1 of 5 PROTOCOL FOR P FRACTIONATION KUZYAKOV LAB 1 20.09.2016 Deejay Maranguit Yakov Kuzyakov Rev. Date Prep. Modify. Check. Approv.
2 of 5 Equipment and Materials 1. 50ml centrifuge tubes with screw caps and refrigerated high-speed centrifuge 2. Shaker, preferably overhead types so that soils do not clump together in the round bottom of the centrifuge tubes 3. Autoclave 4. Plastic vials for storing extracts 5. Whatman no. 40 filter paper Extracting Solutions 1. 0.5 M HCl: dilute 88.5ml conc. HCl to 2L with deionized H 2 O 2. 0.5.M NaHCO 3 (ph8.5): dissolve 84g NaHCO 3 + 1g NaOH in deionized H 2 O and make to 2L 3. 0.1 M NaOH: dissolve 4g NaOH in deionized H 2 O and bring to final volume to 1L 4. 1 M HCl: add 177ml conc. HCl (11.3M) to about 500ml of deionized H 2 O and bring final volume to 2L 5. CHCl 3 6. H 2 O 2 : 30% hydrogen peroxide 7. Concentrated H 2 SO 4 (18M) Extraction Procedure Day1: Weigh duplicate samples 1g soil into a 50ml centrifuge tube Label the sample as sample A & B. Add 30 ml deionized water, and shake overnight (16h, and 30 rpm if using overhead shaker). Day2: Centrifuge soil suspension at 3500 rmp for 15 min. Filter supernatant using whatman no. 42 into a clean vial. Add 30ml NaHCO 3 solution to the sample and shake suspension overnight (16h). Cap the tubes and resuspend soil by handshaking before putting on the mechanical shaker. (sample B) To the other sample, add 1ml CHCl 3, recap tube leave 1h with shaking. Then evaporate CHCl 3 overnight. (sample A) Day3: Centrifuge soil suspension at 3500 rmp for 15min. Filter supernatant using whatman no.42 into a clean vial. Determine inorganic and total P on bicarbonate extract. Then discard the soil (sample B).
3 of 5 To the other sample, add 30ml NaHCO 3 solution to the sample and shake suspension overnight (16h). Cap the tubes and resuspend soil by handshaking before putting on the mechanical shaker. (sample A) (For Sample A) Day4: Centrifuge soil suspension at 3500 rmp for 15 min. Filter supernatant using whatman no. 42 into a clean vial. Determine inorganic and total P on bicarbonate extract. Wash any soil off filter back into the tube using a little 0.1M NaOH. Make volume of NaOH solution to 30ml and shake suspension overnight (16h). Day5: Centrifuge soil suspension at 3500 rmp for 15 min. Filter supernatant using whatman no. 42 into a clean vial. Determine inorganic and total P on NaOH extract. Wash any soil off filter back into the tube using a little 0.1M NaOH. Add 20ml of NaOH solution, centrifuge tube in ice bath or 0 o C and sonicate for 2min.Make to final volume up to 30ml of 0.1M NaOH. Shake overnight (16h). Day6: Centrifuge soil suspension at 3500 rmp for 15min. Filter supernatant using whatman no. 42 into a clean vial. Determine inorganic and total P on sonicated sample. Wash any soil off filter back into the tube using a little 1M HCl. Make volume of 1M HCl solution and shake suspension overnight (16h). Day7: Centrifuge soil suspension at 3500 rmp for 15min. Filter supernatant using whatman no. 42 into a clean vial. Determine inorganic and total P on HCl extract. Digest the soil residue. Add 10ml deionized water to soil residue and disperse the soil. Transfer suspension into 75ml digestion tubes using the minimum amount of water possible to transfer all soil residues, add 5ml conc H 2 SO 4. Heat slowly to evaporate water and allow cooling to hand-warm. Add 0.5ml of 30% H 2 O 2, reheat for 30 mins, during which H 2 O 2 is used up. Repeat H 2 O 2 addition until liquid is clear (usually 10 times). Make sure there is adequate heating after the final H 2 O 2 addition, since residual H 2 O 2 interferes with the P determination. Cool, make to volume (i.e. 25ml), shake and filter into clean vials. Determine P in solution.
4 of 5 Analysis of P in the Extracts Reagents for P Determination 1. Ammonium molybdate: dissolve 40 g ammonium molybdate in deionized H 2 O 2 and bring to volume of 1L. 2. Ascorbic acid: dissolve 26.4 g L-ascorbic acid in deionized H 2 O and bring to a final volume of 0.5L. 3. Antimony potassium tartrate: dissolve 1.454 g antimony potassium tartrate in deionized water and bring to a final volume of 0.5 L. 4. 2.5 M H 2 SO 4 : slowly add 278ml conc. H 2 SO 4 to 1 L of deionized water and bring to final volume of 2L. 5. Color developing reagent: to 250ml 2.5M H 2 SO 4, add 75ml ammonium molybdate solution, then 50 ml ascorbate solution and finally 25 ml of antimony potassium tartrate A. Determination of Inorganic P in 0.5M NaHCO 3 and 0.1 M NaOH Extracts 1. Pipette 10ml solution into a 50ml centrifuge tube 2. Acidify to ph 1.5 and set in fridge for 30 min. a) To acidify 0.5 M NaHCO 3 extract use: ---6ml of 0.9 M H 2 SO 4 b) To acidify 0.1 M NaOH extract use:----- 1.6ml of 0.9 M H 2 SO 4 3. Centrifuge for 10 min at 0 0 C 4. Decant supernatant into a 25 ml vol. flask 5. Rinse tube carefully so as not to disturb the organic matter with a little acidified water and add to the solution in the flask (2 or 3 times) 6. Adjust ph and measure P by the Murphy and Riley method B. Determination of Total P in 0.5 M NaHCO 3, 0.1 M NaOH, and 1.0 M HCl extracts (EPA 1971) Dissolved organic matter is oxidized with ammonium persulfate before P analysis: 1. Pipette 5ml solution into a 25ml vol. flask
5 of 5 2. To 0.5 M NaHCO 3 and 0.1 M NaOH extract add 0.5g ammonium persulfate + 10ml 0.9 M H 2 SO 4 To 1M HCl extract add 0.5g ammonium persulfate + 10ml deionized water 3. Cover with tinfoil and autoclave for 1hr. 4. Cool down and adjust ph and measure P by Murphy and Riley method. ph adjustment and Measuring P by Murphy and Riley Method (1962) 1. Add 3 drops of 2,4-DNP indicator to the extract in the 25ml volumetric flask. a. If yellow, adjust ph by adding 0.25 M H 2 SO 4 dropwise until it goes clear. b. If clear, add 4 M NaOH until solution is yellow, then adjust ph by adding 0.25 M H 2 SO 4 dropwise until it goes clear. c. Add 4ml of reagent B, make to volume with deionized water and mix well d. Allow color to develop for 15 minutes before reading absorbance. References: Chacon, N., Dezzeo, N., 2004. Phosphorus fractions and sorption processes in soil samples taken in a forest-savanna sequence of the Gran Sabana in Southern Venezuela. Biol. Fertil. Soils 40, 14-19. Hedley, M.J., Stewart, J.W.B., Chauhan, B.S., 1982. Changes inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Sci. Soc. Am. J. 46, 970 976. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31-36. Tiessen, H., Stewart, J.W.B., Cole, C.V., 1984. Pathways of phosphorus transformations in soil of differing pedogenesis. Soil Sci. Soc. Am. J. 48, 853 858. Tiessen, H., Moir, J.O., 1993. Characterization of available P by sequential extraction, in: Carter, M.R., (Eds.), Soil sampling and methods of analysis. Canadian Soc. Soil Sci., pp. 75-86.