Background and overview[1]
The most commonly used reagents for the determination of acid phosphatase are phenolphthalein diphosphate or its tetrasodium salt, disodium thymolphthalein monophosphate and disodium naphthylamine monophosphate, which are all classic phosphoric acids used to determine pH. Salt derivatives, usually phosphorous acid, are used in the form of sodium salt. Among these reagents, phenolphthalein diphosphate or its tetrasodium salt is more effective and easy to observe. Compared with other reagents, the cost of this reagent is low, and the toxicity of phenolphthalein released in the medium is low.
Preparation[1]
In a 50g beaker, weigh 8.0g of phenolphthalein and transfer to a three-tube balloon immersed in a crushed ice bath and coarse salt. Then add 12.5ml methylene chloride and 6.3ml phosphorus oxychloride and stir vigorously to obtain a uniform suspension; connect the constant pressure addition funnel containing 6.3ml triethylamine to the three-tube flask so that the reaction temperature does not exceed 4°C. Keep the reaction at room temperature for 6 hours with stirring at a maximum of 4°C. Add a small amount of 10% NaOH solution to evaluate the reaction progress. The test will appear pink. It is possible to keep the reaction at room temperature. If a negative result is observed, i.e. with respect to 10 % NaOH solution is colorless, the test should be repeated.
The methylene chloride was then evaporated to remove, then 100 ml of cold distilled water was added with constant stirring at about 4°C. After releasing all the HCl vapor, a pale yellow precipitate material similar to amber was observed to form, add 40% with stirring NaOH solution until all precipitate is redissolved. Acidify the aqueous solution of phthalate diphosphate with an inorganic acid at pH 1.00 to precipitate the benzoic acid phthalate diphosphate, then separate the substance from the supernatant, and add the acidic diphosphate in a 250 mL beaker. The entire substance of phthalide phosphate is dissolved in a minimum amount of freshly distilled VetecPA grade methanol, a few drops of VetecPA grade formamide are added to increase the solubility, and 1.0mol.L-1 sodium ethoxide solution is added under stirring. , until all precipitation is completed, separate the precipitate of phenylphthalocyanine tetrasodium bisphosphate by filtration, and then wash the tetrasodium phthalate bisphosphate repeatedly with anhydrous ether, repeat this step until the solid is almost dry. Dissolve diphenyl bisphosphate tetrasodium phthalate in the minimum amount of methanol/formamide (5:1), and then slowly add ethanol to reprecipitate the purified phenolphthalein diphosphate. After separation by filtration, phenolphthalein diphosphate is Tetrasodium was kept in a vacuum desiccator.
