Quantification of inorganic phosphate, Pi
written by Dong-Eun Kim
Reagents
1. 10 % Ascorbic acid (keep it in refrigerator at 4 oC)
2. 0.42 % Ammonium molybdate (4H2O) in 1 N H2SO4 (stable at Room Temp.)
(28.6 ml of conc. H2SO4 and 4.2 g of Ammonium Molybdate-4H2O to 1000 ml water)
3. Mix: 1 part of reagent 1 to 6 part of reagent 2 (This mixture need to be kept in an ice
for the day use only)
Procedures
1. Add 0.70 ml of the mix to 0.30 ml of the phosphate containing solution (or to 0.30 ml
of water for the blank) and incubate 20 min at 45 oC or 1hr at 37 oC.
2. Read at 820 nm (0.01 mole of Pi corresponds to be 0.260 Abs reading.)
3. Find a standard curve using phosphate standard solutions.