CN100425618C - Method for continuously separating 5' -nucleoside triphosphate - Google Patents
Method for continuously separating 5' -nucleoside triphosphate Download PDFInfo
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- CN100425618C CN100425618C CNB2006100853847A CN200610085384A CN100425618C CN 100425618 C CN100425618 C CN 100425618C CN B2006100853847 A CNB2006100853847 A CN B2006100853847A CN 200610085384 A CN200610085384 A CN 200610085384A CN 100425618 C CN100425618 C CN 100425618C
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
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Abstract
The invention discloses a method for continuously separating 5' -nucleoside triphosphate. The method adopts a combination technology of a continuous ion exchange system and nanofiltration to separate enzyme-converted 5 '-nucleoside triphosphate, and specifically comprises the steps of pumping enzyme-converted 5' -nucleoside triphosphate clear liquid into the continuous ion exchange system filled with anion exchange resin for adsorption, and pumping inorganic salt solution with the pH of 2-14 and the concentration of 0.001-2M into the continuous ion exchange system at a proper flow rate for impurity washing and elution; and (3) synchronously concentrating and desalting the ion exchange eluent by using a nanofiltration membrane with the molecular weight cutoff of 150-300 daltons, and crystallizing and drying. The invention has the advantages of large carrying capacity of the separation medium, less equipment investment, easy large-scale production and low production cost.
Description
Technical field
The invention belongs to the biological products manufacture field, relate to the method for a kind of continuous separation 5 '-ribonucleoside triphosphote.
Background technology
About the separation method of ucleotides material, before half a century, people have just begun heuristic process, and a kind of styrene type strongly basic anion exchange resin is used for the separation of Nucleotide first, and have reached separating effect preferably.Then people have developed DEAE-dextrane gel, PEI-Mierocrystalline cellulose again, even Zeo-karb is used for the separation of mixture of ribonucleotides.(Cytidine triphosphate biosynthesizing research such as Zhan Guyu, northwest medical magazine [J], 1997,12 (2), it is centrifugal 81-82) to report that it utilizes, ethanol precipitation at first obtains the CTP crude product, behind 732 Hydrogen cationic exchange coloums and two purifying of 711 chlorine type anion-exchange columns, yield is not mentioned, and is still relatively low as can be seen according to the document, by the crystallization of multistep organic solvent, the CTP crystalline content that obtains is 93.4%.Report in the English Patent (No.14043/681968), after the UTP conversion fluid was carried out pre-treatment, at first by chlorine type storng-acid cation exchange resin Dowex2, two-step purifying was carried out by chlorine type acidulous cation resin Duolite A7 in the back.After obtain UTP sodium salt elutriant purity be 97.5%, yield is 72.2%.Qiu Weiran etc. (CN99113707.8) utilize with the conversion of carrageenin konjac polysaccharide fixed yeast and obtain ribonucleoside triphosphote, reaction finishes after product centrifuging, filtrate is handled with 717 anionite-exchange resin, absorption ribonucleoside triphosphote wherein, elutriant is collected with (pH=2) NaCl wash-out of 0.7mol/L in the back, add ethanol, make the ribonucleoside triphosphote precipitation, can obtain target product-ribonucleoside triphosphote after filtration with after the vacuum-drying, do not have the purity and the yield of volume product in the patent.The separating effect of above-mentioned report is not ideal, and purity is not high, and yield is lower.The general more complicated of these separation methods can't satisfy the requirement of its extensive industrialization.
In the production technique of existing NTP, fixed-bed ion exchange technology is widely used.And there is various disadvantages in existing fixed bed ion exchange technique, it is a kind of batch-type operation, steps such as absorption, wash-out, regeneration are carried out in same bed, single batch job cycle is long, discontinuous, the ion exchange resin capacity can not make full use of, thereby chemical actual consumption amount is big, the distinct disadvantage uneconomical, that the big grade of wastewater discharge is this exchanged form.
Summary of the invention
The objective of the invention is to be to overcome cost height, complex operation in present 5 '-ribonucleoside triphosphote sepn process, be difficult for the defective of mass-producing; provide a kind of continuous ionic switching technology to replace general fixed bed switching technology, and separate the method for enzymatic conversion 5 '-ribonucleoside triphosphote in conjunction with nanofiltration.
The present invention is based upon on step separation and the continuous isolating thought, its characteristics are to utilize in the solution between the target product and coexistent impurity the difference in physics, chemistry and biological property, make its in lock out operation, have different rate of mass transfer and (or) equilibrium state, thereby and this novel separation theory of continous way ion exchange technique realize the isolating purpose of 5 '-ribonucleoside triphosphote.
Purpose of the present invention can realize by following technical measures:
The method of a kind of continuous separation 5 '-ribonucleoside triphosphote, this method adopts the combination technique of continuous ionic exchange system and nanofiltration to separate enzymatic conversion 5 '-ribonucleoside triphosphote, concrete grammar is 5 ' of enzymatic conversion-ribonucleoside triphosphote clear liquid to be pumped in the continuous ionic exchange system that anionic ion-exchange resins is housed adsorb, adopt pH2～14, the inorganic salt solution of concentration between 0.001M～2M pumps into the continuous ionic exchange system with suitable flow velocity and washes assorted, wash-out; With molecular weight cut-off is that 150～300 daltonian nanofiltration membrane concentrate ion-exchanging eluent and desalination crystallizing and drying synchronously.
Described method, wherein the continuous ionic exchange system is meant the ion exchange system by the placed in-line operate continuously of many radical ions exchange column, promptly by combined valve with the absorption in the ion exchange process, wash assorted, wash-out and the switching in order between the different workshop sections of regenerating, the adsorption stage ion exchange column is shifted out adsorption stage at once after resin absorption is saturated send into and wash assorted section and wash assorted, washing to shift out at once behind assorted the end and washing assorted section and send into the wash-out section and carry out wash-out, shifting out the wash-out section after wash-out is finished at once sends into RS Regenerator Section and regenerates, shifting out RS Regenerator Section after regeneration is cleaned and to be finished at once sends into adsorption stage and adsorbs, so round-robin operating process, and the state of the 1st radical ion exchange column of each workshop section switches and carries out synchronously, and guarantees to have at least a radical ion exchange column to be in absorption phase.
Described method wherein is meant by 6～60 fixed-bed ion exchangers by the ion exchange system of the placed in-line operate continuously of many radical ions exchange column and forms the ion exchange system of the operate continuously of preferred 12～36 compositions.
Described method, wherein sample concentration is 1～30g/L on the enzymatic conversion clear liquid of 5 '-ribonucleoside triphosphote, preferred 5～25g/L.
Described method, wherein inorganic salt solution is sodium-chlor, ammonium chloride or ammoniumsulphate soln.
Described method, the inorganic salt solution concentration that wherein is used for wash-out is 0.1M～2M.
Described method is that the bronsted lowry acids and bases bronsted lowry of 0.2～2M pumps into by the first order of acid back alkali and needs the regenerated ion exchange column to regenerate with concentration wherein, uses deionized water drip washing after regeneration is finished.
Described method, wherein used acid is hydrochloric acid or sulfuric acid in the regenerative process, alkali is sodium hydroxide or ammoniacal liquor.
Described method, wherein the model of anionic ion-exchange resins is 301 types, 302 types, 303 types, 311 types, 312 types or 313 types.Described method is wherein washed mixture solution pH value 2～8, elute soln pH value 2～6.
Below in conjunction with Figure of description the continuous ionic exchange system is further described:
Feed liquid enters from fixed bed top and finishes the back in each operation and just by the switching of valve the resin column of finishing operation " is moved " next operation, and the per pass operation by operating flow adjusting or the adjusting of resin loadings realizes synchronous operation.Constitute continuously from the friendship system for 16 radical ion exchange columns, according to the characteristics of material and the needs employing adsorption stage of processing condition is 5, washing assorted section is 3, the wash-out section is 3, RS Regenerator Section is 5 a process form, and Fig. 1～17 are in the synoptic diagram of different states respectively in loop cycle for the ion exchange unit that separates 5 '-ribonucleoside triphosphote continuously.5 of adsorption stage in handing over post the 5th to be that protection is whole from the effect of handing over post can not penetrated by upper prop liquid and cause damage from the adsorption stage of handing over post; after handing over post absorption saturated, will be moved out of adsorption stage when the 1st; enter and wash assorted section; become the 3rd post washing general labourer's section; just washing assorted finishing and wash the 1st of assorted section under the control of assorted flow velocity from handing over post washing this moment; enter the wash-out section; become last root post of wash-out workshop section; this moment can by the control elution flow rate make the wash-out section former first also simultaneously wash-out finish; last root that moves into RS Regenerator Section is regenerated; also regenerating for first of RS Regenerator Section finishes, and begins to have adsorbed.Like this by the flow velocity of each workshop section of control, can be so that keep same rhythm to carry out work from four workshop sections of handing over process.
Enzymatic conversion NTP refers to NTP that raw material is obtained through yeast conversion, and this method is a technology known to a person of ordinary skill in the art.
Flow velocity is subjected to each workshop section's condition effect, and principle is to guarantee each workshop section's synchronous operation, and those of ordinary skills can adjust the flow velocity of moving phase according to state of resin.
The present invention has following advantage compared to existing technology:
The present invention uses the continuous ionic switching technology owing to improved the utilization ratio of resin, has reduced resin demand, uses a kind of anionite-exchange resin to carry out separation and purification, and processing step is simple; Elutriant concentration uprises, and washes assorted effectively, has reduced eluent and has washed the consumption of assorted agent; Consumption reduces regenerators such as acid simultaneously, alkali owing to improved utilization ratio, has also reduced discharging sour, alkali simultaneously.
Table 1 fixed bed with continuously from handing over system performance parameter must compare (identical production capacity)
The present invention breaks through the medelling of domestic and international colleague to 5 '-ribonucleoside triphosphote separation and purification mode, and ion-exchange is combined with nanofiltration membrane separation, has saved desalination and spissated step and can directly carry out crystallization purifying.Make that separating and purifying technology of the present invention is simple and easy to do, effect is good, and not only its facility investment, running cost are cheap, and can be according to what of fractional dose, realize from feather weight to tonne separation.All having obtained fabulous result aspect product yield and the quality product, guaranteed product quality by products obtained therefrom of the present invention.
Description of drawings
Fig. 1 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 1.
Fig. 2 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 2.
Fig. 3 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 3.
Fig. 4 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 4.
Fig. 5 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 5.
Fig. 6 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 6.
Fig. 7 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 7.
Fig. 8 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 8.
Fig. 9 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 9.
Figure 10 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 10.
Figure 11 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 11.
Figure 12 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 12.
Figure 13 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 13.
Figure 14 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 14.
Figure 15 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 15.
Figure 16 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 16.
Figure 17 is the synoptic diagram that the continuous ion exchange unit that separates 5 '-ribonucleoside triphosphote of the present invention is in state 17.
Embodiment
Further define the present invention by following embodiment, embodiment is to be the unrestricted the present invention of explanation.Any those of ordinary skill can be understood these embodiments and not limit the present invention in any way in this area, can make suitable modification and without prejudice to essence of the present invention with depart from scope of the present invention.
The continuous ionic exchange system that is adopted in following examples is meant the ion exchange system of the placed in-line operate continuously of many radical ions exchange column, promptly by combined valve with the absorption in the ion exchange process, wash assorted, wash-out and the switching in order between the different workshop sections of regenerating, the adsorption stage ion exchange column is shifted out adsorption stage at once after resin absorption is saturated send into and wash assorted section and wash assorted, washing to shift out at once behind assorted the end and washing assorted section and send into the wash-out section and carry out wash-out, shifting out the wash-out section after wash-out is finished at once sends into RS Regenerator Section and regenerates, shifting out RS Regenerator Section after regeneration is cleaned and to be finished at once sends into adsorption stage and adsorbs, so round-robin operating process, and the state of the 1st radical ion exchange column of each workshop section switches and carries out synchronously, and guarantees have a radical ion exchange column to be in absorption phase at least.
Adopt 16 radical ion exchange columns to constitute continuously from the friendship system, adsorption stage is 5, and washing assorted section is 3, and the wash-out section is 3, and RS Regenerator Section is 5.Every is 1.5 centimetres from handing over post filling 30g resin (311 type) from the diameter of handing over post, highly is 25 centimetres.The processing feed liquid is after 5 '-ribonucleoside triphosphote clear liquid of enzymatic conversion is diluted to 10g/L with deionized water, to regulate material liquid pH value 2.3 with hydrochloric acid, and the absorption flow velocity is 2.5ml/min, and loading capacity is the 0.22g/g wet resin; Washing mixture solution is the 0.075mol/L NaCl solution of pH 5.0, and washing assorted flow velocity is 5ml/min, wash assorted till the inclusion-free (with HPLC detect determine to wash the assorted section first radical ion exchange column inclusion-free flow out for single ion exchange column wash assorted liquid consumption standard); Elutriant is the 1.0mol/LNaCl of pH7.0, elution flow rate is 2ml/min, wash-out (determines that with the HPLC detection wash-out section first radical ion exchange column wash-out is entirely single ion exchange column Xian and takes off liquid consumption standard) fully, the elutriant molecular weight cut-off is the synchronous nanofiltration concentrating and desalinating of 150～300 daltonian nanofiltration membrane post crystallization, drying, obtain purity and be 5 '-ribonucleoside triphosphote of 97.5%, yield is 91.0%.Be that the sodium hydroxide of the hydrochloric acid of 1M and 1M pumps into by the order of acid back alkali earlier and needs the regenerated ion exchange column to regenerate with concentration, use deionized water drip washing after regeneration is finished.
Pending feed liquid is that the polymeric adsorbent model was 301 after 5 '-ribonucleoside triphosphote clear liquid of enzymatic conversion was diluted to 8g/L with deionized water, adopts 16 radical ion exchange columns to constitute continuously from the friendship system, adsorption stage is 4, washing assorted section is 4, and the wash-out section is 3, and RS Regenerator Section is 5.Every from handing over post filling 1000g resin, is 1: 5 from the blade diameter length ratio of handing over post.Regulate material liquid pH value 2.2 with hydrochloric acid, the absorption flow velocity is 20ml/min, and loading capacity is the 0.24g/g wet resin; Washing mixture solution is the 0.05mol/L NaCl solution of pH 2.0, and washing assorted flow velocity is 40ml/min, wash assorted till the inclusion-free (with HPLC detect determine to wash the assorted section first radical ion exchange column inclusion-free flow out for single ion exchange column wash assorted liquid consumption standard); Elutriant is the 1.0mol/L NaCl of pH7.0, elution flow rate is 10ml/min, wash-out (determines that with the HPLC detection wash-out section first radical ion exchange column wash-out is entirely single ion exchange column Xian and takes off liquid consumption standard) fully, the elutriant molecular weight cut-off is the synchronous nanofiltration concentrating and desalinating of 150～300 daltonian nanofiltration membrane post crystallization, drying, obtain purity and be 5 '-ribonucleoside triphosphote of 98.2%, yield is 91.5%.Be that the sodium hydroxide of the hydrochloric acid of 1.0M and 1.0M pumps into by the order of acid back alkali earlier and needs the regenerated ion exchange column to regenerate with concentration, use deionized water drip washing after regeneration is finished.
Pending feed liquid is that the polymeric adsorbent model was 311 after 5 '-ribonucleoside triphosphote clear liquid of enzymatic conversion was diluted to 11g/L with deionized water, adopts 16 radical ion exchange columns to constitute continuously from the friendship system, adsorption stage is 4, washing assorted section is 4, and the wash-out section is 3, and RS Regenerator Section is 5.Every from handing over post filling 1Kg resin, is 1: 5 from the blade diameter length ratio of handing over post.Regulate material liquid pH value 2.4 with hydrochloric acid, the absorption flow velocity is 25ml/min, and loading capacity is the 0.25g/g wet resin; Washing mixture solution is the 0.075mol/L NaCl solution of pH 2.0, and washing assorted flow velocity is 50ml/min, wash assorted till the inclusion-free (with HPLC detect determine to wash the assorted section first radical ion exchange column inclusion-free flow out for single ion exchange column wash assorted liquid consumption standard); Elutriant is the 1.0mol/LNaCl of pH7.0, elution flow rate is 15ml/min, wash-out (determines that with the HPLC detection wash-out section first radical ion exchange column wash-out is entirely single ion exchange column Xian and takes off liquid consumption standard) fully, the elutriant molecular weight cut-off is the synchronous nanofiltration concentrating and desalinating of 150～300 daltonian nanofiltration membrane post crystallization, drying, obtain purity and be 5 '-ribonucleoside triphosphote of 98.3%, yield is 92.0%.Be that the sodium hydroxide of the hydrochloric acid of 1.2M and 1.2M pumps into by the order of acid back alkali earlier and needs the regenerated ion exchange column to regenerate with concentration, use deionized water drip washing after regeneration is finished.
Pending feed liquid is that the polymeric adsorbent model was 301 after 5 '-ribonucleoside triphosphote clear liquid of enzymatic conversion was diluted to 12g/L with deionized water, adopts 16 radical ion exchange columns to constitute continuously from the friendship system, adsorption stage is 5, washing assorted section is 3, and the wash-out section is 3, and RS Regenerator Section is 5.Every from handing over post filling 500g resin, is 1: 5 from the blade diameter length ratio of handing over post.Regulate material liquid pH value 2.5 with hydrochloric acid, the absorption flow velocity is 10ml/min, and loading capacity is the 0.24g/g wet resin; Washing mixture solution is the 0.05mol/L NaCl solution of pH 2.0, and washing assorted flow velocity is 20ml/min, wash assorted till the inclusion-free (with HPLC detect determine to wash the assorted section first radical ion exchange column inclusion-free flow out for single ion exchange column wash assorted liquid consumption standard); Elutriant is the 1.0mol/LNaCl of pH7.0, elution flow rate is 5ml/min, wash-out (determines that with the HPLC detection wash-out section first radical ion exchange column wash-out is entirely single ion exchange column Xian and takes off liquid consumption standard) fully, the elutriant molecular weight cut-off is the synchronous nanofiltration concentrating and desalinating of 150～300 daltonian nanofiltration membrane post crystallization, drying, obtain purity and be 5 '-ribonucleoside triphosphote of 98.3%, yield is 91.4%.Be that the sodium hydroxide of the hydrochloric acid of 1.5M and 1.5M pumps into by the order of acid back alkali earlier and needs the regenerated ion exchange column to regenerate with concentration, use deionized water drip washing after regeneration is finished.
Pending feed liquid is that the polymeric adsorbent model was 311 after 5 '-ribonucleoside triphosphote clear liquid of enzymatic conversion was diluted to 12g/L with deionized water, adopts 16 radical ion exchange columns to constitute continuously from the friendship system, adsorption stage is 4, washing assorted section is 4, and the wash-out section is 3, and RS Regenerator Section is 5.Every from handing over post filling 1Kg resin, is 1: 5 from the blade diameter length ratio of handing over post.Regulate material liquid pH value 2.2 with hydrochloric acid, the absorption flow velocity is 25ml/min, and loading capacity is the 0.25g/g wet resin; Washing mixture solution is the 0.075mol/LNaCl solution of pH 2.0, and washing assorted flow velocity is 50ml/min, wash assorted till the inclusion-free (with HPLC detect determine to wash the assorted section first radical ion exchange column inclusion-free flow out for single ion exchange column wash assorted liquid consumption standard); Elutriant is the 1.0mol/LNaCl of pH7.0, elution flow rate is 15ml/min, wash-out (determines that with the HPLC detection wash-out section first radical ion exchange column wash-out is entirely single ion exchange column Xian and takes off liquid consumption standard) fully, the elutriant molecular weight cut-off is the synchronous nanofiltration concentrating and desalinating of 150～300 daltonian nanofiltration membrane post crystallization, drying, obtain purity and be 5 '-ribonucleoside triphosphote of 98.1%, yield is 92.6%.Be that the sodium hydroxide of the hydrochloric acid of 1.2M and 1.2M pumps into by the order of acid back alkali earlier and needs the regenerated ion exchange column to regenerate with concentration, use deionized water drip washing after regeneration is finished.
Claims (10)
1, the method for a kind of continuous separation 5 '-ribonucleoside triphosphote, it is characterized in that this method adopts the combination technique of continuous ionic exchange system and nanofiltration to separate enzymatic conversion 5 '-ribonucleoside triphosphote, concrete grammar is 5 ' of enzymatic conversion-ribonucleoside triphosphote clear liquid to be pumped in the continuous ionic exchange system that anionic ion-exchange resins is housed adsorb, adopt pH2～14, the inorganic salt solution of concentration between 0.001M～2M pumps into the continuous ionic exchange system with suitable flow velocity and washes assorted, wash-out; With molecular weight cut-off is that 150～300 daltonian nanofiltration membrane concentrate ion-exchanging eluent and desalination crystallizing and drying synchronously.
2, method according to claim 1, it is characterized in that the continuous ionic exchange system is meant the ion exchange system by the placed in-line operate continuously of many radical ions exchange column, promptly by combined valve with the absorption in the ion exchange process, wash assorted, wash-out switches in order with the different workshop sections of regeneration, the adsorption stage ion exchange column is shifted out adsorption stage at once after resin absorption is saturated send into and wash assorted section and wash assorted, washing to shift out at once behind assorted the end and washing assorted section and send into the wash-out section and carry out wash-out, shifting out the wash-out section after wash-out is finished at once sends into RS Regenerator Section and regenerates, shifting out RS Regenerator Section after regeneration is cleaned and to be finished at once sends into adsorption stage and adsorbs, so round-robin operating process, and the state of the 1st radical ion exchange column of each workshop section switches and carries out synchronously, and guarantees to have at least a radical ion exchange column to be in absorption phase.
3, method according to claim 2 is characterized in that ion exchange system by the placed in-line operate continuously of many radical ions exchange column is meant by 6～60 fixed-bed ion exchangers to form.
4, method according to claim 1 is characterized in that sample concentration is 1～30g/L on the enzymatic conversion clear liquid of 5 '-ribonucleoside triphosphote.
5, method according to claim 1 is characterized in that inorganic salt solution is sodium-chlor, ammonium chloride or ammoniumsulphate soln.
6, method according to claim 1, the inorganic salt solution concentration that it is characterized in that being used for wash-out is 0.1M～2M.
7, method according to claim 2 is characterized in that with concentration being that the bronsted lowry acids and bases bronsted lowry of 0.2～2M pumps into by the first order of acid back alkali and needs the regenerated ion exchange column to regenerate, and uses deionized water drip washing after regeneration is finished.
8, method according to claim 7 is characterized in that acid is hydrochloric acid or sulfuric acid, and alkali is sodium hydroxide or ammoniacal liquor.
9, method according to claim 1, the model that it is characterized in that anionic ion-exchange resins are 301 types, 302 types, 303 types, 311 types, 312 types or 313 types.
10, method according to claim 1 is characterized in that washing mixture solution pH value 2～8, elute soln pH value 2～6.
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| 离子交换法分离纯化5‘-三磷酸胞苷. 阮文辉.阮文辉硕士毕业论文. 2004 |
| 离子交换法分离纯化5‘-三磷酸胞苷. 阮文辉.阮文辉硕士毕业论文. 2004 * |
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