Chromatographic separation process for efficient purification of polyunsaturated fatty acids

Abstract

The present invention relates to an improved chromatographic separation process for the purification of polyunsaturated fatty acid (PUFA) products and their derivatives. In particular, the present invention relates to a particularly efficient chromatographic separation process that uses silica having specific physical properties as the adsorbent phase for the purification of PUFAs or their derivatives from a feed mixture.

Description

[Technical field]

[0001] The present invention relates to an improved chromatographic separation process for the purification of polyunsaturated fatty acid (PUFA) products and their derivatives. In particular, the present invention relates to a particularly efficient chromatographic separation process that uses silica having specific physical properties as the adsorbent phase for the purification of PUFAs or their derivatives from a feed mixture. [Background technology]

[0002] Fatty acids, particularly PUFAs and their derivatives, are precursors of biologically important molecules that play important roles in regulating biological functions such as platelet aggregation, inflammation, and immune responses. Thus, PUFAs and their derivatives may be therapeutically useful in treating a wide range of conditions, including central nervous system disorders, neuropathy, including diabetic neuropathy, cardiovascular diseases, and systemic immune system and inflammatory disorders, including inflammatory skin diseases.

[0003] PUFAs are found in natural sources such as vegetable oils and marine oils.However, such PUFAs are often present in such oils in the form of mixtures with saturated fatty acids and many other impurities.Therefore, PUFAs should preferably be purified before nutritional or medicinal use.

[0004] Unfortunately, PUFAs are very fragile. Thus, when heated in the presence of oxygen, they tend to undergo isomerization, peroxidation and oligomerization. Therefore, fractionation and purification of PUFA products to prepare pure fatty acids is difficult. Distillation can cause unacceptable product decomposition even under reduced pressure.

[0005] Chromatographic separation techniques are well known to those of skill in the art. Chromatographic separation techniques, including both fixed bed systems and simulated or actual moving bed systems, are well known to those of skill in the art.

[0006] In a conventional fixed bed chromatography system, the mixture whose components are to be separated permeates into a vessel. The vessel is roughly cylindrical and is typically called a column. The column contains a packing of a porous adsorbent material (commonly called the stationary phase) that exhibits high permeability to fluids. The permeation rate of each component of the mixture depends on the physical properties of that component so that the component exits the column sequentially and selectively. Thus, some of the components tend to adhere strongly to the stationary phase and therefore permeate slowly, while other components tend to adhere weakly and exit the column faster. Many different fixed bed chromatography systems are available and are used for both analytical and industrial production purposes.

[0007] Simulated and real moving bed chromatography are well known techniques well known to those skilled in the art. The principle of operation involves countercurrent movement of a liquid eluent (or desorbent) phase and a solid adsorbent phase. This operation allows for minimal use of solvents and specific adsorbent inventory, thereby making the process economically viable. Such separation techniques have found several applications in diverse fields, including hydrocarbons, industrial chemicals, oils, sugars, and APIs.

[0008] Here, the simulated moving bed chromatography apparatus consists of several individual columns containing adsorbent, connected together in series. The eluent is passed through the columns in a first direction. The feedstock and eluent injection points in the system as well as the separated component collection points are periodically switched by a series of valves or a single multi-position valve. The overall effect is to simulate the operation of a single column containing a moving bed of solid adsorbent, the solid adsorbent moving in a countercurrent direction to the flow of the eluent. Thus, the simulated moving bed system consists of a column, which contains a fixed bed of solid adsorbent through which the eluent is passed, as in a conventional fixed bed system, whereas in the simulated moving bed system the operation is such as to simulate a continuous countercurrent moving bed.

[0009] A typical simulated moving bed chromatography apparatus is illustrated with reference to Figure 1. The concept of a simulated or real moving bed chromatography separation process is explained by considering a vertical chromatography column containing a stationary phase S divided into several sections, more precisely into four overlapping sub-zones I, II, III and IV from the bottom to the top of the column. The eluent is introduced at the bottom in IE by pump P. The mixture of components A and B to be separated is introduced at IA+B between sub-zones II and III. The extract containing mainly B is collected at SB between sub-zones I and II, and the raffinate containing mainly A is collected at SA between sub-zones III and IV.

[0010] In the case of a simulated moving bed system, the pseudo downward movement of the stationary phase S is caused by the movement of the introduction and collection points relative to the solid phase. In the case of a real moving bed system, the pseudo downward movement of the stationary phase S is caused by the movement of the various chromatographic columns relative to the introduction and collection points. In FIG. 1, the eluent flows upwards and the mixture A+B is injected between sub-zones II and III. The components move due to their chromatographic interactions with the stationary phase, for example adsorption in the porous medium. Component B (the slower flowing component), which shows a stronger affinity for the stationary phase, is taken up more slowly by the eluent and follows it with a delay. Component A (the faster flowing component), which shows a weaker affinity for the stationary phase, is taken up easily by the eluent. If the appropriate set of parameters in each sub-zone, especially the flow rates, are properly estimated and controlled, component A, which shows a weaker affinity for the stationary phase, is collected between sub-zones III and IV as the raffinate, and component B, which shows a stronger affinity for the stationary phase, is collected between sub-zones I and II as the extract.

[0011] It will therefore be appreciated that the conventional simulated moving bed system, as shown diagrammatically in FIG. 1, is limited to binary fractionation.

[0012] The advantages of other related so-called simulated moving bed non-conventional operating modes, such as Improved-SMB, Sequential-SMB, Varicol, Powerfeed, Modicon, MCSGP, Outlet Swing Stream-OSS, JO or pseudo SMB, among others, are known and obtained by the skilled person from conventional simulated moving bed processes, as detailed elsewhere (see, for example, Sa Gomes and Rodrigues, Chemical Engineering and Technology Special Issue: Preparative Chromatography and Downstream Processing, 2012, 35(1), 17-34, the contents of which are incorporated herein by reference in their entirety). Processes and apparatus for simulated moving bed chromatography are described in several patents, including U.S. Pat. No. 2,985,589, U.S. Pat. No. 3,696,107, U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533, FR-A-2103302, FR-A-2651148, and FR-A-2651149, all of which are incorporated herein by reference. This subject is also covered in "Preparative and Production Scale Chromatography", edited by Ganetsos and Barker, Marcel Dekker Inc, New York, 1993, all of which are incorporated herein by reference.

[0013] A real moving bed system is similar in operation to a simulated moving bed system, however, rather than switching feed mixture and eluent injection points and separated component collection points by a system of valves or a single multi-position valve, a series of adsorption units (i.e., columns) are instead physically moved relative to the feed and extraction points, and operation is such as to simulate a continuous countercurrent moving bed.

[0014] Processes and apparatus for practical moving bed chromatography are described in several patents, including U.S. Pat. No. 6,979,402, U.S. Pat. No. 5,069,883, and U.S. Pat. No. 4,764,276, which are incorporated herein by reference in their entireties.

[0015] Purification of PUFA products is particularly difficult. Therefore, many suitable raw materials for preparing PUFA products are very complex mixtures, containing many different components that have very similar retention times in chromatographic equipment. Therefore, it is very difficult to separate specific PUFAs from such raw materials. However, a high degree of purity of PUFA products is required, especially for pharmaceutical and nutritional applications. Therefore, distillation is used in the past when high purity PUFA products are required. However, there are significant difficulties in using distillation as a separation technique for sensitive PUFAs, as described above.

[0016] WO2011 / 080503, the entirety of which is incorporated herein by reference, discloses an SMB separation process for recovering PUFA products from raw material mixtures efficiently and with very high purity.The adjustment of this particular equipment is described in WO2013 / 005046, WO2013 / 005047, WO2013 / 005048, WO2013 / 005051, WO2013 / 005052 and WO2014 / 108686 (all of which are incorporated herein by reference).However, it has been found that such chromatographic systems often require large amounts of eluent when operating at low pressure (i.e. less than 20 bar, preferably less than 10 bar). This is in contrast to high performance liquid chromatography (HPLC) systems, which typically operate at higher pressures (i.e., 20-100 bar) and can extract PUFA products from feed mixtures with high productivity and low dilution (i.e., amount) of solvent required. However, operation at high pressures has several drawbacks in large-scale commercial processes. In particular, the stationary phase (e.g., silica) in the chromatography column is placed under increased stress and must be replaced more frequently. Summary of the Invention [Problem to be solved by the invention]

[0017] Therefore, there is a need to provide a more efficient chromatographic separation process for purifying PUFA products from feed mixtures at lower operating pressures so that less eluent is required to extract the PUFA product. [Means for solving the problem]

[0018] The inventors have unexpectedly found that using a silica adsorbent phase with specific physical properties can provide improved resolution of the peaks representing various PUFA products in typical PUFA-containing feedstocks while operating at low pressure.This improved resolution between peaks provides more efficient separation, which requires lower dilution of eluent.It has further been found that lower dilution of eluent can be achieved than when using HPLC process, while also achieving acceptable (relatively, surprisingly good) levels of productivity.

[0019] Accordingly, the present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) Solid adsorbent phase, which is C18 bonded silica wherein the pressure in the one or more chromatography columns is less than 20 bar, and further comprising: (1) the silica has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more; and / or (2) the silica has a carbon loading of 15 to 24 weight percent; and / or (3) Silica is 500m 2 The present invention provides a chromatographic separation process having a surface area of ​​less than or equal to 1 / g.

[0020] In certain embodiments, the present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) a solid adsorbent phase that is C18 bonded silica with a carbon loading of 15-24 wt% wherein the pressure in the one or more chromatography columns is less than 20 bar, and further comprising: (1) the silica has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more; and / or (2) Silica is 500m 2 The present invention provides a chromatographic separation process having a surface area of ​​less than or equal to 1 / g. [Brief description of the drawings]

[0021] [Figure 1] The basic principles of simulated or real moving bed processes for separating binary mixtures are presented. [Diagram 2] FIG. 1 shows a chromatographic separation process that includes two simulated or real moving bed processes for separating EPA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Diagram 3] FIG. 1 shows a chromatographic separation process comprising two simulated or real moving bed processes for separating DHA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Figure 4] FIG. 1 shows a chromatographic separation process that includes two simulated or real moving bed processes for separating EPA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Diagram 5] FIG. 1 shows a chromatographic separation process comprising two simulated or real moving bed processes for separating DHA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Figure 6] FIG. 1 shows a chromatographic separation process that includes two simulated or real moving bed processes for separating EPA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Figure 7]FIG. 1 shows a chromatographic separation process comprising two simulated or real moving bed processes for separating DHA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Figure 8] FIG. 1 shows a chromatographic separation process that includes two simulated or real moving bed processes for separating EPA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Figure 9] FIG. 1 shows a chromatographic separation process that includes two simulated or real moving bed processes for separating EPA from faster and slower moving impurities (i.e., more polar and less polar impurities). [Figure 10] Shown are three ways in which a chromatographic separation step can be carried out, including two simulated or real moving bed processes. [Figure 11] 1 shows a chromatographic separation process for separating EPA from faster and slower running impurities (i.e., more polar and less polar impurities). [Figure 12A] Figure 1 shows the total contribution to particle volume of silica samples as a function of particle size for different C18 silica types: (A) Silica 1 (control silica), two separate batches and Silica 2. (B) Silica 1 (control silica), two separate batches and Silicas 3 and 4. [Figure 12B] Figure 1 shows the total contribution to particle volume of silica samples as a function of particle size for different C18 silica types: (A) Silica 1 (control silica), two separate batches and Silica 2. (B) Silica 1 (control silica), two separate batches and Silicas 3 and 4. [Figure 13] Scanning electron microscope images of (A) Silica 1; (B) Silica 2; and (C) Silica 4 are shown. [Figure 14] FIG. 1 shows pulse studies to demonstrate asymmetry in the EPA peak during purification of EPA-containing feedstock on chromatographic columns using various silicas. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In its most general sense, the present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) A solid adsorbent phase that is C18 bonded silica introducing a feed mixture into a chromatography apparatus comprising one or more chromatography columns comprising:

[0023] Solid Adsorbent Phase The solid adsorbent phase is typically reversed-phase silica. The solid adsorbent phase is typically C18 bonded silica gel. Preferably, the solid adsorbent phase is reversed-phase C18 bonded silica gel. The adsorbent phase is typically non-polar.

[0024] The chromatography device includes one or more chromatography columns, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 columns. In some embodiments, the number of columns is typically one. In other embodiments, the number of columns is typically two or more, preferably four or more, more preferably six or more, even more preferably eight or more, for example 4, 5, 6, 7, 8, 9 or 10 columns. Typically, there are 25 or less columns, preferably 20 or less, more preferably 15 or less. When two or more chromatography columns are used, each chromatography column may contain the same or different adsorbents. Typically, when two or more chromatography columns are used, each column contains the same adsorbent.

[0025] The shape of the solid adsorbent phase material can be, for example, spherical or non-spherical beads, preferably approximately spherical beads.

[0026] C18 bonded silica has specific physical properties.

[0027] In a first embodiment of the present invention, the C18 bonded silica has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more.

[0028] In a second embodiment of the invention, the C18 bonded silica has a carbon loading of 15-24% by weight.

[0029] In a third embodiment of the present invention, the C18 bonded silica is 2 / g or less.

[0030] Each of these specific physical properties of silica has been found to provide increased resolution between different peaks in the separation of PUFA-containing feedstock, with each peak corresponding to a different PUFA product.This therefore allows for more efficient purification of the desired PUFA product in high yield while using less solvent.This reduced use of solvent is more cost-effective and more environmentally friendly for separations carried out on an industrial scale.Furthermore, the ability to achieve these advantages while using relatively large particles of silica allows lower pressures of eluent to be used in the system.This has further advantages in terms of cost savings and reduced equipment life and frequency that the solid adsorbent phase must be replaced.

[0031] Herein, in a first embodiment, the present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) a solid adsorbent phase that is C18-bonded silica with an average particle size of 230-270 μm and a Dv(10) of 160 μm or greater introducing a feed mixture into a chromatography apparatus comprising one or more chromatography columns comprising:

[0032] As defined herein, "mean particle size" refers to the volume moment average (D[4,3], also called the volume weighted mean diameter or De Brouckere mean diameter) of the particles. Dv(10) is the 10th of the particle size in a plot of the cumulative volume distribution of silica particles against increasing particle size. th The Dv(10) value refers to a percentile. The volume moment average is particularly sensitive to the number of coarse particles (i.e., larger size particles) present in a sample, and the Dv(10) value is particularly sensitive to the number of fine particles (i.e., smaller size particles) present in a sample. Thus, taken together, the volume moment average and Dv(10) value represent a useful characterization of both the average particle size and particle size distribution of the silica.

[0033] Volume moment average and Dv(10) are typically measured by laser diffraction, for example using standard method ISO 13320:2020. Details of laser diffraction are described, for example, at https: / / www.malvernpanalytical.com / en / products / technology / light-scattering / laser-diffraction (accessed March 15, 2021), the entire contents of which are incorporated herein by reference.

[0034] It is preferred that the average particle size is fairly large so that low pressure can be used in chromatographic separation.However, it is also preferred that the particle size distribution is narrow, which has been surprisingly found to improve the resolution between different PUFA peaks in the purification of PUFA-containing raw materials and allows less eluent to be used in the separation.In particular, it has been surprisingly found that the reduction or removal of the "tail" of particularly fine particles (i.e. particles with small diameters) from silica samples leads to increased resolution between PUFA peaks.Therefore, it is found that a fairly high average particle size, a high Dv(10) value, and a relatively small difference between the average particle size and the Dv(10) value are all desirable properties of silica for use in this embodiment.

[0035] Here, in the first embodiment, the average particle size is preferably 235 to 265 μm, more preferably 240 to 260 μm, even more preferably 245 to 260 μm, and most preferably 250 to 260 μm.

[0036] In the first embodiment, the silica preferably has a Dv(10) of 165 μm or more, more preferably 170 μm or more, even more preferably 175 μm or more, still more preferably 180 μm or more, and most preferably 185 μm or more. Typically, the silica has a Dv(10) of 225 μm or less, preferably 220 μm or less, more preferably 215 μm or less, and most preferably 210 μm or less. Here, typically, the silica has a Dv(10) of 160 to 225 μm, preferably 165 to 220 μm, more preferably 175 to 215 μm, and most preferably 185 to 210 μm.

[0037] Here, in the first embodiment, the silica preferably has an average particle size of 235 to 265 μm and a Dv(10) of 165 μm or more, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 175 μm or more, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 180 μm or more, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 185 μm or more.

[0038] Here, in the first embodiment, the silica preferably has an average particle size of 235 to 265 μm and a Dv(10) of 225 μm or less, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 220 μm or less, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 215 μm or less, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 210 μm or less.

[0039] Here, in the first embodiment, the silica preferably has an average particle size of 235 to 265 μm and a Dv(10) of 165 to 225 μm, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 175 to 220 μm, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 180 to 215 μm, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 185 to 210 μm.

[0040] In a first embodiment, the silica typically has a carbon loading (%C) of 15-24 wt%, preferably 16-22 wt%, more preferably 16.5-20 wt%, even more preferably 17-19.5 wt%, most preferably 17.5-19 wt%, for example 17.5-18 wt%. Carbon loading is a measure of the % of the solid adsorbent phase that is carbon. Typically, substantially all of the carbon content is derived from C18 functionalization of the silica particles.

[0041] The carbon loading of silica particles is typically measured using combustion analysis, such as the type of method described in standard ISO 21068-2 or a modification thereof, or using the method of Nguyen et al. (Science and Technology Development Journal, 2016, 19(4), 162-166, which is incorporated herein by reference in its entirety).

[0042] In a first embodiment, the silica is typically 500 ml 2 / g, preferably less than 450m 2 / g, more preferably less than 400m 2 / g, most preferably less than 350m 2 Silica typically has a surface area of ​​less than 100 m 2 / g, preferably 150m 2 / g, more preferably 200m 2 / g, and even more preferably 250m 2 / g, most preferably 300m 2 / g. Here, the silica typically has a surface area of ​​100 to 500 m2 / g, preferably 200 to 450m 2 / g, more preferably 250 to 400m 2 / g, most preferably 300 to 350m 2 / g surface area.

[0043] The surface area of ​​silica particles is typically measured by BET surface area analysis, for example by using standard method ISO 9277:2010. In this method, the surface area is determined from adsorption data, typically nitrogen adsorption, using BET theory (Brunauer, Emmett and Teller). The BET method of measuring surface area is described in detail in "Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density" by Lowell, Shields, Thomas and Thommes, Springer, Dordrecht, 2006 (pub: Springer), which is incorporated herein by reference in its entirety.

[0044] In this context, the surface area refers to the silica surface and is measured on bare silica prior to attachment of C18 chains.

[0045] In a second embodiment, the present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) a solid adsorbent phase that is C18 bonded silica with a carbon loading of 15-24 wt% introducing a feed mixture into a chromatography apparatus comprising one or more chromatography columns comprising:

[0046] It has been surprisingly discovered by the inventors that silicas having carbon loadings within this range provide improved resolution between PUFA peaks in the separation of a PUFA product from a feed mixture, regardless of whether the silica contains a "tail" of smaller particles (i.e., whether the Dv(10) value is less than that described in the first embodiment above).

[0047] In a second embodiment, the carbon loading of the silica is preferably 16-22 wt%, more preferably 16.5-20 wt%, even more preferably 17-19 wt%, most preferably 17.5-18 wt%, for example about 17.8 wt% or 17.9 wt%. Typically, substantially all of the carbon content is obtained from C18 functionalization of the silica particles.

[0048] The carbon loading of the silica particles is typically measured as described above in relation to the first embodiment.

[0049] In the second embodiment, typically, the average particle size is 230 to 270 μm, preferably 235 to 265 μm, more preferably 240 to 260 μm, even more preferably 245 to 260 μm, and most preferably 250 to 260 μm.

[0050] In the second embodiment, typically, the Dv(10) value of the silica is 160 μm or more, preferably 165 μm or more, more preferably 170 μm or more, even more preferably 175 μm or more, even more preferably 180 μm or more, and most preferably 185 μm or more. Typically, the silica has a Dv(10) of 225 μm or less, preferably 220 μm or less, more preferably 215 μm or less, and most preferably 210 μm or less. Here, typically, the silica has a Dv(10) of 160 to 225 μm, preferably 165 to 220 μm, more preferably 175 to 215 μm, and most preferably 185 to 210 μm.

[0051] Here, in the second embodiment, the silica typically has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more, preferably has an average particle size of 235 to 265 μm and a Dv(10) of 165 μm or more, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 175 μm or more, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 180 μm or more, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 185 μm or more.

[0052] Here, in the second embodiment, the silica typically has an average particle size of 230 to 270 μm and a Dv(10) of 225 μm or less, preferably has an average particle size of 235 to 265 μm and a Dv(10) of 225 μm or less, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 220 μm or less, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 215 μm or less, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 210 μm or less.

[0053] Here, in the second embodiment, the silica typically has an average particle size of 230 to 270 μm and a Dv(10) of 160 to 225 μm, preferably has an average particle size of 235 to 265 μm and a Dv(10) of 165 to 225 μm, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 175 to 220 μm, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 180 to 215 μm, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 185 to 210 μm.

[0054] The average particle size and the Dv(10) value are typically both measured as described above in relation to the first embodiment.

[0055] In a second embodiment, the silica is typically 500 ml 2 / g, preferably less than 450m 2 / g, more preferably less than 400m 2 / g, most preferably less than 350m 2 Silica typically has a surface area of ​​less than 100 m 2 / g, preferably 150m 2 / g, more preferably 200m 2 / g, and even more preferably 250m 2 / g, most preferably 300m 2 / g. Here, the silica typically has a surface area of ​​100 to 500 m 2 / g, preferably 200 to 450m 2 / g, more preferably 250 to 400m 2 / g, most preferably 300 to 350m 2 / g surface area.

[0056] The surface area of ​​the silica particles is typically measured as described above in relation to the first embodiment.

[0057] In a third embodiment, the present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) 500m 2 A solid adsorbent phase that is C18 bonded silica with a surface area of ​​less than 1 / g introducing a feed mixture into a chromatography apparatus comprising one or more chromatography columns comprising:

[0058] It has been surprisingly found by the present inventors that silica having a surface area within this range provides improved resolution between PUFA peaks in the separation of a PUFA product from a feed mixture.

[0059] In a third embodiment, the silica is preferably 450 ml 2 / g, more preferably less than 400m 2 / g, most preferably less than 350m2 Silica typically has a surface area of ​​less than 100 m 2 / g, preferably 150m 2 / g, more preferably 200m 2 / g, and even more preferably 250m 2 / g, most preferably 300m 2 / g. Here, the silica typically has a surface area of ​​100 to 500 m 2 / g, preferably 200 to 450m 2 / g, more preferably 250 to 400m 2 / g, most preferably 300 to 350m 2 / g surface area.

[0060] The surface area of ​​the silica particles is typically measured as described above in relation to the first embodiment.

[0061] In the third embodiment, typically, the average particle size is 230 to 270 μm, preferably 235 to 265 μm, more preferably 240 to 260 μm, even more preferably 245 to 260 μm, and most preferably 250 to 260 μm.

[0062] In the third embodiment, typically, the Dv(10) value of the silica is 160 μm or more, preferably 165 μm or more, more preferably 170 μm or more, even more preferably 175 μm or more, even more preferably 180 μm or more, and most preferably 185 μm or more. Typically, the silica has a Dv(10) of 225 μm or less, preferably 220 μm or less, more preferably 215 μm or less, and most preferably 210 μm or less. Here, typically, the silica has a Dv(10) of 160 to 225 μm, preferably 170 to 220 μm, more preferably 180 to 215 μm, and most preferably 185 to 210 μm.

[0063] Here, in the third embodiment, the silica typically has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more, preferably has an average particle size of 235 to 265 μm and a Dv(10) of 165 μm or more, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 175 μm or more, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 180 μm or more, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 185 μm or more.

[0064] Here, in the third embodiment, the silica typically has an average particle size of 230 to 270 μm and a Dv(10) of 225 μm or less, preferably has an average particle size of 235 to 265 μm and a Dv(10) of 225 μm or less, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 220 μm or less, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 215 μm or less, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 210 μm or less.

[0065] Here, in the third embodiment, the silica typically has an average particle size of 230 to 270 μm and a Dv(10) of 160 to 225 μm, preferably has an average particle size of 235 to 265 μm and a Dv(10) of 165 to 225 μm, more preferably has an average particle size of 240 to 260 μm and a Dv(10) of 175 to 220 μm, even more preferably has an average particle size of 245 to 260 μm and a Dv(10) of 180 to 215 μm, and most preferably has an average particle size of 250 to 260 μm and a Dv(10) of 185 to 210 μm.

[0066] The average particle size and the Dv(10) value are typically both measured as described above in relation to the first embodiment.

[0067] In a third embodiment, the silica typically has a carbon loading (%C) of 15-24 wt%, preferably 16-22 wt%, more preferably 16.5-20 wt%, even more preferably 17-19 wt%, most preferably 17.5-18 wt%, for example about 17.8 wt% or 17.9 wt%. Typically, substantially all of the carbon content is obtained from C18 functionalization of the silica particles.

[0068] The carbon loading of the silica particles is typically measured as described above in relation to the first embodiment.

[0069] In a preferred embodiment, the silica has the features of the second embodiment in addition to the features of the first and / or third embodiment. Thus, in this preferred embodiment, the invention relates to a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) a solid adsorbent phase that is C18 bonded silica with a carbon loading of 15-24 wt% wherein the pressure in the one or more chromatography columns is less than 20 bar, and further comprising: (1) the silica has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more; and / or (2) Silica is 500m 2 The present invention provides a chromatographic separation process having a surface area of ​​less than or equal to 1 / g.

[0070] In this preferred embodiment, the silica may have the features of the first and second embodiments. Here, the silica typically has an average particle size of 230-270 μm, a Dv(10) of 160 μm or more, and a carbon loading of 15-24 wt%. Preferably, the silica has an average particle size of 235-265 μm, a Dv(10) of 165 μm or more, and a carbon loading of 16-22 wt%. More preferably, the silica has an average particle size of 240-260 μm, a Dv(10) of 175 μm or more, and a carbon loading of 16.5-20 wt%. Even more preferably, the silica has an average particle size of 245-260 μm, a Dv(10) of 180 μm or more, and a carbon loading of 17-19 wt%. Most preferably, the silica has an average particle size of 250-260 μm, a Dv(10) of 185 μm or more, and a carbon loading of 17.5-18.0 wt%.

[0071] In this preferred embodiment, where the silica has the features of the first and second embodiments, typically the silica has an average particle size of 230-270 μm, a Dv(10) of 225 μm or less, and a carbon loading of 15-24 wt%. Preferably, the silica has an average particle size of 235-265 μm, a Dv(10) of 225 μm or less, and a carbon loading of 16-22 wt%. More preferably, the silica has an average particle size of 240-260 μm, a Dv(10) of 220 μm or less, and a carbon loading of 16.5-20 wt%. Even more preferably, the silica has an average particle size of 245-260 μm, a Dv(10) of 215 μm or less, and a carbon loading of 17-19 wt%. Most preferably, the silica has an average particle size of 250-260 μm, a Dv(10) of 210 μm or less, and a carbon loading of 17.5-18.0 wt%.

[0072] In this preferred embodiment, where the silica has the features of the first and second embodiments, typically the silica has an average particle size of 230-270 μm, a Dv(10) of 160-225 μm and a carbon loading of 15-24 wt%. Preferably, the silica has an average particle size of 235-265 μm, a Dv(10) of 165-225 μm and a carbon loading of 16-22 wt%. More preferably, the silica has an average particle size of 240-260 μm, a Dv(10) of 175-220 μm and a carbon loading of 16.5-20 wt%. Even more preferably, the silica has an average particle size of 245-260 μm, a Dv(10) of 180-215 μm and a carbon loading of 17-19 wt%. Most preferably, the silica has an average particle size of 250-260 μm, a Dv(10) of 185-210 μm and a carbon loading of 17.5-18.0 wt %.

[0073] Instead, in this preferred embodiment, the silica has the features of the second and third embodiments, where the silica typically has a carbon loading of 15-24 wt. % and a 500 m 2 Preferably, the silica has a carbon loading of 16-22 wt. % and a total surface area of ​​450 m 2 More preferably, the silica has a carbon loading of 16.5 to 20 wt. % and a total surface area of ​​less than 400 m 2 Even more preferably, the silica has a carbon loading of 17-19 wt. % and a total surface area of ​​less than 400 m 2 Most preferably, the silica has a carbon loading of 17.5 to 18.0 wt. % and a total surface area of ​​less than 350 m 2 / g total surface area.

[0074] In this preferred embodiment, where the silica has the features of the second and third embodiments, typically the silica has a carbon loading of 15-24 wt. % and a 100 m 2 / g. Preferably, the silica has a carbon loading of 16-22 wt. % and a total surface area of ​​150 m 2 More preferably, the silica has a carbon loading of 16.5 to 20 wt. % and a total surface area of ​​200 m 2Even more preferably, the silica has a carbon loading of 17-19 wt. % and a total surface area of ​​250 m 2 Most preferably, the silica has a carbon loading of 17.5 to 18.0 wt. % and a total surface area of ​​300 m 2 / g total surface area.

[0075] In this preferred embodiment, where the silica has the features of the second and third embodiments, typically the silica has a carbon loading of 15-24 wt. % and a carbon content of 100-500 m 2 %. Preferably, the silica has a carbon loading of 16-22 wt. % and a total surface area of ​​150-450 m 2 More preferably, the silica has a carbon loading of 16.5 to 20 wt. % and a total surface area of ​​200 to 450 m 2 / g. Even more preferably, the silica has a carbon loading of 17-19 wt. % and a total surface area of ​​250-400 m 2 Most preferably, the silica has a carbon loading of 17.5 to 18.0 wt. % and a total surface area of ​​300 to 350 m 2 / g total surface area.

[0076] In a particularly preferred embodiment, the silica has all the features of the first, second and third embodiments, where typically, in this particularly preferred embodiment, the silica has an average particle size of 230-270 μm and a Dv(10) of 160 μm or more, a carbon loading of 15-24 wt. %, and a densitometric average particle size of 500 μm or more. 2 Preferably, the silica has an average particle size of 235-265 μm and a Dv(10) of 165 μm or more, a carbon loading of 16-22 wt. % and a total surface area of ​​450 m 2 More preferably, the silica has an average particle size of 240-260 μm and a Dv(10) of 175 μm or more, a carbon loading of 16.5-20 wt. % and a total surface area of ​​400 m 2 Even more preferably, the silica has an average particle size of 245-260 μm and a Dv(10) of 180 μm or more, a carbon loading of 17-19 wt. % and a total surface area of ​​400 m 2Most preferably, the silica has an average particle size of 250-260 μm and a Dv(10) of 185 μm or greater, a carbon loading of 17.5-18.0 wt. %, and a total surface area of ​​350 m 2 / g total surface area.

[0077] In this particularly preferred embodiment, the silica typically has an average particle size of 230-270 μm and a Dv(10) of 225 μm or less, a carbon loading of 15-24 wt. %, and a 100 m 2 Preferably, the silica has an average particle size of 235-265 μm and a Dv(10) of 225 μm or less, a carbon loading of 16-22 wt. %, and a total surface area of ​​150 m 2 More preferably, the silica has an average particle size of 240-260 μm and a Dv(10) of 220 μm or less, a carbon loading of 16.5-20 wt. %, and a total surface area of ​​200 m 2 Even more preferably, the silica has an average particle size of 245-260 μm and a Dv(10) of 215 μm or less, a carbon loading of 17-19 wt. %, and a total surface area of ​​250 m 2 Most preferably, the silica has an average particle size of 250-260 μm and a Dv(10) of 210 μm or less, a carbon loading of 17.5-18.0 wt. %, and a total surface area of ​​300 m 2 / g total surface area.

[0078] In this particularly preferred embodiment, the silica typically has an average particle size of 230-270 μm and a Dv(10) of 160-225 μm, a carbon loading of 15-24 wt. %, and a SiO2 content of 100-500 μm. 2 Preferably, the silica has an average particle size of 235-265 μm and a Dv(10) of 165-225 μm, a carbon loading of 16-22 wt. %, and a total surface area of ​​150-450 m 2 More preferably, the silica has an average particle size of 240-260 μm, a Dv(10) of 175-220 μm, a carbon loading of 16.5-20 wt. %, and a total surface area of ​​200-450 m 2Even more preferably, the silica has an average particle size of 245-260 μm and a Dv(10) of 180-215 μm, a carbon loading of 17-19 wt. %, and a total surface area of ​​250-400 m 2 Most preferably, the silica has an average particle size of 250-260 μm, a Dv(10) of 185-210 μm, a carbon loading of 17.5-18.0 wt. %, and a total surface area of ​​300-350 m 2 / g total surface area.

[0079] In any of the above embodiments (i.e. in any of the first, second or third embodiments including any exemplary preferred or particularly preferred subembodiments thereof), the silica particles are typically porous. Typically, the average pore size of the silica particles is 60-200 Å. Preferably, the average pore size is 65-160 Å, more preferably 70-140 Å, even more preferably 80-130 Å, still more preferably 90-120 Å, most preferably 95-115 Å, for example about 100 Å or about 110 Å.

[0080] In any of the above embodiments, the silica particles typically have a total pore volume of 0.5 to 1.5 cc / g. In a preferred embodiment, the pore volume is 0.6 to 0.84 cc / g, more preferably 0.7 to 0.8 cc / g, and most preferably 0.73 to 0.77 cc / g.

[0081] Total pore volume is typically measured by nitrogen gas adsorption, for example as described in “Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density” by Lowell, Shields, Thomas and Thommes, Springer, Dordrecht, 2006 (pub: Springer), which is incorporated herein by reference in its entirety.

[0082] The average pore diameter d is calculated by the formula d=4 *It is calculated from the total pore volume V and surface area A using V / A, which is the diameter of a hypothetical uniform cylindrical pore with the same volume and area.

[0083] In any of the above embodiments, typically the silica particles have a well-defined particle size distribution such that at least 80% of the particles by number have a diameter greater than 200 μm. Typically at least 80% of the particles by number have a diameter less than 500 μm. Typically at least 80% of the particles by number have a diameter between 200 and 500 μm. Preferably at least 85% of the particles by number have a diameter greater than 200 μm. Typically at least 85% of the particles by number have a diameter less than 500 μm. Typically at least 85% of the particles by number have a diameter between 200 and 500 μm. More preferably at least 90% of the particles by number have a diameter greater than 200 μm. Typically at least 90% of the particles by number have a diameter less than 500 μm. Typically at least 90% of the particles by number have a diameter between 200 and 500 μm. Most preferably, at least 95% of the particles by number have a diameter greater than 200 μm. Typically, at least 95% of the particles by number have a diameter less than 500 μm. Typically, at least 95% of the particles by number have a diameter between 200 and 500 μm.

[0084] In any of the above embodiments, typically the silica particles have a well-defined particle size distribution such that at least 80% of the particles by volume have a diameter greater than 200 μm. Typically, at least 80% of the particles by volume have a diameter less than 500 μm. Typically, at least 80% of the particles by volume have a diameter between 200 and 500 μm. Preferably, at least 85% of the particles by volume have a diameter greater than 200 μm. Typically, at least 85% of the particles by volume have a diameter less than 500 μm. Typically, at least 85% of the particles by volume have a diameter between 200 and 500 μm. More preferably, at least 90% of the particles by volume have a diameter greater than 200 μm. Typically, at least 90% of the particles by volume have a diameter less than 500 μm. Typically, at least 90% of the particles by volume have a diameter between 200 and 500 μm. Most preferably, at least 95% of the particles by volume have a diameter greater than 200 μm. Typically, at least 95% of the particles by volume have a diameter less than 500 μm. Typically, at least 95% of the particles by volume have a diameter between 200 and 500 μm.

[0085] In any of the above embodiments, typically the silica particles have a well-defined particle size distribution such that at least 80% of the particles by mass have a diameter greater than 200 μm. Typically at least 80% of the particles by mass have a diameter less than 500 μm. Typically at least 80% of the particles by mass have a diameter between 200 and 500 μm. Preferably at least 85% of the particles by mass have a diameter greater than 200 μm. Typically at least 85% of the particles by mass have a diameter less than 500 μm. Typically at least 85% of the particles by mass have a diameter between 200 and 500 μm. More preferably at least 90% of the particles by mass have a diameter greater than 200 μm. Typically at least 90% of the particles by mass have a diameter less than 500 μm. Typically at least 90% of the particles by mass have a diameter between 200 and 500 μm. Most preferably, at least 95% of the particles by mass have a diameter greater than 200 μm. Typically, at least 95% of the particles by mass have a diameter less than 500 μm. Typically, at least 95% of the particles by mass have a diameter between 200 and 500 μm.

[0086] In any of the above embodiments, typically the silica particles have a density of 0.71 kg / dm 3 Less than or equal to 0.70 kg / dm 3 Less than or equal to 0.69 kg / dm 3 Less than or equal to 0.68 kg / dm 3 Less than or equal to 0.67 kg / dm 3 Less than or equal to 0.66 kg / dm 3 Typically, silica particles have a bulk (packed bed) density of 0.4 kg / dm 3 More than 0.5kg / dm 3 More preferably, 0.55 kg / dm 3 More preferably, 0.60 kg / dm 3 More preferably, 0.63 kg / dm 3 More than 0.65kg / dm 3 It has a bulk density of not less than 1000 g / m.

[0087] Here, in any of the above embodiments, typically, the silica particles are dispersed in an amount of 0.4 to 0.71 kg / dm 3 , preferably 0.5 to 0.70 kg / dm 3 , more preferably 0.55 to 0.69 kg / dm 3 , and even more preferably 0.60 to 0.68 kg / dm 3 , and even more preferably 0.63 to 0.67 kg / dm 3 , and most preferably 0.65 to 0.66 kg / dm 3 It has a bulk density of

[0088] In any of the above embodiments, typically the silica has a Dv(90) value of 320 μm or more. Dv(90) is the 90th percentile of particle size in a plot of the cumulative volume distribution of silica particles against increasing particle size. th Dv(90) refers to the percentile. Dv(90) is sensitive to the number of coarse particles (i.e. large size particles) present in the sample. Dv(90) can be determined by laser diffraction. Preferably, the Dv(90) value of silica is 325 μm or more, more preferably 330 μm or more, and most preferably 335 μm or more. Typically, silica has a Dv(90) that is 390 μm or less, preferably 370 μm or less, more preferably 350 μm or less, and most preferably 340 μm or less. Here, typically, silica has a Dv(90) of 320 to 290 μm, preferably 325 to 370 μm, more preferably 330 to 350 μm, and most preferably 335 to 340 μm.

[0089] Liquid Eluent Phase The process of the present invention uses an aqueous-organic eluent, i.e. a mixture of water and an organic solvent, in the chromatographic separation. Typically, the eluent is not in a supercritical state. Typically, the eluent is a liquid.

[0090] Typically the organic solvent is selected from alcohols, ethers, esters, ketones and nitriles, with alcohols, ketones and nitriles being preferred.

[0091] Alcohol solvents are well known to those skilled in the art. The alcohol is typically a short-chain alcohol. The alcohol is typically of formula ROH, where R is a linear or branched C1-C6 alkyl group. The C1-C6 alkyl group is preferably unsubstituted. Examples of alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol and t-butanol. Methanol and ethanol are preferred. Methanol is more preferred.

[0092] Ether solvents are well known to those skilled in the art. The ethers are typically short-chain ethers. The ethers are typically of the formula RO-R', where R and R' are the same or different and represent linear or branched C1-C6 alkyl groups. The C1-C6 alkyl groups are preferably unsubstituted. Preferred ethers include diethyl ether, diisopropyl ether and methyl t-butyl ether (MTBE).

[0093] Ester solvents are well known to those skilled in the art. The esters are typically short chain esters. The esters are typically of the formula R-(C=O)O-R', where R and R' are the same or different and represent linear or branched C1-C6 alkyl groups. Preferred esters include methyl acetate and ethyl acetate.

[0094] Ketone solvents are well known to those skilled in the art. Ketones are typically short-chain ketones. Ketones are typically of the formula R-(C=O)-R', where R and R' are the same or different and represent linear or branched C1-C6 alkyl groups. The C1-C6 alkyl groups are preferably unsubstituted. Preferred ketones include acetone, methyl ethyl ketone and methyl isobutyl ketone (MIBK).

[0095] Nitrile solvents are well known to those skilled in the art. The nitrile is typically a short-chain nitrile. The nitrile is typically of the formula R-CN, where R represents a linear or branched C1-C6 alkyl group. The C1-C6 alkyl group is preferably unsubstituted. A preferred nitrile includes acetonitrile.

[0096] Typically, the organic solvent is miscible with water.Preferably, the organic solvent is selected from tetrahydrofuran, isopropyl alcohol, n-propyl alcohol, methanol, ethanol, acetonitrile, 1,4-dioxane, N,N-dimethylformamide and dimethylsulfoxide.Methanol and acetonitrile are particularly preferred organic solvents.

[0097] The ratio between the organic solvent and water in the eluent is not particularly limited. However, typically, the organic solvent:water ratio is 99.9:0.1 to 75:25 parts by volume, preferably 99.5:0.5 to 80:20 parts by volume. When the organic solvent is methanol, the methanol:water ratio is typically 99.9:0.1 to 85:15 parts by volume, preferably 99.5:0.5 to 88:12 parts by volume. When the organic solvent is acetonitrile, the acetonitrile:water ratio is typically 99:1 to 75:25 parts by volume, preferably 96:4 to 80:20 parts by volume.

[0098] PUFA Products and Raw Material Mixtures As used herein, the term "PUFA product" typically refers to a product that contains one or more polyunsaturated fatty acids (PUFAs) and / or derivatives thereof that are nutritionally or medicinally important. Typically, the PUFA product is a single PUFA or derivative thereof. Alternatively, the PUFA product is a mixture of two or more PUFAs or derivatives thereof.

[0099] The term "polyunsaturated fatty acid" (PUFA) refers to a fatty acid containing two or more double bonds. Such PUFAs are well known to those skilled in the art. As used herein, a PUFA "derivative" is a PUFA in the form of a mono-, di-, or triglyceride, ester, phospholipid, amide, lactone, or salt. Mono-, di-, and triglycerides and esters are preferred. Triglycerides and esters are more preferred. Esters are even more preferred. Esters are typically alkyl esters, preferably C1-C6 alkyl esters, more preferably C1-C4 alkyl esters. Examples of esters include methyl and ethyl esters. Ethyl esters are most preferred.

[0100] Typically, the PUFA product is at least one omega-3 or omega-6 PUFA or derivative thereof, preferably at least one omega-3 PUFA or derivative thereof.

[0101] Examples of omega-3 PUFAs include eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). EPA, DPA and DHA are preferred. EPA and DHA are most preferred.

[0102] Examples of omega-6 PUFAs include eicosadienoic acid, gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA), docosadienoic acid, adrenic acid, and docosapentaenoic (omega-6) acid. ARA and GLA are preferred.

[0103] Preferably, the PUFA product is EPA, DHA, a derivative or a mixture thereof. Exemplary derivatives include EPA and DHA mono-, di- and triglycerides and EPA and DHA esters, preferably alkyl esters, such as C1-C4 alkyl esters.

[0104] More preferably, the PUFA product is EPA, DHA or a derivative thereof. Exemplary derivatives include EPA and DHA mono-, di- and triglycerides and EPA and DHA esters, preferably alkyl esters, such as C1-C4 alkyl esters.

[0105] Most preferably, the PUFA product is eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), EPA triglyceride, DHA triglyceride, EPA ethyl ester, or DHA ethyl ester.

[0106] Particularly preferably, the PUFA product is EPA, DHA, EPA ethyl ester or DHA ethyl ester.

[0107] In one embodiment, the PUFA product is EPA and / or EPA ethyl ester (EE).

[0108] In another embodiment, the PUFA product is DHA and / or DHA ethyl ester (EE).

[0109] In yet other embodiments, the PUFA product is a mixture of EPA and DHA and / or EPA EE and DHA EE.

[0110] Typically, the PUFA product is obtained with a purity of more than 80GC-area%, preferably more than 85GC-area%, more preferably more than 90GC-area%, even more preferably more than 95GC-area%, even more preferably more than 97GC-area%, even more preferably more than 98GC-area%, and most preferably more than 99GC-area%, where "GC-area%" is the percentage of the area under the gas chromatogram trace corresponding to the relevant product (herein, the PUFA product).GC-area% can typically be measured using the method for omega-3 fatty acid ethyl esters outlined in the European Pharmacopoeia 6.0, pages 2552-2554 (accessed at http: / / www.uspbpep.com / ep60 / omega-3-acid%20ethyl%20esters%2090%201250e.pdf), the entire contents of which are incorporated herein by reference.

[0111] In the most preferred embodiment, the PUFA product is EPA or an EPA derivative, such as EPA ethyl ester, and is obtained with a purity of more than 90 GC-area%, preferably more than 95 GC-area%, more preferably more than 97 GC-area%, even more preferably more than 98 GC-area%, and even more preferably more than 98.4 GC-area%. Preferably, the PUFA product is EPA or an EPA derivative, such as EPA ethyl ester, and is obtained with a purity of 98-99.5 GC-area%.

[0112] In an alternative preferred embodiment, the PUFA is DHA or a DHA derivative, such as DHA ethyl ester, and is obtained with a purity of more than 80 GC-area%, preferably more than 85 GC-area%, more preferably more than 90 GC-area%, more preferably more than 92 GC-area%, and most preferably more than 95 GC-area%. Preferably, the PUFA product is DHA or a DHA derivative, such as EPA ethyl ester, and is obtained with a purity of 97-99.5 GC-area%.

[0113] Typically, in addition to the PUFA product, a further secondary PUFA product is collected in the chromatographic separation process of the present invention. Preferably, the PUFA product is EPA or a derivative thereof, and the further secondary PUFA product is DHA or a derivative thereof.

[0114] Thus, typically, the process of the present invention is configured to collect a PUFA product that is EPA or a derivative thereof. In such an embodiment, a feed mixture containing EPA, components more polar than EPA, and components less polar than EPA is typically used.

[0115] Instead, the process of the present invention is configured to collect a PUFA product that is DHA or a derivative thereof. In such an embodiment, a feed mixture containing DHA, components more polar than DHA, and components less polar than DHA is typically used.

[0116] Instead, the process is configured to collect a PUFA product that is a concentrated mixture of EPA and DHA or their derivatives. In such an embodiment, a feed mixture is used that contains EPA, DHA, components more polar than EPA and DHA, and components less polar than EPA and DHA.

[0117] Typically, the PUFA product contains 1 GC-area% or less, preferably 0.5 GC-area% or less, more preferably 0.25 GC-area% or less, even more preferably 0.1 GC-area% or less, most preferably 0.01 GC-area% or less of C18 fatty acid impurities, C18 fatty acid mono-, di- and triglyceride impurities and C18 fatty acid alkyl ester impurities.More typically, the PUFA product contains 1 GC-area% or less, preferably 0.5 GC-area% or less, more preferably 0.25 GC-area% or less, even more preferably 0.1 GC-area% or less, most preferably 0.01 GC-area% or less of C18 fatty acid and its derivative impurities.Exemplary C18 fatty acid derivatives are as defined above for PUFA derivatives.As used herein, C18 fatty acid is a C18 aliphatic monocarboxylic acid with a linear or branched hydrocarbon chain. Exemplary C18 fatty acids include stearic acid (C18:0), oleic acid (C18:1n9), vaccenic acid (C18:1n7), linoleic acid (C18:2n6), gamma-linolenic acid / GLA (C18:3n6), alpha-linolenic acid / ALA (C18:3n3) and stearidonic acid / SDA (C18:4n3). In certain preferred embodiments, the PUFA product is substantially free of the above impurities.

[0118] Typically, the PUFA product is not a C18 PUFA, a C18 PUFA mono-, di- or triglyceride or a C18 PUFA alkyl ester. More typically, the PUFA product is not a C18 PUFA or a C18 PUFA derivative. Exemplary C18 PUFAs include linoleic acid (C18:2n6), GLA (C18:3n6) and ALA (C18:3n3).

[0119] In the process of the present invention, the feed mixture is typically (i) a natural or synthetic feedstock containing at least one PUFA product, or (ii) a partially purified feedstock containing at least one PUFA product obtained from partial purification of a natural or synthetic feedstock.Thus, in one embodiment, the feed mixture is a natural or synthetic feedstock containing at least one PUFA product.In another embodiment, the feed mixture is a partially purified feedstock containing at least one PUFA product obtained from partial purification of a natural or synthetic feedstock.

[0120] Feedstock suitable for separation by the process of the present invention are obtained from natural sources including vegetable and animal fats and oils, and synthetic sources including oils obtained from genetically modified plants, animals, and microorganisms including fungi and yeast. Examples include fish oil, algal oil, and microalgal oil, and vegetable oils such as borage oil, echium oil, and evening primrose oil. In one embodiment, the feed mixture is fish oil. In another embodiment, the feed mixture is algal oil. Algal oil and microalgal oil are particularly suitable when the desired PUFA product is EPA, ARA, and / or DHA. Genetically modified yeast are particularly suitable when the desired PUFA product is EPA. Genetically modified plants are particularly suitable when the desired PUFA product is EPA, ARA, and / or DHA.

[0121] In a particularly preferred embodiment, the feedstock is a fish oil or a fish oil derived feedstock. When a fish oil or a fish oil derived feedstock is used, it has been found advantageous that the EPA or EPA ethyl ester PUFA product can be produced by the process of the present invention with a purity of more than 90GC-area%, preferably more than 95GC-area%, more preferably more than 97GC-area%, even more preferably more than 98GC-area%, even more preferably more than 98.4GC-area%, for example 98-99.5GC-area%.

[0122] The feed mixture typically contains the PUFA product and at least one more polar component and at least one less polar component. The less polar component has a stronger adhesion to the adsorbent used in the process of the invention than the PUFA product. In operation, such less polar components typically migrate in the solid adsorbent phase than the liquid eluent phase. The more polar components have a weaker adhesion to the adsorbent used in the process of the invention than the PUFA product. In operation, such more polar components typically migrate in the liquid eluent phase than the solid adsorbent phase. In embodiments of the invention where the chromatographic separation step is performed by real or simulated moving bed chromatography, typically the more polar component will be separated in the raffinate stream and the less polar component will be separated in the extract stream.

[0123] Examples of more and less polar components include: (1) other compounds present in the natural oil (e.g., marine or vegetable oils); (2) by-products formed during storage, refining, and previous concentration steps; and (3) contaminants from solvents or reagents used during previous concentration or purification steps.

[0124] Examples of (1) include other undesirable PUFAs, saturated fatty acids, sterols such as cholesterol, vitamins, and environmental contaminants such as polychlorinated biphenyls (PCBs), polyaromatic hydrocarbon (PAH) pesticides, chlorinated pesticides, dioxins, and heavy metals. PCBs, PAHs, dioxins, and chlorinated pesticides are all highly non-polar components.

[0125] Examples of (2) include isomers and oxidation or degradation products from PUFA production, such as autoxidized polymeric products of fatty acids or their derivatives.

[0126] An example of (3) is urea, which may be added to remove saturated or monounsaturated fatty acids from the feed mixture.

[0127] Preferably, the feedstock mixture is a PUFA-containing marine oil (eg, fish oil), more preferably a marine oil (eg, fish oil) that contains EPA and / or DHA.

[0128] An example feed mixture for preparing concentrated EPA (EE) by the process of the invention contains 50-75% EPA (EE), 0-10% DHA (EE) and other components including other essential omega-3 and omega-6 fatty acids.

[0129] An example feed mixture for preparing concentrated EPA(EE) by the process of the present invention contains 55% EPA(EE), 5% DHA(EE) and other components including other essential omega-3 and omega-6 fatty acids. DHA(EE) is less polar than EPA(EE).

[0130] An example feed mixture for preparing concentrated DHA(EE) by the process of the invention contains 50-75% DHA(EE), 0-10% EPA(EE) and other components including other essential omega-3 and omega-6 fatty acids.

[0131] An example feed mixture for preparing concentrated DHA(EE) by the process of the present invention contains 75% DHA(EE), 7% EPA(EE) and other components including other essential omega-3 and omega-6 fatty acids. EPA(EE) is more polar than DHA(EE).

[0132] An example feedstock mixture for preparing a concentrated mixture of EPA(EE) and DHA(EE) by the process of the invention contains greater than 33% EPA(EE) and greater than 22% DHA(EE).

[0133] The feedstock may be subjected to chemical treatment prior to fractionation by the process of the present invention, for example it may be subjected to glyceride transesterification or glyceride hydrolysis.

[0134] The feedstock may be partially purified prior to fractionation by the process of the present invention, for example, it may be purified by crystallization, molecular or fractional distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodolactonization, supercritical fluid fractionation or chromatography, preferably fixed bed chromatography or simulated or real moving bed chromatography.

[0135] In some embodiments, the feedstock may be both chemically processed and partially refined.

[0136] In other embodiments, the feedstock may be used directly as a feed mixture in the process of the present invention without initial chemical processing steps and partial purification.

[0137] Here, in some embodiments of the present invention, the chromatographic separation process includes directly introducing the feedstock as a feed mixture into a chromatographic device. In these embodiments, the process may result in the purification of the feed mixture to directly obtain the PUFA product. Alternatively, the process may result in the purification of the feed mixture to obtain an intermediate product, which is subjected to further purification to obtain the PUFA product.

[0138] In other embodiments, the chromatographic separation process includes introducing the chemically treated feedstock as a feed mixture into a chromatographic device. In these embodiments, the process may result in the purification of the feed mixture to obtain the PUFA product directly. Alternatively, the process may result in the purification of the feed mixture to obtain an intermediate product, which is subjected to further purification to obtain the PUFA product.

[0139] In other embodiments, the chromatographic separation process includes introducing the partially purified feedstock as a feed mixture into a chromatographic device. In these embodiments, the process may result in the purification of the feed mixture to directly obtain the PUFA product. Alternatively, the process may result in the purification of the feed mixture to obtain an intermediate product, which is subjected to further purification to obtain the PUFA product.

[0140] In other embodiments, the chromatographic separation process involves introducing the partially purified, chemically treated feedstock as a feed mixture into a chromatographic device. In these embodiments, the process may result in the purification of the feed mixture to directly obtain the PUFA product. Alternatively, the process may result in the purification of the feed mixture to obtain an intermediate product, which is subjected to further purification to obtain the PUFA product.

[0141] In any of the above embodiments, the chromatographic separation process results in the preparation of intermediate product, to obtain desired PUFA product from the intermediate product, further purification step is carried out.Typically, said further purification is carried out in one or more chromatographic devices, preferably one or more fixed bed chromatographic devices or one or more real moving bed or simulated moving bed chromatographic devices.More preferably, said further purification comprises flash column chromatography in one or more fixed bed chromatographic devices.

[0142] The PUFA product of the chromatographic separation process of the present invention can be further treated physically or chemically, for example, by treatment with bleaching earth or silica to reduce oxidation by-products or by the addition of antioxidants (such as tocopherol).

[0143] In any embodiment of the present invention that includes two or more chromatographic separation steps to obtain a PUFA product, at least one of the chromatographic separation steps includes a solid sorbent as described herein. Preferably, each of the chromatographic separation steps includes a solid sorbent as described herein.

[0144] In a preferred embodiment, the chromatographic separation process comprises two chromatographic separation steps to obtain PUFA product.Preferably, in this embodiment, both the first and second chromatographic separation steps are carried out in the chromatographic apparatus described herein, i.e., the chromatographic apparatus operates at a pressure of 20 bar or less and comprises the C18 bonded silica described herein as a solid adsorbent phase.However, alternatively, only one of the first and / or second chromatographic separation steps is carried out in the chromatographic apparatus described herein, and the other separation step is carried out in a chromatographic apparatus having a different solid adsorbent phase and / or operating pressure.

[0145] Here, in this embodiment, typically, the first chromatographic separation step can be carried out in a chromatographic device as described herein, i.e. a chromatographic device operating at a pressure of 20 bar or less and comprising, as a solid adsorbent phase, C18-bonded silica as described herein, and the second chromatographic step is carried out in a chromatographic device having a different solid adsorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid adsorbent phase. Alternatively, the first chromatographic separation step is carried out in a chromatographic device as described herein, and the second chromatographic step is carried out in a chromatographic device operating at a pressure above 20 bar. Alternatively, the first chromatographic separation step is carried out in a chromatographic device as described herein, and the second chromatographic step is carried out in a chromatographic device operating at a pressure above 20 bar, and having a different solid adsorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid adsorbent phase.

[0146] Alternatively, in this embodiment, the second chromatographic separation step can be carried out in a chromatographic apparatus as described herein, i.e. a chromatographic apparatus operating at a pressure of 20 bar or less and comprising, as a solid sorbent phase, C18-bonded silica as described herein, and the first chromatographic step is carried out in a chromatographic apparatus having a different solid sorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid sorbent phase. Alternatively, the second chromatographic separation step is carried out in a chromatographic apparatus as described herein, and the first chromatographic step is carried out in a chromatographic apparatus operating at a pressure above 20 bar. Alternatively, the second chromatographic separation step is carried out in a chromatographic apparatus as described herein, and the first chromatographic step is carried out in a chromatographic apparatus operating at a pressure above 20 bar, and having a different solid sorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid sorbent phase.

[0147] Typically, in this embodiment, the first and second chromatographic separation steps are carried out in different chromatographic apparatus. Alternatively, the first and second chromatographic separation steps are carried out in the same chromatographic apparatus.

[0148] The chromatography process of the present invention is typically configured so that the yield of the PUFA product obtained from the feed mixture is greater than 80% by weight, more preferably greater than 90% by weight, even more preferably greater than 95% by weight, and most preferably greater than 98% by weight, based on the total mass of the PUFA product present in the feed mixture.

[0149] Operation of chromatographic separation processes Any known chromatographic device can be used for the chromatographic separation of the present invention. The number of chromatographic columns used in the separation is not particularly limited. The chromatographic device includes one or more chromatographic columns, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 columns. In some embodiments, the number of columns is typically one. In other embodiments, the number of columns is typically two or more, preferably four or more, more preferably six or more, even more preferably eight or more, for example, 4, 5, 6, 7, 8, 9 or 10 columns. Typically, there are 25 or less columns, preferably 20 or less, more preferably 15 or less.

[0150] The dimensions of the columns used are not particularly limited and will depend to some extent on the volume of the raw material mixture to be purified. Those skilled in the art will be able to easily determine the appropriate size column to use. The diameter of each column is typically 50 to 5000 mm, preferably 100 to 2500 mm, more preferably 250 to 1500 mm, and most preferably 250 to 1000 mm. The length of each column is typically 10 to 300 cm, preferably 10 to 200 cm, more preferably 25 to 150 cm, even more preferably 70 to 110 cm, and most preferably 80 to 100 cm.

[0151] Typically, the process of the invention is carried out at a maximum pressure of less than 20 bar. In some embodiments, the process of the invention is carried out at a "medium" pressure of 7 to 20 bar, preferably 7.5 to 15 bar, more preferably 8 to 10 bar. Alternatively, in some embodiments, the process of the invention is carried out at a "low" pressure of 7 bar or less, preferably 1 to 7 bar, more preferably 3 to 6 bar.

[0152] Typically, the process of the present invention is carried out at or above room temperature. Preferably, the process is carried out at a temperature above room temperature.

[0153] Typically, the temperature of at least one of the chromatographic columns through which the feed mixture is passed is above room temperature. More typically, the temperatures of all of the chromatographic columns used are above room temperature.

[0154] As will be understood, when at least one chromatographic column is at a temperature above room temperature, it is the inside of the column that is important for the separation process. Thus, it is typically the eluent and adsorbent inside the chromatographic column that may be at a temperature above room temperature. Of course, it is possible to achieve the required temperature inside the at least one chromatographic column by internal means (e.g., by heating the eluent and / or the feed mixture) and / or by external means (e.g., by heating the outside of the chromatographic column by any known conventional means).

[0155] Typically, the elevated temperature may be achieved by heating the eluent and / or the feed mixture, which has the effect of internally heating the column.

[0156] Here, the temperature of at least one of the chromatographic columns through which the feed mixture is passed can also be measured as the temperature of the eluent. Thus, typically, the temperature of the eluent used in the chromatographic separation is above room temperature.

[0157] Alternatively, the required temperature of at least one of the chromatography columns may be achieved by heating the column. Heating may be accomplished, for example, with an electric heating mantle, a hot water jacket or coil, or by radiant heat lamps. The interior and / or exterior of one or more of the chromatography columns may typically be heated.

[0158] At least one required temperature of the chromatography column may be achieved by heating the column, and / or the aqueous organic solvent eluent, and / or the feed mixture.

[0159] Typically, a temperature above room temperature is greater than 30° C., preferably greater than 35° C., more preferably greater than 40° C., even more preferably greater than 45° C., even more preferably greater than 50° C., even more preferably greater than 55° C., and even more preferably greater than 57° C. A temperature of 56° C. is useful in certain embodiments.

[0160] Typically, the temperature above room temperature is at most 100°C, preferably at most 95°C, more preferably at most 90°C, even more preferably at most 85°C, even more preferably at most 80°C, even more preferably at most 75°C, even more preferably at most 70°C.

[0161] Here, typical temperature ranges are 30 to 100°C, 35 to 95°C, 40 to 90°C, 45 to 85°C, 50 to 80°C, 55 to 75°C, or 57 to 70°C.

[0162] The preferred temperature range is 40 to 70°C, preferably 50 to 67°C, more preferably 56 to 65°C, and even more preferably 57 to 63°C.

[0163] In certain embodiments, a single chromatography column, preferably a single fixed chromatography column, may be used. Separations in this mode are typically performed using known fixed bed chromatography equipment. Separations in this mode may be referred to as "fixed bed" chromatography.

[0164] In other embodiments, two or more chromatographic columns are used. This may involve passing the feed mixture through two or more chromatographic columns, which may be the same or different, arranged in series or parallel. The number of columns used in this embodiment is not particularly limited, but typically does not exceed 30 columns.

[0165] One particular embodiment in which multiple chromatography columns are used is simulated or real moving bed chromatography.

[0166] Simulated and real moving bed chromatography devices are well known to those skilled in the art. Any known simulated or real moving bed chromatography device can be used for the method of the present invention as long as the device is used according to the process of the present invention. Those devices described in US Patent No. 2,985,589, US Patent No. 3,696,107, US Patent No. 3,706,812, US Patent No. 3,761,533, FR-A-2103302, FR-A-2651148, FR-A-2651149, US Patent No. 6,979,402, US Patent No. 5,069,883 and US Patent No. 4,764,276 can all be used when configured according to the process of the present invention. For example, the SMB processes disclosed in WO 2011 / 080503, WO 2013 / 005046, WO 2013 / 005047, WO 2013 / 005048, WO 2013 / 005051, WO 2013 / 005052 and / or WO 2014 / 108686 may be used.

[0167] Typically, a chromatographic separation may involve the use of a single SMB separation step using conventional equipment such as that shown in Figure 1. A separation in this manner may be referred to as a "single-pass" SMB.

[0168] In FIG. 1, countercurrent contact between the liquid and solid phases is formed in a column, which can be divided into four different zones. Zone 1 - everything located between the eluent inlet line and the extract extraction line Zone 2 - everything located between the extract line and the feedstock injection line Zone 3 - everything located between the feedstock injection line and the raffinate extraction line. Zone 4 - everything located between the raffinate extraction line and the eluent injection line.

[0169] Due to the inlet / outlet flow rate, the liquid flow rate varies with the area, so QI , Q II , Q III and Q IV are the flow rates in sections I, II, III and IV, respectively.

[0170] In one embodiment, the feedstock and eluent introduction points are advanced cyclically downstream (in the direction of the main fluid circulation), while the extraction points for the raffinate and extract are advanced simultaneously and according to the same increments (e.g., at least one column).

[0171] Now, in this embodiment, all of the inlet and outlet lines are moved simultaneously for each period ΔT and cycle time, at the end of which their initial positions are Nc * ΔT, whereby Nc is the total number of columns.

[0172] In this embodiment, the inlet / outlet positions are moved simultaneously at regular intervals. The positions of the lines are typically indicated by line (n), which indicates that at a given time a given inlet / outlet line is connected to the inlet of column n. For example, in a 12-column system, feedstock (9) means that the feedstock line is connected to the inlet of column 9, while raffinate (11) means that the raffinate line is connected to the inlet of column 11.

[0173] Using this definition, the system can be represented by El(3) / Ext(6) / Feedstock(9) / Raff(11). For this configuration, the numbers of columns in Zones 1, II, III and IV are 3 / 3 / 2 / 4, respectively. The configuration of the system can then be defined by: Number of column inlets / outlets At time 0: El(3) / Ext(6) / Raw material(9) / Raff(11) 3 / 3 / 2 / 4 After a given time, all of the inlet / outlet positions are moved simultaneously by one column and the system is represented as follows: At time ΔT: El(4) / Ext(7) / Raw Material(10) / Raff(12) 3 / 3 / 2 / 4 After a new period, all of the positions are again moved simultaneously by one column, and the system would then be represented as: 2 * At the time of ΔT: El(5) / Ext(8) / Raw Materials(11) / Raff(1)3 / 3 / 2 / 4 time 2 * At time ΔT, the position of the raffinate has been shifted from position 12 to position 1, which can also be described as position 13 modulo 12 (

[13] 12 ).

[0174] This expression can be generalized to simulate a simulated moving bed containing several columns Nc. If the composition of the simulated moving bed at a given time is El(e) / Ext(x) / Feedstock(f) / Raff(r), then simple reasoning makes it possible to know the number of columns contained in each zone. Area 1:Nb1=[xe] Nc ;Area 2:Nb2=[fx] Nc Area 3:Nb3=[rf] Nc ;Area 4:Nb4=[er] Nc

[0175] The injection and extraction points are changed by one column after a period ΔT and by Nc columns after Nc periods. The number of columns in each zone remains unchanged. Thus, the injection and extraction points are changed by one column after a period ΔT and by Nc columns after Nc periods. * return to their initial positions after ΔT.

[0176] In another embodiment, the chromatographic separation may be performed as described in U.S. Pat. No. 6,136,198 and Sa Gomes and Rodrigues, Chemical Engineering & Technology Special Issue: Preparative Chromatography and Downstream Processing, 2012, 35, 17-34, which are incorporated herein by reference in their entirety. These documents describe non-conventional methods of operating chromatographic separation processes. Typically, in this embodiment, the process may be performed using, inter alia, the so-called Varicol, Powerfeed, ISMB, Modicon, OSS or particle feed / partial waste methods. Preferably, in this embodiment, the process is performed using the Varicol method. This process is a single-pass SMB process with an asynchronous port switch, which may also be referred to as "asynchronous SMB". The Varicol process is described in more detail below.

[0177] The "Varicol" implementation differs from Single-Path SMB in the following ways: (1) The length of the area is not constant; (2) the number of columns per region is not constant over time; and (3) Inlet / outlet lines are not moved simultaneously.

[0178] In the "Varicol" embodiment, improved results can be obtained by not simultaneously moving the inlet and outlet locations of the fluids during the period and cycle time. Improvements include increased purity of the products extracted as extract and as raffinate, and reduced cost of separation.

[0179] In a "Varicol" embodiment, at least one component of the mixture containing it comprises an adsorbent and is purified in a device having a series of chromatography columns or chromatography column sections arranged in series and in a closed loop, whereby the loop comprises at least one feedstock injection point, a raffinate extraction point, an eluent injection point and an extract extraction point, the chromatography zone being determined by the injection points and the extraction points or vice versa, and at the end of a given period, all of the injection and extraction points are changed by one column or column section in a given direction defined relative to the direction of the main fluid flow circulating through the loop, whereby the process is characterized in that during said period, the changes of the different injection and extraction points of the columns or column sections are made at different times such that the length of the zone defined by said different points is variable.

[0180] The period is defined as the minimum time interval ΔT at the end of which each of the inlets and outlets is changed by one column or column section, so that the changes are not made simultaneously for all of the inlets and outlets. * At the end of ΔT, the system had returned to its initial position.

[0181] The adsorbent may in some embodiments be selected from adsorbents such as molecular sieves, e.g. zeolite sieves, or ion exchange resins used in the adsorption process, which may also be stationary phases, reversed phase adsorbents and chiral phases in silica bases.

[0182] In a "Varicol" embodiment, it is possible to effect at least one sequence of the following steps: at a time t1 during said period ΔT, in a given direction, the position of the injection or extraction point is changed for at least one section by one column or column section, so as to increase the length of said section and decrease the length of the section adjacent to said section, then at a time t2 during said period, the position of the injection or extraction point for at least one other section is changed in the same direction by one column or column section, so as to increase the length of said other section and decrease the length of the section adjacent to said other section, the operation being repeated as necessary so that after said period ΔT, the same column configuration as the initial configuration is returned with all changes in the positions of the injection and extraction points of the columns or column sections.

[0183] In one aspect of the "Varicol" embodiment, the lengths of the column sections can be continuously varied, such that an increase in one section is offset by a decrease in the next section (see, e.g., Table 2 of U.S. Pat. No. 6,136,198).

[0184] In another aspect of the "Varicol" embodiment, an increase in the length of a section may be offset by a decrease in the opposite section (see, for example, Table 3 of US Pat. No. 6,136,198).

[0185] During the period, it is possible to perform all of the changes in injection or extraction position with an approximately constant time phase shift and advantageously with a time phase shift at least equal to one quarter of the period.

[0186] In some aspects of the Varicol embodiment, it is possible to vary the location of the injection or extraction points with a non-constant time phase shift during the period.

[0187] In the "Varicol" embodiment, the flow rate of the fluids circulating in a given zone is generally kept approximately constant. It is also advantageous to perform the change in the position of the injection and extraction points in the same direction as the direction of flow in the column or column section. Furthermore, the flow rate of at least one of the fluids circulating in the injection or extraction line can be monitored by the pressure in the device. Preferably, it is the flow rate of the raffinate and / or the extract, whereby the other fluids are then under flow control.

[0188] In the "Varicol" embodiment, it is advantageously possible to use liquids as eluents, but it is also possible to operate with supercritical or subcritical fluids.

[0189] The pressure range at which the product separation is carried out in the "Varicol" embodiment can be from 0.1 to 50 MPa, preferably from 0.5 to 30 MPa. The temperature in the column is generally between 0° C. and 100° C. Typically the number of columns or column sections is less than eight. For values ​​above eight it is highly advantageous to optimize the process by studying the effect of the number and length of columns in each zone combined at the time of change during the cycle.

[0190] The device used in the "Varicol" embodiment comprises several chromatography columns or chromatography column sections containing sorbent and arranged in series and in a closed loop, whereby said loop comprises at least one pump for recirculating the fluid, several fluid injection lines in each column or column section connected to at least one injection pump and several fluid extraction lines in each column or column section connected to at least one extraction pump, at least one valve in each line, whereby said loop defines at least three chromatography zones, whereby each of them is determined by a fluid injection point and a fluid extraction point, whereby the device is characterized in that it comprises means for controlling the change in time of the length of the zone, which are connected to said valves and are suitable for intermittently changing the position of the injection and extraction points by the columns or column sections.

[0191] The valves used are advantageously all-or-none valves.

[0192] As an alternative to the process described above, the chromatographic separation may involve the use of multiple SMB separations.

[0193] In one embodiment, the chromatographic separation may be performed as described in WO 2011 / 080503 and WO 2013 / 005046, the entireties of which are incorporated herein by reference. The preferred process conditions set out in WO 2011 / 080503 and WO 2013 / 005046 are the preferred process conditions for this embodiment and may be incorporated from WO 2011 / 080503 and WO 2013 / 005046.

[0194] The process disclosed in WO2011 / 080503 and WO2013 / 005046 comprises introducing an input stream into a simulated or real moving bed chromatography apparatus having a plurality of linked chromatography columns with an aqueous organic solvent as an eluent, the apparatus having a plurality of zones including at least a first zone and a second zone, each zone having an extract stream and a raffinate stream, in which liquid may be collected from the plurality of linked chromatography columns, and (a) a raffinate stream containing the PUFA product together with more polar components is collected from the columns in the first zone and introduced into a non-adjacent column in the second zone, and / or (b) an extract stream containing the PUFA product together with less polar components is collected from the columns in the second zone and introduced into a non-adjacent column in the first zone, and the PUFA product is separated from the various components of the input stream in each zone. Separation in this manner may be referred to as a "double pass" SMB process.

[0195] In this "double pass" SMB process, the term "zone" refers to a plurality of connected chromatography columns comprising an aqueous organic solvent as eluent and having one or more injection points for an input stream, one or more injection points for water and / or an organic solvent, a raffinate removal stream from which liquid may be collected from the plurality of connected chromatography columns, and an extract removal stream from which liquid may be collected from the plurality of connected chromatography columns. Typically, each zone has only one injection point for the input stream. In one embodiment, each zone has only one injection point for the aqueous organic solvent eluent. In another embodiment, each zone has two or more injection points for water and / or an organic solvent.

[0196] The term "raffinate" is well known to those skilled in the art. In the context of real and simulated moving bed chromatography, it refers to the stream of components that move more rapidly in the liquid eluent phase compared to the solid adsorbent phase. Thus, the raffinate stream is typically enriched in more polar components and depleted in less polar components compared to the input stream.

[0197] The term "extract" is well known to those skilled in the art. In the context of real and simulated moving bed chromatography, it refers to the stream of components that migrate more rapidly in the solid adsorbent phase compared to the liquid eluent phase. Thus, the extract stream is typically enriched in less polar components and depleted in more polar components compared to the input stream.

[0198] As used herein, the term "non-adjacent" refers to columns separated by, for example, one or more columns, preferably three or more columns, more preferably five or more columns, and most preferably about five columns in the same apparatus.

[0199] The "double pass" SMB process is shown in Figure 11. An input stream F containing the PUFA product (B) and the more polar (C) and less polar (A) components is introduced into the top of column 5 in the first zone. An aqueous organic solvent desorbent is introduced into the top of column 1 in the first zone. In the first zone, the less polar component (A) is removed from the bottom of column 2 as extract stream E1. The PUFA product (B) and the more polar component (C) are removed from the bottom of column 7 as raffinate stream R1. The raffinate stream R1 is then introduced into the second zone at the top of column 12. An aqueous organic solvent desorbent is introduced into the top of column 9 in the second zone. In the second zone, the more polar component (C) is removed at the bottom of column 14 as raffinate stream R2. The PUFA product (B) is collected at the bottom of column 10 as extract stream E2.

[0200] In this "double pass" SMB process, the aqueous organic solvent is typically introduced at the top of column 1 in the first section.

[0201] In this "double pass" SMB process, the aqueous organic solvent is typically introduced at the top of column 9 in the second section.

[0202] In this "double pass" SMB process, the input stream is typically introduced at the top of column 5 in the first section.

[0203] In this "double pass" SMB process, a first raffinate stream is typically collected from the bottom of column 7 in a first zone and introduced into the top of column 12 in a second zone. The first raffinate stream may optionally be collected in a vessel prior to being introduced into column 12.

[0204] In this "double pass" SMB process, a first extract stream is typically removed from the bottom of column 2 in the first zone. The first extract stream may optionally be collected in a vessel and a portion reintroduced into the top of column 3 in the first zone. The rate at which liquid collected from the first zone via the extract stream is recycled back into the first zone is the rate at which liquid is pumped from this vessel to the top of column 3.

[0205] In this "double pass" SMB process, a second raffinate stream is typically removed from the bottom of column 14 in the second section.

[0206] In this "double pass" SMB process, a second extract stream is typically collected from the bottom of column 10 in the second zone. This second extract stream typically contains the PUFA product. The second extract stream can optionally be collected in a vessel and a portion reintroduced into the top of column 11 in the second zone. The rate at which the liquid collected from the second zone via the extract stream is recycled back into the second zone is the rate at which the liquid is pumped from this vessel to the top of column 11.

[0207] In this "double pass" SMB process, the rate at which liquid collected from the first zone via the extract stream is recycled back into the first zone is typically faster than the rate at which liquid collected from the second zone via the extract stream is recycled back into the second zone. In this "double pass" SMB process, the eluent is typically substantially the same in each zone.

[0208] In this "double pass" SMB process, the solvent may be recovered by evaporation, membrane or any other method for those skilled in the art of solvent recycle. The solvent may be partially or fully recycled to the SMB as a desorbent after being fully or partially depleted of products or waste products. Thus, in some embodiments, concentration of the product-rich extract / raffinate stream (if applicable) and / or the product-depleted extract / raffinate stream (if applicable) may be performed by evaporation, drying or distillation. Alternatively, concentration of the product-rich extract / raffinate stream (if applicable) and / or the product-depleted extract / raffinate stream (if applicable) may be performed by liquid extraction, membrane, crystallization, adsorption or other solvent recovery techniques.

[0209] Typically, at least one of the first and second chromatographic separation steps comprises at least one, for example one, "double pass" SMB process as defined above.

[0210] In alternative embodiments, the chromatographic separation may be carried out as described in WO 2013 / 005051 and / or WO 2013 / 005052, the entireties of which are incorporated herein by reference. Such embodiments include: (i) in a first SMB step, purifying an input stream in a simulated or real moving bed chromatography apparatus having multiple connected chromatography columns containing an aqueous organic solvent as an eluent to obtain a first product; and (ii) in a second SMB step, purifying the first product obtained in (i) using a simulated or real moving bed chromatography apparatus having multiple connected chromatographic columns containing an aqueous organic solvent as an eluent to obtain a second product. Includes; (a) the first and second SMB steps are performed sequentially in the same chromatography apparatus, the first product being recovered between the first and second SMB steps, and process conditions in the chromatography apparatus being adjusted between the first and second SMB steps such that the PUFA product is separated from different components of the feed mixture at each SMB step; or (b) The first and second SMB steps are carried out in separate first and second chromatographic devices, respectively, and the first product from the first SMB step is introduced into the second chromatographic device, and the PUFA product is separated from different components of the feed mixture in each SMB step. Separation in this manner may be referred to as "back-to-back" SMB.

[0211] In this "back-to-back" SMB process, the term "simulated or real moving bed chromatography apparatus" typically refers to multiple connected chromatography columns that contain aqueous organic solvent as eluent and have one or more injection points for an input stream, one or more injection points for water and / or organic solvent, a raffinate removal stream where liquid may be collected from the multiple connected chromatography columns, and an extract removal stream where liquid may be collected from the multiple connected chromatography columns.

[0212] The chromatography apparatus used in this "back-to-back" SMB process has a single array of serially connected chromatography columns with aqueous organic solvent as the eluent. Typically, each of the chromatography columns is connected to two columns in the apparatus adjacent to the corresponding column. Thus, the output from a given column in the array is connected to the input of an adjacent column in the array, which is downstream with respect to the flow of eluent in the array. Thus, the eluent can flow around the array of connected chromatography columns. Typically, none of the chromatography columns is connected to a non-adjacent column in the apparatus.

[0213] Typically, in this "back-to-back" SMB process, each unit has only one injection point for the input stream. In one embodiment, each unit has only one injection point for the aqueous organic solvent eluent. In another embodiment, each unit has two or more injection points for water and / or organic solvent.

[0214] The number of columns used in each device in this "back-to-back" SMB process is not particularly limited. A person skilled in the art would be able to easily determine the appropriate number of columns to be used. The number of columns is typically 4 or more, preferably 6 or more, more preferably 8 or more, for example 4, 5, 6, 7, 8, 9 or 10 columns. In a preferred embodiment, 5 or 6 columns, more preferably 6 columns are used. In another preferred embodiment, 7 or 8 columns, more preferably 8 columns are used. Typically, there are 25 or less columns, preferably 20 or less, more preferably 15 or less.

[0215] In this "back-to-back" SMB process, the chromatographic devices used in the first and second separation steps typically contain the same number of columns. In certain applications, they may have different numbers of columns.

[0216] In this "back-to-back" SMB process, the columns in the chromatographic apparatus used in the first and second SMB separation steps typically have identical dimensions, but may have different dimensions in certain applications.

[0217] The flow rate into the columns will be limited by the maximum pressure across the column series and will depend on the column dimensions and particle size of the solid phase. One skilled in the art will be able to readily establish the flow rate required for each column size to ensure efficient desorption. Larger diameter columns will generally require higher flow rates to maintain linear flow through the column.

[0218] In this "back-to-back" SMB process, for the typical column sizes outlined above, typically the flow rate of eluent into the chromatographic apparatus used in the first SMB separation step is 50-300 L / min, preferably 100-150 L / min. Typically the flow rate of extract from the chromatographic apparatus used in the first SMB separation step is 5-150 L / min, preferably 25-130 L / min. In embodiments where a portion of the extract from the first SMB separation step is recycled back into the apparatus used in the first SMB separation step, the recycle flow rate is typically 50-100 L / min, preferably about 75 L / min. Typically the flow rate of raffinate from the chromatographic apparatus used in the first SMB separation step is 15-150 L / min, preferably 20-125 L / min. In embodiments in which a portion of the raffinate from the first SMB separation step is recycled back into the apparatus used in the first SMB separation step, the recycle flow rate is typically 20-75 L / min, preferably about 35 L / min.Typically, the flow rate of introduction of the input stream into the chromatographic apparatus used in the first SMB separation step is 0.3-10 L / min, preferably 0.5-7.5 L / min, more preferably 1-4 L / min.

[0219] In this "back-to-back" SMB process, for the typical column sizes outlined above, typically the flow rate of eluent into the chromatographic apparatus used in the second SMB separation step is 50-250 L / min, preferably 100-225 L / min. Typically the flow rate of extract from the chromatographic apparatus used in the second SMB separation step is 25-125 L / min, preferably 50-120 L / min. In embodiments where a portion of the extract from the second SMB separation step is recycled back into the apparatus used in the second SMB separation step, the recycle flow rate is typically 40-90 L / min, preferably 50-75 L / min, more preferably about 60 L / min. Typically the flow rate of raffinate from the chromatographic apparatus used in the second SMB separation step is 25-150 L / min, preferably 50-100 L / min, more preferably about 90 L / min. In embodiments in which a portion of the raffinate from the second SMB separation step is recycled back into the equipment used in the second SMB separation step, the recycle flow rate is typically between 20 and 60 L / min, preferably about 30 L / min.

[0220] As one of skill in the art will appreciate, references to the rate at which liquid is collected or removed via the various extract and raffinate streams refer to the volume of liquid removed in units of time, typically L / min. Similarly, references to the rate at which liquid is recirculated through the apparatus, typically back to an adjacent column in the apparatus, refer to the volume of liquid recirculated in units of time, typically L / min.

[0221] In this "back-to-back" SMB process, actual moving bed chromatography is preferred.

[0222] The step times, i.e., the time between varying the time of injection of the input stream and eluent and the various removal points of the collected fractions, are not particularly limited and will depend on the number and dimensions of the columns used and the flow rates through the apparatus. Those skilled in the art will be able to readily determine appropriate step times for use in the process of the present invention. Step times are typically between 100 and 1000 seconds, preferably between 200 and 800 seconds, more preferably between about 250 and about 750 seconds. In some embodiments, step times of 100 to 400 seconds, preferably between 200 and 300 seconds, more preferably about 250 seconds, are appropriate. In other embodiments, step times of 600 to 900 seconds, preferably between 700 and 800 seconds, more preferably about 750 seconds, are appropriate.

[0223] A "back-to-back" SMB process includes a first and a second SMB separation step.

[0224] These two steps can be easily carried out in a single chromatography device. Thus, in one embodiment, (a) the first and second SMB separation steps are carried out consecutively in the same chromatography device, the first product is collected between the first SMB separation step and the second SMB separation step, and the process conditions in the chromatography device are adjusted between the first SMB separation step and the second SMB separation step so that the PUFA product is separated from the different components of the input stream at each separation step. A preferred embodiment of this "back-to-back" SMB process is shown as Figure 10A. Here, the first SMB separation step (left side) is carried out in an SMB device with eight columns. Between the first SMB separation step and the second SMB separation step, the first product is collected, for example, in a container, and the process conditions in the chromatography device are adjusted so that the PUFA product is separated from the different components of the input stream at each SMB separation step. Then, the second SMB separation step (right side) is carried out in the same SMB device with eight columns.

[0225] In embodiment (a), adjusting the process conditions typically refers to adjusting the process conditions in the equipment as a whole, i.e., physically adjusting the equipment so that the conditions are different. It does not refer to simply reintroducing the first product into a different part of the same equipment where the process conditions may by chance be different.

[0226] Alternatively, first and second separate chromatographic devices can be used for the first and second SMB separation steps. Thus, in another embodiment, (b) the first and second SMB separation steps are carried out in separate first and second chromatographic devices, respectively, and the first product obtained from the first SMB separation step is introduced into the second chromatographic device, and the PUFA product is separated from a different component of the input stream in each SMB separation step.

[0227] In embodiment (b), the two SMB separation steps may be carried out sequentially or simultaneously.

[0228] Thus, in embodiment (b) where two SMB separation steps are performed consecutively, the first and second SMB separation steps are performed consecutively in separate first and second chromatographic devices, respectively, and the first product is recovered between the first and second SMB separation steps, and the process conditions in the first and second SMB chromatographic devices are adjusted so that the PUFA product is separated from the different components of the input stream at each separation step. A preferred embodiment of this "back-to-back" SMB separation process is shown as FIG. 10B. Here, the first SMB separation step (left side) is performed in an SMB device having eight columns, 1 to 8. Between the first SMB separation step and the second SMB separation step, the first product is recovered, for example, in a vessel and then introduced into a second separate SMB device. The second SMB separation step (right side) is performed in a second separate SMB device having eight columns, 9 to 16. The process conditions in the two chromatographic devices are adjusted so that the PUFA product is separated from the different components of the input stream in each SMB separation step.

[0229] In embodiment (b), where two SMB separation steps are carried out simultaneously, the first and second SMB separation steps are carried out in separate first and second chromatographic devices, respectively, and the first product is introduced into the chromatographic device used in the second SMB separation step, and the process conditions in the first and second chromatographic devices are adjusted so that the PUFA product is separated from the different components of the input stream in each SMB separation step. A preferred embodiment of this "back-to-back" SMB separation process is shown as FIG. 10c. Here, the first SMB separation step (left side) is carried out in an SMB device having eight columns, 1 to 8. The first product obtained in the first SMB separation step is then introduced into a second separate chromatographic device used in the second SMB separation step. The first product can be passed from the first SMB separation step to the second SMB separation step directly or indirectly, for example via a vessel. The second SMB separation step (right side) is carried out in a second separate SMB device having eight columns, 9 to 16. The process conditions in the two chromatographic devices are adjusted so that the PUFA product is separated from the different components of the input stream at each separation step.

[0230] In embodiment (b) where two SMB separation steps are carried out simultaneously, the eluent is circulated separately in two separate chromatographic devices. Thus, no eluent is shared between the two separate chromatographic devices except that the eluent may be present as a solvent in the first product purified in the second SMB separation step and introduced into the chromatographic device used in the second SMB separation step. No chromatographic column is shared between the two separate chromatographic devices used in the first and second SMB separation steps.

[0231] In this "back-to-back" SMB process, the aqueous organic solvent eluent may be partially or totally removed after the first product is obtained in the first SMB separation step and before the first product is purified in the second SMB separation step. Alternatively, the first product may be purified in the second SMB separation step without removal of any solvent present.

[0232] As described above, in this "back-to-back" SMB process, the PUFA product is separated from different components of the input stream in each SMB separation step. In embodiment (a), the process conditions of the single SMB device used in both SMB separation steps are adjusted between the first SMB separation step and the second SMB separation step, so that the PUFA product is separated from different components of the input stream in each separation step. In embodiment (b), the process conditions in the two separate chromatographic devices used in the first and second SMB separation steps are set so that the PUFA product is separated from different components of the input stream in each separation step.

[0233] Here, in this "back-to-back" SMB process, the process conditions in the first and second SMB separation steps are varied, including, for example, the size of the columns used, the number of columns used, the packing materials used in the columns, the step times of the SMB apparatus, the temperature of the apparatus, the water:organic solvent ratio of the eluents used in the separation steps, or the flow rates used in the apparatus, particularly the recirculation rates of the liquids collected via the extract or raffinate streams.

[0234] Preferably, in this "back-to-back" SMB process, the process conditions that can be varied are the water:organic solvent ratio of the eluent used in the SMB separation step and / or the recycle rate of liquid collected via the extract or raffinate stream in the SMB separation step. Both of these options are described in more detail below.

[0235] In this "back-to-back" SMB process, the first product obtained from the first SMB separation step is typically enriched in the PUFA product as compared to the input stream.

[0236] In this "back-to-back" SMB process, the first product from the first SMB separation step is then introduced into a chromatographic device used in a second SMB separation step.

[0237] In this "back-to-back" SMB process, the first product is typically collected as a raffinate or extract stream from the chromatographic equipment used in the first SMB separation process.

[0238] Typically in this "back-to-back" SMB process, a first product is collected as a raffinate stream in a first SMB separation step and a second product is collected as an extract stream in a second SMB separation step, where the raffinate stream collected in the first SMB separation step is used as an input stream in the second SMB separation step. The raffinate stream collected in the first SMB separation step typically contains the second product along with more polar components.

[0239] Instead, in this "back-to-back" SMB process, a first product is collected as an extract stream in a first SMB separation step and a second product is collected as a raffinate stream in a second SMB separation step, where the extract stream collected in the first SMB separation step is used as an input stream in the second SMB separation step. The extract stream collected in the first SMB separation step typically contains the second product along with less polar components.

[0240] In this "back-to-back" SMB process, the PUFA product is separated from different components of the input stream at each SMB separation step. Typically, the components separated at each SMB separation step of the process of the present invention have different polarities.

[0241] Preferably, in this "back-to-back" SMB process, the PUFA product is separated from the less polar components of the input stream in a first SMB separation step, and the PUFA product is separated from the more polar components of the input stream in a second SMB separation step.

[0242] Typically, this "back-to-back" SMB process involves: (a) a portion of the extract stream from the apparatus used in the first SMB separation step is recycled back into the apparatus used in the first SMB separation step; and / or (b) a portion of the raffinate stream from the apparatus used in the first SMB separation step is recycled back into the apparatus used in the first SMB separation step; and / or (c) a portion of the extract stream from the apparatus used in the second SMB separation step is recycled back into the apparatus used in the second SMB separation step; and / or (d) A portion of the raffinate stream from the apparatus used in the second SMB separation step is recycled back into the apparatus used in the second SMB separation step.

[0243] Preferably, in this "back-to-back" SMB process: (a) recycling a portion of the extract stream from the apparatus used in the first SMB separation step back into the apparatus used in the first SMB separation step; (b) recycling a portion of the raffinate stream from the apparatus used in the first SMB separation step back into the apparatus used in the first SMB separation step; (c) recycling a portion of the extract stream from the apparatus used in the second SMB separation step back into the apparatus used in the second SMB separation step; (d) A portion of the raffinate stream from the apparatus used in the second SMB separation step is recycled back into the apparatus used in the second SMB separation step.

[0244] Recycle in this "back-to-back" SMB process involves feeding a portion of the extract or raffinate stream from the chromatographic equipment used in the first or second SMB separation step back into the equipment used in that SMB step, typically into an adjacent column that is downstream with respect to the eluent flow in the system.

[0245] In this "back-to-back" SMB process, the rate at which liquid collected via the extract or raffinate stream in the first or second SMB separation step is recycled back into the chromatographic apparatus used in that SMB step is the rate at which liquid collected via that stream is fed back into the apparatus used in that SMB step, typically an adjacent column, i.e., a column downstream relative to the eluent stream in the system.

[0246] This can be seen with reference to Figure 9. The rate of recirculation of the extract in the first SMB separation step is the rate at which the extract collected from the bottom of column 2 of the chromatographic apparatus used in the first SMB separation step is fed to the top of column 3 of the chromatographic apparatus used in the first SMB separation step, i.e. the flow rate of liquid to the top of column 3 of the chromatographic apparatus used in the first SMB separation step.

[0247] In this "back-to-back" SMB process, the rate of recirculation of the extract in the second SMB separation step is the rate at which the extract collected at the bottom of column 2 of the chromatographic apparatus used in the second SMB separation step is fed to the top of column 3 of the chromatographic apparatus used in the second SMB separation step, i.e., the flow rate of liquid to the top of column 3 of the chromatographic apparatus used in the second SMB separation step.

[0248] In this "back-to-back" SMB process, recycle of the extract and / or raffinate stream in the first and / or second SMB separation steps is typically performed by feeding the liquid collected via that stream in that SMB separation step into a vessel and then pumping a quantity of that liquid from the vessel back into the apparatus used in that SMB separation step, typically into an adjacent column. In this case, the rate of recycle of the liquid collected via a particular extract or raffinate stream in the first and / or second SMB separation step back into an adjacent column is the rate at which the liquid is pumped from the vessel back into the chromatography apparatus, typically into an adjacent column.

[0249] As one of ordinary skill in the art will appreciate, in this "back-to-back" SMB process, a quantity of liquid is introduced into the chromatography device via the eluent, and the input stream is balanced with the amount of liquid removed from the device and recirculated back into the device.

[0250] Referring now to FIG. 9, in this “back-to-back” SMB process, for the extract stream, the flow rate (D) of eluent (desorbent) into the chromatographic apparatus used in the first and second SMB separation steps is equal to the rate at which liquid collected via the extract stream in that SMB separation step accumulates in the vessel (E1 and E2) plus the rate at which the extract is recycled back into the chromatographic apparatus used in that particular SMB separation step (D-E1 and D-E2).

[0251] In this "back-to-back" SMB process, for the raffinate stream from an SMB separation step, the rate at which the extract is recycled back into the chromatographic equipment used in that particular SMB separation step (D-E1 and D-E2), plus the rate at which the feedstock is introduced into the chromatographic equipment used in that particular SMB separation step (F and R1), is equal to the rate at which liquid collected via the raffinate stream in that particular SMB separation step accumulates in the vessel (R1 and R2) plus the rate at which the raffinate is recycled back into the chromatographic equipment used in that particular SMB separation step (D+F-E1-R1 and D+R1-E2-R2).

[0252] In this "back-to-back" SMB process, the rate at which collected liquid from a particular extract or raffinate stream from a chromatography device accumulates in a vessel may also be considered to be the net rate of removal of that extract or raffinate stream from that chromatography device.

[0253] Typically, in this "back-to-back" SMB process, the rate at which the liquids collected in the first SMB separation step via the extract and raffinate streams are recirculated back into the equipment used in that separation step is adjusted so that the PUFA products can be separated from different components of the input stream in each SMB separation step.

[0254] Typically, in this "back-to-back" SMB process, the rate at which the liquids collected in the second SMB separation step via the extract and raffinate streams are recirculated back into the equipment used in that SMB separation step is adjusted so that the PUFA products can be separated from the different components of the input stream in each SMB separation step.

[0255] Preferably, in this "back-to-back" SMB process, the rate at which the liquids collected in each SMB separation step via the extract and raffinate streams are recirculated back into the equipment used in that SMB separation step is adjusted so that the PUFA products can be separated from the different components of the input streams in each SMB separation step.

[0256] Typically, in this "back-to-back" SMB process, the rate at which liquid collected via the extract stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is different from the rate at which liquid collected via the extract stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step and / or the rate at which liquid collected via the raffinate stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is different from the rate at which liquid collected via the raffinate stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step.

[0257] Varying the rate at which liquid collected via the extract and / or raffinate streams in the first or second SMB separation steps is recirculated back into the equipment used in that particular SMB separation step has the effect of varying the amount of more and less polar components present in the extract and raffinate streams. Thus, for example, a lower extract recirculation rate will result in fewer less polar components being carried in the raffinate stream in that SMB separation step. A higher extract recirculation rate will result in more less polar components being carried in the raffinate stream in that SMB separation step.

[0258] This can be seen, for example, in Figure 6. The rate at which the liquid collected via the extract stream in the first SMB separation step is recycled back into the chromatographic apparatus used in that SMB separation step (D-E1) will affect the extent to which any of component A is carried into the raffinate stream in the first SMB separation step (R1).

[0259] Typically, in this "back-to-back" SMB process, the rate at which the liquid collected in the first SMB separation step via the extract stream is recycled back into the chromatographic apparatus used in the first SMB separation step is faster than the rate at which the liquid collected in the second SMB separation step via the extract stream is recycled back into the chromatographic apparatus used in the second SMB separation step. Preferably, a raffinate stream containing the second product together with more polar components is collected from the first SMB separation step and purified in the second SMB separation step, and the rate at which the liquid collected in the first SMB separation step via the extract stream is recycled back into the chromatographic apparatus used in the first SMB separation step is faster than the rate at which the liquid collected in the second SMB separation step via the extract stream is recycled back into the chromatographic apparatus used in the second SMB separation step.

[0260] Instead, in this "back-to-back" SMB process, the rate at which liquid collected via the extract stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is slower than the rate at which liquid collected via the extract stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step.

[0261] Typically, in this "back-to-back" SMB process, the rate at which liquid collected via the raffinate stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first separation step is faster than the rate at which liquid collected via the raffinate stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step. Preferably, an extract stream containing the second product together with less polar components is collected from the first SMB separation step and purified in the second SMB separation step, and the rate at which liquid collected via the raffinate stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is faster than the rate at which liquid collected via the raffinate stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step.

[0262] Instead, in this "back-to-back" SMB process, the rate at which liquid collected via the raffinate stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is slower than the rate at which liquid collected via the raffinate stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step.

[0263] In this "back-to-back" SMB process, in which the recycle rates are adjusted so that the PUFA product can be separated from the different components of the input stream at each SMB separation step, the water:organic solvent ratio of the eluent used in each SMB separation step can be the same or different. The typical water:organic solvent ratio of the eluent in each SMB separation step is as defined above.

[0264] Typically, in this "back-to-back" SMB process, the aqueous organic solvent eluent used in each SMB separation step has a different water:organic solvent ratio. The organic solvent used in each SMB separation step is the same. The water:organic solvent ratio used in each SMB separation step is preferably adjusted so that the PUFA product can be separated from the different components of the input stream in each SMB separation step.

[0265] In this "back-to-back" SMB process, the elution strength of the eluent used in each of the SMB separation steps is typically different. Preferably, the elution strength of the eluent used in the first SMB separation step is greater than the elution strength of the eluent used in the second SMB separation step. In practice, this is achieved by varying the relative amounts of water and organic solvent used in each SMB separation step.

[0266] Depending on the choice of organic solvent, they may be stronger desorbents than water. Alternatively, they may be weaker desorbents than water. For example, acetonitrile and alcohol are stronger desorbents than water. Thus, when the aqueous organic solvent is aqueous alcohol or acetonitrile, the amount of alcohol or acetonitrile in the eluent used in the first SMB separation step is typically greater than the amount of alcohol or acetonitrile in the eluent used in the second SMB separation step.

[0267] Typically, in this "back-to-back" SMB process, the water:organic solvent ratio of the eluant in the first SMB separation step is lower than the water:organic solvent ratio of the eluant in the second SMB separation step. Thus, the eluant in the first SMB separation step typically contains more organic solvent than the eluant in the second SMB separation step.

[0268] It will be understood that the ratios of water and organic solvent in each SMB separation step referred to above are average ratios throughout the chromatographic apparatus.

[0269] Typically, in this "back-to-back" SMB process, the water:organic solvent ratio of the eluent in each SMB separation step is controlled by introducing water and / or organic solvent into one or more columns in the chromatographic apparatus used in the SMB separation step. Thus, for example, to achieve a lower water:organic solvent ratio in the first SMB separation step than in the second SMB separation step, water is typically introduced more slowly into the chromatographic apparatus used in the first SMB separation step than in the second SMB separation step.

[0270] Typically, in this "back-to-back" SMB process, an essentially pure organic solvent and essentially pure water may be introduced at different points into the chromatographic apparatus used in each SMB separation step. The relative flow rates of these two streams will determine the overall solvent profile in the chromatographic apparatus. Alternatively, in this "back-to-back" SMB process, various mixtures of organic solvent and water may be introduced at different points into each chromatographic apparatus used in each SMB separation step. It involves introducing two or more different mixtures of organic solvent and water into the chromatographic apparatus used in a particular SMB separation step, each organic solvent / water mixture having a different organic solvent:water ratio. The relative flow rates and relative concentrations of the organic solvent / water mixtures in this "back-to-back" SMB process will determine the overall solvent profile in the chromatographic apparatus used in that SMB separation step.

[0271] Preferably, in this "back-to-back" SMB process: (1) a first product containing the second product along with more polar components is collected as a raffinate stream in a first SMB separation step, and the second product is collected as an extract stream in a second SMB separation step; or (2) The first product, containing the second product along with less polar components, is either collected as an extract stream in a first SMB separation step, and the second product is collected as a raffinate stream in a second SMB separation step.

[0272] Option (1) is preferred for purifying EPA from an input stream.

[0273] Option (1) is shown in Figure 2. An input stream F containing the second product (B) and the more polar (C) and less polar (A) components is purified in a first SMB separation step. In the first SMB separation step, the less polar component (A) is removed as extract stream E1. The second product (B) and the more polar component (C) are collected as raffinate stream R1. Raffinate stream R1 is the first product, which is then purified in a second SMB separation step. In the second SMB separation step, the more polar component (C) is removed as raffinate stream R2. The second product (B) is collected as extract stream E2.

[0274] Option (1) is shown in more detail in Figure 4, which is identical to Figure 2, except that the points of introduction of the organic solvent desorbent (D) and water (W) into each chromatographic device are shown. The organic solvent desorbent (D) and water (W) together constitute the eluent. The (D) phase is typically essentially pure organic solvent, but in certain embodiments may be an organic solvent / water mixture comprising primarily organic solvent. The (W) phase is typically essentially pure water, but in certain embodiments may be an organic solvent / water mixture comprising primarily water, for example a 98% water / 2% methanol mixture.

[0275] A further illustration of option (1) is shown in Figure 6, where there is no separate water injection point, and instead an aqueous organic solvent desorbent is injected at (D).

[0276] In option (1), separation into raffinate and extract streams can be aided by varying the desorption strength of the eluent within each chromatographic device. This can be achieved by introducing the organic solvent (or organic solvent-rich) and aqueous (or aqueous water-rich) components of the eluent at different points in each chromatographic device, where typically the organic solvent is introduced upstream of the extract removal point and the water is introduced to the eluent flow in the system between the extract removal point and the introduction of the feed into the chromatographic device. This is shown in FIG. 4.

[0277] Typically, in option (1), the aqueous organic solvent eluent used in the first SMB separation step contains more organic solvent than the eluent used in the second SMB separation step, i.e. the water:organic solvent ratio in the first SMB separation step is lower than the water:organic solvent ratio in the second SMB separation step.

[0278] In option (1), the SMB separation may be assisted by varying the rate at which liquids collected in the first and second SMB separation steps via the extract and raffinate streams are recycled back into the chromatographic apparatus used in the SMB separation steps.

[0279] Typically, in option (1), the rate at which liquid collected via the extract stream in the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is faster than the rate at which liquid collected via the extract stream in the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step.

[0280] In option (1), the first raffinate stream in the first SMB separation step is typically removed downstream, relative to the eluent stream, of the point of introduction of the input stream into the chromatographic apparatus used in the first SMB separation step.

[0281] In option (1), the first extract stream in the first SMB separation step is typically removed, relative to the eluent stream, upstream of the point of introduction of the input stream into the chromatographic apparatus used in the first SMB separation step.

[0282] In option (1), the second raffinate stream in the second SMB separation step is typically removed relative to the eluent stream downstream of the point of introduction of the first product into the chromatographic apparatus used in the second SMB separation step.

[0283] In option (1), the second extract stream in the second SMB separation step is typically collected, relative to the eluent stream, upstream of the point of introduction of the first product into the chromatographic apparatus used in the second SMB separation step.

[0284] Typically, in option (1), the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the first SMB separation step, relative to the eluent stream, upstream of the removal point of the first extract stream.

[0285] Typically, in option (1), when water is introduced into the chromatographic apparatus used in the first SMB separation step, it is introduced into the chromatographic apparatus used in the first SMB separation step upstream of the point of introduction of the input stream, relative to the eluent stream, but downstream of the point of removal of the first extract stream.

[0286] Typically, in option (1), the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the second SMB separation step, relative to the eluent stream, upstream of the removal point of the second extract stream.

[0287] Typically, in option (1), when water is introduced into the chromatographic apparatus used in the second SMB separation step, it is introduced into the chromatographic apparatus used in the second SMB separation step, relative to the eluent stream, upstream of the point of introduction of the first product, but downstream of the point of removal of the second extract stream.

[0288] Option (2) is preferred for purifying DHA from an input stream.

[0289] Option (2) is shown in Figure 3. The second product (B) and an input stream F containing the more polar (C) and less polar (A) components are purified in a first SMB separation step. In the first SMB separation step, the more polar component (C) is removed as raffinate stream R1. The second product (B) and the less polar component (A) are collected as extract stream E1. Extract stream E1 is the first product, which is then purified in a second SMB separation step. In the second SMB separation step, the less polar component (A) is removed as extract stream E2. The second product (B) is collected as raffinate stream R2.

[0290] Option (2) is shown in more detail in Figure 5, which is identical to Figure 3, except that the points of introduction of the organic solvent desorbent (D) and water (W) into each chromatographic device are shown. As noted above, the (D) phase is typically essentially pure organic solvent, but in certain embodiments may be an organic solvent / water mixture comprising primarily organic solvent. The (W) phase is typically essentially pure water, but in certain embodiments may be an organic solvent / water mixture comprising primarily water, for example a 98% water / 2% methanol mixture.

[0291] A further illustration of option (2) is shown in Figure 7, where there is no separate water injection point; instead, an aqueous organic solvent desorbent is injected at (D).

[0292] Typically, in option (2), the rate at which liquid collected via the raffinate stream in the first SMB separation step is reintroduced into the chromatographic apparatus used in the first SMB separation step is faster than the rate at which liquid collected via the raffinate stream in the second SMB separation step is reintroduced into the chromatographic apparatus used in the second SMB separation step.

[0293] Typically, in option (2), the aqueous organic solvent eluent used in the first SMB separation step contains less organic solvent than the eluent used in the second SMB separation step, i.e. the water:organic solvent ratio in the first SMB separation step is higher than in the second SMB separation step.

[0294] In option (2), the first raffinate stream in the first separation step is typically removed downstream, relative to the eluent stream, of the point of introduction of the input stream into the chromatographic apparatus used in the first SMB separation step.

[0295] In option (2), the first extract stream in the first SMB separation step is typically removed, relative to the eluent stream, upstream of the point of introduction of the input stream into the chromatographic apparatus used in the first SMB separation step.

[0296] In option (2), the second raffinate stream in the second SMB separation step is typically removed relative to the eluent stream downstream of the point of introduction of the first product into the chromatographic apparatus used in the second SMB separation step.

[0297] In option (2), the second extract stream in the second SMB separation step is typically collected, relative to the eluent stream, upstream of the point of introduction of the first product into the chromatographic apparatus used in the second SMB separation step.

[0298] Typically, in option (2), the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the first SMB separation step, relative to the eluent stream, upstream of the removal point of the first extract stream.

[0299] Typically, in option (2), when water is introduced into the chromatographic apparatus used in the first SMB separation step, it is introduced into the chromatographic apparatus used in the first SMB separation step upstream of the point of introduction of the input stream, relative to the eluent stream, but downstream of the point of removal of the first extract stream.

[0300] Typically, in option (2), the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the second SMB separation step, relative to the eluent stream, upstream of the removal point of the second extract stream.

[0301] Typically, in option (2), when water is introduced into the chromatographic apparatus used in the second SMB separation step, it is introduced into the chromatographic apparatus used in the second SMB separation step, relative to the eluent stream, upstream of the point of introduction of the first product, but downstream of the point of removal of the second extract stream.

[0302] In this "back-to-back" SMB process, each of the simulated or real moving bed chromatography apparatuses used in the first and second SMB separation steps preferably consists of eight chromatography columns. These are referred to as columns 1 through 8. In each apparatus, the eight columns are arranged in series such that the bottom of column 1 is connected to the top of column 2, the bottom of column 2 is connected to the top of column 3, etc., the bottom of column 8 is connected to the top of column 1. These connections may be via a holding vessel, optionally with a recycle flow into the next column. The flow of eluent through the system is column 1 through column 2 through column 3, etc. The effective flow of adsorbent through the system is column 8 through column 7 through column 6, etc.

[0303] This is shown in FIG. 8. The input stream F, which contains the second product (B) and the more polar (C) and less polar (A) components, is introduced into the top of column 5 in the chromatographic apparatus used in the first SMB separation step. The organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the first SMB separation step. Water is introduced into the top of column 4 in the chromatographic apparatus used in the first SMB separation step. In the first SMB separation step, the less polar component (A) is removed from the bottom of column 2 as extract stream E1. The second product (B) and the more polar component (C) are removed from the bottom of column 7 as raffinate stream R1. The raffinate stream R1 is the first product, which is then purified in the second SMB separation step by being introduced into the chromatographic apparatus used in the second SMB separation step at the top of column 5. The organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the second SMB separation step. Water is introduced at the top of column 4 in the chromatographic apparatus used in the second SMB separation step, in which the more polar component (C) is removed as raffinate stream R2 at the bottom of column 7. The second product (B) is collected at the bottom of column 2 as extract stream E2.

[0304] In the "back-to-back" SMB process shown in FIG. 8, the organic solvent is typically introduced into the top of column 1 of the chromatographic apparatus used in the first SMB separation step.

[0305] In the "back-to-back" SMB process shown in FIG. 8, water is typically introduced into the top of column 4 of the chromatographic apparatus used in the first SMB separation step.

[0306] In the "back-to-back" SMB process shown in FIG. 8, the organic solvent is typically introduced into the top of column 1 of the chromatographic apparatus used in the second SMB separation step.

[0307] In the "back-to-back" SMB process shown in FIG. 8, the organic solvent is typically introduced at the top of column 4 of the chromatographic apparatus used in the second SMB separation step.

[0308] In a "back-to-back" SMB process, shown in FIG. 8, the input stream is typically introduced into the top of column 5 of the chromatographic apparatus used in the first SMB separation step.

[0309] In the "back-to-back" SMB process shown in Figure 8, a first raffinate stream is typically collected as a first product from the bottom of column 7 of the chromatographic apparatus used in the first SMB separation step. This first product is then purified in a second SMB separation step and typically introduced to the top of column 5 of the chromatographic apparatus used in the second SMB separation step. The first raffinate stream may optionally be collected in a vessel before being purified in the second SMB separation step.

[0310] In the "back-to-back" SMB process shown in Figure 8, a first extract stream is typically removed from the bottom of column 2 of the chromatographic apparatus used in the first SMB separation step. The first extract stream may optionally be collected in a vessel and reintroduced into the top of column 3 of the chromatographic apparatus used in the first SMB separation step.

[0311] In the "back-to-back" SMB process shown in FIG. 8, a second raffinate stream is typically removed from the bottom of column 7 of the chromatographic apparatus used in the second SMB separation step.

[0312] In the "back-to-back" SMB process shown in Figure 8, a second extract stream is typically collected from the bottom of column 2 of the chromatographic apparatus used in the second SMB separation step. This second extract stream typically contains the second product. The second extract stream is optionally collected in a vessel and reintroduced into the top of column 3 of the chromatographic apparatus used in the second SMB separation step.

[0313] In the "back-to-back" SMB process shown in FIG. 8, the eluent used is typically as defined above.

[0314] Typically, in this "back-to-back" SMB process, the water:organic solvent ratio in the chromatographic equipment used in the first SMB separation step is lower than the water:organic solvent ratio in the chromatographic equipment used in the second SMB separation step. Thus, the eluent in the first SMB separation step typically contains more organic solvent than the eluent used in the second SMB separation step.

[0315] In this "back-to-back" SMB process, the water:organic solvent ratio in the first SMB separation step is typically 0.5:99.5 to 1.5:98.5 parts by volume, and the water:organic solvent ratio in the second SMB separation step is typically 2:98 to 6:94 parts by volume.

[0316] In this "back-to-back" SMB process, the apparatus of FIG. 8 is configured as shown in FIG. 10a, although the configurations shown in FIGS. 10b and 10c may also be used.

[0317] This "back-to-back" SMB process is also shown in FIG. 9. The second product (B) and the input stream F containing the more polar (C) and less polar (A) components are introduced into the top of column 5 in the chromatographic apparatus used in the first SMB separation step. The aqueous organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the first SMB separation step. In the first SMB separation step, the less polar component (A) is removed from the bottom of column 2 as extract stream E1. The second product (B) and the more polar component (C) are removed from the bottom of column 7 as raffinate stream R1. The raffinate stream R1 is the first product, which is purified in the second SMB separation step by being introduced into the top of column 4 in the chromatographic apparatus used in the second SMB separation step. The aqueous organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the second SMB separation step. In the second SMB separation step, the more polar component (C) is removed as raffinate stream R2 at the bottom of column 7. The second product (B) is collected as extract stream E2 at the bottom of column 2.

[0318] In the "back-to-back" SMB process shown in FIG. 9, the aqueous organic solvent is typically introduced at the top of column 1 in the chromatographic apparatus used in the first SMB separation step.

[0319] In the "back-to-back" SMB process shown in FIG. 9, the aqueous organic solvent is typically introduced at the top of column 9 in the chromatographic apparatus used in the second SMB separation step.

[0320] In a "back-to-back" SMB process, shown in FIG. 9, the input stream is typically introduced into the top of column 5 in the chromatographic apparatus used in the first SMB separation step.

[0321] In the "back-to-back" SMB process shown in Figure 9, a first raffinate stream is typically collected as a first product from the bottom of column 7 of the chromatographic apparatus used in the first SMB separation step. This first product is then purified in a second SMB separation step and typically introduced to the top of column 5 of the chromatographic apparatus used in the second SMB separation step. The first raffinate stream may optionally be collected in a vessel before being purified in the second SMB separation step.

[0322] In the "back-to-back" SMB process shown in Figure 9, the first extract stream is typically removed from the bottom of column 2 of the chromatographic apparatus used in the first SMB separation step. The first extract stream is optionally collected in a vessel and a portion may be reintroduced into the top of column 3 of the chromatographic apparatus used in the first SMB separation step. The rate of recycle of liquid collected in the first SMB separation step via the extract stream back into the chromatographic apparatus used in the first SMB separation step is the rate at which liquid is pumped from this vessel into the top of column 3.

[0323] In the "back-to-back" SMB process shown in FIG. 9, a second raffinate stream is typically removed from the bottom of column 7 of the chromatographic apparatus used in the first SMB separation step.

[0324] In the "back-to-back" SMB process shown in Figure 9, a second extract stream is typically collected from the bottom of column 2 of the chromatographic apparatus used in the first SMB separation step. This second extract stream typically contains the second product. The second extract stream is optionally collected in a vessel and a portion may be reintroduced into the top of column 3 of the chromatographic apparatus used in the first SMB separation step. The rate of recycle of the liquid collected via the extract stream from the second SMB separation step back into the chromatographic apparatus used in the second SMB separation step is the rate at which liquid is pumped from this vessel into the top of column 3.

[0325] In the "back-to-back" SMB process shown in FIG. 9, the eluent used is as defined above.

[0326] Typically, in this "back-to-back" SMB process, the water:organic solvent ratio in the chromatographic equipment used in the first SMB separation step is lower than the water:organic solvent ratio in the chromatographic equipment used in the second SMB separation step. Thus, the eluent used in the first SMB separation step typically contains more organic solvent than the eluent used in the second SMB separation step.

[0327] In this "back-to-back" SMB process, the water:organic solvent ratio in the first SMB separation step is typically 0.5:99.5 to 1.5:98.5 parts by volume, and the water:organic solvent ratio in the second SMB separation step is typically 2:98 to 6:94 parts by volume.

[0328] In this "back-to-back" SMB process, the rate at which liquid collected via the extract stream from the first SMB separation step is recycled back into the chromatographic apparatus used in the first SMB separation step is typically faster than the rate at which liquid collected via the extract stream from the second SMB separation step is recycled back into the chromatographic apparatus used in the second SMB separation step, in which case the aqueous organic solvent eluent is typically substantially the same for each SMB separation step.

[0329] In this "back-to-back" SMB process, the apparatus of FIG. 9 is configured as shown in FIG. 10a, although the configurations shown in FIGS. 10b and 10c may also be used.

[0330] Typically, the chromatographic separation of the present invention comprises at least one, such as one, "back-to-back" SMB process as defined above.

[0331] In a preferred embodiment, the chromatographic separation process of the present invention comprises two different chromatographic separation steps to obtain the desired PUFA product from the feed mixture. Thus, in this embodiment, the chromatographic separation process comprises: (i) purifying the feed mixture in a first chromatographic separation step using a mixture of water and a first organic solvent as an eluent to obtain an intermediate product; and (ii) purifying the intermediate product in a second chromatographic separation step using a mixture of water and a second organic solvent as an eluent to obtain the PUFA product. Includes.

[0332] Typically, in this embodiment, the second organic solvent is different from the first organic solvent. This so-called "mixed solvent" process is particularly effective in removing C18 impurities from the desired PUFA product and is described in WO 2014 / 108686, the entirety of which is incorporated herein by reference.

[0333] It can be difficult to efficiently remove C18 fatty acids, especially alpha-linolenic acid (ALA) and / or gamma-linolenic acid (GLA), from a feed mixture without using large amounts of hydroalcoholic solvent. Efficient removal of C18 fatty acids is advantageous because many specifications for pharmaceutical and edible oils require low contents of these fatty acids. For example, specifications for certain oils for use in Japan require an ALA content of less than 1% by weight. This "mixed solvent" process is therefore particularly useful because it can be used to efficiently recover PUFA products from a feed mixture while minimizing the amount of C18 fatty acids, such as ALA and / or GLA, present in the resulting product. C18 fatty acids are C18 aliphatic monocarboxylic acids with linear or branched hydrocarbon chains. Exemplary C18 fatty acids include stearic acid (C18:0), oleic acid (C18:1n9), vaccenic acid (C18:1n7), linoleic acid (C18:2n6), gamma-linolenic acid / GLA (C18:3n6), alpha-linolenic acid / ALA (C18:3n3), and stearidonic acid / SDA (C18:4n3).

[0334] For the avoidance of doubt, typically in this "mixed solvent" process, the first chromatographic separation step is carried out in a chromatographic apparatus as described herein, i.e., comprising, as a solid adsorbent phase, C18 bonded silica as described herein. Typically in this "mixed solvent" process, the second chromatographic separation step is carried out in a chromatographic apparatus as described herein, i.e., comprising, as a solid adsorbent phase, C18 bonded silica as described herein.

[0335] Preferably, in this "mixed solvent" process, both the first and second chromatographic separation steps are carried out in a chromatographic apparatus as described herein, i.e., operating at a pressure of less than 20 bar and comprising, as a solid sorbent phase, C18 bonded silica as described herein. However, in an alternative embodiment of this "mixed solvent" process, only one of the first and / or second chromatographic separation steps is carried out in a chromatographic apparatus as described herein, and the other separation step is carried out in a chromatographic apparatus having a different solid sorbent phase and / or operating pressure.

[0336] Thus, in this embodiment of the "mixed solvent" process, typically, the first chromatographic separation step is carried out in a chromatographic apparatus as described herein, i.e., a chromatographic apparatus operating at a pressure of less than 20 bar and comprising, as a solid adsorbent phase, C18-bonded silica as described herein, and the second chromatographic step is carried out in a chromatographic apparatus having a different solid adsorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid adsorbent phase. Alternatively, the first chromatographic separation step is carried out in a chromatographic apparatus as described herein, and the second chromatographic step is carried out in a chromatographic apparatus operating at a pressure of more than 20 bar. Alternatively, the first chromatographic separation step is carried out in a chromatographic apparatus as described herein, and the second chromatographic step is carried out in a chromatographic apparatus operating at a pressure of more than 20 bar, and having a different solid adsorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid adsorbent phase.

[0337] Alternatively, in this embodiment of the "mixed solvent" process, the second chromatographic separation step can also be carried out in a chromatographic apparatus as described herein, i.e., a chromatographic apparatus operating at a pressure of less than 20 bar and including, as a solid adsorbent phase, C18-bonded silica as described herein, and the first chromatographic step is carried out in a chromatographic apparatus having a different solid adsorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid adsorbent phase. Alternatively, the second chromatographic separation step is carried out in a chromatographic apparatus as described herein, and the first chromatographic step is carried out in a chromatographic apparatus operating at a pressure of more than 20 bar. Alternatively, the second chromatographic separation step is carried out in a chromatographic apparatus as described herein, and the first chromatographic step is carried out in a chromatographic apparatus operating at a pressure of more than 20 bar, and having a different solid adsorbent phase, such as alternative silica, such as alternative C18-bonded silica, C8-bonded silica, pure silica, cyano-bonded silica and phenyl-bonded silica or a non-silica-based solid adsorbent phase.

[0338] Typically, in this "mixed solvent" process, the first and second chromatographic separation steps are carried out in different chromatographic apparatus. Alternatively, the first and second chromatographic separation steps are carried out in the same chromatographic apparatus.

[0339] Typically, in this "mixed solvent" process, the second organic solvent has a polarity index that differs from the polarity index of the first organic solvent by no more than 2.0. Thus, if the polarity index of the first organic solvent is P1, the polarity index of the second organic solvent is P2, and |P1-P2| is at most 2.0. The polarity index (P') of a solvent is a well-known measure of how polar a solvent is. A higher polarity index number indicates a more polar solvent. The polarity index is typically determined by measuring the ability of a solvent to interact with various test solutes. More typically, the polarity index (P') of a solvent is as defined in Burdick and Jackson's Solvent Guide (AlliedSignal, 1997), which is incorporated herein by reference in its entirety. Burdick and Jackson rank the solvents by reference to a numerical index that ranks the solvents according to their various polarities. The Burdick and Jackson index is based on the structure of the solvent.

[0340] Preferably, in this "mixed solvent" process, the second organic solvent has a polarity index that differs from the polarity index of the first organic solvent by 0.1 to 2.0. For example, the second organic solvent may have a polarity index that differs from the polarity index of the first organic solvent by at least 0.2, at least 0.3, at least 0.4, at least 0.5, or at least 0.6. For example, the second organic solvent may have a polarity index that differs from the polarity index of the first organic solvent by 1.8 or less, 1.5 or less, 1.3 or less, 1.0 or less, or 0.8 or less. For example, the second organic solvent may have a polarity index that differs from the polarity index of the first organic solvent by 0.2 to 1.8, 0.3 to 1.5, 0.4 to 1.3, 0.5 to 1.0, or 0.6 to 0.8.

[0341] In this "mixed solvent" process, the first and second organic solvents can be any of the organic solvents disclosed herein. Typically, however, the first and second organic solvents are miscible with water. More typically, the first and second organic solvents have a polarity index of 3.9 or greater. Preferably, the first and second organic solvents are selected from tetrahydrofuran, isopropyl alcohol, n-propyl alcohol, methanol, ethanol, acetonitrile, 1,4-dioxane, N,N-dimethylformamide, and dimethylsulfoxide.

[0342] Typically, in this "mixed solvent" process, the first organic solvent:water ratio is 99.9:0.1 to 75:25 parts by volume, preferably 99.5:0.5 to 80:20 parts by volume. When the first organic solvent is methanol, the methanol:water ratio is typically 99.9:0.1 to 85:15 parts by volume, preferably 99.5:0.5 to 88:12 parts by volume. When the first organic solvent is acetonitrile, the acetonitrile:water ratio is typically 99:1 to 75:25 parts by volume, preferably 96:4 to 80:20 parts by volume.

[0343] Typically, in this "mixed solvent" process, the second organic solvent:water ratio is 99.9:0.1 to 75:25 parts by volume, preferably 93:7 to 85:15 parts by volume. When the second organic solvent is methanol, the methanol:water ratio is typically 95:5 to 85:15 parts by volume, preferably 93:7 to 90:10 parts by volume. When the second organic solvent is acetonitrile, the acetonitrile:water ratio is typically 90:10 to 80:20 parts by volume, preferably 88:12 to 85:15 parts by volume.

[0344] Typically, in this "mixed solvent" process, one of the first and second organic solvents is acetonitrile.

[0345] Typically, in this "mixed solvent" process, one of the first and second organic solvents is methanol.

[0346] Preferably, in this "mixed solvent" process, the first and second organic solvents are selected from acetonitrile and methanol. Thus, it is preferred that (i) the first organic solvent is methanol and the second organic solvent is acetonitrile, or (ii) the first organic solvent is acetonitrile and the second organic solvent is methanol.

[0347] More preferably, in this "mixed solvent" process, the first organic solvent is methanol and the second organic solvent is acetonitrile, and (a) the methanol:water ratio is from 99.9:0.1 to 85:15 parts by volume, preferably from 99.5:0.5 to 88:12, and / or (b) the acetonitrile:water ratio is from 90:10 to 80:20 parts by volume, preferably from 88:12 to 85:15 parts by volume. In certain embodiments, it is preferred that (a) the methanol:water ratio is from 91:9 to 93:7 parts by volume, and / or (b) the acetonitrile:water ratio is from 86:14 to 88:12 parts by volume.

[0348] Alternatively, in this "mixed solvent" process, the first organic solvent is acetonitrile and the second organic solvent is methanol, and (a) the acetonitrile:water ratio is from 99:1 to 75:25 parts by volume, preferably from 96:4 to 80:20 parts by volume, and / or (b) the methanol:water ratio is from 95:5 to 85:15 parts by volume, preferably from 93:7 to 90:10 parts by volume. In certain embodiments, it is preferred that (a) the acetonitrile:water ratio is from 86:14 to 88:12 parts by volume, and / or (b) the methanol:water ratio is from 87:13 to 89:11 parts by volume.

[0349] Typically, in this "mixed solvent" process, the first organic solvent is acetonitrile, and the intermediate product has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the feed mixture. Alternatively, in this "mixed solvent" process, the second organic solvent is acetonitrile, and the PUFA product produced in the second separation step has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the intermediate product.

[0350] Preferably, in this "mixed solvent" process, the PUFA product is EPA ethyl ester and (i) the first organic solvent is acetonitrile and the intermediate product has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the feed mixture, or (ii) the second organic solvent is acetonitrile and the PUFA product produced in the second separation step has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the intermediate product.

[0351] More preferably, in this "mixed solvent" process, the PUFA product is EPA ethyl ester and (i) the first organic solvent is acetonitrile and the second organic solvent is methanol and the intermediate product has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the feed mixture, or (ii) the first organic solvent is methanol and the second organic solvent is acetonitrile and the PUFA product produced in the second separation step has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the intermediate product.

[0352] Any known chromatographic apparatus may be used in this "mixed solvent" process. Typically, the first and / or second separation steps are carried out using either a fixed bed chromatographic apparatus as described herein or one or more simulated or actual moving bed chromatographic apparatus.

[0353] Typically, in this "mixed solvent" process, a first chromatographic separation step involves introducing a feed mixture into a fixed bed chromatographic apparatus and a second chromatographic separation step involves introducing an intermediate product into a fixed bed chromatographic apparatus, where typically, in this "mixed solvent" process, the first chromatographic separation step is carried out using a fixed bed chromatographic apparatus and the second chromatographic separation step is carried out using a fixed bed chromatographic apparatus.

[0354] Alternatively, in this "mixed solvent" process, a first chromatographic separation step involves introducing a feed mixture into a fixed bed apparatus and a second chromatographic separation step involves introducing an intermediate product into a simulated or real moving bed chromatographic apparatus, where typically in this "mixed solvent" process, the first chromatographic separation step is carried out using a fixed bed apparatus and the second chromatographic separation step is carried out using a simulated or real moving bed chromatographic apparatus.

[0355] Alternatively, in this "mixed solvent" process, a first chromatographic separation step involves introducing the feed mixture into a simulated or real moving bed chromatographic apparatus and a second chromatographic separation step involves introducing the intermediate product into a fixed bed chromatographic apparatus, where typically in this "mixed solvent" process, the first chromatographic separation step is carried out using a simulated or real moving bed chromatographic apparatus and the second chromatographic separation step is carried out using a fixed bed chromatographic apparatus.

[0356] Alternatively, in this "mixed solvent" process, a first chromatographic separation step involves introducing a feed mixture into a simulated or real moving bed chromatographic apparatus and a second chromatographic separation step involves introducing an intermediate product into a simulated or real moving bed chromatographic apparatus, where typically in this "mixed solvent" process, the first chromatographic separation step is carried out using a simulated or real moving bed chromatographic apparatus and the second chromatographic separation step is carried out using a simulated or real moving bed chromatographic apparatus.

[0357] In this "mixed solvent" process, the first chromatographic separation step can consist of a single chromatographic separation or two or more chromatographic separations, where each separation uses a mixture of water and a first organic solvent as eluent.

[0358] In this "mixed solvent" process, the second chromatographic separation step can consist of a single chromatographic separation or two or more chromatographic separations, where each separation uses a mixture of water and a second organic solvent as eluent.

[0359] In this "mixed solvent" process, the first and second separation steps may be carried out at the same temperature or at different temperatures, preferably at the same temperature.

[0360] Typically, in this "mixed solvent" process, each chromatographic separation step involves passing the feed mixture through one or more chromatographic columns, at least one of which has a temperature above room temperature. More typically, all of the chromatographic columns used have temperatures above room temperature. Preferred temperatures are as described above.

[0361] In this "mixed solvent" process, when the first and / or second chromatographic separation steps are carried out in a simulated moving bed apparatus, at least one of the first and / or second chromatographic separation steps typically comprises at least one, e.g. one, "single pass" SMB step as described above.

[0362] In this "mixed solvent" process, when the first and / or second chromatographic separation steps are carried out in a simulated moving bed apparatus, at least one of the first and / or second chromatographic separation steps typically comprises at least one, e.g. one, "double pass" SMB step as described above.

[0363] In a "mixed solvent" process, references to "input stream" above with respect to the "double pass" mode of operating an SMB separation refer to the feed mixture when said SMB process is used in a first chromatographic separation step, and to the intermediate product when said SMB process is used in a second chromatographic separation step.

[0364] In a "mixed solvent" process, the references above to "aqueous organic solvent" in relation to the "double pass" mode of operating an SMB separation refer to a mixture of water and a first organic solvent when said SMB process is used in a first chromatographic separation step, and to a mixture of water and a second organic solvent when said SMB process is used in a second chromatographic separation step.

[0365] In this "mixed solvent" process, when the first and / or second chromatographic separation steps are carried out in a simulated moving bed apparatus, at least one of the first and / or second chromatographic separation steps typically comprises the "back-to-back" SMB process described above.

[0366] For the avoidance of doubt, in a "mixed solvent" process, if the first chromatographic separation step is a "back-to-back" SMB process along the lines above, the eluent in each of the SMB steps is a mixture of water and a first organic solvent. If the second chromatographic separation step is a "back-to-back" SMB process along the lines above, the eluent in each of the SMB steps is a mixture of water and a second organic solvent.

[0367] In a "mixed solvent" process, references to "input streams" above with respect to the "back-to-back" mode of operating an SMB separation refer to a feed mixture when the SMB process is used in a first chromatographic separation step, and to intermediate products when the SMB process is used in a second chromatographic separation step.

[0368] In a "mixed solvent" process, the references to "aqueous organic solvent" above with respect to the "back-to-back" mode of operating an SMB separation refer to a mixture of water and a first organic solvent when the "back-to-back" SMB process is used in a first chromatographic separation step, and to a mixture of water and a second organic solvent when the "back-to-back" SMB process is used in a second chromatographic separation step. The organic solvent used in the first and second SMB steps is the same. The organic solvent:water ratio used in the first and second SMB steps can be the same or different.

[0369] In a "mixed solvent" process, references above to a "second product" in the "back-to-back" mode of operating an SMB separation refer to an intermediate product when the SMB process is used in a first chromatographic separation step, and to a PUFA product when the SMB process is used in a second chromatographic separation step.

[0370] Preferably, in the "mixed solvent" process, the PUFA product is EPA ethyl ester; (i) the first organic solvent is acetonitrile and the second organic solvent is methanol, the first chromatographic separation step comprises introducing the feed mixture into a fixed bed apparatus and the second chromatographic separation step comprises introducing the intermediate product into a simulated or actual moving bed chromatographic apparatus; or (ii) the first organic solvent is methanol and the second organic solvent is acetonitrile, the first chromatographic separation step comprises introducing the feed mixture into a simulated or real moving bed chromatographic apparatus, and the second chromatographic separation step comprises introducing the intermediate product into a fixed bed chromatographic apparatus.

[0371] Even more preferably, in this "mixed solvent" process, the PUFA product is EPA ethyl ester; (i) the first organic solvent is acetonitrile, the second organic solvent is methanol, the intermediate product has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the feed mixture, the first chromatographic separation step comprises introducing the feed mixture into a fixed bed apparatus, and the second chromatographic separation step comprises introducing the intermediate product into a simulated or actual moving bed chromatographic apparatus; or (ii) the first organic solvent is methanol and the second organic solvent is acetonitrile, the PUFA product produced in the second separation step has a lower concentration of one or more of the C18 fatty acid impurities disclosed above than the intermediate product, the first chromatographic separation step comprises introducing the feed mixture into a simulated or actual moving bed chromatographic apparatus, and the second chromatographic separation step comprises introducing the intermediate product into a fixed bed chromatographic apparatus.

[0372] The following examples illustrate the invention. EXAMPLES

[0373] Example 1: Characterization of different silica particles Four different silicas were selected for testing. The physical parameters of each of these silicas were first measured as described below. The results are listed in Table 1. All silicas were commercially available and obtained from their respective suppliers. Silica 1 was selected as a reference example, and the other three silicas are examples of silicas for use according to the present invention.

[0374] [Table 1]

[0375] The % carbon loading was determined by combustion analysis.

[0376] Surface area is a measure of the surface area of ​​unbonded silica (i.e., not bonded to octadecyl carbon chains). It was determined in porosity experiments by BET surface area analysis.

[0377] Dv(10), D[4,3] and Dv(90) were determined by laser diffraction. Measurements were taken in aqueous suspension using a Malvern Mastersizer 2000 instrument.

[0378] Plots showing the total contribution to particle volume of the silica samples as a function of particle size for various C18 silica types are shown in Figure 12. These are a measure of the particle distribution of each sample.

[0379] FIG. 12A compares the particle distributions of Silica 1 (two separate batches) and Silica 2. Both of these silicas can be observed to contain a significant number of smaller particles with diameters between 10 and 100 μm (the peaks corresponding to these particles are small, but note that the y-axis reflects the total volume contribution of these particles to the entire sample, not the total number of particles of this diameter present; because these particles are small, their contribution to the total volume is small, but their number is large). Thus, these silicas have a broad particle distribution with a noticeable "tail" of smaller particles.

[0380] Figure 12B compares the particle distributions of Silica 1 (two separate batches), Silica 3 and Silica 4. It can be observed that both Silica 3 and Silica 4, in contrast to Silica 1, contain a small number of small particles with diameters between 10 and 100 μm. This is reflected in the higher Dv(10) values ​​reported for Silica 3 and Silica 4 in Table 1 compared to Silica 1. Overall, it can also be seen that Silica 3 and Silica 4 have a much narrower particle size distribution than Silica 1.

[0381] Bulk particle density was determined by recording the weight of material packed into a column of a defined volume.

[0382] Optical microscope images of Silicas 1, 2 and 3 were also obtained and are shown in Figure 13. As can be seen from Figures 13A and 13B, Silicas 1 and 2 contain fractured particles with some particles having small diameters present. In contrast, Silica 3 (Figure 13C) shows no evidence of fractured particles and has a more uniform particle size distribution.

[0383] Example 2: Comparison of peak resolution and pressure drop using various silicas The fish oil derived feedstock (containing about 74 GC-area % EPA and about 10 GC-area % DHA) was pulse-injected onto a single fixed bed chromatography apparatus consisting of three columns in series, each with a diameter of 10 mm and a length of 250 mm. Each of the silicas listed in Table 2 was used here as the stationary phase, and a mixture of methanol:water (90:10) was used as the eluent.

[0384] The operating parameters and flow rates were as follows: Feed mixture pulse: 0.5 mL of 25 wt.% fish oil-derived feedstock dissolved in methanol Eluent feed rate (D1): 7 mL / min of 90:10 vol.% methanol:water.

[0385] The following parameters were measured for each column pulse experiment: · Pressure drop along the column (using the experiment with silica 1 as control); · EPA retention time compared to control experiments; · Selectivity for the αSDA / EPA peaks (i.e. retention time difference); Selectivity for the αDHA / EPA peak; asymmetry of the EPA peak; and Height equivalent theoretical plate (HETP) for the EPA peak.

[0386] The results are shown in Table 2 (each of the silicas used is as per the specifications in Table 1; for brevity, shortened reference names for each silica are used).

[0387] [Table 2]

[0388] All of the silicas tested demonstrated acceptable pressure drops over the length of the chromatographic apparatus compared to the reference. Silicas 2 and 4 provided lower pressure drops over the length of the apparatus than Silica 1, with Silica 4 being particularly advantageous in this regard.

[0389] All of the silicas tested demonstrated acceptable selectivity of the EPA peak over the DHA and SDA peaks. Selectivity was calculated as the ratio of the retention times of the relevant pair of peaks.

[0390] The peak asymmetry (As) of the EPA peak was determined by dividing the peak width after the peak center by the peak width before the peak center at 10% peak height. All of the silicas tested showed improved symmetry of the EPA peak compared to the reference example (Silica 1). Thus, each of these silicas gives improved peak shape of EPA, since a more symmetrical peak means that the "tail" of the EPA peak does not intrude as much into other peaks. This allows for a more efficient separation, since less eluent (i.e. lower eluent dilution) is needed to obtain a high yield of the desired product with a particular purity.

[0391] A graphical representation of the EPA peak asymmetry for each of Silicas 1, 2 and 4, from which the values ​​in Table 2 were calculated, is shown in FIG.

[0392] The HETP value was calculated using the following formula:

number

[0393] Theoretical plates are a mathematical concept since chromatography columns do not contain anything resembling physical distillation plates or other similar features. A column with a higher number of plates is considered to be more efficient; a column with a higher number of theoretical plates will have a narrower peak at a given retention time than a column with a lower N number (i.e., lower HETP). Thus, a lower HETP value indicates a well-resolved peak resulting in higher column efficiency, which means that less eluent is needed to obtain a high yield of a desired product with a particular purity.

[0394] All test silicas 2-4 show improved HETP compared to the control silica 1.

[0395] The following conclusions can be drawn from the data in Tables 1 and 2: Silicas with a more uniform particle size distribution and especially those with fewer small particles (reflected in the Dv(10) values) show improved resolution of the PUFA peaks. This can be seen from a comparison of the HETP values ​​of silicas 2 and 3, which have very similar carbon loadings.

[0396] · Silicas with lower carbon loadings show improved resolution of the PUFA peaks. This can be seen from a comparison of the HETP values ​​for Silicas 1 and 2, both of which have similar particle distributions with a small particle "tail", yet the HETP value for Silica 2 is significantly lower than the HETP value for Silica 1. Similarly, Silicas 3 and 4 have similar particle size distributions, but Silica 4 has a lower carbon loading and improved HETP value.

[0397] Silicas with smaller surface areas show improved resolution of the PUFA peaks. In general, a positive correlation is observed between carbon loading and total surface area.

[0398] Example 3: Comparison of low pressure EPA separation via SMB using silica 4 with HPLC / batch process Three purifications of EPA-containing feedstocks were performed using silica 4 in an SMB process operating under reduced pressure on eight columns, each with a diameter of 1.6 cm and a length of 25 cm, and compared with batch / HPLC separations using various silicas reported in the literature. Productivity and eluent dilution of each process were measured. The results are listed in Table 3.

[0399] Each of the three purifications was performed using either a single pass SMB or a double pass SMB with both a "fast running" fraction (pass 1) and a "slow running" fraction (pass 2) to remove components that run faster and slower than EPA, respectively. The operating parameters for each purification were as follows:

[0400] Test Run A Raw material: EPA ethyl ester content of 92GC-area% Desired product: EPA ethyl ester with 97 GC-area % Process: Single pass SMB Eluent: methanol:water (91:9) Temperature: 40℃ Pressure: <10 bar Feed mixture feed rate (F): 0.5 mL / min of 50 wt% fish oil feedstock in methanol Eluent feed rate (D): 17.3 mL / min Extraction rate: 13.2mL / min Raffinate rate: 4.6mL / min

[0401] Test run B Raw material: EPA ethyl ester content of 74GC-area% Desired product: EPA ethyl ester with 97 GC-area % Process: Double pass SMB Eluent (slow flow fraction): methanol:water (91:9) Eluent (fast flow fraction): methanol:water (90:10) Temperature: 40℃ Pressure: <10 bar

[0402] Path 1 Feed mixture feed rate (F1): 0.5 mL / min of 50 wt% fish oil feedstock in methanol Eluent supply rate (D1): 22.0 mL / min Extract rate (E1): 14.8mL / min Raffinate rate (R1): 7.7mL / min

[0403] Path 2 Feed mixture feed rate (F2): 1.0 mL / min of 50 wt.% raffinate from the slow flowing fraction in methanol Eluent feed rate (D2): 13.5 mL / min Extract rate (E2): 12.3mL / min Raffinate rate (R2): 2.3mL / min

[0404] Test run C Raw material: EPA ethyl ester content of 74GC-area% Desired product: EPA ethyl ester with 97 GC-area % Process: Double pass SMB Eluent (slow flow fraction): methanol:water (90:10) Eluent (fast flow fraction): methanol:water (91:9) Temperature: 40℃ Pressure: <6 bar

[0405] Path 1 Feed mixture feed rate (F1): 0.35 mL / min of 50 wt% fish oil feedstock in methanol Eluent supply rate (D1): 13.9 mL / min Extract accumulation rate (E1): 10.1mL / min Raffinate accumulation rate (R1): 4.2mL / min

[0406] Path 2 Feed mixture feed rate (F2): 0.7 mL / min of 50 wt.% raffinate from the slow flowing fraction in methanol Eluent feed rate (D2): 8.9 mL / min Extraction rate (E2): 8.5mL / min Raffinate rate (R2): 1.1mL / min

[0407] As can be seen from Table 3, the chromatographic separation process of the present invention results in a much more efficient separation requiring substantially reduced dilution of solvent per kg of EPA product, i.e. less solvent than all comparative prior art processes using higher operating pressures. At the same time, reasonable levels of productivity are maintained, which is surprising since it is generally known in the art that at reduced operating pressures, up to a ten-fold decrease in chromatographic separation productivity is typically observed.

[0408] It is also noteworthy that such low level dilution rate and high yield of EPA product can be obtained by using such silica with such large average particle size.It is generally known in the art that larger particle size leads to higher HETP and reduced peak resolution.Therefore, the inventors' discovery that certain types of silica with larger average particle size can be used in PUFA production, further realizing high yield and acceptable productivity, and further providing lower dilution rate of solvent is unexpected.

[0409] [Table 3]

Claims

1. 1. A chromatographic separation process for recovering polyunsaturated fatty acid (PUFA) products from a feed mixture, comprising: (a) a liquid eluent phase that is an aqueous organic solvent; and (b) A solid adsorbent phase that is C18 bonded silica. introducing the feed mixture into a chromatography apparatus comprising one or more chromatography columns comprising: (1) the silica has an average particle size of 230 to 270 μm and a Dv(10) of 160 μm or more; and / or (2) the silica has a carbon loading of 15 to 24 wt. %; and / or (3) The silica is 500 m ２ Chromatographic separation process having a surface area of ​​0.1g or less.

2. the silica has a carbon loading of 15 to 24 wt. %; and said silica has an average particle size of between 230 and 270 μm and a Dv(10) of greater than or equal to 160 μm; and / or - the silica is 500m ２ 10. The chromatographic separation process of claim 1, wherein the surface area of ​​the catalyst is less than or equal to 1000 nm.

3. 2. The chromatographic separation process of claim 1, wherein the silica has an average particle size of 230 to 270 μm, preferably 240 to 260 μm.

4. 2. The chromatographic separation process of claim 1, wherein the silica has a Dv(10) of 160 μm or more and / or a Dv(10) of 225 μm or less, preferably wherein the silica has a Dv(10) of 165 μm or more and / or a Dv(10) of 215 μm or less.

5. 2. The chromatographic separation process of claim 1, wherein the silica has an average particle size of 230-270 μm and a Dv(10) of 160-225 μm, preferably the silica has an average particle size of 240-260 μm and a Dv(10) of 165-215 μm.

6. 10. The chromatographic separation process of claim 1, wherein the silica has a carbon loading of 15 to 24 wt. %.

7. 10. The chromatographic separation process of claim 1, wherein the silica has a carbon loading of 16 to 22 wt. %.

8. The silica is 500 m ２ / g or less and / or a surface area of ​​200 m ２ / g or more, and preferably the silica has a surface area of ​​450 m ２ / g or less surface area and / or 250 m ２ 10. The chromatographic separation process of claim 1, wherein the surface area of ​​the catalyst is 100 / g or greater.

9. The silica is (a) having a Dv(10) of 170 μm or greater; and / or (b) has an average particle size of 250 to 260 μm; and / or (c) having a carbon loading of 17 to 18.5 wt. %; and / or (d) having a pore size of 80 to 140 Å; and / or (e) 0.7kg / dm ３ 2. The chromatographic separation process of claim 1, having a bulk density of:

10. 2. The chromatographic separation process of claim 1, wherein at least 80% of the silica particles by volume have a diameter of 200 to 500 μm.

11. 2. The chromatographic separation process of claim 1, wherein the aqueous organic solvent is a mixture of water and an alcohol, an ether, an ester, a ketone or a nitrile, preferably the aqueous organic solvent is a mixture of water and methanol or a mixture of water and acetonitrile.

12. 2. The chromatographic separation process of claim 1, wherein the pressure in the one or more chromatographic columns is less than 10 bar, preferably less than 6 bar.

13. 2. The chromatographic separation process of claim 1, wherein the chromatographic device is a real moving bed or simulated moving bed (SMB) chromatographic device.

14. 2. The chromatographic separation process of claim 1, wherein the feed mixture is (i) a natural or synthetic feedstock comprising at least one PUFA product, preferably a fish oil feedstock, a vegetable oil feedstock, or a feedstock derived from a microorganism or a fungus, or (ii) a partially purified feedstock comprising at least one PUFA product obtained from partial purification of a natural or synthetic feedstock.

15. The chromatographic separation process of claim 14, wherein the partially purified feedstock is obtained by crystallization, molecular or fractional distillation, urea fractionation, extraction with silver nitrate or other metal salt solutions, iodolactonization, supercritical fluid fractionation or chromatography, preferably fixed bed chromatography or simulated or actual moving bed chromatography.

16. (a) introducing the feed mixture into the chromatography device results in purification of the feed mixture to produce the PUFA product; or 2. The chromatographic separation process of claim 1, wherein (b) introducing the feed mixture into the chromatography device results in purification of the feed mixture to produce an intermediate product, and the intermediate product is subjected to further purification to obtain the PUFA product, preferably the further purification is carried out in a chromatography device, more preferably the further purification is carried out via fixed bed chromatography or simulated or actual moving bed chromatography.

17. The PUFA product comprises a PUFA or a PUFA derivative, wherein the derivative is a mono-, di- or tri-glyceride, ester, phospholipid, amide, lactone or salt of the PUFA, preferably the PUFA product is an ω-3 PUFA or a derivative of an ω-3 PUFA, most preferably (a) the PUFA product is EPA or an EPA derivative, preferably an ester of EPA, more preferably EPA ethyl ester; 2. The chromatographic separation process of claim 1, wherein (b) the PUFA product is DHA or a DHA derivative, preferably an ester of DHA, more preferably DHA ethyl ester.

18. (a) the yield of PUFA product obtained from said chromatographic separation process is greater than 80 wt. %, more preferably greater than 90 wt. %, even more preferably greater than 95 wt. %, and most preferably greater than 98 wt. %, based on the total mass of said PUFA product present in said feed mixture; and / or (b) The chromatographic separation process of claim 1, wherein the PUFA product is produced with a purity of greater than 80% by weight, preferably greater than 85% by weight, more preferably greater than 90% by weight, even more preferably greater than 95% by weight, still more preferably greater than 97% by weight, even more preferably greater than 98% by weight, and most preferably greater than 99% by weight.

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