WO2021036384A1

WO2021036384A1 - Method for purifying vitamin d3

Info

Publication number: WO2021036384A1
Application number: PCT/CN2020/093960
Authority: WO
Inventors: 蔡育森; 余曦; 何剑洋; 王志强; 许德兴; 魏高宁
Original assignee: 厦门金达威维生素有限公司; 厦门金达威集团股份有限公司
Priority date: 2019-08-30
Filing date: 2020-06-02
Publication date: 2021-03-04
Also published as: CN110527700B; CN110527700A

Abstract

Disclosed is a method for purifying vitamin D₃, the method involving: mixing crude vitamin D₃ as a substrate with a weak organic acid or anhydride in a non-aqueous solvent, subjecting same to esterification with lipase column A and extraction with an extractant, concentrating the resulting organic layer, then adding a crystallization solvent to dissolve same, then reducing the temperature for crystallization, and filtering and drying same to obtain a vitamin D₃ ester crystal; re-dissolving same, subjecting same to saponification with lipase column B and extraction, returning the resulting organic layer to the lipase column B for saponification and extracting same, and continuing the cycle until the vitamin D₃ ester residue in the organic layer is < 1 wt%; finally subjecting the organic layer to concentration, crystallization and drying to obtain a fine vitamin ₃ crystal; and treating the crystallization mother liquor for reuse as the substrate.

Description

A kind of vitamin D 3 purification method

Technical field

The present invention relates to the field of vitamins, particularly to a method for the purification of vitamin D _3.

Background technique

Vitamin D ₃ , also known as cholecalciferol, has the physiological functions of promoting intestinal calcium absorption, regulating calcium and phosphorus metabolism, and inducing bone calcium and phosphorus deposition. It can promote bone growth and prevent rickets. Large doses are also used for skin tuberculosis. , Skin and mucous membranes of various types of lupus erythematosus, etc.

_{Vitamin D 3} synthesized by photochemical reaction contains tachysterol, photosterol and residual raw material 7-dehydrocholesterol produced by side reactions. Generally, only 20-30 million IU/g of vitamin D ₃ can be obtained. These by-products are similar in structure. Similar in nature, it is difficult to further separate and purify.

At present, vitamin D ₃ purification methods mainly include chemical methods and column chromatography, and there is also a supercritical CO ₂ column separation method. The chemical method, such as US3157678A, is based on the principle that the illuminated product is made into the corresponding ester, and the difference in solubility in the solvent caused by the difference in the structure of the substance after esterification is used to separate impurities through multiple crystallizations, and then saponified into vitamin D ₃ , and then Further crystallization and purification to obtain food-grade vitamin D ₃ , the esterification reactant is mainly various acid chlorides. To purify vitamin D ₃ by chemical method, the esterification raw materials such as benzoyl chloride or butyryl chloride are highly toxic and produce more difficult-to-treat waste water and waste residue. In addition, the chemical synthesis process is relatively violent, and the yield is also low. The method described in US3157678A is the highest yield of the chemical method recorded in the literature, and the yield of the vitamin D ₃ butyl ester crystallization step is 72%. Column chromatography, such as US3367950A, requires separation by a chromatographic column, eluting with a large amount of solvent to obtain the main section, and then concentrating and replacing the solvent for crystallization to obtain high-unit vitamin D ₃ . Column chromatography requires the use of a large amount of solvent for elution, the subsequent recovery of the solvent is large, the energy consumption is high, the column will also generate more solid waste, and the overall yield is not high, and the industrialization cost is greater than that of the chemical method. Supercritical CO ₂ column separation method, for example, the supercritical method mentioned in CN1240209A is to use supercritical or liquid carbon dioxide through a chromatographic column, select a modified solvent as the mobile phase, and a modified silica gel as a stationary phase for separation. The equipment pressure is 7.0～ 15.0MPa. The supercritical column separation method requires the use of high-pressure equipment above 7MPa, the equipment safety requirements are high, and the more expensive modified silica gel is required, the industrialization cost is high, and it is difficult to industrialize the application.

Summary of the invention

The purpose of the present invention is to provide a method for purifying vitamin D3 with high yield, low cost, and environmental friendliness.

In order to achieve the above objective, the present invention provides a method for purifying vitamin D3, which is characterized in that it comprises the following steps:

S1. Use the _{crude vitamin D 3} as a substrate, mix it with a weak organic acid or anhydride in a non-aqueous solvent, and perform an esterification reaction through lipase column A to obtain an esterified liquid _{containing vitamin D 3 ester;}

S2. Extract the obtained esterified liquid containing vitamin D ₃ ester with an extractant, the residual acid enters the water layer, the vitamin D ₃ ester enters the organic layer, and the organic layer is concentrated to obtain a concentrated vitamin D ₃ ester;

S3. Add the concentrated vitamin D ₃ ester to the crystallization solvent to dissolve and then cool down and crystallize, filter and dry to obtain vitamin D ₃ ester crystals; optionally, the crystalline mother liquor is concentrated, saponified, and thermally isomerized to obtain feed-grade vitamin D ₃ ；

S4. After dissolving the obtained vitamin D ₃ ester crystals in a non-aqueous solvent, the saponification reaction is carried out through the lipase column B to obtain a saponification liquid; the saponification liquid is extracted with water, and the separated organic acid enters the water layer, and the organic layer is heavy Return to the lipase column B to saponify and hydrolyze the remaining vitamin D ₃ ester again, and repeat the cycle of saponification-hydrolysis step 2 to 5 times until the _{residual vitamin D 3} ester in the organic phase is less than 1wt%, then stop the circulation;

S5. Concentrate, crystallize, and dry the organic layer to obtain vitamin D ₃ crystal products;

Optionally, the crystallization mother liquor is used as the substrate of step S1 after being concentrated and thermally isomerized.

_{Further, the content of the crude vitamin D 3} in the step S1 is 1500-3500 IU/g;

Optionally, the weight-volume ratio of the substrate to the non-aqueous phase solvent in the S1 step is 1 g: (5-30) ml, preferably 1 g: 8 ml.

Optionally, in the S1 step and S4 step, the non-aqueous solvent is each independently an alkane, a halogenated hydrocarbon or an aromatic hydrocarbon; preferably, the alkane is selected from n-hexane, pentane, heptane, At least one of cyclohexane and petroleum ether.

Further, in the step S1, the molar ratio of the substrate to the weak organic acid is 1:(1.0-10); preferably, the molar ratio of the substrate to the weak organic acid is 1:1.1;

Optionally, the weak organic acid is butyric acid, valeric acid or lauric acid.

Further, in the step S1, the acid anhydride is n-butyric anhydride, succinic anhydride or valeric anhydride;

Optionally, the molar ratio of the substrate to the acid anhydride is 1:(0.5-5); preferably, the molar ratio of the substrate to the acid anhydride is 1:0.55;

Optionally, the temperature of the esterification reaction is 20-50°C, preferably 35°C;

Optionally, the residence time of the material in the column when passing through the lipase column A is 0.5-5 hours, preferably, 1 hour.

Further, in the step S2, the extractant is water or aqueous methanol; preferably, the volume content of methanol in the aqueous methanol is 0-98%, and more preferably, the volume content of methanol in the aqueous methanol is 95%;

Optionally, the _{feed volume ratio of the extractant to the esterification liquid containing vitamin D 3} ester is (0.2～3):1; preferably, the ratio of the extractant to the esterification liquid _{containing vitamin D 3 ester} The feed volume ratio is 0.8:1.

Further, the extraction operations in the S2 step and the S4 step are performed in an extraction tower.

Further, in the step S3, the crystallization solvent is a ketone or alcohol solvent; preferably, the crystallization solvent is acetone;

Optionally, the crystallization temperature is -5 to -30°C, preferably, the crystallization temperature is -20°C;

Optionally, the ratio of the concentrated vitamin D ₃ ester to the crystallization solvent is 1 g: (0.7-5) ml, preferably 1 g: 1 ml.

Further, in the step S4, the _{mass-volume ratio of the vitamin D 3} ester crystal to the non-aqueous solvent is 1 g: (2-30) ml, preferably 1 g: 5 ml;

Optionally, the temperature of the saponification reaction is 20-50°C, preferably 35°C.

Further, in the step S5, the solvent of the crystallization is methyl formate or ethyl acetate, optionally, the temperature of the crystallization is 0-10°C, and the temperature is kept for more than 3 hours;

Optionally, the mass-volume ratio of the concentrate obtained by concentrating the organic layer to the crystallization solvent is 1 g: (4-20) ml, preferably 1 g: 6 ml.

Further, in the step S1 and the step S4, the lipase in the lipase column A and the lipase column B is an immobilized Candida antarctica lipase B, preferably, each independently is Novozymes 435 immobilized lipase, or Candida Antarctica lipase B treated by the immobilization method disclosed in CN105462952A;

Preferably, in the step S1, the mass ratio of the substrate to the lipase in the lipase column A is 1:(0.2-30), preferably 1:(0.5-2);

Optionally, in the step S4, the _{mass ratio of the vitamin D 3} ester crystal to the lipase in the lipase column B is 1:(0.2-30), preferably 1:(0.5-2).

The substrate material that can be processed in the present invention can be selected in the range of 1500-3500 IU/g. If the content is too low, the effect of esterification and crystallization is poor, and the enzyme activity is likely to decrease quickly, which affects the number of times of lipase application. If the content is too high, esterification may not be required, and it can be purified directly by crystallization.

The present invention improves on the problems of the current chemical synthesis method with relatively low industrialization cost, innovatively adopts the enzyme acylation method to carry out the vitamin D ₃ esterification reaction, and then obtains the higher purity vitamin D ₃ ester through crystallization and purification, and then Utilizing the reversibility of the enzyme acylation reaction, the vitamin D ₃ ester crystal is saponified into vitamin D ₃ by enzyme, and finally high unit vitamin D _{3 is} obtained by crystallization. The invention uses enzymatic method for esterification and saponification, no more toxic raw materials, enzymes can be reused, some raw materials can be recovered, recycled, no solid waste is discharged, conform to the concept of green chemistry, and the enzyme acylation reaction is more gentle , The yield is improved compared with chemical method, and the cost of industrial application is lower.

_{The crystallization yield of vitamin D 3 in the} whole process is 50-75.3%, plus the vitamin D ₃ in each crystallization mother liquor, the recovery rate of pure vitamin D ₃ is 90-97%, that is, the _{loss of vitamin D 3 in the} whole process is 3-10%.

The lipase column A and lipase column B are stainless steel or other solvent-resistant material pipe processing cylindrical columns, the shape is shown in Figure 1 but the size is not limited to the drawings, filter paper and defatted cotton are placed at the inlet and outlet to prevent enzymes from being solution Take out, the lipase used is immobilized Candida antarctica lipase B, for example, it can be Novozymes 435 immobilized lipase (Novozym ^R 435), or the Antarctic pseudolipase treated with the immobilization method disclosed in CN105462952A Silk Lipase B. Lipase is applied more than 40 times.

The vitamin D ₃ crystal detection is based on the 2015 edition of the "Chinese Pharmacopoeia" method to detect specific rotation and absorption coefficient, and the product content is determined based on the "Chinese Pharmacopoeia" general rule 0722 Vitamin D determination method.

The present invention uses lipase catalysts for esterification of vitamin D _3, vitamin D ₃ or a weak organic acid with an excess of the raw material mixed acid anhydride, an esterification reaction using a lipase in a non-aqueous solvent.

The invention uses an extraction tower to extract and remove excess acid from the reaction. After separation, the excess acid can continue to be used for esterification.

The invention utilizes the reversibility of the enzyme esterification reaction, uses lipase in the non-aqueous phase solvent to saponify and hydrolyze vitamin D ₃ ester, and combines the extraction tower to extract the acid produced by the hydrolysis, and the organic phase circulates through the lipase column to promote the reversible reaction Proceed in the direction of hydrolysis, and finally the hydrolysis rate will reach 99% or more. After separation, the extracted organic acid can be applied to the esterification reaction to achieve recycling utilization.

The beneficial effects of the present invention:

1. The present invention creatively adopts the lipase method instead of the original chemical method to _{esterify the raw material vitamin D 3} , avoiding the use of highly toxic and corrosive benzoyl chloride, butyryl chloride and other raw materials.

2. The reaction adopts lipase esterification and saponification, the process is milder than the chemical method, and the yield is obviously improved. Only the esterification and crystallization step has increased by more than 8% compared with the highest 72% in the chemical method.

3. The acid in the water layer of the reaction extraction can be separated and recycled. Compared with the chemical method, there are no difficult-to-treat waste residues and more harmful waste water discharge. Lipase can be recycled many times, which meets the needs of green and environmental protection new industries.

4. Utilizing the reversibility of lipase esterification, combined with extraction tower, circulating column hydrolysis, instead of chemical method using strong alkali for hydrolysis, the process is gentler, and the hydrolyzed acid can be recycled and used to reduce the use of raw materials.

_{5. The unit of the vitamin D 3} crystal product produced by the present invention exceeds 39 million IU/g, and the detection index meets the requirements of the Chinese Pharmacopoeia.

In summary, compared with the chemical method with the lowest industrialization cost, the present invention has higher yield, no high-toxic raw materials, and most of the raw materials can be recycled, avoiding the discharge of waste residues and difficult-to-treat wastewater, and is a greener and more environmentally friendly method. , A lower cost and more economical method for purification _{of vitamin D 3.}

Description of the drawings

Fig. 1 is a schematic diagram of the structure of a lipase column A and a lipase column B according to a specific embodiment of the present invention.

Fig. 2 is a schematic flowchart of a specific embodiment of the present invention.

detailed description

The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings, in which the same or similar reference numerals indicate the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased on the market.

The following embodiments are described and understood with reference to FIGS. 1-2.

In Figure 2, if the density of the non-aqueous solvent used is higher than that of the extractant (for example, the non-aqueous solvent used is a halogenated hydrocarbon), the extraction tower becomes the organic phase, which enters and exits from the top, and the water phase enters and exits from the bottom.

Example 1

_{Put 20g (0.052mol) of crude resinous vitamin D 3} (unit 25.5 million IU/g) into the dissolving kettle, add 5.04g (0.057mol) butyric acid, 160ml n-hexane to dissolve, and use a pump to pass through the water at a flow rate of 0.65ml/min. 20g Novozymes 435 DN10×500 lipase column A (residence time 1h), the column temperature is 35℃, the esterification liquid is obtained, the conversion rate is 99.5%, and the volume flow ratio of methanol water: esterification liquid is 0.8:1 with a pump Pour 95% water-containing methanol and esterification liquid into extraction tower A to obtain the upper organic phase. Control the internal temperature to be less than 35℃ and concentrate to dryness. The n-hexane obtained by concentration can be reused to obtain vitamin D ₃ ester. Use 24ml of acetone. After dissolution, the temperature was slowly lowered to -20°C, and it was allowed to stand for 3 hours, filtered, washed and dried to obtain _{12.79 g of 98.8% vitamin D 3} ester, and the _{total yield of vitamin D 3} ester was 83.8%. After the mother liquor is concentrated and dried, it is saponified and isomerized to obtain feed-grade vitamin D ₃ 8.328 million IU/g, weighing 6.89 g. Vitamin D ₃ ester 12.79g was dissolved in 64ml n-hexane and passed through a DN10×500 lipase column B containing 20g Novozymes 435 at a flow rate of 1ml/min. The column temperature was 35℃. After the column was passed, the pump was pumped into the extraction tower B with water Extraction separation; after extraction, the organic phase passes through lipase column B again at a flow rate of 1ml/min, and the column temperature is 35°C. After the column is passed, the pump is pumped into extraction tower B for extraction and separation with water; after this cycle 3 times, take a sample to detect vitamin D ₃ After the residual ester is less than 1wt%, stop passing the column. After extracting the remaining solution, the organic phase is obtained. After the organic phase is concentrated below 35°C _{, 11.40g of vitamin D 3 is} obtained, the unit is 36.88 million IU/g, the saponification yield is 98.4%, and the n-hexane obtained by concentration Can be reused. Vitamin D _{3 was} dissolved in 69ml of ethyl acetate, filtered through a clean filter, and then slowly cooled and crystallized. After cooling to 5°C, the temperature was kept for 3 hours, filtered, washed with cold ethyl acetate, dried and crystallized to obtain 9.61g of vitamins. D ₃ crystal product, unit 39.95 million IU/g, specific rotation +110°, absorption coefficient 471. After the mother liquor is concentrated and isomerized, _{1.79 g of vitamin D 3} scraps are obtained, with a unit of 20.42 million IU/g, which is applied to the esterification step. _{The crystallization yield of vitamin D 3 in the} whole process is 75.3%, plus the vitamin D ₃ contained in each crystallization mother liquor, the recovery rate of pure vitamin D _{3 is 96.7%.}

Example 2

_{Put 20g (0.052mol) of crude resinous vitamin D 3} (unit 25.5 million IU/g) into the dissolving kettle, add 6.87g (0.078mol) butyric acid, and dissolve with 160ml n-hexane, and use a pump to pass through the water at a flow rate of 0.65ml/min. 20g Novozymes 435 DN10×500 lipase column A (residence time 1h), the column temperature is 35℃, the esterification liquid is obtained, the conversion rate is 99.8%, and the volume flow ratio of methanol water: esterification liquid is 0.8:1 with a pump Pour 95% aqueous methanol and esterification liquid into extraction tower A to extract the upper organic phase. Control the internal temperature to be less than 35°C and concentrate to dry. The concentrated n-hexane can be reused to obtain vitamin D ₃ ester. Use 24ml of acetone. After dissolving, the temperature was slowly lowered to -20°C, and it was allowed to stand for 3 hours, filtered, washed and dried to obtain _{12.50 g of 98.6% vitamin D 3} ester, and the _{total yield of vitamin D 3} ester was 81.8%. After the mother liquor is concentrated and dried, it is saponified and isomerized to obtain feed-grade vitamin D ₃ 8,397,300 IU/g, with a weight of 6.91g. Vitamin D ₃ ester 12.50g is dissolved in 63ml n-hexane and passed through a DN10×500 lipase column B containing 20g Novozymes 435 at a flow rate of 1ml/min. The column temperature is 35℃. After the column is passed, the pump is pumped into the extraction tower B with water Extraction separation; after extraction, the organic phase passes through lipase column B again at a flow rate of 1ml/min, and the column temperature is 35°C. After the column is passed, the pump is pumped into extraction tower B for extraction and separation with water; after this cycle 3 times, take a sample to detect vitamin D ₃ After the residual ester <wt 1%, stop passing the column, extract the remaining solution to obtain the organic phase. After the organic phase is concentrated below 35°C _{, 11.34g of vitamin D 3 is} obtained, the unit is 36.24 million IU/g, and the saponification yield is 98.5%. Alkane can be reused. Vitamin D _{3 was} dissolved by adding 68ml ethyl acetate, filtered through a clean filter, and then slowly cooled to crystallize. After cooling to 5°C, keep it for 3 hours, filter, wash with cold ethyl acetate, dry and crystallize to obtain 9.48g vitamins D ₃ crystal product, unit 39.42 million IU/g, specific rotation +111°, absorption coefficient 473. After the mother liquor is concentrated and isomerized, _{1.86 g of vitamin D 3} scraps are obtained, the unit is 20.07 million IU/g, which is applied to the esterification step. _{The crystallization yield of vitamin D 3 in the} whole process is 73.2%, plus the vitamin D ₃ in each crystallization mother liquor, the recovery rate of pure vitamin D _{3 is 95.0%.}

Example 3

_{Put 20g (0.052mol) of crude resinous vitamin D 3} (unit 25.5 million IU/g) into the dissolving kettle, add 4.52g (0.029mol) butyric anhydride, and dissolve it with 160ml n-hexane. Use a pump to pass through the water at a flow rate of 0.65ml/min. 20g Novozymes 435 DN10×500 lipase column A (residence time 1h), and the column temperature is 35℃, and the esterification liquid is obtained, the conversion rate is 99.6%, and the volume flow ratio of methanol water: esterification liquid is 0.8:1 with a pump. Pour 95% aqueous methanol and esterification liquid into extraction tower A to extract the upper organic phase. Control the internal temperature to be less than 35°C and concentrate to dry. The concentrated n-hexane can be reused to obtain vitamin D ₃ ester. Use 24ml of acetone. After dissolving, the temperature was slowly lowered to -20°C, and it was allowed to stand for 3 hours, filtered, washed and dried to obtain _{12.80 g of 98.8% vitamin D 3} ester, and the _{total yield of vitamin D 3} ester was 83.9%. After the mother liquor is concentrated and dried, it is saponified and isomerized to obtain feed-grade vitamin D ₃ 8.332 million IU/g, weighing 6.89 g. 12.8g vitamin D ₃ ester is dissolved in 64ml n-hexane and passed through the DN10×500 lipase column B containing 20g Novozymes 435 at a flow rate of 1ml/min. The column temperature is 35℃. After the column is passed, the pump is pumped into the extraction tower B with water Extraction separation; after extraction, the organic phase passes through lipase column B again at a flow rate of 1ml/min, and the column temperature is 35°C. After the column is passed, the pump is pumped into extraction tower B for extraction and separation with water; after this cycle 3 times, take a sample to detect vitamin D ₃ After the residual ester is <wt 1%, stop passing the column. After extracting the remaining solution, the organic phase is obtained. After the organic phase is concentrated below 35°C _{, 11.51g of vitamin D 3 is} obtained, the unit is 36.61 million IU/g, and the saponification yield is 98.5%. Alkane can be reused. Vitamin D _{3 was} dissolved in 69ml of ethyl acetate, filtered through a clean filter, and then slowly cooled and crystallized. After cooling to 5°C, the temperature was kept for 3 hours, filtered, washed with cold ethyl acetate, dried and crystallized to obtain 9.62g vitamins D ₃ crystal product, unit 39.7 million IU/g, specific rotation +111°, absorption coefficient 477. After the mother liquor is concentrated and isomerized, _{1.82 g of vitamin D 3} scraps are obtained, with a unit of 20.05 million IU/g, which is applied to the esterification step. _{The crystallization yield of vitamin D 3 in the} whole process is _{75.4%, plus the vitamin D 3} in each crystallization mother liquor, and the recovery rate of pure vitamin D _{3 is 96.9%.}

Example 4

_{Put 20g (0.052mol) of crude resinous vitamin D 3} (unit 25.5 million IU/g) into the dissolving kettle, add 6.58g (0.042mol) butyric anhydride, and dissolve with 160ml n-hexane, and use a pump to pass through the water at a flow rate of 0.65ml/min. 20g Novozymes 435 DN10×500 lipase column A (residence time 1h), the column temperature is 35℃, the esterification liquid is obtained, the conversion rate is 99.8%, and the volume flow ratio of methanol water: esterification liquid is 0.8:1 with a pump Pour 95% aqueous methanol and esterification liquid into extraction tower A to extract the upper organic phase. Control the internal temperature to be less than 35°C and concentrate to dry. The concentrated n-hexane can be reused to obtain vitamin D ₃ ester. Use 24ml of acetone. After dissolution, the temperature was slowly lowered to -20°C, and it was allowed to stand for 3 hours, filtered, washed and dried to obtain _{12.51 g of 98.5% vitamin D 3} ester, and the _{total yield of vitamin D 3} ester was 81.8%. The mother liquor is concentrated and dried and then saponified and isomerized to obtain feed grade vitamin D ₃ 8,207,900 IU/g, weight 6.84g. Vitamin D ₃ ester 12.51g was dissolved in 63ml n-hexane and passed through a DN10×500 lipase column B containing 20g Novozymes 435 at a flow rate of 1ml/min. The column temperature was 35℃. After the column was passed, the pump was pumped into the extraction tower B with water Extraction separation; after extraction, the organic phase passes through lipase column B again at a flow rate of 1ml/min, and the column temperature is 35°C. After the column is passed, the pump is pumped into extraction tower B for extraction and separation with water; after this cycle 3 times, take a sample to detect vitamin D ₃ After the residual ester <wt 1%, stop passing the column, extract the remaining solution to obtain the organic phase. After the organic phase is concentrated below 35°C _{, 11.34g of vitamin D 3 is} obtained, the unit is 36.1 million IU/g, and the saponification yield is 98.2%. Alkane can be reused. Vitamin D _{3 was} dissolved by adding 68ml ethyl acetate. After filtering through a clean filter, it began to slowly cool down and crystallize. After cooling to 5°C, keep it for 3 hours, filter, wash with cold ethyl acetate, dry and crystallize to obtain 9.23g vitamins D ₃ crystal product, unit 39.35 million IU/g, specific rotation +110°, absorption coefficient 474. After the mother liquor is concentrated and isomerized, _{1.96 g of vitamin D 3} scraps are obtained, with a unit of 20.51 million IU/g, which is applied to the esterification step. _{The crystallization yield of vitamin D 3 in the} whole process was 72.4%, plus the vitamin D ₃ in each crystallization mother liquor, the recovery rate of pure vitamin D _{3 was 94.2%.}

Example 5

The esterification solvent used 400ml, and the rest were the same as in Example 1. The esterification yielded _{12.40g of 99.0% vitamin D 3} butyl ester, and finally _{9.43g of vitamin D 3} crystal product, with a unit of 39.56 million IU/g, specific rotation +107°, absorption The coefficient is 480. The total yield of crystallization is 73.3%, and the total recovery of _{vitamin D 3 is 93.9%.}

Example 6

The butyric acid used for esterification was changed to 5.84g (0.057mol) of valeric acid, and the rest was the same as in Example 1. The esterification yielded _{12.03g of 98.8% vitamin D 3} amyl ester, and finally 8.34g of vitamin D ₃ crystals, with a unit of 39.62 million IU/ g, specific rotation +108°, absorption coefficient 469. The total yield of crystallization is 64.8%, and the total recovery of _{vitamin D 3 is 91.9%.}

Example 7

The column temperature was changed to 45°C, and the rest was the same as in Example 1. The esterification yielded _{12.23g of 99.0% vitamin D 3} butyl ester, and finally 9.22g of vitamin D ₃ crystal product, the unit was 39.45 million IU/g, and the specific rotation was +108° , Absorption coefficient 475. The total yield of crystallization is 71.3%, and the total recovery of _{vitamin D 3 is 91.5%.}

Example 8

The dosage of lipase was changed to 10g, and the rest was the same as in Example 1. The esterification conversion rate was 98.5%, and _{12.36g of 99.1% vitamin D 3} butyl ester was obtained; after the saponification and hydrolysis step was cycled 4 times, the test was qualified and finally 9.30g of vitamin D ₃ crystal was obtained. The unit is 39.79 million IU/g, the specific rotation is +110°, and the absorption coefficient is 483. The total yield of crystallization is 72.5%, and the total recovery of _{vitamin D 3 is 93.48%.}

Example 9

The dosage of lipase was changed to 10g, and the rest was the same as in Example 3. The esterification conversion rate was 98.8% compared with Example 1, and _{12.61g of 99.2% vitamin D 3} butyl ester was obtained. After the saponification and hydrolysis step was repeated 4 times, the test was qualified and finally vitamin D _{3 was obtained.} The crystal is 9.51g, the unit is 39.8 million IU/g, the specific rotation is +109°, and the absorption coefficient is 479. The total yield of crystallization is 74.2%, and the total recovery of _{vitamin D 3 is 95.19%.}

In the above embodiment, lipase column A and lipase column B are DN10×500 columns processed with DN10 stainless steel pipes. Filter paper and cotton are placed at the entrance and exit of the column to prevent enzymes from being carried out. The enzyme used is Novozymes 435 lipase and the enzyme activity is 10000 U/g. The enzyme application experiment was performed 40 times in the scheme of Example 1, and the enzyme still had a high efficiency. Lipase column A detected enzyme activity 7230 U/g, lipase column B detected enzyme activity 8243 U/g. The water-washing extraction tower is constructed by laboratory glass tower section and wire mesh packing, and the other steps of filtration and concentration are all commonly used instruments in the laboratory.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art will not depart from the principle and purpose of the present invention. Under the circumstances, changes, modifications, substitutions and modifications can be made to the above-mentioned embodiments within the scope of the present invention.

Claims

A method for purifying vitamin D 3 , which is characterized in that it comprises the following steps:

S1. Use the crude vitamin D 3 as a substrate, mix it with a weak organic acid or anhydride in a non-aqueous solvent, and perform an esterification reaction through lipase column A to obtain an esterified liquid containing vitamin D 3 ester;

S2. Extract the obtained esterified liquid containing vitamin D 3 ester with an extractant, the residual acid enters the water layer, the vitamin D 3 ester enters the organic layer, and the organic layer is concentrated to obtain a concentrated vitamin D 3 ester;

S3. The concentrated vitamin D 3 ester is added to the crystallization solvent to dissolve it and then cooled and crystallized, filtered and dried to obtain vitamin D 3 ester crystals;

S4. After dissolving the obtained vitamin D 3 ester crystals in a non-aqueous solvent, the saponification reaction is carried out through the lipase column B to obtain a saponification liquid; the saponification liquid is extracted with water, and the separated organic acid enters the water layer, and the organic layer is heavy Return to the lipase column B to saponify and hydrolyze the remaining vitamin D 3 ester again, and repeat the cycle of saponification-hydrolysis step 2 to 5 times until the residual vitamin D 3 ester in the organic phase is less than 1wt%, then stop the circulation;

S5. Concentrate, crystallize and dry the organic layer to obtain vitamin D 3 crystal products.
The method for purifying vitamin D 3 according to claim 1, wherein the mother liquor crystallized in step S3 is concentrated, saponified, and thermally isomerized to obtain feed-grade vitamin D 3 .
The method for purifying vitamin D 3 according to claim 1, wherein the mother liquor crystallized in step S5 is used as the substrate of step S1 after being concentrated and thermally isomerized.
The method for purifying vitamin D 3 according to claim 1 , wherein the content of the crude vitamin D 3 in the step S1 is 1500-3500 IU/g.
The method for purifying vitamin D 3 according to claim 1, wherein in the S1 step, the weight-volume ratio of the substrate and the non-aqueous solvent in the S1 step is 1 g: (5-30) ml.
The method for purifying vitamin D 3 according to claim 5, wherein the weight-volume ratio of the substrate and the non-aqueous solvent in the step S1 is 1 g: 8 ml.
The method for purifying vitamin D 3 according to claim 1, wherein in the S1 step, in the S1 step and the S4 step, the non-aqueous solvent is each independently an alkane, a halogenated hydrocarbon or an aromatic hydrocarbon.
8. The method for purifying vitamin D 3 according to claim 7, wherein the alkane is at least one selected from the group consisting of n-hexane, pentane, heptane, cyclohexane and petroleum ether.
The method for purifying vitamin D 3 according to claim 1, wherein in the S1 step, the molar ratio of the substrate to the weak organic acid is 1: (1.0-10).
9. The method for purifying vitamin D 3 according to claim 9, wherein the molar ratio of the substrate to the weak organic acid is 1:1.1.
The method for purifying vitamin D 3 according to claim 10, wherein the weak organic acid is butyric acid, valeric acid or lauric acid.
The method for purifying vitamin D 3 according to claim 1, wherein in step S1, the acid anhydride is n-butyric anhydride, succinic anhydride or valeric anhydride.
The method for purifying vitamin D 3 according to claim 1, wherein in the S1 step, the molar ratio of the substrate to the acid anhydride is 1: (0.5-5).
The method for purifying vitamin D 3 according to claim 13, wherein the molar ratio of the substrate to the acid anhydride is 1:0.55.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S1, the temperature of the esterification reaction is 20-50°C.
The method for purifying vitamin D 3 according to claim 15, wherein the temperature of the esterification reaction is 35°C.
The method for purifying vitamin D 3 according to claim 1, characterized in that, in the step S1, the residence time of the material in the column when passing through the lipase column A is 0.5 to 5 hours.
The method for purifying vitamin D 3 according to claim 17, wherein the residence time of the material in the column when passing through the lipase column A is 1 hour.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S2, the extractant is water or aqueous methanol.
The method for purifying vitamin D 3 according to claim 19, wherein the volume content of methanol in the aqueous methanol is 0-98%.
The method for purifying vitamin D 3 according to claim 20, wherein the volume content of methanol in the aqueous methanol is 95%.
The method for purifying vitamin D 3 according to claim 1, characterized in that, in the step S2, the feed volume ratio of the extractant to the esterified liquid containing the vitamin D 3 ester is (0.2～3):1 .
The method for purifying vitamin D 3 according to claim 22, wherein the feed volume ratio of the extractant to the esterified liquid containing the vitamin D 3 ester is 0.8:1.
The method for purifying vitamin D 3 according to claim 1, wherein the extraction operations in the S2 step and the S4 step are performed in an extraction tower.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S3, the crystallization solvent is a ketone or alcohol solvent.
The method for purifying vitamin D 3 according to claim 25, wherein the crystallization solvent is acetone.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S3, the temperature of the crystallization is -5 to -30°C.
The method for purifying vitamin D 3 according to claim 27, wherein the temperature of the crystallization is -20°C.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S3, the ratio of the concentrated vitamin D 3 ester to the crystallization solvent is 1 g: (0.7-5) ml.
The method for purifying vitamin D 3 according to claim 29, wherein the ratio of the concentrated vitamin D 3 ester to the crystallization solvent is 1 g:1 ml.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S4, the mass-volume ratio of the vitamin D 3 ester crystal and the non-aqueous solvent is 1 g: (2-30) ml.
The method for purifying vitamin D 3 according to claim 31 , wherein the mass volume ratio of the vitamin D 3 ester crystals and the non-aqueous solvent is 1 g: 5 ml.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S4, the temperature of the saponification reaction is 20-50°C.
The method for purifying vitamin D 3 according to claim 33, wherein the temperature of the saponification reaction is 35°C.
The method for purifying vitamin D 3 according to claim 1, wherein in step S5, the solvent for crystallization is methyl formate or ethyl acetate.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S5, the temperature of the crystallization is 0-10°C, and the temperature is kept for more than 3 hours.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S5, the mass-volume ratio of the concentrate obtained by concentrating the organic layer to the crystallization solvent is 1 g: (4-20) ml.
The method for purifying vitamin D 3 according to claim 37, wherein the mass-volume ratio of the concentrate obtained by concentrating the organic layer to the crystallization solvent is 1 g: 6 ml.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S1 and the step S4, the lipase in the lipase column A and the lipase column B is immobilized Candida antarctica Yeast lipase B, or Candida Antarctica lipase B treated with the immobilization method disclosed in CN105462952A.
The method for purifying vitamin D 3 according to claim 39, wherein the lipase in the lipase column A and the lipase column B is independently Novozymes 435 immobilized lipase.
The method for purifying vitamin D 3 according to claim 1, wherein in the step S1, the mass ratio of the substrate to the lipase in the lipase column A is 1:(0.2-30).
The method for purifying vitamin D 3 according to claim 41, wherein the mass ratio of the substrate to the lipase in the lipase column A is 1: (0.5-2).
The method for purifying vitamin D 3 according to claim 1, wherein in the S4 step, the mass ratio of the vitamin D 3 ester crystals and the lipase in the lipase column B is 1:(0.2-30) .
The method for purifying vitamin D 3 according to claim 43 , wherein the mass ratio of the vitamin D 3 ester crystal to the lipase in the lipase column B is 1: (0.5-2).