CN116478035A - Preparation method of high-purity unsaturated fatty acid - Google Patents
Preparation method of high-purity unsaturated fatty acid Download PDFInfo
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- 235000021122 unsaturated fatty acids Nutrition 0.000 title claims abstract description 31
- 150000004670 unsaturated fatty acids Chemical class 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- SECPZKHBENQXJG-FPLPWBNLSA-N palmitoleic acid Chemical compound CCCCCC\C=C/CCCCCCCC(O)=O SECPZKHBENQXJG-FPLPWBNLSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 29
- 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 claims abstract description 22
- 235000020673 eicosapentaenoic acid Nutrition 0.000 claims abstract description 22
- 229960005135 eicosapentaenoic acid Drugs 0.000 claims abstract description 22
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000011068 loading method Methods 0.000 claims abstract description 21
- 239000003480 eluent Substances 0.000 claims abstract description 17
- 235000021319 Palmitoleic acid Nutrition 0.000 claims abstract description 15
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 14
- 235000020669 docosahexaenoic acid Nutrition 0.000 claims abstract description 12
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 9
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 8
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 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 claims description 14
- 239000011550 stock solution Substances 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 11
- 229940090949 docosahexaenoic acid Drugs 0.000 claims description 7
- 238000012856 packing Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000004237 preparative chromatography Methods 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 24
- 238000000926 separation method Methods 0.000 abstract description 17
- 238000011084 recovery Methods 0.000 abstract description 6
- DVSZKTAMJJTWFG-SKCDLICFSA-N (2e,4e,6e,8e,10e,12e)-docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(O)=O DVSZKTAMJJTWFG-SKCDLICFSA-N 0.000 abstract description 5
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 abstract description 5
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 abstract description 5
- 238000000746 purification Methods 0.000 abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
- 238000004458 analytical method Methods 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 239000000945 filler Substances 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000000194 fatty acid Substances 0.000 description 4
- 150000004665 fatty acids Chemical class 0.000 description 4
- 235000021323 fish oil Nutrition 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 235000020778 linoleic acid Nutrition 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000034530 PLAA-associated neurodevelopmental disease Diseases 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 238000012435 analytical chromatography Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention relates to a preparation method of high-purity unsaturated fatty acid. The method utilizes a high-efficiency preparation chromatographic method at a low temperature of between 50 ℃ below zero and 15 ℃ to complete separation and purification, the temperature of an eluent, a chromatographic column and a pipeline is controlled to be +/-2 ℃, a C18 bonding stationary phase is adopted, one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or palmitoleic acid (PLA) is used as a raw material, and methanol water is used for eluting, so that a high-purity product with the purity of more than 99% and controllable key impurities can be obtained. The preparation method of the invention has simple operation, high sample loading and high recovery rate, and meets the requirements of preparation of high-purity unsaturated fatty acid and impurity control.
Description
Technical Field
The invention belongs to the technical field of separation, purification and refining of high-purity unsaturated fatty acid, and relates to a preparation method of high-purity unsaturated fatty acid.
Technical Field
Unsaturated fatty acids mainly comprise alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic Acid (LA), gamma-linolenic acid (GLA) and the like, are indispensable nutrient substances for human bodies, play an important role in resisting blood fat, cancer, inflammation and the like, but have limited self-synthesis capacity, and are mainly supplemented by medicinal and edible sources, so that the unsaturated fatty acids have wide application market in the medical and health care field.
The main sources of unsaturated fatty acid are vegetable oil and fish oil, the content of main components in the raw materials is usually low, and pigments, impurities and the like in the raw materials are removed by a separation and purification process, so that the unsaturated fatty acid can be used as raw materials in the fields of health-care foods, medicines and the like.
The high performance liquid chromatography is a chromatographic separation process for obtaining a purified product by collecting chromatographic effluent substances, has the unique advantages of simple operation, various filler selection, good repeatability, large sample loading quantity, industrialization and the like, and has more application in the purification preparation of fatty acid. Patent document CN109438220B discloses a high performance liquid chromatography method for purifying EPA from fish oil, which can obtain a sample having a liquid phase purity of about 97%. Patent document CN105848748A, CN109401850A, CN107586259B describes a liquid chromatography method for purifying unsaturated fatty acids using a simulated moving bed or an improvement on the basis of a simulated moving bed, which is very advantageous for the purification of mixed fatty acids. The method provides good reference and selection for preparing the high-purity fatty acid, but has the defects of insufficient purity, complex method development, high requirement on equipment design and the like. More importantly, with the deep research and the improvement of the regulation requirements, the limit requirements of unsaturated fatty acid products on key impurities are more and more strict, the current chromatographic method has the problems of insufficient separation degree of target components and key impurities, uncontrollable impurity limit and the like, and the insufficient separation degree of main peaks and impurities can also lead to very low sample loading and recovery rate of the preparation chromatograph, which is not beneficial to the amplified production and industrialization.
The temperature is one of important parameters of chromatographic analysis, and has influence on the solubility of a sample, the viscosity of a mobile phase, the surface property of a filler and the like, so that the retention rule and the selectivity can be changed, and the low-temperature condition mainly obtains special selectivity by enhancing the retention and increasing the separation degree. The low temperature condition has many applications in analytical chromatography, such as excellent effect in separation analysis of thermally unstable and poorly stable substances, enhanced selectivity by low temperature conditions in separation of chiral compounds, and studies of retention behavior of small molecular organic matters under extremely low temperature chromatography using liquid carbon dioxide as mobile phase (doi: 10.1002/jssc.201500251.) and the like. However, the high performance liquid chromatography is generally performed at normal temperature, and there is a disadvantage in temperature control. The unsaturated fatty acid contains a plurality of isomer impurities and impurities with similar structures, has higher requirement on separation degree, and has the advantages that the separation degree between a main peak and the impurities can be obviously increased at low temperature, the peak shape is good, and the high-purity sample can be prepared and meets the limit requirement of the impurities.
Disclosure of Invention
The invention provides a preparation method capable of effectively improving the separation degree between a main peak and impurities and controlling the content of key impurities, which mainly aims at the defects of the existing high-performance liquid phase preparation chromatography in the preparation method of high-purity unsaturated fatty acid.
The invention comprises the following contents:
a preparation method of high-purity unsaturated fatty acid adopts a high-efficiency preparation chromatography method to separate and purify raw materials under a low-temperature condition.
As a preferred embodiment of the present invention, the high performance preparative chromatography method comprises the steps of:
s1: taking an unsaturated fatty acid crude product as a raw material to prepare a sample loading stock solution;
s2: loading the loading stock solution in the step S1 into a chromatographic column;
s3: eluting the chromatographic column with an eluent to obtain an eluent;
s4: and (5) sequentially collecting target fractions from the eluent, and concentrating to obtain a pure product.
As a preferable scheme of the invention, the temperature range of the low-temperature condition is-50 ℃ to 15 ℃;
as a preferred scheme of the invention, the low temperature condition requires controlling the temperature of the eluent, the chromatographic column and the connecting pipeline related to the whole preparation method, and the temperature control precision is +/-2 ℃;
in the step S2, the chromatographic packing for the chromatographic column is an n-alkyl bonded silica gel stationary phase; and/or;
the diameter of the chromatographic column is 10-1000mm, and the height is 50-300mm.
As a preferred embodiment of the present invention, in step S1, the unsaturated fatty acid includes one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or palmitoleic acid (PLA).
As a preferable scheme of the invention, in the step S1, the concentration of the loading stock solution is 10-800mg/mL.
In the step S2, the sample loading amount is 0.1-20% of the mass of the filling material in the chromatographic column;
as a preferable scheme of the invention, in the step S3, the eluent is 80-100% methanol water by volume fraction.
As a preferable mode of the invention, in the step S3, the flow rate of the elution is 80-300cm/h.
The invention has the beneficial effects that: the invention takes the unsaturated fatty acid crude product as the raw material, establishes the method for preparing the high-purity unsaturated fatty acid by high-performance liquid chromatography under the low-temperature condition, increases the separation degree between a main peak and key impurities, can obtain the product with high purity and controllable limited amount of key impurities, and can solve the problem that the limited amount of impurities is uncontrollable in the preparation of the fatty acid.
Drawings
FIG. 1 is a comparison of liquid chromatography analyses of eicosapentaenoic acid of example 1 at different temperatures.
FIG. 2 is a gas chromatography chromatogram of the eicosapentaenoic acid product obtained in example 1, the peak having a retention time of about 1.2min being the solvent peak.
FIG. 3 is a comparison of liquid chromatography analysis of docosahexaenoic acid in example 3 at different temperatures.
FIG. 4 is a comparison of liquid chromatography analysis of palmitoleic acid at various temperatures in example 4.
Detailed Description
A preparation method of high-purity unsaturated fatty acid adopts a high-efficiency preparation chromatography method to separate and purify raw materials under a low-temperature condition. The high-efficiency preparation chromatography method comprises the following steps of:
s1: taking an unsaturated fatty acid crude product as a raw material to prepare a sample loading stock solution;
the unsaturated fatty acid comprises one of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or palmitoleic acid (PLA);
the concentration of the sample stock solution is 10-800mg/mL, and the sample stock solution is prepared by using methanol as a solvent.
S2: loading the loading stock solution in the step S1 into a chromatographic column; the loading amount is 0.1-20% of the mass of the filling material in the chromatographic column.
S3: eluting the chromatographic column with an eluent to obtain an eluent; the eluent is methanol water with the volume fraction of 80-100%; the flow rate of the elution is 80-300cm/h.
The chromatographic packing for the chromatographic column is normal alkyl bonded silica gel stationary phase, preferably C18 bonded stationary phase, and the particle size is 5-30 mu m;
the diameter of the chromatographic column is 10-1000mm, and the height is 50-300mm.
S4: and (5) sequentially collecting target fractions from the eluent, and concentrating to obtain a pure product.
The temperature range of the low-temperature condition is-50 ℃ to 15 ℃; specifically, the low temperature may be, but not limited to, -50 ℃, -40 ℃, -30 ℃, -20 ℃, -10 ℃, -5 ℃, 0 ℃,5 ℃,10 ℃,15 ℃. More specifically, the low temperature condition requires control of the temperature of the eluent, the chromatographic column and the connecting lines involved in the whole preparation method, with a temperature control accuracy of ±2 ℃.
The present invention will be described in further detail with reference to specific examples, but embodiments of the present invention are not limited thereto. Experimental conditions and results of unsaturated fatty acids at different temperatures on analytical columns are added in some examples.
The temperature controls involved in the following examples are: the temperature of the connecting pipeline related to the eluent, the chromatographic column and the whole preparation method is controlled to be +/-2 ℃.
Example 1
This example provides a process for the preparation of the unsaturated fatty acid eicosapentaenoic acid (EPA) which comprises essentially the following:
experimental conditions and results for verifying that the main peak and impurity separation degree are improved at low temperature on the analytical column are as follows:
instrument: a high performance liquid chromatograph;
sample: methanol dissolves EPA samples (fish oil source, gas phase purity 74%) to 50mg/mL;
chromatographic column: c18 (4.6X105 mm,10 μm);
mobile phase: 95 (v/v)% methanol;
flow rate: 0.4mL/min;
and (3) temperature control: 25 ℃,5 ℃;
detection wavelength: 210nm;
sample injection amount: 1 μl;
as shown in FIG. 1, the analysis and comparison chart at 25 ℃ and 5 ℃ shows that the main peak and the front and back impurities at 5 ℃ have better separation degree, which is more beneficial to preparing high-purity samples.
The preparation method of the high-purity EPA comprises the following steps: EPA from fish oil is taken as a raw material (gas phase purity is 74%), 15g of the EPA is dissolved to 400mg/mL by methanol to be taken as a sample stock solution; preparing a chromatographic column which is a C18 bonding stationary phase, wherein the particle size is 10 mu m, the diameter is 50mm, the height is 250mm, and the filler mass is 300g; the experimental process temperature is controlled to be 5 ℃,15g of samples are fully injected, the loading amount is 5% of the mass of the chromatographic packing, the elution is carried out by using 95% methanol water, and the elution flow rate is 140cm/h. Collecting target fraction, concentrating, removing eluting solvent to obtain high purity EPA product with purity of above 99% (product gas chromatography is shown in figure 2), and controlling limit of 0.05% and 0.1% impurities, and recovering target peak with recovery rate of above 90%.
Example 2
This example provides a process for the preparation of the unsaturated fatty acid eicosapentaenoic acid (EPA): controlling the temperature of the experimental process to be-25 ℃, eluting with methanol, and other conditions being the same as in example 1; and (3) collecting target fractions, concentrating and removing the eluting solvent to obtain the high-purity EPA product with the gas chromatography detection purity of more than 99% and controllable limited impurities, and the target peak recovery rate of more than 90%.
Example 3
The embodiment provides a preparation method of unsaturated fatty acid docosahexaenoic acid (DHA), which mainly comprises the following steps:
experimental conditions and results for verifying that the main peak and impurity separation degree are improved at low temperature on the analytical column are as follows:
instrument: a high performance liquid chromatograph;
sample: dissolving the DHA sample to 50mg/mL with methanol;
chromatographic column: c18 (4.6X105 mm,10 μm);
mobile phase: 95 (v/v)% methanol;
flow rate: 0.4mL/min;
and (3) temperature control: 60 ℃,25 ℃ and 10 ℃;
detection wavelength: 210nm;
sample injection amount: 1 μl;
as shown in FIG. 3, the analysis and comparison chart at 60 ℃,25 ℃ and 10 ℃ shows that the lower the temperature is, the better the main peak and the front and rear impurities are separated, which is more beneficial to preparing high purity samples.
The preparation method of the high-purity DHA comprises the following steps: taking DHA as a raw material, taking 10g of DHA and dissolving to 400mg/mL by using methanol to serve as a sample loading stock solution; preparing a chromatographic column which is a C18 bonding stationary phase, wherein the particle size is 10 mu m, the diameter is 50mm, the height is 250mm, and the filler mass is 300g; controlling the temperature of the experimental process to be 10 ℃;10g of sample is fully sampled, the loading amount is 3.3% of the mass of the chromatographic packing, and the sample is eluted by 95% methanol water, and the eluting flow rate is 140cm/h; and collecting the target fraction, concentrating and removing the eluting solvent to obtain the high-purity DHA product with the gas chromatography detection purity of more than 99 percent, and the target peak recovery rate of more than 90 percent.
Example 4
The present embodiment provides a method for preparing unsaturated fatty acid palmitoleic acid (PLA), which mainly includes the following:
experimental conditions and results for verifying that the main peak and impurity separation degree are improved at low temperature on the analytical column are as follows:
instrument: a high performance liquid chromatograph;
sample: dissolving the PLA sample to 65mg/mL with methanol;
chromatographic column: c18 (4.6X105 mm,10 μm);
mobile phase: 95 (v/v)% methanol;
flow rate: 0.4mL/min;
and (3) temperature control: 60 ℃,25 ℃ and 10 ℃;
detection wavelength: 210nm;
sample injection amount: 1 μl;
as shown in FIG. 4, the analysis and comparison chart at 60℃and 25℃and 10℃shows that the main peak and the front and rear impurities have better separation degree with the decrease of the temperature from FIG. 4.
The preparation method of the high-purity PLA comprises the following steps: taking PLA as a raw material, taking 20g of the PLA and dissolving the PLA into 400mg/mL of methanol to serve as a sample stock solution; preparing a chromatographic column which is a C18 bonding stationary phase, wherein the particle size is 10 mu m, the diameter is 30mm, the height is 250mm, and the filler mass is 105g; controlling the temperature of the experimental process to be 10 ℃;30g of sample is fully sampled, the loading amount is 19% of the mass of the chromatographic packing, and the sample is eluted by 95% methanol water, and the eluting flow rate is 140cm/h; and collecting the target fraction, concentrating and removing the eluting solvent to obtain the high-purity PLA product with the gas chromatography detection purity of more than 99 percent, and the target peak recovery rate of more than 90 percent.
The foregoing examples have shown only the preferred embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be pointed out that various other corresponding changes and modifications can be made by those skilled in the art in light of the above description of the technical solution and the idea, and all such changes and modifications are intended to be within the scope of the invention as defined in the appended claims.
Claims (10)
1. A preparation method of high-purity unsaturated fatty acid is characterized in that the raw materials are separated and purified by adopting a high-efficiency preparation chromatography method under the low-temperature condition.
2. The method for producing an unsaturated fatty acid according to claim 1, wherein the high performance preparative chromatography method comprises the steps of:
s1: taking an unsaturated fatty acid crude product as a raw material to prepare a sample loading stock solution;
s2: loading the loading stock solution in the step S1 into a chromatographic column;
s3: eluting the chromatographic column with an eluent to obtain an eluent;
s4: and (5) sequentially collecting target fractions from the eluent, and concentrating to obtain a pure product.
3. The method according to claim 1, wherein the low temperature condition is in a temperature range of-50 ℃ to 15 ℃.
4. The method according to claim 1, wherein the low temperature conditions require control of the temperature of the eluent, the chromatographic column and the connecting lines involved in the whole method, with accuracy of temperature control of
±2℃。
5. The method according to claim 2, wherein in step S2, the chromatographic packing for the chromatographic column is an n-alkyl bonded silica gel stationary phase; and/or;
the diameter of the chromatographic column is 10-1000mm, and the height is 50-300mm.
6. The method according to claim 2, wherein in step S1, the unsaturated fatty acid comprises one of eicosapentaenoic acid, docosahexaenoic acid or palmitoleic acid.
7. The method according to claim 2, wherein in step S1, the concentration of the loading stock solution is 10-800mg/mL.
8. The method according to claim 2, wherein in step S2, the loading amount is 0.1 to 20% of the mass of the packing in the column.
9. The method according to claim 2, wherein in step S3, the eluent is 80-100% methanol water by volume fraction.
10. The method according to claim 2, wherein in step S3, the flow rate of the elution is 80-300cm/h.
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