the flow direction equal to the sum of the lengths of the first and second columns.
• reversed-phase column chromatography is suitable as the column chromatography.
• Any adsorbent of the reversed-phase distribution system can be used as the stationary phase without any particular designation, for example, polymer beads such as polystyrene reticulated with DVB (divinylbenzene), or silica gel bonded with C4, C8, or C18 alkyl groups, particularly an ODS column using octadecylsilyl (ODS).
• Each chromatography column may contain the same or different adsorbents.
• the first column and the second column may contain the same or different adsorbents.
• the first column and the second column contain the same adsorbent.
• the third column when a third column chromatography is performed, the third column may use the same or different adsorbent as the first column or the second column, respectively.
• the stationary phase can be selected from C30, C22, C18, C8, C4, biphenyl, fluorophenyl, hydrophilic interaction liquid chromatography (HILIC) stationary phase, acrylamide, silica, phenylhexyl stationary phase, polar embedded alkyl, fluorophenylpropyl, or any stationary phase known in the art of chromatography. In some embodiments, a chiral stationary phase is used.
• the choice of stationary phase will be apparent to one skilled in the art and may depend on the components to be purified by chromatography. Non-polar components, such as highly unsaturated fatty acid esters, may require the use of a reversed phase stationary phase, such as C18 (ODS).
• ODS octadecyl silica
• More polar components may require a normal phase stationary phase, such as non-bonded silica, amino phase or cyano phase.
• the stationary phase is 1 ⁇ m or more, 20 ⁇ m or more, 40 ⁇ m or more, 60 ⁇ m or more, 80 ⁇ m or more, 100 ⁇ m or more, 1000 ⁇ m or more, 2000 ⁇ m or more, 3000 ⁇ m or more, 4000 ⁇ m or more, or 20 ⁇ m or less, 40 ⁇ m or less, 60 ⁇ m or less, 80 ⁇ m or less, 100 ⁇ m or less, 1000 ⁇ m or less, 2000 ⁇ m or less, 3000 ⁇ m or less, 4
• the present invention includes particles having a median particle size of up to 1,000 ⁇ m, up to 5,000 ⁇ m, or from about 1 ⁇ m to about 20 ⁇ m, from about 20 ⁇ m to about 40 ⁇ m, from about 40 ⁇ m to about 60 ⁇ m, from about 60 ⁇ m to about 80 ⁇ m, from about 80 ⁇ m to about 1000 ⁇ m, from about 1000 ⁇ m to about 2000 ⁇ m, from about 2000 ⁇ m
• the stationary phase comprises particles having a median particle size greater than 50 ⁇ m, greater than 45 ⁇ m, greater than 40 ⁇ m, greater than 35 ⁇ m, greater than 30 ⁇ m, greater than 25 ⁇ m, greater than 20 ⁇ m, greater than 15 ⁇ m, or greater than 10 ⁇ m, and/or less than 5000 ⁇ m, less than 4000 ⁇ m, less than 3000 ⁇ m, less than 2000 ⁇ m, less than 1000 ⁇ m, less than 500 ⁇ m, less than 100 ⁇ m, less than 80 ⁇ m, less than 60 ⁇ m, or between 10 ⁇ m and 5000 ⁇ m, 15 ⁇ m and 4000 ⁇ m, 20 ⁇ m and 3000 ⁇ m, 25 ⁇ m and 2000 ⁇ m, 30 ⁇ m and 1000 ⁇ m, 35 ⁇ m and 500 ⁇ m, 40 ⁇ m and 100 ⁇ m, 45 ⁇ m and 80 ⁇ m, 50 ⁇ m and 60 ⁇ m.
• the median particle size can be measured by laser d
• the stationary phase may be the same or different in the first column and the second column.
• the first column may use, for example, polystyrene reticulated with DVB (divinylbenzene), a C8 alkyl group, or silica gel bonded with octadecylsilyl (ODS)
• the second column may use a stationary phase different from that of the first column, selected from polystyrene reticulated with DVB (divinylbenzene), an alkyl group, or silica gel bonded with octadecylsilyl (ODS).
• the third column when a third column chromatography is performed, the third column may use the same or different stationary phase as the first column or the second column.
• a stationary phase containing particles having the same or different median particle size can be used in the first column and the second column.
• the first column may have a particle size of, for example, 1 ⁇ m or more, 20 ⁇ m or more, 40 ⁇ m or more, 60 ⁇ m or more, 80 ⁇ m or more, 1000 ⁇ m or more, 2000 ⁇ m or more, 3000 ⁇ m or more, 4000 ⁇ m or more, or 20 ⁇ m or less, 40 ⁇ m or less, 60 ⁇ m or less, 80 ⁇ m or less, 1000 ⁇ m or less, 2000 ⁇ m or less, 3000 ⁇ m or less,
• a stationary phase is used that includes particles having a median particle size of 4000 ⁇ m or less, 5000 ⁇ m or less, or from about 1 ⁇ m to about 20 ⁇ m, from about 20 ⁇ m to about 40 ⁇ m, from about 40 ⁇ m to about 60 ⁇ m, from about 60 ⁇ m to about 80
• the thickness is 1 ⁇ m or more, 20 ⁇ m or more, 40 ⁇ m or more, 60 ⁇ m or more, 80 ⁇ m or more, 1000 ⁇ m or more, 2000 ⁇ m or more, 3000 ⁇ m or more, 4000 ⁇ m or more, or 20 ⁇ m or less, 40 ⁇ m or less, 60 ⁇ m or less, 80 ⁇ m or less, 1000 ⁇ m or less, 2000 ⁇ m or less, 3000 ⁇ m or less, 4000 ⁇ m or less, 5000 ⁇ m or less, or about 1 ⁇ m to about A stationary phase different from that of the first column can be used, which includes particles having a median particle size selected from 20 ⁇ m, about 20 ⁇ m to about 40 ⁇ m, about 40 ⁇ m to about 60 ⁇ m, about 60 ⁇ m to about 80 ⁇ m, about 80 ⁇ m to about 1000 ⁇ m, about 1000 ⁇ m to about 2000 ⁇ m, about 2000 ⁇ m to about 3000 ⁇ m, about 3000
• the amount of the packed stationary phase is 1 to 1000 kg, 10 to 900 kg, 20 to 800 kg, 30 to 700 kg, 40 to 600 kg, or 50 to 500 kg by weight.
• the amount of the packed stationary phase in the first column and the amount of the packed stationary phase in the second column may be the same or different.
• the amount of the packed stationary phase in the first column is, for example, 1 to 100 kg, 10 to 100 kg, or 30 to 100 kg.
• the amount of the packed stationary phase in the second column is, for example, 1 to 1000 kg, 10 to 800 g, or 50 to 800 g.
• the ratio of the amount of the packed stationary phase in the first column to the amount of the packed stationary phase in the second column is, when the amount of the packed stationary phase in the first column is 1, 0.5 to 10, 1 to 7, 1.1 to 5, or 2 to 4.5 in the second column.
• the third column when a third column chromatography is performed, the third column may be packed with the same or a different amount of stationary phase as the first column or the second column, respectively.
• the amount of stationary phase packed in the second column is greater than the amount of stationary phase packed in the first column, and is 1.1 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, or 4 times or less, 4.5 times or less, 5 times or less, 6 times or less, 7 times or less, or 10 times or less than the amount of stationary phase packed in the first column.
• the column chromatography includes elution with a mobile phase comprising one or more of water, an organic solvent, or supercritical carbon dioxide.
• the mobile phase comprises methanol.
• the chromatography includes a mobile phase comprising an additive selected from one or more of formic acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide.
• the solvent used as the mobile phase is an organic solvent and is selected from alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane.
• alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. Methanol and ethanol are preferred. Methanol is more preferred.
• Examples of ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE).
• esters include methyl acetate and ethyl acetate.
• ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone (MIBK).
• MIBK methyl isobutyl ketone
• the mobile phase may be the same or different in the first and second column chromatography, but is preferably the same.
• different mobile phases for example, methanol, ethanol, diethyl ether, diisopropyl ether, methyl t-butyl ether, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone (MIBK), acetonitrile, hexanes, and dichloromethane
• a mobile phase different from that of the first column selected from methanol, ethanol, diethyl ether, diisopropyl ether, methyl t-butyl ether, methyl acetate, ethyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone (MIBK), acetonitrile,
• the mobile phase may further include additives, including buffers and pH adjusters.
• the choice of additive may be determined based on the mobile phase used, the stationary phase used, and the components to be purified.
• the mobile phase includes an additive selected from one or more of formic acid, trifluoroacetic acid, heptafluorobutyric acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide.
• the mobile phase may be additive-free.
• the column chromatography includes a mobile phase gradient. In one embodiment of the invention, the column chromatography includes purification of one or more highly unsaturated fatty acid esters.
• the highly unsaturated fatty acid esters include docosahexaenoic acid, crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinoleic acid, eleostearic acid, mead acid, dihomo-gamma-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosate Tolanic acid, adre
• the ester is a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl ester.
• column chromatography includes purification of eicosapentaenoic acid ethyl ester.
• the highly unsaturated fatty acid ester composition contains the desired highly unsaturated fatty acid ester with a purity of about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 86% or more, about 90% or more, about 95% or more, about 96.5% or more, about 98% or more, about 99% or more, about 99.5% or more, about 99.8% or more, or about 99.9% or more, and/or less than about 97%, less than about 98%, less than about 99.0%, less than about 99.95%, or less than about 99.99%.
• 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%.
• the recovery is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.7% or more, and/or less than 95%, less than 97%, less than 98%, less than 99%, less than 99.5%, less than 99.7%, or less than 99.9%.
• the mobile phase may include one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other solvent known in the art.
• the selection of the mobile phase may require consideration of the highly unsaturated fatty acid ester to be purified and the stationary phase to be used.
• a mobile phase should be selected that is sufficiently polar to elute the highly unsaturated fatty acid ester of interest, but not so rapid that elution approaches the solvent front.
• a solvent gradient can be used as the mobile phase during elution.
• the main purpose of gradient elution is to elute the strongly retained analytes from the column faster while the weakly retained analytes elute slower so that the eluted analytes produce well-separated peaks upon detection.
• non-polar components e.g., acetonitrile
• the solvent at the start of the chromatographic elution may contain a high percentage of polar solvent A, e.g., water, selected from about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 0%.
• Solvent B can be a less polar solvent than solvent A, e.g., methanol (when solvent A is water). Solvent B will make up the remaining percentage of the mobile phase. As the column is run and the solvent is eluted through the stationary phase and the column, a gradient will result in a gradual increase in the concentration of solvent B over time.
• a single solvent may be used as the mobile phase.
• the single solvent may be one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other solvent known in the art, either alone or in admixture.
• the rate of increase of solvent B over time can be constant. In some embodiments, there is no gradient and the mobile phase is isocratic during elution. In some embodiments, different rates of increase in the percentage of solvent B at different time ranges within the chromatographic method can be utilized. In some embodiments, the mobile phase can be isocratic for certain time ranges of the chromatographic method and includes a gradient for other time ranges.
• the first column chromatography and the second column chromatography can both be isocratic, both include a gradient, or both can be isocratic over a certain time range and a gradient over another time range.
• the first column chromatography can be isocratic and the second column chromatography can include a gradient, or the first column chromatography can include a gradient and the first column chromatography can be isocratic.
• the third column chromatography can be isocratic or can include a gradient.
• the multiple solvents used in the mobile phase can be stored separately in the mobile phase supply and mixed using a pump before elution through the column.
• the mobile phase supply includes a mobile phase source and a solvent delivery system.
• a solvent delivery system is a pumping device, such as a commercially available column chromatography pump, that provides the solvent or mobile phase to the column.
• Such pumps generally provide pulse-free flow, flow rates in the range of 0.1-100 L/min, precise control of flow rate, generation of high pressure (up to 6000 psi) and corrosion- and solvent-resistant components.
• Reciprocating pumps consist of a small chamber into which the solvent is pumped by the back and forth movement of a motor-driven piston.
• the linear velocity in the first column or the second column may be the same or different.
• the linear velocity in the first column may be faster or slower than the linear velocity in the second column.
• the ratio of the linear velocities in the first and second columns is 0.5 to 2.0, 0.5 to 1.5, or 1 to 1.5 in the second column when the linear velocity in the first column is 1.
• the linear velocities in the first column and the second column are the same.
• the third column when a third column chromatography is performed, the third column can be performed at the same or different linear velocity as the first column or the second column, respectively.
• the column chromatography is performed at room temperature or at a temperature higher than room temperature.
• the method is performed at a temperature higher than room temperature.
• the first and second column chromatography may be performed at the same temperature or at different temperatures, but are preferably at the same temperature.
• the temperature higher than room temperature is 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, or 60°C or lower, 55°C or lower, 50°C or lower, 45°C or lower, or 20 to 60°C, 25 to 55°C, 30 to 50°C, or 35 to 45°C.
• the column chromatography may use two or more columns.
• the column chromatography may be performed using known fixed bed chromatography equipment. Such column chromatography is called fixed bed chromatography.
• the first and second column chromatography are fixed bed chromatography.
• a detector is used to monitor the mobile phase eluting from the column for the presence of the component(s). Detection methods known in the art (e.g., mass spectrometry (MS), UV/Vis absorbance, fluorescence, refractive index, or conductivity) may be used.
• MS mass spectrometry
• UV/Vis absorbance UV/Vis absorbance
• fluorescence fluorescence
• refractive index or conductivity
• any of a variety of standard column chromatography detectors can be used for detection of the eluate immediately after elution from the column.
• each fraction can be individually monitored for the presence of components by analysis, hi some embodiments, the analysis is fatty acid analysis using gas chromatography.
• the eluate from the column is detected as a peak in a chromatogram.
• the retention time of the peak is used to identify the compound, and the height (or area) of the peak is proportional to the amount of eluate in the oil composition.
• "Retention time” is the time required for the eluate to pass through the column, measured from the time of injection (or loading) of the oil composition to the time of elution.
• each eluate of interest will have a characteristic retention time.
• retention of the eluate will vary with variations in eluent, stationary phase, temperature and column chromatography set-up conditions. Thus, retention time of the eluate is compared to the retention time of one or more standard compounds under the same conditions.
• Suitable detectors exhibit good sensitivity, good stability, reproducibility, linear response over several orders of magnitude, short response time when used for quantitative purposes, 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.
• a UV/Vis absorbance detector consisting of a scanning spectrophotometer with grating optics can be used.
• the use of a deuterium source (ultraviolet range, 190-360 nm) either independently or in combination with a tungsten source (visible range, 360-800 nm) provides a simple means of detecting absorbing species as they emerge from the column.
• Photodiode array (PDA) based instruments are ultraviolet/visible absorbance detectors that allow for very rapid collection of data over a selected spectral range. Absorbance spectral data for each chromatographic peak can be collected and stored. The stored data can be compared to the spectra of pure standards from a library. PDA detectors are useful in identifying components that are difficult to separate (overlapping peaks) since the characteristic spectra for each of the unresolved components are likely to be different.
• Fluorescence detectors are useful in detecting analytes that exhibit chemiluminescent properties such as fluorescence or phosphorescence. They are at least an order of magnitude more sensitive than UV absorbance detectors. Fluorescence is observed by detection of the emission radiation, typically at a 90 degree angle to the excitation beam, on a grid. The number of fluorescent species can be enhanced by post-column derivatization (PCD) of the eluted compounds (or pre-column derivatization of the sample itself) with special reagents.
• PCD post-column derivatization
• Refractive index (RI) detectors respond to almost all solutes.
• the difference in the refractive index of the reference mobile phase relative to the column effluent results in the detection of the separated components as peaks on the chromatogram. Due to its extremely high sensitivity to the mobile phase, this detector cannot be used without sufficient pulse attenuation in the LC pump, and it is also not suitable for gradient applications due to the changing mobile phase composition.
• the detection limit is usually lower than that observed with absorbance detectors.
• Conductivity detectors provide sensitive detection of all charged species. They can be used with LC systems for simple and reliable detection of anions, cations, metals, organic acids and surfactants down to ppb levels.
• the addition of chemical suppressors between the column and the conductivity detector serves to reduce the conductivity of the eluent, allowing the use of gradient elution and determination of ppb levels with minimal baseline drift.
• the eluent is converted to its weakly ionized low conductivity acid (e.g. Na2CO3 to carbonic acid ) to reduce background noise.
• the analyte anions are converted to their corresponding high conductivity acid (e.g. NaCl to HCl) to increase the relative analyte signal.
• the purity of the highly unsaturated fatty acid ester 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 based on the entire highly unsaturated fatty acid ester composition.
• the retention time of the target substance is about 0.5 minutes to about 2 minutes, about 2 minutes to about 4 minutes, about 4 minutes to about 6 minutes, about 6 minutes to about 8 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 12 minutes, about 12 minutes to about 14 minutes, about 14 minutes to about 16 minutes, about 16 minutes to about 18 minutes, about 18 minutes to about 20 minutes, about 20 minutes to about 22 minutes, about 22 minutes to about 24 minutes, about 24 minutes to about 26 minutes, about 26 minutes to about 28 minutes, about 28 minutes to about 30 minutes.
• the retention time of the peak top of the target substance in the first column chromatography is shorter than the retention time of the peak top of the target substance in the second column chromatography.
• the retention time of the peak top of the target substance in the first column chromatography 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 time of the peak top of the target substance in the second column chromatography is about 6 minutes to about 8 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 12 minutes, about 12 minutes to about 14 minutes, about 14 minutes to about 16 minutes, about 16 minutes to about 18 minutes, about 18 minutes to about 20 minutes, about 20 minutes to about 22 minutes, about 22 minutes to about 24 minutes, about 24 minutes to about 26 minutes, about 26 minutes to about 28 minutes, about 28 minutes to about 30 minutes, about 30 minutes to about 35 minutes, about 35 minutes to about 40 minutes, about 40 minutes to about 50 minutes, about 45 minutes to about 50 minutes, about 40 minutes to about 60 minutes, about 45 minutes to about 70 minutes, about 45 minutes to about 80 minutes, about 45 minutes to about 90 minutes, about 45 minutes to about 100 minutes, about 45 minutes to about 120 minutes, about 45 minutes to about 140 minutes, about 45 minutes to about 160 minutes, about 45 minutes to about 180 minutes, about 45 minutes to about 190 minutes, about 50 minutes to about 200 minutes, about 50 minutes to about 220 minutes, about 50 minutes to about 240 minutes,
• It can be 2 minutes, about 32 minutes to about 34 minutes, about 34 minutes to about 36 minutes, about 36 minutes to about 38 minutes, or about 38 minutes to about 40 minutes, or less than 40 minutes, less than 38 minutes, less than 36 minutes, less than 34 minutes, less than 32 minutes, less than 30 minutes, less than 28 minutes, less than 26 minutes, less than 24 minutes, less than 22 minutes, less than 20 minutes, less than 18 minutes, less than 16 minutes, less than 14 minutes, less than 12 minutes, less than 10 minutes, or less than 8 minutes.
• an internal standard can be used during analysis by gas chromatography (GC).
• An internal standard can be added to a sample as a reference marker to determine the relative retention time of an analyte relative to the internal standard or to aid in quantification of the analyte.
• the internal standard can be appropriately selected by one skilled in the art to be a compound that is very similar, but not identical, to the target analyte, for example a deuterated derivative of the target analyte.
• the internal standard can then be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the analyte concentration of the standard, where the standard is a sample of known concentration prepared by one skilled in the art to be used as a reference for the unknown analyte sample to be quantified.
• the highly unsaturated fatty acid ester composition is selected from the group consisting of docosahexaenoic acid, crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinoleic acid, eleostearic acid, mead acid, dihomo-gamma-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, osbondoic acid, sardine acid, t
• the highly unsaturated fatty acid ester composition may have a purity of greater than about 70%, greater than about 80%, greater than about 85%, greater than about 86%, greater than about 90%, greater than about 95%, greater than about 96.5%, greater than about 98%, greater than about 99%, greater than about 99.5%, greater than about 99.8%, or greater than about 99.9%. In some embodiments, the highly unsaturated fatty acid ester composition may have a purity of less than about 100%, less than about 99.9999%, less than about 99.5%, less than 99%, less than 98.5%, or less than about 98%.
• the highly unsaturated fatty acid ester composition may 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%, 99.8-100%.
• the highly unsaturated fatty acid ester composition may be obtained with a yield of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%.
• the highly unsaturated fatty acid ester composition may have a yield of less than 100%, less than 95%, less than 90%, or less than 80%.
• the highly unsaturated fatty acid ester composition may have a yield of 50-80%, 55-90%, 60-90%, 65-95%, 70-95%, 75-100%, 80-100%. In some embodiments, the highly unsaturated fatty acid ester composition may have a recovery rate of greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, or greater than 99.7%. In some embodiments, the highly unsaturated fatty acid ester composition may have a recovery rate of less than 100%, less than 99.9999%.
• 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%.
• the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained in the second column chromatography is an EPA ester, for example, EPA ethyl ester, and its purity is 93% by weight or more, 95% by weight or more, 96% by weight or more, 96.5% by weight or more, 97% by weight or more, 97.5% by weight or more, 98% by weight or more, 98.5% by weight or more, 99% by weight or more, or 99.5% by weight or more based on the entire highly unsaturated fatty acid ester composition.
• EPA ester for example, EPA ethyl ester
• the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained in the second column chromatography is a DGLA ester, for example, DGLA ethyl ester, and the purity of the highly unsaturated fatty acid ester composition 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.
• DGLA ester for example, DGLA ethyl ester
• the purity of the highly unsaturated fatty acid ester composition 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,
• the highly unsaturated fatty acid ester contained in the highly unsaturated fatty acid ester composition obtained in the second column chromatography is an ARA ester, for example, ARA ethyl ester, and has a high purity, being 93% by weight or more, 95% by weight or more, 96% by weight or more, 96.5% by weight or more, 97% by weight or more, 97.5% by weight or more, 98% by weight or more, 98.5% by weight or more, 99% by weight or more, or 99.5% by weight or more, based on the entire highly unsaturated fatty acid ester composition.
• ARA ester for example, ARA ethyl ester
• the highly unsaturated fatty acid ester composition obtained in the second column chromatography contains at least one fatty acid ester selected from C18:0, C20:0, C20:1, and C22:0 esters in an amount of 0.6 wt% or less, 0.3 wt% or less, 0.2 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.03 wt% or less, or 0.01 wt% or less based on the total highly unsaturated fatty acid ester composition.
• Example 1 In this test, the shortening of the loading interval was verified in a column model by comparing a single serial column with a double serial column.
• a sample containing EPA-EE, C20:0 ethyl ester, and C20:1 ethyl ester was prepared from crudely refined sardine oil (the preparation method is the same as in Example 2 below).
• the single serial column was a series of columns with an inner diameter of 20 mm and lengths of 300 mm, 500 mm, and 500 mm.
• the double serial column was a first column with an inner diameter of 20 mm and length of 300 mm, and a second column in which two columns with an inner diameter of 20 mm and length of 500 mm were connected in series.
• the column temperature was 40° C.
• the flow rate of the single serial column was 37 mL/min.
• the flow rate of the first column in the double serial column was 32 mL/min, and the flow rate of 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 by UV at 230 nm.
• EPA-EE began to elute 13 minutes after the start of column chromatography, and the end of elution of C20:0 ethyl ester was 24 minutes after the start of elution of EPA-EE.
• the end time of EPA-EE elution from the first column was 3.5 minutes after the start of column chromatography, while in the second column, the end time of EPA-EE elution was 10.5 minutes after the start of column chromatography, and the end time of C20:1 ethyl ester elution was 12 minutes after the start of EPA-EE elution.
• the fatty acid farthest from EPA-EE is C20:0 ethyl ester.
• the fatty acid farthest 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.
• the loading interval on a single column was shortened by half, from 24 minutes to 12 minutes, by introducing two serial columns, proving to be an effective method for reducing the amount of solvent used.
• Example 2 The conditions for column chromatography were as follows.
• the HPLC equipment used was as follows.
• the pump was NP-KX500 (Nihon Seimitsu Kagaku Co., Ltd.)
• the detector was S-3702 (Soma Optical Co., Ltd.)
• the column oven was CO705 (GL Sciences Co., Ltd.).
• the ODS packing 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
• the second column had an inner diameter of 20 mm and a length of 1000 mm.
• the flow rate of the first column was 32 mL/min, and the flow rate of the second column was 37 mL/min.
• the solvent was HPLC grade methanol (Kanto Chemical Co., Ltd.). Detection was UV at 230 nm. 2.4 g of EPA-EE (80%) was loaded.
• EPA-EE (80%) was prepared as follows: Crude sardine oil was subjected to short path distillation (SPD). The SPD-treated oil was subjected to an ethanolysis reaction with ethyl alcohol in the presence of an alkaline catalyst to form fish oil ethyl ester, EPA-EE. The fish oil ethyl ester was subjected to precision distillation to prepare 80% EPA-EE.
• SPD short path distillation
• the SPD-treated oil was subjected to an ethanolysis reaction with ethyl alcohol in the presence of an alkaline catalyst to form fish oil ethyl ester, EPA-EE.
• the fish oil ethyl ester was subjected to precision distillation to prepare 80% EPA-EE.
• the amount of eluted compounds in each fraction was calculated from the GC peak area and the area ratio between the internal standard, and the elution start of EPA-EE (content 93%) was 3 minutes 30 seconds, and the end of elution was 4 minutes 20 seconds, and the composition of related substances at that time was obtained. Therefore, it was decided that the elution range between 3 minutes 30 seconds and 4 minutes 20 seconds from the start of column chromatography would be sent from the first column to the second column.
• the conditions used for the GC analysis were as follows: The instrument was a 7890A Network GC System (Agilent) with a DB-WAX 30 m x 0.25 mm x 0.25 ⁇ m column. The column temperature was 210°C. The injection temperature was 250°C, the split rate was 1:50, and the injection volume was 1 ⁇ L. A 250°C FID detector was used. A carrier gas of helium with a linear velocity of 31 cm/min was used.
• Example 3 The conditions for column chromatography carried out to concentrate DGLA were as follows.
• the columns used were axial compression columns LC-600 type (Kurita Water Industries Ltd.) with an inner diameter of 600 mm, packed with 50 ⁇ m particle size ODS packing material (SP-120-50-ODS-B, Osaka Soda Co., Ltd.) at a packing pressure of 3.5 MPa, with 75 kg in the first column and 115 kg in the second column.
• the length of the first column was 50 cm, and the length of the second column was 80 cm.
• Methanol was used as the mobile phase, and the flow rate was 28 L/min for both the first and second columns.
• Oil composition 1 (DGLA content, 87.7%) was prepared as follows: Microbial oil 1 derived from a Mortierella microorganism containing 37.2% by weight of DGLA in its fatty acid composition was ethyl-esterified with an alkali catalyst in a conventional manner, and the C20 fraction was concentrated by precision distillation to obtain oil composition 1. First, fractionation of the first column was performed. 8.6 kg (4.5% based on the weight of the adsorbent in the first and second columns) of the oil composition 1 (DGLA content, 87.7%) was loaded onto the first column, and fraction (Fr. 1) was fractionated immediately after the end of injection, with Fr. 1 being fractionated for 180 seconds, Fr. 2 to Fr. 19 for 30 seconds each, and Fr.
• Microbial oil 1 derived from a Mortierella microorganism containing 37.2% by weight of DGLA in its fatty acid composition was ethyl-esterified with an alkali catalyst in a conventional manner, and the C20 fraction was concentrated
• ⁇ GC analysis conditions Apparatus: Capillary gas chromatograph GC-2025, Shimadzu Corporation Column: DB-WAX (30 m x 0.25 mm ID, film thickness: 0.25 ⁇ m) Carrier gas: Hydrogen, 1 mL/min Split ratio: 1:30 Column temperature: 230 ° C. (30 min) Inlet temperature: 250°C Detector type and temperature: FID, 250°C Injection volume: 1 ⁇ L
• the elution start of the target DGLA in the first column was 4.5 minutes and the end of elution was 30 minutes, but by deliberately setting the elution start of the target fraction (DGLA content, 92.7%) sent to the second column to 4.5 minutes and the end of elution to 9 minutes, the elution rate of C22:0, which takes a long time to elute, was suppressed to 0.0%.
• the time for transferring the oil composition from the first column to the second column was within the range (4.5 to 9 minutes) specified in the fractionation of the first column described above.
• Fractionation of the eluate from the second column was started immediately after the completion of transferring the oil composition 1 from the first column to the second column, and 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.
• GC analysis was performed in the same manner as in the fractionation test of the first column, and the recovered fractions were specified so that DGLA was 96.5% or more in the fatty acid composition of the recovered fractions, and the fatty acid composition of the recovered fractions at that time was determined.
• Example 4 The conditions for column chromatography performed to enrich ARA were as follows.
• the HPLC column and mobile phase conditions were the same as those in Example 2.
• Oil composition 2 (ARA content, 79.4%) was prepared as follows: Microbial oil 2 derived from a Mortierella microorganism containing 43.6% by weight of ARA in its fatty acid composition was ethyl-esterified with an alkali catalyst in a conventional manner, and the C20 fraction was concentrated by precision distillation to obtain oil composition 2. First, fractionation of the first column was performed. 1.0 kg (0.55% based on the total weight of the adsorbent of the first and second columns) of the oil composition 2 (ARA content, 79.4%) was loaded onto the first column, and fraction (Fr. 1) was fractionated immediately after the end of injection, with Fr. 1 fractionated for 361 seconds, Fr. 2 to Fr. 13 fractionated for 30 seconds, Fr. 14 to Fr.
• the time for transferring from the first column to the second column was set to the range specified in the above-mentioned fractionation of the first column (6.5 to 12 minutes).
• Fractionation of the eluate from the second column was started after the first column was loaded with oil composition 2, and Fr. 1 was fractionated for 1051 seconds, Fr. 2 to Fr. 31 for 10 seconds each, and Fr. 32 to Fr. 33 for 600 seconds each.
• the GC sample prepared by pretreatment in the same manner as in the fractionation test of the first column was analyzed under the GC conditions below, and the recovered fraction was specified so that ARA was 96.5% or more in the fatty acid composition of the recovered fraction, and the fatty acid composition of the recovered fraction at that time was determined.
• ⁇ GC analysis conditions Apparatus: Capillary gas chromatograph GC-2025, Shimadzu Corporation Column: DB-WAX (30 m x 0.25 mm ID, film thickness: 0.25 ⁇ m) Carrier gas: Hydrogen, constant linear velocity control at 31.8 cm/sec Split ratio: 1:30 Column temperature: 210 ° C. (30 min) Inlet temperature: 250°C Detector type and temperature: FID, 250°C Injection volume: 1 ⁇ L

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  • 2024-06-20 WO PCT/JP2024/022429 patent/WO2024262581A1/ja not_active Ceased
  • 2024-06-20 KR KR1020267001436A patent/KR20260025849A/ko active Pending
  • 2024-06-21 TW TW113123091A patent/TW202515985A/zh unknown

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