Phosphoric acid with molybdate ions forms heteropoly molybdophosphate complex H3PO4 + 12H2MoO4 → H3P(Mo3O10)4 +  + 12H2O

The heteropoly complexes are thought to be formed by coordination of molybdate ion with P as the central coordinating atom, oxygen of the molybdate ion with P as the central coordinating atom, the oxygen of the molybdate radical being substituted for that of PO4 .

Analysis of soil P involves 2 steps.

A. Extraction of P with buffered alkaline solution, NaHCO3

B. Determination of P from soil extract.

A. Extraction of P with buffered alkaline solution , NaHCO3

Principle –

The OH and HCO ions in the NaHCO3 solution decrease the concentration or activity of Ca and Al , resulting in increased P solubility in soils. In calcareous soils, the increased calcium phosphate solubility results from the decreased Ca concentratio by the high concentration by the high concentration of CO3 and the precipitation of CaCO3

In acid or neutral soils, the solubility of Al and Fe phosphates increases as increased OH concentration of Al by aluminate complex formation and of Fe by precipitation as the oxide.The increased surface negative charges and /or decreased number of absorption sites on Fe and Al oxide surfaces at high pH levels could be responsible for the desorption of sorbed P as well.

Reagents –

1.H2SO4 , 2.5 M

Add 139mL concentrated H2SO4 to 800 mL water, cool and dilute to 1L.

2.NaOH, 1 M

Dissolve 40g NaOH in water and dilute to 1L

3. NaHCO3, 0.5M

Dissolve 42.0g NaHCO3 in 950mL water, adjust pH with NaOH to 8.5 and dilute to 1L

4. Carbon black

5. p-nitrophenol, 0.25%

Dissolve 0.25g p-nitrophenol in 100mL of water

Procedure for extraction of P from soil

Add 2 g soil and 40mL extracting solution in a 125mL. Erlenmeyer flask. Shake for 30 minutes and filter through Whatman no.42 filter paper. If filtrate is not clear then add half a tea spoon carbon black to filtrate and filtre again through the same filter paper. This treatment is not necessary for most soils when ascorbic acid method is used for most soils when ascorbic acid method is used for P detemination. It means no carbon black is required to be added to most of soils.

B. P determination from soil extract.

Reagents

1.H2SO4 , 2.5 M

Dissolve 70mL concentrated H2SO4 (18M = 36N ) to 500ml water

2. Ammonium molybdate

Dissolve 20g ammonium molybdate- (NH4)6Mo7O24.4H2O in 500 ml water. Store the solution in glass stoppered bottle.

3. Antimony Potassium Tartarate

Dissolve 0.2778g potassium antimony tartrate in 100 mL water.

4. Ascorbic acid

Dissolve 1.76g ascorbic acid in 100 mL water, prepare fresh solution whenever required.

5. Mixed Reagent

Mix throughly 50mL 2.5M H2SO4, 15 mL ammonium molybdate solutions, 30 mL ascorbic acid solution and 5 mL antimony potassium tartrate solution. Prepare fresh reagent at the time of colour development.

6 . H2SO4 3.5 M

Dissolve 194 mL concentrated H2SO4 in water . Cool it and dilute to 1000mL volume .

7. P stock solution 50 mg P L-1

Dissolve 0.2197 gm oven dried (40 degree C ) KH2PO4 in water. Add 25 mL of 3.5M H2SO4 and dilute to 1 L

8. P solution, 5 mg P L-1

Dilute 10 mL of 50 mg P L-1 stock solution to 100 mL

Procedure

  1. Take a suitable aliquot, say 5 mL into a 50 mL volumetric flask , and 5 drops of p-nitrophenol indicator.
  2. Adjust the solution pH by slowly adding 2.5 M H2SO4 down the side of the flask to prevent rapid evolution of CO2 and loss of solution. Then continous addition till the solution become colourless.
  3. Dilute the above content to about 25mL with distilled water.
  4. Add 8 mL mixed reagent. Dilute the solution to 50mL volume and mixed well.
  5. Measure the absorbance at 880 nm on a spectrophotometer after 10 minutes.
  6. Prepare a blank that contains all reagents except P solution.

Preparation of standard curve

  1. Take 0,1, 2, 3, 4, 5, 6, 7 and 8 mL of 5 mg P L-1 solution into nine 50 mL volumetric flasks seperately.
  2. Add 5mL of NaHCO3 0.5 M solution to each flask.
  3. Add 5 drops od p-nitrophenol indicator solution becomes yellow
  4. Adjust the pH with 2.5 , h2so4 by adding drop by drop till the colour becomes just colourless.
  5. Dilute the content to about 40 mL with distilled water.
  6. Add 8 mL of mixed reagent, make up the volume to 50 mL mix well. Blue colour develops.
  7. Here the P concentration between becomes 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 ug p mL-1.
  8. Measure the absorbance at 880 nm after 10 min on a spectrophotometer
  9. Construct a graph by plotting absorbance on ‘Y’ axis vs P concentration ug P mL-1 on X axis .Find out the factor or read the P concentration of sample with respect to absorbance directly from the graph.
  10. Suppose the concentration of P in the coloured solution against the observed absorbance of 0.62 is 0.20ug P mL-1.