CN115010596B - Enrichment method of eicosapentaenoic acid in fish oil raw material - Google Patents
Enrichment method of eicosapentaenoic acid in fish oil raw material Download PDFInfo
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- 235000021323 fish oil Nutrition 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 50
- 235000020673 eicosapentaenoic acid Nutrition 0.000 title claims abstract description 39
- 239000002994 raw material Substances 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
- 239000012535 impurity Substances 0.000 claims abstract description 51
- 238000011068 loading method Methods 0.000 claims abstract description 42
- 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 20
- 239000007864 aqueous solution Substances 0.000 claims abstract description 11
- 238000007599 discharging Methods 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 230000008569 process Effects 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 150000001298 alcohols Chemical class 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- -1 C 4 Alcohols Chemical class 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 9
- 238000000746 purification Methods 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000010924 continuous production Methods 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 abstract description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 14
- 239000012071 phase Substances 0.000 description 13
- 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
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000012856 packing Methods 0.000 description 5
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 4
- 229940090949 docosahexaenoic acid Drugs 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000006073 displacement reaction Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000002881 Colic Diseases 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 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
- 230000006978 adaptation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000003026 cod liver oil Substances 0.000 description 1
- 235000012716 cod liver oil Nutrition 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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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- Chemical & Material Sciences (AREA)
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- Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of material purification, and particularly provides a method for enriching eicosapentaenoic acid in fish oil raw materials. The enrichment method provided by the invention comprises the following steps: feeding fish oil raw materials into a double-column simulated moving bed to sequentially perform loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging; 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 purity more than or equal to 98.5% from the fish oil raw material, and has the advantages of less solvent consumption, high yield and convenience in realizing automatic and continuous production.
Description
Technical Field
The invention relates to the technical field of material 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 polyunsaturated fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) contained in the fish oil have the functions of inhibiting platelet aggregation, resisting thrombus, dilating blood vessels, regulating blood fat, improving the fluidity of biological membranes and the like, and have good curative effects on treating and preventing cardiovascular diseases, diabetes, dermatitis, colic ulcers, tumor inhibition and the like.
At present, the method for separating and purifying the fish oil comprises a urea inclusion method, a low-temperature freezing method, a metal salt precipitation method, a saponification-urea inclusion method, fish oil transesterification, a vacuum distillation method, a supercritical extraction method and the like. However, because the EPA/DHA components contained in the domestic fish oil raw materials are greatly different, and the level of the separation and purification method provided by the method is not high, the quality of the final product is good and is not uniform, and the method is specifically characterized in that: the content of EPA or DHA in the domestic cod liver oil or emulsion product is low, the market competitiveness is not strong and the economic benefit is not high. Thus, there is a need to provide a purification process for 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 a fish oil raw material. The purity of EPA obtained by the enrichment method is more than or equal to 98.5 percent.
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 repeated 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 loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging in sequence;
the loading parameters include: the first chromatographic column and the second chromatographic column are connected in series, the sample loading flow rate is 0.1-3 BV/h, and the sample loading time is 1-30 min;
parameters of the display layer 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 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 speed 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-collecting parameters include: the first chromatographic column and the second chromatographic column are in independent states, and the loading-collecting comprises loading of the first chromatographic column and collecting of the second chromatographic column; the flow rate of the sample on the first chromatographic column is 0.1-3 BV/h, and the sample loading-collecting time is 1-30 min;
the parameters of the second internal loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow speed 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 the impurity removal include: the first chromatographic column and the second chromatographic column are in independent states, and the impurities after discharge comprise impurities after discharge of the first chromatographic column and impurities after discharge of the second chromatographic column; the flow rate of the first chromatographic column discharged impurity reagent and the flow rate of the second chromatographic column discharged impurity reagent are respectively 0.1-5 BV/h, and the time of the second chromatographic column discharged impurity is 20-50 min.
Preferably, the weight percentage of 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 fish oil raw material dissolving reagent is consistent with the flow 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 alcohols in the aqueous alcohol solution are C 1 ～C 4 Alcohols; the 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 chromatographic columns are C8, C16 or C18 chromatographic columns.
Preferably, the particle size of the packing in the first and second chromatographic columns 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 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 repeated 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 loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging in sequence; the loading parameters include: the first chromatographic column and the second chromatographic column are connected in series, the sample loading flow rate is 0.1-3 BV/h, and the sample loading time is 1-30 min; parameters of the display layer 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 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 speed 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-collecting parameters include: the first chromatographic column and the second chromatographic column are in independent states, and the loading-collecting comprises loading of the first chromatographic column and collecting of the second chromatographic column; the flow rate of the sample on the first chromatographic column is 0.1-3 BV/h, and the sample loading-collecting time is 1-30 min; the parameters of the second internal loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow speed 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 the impurity removal include: the first chromatographic column and the second chromatographic column are in independent states, and the impurities after discharge comprise impurities after discharge of the first chromatographic column and impurities after discharge of the second chromatographic column; the flow rate of the first chromatographic column discharged impurity reagent and the flow rate of the second chromatographic column discharged impurity reagent are respectively 0.1-5 BV/h, and the time of the second chromatographic column discharged impurity is 20-50 min. The enrichment method provided by the invention can separate EPA with purity more than or equal to 98.5% from the fish oil raw material, and has the advantages of less solvent consumption, high yield and convenience in realizing automatic and continuous production.
Drawings
FIG. 1 is a schematic diagram of a structure of a double-column simulated moving bed;
FIG. 2 is a schematic diagram showing the connection of a first chromatographic column and a second chromatographic column during loading, spreading, first internal recycle, loading-collecting, second internal recycle 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 repeating times are more than or equal to 3 times.
In the present invention, the raw 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 present invention, the mass percentage of eicosapentaenoic acid in the fish oil raw material is preferably 40 to 90%, more preferably 60 to 80%.
In the present invention, the fish oil raw material is preferably dissolved to obtain a fish oil solution before entering the double-column simulated moving bed for enrichment. In the invention, the reagent for dissolving the fish oil raw material is consistent with the flow of the double-column simulated moving bed; the mobile phase of the double column simulated moving bed will be described later. In the present invention, the concentration of the fish oil raw material in the fish oil solution is preferably 100 to 1000mL/L, more preferably 100 to 500mL/L.
In the invention, the structural schematic diagram of the double-column simulated moving bed is shown in fig. 1. By controlling valves or pumps in the double-column simulated moving bed shown in fig. 1, loading, spreading, first internal circulation, loading-collecting, second internal circulation and post-impurity discharging are realized.
In the present invention, the dual column simulated moving bed comprises a first chromatographic column and a second chromatographic column. In the present invention, the first and second chromatographic columns are preferably C8 chromatographic columns, C16 chromatographic columns or C18 chromatographic columns, and further preferably C18 chromatographic columns. In the present invention, the particle size of the packing in the first and second columns 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 alcohols in the aqueous alcohol solution are preferably C1-C4 alcohols. 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 present invention, the volume fraction of water in the aqueous alcohol solution is preferably 2 to 10%, more preferably 5 to 10%.
In the present invention, the enrichment includes sequentially loading, spreading, first internal circulation, loading-collecting, second internal circulation and post-discharge impurities.
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 rate of the loaded sample is 0.1-3 BV/h, preferably 1-2 BV/h. In the present invention, the loading time is 1 to 30 minutes, preferably 5 to 15 minutes. In the present invention, the connection manner of the first chromatographic column and the second chromatographic column in the loading process is shown in fig. 2.
In the present invention, the parameters of the spreading layer include: the first and second chromatographic columns are preferably connected in series. In the present invention, the reagent of the spread layer is consistent with the flow of the double column simulated moving bed. In the present invention, the flow rate of the reagent for the spreading is 0.1 to 5BV/h, preferably 1 to 3BV/h. In the present invention, the time for spreading the layer is 15 to 40min, preferably 20 to 30min. In the process of the spreading, the connection mode of the first chromatographic column and the second chromatographic column is shown in fig. 2.
In the present invention, the parameters of the first internal 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 present invention, the time of the first internal circulation is 20 to 40 minutes, preferably 25 to 30 minutes. In the present invention, in the process of the first internal circulation, the connection manner of the first chromatographic column and the second chromatographic column is shown in fig. 2.
In the present invention, the loading-collecting parameters include: the first chromatographic column and the second chromatographic column are in independent states. In the present invention, the loading-collecting includes loading onto a first chromatographic column and collecting onto a second chromatographic column. In the invention, the flow rate of the first chromatographic column is 0.1-3 BV/h, preferably 1-2 BV/h. In the present invention, the flow rate of the reagent collected by the second column is preferably 0.1 to 5BV/h, more preferably 1 to 3BV/h. In the present invention, the reagent collected by the second chromatographic column preferably coincides with the flow of the dual column simulated moving bed. In the present invention, the loading-collecting time is 1 to 30 minutes, preferably 5 to 15 minutes. In the process of loading and collecting, the connection mode of the first chromatographic column and the second chromatographic column is shown in fig. 2.
In the present invention, the parameters of the second internal 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 present invention, the second internal circulation time is 5 to 20 minutes, preferably 5 to 15 minutes. In the present invention, in the process of the second internal circulation, the connection manner of the first chromatographic column and the second chromatographic column is shown in fig. 2.
In the present invention, the parameters of the post-impurity removal include: the first chromatographic column and the second chromatographic column are in independent states. In the present invention, the post-displacement impurities include a first column post-displacement impurity and a second column post-displacement impurity. In the invention, the reagent of the first chromatographic column and the reagent of the second chromatographic column are consistent with the flow of the double-column simulated moving bed. In the present invention, the flow rate of the reagent for the first column chromatography and the flow rate of the reagent for the second column chromatography are independently 0.1 to 5BV/h. In the invention, the time for removing the impurities is 20-50 min. In the process of removing the impurities, the connection mode of the first chromatographic column and the second chromatographic column is shown in fig. 2.
In the present invention, the enrichment process is repeated; the repeated times are more than or equal to 3 times; namely, the EPA product with the purity more than or equal to 98.5 can be obtained by repeating the enrichment process for three times in the loading-collecting process.
In the invention, in order to realize automatic and continuous production, the enrichment process can be realized by repeating the enrichment process for a plurality of times (more than or equal to 3 times).
The method for enriching eicosapentaenoic acid in the fish oil raw material provided by the invention is described in detail below with reference to examples, but they should not be construed as limiting the scope of the invention.
Example 1
The fish oil raw material with EPA mass percent of 80% is diluted with 92% methanol water (mobile phase) to be 500mL/L.
The fish oil solution enters 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 250mm multiplied by 10mm, and the packing is C18 with the particle size of 10 mu m.
The enrichment comprises loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging in sequence.
The parameters of loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of a loading material pump is 1mL/min, and the time is 8min.
Parameters of the display layer include: the first chromatographic column and the second chromatographic column are connected in series, the volume fraction of the reagent of the spreading layer is 92% of methanol water, the flow rate of the volume fraction of 92% of methanol water is 3mL/min, and the time of the spreading layer is 23min.
The parameters of the first internal 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 25min.
The loading-collection parameters included: the first chromatographic column and the second chromatographic column are in independent states, and comprise loading of the first chromatographic column and collection of the second chromatographic column, wherein the flow rate of a material pump for loading of the first chromatographic column is 1mL/min; the flow rate of the mobile phase (92% methanol water by volume) collected by the second chromatographic column is 2mL/min; the sample loading-collecting time is 8min; EPA was removed from the second column.
Parameters of the second internal 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 10min.
Parameters of the impurities after the discharge include: the first chromatographic column and the second chromatographic column are in independent states, and comprise first chromatographic column impurity and second chromatographic column impurity, the first chromatographic column impurity and the second chromatographic column impurity reagent are 92% of methanol water in volume fraction, the flow rate of the first chromatographic column impurity reagent is 0.3mL/min, and the flow rate of the second chromatographic column impurity reagent is 3mL/min; the time for removing impurities is 40min.
The system reached equilibrium during the two enrichment procedures.
In the third cycle enrichment process, namely the loading-enrichment process, 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.
EPA content is determined by high performance liquid chromatography, and the liquid phase conditions include:
waters2695 type high performance liquid chromatography; 2698 type DAD detector; column ODS-25 μm,250mm 4.6mm; mobile phase acetonitrile-water solution (90:10); the flow rate is 1mL/min; column temperature is 30 ℃; the sample injection amount is 20 mu L; the EPA content was determined by the 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: and according to the collected extract and the fed raw materials, the EPA purity of the experiment is 98.5%, and the yield is 90.53%.
Example 2
The fish oil raw material with the EPA mass percent of 80% is not diluted and loaded.
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 250mm multiplied by 10mm, and the packing is C18 with the particle size of 10 mu m.
The enrichment comprises loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging in sequence.
The parameters of loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of a loading material pump is 0.3mL/min, and the time is 9min.
Parameters of the display layer include: the first chromatographic column and the second chromatographic column are connected in series, the volume fraction of the reagent of the spreading layer is 92% of methanol water, the flow rate of the volume fraction of 92% of methanol water is 3mL/min, and the time of the spreading layer is 24min.
The parameters of the first internal 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 26min.
The loading-collection parameters included: the first chromatographic column and the second chromatographic column are in independent states, the first chromatographic column loading and the second chromatographic column collecting are included, the flow rate of a material pump for the first chromatographic column loading is 0.3mL/min, the flow rate of a mobile phase (92% methanol water by volume) collected by the second chromatographic column is 1.8mL/min, the loading-collecting time is 9min, and EPA is discharged by the second chromatographic column.
Parameters of the second internal 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 12min.
Parameters of the impurities after the discharge include: the first chromatographic column and the second chromatographic column are in independent states, the first chromatographic column is arranged with the second chromatographic column, the reagent of the first chromatographic column is arranged with the second chromatographic column is arranged with the impurity, the volume fraction of the reagent of the first chromatographic column is 92% methanol water, the flow rate of the reagent of the first chromatographic column is 0.3mL/min, the flow rate of the reagent of the second chromatographic column is arranged with the second chromatographic column is 3mL/min, and the time of the second chromatographic column is 35min.
The system reached equilibrium during the two enrichment procedures.
In the third cycle enrichment process, namely the loading-enrichment process, 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.
The result was 98.5% EPA purity and 98% EPA yield.
Example 3
The fish oil raw material with EPA mass percent of 80% is diluted with 92% methanol water (mobile phase) to 300mL/L.
The fish oil solution enters 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 250mm multiplied by 10mm, and the packing is C18 with the particle size of 10 mu m.
The enrichment comprises loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging in sequence.
The parameters of loading include: the first chromatographic column and the second chromatographic column are connected in series, the flow rate of a loading material pump is 2mL/min, and the time is 10min.
Parameters of the display layer include: the first chromatographic column and the second chromatographic column are connected in series, the volume fraction of the reagent of the spreading layer is 92% of methanol water, the flow rate of the volume fraction of 92% of methanol water is 3mL/min, and the time of the spreading layer is 24min.
The parameters of the first internal 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 22min.
The loading-collection parameters included: the first chromatographic column and the second chromatographic column are in independent states, and the first chromatographic column loading and the second chromatographic column collecting are included; the flow rate of a material pump for loading the first chromatographic column material is 2mL/min, the flow rate of a mobile phase (92% methanol water by volume fraction) collected by the second chromatographic column is 1.5mL/min, the loading-collecting time is 10min, and the second chromatographic column enters the mobile phase and exits EPA.
Parameters of the second internal 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 13min.
Parameters of the impurities after the discharge include: the first chromatographic column and the second chromatographic column are in independent states, the first chromatographic column is arranged with the second chromatographic column, the reagent of the first chromatographic column is arranged with the second chromatographic column, the volume fraction of the reagent of the first chromatographic column is 92% methanol water, the flow rate of the reagent of the first chromatographic column is 0.25mL/min, the flow rate of the second chromatographic column is 3mL/min, and the time of the second chromatographic column is 45min.
The system reached equilibrium during the two enrichment procedures.
In the third cycle enrichment process, namely the loading-enrichment process, 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 merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (1)
1. The enrichment method of 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 repeated 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 loading, spreading, first internal circulation, loading-collecting, second internal circulation and impurity discharging in sequence;
the loading parameters include: the first chromatographic column and the second chromatographic column are connected in series, the sample loading flow rate is 0.1-3 BV/h, and the sample loading time is 1-30 min;
parameters of the display layer include: the first chromatographic column and the second chromatographic column are connected in series, the reagent of the spreading layer is consistent with the flowing phase, the flow velocity 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 speed 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-collecting parameters include: the first chromatographic column and the second chromatographic column are in independent states, and the loading-collecting comprises loading of the first chromatographic column and collecting of the second chromatographic column; the flow rate of the sample on the first chromatographic column is 0.1-3 BV/h, and the sample loading-collecting time is 1-30 min;
the parameters of the second internal loop include: the first chromatographic column and the second chromatographic column are connected end to form a closed loop, the flow speed 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 the impurity removal include: the first chromatographic column and the second chromatographic column are in independent states, and the impurities after discharge comprise impurities after discharge of the first chromatographic column and impurities after discharge of the second chromatographic column; the flow rate of the first chromatographic column discharged impurity reagent and the flow rate of the second chromatographic column discharged impurity reagent are respectively 0.1-5 BV/h, and the time of the second chromatographic column discharged impurity is 20-50 min;
the mass percentage of eicosapentaenoic acid in the fish oil raw material is 40-90%;
before the fish oil raw material enters a 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 fish oil raw material dissolving reagent is consistent with the mobile phase of the double-column simulated moving bed; the concentration of the fish oil raw material in the fish oil solution is 100-1000 mL/L;
the alcohols in the alcohol aqueous solution are C 1 ～C 4 Alcohols;
the C is 1 ～C 4 Alcohols include one or more of methanol, ethanol, and isopropanol;
the volume fraction of water in the alcohol aqueous solution is 2-10%;
the first chromatographic column and the second chromatographic column are C8 chromatographic column, C16 chromatographic column or C18 chromatographic column;
the particle size of the filler in the first chromatographic column and the second chromatographic column is 10-50 mu m;
the flow rate of the reagent collected by the second chromatographic column is 0.1-5 BV/h.
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| PCT/CN2022/129279 WO2024000977A1 (en) | 2022-07-01 | 2022-11-02 | Enrichment method for eicosapentaenoic acid in fish oil raw material |
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| CN115010596B (en) * | 2022-07-01 | 2024-01-30 | 江苏汉邦科技股份有限公司 | Enrichment method of eicosapentaenoic acid in fish oil raw material |
| CN118271420A (en) * | 2024-03-08 | 2024-07-02 | 江苏汉邦科技股份有限公司 | GLP-1 analogue purification method |
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