Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography

A technology of microcystin and column chromatography, which is applied in the field of analytical chemistry, can solve the problems of expensive instruments and separation columns, inability to meet the requirements of batch preparation of pure MCs, and difficulty in realizing it, and achieve good reproducible results

Inactive Publication Date: 2010-06-23
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can obtain high-purity MCs, the instruments and separation columns are expensive and difficult to realize under general laboratory conditions, and the amount of MCs purified each time is also very small, which cannot meet the requirements for batch preparation of pure MCs

Method used

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  • Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography
  • Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography
  • Method for purifying microcystins MCLR through extraction and normal-pressure column chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Take Microcystis aeruginosa (Microcystis aeruginosa) suspension 2500mL, centrifuge at 4800r / min for 10min, discard the supernatant, wash and centrifuge with ultrapure water, discard the supernatant to obtain the algae cell residue, and use Ultrasonic cell disruptor for cell disruption;

[0025] 2. Use 25mL80% methanol to shake and extract the algae cell suspension in step 1 for 4h, centrifuge at 4800r / min for 10min, and collect the supernatant (the liquid chromatogram of the crude extract is as follows: figure 1 shown);

[0026] 3. Repeat step 2 once;

[0027] 4. Combine the supernatants extracted twice in step 2 and step 3 to obtain the crude extract of MCLR, and remove methanol by rotary evaporation at 30°C;

[0028] 5. Carry out liquid-liquid extraction to the aqueous solution of step 4 with chloroform, water phase: chloroform=2: 1, extract the lower layer water phase (MCLR water sample scanning picture before and after liquid-liquid extraction is as figure 2 ...

Embodiment 2-3

[0034] Except using dichloromethane and trichloromethane for liquid-liquid extraction respectively, other steps are the same as the method described in the examples to extract MCLR.

Embodiment 4

[0036] 1. Get 2500 mL of Microcystis aeruginosa suspension, centrifuge at 4800r / min for 10 min, discard the supernatant, wash and centrifuge with ultrapure water, discard the supernatant to obtain the algae cell residue, and use Repeated freeze-thaw method for cell disruption;

[0037] 2. Use 25 mL of 40% methanol to shake and extract the algae cell suspension after freezing and thawing in step 1 for 4 hours, centrifuge at 4800 r / min for 10 minutes, and collect the supernatant;

[0038] 3. Repeat step 2 once;

[0039] 4. Combine the supernatants extracted twice in step 2 and step 3 to obtain the MCLR crude extract, and remove methanol by rotary evaporation at 30°C;

[0040] 5. Carry out liquid-liquid extraction to the aqueous solution of step 4 with n-hexane, water phase: n-hexane=2:1, extract the lower aqueous phase;

[0041] 6. Repeat step 5 three times;

[0042] 7. Activate the C18 solid-phase extraction (SPE) cartridge (1000mg) with 10mL methanol first, then wash the ca...

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Abstract

The invention relates to the field of analytical chemistry, in particular to a method for purifying microcystins MCLR through extraction and normal-pressure column chromatography. The method comprises the following steps: 1) crushing algae cells; 2) performing crude extraction with methanol; 3) performing liquid-liquid extraction; 4) performing C18 solid-phase extraction; 5) performing Sephadex LH-20 gel chromatography and collecting MCLR activity peaks; and 6) performing DEAE ion-exchange chromatography, collecting MCLR activity peaks and obtaining pure MCLR. The method provided by the invention has the advantages of simplicity, effectiveness, economical efficiency, implementation capability under common experimental conditions and relative stability, and can prepare milligram-grade pure MCLR of which the purity is proved to be up to over 85 percent by HPLC analysis.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a method for extracting and purifying microcystin MCLR by normal pressure column chromatography. Background technique [0002] The frequent occurrence of algal blooms has been exacerbated by the increasingly serious eutrophication of surface water bodies in my country. When algal blooms occur, algae proliferate and decay in large numbers, resulting in foul smell of water, reduced transparency of water body, and affecting dissolved oxygen in water body, causing serious deterioration of aquatic ecology. After the death of certain algae (such as cyanobacteria), the algal toxins released due to cell lysis have a great impact on human health. Microcystins (MCs) are a type of algal toxins that appear frequently, produce large amounts, and cause serious damage in cyanobacterial bloom pollution. Subunit binding, thereby inhibiting their activity and promoting tumorigenesis. In view...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06
Inventor 梁文艳王金丽陈莉
Owner BEIJING FORESTRY UNIVERSITY
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