CN101648984A - Method for separating and purifying 5'-cytidylic acid and derivatives thereof - Google Patents
Method for separating and purifying 5'-cytidylic acid and derivatives thereof Download PDFInfo
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- CN101648984A CN101648984A CN200810044191A CN200810044191A CN101648984A CN 101648984 A CN101648984 A CN 101648984A CN 200810044191 A CN200810044191 A CN 200810044191A CN 200810044191 A CN200810044191 A CN 200810044191A CN 101648984 A CN101648984 A CN 101648984A
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Abstract
The invention provides a method for separating and purifying 5'-cytidylic acid and derivatives thereof. The nanofiltration membrane separation technology is applied to the separation and purificationof the 5'-cytidylic acid and the derivatives thereof, and the separating and purifying process comprises desalting operation and concentrating operation. The method has the advantages of no phase change, no chemical reaction, no damage to bioactivity, no need of heating and normal-temperature operation in the separating process, simple steps, high selectivity, low energy consumption and the like.
Description
Technical field
The present invention relates to the separation purification method of a kind of 5 '-cytidylic acid and derivative thereof.
Background technology
5 '-cytidylic acid can be used as in raw material, intermediate and the foodstuff additive etc. of producing nucleotide drug, as be used for the raw material of synthetic cytidine (CTP), Polyinosinic-polycytidylic acid (PolyIC), my cytidine (Ara-C), 2 ', 3 '-zalcitabine (DDC), cytidine diphosphate (CDP-choline) and mix afterwards as healthcare products and foodstuff additive with other Nucleotide.
In the production process of 5 '-cytidylic acid, can produce a large amount of salt, purity to product has bigger influence, and especially a large amount of salt has significantly reduced the rate of recovery and reclaimed the quality of product in mother liquor reclaims, so be vital for the desalting and purifying of 5 '-cytidylic acid.
Existing desalting technology has condensing crystal method, solvent method, charcoal post exchange process etc., but they all exist some shortcomings:
1, the condensing crystal method is to remove by evaporation to desolvate to improve strength of solution, and the solubleness of solute descends in the solution, and solute just can be separated out.But the condensing crystal method can only realize partially desalted, and removes the sodium-chlor poor effect, if it is too high to be concentrated into concentration, 5 '-cytidylic acid in the solution also can be separated out in the lump, influences recovery rate.
2, solvent method is to utilize the minimum principle of the solubleness of solute in organic solvent, adds certain amount of organic solvent in solution, and to reduce the solubleness of solute in solution, solute just can be separated out.Also there is the similar shortcoming of condensing crystal method in solvent method, and organic solvent loss in use is bigger, and the solvent recuperation cost is higher.
3, charcoal post exchange process is that the characteristic of not adsorbing inorganics by the charcoal absorption organism reaches the purpose of separating desalination.The elementary operation process is: absorption, washing, wash-out, condensing crystal etc.Absorption be with solution stream through activated carbon column, organism is by charcoal absorption, inorganics then flows out with water.Washing is that water is with the debris flush away.Wash-out is that the organism desorption that utilizes suitable eluent will be adsorbed on the gac gets off.Concentrating is that the elutriant that concentration is lower evaporates except that desolvating, and to improve concentration, carries out crystallization with suitable manner again, also will decolour in case of necessity.Though this method is feasible theoretically, there is a lot of shortcomings, oversize such as operating process, equipment scale is huge, and the production cycle is long, the production cost height, and all to pass through acid-alkali regeneration at every turn, can produce a large amount of waste water and useless charcoal.
Membrane separation technique is an emerging isolation technique that has grown up since the sixties in 20th century, is a kind ofly to utilize one special to have a film of selecting performance, try hard to recommend outside moving down to mixture separate, purification, spissated a kind of separation method.According to the component difference of holding back, usually the membrane sepn process is divided into micro-filtration (MF), ultrafiltration (UF), nanofiltration (NF), reverse osmosis (RO), dialysis (D), electrodialysis (ED), gas delivery (GP), infiltration gasification (PV) etc.Wherein nanofiltration (Nanofiltration) is a kind of novel pressure-actuated membrane separation technique between reverse osmosis and ultrafiltration that grows up late 1980s, have good cutoff performance for molecular weight between the organism more than 200～2000, relatively low for the rejection of univalent ion and small-molecule substance.
Summary of the invention
The separation purification method of a kind of 5 '-cytidylic acid provided by the invention and derivative thereof, the nanofiltration membrane separation technology is applied to the separation and the purifying of 5 '-cytidylic acid and derivative thereof, have that sepn process does not undergo phase transition, do not take place any chemical reaction, does not destroy biological activity, need not heating and normal-temperature operation, step is simple, selectivity is high, low power consumption and other advantages, be particularly suitable for heat-sensitive substance for example fruit juice, protein, polypeptide, amino acid, medicine etc. fractional separation and concentrate.
In order to achieve the above object, the invention provides the separation purification method of a kind of 5 '-cytidylic acid and derivative thereof, the nanofiltration membrane separation technology is applied to the separation and the purifying of 5 '-cytidylic acid and derivative thereof, the method includes the steps of:
Step 1, prepare solution to be separated;
Solution to be separated should reach following requirement: solution should be as clear as crystal, do not contain foreign matter (should filter in advance if any foreign matter or muddiness), and concentration is below 20%, and pH value 2～11 requires dechlorination to handle;
Step 2, solution to be separated is poured in the material fluid bath;
Step 3, carry out desalination operation;
Step 3.1, operational outfit, pressure regulating device is regulated pressure to 1.0～2.0MPa, makes solution to be separated pass through nanofiltration membrane;
Step 3.2, collection penetrating fluid, trapped fluid returns liquid bath;
Step 3.3, control material concentration and keep the liquid level of material in feed liquid is poor, that is, the feed liquid in feed liquid is poor reduces how much weight (be permeate collection how much weight), just adds the distilled water of identical weight;
Step 3.4, measure specific conductivity and absorbancy (OD), and measure the interior temperature of charge of material fluid bath every getting penetrating fluid and the feed liquid concentrated solution in poor in 30～120 minutes respectively;
Step 3.5, continuous record for 3～6 times feed liquid poor in the specific conductivity of concentrated solution during less than 1000～10000 μ s/cm desalination finish;
Step 4, carry out concentration operation;
Step 4.1, material in the material fluid bath is supplied initial weight when carrying out step 2;
Step 4.2, operational outfit, pressure regulating device is regulated pressure to 1.0～2.0MPa, makes solution to be separated pass through nanofiltration membrane;
Step 4.3, collection penetrating fluid, trapped fluid returns liquid bath;
Step 4.4, every measured respectively in 30～120 minutes penetrating fluid and feed liquid poor in the specific conductivity and the absorbancy of concentrated solution, and the interior temperature of charge of material fluid bath;
Step 4.5, flow velocity are lower than 10～50L/h or the interior liquor capacity of material fluid bath just stopped concentration operation at remaining 1/5～1/3 o'clock.
In the separation and purification process of above-mentioned 5 '-cytidylic acid and derivative thereof, the material fluid bath chuck makes temperature be controlled at 20～50 ℃ with cooling water temperature.
The separation purification method of a kind of 5 '-cytidylic acid provided by the invention and derivative thereof, the nanofiltration membrane separation technology is applied to the desalination of 5 '-cytidylic acid and concentrates is very feasible, and is applicable to suitability for industrialized production.
Embodiment
Below specify preferred embodiment of the present invention:
1, reagent:
1), 5 '-cytidylic acid (Shanghai Sepal Chemical Co., Ltd.'s production), purity is greater than 98%;
2), sodium hydroxide, hydrochloric acid, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, be analytical pure;
3), distilled water, specific conductivity is less than 10 μ s/cm;
4), contrast solution:
20%5 '-cytidylic acid solution is transferred pH5 with 10% sodium hydroxide solution;
10%5 '-cytidylic acid solution is transferred pH5 with 10% sodium hydroxide solution;
4%5 '-cytidylic acid solution is transferred pH5 with 10% sodium hydroxide solution;
1% sodium chloride solution;
2, instrument:
DDS-IIA type electric conductivity instrument (Shanghai thunder magnetic instrument plant) is measured specific conductivity;
PHS-2F type pH meter (Shanghai thunder magnetic instrument plant) is measured the pH value;
757 type ultraviolet-visible spectrophotometers (Shanghai analytical instrument factory) are measured absorbancy;
LC-10ATVP high performance liquid chromatograph (Tianjin, island instrument company) is measured purity (HPLC);
3, experimental installation:
Nanofiltration membrane element NF 2540;
Embodiment 1
Step 1, prepare solution to be separated;
Get 5 '-cytidylic acid 1.5kg, sodium-chlor 1.5kg, SODIUM PHOSPHATE, MONOBASIC 1kg, adding distil water 35kg is transferred to 5.0 ± 0.1 back adding distil water ad pond om 38kg with 30% sodium hydroxide solution with the pH value;
Step 2, solution to be separated is poured in the material fluid bath;
Step 3, carry out desalination operation;
Step 3.1, operational outfit, pressure regulating device is regulated pressure to 1.2～1.5MPa, makes solution to be separated pass through nanofiltration membrane;
Step 3.2, collection penetrating fluid, trapped fluid returns liquid bath;
Step 3.3, control material concentration and keep the liquid level of material in feed liquid is poor, that is, add distilled water 10kg when the feed liquid in feed liquid is poor reduces weight 10kg (being permeate collection 10kg);
Step 3.4, every 30 minutes, get penetrating fluid and the feed liquid concentrated solution in poor respectively and measure specific conductivity and absorbancy (OD), and measure the temperature of charge in the material fluid bath;
Step 3.5, continuous record for three times feed liquid poor in the specific conductivity of concentrated solution during less than 10000 μ s/cm desalination finish;
Step 4, carry out concentration operation;
Step 4.1, material in the material fluid bath is supplied initial weight when carrying out step 2;
Step 4.2, operational outfit, pressure regulating device is regulated pressure to 1.2～1.5MPa, makes solution to be separated pass through nanofiltration membrane;
Step 4.3, collection penetrating fluid, trapped fluid returns liquid bath;
Step 4.4, the every rising 1% of concentration (30～120 minutes), measure respectively penetrating fluid and feed liquid poor in the specific conductivity and the absorbancy of concentrated solution, and the temperature of charge in the material fluid bath;
Step 4.5, flow velocity stop concentration operation when being lower than 10L/h or weighing less than 12Kg.
Experimental result is with reference to following table 1:
Embodiment 2
Step 1, prepare solution to be separated;
Get the water 15Kg after the extracting and demixing in the rough synthetic production in 5 '-cytidylic acid workshop, adding distil water 22Kg dilution, with 30% sodium hydroxide solution the pH value is transferred to 5.0 ± 0.1, contains 5 '-cytidylic acid 2.65kg in this solution approximately, inorganic salt mainly are sodium-chlor and SODIUM PHOSPHATE, MONOBASIC;
Step 2, solution to be separated is poured in the material fluid bath;
Step 3, carry out desalination operation;
Step 3.1, operational outfit, pressure regulating device is regulated pressure to 1.2～1.5MPa, makes solution to be separated pass through nanofiltration membrane;
Step 3.2, collection penetrating fluid, trapped fluid returns liquid bath;
Step 3.3, control material concentration and keep the liquid level of material in feed liquid is poor, that is, add distilled water 10kg when the feed liquid in feed liquid is poor reduces weight 10kg (being permeate collection 10kg);
Step 3.4, every 30 minutes, get penetrating fluid and the feed liquid concentrated solution in poor respectively and measure specific conductivity and absorbancy (OD), and measure the temperature of charge in the material fluid bath;
Step 3.5, continuous record for three times feed liquid poor in the specific conductivity of concentrated solution during less than 10000 μ s/cm desalination finish;
Step 4, carry out concentration operation;
Step 4.1, material in the material fluid bath is supplied initial weight when carrying out step 2;
Step 4.2, operational outfit, pressure regulating device is regulated pressure to 1.2～1.5MPa, makes solution to be separated pass through nanofiltration membrane;
Step 4.3, collection penetrating fluid, trapped fluid returns liquid bath;
Step 4.4, the every rising 1% of concentration (30～120 minutes), measure respectively penetrating fluid and feed liquid poor in the specific conductivity and the absorbancy of concentrated solution, and the temperature of charge in the material fluid bath;
Step 4.5, flow velocity stop concentration operation when being lower than 10L/h or weighing less than 12Kg.
Experimental result is with reference to following table 2:
4, discussion of results:
1), the nanofiltration membrane separation technology is applied to the separation of 5 '-cytidylic acid and derivative thereof and the desalting effect in the purifying
Measure the specific conductivity of contrast solution, obtain as follows:
20%5 '-cytidylic acid solution (pH5) specific conductivity is about 8500 μ s/cm
10%5 '-cytidylic acid solution (pH5) specific conductivity is about 7800 μ s/cm
4%5 '-cytidylic acid solution (pH5) specific conductivity is about 7000 μ s/cm;
1% sodium chloride solution specific conductivity is about 7600 μ s/cm;
So this experiment with record for continuous three times feed liquid poor in the specific conductivity of concentrated solution be the terminal point that desalination is operated less than 10000 μ s/cm, be considered as inorganic salt and remove substantially.
Find out the specific conductivity of the specific conductivity of concentrated solution near contrast solution from table 1 and table 2, specific conductivity shows that sodium-chlor is lower than 1% in the penetrating fluid, and promptly ratio of desalinization reaches more than 99%, and inorganic salt remove substantially, and desalting effect clearly.
2), the nanofiltration membrane separation technology is applied to the effect of holding back in the separation of 5 '-cytidylic acid and derivative thereof and the purifying
The effect of holding back of nanofiltration is represented with rejection, calculates rejection by solution absorbency before and after the determination experiment and weight.
Method of calculation are: rejection=(absorbancy * weight of experiment back material)/(absorbancy * weight of material before the experiment) * 100%.
The result shows that the rejection of embodiment 1 is 98.44%, and the rejection of embodiment 2 is 97.84%, and it is very good to hold back effect.
3), the nanofiltration membrane separation technology is applied to the separation of 5 '-cytidylic acid and derivative thereof and the purification effect in the purifying
Before and after the experiment, respectively solution is carried out high pressure liquid chromatographic analysis, the results are shown in following table 3:
| Purity before the experiment | Experiment back purity | |
| Embodiment 1 | ??98.18％ | ??98.76％ |
| Embodiment 2 | ??93.54％ | ??97.43％ |
The result shows that behind the nanofiltration membrane separation purifying, the purity of solution obviously rises, and purification effect is obvious.
4), the nanofiltration membrane separation technology is applied to the separation of 5 '-cytidylic acid and derivative thereof and the concentrated effect in the purifying
Concentration owing to material in the nanofiltration concentration process is more and more higher, the flux of film is also more and more lower, the concentration that found through experiments for the nanofiltration under 1.5MPa pressure of 5 '-cytidylic acid can reach more than 20%, higher if desired concentration, only need heighten pressure and get final product, the concentrated effect that this shows nanofiltration also is extraordinary.
Advantages such as spissated method is a lot, and commonly used is that reduction vaporization concentrates, and nanofiltration concentrates and do not have more and need heat by contrast, does not destroy this body structure of material, and is simple to operate, and energy consumption is low.
5), pressure is for the nanofiltration Effect on Performance
Nanofiltration belongs to pressure-driven type membrane separation technique, and under the constant situation of concentration, the flux of the high more film of pressure is big more, and what just see through is fast more.The flux that causes for the equilibrium concentration rising descends and must improve the pressure that moves in good time, to guarantee suitable flux.But pressure is not high more good more, and too high pressure can make the life-span of film descend, and rejection also can descend, and our selected pressure-controlling is proper at 1.0～2.0MPa by experiment, can not produce any influence.
6), temperature is for the nanofiltration Effect on Performance
Because the operation of pump and the infiltration of film can make temperature of charge rise, temperature rises can influence the effect of membrane sepn, and holding back effect can descend to some extent, so should be at chuck with the subcooling cooling water circulation, to guarantee to expect that temperature carries out under lower situation, operating temperature is decided to be 20～50 ℃.
Claims (6)
1. the separation purification method of 5 '-cytidylic acid and derivative thereof is characterized in that, the nanofiltration membrane separation technology is applied to the separation and the purifying of 5 '-cytidylic acid and derivative thereof, and the method includes the steps of:
Step 1, prepare solution to be separated;
Step 2, solution to be separated is poured in the material fluid bath;
Step 3, carry out desalination operation; In this step, comprise the step of solution to be separated by nanofiltration membrane;
Step 4, carry out concentration operation; In this step, comprise the step of solution to be separated by nanofiltration membrane.
2. the separation purification method of 5 '-cytidylic acid as claimed in claim 1 and derivative thereof, it is characterized in that, in the described step 1, solution to be separated should reach following requirement: solution should be as clear as crystal, do not contain foreign matter, should filter in advance if any foreign matter or muddiness, concentration is below 20%, pH value 2～11 requires dechlorination to handle.
3. the separation purification method of 5 '-cytidylic acid as claimed in claim 1 and derivative thereof is characterized in that, described step 3 comprises following steps:
Step 3.1, operational outfit, pressure regulating device is regulated pressure to 1.0～2.0MPa, makes solution to be separated pass through nanofiltration membrane;
Step 3.2, collection penetrating fluid, trapped fluid returns liquid bath;
Step 3.3, control material concentration and keep the liquid level of material in feed liquid is poor, that is, how much weight is the feed liquid in feed liquid is poor reduce, and just adds the distilled water of identical weight;
Step 3.4, measure specific conductivity and absorbancy, and measure the interior temperature of charge of material fluid bath every getting penetrating fluid and the feed liquid concentrated solution in poor in 30～120 minutes respectively;
Step 3.5, continuous record for 3～6 times feed liquid poor in the specific conductivity of concentrated solution during less than 1000～10000 μ s/cm desalination finish.
4. the separation purification method of 5 '-cytidylic acid as claimed in claim 1 and derivative thereof is characterized in that, described step 4 comprises following steps:
Step 4.1, material in the material fluid bath is supplied initial weight when carrying out step 2;
Step 4.2, operational outfit, pressure regulating device is regulated pressure to 1.0～2.0MPa, makes solution to be separated pass through nanofiltration membrane;
Step 4.3, collection penetrating fluid, trapped fluid returns liquid bath;
Step 4.4, every measured respectively in 30～120 minutes penetrating fluid and feed liquid poor in the specific conductivity and the absorbancy of concentrated solution, and the interior temperature of charge of material fluid bath;
Step 4.5, flow velocity are lower than 10～50L/h or the interior liquor capacity of material fluid bath just stopped concentration operation at remaining 1/5～1/3 o'clock.
5. as the separation purification method of claim 3 or 4 described 5 '-cytidylic acids and derivative thereof, it is characterized in that, in the separation and purification process of 5 '-cytidylic acid and derivative thereof, the material fluid bath chuck makes temperature be controlled at 20～50 ℃ with water coolant or chilled brine cooling.
6. one kind is applied to the nanofiltration membrane separation technology new purposes that 5 '-cytidylic acid and derivative thereof are carried out separation and purification.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108440624A (en) * | 2018-04-24 | 2018-08-24 | 苏州华赛生物工程技术有限公司 | A kind of method that environment-friendly type isolates and purifies cytidine from microbial fermentation solution |
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2008
- 2008-12-25 CN CN200810044191A patent/CN101648984A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108440624A (en) * | 2018-04-24 | 2018-08-24 | 苏州华赛生物工程技术有限公司 | A kind of method that environment-friendly type isolates and purifies cytidine from microbial fermentation solution |
| CN108440624B (en) * | 2018-04-24 | 2023-01-31 | 苏州华赛生物工程技术有限公司 | Environment-friendly method for separating and purifying cytidine from microbial fermentation broth |
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Application publication date: 20100217 |