In this article we will discuss about the colorimetric method for estimation of phosphorus in plants.
Principle:
When ammonium molybdate solution is added to a solution of phosphate containing conc. H2SO4 it produces a yellow crystalline precipitation of ammonium phospho-molybdate. Phospho-molybdate reacts with amino-naphthol-sulphonic acid and produces a molybdenum complex which forms a blue- coloured solution.
The appearance of colour is due to the formation of a coloured complex in the reaction mixture. The intensity of the colour against known concentration is plotted on a graph paper to prepare a standard curve. By comparing the intensity of the colour of unknown samples with the standard curve, the conc. of phosphate in the unknown sample can be estimated.
The reaction processes are:
Na2HP04 + 12 (NH4)2MO4 + H2SO4
(Sodium mono-hydrogen phosphate) (ammonium molybdate)
↓
(NH4)3PO4MOO3H2O + H2SO4 + (NH4)2SO4 + Na2SO4 + H2O
(Ammonium-phospho-molybdate) + Amino-naphthol sulphonic acid
↓
(Blue coloured molybdenum complex)
Requirements:
(a) Reagents:
1. KH2PO4 solution (10 µgm. of phosphorus per ml. of soln.)
2. 10 n sulphuric acid.
3. 2.5% Ammonium molybdate.
4. Amino-napthol sulphonic acid.
(b) Glass-wares and instruments:
1. Test tubes, pipettes, beakers, etc.
2. Colorimeter.
(c) Preparation of standard phosphate solution:
11 µgm. KH2PO4 salt is dissolved in dist. water and the final volume is made up to 250 in a volumetric flask. The phosphate concentration of the solution is 10 µgm/ml.
Test Procedure:
The following experimental sets are made for estimation:
Set I — 1 ml of KH2PO4 soln. (10 µgm. of phosphorus) + 4 ml of dist. water.
Set II — 2 ml of KH2PO4 soln. (20 µgm. of phosphorus) + 3 ml of dist. water.
Set III — 3 ml of KH2PO4 soln. (30 µgm. of phosphorus) + 2 ml of dist. water.
Set IV — 4 ml of KH2PO4 soln. (40 µgm. of phosphorus) + 1 ml of dist. water.
Set V — 5 ml of KH2PO4 soln. (50 µgm. of phosphorus) + No dist. water.
1. A replica of five for each set is prepared.
2. Then 0.4 ml of 10 NH2SO4, 0.8 ml of 2.5% ammonium molybdate and 0.4 ml of amino naphthol sulphonic acid are added one after another to each experimental set.
3. Blue colour develops in each tube within 15-20 minutes.
4. A control blank set is also prepared without the addition of phosphate soln. instead 5 ml dist. water taken in the experimental tube.
5. The intensity of the colour of the reaction mixture is measured by colorimeter at 570 nm and the results are plotted on graph paper against known conc. of phosphorous to prepare a standard curve.
6. The concentration of phosphorus in phosphate soln. of unknown concentration can be determined by developing coloured solutions and estimating their intensities by Colorimeter. With the help of the standard curve the conc. of such soln. is also calculated.