Method and device for separating ethyl eicosapentaenoate

Abstract

The invention provides a method and a device for separating ethyl eicosapentaenoate. The separation method comprises the steps of loading a sample, washing impurities before washing, eluting and washing impurities after washing in sequence, and is carried out in a double-column simulated moving bed. According to the process, the elution capacity of the eluant during elution of the post-impurity is creatively improved, so that the post-impurity with higher adsorption capacity with the filler can be eluted by using a small amount of eluant, meanwhile, the regeneration of the chromatographic column is realized, and after the post-impurity elution is completed, only a small amount of low-concentration eluant is required to be properly balanced, so that the sample can be re-loaded, and the operation is simple and convenient. And a new round of preparation process is started. In the process, the difference of the adsorption capacity between different components and silica gel filler is fully considered, corresponding first eluent and second eluent are selected, and front impurities, middle components, positive components and rear impurities are sequentially eluted, so that the elution efficiency is maximized, the use amount of the used eluent is greatly reduced, the system operation time is shortened, and the preparation efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of medicine and pharmaceuticals, and in particular to a method and a device for separating eicosapentaenoic acid ethyl ester. Background Art

[0002] As people pay more and more attention to their own health, the public begins to take more nutritional and healthy dietary supplements, among which polyunsaturated fatty acid (PUFA) products occupy an important position. Polyunsaturated fatty acids refer to fatty acids with at least two double bonds in their molecular structure, and exist in the form of fatty acid ethyl esters, fatty acid methyl esters, fatty acid glycerides, and free fatty acids.

[0003] Polyunsaturated fatty acids (PUFA) are an important material basis for body metabolism, especially for infant brain development. They are components of cell membranes and play a major role in maintaining cell membrane fluidity, promoting cholesterol esterification, reducing cholesterol and triglycerides, reducing blood viscosity, improving blood circulation and other physiological functions. They also have the effects of improving human thinking and enhancing memory. The human body cannot synthesize polyunsaturated fatty acids (PUFA) by itself and must be obtained through diet.

[0004] There are many types of polyunsaturated fatty acids (PUFA), mainly including ω-3 polyunsaturated fatty acids (PUFA) (ω-3PUFA), ω-6 polyunsaturated fatty acids (PUFA) (ω-6PUFA), ω-9 polyunsaturated fatty acids (PUFA) (ω-9PUFA) and other forms, such as α-linolenic acid (ALA), eicosapentaenoic acid (EPA), henicosapentaenoic acid (HPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), linoleic acid (LA), conjugated linoleic acid (CLA), γ-linolenic acid (GLA), arachidonic acid (AA), etc. Among them, ω-3 polyunsaturated fatty acids (ω-3PUFA) represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the most well-known and accepted by the public. They can be used together with a low-fat diet to reduce hypertriglyceridemia.

[0005] Generally, crude fish oil (referred to as crude fish oil or raw fish oil) with a content of about 30% can be obtained from various marine raw materials by squeezing, extraction and other means, wherein EPA and DHA are usually present in the form of triglycerides, and a small amount is present in the form of wax esters and phospholipids. Since other fatty acid molecules are also connected to the long chain of triglycerides, it is difficult to obtain a certain specific component with a high content when it is purified in the form of triglycerides. Therefore, before separation and purification, it is often ethyl esterified and converted into the form of EPA ethyl ester (EPA-EE) and DHA ethyl ester (DHA-EE) to separate the active ingredients (referred to as ethyl esterification), and then through a series of means, a crude product with a total content of EPA and DHA of more than 70% is obtained (referred to as initial extraction). However, the mechanisms of action of EPA and DHA on the human body are different and affect each other, so it is often necessary to further separate the two to obtain a product with a high content of a certain specific fatty acid. For this reason, it is necessary to further increase the content of EPA or DHA in the refining process to more than 90% (referred to as high-purity extraction).

[0006] The drug developed by Amarin Corporation in the United States for the treatment of hypertriglyceridemia and dyslipidemia uses eicosapentaenoic acid ethyl ester (EPA-EE) with a purity greater than 96.5%. It is the only prescription omega-3-acid ethyl ester drug for the treatment of hypertriglyceridemia and dyslipidemia in the United States and has good therapeutic effects.

[0007] High-purity eicosapentaenoic acid ethyl ester is mainly obtained by purifying ethyl ester fish oil. Currently, the methods for separating and purifying EPA-EE from low-content ethyl ester fish oil mainly include the following: urea inclusion method, low-temperature freezing method, metal salt precipitation method, vacuum distillation method, supercritical extraction method, high performance liquid chromatography and column chromatography.

[0008] In the prior art, there are a large number of studies on purification of high-purity eicosapentaenoic acid ethyl ester from the aspects of process or equipment innovation. Among them, CN114349638A discloses a method for purifying omega-3-acid ethyl ester in ethyl ester fish oil, wherein the ethyl ester fish oil is first subjected to sequential simulated moving bed chromatography purification to obtain a crude product, and then the crude product is separated by supercritical liquid chromatography to obtain high-purity omega-3-acid ethyl ester. The process requires the use of simulated moving bed chromatography and supercritical liquid chromatography, which are inefficient and result in high product costs.

[0009] CN113831244A describes a set of equipment and process for purifying high-purity EPA-EE. The equipment includes several chromatographic columns and several preparation bottles, and the sample is loaded and purified for multiple times, which has low efficiency, especially the efficiency is lower when the sample is loaded and purified twice, and the process is not economical.

[0010] CN113698984A invented a method for separating and purifying eicosapentaenoic acid in fish oil, wherein the crude fish oil is chromatographed on a microsphere chromatography column to obtain high-content eicosapentaenoic acid ethyl ester. However, since a low-pressure column is used, the sample loading amount is low and the amount of solvent used is large, which is not suitable for industrial production.

[0011] CN110709496A discloses a method for producing a composition containing highly unsaturated fatty acids. According to the specific adsorption of double bonds, an aqueous solution of silver salt is used for refining. The process includes steps such as silver salt refining and recovery. The silver salt is easily lost, which poses a risk in terms of food safety. In addition, the output is small and the cost is high.

[0012] CN108640840B provides a chromatographic method and equipment for purifying eicosapentaenoic acid ethyl ester. The equipment is a multi-column continuous chromatography system, which is divided into two major areas: a coarse separation area and a fine separation area. Each area is further divided into 5 sub-areas. The system is very complex and large, with low efficiency, and is not suitable for industrial production.

[0013] CN15015458A provides a split flow chromatography system and a method for preparing ethyl eicosapentaenoate using the split flow chromatography system. The system contains two preparative chromatography columns and two sets of detectors, wherein the first chromatography column is used for loading and displaying pre-impurities, and the second chromatography column is used for displaying pre-impurities and eluting positive fractions. The first chromatography column can be connected in series with the second chromatography column, but the second chromatography column cannot be connected in series with the first chromatography column. During the operation, the polarity differences of pre-impurities, positive fractions, post-impurities, etc. are not taken into account, and only one concentration of eluent is used, resulting in the two chromatography columns involving the use of a large amount of eluent for regeneration and balancing.

[0014] In general, the previous technology for preparing high-content eicosapentaenoic acid ethyl ester has several defects: either the equipment structure is complex, the system is large, and the operation is difficult during operation, which is not suitable for large-scale industrial production; or a low-pressure column is used, the sample amount is small during preparation, the amount of eluent used is large, the cost of the produced product is high, and the economy is not strong; or in the double-column process, on the one hand, the difference in adsorption capacity of the front impurities, positive separation, and rear impurities is not taken into account, and the difficulty of elution or regeneration on the chromatographic column is different, and only one concentration (polarity) of eluent is used, resulting in too much eluent when eluting the front impurities and rear impurities, which not only causes a large amount of solvent consumption, but also increases the subsequent solvent recovery and processing volume accordingly, and the operation time is prolonged, reducing production efficiency; on the other hand, the functions of the two preparation columns are fixed and single, and effective switching cannot be achieved, thereby failing to achieve fully continuous production, further affecting the improvement of production efficiency.

[0015] In view of the defects in the prior art, it is necessary to find a process that can efficiently prepare eicosapentaenoic acid ethyl ester and is suitable for industrial production. Summary of the invention

[0016] The main purpose of the present invention is to provide a method and device for separating eicosapentaenoic acid ethyl ester, so as to solve the problems of complicated eicosapentaenoic acid ethyl ester separation process and large amount of eluent solvent in the prior art.

[0017] In order to achieve the above-mentioned object, according to one aspect of the present invention, a separation method of eicosapentaenoic acid ethyl ester is provided, the separation method comprising the steps of loading, pre-washing impurities, elution and post-washing impurities which are carried out in sequence, and the steps are carried out in a double-column simulated moving bed; wherein, in the loading step, the first chromatographic column and the second chromatographic column of the double-column simulated moving bed are in an independent state, the raw material is loaded onto the first chromatographic column, and the second chromatographic column of the double-column simulated moving bed is balanced with the first eluent at the same time; in the pre-washing impurities step, the first chromatographic column and the second chromatographic column are in a series state, the first eluent is input into the first chromatographic column, and the pre-impurities are collected at the end of the second chromatographic column; in the elution step, the first chromatographic column and the second chromatographic column are in a series state, the first eluent is input into the first chromatographic column, and the intermediate components are collected at the end of the second chromatographic column; in the post-washing impurities step, the first chromatographic column and the second chromatographic column are in an independent state, the second eluent is input into the first chromatographic column to elute and remove the post-impurities, the first eluent is input into the second chromatographic column, and the eicosapentaenoic acid ethyl ester solution is collected at the end; the elution capacity of the second eluent is greater than the elution capacity of the first eluent.

[0018] Furthermore, the steps of sample loading, pre-washing, elution and post-washing are repeated, and the functions of the first chromatographic column and the second chromatographic column are switched.

[0019] Furthermore, the liquid flowing out from the ends of the first chromatographic column and the second chromatographic column is detected online; preferably, an ultraviolet spectrophotometer is used for the online detection;

[0020] Preferably, in the pre-washing impurity step, when ethyl eicosapentaenoate flows out from the end of the second chromatographic column, the pre-washing impurity step is finished;

[0021] Preferably, in the pre-washing step, when no more eicosapentaenoic acid ethyl ester flows out from the end of the first chromatographic column, the elution step is finished.

[0022] Furthermore, the first chromatographic column and the second chromatographic column are each independently selected from any one of a C8 chromatographic column, a C18 chromatographic column and a C30 chromatographic column; preferably, the fillers of the first chromatographic column and the second chromatographic column are silica-based fillers; preferably, the particle size of the silica-based fillers is 5-100 μm, more preferably 10-50 μm.

[0023] Further, the first eluent and the second eluent are methanol-water solution, or the first eluent is methanol-water solution and the second eluent is methanol;

[0024] Preferably, the volume concentration of methanol in the second eluent differs from the volume concentration of methanol in the first eluent by 2% to 12%, preferably by 3% to 8%;

[0025] Preferably, the volume concentration of methanol in the first eluent is 88% to 97%;

[0026] Preferably, the volume concentration of methanol in the second eluent is 95% to 100%.

[0027] Furthermore, the flow rates of the first eluent and the second eluent are 15-25 BV / hr, preferably 18-20 BV / hr.

[0028] Further, the time of the pre-washing miscellaneous process is 8-12 minutes, preferably 8-10 minutes;

[0029] Preferably, the elution time is 3-8 minutes, preferably 5-6 minutes;

[0030] Preferably, the washing time is 8-15 minutes, preferably 10-12 minutes.

[0031] Furthermore, the mass content of eicosapentaenoic acid ethyl ester in the raw material is above 60%, preferably above 70%, and more preferably above 80%.

[0032] Further, the loading amount of the raw material is 10-20% of the mass of the filler material of the first chromatographic column;

[0033] Preferably, the raw material is dissolved in the first eluent when loading the sample, and the concentration of eicosapentaenoic acid ethyl ester is 2 to 12 g / 100 mL.

[0034] According to another aspect of the present invention, a separation device for eicosapentaenoic acid ethyl is provided, and the separation device can obtain eicosapentaenoic acid ethyl by using any of the above separation methods.

[0035] The technical scheme of the present invention is applied, and the complexity of the raw material composition is fully considered. There are fatty acid ethyl esters with shorter carbon chains or fewer double bonds, such as C16:0, C18:0, C18:1, C18:2, C20:0, C20:1, etc., and the adsorption capacity between these fatty acid ethyl esters and silica gel is weak, and they are often easily eluted out of the chromatographic column first, which is the front impurity. Some fatty acid ethyl esters and eicosapentaenoic acid ethyl ester are very similar to the silica gel adsorption capacity, such as C20:4n-3 and C20:4n-6, and the separation difficulty is relatively large. During elution, they are often in the front section of elution and form a cross section with EPA-EE. This part is an intermediate component and needs to be collected and processed separately. In addition to the above-mentioned fatty acid ethyl esters, there are also other fatty acid ethyl esters such as DHA-EE, C24:1, HPA-EE, DPA-EE, and a very small amount of free fatty acids, soaps, etc., which often form stronger adsorption with silica gel, are not easy to elute, and are the back impurities.

[0036] In the process of the present invention, the elution capacity of the eluent when eluting the rear impurities is creatively increased, so that the rear impurities with strong adsorption capacity to the filler can be eluted with a smaller amount of eluent, and the regeneration of the chromatographic column can be achieved at the same time. After the rear impurities are eluted, only a small amount of low-concentration eluent is needed to balance appropriately, and then the sample can be reloaded to start a new round of preparation process. In the process of the invention, the difference in adsorption capacity between different components and silica gel fillers is fully considered, and the corresponding first eluent and second eluent are selected to elute the front impurities, intermediate components, positive components and rear impurities in turn, so as to maximize the elution efficiency, greatly reduce the amount of eluent used, shorten the system operation time, and improve the preparation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0038] Figure 1 A schematic structural diagram of a double-column simulated moving bed according to Example 1 of the present invention is shown.

[0039] Among them, the above-mentioned drawings include the following drawing marks: 001, feed pump; 002, elution pump B; 003, elution pump A; 100, first chromatographic column, 200, second chromatographic column; 11, first chromatographic column control valve A; 12, first chromatographic column control valve B, 13, first chromatographic column control valve C; 14, first chromatographic column control valve D; 15, first chromatographic column control valve E; 16, first chromatographic column control valve F; 21, second chromatographic column control valve A; 22, second chromatographic column control valve B, 23, second chromatographic column control valve C; 24, second chromatographic column control valve D; 25, second chromatographic column control valve E; 26, second chromatographic column control valve F. DETAILED DESCRIPTION

[0040] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0041] In addition to EPA-EE, other fatty acid ethyl esters and a small amount of free fatty acids, fatty acid soaps, waxes and other components with strong polarity are often present in the raw materials for extracting EPA-EE. In order to increase the content of the effective ingredient EPA-EE, these components must be removed as completely as possible. Although these components are small in quantity, they have strong adsorption to silica gel fillers, and it is very difficult to completely elute them from the chromatographic column. In the prior art, a large amount of eluents with the same polarity as the pre-wash impurities and the positive fraction are often used for elution. In order to elute this part of the impurities, the eluent used may even reach more than 60% of the total eluent dosage, or even more. Even such a large amount of eluent dosage cannot completely elute the impurities, resulting in a small amount of incompletely eluted impurity components being eluted into the positive fraction in the subsequent sample loading and elution process to form impurities; not only is it not conducive to improving the purity of the product, but also increases the running time of the chromatographic system and reduces the running efficiency. In order to solve this problem, the present application provides a method and device for separating eicosapentaenoic acid ethyl ester.

[0042] According to another typical embodiment of the present application, a separation method of eicosapentaenoic acid ethyl ester is provided, which separation method comprises the steps of loading, pre-washing, eluting and post-washing in sequence, and is carried out in a double-column simulated moving bed; wherein, in the loading step, the first chromatographic column and the second chromatographic column of the double-column simulated moving bed are in an independent state, the raw material is loaded onto the first chromatographic column, and the second chromatographic column of the double-column simulated moving bed is balanced with the first eluent; in the pre-washing step, the first chromatographic column and the second chromatographic column are in a series state, and the first eluting liquid is input into the first chromatographic column. The first chromatographic column and the second chromatographic column are connected in series, the first eluent is input into the first chromatographic column, and the intermediate components are collected at the end of the second chromatographic column; in the post-washing impurity process, the first chromatographic column and the second chromatographic column are independent, the second eluent is input into the first chromatographic column to elute and remove the post-wash impurities, the first eluent is input into the second chromatographic column, and the eicosapentaenoic acid ethyl ester solution is collected at the end; the elution capacity of the second eluent is greater than that of the first eluent; the sample loading, pre-washing impurities, elution and post-washing impurities processes are repeated.

[0043] The separation method of eicosapentaenoic acid ethyl ester of the present application takes full account of the complexity of raw material composition, and has existing or carbon chain is shorter, or the fatty acid ethyl ester with less double bond number, such as C16:0, C18:0, C18:1, C18:2, C20:0, C20:1, etc., and the adsorption capacity between this part of fatty acid ethyl ester and silica gel is weak, and it is often easy to be eluted out of the chromatographic column first, which is the front impurity. Some fatty acid ethyl esters and eicosapentaenoic acid ethyl ester and silica gel adsorption capacity are very close, such as C20:4n-3, C20:4n-6, and the separation difficulty is larger. During elution, they are often in the front section of elution, and form a cross section with EPA-EE. This part is an intermediate component and needs to be collected and processed separately. In addition to the above-mentioned fatty acid ethyl ester, there are also other fatty acid ethyl esters such as DHA-EE, C24:1, HPA-EE, DPA-EE, and a very small amount of free fatty acids, soaps, etc., which often form stronger adsorption with silica gel, are not easy to elute, and are the rear impurities.

[0044] In the process of the present invention, the elution capacity of the eluent when eluting the rear impurities is creatively increased, so that the rear impurities with strong adsorption capacity to the filler can be eluted with a smaller amount of eluent, and the regeneration of the chromatographic column can be achieved at the same time. After the rear impurities are eluted, only a small amount of low-concentration eluent is needed to balance appropriately, and then the sample can be reloaded to start a new round of preparation process. In the process of the invention, the difference in adsorption capacity between different components and silica gel fillers is fully considered, and the corresponding first eluent and second eluent are selected to elute the front impurities, intermediate components, positive components and rear impurities in turn, so as to maximize the elution efficiency, greatly reduce the amount of eluent used, shorten the system operation time, and improve the preparation efficiency.

[0045] In addition to achieving precise elution and separation of pre-impurities, positive separation and post-impurities with different adsorption forces by utilizing eluents with different elution capacities, thereby greatly reducing the amount of eluent used, the seamless cycle of sample loading, pre-washing, elution and post-washing can also be achieved by switching the dual-column functions in the process.

[0046] In some typical embodiments of the present application, the steps of loading, washing before impurities, eluting and washing after impurities are repeated, and the functions of the first chromatographic column and the second chromatographic column are switched. That is to say, in the previous round of preparation process, the first chromatographic column is loaded, and after the washing before impurities and elution steps, the positive fraction is transferred to the second chromatographic column and finally eluted with the first eluent, and the impurities with larger adsorption power remain in the first chromatographic column, and are eluted and regenerated by the second eluent with higher elution power. In the subsequent next round of preparation process, the second chromatographic column is loaded, and the first chromatographic column is balanced with the first eluent at the same time, and after the washing before impurities and elution steps, the positive fraction is transferred to the first chromatographic column and finally eluted with the first eluent, and the impurities with larger adsorption power remain in the second chromatographic column, and are eluted and regenerated by the second eluent with higher elution power. In this way, the first chromatographic column and the second chromatographic column are constantly switched in function, and compared with the single column and the double column process in the prior art, the amount of eluent used is greatly reduced, and the efficiency is greatly improved.

[0047] In some typical embodiments of the present application, in order to achieve a better elution effect, the eluent composition of the chromatographic column is monitored, and the liquid flowing out from the ends of the first chromatographic column and the second chromatographic column is detected online; preferably, an ultraviolet spectrophotometer is used for online detection. In some embodiments of the present application, the end point of certain processes is determined by whether the liquid flowing out from the ends of the first chromatographic column and the second chromatographic column contains ethyl eicosapentaenoic acid, for example, in the pre-washing miscellaneous process, when ethyl eicosapentaenoic acid flows out from the end of the second chromatographic column, the pre-washing miscellaneous process ends; in the pre-washing miscellaneous process, when no ethyl eicosapentaenoic acid flows out from the end of the first chromatographic column, the elution process ends. Since the feature of whether ethyl eicosapentaenoic acid flows out from the end of the chromatographic column is very convenient for detection and identification, the above method is used as a judgment indicator of the process, which is easy to implement and accurately controlled, and has a significant effect on improving the recovery rate and purity of the target product.

[0048] In order to further improve the separation efficiency of the target product, in some embodiments of the present application, the time for the pre-washing impurity process is 8-12 minutes, preferably 8-10 minutes; in some embodiments of the present application, the elution time is 3-8 minutes, preferably 5-6 minutes; in some embodiments of the present application, the post-washing impurity time is 8-15 minutes, preferably 10-12 minutes.

[0049] In some embodiments of the present application, the first chromatographic column and the second chromatographic column are independently selected from any one of a C8 chromatographic column, a C18 chromatographic column and a C30 chromatographic column; preferably, the fillers of the first chromatographic column and the second chromatographic column are silica matrix fillers, and the silica matrix fillers include but are not limited to a silica skeleton and C8-C30 alkanes bonded to the surface of the silica skeleton. Preferably, the particle size of the silica matrix filler is 5-100 μm, more preferably 10-50 μm, and further preferably 10-25 μm.

[0050] In some typical embodiments of the present application, the first eluent and the second eluent are methanol-water solution, or the first eluent is methanol-water solution and the second eluent is methanol, and the elution capacity of the eluent is adjusted by adjusting the ratio of methanol and water in the eluent. In order to better balance the elution effect and the ability to remove impurities, it is preferred that the volume concentration of methanol in the second eluent differs from the volume concentration of methanol in the first eluent by 2% to 12%, more preferably 3% to 8%, and further preferably 3% to 5%. In some embodiments of the present application, the volume concentration of methanol in the first eluent is 88% to 97%; preferably, the volume concentration of methanol in the second eluent is 95% to 100%. The first eluent in each process may be the same or different. When different, it is preferred that the second eluent has a higher methanol content than the first eluent to meet the above range. Preferably, the first eluent used in each process is the same, which is not only convenient for the implementation of the process, but also easier to control the process.

[0051] In some embodiments of the present application, in order to better balance the elution effect and product purity of the eluent and further improve the separation efficiency of eicosapentaenoic acid ethyl ester, the flow rate of the first eluent and the second eluent is 15-25BV / hr, preferably 18-20BV / hr, which better balances the purity, recovery rate and elution efficiency of the product.

[0052] The raw material of the above separation method can be fish oil raw material. The higher the eicosapentaenoic acid ethyl ester content in the raw material, the higher the separation efficiency. For example, the mass content of eicosapentaenoic acid ethyl ester in the raw material can be above 60%, preferably above 70%, and more preferably above 80%.

[0053] In some embodiments of the present application, the loading amount of the raw material is 10-20% of the mass of the first chromatographic column filler, that is, the mass of eicosapentaenoic acid ethyl ester in the loading raw material is 10-20% of the mass of the chromatographic column filler. Preferably, the raw material is dissolved in the first eluent when loading, and the concentration of eicosapentaenoic acid ethyl ester is 2-12g / 100mL, that is, the content of eicosapentaenoic acid ethyl ester in every 100mL solution is 2-12g. For example, the concentration of eicosapentaenoic acid ethyl ester is 2g / 100mL, 3g / 100mL, 4g / 100mL, 5g / 100mL, 6g / 100mL, 7g / 100mL, 8g / 100mL, 9g / 100mL, 10g / 100mL, 11g / 100mL, 12g / 100mL or any range value between the two. In some embodiments of the present application, the loading time is 6-10 minutes, preferably 8 minutes.

[0054] After collecting the positive fraction eluate, the eluate is concentrated and recovered to a constant weight to obtain eicosapentaenoic acid ethyl ester. In some embodiments of the present application, the mass content of EPA-EE is above 97.5%, the total recovery rate of EPA-EE is greater than 90%, and the content of a single impurity is less than 0.5wt%.

[0055] According to another typical embodiment of the present application, a separation device for eicosapentaenoic acid ethyl is provided, and the separation device can obtain eicosapentaenoic acid ethyl by using any of the above separation methods.

[0056] The ethyl eicosapentaenoic acid separated by the separation process of the present application creatively increases the elution capacity of the eluent when eluting the rear impurities, so that the rear impurities with strong adsorption capacity to the filler can be eluted with a smaller amount of eluent, and the regeneration of the chromatographic column can be achieved at the same time. After the elution of the rear impurities is completed, only a small amount of low-concentration eluent is needed to properly balance, and the sample can be reloaded to start a new round of preparation process. The invention process fully considers the difference in adsorption capacity between different components and silica gel fillers, and selects the corresponding first eluent and second eluent, and sequentially elutes the front impurities, intermediate components, positive components and rear impurities to maximize the elution efficiency, greatly reduce the amount of eluent used, shorten the system operation time, improve the preparation efficiency, and significantly reduce the production cost of ethyl eicosapentaenoic acid.

[0057] The beneficial effects that can be achieved by the present application will be further illustrated below in combination with embodiments and comparative examples.

[0058] Example 1

[0059] Use Figure 1 The apparatus shown performs the separation of eicosapentaenoic acid ethyl ester.

[0060] The first chromatographic column 100 and the second chromatographic column 200 are both C18 chromatographic columns (size 50mm*250mm, filler is C18 silica gel filler, particle size 10μm), the EPA-EE content in the raw material is 83.2wt%, the raw material is dissolved in eluent A, the concentration is 8g / 100mL, methanol: water in eluent A = 96:4 (v / v), and eluent B is anhydrous methanol. In the whole process, the eluent flow rate is 18BV / hr.

[0061] 1). Loading. Close the valves of the first chromatographic column control valve F16 and the second chromatographic column control valve A 21, so that the first chromatographic column 100 and the second chromatographic column 200 are in an independent state. The eluent A that dissolves the raw material enters the first chromatographic column 100 through the feed pump 001 and the first chromatographic column control valve B12, and the first chromatographic column control valve E15 is opened to discharge the front impurities. At the same time, the eluent A passes through the elution pump A 003, enters the second chromatographic column 200 for balancing through the second chromatographic column control valve D 24, and the discharged liquid passes through the second chromatographic column control valve F 26 to remove the rear impurities collection tank. The loading and balancing time is 8 minutes, and the loading amount is 15.2% of the filler mass.

[0062] 2) Wash the pre-impurities. After the loading is completed, close the valves of the first chromatographic column control valve B12, the first chromatographic column control valve E15, and the second chromatographic column control valve D24, open the first chromatographic column control valve D14 to allow the eluent A to enter the first chromatographic column 100, open the first chromatographic column control valve F16 and the second chromatographic column control valve A21 to connect the first chromatographic column 100 and the second chromatographic column 200 in series, open the second chromatographic column control valve A21, and the eluent passes through the second chromatographic column 200 and the UV detector connected thereto, and the pre-impurities are collected through the second chromatographic column control valve F26. When the UV detector after the second chromatographic column 200 detects that EPA-EE flows out from the end of the second chromatographic column 200, stop collecting the pre-impurities. The pre-impurity washing time is 8.1 minutes.

[0063] 3). Elution. Continue to use eluent A for elution. The first chromatographic column 100 and the second chromatographic column 200 are still in series connection. The second chromatographic column control valve F26 collects not the fore impurities but the intermediate components. The elution is stopped when the UV detector connected to the chromatographic column 1 does not detect EPA-EE. The elution time is 5.3 minutes.

[0064] 4) Wash the impurities. Close the first chromatographic column control valve F16 and the second chromatographic column control valve A21, and the first chromatographic column 100 and the second chromatographic column 200 are in an independent state. Close the first chromatographic column control valve D14, open the first chromatographic column control valve C13 and the first chromatographic column control valve E15, and inject the eluent B into the first chromatographic column 100 through the elution pump B002, and collect the impurities through the first chromatographic column control valve E15. At the same time, open the second chromatographic column control valve D24, elute the positive fraction accumulated in the second chromatographic column 200 with the eluent A, and collect it through the second chromatographic column control valve F26. When no impurity peaks are detected in the UV detector after the first chromatographic column 100, the elution is stopped, and the elution time is 12.2 minutes. The time for collecting the positive fraction in the second chromatographic column 200 does not need to be 12.2 minutes. After 4.6 minutes, EPA-EE is no longer detected in the UV detector connected to the second chromatographic column 200. However, in order to ensure the continuous circulation of the system, after the collection of the positive fraction in the second chromatographic column 200 is completed, it is still equilibrated with eluent A for 7.6 minutes.

[0065] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 99.4%, the recovery rate was 90.6%, and the maximum single impurity ratio was 0.32%.

[0066] The cycle steps 1) to 4) are repeated, except that the functions of the first chromatographic column 100 and the second chromatographic column 200 are switched, that is, the sample is loaded onto the second chromatographic column 200, the first chromatographic column 100 is used to remove the front impurities, collect the intermediate components, collect the positive fraction, and finally elute the rear impurities from the second chromatographic column 200 with the eluent B, and the second chromatographic column 200 is regenerated at the same time. During this cycle, the first chromatographic column control valve F16 and the second chromatographic column control valve A 21 are in a closed state, and the first chromatographic column 100 and the second chromatographic column 200 are in an independent or series state according to the closed or open state of the second chromatographic column control valve E25 and the first chromatographic column control valve A 11. The eluent in the positive fraction collected in the cycle step is concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reaches 99.5%, the recovery rate is 91.2%, and the maximum single impurity ratio is 0.33%.

[0067] Example 2

[0068] The first chromatographic column 100 and the second chromatographic column 200 are both C8 chromatographic columns (size 50mm*250mm, filler is C8 silica gel filler, particle size 50μm), the EPA-EE content in the raw material is 61.8wt%, the sample is dissolved in eluent A, the concentration is 10g / 100mL, methanol: water in eluent A = 88:12 (v / v), methanol: water in eluent B = 95:5 (v / v). The eluent flow rate during loading and elution is 15BV / hr. The basic operations such as loading, pre-washing impurities, elution, and post-washing impurities are the same as in Example 1.

[0069] The loading time was 10 minutes, the loading amount was 20.8% of the filler mass, the time before washing was 12.3 minutes, the elution time was 7.8 minutes, and the time after washing and collecting the positive fraction was 15.4 minutes.

[0070] The EPA-EE monomer product was obtained by recovering the collected eluent from the positive fraction, wherein the mass content of EPA-EE was 96.7%, the recovery rate was 93.1%, and the maximum single impurity ratio was less than 1.3wt%.

[0071] Example 3

[0072] The first chromatographic column 100 and the second chromatographic column 200 are both C30 chromatographic columns (size 50mm*250mm, filler is C30 silica gel filler, particle size 25μm), the EPA-EE content in the raw material is 72.4wt%, the sample is dissolved in eluent A, the concentration is 4.8g / 100mL, methanol: water in eluent A = 97:3 (v / v), and eluent B is anhydrous methanol. The eluent flow rate during loading and elution is 25BV / hr. The basic operations such as loading, pre-washing impurities, elution, and post-washing impurities are the same as in Example 1.

[0073] The loading time was 10 minutes, the loading amount was 10% of the filler mass, the time before washing was 10.4 minutes, the elution time was 5.9 minutes, and the time after washing and collecting the positive fraction was 10.3 minutes.

[0074] The EPA-EE monomer product was obtained by recovering the collected eluent from the positive fraction, wherein the mass content of EPA-EE was 97.9%, the recovery rate was 89.8%, and the maximum single impurity ratio was less than 0.8wt%.

[0075] Example 4

[0076] The first chromatographic column 100 and the second chromatographic column 200 are both C18 chromatographic columns (size 50mm*250mm, filler is C18 silica gel filler, particle size 30μm), the EPA-EE content in the raw material is 81.2wt%, the sample is dissolved in eluent A, the concentration is 10g / 100mL, methanol: water in eluent A = 92:8 (v / v), methanol: water in eluent B = 98:2 (v / v). The eluent flow rate during loading and elution is 20BV / hr. The basic operations such as loading, pre-washing impurities, elution, and post-washing impurities are the same as in Example 1.

[0077] The loading time was 6 minutes, the loading amount was 16.6% of the filler mass, the time before washing was 8.4 minutes, the elution time was 3.4 minutes, and the time after washing and collecting the positive fraction was 8.3 minutes.

[0078] After the collected eluent from the positive fraction is recovered, an EPA-EE monomer product is obtained, wherein the EPA-EE mass content is 98.6%, the recovery rate is 91.4%, and the maximum single impurity ratio is less than 0.6wt%.

[0079] Example 5

[0080] The only difference from Example 1 is that when the cycle steps 1) to 4) are repeated, the functions of the first chromatographic column 100 and the second chromatographic column 200 remain unchanged, which is exactly the same as the previous cycle. The positive fraction eluate collected in the cycle step is concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reaches 96.5%, the recovery rate is 68%, and the maximum single impurity ratio is 0.57%.

[0081] Example 6

[0082] The only difference from Example 1 is that in step 3), after 3 minutes of elution, when the UV detector connected to the first chromatographic column 100 can still detect EPA-EE, the process is stopped.

[0083] The time for washing impurities and collecting the positive fraction was 21.3 minutes. The eluent in the collected positive fraction was recovered and concentrated to constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 97.2%, the recovery rate was 72.3%, and the maximum single impurity ratio was 0.54%.

[0084] Example 7

[0085] The only difference from Example 1 is that in steps 1) to 4), the flow rate of the eluent is 15 BV / hr. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as in Example 1.

[0086] Among them, the loading time is 9.5 minutes, the loading amount is 20.8% of the filler mass, the pre-washing impurity time is 10 minutes, the elution time is 6.4 minutes, and the post-washing impurity and collection time is 14.5 minutes.

[0087] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 98.7%, the recovery rate was 84.5%, and the maximum single impurity ratio was 0.29%.

[0088] Example 8

[0089] The only difference from Example 1 is that in steps 1) to 4), the flow rate of the eluent is 25 BV / hr. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as in Example 1.

[0090] Among them, the loading time is 5.8 minutes, the loading amount is 20.8% of the filler mass, the pre-washing impurity time is 5.9 minutes, the elution time is 3.8 minutes, and the post-washing impurity and collection time is 8.8 minutes.

[0091] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 96.7%, the recovery rate was 83.9%, and the maximum single impurity ratio was 0.41%.

[0092] Example 9

[0093] The only difference from Example 1 is that in steps 1) to 4), the flow rate of the eluent is 8 BV / hr. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as in Example 1.

[0094] Among them, the loading time is 18 minutes, the loading amount is 20.8% of the filler mass, the time before washing is 18.2 minutes, the elution time is 11.9 minutes, and the time after washing and collecting the positive fraction is 27.5 minutes.

[0095] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 99.3%, the recovery rate was 92.1%, and the maximum single impurity ratio was 0.27%.

[0096] Example 10

[0097] The only difference from Example 1 is that the sample loading amount is 10 g / 100 mL of the filler mass. The basic operations of sample loading, pre-washing, elution, and post-washing are the same as those in Example 1.

[0098] Among them, the loading time is 5.3 minutes, the pre-wash time is 7.9 minutes, the elution time is 3.7 minutes, and the post-wash and collection time is 7.5 minutes.

[0099] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 99.5%, the recovery rate was 92.5%, and the maximum single impurity ratio was 0.25%.

[0100] Embodiment 11

[0101] The only difference from Example 1 is that the sample is dissolved in eluent A, and the concentration of EPA-EE is 12 g / 100 mL. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as in Example 1.

[0102] Among them, the loading time is 6 minutes, the pre-wash time is 8.0 minutes, the elution time is 5.3 minutes, and the post-wash and collection time is 12.3 minutes.

[0103] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 98.9%, the recovery rate was 89.8%, and the maximum single impurity ratio was 0.35%.

[0104] Example 12

[0105] The only difference from Example 1 is that methanol:water=96:4 (v / v) in eluent A and methanol:water=98:2 (v / v) in eluent B. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as in Example 1.

[0106] Among them, the loading time is 8 minutes, the pre-wash time is 8.1 minutes, the elution time is 5.4 minutes, and the post-wash and collection time is 15.7 minutes.

[0107] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 99.2%, the recovery rate was 90.8%, and the maximum single impurity ratio was 0.41%.

[0108] Embodiment 13

[0109] The only difference from Example 1 is that methanol:water=96:4 (v / v) in eluent A and methanol:water=97:3 (v / v) in eluent B. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as in Example 1.

[0110] Among them, the loading time is 8 minutes, the pre-wash time is 8.1 minutes, the elution time is 5.4 minutes, and the post-wash and collection time is 18.5 minutes.

[0111] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 98.9%, the recovery rate was 90.4%, and the maximum single impurity ratio was 0.51%.

[0112] Embodiment 14

[0113] The only difference from Example 1 is that the ratio of methanol to water in eluent A is 88:12 (v / v), and eluent B is anhydrous methanol. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as those in Example 1.

[0114] Among them, the loading time is 8 minutes, the pre-wash time is 9.4 minutes, the elution time is 6.2 minutes, and the post-wash and collection time is 27.2 minutes.

[0115] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 99.6%, the recovery rate was 94.2%, and the maximum single impurity ratio was 0.19%.

[0116] Embodiment 15

[0117] The only difference from Example 1 is that the ratio of methanol to water in eluent A is 85:15 (v / v), and eluent B is anhydrous methanol. The basic operations such as sample loading, pre-washing, elution, and post-washing are the same as those in Example 1.

[0118] Among them, the loading time is 9.2 minutes, the pre-wash time is 11.2 minutes, the elution time is 7.5 minutes, and the post-wash and collection time is 31.5 minutes.

[0119] The collected eluate from the positive fraction was concentrated to a constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 99.6%, the recovery rate was 95.4%, and the maximum single impurity ratio was 0.16%.

[0120] Comparative Example 1

[0121] The difference from Example 1 is that the methanol concentration of eluent B is the same as that of eluent A, which is 96% (V / V).

[0122] The loading, washing of pre-impurities and elution processes are all the same as those in Example 1. When washing post-impurities, a tiny impurity peak is still detected in the UV detector connected to the first chromatographic column 100 after 56 minutes, and the post-impurities are still not completely eluted. After entering the next loading cycle, an impurity peak with an area ratio of 0.09% and a retention time longer than EPA-EE appears in the obtained positive fraction, indicating that the post-impurities are not completely eluted in the previous cycle and are carried into the positive fraction of the next cycle.

[0123] It can be seen from the comparative examples that the impurities are not eluted by selecting an eluent with stronger elution ability during the elution process, which not only greatly prolongs the operation time and greatly increases the amount of eluent used, but also reduces the quality of the final product.

[0124] The difference between Comparative Example 2 and Example 1 is that after step 2), step 4) post-washing process is directly performed.

[0125] The time for washing impurities and collecting the positive fraction was 15.1 minutes. The eluent in the collected positive fraction was recovered and concentrated to constant weight to obtain an EPA-EE product, wherein the EPA-EE content reached 89.8%, the recovery rate was 94.5%, and the maximum single impurity ratio was 0.89%.

[0126] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the process creatively increases the elution capacity of the eluent when eluting the rear impurities, so that the rear impurities with strong adsorption capacity of the filler can be eluted with a smaller amount of eluent, and the regeneration of the chromatographic column can be achieved at the same time. After the elution of the rear impurities is completed, only a small amount of low-concentration eluent is needed to properly balance, and the sample can be reloaded to start a new round of preparation process. The invention process fully considers the difference in adsorption capacity between different components and silica gel fillers, and selects the corresponding first eluent and second eluent to elute the front impurities, intermediate components, positive fractions and rear impurities in turn to maximize the elution efficiency, greatly reduce the amount of eluent used, shorten the system operation time, and improve the preparation efficiency. In addition to using eluents with different elution capacities to achieve accurate elution and separation of front impurities, positive fractions and rear impurities with different adsorption forces, greatly reducing the amount of eluent used, the process can also achieve seamless circulation of loading, washing front impurities, eluting and washing rear impurities through switching of the double column functions.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for separating eicosapentaenoic acid ethyl ester, It is characterized in that The method comprises the steps of sample loading, pre-washing, elution and post-washing in sequence, and is carried out in a double-column simulated moving bed; wherein, In the loading step, the first chromatographic column and the second chromatographic column of the double-column simulated moving bed are in an independent state, the raw material is loaded onto the first chromatographic column, and the second chromatographic column of the double-column simulated moving bed is balanced with the first eluent; In the step of washing the impurities, the first chromatographic column and the second chromatographic column are connected in series, the first eluent is input into the first chromatographic column, and the impurities are collected at the end of the second chromatographic column; In the elution process, the first chromatographic column and the second chromatographic column are connected in series, the first eluent is input into the first chromatographic column, and the intermediate components are collected at the end of the second chromatographic column; In the post-impurity washing process, the first chromatographic column and the second chromatographic column are in an independent state, the second eluent is introduced into the first chromatographic column to elute and remove impurities, the first eluent is introduced into the second chromatographic column, and the eicosapentaenoic acid ethyl ester solution is collected at the end; the elution capacity of the second eluent is greater than the elution capacity of the first eluent.

2. The separation method according to claim 1, It is characterized in that The steps of sample loading, pre-washing, elution and post-washing are repeated, and the functions of the first chromatographic column and the second chromatographic column are switched.

3. The separation method according to claim 1, It is characterized in that Performing online detection on the liquid flowing out of the ends of the first chromatographic column and the second chromatographic column; preferably, an ultraviolet spectroscopic detector is used for the online detection; Preferably, in the pre-washing impurity step, when ethyl eicosapentaenoate flows out from the end of the second chromatographic column, the pre-washing impurity step is finished; Preferably, in the pre-washing step, when no eicosapentaenoic acid ethyl ester flows out from the end of the first chromatographic column, the elution step ends.

4. The separation method according to claim 1, It is characterized in that The first chromatographic column and the second chromatographic column are each independently selected from any one of a C8 chromatographic column, a C18 chromatographic column and a C30 chromatographic column; preferably, the fillers of the first chromatographic column and the second chromatographic column are silica-based fillers; preferably, the particle size of the silica-based fillers is 5-100 μm, more preferably 10-50 μm.

5. The separation method according to claim 1, It is characterized in that The first eluent and the second eluent are methanol-water solutions, or the first eluent is methanol-water solutions and the second eluent is methanol; Preferably, the volume concentration of methanol in the second eluent differs from that in the first eluent by 2% to 12%, preferably by 3% to 8%; Preferably, the volume concentration of methanol in the first eluent is 88% to 97%; Preferably, the volume concentration of methanol in the second eluent is 95% to 100%.

6. The separation method according to claim 1, It is characterized in that The flow rates of the first eluent and the second eluent are 15-25 BV / hr, preferably 18-20 BV / hr.

7. The separation method according to claim 6, It is characterized in that The time of the pre-washing miscellaneous process is 8-12 minutes, preferably 8-10 minutes; Preferably, the elution time is 3-8 minutes, preferably 5-6 minutes; Preferably, the post-washing time is 8-15 minutes, preferably 10-12 minutes.

8. The separation method according to claim 1, It is characterized in that The mass content of eicosapentaenoic acid ethyl ester in the raw material is above 60%, preferably above 70%, and more preferably above 80%.

9. The separation method according to claim 8, It is characterized in that The loading amount of the raw material is 10-20% of the mass of the first chromatographic column filler; Preferably, the raw material is dissolved in the first eluent when loading the sample, and the concentration of eicosapentaenoic acid ethyl ester is 2 to 12 g / 100 mL.

10. A separation device for eicosapentaenoic acid ethyl ester, It is characterized in that Eicosapentaenoic acid ethyl ester can be obtained by the separation method described in any one of claims 1 to 9.

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