Reagents The following reagents are required: Disodium p-nitrophenyl phosphate,
ID: 1054597 • Letter: R
Question
Reagents The following reagents are required: Disodium p-nitrophenyl phosphate, 19 mM, 4 mg/ml (Allied Chemical Co.) mdash the substrate is made up using the appropriate buffer; p-Nitrophenol, 0.68 mM, 0.095 mg/ml (Eastman Organic Chemicals) mdash the molar extinction coefficient for p-nitrophenol is 17, 500 at 410 nm (15); Citric acid-Sodium citrate buffer, 0.3 M, pH 5.6, approximately 44 ml of sodium citrate with 6 ml of citric acid (16); Trichloroacetic acid, 30%, 300 g/l (Mallinckrodt Chemical Works); Sodium hydroxide. 1 N, 40 g/1 (Mallinckrodt Chemical Works); Magnesium chloride, 0.048 M, 9.7 g/1 (Mallinckrodt Chemical Works).Explanation / Answer
preperation of disodium p-nitrophenyl phosphate:
place 200ml of dry pyridine containing 30ml of POCl3 in 1000ml two neck flask , fitted for stirring and with a 125ml separatory funnel. While cooling in an ice bath, add dropwise with stirring 100 ml of dry pyridine containing 27.8 gm of p-nirophenol .
after 30 minutes, pour the reaction mixture with stirring into 400ml of cold H2O. bring the mixture to pH 7-8 with sodium hydroxide solution . extract the dry residue with 600, 300 and 200ml. portions of boiling 87% ethyl alchol , filtering while hot. filter and wash with cold 87% alcohol, followed with absolute alchol with ether.
upon drying in desiccator, a product light in colour is obtained. the yield is 28-30gm. purity 80-85%. although the preperation becomes yellower with time owing to the lberation of p-Nitrophenyl.the molar extinction coefficient of p-nirophenol is 17,500 at 410nm and act as buffer at pH is 9-10. it is stable under the conditions used fior acid hydrolysis.
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