pH indicator 4-Nitrophenol can be used as a
pH indicator. A solution of 4-nitrophenol appears colorless below pH 5.4 and yellow above pH 7.5. This color-changing property makes this compound useful as a
pH indicator. The yellow color of the 4-nitrophenolate form (or 4-nitro
phenoxide) is due to a maximum of absorbance at ( = in strong alkali). In contrast, 4-nitrophenol has a weak absorbance at ( = ).
Other uses • 4-Nitrophenol is an intermediate in the synthesis of
paracetamol. It is reduced to
4-aminophenol, then acetylated with
acetic anhydride. • 4-Nitrophenol is used as the precursor for the preparation of
phenetidine and
acetophenetidine, indicators, and raw materials for fungicides.
Bioaccumulation of this compound rarely occurs. • In
peptide synthesis,
carboxylate ester derivatives of 4-nitrophenol may serve as activated components for construction of
amide moieties.
Uses of derivatives In the laboratory, it is used to detect the presence of
alkaline phosphatase activity by hydrolysis of
PNPP. In basic conditions, presence of hydrolytic enzymes will turn reaction vessel yellow. 4-Nitrophenol is a product of the enzymatic cleavage of several synthetic substrates such as 4-nitrophenyl phosphate (used as a substrate for
alkaline phosphatase), 4-nitrophenyl acetate (for
carbonic anhydrase), 4-nitrophenyl-β--glucopyranoside and other sugar derivatives which are used to assay various
glycosidase enzymes. Amounts of 4-nitrophenol produced by a particular
enzyme in the presence of its corresponding substrate can be measured with a
spectrophotometer at or around and used as a proxy measurement for the amount of the enzyme activity in the sample. Accurate measurement of enzyme activity requires that the 4-nitrophenol product is fully deprotonated, existing as 4-nitrophenolate, given the weak absorbance of 4-nitrophenol at . Complete ionization of the alcohol functional group affects the conjugation of the pi bonds on the compound. A lone pair from the oxygen can be delocalized via conjugation to the benzene ring and nitro group. Since the length of conjugated systems affects the color of organic compounds, this ionization change causes the 4-nitrophenol to turn yellow when fully deprotonated and existing as 4-nitrophenolate. A common mistake in measuring enzyme activity using these substrates is to perform the assays at neutral or acidic pH without considering that only part of the chromophoric product is ionized. The problem can be overcome by stopping the reaction with
sodium hydroxide (NaOH) or other strong base, which converts all product into 4-nitrophenoxide; final pH must be >
ca. 9.2 to ensure more than 99% of the product is ionised. Alternatively enzyme activity can be measured at , the
isosbestic point for 4-nitrophenol/4-nitrophenoxide. ==Toxicity==