Filler for chromatography

Abstract

Provided is a method for producing a desired highly unsaturated fatty acid ester composition efficiently and in high purity. Provided is a method for producing a highly unsaturated fatty acid ester composition, the method comprising purifying a crude composition containing a highly unsaturated fatty acid ester by column chromatography using an ODS silica gel filler, wherein the ODS silica gel filler has an average pore size of 8.5 to 11.9 nm and an average particle size of 15 to 229 μm.

Description

Chromatography packing materials

[0001] The present invention relates to a packing material, specifically an ODS silica gel packing material, used in column chromatography for producing a composition of highly unsaturated fatty acid (PUFA) esters.

[0002] PUFAs are used as medicines and health foods. However, since chemical synthesis of PUFAs is difficult, they are generally produced by extraction from natural sources such as vegetable oils and marine oils that are rich in PUFAs.

[0003] Conventionally, a separation method using column chromatography has been known as a method for separating a specific fatty acid from a mixture containing multiple types of free fatty acids or their esters (Patent Documents 1 and 2). In column chromatography, a mixture containing the components to be separated permeates through a porous, adsorbent packing material in a column. Since each component in the mixture has a different permeation rate depending on its physical properties, it is sequentially and selectively discharged from the column. Octadecylsilylated silica gel (ODS silica gel) packing material is widely used as a packing material for liquid chromatography.

[0004] In order to purify PUFAs and their esters to high purity with high efficiency and low cost, development of a suitable ODS silica gel packing material has been underway (Patent Documents 1 and 2). In addition, a method for improving the performance of ODS silica gel packing materials by end-capping has been reported (Patent Documents 3 and 4).

[0005] International Publication No. 2023 / 111317 Japanese Patent Laid-Open No. 10-310555 Japanese Patent Laid-Open No. 2008-232802 Japanese Patent Laid-Open No. 2002-022721

[0006] There remains a need for an ODS silica gel packing material that can efficiently separate the target highly unsaturated fatty acid ester composition with high purity.

[0007] The inventors of the present invention have found that, in column chromatography, by using an ODS silica gel filler having a specific specific surface area, average pore diameter, carbon content, average particle diameter, carbon density or uniformity coefficient, the object PUFAs can be efficiently produced in response to the above problems, and thus completed the present invention.

[0008] The gist of the present invention is a method for producing a highly unsaturated fatty acid ester composition using the following ODS silica gel filler.

[0009] A method for producing a highly unsaturated fatty acid ester composition, comprising purifying a crude composition containing a highly unsaturated fatty acid ester by column chromatography using an ODS silica gel filler, wherein the ODS silica gel filler has an average pore diameter of 8.5 to 11.9 nm and an average particle diameter of 15 to 229 μm. [[1-2]] The method according to [1-1], wherein the ODS silica gel filler has a specific surface area of 400 to 480 m ２ / g. [[1-3]] The method according to [1-1] or [1-2], wherein the ODS silica gel filler has a carbon content of 13% or more and less than 15%. [[1-4]] The method according to any one of [1-1] to [1-3], wherein the ODS silica gel filler has a carbon density of 0.030 to 0.040. [[1-5]] The method according to any one of [1-1] to [1-4], wherein the ODS silica gel filler has a uniformity coefficient (D40 / D90) of 1.00 to 1.40.

[0010] [1-6] The method according to any one of [1-1] to [1-5], wherein the column chromatography uses methanol or ethanol containing 5% or less water as an eluent. [1-7] The method according to any one of [1-1] to [1-6], wherein the crude composition is obtained by distilling a raw material composition. [1-8] The method according to [1-7], wherein the raw material composition is obtained by esterifying oil obtained from a genetically modified plant, animal, or microorganism. [1-9] The method according to any one of [1-1] to [1-8], wherein the crude composition comprises 50 area % or more of highly unsaturated fatty acid esters. [1-10] The method according to any one of [1-1] to [1-8], wherein the crude composition comprises highly unsaturated fatty acid esters having three or more double bonds and monounsaturated fatty acids. [1-11] The method according to [1-10], wherein the crude composition further comprises saturated fatty acids. [1-12] The method according to [1-10], wherein the crude composition further contains 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. [1-13] The method according to any one of [1-1] to [1-12], wherein the crude composition contains eicosapentaenoic acid (EPA) esters at 78 area% or more, 70 area% or more, 60 area% or more, or 50 area% or more. [1-14] The method according to any one of [1-1] to [1-13], wherein the crude composition contains EPA esters, C20:4n-6 esters, and C18:4n-3 esters at a combined total of 80 area% or more, 70 area% or more, or 60 area% or more. [1-15] The method according to any one of [1-1] to [1-14], wherein the crude composition contains 87 area % or more, 85 area % or more, 80 area % or more, or 70 area % or more of highly unsaturated fatty acid esters having three or more double bonds. [1-16] The method according to any one of [1-1] to [1-15], wherein the highly unsaturated fatty acid ester composition contains 95% or more highly unsaturated fatty acid esters. [1-17] The method according to any one of [1-1] to [1-16], wherein the highly unsaturated fatty acid ester composition contains EPA esters with a purity of 95% or more.[1-18] The method according to any one of [1-1] to [1-17], wherein arachidonic acid (ARA) esters are separated from EPA esters by column chromatography.

[0011] [1-19] The method according to any one of [1-18], wherein the degree of resolution, calculated by dividing the difference in retention time between the ARA ester and the EPA ester separated by column chromatography by the average width of the peaks of the ARA ester and the EPA ester, is 1.15 or more. [1-20] The ester is C １－６ [1-21] The method according to any one of [1-9] to [1-22], wherein the ODS silica gel filler is end-capped.

[0012] [1-23] The method according to any one of [1-1] to [1-22], wherein the column chromatography is a batch type. [1-24] The method according to any one of [1-1] to [1-23], wherein each of the one or more columns used in the column chromatography has a length of 100 to 2000 mm. [1-25] The method according to any one of [1-1] to [1-24], wherein each of the one or more columns used in the column chromatography has an inner diameter of 10 to 1000 mm. [1-26] The method according to any one of [1-1] to [1-25], wherein the ODS silica gel packing has an average pore diameter of 8.5 to 11.5 nm and an average particle diameter of 15 to 60 μm.

[0013] [2-1] An octadecylsilane (ODS) silica gel packing material for use in packing a column in column chromatography purification in the production of a highly unsaturated fatty acid ester composition, the ODS silica gel packing material having an average pore diameter of 8.5 to 11.9 nm and an average particle diameter of 15 to 229 μm. [2-2] ２The ODS silica gel packing material according to [2-1], having a specific surface area of ​​1.00 to 1.40. [2-3] The ODS silica gel packing material according to [2-1] or [2-2], having a carbon content of 13% or more and less than 15%. [2-4] The ODS silica gel packing material according to any one of [2-1] to [2-3], further having a carbon density of 0.030 to 0.040. [2-5] The ODS silica gel packing material according to any one of [2-1] to [2-4], having a uniformity coefficient (D40 / D90) of 1.00 to 1.40.

[0014] [2-6] The ODS silica gel packing according to any one of [2-1] to [2-5], which has been end-capped. [2-7] A column for purifying a highly unsaturated fatty acid ester composition, packed with the ODS silica gel packing according to any one of [2-1] to [2-6]. [2-8] An analytical device equipped with the column for purifying a highly unsaturated fatty acid ester composition according to [2-7]. [2-9] The ODS silica gel packing according to any one of [2-1] to [2-6], which has an average pore diameter of 8.5 to 11.5 nm and an average particle diameter of 15 to 60 μm.

[0015] Figure 1 is a graph showing the elution profiles of EPA-E and C20:4n-6 ethyl ester in Example 1. Figure 2 shows the formula for calculating the resolution of two peaks. Figure 3 shows how to calculate the symmetry factor.

[0016] The present invention will be described in more detail below. The following abbreviations may be used in the present specification: PUFA: highly unsaturated fatty acid DHA: docosahexaenoic acid DHA-E: docosahexaenoic acid ethyl ester EPA: eicosapentaenoic acid EPA-E: eicosapentaenoic acid ethyl ester DPA: docosapentaenoic acid DPA-E: docosapentaenoic acid ethyl ester ARA: arachidonic acid ARA-E: arachidonic acid ethyl ester GLA: gamma-linolenic acid GLA-E: gamma-linolenic acid ethyl ester DGLA: dihomo-gamma-linolenic acid DGLA-E: dihomo-gamma-linolenic acid ethyl ester

[0017] Composition As used herein, the term "crude composition" refers to a composition containing one or more ester derivatives of PUFAs. The crude composition can be obtained from a raw material containing PUFAs as constituent fatty acids. For example, suitable crude compositions may be obtained from raw material compositions obtained by esterifying natural oils and fats, including plant and animal oils and fats, as well as from raw material compositions obtained by esterifying oils obtained from genetically modified plants, animals, and microorganisms, including yeast and filamentous fungi. For example, a composition obtained by esterifying oils obtained from microorganisms of the genus Mortierella, such as filamentous fungi, can be used. Examples include compositions obtained by ethyl esterification of fish oil, algae oil, and microalgae oil, as well as vegetable oils such as borage oil, scutellaria oil, and evening primrose oil. In one embodiment, the raw material composition is a composition obtained by ethyl esterification of fish oil. In another embodiment, the raw material composition is a composition obtained by ethyl esterification of algal oil.

[0018] In one embodiment of the present invention, the crude composition typically contains PUFA and at least one fatty acid and / or its ester. In one aspect, the crude composition contains 50 area % or more highly unsaturated fatty acid esters. In another aspect of the present invention, a composition containing highly unsaturated fatty acid esters obtained by distilling a raw material composition can be used as the crude composition. In one embodiment of the present invention, a composition containing 75 area % or more EPA ethyl esters obtained by precision distillation of fatty acid ethyl esters at a vacuum of 0.2 Torr or less and a temperature of 190°C or less for the entire column can be used as the crude composition.

[0019] In one embodiment of the present invention, the range of PUFAs separated and purified as ester derivatives is not particularly limited. From the viewpoint of application as pharmaceuticals or functional foods, naturally occurring PUFAs are preferred. For example, PUFAs having 18 or more carbon atoms and three or more double bonds are included, specifically DHA, DPA, EPA, ARA, GLA, DGLA, C18:3, C19:4, C20:4, and C21:5.

[0020] In one embodiment of the present invention, the source of PUFA is not particularly limited as long as it contains the target substance. It can be selected from animal oils such as fish oil and liver oil, vegetable oils such as fruit oil, and microbial oils derived from fungi and algae, depending on the target substance. For example, as mentioned above, DHA, DPA, and EPA are known to be found in large amounts in fish oil. Furthermore, GLA is known to be found in large amounts in evening primrose extract. The selection of an appropriate source is easy for those skilled in the art.

[0021] It is known that oils and fats containing one or more of DHA, EPA, ARA, etc. can be obtained by culturing certain microorganisms, particularly marine fungi or algae (see, for example, Japanese Patent Application Laid-Open Nos. 7-87988, 7-8268, 7-75556, and 1-304892). These microbial oils and fats often have a simpler fatty acid composition than fish oils and the like, and therefore the separation and purification method of the present invention can be applied more efficiently. Therefore, cultured cells of microorganisms that produce PUFAs are an example of a preferred supply source.

[0022] It has been reported that fungi such as those belonging to the genera Thraustochytrium and Schizochytrium accumulate lipids containing DHA and DPA in their cells (J. Am. Oil Chem. Soc., Vol. 73, No. 11, p. 1421 (1996)). For example, when highly purifying DHA and / or DPA, cultured cells of the above-mentioned microorganisms and microorganisms of similar species can be advantageously used. Of course, the cultured cells are not limited to those exemplified above, and any cells derived from a bacterial species capable of producing DHA may be used.

[0023] In one aspect, culture conditions such as medium composition, temperature, and pH are selected depending on the type of microorganism to be cultured. Selection of appropriate culture conditions is easily within the skill of a person skilled in the art. For example, the genera Thraustochytrium and Schizochytrium accumulate lipid components including DHA and DPA when cultured in a liquid medium containing 50% artificial seawater and based on glucose and corn starch liquor at a temperature near room temperature, a weakly acidic pH, and under aerobic conditions.

[0024] In one aspect, the cultured cells of the microorganism are dried as needed, and then crushed or otherwise treated according to a conventional method, and the lipids are extracted. When other sources such as animal and vegetable oils are used, lipids can be extracted after pretreatment according to the type of source.

[0025] In one aspect of the present invention, a composition containing PUFA esters obtained by esterifying lipids extracted from the above-mentioned sources is used as the crude composition. The ester group of the PUFA ester is typically an alkyl group. However, it may also be an alkenyl group such as vinyl, an aryl group such as phenyl, or an arylalkyl group such as benzyl. Examples of the alkyl group include alkyl groups having 1 to 6 carbon atoms, and alkyl groups having 1 to 4 carbon atoms. Examples of pharmaceuticals or functional foods to be administered directly to humans include ethyl esters.

[0026] Appropriate reaction conditions for esterification are known to those skilled in the art. For example, after dissolving the lipid in an organic solvent such as hexane, an alkali (e.g., ethanol containing 1N potassium hydroxide) is added and esterification can be carried out at a temperature of about 30 to 80°C. After separating the organic phase, the mixture is concentrated according to a conventional method to obtain a raw material composition containing the ester derivative.

[0027] In one aspect of the present invention, a composition containing a highly unsaturated fatty acid ester obtained by the above-mentioned esterification reaction can be subjected to HPLC as a crude composition without a preliminary purification procedure. In one embodiment of the present invention, after preparing a crude composition containing a highly unsaturated fatty acid ester obtained by the esterification reaction, the target highly unsaturated fatty acid ester composition is obtained by a single HPLC procedure without a preliminary purification procedure.

[0028] In one embodiment of the present invention, a crude composition comprises polyunsaturated fatty acid (PUFA) esters. In one aspect, the crude composition comprises, for example, PUFA esters having three or more double bonds and monounsaturated fatty acid esters, or PUFA esters having three or more double bonds, monounsaturated fatty acid esters, and saturated fatty acid esters. In one aspect, the crude composition contains, as PUFA esters, for example, EPA esters at 78 area% or more, 75 area% or more, 70 area% or more, 60 area% or more, or 50 area% or more. Furthermore, the crude composition contains, for example, EPA esters, C20:4n-6 esters, and C18:4n-3 esters combined at 80 area% or more, 70 area% or more, or 60 area% or more. Furthermore, the crude composition contains PUFAs having three or more double bonds at 87 area% or more, 85 area% or more, 80 area% or more, or 70 area% or more.

[0029] In one embodiment of the present invention, the highly unsaturated fatty acid ester composition obtained by the method of the present invention contains a target PUFA ester at a higher concentration than the crude composition. The target PUFA ester can be an ester, for example, an ethyl ester, of any of EPA, DHA, DGLA, and ARA.

[0030] As used herein, the term "fatty acid" refers to long-chain aliphatic carboxylic acids of various chain lengths, ranging from C12 to C22 (where the number refers to the total number of carbon atoms in the chain). The predominant chain length is C16 to C22. Fatty acid structures can be represented using a simple "X:Y" notation, where X is the total number of carbon atoms in the particular fatty acid and Y is the number of double bonds. For example, a saturated fatty acid with 20 carbon atoms may be represented as "C20:0," a monounsaturated fatty acid with 18 carbon atoms may be represented as "C18:1," etc., and ARA may be represented as "C20:4,n-6," etc. The "n-" indicates the position of the double bond beginning from the methyl end of the fatty acid; for example, "n-6" indicates that the double bond begins at the sixth position counting from the methyl end of the fatty acid. This method is well known to those skilled in the art, and fatty acids represented according to this method can be easily identified by those skilled in the art.

[0031] Fatty acids are carboxylic acids with long aliphatic chains, which can be either saturated or unsaturated. Fatty acids are usually produced industrially by hydrolysis of triglycerides or phospholipids from natural sources. Some are also produced synthetically. Regardless of the method of production, purification methods are required to obtain pure products for food, cosmetic, or industrial use.

[0032] As used herein, the term "polyunsaturated fatty acid (PUFA)" refers to a fatty acid with more than one double bond. The PUFA herein may be in the form of an ester. The ester is typically an alkyl ester, e.g., C １ -C ６ Alkyl ester, or C １ -C ４ It is an alkyl ester. An example of an ester is an ethyl ester.

[0033] In one aspect of the invention, the PUFAs include EPA, DPA, DHA, DGLA, ARA, stearidonic acid, C18:3, C19:4, C20:4, and C21:5.

[0034] Typical fatty acid esters are similar to those defined for PUFAs above. In one aspect, the crude composition comprises at least one of a fatty acid having 18 to 22 carbon atoms and a fatty acid ethyl ester. In one aspect, the crude composition comprises a PUFA and at least one fatty acid selected from C18:0 (stearic acid), C20:0 (arachidic acid), C20:1 (eicosenoic acid), C22:0 (behenic acid), and esters thereof. In one aspect, the crude composition comprises the fatty acid having 18 to 22 carbon atoms and the fatty acid ethyl ester in an amount of 50 area % or more, 40 area % or more, 30 area % or more, or 20 area % or more. In one embodiment of the present invention, the at least one fatty acid ester described above is removed by column chromatography. By the purification method of the present invention, the amount of the fatty acid having 18 to 22 carbon atoms and the fatty acid ethyl ester is reduced to 5 area % or less.

[0035] Column Chromatography As used herein, "column chromatography" refers to a method of separating substances by utilizing differences in the adsorption capacity of the substances to a packing material contained in a column. In one embodiment, column chromatography is performed by utilizing 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(s) composed of finely divided substances and / or materials with capillary channels. 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, HPLC, simulated moving bed chromatography, actual moving bed chromatography, and supercritical fluid chromatography (SFC).

[0036] In one embodiment of the present invention, column chromatography is performed in a batch mode. The batch mode column chromatography in one embodiment of the present invention is characterized by having a simpler configuration and being easier to introduce than simulated moving bed column chromatography.

[0037] The terms "non-polar" and "polar" to describe a mobile phase or a highly unsaturated fatty acid ester can be used relative to one another. For example, "non-polar" can refer to the least polar solvent in the mobile phase, while "polar" can refer to a solvent in the mobile phase that is more polar than the "non-polar" solvent.

[0038] In one aspect of the invention, column chromatography comprises passing the crude composition through one or more columns, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 columns. When two or more columns are used, they can be used connected in series or in parallel. Any known column can be used in the claimed methods.

[0039] In one aspect of the invention, the length of the column is 25 mm or more, 50 mm or more, 100 mm or more, 200 mm or more, or 8000 mm or less, 4000 mm or less, 2000 mm or less, 1500 mm or less, 1200 mm or less, or 25 to 8000 mm, 25 to 4000 mm, 25 to 2000 mm, 100 to 2000 mm, 200 to 2000 mm, 200 to 1500 mm, or 200 to 1200 mm. One or more columns can be the same or different lengths.

[0040] In one embodiment of the present invention, the column is cylindrical having an outer diameter, an inner diameter, and a length. In certain embodiments, the column is a preparative chromatography column. Preparative chromatography columns can have an inner diameter of 5 mm or more, 10 mm or more, 20 mm or more, 50 mm or more, 100 mm or more, or 4000 mm or less, 2000 mm or less, 1000 mm or less, 800 mm or less, or 5 to 4000 mm, 10 to 2000 mm, 10 to 1000 mm, 20 to 1000 mm, or 50 to 800 mm, and a length of 100 to 5000 mm, 20 to 2000 mm, 100 to 2000 mm, or 100 to 1500 mm. In certain embodiments, the column is an analytical chromatography column. Analytical columns can have an inner diameter of 1 to 100 mm and a length of 10 to 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. One or more columns can have the same or different outer or inner diameters.

[0041] In one aspect of the present invention, the parameters representing the performance of the ODS silica gel packing are calculated based on a graph in which the vertical axis represents the elution amount of the target substance and the horizontal axis represents the retention time. Examples of the parameters include resolution, symmetry factor, productivity, and HETP. Unless otherwise specified in this specification, these parameters are calculated based on the graph.

[0042] In one embodiment of the present invention, the elution amount of the target substance in the graph can be measured by performing column chromatography using an ODS silica gel packing material, obtaining fractions, and quantitatively analyzing the target substance contained in each fraction. Examples of quantitative analysis methods include GC analysis. In one embodiment, the chromatography involves collecting fractions every predetermined time (e.g., 5 seconds, 10 seconds, 20 seconds, or 30 seconds) after the start of elution of the target substance has been confirmed by observing absorbance (e.g., UV absorption, refractive index, etc.), and continuing fraction collection until the elution of the target substance has ended. The time for fraction collection can be appropriately determined by one skilled in the art based on the amount of packing material used in the column, the flow rate of the eluent, the load of the substance to be applied to the column, etc.

[0043] As used herein, "peak" refers to a peak in a graph showing retention time on the horizontal axis and the elution amount of the target substance (elution amount based on quantitative analysis results or signal intensity measured by chromatography) on the vertical axis. After the target substance starts to elute, the elution amount value shown in the graph increases, indicating a "peak." As used herein, "chromatogram" refers to a graph showing signal intensity (UV absorption, refractive index, etc.) measured by chromatography on the vertical axis and retention time on the horizontal axis.

[0044] As used herein, "peak top" refers to the point at which the value of each peak is maximum. As used herein, "peak width" refers to the width from the start point to the end point of the peak. The width from the start point to the end point of the peak is the time from the start to the end of the elution of the target substance in the graph, and is shown as W in Figures 2 and 3. １ , W ２ , and W.

[0045] As used herein, "separation" refers to a process characterized by the spatial separation of components of a composition containing highly unsaturated fatty acid esters due to their differential distribution between phases in relative motion (e.g., a mobile phase and a stationary phase). Separation results from loading a sample onto a column and subsequent elution from the column.

[0046] As used herein, "fractionation" refers to a separation process in which a volume of a mixture is divided into several smaller volumes during a phase transition, where the composition varies along 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 phases.

[0047] As used herein, the term "fraction" refers to a crude composition loaded onto a column and subjected to chromatography, and an eluent received in the column for developing and eluting the crude composition, which is collected in small amounts at specific time intervals after elution from the column. As used herein, "fractionation" refers to the process of obtaining a fraction.

[0048] In one aspect of the present invention, the sample is a crude composition. In one embodiment, the sample is loaded onto the top of the stationary phase of a packed column, where "top" refers to the end of the stationary phase that first receives the mobile phase as it elutes through the column. The crude composition can be subjected to column chromatography by injector, pump, or direct application of the sample onto the top of the stationary phase. The sample can be mixed with a minimal amount of mobile phase or other solvent for loading.

[0049] As used herein, "elution" means that a component loaded onto a column passes through the column and flows out as a solution in the mobile phase. As used herein, "eluent" means a liquid used in chromatography as a mobile phase that contacts the stationary phase and passes through the gaps or surface of the stationary phase. In chromatography, the eluent is used to develop and elute components such as PUFAs or ester derivatives thereof that are adsorbed to the column. As used herein, the eluent is sometimes referred to as the mobile phase.

[0050] In one embodiment of the present invention, the eluate from the column is collected together with the PUFAs or ester derivatives thereof as fractions. The fractions are also called eluates and may contain components such as the eluate and the PUFAs or ester derivatives thereof.

[0051] As used herein, the term "eluent" refers to the mobile phase eluted from the column, which may contain components contained in the crude composition loaded onto the column. In one embodiment, the mobile phase eluted from the column is a solution containing components contained in the crude composition.

[0052] As used herein, the term "eluate" refers to components discharged from a column in column chromatography. As used herein, the term "components" refers to components contained in a crude composition subjected to column chromatography, and includes highly unsaturated fatty acid esters and fatty acid esters.

[0053] As used herein, "biphenyl," "C30," "C22," "C18," "C8," "C5," and "C4" refer to functional groups present on the column packing material (stationary phase). For example, a biphenyl column exposes materials flowing through the column to unsubstituted biphenyl groups, while a C18 column exposes materials flowing through the column (e.g., mobile phase and components) to unsubstituted straight- or branched-chain 18-carbon alkyl groups.

[0054] As used herein, "chromatographic conditions" refers to the parameters under which column chromatography is operated, including, for example, packing pressure, mobile and stationary phase composition, slurry concentration, pressure at which the column is operated, column temperature, mobile phase temperature, mobile phase gradient, mobile phase flow rate, column type used, detection instrumentation and parameters used, sample preparation protocol employed, sedimentation time and pressure at which sedimentation is performed, and stand time and pressure at which stand is performed.

[0055] As used herein, "gradient" refers to a change in mobile phase composition over time while column chromatography is performed. The composition of the mobile phase can change as the solvent is eluted through the column. Different mobile phases can be added at increasing percentages over time during elution.

[0056] As used herein, "purity" refers to a ratio indicating the content of a major component of a composition, and can be calculated, for example, from the results of GC or HPLC measurement using an internal standard. In one embodiment of the present specification, the major component is a highly unsaturated fatty acid ester, such as an EPA ester, a DGLA ester, or an ARA ester.

[0057] As used herein, the term "target product" refers to a target highly unsaturated fatty acid ester, such as an EPA ester, a DGLA ester, or an ARA ester, obtained from a single fraction or a combination of multiple fractions obtained by elution and fractionation.

[0058] In one embodiment of the present invention, the column chromatography is reversed-phase column chromatography. In one embodiment of the present invention, the column chromatography uses the packing material of the present invention as a stationary phase.

[0059] In one embodiment of the present invention, a variety of different types of octadecyl silica (ODS) can be used as the stationary phase, including fully end-capped, partially end-capped, and base-deactivated.

[0060] In one embodiment of the invention, the amount of 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.

[0061] In one embodiment of the present invention, the column chromatography comprises elution with a mobile phase comprising one or more of water, an organic solvent, or supercritical carbon dioxide. In some embodiments of these methods, the mobile phase can comprise 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 used. For reversed-phase stationary phases for non-polar highly unsaturated fatty acid esters, a polar 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.

[0062] In one embodiment of the present invention, the solvent used as the mobile phase is an organic solvent selected from alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane. Examples of alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. Methanol and ethanol are preferred. Methanol is more preferred. Examples of ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE). Examples of esters include methyl acetate and ethyl acetate. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone (MIBK). An example of a nitrile is acetonitrile.

[0063] In one embodiment of the present invention, the mobile phase may further contain additives, including buffers and pH adjusters. The choice of additive may be determined based on the mobile phase used, the stationary phase used, and the component to be purified. In some embodiments, the mobile phase contains an additive selected from one or more of formic acid, trifluoroacetic acid, heptafluorobutyric acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide. In some embodiments, the mobile phase may be additive-free.

[0064] In one aspect of the invention, the column chromatography comprises a mobile phase gradient. In one aspect of the invention, the column chromatography comprises the purification of one or more highly unsaturated fatty acid esters. In one embodiment of the present invention, the highly unsaturated fatty acid ester is selected from the group consisting of docosahexaenoic acid, crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosapentaenoic acid, and herring acid. acid), propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceric acid, silicic acid, geddic acid and polyunsaturated fatty acids selected from one or more of: hexatriacontylic acid, celloplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid. In one aspect of the invention, the ester is a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl ester.In one embodiment of the invention, column chromatography comprises purification of eicosapentaenoic acid ethyl ester.

[0065] In some embodiments, the crude composition contains the desired highly unsaturated fatty acid ester at a purity of 50% or more, 60% or more, 70% or more, 80% or more, and / or less than 85%, less than 90%, or less than 95%. In some embodiments, the highly unsaturated fatty acid ester composition purified by the methods of the present invention contains the desired highly unsaturated fatty acid ester at a purity of 85% or more, 86% or more, 90% or more, 95% or more, 96.5% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more, and / or less than 97%, less than 98%, less than 99.0%, less than 99.95%, or less than 99.99%.

[0066] In some embodiments, a solvent gradient can be used as the mobile phase during elution. The primary purpose of gradient elution is to elute strongly retained components from the column faster while weakly retained components elute more slowly, so that the eluted components produce well-separated peaks on a chromatogram upon detection. For example, in reversed-phase chromatography, starting with a low content of nonpolar solvent in the eluent allows weakly retained components to separate. Strongly retained components will remain on the adsorbent surface at the top of the column or will migrate very slowly. Increasing the amount of nonpolar components (e.g., acetonitrile) in the eluent allows strongly retained components to migrate faster due to steadily increasing competition for adsorption sites by the nonpolar solvent.

[0067] Thus, in reversed-phase chromatography used for non-polar highly unsaturated fatty acid esters, the solvent at the beginning of the chromatography elution may contain a high percentage of polar solvent A, e.g., water, selected from 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Solvent B can be a solvent less polar 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 elutes through the stationary phase and the column, a gradient will result in a gradual increase in the concentration of solvent B over time. In some embodiments, a single solvent composition can also be used as the mobile phase. In some embodiments, a single solvent may be used, alone or in combination, such as one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other solvent known in the art.

[0068] In some embodiments, the rate of increase of solvent B over time can be constant. In some embodiments, there is no gradient and the mobile phase is isocratic during elution. In some embodiments, different rates of increase in the percentage of solvent B at different time ranges within the chromatography can be utilized. In some embodiments, the mobile phase can be isocratic during certain time ranges of the chromatography and include a gradient during other time ranges.

[0069] In one embodiment of the present invention, multiple solvents used in the mobile phase can be stored separately in a mobile phase supply and mixed using a pump before elution through the column. The mobile phase supply includes a mobile phase source and a solvent delivery system. Such a solvent delivery system is a pumping device, such as a commercially available column chromatography pump, that provides the solvent or mobile phase to the column. Such pumps generally provide pulse-free flow, flow rates ranging from 0.1 to 100 L / min, precise control of flow rate, high pressure (up to 6000 psi), and corrosion- and solvent-resistant components. Reciprocating pumps consist of a small chamber into which the solvent is pumped by the back-and-forth motion of a motor-driven piston. Two check valves, which alternately open and close, control the direction and flow of the solvent into and out of the cylinder. Single-piston pumps use specially designed cams to enable very rapid refill times and produce a more continuous flow. The disadvantages of pulsed flow with reciprocating pumps are often overcome by using a pulse damper. The use of a dual piston pump, which operates with pistons moving out of phase with each other, provides a rational solution for pulse-free fluid delivery.

[0070] Linear velocity in a column represents the speed at which a fluid passes through the cross section of the column. Linear velocity (m / time) = Flow rate (m ３ / time) / column cross-sectional area (m ２ )) can be 0.2 to 20.0 m / hr, 1.0 to 15.0 m / hr, 1.0 to 10.0 m / hr, 1.5 to 10.0 m / hr, or 2.0 to 9.0 m / hr. In one embodiment, the linear velocity is 4.0 to 9.0 m / hr.

[0071] In one embodiment of the present invention, the column chromatography is carried out at room temperature or above room temperature. Preferably, the method is carried out at a temperature above room temperature. The first and second column chromatography steps can be carried out at the same temperature or at different temperatures, but are preferably at the same temperature.

[0072] In one embodiment of the present invention, the temperature higher than room temperature is 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, or 60°C or lower, 55°C or lower, 50°C or lower, 45°C or lower, or 20 to 60°C, 25 to 55°C, 30 to 50°C, or 35 to 45°C.

[0073] In one embodiment of the present invention, column chromatography can be performed using two or more columns. Column chromatography can be performed using a known fixed-bed chromatography device. Such column chromatography is called fixed-bed chromatography.

[0074] In one embodiment of the present invention, an analytical device equipped with a column for purifying a highly unsaturated fatty acid ester composition can include a chromatography column, a pump, and a sample injection section. In some embodiments, a detector is used to monitor the mobile phase eluting from the column for the presence of a component or components. Detection methods known in the art (e.g., mass spectrometry (MS), UV / Vis absorbance, fluorescence, refractive index, or conductivity) can be used.

[0075] In other embodiments, any of a variety of standard column chromatography detectors can be used to detect the eluate immediately after elution from the column. In other embodiments, each fraction can be individually monitored for the presence of components by analysis. In some embodiments, the analysis is fatty acid analysis using gas chromatography.

[0076] In some embodiments, the eluate from the column is detected as a peak in a chromatogram. The retention time of the peak on the chromatogram is used to identify the compound, and the height (or area) of the peak on the chromatogram is proportional to the amount of eluate in the crude composition. "Retention time" is the time required for the eluate to pass through the column and is measured from the time of injection (or loading) of the crude composition to the time of elution. Ideally, each eluate of interest will have a characteristic retention time. However, the retention of the eluate will vary depending on the eluent, stationary phase, temperature, and column chromatography settings. Therefore, the retention time of the eluate is compared 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 when used for quantitative purposes, short response time, and ease of operation. Such detectors include, but are not limited to, UV / Vis absorbance detectors, photodiode array detectors, fluorescence detectors, refractive index detectors, and conductivity detectors.

[0077] In one embodiment of the invention, 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.

[0078] 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 (peaks on overlapping chromatograms) because the characteristic spectra for each of the unresolved components are likely to be different.

[0079] Fluorescence detectors are useful for detecting compounds that exhibit chemiluminescent properties such as fluorescence or phosphorescence. They are at least an order of magnitude more sensitive than UV absorbance detectors. Fluorescence is typically observed by detecting grating-separated emission radiation at a 90-degree angle relative to the excitation beam. The number of fluorescent species can be enhanced by post-column derivatization (PCD) of the eluted compounds (or pre-column derivatization of the sample itself) with special reagents.

[0080] Refractive index (RI) detectors respond to almost all solutes. Differences in the refractive index of the reference mobile phase relative to the column effluent result in the detection of separated components as peaks on the chromatogram. Due to its extremely high sensitivity to the mobile phase, this detector cannot be used without sufficient pulse attenuation in the LC pump, and it is also not suitable for gradient applications due to the varying mobile phase composition. The detection limit is usually lower than that observed with absorbance detectors.

[0081] Conductivity detectors offer sensitive detection of all charged species. They can be used with LC systems for simple and reliable detection of anions, cations, metals, organic acids, and surfactants down to ppb levels. The addition of a chemical suppressant between the column and the conductivity detector serves to reduce the conductivity of the eluate, allowing the use of gradient elution and ppb-level determination with minimal baseline drift. For typical determination of low levels of anions, the eluate is diluted with a weakly ionized, low-conductivity acid (e.g., Na ２ CO ３ Concurrently, component anions are converted to their corresponding high conductivity acids (e.g., NaCl to HCl), increasing the relative component signals.

[0082] In one aspect of the invention, the retention time of the target can be between 0.5 minutes and 2 minutes, between 2 minutes and 4 minutes, between 4 minutes and 6 minutes, between 6 minutes and 8 minutes, between 8 minutes and 10 minutes, between 10 minutes and 12 minutes, between 12 minutes and 14 minutes, between 14 minutes and 16 minutes, between 16 minutes and 18 minutes, between 18 minutes and 20 minutes, between 20 minutes and 22 minutes, between 22 minutes and 24 minutes, between 24 minutes and 26 minutes, between 26 minutes and 28 minutes, between 28 minutes and 30 minutes, between 30 minutes and 32 minutes, between 32 minutes and 34 minutes, between 34 minutes and 36 minutes, between 36 minutes and 38 minutes, or between 38 minutes and 40 minutes, or greater than 0.5 minutes, greater than 1 minute, greater than 1.5 minutes, greater than 2 minutes, or greater than 5 minutes, and / or less than 50 minutes, less than 40 minutes, less than 30 minutes, or less than 20 minutes.

[0083] In one aspect of the present invention, an internal standard can be used during GC analysis.The internal standard can be added to a sample as a reference marker to determine the relative retention time of a component relative to the internal standard or to aid in the quantification of the component.The internal standard can be appropriately selected by those skilled in the art to be a compound that is very similar to, but not identical to, the target component, such as a deuterated derivative of the target component.When used for quantification purposes, the internal standard can then be used for calibration by plotting the ratio of the component signal to the internal standard signal as a function of the standard component concentration, where the standard is a sample of known concentration prepared by those skilled in the art to be used as a reference for the unknown component sample to be quantified.

[0084] In one embodiment, the highly unsaturated fatty acid ester composition may comprise any of docosahexaenoic acid, crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, osbondo acid, sardine acid, tetracosapentaenoic acid, herring acid, propionic acid, butyric acid, valeric acid, and caproic acid. , enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboseric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, russellic acid, silicic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, tetracontylic acid, or an ester thereof.

[0085] In one embodiment of the present invention, the packing material is preferably used as a packing material for column chromatography, particularly as a packing material for high-performance liquid chromatography or supercritical fluid chromatography, but is not limited thereto. For example, the packing material may be used as a carrier or adsorbent for thin-layer chromatography, or in the form of a filter aid or filter.

[0086] In one embodiment of the present invention, the packing material can be packed into the column as a slurry with a liquid or as a powder. In one embodiment of the present invention, the packing material is an ODS silica gel packing material. In one embodiment of the present invention, the packing material is spherical or non-spherical beads.

[0087] As used herein, the "specific surface area" of a filler refers to the surface area per unit mass of a substance. Furthermore, the "average pore diameter" of a filler refers to the diameter of the filler's pores, calculated by the formula d = 4 * V / A, where V is the total pore volume and A is the surface area. This is the diameter of a hypothetical uniform cylindrical pore with the same volume and area. As used herein, the specific surface area and average pore diameter are calculated by a common method, such as the BET method, i.e., the standard method of ISO 9277:2010. This method is based on the BET theory (Brunauer, Emmett, and Teller), and is typically determined based on nitrogen adsorption data. Measurement of specific surface area by the BET method is described in detail in "Characterization of Porous Solids and Powders: Surface Area, Pore Size, and Density" by Lowell, Shields, Thomas, and Thommes, Springer, Dordrecht, 2006.

[0088] In one embodiment of the present invention, the specific surface area of ​​the filler is 300 m ２ / g or more, 350m ２ / g or more, or 400m ２ / g or more, or 600m ２ / g or less, 550m ２ / g or less, or 500m ２ / g or less, or 300 to 600m ２ / g, 350-550m ２ / g or 400-500m ２ / g.

[0089] In one aspect of the invention, the average pore size of the filler can be 7.0 nm or more, 8.0 nm or more, or 8.5 nm or more, or 13.0 nm or less, 12.0 nm or less, 11.9 nm or less, 11.7 nm or less, or 11.5 nm or less, or 7 to 13 nm, 8 to 12 nm, 8.5 to 11.9 nm, 8.5 to 11.7 nm, or 8.5 to 11.5 nm.

[0090] As used herein, the "total pore volume" of a filler refers to the volume of pores contained in 1 g of a porous material. The total pore volume is generally measured by nitrogen gas adsorption. Measurement by nitrogen gas adsorption is described in detail in "Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density" by Lowell, Shields, Thomas and Thommes, Springer, Dordrecht, 2006.

[0091] In one embodiment of the present invention, the total pore volume of the filler can be 0.9 mL / g or more, 1.0 mL / g or more, or 1.3 mL / g or less, 1.2 mL / g or less, or 0.9 to 1.3 mL / g, 1.0 to 1.2 mL / g.

[0092] In this specification, the "pore size distribution" of a filler refers to the distribution of pore sizes in the pore structure of a porous solid, and gas adsorption and mercury intrusion methods are often used to measure this.

[0093] In one embodiment of the present invention, the pore size distribution of the filler may be such that, when the average particle size is 50 μm, at least 90% of the particles have a particle size of 30 μm to 70 μm, and when the average particle size is 20 μm, at least 90% of the particles have a particle size of 10 μm to 30 μm.

[0094] As used herein, the "carbon content" of a filler refers to the weight percent of carbon in the filler. Generally, substantially all of the carbon content is obtained by C18 functionalization of silica particles. The carbon content in this specification is calculated by performing elemental analysis by the combustion method using an organic elemental analyzer (Microcoder JM11; J-Science Co., Ltd.). The measurement method by the combustion method is described in ISO 21068-2.

[0095] In one aspect of the invention, the carbon content can be 12.0 wt% or more, 13.0 wt% or more, or 14.0 wt% or more, and 16.5 wt% or less, 16.0 wt% or less, or 15.5 wt% or less, or 12.0 to 16.5 wt%, 13.0 to 16.0 wt%, or 14 to 15.5 wt%.

[0096] In this specification, the "carbon density" of a filler is calculated as the carbon content of the filler / specific surface area. In one embodiment of the present invention, the carbon density of the filler is 0.0002 wt% / (g / m ２ ) or more, 0.0003% by weight / (g / m ２ ) or more, or 0.00045 wt% (g / m ２ ) or less, 0.00040% by weight (g / m ２ ) or less, or 0.0002 to 0.00045 wt% (g / m ２ ), 0.0003 to 0.0004% by weight (g / m ２ ) can be.

[0097] In this specification, the "average particle size" of a filler refers to the median diameter (D50) of the particle size distribution of the filler expressed as a cumulative distribution, i.e., the diameter at which the larger and smaller particles are equal when the powder is divided into two. The particle size of the filler is essentially determined by the particle size of the porous particles that serve as the carrier. Dv(10) refers to the 10% diameter of the volume-based cumulative distribution of silica particles. The average particle size and Dv(10) of the filler in this specification are calculated based on the sieve method specified in JIS K0069 using a sieve (diameter 75 mm, JIS Z8801 compliant).

[0098] In one aspect of the invention, the average particle size of the filler can be 15 μm or more, 20 μm or more, or 30 μm or more, or 100 μm or less, 90 μm or less, or 80 μm or less, or 15 to 100 μm, 20 to 90 μm, or 30 to 80 μm.

[0099] If the average particle size of the packing material is equal to or greater than the lower limit, the pressure loss when the packing material is packed into a column and a liquid is passed through the column is reduced, thereby increasing the liquid passing rate and improving the productivity of the separation process.On the other hand, if the average particle size is equal to or less than the upper limit, the efficiency of the column is not reduced and separation performance can be maintained.

[0100] In this specification, the Dv(10) of a filler refers to the size of the particle at 10% when the particles are arranged from the largest to the smallest on a volume basis. In one embodiment of the present invention, the Dv(10) of the filler can be 10 μm or more, 15 μm or more, 20 μm or more, or 25 μm or more, or 90 μm or less, 80 μm or less, 70 μm or less, or 60 μm or less, or 10 to 90 μm, 15 to 80 μm, 20 to 70 μm, or 35 to 60 μm.

[0101] In this specification, the Dv(90) of a filler indicates the size of the particle at 90% when the particles are arranged from the largest to the smallest on a volume basis. In one embodiment of the present invention, the Dv(90) of the filler is 5 μm or more, 10 μm or more, 12 μm or more, or 14 μm or more, or 229 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, or 40 μm or less, or 5 to 229 μm, 5 to 150 μm, 5 to 100 μm, 5 to 90 μm, 5 to 80 μm, It can be 5 to 70 μm, 10 to 229 μm, 10 to 150 μm, 10 to 100 μm, 10 to 60 μm, 12 to 229 μm, 12 to 150 μm, 12 to 100 μm, 12 to 60 μm, 12 to 50 μm, 14 to 220 μm, 14 to 150 μm, 14 to 100 μm, 14 to 60 μm, 14 to 40 μm, 15 to 229 μm, 15 to 150 μm, 15 to 100 μm, or 15 to 60 μm.

[0102] In this specification, the "uniformity coefficient" of the particle size of a filler is the ratio of the particle size (D40) at which the particle size distribution of the filler is 40% when expressed as a cumulative distribution to the particle size (D90) at which the particle size distribution is 90%. Therefore, the smaller the uniformity coefficient, the more uniform the particle sizes are, and when all the particles have the same particle size, the uniformity coefficient is 1.0.

[0103] In one embodiment of the present invention, the particle size uniformity coefficient (D40 / D90) of the filler can be 1.10 or more, 1.15 or more, or 1.20 or more, or 1.50 or less, 1.45 or less, or 1.40 or less, or 1.10 to 1.50, 1.15 to 1.45, or 1.20 to 1.40. In one embodiment of the present invention, the filler may be used after end-capping the remaining active groups with a different modification structure. Furthermore, during the end-capping process, the fine particles may be substituted with a dry gas. The end-capping structure may be any structure capable of reacting with the remaining active groups on the particles. Examples of suitable end-capping structures include, but are not limited to, alkylsilyl groups, acyl groups, alkoxyl groups, and alkylamino groups, which have small excluded volumes.

[0104] In one embodiment of the present invention, examples of suitable end-capping agents that can be used include disilazane compounds such as hexamethyldisilazane, hydrogen silane compounds such as diethylmethylsilane and triethylsilane, alkoxysilane compounds such as trimethylmethoxysilane, siloxane compounds such as pentamethyldisiloxane, hexamethylcyclotrisiloxane, and 1,1,3,3-tetramethyldisiloxane, and chlorosilane compounds such as trimethylchlorosilane.

[0105] Certain ranges are provided herein by numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically stated number, an unstated number that is close to or approaching the stated number can be a number that provides a substantial equivalent of the specifically stated number in the context in which it is given. In some embodiments, about can refer to ±5%, ±2.5%, or ±1% of the number to which it refers.

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0107] Where a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the scope of the invention, and where one or both limits are included, subject to any specifically excluded limits, ranges excluding either or both of those included limits are also included in the invention.

[0108] The present invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0109] In the following examples, elemental analysis was performed by the combustion method using an organic elemental analyzer (Microcorder JM11; J-Science Co., Ltd.), and the carbon content was calculated. Furthermore, the specific surface area and average pore size of the silica gel were measured by the BET method (multipoint method) using an automatic specific surface area / pore size distribution analyzer (TriStar-3000; Micromeritics). Furthermore, the average particle size and uniformity coefficient were calculated by the sieve method (JIS K 0069) using a sieve (diameter 75 mm, JIS Z8801 compliant).

[0110] Example 1: Using a three-neck flask, Daisogel SP-100-50 (average particle size 51.6 μm, uniformity coefficient (D40 / D90) 1.30, pore size 8.7 nm, specific surface area 467 m) was dissolved in water. ２ 100 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daiso Gel SP-100-50-ODS-Z (carbon content: 14.9%).

[0111] The HPLC equipment used was as follows: NP-KX500 pump (Nihon Seimitsu Kagaku Co., Ltd.), S-3702 detector (Soma Optical Co., Ltd.), and CO705 column oven (GL Sciences Inc.). The prepared Daiso Gel SP-100-50-ODS-Z (Osaka Soda Co., Ltd.) was packed into a column. The column size was 20 mm inner diameter, and three columns were used in combination: one 300 mm long column and two 500 mm long columns. The column flow rate was 37 mL / min. The mobile phase was 2.5% (v / v) aqueous methanol. The methanol was HPLC-grade methanol (Kanto Chemical Co., Ltd.). The water was distilled water. Detection was by UV at 230 nm.

[0112] A highly unsaturated fatty acid ester composition containing EPA-E (80 area %) was prepared as follows. Crude sardine oil was subjected to short path distillation (SPD). The SPD-treated oil was subjected to ethanolysis reaction with ethyl alcohol in the presence of an alkali catalyst to obtain fish oil ethyl ester. The fish oil ethyl ester was subjected to precision distillation to prepare 80 area % EPA-E. The composition of the crude composition is shown in Tables 1 and 2.

[0113]

[0114]

[0115] 3.1 g of EPA-E (80 area %) from Sample 1 was loaded onto the column, and fraction (Fr. 0) was fractionated from immediately after injection until the UV increased, followed by fractions from Fr. 1 to 38, which were fractionated every 10 seconds, and then Fr. 39, which was fractionated for 35 minutes. Methanol was removed from the collected fractions using a vacuum evaporator, and a 1 mg / mL hexane solution of C23:0 methyl ester was added as an internal standard, followed by GC analysis.

[0116] The conditions used for GC analysis were as follows: The instrument was a 7890A Network GC System (Agilent) with a DB-WAX 30 m x 0.25 mm x 0.25 μm column. The column temperature was 210°C. The injection temperature was 250°C, the split rate was 1:50, and the injection volume was 1 μL. A 250°C FID detector was used. A carrier gas of helium with a linear velocity of 31 cm / min was used.

[0117] The amount of eluted compound in each fraction was calculated from the area ratio between the GC peak area and the internal standard. The horizontal axis represents elution time, the first vertical axis represents the elution amount of EPA-E, and the second vertical axis represents the elution amount of C20:4n-6 ethyl ester (Figure 1). The two peaks were plotted with the same peak height. The resolution between EPA-E and C20:4n-6 ethyl ester was determined from Figure 1. Resolution R ｓ is the interval (difference in retention time) t' between the two peaks in Figure 1 Ｒ２ -t' Ｒ１ (min) is the peak width W of the two peaks １ , W ２ The resolution is shown in Figure 2. The resolution between EPA-E and C20:4n-3 ethyl ester and the resolution between EPA-E and C18:4n-3 ethyl ester were determined in the same manner.

[0118] Furthermore, the symmetry coefficient of the EPA-E peak was calculated. The symmetry coefficient, also called peak symmetry or tailing coefficient, is a value that indicates the degree of symmetry of the peak and is given by the following formula:

[0119]

[0120] If the peak has good left-right symmetry, the symmetry coefficient will be close to 1. A value smaller than 1 indicates leading, and a value larger than 1 indicates tailing. ０．０５ or S), where W ０．０５ｈ is the peak width at a position 1 / 20 of the peak height from the peak baseline. ０．０５ｈ is the distance between a perpendicular line drawn from the peak apex and a horizontal line (W) drawn at 1 / 20 of the peak height from the peak baseline. ０．０５ｈ ) along the horizontal line from the leading edge of the peak to the vertical line as it intersects the

[0121] To evaluate productivity, the resolution between EPA-E and C20:4n-6 ethyl ester was divided by the difference in peak top time between EPA-E and C20:4n-6 ethyl ester. A larger value indicates higher productivity.

[0122] Furthermore, the number of theoretical plates (N) and HETP (Height Equivalent to a Theoretical Plate) were calculated using a graph plotting the elution time (unit: minutes) on the horizontal axis and the elution amount of EPA-E on the vertical axis. The number of theoretical plates was calculated using the tangent method. HETP represents the packing height that provides the same performance as one theoretical plate, and the smaller the value, the better the column's performance. N = 16 (t R / W) 2 t R : Retention time (min) W: Peak width at baseline (min) HETP = L / N L: Column length (mm)

[0123] Comparative Example 1: Using a three-neck flask, Daisogel SP-120-50 (average particle size 51.3 μm, uniformity coefficient D40 / D90 1.35, pore size 12.7 nm, specific surface area 334 m ２120-50-ODS-B (120-50 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and the mixture was heated and refluxed for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the mixture was refluxed for 3 hours to complete the reaction. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daisogel SP-120-50-ODS-B (carbon content: 14.1%). Daisogel SP-120-50-ODS-B was also packed into a column in the same manner as in Example 1, and a fractionation test was performed.

[0124] The ODS physical property values ​​of Example 1 and Comparative Example 1, the degree of resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, and the symmetry factor of EPA-E are shown in Table 3. Example 1, which had a larger specific surface area and a smaller average pore diameter, had a higher degree of resolution and a symmetry factor closer to 1.

[0125]

[0126] Example 2 The same Daiso Gel SP-100-50-ODS-Z as in Example 1 was used.

[0127] Comparative Example 2: Using a three-neck flask, Daisogel SP-120-50 (average particle size 50.9 μm, uniformity coefficient (D40 / D90) 1.34, pore size 13.5 nm, specific surface area 315 m) was used. ２ 1 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daisogel SP-120-50-ODS-B (carbon content: 14.3%).

[0128] In Example 2 and Comparative Example 2, fractionation tests were carried out in the same manner as in Example 1, except that the mobile phase was 100% methanol. The resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, as well as the symmetry factor of EPA-E, were determined. Table 4 shows the ODS physical property values, resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, and the symmetry factor of EPA-E for Example 2 and Comparative Example 2. Example 2, which had a larger specific surface area and a smaller average pore diameter, had a higher resolution and a symmetry factor closer to 1.

[0129]

[0130] Example 3: Using a three-neck flask, Daisogel SP-100-20P (average particle size 18.5 μm, uniformity coefficient (D40 / D90) 1.37, pore size 9.6 nm, specific surface area 432 m) was dissolved in water. ２ 100 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daiso Gel SP-100-20-ODS-Z (carbon content: 14.9%).

[0131] Comparative Example 3: Using a three-neck flask, Daisogel SP-120-20P (average particle size 19.7 μm, uniformity coefficient (D40 / D90) 1.33, pore size 13.5 nm, specific surface area 315 m) was used. ２120 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daisogel SP-120-20-ODS-BP (carbon content: 14.8%).

[0132] The particles of Example 3 and Comparative Example 3, each having a particle size of 20 μm, were packed into a column under the same conditions as in Example 2, and a fractionation test was performed to determine the resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, as well as the symmetry factor of EPA-E. The ODS physical property values, resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, and the symmetry factor of EPA-E for Example 3 and Comparative Example 3 are shown in Table 5. Even with a particle size of 20 μm, Example 3, which has a larger specific surface area and a smaller average pore size, had a higher resolution and a symmetry factor closer to 1.

[0133]

[0134] Example 4: Using a three-neck flask, Daisogel SP-100-50 (average particle size 52.0 μm, uniformity coefficient (D40 / D90) 1.37, pore size 9.0 nm, specific surface area 452 m) was used. ２ 100 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daisogel SP-100-50-ODS-P (carbon content: 16.1%).

[0135] The carbon content of Example 4 was slightly higher than that of Example 2, but other physical properties were similar. Example 4 was packed into a column in the same manner as Example 2, and a fractionation test was carried out to determine the degree of resolution between EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and 1C18:4n-3 ethyl ester, as well as the symmetry factor of EPA-E. As shown in Table 6, the degree of resolution at a carbon content of 16.1% was similar to that of Example 2, but the symmetry factor of EPA-E in Example 2 was closer to 1.

[0136]

[0137] Example 5: Using a three-neck flask, Daisogel SP-100-50 (average particle size 46.2 μm, uniformity coefficient (D40 / D90) 1.22, pore size 8.7 nm, specific surface area 466 m) was used. ２ 100 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daiso Gel SP-100-50-ODS-Z (carbon content: 14.2%).

[0138] Comparative Example 4: Using a three-neck flask, Daisogel SP-100-50 (average particle size 46.2 μm, uniformity coefficient (D40 / D90) 1.22, pore size 8.7 nm, specific surface area 466 m) was used. ２ 100 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daiso Gel SP-100-50-ODS-Z (carbon content: 12.6%).

[0139] Comparative Example 5: Using a three-neck flask, Daisogel SP-100-50 (average particle size 46.2 μm, uniformity coefficient (D40 / D90) 1.22, pore size 8.7 nm, specific surface area 466 m) was used. ２ 100 / g) was subjected to azeotropic dehydration in a slurry solvent, toluene, under a nitrogen atmosphere, and then octadecyldimethylchlorosilane and pyridine were added and heated to reflux for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the reaction was completed by refluxing for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daiso Gel SP-100-50-ODS-Z (carbon content: 11.4%).

[0140] The columns packed with the ODS prepared in Example 5 and Comparative Examples 4 and 5 had an inner diameter of 10 mm and a length of 250 mm. A fractionation test was conducted using the same apparatus as in Example 2. In Example 5 and Comparative Examples 4 and 5, Sample 2, EPA-E (80 area%), was used. However, the load of EPA-E (80 area%) was 0.13 g, and the flow rate was 2 mL / min. A fractionation test was conducted to determine the resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, as well as the symmetry factor of EPA-E. Table 7 shows the ODS physical properties, resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, and the symmetry factor of EPA-E for Example 5 and Comparative Examples 4 and 5. Example 5 differs from Comparative Examples 4 and 5 in carbon content. Comparative Examples 4 and 5, which had low carbon contents, had lower separation degrees than Example 5.

[0141]

[0142] Example 6: Using a three-neck flask, Daisogel SP-100-50 (average particle size 49.6 μm, uniformity coefficient (D40 / D90) 1.19, pore size 9.2 nm, specific surface area 434 m) was used. ２100-50-ODS-Z (carbon content: 15.0%) was obtained by azeotropic dehydration of 100-50-ODS-Z (Osaka Soda Co., Ltd.) in a toluene slurry solvent under a nitrogen atmosphere, followed by the addition of octadecyldimethylchlorosilane and pyridine, followed by heating and refluxing for 3 hours. After cooling to room temperature, trimethylchlorosilane and pyridine were added for end-capping, and the mixture was refluxed for 3 hours to complete the reaction. After the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with methanol and toluene. It was then dried under reduced pressure at 70°C for 24 hours to obtain Daiso Gel SP-100-50-ODS-Z (carbon content: 15.0%). A packed column of Daiso Gel SP-100-50-ODS-Z (Osaka Soda Co., Ltd.), which has a uniformity coefficient (D40 / D90) close to 1, was tested in the same manner as in Example 2, and a fractionation test was performed using Sample 3 EPA-E. However, the load was 1.65 g. The resolution of EPA-E from C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, as well as the symmetry factor of EPA-E, were determined.

[0143] Example 7 A fractionation test was performed on Daisogel SP-100-50-ODS-Z of Example 2, with a loading of 1.65 g of EPA-E in Sample 3. Table 8 shows the physical properties of the ODS of Examples 6 and 7, the resolution of EPA-E and C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, and the symmetry coefficient of EPA-E. Example 6 and Example 7 had similar physical properties except for the uniformity coefficient (D40 / D90), but Example 6, whose uniformity coefficient (D40 / D90) was closer to 1, had a higher resolution.

[0144]

Claims

1. A method for producing a highly unsaturated fatty acid ester composition, comprising purifying a crude composition containing highly unsaturated fatty acid esters by column chromatography using an ODS silica gel packing material, wherein the ODS silica gel packing material has an average pore diameter of 8.5 to 11.9 nm and an average particle diameter of 15 to 229 μm.

2. ODS silica gel packing material is 400-480m ２ The method of claim 1, wherein the specific surface area is 0.05 to 0.15 g.

3. The method of claim 1 or 2, wherein the ODS silica gel packing has a carbon content of 13% or more and less than 15%.

4. The method of any one of claims 1 to 3, wherein the ODS silica gel packing has a carbon density of 0.030 to 0.

040.

5. The method according to any one of claims 1 to 4, wherein the ODS silica gel packing has a uniformity coefficient (D40 / D90) of 1.00 to 1.

40.

6. The method according to any one of claims 1 to 5, wherein the column chromatography uses methanol or ethanol containing 5% or less water as an eluent.

7. The method according to any one of claims 1 to 6, wherein the highly unsaturated fatty acid ester composition contains EPA esters with a purity of 95% or more.

8. The method according to any one of claims 1 to 7, wherein arachidonic acid (ARA) esters are separated from eicosapentaenoic acid (EPA) esters in column chromatography.

9. The method according to claim 8, wherein the degree of resolution obtained by dividing the difference in retention time between ARA esters and EPA esters separated in column chromatography by the average width of the peaks of ARA esters and EPA esters is 1.15 or more.

10. The method according to any one of claims 1 to 9, wherein the highly unsaturated fatty acid ester is a highly unsaturated fatty acid ethyl ester.

11. The method according to any one of claims 1 to 10, wherein the ODS silica gel packing is end-capped.

12. The method according to any one of claims 1 to 11, wherein the column chromatography is a batch process.

13. The method according to any one of claims 1 to 12, wherein each of the one or more columns used in the column chromatography has a length of 100 to 2000 mm.

14. The method according to any one of claims 1 to 13, wherein each of the one or more columns used in the column chromatography has an inner diameter of 10 to 1000 mm.

15. An octadecylsilane (ODS) silica gel packing material for use in packing a column in column chromatography purification in the production of a highly unsaturated fatty acid ester composition, the ODS silica gel packing material having an average pore diameter of 8.5 to 11.9 nm and an average particle diameter of 15 to 229 μm.

16. 400-480m ２ 16. The ODS silica gel packing material according to claim 15, having a specific surface area of ​​1 / g.

17. The ODS silica gel packing material according to claim 15 or 16, having a carbon content of 13% or more and less than 15%.

18. The ODS silica gel packing material of any one of claims 15 to 17, having a carbon density of 0.030 to 0.

040.

19. The ODS silica gel packing material according to any one of claims 15 to 18, having a uniformity coefficient (D40 / D90) of 1.00 to 1.

40.

20. The ODS silica gel packing material according to any one of claims 15 to 19, which has been end-capped.

21. A column for purifying a highly unsaturated fatty acid ester composition, packed with the ODS silica gel packing material according to any one of claims 15 to 20.

22. An analytical device equipped with the column for purifying the highly unsaturated fatty acid ester composition of claim 21.

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