Simulated moving bed type chromatographic separation method

Abstract

To provide a composition comprising a polyunsaturated fatty acid (PUFA) product.SOLUTION: A composition comprises a PUFA product, where (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.89 μg / kg, and / or (b) the total amount of dioxins, furans, dibenzo-para-dioxins and polychlorinated dibenzofurans in the composition is up to 0.35 pg / g, and / or (c) the total amount of polychlorinated biphenyls in the composition is up to 0.0035 mg / kg, and / or (d) the total amount of dioxins, furans, dibenzo-para-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls in the composition is up to 1 pg / g.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an improved chromatographic fractionation method for purifying polyunsaturated fatty acids (PUFAs) and their derivatives. In particular, the present invention relates to an improved simulated or moving bed chromatographic separation method for purifying PUFAs and their derivatives.

Background Art

[0002] Fatty acids, particularly PUFAs, and their derivatives are precursors of biologically important molecules that play important roles in regulating biological functions such as blood coagulation, inflammation, and immune response. Thus, PUFAs and their derivatives may be therapeutically useful in treating a wide range of pathological conditions including CNS conditions; neuropathies including diabetic neuropathy; cardiovascular diseases; and systemic immune and inflammatory conditions including inflammatory skin diseases. PUFAs are found in natural sources such as vegetable oils and fish oils. However, such PUFAs are often present in admixture with saturated fatty acids and many other impurities in such oils. Thus, PUFAs are desirably purified before use for nutritional or pharmaceutical applications.

[0003] Unfortunately, PUFAs are extremely labile. Thus, they are prone to isomerization, peroxidation, and oligomerization when heated in the presence of oxygen. Thus, the fractionation and purification of PUFA products to produce pure fatty acids is difficult. Distillation under vacuum can also result in unacceptable product degradation.

[0004]

[0005] Simulated and real moving bed chromatography are well-known prior arts to those skilled in the art. The principle of operation involves the counter-current movement of a liquid eluent phase and a solid adsorbent phase. This operation allows for the minimal use of solvents and makes this method economically viable. Such separation techniques have been used in several applications in various fields including hydrocarbons, industrial chemicals, oils, sugars, and APIs. Such separation techniques have also been used to purify PUFAs and their derivatives.

[0006] As is well known, in a conventional fixed bed chromatography system, the mixture to be separated permeates a vessel. The vessel is generally cylindrical and is typically referred to as a column. The column contains a packing of a porous material (generally referred to as the stationary phase) that exhibits a high liquid permeability. The penetration rate of each component of the mixture depends on the physical characteristics of the component such that the components emerge from the column continuously and selectively. Thus, some components tend to bind strongly to the stationary phase and penetrate slowly, while other components tend to bind weakly and exit the column more rapidly. Many different fixed bed chromatography systems have been proposed and are used for both analytical and industrial production purposes.

[0007] In contrast, a simulated moving bed system consists of several individual columns containing adsorbent, connected in series with each other. The eluent is passed through the columns in a first direction. The injection points of the feedstock and the eluent, as well as the collection points of the separated components within the system, are periodically shifted by a series of valves. The overall effect is to simulate the operation of a single column containing a moving bed of solid adsorbent. Therefore, the simulated moving bed system, like the conventional fixed bed system, The adsorbent is passed through a column containing a fixed bed of solid adsorbent, but in a simulated moving bed system The operation is carried out to simulate a continuous countercurrent moving bed.

[0008] A method and apparatus for simulated moving bed chromatography is incorporated herein by reference in its entirety. U.S. Patent Nos. 2,985,589 and 3,696,107, both of which are incorporated herein by reference. , 3,706,812, 3,761,533, French Patent Application Publication No. 2103 302(A), 2651148(A) and 2651149(A). This subject matter is described in several patents, the entireties of which are incorporated herein by reference. "Preparative and Production Scale Chromatography," by Ganetsos and Bar This is also covered in detail in "Theoretical and Physical Sciences of the 1990s: A Case Study of the 1990s," edited by Marcel Dekker, Marcel Dekker Inc, New York, 1993.

[0009] A real moving bed system is similar in operation to a simulated moving bed system. However, the feed mixture Instead of shifting the injection point of the eluent and the collection point of the separated components by a system of valves, Instead, a series of adsorption units (i.e., columns) are physically moved relative to the feed and extraction points. Again, operation is performed to simulate a continuous countercurrent moving bed.

[0010] The method and apparatus for practical moving bed chromatography are incorporated herein by reference in their entirety. Nos. 6,979,402, 5,069,883, and 6,069,883, which are incorporated herein by reference. and 4,764,276.

[0011] The simulated and true moving bed technologies are generally only suitable for separating binary mixtures. . Thus, the more polar component will move with the eluent and be collected as the raffinate stream while the less polar component will move with the adsorbent and be collected as the extract stream. Therefore, it is difficult to separate the desired product from a crude mixture containing both polar and nonpolar impurities using simulated or true moving bed technology. This limits the applicability of such technology, for example, in the purification of PUFA products from fish oil.

[0012] Therefore, conventionally, when separating PUFAs from natural oils using simulated or true moving bed technology, it is generally necessary to first subject the natural oil to a preliminary separation step (e.g., fixed column chromatography) before purifying the intermediate product obtained using simulated or true moving bed technology. (see, for example, European Patent Application Publication No. 0697034(A)). Typically, the first purification step removes polar or nonpolar components, essentially producing a binary mixture, which is then subjected to moving bed chromatography.

[0013] Referring to Figure 1, a method for separating this binary mixture is shown. The concept of the simulated or true continuous countercurrent chromatographic separation method is explained by considering a vertical chromatographic column containing a stationary phase S divided into four superimposed small zones I, II, III, and IV extending from the bottom to the top of the column, more precisely. The eluent is introduced at the bottom in IE by pump P. A mixture of components A and B to be separated is introduced at IA + B between small zones II and III. The extract mainly containing B is in small zone ​ The raffinate collected at SB between zone I and narrow zone II and mainly containing A is in narrow zone II is collected at SA between zone I and narrow zone IV.

[0014] In the case of a simulated moving bed system, the simulated downward movement of the stationary phase S is caused by the movement of the introduction point and the collection point with respect to the solid phase. In the case of a true moving bed system, the downward movement of the stationary phase S is caused by the movement of various chromatographic columns with respect to the introduction point and the collection point. In FIG. 1, the eluent flows upward, and the mixture A + B is injected between narrow zone II and narrow zone III. Their components move according to their chromatographic phase interactions with the stationary phase , for example, adsorption onto a porous medium. Component B, which shows a stronger affinity for the stationary phase (the component that flows more slowly), is carried along more slowly by the eluent and will lag behind and follow later. Component A, which shows a weaker affinity for the stationary phase (the component that flows more rapidly), will be carried along more easily by the eluent. When an appropriate set of parameters , especially the flow rates in each zone, are accurately estimated and controlled, component A, which shows a weaker affinity for the stationary phase, is collected as raffinate between narrow zone III and narrow zone IV, and component B, which shows a stronger affinity for the stationary phase, is collected as extract between narrow zone I and narrow zone II .

[0015] Therefore, it will be understood that the conventional moving bed system schematically shown in FIG. 1 is limited to the separation of two components .

Summary of the Invention

Problems to be Solved by the Invention

[0016] Therefore, there is a need for a single simulated or true moving bed chromatography separation method that can separate PUFAs or their derivatives from both more rapidly flowing components and more slowly flowing components (i.e., more polar impurities and less polar impurities) to produce essentially pure PUFAs or their derivatives. Further, it is desirable that the method use an inexpensive eluent that operates under standard temperature and pressure conditions.

Means for Solving the Problem

[0017] wherein the apparatus has a plurality of zones including at least a first zone and a second zone, each zone having an extract stream and a raffinate stream from which liquid from the plurality of connected chromatographic columns can be collected, and (a) the raffinate stream containing the PUFA product together with the more polar components is collected from the columns in the first zone and introduced into non-adjacent columns in the second zone, and / or (b) the extract stream containing the PUFA product together with the less polar components is collected from the columns in the second zone and introduced into non-adjacent columns in the first zone, and the PUFA product is separated from different components of the feed mixture in each zone. The present invention provides a chromatographic separation method. ​​​​​​​​​​​

[0018] PUFA products obtained by the method of the present invention are also provided.

[0019] PUFA products produced by the method of the present invention are produced in high yields and have high purity. Furthermore, the content of specific impurities typically resulting from the distillation of PUFAs is very low. As used herein, the term "isomeric impurities" is used to typically represent impurities generated through the distillation of PUFA-containing natural oils. These include PUFA isomers, peroxidation products, and oligomerization products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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Mode for Carrying Out the Invention

[0021] The term "polyunsaturated fatty acid" (PUFA) refers to a fatty acid containing more than one double bond. Such PUFAs are well known to those skilled in the art. As used herein, a PUFA derivative is a PUFA in the form of a mono, di or triglyceride, ester, phospholipid, amide, lactone or salt. Triglycerides and esters are preferred. Esters are more preferred. Esters are typically alkyl esters, preferably C1-C6 alkyl esters, more preferably C1-C4 alkyl esters. Examples of esters include methyl esters and ethyl esters. Ethyl ester is most preferred. As used herein, the term "PUFA product" typically refers to a product containing one or more polyunsaturated fatty acids (PUFAs) and / or their derivatives that have nutritional or pharmaceutical significance. Typically, a PUFA product is a single PUFA or its derivative. Alternatively, a PUFA product is a mixture of two or more, for example two, PUFAs or their derivatives. As used herein, the term "zone" refers to a plurality of connected chromatography columns, the plurality of connected chromatography columns containing aqueous alcohol as an eluent, one or more injection points for the feed mixture stream, one or more injection points for water and / or alcohol, a raffinate split stream from which liquid from the plurality of connected chromatography columns can be collected, and an extract split stream from which liquid from the plurality of connected chromatography columns can be collected.

[0022]

[0023] ​​​​​​​​​​​​​​​​ It has. Typically, each zone has only one injection point for the feed mixture. In one embodiment, each zone has only one injection point for the aqueous alcohol eluent. In another embodiment, each zone has two or more injection points for water and / or alcohol.

[0024] The term "raffinate" is well known to those skilled in the art. In the context of true and simulated moving bed chromatography, it refers to the flow of components that move more rapidly with the liquid eluent phase than with the solid adsorbent phase. Thus, the raffinate stream typically contains a higher abundance of more polar components and a

[0025] lack of less polar components compared to the feed stream. The term "extract" is well known to those skilled in the art. In the context of true and simulated moving bed chromatography, it refers to the flow of components that move more rapidly with the solid adsorbent phase than with the liquid eluent phase. Thus, the

[0026] extract stream typically contains a higher abundance of less polar components and a lack of more polar components compared to the feed stream. As used herein, the term "non-adjacent" when applied to columns in the same

[0027] apparatus refers to columns separated by one or more columns, preferably three or more columns, more preferably five or more columns, and most preferably about five columns. Thus, when a raffinate stream containing a PUFA product along with more Yes. (b) An extract stream containing the PUFA product together with a less polar component is collected from the column in the second band and introduced into an adjacent column in the first band. In this case, the extract stream collected from the second band is the feed mixture in the first band.

[0028] Typically, the PUFA product comprises at least one ω-3 or ω-6 PUFA, preferably at least one ω-3 PUFA. Examples of ω-3 PUFAs include alpha -linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). SDA, EPA, DPA and DHA are preferred. EPA and DHA are more preferred. Examples of ω-6 PUFAs include linoleic acid (LA), gamma-linolenic acid (GLA), eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA), docosadienoic acid, adrenic acid and docosapentaenoic (ω-6) acid. LA, ARA, GLA and DGLA are preferred.

[0029] In one embodiment, the PUFA product is EPA and / or EPA ethyl ester (EE).

[0030] In another embodiment, the PUFA product is DHA and / or EPA ethyl ester (EE).

[0031] In yet a further embodiment, the PUFA product is a mixture of EPA and DHA and / or E PA EE and DHA EE.

[0032] Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA. Suitable feed mixtures for fractionation by the method of the present invention can be obtained from natural sources including plant and animal fats and oils, and synthetic sources including oils obtained from genetically engineered plants, animals, and microorganisms including yeast. Examples include fish oil, algal oil and microalgal oil, and vegetable oils such as borage oil, evening primrose oil and echium oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil is particularly suitable where the desired PUFA product is EPA and / or DHA. Genetically engineered safflower oil is particularly suitable where the desired PUFA product is GLA. Genetically engineered yeast is particularly suitable where the desired PUFA product is EPA.

[0033] The feed mixture may be chemically treated prior to fractionation by the method of the present invention. For example, it may be subjected to glyceride transesterification or glyceride hydrolysis, followed optionally by crystallization, molecular distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodraction or supercritical fluid fractionation or other selective treatments. The feed mixture may be chemically treated prior to fractionation by the method of the present invention. For example, it may be subjected to glyceride transesterification or glyceride hydrolysis, followed optionally by crystallization, molecular distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodraction or supercritical fluid fractionation or other selective treatments. The feed mixture may be chemically treated prior to fractionation by the method of the present invention. For example, it may be subjected to glyceride transesterification or glyceride hydrolysis, followed optionally by crystallization, molecular distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodraction or supercritical fluid fractionation or other selective treatments. The feed mixture may be chemically treated prior to fractionation by the method of the present invention. For example, it may be subjected to glyceride transesterification or glyceride hydrolysis, followed optionally by crystallization, molecular distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodraction or supercritical fluid fractionation or other selective treatments.

[0034] The feed mixture typically contains a PUFA product, at least one more polar component, and at least one less polar component. The less polar component has a stronger adhesion to the adsorbent used in the method of the present invention than the PUFA product. Throughout the operation, such less polar components typically move with both the solid adsorbent phase rather than the liquid eluent phase. The more polar component has a lower adhesion to the adsorbent used in the method of the present invention than the PUFA product. The feed mixture typically contains a PUFA product, at least one more polar component, and at least one less polar component. The less polar component has a stronger adhesion to the adsorbent used in the method of the present invention than the PUFA product. Throughout the operation, such less polar components typically move with both the solid adsorbent phase rather than the liquid eluent phase. The more polar component has a lower adhesion to the adsorbent used in the method of the present invention than the PUFA product. The feed mixture typically contains a PUFA product, at least one more polar component, and at least one less polar component. The less polar component has a stronger adhesion to the adsorbent used in the method of the present invention than the PUFA product. Throughout the operation, such less polar components typically move with both the solid adsorbent phase rather than the liquid eluent phase. The more polar component has a lower adhesion to the adsorbent used in the method of the present invention than the PUFA product. The feed mixture typically contains a PUFA product, at least one more polar component, and at least one less polar component. The less polar component has a stronger adhesion to the adsorbent used in the method of the present invention than the PUFA product. Throughout the operation, such less polar components typically move with both the solid adsorbent phase rather than the liquid eluent phase. The more polar component has a lower adhesion to the adsorbent used in the method of the present invention than the PUFA product. The feed mixture typically contains a PUFA product, at least one more polar component, and at least one less polar component. The less polar component has a stronger adhesion to the adsorbent used in the method of the present invention than the PUFA product. Throughout the operation, such less polar components typically move with both the solid adsorbent phase rather than the liquid eluent phase. The more polar component has a lower adhesion to the adsorbent used in the method of the present invention than the PUFA product. It has weak adhesion to the adsorbent. Through the operation, such more polar components are typically not in the solid adsorption phase but move with the liquid eluent phase. Generally, the more polar components will be separated into the raffinate stream and the less polar components will be separated into the extract stream.

[0035] Examples of the more polar components and the less polar components include (1) other compounds present in natural oils (e.g., fish oil or vegetable oil), (2) by-products formed during the storage process, purification process, and previous concentration process, and (3) contaminants from solvents or reagents used during the previous concentration process or purification process.

[0036] Examples of (1) include other undesirable PUFAs; saturated fatty acids; sterols such as cholesterol; vitamins; and environmental contaminants such as polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pesticides, chlorinated pesticides, dioxins, and heavy metals. PCBs, PAHs, dioxins, and chlorinated pesticides are all highly non-polar components.

[0037] Examples of (2) include isomers and oxidation or decomposition products of PUFA products, such as auto-acid value polymer products of fatty acids or their derivatives.

[0038] Examples of (3) include urea that can be added to remove saturated or monounsaturated fatty acids from the feed mixture.

[0039] Preferably, the feed mixture is a PUFA-containing fish oil, more preferably a fish oil containing EPA and / or DHA.

[0040] ​​​A typical feed mixture for preparing high-concentration EPA by the method of the present invention is 50 to 7 5% EPA, 0 to 10% DHA, and other components including other essential ω-3 and ω-6 fatty acids are included.

[0041] A preferred feed mixture for preparing high-concentration EPA by the method of the present invention is 55% E PA, 5% DHA, and other components including other essential ω-3 and ω-6 fatty acids are included. DHA is less polar than EPA.

[0042] A typical feed mixture for preparing high-concentration DHA by the method of the present invention is 50 to 7 5% DHA, 0 to 10% EPA, and other components including other essential ω-3 and ω-6 fatty acids are included.

[0043] A preferred feed mixture for preparing high-concentration DHA by the method of the present invention is 75% D HA, 7% EPA, and other components including other essential ω-3 and ω-6 fatty acids are included. EPA is more polar than DHA.

[0044] A typical feed mixture for preparing a high-concentration mixture of EPA and DHA by the method of the present invention contains more than 33% EPA and more than 22% DHA.

[0045] The method of the present invention requires a plurality of bands in the chromatography apparatus. Typically, two or more bands are used. The number of bands is not particularly limited, but generally there are 2 to 5 bands. Preferably, there are 2 or 3 bands, and more preferably 2 bands.

[0046] Typically, the components separated in each band of the apparatus used in the method of the present invention have different polarities​​ has properties.

[0047] Typically, a) the aqueous alcohol eluent present in each zone has a different water: alcohol ratio and / or (b) the rate at which the liquid collected via the extract stream and the raffinate stream in each zone is recycled within the same zone is adjusted so that the PUFA product can be separated from the different components of the feed mixture in each zone. from the different components of the feed mixture in each zone. is adjusted.

[0048] When the apparatus used in the method of the present invention has two zones, the present invention typically is a chromatographic separation method for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, the method comprising introducing the feed mixture into a simulated or moving bed chromatographic apparatus having a plurality of connected chromatography columns containing an aqueous alcohol as an eluent wherein the apparatus has a first zone and a second zone, each zone having an extract stream and a raffinate stream capable of collecting the liquid from the plurality of connected chromatography columns, and (a) the raffinate stream containing the PUFA product together with the more polar components is collected from the column in the first zone and introduced into a non-adjacent column in the second zone and / or (b) the extract stream containing the PUFA product together with the less polar components is collected from the column in the second zone and introduced into a non-adjacent column in the first zone, wherein in the first zone, the PUFA product is separated from the less polar components of the feed mixture and in the second zone, the PUFA product is separated from the more polar components of the feed mixture. and a raffinate stream, and (a) the raffinate stream containing the PUFA product together with the more polar components is collected from the column in the first zone and introduced into a non-adjacent column in the second zone and / or (b) the extract stream containing the PUFA product together with the less polar components is collected from the column in the second zone and introduced into a non-adjacent column in the first zone, wherein in the first zone, the PUFA product is separated from the less polar components of the feed mixture and in the second zone, the PUFA product is separated from the more polar components of the feed mixture. and introduced into a non-adjacent column in the first zone, wherein in the first zone, the PUFA product is separated from the less polar components of the feed mixture and in the second zone, the PUFA product is separated from the more polar components of the feed mixture. is separated.

[0049] Typically, when the apparatus used in the method of the present invention contains two zones, the first zone The eluent in the first zone contains more alcohol than the eluent in the second zone, and the second zone is downstream of the first zone with respect to the flow of the eluent in the system. Thus , the eluent in the system typically moves from the first zone to the second zone. Conversely , the solid adsorbent phase typically moves from the second zone to the first zone. Typically, the two zones do not overlap. That is, there is no chromatographic column in both zones.

[0050] In a further embodiment of the present invention, the apparatus has a first zone, a second zone and a third zone . The water: alcohol ratio of the aqueous alcohol eluent present in the first, second and third zones is typically different. As will be apparent to those skilled in the art, this results in the ability to remove impurities having different polarities in each zone.

[0051] Preferably, when the apparatus has three zones, the eluent in the first zone contains more alcohol than the eluents in the second zone and the third zone, and the first zone is upstream of the second zone and the third zone with respect to the flow of the eluent in the system. Typically , the eluent in the second zone contains more alcohol than the eluent in the third zone , and the second zone is upstream of the third zone with respect to the flow of the eluent in the system. Typically, in the first zone, the PUFA product is separated from components of a feed mixture that are less polar than the PUFA product. Typically, in the second zone, the PU FA product is separated from components of a feed mixture that are less polar than the PUFA product but more polar than the components separated in the first zone. Typically, in the third zone, the PUF ​​​​​ The A product is separated from components of a feed mixture that are more polar than the PUFA product.

[0052] In a further embodiment, in the first zone, the PUFA product is separated from components of a feed mixture that are less polar than the PUFA product, and in the second zone, the PUFA product is separated from components of a feed mixture that are more polar than the PUFA product, and in the third zone, the PUFA product is separated from components of a feed mixture that are more polar than the PUFA product and more polar than the components separated in the second zone. In the third zone, the PUFA product is separated from components of a feed mixture that are more polar than the components separated in the second zone and more polar than the PUFA product. Such a configuration having three zones is suitable for separating EPA and DHA from a mixture containing impurities that are less polar than DHA and EPA and also containing impurities that are more polar than EPA. In the first zone, components that are less polar than DHA and EPA are removed as an extract stream, and a raffinate stream containing DHA, EPA, and components that are more polar than EPA is collected and introduced into the second zone. In the second zone, DHA is removed as an extract stream, and a raffinate stream containing EPA and components that are more polar than EPA is collected and introduced into the third zone. In the third zone, components that are more polar than EPA are removed as a raffinate stream, and purified EPA is collected as an extract stream. In this embodiment, the purified EPA is a purified PUFA product. Such a configuration has the advantage that secondary PUFA can also be collected. In this case, the secondary PUFA is the DHA collected as an extract stream from the second zone.

[0053] In the chromatographic separation method of the present invention, typically, in addition to the PUFA product, In the first zone, components that are less polar than DHA and EPA are removed as an extract stream, and a raffinate stream containing DHA, EPA, and components that are more polar than EPA is collected and introduced into the second zone. In the first zone, components that are less polar than DHA and EPA are removed as an extract stream, and a raffinate stream containing DHA, EPA, and components that are more polar than EPA is collected and introduced into the second zone. In the second zone, DHA is removed as an extract stream, and a raffinate stream containing EPA and components that are more polar than EPA is collected and introduced into the third zone. In the second zone, DHA is removed as an extract stream, and a raffinate stream containing EPA and components that are more polar than EPA is collected and introduced into the third zone. In the third zone, components that are more polar than EPA are removed as a raffinate stream, and purified EPA is collected as an extract stream. In the third zone, components that are more polar than EPA are removed as a raffinate stream, and purified EPA is collected as an extract stream. In this embodiment, the purified EPA is a purified PUFA product. Such a configuration has the advantage that secondary PUFA can also be collected. In this case, the secondary PUFA is the DHA collected as an extract stream from the second zone.

[0054] In the chromatographic separation method of the present invention, typically, in addition to the PUFA product, Secondary PUFA products are collected. Preferably, the PUFA product is EPA and a further secondary PUFA product is DHA.

[0055] In a further embodiment of the invention, the apparatus is configured to collect a PUFA product which is a high-concentration mixture of EPA and DHA. Accordingly, a feed mixture containing EPA, DHA, components more polar than EPA and DHA, and components less polar than EPA and DHA is used. In a first zone, materials less polar than EPA and DHA are removed. In a second zone, materials more polar than EPA and DHA are removed and a high-concentration mixture of EPA and DHA is collected as the PUFA product. EPA, DHA, components more polar than EPA and DHA, and components less polar than EPA and DHA are used. In a first zone, materials less polar than EPA and DHA are removed. In a second zone, materials more polar than EPA and DHA are removed and a high-concentration mixture of EPA and DHA is collected as the PUFA product. and a high-concentration mixture of EPA and DHA is collected as the PUFA product. In a first zone, materials less polar than EPA and DHA are removed. In a second zone, materials more polar than EPA and DHA are removed and a high-concentration mixture of EPA and DHA is collected as the PUFA product. EPA and DHA is collected as the PUFA product.

[0056] If the method of the invention is characterized by a plurality, in particular two zones, any known simulated or true moving bed chromatography apparatus can be utilized for the purposes of the method of the invention. The apparatuses described in U.S. Patent Nos. 2,985,589, 3,696,107, 3,706,812, 3,761,533, French Patent Application Publication Nos. 2103302(A), 2651148(A), 2651149(A), U.S. Patent Nos. 6,979,402, 5,069,883 and 4,764,276 can all be used if they are configured according to the method of the invention. 2, 3,761,533, French Patent Application Publication Nos. 2103302(A), 2 651148(A), 2651149(A), U.S. Patent Nos. 6,979,402 5,069,883 and 4,764,276 can all be used if they are configured according to the method of the invention.

[0057]

[0057] The number of columns used in the apparatus is not particularly limited. One skilled in the art will be able to readily determine the appropriate number of columns to use. The number of columns is typically 8 or more, preferably 15 or more. In a more preferred embodiment, 15 or 16 columns are used. are used. In another more preferred embodiment, 19 or 20 columns are used . In other more preferred embodiments, 30 or more columns are used. Typically, there are 50 or fewer, preferably 40 or fewer columns.

[0058] Each band is typically composed of approximately an equal share of the total number of columns. Thus, in the case of an apparatus composed of two bands, each band is composed of approximately half of the total number of chromatographic columns in the system. Thus, the first band typically contains 4 or more, preferably 8 or more, more preferably about 8 columns. The second band typically contains 4 or more columns, preferably 7 or more, more preferably 7 or 8 columns.

[0059] The dimensions of the columns used in the apparatus are not particularly limited and will depend on the volume of the feedstock to be purified . A person skilled in the art will be able to easily determine the appropriate size of the columns to be used . The diameter of each column is typically from 10 to 500 mm, preferably from 25 to 250 mm, more preferably from 50 to 100 mm, and most preferably from 70 to 80 mm. The length of each column is typically from 10 to 200 cm, preferably from 25 to 1 50 cm, more preferably from 70 to 110 cm, and most preferably from 80 to 100 cm .

[0060] The columns in each band typically have the same dimensions, but may have different dimensions depending on the application .

[0061] The flow rate to the columns is limited by the maximum pressure of the series of columns and depends on the column dimensions and the solid It will depend on the particle size of the phase. A person skilled in the art will be able to easily determine the required flow rate according to each column dimension in order to desorb efficiently. As the diameter of the column increases, generally, it is necessary to increase the flow rate to maintain a linear flow in the column.

[0062] In a typical column size and an apparatus having two zones schematically shown above, typically, the flow rate of the eluent to the first zone is from 1 to 4.5 L / min, preferably from 1.5 to 2.5 L / min. Typically, the flow rate of the eluent from the first zone is from 0.1 to 2.5 L / min, preferably from 0.5 to 2.25 L / min. In an embodiment where a part of the extractant is recycled within the first zone from the first zone, the recycling flow rate is typically from 0.7 to 1 .4 L / min, preferably about 1 L / min. Typically, the flow rate of the raffinate from the first zone is from 0.2 to 2.5 L / min, preferably from 0.3 to 2.0 L / min. In an embodiment where a part of the raffinate from the first zone is recycled within the first zone, the recycling flow rate is typically from 0.3 to 1.0 L / min, preferably about 0.5 L / min. Typically, the flow rate of the introduction of the feed mixture to the first zone is from 5 to 150 mL / min, preferably from 10 to 10 0 mL / min, more preferably from 20 to 60 mL / min.

[0063] In a typical column size and an apparatus having two zones schematically shown above, typically, the flow rate of the eluent to the second zone is from 1 to 4 L / min, preferably from 1.5 to 3. 5 L / min. Typically, the flow rate of the eluent from the second zone is from 0.5 to 2 L / min, preferably from 0.7 to 1.9 L / min. In an embodiment where a part of the eluent is recycled within the second zone from the second zone In an embodiment where it is recycled, the flow rate of the recycle is typically from 0.6 to 1.4 L / min , preferably from 0.7 to 1.1 L / min, more preferably about 0.9 L / min. Typically , the flow rate of the raffinate from the second zone is from 0.5 to 2.5 L / min, preferably from 0.7 to 1.8 L / min, more preferably about 1.4 L / min.

[0064] As will be understood by those skilled in the art, reference to the flow rate at which liquid is collected or removed via the various extract and raffinate streams refers to the volume of liquid removed over a period of time, typically L / min. Similarly, reference to the rate at which liquid is recycled to adjacent columns in the same zone, typically in the same zone, refers to the volume of liquid recycled over a period of time, typically L / min.

[0065] Typically, a portion of one or more of the extract stream from the first zone, the raffinate stream from the first zone, the extract stream from the second zone and the raffinate stream from the second zone is recycled to adjacent columns in the same zone , typically in the same zone. This recycling is different from the supply of an extract or raffinate stream to non - adjacent columns in another zone. Rather, recycling involves supplying a portion of an extract or raffinate stream from one zone to adjacent columns in the same zone

[0066] , typically in the same zone. Rather, recycling involves supplying a portion of an extract or raffinate stream from one zone to adjacent columns in the same zone , typically in the same zone.

[0067] The rate at which liquid collected via an extract or raffinate stream from the first or second zone is recycled within the same zone is the rate at which the liquid collected via that stream is returned to adjacent columns in the same zone, typically in the same zone. This can be understood with reference to Figure 9. The rate of recycling of the extract in the first zone is from the bottom of column 2 to the top of column 2, and the rate of recycling of the raffinate in the first zone is from the bottom of column 3 to the top of column 3. The rate of recycling of the extract in the second zone is from the bottom of column 4 The rate at which the collected extract is supplied to the top of column 3, i.e., the liquid flow rate to the top of column 3. is. The rate of recirculation of the extract in the second zone is the rate at which the extract collected at the bottom of column 10 is supplied to the top of column 11, i.e., the liquid flow rate to the top of column 11.

[0068] Recirculation of the extract stream and / or raffinate stream is typically carried out by feeding the liquid collected through that stream to a container and then pumping a certain amount of that liquid back from the container to the same zone. In this case, the rate of recirculation of the liquid collected through a particular extract stream or raffinate stream to an adjacent column, typically in the same zone, is the rate at which the liquid is pumped back from the container to the same zone, typically an adjacent column. As will be understood by those skilled in the art, the amount of liquid introduced into a zone through the eluent stream and feed stream is balanced with the amount of liquid removed from a zone and recirculated within the same zone. Thus, referring to FIG. 9, for the extract stream, the flow rate (D) of the eluent (desorbent) to the first or second zone is such that the rate (E1 / E2) at which the liquid collected from that zone through the extract stream accumulates in the container, plus the rate (D - E1 / D - E2) at which the extract is recirculated within the same zone, is equal to the rate. For the raffinate stream in a zone, the rate (D - E1 / D - E2) at which the extract is recirculated within a zone, plus the rate

[0069] (F / R1) at which the feed is introduced into a zone, is equal to the rate (R1 / R2) at which the liquid collected from that zone through the raffinate stream accumulates in the container, plus the rate (D + F - E1 - R1 / D + R1 - E2 - R2) at which the raffinate is recirculated within the same zone. The flow rate (D) of the eluent (desorbent) to the first or second zone is such that the rate (E1 / E2) at which the liquid collected from that zone through the extract stream accumulates in the container, plus the rate (D - E1 / D - E2) at which the extract is recirculated within the same zone, is equal to the rate. For the raffinate stream in a zone, the rate (D - E1 / D - E2) at which the extract is recirculated within a zone, plus the rate (F / R1) at which the feed is introduced into a zone, is equal to the rate (R1 / R2) at which the liquid collected from that zone through the raffinate stream accumulates in the container, plus the rate (D + F - E1 - R1 / D + R1 - E2 - R2) at which the raffinate is recirculated within the same zone. (F / R1) at which the feed is introduced into a zone, is equal to the rate (R1 / R2) at which the liquid collected from that zone through the raffinate stream accumulates in the container, plus the rate (D + F - E1 - R1 / D + R1 - E2 - R2) at which the raffinate is recirculated within the same zone. ​​​

[0070] Liquid collected from a specific extract stream or raffinate stream from a certain band accumulates in the container The rate can be considered to be the net rate at which the extract stream or raffinate stream is removed from that band as well.

[0071] Typically, the rate at which liquid collected from the first band via the extract stream is recirculated within the first band is different from the rate at which liquid collected from the second band via the extract stream is recirculated within the second band and / or the rate at which liquid collected from the first band via the raffinate stream is recirculated within the first band is different from the rate at which liquid collected from the second band via the raffinate stream is recirculated within the second band band.

[0072] Changing the rate at which liquid collected via the extract stream and / or raffinate stream in each band is recirculated within the same band has the effect of changing the amounts of more polar and less polar components present in other extract streams and raffinate streams. Thus, for example when the recirculation rate of the extract is low, the amount of less polar components in that band carried by the raffinate stream in that band decreases. When the recirculation rate of the extract is high, the amount of less polar components in that band carried by the raffinate stream in that band increases. This can be understood, for example, in the specific embodiment of the present invention shown in FIG. 6. The rate (D-E1) at which liquid collected from the first band via the extract stream is recirculated within the same band affects the degree (R1) to which component A is carried by the raffinate stream in the first band band. This can be understood, for example, in the specific embodiment of the present invention shown in FIG. 6. The rate (D-E1) at which liquid collected from the first band via the extract stream is recirculated within the same band affects the degree (R1) to which component A is carried by the raffinate stream in the first band band.

[0073] Typically, the rate at which liquid collected from the first band via the extract stream is recirculated within the first band The velocity at which is recycled is faster than the velocity at which the liquid collected through the extract stream from the second zone is recycled within the second zone. Preferably, the raffinate stream containing the PUFA product together with the more polar components is collected from the column in the first zone and introduced into a non - adjacent column in the second zone, and the velocity at which the liquid collected through the extract stream from the first zone is recycled within the first zone is faster than the velocity at which the liquid collected through the extract stream from the second zone is recycled within the second zone.

[0074] Alternatively, the velocity at which the liquid collected through the extract stream from the first zone is recycled within the first zone is slower than the velocity at which the liquid collected through the extract stream from the second zone is recycled within the second zone.

[0075] Typically, the velocity at which the liquid collected through the raffinate stream from the second zone is recycled within the second zone is faster than the velocity at which the liquid collected through the raffinate stream from the first zone is recycled within the first zone. Preferably, the extract stream containing the PUFA product together with the less polar components is collected from the column in the second zone and introduced into a non - adjacent column in the first zone, and the velocity at which the liquid collected through the raffinate stream from the second zone is recycled within the second zone is faster than the velocity at which the liquid collected through the raffinate stream from the first zone is recycled within the first zone.

[0076] Alternatively, the velocity at which the liquid collected through the raffinate stream from the second zone is recycled within the second zone is slower than the velocity at which the liquid collected through the raffinate stream from the first zone is recycled within the first zone.

[0077] ​​​​​​​​​​​​​​​The step time, i.e., the time for shifting the injection points of the feed mixture and the eluent and the various branching points of the recovered fractions is not particularly limited and will depend on the number and size of the columns used, the method, and the flow rate through the apparatus. A person skilled in the art will be able to easily determine the appropriate step time to be used in the method of the present invention. The step time is typically from 100 to 1000 seconds, preferably from 200 to 800 seconds, more preferably from about 250 to about 750 seconds. In some embodiments, a step time of 100 to 400 seconds, preferably 200 to 300 seconds, more preferably about 250 seconds is suitable. In other embodiments a step time of 600 to 900 seconds, preferably 700 to 800 seconds, more preferably about 750 seconds is suitable.

[0078] In the method of the present invention, true moving bed chromatography is preferred.

[0079] Conventional adsorbents known in such technical fields for true and simulated moving bed systems can be used in the method of the present invention. Each column for chromatography may contain the same adsorbent or different adsorbents. Typically, each column contains the same adsorbent. Examples of such commonly used materials are polymeric beads, preferably polystyrene reticulated with DVB (divinylbenzene); and silica gel, preferably reverse-phase bonded silica gel containing C8 or C18 alkane, especially C18 alkane. C18-bonded reverse-phase silica gel is preferred. The adsorbent used in the method of the present invention is preferably non-polar. The form of the adsorbent stationary phase material is, for example, spherical or non-spherical beads, preferably substantially spherical

[0080] ​Beads may also be used. Such beads typically have a diameter of from 40 to 500 microns, preferably from 100 to 500 microns, more preferably from 250 to 500 microns, still more preferably from 250 to 400 microns, and most preferably from 250 to 350 microns. These preferred particle sizes are somewhat larger than those of the beads conventionally used in simulated and moving bed processes. By using larger particles, the pressure of the eluent used in the system can be reduced. This has advantages in terms of cost savings, efficiency and the life of the apparatus. Surprisingly, adsorbent beads of large particle size can be used in the process of the present invention (along with their attendant advantages) without causing a decrease in resolution.

[0081] The adsorbent typically has a pore size of from 10 to 50 nm, preferably from 15 to 45 nm, more preferably from 20 to 40 nm, and most preferably from 25 to 35 nm.

[0082] The eluent used in the process of the present invention is an aqueous alcohol. An aqueous alcohol typically contains water and one or more short-chain alcohols. Short-chain alcohols typically have from 1 to 6 carbon atoms. Examples of suitable alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s- butanol and t-butanol. Methanol and ethanol are suitable and methanol is more suitable.

[0083] Typically, the eluent is not in a supercritical state. Typically, the eluent is a liquid.

[0084] Typically, the average water: alcohol ratio of the eluent throughout the apparatus is from 0.1:99.9 or from 9:91 parts by volume, preferably from 0.25:99.75 to 7:93 parts by volume, more preferably from 0.5:99.5 to 6:94 parts by volume.

[0085] Typically, the elution power of the eluent varies in each of the bands. Preferably, the elution power of the eluent in the first band is greater than the elution power of the eluent in the second band and subsequent bands. In fact, this is achieved by varying the relative amounts of water and alcohol in each band. Alcohol is generally a more powerful desorbent than water. Thus, the amount of alcohol in the eluent of the first band is typically greater than the amount of alcohol in the eluent of the second band and subsequent bands.

[0086] In embodiments where the aqueous alcohol present in each band has a different water - alcohol content, the water: alcohol ratio of the eluent in the first band is typically from 0:100 to 5: 95 parts by volume, preferably from 0.1:99.9 to 2.5:97.5 parts by volume, more preferably from 0.25:99.75 to 2:98 parts by volume, most preferably from 0.5:99.5 to 1.5 :98.5 parts by volume. In these embodiments, the water: alcohol ratio of the eluent in the second band is typically from 3:97 to 7:93 parts by volume, preferably from 4:96 to 6: 94 parts by volume, more preferably from 4.5:95.5 to 5.5:94.5 parts by volume.

[0087] In a particularly preferred embodiment where the aqueous alcohol present in each band has a different water - alcohol content, the water: alcohol ratio of the eluent in the first band is 0.5:99.5 or ​​​​​​It is a 1.5:98.5 volume part, and the water: alcohol ratio of the eluent in the second band is 4 .5:95.5 to 5.5:94.5 volume parts.

[0088] In the embodiments where the speed at which the liquid collected via the extract stream and the raffinate stream in each band is recycled within the same band is adjusted such that the PUFA product can be separated from different components of the feed mixture in each band, the water: alcohol ratio of the eluent in each band may be the same or different. Typically, the water: alcohol ratio of the eluent in each band is from 0.5:99.5 to 5.5:94.5 volume parts. In one embodiment, the water: alcohol ratio of the eluent in the first band is smaller than the water: alcohol ratio of the eluent in the second band. In another embodiment, the water: alcohol ratio of the eluent in the first band is greater than the water: alcohol ratio of the eluent in the second band. In a further embodiment, the water: alcohol ratio of the eluent in the first band is the same as the water: alcohol ratio of the eluent in the second band. It will be understood that the ratios of water and alcohol in each of the bands listed above are the average ratios within the total of the bands . Typically, the water: alcohol ratio of the eluent in each band is controlled by introducing water and / or alcohol into one or more columns in those bands. Thus, for example, to achieve a smaller water: alcohol ratio in the first band than in the second band, water is typically introduced into the first band more slowly than into the second band . In some embodiments, essentially pure alcohol and essentially

[0089] It will be understood that the ratios of water and alcohol in each of the bands listed above are the average ratios within the total of the bands .

[0090] Typically, the water: alcohol ratio of the eluent in each band is controlled by introducing water and / or alcohol into one or more columns in those bands. Thus, for example, to achieve a smaller water: alcohol ratio in the first band than in the second band, water is typically introduced into the first band more slowly than into the second band . Thus, for example, to achieve a smaller water: alcohol ratio in the first band than in the second band, water is typically introduced into the first band more slowly than into the second band . To achieve a smaller water: alcohol ratio in the first band than in the second band, water is typically introduced into the first band more slowly than into the second band . In some embodiments, essentially pure alcohol and essentially Pure water can be introduced at different points in each zone. The relative flow rates of these two liquid streams will determine the overall solvent profile of the zone. In other embodiments, different alcohol / water mixtures can be introduced at different points in each zone. This includes introducing into the zone two or more different alcohol / water mixtures having different alcohol:water ratios for each alcohol / water mixture. The relative flow rates and relative concentrations of the alcohol / water mixtures in this embodiment will determine the overall solvent profile of the zone. In other embodiments where the water:alcohol ratio of the eluent in each zone is the same, the same alcohol / water mixture is introduced into each zone.

[0091] Typically, the method of the present invention is carried out at 15 to 55 °C, preferably 20 to 40 °C, more preferably about 30 °C. Thus, the method is typically carried out at room temperature, but may also be carried out at elevated temperatures.

[0092] The method of the present invention includes introducing a feed stream into one zone (e.g., the first zone), collecting a first intermediate stream rich in PUFA product, and introducing the first intermediate stream into another zone (e.g., the second zone). Thus, when the apparatus has two zones, the method includes (a) collecting the first intermediate stream from the first zone and introducing it into the second zone, or (b) collecting the first intermediate stream from the second zone and introducing it into the first zone. In this way, the PUFA product can be separated from both the more polar components and the less polar components in a single method.

[0093] ​​​​​​​​​​​​Collect the raffinate stream containing the PUFA product together with the more polar component in the first zone from the column in and introduce it into a non-adjacent column in the second zone, or (b ) collect the extract stream containing the PUFA product together with the less polar component in the second zone from the column in and introduce it into a non-adjacent column in the first zone. In a particularly preferred embodiment, the apparatus has two zones and the method of the present invention comprises (i) introducing the feed mixture into the first zone and removing a first raffinate stream rich in the PUFA product and a first extract stream lacking the PUFA product, and

[0094] (ii) introducing the first raffinate stream into the second zone, removing a second raffinate stream lacking the PUFA product, and collecting the second extract stream to obtain the PUFA product. This particularly preferred embodiment is suitable for purifying EPA from the feed mixture. This particularly preferred embodiment is shown in Figure 2. A feed mixture F containing the PUFA product (B), as well as a more polar component (C) and a less polar component (A) is introduced into the first zone. In the first zone, the less polar component (A) is removed as the extract stream E1. The PUFA product (B) and the more polar component (C) are removed as the raffinate stream R1. The raffinate stream R1 is then introduced into the second zone.

[0095] In the second zone, the more polar component (C) is removed as the raffinate stream R2.

[0096] The PUFA product (B) is collected as the extract stream E2.

[0097]

[0097] ​​This embodiment is shown in detail in FIG. 4. FIG. 4 is the same as FIG. 2 except that the introduction points of the alcohol desorbent (D) and water (W) into each zone are shown. The alcohol desorbent (D) and water (W) together constitute the eluent. The (D) phase is typically substantially pure alcohol, but in some embodiments may be an alcohol / water mixture predominantly containing alcohol. The (W) phase is typically substantially pure water, but in some embodiments may be an alcohol / water mixture predominantly containing water, such as a mixture of 98% water and 2% methanol. Although it is a pure alcohol in nature, in some embodiments, it may be an alcohol / water mixture mainly containing alcohol. The (W) phase is typically substantially pure water, but in some embodiments, it may be an alcohol / water mixture mainly containing water, for example, a mixture of 98% water and 2% methanol.

[0098] A further example of this particularly preferred embodiment is shown in FIG. 6. Here, there is no individual water injection point, but instead an aqueous alcohol desorbent is injected into (D).

[0099] By varying the desorbing power of the eluent within each zone, separation into the raffinate stream and the extract stream can be facilitated. This can be achieved by introducing the alcohol (or alcohol-rich) component and the water (or water-rich) component of the eluent at different points within each zone. Thus, typically, for the flow of the eluent in the system, alcohol is introduced upstream of the extract branch point, and water is introduced between the extract branch point and the introduction point of the feed to the zone. This is shown in FIG. 4. By changing the desorbing power of the eluent within each zone, separation into the raffinate stream and the extract stream can be promoted. This can be achieved by introducing the alcohol (or alcohol-rich) component and the water (or water-rich) component of the eluent at different points within each zone. Therefore, typically, for the flow of the eluent in the system, alcohol is introduced upstream of the extract branch point, and water is introduced between the extract branch point and the introduction point of the feed to the zone. This is shown in FIG. 4.

[0100] Alternatively, separation can be facilitated by changing the rate at which the liquid collected from the two zones via the extract stream and the raffinate stream is recycled within the same zone.

[0101] Typically, in this particularly preferred embodiment, the liquid collected from the first zone via the extract stream The rate at which the liquid being recycled within the first zone is collected via the extract stream from the second zone is faster than the rate at which the liquid being recycled within the second zone, or the water:alcohol ratio of the eluent in the first zone is less than that in the second zone.

[0102] In this particularly preferred embodiment, the first raffinate stream in the first zone is typically , downstream of the point of introduction of the feed mixture into the first zone with respect to the flow of the eluent in the first zone and is removed.

[0103] In this particularly preferred embodiment, the first extract stream in the first zone is typically , upstream of the point of introduction of the feed mixture into the first zone with respect to the flow of the eluent in the first zone and is removed.

[0104] In this particularly preferred embodiment, the second raffinate stream in the second zone is typically , downstream of the point of introduction of the first raffinate stream into the second zone with respect to the flow of the eluent in the second zone and is removed.

[0105] In this particularly preferred embodiment, the second extract stream in the second zone is typically , upstream of the point of introduction of the first raffinate stream into the second zone with respect to the flow of the eluent in the second zone and is collected.

[0106] Typically, in this particularly preferred embodiment, the alcohol or aqueous alcohol is introduced into the first zone upstream of the point of removal of the first extract stream with respect to the flow of the eluent in the first zone within the first zone.

[0107] Typically, in this particularly preferred embodiment, when water is introduced into the first zone , water is upstream of the introduction point of the feed mixture with respect to the flow of the eluent in the first zone but is introduced into the first zone downstream of the removal point of the first extract stream.

[0108] Typically, in this particularly preferred embodiment, the alcohol or aqueous alcohol is introduced into the second zone upstream of the removal point of the second extract stream with respect to the flow of the eluent in the second zone and within the second zone.

[0109] Typically, in this particularly preferred embodiment, when water is introduced into the second zone , water is upstream of the introduction point of the first raffinate stream with respect to the flow of the eluent in the second zone but is introduced into the second zone downstream of the removal point of the second extract stream.

[0110] In another particularly preferred embodiment, the apparatus has two zones and the method comprises (i) introducing the feed mixture into the second zone and removing a first raffinate stream lacking the PUFA product, and a first extract stream rich in the PUFA product, and (ii) introducing the first extract stream into the first zone, removing a second extract stream lacking the PUFA product, and collecting the second raffinate stream to obtain the PUFA product .

[0111] This particularly preferred embodiment is suitable for purifying DHA from the feed mixture.

[0112] This embodiment is shown in Figure 3. A feed mixture F containing the PUFA product (B), as well as more polar component (C) and less polar component (A) is introduced into the second zone. In the second zone, the more polar component (C) is removed as the raffinate stream R1. PUF Product (B) and a less polar component (A) are collected as the extract stream E1. Next, the extract stream E1 is introduced into the first zone. In the first zone, the less polar component (A) is removed as the extract stream E2. The PUFA product (B) is collected as the raffinate stream R2.

[0113] This embodiment is shown in more detail in FIG. 5. FIG. 5 is the same as FIG. 3 except that the introduction points of the short-chain alcohol desorbent (D) and water (W) into each zone are shown. As described above, the (D) phase is typically essentially pure alcohol, but in some embodiments it may be an alcohol / water mixture mainly containing alcohol. The (W) phase is typically essentially pure water, but may be an alcohol / water mixture mainly containing water, for example a mixture of 98% water and 2% methanol.

[0114] A further example of this particularly preferred embodiment is shown in FIG. 7. Here, there is no individual water injection point, but instead an aqueous alcohol desorbent is injected into (D).

[0115] Typically, in this embodiment, the rate at which the liquid collected from the raffinate stream in the second zone is reintroduced into the second zone is faster than the rate at which the liquid collected from the raffinate stream in the first zone is reintroduced into the first zone, or the water:alcohol ratio of the eluent in the first zone is lower than that in the second zone.

[0116] In this second particularly preferred embodiment, the first raffinate stream in the second zone is typically removed downstream of the introduction point of the feed mixture into the second zone with respect to the flow of the eluent in the second zone. ​

[0117] In this second particularly preferred embodiment, the first extract stream in the second zone is typically collected upstream of the point of introduction of the feed mixture into the second zone with respect to the flow of the eluent in the second zone.

[0118] In this second particularly preferred embodiment, the second raffinate stream in the first zone is typically collected downstream of the point of introduction of the first extract stream into the first zone with respect to the flow of the eluent in the first zone.

[0119] In this second particularly preferred embodiment, the second extract stream in the first zone is typically removed upstream of the point of introduction of the first extract stream into the first zone with respect to the flow of the eluent in the first zone.

[0120] Typically, in this second particularly preferred embodiment, the alcohol or aqueous alcohol is introduced into the second zone upstream of the point of removal of the first extract stream with respect to the flow of the eluent in the second zone.

[0121] Typically, when water is introduced into the second zone in this second particularly preferred embodiment, the water is introduced into the second zone upstream of the point of introduction of the feed mixture but downstream of the point of removal of the first extract stream with respect to the flow of the eluent in the second zone.

[0122] Typically, in this second particularly preferred embodiment, the alcohol or aqueous alcohol is introduced into the first zone upstream of the point of removal of the second extract stream with respect to the flow of the eluent in the first zone.

[0123] Typically, in this second particularly preferred embodiment, water is introduced into the first zone. In the case where this occurs, the water is upstream of the introduction point of the first raffinate stream with respect to the flow of the eluent in the first zone. It is a stream, but is introduced into the first zone downstream of the removal point of the second extract stream.

[0124] In a preferred embodiment of the present invention, the simulated or true moving bed chromatography apparatus comprises 15 chromatography columns. These are referred to as columns 1 to 15. The 15 columns are arranged in series such that the bottom of column 1 is connected to the top of column 2, and the bottom of column 2 is connected to the top of column 3, and so on. This may be via a holding vessel in some cases, and the recycle stream may be allowed to flow into the next column. The flow of the eluent through the system is from column 1 to column 2, from column 2 to column 3, and so on. The flow of the adsorbent through the system is from column 15 to column 14, from column 14 to column 13, and so on. The flow of the eluent through the system is from column 1 to column 2, from column 2 to column 3, and so on. The flow of the adsorbent through the system is from column 15 to column 14, from column 14 to column 13, and so on. 14 to column 13, and so on.

[0125] In the most preferred embodiment, the first zone typically consists of 8 adjacent columns connected as described above, i.e., columns 1 to 8. In this most preferred embodiment, the second zone typically consists of 7 columns connected as described above, i.e., columns 9 to 15. To avoid any ambiguity, the bottom of column 8 in the first zone is connected to the top of column 9 in the second zone. In the most preferred embodiment, the second zone typically consists of 7 columns connected as described above, i.e., columns 9 to 15. To avoid any ambiguity, the bottom of column 8 in the first zone is connected to the top of column 9 in the second zone. i.e., columns 9 to 15. To avoid any ambiguity, the bottom of column 8 in the first zone is connected to the top of column 9 in the second zone.

[0126] The most preferred embodiment is shown in Figure 8. A feed mixture F containing the PUFA product (B), as well as the more polar component (C) and the less polar component (A), is fed to column 1 in the first zone. It is introduced at the top of the ram 5. The alcohol desorbent is introduced at the top of column 1 in the first band. It is introduced. Water is introduced at the top of column 4 in the first band. In the first band, the less polar component (A) is removed as the extract stream E1 from the bottom of column 2. The PUFA product (B) and the more polar component (C) are removed as the raffinate stream R1 from the bottom of column 7. Then, the raffinate stream R1 is introduced at the top of column 13 in the second band. The alcohol desorbent is introduced at the top of column 9 in the second band. Water is introduced at the top of column 12 in the second band. In the second band, the more polar component (C) is removed as the raffinate stream R2 at the bottom of column 15. The PUFA product (B) is collected as the extract stream E2 at the bottom of column 10.

[0127] In this most preferred embodiment, the alcohol is typically introduced at the top of column 1 in the first band.

[0128] In this most preferred embodiment, the water is typically introduced at the top of column 4 in the first band.

[0129] In this most preferred embodiment, the alcohol is typically introduced at the top of column 9 in the second band.

[0130] In this most preferred embodiment, the alcohol is typically introduced at the top of column 12 in the second band.

[0131] In this most preferred embodiment, the feed stream is typically introduced at the top of column 5 in the first band.

[0132] ​​​​​​ In this most preferred embodiment, the first raffinate stream is typically collected from the bottom of column 7 in the first zone and introduced to the top of column 13 in the second zone. The first raffinate stream may optionally be collected in a vessel before being introduced to column 13.

[0133] In this most preferred embodiment, the first extract stream is typically removed from the bottom of column 2 in the first zone. The first extract stream may optionally be collected in a vessel and reintroduced to the top of column 3 in the first zone.

[0134] In this most preferred embodiment, the second raffinate stream is typically removed from the bottom of column 15 in the second zone.

[0135] In this most preferred embodiment, the second extract stream is typically collected from the bottom of column 10 in the second zone. This second extract stream typically contains the purified PUFA product. The second extract stream may optionally be collected in a vessel and reintroduced to the top of column 11 in the second zone.

[0136] Typically, in this most preferred embodiment, the water:alcohol ratio in the first zone is less than the water:alcohol ratio in the second zone.

[0137] A further most preferred embodiment is shown in FIG. 9. A feed mixture F containing the PUFA product (B), as well as more polar component (C) and less polar component (A), is introduced to the top of column 5 in the first zone. An aqueous alcohol desorbent is introduced to column It is introduced at the top of column 1. In the first band, the less polar component (A) is in column 2 is removed as the extract stream E1 from the bottom. The PUFA product (B) and the more polar component (C) are removed as the raffinate stream R1 from the bottom of column 7. Then, the raffinate stream R 1 is introduced at the top of column 12 in the second band. An aqueous alcohol desorbent is introduced at the top of column 9 in the second band. In the second band, the more polar component (C) is removed as the raffinate stream R2 at the bottom of column 14. The PUFA product (B) is in the column is collected as the extract stream E2 at the bottom of column 10.

[0138] In this most preferred embodiment, the aqueous alcohol is typically introduced at the top of column 1 in the first band .

[0139] In this most preferred embodiment, the aqueous alcohol is typically introduced at the top of column 9 in the second band .

[0140] In this most preferred embodiment, the feed stream is typically introduced at the top of column 5 in the first band .

[0141] In this most preferred embodiment, the first raffinate stream is typically collected from the bottom of column 7 in the first band and introduced at the top of column 12 in the second band. The first raffinate stream may optionally be collected in a vessel before being introduced into column 12.

[0142] In this most preferred embodiment, the first extract stream is typically removed from the bottom of column 2 in the first band. The first extract stream may optionally be collected in a vessel and One portion may be reintroduced to the top of column 3 in the first band. The liquid collected via the extract flow is recycled within the first band at a rate that is the rate at which the liquid is pumped to the top of column 3 from this vessel.

[0143] In this most preferred embodiment, the second raffinate stream is typically removed from the bottom of column 14 in the second band.

[0144] In this most preferred embodiment, the second extract stream is typically collected from the bottom of column 10 in the second band. This second extract stream typically contains the purified PUFA product. The second extract stream may optionally be collected within the vessel and a portion may be reintroduced to the top of column 11 in the second band. The rate at which the liquid collected via the extract stream from the second band is recycled within the second band is the rate at which the liquid is pumped to the top of column 11 from this vessel.

[0145] In this most preferred embodiment, the rate at which the liquid collected via the extract stream from the first band is recycled within the first band is typically faster than the rate at which the liquid collected via the extract stream from the second band is recycled within the second band.

[0146] In this most preferred embodiment, the aqueous alcohol eluent is substantially the same in each band.

[0147] In a further preferred embodiment of the present invention, the simulated or moving bed chromatography apparatus consists of 19 chromatography columns. These are referred to as columns 1 to 19. ​They are. Those 15 columns are arranged in series such that the bottom of column 1 is connected to the top of column 2, and the bottom of column 2 is connected to the top of column 3, and so on. The flow of the eluent through the system flows from column 1 to column 2, and from column 2 to column 3. The flow of the adsorbent through the system flows from column 19 to column 18, and from column 18 to column 17. In this embodiment, the first band typically consists of 10 adjacent columns connected as described above, i.e., columns 1 to 10. The second band typically consists of 8 columns connected as described above, i.e., columns 11 to 19. This further preferred embodiment is shown in FIG. 10. A feed mixture F containing a PUFA product (B), as well as more polar components (C) and less polar components (A and A') is introduced at the top of column 7 in the first band. A first desorbent (D1) containing 100% alcohol is introduced at the top of column 1 in the first band. A second desorbent (D2) containing a water / alcohol mixture (preferably 2% methanol and 98% water) is introduced at the top of column 5 in the first band. In the first band, the less polar components (A') and (A) are removed as extract streams E1' and E1 from the bottoms of columns 1 and 4, respectively. The PUFA product (B) and the more polar component (C) are removed as raffinate stream R1 from the bottom of column 10. Then, the raffinate stream R1 is introduced at the top of column 17 in the second band. A water / alcohol mixture (preferably 2% methanol and 98 % water) is introduced at the top of column 18 in the second band. In the second band, the more polar component (C) is removed as extract stream E2 from the bottom of column 18. The less polar components (A and A') are removed as raffinate stream R2 from the bottom of column 19.

[0148] In this embodiment, the first band typically consists of 10 adjacent columns connected as described above, i.e., columns 1 to 10. The second band typically consists of 8 columns connected as described above, i.e., columns 11 to 19.

[0149] This further preferred embodiment is shown in FIG. 10. A feed mixture F containing a PUFA product (B), as well as more polar components (C) and less polar components (A and A') is introduced at the top of column 7 in the first band. A first desorbent (D1) containing 100% alcohol is introduced at the top of column 1 in the first band. A second desorbent (D2) containing a water / alcohol mixture (preferably 2% methanol and 98% water) is introduced at the top of column 5 in the first band. In the first band, the less polar components (A') and (A) are removed as extract streams E1' and E1 from the bottoms of columns 1 and 4, respectively. The PUFA product (B) and the more polar component (C) are removed as raffinate stream R1 from the bottom of column 10. Then, the raffinate stream R1 is introduced at the top of column 17 in the second band. A water / alcohol mixture (preferably 2% methanol and 98 % water) is introduced at the top of column 18 in the second band. In the second band, the more polar component (C) is removed as extract stream E2 from the bottom of column 18. The less polar components (A and A') are removed as raffinate stream R2 from the bottom of column 19. A second desorbent (D2) containing % water) is introduced at the top of column 11 in the second zone. In the second zone, the more polar component (C) is removed as raffinate stream R2 at the bottom of column 19. The PUFA product (B) is collected as extract stream E2 at the bottom of column 14.

[0150] In this preferred embodiment, the alcohol is typically introduced at the top of column 1 in the first zone.

[0151] In this preferred embodiment, a mixture of 2% MeOH and 98% water is typically introduced at the top of column 5 in the first zone.

[0152] In this preferred embodiment, a mixture of 2% MeOH and 98% water is typically introduced at the top of column 11 in the second zone.

[0153] In this preferred embodiment, the feed stream is typically introduced at the top of column 7 in the first zone.

[0154] In this preferred embodiment, the first raffinate stream is typically collected from the bottom of column 10 in the first zone and introduced at the top of column 17 in the second zone. The first raffinate stream may optionally be collected in a vessel before being introduced into column 17.

[0155] In this preferred embodiment, the extract stream is typically removed from the bottoms of columns 1 and 4 in the first zone. The extract stream collected from the bottom of column 4 may optionally be collected in a vessel and reintroduced at the top of column 5 in the first zone.

[0156] ​​In this preferred embodiment, the second raffinate stream is typically the ca in the second zone removed from the bottom of column 19.

[0157] In this preferred embodiment, the second extract stream is typically the ca in the second zone collected from the bottom of column 14. This second extract stream typically contains purified PUFA product matter. The second extract stream may optionally be collected in a vessel and reintroduced to the top of column 15 in the second zone .

[0158] Typically, in this most preferred embodiment, the water:alcohol ratio in the first zone is less than the water:alcohol ratio in the second zone.

[0159] The method of the present invention enables the achievement of a much higher purity PUFA product than when using conventional chromatographic techniques. The PUFA product produced by the method of the present invention also has a particularly advantageous impurity profile that is completely different from that found in oils prepared by known techniques. Thus, the present invention also relates to a composition comprising a PUFA product, for example, a PUFA product obtained by the method of the present invention . Accordingly, in one embodiment, the present invention also provides a composition comprising a PUFA product

[0160] wherein the PUFA product is EPA and the PUFA product is present in an amount greater than 93% by weight and the total content of ω-6 polyunsaturated fatty acids is up to 0.40% by weight. As used herein, the weight % of a component is relative to the total weight of the composition

[0161] .

[0162] ​PUFA products and ω-6 PUFAs may optionally be in the form of their alkyl esters, typically in the form of ethyl esters. Preferably, the EPA PUFA product is in the form of its ethyl ester.

[0163] Typically, in this embodiment, the EPA PUFA product is present in an amount greater than 94% by weight , preferably greater than 95% by weight, more preferably greater than 96% by weight, even more preferably greater than 97% by weight, and most preferably greater than 98% by weight.

[0164] The total content of ω-6 polyunsaturated fatty acids in this embodiment is up to 0.40% by weight . Thus, typically, the composition contains ω-6 polyunsaturated fatty acids in an amount up to this amount . Typically, the total content of ω-6 polyunsaturated fatty acids is up to 0.35% by weight, preferably up to 0.3% by weight, more preferably up to 0.25% by weight, and most preferably up to 0.22% by weight . Typically, the total content of ω-6 polyunsaturated fatty acids is 0.05% by weight or more, preferably 0.1% by weight or more.

[0165] Typically, in this embodiment, the content of arachidonic acid is up to 0.25% by weight, preferably up to 0.24% by weight, more preferably up to 0.23% by weight, and most preferably up to 0. 22% by weight. Thus, typically, the composition contains arachidonic acid in an amount up to these amounts . Typically, the total content of arachidonic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0166] Typically, in this embodiment, the total content of ω-3 polyunsaturated fatty acids is 97% by weight Exceeds, preferably exceeds 97.5% by weight, more preferably exceeds 97.9% by weight. In one In some embodiments, the total content of ω-3 polyunsaturated fatty acids exceeds 99% by weight.

[0167] Typically, in this embodiment, the total content of DHA is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight, and most preferably up to 0.2% by weight. Therefore, typically, the composition contains DHA in amounts up to these amounts. Typically the total content of DHA is 0.05% by weight or more, preferably 0.1% by weight or more.

[0168] Typically, in this embodiment, the total content of DHA is up to 0.2% by weight, preferably up to 0.175% by weight, more preferably up to 0.16% by weight. Therefore, typically the composition contains DHA in amounts up to these amounts. Typically, the total content of DHA is 0.05% by weight or more, preferably 0.1% by weight or more.

[0169] Typically, in this embodiment, the total content of α-linolenic acid is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight. Therefore, typically the composition contains α-linolenic acid in amounts up to these amounts. Typically, the total content of α-linolen ic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0170] Typically, in this embodiment, the total content of α-linolenic acid is up to 0.35% by weight preferably up to 0.3% by weight, more preferably up to 0.29% by weight. Therefore typically, the composition contains α-linolenic acid in amounts up to these amounts. Typically, α The total content of α-linolenic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0171] Typically, in this embodiment, the total content of stearidonic acid is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight. Therefore, typically the composition contains stearidonic acid in amounts up to these amounts. Typically, the total content of stearidonic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0172] Typically, in this embodiment, the total content of stearidonic acid is up to 0.4% by weight, preferably up to 0.35% by weight, more preferably up to 0.34% by weight. Therefore typically the composition contains stearidonic acid in amounts up to these amounts. Typically, the total content of stearidonic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0173] Typically, in this embodiment, the total content of eicosatetraenoic acid is up to 1% by weight preferably up to 0.75% by weight, more preferably up to 0.5% by weight. Therefore typically the composition contains eicosatetraenoic acid in amounts up to these amounts. Typically the total content of eicosatetraenoic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0174] Typically, in this embodiment, the total content of eicosatetraenoic acid is up to 0.5% by weight preferably up to 0.475% by weight, more preferably up to 0.46% by weight. Thus typically the composition contains eicosatetraenoic acid in amounts up to these amounts. Typically, the total content of eicosatetraenoic acid is 0.05% by weight or more, preferably 0. 1% by weight or more.

[0175] Typically, in the present embodiment, the total content of docosapentaenoic acid is up to 1% by weight, preferably up to 0.6% by weight, more preferably up to 0.3% by weight. Therefore, typically the composition contains docosapentaenoic acid in an amount up to these amounts. Typically, the total content of docosapentaenoic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0176] Typically, in the present embodiment, the total content of docosapentaenoic acid is up to 0.4% by weight, preferably up to 0.35% by weight, more preferably up to 0.33% by weight. Therefore, typically the composition contains docosapentaenoic acid in an amount up to these amounts. Typically, the total content of docosapentaenoic acid is 0.05% by weight or more, preferably 0.1% by weight or more.

[0177] In the present embodiment, the composition preferably contains more than 96.5% by weight of EPA, 1% by weight or less of DHA, 1% by weight or less of α-linolenic acid, 1% by weight or less of stearidonic acid, 1% by weight or less of eicosatetraenoic acid, 1% by weight or less of docosapentaenoic acid, and 0.25% by weight or less of arachidonic acid.

[0178] In the present embodiment, the composition preferably contains more than 96.5% by weight of EPA, 0.2% by weight or less of DHA, 0.3% by weight or less of α-linolenic acid, 0.4% by weight or less of stearidonic acid, 0.5% by weight or less of eicosatetraenoic acid, 0.35% by weight or less of docosapentaenoic acid. ​It contains eicosapentaenoic acid and up to 0.25% by weight of arachidonic acid.

[0179] In this embodiment, the composition preferably contains 96.5 to 99% by weight of EPA, up to 0.6% by weight of DHA, up to 0.6% by weight of α-linolenic acid, 0.15 to 0.6 % by weight of stearidonic acid, 0.1 to 0.75% by weight of eicosatetraenoic acid, 0.6% by weight of docosapentaenoic acid and up to 0.6% by weight of arachidonic acid.

[0180] In this embodiment, the composition preferably contains 96.5 to 99% by weight of EPA, up to 0.2% by weight of DHA, up to 0.3% by weight of α-linolenic acid, 0.15 to 0.4 % by weight of stearidonic acid, 0.1 to 0.5% by weight of eicosatetraenoic acid, 0.35% by weight of docosapentaenoic acid and up to 0.25% by weight of arachidonic acid.

[0181] In this embodiment, the composition most preferably contains 98 to 99% by weight of EPA, 0. 1 to 0.3% by weight of DHA, 0.3 to 0.35% by weight of stearidonic acid, 0.1 to 0.3% by weight of eicosatetraenoic acid and 0.3 to 0.35% by weight of docosapentaenoic acid.

[0182] In this embodiment, the composition most preferably contains 96.5 to 99% by weight of EPA, 0.1 to 0.5% by weight of DHA, 0.1 to 0.5% by weight of stearidonic acid, 0.1 to 0.5% by weight of eicosatetraenoic acid, 0.1 to 0.5% by weight of docosapentaenoic acid and 0.1 to 0.3% by weight of arachidonic acid.

[0183] In this embodiment, the composition most preferably contains 98 to 99% by weight of EPA, 0. From 1 to 0.2% by weight of DHA, from 0.3 to 0.35% by weight of stearidonic acid, from 0.1 to 0.2% by weight of eicosatetraenoic acid and from 0.3 to 0.35% by weight of docosapentaenoic acid.

[0184] In this embodiment, the composition most preferably comprises from 96.5 to 97.5% by weight of EPA A, from 0.25 to 0.35% by weight of α-linolenic acid, from 0.18 to 0.24% by weight of stearic idonic acid, from 0.4 to 0.46% by weight of eicosatetraenoic acid and from 0.15 to 0. 25% by weight of arachidonic acid.

[0185] Typically, in this embodiment, the content of isomeric impurities is up to 1.5% by weight. Typically, the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25% by weight, even more preferably up to 0.25% by weight, and most preferably up to 0 .1% by weight.

[0186] In a further embodiment, the invention also provides a composition comprising a PUFA product, wherein the PUFA product is a mixture of EPA and DHA, and (i) the total content of EPA and DHA is 80% by weight or more, (ii) the content of EPA is from 41 to 60% by weight, and the content of DHA is from 16 to 48% by weight, and (iii) the total content of ω-3 polyunsaturated fatty acids is 9 4% by weight or more and / or the total content of ω-6 polyunsaturated fatty acids is up to 4% by weight of the composition.

[0187] The PUFA product, ω-3 and ω-6 PUFAs may optionally be in the form of their alkyl esters, typically ethyl esters. Preferably, the EPA / DHA PUFA product The product is in the form of its ethyl ester.

[0188] Thus, in this further embodiment, the composition is typically a composition comprising a PUFA product wherein the PUFA product is a mixture of EPA and DHA, and (i) the total content of EPA and DHA is 80% by weight or more, (ii) the content of EPA is from 41 to 60 % by weight, the content of DHA is from 16 to 48% by weight, and (iii) the total content of ω-3 polyunsaturated fatty acids is 94% by weight or more.

[0189] Alternatively, in this further embodiment, the composition is a composition comprising a PUFA product wherein the PUFA product is a mixture of EPA and DHA, and (i) the total content of EPA and DHA is 80% by weight or more, (ii) the content of EPA is from 41 to 60% by weight, the content of DHA is from 16 to 48% by weight, and (iii) the total content of ω-6 polyunsaturated fatty acids is up to 4% by weight.

[0190] Typically, in this further embodiment, the total content of EPA and DHA is 82% by weight or more, preferably 83% by weight or more, more preferably 84% by weight or more, even more preferably 85% by weight or more, and most preferably 86% by weight or more.

[0191] Typically, in this further embodiment, the content of EPA is from 41 to 60% by weight preferably from 45 to 60% by weight, more preferably from 47 to 60% by weight, even more preferably from 47 to 57% by weight, and most preferably from 50 to 55% by weight.

[0192] Typically, in this further embodiment, the content of DHA is from 16 to 48% by weight , preferably 20 to 45% by weight, more preferably 25 to 42% by weight, even more preferably 28 to 38% by weight, most preferably 30 to 35% by weight.

[0193] Typically, in this further embodiment, the total content of ω-3 polyunsaturated fatty acids is 94% by weight or more, preferably 95% by weight or more, more preferably 96% by weight or more, most preferably 97% by weight or more.

[0194] Typically, in this further embodiment, the total content of α-linolenic acid is up to 0.4% by weight, preferably up to 0.35% by weight, more preferably up to 0.31% by weight. Thus, typically, the composition contains α-linolenic acid in amounts up to these amounts. Typically, the total content of α-linolenic acid is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 0.2% by weight or more, even more preferably 0.2 to 0.4% by weight. .

[0195] Typically, in this further embodiment, the total content of stearidonic acid is up to 1.9% by weight, preferably up to 1.5% by weight, more preferably up to 1.25% by weight. Thus, typically, the composition contains stearidonic acid in amounts up to these amounts. Typically, the total content of stearidonic acid is 0.05% by weight or more, preferably 0.1% by weight or more .

[0196] Typically, in this further embodiment, the total content of eicosatetraenoic acid is up to 2 .0% by weight, preferably up to 1.9% by weight. Thus, typically, the composition contains eicosatetraenoic acid in amounts up to these amounts. Typically, eicosatetraenoic The total content of the acid is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 1.0% by weight or more, even more preferably from 1.0 to 1.9% by weight.

[0197] Typically, in this further embodiment, the total content of eicosatetraenoic acid is 3 .0% by weight or less, preferably 2.75% by weight or less. Thus, typically, the composition contains eicosatetraenoic acid in amounts up to these amounts. Typically, the total content of eicosatetraenoic acid is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 2% by weight or more, even more preferably from 2 to 2.75% by weight.

[0198] Typically, in this further embodiment, the total content of docosapentaenoic acid is 6-fold weight % or less, preferably 5.5% by weight or less, more preferably 5.25% by weight or less. Thus the composition typically contains docosapentaenoic acid in amounts up to these amounts. Typically the total content of docosapentaenoic acid is 0.05% by weight or more, preferably 0.1 weight % or more, more preferably 4% by weight or more, even more preferably from 4 to 5.25% by weight is.

[0199] The total content of ω-6 polyunsaturated fatty acids in this further embodiment is typically 4-fold weight % or less. Thus, typically, the composition contains ω-6 polyunsaturated fatty acids in amounts up to these amounts. Typically, the total content of ω-6 polyunsaturated fatty acids is 3.75 weight % or less, preferably 3.5% by weight or less, more preferably 3.25% by weight or less, even more preferably 3% by weight or less, most preferably 2.85% by weight or less. Typically, ω- The total content of polyunsaturated fatty acids is 0.05% by weight or more, preferably 0.1% by weight or more. It is.

[0200] Typically, in this further embodiment, the total content of linoleic acid is up to 0.5% by weight, preferably up to 0.4% by weight, more preferably up to 0.25% by weight. Thus, typically, the composition contains linoleic acid in amounts up to these amounts. Typically, the total content of linoleic acid is 0.05% by weight or more, preferably 0.1% by weight or more, more preferably 0.15% by weight or more, and even more preferably from 0.15 to 0.25% by weight.

[0201] Typically, in this further embodiment, the total content of gamma-linolenic acid is 0. up to 19% by weight, preferably up to 0.15% by weight, more preferably up to 0.1% by weight. Thus, typically, the composition contains gamma-linolenic acid in amounts up to these amounts. Typically, the total content of gamma-linolenic acid is 0.05% by weight or more, preferably 0 .1% by weight or more.

[0202] Typically, in this further embodiment, the total content of dihomo-gamma-linolenic acid is up to 0.1% by weight. Thus, typically, the composition contains dihomo-gamma-linolenic acid in amounts up to these amounts. Typically, the total content of dihomo-gamma-linolenic acid is 0.05% by weight or more.

[0203] Typically, in this further embodiment, the total content of arachidonic acid is up to 2.5% by weight, preferably up to 2.25% by weight, more preferably up to 2.1% by weight. Thus, Thus, typically, the composition contains arachidonic acid in amounts up to these amounts. Typically , the total arachidonic acid content is 0.05% by weight or more, preferably 0.1% by weight or more .

[0204] Typically, in this further embodiment, the total adrenic acid content is 0.1% by weight or less. Thus, typically, the composition contains adrenic acid in amounts up to this amount. Typically, the total adrenic acid content is 0.05% by weight or more.

[0205] Typically, in this further embodiment, the total docosapentaenoic (ω-6) acid content is up to 0.9% by weight, preferably up to 0.75% by weight, more preferably up to 0.65% by weight . Thus, typically, the composition contains docosapentaenoic (ω-6) acid in amounts up to these amounts. Typically, the total docosapentaenoic (ω-6) acid content is 0 .05% by weight or more, preferably 0.1% by weight or more.

[0206] In this further embodiment, the composition preferably contains 50 to 55% by weight of EPA, 30 to 35% by weight of DHA, up to 0.4% by weight of α-linolenic acid, up to 1.25% by weight of stearidonic acid, up to 1.9% by weight of eicosatetraenoic acid, up to 2.75% by weight of Hen eicosapentaenoic acid, up to 5.25% by weight of docosapentaenoic acid, up to 0.25% by weight of linoleic acid, up to 0.1% by weight of gamma-linolenic acid, up to 0.1% by weight of dihomo-gamma-linolenic acid, up to 2.1% by weight of arachidonic acid, up to 0.1% by weight of adrenic acid and up to 0.75% by weight of docosapentaenoic (ω-6) acid.

[0207] In this further embodiment, the composition more preferably comprises from 50 to 55% by weight of EPA, from 30 to 35% by weight of DHA, from 0.2 to 0.4% by weight of α-linolenic acid, up to 1.25% by weight of stearidonic acid, from 1.0 to 1.9% by weight of eicosatetraenoic acid, from 2 to 2.75% by weight of Hen eicosapentaenoic acid, from 4 to 5.25% by weight of docosapentaenoic acid, from 0.15 to 0.25% by weight of linoleic acid, up to 0.1% by weight of gamma-linolenic acid, up to 0.1% by weight of dihomo-gamma-linolenic acid, up to 2.1% by weight of arachidonic acid, up to 0.1% by weight of adrenic acid and up to 0.75% by weight of docosapentaenoic (ω-6) acid.

[0208] Typically, in this further embodiment, the content of isomeric impurities is up to 1.5% by weight Typically, the content of isomeric impurities is up to 1% by weight, preferably up to 0.5% by weight up to, more preferably up to 0.25% by weight, even more preferably up to 0.25% by weight and most preferably up to 0.1% by weight.

[0209] The inventors have also surprisingly found that it is possible to produce an oil with reduced environmental contaminants compared to known oils. Thus, in yet a further embodiment, the invention also provides a composition comprising a PUFA product as described herein, wherein (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.89 μg / kg, (b) the total amount of dioxins, furans, dibenzo-para-dioxins and polychlorinated dibenzofurans is up to 0.35 pg / g, (c) the total amount of polychlorinated biphenyls is up to 0.0035 mg / kg and / or (d) the total amount of dioxins, furans, dibenzo-para-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 1 pg / g. ​​​​​​

[0210] Typically, the present invention is a composition comprising the PUFA product described herein, wherein (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.89 μg / kg, (b) the total amount of dioxins, furans, dibenzo - para - dioxins and polychlorinated dibenzofurans is up to 0.35 pg / g, and / or (d) the total amount of dioxins, furans and dioxin - like polychlorinated biphenyls is up to 1 pg / g.

[0211] In still further embodiments, the total amount of polyaromatic hydrocarbons in the composition is 0.89 μg / kg or less. Thus, typically, the composition contains an amount of polyaromatic hydrocarbons up to this amount. Typically, the total amount of polyaromatic hydrocarbons in the composition is 0.85 μg / kg or less, preferably 0.8 μg / kg or less, more preferably 0.7 μg / kg or less, even more preferably 0.6 μg / kg or less, even more preferably 0.5 μg / kg or less, even more preferably 0.4 μg / kg or less, even more preferably 0.3 μg / kg or less, even more preferably 0.2 μg / kg or less, even more preferably 0.1 μg / kg or less, even more preferably 0.05 μg / kg or less, and most preferably 0.05 μg / kg or less.

[0212] Typical polyaromatic hydrocarbons are well known to those skilled in the art and include acenaphthene, acenaphthylene, anthracene, benz[a]anthracene, benzo[a]pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, dibenz(ah)anthracene, fluoranthene, ​Anthracene, fluorine, indeno(1,2,3-cd)pyrene, phenanthrene, pyrene, coronene, corannulene, tetracene, naphthalene, pentacene, triphenylene and o barene are included. Typically, the amounts shown above refer to the content of benzo[a]pyrene .

[0213] In this further embodiment, the total amount of dioxins, furans, dibenzo-p-dioxins and polychlorinated dibenzofurans is up to 0.35 pg / g. Thus , typically, the composition contains an amount of dioxins, furans, dibenzo-p-dioxins and polychlorinated dibenzofurans up to this amount. Typically, the total amount of dioxins, furans, di benzo-p-dioxins and polychlorinated dibenzofurans is up to 0.325 pg / g, preferably up to 0.3 pg / g, more preferably up to 0.275 pg / g, further more preferably up to 0.25 pg / g, even more preferably up to 0.225 pg / g and, even more preferably, up to 0.2 pg / g, most preferably up to 0.185 pg / g . These amounts are expressed in World Health Organization (WHO ) toxicity equivalents using the WHO toxicity equivalence factors (TEF). The WHO toxicity equivalence factors are well known to those skilled in the art.

[0214] Dioxins, furans, dibenzo-p-dioxins (PCDD) and polychlorinated di benzofurans (PCDF) are well known to those skilled in the art. Typically, these are all incorporated herein by reference in the Community Regulations (EC) No. 1881 / 2006 and 1883 / 2006.

[0215] As defined in Community Regulations (EC) No. 1881 / 2006 and 1883 / 2006 The PCDDs, PCDFs and dioxin-like PCBs, and their TEF values are as follows follows.

[0216]

Table 1

[0217] Typically, the amounts of PCDDs, PCDFs and dioxin-like PCBs are measured according to the methods described in Community Regulations (EC ) No. 1881 / 2006 and 1883 / 2006 determined.

[0218] Furthermore, the total amount of polychlorinated biphenyls in a further embodiment is up to 0.0035 mg / kg Thus, typically, the composition contains an amount of polychlorinated biphenyl up to this amount. Typically, the total amount of polychlorinated biphenyls is up to 0.003 mg / kg, preferably up to 0.0025 mg / kg, more preferably up to 0.002 mg / kg, further more preferably up to 0.0015 mg / kg, even more preferably up to 0.001 mg / kg, even more preferably up to 0.00075 mg / kg, most preferably up to 0. 0007 mg / kg.

[0219] Polychlorinated biphenyls (PCBs) are well known in the art and include biphenyl, monochloro biphenyl, dichlorobiphenyl, trichlorobiphenyl, tetrachlorobiphenyl , pentachlorobiphenyl, hexachlorobiphenyl, heptachlorobiphenyl, octa chlorobiphenyl, nonachlorobiphenyl and decachlorobiphenyl.

[0220] Dioxins, furans, dibenzo-para-dioxins in this further embodiment , the total amount of polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is 1 pg / g or less. Thus, typically, the composition contains an amount of dioxins, furans , dibenzo-para-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls up to this amount. Typically, the total amount of dioxins, furans, dibenzo-para-dioxins , polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.75 pg / g, preferably up to 0.5 pg / g, more preferably up to 0.45 pg / g, even more preferably up to 0.4 pg / g, even more preferably up to 0.35 pg / g, and most preferably up to 0.3 pg / g.

[0221] Dioxins, furans, dibenzo-para-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls are well known to those skilled in the art . Typically, these are as defined in Community Regulations (EC) No. 1881 / 2006 and 1883 / 2006, which are incorporated herein by reference in their entirety .

[0222] PCDDs, PCDFs and dioxin-like PCBs as defined in Community Regulations (EC) No. 1881 / 2006 and 1883 / 2006, and their TEF values are as described above .

[0223] In this further embodiment, preferably, (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 μg / kg, and (b) dioxins, furans, dibenzo ... ​​The total amount of non - para - dioxin and polychlorinated dibenzofuran is up to 0.2 pg / g and (c) the total amount of polychlorinated biphenyls is up to 0.0015 mg / kg, and / or (d) the total amount of dioxins, furans, dibenzo - para - dioxins, polychlorinated dibenz ofurans and dioxin - like polychlorinated biphenyls is up to 0.3 pg / g .

[0224] In this still further embodiment, preferably, (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 μg / kg, (b) the total amount of dioxins, furans, dibenzo no - para - dioxins and polychlorinated dibenzofurans is up to 0.2 pg / g and / or (d) the total amount of dioxins, furans, dibenzo - para - dioxins, polychlorinated dibenz ofurans and dioxin - like polychlorinated biphenyls is up to 0.3 pg / g and (d) the total amount of dioxins, furans, dibenzo - para - dioxins, polychlorinated dibenzofurans and dioxin - like polychlorinated biphenyls is up to 0.3 pg / g .

[0225] In this still further embodiment, more preferably, (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 μg / kg, (b) the total amount of dioxins, furans, dibenzo no - para - dioxins and polychlorinated dibenzofurans is up to 0.2 pg / g and (c) the total amount of polychlorinated biphenyls is up to 0.0015 mg / kg, and (d) the total amount of dioxins, furans, dibenzo - para - dioxins, polychlorinated dibenzof urans and dioxin - like polychlorinated biphenyls is up to 0.3 pg / g .

[0226] In this still further embodiment, more preferably, (a) the total amount of polyaromatic hydrocarbons in the composition is up to 0.05 μg / kg, (b) the total amount of dioxins, furans, dibenzo no - para - dioxins and polychlorinated dibenzofurans is up to 0.2 pg / g The total amount of dioxino - para - dioxin and polychlorinated dibenzofurans is up to 0.2 pg / g, and (d) the total amount of dioxins, furans, dioxino - para - dioxin, polychlorinated dibenzofurans and dioxin - like polychlorinated biphenyls is up to 0.3 pg / g.

[0227] Typically, in this still further embodiment, the content of isomeric impurities is up to 1.5 weight percent. Typically, the content of isomeric impurities is up to 1 weight percent, preferably 0. 5 weight percent, more preferably up to 0.25 weight percent, even more preferably up to 0.25 weight percent, and most preferably up to 0.1 weight percent.

[0228] The inventors have also found that it is possible to produce highly pure oils that avoid the problems of isomerization, peroxidation, and oligomerization associated with distilled oils. The amount of isomeric impurities present in the PUFA products of the present invention will depend on the amount of isomeric impurities present in the feed mixture. However, importantly, unlike distillation, the amount of isomeric impurities is not increased by the method of the present invention. Thus, the limit of the content of isomers in the PUFA products is the isomer content of the starting material. If no isomers are present in the starting material, then substantially no isomers will be present in the resulting PUFA products.

[0229] Thus, in one embodiment, the chromatographic separation method of the present invention does not substantially increase the amount of isomeric impurities in the PUFA products as compared to the amount of isomeric impurities present in the feed mixture. "Substantially increase" typically means up to 10 ​is 5% by weight or less, more preferably 3% by weight or less, even more preferably 1% by weight or less, even more preferably 0.5% by weight or less, and most preferably 0.1% by weight or less of an increase. It is understood to be such.

[0230] Accordingly, in yet a further embodiment, the present invention also provides a composition comprising a PUFA product, the composition having an isomer impurity content of up to 1.5% by weight. Typically, the composition contains an amount of isomer impurity up to this amount. Typically, the isomer impurity content is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25 % by weight, and most preferably up to 0.1% by weight. Isomerization is particularly problematic in the preparation of high-purity DHA by distillation because higher temperatures are required for separation. Typically, the PUFA product is DHA, optionally in the form of its ethyl ester. Typically, the composition contains more than 85% by weight, preferably more than 90% by weight, more preferably 92. 5% by weight, and most preferably more than 95% by weight of the PUFA product. Preferably, the composition contains more than 85% by weight, preferably more than 90% by weight, more preferably 92.5 % by weight, and most preferably more than 95% by weight of DHA, optionally in the form of its ethyl ester. In this embodiment, the composition typically contains DHA as the PUFA product in an amount of more than 95% by weight, optionally in the form of its ethyl ester, and the isomer impurity content is up to 1% by weight, preferably up to 0.5% by weight, more preferably up to 0.25% by weight, and most preferably up to 0.1% by weight. % by weight, and most preferably up to 0.1% by weight.

[0231] The improved method of the present invention removes both more polar impurities and less polar impurities. Since it can be removed in a single process, it is possible to efficiently achieve a much higher purity PUFA product. This enables the following.

[0232] The PUFA product of the present invention typically has a purity exceeding 80% by weight, preferably exceeding 85% by weight, more preferably exceeding 90% by weight, even more preferably exceeding 95% by weight, still more preferably exceeding 97% by weight, and most preferably exceeding 99% by weight. When the PUFA product is a single PUFA or its derivative, the above concentration refers to the concentration of that PU FA or derivative. When the PUFA product is two or more, for example, a mixture of two PUFAs or their derivatives, the above concentration refers to the combined concentration of those PUFAs or their derivatives.

[0233] The method of the present invention also avoids problems of isomerization, peroxidation, and oligomerization associated with distilled oils. The PUFA product of the present invention typically has an isomer impurity content of less than 5% by weight, preferably less than 3% by weight, more preferably less than 1% by weight. As described above, isomer impurities include PUFA isomers, peroxidation products, and oligomerization products. PU FA isomers include positional isomers and / or geometric isomers. Examples of positional isomers and / or geometric isomers of EPA include 17E-EPA, 5E-EPA, 5E,8E-EPA , 8E,11E-EPA, 5E,14E-EPA, and 5E,8E,11E,17E-E PA. Such isomers are described in more detail in Wijesundera, R.C. et al., Journal of the American Oil Chemists' Society, 1989, Vol. 66, No. 12, pp. 182 2-1830, which is incorporated herein by reference in its entirety. ​​

[0234] In fact, the method of the present invention will generally be controlled by a computer. Thus the present invention also provides a computer program for controlling the chromatograph apparatus described herein, which, when executed, contains code means for instructing the apparatus to implement the method of the present invention and to provide a computer program containing code means for instructing the apparatus to implement the method of the present invention.

[0235] The following examples illustrate the present invention.

Example

[0236] A simulated moving bed chromatography system schematically shown in FIG. 8 is used to fractionate a fish oil-derived raw material (55 wt% EPA EE, 5 wt% DHA EE ) using bonded C18 silica gel (particle size 3 00 μm) as the stationary phase and aqueous methanol as the eluent. As shown in FIG. 8, 15 columns (diameter: 76.29 mm, length: ) are connected in series. 914.40 mm).

[0237] The operating parameters and flow rates are as follows for 8 different cases. In the following conditions, EPA EE is produced with a high purity (85 to 98% by GC FAMES ). The GC FAMES traces of the extract and raffinate of zone 1, and the extract and raffinate of zone 2 are shown in FIGS. 11 and 12, respectively.

[0238] (Example 1a) Step time: 750 seconds Cycle time: 200 minutes Raw material (F) supply rate: 70 ml / min Desorbent (D) supply rate: 850 ml / min Extract rate: 425 ml / min Raffinate rate: 495 ml / min ​​​

[0239] (Example 1b) Step time: 250 seconds Cycle time: 66.67 minutes Feed rate of raw material (F): 210 ml / min Feed rate of desorbent (D): 2550 ml / min Extract rate: 1275 ml / min Residue rate: 1485 ml / min

[0240] (Example 1c) Step time: 500 seconds Cycle time: 133.33 minutes Feed rate of raw material (F): 25 ml / min Desorbent feed rate in the first zone (D1): 2050 ml / min Extract container accumulation rate in the first zone (E1): 1125 ml / min Extract recirculation rate in the first zone (D1 - E1): 925 ml / min Residue rate in the first zone (R1): 950 ml / min Desorbent feed rate in the second zone (D2): 1700 ml / min Extract container accumulation rate in the second zone (E2): 900 ml / min Extract recirculation rate in the second zone (D2 - E2): 800 ml / min Residue rate in the second zone (R2): 800 ml / min

[0241] (Example 1d) Step time: 250 seconds Cycle time: 66.67 minutes Feed rate of raw material (F): 50 ml / min Desorbent feed rate in the first zone (D1): 4125 ml / min Extract container accumulation rate in the first zone (E1): 2250 ml / min Extract recirculation rate in the first zone (D1 - E1): 1875 ml / min Residue rate in the first zone (R1): 1925 ml / min Desorbent supply rate (D2) in the second zone: 3375 ml / min Extract container accumulation rate (E2) in the second zone: 1800 ml / min Extract recirculation rate (D2 - E2) in the second zone: 1575 ml / min Residual extraction liquid rate (R2) in the second zone: 1575 ml / min

[0242] (Example 1e) Step time: 500 seconds Cycle time: 133.33 minutes Raw material (F) supply rate: 50 ml / min Desorbent supply rate (D1) in the first zone: 4000 ml / min Extract container accumulation rate (E1) in the first zone: 2250 ml / min Extract recirculation rate (D1 - E1) in the first zone: 1750 ml / min Residual extraction liquid rate (R1) in the first zone: 1800 ml / min Desorbent supply rate (D2) in the second zone: 3200 ml / min Net extract accumulation rate (E2) in the second zone: 1750 ml / min Extract recirculation rate (D2 - E2) in the second zone: 1450 ml / min Residual extraction liquid rate (R2) in the second zone: 1450 ml / min

[0243] (Example 1f) Step time: 250 seconds Cycle time: 66.67 minutes Raw material (F) supply rate: 100 ml / min Desorbent supply rate (D1) in the first zone: 4050 ml / min Extract container accumulation rate (E1) in the first zone: 2100 ml / min Extract recirculation rate (D1 - E1) in the first zone: 1950 ml / min Residual extraction liquid rate (R1) in the first zone: 2050 ml / min Desorbent supply rate (D2) in the second zone: 3300 ml / min Net extraction liquid accumulation rate (E2) in the second band: 1700 ml / min Extraction liquid recirculation rate (D2 - E2) in the second band: 1600 ml / min Residual extraction liquid rate (R2) in the second band: 1600 ml / min

[0244] (Example 1g) Step time: 500 seconds Cycle time: 133.33 minutes Raw material (F) supply rate: 25 ml / min Desorbent supply rate (D1) in the first band: 1275 ml / min Extraction liquid container accumulation rate (E1) in the first band: 750 ml / min Extraction liquid recirculation rate (D1 - E1) in the first band: 550 ml / min Residual extraction liquid rate (R1) in the first band: 575 ml / min Desorbent supply rate (D2) in the second band: 1275 ml / min Net extraction liquid accumulation rate (E2) in the second band: 950 ml / min Extraction liquid recirculation rate (D2 - E2) in the second band: 325 ml / min Residual extraction liquid rate (R2) in the second band: 325 ml / min

[0245] (Example 1h) Step time: 250 seconds Cycle time: 66.67 minutes Raw material (F) supply rate: 50 ml / min Desorbent supply rate (D1) in the first band: 2550 ml / min Extraction liquid container accumulation rate (E1) in the first band: 1500 ml / min Extraction liquid recirculation rate (D1 - E1) in the first band: 950 ml / min Residual extraction liquid rate (R1) in the first band: 1000 ml / min Desorbent supply rate (D2) in the second band: 2000 ml / min Net extraction liquid accumulation rate (E2) in the second band: 900 ml / min Extraction liquid recirculation rate in the second zone (D2-E2): 600 ml / min Raffinate rate in the second zone (R2): 600 ml / min EXAMPLES

[0246] Eicosatetraenoic acid ethyl ester (ETA EE), EPA EE, and their isomers The fish oil-derived raw material containing the body and DHA EE is processed by the system shown in FIG. The stationary phase was bonded C18 silica gel (particle size 40-60 μm) and the eluent was aqueous methanol. The separation was carried out using a moving bed chromatography system using a nitrile. As shown, 19 columns (diameter: 10 mm, length: 250 mm) are connected in series. do.

[0247] The operating parameters and flow rates are as follows:

[0248] Cycle time: 600 seconds Feed rate of raw material (F): 0.5 ml / min. Desorbent (D1, 100% methanol) to the first zone Supply rate of 10ml / min Desorbent (D2, 99% methanol / 1% water) feed rate to the first zone: 6 ml / min Extraction rate from the first zone (E1'): 3 ml / min Extraction rate from the first zone (E1): 1.9 ml / min Raffinate rate from first zone (R1): 4.6 ml / min Desorbent (D2, 97% methanol / 3% water) feed rate to the second zone: 6 ml / min Extraction rate from the second zone (E2): 2.4 ml / min Raffinate rate from second zone (R2): 4.6 ml / min

[0249] Again, EPA EE is of high purity (>90% by weight, >95% by weight). It was produced with a purity exceeding 98% by weight.

Example

[0250] Using the system schematically shown in FIG. 8, a bonded C18 silica gel (particle size 3 00 μm, particle porosity 150 Å) as the stationary phase and aqueous methanol as the eluent was used in a simulated moving bed chromatography system to fractionate a fish oil-derived raw material (55 wt% EPA EE, 5 wt% DHA EE). As shown in FIG. 8, 15 columns (diameter: 10 mm, length: 250 mm) were connected in series.

[0251] The operating parameters and flow rates are as follows.

[0252] Cycle time: 380 seconds Raw material (F) supply rate: 0.5 ml / min Supply rate of desorbent (D, 98.5% methanol / 1.5% water) to the first zone: 9 m l / min Supply rate of water-rich phase (W, 85% methanol / 15% water) to the first zone: 3.1 ml / min Extract (E1) rate from the first zone: 4 ml / min Residue (R1) rate from the first zone: 8.6 ml / min Supply rate of desorbent (D, 97% methanol / 3% water) to the second zone: 10.8 ml / min Supply rate of water-rich phase (W, 85% methanol / 15% water) to the second zone: 3.1 ml / min Extract (E2) rate from the second zone: 4.1 ml / min Residue (R2) rate from the second zone: 10.3 ml / min

[0253] EPA EE was produced with a high purity (>95% purity). The GC trace of the product is shown in FIG. 13.

Example

[0254] Using the system schematically shown in Fig. 8, a preparative moving bed chromatography system using bonded C18 silica gel (particle size 3 00 μm) as the stationary phase and aqueous methanol as the eluent is used to fractionate a fish oil-derived raw material (70 wt% DHA EE, 7 wt% EPA EE ). As shown in Fig. 8, 15 columns (diameter: 76.29 mm, length: 914.40 mm) are connected in series. The operating parameters and flow rates are as follows.

[0255] The operating parameters and flow rates are as follows.

[0256] Step time: 600 seconds Cycle time: 160 minutes Feed (F) supply rate: 25 ml / min Desorbent supply rate (D1) in the first zone: 2062.5 ml / min Extract rate (E1) in the first zone: 900 ml / min Raffinate rate (R1) in the first zone: 1187.5 ml / min Desorbent supply rate (D2) in the second zone: 1500 ml / min Extract rate (E2) in the second zone: 450 ml / min Raffinate rate (R2) in the second zone: 1050 ml / min

[0257] DHA EE is produced with a high purity (> 97% by GC FAMES). The GC FAMES trace of the extract in zone 2 is shown in Fig. 14.

Example

[0258] Using the system schematically shown in Fig. 8, a preparative moving bed chromatography system using bonded C18 silica gel (particle size 3 00 μm) as the stationary phase and aqueous methanol as the eluent is used Use a stem to fractionate a fish oil-derived raw material (33 wt% EPA EE, 22 wt% DHA E E). As shown in Figure 8, connect 15 columns (diameter: 76.29 mm, length : 914.40 mm) in series.

[0259] The operating parameters and flow rates are as follows.

[0260] Step time: 380 seconds Cycle time: 101.33 minutes Raw material (F) supply rate: 40 ml / min Desorbent supply rate (D1) in the first band: 1950 ml / min Extract rate (E1) in the first band: 825 ml / min Residue rate (R1) in the first band: 1165 ml / min Desorbent supply rate (D2) in the second band: 1425 ml / min Extract rate (E2) in the second band: 787.5 ml / min Residue rate (R2) in the second band: 637.5 ml / min

[0261] A mixture of EPA EE and DHA EE is produced with a high purity (total of EPA EE and DHA EE > 80%).

Example

[0262] An experiment was conducted to compare the amounts of environmental contaminants present in two PUFA products according to the present invention with the amounts of environmental contaminants present in similar oils prepared by distillation. Those The contaminant profiles of the oils are shown in Table 1 below.

[0263]

Table 2

Example

[0264] An experiment was conducted to measure the amount of isomeric impurities present in the oil prepared according to the present invention, compared to an equal amount of oil prepared by distillation.

[0265] The GC trace of the DHA-rich oil prepared according to the present invention is shown in FIG. 14. No trace of isomeric impurities is present in the GC trace.

[0266] The GC trace of the oil prepared by distillation is shown in FIG. 15. Four peaks with an elution time longer than the DHA peak correspond to DHA isomers. It can be understood from the GC trace that the oil prepared by distillation contains about 1.5 wt% of isomeric impurities.

Example

[0267] Two EPA-rich products of the method of the present invention were compared with an EPA-rich oil produced by distillation. The analysis results (wt%) of their PUFA components are shown below.

[0268]

Table 3

Example

[0269] An EPA / DHA-rich product of the method of the present invention was compared with an EPA / DHA-rich oil produced by distillation. The analysis results (wt%) of their PUFA components are shown below.

[0270]

Table 4

Explanation of Signs

[0271] Component less polar than A B PUFA product Component with higher polarity than C D Alcohol desorbent E1, E2 Extract streams R1, R2 Raffinate streams W Water

Claims

**Claim 1** A composition comprising a PUFA product, wherein (a) the total amount of polyaromatic hydrocarbons in the composition is 0.89 μg / kg or less, and / or (b) the total amount of dioxins, furans, dibenzo-para-dioxins and polychlorinated dibenzofurans in the composition is 0.35 pg / g or less, and / or (c) the total amount of polychlorinated biphenyls in the composition is 0.0035 mg / kg or less, and / or (d) the total amount of dioxins, furans, dibenzo-para-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls in the composition is 1 pg / g or less, and further the composition contains more than 85% by weight of the PUFA product, and the content of isomeric impurities in the composition is 1% by weight or less, wherein the isomeric impurities are positional isomers and / or geometric isomers of the PUFA product. **Claim 2** The composition according to claim 1, wherein the PUFA product is EPA. **Claim 3** The composition according to claim 1, wherein the PUFA product is DHA. **Claim 4** (a) the total amount of polyaromatic hydrocarbons in the composition is 0.05 μg / kg or less, and / or (b) the total amount of dioxins, furans, dibenzo-para-dioxins and polychlorinated dibenzofurans in the composition is 0.2 pg / g or less, and / or (c) the total amount of polychlorinated biphenyls in the composition is 0.0015 mg / kg or less, and / or (d) the total amount of dioxins, furans, dibenzo-para-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls in the composition is 0.3 pg / g or less, the composition according to claim 1. **Claim 5** The composition according to claim 1, wherein the content of isomeric impurities is 0.5% by weight or less. **Claim 6** The composition according to claim 1, wherein the content of isomeric impurities is 0.25% by weight or less. **Claim 7** The composition according to claim 1, wherein the content of isomeric impurities is 0.1% by weight or less.

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