CN113045632B - Method for purifying microcystin MC-LR in batches - Google Patents
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Abstract
A method for purifying microcystin MC-LR in batches aims at overcoming the defects that in the prior art, microcystin samples obtained from natural water are high in complexity and high in impurity interference, the obtained microcystin content is low, and the preparation process of microcystin needs to be adjusted continuously due to poor sample repeatability, and the method extracts and purifies microcystin MC-LR from microcystin aeruginosa and comprises the following steps: firstly, carrying out laboratory culture on microcystis aeruginosa; secondly, crude extraction of microcystin MC-LR; thirdly, removing impurities in the crude extract by adopting a chromatographic column with MCI GEL CHP20P as a filler to obtain primarily purified microcystins; and fourthly, further purifying the primarily purified microcystins by preparative HPLC to obtain microcystins MC-LR with the chromatographic purity of more than 95 percent. The microcystin obtained by the invention has high purity, can realize batch preparation, and lays a material foundation for various subsequent researches on microcystin.
Description
Technical Field
The invention relates to the technical field of biochemical separation, in particular to a batch purification method of microcystin MC-LR.
Background
The continuous outbreak of the global cyanobacterial bloom arouses the high attention of domestic and foreign scholars to the cyanobacterial toxin in the polluted water body, and carries out extensive and intensive research on the toxic effect of the cyanobacterial toxin, and the research result shows that a series of toxic effects are caused by aquatic animals or human beings contacting or drinking the toxic algae-producing water source for a long time. Most of the research on the microcystins is based on sampling of naturally polluted water or laboratory simulation experiments by adopting experimental animals, and most of the adopted toxicity attacking experiment samples are concentrated algae powder or polluted water body extracts, although the field sampling can obtain large biomass, but the sample complexity is high, the impurity interference is large, the experimental result is influenced by various unknown factors, the content of the obtained algae toxin is low, the sample repeatability is poor, the algae toxin preparation and purification process can not be directly suitable for different batches of samples, the scheme optimization is required to be carried out in a targeted manner every time, therefore, the pure cultured microcystis in a laboratory is required to extract the microcystin, and a batch purification method of microcystin MC-LR is needed to be provided, which can purify microcystin in batch, and the obtained microcystins have high purity, thereby laying a material foundation for the subsequent development of various researches on the microcystins.
Disclosure of Invention
The invention overcomes the defects that a microcystin sample obtained from a natural water body in the prior art is high in complexity, high in impurity interference, low in content of obtained microcystin and poor in sample repeatability, and the preparation process of the microcystin needs to be adjusted continuously, and provides a batch purification method of microcystin MC-LR.
The invention adopts the technical scheme that the purpose of the invention is realized by: a method for purifying microcystin MC-LR in batches is used for extracting and purifying microcystin MC-LR from Microcystis aeruginosa and comprises the following steps: step one, laboratory culture of microcystis aeruginosa: the aerated culture of the Microcystis aeruginosa is carried out by adopting BG11 culture medium, and the Microcystis aeruginosa is Microcystis aeruginosa FACHB-905.
Step two, crude extraction of microcystin MC-LR: and (3) carrying out cell wall breaking treatment on the microcystis aeruginosa obtained by laboratory culture in the step one, and extracting by using an organic solvent to obtain a crude extract of microcystin MC-LR.
Step three, impurity removal of the crude extract of microcystin MC-LR: and (3) purifying the crude extract obtained in the step two by adopting a chromatographic column with MCI GEL CHP20P as a filler, and removing impurities in the crude extract to obtain primarily purified microcystin MC-LR.
Step four, preparative HPLC purification of microcystin MC-LR: and (3) further purifying the primarily purified microcystin MC-LR obtained in the step three through primary separation and secondary preparation to obtain the microcystin MC-LR with the chromatographic purity of more than 95%.
The initial separation conditions were as follows: mobile phase A: h2O +0.1% TFA, mobile phase B: ACN +0.1% TFA; flow rate: 8 mL/min; detection wavelength: 238 nm; the sample volume is 300 mL; a detector: a visible ultraviolet spectroscopic detector; chromatographic separation gradient conditions: 0 min, 100% of mobile phase A, 35 min, 90% of mobile phase A, 39 min, 50% of mobile phase A, 43 min, 0% of mobile phase A, 47 min and 100% of mobile phase A.
The secondary preparation conditions were as follows: mobile phase A: h2O +0.1% TFA, mobile phase B: ACN +0.1% TFA; flow rate: 15 mL/min; detection wavelength: 238 nm; the sample injection amount is 30 mL; a detector: a visible ultraviolet spectroscopic detector; chromatographic separation gradient conditions: 0 min, mobile phase A90%, 5 min, mobile phase A70%, 13 min, mobile phase A65%, 13.5 min, mobile phase A65%, 14 min, mobile phase A10%, 15 min, mobile phase A10%, 15.5 min, mobile phase A90%.
Further, the formula of the BG11 culture medium in the first step is as follows: NaNO3 1.5 g/L，K2HPO4•3H2O 0.04 g/L，MgSO4•7H2O 0.075 g/L，CaCl2.2H20.036 g/L of O, 0.006 g/L of citric acid, 0.006 g/L of ferric ammonium citrate, 0.001 g/L of EDTA, and Na2CO30.02 g/L, 1mL of A5+ Co stock solution, and 1000 mL of distilled water. Wherein the A5+ Co stock solution comprises the following components: h3BO3 2.86 g，MnCl2•2H2O 1.81 g，ZnSO4•7H2O 0.222 g，CuSO4•5H2O 0.079 g，NaMoO4•2H2O 0.390 g，Co(NO3)2•6H2O0.049 g and distilled water 1000 mL.
Further, the organic solvent in the second step is methanol.
Further, the second step of crude extraction of microcystin MC-LR comprises the following steps: step a, carrying out centrifugal sedimentation on 15L-25L of microcystis aeruginosa algae liquid at the rotating speed of 3000-8000 r/min for 10-20 min, then discarding the supernatant, collecting algae cells, then resuspending the algae cells with deionized water, fully oscillating and shaking up to obtain microcystis aeruginosa algae slurry; step b, taking the algae slurry of the microcystis aeruginosa obtained in the step a, centrifuging for 10-20 min at the rotating speed of 10000-; step c, adding 100 plus 200 mL of 50% -70% methanol solution into the residue obtained after centrifugation in the step b, magnetically stirring for 0.5-2 h, then centrifuging at the rotating speed of 5000 plus 10000 r/min for 10-20 min, and collecting the supernatant II; d, repeatedly extracting the residue obtained after centrifugation in the step c by using a methanol solution twice according to the operation in the step c, discarding the residue to obtain a supernatant III and a supernatant IV, and combining the supernatant III and the supernatant II obtained in the steps b and c to obtain a microcystin stock solution; e, performing vacuum rotary evaporation on the microcystin stock solution obtained in the step d at the temperature of 50 ℃ to remove methanol in the solution; and f, centrifuging the microcystin stock solution concentrated in the step e for 10-20 min at the rotating speed of 10000-.
Furthermore, the filler MCI GEL CHP20P of the chromatographic column in the third step is a polymer taking polystyrene and divinyl copolymer as a matrix.
Further, the filler MCI GEL CHP20P is pretreated before use by the following steps: 1) soaking: soaking in anhydrous ethanol for 24 h; 2) cleaning: packing the column with ethanol by a wet method, cleaning the column with ethanol until the effluent is colorless, and then washing the column with distilled water to remove the ethanol; 3) soaking in 2-10% HCl solution for 3-6h, and washing with distilled water to neutrality; 4) soaking in 1-5% NaOH solution for 2-6 hr, and washing with distilled water to neutral.
Further, the filler MCI GEL CHP20P is stored in 20% ethanol solution.
Further, the conditions of purifying the crude extract of microcystin MC-LR by using a chromatographic column with MCI GEL CHP20P as a filler in the third step are as follows: gradient elution is carried out by adopting methanol water solution, and the elution flow rate is 1 mL/min.
Further, in the third step, the step of purifying the crude extract of microcystin MC-LR by using a chromatographic column with MCI GEL CHP20P as a filler comprises the following steps: 1) eluting the column with 20-30% methanol solution to remove impurities in the crude extract; 2) eluting toxin with 70-90% methanol solution for 3-4 times; 3) collecting eluate by stages, and concentrating on rotary evaporator to obtain primarily purified microcystin MC-LR.
The invention has the beneficial effects that: (1) the method for purifying the microcystis biomass cultured in a laboratory in batches has the advantages of simple and convenient operation, low purification difficulty, high efficiency and the like.
(2) The invention utilizes the microcystis cultured in a laboratory to carry out the batch purification of the algae toxin, compared with the field natural toxin-producing microcystis, the biomass reproducibility of the algae cultured in the laboratory is good, the toxin-producing type of single algae is less, the extraction and purification process of the algae toxin is relatively simple, and a large amount of algae cells can be obtained in a short time through the large-batch aeration culture in the laboratory, so that the invention is suitable for the batch preparation of the algae toxin and lays a material foundation for developing various researches on the algae toxin in the future.
(3) According to the invention, the MCI GEL CHP20P is adopted as the chromatographic column of the filler to remove the foreign protein and other impurities in the crude extract of the microcystin, and the MCI GEL CHP20P can well separate substances similar to the microcystin, so that the purification rate of the microcystin is obviously improved.
(4) MCI GEL CHP20P filler has high resolution, high loading, high flow rate, low back pressure, easy self-filling and amplification, can be directly transferred from laboratory scientific research to process development, and is suitable for batch purification of microcystins.
(5) The method adopts gradient elution to purify the microcystin by preparative HPLC (high performance liquid chromatography), effectively realizes the separation of microcystin and impurities by optimizing the mobile phase ratio and the gradient elution procedure, and improves the purification effect; in addition, the secondary preparation condition is further optimized on the basis of primary separation, the preparation and purification time is shortened by adjusting the gradient change speed of the mobile phase, and the purification efficiency and the separation rate can be obviously improved.
(6) The purity of the microcystin MC-LR obtained by the invention can reach 95%.
The invention is further described with reference to the following detailed description and accompanying drawings.
Drawings
FIG. 1 is a chromatogram of the crude extractive solution of microcystin MC-LR after impurity removal.
FIG. 2 is a chromatogram of MC-LR purified by preparative HPLC.
Detailed Description
The invention relates to a batch purification method of microcystin MC-LR, which extracts and purifies microcystin MC-LR from Microcystis aeruginosa and comprises the following steps: step one, laboratory culture of microcystis aeruginosa: adopting BG11 culture medium to perform aerated culture of Microcystis aeruginosa FACHB-905.
The formula of the BG11 culture medium is as follows: NaNO3 1.5 g/L，K2HPO4•3H2O 0.04 g/L，MgSO4•7H2O 0.075 g/L，CaCl2.2H20.036 g/L of O, 0.006 g/L of citric acid, 0.006 g/L of ferric ammonium citrate, 0.001 g/L of EDTA, and Na2CO30.02 g/L, 1mL of A5+ Co stock solution, and 1000 mL of distilled water. Wherein the A5+ Co stock solution comprises the following components: h3BO3 2.86 g，MnCl2•2H2O 1.81 g，ZnSO4•7H2O 0.222 g，CuSO4•5H2O 0.079 g，NaMoO4•2H2O 0.390 g，Co(NO3)2•6H2O0.049 g and distilled water 1000 mL. A large amount of algae cells can be obtained in a short time through large-batch aeration culture in a laboratory.
Step two, crude extraction of microcystin MC-LR: and (3) carrying out cell wall breaking treatment on the microcystis aeruginosa obtained by laboratory culture in the step one, and extracting by using an organic solvent to obtain a crude extract of microcystin MC-LR. The organic solvent for extracting the crude extract of microcystin is methanol, and 50-70% methanol solution is generally selected, and 60% methanol solution is preferably used. The crude extraction of the microcystin comprises the following steps: centrifuging and settling 21L of algae solution for 15 min at a rotation speed of 5000 r/min, discarding the supernatant, and collecting algae cells; resuspending algae cells with deionized water, shaking, collecting 3 parts of 30 mL algae slurry, and boiling in water bath for 10 min, 20 min, and 30 min; centrifuging at the rotating speed of 12000 r/min for 15 min, and collecting supernatant I; adding 150 mL of 60% methanol solution into the residue, and stirring for 60 min on a magnetic stirrer; centrifuging at 8000 r/min for 15 min, and collecting supernatant II; extracting with methanol solution twice to obtain supernatants III and IV, mixing the supernatants I, II, III and IV to obtain microcapsule algae stock solution, and discarding residue; carrying out vacuum rotary evaporation on the microcystis stock solution at 50 ℃ to remove methanol in the solution; centrifuging the primary concentrated microcystin stock solution for 15 min, 12000 r/min, filtering with 0.22 μm filter membrane, and collecting filtrate to obtain crude extract of microcystin MC-LR.
The invention uses wet algae slurry, compared with dry algae powder, the invention omits the part of dry algae slurry, and greatly saves the extraction process and the extraction cost of microcystin. And the crushing of the microcystis cells is carried out by adopting a boiling water bath mode, so that not only can a higher extraction rate of the microcystis be obtained, but also the energy consumption is low, and the sample amount of single treatment is large, thereby being very suitable for the batch preparation of the microcystin.
Step three, impurity removal of the crude extract of microcystin MC-LR: and (3) purifying the crude extract obtained in the step two by adopting a chromatographic column with MCI GEL CHP20P as a filler, and removing impurities in the crude extract to obtain primarily purified microcystin MC-LR. Wherein, the filler MCI GEL CHP20P is a polymer taking polystyrene and divinyl copolymer as a matrix, and is pretreated by the following steps before use: 1) soaking: soaking in absolute ethyl alcohol for 24 h; 2) cleaning: packing the column with ethanol by wet method, then washing with ethanol until the effluent is colorless, and washing with distilled water to remove ethanol; 3) soaking the mixture in 5% HCl solution for 4h, and washing the mixture with distilled water to be neutral; 4) soaking the mixture in 2% NaOH solution for 4h, and washing the mixture with distilled water to be neutral; when the filler MCI GEL CHP20P is not used for a long time, the filler is preserved in 20% ethanol solution.
MCI GEL CHP20P filler is spherical particles with narrow particle size distribution, the particle size distribution is 35-75 μm and 75-150 μm, the smaller particles can ensure higher chromatographic separation performance, the column packed with MCI GEL CHP20P has high efficiency, and can well separate substances similar to microcystin, thereby obviously improving the purification rate of the microcystin and obtaining high-purity pure product of the microcystin.
The polymer base material filler can keep the structure and the stability of the sphere in extreme acid-base solutions and organic solvents, so that MCI GEL CHP20P has wide pH application range and compatible pH 0-14, can be used in strong acid and strong alkali media, and can also be cleaned and disinfected in situ by using the strong acid and strong alkali solutions. The microcapsule microcystin purifying preparation method has the advantages of long service life, repeated utilization, simple required equipment, high resolution of MCI GEL CHP20P filler, high loading capacity, high flow rate, low back pressure and easy self-filling and amplification, can be directly transited from laboratory scientific research to process development, and is suitable for batch purification preparation of the microcystin.
In the experiment of purifying the crude extract of microcystin MC-LR by using a chromatographic column with MCI GEL CHP20P as a filler in the third step, in order to obtain a preferable elution process, gradient elution is firstly carried out by using 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% methanol aqueous solutions, the elution flow rate is 1mL/min, effluent liquid is collected in sections, and the preferable elution process is determined by detecting the content of microcystin MC-LR in the eluate. According to the comparison of the data in the experiment, the preferable conditions for purifying the crude extract of microcystin MC-LR by using the chromatographic column with MCI GEL CHP20P as the packing are as follows: gradient elution is carried out by adopting methanol water solution, and the elution flow rate is 1 mL/min. The preferred purification steps are: 1) eluting the column with 20-30% methanol solution to remove impurities in the crude extract; 2) eluting toxin with 70-90% methanol solution for 3-4 times; 3) collecting eluate by stages, and concentrating on rotary evaporator to obtain primarily purified microcystin MC-LR.
Step four, purifying the microcystin MC-LR by preparative chromatography: and (3) further purifying the primarily purified microcystin MC-LR obtained in the step three through primary separation and secondary preparation to obtain the microcystin MC-LR with the chromatographic purity of more than 95%.
The initial separation conditions were as follows: mobile phase A: h2O +0.1% TFA, mobile phase B: ACN +0.1% TFA; flow rate: 8 mL/min; detection wavelength: 238 nm; the sample volume is 300 mL; a detector: a visible ultraviolet spectroscopic detector; gradient of chromatographic separationConditions are as follows: 0 min, 100% of mobile phase A, 35 min, 90% of mobile phase A, 39 min, 50% of mobile phase A, 43 min, 0% of mobile phase A, 47 min and 100% of mobile phase A.
The secondary preparation conditions were as follows: mobile phase A: h2O +0.1% TFA, mobile phase B: ACN +0.1% TFA; flow rate: 15 mL/min; detection wavelength: 238 nm; the sample injection amount is 30 mL; a detector: a visible ultraviolet spectroscopic detector; chromatographic separation gradient conditions: 0 min, mobile phase A90%, 5 min, mobile phase A70%, 13 min, mobile phase A65%, 13.5 min, mobile phase A65%, 14 min, mobile phase A10%, 15 min, mobile phase A10%, 15.5 min, mobile phase A90%.
In the fourth step, gradient elution is adopted to carry out preparative HPLC purification of microcystins, and by optimizing the mobile phase ratio and the gradient elution procedure, separation of microcystins and impurities can be effectively realized, and the purification effect is improved. In addition, the secondary preparation condition is further optimized on the basis of primary separation, the preparation and purification time is shortened by adjusting the gradient change speed of the mobile phase, and the purification efficiency and the separation rate can be obviously improved.
After preparative HPLC purification, purified microcystin MC-LR is obtained, and the crude extract of microcystin after impurity removal and the microcystin after preparative HPLC purification are compared and analyzed by chromatogram map, the results are as follows: the chromatogram (figure 1) after the impurity removal of the crude extract of microcystin can be seen: only 3 main peaks are shown on the chromatogram, the impurity peaks are few, and the response value is low, which indicates that the primary purification of microcystins can be achieved after the microcystins are subjected to wall breaking by boiling water bath, extraction by 60% methanol, concentration by a rotary evaporator and impurity removal by MCI GEL CHP20P chromatographic columns, and the subsequent purification work is simplified.
After the primarily purified microcystin MC-LR is purified by preparative HPLC, only one chromatographic peak can be seen from a detected chromatogram (figure 2), which indicates that the sample purified by the experiment only contains a single component and is relatively pure. The purified sample can be basically determined to be MC-LR through the retention time of the existing standard sample; for further verification, the mixed solution of the purified sample and the standard sample is injected, and the chromatogram has only one toxin peak, which indicates that the sample has only a single microcystin. The purity was found to be 95% by area normalization.
Claims (9)
1. A method for purifying microcystin MC-LR in batches is used for extracting and purifying microcystin MC-LR from microcystis aeruginosa and is characterized by comprising the following steps:
step one, laboratory culture of microcystis aeruginosa: adopting BG11 culture medium to perform aeration culture of Microcystis aeruginosa FACHB-905;
step two, crude extraction of microcystin MC-LR: carrying out cell wall breaking treatment on the microcystis aeruginosa obtained by laboratory culture in the step one, and extracting by using an organic solvent to obtain a crude extract of microcystin MC-LR;
step three, impurity removal of the crude extract of microcystin MC-LR: purifying the crude extract obtained in the second step by using a chromatographic column with MCI GEL CHP20P as a filler, and removing impurities in the crude extract to obtain primarily purified microcystin MC-LR;
step four, preparative HPLC purification of microcystin MC-LR: further purifying the primarily purified microcystin MC-LR obtained in the third step through primary separation and secondary preparation to obtain microcystin MC-LR with the chromatographic purity of more than 95%;
the initial separation conditions were as follows: mobile phase A: h2O +0.1% TFA, mobile phase B: ACN +0.1% TFA;
flow rate: 8 mL/min; detection wavelength: 238 nm; the sample volume is 300 mL; a detector: a visible ultraviolet spectroscopic detector;
chromatographic separation gradient conditions: 0 min, 100% of mobile phase A, 35 min, 90% of mobile phase A, 39 min, 50% of mobile phase A, 43 min, 0% of mobile phase A, 47 min and 100% of mobile phase A;
the secondary preparation conditions were as follows: mobile phase A: h2O +0.1% TFA, mobile phase B: ACN +0.1% TFA;
flow rate: 15 mL/min; detection wavelength: 238 nm; the sample injection amount is 30 mL; a detector: a visible ultraviolet spectroscopic detector;
chromatographic separation gradient conditions: 0 min, mobile phase A90%, 5 min, mobile phase A70%, 13 min, mobile phase A65%, 13.5 min, mobile phase A65%, 14 min, mobile phase A10%, 15 min, mobile phase A10%, 15.5 min, mobile phase A90%.
2. The method for batch purification of microcystin MC-LR as recited in claim 1, wherein the formula of BG11 medium in the first step is: NaNO3 1.5 g/L，K2HPO4•3H2O 0.04 g/L，MgSO4•7H2O 0.075 g/L，CaCl2.2H20.036 g/L of O, 0.006 g/L of citric acid, 0.006 g/L of ferric ammonium citrate, 0.001 g/L of EDTA, and Na2CO30.02 g/L, 1mL of A5+ Co stock solution and 1000 mL of distilled water;
wherein the A5+ Co stock solution comprises the following components: h3BO3 2.86 g，MnCl2•2H2O 1.81 g，ZnSO4•7H2O 0.222 g，CuSO4•5H2O 0.079 g，NaMoO4•2H2O 0.390 g，Co(NO3)2•6H2O0.049 g and distilled water 1000 mL.
3. The method of claim 1, wherein the organic solvent used in step two is methanol.
4. The method for batch purification of microcystin MC-LR as claimed in claim 1 or 3, wherein the crude extraction of microcystin MC-LR in step two comprises the following steps:
step a, carrying out centrifugal sedimentation on 15L-25L of microcystis aeruginosa algae liquid at the rotating speed of 3000-8000 r/min for 10-20 min, then discarding the supernatant, collecting algae cells, then resuspending the algae cells with deionized water, fully oscillating and shaking up to obtain microcystis aeruginosa algae slurry;
step b, taking the algae slurry of the microcystis aeruginosa obtained in the step a, centrifuging for 10-20 min at the rotating speed of 10000-;
step c, adding 100 plus 200 mL of 50% -70% methanol solution into the residue obtained after centrifugation in the step b, magnetically stirring for 0.5-2 h, then centrifuging at the rotating speed of 5000 plus 10000 r/min for 10-20 min, and collecting the supernatant II;
d, repeatedly extracting the residue obtained after centrifugation in the step c by using a methanol solution twice according to the operation in the step c, discarding the residue to obtain a supernatant III and a supernatant IV, and combining the supernatant III and the supernatant II obtained in the steps b and c to obtain a microcystin stock solution;
e, performing vacuum rotary evaporation on the microcystin stock solution obtained in the step d at the temperature of 50 ℃ to remove methanol in the solution;
and f, centrifuging the microcystin stock solution concentrated in the step e for 10-20 min at the rotating speed of 10000-.
5. The method of claim 1, wherein the filler MCI GEL CHP20P of the chromatography column in step three is a polymer based on polystyrene and divinyl copolymer.
6. The batch purification method of microcystin MC-LR as claimed in claim 5, wherein said filler MCI GEL CHP20P is pretreated by the following steps before use:
1) soaking: soaking in anhydrous ethanol for 24 h;
2) cleaning: packing the column with ethanol by a wet method, cleaning the column with ethanol until the effluent is colorless, and then washing the column with distilled water to remove the ethanol;
3) soaking in 2-10% HCl solution for 3-6h, and washing with distilled water to neutrality;
4) soaking in 1-5% NaOH solution for 2-6 hr, and washing with distilled water to neutral.
7. The method for batch purification of microcystin MC-LR according to claim 5 or 6, wherein the filler MCI GEL CHP20P is stored in 20% ethanol solution.
8. The method of claim 1, wherein the conditions for purifying the crude extract of microcystin MC-LR using MCI GEL CHP20P as the packing in the third step are as follows: gradient elution is carried out by adopting methanol water solution, and the elution flow rate is 1 mL/min.
9. The method of claim 8, wherein the step of purifying the crude extract of microcystin MC-LR using MCI GEL CHP20P as a packing column comprises the steps of:
1) eluting the column with 20-30% methanol solution to remove impurities in the crude extract;
2) eluting toxin with 70-90% methanol solution for 3-4 times;
3) collecting eluate by stages, and concentrating on rotary evaporator to obtain primarily purified microcystin MC-LR.
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| CN101955517B (en) * | 2010-10-29 | 2012-12-12 | 中国科学院水生生物研究所 | Method for extracting and purifying microcystin |
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