Purification method of vitamin B12

Abstract

The invention discloses a purification method of vitamin B12, which comprises the following steps: column chromatography: adding a vitamin B12 crude product into a methanol-containing dichloromethane solution, and fully dissolving; selecting silica gel as a stationary phase, filling the stationary phase into a chromatographic column, and loading the vitamin B12 solution to the chromatographic column; eluting by using a mixed solution of methanol and dichloromethane as a mobile phase; through TLC detection, a mixed solution of dichloromethane and methanol is used as a developing solvent, and a mobile phase is collected; spin-drying the mobile phase; recrystallization: adding the vitamin B12 column passing product into an acetone aqueous solution, heating and stirring until the vitamin B12 column passing product is completely dissolved, filtering the solution, controlling the temperature, dropwise adding acetone, controlling the temperature and stirring for a preset time after dropwise adding, filtering to obtain vitamin B12 crystals, and performing vacuum drying to obtain a vitamin B12 finished product. The method is easy and convenient to operate and low in cost, the yield and purity of the vitamin B12 can be effectively improved, the weight yield is 75% or above, the purity is 99% or above, and the method is very suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of purification methods, and in particular to a method for purifying vitamin B12. Background Art

[0002] Vitamin B12 is called Vitamin B12 in English, CAS NO: 68-19-9, chemical name: Coα-[α-(5,6-dimethylbenzimidazole)]-Coβ cyanocobalamide, molecular formula is C 63 H 88 CoN 14 O 14 P, molecular weight is 1355.38, structural formula is as follows:

[0003]

[0004] Vitamin B12, also known as cyanocobalamin, is an essential vitamin for the human body. Vitamin B12 in nature is synthesized by microorganisms. The main physiological function of vitamin B12 is to participate in the production of bone marrow red blood cells, prevent pernicious anemia, and prevent brain nerve damage. Vitamin B12 is widely used in medicines and foods, such as vitamin B12 injection, eye drops, baby food, etc. There is a lot of room for development in my country's vitamin B12 market.

[0005] Vitamin B12 is the most complex small molecule synthesized by organisms in nature. Currently, vitamin B12 is mainly obtained by bacterial culture and fermentation in industry. The existing purification process mainly uses macroporous resin adsorption, desorption and other processes to improve the purity of vitamin B12. Patent Publication No. CN110563778A discloses a method for preparing vitamin B12. After the vitamin B12 conversion liquid is adsorbed, washed and desorbed by resin, a vitamin B12 purified liquid is obtained. After concentration, crystallization, suction filtration and drying, vitamin B12 is obtained. The yield and purity of this method are not high, and the product has the risk of residual resin residues. Patent Publication No. CN113563396A discloses a method for preparing high-purity vitamin B12. The method uses octadecylsilane bonded silica gel as a stationary phase and uses preparative high-performance liquid chromatography to separate and purify vitamin B12 to obtain a product with a purity of more than 99.5%. However, the equipment and stationary phase used in this method are expensive, and the sample loading amount is small, making it difficult to achieve large-scale production. Summary of the invention

[0006] In order to overcome the defects of low yield, low purity, high cost and difficulty in large-scale production of vitamin B12 in the purification process in the prior art, the purpose of the present invention is to provide a method for purifying vitamin B12, which is not only simple to operate and low cost, but also can effectively improve the yield and purity of vitamin B12, with a weight yield of more than 75% and a purity of more than 99%, which is very suitable for large-scale industrial production.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] A method for purifying vitamin B12, comprising:

[0009] 1) Column pass steps:

[0010] 1-1) Raw material preparation: Add crude vitamin B12 into an appropriate amount of dichloromethane solution containing methanol and fully dissolve it to obtain a vitamin B12 solution;

[0011] 1-2) Column loading and sample loading: Silica gel is selected as the stationary phase, loaded into the chromatography column, and the dissolved vitamin B12 solution is loaded onto the chromatography column;

[0012] 1-3) Elution and collection: Use a mixed solution of methanol and dichloromethane as the mobile phase for elution; use TLC (thin layer chromatography) detection, use a mixed solution of dichloromethane and methanol with a volume ratio of 3:1 as the developing solvent, monitor the outflow of vitamin B12, and collect the mobile phase segment containing vitamin B12;

[0013] 1-4) Concentration: The collected mobile phase is spun dry to obtain the vitamin B12 column product;

[0014] 2) Recrystallization step:

[0015] 2-1) Dissolving: Add the vitamin B12 column product into an acetone aqueous solution, heat and stir until it is completely dissolved to obtain a solution;

[0016] 2-2) Filtration and crystallization: After the solution obtained in step 2-1) is filtered through a filter membrane, the filtrate is temperature-controlled to 20-30° C., and acetone is slowly added dropwise to induce crystallization. After the addition is complete, the temperature is continued to be controlled and stirred for a preset time, and then filtered to obtain vitamin B12 crystals;

[0017] 2-3) Drying: The vitamin B12 crystals are vacuum dried to obtain the finished vitamin B12 product.

[0018] Furthermore, in the step 1-1) of preparing raw materials, the volume ratio of methanol to dichloromethane in the dichloromethane solution containing methanol is 1:(3-5).

[0019] Furthermore, in 1-2) the column loading and sample loading steps, the mesh size of the silica gel is 100-300 mesh, preferably 200-300 mesh; the amount of the silica gel used is 30-50 times the weight of the crude vitamin B12, preferably 30-40 times.

[0020] Furthermore, in 1-2) the column packing and sample loading steps, the diameter-to-height ratio of the silica gel column is 1:5-1:10.

[0021] Furthermore, in 1-3) elution and collection, in the mixed solution of methanol and dichloromethane used as the mobile phase, the volume ratio of methanol to dichloromethane is 1:(10-20).

[0022] Furthermore, in the dissolution step 2-1), the volume ratio of acetone to water in the acetone aqueous solution is 1:(1-2), preferably 1:1, the amount of acetone aqueous solution is 10-15 times the amount of the vitamin B12 column product, and the dissolution temperature is 30-50°C.

[0023] Furthermore, in the filtration and crystallization step 2-2), the amount of acetone used is 20-50 times, preferably 30-40 times, the weight of the vitamin B12 column product.

[0024] Furthermore, in the filtration and crystallization step 2-2), the acetone addition time is controlled to be 1-1.5 hours, and the crystallization temperature is controlled to be 10-15°C.

[0025] Furthermore, in the filtration and crystallization step 2-2), after the dropwise addition is completed, the temperature is continued to be controlled to 15° C. and stirred for 3 hours.

[0026] Furthermore, in the drying step 2-3), the vitamin B12 crystals are vacuum dried at a temperature of 50°C.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention significantly improves the yield and purity of vitamin B12 by optimizing the column and recrystallization steps. Silica gel is used as the stationary phase, and a relatively cheap solvent system is used to reduce production costs. The method is simple to operate and can be easily scaled up to industrial production scale to meet market demand. The problem of resin residue is avoided, and the safety and purity of the product are improved.

[0029] 1. Improve the yield and purity: The purification method of the present invention significantly improves the yield and purity of vitamin B12 by optimizing the column step and the recrystallization step. Compared with the traditional macroporous resin adsorption method, this method can effectively reduce product loss and avoid the problem of resin residue, thereby improving the purity and quality of the final product. In the column step, silica gel as a stationary phase has good adsorption properties and can selectively adsorb vitamin B12, while the precise control of the mobile phase ensures the effective removal of impurities. The recrystallization step further purifies vitamin B12 by controlling the solvent ratio, temperature and crystallization conditions to obtain high-purity crystals.

[0030] 2. Reduce costs: The silica gel stationary phase and solvents such as methanol, dichloromethane, and acetone used in the present invention are relatively cheap, and the entire purification process does not require the use of expensive equipment and complicated operations, thereby significantly reducing production costs. Silica gel as a stationary phase is not only low in cost, but also easy to regenerate and reuse, reducing the cost of the stationary phase. At the same time, solvents such as methanol, dichloromethane, and acetone are all common chemical raw materials, relatively low in price, and easy to obtain and handle. A mixed solution of dichloromethane and methanol in a volume ratio of 3:1 is used as a developing agent, which has the advantages of good solubility, good separation effect, high stability, and relatively high safety.

[0031] 3. Suitable for large-scale production: The purification method of the present invention is simple to operate and easy to control, and each step has good reproducibility and stability, which is very suitable for scaling up to industrial production scale. The column step and the recrystallization step are both common chemical separation and purification technologies with mature operating experience and equipment support. By accurately controlling the conditions and parameters of each step, the stability and controllability of the production process can be ensured, thereby meeting the needs of large-scale production.

[0032] 4. High product safety: The purification method of the present invention avoids the problem of resin residue, thereby improving the safety and purity of the product. This is particularly important for the pharmaceutical and food industries, as residues may affect the quality and safety of the product. The traditional macroporous resin adsorption method has the problem of resin residue, while the present invention uses silica gel as the stationary phase and removes impurities and residues through precise elution and recrystallization steps, thereby ensuring the purity and safety of the product.

[0033] In addition, relative to the macroporous resin purification process, the purity of the vitamin B12 obtained by the purification method of the present invention is higher, and the purity of the crude vitamin B12 obtained by the purification method of the present invention is about 90%. After purification, the finished vitamin B12 product is obtained, and the purity reaches more than 99% (according to the 2020 version of the Chinese Pharmacopoeia), which can meet the market's ever-increasing quality requirements. Relative to the preparative chromatography process, the purification method of the present invention has low equipment and consumables costs and high yields. The weight yield of the purification method of the present invention is more than 75%, which is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the liquid chromatogram of vitamin B12 in Example 1.

[0035] Figure 2 This is the liquid chromatogram of vitamin B12 in Example 2. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment. Unless otherwise specified, the materials and equipment used in this embodiment can be purchased from the market.

[0037] A method for purifying vitamin B12, comprising:

[0038] 1) Column pass steps:

[0039] 1-1) Raw material preparation: Add crude vitamin B12 into an appropriate amount of dichloromethane solution containing methanol and fully dissolve it to obtain a vitamin B12 solution;

[0040] 1-2) Column loading and sample loading: Silica gel is selected as the stationary phase, loaded into the chromatography column, and the dissolved vitamin B12 solution is loaded onto the chromatography column;

[0041] 1-3) Elution and collection: Use a mixed solution of methanol and dichloromethane as the mobile phase for elution; use TLC (thin layer chromatography) detection, use a mixed solution of dichloromethane and methanol with a volume ratio of 3:1 as the developing solvent, monitor the outflow of vitamin B12, and collect the mobile phase segment containing vitamin B12;

[0042] 1-4) Concentration: The collected mobile phase is spun dry to obtain the vitamin B12 column product;

[0043] 2) Recrystallization step:

[0044] 2-1) Dissolving: Add the vitamin B12 column product into an acetone aqueous solution, heat and stir until it is completely dissolved to obtain a solution;

[0045] 2-2) Filtration and crystallization: After the solution obtained in step 2-1) is filtered through a filter membrane, the filtrate is temperature-controlled to 20-30° C., and acetone is slowly added dropwise to induce crystallization. After the addition is complete, the temperature is continued to be controlled and stirred for a preset time, and then filtered to obtain vitamin B12 crystals;

[0046] 2-3) Drying: The vitamin B12 crystals are vacuum dried to obtain the finished vitamin B12 product.

[0047] The present invention significantly improves the yield and purity of vitamin B12 by optimizing the column and recrystallization steps. Silica gel is used as the stationary phase, and a relatively cheap solvent system is used to reduce production costs. The method is simple to operate and can be easily scaled up to industrial production scale to meet market demand. The problem of resin residue is avoided, and the safety and purity of the product are improved.

[0048] 1. Improve the yield and purity: The purification method of the present invention significantly improves the yield and purity of vitamin B12 by optimizing the column step and the recrystallization step. Compared with the traditional macroporous resin adsorption method, this method can effectively reduce product loss and avoid the problem of resin residue, thereby improving the purity and quality of the final product. In the column step, silica gel as a stationary phase has good adsorption properties and can selectively adsorb vitamin B12, while the precise control of the mobile phase ensures the effective removal of impurities. The recrystallization step further purifies vitamin B12 by controlling the solvent ratio, temperature and crystallization conditions to obtain high-purity crystals.

[0049] 2. Reduce costs: The silica gel stationary phase and solvents such as methanol, dichloromethane, and acetone used in the present invention are relatively cheap, and the entire purification process does not require the use of expensive equipment and complicated operations, thereby significantly reducing production costs. Silica gel as a stationary phase is not only low in cost, but also easy to regenerate and reuse, reducing the cost of the stationary phase. At the same time, solvents such as methanol, dichloromethane, and acetone are all common chemical raw materials, relatively low in price, and easy to obtain and handle. A mixed solution of dichloromethane and methanol in a volume ratio of 3:1 is used as a developing agent, which has the advantages of good solubility, good separation effect, high stability, and relatively high safety.

[0050] 3. Suitable for large-scale production: The purification method of the present invention is simple to operate and easy to control, and each step has good reproducibility and stability, which is very suitable for scaling up to industrial production scale. The column step and the recrystallization step are both common chemical separation and purification technologies with mature operating experience and equipment support. By accurately controlling the conditions and parameters of each step, the stability and controllability of the production process can be ensured, thereby meeting the needs of large-scale production.

[0051] 4. High product safety: The purification method of the present invention avoids the problem of resin residue, thereby improving the safety and purity of the product. This is particularly important for the pharmaceutical and food industries, as residues may affect the quality and safety of the product. The traditional macroporous resin adsorption method has the problem of resin residue, while the present invention uses silica gel as the stationary phase and removes impurities and residues through precise elution and recrystallization steps, thereby ensuring the purity and safety of the product.

[0052] In addition, relative to the macroporous resin purification process, the purity of the vitamin B12 obtained by the purification method of the present invention is higher, and the purity of the crude vitamin B12 obtained by the purification method of the present invention is about 90%. After purification, the finished vitamin B12 product is obtained, and the purity reaches more than 99% (according to the 2020 version of the Chinese Pharmacopoeia), which can meet the market's ever-increasing quality requirements. Relative to the preparative chromatography process, the purification method of the present invention has low equipment and consumables costs and high yields. The weight yield of the purification method of the present invention is more than 75%, which is suitable for large-scale production.

[0053] As a preferred embodiment, in the step 1-1) of preparing raw materials, the volume ratio of methanol to dichloromethane in the dichloromethane solution containing methanol is 1:(3-5). The ratio of 1:(3-5) can ensure that vitamin B12 is fully dissolved in the solution, while avoiding unnecessary dissolution or interaction between solvents that may be caused by excessive methanol content, thereby affecting the subsequent purification step.

[0054] As a preferred embodiment, in 1-2) column loading and sample loading steps, the mesh number of the silica gel is 100-300 mesh, preferably 200-300 mesh; the silica gel particles selected within this range are of moderate size, which not only ensures sufficient specific surface area for adsorbing vitamin B12, but also avoids fluidity and operability problems that may be caused by too small particles. The amount of silica gel used is 30-50 times the weight of the crude vitamin B12, preferably 30-40 times. This dosage range ensures that the silica gel can fully adsorb vitamin B12, while avoiding the increased cost and subsequent processing difficulties that may be caused by excessive use of silica gel.

[0055] As a preferred embodiment, in 1-2) the column packing and sample loading steps, the diameter-to-height ratio of the silica gel column is 1:5-1:10. An appropriate diameter-to-height ratio helps to form a stable mobile phase flow pattern, reduce eddy currents and diffusion phenomena, and thus improve the separation effect. A diameter-to-height ratio that is too small may cause the mobile phase flow pattern to be unstable and affect the separation efficiency; while a diameter-to-height ratio that is too large may increase the dead volume of the column and reduce the degree of separation. An appropriate diameter-to-height ratio can take into account both the processing volume and the separation speed while ensuring the separation effect. A diameter-to-height ratio that is too small may result in limited processing volume and too slow a separation speed; while a diameter-to-height ratio that is too large may increase the difficulty of filling the column and the elution time. Within this diameter-to-height ratio range, the column can be fully utilized, and the distribution of silica gel particles is relatively uniform, which is conducive to the uniform distribution and effective separation of the sample in the column.

[0056] As a preferred embodiment, in 1-3) elution and collection, in the mixed solution of methanol and dichloromethane as the mobile phase, the volume ratio of methanol to dichloromethane is 1: (10-20). The mixing ratio of methanol and dichloromethane directly affects the elution behavior of the mixture on the silica gel column. Within this specific ratio range, the different components in the mixture can be effectively separated according to the difference in their adsorption force with the silica gel column. Methanol has a strong polarity and can destroy part of the adsorption force between vitamin B12 and the silica gel column, thereby promoting its elution; while dichloromethane has a weak polarity and helps to maintain a certain elution selectivity. The appropriate ratio of methanol and dichloromethane can ensure that vitamin B12 will not be over-eluted and lost during the elution process, nor will it remain on the silica gel column due to incomplete elution. This helps to improve the purity and yield of the final product and reduce the interference of impurities.

[0057] As a preferred embodiment, in the dissolving step 2-1), the volume ratio of acetone to water in the acetone aqueous solution is 1:(1-2), preferably 1:1, which can ensure good solubility of vitamin B12 in the acetone aqueous solution. Acetone, as an organic solvent, can increase the solubility of vitamin B12; and an appropriate amount of water helps to adjust the polarity and viscosity of the solution, and promotes the dissolution and dispersion of vitamin B12.

[0058] The amount of acetone aqueous solution used is 10-15 times the amount of vitamin B12 column product. This ratio ensures that there is enough solvent to dissolve vitamin B12, avoiding the problem of incomplete dissolution or too slow dissolution speed caused by insufficient solvent. At the same time, it also avoids excessive use of solvent and reduces production costs.

[0059] The dissolution temperature is 30-50℃. This temperature range will neither cause the decomposition or degradation of vitamin B12 due to excessively high temperature, nor affect its dissolution rate and solubility due to excessively low temperature. The appropriate dissolution temperature helps maintain the stability and activity of vitamin B12.

[0060] As a preferred embodiment, in 2-2) the filtration and crystallization step, the amount of acetone is 20-50 times, preferably 30-40 times, the weight of the vitamin B12 column product. Acetone is used as a solvent, and its amount directly affects the crystallization speed and efficiency of vitamin B12. When the amount of acetone is too little, it may lead to incomplete crystallization or too slow crystallization speed; while too much may increase the difficulty and cost of subsequent processing. Therefore, choosing an appropriate amount of acetone is crucial to improving the crystallization efficiency. An appropriate amount of acetone helps to effectively precipitate vitamin B12 from the solution while reducing the carryover and residue of impurities. By controlling the amount of acetone, the purity of vitamin B12 can be optimized to a certain extent, reducing the burden of subsequent purification steps.

[0061] As a preferred embodiment, in the filtration and crystallization step 2-2), the acetone addition time is controlled to be 1-1.5 hours. The acetone addition time is controlled to be 1-1.5 hours, which can ensure that the acetone is fully in contact with the vitamin B12 in the solution and gradually changes the composition of the solution, thereby facilitating the orderly arrangement and crystallization of the vitamin B12 molecules. Too fast a drop speed may cause a sharp change in the solution composition, which is not conducive to the uniform crystallization of vitamin B12; while too slow a drop speed may prolong the crystallization process and reduce production efficiency.

[0062] The crystallization temperature is controlled at 10-15°C. This ensures that vitamin B12 crystallizes at a suitable temperature, avoiding decomposition or degradation caused by too high a temperature, and slow or incomplete crystallization caused by too low a temperature. This temperature range helps the orderly arrangement of vitamin B12 molecules and the growth of crystals, thereby improving crystallization efficiency and purity.

[0063] As a preferred embodiment, in 2-2) the filtering and crystallization step, after the dropwise addition is completed, the temperature is continued to be controlled to 15°C and stirred for 3 hours. By continuing to control the temperature and stirring operation, it can be ensured that vitamin B12 is fully crystallized under suitable conditions, thereby improving the crystallization efficiency. This helps to shorten the production cycle and improve production efficiency. Stable temperature and stirring operation are conducive to the uniform growth and dispersion of vitamin B12 crystals, thereby obtaining a crystalline product of better quality. This has an important impact on the subsequent recrystallization step and the purity and performance of the final product.

[0064] As a preferred embodiment, in the drying step 2-3), the vitamin B12 crystals are vacuum dried at a temperature of 50°C. In the vacuum drying process, the choice of temperature is crucial. For vitamin B12 crystals, a temperature of 50°C is an ideal choice. This temperature is neither too high to cause damage to the crystal structure or loss of active ingredients, nor too low to affect the drying efficiency. At the same time, a temperature of 50°C can also inhibit the growth of bacteria to a certain extent, ensuring the sanitary quality of the product.

[0065] Embodiment 1:

[0066] A method for purifying vitamin B12 specifically comprises the following steps:

[0067] 1) Column pass steps:

[0068] 1-1) Raw material preparation: 500 g of crude vitamin B12 (purity 89.8%) was added to a dichloromethane solution containing methanol (the volume ratio of methanol to dichloromethane was 1:5) at a certain weight-to-volume ratio, and fully dissolved to obtain a vitamin B12 solution.

[0069] 1-2) Column loading and sample loading: Use 200 mesh, 15 kg silica gel as the stationary phase, load it into the chromatography column (diameter-to-height ratio is 1:8), and load the dissolved vitamin B12 solution onto the chromatography column.

[0070] 1-3) Elution and collection: Use a mixed solution of methanol and dichloromethane (volume ratio of 1:20) as the mobile phase for elution. Monitor the outflow of vitamin B12 through TLC detection, and collect the mobile phase segment containing vitamin B12.

[0071] 1-4) Concentration: The collected mobile phase was spin-dried, 500 g of acetone was added and spin-dried again to obtain 450 g of vitamin B12 column product.

[0072] 2) Recrystallization step:

[0073] 2-1) Dissolution: Add 450g of vitamin B12 column product into 4.5kg of acetone aqueous solution (the volume ratio of acetone to water is 1:1), heat and stir until completely dissolved. The dissolution temperature is 40°C, and stir and dissolve for 30 minutes.

[0074] 2-2) Filtration and crystallization: After filtering the solution through a filter membrane, the filtrate is temperature controlled to 25°C, and 14 kg of acetone is slowly added dropwise for 1 hour to induce crystallization. After the addition is completed, the temperature is continued to be controlled to 15°C, stirred for 3 hours, and then filtered to obtain vitamin B12 crystals.

[0075] 2-3) Drying: The vitamin B12 crystals were vacuum dried at 50°C to obtain 388 g of finished vitamin B12 product.

[0076] In this embodiment, weight yield = weight of finished vitamin B12 product ÷ crude vitamin B12 × 100%; weight yield = 388 ÷ 500 × 100% = 77.6%.

[0077] Reference Figure 1 The purity is 99.1% according to the Chinese Pharmacopoeia method.

[0078] Embodiment 2:

[0079] A method for purifying vitamin B12 specifically comprises the following steps:

[0080] 1) Column pass steps:

[0081] 1-1) Raw material preparation: 500 g of crude vitamin B12 (purity 89.8%) was added to a dichloromethane solution containing methanol (the volume ratio of methanol to dichloromethane was 1:3) at a certain weight-to-volume ratio, and fully dissolved to obtain a vitamin B12 solution.

[0082] 1-2) Column loading and sample loading: Use 300 mesh, 20 kg silica gel as the stationary phase, load it into the chromatography column (diameter-to-height ratio is 1:10), and load the dissolved vitamin B12 solution onto the chromatography column.

[0083] 1-3) Elution and collection: Use a mixed solution of methanol and dichloromethane (volume ratio of 1:10) as the mobile phase for elution. Monitor the outflow of vitamin B12 through TLC detection, and collect the mobile phase segment containing vitamin B12.

[0084] 1-4) Concentration: The collected mobile phase was spin-dried, 500 g of acetone was added and spin-dried again to obtain 441 g of vitamin B12 column product.

[0085] 2) Recrystallization step:

[0086] 2-1) Dissolution: Add 441 g of vitamin B12 column product into 4.4 kg of acetone aqueous solution (volume ratio of acetone to water is 1:1), heat and stir until completely dissolved. The dissolution temperature is 45°C, and stir and dissolve for 30 minutes.

[0087] 2-2) Filtration and crystallization: After filtering the solution through a filter membrane, the filtrate is temperature controlled to 28°C, and 15 kg of acetone is slowly added dropwise for 1.5 hours to induce crystallization. After the addition is completed, the temperature is continued to be controlled to 10°C, stirred for 3 hours, and then filtered to obtain vitamin B12 crystals.

[0088] 2-3) Drying: The vitamin B12 crystals were vacuum dried at 50°C to obtain 279 g of finished vitamin B12 product.

[0089] In this embodiment, weight yield = weight of finished vitamin B12 product ÷ crude vitamin B12 × 100%; weight yield = 379 ÷ 500 × 100% = 75.8%.

[0090] Reference Figure 2 The purity is 99.3% according to the Chinese Pharmacopoeia method.

[0091] The above implementation modes are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for purifying vitamin B12, characterized in that: include: 1) Column pass steps: 1-1) Raw material preparation: Add crude vitamin B12 into an appropriate amount of dichloromethane solution containing methanol and fully dissolve it to obtain a vitamin B12 solution; 1-2) Column loading and sample loading: Silica gel is selected as the stationary phase, loaded into the chromatography column, and the dissolved vitamin B12 solution is loaded onto the chromatography column; 1-3) Elution and collection: Use a mixed solution of methanol and dichloromethane as the mobile phase for elution; use TLC detection, use a mixed solution of dichloromethane and methanol with a volume ratio of 3:1 as the developing solvent, monitor the outflow of vitamin B12, and collect the mobile phase segment containing vitamin B12; 1-4) Concentration: The collected mobile phase is spun dry to obtain the vitamin B12 column product; 2) Recrystallization step: 2-1) Dissolving: Add the vitamin B12 column product into an acetone aqueous solution, heat and stir until it is completely dissolved to obtain a solution; 2-2) Filtration and crystallization: After the solution obtained in step 2-1) is filtered through a filter membrane, the filtrate is temperature-controlled to 20-30° C., and acetone is slowly added dropwise to induce crystallization. After the addition is complete, the temperature is continued to be controlled and stirred for a preset time, and then filtered to obtain vitamin B12 crystals; 2-3) Drying: The vitamin B12 crystals are vacuum dried to obtain the finished vitamin B12 product.

2. The method for purifying vitamin B12 according to claim 1, characterized in that 1-1) In the raw material preparation step, the volume ratio of methanol to dichloromethane in the dichloromethane solution containing methanol is 1:(3-5).

3. The method for purifying vitamin B12 according to claim 1, characterized in that 1-2) In the column packing and sample loading steps, the mesh size of the silica gel is 100-300 mesh; the amount of the silica gel used is 30-50 times the weight of the crude vitamin B12.

4. The method for purifying vitamin B12 according to claim 1, characterized in that 1-2) In the column packing and sample loading steps, the diameter-to-height ratio of the silica gel column is 1:5-1:

10.

5. The method for purifying vitamin B12 according to claim 1, characterized in that: 1-3) During elution and collection, the mobile phase is a mixed solution of methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is 1:(10-20).

6. The method for purifying vitamin B12 according to claim 1, characterized in that: 2-1) In the dissolution step, the volume ratio of acetone to water in the acetone aqueous solution is 1:(1-2), the amount of acetone aqueous solution used is 10-15 times the amount of the vitamin B12 column product, and the dissolution temperature is 30-50°C.

7. The method for purifying vitamin B12 according to claim 1, characterized in that: 2-2) In the filtration and crystallization steps, the amount of acetone used is 20-50 times the weight of the vitamin B12 column product.

8. The method for purifying vitamin B12 according to claim 1, characterized in that: 2-2) In the filtering and crystallization steps, the acetone addition time is controlled to be 1-1.5 hours, and the crystallization temperature is controlled to be 10-15°C.

9. The method for purifying vitamin B12 according to claim 1, characterized in that: 2-2) In the filtration and crystallization step, after the dropwise addition is completed, the temperature is continued to be controlled to 15° C. and stirred for 3 hours.

10. The method for purifying vitamin B12 according to claim 1, characterized in that: 2-3) In the drying step, the vitamin B12 crystals are dried in vacuum at 50°C.

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