WO2013083482A1

WO2013083482A1 - Chromatographic method for the separation of fatty acid mixtures

Info

Publication number: WO2013083482A1
Application number: PCT/EP2012/074047
Authority: WO
Inventors: Thomas Müller-Späth; Lars Aumann; Guido Ströhlein; Michael Bavand
Original assignee: Chromacon Ag
Priority date: 2011-12-05
Filing date: 2012-11-30
Publication date: 2013-06-13

Abstract

The invention relates to a process for purification of a fatty acid containing, multi-component mixture (F) by means of at least two individual chromatographic columns through which the mixture (F) is fed by means of at least one solvent (s). According to the invention, the multi- component mixture (F) at least comprises weakly adsorbing impurities (W), a first target product (P1)to be purified, an intermediate impurity (S1), a second target product (P2) to be purified, and strongly adsorbing impurities (S2), the first target product (P1) is first fatty acid, e.g. eicosapentaenoic acid (EPA), or a derivative thereof, and the second target product (P2) is a second fatty acid different from the first target product (P1), or a derivative of said second fatty acid. For the purification a multicolumn countercurrent purification chromatography is used, and in this multicolumn countercurrent purification chromatography at least one outlet thereof is further separated by using outlet fractionation.

Description

TITLE

Chromatographic method for the separation of fatty acid mixtures

TECHNICAL FIELD

The present invention relates to a process for purification of a fatty acid or fatty acid derivative containing, multi-component mixture by means of at least two individual chromatographic columns through which the mixture is fed by means of at least one solvent.

PRIOR ART

Fatty acids obtained from natural oil or fat play an increasingly important role in various aspects of human society such as nutrition, health, energy supply and transportation. Polyunsaturated fatty acids (PUFAs) have positive effects on human health and some of them are an essential part of human nutrition. Especially polyunsaturated omega 3- fatty acids and the ratio of omega 6-PUFAs to omega 3-PUFAs have been mentioned in this context. In disease prevention omega 3- PUFAs have been shown to reduce the risk of a number of diseases including cardiovascular diseases and arteriosclerosis. Since the diet in many western countries is poor in omega 3-PUFAs, omega 3-PUFAs are used as food additives. Omega 3- PUFAs are also used in therapeutic applications to treat a number of chronic diseases such as Alzheimer's disease, cardiovascular diseases, and diabetes. Both PUFAs in their natural triglyceride form and PUFA-esters have been shown to be effective pharmaceuticals.

In the field of energy supply and transportation, biodiesel fuels, produced from natural oils and fats, have drawn increased attention as source of renewable energy as the global supply of fossil fuels is limited.

Different organisms can serve as sources for natural oils and fats such as plants, micro- and macro-algae, and fish. Depending on the organism, large differences are observed in the fatty acid composition. For instance, while olive oil typically contains more than 70% mono-unsaturated fatty acids (MUFAs) and less than 10% PUFAs, soybean oil contains around 25% MUFAs and more than 50% PUFAs. The oil derived from marine sources such as fish or algae is typically composed of more than 10% omega-3 PUFAs while the amount of omega-3 PUFAs found in land-based plants such as sunflowers and palms is typically below 10%.

Two of the main PUFAs of interest, eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA) are almost exclusively produced from marine oils, thus oils obtained from fish, microalgae or macroalgae. For certain pharmaceutical applications, and to achieve a maximum specific activity, a maximization of the purity with respect to a single PUFA or PUFA ester such as eicosapentaenoic acid (EPA) or as eicosapentaenoic acid (EPA) ethyl ester, respectively, is desired. Typically, the desired purity is at least 90%.

The main constituents of biodiesel are MUFAs and saturated fatty acids. A low PUFA content is desired in biodiesel, since PUFAs are susceptible to oxidative degradation and form breakdown products during long-term storage, spoiling the biodiesel. Thus PUFAs have to be removed during the biodiesel refining process. For biodiesel, the EN 14214 specification limits the PUFA content allowed.

Natural oils and fats consist mainly of triglycerides but contain also free fatty acids, monoglycerides, diglycerides and unsaponifiable lipids.

The term "microalgae" in this context includes prokaryotic microorganisms such as Cyanobacteria (Cyanophycea) and eukaryotic photosynthetic microorganisms that are unicellular or have a simple multicellular structure such as green algae and phytoplankton. The term "oil" in this context includes liquid mixtures containing triglycerides and more specifically relates to mixtures obtained from fish, microalgae, plants, fungi or yeast.

The terms "natural oil" and "natural fat" relate to oil and fat that is produced from renewable, non-fossilized organisms such as plants, fish and algae and are to be seen in contrast to oil obtained from fossil sources. It explicitly includes oil and fat that is produced by means of genetically modified organisms.

The term "biodiesel" relates to liquid fuel for combustion produced from natural oils and fats.

The term "supercritical fluid" relates to a fluid with a pressure and temperature beyond its critical pressure and temperature, respectively.

The production of oil from fish typically includes steps of cooking of the raw material such as fish heads, pressing of the cooked material and recovery of the oil from the emulsion by means of centrifugation or filtration. Alternatively, the oil may be extracted from dried fish material by fluids such as organic solvents or supercritical fluids, e.g. C0₂ in a supercritical fluid extraction (SFE) step.

The production of oil from microalgae up to the point where the purification of the fatty acids starts typically includes a cultivation in open ponds or bioreactors, a harvest step, a dewatering or drying step, a cell disruption step, an oil extraction step and a transesterification step. In cases where the oils are secreted by the microalgae in the surrounding environment, the steps of dewatering, drying, cell disruption and extraction can be replaced in part or in total by a filtration, centrifugation or skimming step to separate the oil from water and cells.

Also the direct saponification of oils contained in wet microalgae using lye and alcohol, without a cell disruption step, has been described.

Moreover, the direct ("in situ") transesterification of dried, milled and not pre-extracted oil seeds has been described, reducing the number of process steps.

In most cases, independent of their origin (i.e. plants, fish etc.) and their purpose (i.e. use as biofuels or food additives, pharmaceuticals), the oils are subjected to a transesterification step to facilitate further processing. In the transesterification step, the triglycerides are converted into fatty acid esters by reaction with alcohols. In the case of food additives and pharmaceuticals, mostly ethanol is applied to produce fatty acid ethyl esters and in the case of biodiesel, mostly methanol is used to form fatty acid methyl esters. Following the transesterification step, the desired fatty acids have to be refined prior to application as pharmaceuticals, nutrition or as biodiesel, respectively. Since natural oils and fats contain a variety of fatty acids that are similar in their physico-chemical properties, i.e. the number of carbon atoms and the number of double bonds, it is difficult to isolate a single fatty acid or fatty acid ester species with high recovery and purity at the same time.

After transesterification, typically the fatty acid esters are concentrated by fractional or molecular distillation.

The refining of fatty acid esters typically includes steps of degumming for the separation of phospholipids, deacidification for removal of free fatty acids, bleaching for the adsorption of pigments, deodorization for removal of smell and physical adsorption on activated carbon for the removal of dioxins and polychlorinated biphenyls (PCBs).

For the final purification of PUFAs or esters thereof, a number of processes have been suggested. Frequently, reverse phase chromatography is used in at least one of the process steps. Other available methods include enzymatic splitting, low-temperature crystallization, supercritical fluid extraction and urea complexation. In fish oil processing, urea complexation removes MUFAs andsaturated fatty acids yielding PUFA concentrates with more than 90% PUFA purity. The method works for free fatty acids and for fatty acid esters. The method described in EP 0 610 506 Bl comprises the distilling of a mixture containing EPA or EPA-ester under low pressure using a plurality of distillation columns, followed by a reversed phase chromatography step using a silica gel carrying immobilized octadecyl groups (ODS) as packing material in order to obtain a purity of EPA of larger 90%. The drawback of this method is the high cost of the packing material.

The method described in US 6,433,201 B2 comprises the chromatographic fractionation of a mixture containing EPA ethyl ester on a column filled with silica particles followed by a precision distillation to obtain a purity of EPA ethyl ester of larger 95% purity.

The general drawback of methods that include distillation is that high operating temperatures are required even when carried out under low pressure causing product oxidation, denaturation by polymerization and isomerization.

In addition, a process using a chromatographic column packing carrying immobilized silver has been described for the purification of EPA ethyl esters (JP 9-151390). Although purities larger 95% can be obtained using this type of chromatography, the cost of the column packing is high and the risk of product contamination by silver is problematic.

A method for the chromatographic purification of docosahexaenoic acid (DHA) and docosapentaenoic acid (DP A) in their ethyl ester forms has been described. The process uses octadecyl silica as column packing and produces DHA and DPA ethyl esters with purities of larger 99% but with yields of only 23.3% (DHA- ethyl ester) and 79.6% (DPA- ethyl ester) using a methanol / water mixture as solvent.

An analytical method for separating non-esterified, free fatty acid using reversed phase chromatography and an acetonitrile/water mixture with phosphoric acid as solvent has been described. In another case the use of a acetonitrile/ tetrahydrofuran (THF) / water mixture as solvent for the separation of free fatty acids has been reported. The use of so called high speed countercurrent chromatography (HSCCC) has been reported for the refining of fish oil. However, since this process does not contain a solid stationary phase it should be considered an extraction process. The process appears to be capable of refining a mixture of PUFA ethyl esters, but the data show that it is not possible to resolve single PUFAs. Furthermore, the process is not scalable in size.

US 5719302 describes the use of a combination of single column batch chromatography and simulated moving bed (SMB) continuous chromatography with octadecyl-bonded silica as stationary phase and mixtures of organic solvents and water as mobile phases. In a first step, the single column chromatography was operated to produce four fractions out of which two are subjected to purification by SMB. With SMB, two fractions, one rich in EPA-ethyl ester and one rich in DHA-ethyl ester were produced. The purities of these fractions were 80% and 66%, respectively. These purities do not reflect the state-of-the-art in pharmaceutical manufacturing that allows for production of the single PUFAs with purities above 90%.

The beneficial properties of supercritical fluids are exploited in PUFA production for extraction (supercritical fluid extraction, SFE), fractionation (supercritical fluid fractionation, SFF) and purification by chromatography (supercritical fluid chromatography, SFC). A commonly used supercritical fluid is C0₂, which has the advantages of having a low critical temperature and being cost-effective, non- toxic, nonflammable and easily removable.

SFF has been described for the separation of fish oil ethyl esters into an extract fraction containing ethyl esters of fatty acids with carbon numbers lower than 20 and into a raffinate fraction containing ethyl esters of fatty acids with carbon numbers of 20 and higher, including EPA and DHA, with a yield of 95% and a purity of greater 95%.

Since the SFF processes described in the literature can separate PUFA ethyl esters only based on their carbon number they have to be used in combination with other techniques in order to isolate the desired PUFA ethyl esters like EPA ethyl ester and DHA ethyl ester. Regarding SFC, in the literature, the use of silica gels with and without attachment of aliphatic groups such as octadecyl groups, has been described. SFC for PUFA purification has been demonstrated in most cases in discontinuous, single column batch mode.

Others have described the operation of single column SFC in laboratory and pilot scale for the production of an EPA ethyl ester. They obtained EPA ethyl ester with 95% purity at 11% yield and 90% purity at 43% yield, in a laboratory scale and EPA ethyl ester with a purity of 93% purity and 24.6% yield in a pilot scale.

The use of simulated moving bed (SMB) continuous chromatography in combination with supercritical C0₂ for the purification of PUFA ethyl esters has also been described. In a first step, SFC in single column mode was used to prepare two fractions, one high in DHA- ester content and the other one high in EPA-ester content. Both fractions were then fed at different positions into an SMB system which consisted of 8 columns. The single column process and the SMB process both operated using supercritical C0₂ as mobile phase and octadecyl-silica as stationary phase. The overall yield of the combined process was reported to be > 99% for both EPA-ester and DHA-ester. The purity of the final EPA-ester rich fraction was 92%, while the purity of the DHA-ester rich fraction was 85%.

A drawback of all methods using supercritical fluids are high pressures involved which require specialized equipment and high capital investment.

For removal of free fatty acids in biodiesel production, often a step using acid esterification is used. For biodiesel refining, mainly a simplified scheme has been reported that uses the following methods: Warm water washing, dry washing using an ion exchange resin or dry washing by membrane extraction using hollow fibers (not yet implemented commercially). Since both main side products from the transesterification, glycerol and alcohol are highly soluble in water, the water washing is the most common method. Furthermore this method effectively removes soaps and residual transesterification catalyst. Finally, water from biodiesel is removed by evaporation in a drying step. Optionally the biodiesel is processed in another distillation step in order to remove odor, color and sulfur.

Except for SMB, the use of continuous countercurrent chromatography has not been reported for the purification of fatty acid esters. A drawback of SMB is its limitation to the separation of two fractions while in cases of fatty acid or fatty acid ester purification a ternary separation is required. Pseudo-ternary separations have been reported for SMB, producing also more than two fractions, but this scenario assumes that the components contained in the third fraction have significantly different physicochemical properties than the other components of the mixture. Thus, it is not possible with SMB to isolate single fatty acids or fatty acid esters with high purity from a complex fatty acids or fatty acid ester-mixture in which the desired fatty acids or fatty acid ester species represent components which have intermediate physicochemical properties compared to the impurities. Therefore, the purities of the products, produced using SMB, are limited and strongly depend on the feed quality.

Summarizing, a single SMB process cannot be used to isolate a product overlapping with early and late eluting impurities and cannot be used for the production of single fatty acids with a purity of >90%.

SUMMARY OF THE INVENTION

In contrast to SMB, Multicolumn Countercurrent Solvent Purification, in particular Multicolumn Countercurrent Solvent Gradient Purification (MCSGP, see e.g. WO 2006/116886 and WO 2010/079060A1 as well as Aumann L, Morbidelli M. 2007; A continuous multicolumn countercurrent solvent gradient MCSGP process; Biotechnology and Bioengineering 98 (5): 1043-1055) is a continuous countercurrent chromatographic process that is capable of purifying single components from complex mixtures with similar physicochemical properties. Due to the countercurrent movement between the mobile and stationary phases and the internal recycling of impure side fractions, the desired product species can be isolated achieving high purity and high yield simultaneously, even if the corresponding single column chromatogram from which the MCSGP operating parameters are derived shows a strong overlap of the product and early and late eluting impurity peaks. Application examples for MCSGP include the purification of peptides produced by chemical synthesis or the separation of monoclonal antibody variants. MCSGP is scalable to an industrial scale.

The object of the present invention is to provide a further improvement in particular to the processes according to WO 2006/116886 and WO 2010/079060A1, which are used to isolate one fraction of components with intermediate adsorptive properties (WO 2006/116886) or multiple fractions of components with intermediate adsorptive properties (WO 2010/079060A1) from a complex multi-component mixture. The invention relates to a major amendment to the 2-column and multicolumn embodiments that allows for the isolation of additional fractions of components with intermediate adsorptive properties without increasing the number of chromatographic columns in the process. The invention furthermore relates to the specific solvent conditions that are required for the amendment to be effective.

According to the state-of-the-art it has not been demonstrated that certain fatty acids or fatty acid esters can be purified with high yield of larger 90% and high purity of larger 90% from a crude mixture at a production scale. The methods investigated so far included distillation, extraction, complexation, crystallization, chromatography using organic solvents and supercritical fluids.

Surprisingly, it has been found that two fatty acid ester species of interest, namely EPA ethyl ester or DHA ethyl ester can be isolated from a crude mixture of fatty acid esters simultaneously with a purity of > 90% using Multicolumn Countercurrent Solvent Purification, in particular Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) when combined with compositions of organic solvents in combination and/or a sub-fractionation of either the outlet for the weakly adsorbing components (δ-section, see Fig 1) or the outlet for the strongly adsorbing components (γ-section, see Figs 2-4). The first fatty acid or fatty acid ester species of interest, the component called first target product PI in the following, respectively, is obtained with purity of > 90% and a yield of > 90%. A high purity fraction (> 90%) of the second fatty acid or fatty acid ester species of interest, the component called second target product P2 in the following, respectively, can be obtained in particular if subfractionation of a defined Multicolumn Countercurrent Solvent Gradient Purification process such as MCSGP outlet is applied. In applying subfractionation, P2 is isolated also with a purity of > 90%. The principle of the subfractionation is outlined schematically in Figure 1 where a purification process with six columns together with its internal profiles, adapted from e.g. Fig. 9, WO 2006/116886 is shown in combination with the subfractionation of the γ section (column 1). By means of the subfractionation a pure fraction of the second target product P2 can be obtained from column 1 in addition to the pure first target product PI that is obtained from column 3. Without the subfractionation, the second target product P2 is contaminated with the impurities SI and S2 and can be obtained only with low purity.

To consider subfractionation of an outlet of such a process is something that would not have been considered by the person skilled in the art in the field, since SMB processes, and also correspondingly MCSGP processes, are tailored to be adapted to a specific separation process by corresponding structuring of the switching intervals, gradients, flow rates, solvents etc. in order to avoid loss of useful product by complete recycling of fractions overlapping with desired product fractions. Therefore subfractionation, while admittedly well known from the field of conventional batch chromatography, would not be considered as an option of an outlet of such a countercurrent process since countercurrent processes are exactly adapted to avoid sub fractionation. However it was now found that for specific separation problems like the one given here, where for example the first target product is EPA ethyl ester, which is to be made available at very high purity, and is to be separated from a starting mixture from a second fatty acid component of interest, namely preferably DHA ethyl ester, subfractionation enables isolation of PI and P2 with high purity. In the elution profile of the corresponding mixture EPA ethyl ester is separated from such a second component of interest, namely in particular DHA ethyl ester, by intermediate impurities SI, the second fatty acid component however does not have to be provided at the same high purity and/or yield as EPA ethyl ester and correspondingly additional column separation is not justified, in particular for an industrial process. It was surprisingly found that a very stable, robust and economically viable separation process can be realized if for the isolation of the fraction comprising the second fatty acid component DHA ethyl ester can be made available by providing subfractionation on the outlet of one of the columns in such a process.

The concept of the subfractionation can be used also for the outlet of the δ section (column 5) depending on the position of the second target product P2 with respect to the first target product PI in the chromatogram (earlier or later eluting, sub-fractionation of δ section or γ section, respectively).

In an analogous manner, the subfractionation method can be applied to the embodiments of the processes presented in WO 2010/079060A1, for instance to the δ and γ sections of the embodiments shown in Figure 5 and Figure 6.

The composition of the fluids used in the MCSGP process is critical to the result of the sub-fractionation. During the sub-fractionation period separation conditions have to remain within a certain range of organic modifier contents to isolate both fatty acid or fatty acid ethyl ester species PI and P2, respectively, with the desired purity of larger 90%. The best results are obtained if the process in section γ is operated isocratically at the final modifier concentration of the previous column (column 2), see Figure 2 and 3. Once the P2 fraction has been eluted from the process, the modifier concentration may be changed in order to clean the column, as shown in Figure 2 and 3.

The purification method can be carried out without pretreatment using a single MCSGP step in order to obtain product with the desired purity. Depending on the feed mixture quality a simple pretreatment can be used to decrease the amount of key impurities and to ensure that the isolation of the fatty acid species of interest with the desired purity can be carried out at a high yield.

More generally speaking, and as outlined in the appended claims, the corresponding invention relates to a process for purification of a fatty acid or fatty acid derivative containing, multi-component mixture F by means of at least two individual chromatographic columns through which the mixture F is fed by means of at least one solvent s. The multi-component mixture F at least comprises a weakly adsorbing impurity W, a first target product PI to be purified, an intermediate impurity SI, a second target product P2 to be purified, and a strongly adsorbing impurity S2. Between the first target product and the second target product there is, in the elution profile, therefore located an intermediate impurity which is not to be in the fraction of the first target product and not in the fraction of the second target product.

In fatty acid or fatty acid derivative purification from natural origin (i.e. non-fossil based) oils such as fish or algae oils, The expression "natural origin oil" includes oils derived from genetically modified organisms, normally, the first target product PI is eicosapentaenoic acid (EPA), or a derivative thereof, and the second target product P2 is a second fatty acid (e.g. docosahexanoic acid DHA) different from the first target product PI, or a derivative of said second fatty acid. However it is also possible that the first target product PI is e.g. docosahexanoic acid DHA or a derivative thereof, and the second target product P2 is a second fatty acid (e.g. eicosapentaenoic acid EPA) different from the first target product PI, or a derivative of said second fatty acid.

As mentioned above, in accordance with the present invention, for the purification of such a mixture a multicolumn countercurrent purification chromatography is used. In this multicolumn countercurrent purification chromatography preferentially at least one outlet thereof is further separated using outlet fractionation.

Multicolumn countercurrent purification chromatography in this context is to be understood as a process according to WO 2006/116886 or also WO 2010/079060. With respect to this process therefore the disclosure of these two documents is expressly included.

In the case that, apart from the first fatty acid component PI, the second fatty acid component P2 has to be provided with similarly or equally high purity and yield as PI and a third fatty acid component P3 is present in the feed mixture that is flanked by the impurities S2 and S3 and does not have to be provided at the same high purity and/or yield as the previous two components PI and P2, additional column separation may be carried out to isolate P2 with high purity and yield. P3 may then be made available by providing sub-fractionation to the outlet for P3.

In the case that, apart from the first fatty acid component PI, at least one additional fatty acid component has to be provided with high purity and yield and a last fatty acid component is present in the feed mixture that is flanked by impurities and does not have to be provided at the same high purity and/or yield as the previous components, additional column separation may be carried out to isolate the additional components with high purity and yield. The last component may then be made available by providing sub-fractionation to the outlet for the last component.

According to a first preferred embodiment therefore in the multicolumn countercurrent purification chromatography process columns are run:

in at least one batch mode position in which the outlet of one column is used to collect first target product PI as well as

in at least one interconnected mode position, wherein the outlet of at least one section is fluidly connected with the inlet of at least one other section,

wherein said batch mode and said interconnected mode are either realized synchronously or sequentially,

and wherein after or within a switch time t* the columns are moved in their positions in a counter direction to the general direction of flow of the solvent.

The columns are preferably grouped into at least four sections α,β,γ,δ, wherein each section α,β,γ,δ comprises at least one column with the proviso that the function of several sections can be fulfilled sequentially and be realized by single columns, namely

a first section is provided with at least one inlet of solvent s and at least one outlet for the first target product PI, such that it washes the first target product PI out of the system, but keeps the intermediate impurities SI as well as possibly the second target product P2 and the strongly adsorbing impurities S2 inside the section a,

a second section β is provided with at least one inlet of solvent s and at least one outlet connected to an inlet of a fourth section δ, such that it washes the first target product PI, which is contaminated with intermediate impurities SI as well as possibly with second target product P2 and strongly adsorbing impurities S2 into the fourth section δ through said outlet, but keeps the intermediate impurities SI as well as possibly with second target product P2 and strongly adsorbing impurities S2 inside the second section β,

a third section γ is provided with at least one inlet of solvent s and an outlet for intermediate impurities SI, second target product P2 and strongly adsorbing impurities S2, such that it washes the intermediate impurities SI, second target product P2 and strongly adsorbing impurities S2 through said outlet out of the system and cleans the chromatographic columns,

the fourth section δ is provided with at least one inlet to receive output of the outlet of the second section β as well as at least one inlet for feeding in the multi-component mixture F and at least one outlet for weakly adsorbing impurities W, such that it washes the weakly adsorbing impurities W out of the system, but keeps the first target product PI inside the section δ.

The functions of the sections can either be fulfilled synchronously or sequentially, and after or within a switch time t*. the last column from the first section a is moved to the first position of the second section β, the last column of the second section β is moved to the first position of the third section γ, the last column of the third section γ is moved to the first position of the fourth section δ and the last column of the fourth section δ is moved to become the first column of the first section a.

If groupings of the sections ;β;γ;δ / 5_g,6_f,5_r are realized by single columns, the functions of individual sections ;β;γ;δ / δ_§,δ_ί,δ_Γ can also be fulfilled sequentially with alternating steps of interconnected mode and steps with batch mode within one switch time t*.

According to yet another preferred embodiment, the outlet of the third section γ is provided with outlet fractionation into at least three fractions for intermediate impurities SI, second target product P2 and strongly adsorbing impurities S2, respectively.

Preferably the outlet fractionation is controlled such that in a time interval a first fraction is isolated essentially comprising intermediate impurities SI and optionally some of the second target product P2, in a second subsequent time interval a second fraction is isolated essentially only comprising second target product P2, and in a third subsequent time interval a third fraction is isolated essentially comprising strongly adsorbing impurities S2 and optionally some of the second target product P2. Further preferably the third time interval can be split into two sub-intervals, first sub-interval for the isolation of some of the second target product P2 and some of the strongly adsorbing impurities S2, and the second sub-interval for the isolation of the strongly adsorbing impurities S2.

The time intervals for the outlet fractionation can be set such as to lead to a purity of the second target product P2 in the corresponding fractionation of at least 80%, preferably of at least 85%, most preferably of at least 90%.

In the following further embodiments shall be described in the context of figures 8 and 9. In this context it is to be pointed out that the processes which in the following will be described in the context of figures 8 and 9 (and if required also in combination with other further embodiments as defined above and below) can be carried out in the context of purification of fatty acid or fatty acid derivatives, however they can also generally be applied to the separation of other multicomponent mixtures, such as e.g. mixtures of peptides or proteins, derivatives thereof, amino acids in general, DNA fragments and the like, by means of at least 2 individual chromatographic columns through which the mixture is fed by means of at least one solvent. Furthermore while in the context of these embodiments it is possible to work with additional outlet fractionation of an outlet of one section, typically and preferably an outlet of the third section (γ), this is not necessary in all cases. Therefore while this is a limitation in the claim 1 as attached, this is not necessary and mandatory for these processes of figures 8 and 9. So in other words the processes described in the following in the context of figures 8 and 9 are independent inventions as such which can be carried out in the context of the subject matter as defined in claim 1, however they can also be more generally applied as outlined just above, so without the limitation to fatty acid or fatty acid derivatives separations and without outlet fractionation. Correspondingly according to a further preferred embodiment (schematically illustrated in Figure 8) the multicolumn countercurrent purification chromatography process columns can be run using a specific scheme:

The columns are preferably grouped into at least four sections α,β,γ,δ, wherein each section ,β,γ,δ comprises at least one column with the proviso that the function of several sections can be fulfilled sequentially and be realized by single columns, namely

a first section is provided with at least one inlet of solvent s and at least one outlet for the first target product PI, such that it washes the first target product PI out of the system, but keeps the remaining components inside the section a,

a second section β is provided with at least one inlet of solvent s and at least one outlet connected to an inlet of a fourth section δ, such that it washes the first target product PI, which is contaminated with intermediate impurities SI into the fourth section δ, washes the impurities SI out of the system, washes the second target product P2 that is contaminated with strongly adsorbing impurities SI into the fourth section δ, washes the target product P2 out of the system, washes the second target product P2 that is contaminated with strongly adsorbing impurities S2 into the fourth section δ but keeps the remaining components inside the second section β,

a third section γ is provided with at least one inlet of solvent s and an outlet for intermediate impurities S2, third target product P3 and strongly adsorbing impurities S3, such that it washes the remaining intermediate impurities S2, third target product P3 and strongly adsorbing impurities S3 through said outlet out of the system and cleans the chromatographic columns,

the fourth section δ is provided with at least one inlet to receive output of the outlet of the second section β as well as at least one inlet for feeding in the multi-component mixture F and at least one outlet for weakly adsorbing impurities W, such that it washes the weakly adsorbing impurities W out of the system, but keeps the first target product PI inside the section δ. Preferably at least one outlet of this section is subjected to outlet fractionation. The functions of the sections can either be fulfilled synchronously or sequentially, and after or within a switch time t*.

the last column from the first section a is moved to the first position of the second section β, the last column of the second section β is moved to the first position of the third section γ, the last column of the third section γ is moved to the first position of the fourth section δ and the last column of the fourth section δ is moved to become the first column of the first section a.

If groupings of the sections α;β;γ;δ / 5_g,6f₅5r are realized by single columns, the functions of individual sections ;β;γ;δ / δ_δ,δ_ί,δ_Γ can also be fulfilled sequentially with alternating steps of interconnected mode and steps with batch mode within one switch time t*.

According to yet another preferred embodiment, the outlet of the third section γ is provided with outlet fractionation into at least three fractions for intermediate impurities S2, third target product P3 and strongly adsorbing impurities S3, respectively.

Preferably the outlet fractionation is controlled such that in a time interval a first fraction is isolated essentially comprising intermediate impurities S2 and optionally some of the third target product P3, in a second subsequent time interval a second fraction is isolated essentially only comprising third target product P3, and in a third subsequent time interval a third fraction is isolated essentially comprising strongly adsorbing impurities S3 and optionally some of the third target product P3. Further preferably the third time interval can be split into two sub-intervals, first sub-interval for the isolation of some of the third target product P3 and some of the strongly adsorbing impurities S3, and the second sub- interval for the isolation of the strongly adsorbing impurities S3.

The time intervals for the outlet fractionation can be set such as to lead to a purity of the third target product P3 in the corresponding fractionation of at least 80%, preferably of at least 85%, most preferably of at least 90%.

As one can see from the setup according to Figure 9, the second section (β) may comprise at least three columns grouped into three sub-sections (β_ΐ5 β₂, β₃), wherein the first subsection (β comprises at least one inlet for feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (5_g) of the fourth section, most preferably into a first sub-sub-section (5_gl) of the second sub-section (6_g) of the fourth section, wherein the second sub-section (β₂) comprises at least one inlet feeding in solvent and at least one outlet for washing out product (P2), wherein the third sub-section (β₃) comprises at least one inlet feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (5_g) of the fourth section, most preferably into a third sub-sub-section (5_g3) of the second sub-section (5_g) of the fourth section. In this case, after or within a switch time (t*) a last column from the first sub-section _\) is moved to the first position of the second sub-section (β₂), the last column of the second sub-section (β₂) is moved to the first position of the third subsection (β₃), the last column of the third sub-section (β₃) is moved to the first position of the third section (γ) and the last column of the fourth section, preferably of the second sub- section (5_g) thereof, is moved to become the first column of the first section a and the last column of the first section a is moved to become the first column of the first sub-section (βι)·

The functions of the sections (β_ΐ5 β₂, β₃) are either fulfilled synchronously or sequentially, preferably the functions are taken by two columns only as illustrated in Figure 9.

As one can see from the setup according to Figure 8, the second section (β) may comprise even at least five columns grouped into five sub-sections (β_ΐ5 β₂, β₃, β₄, β₅), wherein the first sub-section (β comprises at least one inlet for feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (6_g) of the fourth section, most preferably into a first sub-sub-section (6_gl) of the second sub-section (5_g) of the fourth section, wherein the second sub-section (β₂) comprises at least one inlet feeding in solvent and at least one outlet for washing out component (SI), wherein the third sub-section (β₃) comprises at least one inlet feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (5_g) of the fourth section, most preferably into a third sub-sub-section (6_g3) of the second sub-section (6_g) of the fourth section, wherein the fourth sub-section (β₄) comprises at least one inlet feeding in solvent and at least one outlet for washing out product (P2), wherein the fifth sub-section (β₅) comprises at least one inlet feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (6_g) of the fourth section, most preferably into a fifth sub-subsection (6_g5) of the second sub-section (6_g) of the fourth section.

In this case, after or within a switch time (t*) a last column from the first sub-section (β is moved to the first position of the second sub-section (β₂), the last column of the second sub-section (β₂) is moved to the first position of the third sub-section (β₃), the last column of the third sub-section (β₃) is moved to the first position of the fourth sub-section (β₄), the last column of the fourth sub-section (β₄) is moved to the first position of the fifth sub- section (β₅), the last column of the fifth sub-section (β₅) is moved to the first position of the third section (γ, the last column of the fourth section, preferably of the second subsection (5_g) thereof, is moved to become the first column of the first section a and the last column of the first section a is moved to become the first column of the first sub-section (βι)·

The functions of the sections (β_ΐ5 β₂, β₃, β₄, β₅) are preferably either fulfilled synchronously or sequentially, preferably the functions are taken by two columns only as illustrated in Figure 8.

As one can see from the setup according to Figure 9, the second sub- section (5_g) may comprise at least four columns grouped into five sub-sub-sections (6_gl, 6_g2, 5_g3, 6_g4, 5_g5), wherein the first sub-sub-section (5_gl) comprises at least one inlet for taking up outlet from the second section (β), preferably from a first subsection (β thereof, and at least one outlet for washing out product or waste (W), wherein the second sub-sub-section (6_g2) comprises at least one inlet feeding in feed (F*) and/or solvent and at least one outlet for washing out product or waste (W), wherein the third sub-sub-section (6_g3) comprises at least one inlet for taking up outlet from the second section (β), preferably from a third subsection (β₃) thereof, and at least one outlet for washing out product or waste (W), wherein the fourth sub-sub-section (5_g4) comprises at least one inlet feeding in feed (F*) and/or solvent and at least one outlet for washing out product or waste (W), and wherein the fifth sub-sub-section (6_g5) comprises at least one inlet for solvent and at least one outlet to the first sub-section (¾_·).

In this case, after or within a switch time (t*) a last column from the first sub-sub-section (5_gl) is moved to the first position of the second sub-sub-section (6_g2), the last column of the second sub-sub-section (6_g2) is moved to the first position of the third sub-sub-section (5_g3), the last column of the third sub-sub-section (5_g3) is moved to the first position of the fourth sub-sub-section (5_g4), the last column of the fourth sub-sub-section (6_g4) is moved to the first position of the fifth sub-sub-section (5_g5) and the last column of the fifth sub-subsection (5_g5) is moved to the first column of the first section (a) and the last column of the first sub-section (5_f) is moved to become a first column of the first sub-sub-section (6_gl). Again, preferably the functions of the sections (6_gl, 6_g2, 5_g3, 6_g4, 5_g5) are either fulfilled synchronously or sequentially, preferably the functions are taken by two columns only as illustrated in Figure 9.

As one can see from the setup according to Figure 8, the second sub-section (5_g) may comprise at least seven columns grouped into six sub-sub-sections (5_gi, 6_g2, 6_g3, 5_g4, 6_g5, 5_g6, 5_g7), wherein the first sub-sub-section (6_gl) comprises at least one inlet for taking up outlet from the second section (β), preferably from a first subsection (β thereof, and at least one outlet for washing out product or waste (W), wherein the second sub-sub-section (5_g2) comprises at least one inlet feeding in feed (F*) and/or solvent and at least one outlet for washing out product or waste (W), wherein the third sub-sub-section (5_g3) comprises at least one inlet for taking up outlet from the second section (β), preferably from a third subsection (β₃) thereof, and at least one outlet for washing out product or waste (W), wherein the fourth sub-sub-section (5_g4) comprises at least one inlet feeding in feed (F*) and/or solvent and at least one outlet for washing out product or waste (W), wherein the fifth sub-sub-section (6_g5) comprises at least one inlet for taking up outlet from the second section (β), preferably from a fifth subsection (β₅) thereof, and at least one outlet for washing out product or waste (W), wherein the sixth sub-sub-section (6_g ) comprises at least one inlet feeding in feed (F*) and/or solvent and at least one outlet for washing out product or waste (W), and wherein the seventh sub-sub-section (5_g7) comprises at least one inlet for solvent and at least one outlet to the first sub-section (δ_Γ). After or within a switch time (t*) a last column from the first sub-sub-section (5_gl) is moved to the first position of the second sub-sub-section (6_g2), the last column of the second sub-sub-section (6_g2) is moved to the first position of the third sub-sub-section (5_g3), the last column of the third sub-sub-section (6_g3) is moved to the first position of the fourth sub-sub-section (6_g4), the last column of the fourth sub-sub-section (5_g4) is moved to the first position of the fifth sub-sub-section (5_g5), the last column of the fifth sub-subsection (6_g5) is moved to the first position of the sixth sub-sub-section (5_g6), the last column of the sixth sub-sub-section (5_g6) is moved to the first position of the seventh sub-sub- section (6_g7), the last column of the seventh sub- sub-section (6_g7) is moved to the first position of the first section (a) and the last column of the first sub-section (5_f) is moved to become a first column of the first sub-sub-section (6_gl). Again the functions of the sections (5_gl, 6_g2, 5_g3, 6_g4, 8_g5, 5_g6,6_g7) are either fulfilled synchronously or sequentially, and preferably by 2 columns only as given in Figure 8.

According to yet another preferred embodiment, the fourth section δ comprises at least three columns grouped into three sub-sections ¾ 6_g, ¾, wherein

the first sub-section ¾ comprises at least one inlet for feeding in the multi-component mixture F, preferably at a flow rate lower than the overall flow rate in the system, and at least one outlet either for direct removal of weakly adsorbing impurities W out of the system or into an inlet of the third sub-section wherein

the second sub-section 5_g comprises at least one inlet for taking up output of the second section β and at least one outlet connected to at least one input of the third sub-section 6_r, wherein

the third sub- section ¾- comprises at least one inlet for taking up output of the second sub- section 6_g and possibly at least one inlet for taking up output of the first sub-section ¾ and at least one outlet,

wherein after or within a switch time t* a column from the first sub-section 6f is moved to the first position of the second sub-section 6_g, the last column of the second sub-section 6_g is moved to the first position of the first section a, the last column of the third section γ is moved to the first position of the third sub-section 6_r and the last column of the third subsection δ, is moved to become a column of the first sub-section 6_t, and wherein the functions of the sections 6_f, 5_g, δ_Γ are either fulfilled synchronously or sequentially.

Pairs of sequential functions of the sections α;β;γ; 5 / 6_g,5_f,6_r can be combined within one column, and wherein within one switch time steps of interconnected mode and steps with batch mode, fulfilling those functions in sequential manner, alternate.

The fourth section 6 ma comprise three sub-sections 5_f, 5_g, δ_Γ as given above, and in the full system four columns can be used provided, these four columns being connected sequentially in a step CCL of interconnected mode within a first fraction of one switch time, and being driven in a batch step BL for taking out individual fractions W,P1,S1,P2,S2 of the multi-component mixture F within a second fraction of the switch time, wherein in this batch step BL one of the columns has a flow rate close to or equal to zero.

The system can also be comprised of two columns only (see in particular Fig 4), wherein the fourth section δ comprises three sub-sections δ,-, 5_g, δ_Γ according to claim 5, wherein the two columns are

in a first part of the switch time connected in series for continuous elution while by means of the outlet weakly adsorbing impurities W are collected, wherein

in a second part of the switch time the columns are driven in batch mode for collecting the first target product PI on the upstream column and weakly adsorbing impurities W on the downstream column while at the same time feeding the multi-component mixture F into the downstream column, wherein

in a third part of the switch time the columns are connected in series for continuous elution while by means of the outlet weakly adsorbing impurities W are collected, and wherein in a fourth part of the switch time the columns are driven in batch mode for collecting the intermediate impurities SI, second target product P2 and strongly adsorbing impurities S2 on the upstream column wherein the outlet thereof is further separated by using outlet fractionation to separate intermediate impurities SI, second target product P2 and strongly adsorbing impurities S2, respectively, and weakly adsorbing impurities W on the downstream column,

wherein after each switch time of the positions of the two columns are interchanged.

As mentioned above, as solvent a mixture of water and an organic solvent is preferably used, the organic solvent preferably being selected from the group of methanol, ethanol, acetonitrile or a combination thereof, is used. According to yet another preferred embodiment, the first section a, the second section β and the fourth section δ are operated with varying ratio of water and organic solvent, the organic solvent in the sections being adapted to be in the range of 40-100 weight percent, preferably in the range of 70-100 weight percent. Further preferably the fourth section δ comprises three sub-sections ¾, 6_g, δ_Γ as outlined above , and only one sub-section 5_g thereof being operated with varying ratio of water and organic solvent.

The first section a, the second section β and the fourth section δ are, according to yet another preferred embodiment, operated under gradient conditions, wherein if the fourth section δ comprises three sub-sections 6_f, 6_g, δ_Γ as outlined above, only one sub-section 5_g thereof is operated with varying ratio of water and organic solvent, and the organic solvent proportion is essentially continuously increasing from the corresponding sub-section δ_δ to the first section a and the second section β, preferably starting at a proportion of organic solvent in the range of 40-90 weight percent, preferably 60-85 weight percent and ending at a proportion of organic solvent in the range of 70-100 weight percent, preferably 70-85 weight percent.

According to yet another preferred embodiment, the first section a, the second section β and the fourth section δ are operated under step gradient conditions, wherein if the fourth section δ comprises three sub-sections δ_ί, 6_g, δ_Γ as outlined above, sub-section _g and the first section a is operated with a first, preferably constant ratio of water to organic solvent (a gradient is however also possible in this interval), and the second section β being operated with a second, preferably constant ratio of water to organic solvent different from the first ratio. Preferably the first ratio in this case is smaller than the second ratio, and more preferably the first ratio is such that the proportion of organic solvent is in the range of 70-100 weight percent, preferably 75-90 weight percent, and the second ratio is such that the proportion of organic solvent is in the range of 50-85 weight percent, preferably 60-80 weight percent.

As mentioned above, the process can be operated under gradient conditions or can be operated under step gradient conditions with specific ratios of water to organic solvent in the context of separation of the above mentioned specific fatty acid mixtures. It should be noted that this operation using gradients or step gradients under these conditions is possible also without subfractionation and represents a separate and at least partly independent invention of its own merits. Therefore the present invention also independently of the above mentioned invention including subfractionation relates to a process for purification of a fatty acid or fatty acid derivative containing, multi-component mixture F by means of at least two individual chromatographic columns through which the mixture F is fed by means of at least one solvent s. The multi-component mixture F at least comprises a weakly adsorbing impurity W, a first target product PI to be purified, an intermediate impurity SI, a second target product P2 to be purified, and a strongly adsorbing impurity S2. Between the first target product and the second target product there is, in the elution profile, therefore located an intermediate impurity which is not to be in the fraction of the first target product and not in the fraction of the second target product. As mentioned above, in accordance with this second aspect of this second invention, for the purification of such a mixture a multicolumn countercurrent purification chromatography is used. In this multicolumn countercurrent purification chromatography in addition to that, operation under gradient conditions or operation under step gradient conditions with specific ratios of water to organic solvent (preferably of the mentioned specific type) in the context of separation of the above mentioned specific fatty acid mixtures is employed. This does not exclude combination with subfractionation, but subfractionation is not a mandatory element of this process anymore. As for the rest of the parameters the second invention can be combined with any of the above-mentioned and below mentioned preferred embodiments in the context of this second aspect of the invention.

As already mentioned above, typically the second target product is docosahexanoic acid DHA or a derivative thereof.

The derivative of the eicosapentaenoic acid EPA, and/or in case of docosahexanoic acid DHA is a corresponding mono- or di-glyceride, or preferably a triglyceride, an alkyl ester, preferably a methyl ester or an ethyl ester.

The stationary phase is preferably a silica-based or polymeric stationary phase.

The temperature used during the purification is preferably in the range of 4°C to 60°C.

Further preferably the process is adapted such that the productivity for each product is at least 1.0 gram of product per hour per liter of stationary phase for a product purity of at least 90%.

The multi-component mixture F is for example a fish oil ethyl ester mixture, however also other sources of fatty acid mixtures are possible, for example vegetable sources or algae sources. Such a mixture is preferably fed with at most 50 gram, preferably at most 40 gram or at most 30 gram natural origin, non-fossil based oil alkyl (e.g. ethyl) ester mixture such as fish oil ethyl ester per liter of stationary phase volume in the loading column. In a preferred embodiment of the method, the feed mixture purity with respect to the desired species is larger 50%, more preferably larger 60% and even more preferably larger 70%. The feed mixture consists of either fatty acid esters, fatty acid ethyl esters or fatty acid methyl esters.

In a preferred embodiment of the method, the packing material of the chromatographic columns of the pretreatment step and/or the MCSGP step is silica based and functionalized with aliphatic chains such as octadecyl residues. The material preferably has an average particle size in the range of 1 μηι to ΙΟΟμιη.

In another preferred embodiment, the stationary phase material is silica based and not functionalized.

The outlet fractionation can be carried out by means of valves, including systems of valves and multiposition valves or by automated or manual fractionation. Another embodiment includes the fractionation by means of a robotic fraction collector. The fraction size is not required to be fixed; however it is recommended to carry out an initial fractionation with small fraction size and to analyze the fractions in order to determine the regions of the highest purity of P2 as outlined in the examples.

In the following, a broad P2 fraction may be collected in subsequent multicolumn countercurrent purification chromatography runs based on the outcome of the analytical results. In a preferred embodiment, fractions of > 90.0% purity are chosen for the P2 product pool. In a preferred embodiment the fluids used as carrier and solvent for the mixture to be separated are of organic nature or a mixture of organic solvents or water. In another preferred embodiment, the fluids comprise the organic modifiers acetonitrile, ethanol, or methanol. The organic modifier concentration is in the range of 40-100 weight- %. In another embodiment the organic modifier content is in the range of 80-100 weight- %. In a preferred embodiment, the organic modifier concentration is not changed during the operation of the multicolumn countercurrent purification chromatography process. In another preferred embodiment, the multicolumn countercurrent purification chromatography process is operated using a stepwise or linear gradient of organic modifier concentration within the range specified above.

In a preferred embodiment, during subfractionation for the collection of P2, the modifier concentration in the solvent is continued according to the conditions used during the elution of PI. Thus, if a linear gradient is operated during the elution of PI, the gradient is continued with the same slope during the elution and sub-fractionation of P2. If the process is operated isocratically during the elution of PI, it is continued to operate isocratically during the elution of P2.

In a preferred embodiment of the process, the modifier concentration is changed to a lower level during the elution of P2 than during the product elution of PI .

The decrease of the modifier concentration increases the selectivity of the components and thus the resolution, allow for isolation of P2 with higher yield for a given purity compared to constant isocratic conditions in the sections of the MCSGP process involved in the separation (5_g, α, β) or compared to positive linear gradient conditions in the sections of the multicolumn countercurrent purification chromatography process involved in the separation (6_g, α, β). A preferred point in time for the change of the modifier concentration to a lower level is at the termination time point of the collection of PI (section a)

The modifier decrease preferably is by 5%-20% whereby larger values are preferred. The decrease in modifier concentration prior to the subfractionation start is schematically outlined in Figure 2.

In another preferred embodiment a negative gradient is operated within the range specified above during the collection of P2. Also in this mode the preferred starting point of the negative gradient corresponds to termination time point of the collection of PI.

In another preferred embodiment, the fluid is a supercritical fluid such as supercritical C0₂ or Propane.

In a further embodiment, the fluid comprises a supercritical fluid such as supercritical C0₂ and a modifier, such as an organic fluid which is completely miscible with the supercritical fluid, for example methanol, ethanol, 2-propanol, acetonitrile and chloroform. In a preferred embodiment, the pressure and the temperature within the multicolumn countercurrent purification chromatography process are constant and in the range of 40- 90°C and 100-300 bar, respectively. In another preferred embodiment, the temperature is kept constant and the pressure is varied within the different sections of the process in the range specified above.

In a preferred embodiment, the conditioning step is a washing step, a dilution step, a distillation step, an extraction step, a crystallization step or a complexation step.

After the conditioning step, the feed mixture or parts thereof are applied to the multicolumn countercurrent purification chromatography process for purification of one or more of the fatty acid or fatty acid derivate species.

Further embodiments of the invention are laid down in the dependent claims. The method described by the present invention enables the purification of fatty acids or esters thereof from a mixture of fatty acids or esters thereof with high yield and high purity simultaneously. It significantly increases the throughput and has a low demand of expensive chromatographic column packing material. The process is scalable to an industrial scale.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1 shows a schematic of the fractionation method applied to the γ section of the process as described in WO 2006/116886, Fig 8; the arrows with full heads indicate the direction of the column switching; the arrows with split heads indicate the outlet fractionation into n fractions (where n is a natural number, in this case n = 4); the captions below the fractions indicate the main component in the respective fraction, e.g. SI;

Fig. 2 shows a schematic of the fractionation method applied to the process described in WO 2006/116886, Fig 9; the upper part of the Figure shows the six sections of the process numbered 1 to 6 while the lower part of the chromatogram shows the internal profile of the cyclic process with gradient elution during the target fraction phase;

Fig. 3 shows a schematic of the fractionation method applied to the process described in WO 2006/116886, Fig 8; the upper part of the Figure shows the six sections of the process numbered 1 to 6 while the lower part of the chromatogram shows the internal profile of the cyclic process with step gradient elution during the target fraction phase;

Fig. 4 shows a schematic of the fractionation method applied to the process described in WO 2006/116886, Fig 28; the upper part of the Figure shows the sections of the process while the lower part of the chromatogram shows the internal profile of the cyclic process. Figure 4a shows a process with a linear gradient elution during the PI recycling and collection phases ( , β, 5g) and Figure 4b shows a process with isocratic conditions during the PI recycling and collection phases (α, β, 5g). Figure 4c shows a more comprehensive representation of Figure 4b, where the tasks of the process and the corresponding sections in the chromatogram are arranged in a clearer way. In all three cases, an isocratic elution of the P2 sub- fractionation phase is shown.

shows the UV absorbance at 225 nm recorded at the outlet of the second column of the MCSGP device (continuous line); the vertical dashed lines indicate the individual cycles of the cyclic process;

shows a zoom of Figure 1; UV absorbance at 225 nm recorded at the outlet of the second column of an MCSGP device in operation (continuous line); the vertical dashed lines indicate individual cycles of the cyclic process; the collection intervals for the product fraction that contains the purified EPA ethyl ester (EPA-EE) are marked by rectangles; the DHA ethyl ester (DHA- EE) peaks are indicated by arrows;

shows an overlay of analytical chromatograms of the raw fish oil ethyl ester mixture (dotted line), the pre-conditioned fish oil ethyl ester mixture (after extraction, dashed line) and the MCSGP-purified fish oil ethyl ester (full line); the key impurities are indicated by parentheses; and

shows a schematic of a further fractionation method based on a further development of the process as described in WO 2006/116886, Fig 28, for the purification of three components of interest PI, P2, P3; wherein the upper part of the Figure shows the sections of the process while the lower part of the chromatogram shows the internal profile of the process; a process with linear gradient conditions during the PI, P2 and P3 recycling and collection phases (α, β, 5g) is shown, whereinimpure side-fractions of PI and P2 are internally recycled in phase CC1, CC2 and in phases CC3, CC4, respectively; P3 is recovered by sub-fractionation; and

shows yet another a schematic of a further fractionation method based on a further development of the process as described in WO 2006/116886, using 2 columns for the purification of PI and P2 in the absence of an intermediate impurity and where there are less BL and CC zones than in the process as illustrated in Figure 8.

DESCRIPTION OF PREFERRED EMBODIMENTS Figure 1 shows a schematic of the fractionation method applied to the γ section of the six- section multicolumn process as described in WO 2006/116886, e.g. inFig 8, 9 or 21 thereof. The greek nomenclature (α;β;γ;δ₈,δί,δτ) refers to the sections in a general sense, whereby each section may comprise multiple columns. The numbering of the chromatographic column positions (1,2,3,4,5,6) refers to individual tasks. The dashed arrows with the full heads indicate the direction of column switching. The columns switch positions in the direction 1 - 2 -> 3 - 4 - 5 ->6 -M ... One "cycle" is complete after a chromatographic column has switched until it has reached is original position. A typical multicolumn chromatography experiment operates over multiple cycles. At any given point in time, the column positions (1,2,3,4,5,6) may be occupied or not depending on the number of columns used in the process. In a six-column embodiment of the process as schematically illustrated in Figure 1 , each column position is occupied by one column at any given point in time (except for the very short time interval in which the columns switch positions). In the three-column embodiment of the process as schematically illustrated in Figure 2 either odd numbered positions (1,3,5) or even numbered positions (2,4,6) are occupied at any given point in time. Note that the odd numbered positions (1,3,5) are positions in "batch mode", i.e. they have solvent inlets and outlets which have no fluid connection with other positions. In contrast, the even numbered positions (2,4,6) are in "interconnected mode", i.e. they have a fluid connection that serves for internal recycling of solvents and components to be separated. The directions of the solvent flow in the interconnected mode is 2 -> 4 and 4 - 6. The feed mixture is injected into the column(s) in position 5. In the separation case of EPA ethyl ester (target product PI) and DHA ethyl ester (target product P2), the following components are eluted from the positions in batch mode:

From position 5, weakly adsorbing components, summarized as "W", are eluted

from position 3 target product PI is eluted

from position 1 the side component SI, target product P2 and side components SI are eluted.

By fractionation of the outlet of position 1, fractions rich in SI, P2 and S2 are obtained. In the figure, the arrows with split heads indicate the outlet fractionation into n fractions (where n is a natural number, in this case n = 4); the captions below the fractions indicate the main component in the respective fraction, e.g. SI; It is possible that also two adjacent fractions are rich in the same component. The number of fractions is flexible and can be tailored to the feed mixture to maximize the yield for a certain desired purity of the product P2. In the case of the EPA / DHA ethyl ester purification it is recommended to first perform an experimental run with a fine fractionation (n > 10) and to determine the purity and the concentration of the fractions by offline analysis. From the results of the offline analysis, it can be determined which fractions to pool in subsequent MCSGP runs to maximize the yield for a desired purity of P2. In subsequent experiments, the fractionation interval can be set from the beginning in such way, that this P2 pool is collected directly. Figure 2 shows a schematic of the fractionation method applied to the process described in WO 2006/116886, e.g. in Fig. 9 thereof; the upper part of the figure shows the six sections (a; ;y;6g,5_f,5_r). The section borders are indicated by the vertical dashed lines.

In the following example, we first assume that each section is occupied with one column. The columns are numbered 1 to 6, according to Figure 1, Figure 2 is actually just another representation of the process of Fig. 1. The solid arrows indicate fluid connections, inlets and outlets. The dashed arrows indicate optional fluid outlets of the γ section that are used sequentially as the subfractionation progresses.

The lower part of the figure shows the internal profile of the multicolumn cyclic process with gradient elution during the PI separation phase (sections α;β;δ_δ) in cyclic steady state at the beginning of a "switch" of duration t*. The internal chromatogram resembles a single column batch chromatogram. In fact the MCSGP process parameters are derived from a single column batch chromatogram that has been divided into different sections according to the presence of the components W, PI, SI, P2, S2 (detailed operating parameter determination procedure described in WO 2006/116886): in section 5_g, both W and PI (overlapping) are present; in section a pure PI is present, in section β PI and SI (overlapping) are present and in section γ SI, P2 and S2 (overlapping) are present.

Section 6_f corresponds to the feeding section and δ_Γ corresponds to a section of column equilibration. The sections of the batch chromatogram correspond to sections of the MCSGP process.

In the interconnected state, denoted with "CC", a fluid connection is present between positions 2 and 4 that allows for internal recycling of the product PI overlapping with SI from one column to the other (section β). At the same time, in the interconnected state, a fluid connection is present between positions 4 and 6 that allows for internal recycling of the target product PI overlapping with W (section δ₄). By means of the internal recycling, impure side fractions containing valuable PI (EPA-ethyl ester in the case of purification of fatty acid ethyl esters from fish- or algae-oil) are kept inside the system and are re- separated, so that the target product PI is not lost and a high yield of PI can be obtained. On the other hand, only pure PI is withdrawn from the system (section a), so a high product purity and high product yield of PI can be obtained simultaneously.

In the above-mentioned fatty acid ethyl ester purification case also a valuable second product P2 (DHA-ethyl ester) is present in the feed mixture. The internal chromatogram shows that P2 is flanked by intermediate impurities S 1 and strongly adsorbing impurities S2. In an MCSGP process according to WO 2006/116886, SI, P2 and S2 would be collected together and P2 would be recovered only with very low purity. By means of outlet fractionation into multiple fractions, first a fraction rich in impurity SI is obtained. This fraction may contain also partially P2. Next, a high purity fraction of P2 is obtained. Finally a fraction rich in S2 is obtained, that may contain also partially P2. By broadening the width of the P2 fraction, one can obtain P2 with higher yield and lower purity. By narrowing the width of the P2 fraction one can obtain P2 with higher purity and lower yield. Typically the size of the P2 fraction is selected such that a pre-defined purity specification is fulfilled, e.g. a purity of > 90.0%.

The dashed thick line in the chromatogram indicates the modifier gradient which is generated by varying over time the fluid composition of the streams that enter the columns. The modifier concentration in each section can be changed individually through pumps supplying the inlet of each section. The figure shows a gradient that increases linearly over time through the sections where the actual purification of PI takes place (sections 5_g; ;P). In the subsequent γ section the linear gradient is discontinued and the process is operated in isocratic mode (constant modifier concentration) instead. By using isocratic conditions the elution of the components from the γ section is slowed down and a better resolution of SI, P2, S2 is obtained, allowing for a better sub-fractionation in a sense that a broader P2 fraction can be collected and more high purity P2 is recovered in this fraction. For the isocratic elution in section yalso a lower modifier concentration than the final modifier concentration of section β may be chosen in order to improve the resolution of SI, P2, S2 even further. It is preferred to use to (lower) isocratic conditions readily in section β as indicated in figure 3. In sections δ_Γ and5_f typically modifier concentrations are used that are sufficiently low to cause adsorption of PI and P2 in those sections.

Figure 3 shows a schematic of the fractionation method applied to the process that has been introduced in Figure 2. The only differences are the modifier conditions which in this case are isocratic at a relatively high modifier concentration in sections 5_g and a and isocratic at a lower modifier concentration in sections β and γ, so a step gradient is used at the transition between phases a and β. As mentioned in the description of figure 2, the lower modifier concentration improves the resolution of SI, P2, S2 in the γ-section and enables a better subfractionation. The examples as illustrated in Figures 2 and 3 can also be operated with three columns only, wherein the columns switch consecutively between the CC and the BL to perform all the functions of the sections i.e. the three BL and the three CC tasks are carried out sequentially instead of in parallel.

Fig. 4 shows a schematic of the fractionation method applied to the process as e.g. described in WO 2006/116886, Fig 28; two different modifier conditions are shown in Figure 4a and Figure 4b, respectively. The upper parts of the figures show the sections of the process (α;β;γ;δ_§,δ_ί,δ_Γ) while the lower parts of the figures show the internal profiles of the process with a linear gradient elution in the range where the target components elute (Figure 4a) and with isocratic conditions in the range where the target components elute (Figure 4b). In both figures, the section borders are indicated by the vertical dashed lines. Figure 4c shows a different representation of Figure 4b where the elution of the components from the columns is matching the corresponding regions of the schematic chromatogram in phase δ„ e.g. the elution of pure W from the downstream column corresponds to the region of pure W in schematic chromatogram at the bottom of the figure. This representation facilitates the understanding of the determination of the MCSGP process operating parameters.

In the following example, we assume that the MCSGP process is operated with two columns, numbered 1 and 2. The solid arrows indicate fluid connections, inlets and outlets. The dashed arrows indicate optional fluid outlets of the γ section that are used sequentially as the fractionation progresses. The lower part of the figures shows the internal profile of the multicolumn cyclic process with gradient elution during the PI separation phase (sections α;β;δ_δ) in cyclic steady state at the beginning of a "switch" of duration t* as introduced in the description of figure 2.

Since the process is operated with only two columns, in the interconnected state internal recycling tasks of W, PI recycling and PI, SI recycling have to be carried out sequentially rather than in parallel as in a 6- or 3- column MCSGP process. For this reason, during each switch two interconnected states exist: a first one for the internal recycling of W, PI from section 5_g to section δ_Γ (first configuration of columns in figures 4a and 4b), called CC1, and a second one for the internal recycling of PI, SI from section β ίο δ_δ (third configuration of columns in figures 4a, 4b, and 4c), called CC2.

Similarly, since the process is operated with two columns, in the batch state the tasks of feeding (including the elution of W), the collection of PI and the outlet fractionation of section γ for the collection of P2 have to be carried out sequentially rather than in parallel. Thus during each switch two batch states exist: a first batch state for the injection of the feed mixture (including the elution of W) into one column and in parallel the collection of Plfrom the other column (sections 6_f and a), and a second batch state for the collection of P2 from section γ and the elution of W from 6_g .The first batch state is called "BL1" and corresponds to the second column configuration in the figure and the second batch state is called "BL2" and corresponds to the fourth column configuration in the figures 4a, 4b, and 4c.

Independent of the fact that the process tasks are operated in a sequential manner rather than in parallel, the tasks themselves are identical to the ones outlined in the description of figure 2.

The 2-column MCSGP process comprises two "switches" per cycle. Each switch consists of CC1, BL1, CC2, BL2 intervals. In the first switch, column 1 is downstream of column 2 and in the second switch column 2 is downstream of column 1.

Also in the 2-column MCSGP process varying modifier concentrations can be supplied to each column in order to provide gradients or isocratic conditions.

In figure 4a, as in figure 2, a linear gradient is shown schematically that extends over the sections involved in the purification of PI (sections 6_g;a;P). During the purification of P2 (section γ), isocratic conditions are indicated.

In figure 4b, isocratic conditions that extend through all sections involved in the purification of PI and P2 are indicated (sections 5_g;a$;y). For column cleaning, an elevated modifier concentration is indicated.

Figure 4c shows a more comprehensive representation of Figure 4b, where the 5_g tasks of the process and the corresponding 5_g sections in the chromatogram are arranged in a different way.

Fig. 8 shows a schematic of the fractionation method applied and based on the process as e.g. described in WO 2006/116886, Fig 28, for the separation of two target components PI and P2 with high yield and high purity and the isolation of a third component P3 by sub- fractionation using a linear modifier gradient. The upper part of the figure shows the sections of the process (α;β;γ;δ_§,δί,δτ) while the lower part of the figure shows the internal profile of the process with a linear gradient elution in the range where the target components elute. The section borders are indicated by the vertical dashed lines.

Since the process is operated with only two columns, in the interconnected state internal recycling tasks of W/Pl recycling, Pl/Sl, S1/P2 and P2/S2 recycling are carried out sequentially rather than in parallel as in a multi-column MCSGP process (WO/2010/079060). For this reason, during each switch four interconnected states exist: a first one for the internal recycling of W, PI from section 5g to section 5r (first configuration of columns in figure 8), called CC1, a second one for the internal recycling of PI, SI from section β to 6_g (third configuration of columns in figure 8), called CC2, a third one for the internal recycling of SI, P2 from section β to 6_g (fifth configuration of columns in figure 8), called CC3, and a fourth one for the internal recycling of P2, S2 from section β to 5_g (seventh configuration of columns in figure 8), called CC4.

Similarly, since the process is operated with two columns, in the batch state the tasks of feeding (including the elution of W), the collection of PI, the collection of P2 and the outlet fractionation of section γ for the collection of P3 have to be carried out sequentially rather than in parallel. Thus during each switch four batch states exist: a first batch state for the injection of the feed mixture (including the elution of W) into one column and in parallel the collection of Plfrom the other column (sections 5f and a), a second batch state for the elution of W and the collection or removal of the intermediate impurity SI (sections 8_f and β), a third batch state for the elution of W and the collection of P2 (sections ¾ and β), and a fourth batch state for the elution of W and the collection of P3 (sections 6_f and γ). Optionally, if the removal of W has already been achieved during the ¾■ or 5f phase, the column that is not used for washing out SI and P2 can carry out washing steps or be inactive. The first batch state is called "BL1" and corresponds to the second column configuration in the figure, the second batch state is called "BL2" and corresponds to the fourth column configuration in figure 8, the third batch state is called "BL3" and corresponds to the sixth column configuration in figure 8, and the fourth batch state is called "BL4" and corresponds to the eighth column configuration. Independent of the fact that the process tasks are operated in a sequential manner rather than in parallel, the tasks themselves are identical to the ones outlined in the description of figure 2.

The 2-column MCSGP process as shown in Figure 8 comprises two "switches" per cycle. Each switch consists of CC1, BL1, CC2, BL2, CC3, BL3, CC4, BL4 intervals. In the first switch, column 1 is downstream of column 2 and in the second switch column 2 is downstream of column 1. In Figure 8, only the first switch is shown for the sake of simplicity.

In figure 8, as in figure 2, a linear gradient is shown schematically that extends over the sections involved in the purification of PI, P2 and P3 (sections δ_§;α;β; γ). For column cleaning, an elevated modifier concentration is indicated. However, the process can be run in an identical manner with isocratic conditions in the sections involved in the purification of PI, P2 and P3. The feed flow F is complemented with an asterisk "*", which indicates that the feed solution can be applied either in BL1, BL2, BL3, BL4 or in multiple positions.

Fig 9. shows yet another a schematic of a fractionation method using 2 columns and where there are less BL and CL zones than in the process as illustrated in Figure 8. While not specifically illustrated, here the outlet of column 2 in BL3 can be subjected to outlet fractionation. In the alternative or in addition to that the outlet of column 2 in BL2 and/or in BL1 can be subjected to outlet fractionation.

In the cases shown in figures 4a , figure 4b and figure 4c, the argumentation regarding the effect of the modifier conditions on the separation of W, PI, SI, P2, S2 and on the subfractionation is identical to the one provided in the description of figure 2.

Example 1: Sample analysis

Sample analysis was performed using an Agilent 1100 series instrument (Agilent, Santa Clara, CA, USA). The analytical column was of brand Kromasil C18-100A-3u 250 x 4.6mm (adapted from Fournier V, Juaneda P, Destaillats F, Dionisi F, Lambelet P, Sebedio JL, Berdeaux O. 2006. Analysis of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization. Journal of Chromatography A. Volume 1129, Issue 1, pages 21-28). As mobile phases de-ionized water was used as solvent A and pure acetonitrile was used as solvent B. A gradient was run from 85% B to 99% B in 26 min at a flow rate of 0.5 mL /min. Absorbance detection was done at 220 nm. Figure 7 shows an overlay of the analytical chromatograms of the original fish oil ethyl ester mixture, the water phase of the extraction process (potential feed for MCSGP) and the MCSGP purified EPA-EE, as obtained by the method described in example 1. The key impurities, which are significantly depleted by the extraction procedure, are indicated by parentheses in the chromatogram overlay. It is worth noting, that also the DHA-EE content is decreased by the extraction procedure.

Example 2: Purification of EPA ethyl ester from esterified crude fish oil

A crude fish oil ethyl ester mixture (Ethyl-all cis-5,8,ll,14,17-Eicosapentaenoate > 65.0%, TCI Europe N.V. Belgium) containing 68.6% EPA ethyl ester (EPA-EE) and 17% DHA ethyl ester (DHA-EE) was fed to a 2-column MCSGP device. The MCSGP process was run using two 15 cm x 0.46 cm steel columns packed with octadecyl-functionalized- silica (ODS) as stationary phase. As solvent A a mixture of 75.0 weight-% acetonitrile and 25.0 weight % water was used and as solvent B a mixture of 95.0 weight-% acetonitrile and 5.0 weight % water was used. The MCSGP equipment was assembled from standard components manufactured by Knauer GmbH Berlin, namely Smartline pumps 40P, multiposition-valves V6 , UV detectors 40D and pH/cond sensor 2900.The MCSGP process was operated in gradient mode with a negative gradient from 91.0 weight-% acetonitrile to 83.6 weight-% acetonitrile within each cycle (80 vol-% B to 43 vol-% B). The cycle time of the MCSGP process was 53 min, and one product sample was taken per cycle. Figure 5 illustrates the cyclically continuous operation of the MCSGP process by means of the repetitive UV signals that were recorded at the outlet of the second column of the MCSGP device. A zoom of the repetitive pattern of the UV signals is provided in Figure 6. In Figure 6, also the product collection interval of the MCSGP process that withdraws a part of peak that corresponds to the EPA ethyl ester (EPA-EE, PI) as product fraction is indicated. Moreover, the position of the DHA-EE (P2) peak is shown. The purity of the EPA-EE product fractions was determined by offline analysis. From the MCSGP process, EPA-EE with a purity of 97.0% and a yield of 93% was obtained. For the sub-fractionation of the outlet of section γ, isocratic operation was continued at 83.6% acetonitrile. Fractions of 1 mL per fraction were collected using Foxy Rl fraction collector (Teledyne Inc.) and subjected to offline analysis using the method described in example 1. Based on the analytical results in following reproductive experiments with the same MCSGP operating parameters the fraction size was adjusted in order to collect fractions that had been previously analyzed to have > 90% purity as a P2 pool. For the pool collection, a multiposition valve was used and the fractionation was done automatically. The product pool corresponded to 91% purity and 61% yield of DHA-EE (P2).

Example la: Purification of EPA ethyl ester from esterifled crude fish oil

The crude fish oil ethyl ester mixture of example 1 was fed to a 2-column MCSGP device. The MCSGP process was run using the columns and the solvents of example 1.

In contrast to example 1, the MCSGP process was operated in isocratic mode at 75 weight- % acetonitrile (0 vol-% buffer B). The cycle time of the MCSGP process was 40 min and samples of the EPA-EE-rich fractions from the MCSGP process were taken. All EPA-EE was eluted in the stream for the strongly adsorbing components and there was some separation of EPA-EE and DHA-EE. The purity of these fractions was determined by offline analysis according to example 1. In this case, the yield of EPA-EE was 72% and the purity was only 76%, which is far below the usually desired purity of at least 90%.

Example 2: Purification of EPA ethyl ester from esterifled crude fish oil

A crude fish oil ethyl ester mixture (Ethyl-all cis-5,8,1 1 ,14,17-Eicosapentaenoate > 65.0%, TCI Europe N.V. Belgium) containing 68.6% EPA ethyl ester (EPA-EE) and 17% DHA ethyl ester (DHA-EE) was fed to a 2-column MCSGP device. The MCSGP process was run using two 15 cm x 0.46 cm steel columns packed with octadecyl-functionalized- silica (ODS) as stationary phase. As solvent A a mixture of 85 weight-% ethanol and 15 weight % water was used.

The MCSGP process was operated in isocratic mode at 85 weight-% ethanol. The cycle time of the MCSGP process was 20 min, and one product sample was taken per cycle. The purity of the EPA-EE product fractions was determined by offline analysis according to example 1. From the MCSGP process, EPA-EE with a purity of >95% and a yield of >92% was achieved. For the sub-fractionation of the outlet of section γ, isocratic operation was continued at 80 weight-% Ethanol. The moment of switching the solvent composition to a lower level was provided by the peak maximum of the EPA-EE peak, visible in the internal chromatogram of the MCSGP process (see Fig 7). Fractions of 0.5 mL/fraction were collected and subjected to offline analysis. By pooling only fractions of > 90.0% purity according to the analytical results a DHA-EE was obtained with 92% purity and 65% yield.

Example 3: Pretreatment of esterifled crude fish oil

The crude fish oil ethyl ester mixture of example 1 was subjected to liquid-liquid extraction as a conditioning step.

The extraction was carried out by mixing 100 parts of Acetonitrile and 10 parts of water, adding 33 parts of fish oil ethyl ester mixture and allowing 2 hrs for phase separation. After phase separation, the water-phase, which contained also the EPA-EE, had a decreased content of the key impurities (see example 1 and Figure 5). The key impurities were decreased from 11.4% to 8.0% by the extraction procedure. Accordingly, a higher key impurity content of 12.6% was measured in the oily phase. The use of a feed stock for MCSGP pre-treated by extraction as described here or by other means that reduce the key impurity content, can lead to an improved performance of the MCSGP processing step. For instance, a higher yield can be obtained under the same purity constraint.

List of Reference Signs

W weakly adsorbing impurities S3 very strongly adsorbing

PI first target product impurities

P2 second target product CC interconnected state

P3 third target products 1 BL batch state

intermediate impurities

S2 strongly adsorbing impurities

Claims

1. A process for purification of a fatty acid and/or fatty acid derivative containing, multi-component mixture (F) by means of at least two individual chromatographic columns through which the mixture (F) is fed by means of at least one solvent (s), wherein the multi-component mixture (F) at least comprises weakly adsorbing impurities (W), a first target product (PI) to be purified, an intermediate impurity

(51) , a second target product (P2) to be purified, and strongly adsorbing impurities

(52) ,

wherein the first target product (PI) is a first fatty acid, or a derivative thereof, wherein the second target product (P2) is a second fatty acid different from the first target product (PI), or a derivative of said second fatty acid,

wherein for the purification multicolumn countercurrent purification chromatography is used,

and wherein in this multicolumn countercurrent purification chromatography at least one outlet thereof is subjected to outlet fractionation.

2. A process according to claim 1, wherein in the multicolumn countercurrent purification chromatography process columns are run:

in at least one batch mode position in which the outlet of one column is used to collect first target product (PI) as well as

wherein after or within a switch time (t*) the columns are moved in their positions in a counter direction to the general direction of flow of the solvent, wherein the columns are grouped into at least four sections ( ,β,γ,δ), wherein each section (α,β,γ,δ) comprises at least one column with the proviso that the function of several sections can be fulfilled sequentially and be realized by single columns, a first section (a) is provided with at least one inlet of solvent (s) and at least one outlet for the first target product (PI), such that it washes the first target product (PI) out of the system, but keeps the intermediate impurities (SI) as well as the second target product (P2) and the strongly adsorbing impurities (S2) inside the section (a), a second section (β) is provided with at least one inlet of solvent (s) and at least one outlet connected to an inlet of a fourth section (δ), such that it washes the first target product (PI), which is contaminated with intermediate impurities (SI) as well as possibly with second target product (P2) and strongly adsorbing impurities (S2) into the fourth section (δ) through said outlet, but keeps the remaining part of the intermediate impurities (SI) as well as possibly the remaining part of the second target product (P2) and the strongly adsorbing impurities (S2) inside the second section (β), a third section (γ) is provided with at least one inlet of solvent (s) and an outlet for intermediate impurities (SI), second target product (P2) and strongly adsorbing impurities (S2), such that it washes the intermediate impurities (SI), second target product (P2) and strongly adsorbing impurities (S2) through said outlet out of the system and cleans the chromatographic column(s), the fourth section (δ) is provided with at least one inlet to receive output of the outlet of the second section (β) as well as at least one inlet for feeding in the multi- component mixture (F) and at least one outlet for weakly adsorbing impurities (W), such that it washes the weakly adsorbing impurities (W) out of the system, but keeps the first target product (PI) inside the section (δ), wherein it is possible that feeding in the multi-component mixture (F) takes place not with every column when taking the function of the fourth section (δ), wherein preferably in this case only one physical column, each time when taking the function of the fourth section (δ), is fed with multi-component mixture (F), wherein the functions of the sections are either fulfilled synchronously or sequentially, and

wherein after or within a switch time (t*) the last column from the first section (a) is moved to the first position of the second section (β), the last column of the second section (β) is moved to the first position of the third section (γ), the last column of the third section (γ) is moved to the first position of the fourth section (δ) and the last column of the fourth section (δ) is moved to become the first column of the first section (a), wherein if groupings of the sections (α;β;γ;δ / 5_g,6_f,5_r) are realized by single columns, the functions of individual sections ( ;β;γ;δ / 6_g,6_f,6_r) are fulfilled sequentially with alternating steps of interconnected mode and steps with batch mode within one switch time (t*).

3. A process according to claim 2, wherein the outlet of the third section (γ) is provided with outlet fractionation into at least three fractions for intermediate impurities (SI), the second target product (P2) and strongly adsorbing impurities (S2), respectively, wherein preferably the outlet fractionation is controlled such that in a time interval a first fraction is isolated essentially comprising intermediate impurities (SI) and optionally some of the second target product (P2), in a second subsequent time interval a second fraction is isolated essentially only comprising second target product (P2), and in a third subsequent time interval a third fraction is isolated essentially comprising strongly adsorbing impurities (S2) and optionally some of the second target product (P2), wherein further preferably at least one of said time intervals can be split into two or more sub-intervals, preferably in case of splitting the last time interval into a first sub-interval for the isolation of some of the second target product (P2) and some of the strongly adsorbing impurities (S2), and into a second sub-interval for the isolation of the strongly adsorbing impurities (S2).

4. A process according to claim 2, wherein the outlet of the first section (a) is provided with outlet fractionation into at least three fractions for weakly adsorbing impurities (W), the first target product (PI) and intermediate impurities (SI), respectively, wherein preferably the outlet fractionation is controlled such that in a time interval a first fraction is isolated essentially comprising weakly adsorbing impurities (W) and optionally some of the first target product (PI), in a second subsequent time interval a second fraction is isolated essentially only comprising first target product (PI), and in a third subsequent time interval a third fraction is isolated essentially comprising intermediate impurities (SI) and optionally some of the first target product (PI), wherein further preferably at least one of said time intervals can be split into two or more sub-intervals, preferably in case of splitting the last time interval into a first sub-interval for the isolation of some of the first target product (PI) and some of the intermediate impurities (SI), and into a second sub-interval for the isolation of the intermediate impurities (SI).

5. A process according to claim 3 or 4, wherein the time intervals for the outlet fractionation are set such as to lead to a purity of the first (PI) or respectively the second target product (P2) in the corresponding fractionation of at least 80%, preferably of at least 85%, most preferably of at least 90%.

6. A process according to any of the preceding claims 2-5, wherein the fourth section (δ) comprises at least three columns grouped into three sub-sections (¾, 5_g, 6_r), wherein the first sub-section (¾^■) comprises at least one inlet for feeding in the multi- component mixture (F), preferably at a flow rate lower than the overall flow rate in the system, and at least one outlet either for direct removal of weakly adsorbing impurities (W) out of the system or into an inlet of the third sub-section (¾■), wherein the second sub-section (8_g) comprises at least one inlet for taking up output of the second section (β) and at least one outlet connected to at least one input of the third sub-section (5_r), wherein the third sub-section (δ_Γ) comprises at least one inlet for taking up output of the second sub-section (6_g) and possibly at least one inlet for taking up output of the first sub-section (¾), and at least one outlet, wherein after or within a switch time (t*) a column from the first sub-section (δ ) is moved to the first position of the second sub-section (5_g), the last column of the second sub-section (5_g) is moved to the first position of the first section (a), the last column of the third section (γ) is moved to the first position of the third sub-section (δ_Γ) and the last column of the third sub-section (5_r) is moved to become a column of the first sub-section (5_f), and wherein the functions of the sections (¾, 6_g, δ_Γ) are either fulfilled synchronously or sequentially.

7. A process according to claim 6, wherein pairs of sequential functions of the sections (α;β;γ; δ / 6_g,6_f,5_r) are combined within one column, and wherein within one switch time steps of interconnected mode and steps with batch mode, fulfilling those functions in sequential manner, alternate.

8. A process according to any of the preceding claims 2-7, wherein the fourth section (δ) comprises three sub-sections (¾, δ_§, δ_Γ) according to claim 6, and wherein in the full system four columns are provided, these four columns being connected sequentially in a step (CC) of interconnected mode within a first fraction of one switch time, and being driven in a batch step (BL) for taking out individual fractions (W,P1,S1,P2,S2) of the multi-component mixture (F) within a second fraction of the switch time, wherein in this batch step (BL) one of the columns has a flow rate close to or equal to zero.

9. A process according to any of the preceding claims 2-5, wherein the system is comprised of two columns, wherein the fourth section (δ) comprises three subsections (8_f, 5_g, δ_Γ) according to claim 6, wherein the two columns are in a first part of the switch time connected in series for continuous elution while by means of the outlet weakly adsorbing impurities (W) are collected (δ_δ, ¾·), wherein in a second part of the switch time the columns are driven in batch mode for collecting the first target product (PI) on the upstream column and weakly adsorbing impurities (W) on the downstream column while at the same time feeding the multi-component mixture (F) into the downstream column (α,δί) wherein

in a third part of the switch time the columns are connected in series for continuous elution while by means of the outlet weakly adsorbing impurities (W) are collected (β, δ₈), and wherein

in a fourth part of the switch time the columns are driven in batch mode for collecting the intermediate impurities (SI), second target product (P2) and strongly adsorbing impurities (S2) on the upstream column wherein the outlet thereof is further separated using outlet fractionation to separate intermediate impurities (SI), second target product (P2) and strongly adsorbing impurities (S2), respectively, and the downstream column is either inactive or used to collect weakly adsorbing impurities (W), wherein after each switch time of the positions of the two columns are interchanged.

10. A process according to any of the preceding claims, wherein as solvent a mixture comprising, essentially consisting, or consisting of water and an organic solvent, preferably selected from the group of methanol, ethanol, acetonitrile or combination thereof, is used, and wherein the first section (a), the second section (β) and the fourth section (δ) are preferably operated with varying ratio of water and organic solvent, the organic solvent in the sections being adapted to be in the range of 40- 100 weight percent, preferably in the range of 70-100 weight percent, and wherein preferably the fourth section (δ) comprises three sub-sections (5_f, 6_g, δ_Γ) according to claim 5, and only one sub-section (5_g) thereof being operated with varying ratio of water and organic solvent.

11. A process according to claim 10, wherein the first section (a), the second section (β) and the fourth section (δ) are operated under gradient conditions, wherein preferably the fourth section (δ) comprises three sub-sections (¾, 5_g, 8_r) according to claim 5, only one sub-section (6_g) thereof being operated with varying ratio of water and organic solvent, and wherein the organic solvent proportion is essentially continuously increasing from the corresponding sub-section (6_g) to the first section (a) and the second section (β), preferably starting at a proportion of organic solvent in the range of 40-90 weight percent, preferably 60-85 weight percent and ending at a proportion of organic solvent in the range of 70-100 weight percent, preferably 70-85 weight percent.

12. A process according to claim 10 or 11, wherein the first section (a), the second section (β) and the fourth section (δ) are operated under step gradient conditions, wherein preferably the fourth section (δ) comprises three sub-sections (5_f, 6_g, δ_Γ) according to claim 5, sub-section (5_g) and the first section (a) being operated with a first, preferably constant ratio of water to organic solvent, and the second section (β) being operated with a second preferably constant ratio of water to organic solvent different from the first ratio, wherein preferably the first ratio is smaller than the second ratio, and wherein more preferably the first ratio is such that the proportion of organic solvent is in the range of 70-100 weight percent, preferably 75-90 weight percent, and the second ratio is such that the proportion of organic solvent is in the range of 50-85 weight percent, preferably 60-80 weight percent.

13. A process according to any of the preceding claims, wherein the first target product is (PI) eicosapentaenoic acid (EPA) or docosahexanoic acid (DHA) or a respective derivative thereof and the second target product (P2) is respectively docosahexanoic acid (DHA) or eicosapentaenoic acid (EPA) or a respective derivative thereof.

14. A process according to any of the preceding claims, wherein the derivative of the first and/or second fatty acid, in particular of eicosapentaenoic acid (EPA), and/or of docosahexanoic acid (DHA), is a corresponding mono-, di-, or preferably a triglyceride, ; or an alkyl ester, preferably ethyl ester or a methyl ester.

15. A process according to any of the preceding claims, wherein the stationary phase is a silica-based or polymeric stationary phase, and/or wherein the temperature used during the purification is in the range of 4°C to 60°C, preferably such that to the productivity for each product is at least 1.0 gram of product per hour per liter of stationary phase for a product purity of at least 90%.

16. A process according to any of the preceding claims, wherein the multi-component mixture (F) is a natural origin, non-fossil based oil ethyl ester mixture, preferably a fish oil or algae oil ethyl ester mixture, which is fed with at most 50 gram, preferably at most 40 gram or at most 30 gram oil ethyl ester per litre of stationary phase volume in the loading column.

17. A process according to any of the preceding claims, wherein as the solvent (s) a supercritical fluid used, wherein preferably the fluid used is supercritical C0₂, preferably in combination with an organic modifiers, preferably selected from the group consisting of: methanol, ethanol, acetonitril or combinations thereof, more preferably employed in the temperature range of 40 to 90°C and in the pressure range of 100 to 300 bar in the part of the process where the separation takes place.

A process according to any of the preceding claims, wherein in the multicolumn countercurrent purification chromatography process columns are run:

and wherein after or within a switch time t* the columns are moved in their positions in a counter direction to the general direction of flow of the solvent, wherein the columns are grouped into at least four sections ,β,γ,δ, wherein each section α,β,γ,δ comprises at least one column with the proviso that the function of several sections can be fulfilled sequentially and be realized by single columns, a first section a is provided with at least one inlet of solvent s and at least one outlet for the first target product PI, such that it washes the first target product PI out of the system, but keeps the remaining components inside the section a, a second section β is provided with at least one inlet of solvent s and at least one outlet connected to an inlet of a fourth section δ, such that it washes the first target product PI, which is contaminated with intermediate impurities SI into the fourth section δ, washes the impurities SI out of the system washes the second target product P2 that is contaminated with strongly adsorbing impurities SI into the fourth section δ, washes the target product P2 out of the system, washes the second target product P2 that is contaminated with strongly adsorbing impurities S2 into the fourth section δ, but keeps the remaining components inside the second section β.

a third section γ is provided with at least one inlet of solvent s and an outlet for intermediate impurities S2, third target product P3 and strongly adsorbing impurities S3, such that it washes the intermediate impurities S2, third target product P3 and strongly adsorbing impurities S3 through said outlet out of the system and cleans the chromatographic columns, and wherein preferably at least one outlet thereof is subjected to outlet fractionation the fourth section δ is provided with at least one inlet to receive output of the outlet of the second section β as well as at least one inlet for feeding in the multi- component mixture F and at least one outlet for weakly adsorbing impurities W, such that it washes the weakly adsorbing impurities W out of the system, but keeps the first target product PI inside the section δ, wherein the functions of the sections can either be fulfilled synchronously or sequentially, and

wherein after or within a switch time t* the last column from the first section a is moved to the first position of the second section β, the last column of the second section β is moved to the first position of the third section γ, the last column of the third section γ is moved to the first position of the fourth section δ and the last column of the fourth section δ is moved to become the first column of the first section , wherein if groupings of the sections α;β;γ;δ / δ_?,δι,δ_Γ are realized by single columns, the functions of individual sections α;β;γ;δ / 6_g,¾,6_r can be fulfilled sequentially with alternating steps of interconnected mode and steps with batch mode within one switch time t*.

A process according to any of the preceding claims 2-18, wherein the second section (β) comprises at least three columns grouped into three sub-sections (βι, β₂, β₃), wherein

the first sub-section (β comprises at least one inlet for feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (5_g) of the fourth section, most preferably into a first sub-subsection (6_gi) of the second sub-section (5_g) of the fourth section, wherein the second sub-section (β₂) comprises at least one inlet feeding in solvent and at least one outlet for washing out product (P2), wherein the third sub-section (β₃) comprises at least one inlet feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (6_g) of the fourth section, most preferably into a third sub-sub-section (6_g3) of the second sub-section (5_g) of the fourth section, wherein after or within a switch time (t*) the last column from the first sub-section (β is moved to the first position of the second sub-section (β₂), the last column of the second sub-section (β₂) is moved to the first position of the third sub-section (β₃), the last column of the third sub-section (β₃) is moved to the first position of the third section (γ) and the last column of the fourth section, preferably of the second sub-section (5_g) thereof, is moved to become the first column of the first section a and the last column of the first section a is moved to become the first column of the first sub-section (β , and wherein the functions of the sections (βι, β₂, β₃) are either fulfilled synchronously or sequentially, or wherein the second section (β) comprises at least five columns grouped into five sub-sections (β_1; β₂, β₃, β₄, β₅), wherein the first sub-section (βι) comprises at least one inlet for feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (6_g) of the fourth section, most preferably into a first sub-subsection (6_gl) of the second sub-section (6_g) of the fourth section, wherein the second sub-section (β₂) comprises at least one inlet feeding in solvent and at least one outlet for washing out the component (SI), wherein

the third sub-section (β₃) comprises at least one inlet feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (6_g) of the fourth section, most preferably into a third sub-sub-section (6_g3) of the second sub-section (6_g) of the fourth section, wherein the fourth sub-section (β₄) comprises at least one inlet feeding in solvent and at least one outlet for washing out product (P2), wherein

the fifth sub-section (β₅) comprises at least one inlet feeding in solvent, and at least one outlet into an inlet of the fourth section (δ), preferably into an inlet of a second sub-section (6_g) of the fourth section, most preferably into a fifth sub-sub-section (5_g5) of the second sub-section (6_g) of the fourth section, wherein after or within a switch time (t*) a last column from the first sub-section (β is moved to the first position of the second sub-section (β₂), the last column of the second sub-section (β₂) is moved to the first position of the third sub-section (β₃), the last column of the third sub-section (β₃) is moved to the first position of the fourth sub-section (β₄), the last column of the fourth sub-section (β₄) is moved to the first position of the fifth sub-section (β₅), the last column of the fifth subsection (β₅) is moved to the first position of the third section (γ), the last column of the fourth section, preferably of the second sub-section (6_g) thereof, is moved to become the first column of the first section a and the last column of the first section a is moved to become the first column of the first sub-section (βι), and wherein the functions of the sections (βι, β₂, β₃, β₄, β₅) are either fulfilled synchronously or sequentially.

A process according to any of the preceding claims 2-18, in combination with claim 6, wherein the second sub-section (6_g) comprises at least four columns grouped into five sub-sub-sections (5_gl, 6_g2, 6_g3, 6_g4, 5_g5), wherein the first sub-sub-section (5_gl) comprises at least one inlet for taking up outlet from the second section (β), preferably from a first subsection (β thereof, and at least one outlet for washing out product or waste (W), wherein the second sub-sub-section (5_g2) comprises at least one inlet feeding in feed or solvent (F*) and at least one outlet for washing out product or waste (W), wherein the third sub-sub-section (6_g3) comprises at least one inlet for taking up outlet from the second section (β), preferably from a third subsection (β₃) thereof, and at least one outlet for washing out product or waste (W), wherein the fourth sub-sub-section (5_g4) comprises at least one inlet feeding in feed or solvent (F*) and at least one outlet for washing out product or waste (W), the fifth sub-sub-section (δ_§5) comprises at least one inlet for solvent and at least one outlet to the first sub-section (δ_Γ), wherein after or within a switch time (t*) a last column from the first sub- sub-section (6_gl) is moved to the first position of the second sub-sub-section (5_g2), the last column of the second sub-sub-section (6_g2) is moved to the first position of the third sub-sub-section (6_g3), the last column of the third sub-sub-section (5_g3) is moved to the first position of the fourth sub-subsection (5_g4), the last column of the fourth sub-sub-section (5_g4) is moved to the first position of the fifth sub-sub-section (6_g5) and the last column of the fifth sub-subsection (6g₅) is moved to the first section (a) and the last column of the first subsection (5_f) is moved to become a first column of the first sub-sub-section (6_gl), and wherein the functions of the sections (5_gl, 5_g2, 6_g3, 5_g4, 6_gs) are either fulfilled synchronously or sequentially, or wherein second sub-section (6_g) comprises at least seven columns grouped into seven sub-sub-sections (6_gl, 6_g2, 5_g3, 6_g4, 6_g5, 5_g , 5_g7), wherein the first sub-sub-section (6_gl) comprises at least one inlet for taking up outlet from the second section (β), preferably from a first subsection (β thereof, and at least one outlet for washing out product or waste (W), wherein the second sub-sub-section (5_g2) comprises at least one inlet feeding in feed or solvent (F*) and at least one outlet for washing out product or waste (W), wherein the third sub-sub-section (6_g3) comprises at least one inlet for taking up outlet from the second section (β), preferably from a third subsection (β₃) thereof, and at least one outlet for washing out product or waste (W), wherein the fourth sub-sub-section (5_g4) comprises at least one inlet feeding in feed or solvent (F*) and at least one outlet for washing out product or waste (W), wherein the fifth sub-sub-section (6_g5) comprises at least one inlet for taking up outlet from the second section (β), preferably from a fifth subsection (β₅) thereof, and at least one outlet for washing out product or waste (W), wherein the sixth sub-sub-section (5_g6) comprises at least one inlet feeding in feed or solvent (F*) and at least one outlet for washing out product or waste (W), the seventh sub-sub-section (6_g7) comprises at least one inlet for solvent and at least one outlet to the first sub-section (δ_Γ), wherein after or within a switch time (t*) a last column from the first sub-sub-section (6_gl) is moved to the first position of the second sub-sub-section (6_g2), the last column of the second sub-sub-section (6_g2) is moved to the first position of the third sub-sub-section (6_g3), the last column of the third sub-sub-section (5_g3) is moved to the first position of the fourth sub-subsection (5_g4), the last column of the fourth sub-sub-section (8_g4) is moved to the first position of the fifth sub-sub-section (6_gs), the last column of the fifth sub-subsection (6_g5) is moved to the first position of the sixth sub-sub-section (6_g6), the last column of the sixth sub-sub-section (5_g6) is moved to the first position of the seventh sub-sub-section (6_g7), the last column of the seventh sub-sub-section (6_g7) is moved to the first position of the first section (a) and the last column of the first sub-section (6_t) is moved to become a first column of the first sub-sub-section (6_gl), and wherein the functions of the sections (6_gl, 6_g2, 5_g3, 6_g4, 8_g5, 6_g6, 5_g7) are either fulfilled synchronously or sequentially.