A kind of vitamin D3Method of purification
阅读说明：本技术 一种维生素d3的提纯方法 (A kind of vitamin D3Method of purification ) 是由 蔡育森 余曦 何剑洋 王志强 许德兴 魏高宁 于 2019-08-30 设计创作，主要内容包括：本发明公开了一种维生素D_3的提纯方法。具体为：以维生素D_3粗品为底物,在非水相溶剂中与弱有机酸或酸酐混合,通过脂肪酶柱A酯化,萃取剂萃取,将得到的有机层进行浓缩,然后加入结晶溶剂溶解后降温结晶,过滤、烘干得到维生素D_3酯结晶；再溶解后,通过脂肪酶柱B皂化,萃取,将得到的有机层重回脂肪酶柱B皂化,萃取,直至有机相中维生素D_3酯残存＜1wt％后,停止循环；最后将有机层进行浓缩、结晶、烘干,得维生素D_3结晶精品；结晶的母液处理后作为底物重新利用。所述方法收率更高,无高毒原料使用,原料可大部分循环使用,避免废渣和难处理废水的排放,是一种更绿色环保,成本更低更经济的维生素D_3提纯方法。(The invention discloses a kind of vitamin Ds 3 Method of purification.Specifically: with vitamin D 3 Crude product is substrate, is mixed in Examples of non-aqueous solvents with weak organic acid or acid anhydrides, is esterified by lipase column A, extractant extraction, and obtained organic layer is concentrated, and decrease temperature crystalline after recrystallisation solvent dissolution is then added, and filtering, drying obtain vitamin D 3 Ester crystallization；It after redissolution, is saponified by lipase column B, extraction, obtained organic layer is returned into lipase column B saponification, extraction, until vitamin D in organic phase 3 After ester remaining < 1wt%, stop circulation；Finally organic layer is concentrated, crystallized, is dried, vitamin D is obtained 3 Crystallize fine work；It is re-used after the mother liquid disposal of crystallization as substrate.The method yield is higher, and no high poison raw material uses, and raw material can largely be recycled, avoid the discharge of waste residue and difficult waste water, be a kind of more environmentally protective, the lower more economical vitamin D of cost 3 Method of purification.)
1. a kind of vitamin D3Method of purification, which comprises the steps of:
S1. with vitamin D3Crude product is substrate, is mixed in Examples of non-aqueous solvents with weak organic acid or acid anhydrides, by lipase column A into Row esterification is obtained containing vitamin D3The esterifying liquid of ester；
S2. gained is contained into vitamin D3The esterifying liquid of ester is extracted with extractant, and residual acid enters water layer, vitamin D3Ester into Enter organic layer, organic layer is concentrated to give to the vitamin D of concentration3Ester；
S3. by the vitamin D of concentration3Decrease temperature crystalline after recrystallisation solvent dissolution is added in ester, and filtering, drying obtain vitamin D3Ester knot It is brilliant；Optional, the mother liquor of crystallization is concentrated, is saponified, obtains feed grade vitamin D after hot isomery3；
S4. by resulting vitamin D3After ester crystallization is dissolved with Examples of non-aqueous solvents, saponification is carried out by lipase column B, obtains soap Change liquid；Saponification liquor is extracted with water, the organic acid isolated enters water layer, and organic layer is returned lipase column B and is saponified again Hydrolyze the vitamin D of remaining3Ester, repetitive cycling saponification-hydrolysis step 2~5 time are up to vitamin D in organic phase3Ester remaining < After 1wt%, stop circulation；
S5. organic layer is concentrated, crystallized, dried, obtain vitamin D3Crystallize fine work；
Optional, the mother liquor of crystallization is concentrated, the substrate after hot isomery as S1 step utilizes.
2. vitamin D as described in claim 13Method of purification, which is characterized in that the vitamin D in the S1 step3Crude product Content be 1500-3500IU/g；
Optional, the w/v of substrate and Examples of non-aqueous solvents in the S1 step is 1g:(5~30) ml, preferably 1g: 8ml。
Optional, in the S1 step and S4 step, the Examples of non-aqueous solvents is each independently alkane, halogenated hydrocarbons or fragrance Hydrocarbon；Preferably, the alkane is selected from least one of n-hexane, pentane, heptane, hexamethylene and petroleum ether.
3. vitamin D as described in claim 13Method of purification, which is characterized in that in the S1 step, substrate and weak organic acid Molar ratio be 1:(1.0~10)；Preferably, the molar ratio of the substrate and weak organic acid is 1:1.1；
Optional, the weak organic acid is butyric acid, valeric acid or lauric acid.
4. vitamin D as described in claim 13Method of purification, which is characterized in that in the S1 step, the acid anhydrides is positive fourth Acid anhydrides, succinic anhydride or valeric anhydride；
Optional, the molar ratio of the substrate and acid anhydrides is 1:(0.5~5)；Preferably, the molar ratio of the substrate and acid anhydrides is 1:0.55；
Optional, the temperature of the esterification is 20~50 DEG C, preferably 35 DEG C；
Optional, residence time of the material in column is 0.5~5 hour when the column A by lipase, it is preferred that small for 1 When.
5. vitamin D as described in claim 13Method of purification, which is characterized in that in the S2 step, the extractant be water Or aqueous methanol；Preferably, the volume content of methanol is 0~98% in the aqueous methanol, it is furthermore preferred that the aqueous methanol The volume content of middle methanol is 95%；
It is optional, the extractant with contain vitamin D3The input material volume ratio of the esterifying liquid of ester is (0.2~3): 1；Preferably, institute It states extractant and contains vitamin D3The input material volume ratio of the esterifying liquid of ester is 0.8:1.
6. vitamin D as described in claim 13Method of purification, which is characterized in that the extraction in the S2 step and S4 step Operation carries out in extraction tower.
7. vitamin D as described in claim 13Method of purification, which is characterized in that in the S3 step, the recrystallisation solvent is Ketone or alcohols solvent；Preferably, the recrystallisation solvent is acetone；
Optional, the temperature of the crystallization is -5~-30 DEG C, it is preferred that the temperature of the crystallization is -20 DEG C；
Optional, the vitamin D of concentration3The ratio of ester and recrystallisation solvent is 1g:(0.7~5) ml, preferably 1g:1ml.
8. vitamin D as described in claim 13Method of purification, which is characterized in that in the S4 step, the vitamin D3Ester The mass volume ratio example of crystallization and Examples of non-aqueous solvents is 1g:(2~30) ml, preferably 1g:5ml；
Optional, the temperature of the saponification is 20~50 DEG C, preferably 35 DEG C.
9. vitamin D as described in claim 13Method of purification, which is characterized in that in the S5 step, the solvent of the crystallization For methyl formate or ethyl acetate；
Optional, the temperature of the crystallization is 0~10 DEG C, heat preservation 3 hours or more；
Optional, the organic layer carries out that resulting concentrate and the mass volume ratio example of recrystallisation solvent is concentrated to be 1g:(4~20) Ml, preferably 1g:6ml.
10. vitamin D as described in claim 13Method of purification, which is characterized in that it is described in the S1 step and S4 step Lipase in lipase column A and lipase column B is the candida antarctica lipase B handled through immobilization, it is preferred that respectively It independently is Novi and believes 435 immobilized lipases, or the South Pole using the processing of process for fixation disclosed in CN105462952A False silk lipase B；
Preferably, in the S1 step, the mass ratio of the substrate and the lipase in lipase column A is 1:(0.2~30), it is excellent Select 1:(0.5-2)；
Optional, in the S4 step, the vitamin D3Ester crystallization and the mass ratio of the lipase in lipase column B are 1: (0.2~30), preferably 1:(0.5-2).
Technical field
The present invention relates to vitamin field more particularly to a kind of vitamin Ds3Method of purification.
Background technique
Vitamin D3Also known as cholecalciferol, there is the metabolism for promoting intestinal calcium absorption, adjusting calcium and phosphorus, induction sclerotin calcium The physiological function of phosphorus calmness can promote bone growth, prevent rickets, and it is each that large dosage is also used for cutaneous tuberculosis, skin and mucous membrane Type lupus erythematosus etc..
The vitamin D of photochemical reaction synthesis3Tachysterol, lumisterol and the residual raw materials 7- dehydrogenation generated containing side reaction Cholesterol is generally only capable of obtaining the vitamin D of ten thousand IU/g of 2000-30003, for these by-products because structure is similar, property is close, compared with Difficult further separation, purification.
Vitamin D at present3Method of purification mainly has chemical method and column chromatography, and in addition there are also supercritical COs2Post separation method. Chemical method, such as US3157678A, principle are illumination product to be made corresponding ester, are caused using structure of matter difference after esterification Dissolubility difference in a solvent, impurity is separated by multiple crystallization, resaponifying is vitamin D3, then further tie Crystalline substance purification obtains food-grade vitamin D3, esterification agent is mainly all kinds of acyl chlorides.Vitamin D is purified using chemical method3, used Esterification feed chlorobenzoyl chloride or the material toxicities such as butyl chloride it is big, generate more intractable waste water and waste residue in the process, in addition Chemical method synthesis process is relatively violent, and yield is also relatively low, and US3157678A the method is that document records chemical method highest yield, Accomplish vitamin D3Butyl ester crystallisation step yield is 72%.Column chromatography, such as US3367950A are needed with chromatographic column point From, carried out affording principal piece with a large amount of solvents, then be concentrated replacement solvent crystallized, obtain high unit vitamin D3.Column layer Analysis method needs are eluted using a large amount of solvent, and subsequent recovery quantity of solvent is larger, and energy consumption is high, and more consolidate can also be generated by crossing column It is useless, and whole yield is not high, industrialization cost is greater than chemical method.Supercritical CO2Post separation method, such as mentioned in CN1240209A Overcritical rule be by chromatographic column using overcritical or liquid carbon dioxide, select modified solvent to make mobile phase, modified silicon Glue is separated as stationary phase, and equipment pressure is in 7.0~15.0MPa.Overcritical post separation method needs the height using 7MPa or more Equipment is pressed, device security requires modified silica-gel that is high, and needing more expensive, industrializes at high cost, it is difficult to industrial applications.
Summary of the invention
The purpose of the present invention is to provide a kind of high yield, low cost, the methods of purification of environmental-friendly vitamine D3.
To achieve the above object, the present invention provides a kind of method of purification of vitamine D3, which is characterized in that including walking as follows It is rapid:
S1. with vitamin D3Crude product is substrate, is mixed in Examples of non-aqueous solvents with weak organic acid or acid anhydrides, and lipase is passed through Column A carries out esterification, obtains containing vitamin D3The esterifying liquid of ester；
S2. gained is contained into vitamin D3The esterifying liquid of ester is extracted with extractant, and residual acid enters water layer, vitamin D3 Ester enters organic layer, and organic layer is concentrated to give to the vitamin D of concentration3Ester；
S3. by the vitamin D of concentration3Decrease temperature crystalline after recrystallisation solvent dissolution is added in ester, and filtering, drying obtain vitamin D3 Ester crystallization；Optional, the mother liquor of crystallization is concentrated, is saponified, obtains feed grade vitamin D after hot isomery3；
S4. by resulting vitamin D3After ester crystallization is dissolved with Examples of non-aqueous solvents, be saponified by lipase column B anti- It answers, obtains saponification liquor；Saponification liquor is extracted with water, the organic acid isolated enters water layer, and organic layer returns lipase column B again The vitamin D of secondary saponification remaining3Ester, repetitive cycling saponification-hydrolysis step 2~5 time are up to vitamin D in organic phase3Ester After remaining < 1wt%, stop circulation；
S5. organic layer is concentrated, crystallized, dried, obtain vitamin D3Crystallize fine work；
Optional, the mother liquor of crystallization is concentrated, the substrate after hot isomery as S1 step utilizes.
Further, the vitamin D in the S1 step3The content of crude product is 1500-3500IU/g；
Optional, the w/v of substrate and Examples of non-aqueous solvents in the S1 step is 1g:(5~30) ml, preferably 1g:8ml.
It is optional, in the S1 step and S4 step, the Examples of non-aqueous solvents be each independently alkane, halogenated hydrocarbons or Aromatic hydrocarbon；Preferably, the alkane is selected from least one of n-hexane, pentane, heptane, hexamethylene and petroleum ether.
Further, in the S1 step, the molar ratio of substrate and weak organic acid is 1:(1.0~10)；Preferably, the bottom The molar ratio of object and weak organic acid is 1:1.1；
Optional, the weak organic acid is butyric acid, valeric acid or lauric acid.
Further, in the S1 step, the acid anhydrides is n butanoic anhydride, succinic anhydride or valeric anhydride；
Optional, the molar ratio of the substrate and acid anhydrides is 1:(0.5~5)；Preferably, mole of the substrate and acid anhydrides Than for 1:0.55；
Optional, the temperature of the esterification is 20~50 DEG C, preferably 35 DEG C；
Optional, residence time of the material in column is 0.5~5 hour when the column A by lipase, it is preferred that is 1 Hour.
Further, in the S2 step, the extractant is water or aqueous methanol；Preferably, first in the aqueous methanol The volume content of alcohol is 0~98%, it is furthermore preferred that the volume content of methanol is 95% in the aqueous methanol；
It is optional, the extractant with contain vitamin D3The input material volume ratio of the esterifying liquid of ester is (0.2~3): 1；It is preferred that , the extractant with contain vitamin D3The input material volume ratio of the esterifying liquid of ester is 0.8:1.
Further, the extracting operation in the S2 step and S4 step carries out in extraction tower.
Further, in the S3 step, the recrystallisation solvent is ketone or alcohols solvent；Preferably, the recrystallisation solvent For acetone；
Optional, the temperature of the crystallization is -5~-30 DEG C, it is preferred that the temperature of the crystallization is -20 DEG C；
Optional, the vitamin D of concentration3The ratio of ester and recrystallisation solvent is 1g:(0.7~5) ml, preferably 1g:1ml.
Further, in the S4 step, the vitamin D3Ester crystallization and the mass volume ratio example of Examples of non-aqueous solvents are 1g: (2~30) ml, preferably 1g:5ml；
Optional, the temperature of the saponification is 20~50 DEG C, preferably 35 DEG C.
Further, in the S5 step, the solvent of the crystallization is methyl formate or ethyl acetate, optional, it is described The temperature of crystallization is 0~10 DEG C, heat preservation 3 hours or more；
Optional, the organic layer carries out that resulting concentrate and the mass volume ratio example of recrystallisation solvent is concentrated to be 1g:(4 ~20) ml, preferably 1g:6ml.
Further, in the S1 step and S4 step, the lipase in the lipase column A and lipase column B is through solid The candida antarctica lipase B of fixedization processing, it is preferred that be each independently Novi and believe 435 immobilized lipases, or use The South Pole vacation silk lipase B of the processing of process for fixation disclosed in CN105462952A；
Preferably, in the S1 step, the mass ratio of the lipase in the substrate and lipase column A be 1:(0.2~ 30), preferably 1:(0.5-2)；
Optional, in the S4 step, the vitamin D3Ester is crystallized is with the mass ratio of the lipase in lipase column B 1:(0.2~30), preferably 1:(0.5-2).
The accessible substrate raw material of the present invention is chosen as 1500-3500IU/g range.The too low esterification crystallization effect of content is poor, And be easy to decline enzyme activity comparatively fast, it influences lipase and applies number, content is too high, may not be needed to be esterified, can be straight by crystallizing Connect purification.
There are problems to improve for the current chemical synthesis for industrializing advantage of lower cost by the present invention, innovatively Vitamin D is carried out using the acylated method of enzyme3Then esterification obtains the higher vitamin D of purity by crystallization and purification3Ester connects Using enzyme acylation reaction invertibity, by vitamin D3The saponification of ester crystallization enzyme is vitamin D3, finally crystallization obtains high unit Vitamin D3.The present invention is esterified using enzyme process, is saponified, and does not use the big raw material of toxicity, and enzyme is reusable, part Raw material can recycle, and recycle, and no solid waste discharge meets green chemical concept, due to enzyme acylation reaction milder, yield compared with Chemical method increases, and industrial applications cost is lower.
Overall process vitamin D3Crystallization yield 50~75.3%, in addition the vitamin D in each crystalline mother solution3, pure vitamin D3The rate of recovery is 90~97%, i.e. overall process vitamin D3Loss 3~10%.
The lipase column A and lipase column B be stainless steel or other solvent resistant material pipeline processing cylindrical columns, outside Shape is as shown in Fig. 1 but size is not limited to attached drawing mark, and inlet and outlet, which place filter paper and degreasing cotton, prevents enzyme from being taken out of by solution, institute It is the candida antarctica lipase B handled through immobilization with lipase, such as can believes 435 immobilized lipases for Novi (NovozymR435) South Pole vacation silk lipase B, or using process for fixation disclosed in CN105462952A handled.Rouge Fat enzyme applies number up to 40 times or more.
The vitamin D3Crystallization detection is according to version " Chinese Pharmacopoeia " method detection specific rotations in 2015 and absorption coefficient, root Product content is measured according to 0722 vitamin D measuring method of " Chinese Pharmacopoeia " general rule.
The present invention carries out vitamin D using Lipids Enzymatic3Esterification, the vitamin D in Examples of non-aqueous solvents3Raw material and excess Weak organic acid or acid anhydrides mixing, carry out esterification using lipase.
The present invention reacts extra acid using extraction tower extraction removal.Extra acid can continue set for being esterified after separation.
The present invention utilizes the invertibity of enzyme esterification, and lipase saponification vitamin D is used in Examples of non-aqueous solvents3 Ester, and the acid for hydrolyzing output is extracted in conjunction with extraction tower, organic phase cycles through lipase column, promotes reversible reaction towards hydrolysis side To progress, finally make percent hydrolysis up to 99% or more.The organic acid extracted can be applied to esterification after separation, reach cycle It utilizes.
Beneficial effects of the present invention:
1, the use Lipids Enzymatic of the invention substitutes former chemical method to raw material vitamin D3It is esterified, avoids making With raw materials such as high poison, the chlorobenzoyl chloride of strong corrosive, butyl chlorides.
2, reaction is using esterification by lipase reaction, saponification, and process is mild compared with chemical method, and yield significantly improves.Only esterification crystallization step Suddenly highest compared with chemical method 72% 8% or more is improved.
3, recycle and use after acid is separable in the water layer of reaction, extraction, relative to chemical method, without intractable waste residue and More harmful discharge of wastewater, lipase can be recycled for multiple times, and meet environmentally protective new industrial requirement.
4, using the invertibity of esterification by lipase reaction, in conjunction with extraction tower, be circulated throughout column hydrolysis, substitution chemical method using highly basic into The acid of row hydrolysis, process milder, hydrolysis is recyclable, reduces raw material and uses.
5, the vitamin D of output of the present invention3The unit of crystallization fine work is more than 39,000,000 IU/g, and Testing index meets " China Pharmacopeia " it requires.
In conclusion comparing the minimum chemical method of original industrialization cost, yield of the present invention is higher, and no high poison raw material makes With, raw material can largely be recycled, and avoid the discharge of waste residue and difficult waste water, be it is a kind of more environmentally protective, cost is lower More economical vitamin D3Method of purification.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the lipase column A and lipase column B of a specific embodiment of the invention.
Fig. 2 is the general flow chart of a specific embodiment of the invention.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.Embodiment In particular technique or condition person is not specified, described technology or conditions or according to the description of product according to the literature in the art Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Following embodiment combination attached drawing 1-2 is explained and appreciated.
In Fig. 2 according to Examples of non-aqueous solvents density be higher than extractant when (if the Examples of non-aqueous solvents that uses is halogenated hydrocarbons), Extraction tower becomes organic phase upper entering and lower leaving, water phase bottom in and top out.