WO2025225709A1 - Filler for chromatography - Google Patents

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

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.

PUFAs are used as medicines and health foods. PUFAs are difficult to synthesize chemically, so they are generally produced by extraction from natural materials such as vegetable oils and marine oils that are rich in PUFAs.

Column chromatography has been known as a method for separating specific fatty acids 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 porous, adsorbent packing materials inside a column. Each component in the mixture has a different permeation rate depending on its physical properties, and so is selectively discharged from the column in sequence. Octadecylsilylated silica gel (ODS silica gel) packing material is widely used as a packing material for liquid chromatography.

In order to purify PUFAs and their esters to high purity with high efficiency and low cost, efforts are underway to develop suitable ODS silica gel packing materials (Patent Documents 1 and 2). Furthermore, methods for improving the performance of ODS silica gel packing materials by end-capping have been reported (Patent Documents 3 and 4).

International Publication No. 2023/111317 Japanese Patent Application Publication No. 10-310555 Special Publication No. 2008-232802 Japanese Patent Application Laid-Open No. 2002-022721

There remains a need for ODS silica gel packing materials that can efficiently separate the desired highly unsaturated fatty acid ester composition with high purity.

The inventors of the present invention discovered that by using an ODS silica gel packing material with a specific specific surface area, average pore size, carbon content, average particle size, carbon density, or uniformity coefficient in column chromatography, it is possible to address the above issues and efficiently produce the desired PUFAs, thereby completing the present invention.

The present invention relates to a method for producing a highly unsaturated fatty acid ester composition using the following ODS silica gel filler.

[1-1] 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,
ODS silica gel packing material,
an average pore size of 8.5 to 11.9 nm, and an average particle size of 15 to 229 μm.
[1-2] The method according to [1-1], wherein the ODS silica gel packing 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 packing 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.

[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 of 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 oils obtained from genetically modified plants, animals, and microorganisms.
[1-9] The method according to any one of [1-1] to [1-8], wherein the crude composition contains 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 contains a highly unsaturated fatty acid ester having three or more double bonds and a monounsaturated fatty acid.
[1-11] The method according to [1-10], wherein the crude composition further contains 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 in an amount of 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 in a total amount 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 ester 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.

[1-19] The method according to any one of [1-18], wherein the resolution obtained 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 method according to any one of [1-1] to [1-19], wherein the ester is a C _1-6 alkyl ester.
[1-21] The method according to [1-9], wherein the highly unsaturated fatty acid ester is a highly unsaturated fatty acid ethyl ester.
[1-22] The method according to any one of [1-1] to [1-21], wherein the ODS silica gel packing is end-capped.

[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] ODS silica gel packing material,
The method according to any one of [1-1] to [1-25], having an average pore diameter of 8.5 to 11.5 nm and an average particle diameter of 15 to 60 μm.

[2-1] An octadecylsilane (ODS) silica gel packing material for use in packing a column in purification by column chromatography in the production of a highly unsaturated fatty acid ester composition,
An ODS silica gel packing material having an average pore size of 8.5 to 11.9 nm and an average particle size of 15 to 229 μm.
[2-2] The ODS silica gel packing material according to [2-1], which has a specific surface area of 400 to 480 m ² /g.
[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.

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

FIG. 1 is a graph showing the elution behavior of EPA-E and C20:4n-6 ethyl ester in Example 1. FIG. 2 shows the formula for calculating the degree of resolution of two peaks. FIG. 3 shows how to determine the symmetry coefficient.

The present invention will be described in more detail below.
The following abbreviations may be used in this 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: γ-linolenic acid GLA-E: γ-linolenic acid ethyl ester DGLA: dihomo-γ-linolenic acid DGLA-E: dihomo-γ-linolenic acid ethyl ester

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.

In one embodiment of the present invention, the crude composition typically comprises PUFAs and at least one fatty acid and/or ester thereof. In one aspect, the crude composition comprises 50 area % or more highly unsaturated fatty acid esters.
In one aspect of the present invention, a composition containing a highly unsaturated fatty acid ester 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 of EPA ethyl ester obtained by precision distillation of ethyl esters of fatty acids at a vacuum of 0.2 Torr or less and a temperature of 190°C or less in the total column can be used as the crude composition.

In one aspect of the present invention, the range of PUFAs that are separated and purified as ester derivatives is not particularly limited. From the perspective of application as pharmaceuticals or functional foods, naturally occurring PUFAs are preferred. Examples include PUFAs with 18 or more carbon atoms and three or more double bonds, such as DHA, DPA, EPA, ARA, GLA, DGLA, C18:3, C19:4, C20:4, and C21:5.

In one aspect of the present invention, the source of PUFA is not particularly limited as long as it contains the target substance. It can be selected according to the target substance from animal oils such as fish oil and cod liver oil, vegetable oils such as fruit oil, and microbial oils derived from fungi and algae. 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. Selecting an appropriate source is within the skill of a person skilled in the art.

It is known that oils 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 often have a simpler fatty acid composition than fish oils, etc., 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 source.

Fungi such as those of the genera Thraustochytrium and Schizochytrium have been reported to accumulate lipids containing DHA and DPA within 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 microorganisms and similar species of microorganisms can be advantageously used. Of course, the cultured cells are not limited to those exemplified, and any cell derived from a bacterial species that produces DHA can be used.

In one aspect, culture conditions such as medium composition, temperature, and pH are selected depending on the type of microorganism being cultured. Selecting appropriate culture conditions is easy for those 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.

In one aspect, cultured microbial cells are dried as needed, and then subjected to operations such as crushing the cells in accordance with standard methods, after which lipids are extracted. When using other sources such as animal or vegetable oils, lipids can also be extracted after pretreatment appropriate for the type of source.

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 alkyl groups 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.

Appropriate reaction conditions for esterification are well known to those skilled in the art. For example, lipids can be dissolved in an organic solvent such as hexane, followed by the addition of an alkali (e.g., ethanol containing 1N potassium hydroxide), and esterification can be carried out at a temperature of approximately 30-80°C. After separating the organic phase, it can be concentrated using standard methods to obtain a raw material composition containing the ester derivative.

In one aspect of the present invention, a composition containing highly unsaturated fatty acid esters obtained by the above-described esterification reaction can be subjected to HPLC as a crude composition without any preliminary purification procedures. In one embodiment of the present invention, after preparing a crude composition containing highly unsaturated fatty acid esters obtained by the esterification reaction, the desired highly unsaturated fatty acid ester composition is obtained by a single HPLC procedure without any preliminary purification procedures.

In one embodiment of the present invention, the 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.

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

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 at which the double bond begins, counting 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.

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 a pure product for food, cosmetic, or industrial use.

As used herein, the term "highly unsaturated fatty acid (PUFA)" refers to a fatty acid with more than one double bond. The PUFAs herein can be in the form of an ester. The ester is typically an alkyl ester, for example, a _C1 - _C6 alkyl ester, or a _C1 - _C4 alkyl ester. Examples of esters include ethyl esters.

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

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 a 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. Purification in the method of the present invention reduces the amount of the fatty acid having 18 to 22 carbon atoms and a fatty acid ethyl ester to 5 area % or less.

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).

In one embodiment of the present invention, column chromatography is performed in a batch mode. Compared to simulated moving bed column chromatography, the batch mode column chromatography in one embodiment of the present invention is characterized by a simpler configuration and easier implementation.

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.

In one embodiment of the present invention, column chromatography involves 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 connected in series or in parallel. Any known column can be used in the claimed method.

In one embodiment of the present invention, the column length 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.

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 an inner diameter of 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.

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

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 ODS silica gel packing, 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 observing absorbance (e.g., UV absorption, refractive index, etc.) to confirm the start of elution of the target substance, and then collecting fractions every predetermined time (e.g., 5 seconds, 10 seconds, 20 seconds, or 30 seconds), and continuing to collect fractions until elution of the target substance is complete. The time for collecting fractions can be determined appropriately 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 applied to the column, etc.

In this specification, "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 begins to elute, the elution amount value shown in the graph increases, indicating a "peak." In this specification, "chromatogram" refers to a graph showing signal intensity (UV absorption, refractive index, etc.) measured in chromatography on the vertical axis and retention time on the horizontal axis.

In this specification, the term "peak top" refers to the point at which the value of each peak is maximum.
As used herein, the term "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 elution of the target substance in the graph, and is represented as W ₁ , W ₂ , and W in Figures 2 and 3.

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

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.

As used herein, "fraction" refers to the crude composition loaded onto a column and subjected to chromatography, and the eluent contained 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.

In one aspect of the 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" is the end of the stationary phase that first receives 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.

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" refers to a liquid used in chromatography as a mobile phase, which is a phase that contacts the stationary phase and passes through the gaps or surface of the stationary phase. The eluent is used in chromatography to develop and elute components such as PUFAs or their ester derivatives that are adsorbed to a column. As used herein, the eluent is sometimes referred to as the mobile phase.

In one embodiment of the present invention, the eluate from the column is collected as a fraction together with the PUFA or its ester derivative. The fraction is also called an eluate, and may contain components such as the eluate and the PUFA or its ester derivative.

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.

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

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.

As used herein, "chromatographic conditions" refers to 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, and settling time and pressure at which settling is performed.

As used herein, "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 at increasing percentages over time during elution.

As used herein, "purity" refers to the ratio indicating the content of the main 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 main component is a highly unsaturated fatty acid ester, for example, EPA ester, DGLA ester, or ARA ester.

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

In one embodiment of the present invention, the column chromatography is reverse-phase column chromatography. In one embodiment of the present invention, the column chromatography uses the packing material of the present invention as the stationary phase.

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.

In one embodiment of the present invention, the amount of packed stationary phase is, by weight, 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.

In one embodiment of the invention, column chromatography involves 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 can 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.

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.

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.

In one embodiment of the present invention, the column chromatography comprises a mobile phase gradient. In one embodiment of the present 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 esters are 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, and eicosate. Tetracosapentaenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosapentaenoic acid, herring 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, geddi The eicosapentaenoic acid ethyl ester is an ester of a highly unsaturated fatty acid selected from one or more of geddic acid, celloplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid. In one embodiment of the present invention, the ester is a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl ester. In one embodiment of the present invention, column chromatography comprises purifying the eicosapentaenoic acid ethyl ester.

In some embodiments, the crude composition contains the desired highly unsaturated fatty acid ester with 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 method of the present invention contains the desired highly unsaturated fatty acid ester with 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%.

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.

Thus, in reversed-phase chromatography used for non-polar highly unsaturated fatty acid esters, the solvent at the start of chromatographic 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 may be a solvent less polar than solvent A, e.g., methanol (when solvent A is water). Solvent B will constitute the remaining percentage of the mobile phase. As the column is run and the solvent 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. In some embodiments, a single solvent composition may be used as the mobile phase. In some embodiments, 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 combination.

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 for certain time ranges of the chromatography and include a gradient for other time ranges.

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 dual piston pumps, which operate with pistons moving out of phase with each other, provides a rational solution for pulse-free fluid delivery.

The linear velocity in a column refers to the speed at which a fluid passes through the cross section of the column. The linear velocity (linear velocity (m/hr) = flow rate (m ³ /hr) / 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.

In one embodiment of the present invention, the column chromatography is performed at room temperature or at a temperature higher than room temperature. Preferably, the method is performed at a temperature higher than room temperature. The first and second column chromatography steps can be performed at the same temperature or at different temperatures, but are preferably performed at the same temperature.

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.

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

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, such as mass spectrometry (MS), UV/Vis absorbance, fluorescence, refractive index, or conductivity, can be used.

In other embodiments, any of a variety of standard column chromatography detectors can be used to detect the effluent immediately after elution from the column.
In other embodiments, 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.

In some embodiments, eluates from the column are detected as peaks 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 an 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, eluate retention varies with variations in the eluent, stationary phase, temperature, and column chromatography setup. Therefore, the retention time of an eluate is compared to the retention time of one or more standard compounds under identical conditions. Suitable detectors exhibit good sensitivity, good stability, reproducibility, linear response over several orders of magnitude when used for quantitative purposes, short response times, 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.

In one embodiment of the present 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) 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 across 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.

Fluorescence detectors are useful in 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 observed by detecting grating-separated emission radiation, typically at a 90-degree angle to the excitation beam. The number of fluorescent species can be enhanced by post-column derivatization (PCD) of the eluted compounds (or pre-column derivatization of the sample itself) with special reagents.

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.

Conductivity detectors provide highly 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 the ppb level. Adding a chemical suppressor between the column and the conductivity detector reduces the conductivity of the eluent, allowing for the use of gradient elution and ppb-level determinations with minimal baseline drift. For typical determination of low levels of anions, the eluent is converted to its weakly ionized, low-conductivity acid (e.g., _Na2CO3 to carbonate) to reduce background noise. Concurrently, component anions are converted to their corresponding high-conductivity acids (e.g., _NaCl to HCl), increasing the relative component signal.

In one embodiment of the present invention, the retention time of the target substance can be 0.5 to 2 minutes, 2 to 4 minutes, 4 to 6 minutes, 6 to 8 minutes, 8 to 10 minutes, 10 to 12 minutes, 12 to 14 minutes, 14 to 16 minutes, 16 to 18 minutes, 18 to 20 minutes, 20 to 22 minutes, 22 to 24 minutes, 24 to 26 minutes, 26 to 28 minutes, 28 to 30 minutes, 30 to 32 minutes, 32 to 34 minutes, 34 to 36 minutes, 36 to 38 minutes, or 38 to 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.

In one embodiment of the present invention, an internal standard can be used during analysis by GC. An 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 one skilled in the art to be a compound that is very similar, but not identical, to the target component, for example, 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 one skilled in the art to be used as a reference for the unknown component sample to be quantified.

In one embodiment, the highly unsaturated fatty acid ester composition comprises 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, caproic acid The acid is or contains enanthic, caprylic, pelargonic, capric, undecylic, lauric, tridecylic, myristic, pentadecylic, palmitic, margaric, stearic, nonadecylic, arachidic, heneicosylic, behenic, tricosylic, lignoceric, pentacosylic, cerotic, carboseric, montanic, nonacosylic, melissic, hentriacontylic, russellic, silicic, gedic, ceroplastic, hexatriacontylic, heptatriacontylic, octatriacontylic, nonatriacontylic, or tetracontylic acid, or an ester thereof.

Filling Material: In one embodiment of the present invention, the filling material is preferably used as a filling material for column chromatography, particularly as a filling material for high-performance liquid chromatography or supercritical fluid chromatography, but is not limited to this. For example, it may be used as a support or adsorbent for thin-layer chromatography, or in the form of a filter aid or filter.

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 invention, the filler is a spherical or non-spherical bead.

In this specification, the "specific surface area" of a filler refers to the surface area per unit mass of a substance. Furthermore, in this specification, 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. In this specification, the specific surface area and average pore diameter are calculated by a common method, for example, the BET method, i.e., in accordance with 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 using 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.

In one embodiment of the present invention, the specific surface area of the filler can be 300 m ² /g or more, 350 ^{m 2} /g or more, or 400 m ² /g or more, or 600 m ^{2 /g or less, 550 m 2 /} g or less, or 500 m ² /g or less, or 300 to 600 m 2 /g, 350 to 550 m ² /g, or 400 to 500 m ² /g.

In one embodiment of the present invention, the average pore diameter 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.

In this specification, the "total pore volume" of a filler refers to the volume of pores contained in 1 g of porous material. 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.

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.

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 porosimetry are commonly used methods for measurement.

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

In this specification, the "carbon content" of a filler refers to the weight percentage 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 (Microcorder JM11; J-Science Co., Ltd.). The measurement method using the combustion method is described in ISO 21068-2.

In one embodiment of the present invention, the carbon content can be 12.0% by weight or more, 13.0% by weight or more, or 14.0% by weight or more, and 16.5% by weight or less, 16.0% by weight or less, or 15.5% by weight or less, or 12.0 to 16.5% by weight, 13.0 to 16.0% by weight, or 14 to 15.5% by weight.

As used herein, the "carbon density" of a filler is calculated as the carbon content/specific surface area of the filler.
In one embodiment of the invention, the carbon density of the filler can be 0.0002 wt%/(g/m ² ) or more, 0.0003 wt%/(g/m ² ) or more, or 0.00045 wt% (g/m ² ) or less, 0.00040 wt% (g/m ² ) or less, or 0.0002 to 0.00045 wt% (g/m ² ), 0.0003 to 0.0004 wt% (g/m ² ).

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 (75 mm diameter, JIS Z8801 compliant).

In one embodiment of the present 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.

If the average particle size of the packing material is above the lower limit above, the pressure loss when packed into a column and liquid is passed through it will be small, allowing for a higher liquid passage speed and improving the productivity of the separation process. On the other hand, if the average particle size is below the upper limit above, separation performance can be maintained without a decrease in column efficiency.

In this specification, the Dv(10) of a filler indicates the size of the particle at 10% when the particles are arranged from the largest on a volume basis.
In one aspect of the 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.

In this specification, the Dv(90) of a filler indicates the size of the particles 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.

In this specification, the "uniformity coefficient" of filler particle size is the ratio of the particle size (D40) at which the particle size distribution of the filler expressed as a cumulative distribution is 40% 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; when all particles have the same particle size, the uniformity coefficient is 1.0.

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, the end-capping may involve substituting the fine particles with a dry gas. The end-capping structure may be any structure that can react 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.

In one embodiment of the present invention, suitable end-capping agents 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.

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 near or approximately the number preceded by the term. In determining whether a number is near or approximately a specifically recited number, an unrecited number that is near or approaching the recited number can be a number that provides a substantial equivalent amount of the specifically recited number in the context in which it is given. In certain embodiments, about can refer to ±5%, ±2.5%, or ±1% of the number to which it refers.

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. 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.

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 scope of 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.

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 is intended to be limited only by the appended claims.

In the following examples, elemental analysis was performed by the combustion method using an organic elemental analyzer (Microcorder JM11; J-Science Co., Ltd.) to calculate the carbon content. Additionally, the specific surface area and average pore diameter 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 (75 mm diameter, JIS Z8801 compliant).

Example 1
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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%).

The HPLC equipment used was as follows: the pump was an NP-KX500 (Nihon Seimitsu Kagaku Co., Ltd.), the detector was an S-3702 (Soma Optical Co., Ltd.), and the column oven was a CO705 (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 an inner diameter of 20 mm, and a total of three columns were used connected together: one 300 mm long column and two 500 mm long columns. The column flow rate was 37 mL/min. The mobile phase used 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.

A highly unsaturated fatty acid ester composition containing EPA-E (80% by area) 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 alkali catalyst to obtain fish oil ethyl ester. The fish oil ethyl ester was then subjected to precision distillation to prepare 80% by area EPA-E. The composition of the crude composition is shown in Tables 1 and 2.

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 1 to 38 every 10 seconds, and then Fraction 39 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 before analysis by GC.

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. Helium carrier gas with a linear velocity of 31 cm/min was used.

The amount of eluted compounds 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 height.
The resolution between EPA-E and C20:4n-6 ethyl ester was determined from Figure 1. The resolution _Rs indicates how many times the interval (difference in retention times) _t'R2 - _t'R1 (minutes) between the two peaks in Figure 1 is multiplied by the average value (minutes) of the peak widths _W1 and _W2 of the two peaks. The method for determining this 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 a similar manner.

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

If the peak has good left-right symmetry, the symmetry coefficient will be close to 1; a value less than 1 indicates leading, and a value greater than 1 indicates tailing. Figure 3 shows a peak with the variables used to calculate the symmetry coefficient (As _0.05 or S). Here, W _0.05h is the peak width at a position 1/20 of the peak height from the peak baseline. Also, f or A _0.05h is the horizontal distance from the leading edge of the peak to the vertical line (W _0.05h ) drawn from the peak apex when the vertical line intersects with the horizontal line drawn at 1/20 of the peak height from the peak baseline.

In addition, to evaluate productivity, the degree of resolution between EPA-E and C20:4n-6 ethyl ester was calculated by dividing it by the difference in peak top times for EPA-E and C20:4n-6 ethyl ester. A higher value indicates higher productivity.

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 performance equivalent to one theoretical plate, and a smaller value indicates a better column performance.
N = 16 (t _R /W) ²
_tR : Retention time (minutes)
W: Peak width at baseline (min)
HETP＝L/N
L: column length (mm)

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 ² /g) was azeotropically dehydrated in a slurry solvent toluene under a nitrogen atmosphere, followed by the addition of octadecyldimethylchlorosilane and pyridine and heating 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: 14.1%). 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.

Table 3 shows the ODS physical properties of Example 1 and Comparative Example 1, the degree of separation of EPA-E from C20:4n-6 ethyl ester, C20:4n-3 ethyl ester, and C18:4n-3 ethyl ester, and the symmetry coefficient of EPA-E. Example 1, which has a larger specific surface area and a smaller average pore diameter, had a higher degree of separation and a symmetry coefficient closer to 1.

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

Comparative Example 2
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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-120-50-ODS-B (carbon content: 14.3%).

In Example 2 and Comparative Example 2, the fractionation test was carried out in the same manner as in Example 1. However, the mobile phase was 100% methanol.
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.
The ODS physical property values, 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 for Example 2 and Comparative Example 2 are shown in Table 4. Example 2, which had a larger specific surface area and a smaller average pore diameter, had a higher resolution and a symmetry coefficient closer to 1.

Example 3
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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%).

Comparative Example 3
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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-120-20-ODS-BP (carbon content: 14.8%).

The 20 μm particle size particles of Example 3 and Comparative Example 3 were also packed into a column under the same conditions as in Example 2, and a fractionation test was carried out to determine 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.
The ODS physical property values, 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 for Example 3 and Comparative Example 3 are shown in Table 5. Even with a particle diameter of 20 μm, Example 3, which has a larger specific surface area and a smaller average pore diameter, has a higher resolution and a symmetry coefficient closer to 1.

Example 4
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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-P (carbon content: 16.1%).

Example 4 had a slightly higher carbon content than 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 conducted 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 was closer to 1 in Example 2.

Example 5
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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%).

Comparative Example 4
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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%).

Comparative Example 5
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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%).

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. Fractionation tests were 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 amount of EPA-E (80 area %) was 0.13 g, and the flow rate was 2 mL/min. Fractionation tests were conducted to determine 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.
Table 7 shows the ODS physical property values, the resolution between 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 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 have low carbon contents, had lower resolution than Example 5.

Example 6
Using a three-neck flask, Daiso Gel 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 ² /g) was azeotropically dehydrated 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 completion of the reaction, 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 column packed with Daisogel 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 carried out using EPA-E from Sample 3. 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 coefficient of EPA-E, were determined.

Example 7
A fractionation test was carried out on Daisogel SP-100-50-ODS-Z of Example 2, with the loading amount of EPA-E of Sample 3 set to 1.65 g.
The ODS physical properties 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 are shown in Table 8. 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.

Claims (22)

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,
ODS silica gel packing material,
an average pore diameter of 8.5 to 11.9 nm, and an average particle diameter of 15 to 229 μm;
A method comprising: The method of claim 1, wherein the ODS silica gel packing has a specific surface area of 400 to 480 m ² /g. The method of claim 1 or 2, wherein the ODS silica gel filler has a carbon content of 13% or more and less than 15%. The method according to any one of claims 1 to 3, wherein the ODS silica gel filler has a carbon density of 0.030 to 0.040. The method according to any one of claims 1 to 4, wherein the ODS silica gel filler has a uniformity coefficient (D40/D90) of 1.00 to 1.40. 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. 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. The method of any one of claims 1 to 7, wherein arachidonic acid (ARA) esters are separated from eicosapentaenoic acid (EPA) esters by column chromatography. The method of 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 peak width of ARA esters and EPA esters is 1.15 or greater. 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. The method according to any one of claims 1 to 10, wherein the ODS silica gel filler is end-capped. The method according to any one of claims 1 to 11, wherein the column chromatography is batchwise. The method according to any one of claims 1 to 12, wherein each of the one or more columns used in column chromatography has a length of 100 to 2000 mm. The method according to any one of claims 1 to 13, wherein each of the one or more columns used in column chromatography has an inner diameter of 10 to 1000 mm. 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, comprising:
an average pore diameter of 8.5 to 11.9 nm, and an average particle diameter of 15 to 229 μm;
The ODS silica gel packing material has the following structure: 16. The ODS silica gel packing material according to claim 15, having a specific surface area of 400 to 480 m ² /g. The ODS silica gel filler according to claim 15 or 16, having a carbon content of 13% or more and less than 15%. The ODS silica gel filler according to any one of claims 15 to 17, having a carbon density of 0.030 to 0.040. 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. The ODS silica gel packing material according to any one of claims 15 to 19, which has been end-capped. A column for purifying highly unsaturated fatty acid ester compositions, packed with the ODS silica gel packing material described in any one of claims 15 to 20. An analytical device equipped with the column for purifying the highly unsaturated fatty acid ester composition of claim 21.