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DETERMINATION OF PHOSPHOROUS IN SOIL

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PHOSPHOROUS DETERMINATION IN SOIL BY VARIOUS METHODS

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DETERMINATION OF PHOSPHOROUS IN SOIL

  1. 1. 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
  2. 2.  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.
  3. 3.  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.
  4. 4. 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.
  5. 5. • 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.
  6. 6. 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).
  7. 7. 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.
  8. 8. 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%)
  9. 9. 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.
  10. 10. 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.
  11. 11. 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.
  12. 12.  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.
  13. 13. Reagents:  Sodium bi carbonate (0.5N) of 8.5 pH  Ammonium molybdate (1.5%)  Darco –G 60  Stannous chloride stock solution
  14. 14. 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.
  15. 15. 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.
  16. 16. 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.
  17. 17. 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.
  18. 18. 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.
  19. 19. 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.
  20. 20. 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
  21. 21. SLOPE= Y/X Graph ppm=slope x conc. (spectro photometer reading) Preparation of standard curve
  22. 22. 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

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