Principles of the Reaction

p-Nitrophenylphosphate is hydrolyzed to form p-nitrophenol and phosphate in the presence of magnesium ions and alkaline phosphatase in a 2-amino-2-methyl-1-propanol [AMP] buffer.

                                           alk
                                          phos
p-nitrophenylphosphate -> p-nitrophenol + phosphate
                                         Mg+2                  (yellow)

The rate of appearance of p-nitrophenol measured at 405 nm is directly proportional to the alkaline phosphatase activity present in the original sample.