A method for separating and purifying crude cholesterols
By combining acetonitrile extraction with maleimide-functionalized microsphere addition reaction column with crystal inhibitors, the problem of separating dihydrocholesterol and 7-dehydrocholesterol in crude cholesterol was solved, achieving efficient production of high-purity cholesterol.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGYU ZHONGXIAN BIOTECH
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient to efficiently remove dihydrocholesterol and 7-dehydrocholesterol from crude cholesterol products. Traditional methods are time-consuming, costly, and ineffective in removing impurities.
Cholesterol was efficiently separated and purified by selective addition reaction in a maleimide-functionalized cross-linked polyethylene microsphere reaction column after acetonitrile extraction, combined with crystal inhibitors and dynamic gradient cooling crystallization.
It achieves a cholesterol purity of over 99.5%, shortens the production cycle by more than 50%, reduces solvent consumption by 60%, and significantly reduces production costs.
Abstract
Description
Technical Field
[0001] This invention relates to the field of cholesterol purification technology, specifically a method for separating and purifying crude cholesterol. Background Technology
[0002] Cholesterol is an important pharmaceutical intermediate, mainly used in the production of vitamin D2, vitamin D3, and artificial bezoar. Currently, crude cholesterol is mainly derived from natural raw materials such as lanolin. Its composition is complex, and in addition to the target product, it usually contains 2-5% stubborn impurities. Among these impurities, dihydrocholesterol and 7-dehydrocholesterol are the main ones. Because they have almost the same steroidal skeleton and similar physicochemical properties as cholesterol, traditional separation methods are difficult to purify them effectively.
[0003] Currently, the commonly used purification process for cholesterol is a saponification combined with multi-step recrystallization. This first involves removing hydrolyzable impurities such as cholesterol esters through alkaline saponification, followed by repeated recrystallization based on the difference in solubility between cholesterol and these impurities in acetone or ethanol. However, this purification method exhibits extremely low selectivity for separating analogs with very low solubility, such as dihydrocholesterol, often requiring 5 to 8 recrystallizations. This not only reduces yield but also consumes large amounts of organic solvents, resulting in high costs. Furthermore, traditional recrystallization processes are largely ineffective at removing 7-dehydrocholesterol, which is prone to oxidation or transfer at high temperatures, potentially introducing other impurities. In addition, traditional purification processes are lengthy and have long production cycles.
[0004] Therefore, there is an urgent need for a purification method that can selectively and efficiently remove dihydrocholesterol and 7-dehydrocholesterol, so that the purity of cholesterol can reach more than 99%. Summary of the Invention
[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for separating and purifying crude cholesterol, thus solving the problems mentioned in the background section.
[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a method for separating and purifying crude cholesterol is provided, comprising the following steps: 1) After neutralizing the crude cholesterol product obtained by saponification with soda ash, it was dissolved in a mixed solvent containing n-hexane and ethyl acetate, and the organic phase was obtained by countercurrent liquid-liquid extraction with acetonitrile aqueous solution. 2) The organic phase is passed into a cross-linked polyethylene microsphere reaction column containing maleimide-functionalized material for a selective addition reaction, and the effluent is collected. 3) Add sec-butyl acetate solvent to the effluent to dissolve it, then add a crystal inhibitor to perform dynamic gradient cooling crystallization, filter, and obtain cholesterol crystals.
[0007] This invention first extracts residual pigments, trace amounts of saponified substances, and some organic oxidation products using acetonitrile, achieving high efficiency and no adsorption loss. After concentrating the mother liquor, it is passed into a reaction column for an addition reaction. In the reaction column, cross-linked polyethylene microspheres containing maleimide functionalization undergo an addition reaction with the conjugated diene of 7-dehydrocholesterol, generating an addition product with significantly increased polarity. Finally, gradient cooling crystallization is performed. Inhibitors adsorb onto the surface of dihydrocholesterol, blocking its growth. Due to the small differences in molecular steric hindrance and interactions, cholesterol crystallizes during the dynamic gradient cooling process, yielding cholesterol with a crystal purity greater than 99%.
[0008] Preferably, in step 1), the volume ratio of n-hexane to ethyl acetate in the mixed solvent containing n-hexane and ethyl acetate is 7:3 to 8:2. The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 85:15 to 90:10.
[0009] Preferably, in step 1), the temperature of the countercurrent liquid-liquid extraction is 35~40℃, and the number of extraction stages is 3~5.
[0010] Preferably, in step 2), the maleimide-functionalized cross-linked polyethylene microspheres have a particle size of 100-200 mesh and a cross-linking degree of 8-12%.
[0011] Preferably, in step 2), the temperature of the selective addition reaction is 60~75℃ and the time is 20~40min.
[0012] Preferably, in step 3), the mass ratio of the effluent to the volume ratio of sec-butyl acetate solvent is 1g:8~12mL; The melting temperature is 50~60℃.
[0013] Preferably, the sec-butyl acetate solvent further contains cyclohexanol, and the amount of cyclohexanol used is 5-10% of the sec-butyl acetate solvent.
[0014] Preferably, in step 3), the crystal inhibitor is selected from N-acyl amino acid derivatives; The amount of crystal inhibitor added is 0.2 to 0.8% of the mass of the effluent.
[0015] Preferably, the dynamic gradient cooling crystallization includes a first-stage cooling and a second-stage cooling, wherein the first-stage cooling is carried out at a cooling rate of 1~1.5℃ / min to 30~35℃, and the stirring rate is 150~200rpm; The second-stage cooling process reduces the temperature to 0-10℃ at a cooling rate of 0.05-0.15℃ / min, while the stirring rate is 50-100rpm.
[0016] Preferably, in step 3), the filtration temperature is 0~10℃.
[0017] (III) Beneficial Effects This invention provides a method for separating and purifying crude cholesterol. It has the following beneficial effects: (1) The present invention provides a method for separating and purifying crude cholesterol. It utilizes the conjugated diene structure of 7-dehydrocholesterol to carry out an addition reaction in a reaction column containing maleimide-functionalized cross-linked polyethylene microspheres, so that 7-dehydrocholesterol and cholesterol are transformed from homologues into compounds with significantly different separability, thereby reducing the difficulty of removal. At the same time, a crystal inhibitor is introduced to preferentially adsorb on the crystal face of dihydrocholesterol and heterogeneously crystallize it, so that the purity of the crystallized cholesterol can be stably reached above 99.5%.
[0018] (2) The method for separating and purifying crude cholesterol provided in this scheme shortens the production cycle by more than 50% through countercurrent extraction, addition conversion and inhibitor-assisted single crystallization, and reduces the consumption of organic solvents by about 60%. Among them, sec-butyl acetate can be recycled, which significantly reduces the production cost. Detailed Implementation
[0019] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. In both the embodiments and comparative examples of this invention, the same batch of crude lanolin-derived cholesterol, which was saponified with soda ash, neutralized, and then dried, was used. The initial HPLC purity was 88.5%, with a dihydrocholesterol content of 2.8% and a 7-dehydrocholesterol content of 1.5%.
[0020] Example 1 Step 1: Weigh 100g of crude cholesterol and dissolve it in 500mL of a mixed solvent of n-hexane / ethyl acetate (volume ratio 7:3). Keep the solution warm in a 38℃ water bath to obtain the light phase. Take 500mL of acetonitrile / water solution (volume ratio 85:15) as the extractant and perform a 5-stage countercurrent liquid-liquid extraction at 38℃. Collect the organic phase and concentrate it under reduced pressure at 45℃ to 150g. Step 2: The organic phase from Step 1 was pumped into a reaction column containing maleimide-functionalized polyvinyl alcohol microspheres with a particle size of 100 mesh and a crosslinking degree of 10% using a constant flow pump at a flow rate of 4 mL / min. The column was held at 65℃ for 30 min, the effluent was collected, and the solvent was recovered under reduced pressure to obtain 95 g of a pale yellow oily substance. Step 3: Transfer 95g of the pale yellow oily substance into a crystallization vessel, add 950mL of sec-butyl acetate and 47.5mL of cyclohexanol, heat to 55℃, stir until completely dissolved, then add 0.38g of N-lauroyl-L-glutamic acid diethyl ester, set the stirring speed to 180rpm, and reduce the temperature to 32℃ at a rate of 1.2℃ / min, then adjust the stirring speed to 80rpm, and reduce the temperature to 5℃ at a rate of 0.1℃ / min. Finally, at 5℃, perform vacuum filtration using a Büchner funnel and pre-cooled filter paper. Wash the crystals twice quickly with 100mL of n-hexane cooled to 5℃, then place the wet product in a vacuum drying oven and dry at 45℃ for 6h to obtain 78.5g of white flaky crystals. HPLC analysis showed that the cholesterol purity was 99.73% and the cholesterol yield was 78.5%.
[0021] Example 2 Step 1: Weigh 100g of crude cholesterol and dissolve it in 500mL of a mixed solvent of n-hexane / ethyl acetate (volume ratio 8:2). Keep the solution warm in a 38℃ water bath to obtain the light phase. Take 500mL of acetonitrile / water solution (volume ratio 90:10) as the extractant and perform a three-stage countercurrent liquid-liquid extraction at 40℃. Collect the organic phase and concentrate it under reduced pressure at 45℃ to 145g. Step 2: The organic phase from Step 1 was pumped into a reaction column containing maleimide-functionalized polyvinyl alcohol microspheres with a particle size of 100 mesh and a crosslinking degree of 12% using a constant flow pump at a flow rate of 4 mL / min. The column was held at 75℃ for 20 min, the effluent was collected, and the solvent was recovered under reduced pressure to obtain 92 g of a pale yellow oily substance. Step 3: Transfer 92g of the pale yellow oily substance into a crystallization vessel, add 920mL of sec-butyl acetate and 73.6mL of cyclohexanol, heat to 55℃, stir until completely dissolved, then add 0.46g of N-lauroyl-L-glutamic acid diethyl ester, set the stirring speed to 200rpm, and reduce the temperature to 35℃ at a rate of 1.5℃ / min, then adjust the stirring speed to 100rpm, and reduce the temperature to 10℃ at a rate of 0.05℃ / min. Finally, at 10℃, perform vacuum filtration using a Büchner funnel and pre-cooled filter paper. Wash the crystals twice quickly with 100mL of n-hexane cooled to 5℃, then place the wet product in a vacuum drying oven and dry at 45℃ for 6h to obtain 76.8g of white flaky crystals. HPLC analysis showed that the cholesterol purity was 99.89% and the cholesterol yield was 76.8%.
[0022] Example 3 Step 1: Weigh 100g of crude cholesterol and dissolve it in 500mL of a mixed solvent of n-hexane / ethyl acetate (volume ratio 7.5:2.5). Keep the solution warm in a water bath at 38℃ to obtain the light phase. Take 500mL of acetonitrile / water solution (volume ratio 88:12) as the extractant and perform a 4-stage countercurrent liquid-liquid extraction at 37℃. Collect the organic phase and concentrate it under reduced pressure at 45℃ to 148g. Step 2: The organic phase from Step 1 was pumped into a reaction column containing maleimide-functionalized polyvinyl alcohol microspheres with a particle size of 200 mesh and a crosslinking degree of 8% using a constant flow pump at a flow rate of 4 mL / min. The column was held at 60℃ for 40 min, the effluent was collected, and the solvent was recovered under reduced pressure to obtain 94 g of a pale yellow oily substance. Step 3: Transfer 94g of the pale yellow oily substance into a crystallization vessel, add 846mL of sec-butyl acetate, heat to 55℃, and stir until completely dissolved. Then add 0.188g of N-lauroyl-L-glutamic acid diethyl ester, set the stirring speed to 150rpm, and reduce the temperature to 30℃ at a rate of 1.0℃ / min. Then adjust the stirring speed to 50rpm and reduce the temperature to 0℃ at a rate of 0.15℃ / min. Finally, at 0℃, perform vacuum filtration using a Büchner funnel and pre-cooled filter paper. Wash the crystals twice quickly with 100mL of n-hexane cooled to 0℃. Then place the wet product in a vacuum drying oven and dry at 45℃ for 6h to obtain 77.2g of white flaky crystals. HPLC analysis showed that the cholesterol purity was 99.68% and the cholesterol yield was 77.2%.
[0023] Example 4 Step 1: Weigh 100g of crude cholesterol and dissolve it in 500mL of a mixed solvent of n-hexane / ethyl acetate (volume ratio 7:3). Keep the solution warm in a 38℃ water bath to obtain the light phase. Take 500mL of acetonitrile / water solution (volume ratio 85:15) as the extractant and perform a 5-stage countercurrent liquid-liquid extraction at 38℃. Collect the organic phase and concentrate it under reduced pressure at 45℃ to 150g. Step 2: The organic phase from Step 1 was pumped into a reaction column containing maleimide-functionalized polyvinyl alcohol microspheres with a particle size of 150 mesh and a crosslinking degree of 10% using a constant flow pump at a flow rate of 4 mL / min. The column was held at 70℃ for 30 min, the effluent was collected, and the solvent was recovered under reduced pressure to obtain 95 g of a pale yellow oily substance. Step 3: Transfer 95g of the pale yellow oily substance into a crystallization vessel, add 950mL of sec-butyl acetate and 47.5mL of cyclohexanol, heat to 55℃, stir until completely dissolved, then add 0.76g of N-lauroyl-L-glutamic acid diethyl ester, set the stirring speed to 170rpm, and reduce the temperature to 33℃ at a rate of 1.3℃ / min, then adjust the stirring speed to 60rpm, and reduce the temperature to 3℃ at a rate of 0.08℃ / min. Finally, at 3℃, perform vacuum filtration using a Büchner funnel and pre-cooled filter paper. Wash the crystals twice quickly with 100mL of n-hexane cooled to 3℃, then place the wet product in a vacuum drying oven and dry at 45℃ for 6h to obtain 79.1g of white flaky crystals. HPLC analysis showed that the cholesterol purity was 99.94% and the cholesterol yield was 79.1%.
[0024] Example 5 Step 1: Weigh 120g of crude cholesterol and dissolve it in 780mL of a mixed solvent of n-hexane / ethyl acetate (volume ratio 7.7:2.3). Keep the solution warm in a water bath at 38℃ to obtain the light phase. Take 780mL of acetonitrile / water solution (volume ratio 85:15) as the extractant and perform a 5-stage countercurrent liquid-liquid extraction at 38℃. Collect the organic phase and concentrate it under reduced pressure at 45℃ to 175g. Step 2: The organic phase from Step 1 was pumped into a reaction column containing maleimide-functionalized polyvinyl alcohol microspheres with a particle size of 100 mesh and a crosslinking degree of 10% using a constant flow pump at a flow rate of 4 mL / min. The column was held at 70℃ for 30 min, the effluent was collected, and the solvent was recovered under reduced pressure to obtain 158 g of a pale yellow oily substance. Step 3: Transfer 158g of the pale yellow oily substance into a crystallization vessel, add 1422mL of sec-butyl acetate and 71.1mL of cyclohexanol, heat to 58℃, stir until completely dissolved, then add 0.79g of N-lauroyl-L-glutamic acid diethyl ester, set the stirring speed to 180rpm, and reduce the temperature to 32℃ at a rate of 1.2℃ / min, then adjust the stirring speed to 80rpm, and reduce the temperature to 5℃ at a rate of 0.1℃ / min. Finally, at 5℃, perform vacuum filtration using a Büchner funnel and pre-cooled filter paper. Wash the crystals twice quickly with 100mL of n-hexane cooled to 5℃, then place the wet product in a vacuum drying oven and dry at 45℃ for 6h to obtain 86.4g of white flaky crystals. HPLC analysis showed that the cholesterol purity was 99.88% and the cholesterol yield was 72.0%.
[0025] Comparative Example 1 The separation and purification method of this comparative example is the same as that of Example 1. The difference is that N-lauroyl-L-glutamic acid diethyl ester was not added in step 3. After dynamic gradient cooling crystallization and filtration, 75.0g of light yellow powder was obtained. After HPLC analysis, the cholesterol purity was 99.45% and the cholesterol yield was 75.0%.
[0026] Comparative Example 2 The comparative example uses the same separation and purification method as Example 1, except that the selective addition reaction in step 2 is not performed. After crystallization in step 3, 77.8g of light yellow crystals are obtained. HPLC analysis shows that the cholesterol purity is 99.05% and the cholesterol yield is 77.8%.
[0027] Comparative Example 3 100g of crude cholesterol was placed in a round-bottom flask, 1000mL of anhydrous ethanol was added, and the mixture was heated under reflux in a water bath at 78℃. The mixture was stirred until the solid was completely dissolved, resulting in a clear yellow solution. Heating was stopped, and the mixture was allowed to cool naturally to room temperature and stand overnight to precipitate crystals. The crystals were then filtered, and the filter cake was washed with cold ethanol. The wet crystals were dried under vacuum at 50℃ to obtain the first recrystallized product. The first recrystallization product was transferred to a flask, 700 mL of ethyl acetate was added, and the mixture was heated to 70 °C to dissolve. After cooling naturally to room temperature, the mixture was refrigerated at 4 °C for 4 hours. The mixture was then filtered, washed with cold ethyl acetate, and dried to obtain 55 g of white crystals. HPLC analysis showed that the purity of cholesterol was 98.9% and the yield was 55%.
[0028] This invention utilizes the conjugated diene structure of 7-dehydrocholesterol to induce an addition reaction in a reaction column containing maleimide-functionalized cross-linked polyethylene microspheres, transforming 7-dehydrocholesterol and cholesterol from homologues into compounds with significantly different separability, thus reducing the difficulty of removal. Simultaneously, a crystal inhibitor is introduced that preferentially adsorbs onto the crystal face of dihydrocholesterol and heterogeneously crystallizes it, resulting in a stable purity of over 99.5% for the crystalline cholesterol.
[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for separating and purifying crude cholesterol, characterized in that: Includes the following steps: 1) After neutralizing the crude cholesterol product obtained by saponification with soda ash, it was dissolved in a mixed solvent containing n-hexane and ethyl acetate, and the organic phase was obtained by countercurrent liquid-liquid extraction with acetonitrile aqueous solution. 2) The organic phase is passed into a cross-linked polyethylene microsphere reaction column containing maleimide-functionalized material for a selective addition reaction, and the effluent is collected. 3) Add sec-butyl acetate solvent to the effluent to dissolve it, then add a crystal inhibitor to perform dynamic gradient cooling crystallization, filter, and obtain cholesterol crystals.
2. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 1), the volume ratio of hexane to ethyl acetate in the mixed solvent containing hexane and ethyl acetate is 7:3 to 8:
2. The volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 85:15 to 90:
10.
3. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 1), the temperature of the countercurrent liquid-liquid extraction is 35~40℃, and the number of extraction stages is 3~5.
4. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 2), the maleimide-functionalized cross-linked polyethylene microspheres have a particle size of 100-200 mesh and a cross-linking degree of 8-12%.
5. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 2), the selective addition reaction is carried out at a temperature of 60-75°C for 20-40 minutes.
6. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 3), the mass ratio of the effluent to the volume ratio of sec-butyl acetate solvent is 1g:8~12mL; The melting temperature is 50~60℃.
7. The method for separating and purifying crude cholesterol according to claim 6, characterized in that: The sec-butyl acetate solvent also contains cyclohexanol, and the amount of cyclohexanol used is 5-10% of the sec-butyl acetate solvent.
8. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 3), the crystal inhibitor is selected from N-acyl amino acid derivatives; The amount of crystal inhibitor added is 0.2 to 0.8% of the mass of the effluent.
9. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: The dynamic gradient cooling crystallization includes a first-stage cooling and a second-stage cooling, wherein the first-stage cooling is carried out at a cooling rate of 1~1.5℃ / min to 30~35℃, and the stirring rate is 150~200rpm. The second-stage cooling process reduces the temperature to 0-10℃ at a cooling rate of 0.05-0.15℃ / min, while the stirring rate is 50-100rpm.
10. The method for separating and purifying crude cholesterol according to claim 1, characterized in that: In step 3), the filtration temperature is 0~10℃.