 4-Nitrophenyl phosphate

4-Nitrophenyl phosphate is a phosphate monoester in which a phosphate oxygen joins the aromatic group of 4-Nitrophenol. The neutral acid has formula ${\mathrm{C}}_6{\mathrm{H}}_6\mathrm{N}{\mathrm{O}}_6\mathrm{P}$; a salt or hydrated reagent has a different complete formula. The “4” places the nitro group opposite the oxygen-linked substituent on the ring.^[ref]

Alkaline phosphatase can hydrolyze 4-Nitrophenyl phosphate, releasing 4-Nitrophenol and inorganic phosphate. In the alkaline assay mixture the colored anionic product permits measurement with a spectrophotometer at $405\mathrm{nm}$. The manufacturer's calculation subtracts a blank and specifies a coefficient of $18.5\mathrm{m}{\mathrm{M}}^{-1}\mathrm{c}{\mathrm{m}}^{-1}$ under its stated conditions.^[ref]

For an original endpoint example, suppose blank-corrected absorbance is $0.370$ through an optical path length of $0.50\mathrm{cm}$, with that coefficient applicable. The Beer-Lambert law givesThis is $40\mu \mathrm{M}$ product. For an initial substrate concentration of $100\mu \mathrm{M}$, one-to-one conversion with no other product source would mean 40-percent conversion. These numbers are an illustrative model, not measured data from the cited assay.

The interpretation requires a suitable blank correction, known optical path length and stable measurement conditions. Changing pH changes the product's ionization and optical response. An endpoint measures accumulated product; estimating catalytic activity requires a time-dependent measurement and the corresponding calibration.