PHOSPHOROUS DETERMINATION IN SOIL BY VARIOUS METHODS

2. NRM-401 (0+5), SOIL HEALTH CLINIC
COURSE TEACHERS:
Dr.H.C.PRAKASH
Dr.SARALA KUMARIBY:
NARENDRA.M.S
ALB-1081
Topic: DETERMINATION OF AVAILABLE P205 IN SOIL

3.  There 17 essential nutrients required by the plants for their
growth and development.
 Phosphorous is the second fertilizer element and it is an
essential constituent of every living cell and for the nutrition
of plant and animal.
 It takes active part in all type of metabolism of plant.
 It is an essential constituent of majority of enzyme and also
structural component of membrane system of cell, the
chloroplast s and mitochondria.
 It is intimately associated with the life process.

4.  The available phosphorous in a soil represents a fraction of
total phosphorous which is susceptible to plant uptake during
their growth. Plants take up phosphorous in H2PO4- and
HPO42- forms, but the water soluble phosphorous at any one
time is very small.
 The phosphorous exists in soil in both organic & inorganic
fraction.
 The organic fractions of phosphorous (phytin, nucleic acids &
inositol phosphates) do not contribute to the phosphorous
nutrition of the plants in our soils as they are low in organic
matter.
 Among the inorganic fractions Ca, Fe &Al phosphates are in a
dynamic equilibrium with water soluble phosphorous. There
fore, a measure of these in organic phosphorous fractions is
considered a useful index of the available phosphorous in the
soils.

5. There are two distinct phases in soil phosphorous determination:
1. Extraction of phosphorous
( bringing the soil phosphorous in to the solution):
• The choice of the extractant depends on the
nature of the soil and the kinds of phosphorous
compounds found in the soil.
• First of all a preliminary trail has to be conducted
to get information on the nature of the extractant
to be used for a particular soil as the nature of the
phosphate compounds vary with the type of soils.

6. • As different extractants
extract different types
of phosphorous
compounds present in
the soil, selection of
proper extractant is
essential. Also there
should be proper
correlation between soil
test values & crop
uptake.

7. 2. Quantification of the extracted phosphorous :
o The determination of available phosphorous is done by
Bray’s method for acid soils and Olsen’s method for
neutral, alkaline and calcareous soils and is normally
followed for soils having high CEC and high base
saturation with very low free CaCO3.
o The modified Olsen’s method is appropriate for soils
having pH more than 6.5 and in areas where soils have a
wide range of pH up to 9( both below and above6.5 pH).

8. There are mainly two methods for determination available
phosphorous in soils based on the pH and type of soil.
They are
1. Bray’s No. 1 method ( for acid soils )
2. Olsen’s method (for neutral, alkaline and calcareous soils )
PRINCIPLE: the combination of HCl and NH4F is designed to extract
easily acid soluble forms of phosphorous, largely Ca-phosphates
and Al & Fe phosphates. The NH4F dissolves Al, Fe and Mn
phosphates by forming complex ion with these metal ions in acid
solution and there by releases phosphate ion in to the solution. In
presence of chloromolybdic acid in an acid medium, the phosphate
ion forms a heteropoly complex compound of phosphorous, on
reduction, imparts blue colour to the solution. The intensity of blue
colour can be measured at 660 nm, using a SPECTRO PHOTO
METER.

9. REAGENTS REQUIRED:
 Bray’s No.1 (0.03 N NH4F + 0.025 N HCl)
 Ammonium molybdate (1.5%)
(Dickman & Bray’s reagent)
 Std. phosphorous solution (5ppm)
 Stannous chloride stock solution (40%)

10. Procedure :
1. Weigh 5 g of soil in to a 250ml conical flask.
2. Add 50 ml of Bray’s extractant, mix and shake the
contents for 5 min and filter.
3. Take 5 ml of the filtrate in to 25 ml volumetric flask and
add 5 ml of ammonium molybdate solution. Mix well
until the evolution of CO2 ceases. If more aliquot has to
be taken for analysis fluoride interference has to be
eliminated by adding 75 ml of 0.5% boric acid to 5 ml
extract.
4. Add about 10 ml distilled water washing the neck of the
flask to remove the adhering molybdate.

11. 5. Add about 1 ml of working stannous chloride solution
and make up the volume to the mark with distilled water.
6. In the mean time switch on the colorimeter and adjust
the wave length to 660 nm, allow to warm up for about
10-15 minutes.
7. Adjust the pointer on the galvanometer and adjust the
wave length to 660 nm, allow to warm up for about
10-15 minutes.
8.Rinse the colorimeter tube (cuvette) with the blank
solution then pour suitable volume. Place the tube in the
cuvette and turn the 100 set knob to read 100.
Pour the test solution into another colorimeter tube and
read the percentage transmission or the absorbance.

12. Preparation of the standard curve
Pipette out 0, 0.5, 1 , 2 , 3 , 4 and 5 ml of 5 ppm of
P solution in different 25 ml volumetric flasks.
Then proceed to develop colour as done for the
test sample and record the absorbance or
transmittance.
Plot the absorbance values on Y-axis and
concentration on X-axis and draw a line to pass
through maximum number of points.
From the graph, calculate the concentration ‘C’ at
OD of 0.1 and is used in further quantification of
soil phosphorous.

15.  The phosphorous in neutral, alkaline and
calcareous soil is extracted with sodium bi
carbonate which is designed to extract largely
calcium phosphates.
In presence of chloro-molybdic acid in a acidic
medium, the phosphate ion forms a hetero
poly complex compound of phosphorous, on
reduction, imparts blue colour to the solution.
The intensity of blue colour can be measured
at 660 nm, using a spectro photo meter.

16. Reagents:
 Sodium bi carbonate (0.5N) of 8.5 pH
 Ammonium molybdate (1.5%)
 Darco –G 60
 Stannous chloride stock solution

17. Procedure:
Weigh 5 g of soil in to a 250ml conical flask.
Add a pinch of phosphorous free Darco-G 60 & 50 ml of
0.5N sodium bi carbonate solution ( soil:solution-1:10).
Shake and filter the contents using whatman no.1 filter
paper (shake the solution before pouring suspension in to
funnel).
Take 5 ml of the filtrate in to 25 ml volumetric flask and
add 5 ml of ammonium molybdate solution. Mix well until
the evolution of CO2 ceases.
Add about 10 ml of distilled water washing the neck of the
flask to remove the adhering molybdate.
Add about 1 ml of working stannous chloride solution and
make the volume to the mark with distilled water.

18. Ascorbic acid method for
developing colour
Ascorbic acid reagent can be used for developing the colour
in phosphorous estimation in place of stannous chloride.
REAGENTS:
A). REAGENT-A: dissolve 12 g
of ammonium molybdate in
250 ml distilled water and of
antimony potassium tartarate
in 250 ml distilled water
separately. Add both reagents
1000 ml of 5N H2SO4. mix
thoroughly and make up to 2
L. store in Pyrex glass bottle in
cool dark place.

19. B). REAGENT-B: dissolve
1.056g of ascorbic acid
in 200 ml of REAGENT-A
and mix. This reagent
should be prepared
fresh as and when
required.

20. Procedure:
1) Pipette out 5 ml aliquot (Bray’s or Olsen's extract) in a
25 ml volumetric flask.
2) Acidify with 5 N H2SO4 to pH 5.
3) Determine the volume of acid required to bring the
solution to pH by using Para-nitro phenol (gives yellow
colour at pH 5).
4) Add 20 ml of distilled water and 4 ml of Reagent-B.
5) Read the intensity of blue colour at 730 nm after 10
minutes.

21. Precautions:
Test cuvette should be handled from the top rather than
from the lower part and it should be wiped clean with a
piece of tissue paper before putting it in the cuvette
holder.
The filtrate should be colourless, if not add more of
Darco-G 60 and filter.
Working SnCl2 solution should be prepared fresh after
2-3 hours.
The blue colour starts fainting after about 15 minutes,
hence the reading should be taken with in 10 minutes of
developing colour.

22. If the intensity of the colour is too high, take a
smaller amount of Aliquot and develop colour.
Remember that the colour solution can not be
diluted.
Temperature and shaking speed increases the
phosphorous extracted by Olsen's extractant. So they
have to be regulated.
For quality analysis, a minimum of one reference and
one blank should be included in a batch of 25
samples. Duplicates are done on approximately 5 %
of samples.

23. OBSERVATIONS
 wt. Of sample- 5 g
 Vol. of the extractant- 50 ml
 Vol. of aliquot (filtrate) taken – 5 ml
 Final volume after development of colour- 25 ml
 Transmittance of the test solution- ‘T’
 Conc. Of phosphorous read from the std. curve- ‘X’ ppm
 Avail. Phosphorous (ppm)- X x 50
 Avail. Phosphorous (Kg/ha)- ppm x 2.24

24. SLOPE= Y/X
Graph ppm=slope x conc. (spectro photometer reading)
Preparation of standard curve

25. INTERPRETATION
Sl. No Avail. P2O5 (Kg/ha) Ratings
1 <22.9 Low
2 22.9-56.33 Medium
3 >56.33 High
Avail. P2O5 graph ppm x vol. of extractant x vol. made x 2x106 x2.29
(Kg/ha)= -------------------------------------------------------------------------------
106 x wt. of soil x aliquot taken
Calculate the available phosphorous by using the formula