Preparation method of biochemical detection reagent of chlorinated tetrazolium salts

Abstract

The invention relates to a preparation method of a biochemical detection reagent of chlorinated tetrazolium salts. In the preparation method, formazan is used as a reactant and is oxidized into a chlorinated tetrazolium salt by adopting a hydrogen chloride / hydrogen peroxide system. The preparation method specifically comprises the following steps of: 1) dissolving formazan into the concentrated hydrochloric acid / methanol mixed liquid, 2) dropwise adding aqueous hydrogen peroxide solution under the condition that the dropwise adding temperature range is minus 10 DEG C to 15 DEG C, and stirring at the room temperature after dropwise adding, and 3) removing methanol by rotary evaporation, extracting with water-insoluble organic solvent and drying, adding anhydrous ethyl ether to separate out a crude product, and recrystallizing the mixed solvent to obtain the chlorinated tetrazolium salt refined product. The preparation method provided by the invention has the advantages of improving the yield at last, being simple in treatment, having no heavy metal residual in the product and being green in the preparation process.

Description

technical field [0001] The invention relates to a tetrazolium chloride salt compound, which can be used in the fields of cell proliferation detection, dehydrogenase detection and the like. Background technique [0002] Tetrazolium chloride salt is an organic salt with a five-membered tetrazole ring structure, in which the substituents on the ring are mostly benzene rings and substituted benzene rings, and the anion is chloride ion. These compounds are colorless solids, and their solutions only absorb in the ultraviolet region. Because their redox potential is similar to that in biological systems, they can generate formazan compounds under the action of cellular dehydrogenases. ), resulting in absorption in the visible region. Therefore, it is often used as a coloring agent for biological cells to qualitatively observe and quantitatively detect the activity of microorganisms and cells, and because tetrazolium salts and formazan are non-toxic, they can be directly used in th...

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Problems solved by technology

[0009] The conventional method for preparing tetrazolium chloride salt from formazan one-step reaction has: lead tetraacetate oxidation method [Chem.Revs, 1955 (55): 355-483], this method uses lead tetraacetate as oxidant, formsformazolium in the presence of hydrogen chloride Oxidation of tetrazolium chloride to tetrazolium chloride salt, because lead tetraacetate itself is reduced to divalent lead ion, there is a shortcoming of heavy metal residue in the tetrazolium chloride salt product, which will produce toxicity when used in live cell or tissue detection. And this method produces more lead-containing wastewater

[0010] Isopentyl nitrite oxidation method [Talanta, 44 (1997), 1299-1305], this method usually is to reflux formazan with isopentyl nitrite in acetic acid solvent, and the reaction temperature is high, because formazan is at high temperature Unstable, partially decomposed, resulting in more by-products, which has the disadvantages of complex post-treatment and poor yield of tetrazolium chloride salt products

[0011] Tert-butyl hypochlorite oxidation method [J Am Chem Soc, 1954, 76 (6): 1695-1695], this method is that formazan is dissolved in dioxane and glacial acetic acid mixed solvent, reflux after heating, cooling Precipitate jelly, add chloroform to dissolve and then add acetone to precipitate product. The disadvantage of this method is that the mixed solvent of dioxane and glacial acetic acid is used in a large amount, which is difficult to separate and recover. The precipitated jelly sticks to the wall in the kettle, which is difficult to handle. Suitable for industrial production

[0012] Chlorine oxidation method [Chinese Journal of Pharmaceutical Industry, 2006,37 (1): 10-11], this method is to pass into chlorine gas in the methanol solution of formazan to prepare, the shortcoming of this method is that chlorine gas will be in tetrazole in reaction process Aromatic ring chlorinated by-products are produced on the aromatic ring substituent of the salt, which is difficult to purify and the yield is low

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