EXPERIMENT NO: 02

DETERMINATION OF THE CONCENTRATION OF PHOSPHATE IN A SOIL SAMPLE (TOTAL,

INORGANIC AND ORGANIC)

Introduction
Phosphatic fertilizers are added to soil for cultivation, as plant available fraction of soil phosphorous is
low. Generally excessive phosphatic fertilizer application does not bring harmful effects to plants.
However that results undesirable concentrations of phosphorous, for in drainage waters. The risk of
leaching phosphorous to grand water can be lessened by reducing of application of phosphorous to soils
containing high level plant available phosphorous, soil P-index for available phosphorous can be
determined in the laboratory. For many crops critical levels of soil P- indices are known. Phosphate
fertilizers should be applied to soils only if the current soil P index is below the critical value.
The determination of available phosphorous involves measurement by chemical means that portion of
the total soil phosphorous which can be utilized by plants. Different methods use different solutions as
extractants.
The concentration of P in a soil extract is generally determined by adding a soluble molybdate and a
reducing agent under precise conditions of acidity and concentrations of molybdate and reluctant. The
blue colour that develops afterwards is related to the quantity of orthophosphate ion.
The molybdenum blue colour develops at a rate depending on the temperature and the stannous ion
concentration. It is stable for a certain limited period and then begins to fade somewhat rapidly. Thus the
analysis of the soil extract should be carried out under constant temperature conditions and the depth of
the colour must be read within the period of stability. As with the extraction of phosphorous from soil
rigorous standards must be established to give consistent & accurate results for the determination of
phosphate ion In soil extracts.

Procedure

(1) Modified Truog Method for Determination of Inorganic Phosphate.

This procedure involves removal of adsorbed phosphate from soil by an extracting solution. In an
acid solution, soluble orthophosphates combine with molybdate forming heteropolymolybdophosphoric
acid which upon reduction forms a blue colored soluble complex. This colored complex is the basis for the
colorimetric determination, the depth of the colour being proportional to the amount of phosphate
present.

(A) Preparation of reagents

(1) Truog Extracting Solution
Dissolve appropriate weight of (NH4)2SO4 in 0.01 M H2SO4 (which is prepared using D.I. water) to
obtain as 0.02 M solution.

(2) Molybdate reagent, ( 2.5 % )

Dissolve 26.54 g of (NH4)6Mo7O24.4H2O in 200 ml of D.I water, previously warmed to 60°C. Filter if
necessary. Dilute 280 ml of conc.H2SO4 (P & As free) to 750 ml with D.I. water. After both solutions
have cooled, add the ammonium molybdate solution slowly with shaking, to the sulfuric acid
solution. After the combined solution has cooled to room temperature, dilute to exactly l liter. This
is a 5M H2SO4 solution, containing 2.5 g of (NH4)6Mo7O24.4 H2O per 100 ml. Store in an acid-weathered
amber glass bottle.
(3) Stannous Chloride solution
Dissolve 2.5 g of SnC12.2H2O in 10 ml conc. HCl and dilute (with rapid Stirring) to 100 mL with D.I.
water. Filter if necessary. Add a few pieces of metallic tin (Sn). Store in an amber coloured bottle.
• Keep this solution in a refrigerator. Prepare a fresh solution at least once every 3 weeks or until the
solution becomes cloudy.
• Direct contact between the molybdate reagent and the SnCI2 solution will cause formation of blue
colour even in the absence of phosphorous:

(4) Standard Phosphorous solution

Dissolve 0.4394 g of KH2PO4 (dried at 1000C ) in 1000 ml of 0.01 M H2SO4, to obtain
100 ppm phosphorous solution. Then dilute this solution 25 times, using 0.01 M H2SO4 obtain 4ppm
phosphorous.
Note : All the solutions must be clear, in order to measure absorbance

(B) Preparation of Calibration Curve

(1) Transfer 5.00 mI, 10.00 m1, 15.00 ml, 20.00 ml, 25.00 ml of the 4 ppm standard into 100 ml volumetric
flasks. Add the extracting solution until the flasks are about half full.
(2) Into each flask pipette 2.00 ml of molybdate reagent and mix well.
(3) Pipette 2.0ml of stannous chloride solution into each flask. make up to the mark with the extracting
solution and mix thoroughly.
* Since colour development just after the addition of stannous chloride solution. familiarize
yourself with the operation of spectrophotometer before that. If in doubt consult a demonstrator.
(4) Leave to stand for 4 minutes and measure the absorbance at 660 nm. Use the extracting solution as
the blank, to set zero absorbance.
• The time period is somewhat critical, so try to stick that as closely as possible.
• Recording the absorbance reading to first 2 decimal points, is sufficient
(5) When absorbance of the standard samples have been measured, make a plot of absorbance vs
concentration of Phosphorous (in ppm ).

(C) Measurement of Soil Samples

(1) Measure 3.0 g of soil sample (air dried and finely ground) into a reagent bottle.
(2) Add l00.0ml of the extracting solution,
(3) Stopper and shake about 30 minutes.
(4) Allow to stand for 15 minutes and filter through a folded filter paper.
(5) Transfer 50.00 ml aliquot into a l00 mL volumetric flask,
(6) Add the reagents and measure the absorbance as described under preparation of calibration curve
starting from step 2.

Results & Calculations

(1) Plot a calibration graph of absorbance vs. ppm of phosphorous and draw the best straight line as
determined by the method of least squares.
(2) Find the phosphorous concentration of soil extract using the corresponding absorbance reading
and dilution factor of 2.
(3) For 3.0 g soil and 100 ml extracting solution, the corresponding factor to convert P in solution to
P in soil is 100/3.'I'herefore calculate ppm P in soil using the following, equation.
ppm P in soil = ppm P (from calibration curve) x 2 x 100/3
(4) Using this value calculate ppm in soil.
(2) Total Phosphate Determination

(1) Take 25 ml of the soil extract solution (in part C step 4) in a flask and evaporate to dryness. Cool
and dissolve the residue in 1 ml of perchloric acid.
(2) Heat the flask gently so that the content becomes colorless. Cool and add 10 ml of distilled water
and 2 drops of phenolphthalein indicator.
(3) Titrate against sodium hydroxide solution till the appearance of light pink color takes place.
(4) Make up the volume to 25 ml by adding distilled water. Add 1 ml ammonium molybdate solution
and 3 drops of stannous chloride solution. A blue color will appear.
(5) Wait for 5 minutes and record the absorbance on spectrophotometer at 690 nm.
(6) Run simultaneously distilled water blank in similar manner.
(7) Process the standard phosphorous solution of different concentration in a similar manner and
plot a standard curve between absorbance and concentration of standard phosphorous solutions.

Results & Calculations

(1) Deduce the total phosphorous content of sample by comparing its absorbance with standard
curve and express the result in mg/liter.
(2) Deduce organic phosphate content using the following relationship

Organic phosphate (mg/liter) = TP - IP

Where TP= Total phosphate (mg/liter)
IP = Inorganic phosphate (mg/liter)

Discussion
1) There are four types of phosphate are available in the soil sample namely,
a) Total phosphate
b) Inorganic phosphate
c) Organic phosphate
d) Plant-available or exchangeable phosphate
Briefly explain these phosphates?

2) Several substances can interfere with the phosphorus analysis. Explain briefly.

3) Some soft drinks contains higher amount of phosphates, discuss the potential health effects of
consuming soft drinks.