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Estimation of total Phosphorus (Colorimetric Method)
Aim
To estimate the percentage of phosphorus in the triple acid extract of the given sample.
Principle
The heteropoly compound produced by the reaction between vanadomolybdate and phosphate radical in nitric acid medium is yellow in colour. The intensity of the yellow colour is read in the colorimeter with a maximum between 400 - 490 nm.
Reactions
H3 PO4 + 12 (NH4)2 Mo O4 + 21 NH3 → (NH4)3 PO4 + 12MoO3 + 21 NH4 NO3 + 12 H2O.
Materials required
1. Volumetric flask 25 ml, 2. 5 ml pipette 3. Spectrophotometer,
Bartons Reagent
Solution A : Dissolve 25 g of Ammonium molybdate in 400 ml of distilled water.
Solution B:1. Dissolve 1.25 g of ammonium metavanadate in 300 ml of concentrated Nitric acid. Add solution A to solution B and make up the volume to one litre
2. P Standards ranging from 0 to 20 ppm.
Procedure
Preparation of Standard Curve
Dissolve 0.2195 g of pure KH2PO4 in 400 ml of distilled water. Then add 25 ml of 1N H2SO4 and make up the volume to 1000 ml to get 50 ppm of P (50 g/ml). From this stock solution, prepare phosphorus standard as can be seen in the following table.
Sl.No.
Standard required (ppm)
Volume to be pipetted from 50 ppm
Volume made up to
1.
1
0.5 ml
25 ml
2.
2
1.0 ml
25 ml
3
3
1.5 ml
25 ml
4.
4
2.0 ml
25 ml
5
5
2.5 ml
25 ml
6
6
3.0 ml
25 ml
7.
7
3.5 ml
25 ml
8.
8
4.0 ml
25 ml
9.
9
4.5 ml
25 ml
10.
10
5.0
25 ml
Develop the yellow colour by adding 5 ml of Bartons reagent in each solution and make up the volume to 25 ml. Note the readings in spectrophotometer to construct a curve against the quantity of P present as g in each standard.
Pipette out 5 ml of the clear aliquot of diacid extract to a 25 ml volumetric flask. Neutralize the extract with ammonia using red litmus paper and again with HNO3 acid. Add 5 ml of Barton's reagent, shake well and make up the volume to 25 ml with distilled water. Read the intensity of the yellow colour in a spectrophotometer at 420 nm. Refer the meter reading with the standard curve. From the Standard curve, the content in ppm of the plant sample is obtained. Calculate the percentage of P content in plant sample.
Observation and calculation
Weight of the sample taken
=
'W' g (0.5/1.0)
Volume of diacid extract
=
'V' ml (250/100/50)
Aliquot taken for colour development
=
5 ml
Spectrophotometer reading
=
O.D. / T %
Corresponding ppm from Standard graph
=
X ppm
On Moisture free basis
=
Where M = Moisture content of plant sample.
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