CN115010596A - Method for enriching eicosapentaenoic acid in fish oil raw material - Google Patents
Method for enriching eicosapentaenoic acid in fish oil raw material Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 55
- 235000021323 fish oil Nutrition 0.000 title claims abstract description 51
- 235000020673 eicosapentaenoic acid Nutrition 0.000 title claims abstract description 39
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 title claims abstract description 38
- 229960005135 eicosapentaenoic acid Drugs 0.000 title claims abstract description 38
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 239000002994 raw material Substances 0.000 title claims abstract description 38
- 239000012535 impurity Substances 0.000 claims abstract description 66
- 238000011068 loading method Methods 0.000 claims abstract description 46
- 230000007480 spreading Effects 0.000 claims abstract description 29
- 238000003892 spreading Methods 0.000 claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000007864 aqueous solution Substances 0.000 claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 230000008569 process Effects 0.000 claims description 21
- 238000004587 chromatography analysis Methods 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- -1 C 4 Alcohols Chemical class 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 3
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- 239000012071 phase Substances 0.000 description 18
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 14
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 4
- 229940090949 docosahexaenoic acid Drugs 0.000 description 4
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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Abstract
The invention belongs to the technical field of substance purification, and particularly provides a method for enriching eicosapentaenoic acid in a fish oil raw material. The enrichment method provided by the invention comprises the following steps: feeding the fish oil raw material into a double-column simulated moving bed to sequentially carry out sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and impurity discharge; the double-column simulated moving bed comprises a first chromatographic column and a second chromatographic column; the mobile phase of the double-column simulated moving bed is an alcohol aqueous solution. The enrichment method provided by the invention can separate EPA with the purity of more than or equal to 98.5% from the fish oil raw material, has the advantages of small solvent dosage and high yield, and is convenient for realizing automatic and continuous production.
Description
Technical Field
The invention relates to the technical field of substance purification, in particular to a method for enriching eicosapentaenoic acid in fish oil raw materials.
Background
The fish oil is a natural health food with high utilization value, and the polyunsaturated fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) contained in the fish oil have the functions of inhibiting platelet aggregation, resisting thrombus, relaxing blood vessels, regulating blood fat, improving the fluidity of biological membranes and the like, and have better curative effects on treating and preventing cardiovascular diseases, diabetes, dermatitis, large intestine ulcer, inhibiting tumors and the like.
At present, methods for separating and purifying fish oil include urea inclusion method, low-temperature freezing method, metal salt precipitation method, saponification-urea inclusion method, fish oil cross-esterification, vacuum distillation method, supercritical extraction method and the like. However, the difference of EPA/DHA components contained in domestic fish oil raw materials is large, and the level of the separation and purification method provided by the method is not high, so that the final product quality is not uniform, and the method is specifically represented as follows: the EPA or DHA content in the domestic cod-liver oil or emulsion product is low, the market competitiveness is not strong, and the economic benefit is not high. Therefore, there is a need to provide a process for purifying fish oil products with high EPA content.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for enriching eicosapentaenoic acid in fish oil raw materials. The purity of EPA obtained by the enrichment method provided by the invention is more than or equal to 98.5%.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for enriching eicosapentaenoic acid in fish oil raw materials, which comprises the following steps:
feeding the fish oil raw material into a double-column simulated moving bed for enrichment;
repeating the enrichment process;
the repetition times are more than or equal to 3 times;
the double-column simulated moving bed comprises a first chromatographic column and a second chromatographic column;
the mobile phase of the double-column simulated moving bed is an alcohol aqueous solution;
the enrichment comprises the steps of sequentially carrying out sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and post impurity removal;
the loading parameters include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of the sample loading is 0.1-3 BV/h, and the sample loading time is 1-30 min;
the parameters of the layup include: the first chromatographic column and the second chromatographic column are connected in series, the reagent of the spreading layer is consistent with the mobile phase, the flow speed of the reagent of the spreading layer is 0.1-5 BV/h, and the time of the spreading layer is 15-40 min;
the parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the first internal circulation is 0.1-5 BV/h, and the time of the first internal circulation is 20-40 min;
the loading-collection parameters include: the first chromatographic column and the second chromatographic column are in an independent state, and the sample loading-collection comprises first chromatographic column sample loading and second chromatographic column collection; the flow rate of the first chromatographic column sample loading is 0.1-3 BV/h, and the sample loading-collecting time is 1-30 min;
the parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the second internal circulation is 0.1-5 BV/h, and the time of the second internal circulation is 5-20 min;
the parameters of post-draining impurities include: the first chromatographic column and the second chromatographic column are in an independent state, and the post-column impurity removal comprises a first chromatographic column post-column impurity removal and a second chromatographic column post-column impurity removal; the reagent for removing the impurities in the first chromatographic column and the reagent for removing the impurities in the second chromatographic column are consistent with the mobile phase, the flow rate of the reagent for removing the impurities in the first chromatographic column and the flow rate of the reagent for removing the impurities in the second chromatographic column are independently 0.1-5 BV/h, and the time for removing the impurities is 20-50 min.
Preferably, the weight percentage content of the eicosapentaenoic acid in the fish oil raw material is 40-90%.
Preferably, before the fish oil raw material enters the double-column simulated moving bed for enrichment, the method comprises the steps of dissolving the fish oil raw material to obtain a fish oil solution; the reagent for dissolving the fish oil raw material is consistent with the mobile phase of the double-column simulated moving bed.
Preferably, the concentration of the fish oil raw material in the fish oil solution is 100-1000 mL/L.
Preferably, the alcohol in the alcohol aqueous solution is C 1 ～C 4 Alcohols; said C is 1 ～C 4 The alcohols include one or more of methanol, ethanol, and isopropanol.
Preferably, the volume fraction of water in the alcohol aqueous solution is 2-10%.
Preferably, the first and second chromatography columns are C8 chromatography columns, C16 chromatography columns or C18 chromatography columns.
Preferably, the particle size of the filler in the first chromatographic column and the second chromatographic column is 10-50 μm.
Preferably, the flow rate of the reagent collected by the second chromatographic column is 0.1-5 BV/h.
The invention provides an enrichment method of eicosapentaenoic acid in fish oil raw materials, which comprises the following steps: feeding the fish oil raw material into a double-column simulated moving bed for enrichment; repeating the enrichment process; the repetition times are more than or equal to 3 times; the double-column simulated moving bed comprises a first chromatographic column and a second chromatographic column; the mobile phase of the double-column simulated moving bed is an alcohol aqueous solution; the enrichment comprises the steps of sequentially carrying out sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and post-impurity discharge; the loading parameters include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of the sample loading is 0.1-3 BV/h, and the sample loading time is 1-30 min; the parameters of the layup include: the first chromatographic column and the second chromatographic column are connected in series, the reagent of the spreading layer is consistent with the mobile phase, the flow rate of the reagent of the spreading layer is 0.1-5 BV/h, and the time of spreading the layer is 15-40 min; the parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the first internal circulation is 0.1-5 BV/h, and the time of the first internal circulation is 20-40 min; the loading-collection parameters include: the first chromatographic column and the second chromatographic column are in an independent state, and the sample loading-collection comprises first chromatographic column sample loading and second chromatographic column collection; the flow rate of the first chromatographic column sample loading is 0.1-3 BV/h, and the sample loading-collecting time is 1-30 min; the parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the second internal circulation is 0.1-5 BV/h, and the time of the second internal circulation is 5-20 min; the parameters of post-draining impurities include: the first chromatographic column and the second chromatographic column are in an independent state, and the post-column impurity removal comprises a first chromatographic column post-column impurity removal and a second chromatographic column post-column impurity removal; and the flow rate of the reagent for removing the post-impurity of the first chromatographic column and the flow rate of the reagent for removing the post-impurity of the second chromatographic column are respectively 0.1-5 BV/h, and the time for removing the post-impurity is 20-50 min. The enrichment method provided by the invention can separate EPA with the purity of more than or equal to 98.5% from the fish oil raw material, has the advantages of small solvent dosage and high yield, and is convenient for realizing automatic and continuous production.
Drawings
FIG. 1 is a schematic structural diagram of a two-column simulated moving bed;
FIG. 2 is a schematic view showing the connection of a first chromatographic column and a second chromatographic column in the processes of loading, spreading, first internal circulation, loading-collection, second internal circulation and post-impurity removal;
FIG. 3 is a liquid chromatogram of EPA obtained in example 1.
Detailed Description
The invention provides a method for enriching eicosapentaenoic acid in fish oil raw materials, which comprises the following steps:
feeding the fish oil raw material into a double-column simulated moving bed for enrichment;
repeating the enrichment process;
the repetition times are more than or equal to 3 times.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The invention enriches the fish oil raw material in a double-column simulated moving bed.
In the invention, the weight percentage content of eicosapentaenoic acid in the fish oil raw material is preferably 40-90%, and more preferably 60-80%.
In the invention, before the fish oil raw material enters the double-column simulated moving bed for enrichment, the fish oil raw material is preferably dissolved to obtain a fish oil solution. In the invention, the reagent for dissolving the fish oil raw material is consistent with the mobile phase of the double-column simulated moving bed; the mobile phase of the two-column simulated moving bed is described later. In the invention, the concentration of the fish oil raw material in the fish oil solution is preferably 100-1000 mL/L, and more preferably 100-500 mL/L.
In the present invention, a schematic structure of the two-column simulated moving bed is shown in fig. 1. By controlling the valves or pumps in the dual-column simulated moving bed shown in fig. 1, sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and impurity removal are realized.
In the present invention, the two-column simulated moving bed includes a first chromatography column and a second chromatography column. In the present invention, the first chromatography column and the second chromatography column are preferably a C8 chromatography column, a C16 chromatography column, or a C18 chromatography column, and more preferably a C18 chromatography column. In the present invention, the particle size of the filler in the first chromatographic column and the second chromatographic column is preferably 10 to 50 μm.
In the invention, the mobile phase of the double-column simulated moving bed is an alcohol aqueous solution. In the present invention, the alcohol in the alcohol aqueous solution is preferably a C1-C4 alcohol. In the present invention, the C1 to C4 alcohols preferably include one or more of methanol, ethanol, and isopropanol, and more preferably methanol. In the invention, the volume fraction of water in the alcohol aqueous solution is preferably 2-10%, and more preferably 5-10%.
In the invention, the enrichment comprises the steps of sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and impurity discharge in sequence.
In the present invention, the loading parameters include: the first chromatographic column and the second chromatographic column are connected in series. In the invention, the flow speed of the sample loading is 0.1-3 BV/h, preferably 1-2 BV/h. In the invention, the sample loading time is 1-30 min, preferably 5-15 min. In the present invention, the connection manner of the first chromatographic column and the second chromatographic column in the loading process is as shown in fig. 2.
In the present invention, the parameters of the spread layer include: the first chromatography column and the second chromatography column are preferably connected in series. In the present invention, the reagent of the spreading layer is in accordance with the flow of the two-column simulated moving bed. In the invention, the flow speed of the reagent for spreading the layer is 0.1-5 BV/h, preferably 1-3 BV/h. In the invention, the time for spreading the layer is 15-40 min, preferably 20-30 min. In the present invention, in the process of layer spreading, the connection manner of the first chromatographic column and the second chromatographic column is as shown in fig. 2.
In the present invention, the parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are preferably connected end to form a closed loop. In the invention, the flow rate of the first internal circulation is 0.1-5 BV/h. In the invention, the time of the first internal circulation is 20-40 min, preferably 25-30 min. In the present invention, the connection manner of the first chromatographic column and the second chromatographic column in the process of the first internal cycle is as shown in fig. 2.
In the present invention, the parameters of the sample-collection include: the first chromatographic column and the second chromatographic column are in an independent state. In the present invention, the loading-collecting comprises a first chromatographic column loading and a second chromatographic column collecting. In the invention, the flow rate of the first chromatographic column sample loading is 0.1-3 BV/h, preferably 1-2 BV/h. In the invention, the flow rate of the reagent collected by the second chromatographic column is preferably 0.1-5 BV/h, and more preferably 1-3 BV/h. In the present invention, the reagent collected by the second chromatographic column is preferably in accordance with the mobile phase of the two-column simulated moving bed. In the invention, the time for loading and collecting is 1-30 min, preferably 5-15 min. In the present invention, the connection manner of the first chromatographic column and the second chromatographic column in the sample loading-collecting process is shown in fig. 2.
In the present invention, the parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop. In the invention, the flow rate of the second internal circulation is 0.1-5 BV/h. In the invention, the time of the second internal circulation is 5-20 min, preferably 5-15 min. In the present invention, the connection mode of the first chromatographic column and the second chromatographic column during the second internal cycle is as shown in fig. 2.
In the present invention, the parameters of post-exclusion include: the first chromatographic column and the second chromatographic column are in an independent state. In the present invention, the post-row impurity includes a first column post-row impurity and a second column post-row impurity. In the invention, the reagents of the first chromatographic column row impurity and the second chromatographic column row impurity are consistent with the mobile phase of the double-column simulated moving bed. In the invention, the flow rate of the reagent mixed after the first chromatographic column and the flow rate of the reagent mixed after the second chromatographic column are independently 0.1-5 BV/h. In the invention, the time for removing the impurities is 20-50 min. In the process of removing the post impurity, the connection mode of the first chromatographic column and the second chromatographic column is shown in fig. 2.
In the present invention, the process of enrichment is repeated; the repetition times are more than or equal to 3 times; namely repeating the enrichment process three times, and then collecting the EPA product with the purity more than or equal to 98.5 in the sample loading-collecting process.
In the invention, in order to realize automatic and continuous production, the process of repeatedly enriching for more than or equal to 3 times can be realized.
The method for enriching eicosapentaenoic acid in fish oil raw material provided by the present invention is described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The fish oil raw material with the EPA content of 80% by mass is diluted by methanol water (mobile phase) with the volume fraction of 92% to be 500 mL/L.
Feeding the fish oil solution into a double-column simulated moving bed for enrichment;
the double-column simulated moving bed has 2 chromatographic columns, namely a first chromatographic column and a second chromatographic column, wherein the sizes of the first chromatographic column and the second chromatographic column are both 250mm multiplied by 10mm, and the packing is C18 with the particle size of 10 mu m.
The enrichment comprises the steps of sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and impurity discharge in sequence.
The parameters of the sample loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of a material pump for loading is 1mL/min, and the time is 8 min.
The parameters of the layup include: the first chromatographic column and the second chromatographic column are connected in series, the reagent for spreading is the methanol-water with the volume fraction of 92%, the flow rate of the methanol-water with the volume fraction of 92% is 3mL/min, and the time for spreading is 23 min.
The parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the circulating pump is 3mL/min, and the time is 25 min.
The parameters of the loading-collection include: the first chromatographic column and the second chromatographic column are in an independent state, and comprise a first chromatographic column sample loading and a second chromatographic column collection, wherein the material pump flow rate of the first chromatographic column sample loading is 1 mL/min; the flow rate of the mobile phase (the volume fraction of which is 92 percent methanol water) collected by the second chromatographic column is 2 mL/min; the time for sample loading and collection is 8 min; EPA is shown in the second chromatographic column.
The parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the circulating pump is 3mL/min, and the time is 10 min.
Parameters for post-exclusion include: the first chromatographic column and the second chromatographic column are in an independent state and comprise first chromatographic column row back impurities and second chromatographic column row back impurities, reagents of the first chromatographic column row back impurities and the second chromatographic column row back impurities are methanol water with the volume fraction of 92%, the flow rate of the reagents of the first chromatographic column row back impurities is 0.3mL/min, and the flow rate of the reagents of the second chromatographic column row back impurities is 3 mL/min; the time for impurity removal is 40 min.
During the repetition of the two enrichments, the system reached equilibrium.
In the third cycle enrichment process, namely the sample loading-enrichment process, an EPA methanol aqueous solution is obtained at an extraction port of the second chromatographic column, and impurities are collected at a raffinate port; analysis was performed by high performance liquid chromatography.
Determining EPA content by high performance liquid chromatography, wherein the liquid phase conditions comprise:
waters model 2695 high performance liquid chromatography; model 2698 DAD detector; chromatographic column ODS-25 μm, 250mm 4.6 mm; mobile phase acetonitrile-water solution (90: 10); the flow rate is 1 mL/min; the column temperature is 30 ℃; the sample volume is 20 mu L; the EPA content is determined by an external standard method.
The resulting liquid chromatogram is shown in FIG. 3. Specific chromatographic information is shown in table 1.
TABLE 1 specific chromatographic information
| Retention time (min) | Peak area (pA. s) | Peak area% | Start time (min) |
| 17.162 | 7.335 | 0.070 | 17.012 |
| 18.203 | 8.672 | 0.083 | 17.994 |
| 18.802 | 10353.061 | 99.038 | 18.339 |
| 19.006 | 17.873 | 0.171 | 18.923 |
| 19.244 | 12.881 | 0.123 | 19.151 |
| 19.377 | 31.174 | 0.298 | 19.294 |
| 20.325 | 2.157 | 0.021 | 20.224 |
| 20.633 | 5.791 | 0.055 | 20.436 |
| 21.162 | 6.269 | 0.060 | 21.012 |
| 21.391 | 1.478 | 0.014 | 21.301 |
As can be seen from fig. 3 and table 1: according to the calculation of collected extract and the raw materials, the purity of the EPA of the experiment is 98.5 percent, and the yield is 90.53 percent.
Example 2
And (3) not diluting and loading the fish oil raw material with the weight percentage of EPA of 80%.
Feeding the fish oil raw material into a double-column simulated moving bed for enrichment;
the double-column simulated moving bed has 2 chromatographic columns, namely a first chromatographic column and a second chromatographic column, wherein the sizes of the first chromatographic column and the second chromatographic column are both 250mm multiplied by 10mm, and the packing is C18 with the particle size of 10 mu m.
The enrichment comprises the steps of sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and impurity discharge in sequence.
The parameters of the sample loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of a material pump for loading is 0.3mL/min, and the time is 9 min.
The parameters of the layup include: the first chromatographic column and the second chromatographic column are connected in series, the reagent for spreading is the methanol water with the volume fraction of 92%, the flow rate of the methanol water with the volume fraction of 92% is 3mL/min, and the time for spreading is 24 min.
The parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the circulating pump is 3mL/min, and the time is 26 min.
The parameters of the loading-collection include: the first chromatographic column and the second chromatographic column are in an independent state and comprise a first chromatographic column sample feeding and a second chromatographic column collection, the material pump flow rate of the first chromatographic column sample feeding is 0.3mL/min, the flow rate of a mobile phase (the volume fraction is 92% methanol water) collected by the second chromatographic column is 1.8mL/min, the sample feeding-collection time is 9min, and EPA is obtained by the second chromatographic column.
The parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the circulating pump is 3mL/min, and the time is 12 min.
Parameters for post-exclusion include: the first chromatographic column and the second chromatographic column are in an independent state and comprise first chromatographic column row back impurities and second chromatographic column row back impurities, reagents of the first chromatographic column row back impurities and the second chromatographic column row back impurities are methanol water with the volume fraction of 92%, the flow rate of the reagents of the first chromatographic column row back impurities is 0.3mL/min, the flow rate of the reagents of the second chromatographic column row back impurities is 3mL/min, and the time for discharging the back impurities is 35 min.
During the repetition of the two enrichments, the system reached equilibrium.
In the third cycle enrichment process, namely the sample loading-enrichment process, an EPA methanol aqueous solution is obtained at an extraction port of the second chromatographic column, and impurities are collected at a raffinate port; analysis was performed by high performance liquid chromatography.
As a result, the EPA purity was 98.5% and the EPA yield was 98%.
Example 3
The fish oil raw material with the EPA content of 80% by mass is diluted by methanol water (mobile phase) with the volume fraction of 92% to be 300 mL/L.
Feeding the fish oil solution into a double-column simulated moving bed for enrichment;
the double-column simulated moving bed has 2 chromatographic columns, namely a first chromatographic column and a second chromatographic column, wherein the sizes of the first chromatographic column and the second chromatographic column are both 250mm multiplied by 10mm, and the packing is C18 with the particle size of 10 mu m.
The enrichment comprises the steps of sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and impurity discharge in sequence.
The parameters of the sample loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of a material pump for loading is 2mL/min, and the time is 10 min.
The parameters of the layup include: the first chromatographic column and the second chromatographic column are connected in series, the reagent for spreading is the methanol water with the volume fraction of 92%, the flow rate of the methanol water with the volume fraction of 92% is 3mL/min, and the time for spreading is 24 min.
The parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the circulating pump is 3mL/min, and the time is 22 min.
The parameters of the loading-collection include: the first chromatographic column and the second chromatographic column are in an independent state and comprise a first chromatographic column sample and a second chromatographic column collection; the flow rate of a material pump for loading the material of the first chromatographic column is 2mL/min, the flow rate of a mobile phase (the volume fraction of the methanol water is 92%) collected by the second chromatographic column is 1.5mL/min, the time for loading-collecting is 10min, and the EPA flows out from the mobile phase in the second chromatographic column.
The parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the circulating pump is 3mL/min, and the time is 13 min.
Parameters for post-exclusion include: the first chromatographic column and the second chromatographic column are in an independent state and comprise first chromatographic column row back impurities and second chromatographic column row back impurities, reagents of the first chromatographic column row back impurities and the second chromatographic column row back impurities are methanol water with the volume fraction of 92%, the flow rate of the reagents of the first chromatographic column row back impurities is 0.25mL/min, the flow rate of the reagents of the second chromatographic column row back impurities is 3mL/min, and the time for discharging the back impurities is 45 min.
During the repetition of the two enrichments, the system reached equilibrium.
In the third cycle enrichment process, namely the sample loading-enrichment process, an EPA methanol aqueous solution is obtained at an extraction port of the second chromatographic column, and impurities are collected at a raffinate port; analysis was performed by high performance liquid chromatography.
As a result, the EPA purity was 98.5% and the EPA yield was 80%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. The method for enriching eicosapentaenoic acid in the fish oil raw material is characterized by comprising the following steps:
feeding the fish oil raw material into a double-column simulated moving bed for enrichment;
repeating the enrichment process;
the repetition times are more than or equal to 3 times;
the double-column simulated moving bed comprises a first chromatographic column and a second chromatographic column;
the mobile phase of the double-column simulated moving bed is an alcohol aqueous solution;
the enrichment comprises the steps of sequentially carrying out sample loading, layer spreading, first internal circulation, sample loading-collection, second internal circulation and post impurity removal;
the parameters of the sample loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of the sample loading is 0.1-3 BV/h, and the sample loading time is 1-30 min;
the parameters of the layup include: the first chromatographic column and the second chromatographic column are connected in series, the reagent of the spreading layer is consistent with the mobile phase, the flow speed of the reagent of the spreading layer is 0.1-5 BV/h, and the time of the spreading layer is 15-40 min;
the parameters of the first inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the first internal circulation is 0.1-5 BV/h, and the time of the first internal circulation is 20-40 min;
the loading-collection parameters include: the first chromatographic column and the second chromatographic column are in an independent state, and the sample loading-collection comprises first chromatographic column sample loading and second chromatographic column collection; the flow rate of the sample loading of the first chromatographic column is 0.1-3 BV/h, and the time of sample loading-collection is 1-30 min;
the parameters of the second inner loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow rate of the second internal circulation is 0.1-5 BV/h, and the time of the second internal circulation is 5-20 min;
the parameters of post-draining impurities include: the first chromatographic column and the second chromatographic column are in an independent state, and the post-column impurity removal comprises a first chromatographic column post-column impurity removal and a second chromatographic column post-column impurity removal; the reagent for removing the impurities in the first chromatographic column and the reagent for removing the impurities in the second chromatographic column are consistent with the mobile phase, the flow rate of the reagent for removing the impurities in the first chromatographic column and the flow rate of the reagent for removing the impurities in the second chromatographic column are independently 0.1-5 BV/h, and the time for removing the impurities is 20-50 min.
2. The enrichment method according to claim 1, wherein the eicosapentaenoic acid is present in the fish oil feedstock in an amount of 40 to 90% by weight.
3. The enrichment method of claim 1, wherein before the fish oil raw material enters the double-column simulated moving bed for enrichment, the method comprises the steps of dissolving the fish oil raw material to obtain a fish oil solution; the reagent for dissolving the fish oil raw material is consistent with the mobile phase of the double-column simulated moving bed.
4. The enrichment method according to claim 3, wherein the concentration of the fish oil raw material in the fish oil solution is 100-1000 mL/L.
5. The method according to claim 1, wherein the alcohol in the aqueous alcohol solution is C 1 ～C 4 Alcohols; said C is 1 ～C 4 The alcohols include one or more of methanol, ethanol, and isopropanol.
6. The enrichment method according to claim 1 or 5, wherein the volume fraction of water in the alcohol aqueous solution is 2-10%.
7. The enrichment method of claim 1, wherein the first and second chromatography columns are C8, C16 or C18 chromatography columns.
8. The enrichment method according to claim 7, wherein the particle size of the filler in the first chromatographic column and the second chromatographic column is 10 to 50 μm.
9. The enrichment method according to claim 1, wherein the flow rate of the collected reagent in the second chromatographic column is 0.1-5 BV/h.
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| CN112592268A (en) * | 2020-12-18 | 2021-04-02 | 江苏汉邦科技有限公司 | Method for separating EPA (eicosapentaenoic acid) in fish oil by using continuous chromatographic system |
| WO2024000977A1 (en) * | 2022-07-01 | 2024-01-04 | 江苏汉邦科技股份有限公司 | Enrichment method for eicosapentaenoic acid in fish oil raw material |
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