Method for separating and purifying microcystin

A microcystin, separation and purification technology, applied in the field of analytical chemistry, can solve the problems of high price, achieve the effect of low maintenance cost, simple process, and easy popularization

Inactive Publication Date: 2009-11-25
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, microcystins can only be isolated and purified from cyanobacteria cultivated in the wild or indoors, which is expensive and currently mainly depends on imports
There are not many pate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0012] Example 1

[0013] Step 1: Weigh 1.0g of cyanobacteria powder (Yunnan Delinhai Biotechnology Co., Ltd.) in a 50ml centrifuge tube, add 30ml of 5% acetic acid, sonicate in an ultrasonic water bath (power 100w, frequency 40KHz) at room temperature for 30min, centrifuge at 10000rpm for 15min, and pour out Supernatant. Add 30 ml of 5% acetic acid to the residue, repeat the above operation, and combine the two supernatants.

[0014] Step 2: Equilibrate the SPE column (5g / 20ml, Beijing Kanglin Technology Co., Ltd.) with 50ml methanol and 50ml 5% acetic acid successively, then pass the supernatant from step 1 through the SPE column, wash with 50ml30% methanol, and 75ml It was eluted with 0.1% TFA (trifluoroacetic acid) methanol, and the eluate was concentrated by rotary evaporation in a water bath at 35°C to near dryness, and dissolved with 2.5 ml of 50% methanol.

[0015] Step 3: Put the mobile phase storage tank (4L, the mobile phase is composed of water, methanol, and trifluoro...

Example Embodiment

[0018] Example 2

[0019] Step 1: Indoor cultured Microcystis FACHB905 (purchased by China Freshwater Algae Seed Bank) culture solution (cultured in BG-11 medium at a temperature of 28°C, a light intensity of 2000lx, and a light-to-dark ratio of 12:12 for 20 days. 1.56mg / ml) 500ml, centrifuged at 15000rpm for 30min. Collect the algae cells, after repeated freezing and thawing in liquid nitrogen, add 25ml of 5% acetic acid, sonicate in an ultrasonic water bath (power 100w, frequency 40KHz) at room temperature for 30min, centrifuge at 10000rpm for 15min, and pour out the supernatant. Add 25ml of 5% acetic acid to the residue, repeat the above operation, and combine the two supernatants.

[0020] Step 2: Equilibrate the SPE column (5g / 20ml, Beijing Kanglin Technology Co., Ltd.) with 50ml methanol and 50ml 5% acetic acid successively, then pass the supernatant from step 1 through the SPE column, wash with 50ml40% methanol, and 100ml It is eluted with methanol containing 0.1% TFA (trif...

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Abstract

The invention relates to a method for separating and purifying the microcystin, belonging to the analytical chemistry technology field. The method comprises following steps: extracting the blue algae producing the microcystin with the solvent in 1:20-80, purifying the extract through a SPE column, separating and purifying the microcystin by a column chromatography system composed of a constant flow pump, a C18 chromatography column and an automatic collector. The mobile phase is composed of 35-50% of water, 50-65% of methanol and 0.05-0.1% of acid. The purified microcystin RR(MC-RR) or microcystin-LR(MC-LR) is analyzed by a HPLC. The method has simple art, low costs for operation and maintenance and easy popularization. The purity of the purified microcystin is more than 95%.

Description

technical field [0001] The invention relates to a method for separating and purifying microcystin, belonging to the technical field of analytical chemistry. Background technique [0002] Microcystins (MCs) are a class of cyclic peptide toxins with hepatotoxicity, and are the most common cyanotoxins in freshwater blooms. ), Oscillatoria (or Planktothrix)), Nostoc, Anabaenopsis, Hapalosiphon, etc. The presence of microcystins in water has led to the death of wild animals and domestic animals worldwide. Recent studies have shown that microcystins are a potential threat to human health. The acute and chronic toxicity of microcystins has prompted extensive research on its detection methods, toxicology, detoxification in drinking water, accumulation in aquatic organisms, migration and degradation in the environment, all of which require purified microcystins. Cystin. [0003] Microcystin consists of 7 amino acids with a molecular weight of 800-1100Da. Its structure is character...

Claims

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Application Information

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IPC IPC(8): C07K7/56C07K1/16C07K1/14
Inventor 耿志明王澎刘蔼民
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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