Alkaline phosphatases (FAL) catalyze the hydrolysis of 4-nitrophenyl phosphate (4-NFF) to form 4-nitrophenol and inorganic phosphate, with the alkaline buffer acting as a phosphate acceptor. The reaction is kinetically monitored at 405 nm based on the rate of 4-nitrophenol formation, proportional to the FAL activity present in the sample.