Purification method of high-purity phospholipid
By using methods such as heating and stirring, acid-adjusted pH, centrifugation, and gradient elution in chromatography columns, the problems of low phospholipid purification efficiency and large solvent consumption were solved, achieving the preparation of high-purity phospholipids and saving eluent.
Patent Information
- Application Number
- CN202510965750.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-14
- Publication Date
- 2025-10-31
AI Technical Summary
Existing technologies suffer from low phospholipid purification efficiency, high solvent consumption, and high costs.
The method of heating and stirring, acid adjustment of pH, centrifugation and gradient elution of chromatography column is adopted. The centrifugation technique is combined with the method of primary purification of phospholipids to reduce the amount of eluent used and improve the purity through gradient elution.
It improves the purity of phospholipids, meets the needs of high-end applications, and reduces the consumption of eluent and the pressure of recycling.
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Figure CN120865280A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of purification technology, and in particular to a method for purifying high-purity phospholipids. Background Technology
[0002] Phospholipids, also known as phospholipids or phospholipid derivatives, are lipids containing phosphoric acid and belong to the category of complex lipids. Phospholipids are the main components of biological membranes, and are divided into two main categories: glycerophospholipids and sphingomyelins, which are composed of glycerol and sphingosine, respectively. Phospholipids are amphoteric molecules, with one end being a hydrophilic nitrogen- or phosphorus-containing head and the other end being a hydrophobic (lipophilic) long hydrocarbon chain. For this reason, the hydrophilic ends of phospholipid molecules are close to each other, and the hydrophobic ends are close to each other. They often form the phospholipid bilayer, i.e., the structure of the cell membrane, together with other molecules such as proteins, glycolipids, and cholesterol.
[0003] Using soybean oil residue is a common industrial process for preparing lecithin. Lecithin, extracted from soybean oil residue, contains choline, vitamins, minerals, linolenic acid, and alpha-linolenic acid. Choline is the main component of soybean lecithin and is an essential nutrient for the human body. It has emulsifying, antioxidant, cholesterol-lowering, and blood lipid-regulating effects, as well as nutritional and health benefits such as delaying aging, enhancing memory, and preventing cardiovascular and cerebrovascular diseases. It is currently widely used in the food, cosmetics, and feed industries.
[0004] Phospholipids prepared from soybean oil residue are usually of low purity and require further purification to meet higher-end application requirements. Existing methods for phospholipid purification include solvent washing, which has low purification efficiency and low final purity; column chromatography, which requires a large amount of solvent; and supercritical CO2 extraction, which, although producing high-purity phospholipids, involves large upfront investment and high costs. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the defects of the prior art and provide a method for purifying high-purity phospholipids.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention discloses a method for purifying high-purity phospholipids, comprising the following steps: Step 1: Heat the soybean oil foot to 60-70°C and stir it at 100-300 rpm for 30-90 minutes until it is uniform, then remove the impurities. Step 2: Add acid to the heated and stirred soybean oil residue, and adjust its pH to 4-5 while maintaining a constant temperature of 60-70℃. Step 3: Mix soybean oil residue with water in a mass ratio of 1:1-2, and stir at 200-400 rpm for 30-60 minutes at a constant temperature of 50-70℃ to form phospholipid colloid. Step 4: Transfer the obtained phospholipid colloid to a centrifuge for centrifugation, collect the middle layer after centrifugation to obtain the phospholipid-containing colloid, recover the upper layer of clear neutral oil, and discharge and treat the lower layer of wastewater. Step 5: Transfer the collected phospholipid-containing colloid to a chromatography column for purification to obtain high-purity phospholipids.
[0007] As a preferred embodiment of the present invention, in step 1, the soybean oil foot is heated to 70°C and stirred at 300 rpm for 90 minutes until uniform, and then impurities are removed by a 50-mesh filter.
[0008] As a preferred embodiment of the present invention, in step 2, the soybean oil residue is placed in a container at a constant temperature of 70°C, and 0.3% citric acid is added to adjust its pH to 5.
[0009] As a preferred embodiment of the present invention, in step 3, water is added to the container containing soybean oil residue at a mass ratio of 1:1 to water, and the mixture is stirred at 200 rpm for 60 minutes at a constant temperature of 70°C.
[0010] As a preferred embodiment of the present invention, in step 4, the phospholipid colloid is centrifuged in a centrifuge at a temperature of 70°C and a centrifugal force of 3000g.
[0011] As a preferred embodiment of the present invention, step 5 includes the following sub-steps: Phospholipid-containing colloids are dissolved in polar solvents and diluted to obtain an organic phase; The organic phase was passed into a wet-packed chromatography column and then subjected to gradient elution. The eluent produced at each stage was collected separately.
[0012] As a preferred embodiment of the present invention, the polar solvent in step 5 is dichloromethane with a concentration of 5%-10%; The chromatography column used is a silica gel column with a particle size of 60-200 mesh and a height-to-diameter ratio of 1:10-20.
[0013] As a preferred embodiment of the present invention, the gradient elution in step 5 includes: First-stage elution: the first eluent is hexane:diethyl ether = 3-9:1, and the column is washed with the solution at a flow rate of 1-3 mL / min to obtain the first effluent. The chromatography column was washed with chloroform as the second eluent and the column was washed at a flow rate of 1-4 mL / min to obtain the second effluent. The chromatography column was washed with a three-stage elution process, with the third eluent being chloroform:methanol = 3-9:1, and the column was washed at a flow rate of 1-5 mL / min to obtain the third effluent. The chromatography column was eluted in four stages. The fourth eluent was chloroform:methanol:water = 5-13:1-7:1. The column was then washed with the eluent at a flow rate of 1-5 mL / min to obtain the fourth effluent.
[0014] As a preferred embodiment of the present invention, the first effluent, the second effluent, the third effluent and the fourth effluent are tested respectively, and the eluent containing a high concentration of phospholipids is fractionated to obtain high-purity phospholipids.
[0015] As a preferred embodiment of the present invention, the collected first effluent is filtered and distilled, and then used as the first eluent to wash the chromatography column in the next elution.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This method first uses centrifugation to perform primary purification of phospholipids. The colloid containing phospholipids with improved purity is then used to prepare the mobile phase for elution, which consumes less eluent and reduces the pressure of eluent recovery and treatment. 2. Phospholipids purified by centrifugation have fewer impurities, and the first effluent produced by elution can be directly fed into the chromatography column and reused as the first eluent after treatment, thereby reducing the pressure of eluent recycling. 3. The phospholipids purified by this method have high purity, which can meet the needs of high-end applications. Attached Figure Description
[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a flowchart of the steps of the present invention. Detailed Implementation
[0018] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.
[0019] In the attached diagram, all identical reference numerals refer to the same components.
[0020] like Figure 1 As shown, the present invention provides a method for purifying high-purity phospholipids, comprising the following steps: Step 1: Heat the soybean oil foot to 60-70°C and stir it at 100-300 rpm for 30-90 minutes until it is uniform, then remove the impurities. Step 2: Add acid to the heated and stirred soybean oil residue, and adjust its pH to 4-5 while maintaining a constant temperature of 60-70℃. Step 3: Mix soybean oil residue with water in a mass ratio of 1:1-2, and stir at 200-400 rpm for 30-60 minutes at a constant temperature of 50-70℃ to form phospholipid colloid. Step 4: Transfer the obtained phospholipid colloid to a centrifuge for centrifugation, collect the middle layer after centrifugation to obtain the phospholipid-containing colloid, recover the upper layer of clear neutral oil, and discharge and treat the lower layer of wastewater. Step 5: Transfer the collected phospholipid-containing colloid to a chromatography column for purification to obtain high-purity phospholipids.
[0021] Example 1: Furthermore, in step 1, the soybean oil foot is heated to 70°C and stirred at 300 rpm for 90 minutes until homogeneous, and then filtered through a 50-mesh filter to remove impurities.
[0022] Furthermore, in step 2, the soybean oil residue is placed in a container at a constant temperature of 70°C, and 0.3% citric acid is added to adjust its pH to 5.
[0023] Furthermore, in step 3, water is added to the container containing soybean oil residue at a mass ratio of 1:1 to water, and the mixture is stirred at 200 rpm for 60 minutes at a constant temperature of 70°C.
[0024] Furthermore, in step 4, the phospholipid colloid is centrifuged in a centrifuge at a temperature of 70°C and a centrifugal force of 3000g.
[0025] The collected phospholipid-containing gel A.
[0026] Example 2: Furthermore, in step 1, the soybean oil foot is heated to 60°C and stirred at 100 rpm for 30 minutes until homogeneous, and then filtered through a 50-mesh filter to remove impurities.
[0027] Furthermore, in step 2, the soybean oil residue is placed in a container at a constant temperature of 70°C, and 0.1% citric acid is added to adjust its pH to 4.
[0028] Furthermore, in step 3, water is added to the container containing soybean oil residue at a mass ratio of 1:2 (soybean oil residue to water), and the mixture is stirred at 400 rpm for 30 minutes at a constant temperature of 70°C.
[0029] Furthermore, in step 4, the phospholipid colloid is centrifuged in a centrifuge at a temperature of 60°C and a centrifugal force of 3000g.
[0030] Collected phospholipid-containing gel B.
[0031] Example 3: Furthermore, in step 1, the soybean oil foot is heated to 65°C and stirred at 250 rpm for 60 minutes until homogeneous, and then filtered through a 50-mesh filter to remove impurities.
[0032] Furthermore, in step 2, the soybean oil residue is placed in a container at a constant temperature of 65°C, and 0.2% citric acid is added to adjust its pH to 5.
[0033] Furthermore, in step 3, water is added to the container containing soybean oil residue at a mass ratio of 1:1 to water, and the mixture is stirred at 300 rpm for 45 minutes at a constant temperature of 70°C.
[0034] Furthermore, in step 4, the phospholipid colloid is centrifuged in a centrifuge at a temperature of 65°C and a centrifugal force of 3000g.
[0035] Collected phospholipid-containing colloidal C.
[0036] The collected phospholipid-containing colloids A, B, and C were analyzed by thin-film chromatography and HPLC, and the phospholipid content was obtained by combining the analytical results, as shown in Table 1 below.
[0037] Table 1
[0038] As can be seen from Table 1, the phospholipid-containing gel prepared by the method of Example 1 not only has a high yield but also a high phospholipid content, making it suitable as a base raw material for subsequent purification.
[0039] Furthermore, step 5 includes the following sub-steps: Phospholipid-containing colloids are dissolved in polar solvents and diluted to obtain an organic phase; The organic phase was passed into a wet-packed chromatography column and then subjected to gradient elution. The eluent produced at each stage was collected separately.
[0040] Furthermore, the polar solvent in step 5 is dichloromethane with a concentration of 5%-10%; The chromatography column used is a silica gel column with a particle size of 60-200 mesh and a height-to-diameter ratio of 1:10-20.
[0041] Furthermore, the gradient elution in step 5 includes: First-stage elution: the first eluent is hexane:diethyl ether = 3-9:1, and the column is washed with the solution at a flow rate of 1-3 mL / min to obtain the first effluent. Secondary elution was performed, with chloroform as the second eluent, and the column was washed at a flow rate of 1-4 mL / min to obtain the second effluent. The chromatography column was washed with a three-stage elution process, with the third eluent being chloroform:methanol = 3-9:1, and the column was washed at a flow rate of 1-5 mL / min to obtain the third effluent. The chromatography column was eluted in four stages. The fourth eluent was chloroform:methanol:water = 5-13:1-7:1. The column was then washed with the eluent at a flow rate of 1-5 mL / min to obtain the fourth effluent.
[0042] Furthermore, the first, second, third, and fourth effluents were tested separately, and the eluent containing a high concentration of phospholipids was fractionated to obtain high-purity phospholipids.
[0043] All of the following are based on phospholipid-containing gelatin A.
[0044] Example 4: 100 mL of phospholipid-containing colloidal A was mixed with 5% dichloromethane at a weight ratio of 1:9 to obtain an organic phase. The organic phase was then passed into a 200-mesh silica gel column packed using a wet packing method. The silica gel column had a height of 200 cm and a diameter of 10 cm.
[0045] 1. Use 2000 mL of the first eluent obtained by mixing n-hexane and diethyl ether in a mass ratio of 9:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2400 mL of the first eluent.
[0046] 2. Use 2000 mL of chloroform as the second eluent to wash the chromatography column at a rate of 4 mL / min, and collect the first 2200 mL of the second eluent.
[0047] 3. Use 2000 mL of the third eluent obtained by mixing chloroform and methanol in a mass ratio of 9:1 to wash the chromatography column at a rate of 5 mL / min, and collect the first 2200 mL of the third eluent.
[0048] 4. Use 2000 mL of the fourth eluent obtained by mixing chloroform, methanol and water in a mass ratio of 12:7:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2200 mL of the fourth eluent.
[0049] Two sets of 50 mL samples were taken from the first, second, third, and fourth effluents, and analyzed by thin-layer chromatography and HPLC, respectively. The results from both sides were combined, and the effluents were distilled to obtain high-purity phospholipids based on the results.
[0050] Example 5: 100 mL of phospholipid-containing colloidal A was mixed with 10% dichloromethane at a weight ratio of 1:4 to obtain an organic phase. The organic phase was then passed into a 60-mesh silica gel column packed using a wet packing method. The silica gel column had a height of 200 cm and a diameter of 10 cm.
[0051] 1. Use 2000 mL of the first eluent obtained by mixing n-hexane and diethyl ether in a mass ratio of 4:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2400 mL of the first eluent.
[0052] 2. Use 2000 mL of chloroform as the second eluent to wash the chromatography column at a rate of 4 mL / min, and collect the first 2200 mL of the second eluent.
[0053] 3. Use 2000 mL of the third eluent obtained by mixing chloroform and methanol in a mass ratio of 4:1 to wash the chromatography column at a flow rate of 5 mL / min, and collect the first 2200 mL of the third eluent.
[0054] 4. Use 2000 mL of the fourth eluent obtained by mixing chloroform, methanol and water in a mass ratio of 7:2:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2200 mL of the fourth eluent.
[0055] Two sets of 50 mL samples were taken from the first, second, third, and fourth effluents, and analyzed by thin-layer chromatography and HPLC, respectively. The results from both sides were combined, and the effluents were distilled to obtain high-purity phospholipids based on the results.
[0056] Example 6: 100 mL of phospholipid-containing colloidal A was mixed with 5% dichloromethane at a weight ratio of 1:9 to obtain an organic phase. The organic phase was then passed into a 200-mesh silica gel column packed using a wet packing method. The silica gel column had a height of 200 cm and a diameter of 10 cm.
[0057] 1. Use 2000 mL of the first eluent obtained by mixing n-hexane and diethyl ether at a mass ratio of 9:1 to wash the chromatography column at a rate of 1 mL / min, and collect the first 2400 mL of the first eluent.
[0058] 2. Use 2000 mL of chloroform as the second eluent to wash the chromatography column at a rate of 1 mL / min, and collect the first 2200 mL of the second eluent.
[0059] 3. Use 2000 mL of the third eluent obtained by mixing chloroform and methanol in a mass ratio of 9:1 to wash the chromatography column at a rate of 1 mL / min, and collect the first 2200 mL of the third eluent.
[0060] 4. Use 2000 mL of the fourth eluent obtained by mixing chloroform, methanol and water in a mass ratio of 12:7:1 to wash the chromatography column at a rate of 1 mL / min, and collect the first 2200 mL of the fourth eluent.
[0061] Two sets of 50 mL samples were taken from the first, second, third, and fourth effluents, and analyzed by thin-layer chromatography and HPLC, respectively. The results from both sides were combined, and the effluents were distilled to obtain high-purity phospholipids based on the results.
[0062] Comparative Example 1: 100 mL of phospholipid colloid (without centrifugation) was mixed with 5% dichloromethane at a weight ratio of 1:9 to obtain an organic phase. The organic phase was then passed into a 200-mesh silica gel column packed by wet packing. The silica gel column was 200 cm high and 10 cm in diameter.
[0063] 1. Use 2000 mL of the first eluent obtained by mixing n-hexane and diethyl ether in a mass ratio of 9:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2400 mL of the first eluent.
[0064] 2. Use 2000 mL of chloroform as the second eluent to wash the chromatography column at a rate of 4 mL / min, and collect the first 2200 mL of the second eluent.
[0065] 3. Use 2000 mL of the third eluent obtained by mixing chloroform and methanol in a mass ratio of 9:1 to wash the chromatography column at a rate of 5 mL / min, and collect the first 2200 mL of the third eluent.
[0066] 4. Use 2000 mL of the fourth eluent obtained by mixing chloroform, methanol and water in a mass ratio of 12:7:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2200 mL of the fourth eluent.
[0067] Two sets of 50 mL samples were taken from the first, second, third, and fourth effluents, and analyzed by thin-layer chromatography and HPLC, respectively. The results from both sides were combined, and the effluents were distilled to obtain high-purity phospholipids based on the results.
[0068] Comparative Example 2: 100 mL of phospholipid colloid (without centrifugation) was mixed with 10% dichloromethane at a weight ratio of 1:4 to obtain an organic phase. The organic phase was then passed into a 60-mesh silica gel column packed by wet packing. The silica gel column was 200 cm high and 10 cm in diameter.
[0069] 1. Use 2000 mL of the first eluent obtained by mixing n-hexane and diethyl ether in a mass ratio of 4:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2400 mL of the first eluent.
[0070] 2. Use 2000 mL of chloroform as the second eluent to wash the chromatography column at a rate of 4 mL / min, and collect the first 2200 mL of the second eluent.
[0071] 3. Use 2000 mL of the third eluent obtained by mixing chloroform and methanol in a mass ratio of 4:1 to wash the chromatography column at a flow rate of 5 mL / min, and collect the first 2200 mL of the third eluent.
[0072] 4. Use 2000 mL of the fourth eluent obtained by mixing chloroform, methanol and water in a mass ratio of 7:2:1 to wash the chromatography column at a rate of 3 mL / min, and collect the first 2200 mL of the fourth eluent.
[0073] Two sets of 50 mL samples were taken from the first, second, third, and fourth effluents, and analyzed by thin-layer chromatography and HPLC, respectively. The results from both sides were combined, and the effluents were distilled to obtain high-purity phospholipids based on the results.
[0074] Comparative Example 3: 100 mL of phospholipid colloid (without centrifugation) was mixed with 5% dichloromethane at a weight ratio of 1:9 to obtain an organic phase. The organic phase was then passed into a 200-mesh silica gel column packed by wet packing. The silica gel column was 200 cm high and 10 cm in diameter.
[0075] 1. Use 2000 mL of the first eluent obtained by mixing n-hexane and diethyl ether at a mass ratio of 9:1 to wash the chromatography column at a rate of 1 mL / min, and collect the first 2400 mL of the first eluent.
[0076] 2. Use 2000 mL of chloroform as the second eluent to wash the chromatography column at a rate of 1 mL / min, and collect the first 2200 mL of the second eluent.
[0077] 3. Use 2000 mL of the third eluent obtained by mixing chloroform and methanol in a mass ratio of 9:1 to wash the chromatography column at a rate of 1 mL / min, and collect the first 2200 mL of the third eluent.
[0078] 4. Use 2000 mL of the fourth eluent obtained by mixing chloroform, methanol and water in a mass ratio of 12:7:1 to wash the chromatography column at a rate of 1 mL / min, and collect the first 2200 mL of the fourth eluent.
[0079] Two sets of 50 mL samples were taken from the first, second, third, and fourth effluents, and analyzed by thin-layer chromatography and HPLC, respectively. The results from both sides were combined, and the effluents were distilled to obtain high-purity phospholipids based on the results.
[0080] The detection results of Examples 4-6 and Comparative Examples 1-3 are shown in Table 2.
[0081] Table 2
[0082] Table 2 shows that the phospholipid yield and phospholipid content obtained in Example 4 are the highest. The phospholipid content obtained in Examples 4-6 is higher than that in Comparative Examples 1-3, and the amount of solvent used is also less, which reduces the pressure of eluent recovery treatment. It can be seen from Example 5 and Comparative Example 2 that an excessively high solvent ratio will affect the yield and phospholipid content. It can be seen from Example 6 and Comparative Example 3 that an excessively slow elution speed will lead to a decrease in yield and phospholipid content.
[0083] Furthermore, the collected first effluent is filtered and distilled, and then used as the first eluent to wash the chromatography column during the next elution.
[0084] The rinsing effect did not decrease significantly within 3 repetitions of this step, but the rinsing effect decreased significantly after 3 repetitions. This indicates that the first eluent can be reused 3 times, thereby reducing the pressure on eluent recycling.
[0085] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for purifying high-purity phospholipids, characterized in that, Includes the following steps: Step 1: Heat the soybean oil foot to 60-70°C and stir it at 100-300 rpm for 30-90 minutes until it is uniform, then remove the impurities. Step 2: Add acid to the heated and stirred soybean oil residue, and adjust its pH to 4-5 while maintaining a constant temperature of 60-70℃. Step 3: Mix soybean oil residue with water in a mass ratio of 1:1-2, and stir at 200-400 rpm for 30-60 minutes at a constant temperature of 50-70℃ to form phospholipid colloid. Step 4: Transfer the obtained phospholipid colloid to a centrifuge for centrifugation, collect the middle layer after centrifugation to obtain the phospholipid-containing colloid, recover the upper layer of clear neutral oil, and discharge and treat the lower layer of wastewater. Step 5: Transfer the collected phospholipid-containing colloid to a chromatography column for purification to obtain high-purity phospholipids.
2. The method for purifying high-purity phospholipids according to claim 1, characterized in that, In step 1, the soybean oil foot is heated to 70°C and stirred at 300 rpm for 90 minutes until homogeneous, and then filtered through a 50-mesh filter to remove impurities.
3. The method for purifying high-purity phospholipids according to claim 1, characterized in that, In step 2, the soybean oil residue is placed in a container at a constant temperature of 70°C, and 0.3% citric acid is added to adjust its pH to 5.
4. The method for purifying high-purity phospholipids according to claim 1, characterized in that, In step 3, water is added to the container containing soybean oil residue at a mass ratio of 1:1 (soybean oil residue to water), and the mixture is stirred at 200 rpm for 60 minutes at a constant temperature of 70°C.
5. The method for purifying high-purity phospholipids according to claim 1, characterized in that, In step 4, the phospholipid colloid is centrifuged in a centrifuge at a temperature of 70°C and a centrifugal force of 3000g.
6. A method for purifying high-purity phospholipids according to any one of claims 1-5, characterized in that, Step 5 includes the following sub-steps: Phospholipid-containing colloids are dissolved in polar solvents and diluted to obtain an organic phase; The organic phase was passed into a wet-packed chromatography column and then subjected to gradient elution. The eluent produced at each stage was collected separately.
7. The method for purifying high-purity phospholipids according to claim 6, characterized in that, The polar solvent in step 5 is dichloromethane with a concentration of 5%-10%; The chromatography column used is a silica gel column with a particle size of 60-200 mesh and a height-to-diameter ratio of 1:10-20.
8. The method for purifying high-purity phospholipids according to claim 7, characterized in that, The gradient elution in step 5 includes: First-stage elution: the first eluent is hexane:diethyl ether = 3-9:1, and the column is washed with the solution at a flow rate of 1-3 mL / min to obtain the first effluent. Secondary elution was performed, with chloroform as the second eluent, and the column was washed at a flow rate of 1-4 mL / min to obtain the second effluent. Three-stage elution, the third eluent is chloroform:methanol = 3-9:1, and the column is washed with the third eluent at a flow rate of 1-5 mL / min to obtain the third effluent; The chromatography column was eluted in four stages. The fourth eluent was chloroform:methanol:water = 5-13:1-7:
1. The column was then washed with the eluent at a flow rate of 1-5 mL / min to obtain the fourth effluent.
9. The method for purifying high-purity phospholipids according to claim 8, characterized in that, The first, second, third, and fourth effluents were tested separately, and the eluent containing a high concentration of phospholipids was fractionated to obtain high-purity phospholipids.
10. The method for purifying high-purity phospholipids according to claim 9, characterized in that, The collected first eluent is filtered and distilled, and then used as the first eluent to wash the chromatography column in the next elution.