CN1687114A - Method for purifying microcysin LR and RR synchronously - Google Patents
Method for purifying microcysin LR and RR synchronously Download PDFInfo
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- CN1687114A CN1687114A CN 200510024573 CN200510024573A CN1687114A CN 1687114 A CN1687114 A CN 1687114A CN 200510024573 CN200510024573 CN 200510024573 CN 200510024573 A CN200510024573 A CN 200510024573A CN 1687114 A CN1687114 A CN 1687114A
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Abstract
The present invention relates to a technique for simultaneously separating and purifying microcystin LR and RR by utilizing multistep chromatography. Said technique includes the following steps: using 5% acetic acid to treat cyanophycean water bloom sample, making the extract pass through ODS column, and making 90% methyl alcohol eluent be passed through first silica gel chromatographic column to obtain components of LR and RR, making said two components be respectively passed through second silica gel chromatographic column, and making LR be passed through preparation HPLC and making RR be passed through ion exchange chromatograph, finally desalting by using C18 small column. After HPLC detection the purity of LR and RR is greater than or equal to 90%.
Description
Technical field:
The present invention relates to a kind of method of from Microcystis aeruginosa, extracting purifying microcysin LR and RR simultaneously.
Background technology:
Microcystin is ubiquitous seven peptide toxin in the eutrophication water, this toxoid is the short tumour material of the strongest okadaic acid class found so far, human health has been constituted very big threat, all kinds of researchs of carrying out around Microcystin comprise the mensuration of mechanism that its produces, environmental concentration, effectively remove the full appreciation of measure and toxic effect, and all correlative studys all depend on the acquisition of Microcystin standard substance.
Owing to have two kinds of variable amino acid in the structure, thereby 70 kinds of congeners are arranged approximately, this makes that the component of the Microcystin that exists in the water surrounding is very complicated.That relative content is higher, it is more to exist, toxicity is maximum is microcysin LR and RR, and two kinds of variable compositions wherein are respectively leucine (L)-arginine (R) and arginine (R)-arginine (R).The source of standard substance LR and RR mainly is external chemical reagents corporation, and costing an arm and a leg in the past is important limiting factor, now because toxin is classified as the biotoxin material, more therefore is subjected to the control of some non-science factors and economic factors.Therefore, the acquisition of standard substance becomes the restraining factors that all kinds of correlative studys are carried out in restriction.In the preparation of Microcystin standard substance, can adopt multiple chromatographic technique, main difficult point is how multiple toxin in the Microcystis aeruginosa to be separated and combined separation and purification technology how.From Microcystis aeruginosa, extract at present the purified toxins method have multiple, but all can not from Microcystis aeruginosa, extract highly purified microcysin LR and RR simultaneously.
Summary of the invention:
The invention provides a kind of reasonable combination and utilize the technology of multistep chromatography while separation and purification microcysin LR and RR, be suitable for preparing simultaneously a milligram level microcysin LR and a RR, the gained Microcystin detects purity 〉=90% through HPLC.Present method cost is low and need not too high device requirement.
Technical scheme of the present invention is as follows:
A. blue-green alga bloom sample adds 5% acetum of 50 times of its weight, magnetic agitation 30 minutes, and centrifugal 20 minutes of 10000g gets supernatant, and precipitation repeats said extracted 2 times, merges the extraction supernatant three times, uses the GF/C membrane filtration.
The B.ODS post, 5 * 50cm uses methyl alcohol, 20% methyl alcohol, water treatment respectively.Filtrate in the A step is crossed post, and 90% methanol-eluted fractions is used in water, 20% methyl alcohol drip washing respectively more then, collects elutriant.
C. elutriant is removed organic solvent with rotation concentration and evaporation instrument, obtains extract of crude toxin, with thin-layer chromatography elution fraction is identified, confirms wherein to contain toxin LR and RR.
D. silicagel column 1.5 * 80cm, use the developing agent chloroform: methyl alcohol: water (65: 35: 10) dress post and balance, extract of crude toxin dissolves, is added on the post with developing agent, use the developing agent wash-out, fraction collection, with thin-layer chromatography elution fraction is identified, merged the component that contains LR and RR respectively, the rotation concentrate drying.Through this step, toxin LR is separated with RR, obtains LR component and RR component.
The E1.LR component is silica gel column chromatography for the second time, post 1.0 * 35cm, use the developing agent vinyl acetic monomer: Virahol: water (5: 3: 4) balance, the LR component of D step behind concentrate drying dissolved with developing agent, is added on the post, uses the developing agent wash-out, fraction collection, with thin-layer chromatography elution fraction is identified, merged the component that contains single component, the rotation concentrate drying.Through this step, the impurity in the toxin LR component is further removed.
The E2.RR component is silica gel column chromatography for the second time, post 1.0 * 35cm, use the developing agent vinyl acetic monomer: Virahol: water (5: 3: 4) balance, the RR component of D step behind concentrate drying dissolved with developing agent, is added on the post, uses the developing agent wash-out, fraction collection, with thin-layer chromatography elution fraction is identified, merged the component that contains single component, the rotation concentrate drying.Through this step, the impurity in the toxin LR component is further removed.
The toxin LR component dissolve with methanol of F1.E1 step, preparation HPLC, moving phase consists of the 0.02M ammonium acetate: second eyeball (75: 25), and the toxin of further that LR is close with other structure low levels separates, and the LR that obtains measures purity 〉=90% through HPLC-UV/DAD.
The toxin component dissolve with methanol of F2.E2 step, ion exchange column is further removed the impurity in the RR component, and the RR that obtains measures purity 〉=90% through HPLC-UV/DAD.
G. above-mentioned prepared product is used the desalination of C18 pillar respectively.
Claims (4)
1, the purification process of a kind of microcysin LR and RR is characterized in that, operation steps is carried out in the following order:
A. blue-green alga bloom sample adds 5% acetum of 50 times of its weight, magnetic agitation 30 minutes, and centrifugal 20 minutes of 10000g gets supernatant, and precipitation repeats said extracted 2 times, merges the extraction supernatant three times, uses the GF/C membrane filtration.
The B.ODS post, 5 * 60cm uses methyl alcohol, 20% methyl alcohol, water treatment respectively.Filtrate in the A step is crossed post, and 90% methanol-eluted fractions is used in water, 20% methyl alcohol drip washing respectively more then, collects elutriant.
C. elutriant is removed organic solvent with rotation concentration and evaporation instrument, obtains extract of crude toxin, with thin-layer chromatography elution fraction is identified, confirms wherein to contain toxin LR and RR.
D. silicagel column 1.5 * 80cm, use the developing agent chloroform: methyl alcohol: water (65: 35: 10) dress post and balance, extract of crude toxin dissolves, is added on the post with developing agent, use the developing agent wash-out, fraction collection, with thin-layer chromatography elution fraction is identified, merged the component that contains LR and RR respectively, the rotation concentrate drying.Through this step, toxin LR is separated with RR, obtains LR component and RR component.
The E1.LR component is silica gel column chromatography for the second time, post 1.0 * 35cm, use the developing agent vinyl acetic monomer: Virahol: water (5: 3: 4) balance, the LR component of D step behind concentrate drying dissolved with developing agent, is added on the post, uses the developing agent wash-out, fraction collection, with thin-layer chromatography elution fraction is identified, merged the component that contains single component, the rotation concentrate drying.Through this step, the impurity in the toxin LR component is further removed.
The E2.RR component is silica gel column chromatography for the second time, post 1.0 * 35cm, use the developing agent vinyl acetic monomer: Virahol: water (5: 3: 4) balance, the RR component of D step behind concentrate drying dissolved with developing agent, is added on the post, uses the developing agent wash-out, fraction collection, with thin-layer chromatography elution fraction is identified, merged the component that contains single component, the rotation concentrate drying.Through this step, the impurity in the toxin LR component is further removed.
The toxin LR component dissolve with methanol of F1.E1 step, preparation HPLC, moving phase consists of the 0.02M ammonium acetate: second eyeball (75: 25), and the toxin of further that LR is close with other structure low levels separates, and the LR that obtains measures purity 〉=90% through HPLC-UV/DAD.
The toxin component dissolve with methanol of F2.E2 step, ion exchange column is further removed the impurity in the RR component, and the RR that obtains measures purity 〉=90% through HPLC-UV/DAD.
G. above-mentioned prepared product is used the desalination of C18 pillar respectively.
2, the purification process of a kind of microcysin LR according to claim 1 and RR is characterized in that, an extract is crossed the post processing and can be obtained purity LR and RR all 〉=90% simultaneously.
3, the purification process of a kind of microcysin LR according to claim 1 and RR is characterized in that, adopts the silicagel column of relatively economical, forms the optimization that reaches chromatographic separation condition by changing twice developing agent.
4, the purification process of a kind of microcysin LR according to claim 1 and RR is characterized in that, adopts thin-layer chromatography sepn process to be monitored in real time and the purity evaluation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510024573 CN1687114A (en) | 2005-03-24 | 2005-03-24 | Method for purifying microcysin LR and RR synchronously |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200510024573 CN1687114A (en) | 2005-03-24 | 2005-03-24 | Method for purifying microcysin LR and RR synchronously |
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| CN1687114A true CN1687114A (en) | 2005-10-26 |
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| CN 200510024573 Pending CN1687114A (en) | 2005-03-24 | 2005-03-24 | Method for purifying microcysin LR and RR synchronously |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101446575B (en) * | 2008-12-29 | 2011-06-22 | 无锡市疾病预防控制中心 | Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column |
| CN101750460B (en) * | 2008-12-03 | 2012-09-19 | 北京林业大学 | Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography |
| CN103048398A (en) * | 2012-12-05 | 2013-04-17 | 上海应用技术学院 | Method for determining microcystin MC-LR in water body |
| CN103163001A (en) * | 2013-03-27 | 2013-06-19 | 云南省环境监测中心站 | Method for extracting and purifying microcystic toxins LR and RR by taking cyanobacterial bloom in Dian Lake as raw material |
-
2005
- 2005-03-24 CN CN 200510024573 patent/CN1687114A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750460B (en) * | 2008-12-03 | 2012-09-19 | 北京林业大学 | Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography |
| CN101446575B (en) * | 2008-12-29 | 2011-06-22 | 无锡市疾病预防控制中心 | Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column |
| CN103048398A (en) * | 2012-12-05 | 2013-04-17 | 上海应用技术学院 | Method for determining microcystin MC-LR in water body |
| CN103163001A (en) * | 2013-03-27 | 2013-06-19 | 云南省环境监测中心站 | Method for extracting and purifying microcystic toxins LR and RR by taking cyanobacterial bloom in Dian Lake as raw material |
| CN103163001B (en) * | 2013-03-27 | 2016-05-18 | 云南省环境监测中心站 | A kind of method of extracting purifying microcysin LR, RR taking Dian Chi blue-green alga bloom as raw material |
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