WO2012055253A1 - Regeneration method of silica gel for chromatographing coenzyme q10 - Google Patents

Abstract

A regeneration method of chromatographic silica gel, especially a regeneration method of silica gel for chromatographing coenzyme Q10 is provided, wherein a high polar solvent is fed from the bottom of the chromatographic column to wash the column in the reverse direction. The treated chromatographic column can be repeatedly used to separate and purify coenzyme Q10. The regeneration method allows the repeated use of the chromatographic column for over 20 times, even over 100 times, while maintaining a good chromatographic effect. The method also has decreased production cost and labor intensity, and is safe and environment-friendly.

Description

 Method for regenerating coenzyme Q10 chromatography silica gel

 The invention belongs to the field of enzymology and organic chemistry, and relates to a method for regenerating chromatographic silica gel, in particular to a method for regenerating coenzyme Q10 chromatography silica gel. Background technique

 Coenzyme Q10 (Ubiquinone, Coenzyme Q10, CoQI O) plays an important role in the transport of electrons and protons in the oxidative respiratory chain. It is the key to the formation of ATP, which protects and restores the integrity of biofilm structure, stabilizes membrane potential and enhances immune response. The role is widely used in the adjuvant treatment of heart failure, coronary heart disease, hypertension, diabetes, cancer, AIDS, acute and chronic hepatitis, Parkinson's disease and other diseases. In addition, Coenzyme Q10 is widely used in food, health care products and cosmetics industries, and the market prospect is very broad.

 The traditional coenzyme Q10 production methods at home and abroad mainly include animal and plant tissue extraction methods, chemical synthesis methods and microbial fermentation methods. The extraction method of animal and plant tissue is limited by the content of coenzyme Q10 in the raw material, and the extraction yield is low and the cost is high. The chemical synthesis method produces cis and trans isomerism during synthesis, and a mixture of various isomers is obtained, which needs to be separated. The synthesis cost is increased, and some toxic reagents are added during chemical synthesis, which also puts forward higher requirements for purification to obtain clinically applicable products; microbial fermentation method is a new production method which has emerged in recent years, because microbial cells are easy to grow in large scale. The product is completely natural in all-trans configuration, has high bioavailability, and is safe and efficient relative to chemical synthesis. There is almost no chemical poison residue in the product, easy to separate and purify, and the clinical curative effect is good, which is the most promising prospect. Production method.

The microbial fermentation method produces coenzyme Q10, and the purification process generally adopts the following process: After the fermentation liquid is filtered, the filter residue is dried and granulated to obtain coenzyme Q10 powder, and the powder is then soaked and extracted with an organic solvent. The extract is concentrated to a certain content and then subjected to silica gel column chromatography. The petroleum ether and the acetone mixed solvent are eluted, and the eluate is concentrated and concentrated under reduced pressure, and crystallized with anhydrous ethanol, and dried by filtration to obtain a crude crystal of coenzyme Q10.

 As a kind of adsorbent, silica gel has a large specific surface area and strong adsorption capacity, and is widely used in column chromatography separation and purification in various industries such as medicine and chemical industry. During the column stratification, silica gel will adsorb some highly polar impurities, block the pores of the particles, and reduce their activity. The production cost of silica gel is also relatively high, and its production process pollutes the environment.

 In the purification process of coenzyme Q10, the column chromatography process accounts for more than 50% of the cost, and the silica gel consumption and solvent loss ^ A occupy the most important cost, making the production cost of coenzyme Q10 high.

 Chinese Patent Publication No. CN 101445435A describes a cleaning and purification process of coenzyme Q10: Coenzyme Q10 is dissolved in an organic solvent, and is packed in a high pressure liquid phase preparative column or a medium pressure silica gel packed column, eluted with an eluent, and collected in stages to obtain a high Purified coenzyme Q10 product. The packed silica gel is activated at a high temperature of 200 - 300 ° C and reused 10 - 200 times. The method for regenerating silica gel adopts a high-temperature activation method, and the method has high energy consumption, and each time the column chromatography process is completed, the silica gel needs to be removed from the column, which not only has high labor intensity, but also causes solvent evaporation, Safety problems such as static ignition occur, and solvent loss is also large. Although the high-temperature calcination method can remove the organic impurities in the silica gel, it also changes the internal pore structure of the silica gel, resulting in a decrease in the adsorption capacity of the silica gel.

 U.S. Patent No. 2007/0025976 A1 discloses a purification process for coenzyme Q10, mentioning a method for regenerating silica gel: eluting with 1 column volume of ethanol at room temperature, and then using 4 - 5 column volumes of hexane at 50 Elution at °C.

 In addition, there is a patent document reporting a method of regenerating a silica gel column after preparing other products.

However, the related patent documents report on the regeneration method of the silica gel column (especially the silica gel column after the preparation of the coenzyme Q10) are all based on the method of positive column washing, and the number of times the silica gel is repeatedly used is small. Summary of the invention

 The inventor discovered a coenzyme by creative labor and a large number of experiments.

The regeneration method of Q10 chromatography silica gel, and surprisingly found that the silica gel column after separation and purification of coenzyme Q10 can be reused after being treated by the method of the invention (ie, for chromatography of coenzyme Q10), and thus used-regenerated The number of cycles can be up to 20 or even more than 100 times, while the chromatographic yield can be maintained at 90% or 95% or even 98%. Thus, the following invention is provided: A method for regenerating a coenzyme Q10 chromatography silica gel, which comprises washing a column in a reverse direction from a bottom of a column with a highly polar solvent.

 Without being bound by theory, one possible reason is that silica gel column chromatography itself is a normal phase chromatography. The weakly polar substances are eluted first, and the strong polar impurities are adsorbed on the column head. Columns, when the strong polar impurities are eluted downward, will again contaminate the silica gel without adsorbing strong polar impurities, thus causing the use of silica gel to be greatly reduced.

 A regeneration method according to any one of the present invention, characterized by any one or more of the following items (1) to (4):

 (1) The highly polar solvent is one or more selected from the group consisting of ethyl acetate, acetone, methyl ethyl ketone, ethanol (preferably acetone), or a mixture of a highly polar solvent and a low polar solvent, wherein The polar solvent accounts for more than 50% by volume of the mixed liquid, and is preferably greater than 90%;

 (2) The high polar solvent is used in an amount of from 1 to 5 column volumes, preferably from 2-3 times column volume;

 The volume of the column is 0. 5 - 2 times the column volume / hour, preferably 0.5 - 1 column volume / hour;

 (4) The temperature of the highly polar solvent used for column washing is 20 - 50.

A regeneration method according to any one of the present invention, characterized in that Afterwards, a low polarity solvent is introduced from the top of the column for forward equilibrium.

 A regeneration method according to any one of the inventions, characterized by the following (1)

- Any one or more of (4):

 (1) The low-polar solvent is selected from one or more of petroleum ether, pentane, n-hexane, and heptane, or a mixture of a highly polar solvent and a low-polar solvent, wherein the low-polar solvent accounts for The volume of the mixed solution is greater than 50%, and preferably greater than 90%;

 (2) The low polar solvent is used in an amount of from 1 to 5 column volumes, preferably from 2 to 4 column volumes, more preferably from 3 column volumes;

 (3) the equilibrium flow rate is 0.5 - 2 column volumes / hour, preferably 0.5 - 1 column volume / hour;

 (4) The temperature of the lower-grade solvent used for the balance is 20*€ - 50*€.

 The coenzyme Q10 chromatography silica gel subjected to the regeneration treatment according to the method described in any one of the above is used for separating and purifying the coenzyme Q10, and the "regeneration-use" process is carried out one or more times; preferably, 15 to 25 times, More preferably, it is carried out 20 to 25 times, and more preferably 20 times. This allows the column to be reused more than 20 times. The "regeneration" refers to a regeneration treatment according to the method described in any one of the above, wherein the "use" refers to the separation of the coenzyme Q10 chromatography silica gel after the regeneration treatment according to the method described above. Purification of coenzyme Q10. The "use" step may be a method commonly used in the art, for example, referring to the product specification or the steps shown in the embodiment 1.

 Whenever the column is reused 15-25 times (preferably 20 times), the regeneration method is carried out according to the following steps 1) - 4):

 1) Wash the column one or more times with hydrogen peroxide in the opposite direction;

 2) Washing the column one or more times with the opposite direction of water;

3) Remove the silica gel and dry it at 1001C - 150X until the moisture is less than 5% (w/w); 4) Repack the column. The method can achieve the use of silica gel more than 100 times.

 The regeneration method according to any one of the present invention, wherein the pH of the hydrogen peroxide in the step 1) is 9 - 11; preferably 10. 5 - 11. The pH of the hydrogen peroxide can be adjusted with K0H or NaOH.

 Step 1) The action of the hydrogen peroxide is to remove the oxidized impurities of the impurities adsorbed in the silica gel. Since hydrogen peroxide is alkaline, it is necessary to replace the alkaline hydrogen peroxide with hot water, otherwise it will remain in the silica gel column and affect the next chromatography. Hydrogen peroxide and hot water can effectively remove the strong polar impurities that are difficult to elute from organic solvents.

 The regeneration method according to any one of the present invention, wherein the concentration of the hydrogen peroxide in the step 1) is from 3 % to 20% (w/w); preferably from 5% to 10% (w/w).

 The regeneration method according to any one of the invention, wherein the water in the step 2) is preferably deionized water.

 The regeneration method according to any one of the invention, wherein, in steps 1) and 2), the amount of hydrogen peroxide and water is independently from 1 to 5 column volumes; preferably two column volumes.

 The regeneration method according to any one of the present invention, wherein, in the steps 1) and 2), the temperature of the hydrogen peroxide and the water is independently 50*€ - Ι ΟΟ; preferably 70 - 80*€.

The regeneration method according to any one of the present invention, wherein the drying in the step 3) is atmospheric drying or reduced pressure drying, and preferably drying under reduced pressure. In the present invention, the term "silica gel" or "silica gel column" or "silica gel column" has the same meaning unless specifically stated otherwise, specifically, a silica gel column after separation and purification of coenzyme Q10. In one embodiment of the invention, the inner diameter is 25 - 500 mm, the column length is 300 - 5000 mm, and the silica gel mesh is 60 - 400 mesh; in a specific embodiment of the invention, the inner diameter is 400 legs and the column length is 4000 legs. Silicone 200Kg (200 - 300 mesh). 5 - The column pressure is 0. 1 - 2 MPa.

 In the present invention, unless otherwise specified, the term "Coenzyme Q10 Chromatography Silica Gel" refers to a silica gel column after separation and purification of Coenzyme Q10; specifically, the sample solution is an extract extracted from Coenzyme Q10 powder by an organic solvent. The organic solvent may be one or more of petroleum ether, pentane, n-hexane, heptane, preferably petroleum ether and or n-hexane.

 In the present invention, the term "highly polar solvent" means a solvent having a solvent polarity index of > 4. 0. Specifically, the highly polar solvent is one or more selected from the group consisting of ethyl acetate, acetone, methyl ethyl ketone, and ethanol, or a mixture of a high polar solvent and a low polar solvent, wherein the high polar solvent accounts for The volume of the mixed liquor is greater than 50%, and preferably greater than 90%, more preferably 100%.

 In the present invention, the term "low-polar solvent" means a solvent having a solvent polarity index < 0.2. Specifically, the low-polar solvent is selected from one or more of petroleum ether, pentane, n-hexane, and heptane, or a mixture of a highly polar solvent and a low-polar solvent, wherein the low-polar solvent accounts for The volume of the mixed liquor is greater than 50%, and preferably greater than 90%, more preferably 100%.

 In the present invention, the calculation method of the chromatographic yield: the chromatographic solution containing the coenzyme Q10 is collected in stages, and the chromatographic solution containing only the coenzyme Q10 spot is combined by TLC, and the coenzyme Q10 in the chromatographic solution is detected by HPLC. External standard content and area normalized content, chromatographic yield = (chromatography volume X chromatography liquid external standard content) I (loading volume X sample liquid content) X picture.

In the present invention, the effect of the repeated use of the silica gel is judged: comparing the chromatographic yields under the same loading amount, eluent, and elution conditions, when the chromatographic yield is greater than or equal to 90%, it is judged that the re-use effect is higher. Preferably, when the chromatographic yield is less than 90%, it is judged that the repeated use effect is deteriorated. Advantageous effects of the invention

 1. Adopting the reverse column washing technology to effectively prevent the strong polar impurities of the adsorption column head from contaminating other silica gels during the forward washing process, so that the chromatography silica gel can be reused more than 100 times, which greatly reduces the consumption of chromatography silica gel, thereby reducing production. Cost

 2. Adopt column-washing column to achieve continuous production, reduce the number of disassembly and assembly columns, and achieve a fully enclosed system throughout the production process, which not only reduces labor intensity, but also reduces solvent volatilization and achieves the purpose of clean production;

 3. Online washing column is used to replace the traditional in-pot stirring and washing. The solvent concentration gradient is large during washing, which significantly improves the efficiency of column washing. DRAWINGS

 Fig. 1: Schematic diagram of the silica gel regeneration process. detailed description

 The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. If no specific conditions are specified in the examples, they are carried out according to the general conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all commercially available products. Example 1 : Chromatographic effect of a new silica gel column (first use)

 The inner diameter of the column is 400 legs, the column length is 4000 legs, and the silica gel is 200Kg (200 ~ 300 mesh). The column is packed by wet method, and the column is hammered with a rubber hammer to make the silica gel tightly packed. The packed column was equilibrated with petroleum ether for 2 hours.

Take 300L Coenzyme Q10 extract (concentrated with petroleum ether from coenzyme Q10 powder, content 30mg/ml), through silica gel column chromatography on a plunger metering pump After completion, elute with petroleum ether/acetone (20/1) mixed solvent, collect Coenzyme Q10 chromatography solution by TLC, combine qualified chromatographic solution, and sample the HPLC to detect the content of coenzyme Q10 in the chromatographic solution. The yield of the HPLC method of the main peak of the chromatographic solution was 98.12%. Example 2: Chromatographic effect of a silica gel column after regeneration (after a "regeneration-use" process)

 Example 1 After eluting the column after coenzyme Q10, 1500 L of acetone 30 was pumped from the bottom of the column at a flow rate of 500 L/h to wash the column in the opposite direction until the top of the column was colorless, and then 500 L/ from the top of the column. The flow rate of h is pumped into a petroleum ether equilibrium column at a temperature of 1500 L. The chromatographic yield was 94.8%, chromatographic. The chromatographic yield of the recombination was 94.8%, and the chromatographic yield of the recombination was 94.8%. 10%。 The liquid primary peak HPLC area normalized content of 98. 10%. Example 3: Chromatographic effect of a silica gel column after regeneration (after 20 "regeneration-use" processes)

 The silica gel chromatography column was repeatedly used according to the column washing method and the chromatographic method of Example 2. According to the sample loading, elution method and collection method of the same example, the 20th chromatographic yield was 93. 8%。 The HPLC content of the main peak of the chromatographic solution was 98.7%. Example 4: Chromatographic effect of a silica gel column after regeneration (after 21 "regeneration-use" processes)

The silica gel column used in Example 3 was used 20 times, and was pumped from the bottom of the column to a pH of 1:1, a concentration of 5%, and a temperature of 80. (, the amount of 1000L of hydrogen peroxide, and then pumped into 1000L temperature 80 ^e C hot water wash, after washing, use N ₂ to dry the water, remove the silica gel, placed in a 150 Ό drier vacuum drying to silica gel moisture less than 5 %. The crystallization, the chromatographic yield was 94.5%, the layer was subjected to the 21st chromatography. The chromatography yield was 94.5%. The HPLC main area of the main peak of the liquid separation is 98. 08% ₀ Example 5: The chromatographic effect of the silica gel column after regeneration (after 100 "regeneration-use" processes)

 The silica gel column of Example 4 was used, and the regeneration method combined with Example 2 and Example 4 (i.e., 20 "regeneration-use" processes per the regeneration method of Example 2 was carried out, and the regeneration method of Example 4 was repeated once. 05%。 The chromatographic solution of the main peak HPLC area normalized content of 98. 05%, the chromatographic yield of the main peak of the chromatographic solution is 98.05%. . Comparative Example 1: Chromatographic effect of silica gel column using positive direction column regeneration treatment. Silica gel was chromatographed once in the same manner as in Example 1. Pumping 3 times column volume of acetone from the top of the column for forward washing to the bottom of the column The solution was colorless and was pumped from the top of the column to a column of 3 volumes of petroleum ether. The lysate of the chromatographic peak was HPLC. The chromatographic yield was 93.8%, and the chromatographic peak was analyzed by HPLC. The chromatographic yield was 93.8%. 10%。 The area normalized content of 98. 10%. The singularity of the HPLC method is 98.2%, repeating. The effect of use is worse. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

Rights request A method for regenerating a coenzyme Q10 chromatography silica gel, which comprises washing a column in the opposite direction from a highly polar solvent at the bottom of the column.  2. The method according to claim 1, characterized by any one or more of the following items (1) - (4):  (1) The highly polar solvent is one or more selected from the group consisting of ethyl acetate, acetone, methyl ethyl ketone, and ethanol, or a mixture of a highly polar solvent and a low polar solvent, wherein the high polar solvent accounts for The volume of the mixed solution is greater than 50%;  (2) the high polar solvent is used in an amount of from 1 to 5 column volumes;  (3) the flow rate of the washing column is 0.5 - 2 column volumes / hour;  (4) The temperature of the highly polar solvent used is 20*€ - 50*€.  The method according to any one of claims 1 or 2, characterized in that after the column is washed, a low polarity solvent is introduced from the column for positive equilibrium.  4. The method according to claim 3, characterized by any one or more of the following items (1) - (4):  (1) The low-polar solvent is selected from one or more of petroleum ether, pentane, n-hexane, and heptane, or a mixture of a highly polar solvent and a low-polar solvent, wherein the low-polar solvent accounts for The volume of the mixed solution is greater than 50%;  (2) the low polar solvent is used in an amount of from 1 to 5 column volumes;  (3) the equilibrium flow rate is 0.5 - 2 column volumes / hour;  (4) The temperature of the lower-grade solvent used is 50. The method according to any one of claims 1 to 4, wherein the coenzyme Q10 chromatography silica gel subjected to regeneration treatment according to the method of any one of claims 1 to 4 is used for separation and purification of coenzyme Q10 And performing such a "regeneration-use" process one or more times; preferably 15-25 times, more preferably 20-25 More preferably, it is 20 times.  6. The method of claim 5 further comprising the steps of:  1) Wash the column one or more times with hydrogen peroxide in the opposite direction;  2) Washing the column one or more times with the opposite direction of water;  3) Remove the silica gel and dry it at 1001C - 150X until the moisture is less than 5% (w/w); 4) Repack the column.  7. The method according to claim 6, wherein the pH of the hydrogen peroxide in step 1) is 9-11.  8. The method according to claim 6, wherein the concentration of hydrogen peroxide in step 1) is from 3% to 20% (w/w).  9. The method according to claim 6, wherein, in steps 1) and 2), the amount of hydrogen peroxide and water is independently from 1 to 5 column volumes.  10. The method according to claim 6, wherein in steps 1) and 2), the temperature of the hydrogen peroxide and the water are independently 50*€ - 100X.