CN113563178B - Separation and purification method of docosahexaenoic acid in fish oil - Google Patents
Separation and purification method of docosahexaenoic acid in fish oil Download PDFInfo
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- CN113563178B CN113563178B CN202110994288.9A CN202110994288A CN113563178B CN 113563178 B CN113563178 B CN 113563178B CN 202110994288 A CN202110994288 A CN 202110994288A CN 113563178 B CN113563178 B CN 113563178B
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- 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 title claims abstract description 152
- 235000020669 docosahexaenoic acid Nutrition 0.000 title claims abstract description 83
- 229940090949 docosahexaenoic acid Drugs 0.000 title claims abstract description 74
- 235000021323 fish oil Nutrition 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 46
- 238000000746 purification Methods 0.000 title claims abstract description 28
- 238000000926 separation method Methods 0.000 title claims abstract description 18
- 239000003960 organic solvent Substances 0.000 claims abstract description 24
- 239000004005 microsphere Substances 0.000 claims abstract description 18
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 14
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 238000011068 loading method Methods 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 33
- 239000011148 porous material Substances 0.000 claims description 14
- 239000012535 impurity Substances 0.000 claims description 11
- 239000002245 particle Substances 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 239000012043 crude product Substances 0.000 claims description 6
- 238000012856 packing Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 1
- 230000005526 G1 to G0 transition Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 37
- 239000012071 phase Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000003480 eluent Substances 0.000 description 8
- 238000004817 gas chromatography Methods 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000011067 equilibration Methods 0.000 description 5
- 239000004519 grease Substances 0.000 description 5
- 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 description 4
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 description 4
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011097 chromatography purification Methods 0.000 description 3
- ITNKVODZACVXDS-YNUSHXQLSA-N ethyl (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate Chemical compound CCOC(=O)CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC ITNKVODZACVXDS-YNUSHXQLSA-N 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000000199 molecular distillation Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- YUFFSWGQGVEMMI-JLNKQSITSA-N (7Z,10Z,13Z,16Z,19Z)-docosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCCCC(O)=O YUFFSWGQGVEMMI-JLNKQSITSA-N 0.000 description 2
- 241001474374 Blennius Species 0.000 description 2
- 235000021294 Docosapentaenoic acid Nutrition 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 239000000344 soap Substances 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000199912 Crypthecodinium cohnii Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000233671 Schizochytrium Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- FKCBLVCOSCZFHV-UHFFFAOYSA-N acetonitrile;ethanol Chemical compound CCO.CC#N FKCBLVCOSCZFHV-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- VCSQUSNNIFZJAP-AAQCHOMXSA-N ethyl (7Z,10Z,13Z,16Z,19Z)-docosapentaenoate Chemical compound CCOC(=O)CCCCC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC VCSQUSNNIFZJAP-AAQCHOMXSA-N 0.000 description 1
- -1 ethyl DHA Chemical compound 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 150000004291 polyenes Chemical class 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 239000008149 soap solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 125000005457 triglyceride group Chemical group 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 238000005406 washing Methods 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
- C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
- C07C57/03—Monocarboxylic acids
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)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention provides a separation and purification method of docosahexaenoic acid in fish oil. The method for separating and purifying the docosahexaenoic acid in the fish oil comprises the following steps: 1) Dissolving and filtering the crude fish oil to obtain a fish oil solution; 2) Loading the fish oil solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the fish oil solution by adopting pure water and an organic solvent as flowing phases; 3) And (3) collecting the fish oil solution with the target peak value after chromatography and elution in the step (2) in a sectional manner, and summarizing the component solution meeting the requirements to obtain the purified docosahexaenoic acid. The method for separating and purifying the docosahexaenoic acid in the fish oil only needs one-step chromatography to separate and purify the DHA in the fish oil, has high purity, high and stable yield, simple and convenient operation, can recycle the used stationary phase, saves less mobile phasor, and greatly reduces the cost.
Description
Technical Field
The invention belongs to the technical field of separation and purification, relates to a separation and purification method of docosahexaenoic acid, and particularly relates to a separation and purification method of docosahexaenoic acid in fish oil.
Background
DHA (docosahexaenoicacid, DHA), commonly known as brain gold, is an unsaturated fatty acid which is very important for human body, DHA is a main component for growth and maintenance of cells in nervous system, is an important constituent component of brain and retina, has a content of up to 20% in cerebral cortex of human body and has a proportion of about 50% in retina of eyes, and is important for intelligence and vision development of fetal infants. The good DHA product should have the characteristics of nature, safety and good absorption, and is rich in various nutrients.
Wherein, the structural formula of DHA is as follows:
Early studies showed that DHA is normally present in the form of triglycerides in nature. Marine fish, certain seaweeds or lower fungi are important sources of DHA. For example, fish oil DHA is present in the form of triglycerides, but the content is low, usually 5% -14%. Humans have long been subjected to scientific research and experimentation to obtain DHA. The DHA in the early market is extracted from fish oil or seaweed by an organic solvent, and the structure of the DHA is not changed by the organic solvent adopted in the early stage, so that the DHA extracted by the organic solvent in the early stage is triglyceride DHA existing in a natural form, but the extraction efficiency is low.
In order to improve the extraction efficiency and obtain higher content and more DHA, the methods such as a low-temperature fractionation method, a solvent extraction method, a urea inclusion method, a molecular distillation method, a supercritical gas extraction method and the like are invented. Taking solvent extraction as an example, ethanol, fish oil and sodium hydroxide (NaOH) are mixed according to a certain proportion, and then heated to saponify the fish oil. And (5) carrying out filter pressing on the saponified mixed solution to obtain soap liquid and soap particles respectively. Adding sulfuric acid (H 2SO4) into the soap solution under stirring, separating the upper layer crude fatty acid ethanol mixed solution, heating again to recover ethanol, and repeatedly washing with water to obtain refined fish oil with higher DHA content. The efficiency of DHA extraction by this method is significantly improved, but the DHA obtained is no longer of the triglyceride type, which occurs in its natural form, but of the ethyl type. A significant portion of the DHA preparations currently on the market are ethyl ester DHA.
For example, CN103864614a discloses a method for separating and purifying DHA from DHA grease produced by microbial fermentation, comprising the following steps: (1) taking DHA grease and performing ethyl esterification; (2) Performing primary separation on the ethyl esterified DHA grease by adopting a urea embedding-gradient freezing crystallization method; (3) Molecular distillation is carried out on the primarily separated fatty acid ethyl ester for re-separation, and DHA ethyl ester with high purity is prepared. The method based on the coupling of various separation and purification means obviously improves DHA purification, and DHA grease produced by the Crypthecodinium cohnii is taken as a raw material to prepare DHA ethyl ester with purity more than 98 percent; DHA grease produced by schizochytrium is used as a raw material to prepare a product with DHA ethyl ester plus DPA ethyl ester more than 98 percent, and the ethyl DHA is obtained by the method.
CN1757395A discloses a purified adeps Phocae Vitulinae and its preparation method, the method esterifies natural adeps Phocae Vitulinae, then uses multi-stage molecular distillation purification technology to purify the esterified adeps Phocae Vitulinae and obtain purified adeps Phocae Vitulinae, the total content of three omega-3 polyene fatty acids-eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in the obtained purified adeps Phocae Vitulinae is obviously improved, at the same time the specific fishy smell of adeps Phocae Vitulinae is basically removed, and the physical properties are greatly improved. However, the purity and yield of DHA obtained by the method are both to be improved.
Therefore, it is necessary to provide a method for efficiently separating and purifying docosahexaenoic acid in fish oil.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a separation and purification method of docosahexaenoic acid in fish oil, which can meet the requirement of purity of 97% only by one-step chromatography purification, has high and stable purification yield, is simple and convenient, can be used for large-scale production, and greatly reduces the production cost.
To achieve the purpose, the invention adopts the following technical scheme:
a separation and purification method of docosahexaenoic acid in fish oil comprises the following steps:
1) Dissolving and filtering a fish oil crude product with DHA purity of 65-70%, so as to obtain a fish oil solution with DHA concentration of 5-9 mg/mL;
2) Loading the fish oil solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting with pure water and an organic solvent as flowing relative to the fish oil solution, wherein the volume percentage of the organic solvent in the mobile phase is 85-86%;
3) And (3) collecting the fish oil solution with the target peak value after chromatography and elution in the step (2) in a sectional manner, and summarizing the component solution meeting the requirements to obtain the purified docosahexaenoic acid.
In the step 2), the model number of the UniSil microsphere is C18UniSil microsphere. The C18UniSil microsphere has a particle diameter of 10-30 μm and a pore diameterThe polymer microsphere has strictly controlled particle size and pore diameter structure, is monodisperse and has pore channel structure, so that the polymer microsphere has good pertinence when being used as chromatographic packing. Preferably, the particle size of the C18UniSil microsphere is 10 μm.
In the step 2), the organic solvent is one of methanol, ethanol or acetonitrile; ethanol is preferred.
In step 1), the DHA purity of the crude fish oil is 65-70%, such as 65%, 66%, 67%, 68%, 69% or 70%, etc.; the concentration of DHA in the fish oil solution is 5-9mg/mL, for example, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, etc.
In the step 2), the volume ratio of the pure water to the organic solvent in the mobile phase is (14-15): 85-86.
The invention adopts the organic solvent and the purified water as the mobile phase, and can complete the DHA elution only by 8-12 column volumes, so the mobile phase used in the process is safe, pollution-free and low in cost, the used mobile phase quantity is less, and the purification period is shorter. Wherein the packing volume in the present invention is 490mL, i.e., the column volume is 490mL.
In the step 2), the specific process of elution is as follows: eluting with a flowing organic solvent with a volume percentage of 85-86% relative to the fish oil solution for 50-70min, such as 50min、51min、52min、53min、54min、55min、56min、57min、58min、59min、60min、61min、62min、63min、64min、65min、66min、67min、68min、69min or 70 min; the flow rate of the mobile phase is 70-80mL/min, such as 70mL/min, 71mL/min, 72mL/min, 73mL/min, 74mL/min, 75mL/min, 76mL/min, 77mL/min, 78mL/min, 79mL/min or 80mL/min, etc.
The step 2) also comprises the step of carrying out column pretreatment on the chromatographic column before loading.
The specific process of the column pretreatment is as follows: the pure ethanol is adopted to remove impurities from the chromatographic column, and then the flow with the volume percentage of the organic solvent of 85-86 percent is adopted to balance the chromatographic column after the impurity removal.
In step 1), the filtration is performed using a filter membrane having a pore size of 0.3 to 0.5. Mu.m, for example, a filter membrane having a pore size of 0.3. Mu.m, 0.35. Mu.m, 0.4. Mu.m, 0.45. Mu.m, 0.5. Mu.m, etc.
As a preferred scheme of the invention, the separation and purification method of docosahexaenoic acid in fish oil comprises the following steps:
1) Dissolving the crude product of the fish oil with the DHA purity of 65-70%, and filtering by adopting a filter membrane with the pore diameter of 0.3-0.5 mu m to obtain a fish oil solution with the DHA concentration of 5-9 mg/mL;
2) Removing impurities from a chromatographic column filled with C18UniSil microspheres by using pure ethanol, balancing the chromatographic column subjected to impurity removal by using a flow with the volume percentage of an organic solvent of 85-86%, loading the fish oil solution obtained in the step 1) into the chromatographic column subjected to balance treatment for chromatography, eluting the fish oil solution by using a flow with the volume percentage of the organic solvent of 85-86%, wherein the eluting time is 50-70min, and the flow rate of a mobile phase is 70-80mL/min;
3) And (3) collecting the fish oil solution with the target peak value after chromatography and elution in the step (2) in a sectional manner, and summarizing the component solution meeting the requirements to obtain the purified docosahexaenoic acid.
The second object of the present invention is to provide docosahexaenoic acid obtained by the separation and purification method according to one of the first objects.
Compared with the prior art, the invention has the beneficial effects that:
The separation and purification method of docosahexaenoic acid in fish oil provided by the invention only needs one-step chromatography to separate and purify DHA in fish oil, has the advantages of high purity, high and stable yield, simple and convenient operation, the stationary phase used can be recycled, the mobile phase quantity used is less, the organic solvent and the purified water are adopted as the mobile phase, and the elution of DHA can be completed only by 8-12 column volumes, so that the mobile phase used in the process is safe, pollution-free and low in cost, the mobile phase quantity used is less, the purification period is shorter, and the cost is greatly reduced. Specifically, the purity of the product can be more than 98% by only one-step chromatographic purification, and the purification yield is more than 95%.
Drawings
FIG. 1 is a scanning electron micrograph of a C18UniSil microsphere used in example 1 of the present invention;
FIG. 2 is a gas chromatographic analysis of docosahexaenoic acid (DHA) in fish oil before purification according to example 1 of the present invention;
FIG. 3 shows a gas chromatographic analysis of docosahexaenoic acid (DHA) in fish oil purified according to example 1 of the present invention.
Detailed Description
The technical scheme of the invention is further described below by means of specific embodiments with reference to the accompanying drawings 1-3.
The various starting materials of the present invention are commercially available, or may be prepared according to methods conventional in the art, unless specifically indicated.
Example 1
1) Taking a fish oil crude product with DHA purity of 68%, and adding 85% ethanol solution, wherein DHA content in the solution is 7mg/mL; after the solution is clarified, filtering by a filter membrane with the pore diameter of 0.45 mu m, and collecting filtrate for later use;
2) Using a 50X 250mm column, uniSilC18 (manufactured by Soy micro technology Co., ltd., particle size of 10 μm, pore size) ) As the packing of the chromatographic column, the column loading volume is 490mL; pre-treating the chromatographic column before the column, removing impurities by using pure ethanol, and balancing the purified chromatographic column by using flow with the ethanol volume percentage of 85 percent; then adopting the flow of which the volume percentage of ethanol is 85 percent to carry out elution for 60 minutes relative to the fish oil solution, and controlling the flow rate at 75mL/min;
3) And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 98.5%, and the yield is 96.5%.
Among them, fig. 1 is a scanning electron microscope photograph of C18UniSil microsphere used in example 1, and it can be seen that the C18UniSil microsphere has a strictly controlled particle size and pore size structure, and is a monodisperse microsphere having a pore structure. FIG. 2 shows the gas chromatographic analysis of docosahexaenoic acid in fish oil before purification, and certain impurities are found. FIG. 3 shows the gas chromatographic analysis of docosahexaenoic acid in purified fish oil, showing very few impurities and very few peaks.
Example 2
1) Taking a fish oil crude product with DHA purity of 68%, and adding 86% ethanol solution, wherein DHA content in the solution is 7mg/mL; after the solution is clarified, filtering by a filter membrane with the pore diameter of 0.45 mu m, and collecting filtrate for later use;
2) Using a 50X 250mm column, uniSilC18 (manufactured by Soy micro technology Co., ltd., particle size of 10 μm, pore size) ) As the packing of the chromatographic column, the column loading volume is 490mL; pre-treating the chromatographic column before purifying with high concentration organic phase, and balancing the purified chromatographic column with 86% ethanol flow; then adopting 86% ethanol to flow for eluting with respect to the fish oil solution for 60min, and controlling the flow rate at 75ml/min;
3) And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 98.45%, and the yield is 95.9%.
Example 3
This example differs from example 1 in that no column pretreatment, i.e., no purification and equilibration of the column, was performed, and the other steps were the same as in example 1.
And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 98.35%, and the yield is 95.4%.
Example 4
This example differs from example 1 in that the organic solvent in the mobile phase used for the equilibration and elution treatments is methanol, and the other is the same as example 1.
And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 98.43%, and the yield is 95.1%.
Comparative example 1
This comparative example differs from example 1 in that the packing phase in the column is 15. Mu.mC 18 of the commercial non-uniform particle size distribution, otherwise identical to example 1.
And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 98.12%, and the yield is 90.1%.
Comparative example 2
The comparative example is a conventional DHA purification method, and a molecular rectification method is directly adopted.
The purity of DHA in the fraction was 80.5%.
Comparative example 3
This comparative example differs from example 1 in that the mobile phase used for the equilibration and elution treatments was pure methanol, otherwise identical to that of example 1.
And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 70%, and the yield is 98%.
Comparative example 4
This comparative example differs from example 1 in that the mobile phase used for the equilibration and elution treatments was 50% by volume of the organic solvent, the remainder being the same as example 1.
And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 98.76%, and the yield is 80%.
Comparative example 5
This comparative example is different from example 1 in that the mobile phase used for the equilibration treatment and the elution treatment is 95% by volume of the organic solvent, and the other is the same as example 1.
And collecting the solution of the target peak value in sections, summarizing the component solutions meeting the requirements, and analyzing by gas chromatography, wherein the purity of DHA in the eluent is 97.5%, and the yield is 96.3%.
The method is used for deep and precise purification of docosahexaenoic acid (DHA) in fish oil, the requirement of DHA purity of >98% can be met only by one-step chromatographic purification, the purification yield is >95% and stable, and meanwhile, the purification and separation method is simple and convenient, can be used for large-scale production, and greatly reduces the production cost.
The detailed process equipment and process flow of the present invention are described by the above embodiments, but the present invention is not limited to, i.e., it does not mean that the present invention must be practiced depending on the detailed process equipment and process flow. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (7)
1. The separation and purification method of docosahexaenoic acid in fish oil is characterized by comprising the following steps:
1) Dissolving and filtering a fish oil crude product with DHA purity of 65-70%, so as to obtain a fish oil solution with DHA concentration of 5-9 mg/mL;
2) Loading the fish oil solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting with pure water and an organic solvent as flowing relative to the fish oil solution, wherein the volume percentage of the organic solvent in the mobile phase is 85-86%;
3) Collecting fish oil solution with target peak value after chromatography and elution in step 2) in a sectional way, and summarizing component liquid meeting the requirements to obtain purified docosahexaenoic acid;
In the step 2), the model number of the UniSil microsphere is C18UniSil microsphere;
The particle size of the C18UniSil microsphere is 10 mu m;
The organic solvent is ethanol.
2. The method according to claim 1, wherein in the step 2), the volume of the packing material contained in the column is 1 column volume, and the amount of the mobile phase is 8 to 12 column volumes.
3. The method according to claim 1, wherein in step 2), the specific elution process is as follows: eluting with 85-86% organic solvent by volume ratio of mobile phase to fish oil solution for 50-70min, and flowing speed of mobile phase is 70-80mL/min.
4. The method according to claim 1, wherein the step 2) further comprises a step of subjecting the chromatographic column to a column pretreatment before loading.
5. The method according to claim 4, wherein the specific process of the column pretreatment is as follows: ethanol is adopted to remove impurities from the chromatographic column, and then the flow with the volume percentage of the organic solvent of 85-86 percent is adopted to balance the chromatographic column after the impurities are removed.
6. The method according to claim 1, wherein in the step 1), the filtration is performed by using a filter membrane having a pore size of 0.3 to 0.5. Mu.m.
7. The separation and purification method according to claim 1, wherein the separation and purification method comprises the steps of:
1) Dissolving the crude product of the fish oil with the DHA purity of 65-70%, and filtering by adopting a filter membrane with the pore diameter of 0.3-0.5 mu m to obtain a fish oil solution with the DHA concentration of 5-9 mg/mL;
2) Removing impurities from a chromatographic column filled with C18UniSil microspheres by adopting ethanol, balancing the chromatographic column subjected to impurity removal by adopting a flow with the volume percentage of an organic solvent of 85-86%, loading the fish oil solution obtained in the step 1) into the chromatographic column subjected to balance treatment for chromatography, eluting the fish oil solution by adopting a flow with the volume percentage of the organic solvent of 85-86%, wherein the eluting time is 50-70min, and the flow rate of a mobile phase is 70-80mL/min;
3) And (3) collecting the fish oil solution with the target peak value after chromatography and elution in the step (2) in a sectional manner, and summarizing the component solution meeting the requirements to obtain the purified docosahexaenoic acid.
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