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Soil and Plant Phosphorus Protocol

Authors

Christopher Buyarski and Sandra Brovold

Updated

19 September 2018

Goals

Measure phosphorus from dried, soil and plant samples through sulfuric acid digestion.

Reagents

  1. 10 N H2SO4: In a clean 500 mL volumetric flask, add ~200 mL Nanopure, place in an ice bath then add 138.9 mL H2SO4 and fill to 500 mL with Nanopure. Always add acid to water that is in an ice bath

  2. MOLYBDATE REAGENT: In a clean 1 L volumetric flask dissolve 0.208 g Antimony Potassium - Tartrate and 9.6 g Ammonium Heptamolybdate 4-hydrate in Nanopure. Fill to 1 L.

  3. ASCORBIC ACID: 20 g/L (1g/50mL) Make fresh daily

  4. APPLE STANDARD = 0.159% P

Template

Phosphorus Template

Method

  1. Use acid soaked glassware for all steps. This includes:

    • 4 beakers to hold reagents and Nanopure
    • Pipette tips
    • Borosilcate test tubes. Weigh the clean, dry, and empty test tubes. Record the weight. Including standards and blanks
    • Test tube caps if using Autoclave (see step 6)
    • 1cm Glass or Quartz cuvette
    • 15 mL Falcon centrifuge tubes
    • APPLE NIST 1515
    • RAL Check soil Standard (for soils)

  2. Pre-weigh samples and standards.

    • APPLE STANDARD (NIST 1515) can be placed in an aluminum foil boat and dried in 60°C oven overnight. Let cool to room temperature in desiccator before weighing.
    • WEIGHING-- The first step is to weigh/record all of the test tubes. Then place a small weigh tin on the microbalance and tare scale. For the standards (APPLE NIST 1515), weigh out 8-10 different weights in the range of 0.2 to 9 mg and place it in the bottom of test tubes. For samples, weigh out between 3-5 mg depending on how much P are in your samples. After samples are weighed and placed into tubes, reweigh/record tins to see how much residual sample remains.

  3. Ash samples, standards, and blanks in muffle furnace at 300°C for 30 minutes then at 550°C for 2 hours. Remove from furnace and let cool to room temperature before continuing. (ash in borosilcate tubes only e.g.- no lids.)

  4. Add 0.4 mL 10 N H2SO4. Vortex. Make sure tubes get mixed all the way to the bottom.

  5. Add 5 mL Nanopure. Vortex.

  6. Heat samples in water bath at 100°C for 30 minutes (no lids). Remove from heat and allow samples to cool to room temperature. (~ 30 minutes) Or autoclave CAPPED samples on the liquid cycle at 121°C with an exposure time of 30 minutes.

  7. Add 1 mL molybdate reagent. Vortex.

  8. Add 0.4 mL ascorbic acid solution. Vortex.

  9. Fill tubes to 10 mL with Nanopure. (e.g.- add 3.2 mL of Nanopure) Vortex. Weigh tubes containing sample/standards plus all liquids and record weight. The difference between empty vials and full vials will give you the exact volume.

  10. Measure at 880 nm after 1 hour, but within 3 hours. Currently using Cary 50 Bio UV-Visible spectrophotometer with 1 cm cells on fourth floor labs (Finlay and Cotner).

**FOR SOIL SAMPLES 10. Pour liquid into 15ml Falcon Centrifuge tubes.

**FOR SOIL SAMPLES 11. Centrifuge Falcon tubes at 2500 rpm’s for 10 mins

Hazardous Waste Handling

Sulfuric acid: 0.443% Antimony potassium tartrate: 0.0021% Ammonium molybdate: 0.096% Ascorbic acid: 0.19% Water: 99.27%

Calculations

% P dried weight calculations: \({(concentration\;of\;sample\;(abs)\;*\;final\;volume\;of\;sample\;in\;L\;*\;mass\;of\;P\;(31)) \over (dried\;weight\;of\;sample\;*\;1000))} * 100\)

For example:

  • 0.0359 mg sample gives 1.594 uM conc. when filled to 10 mL.
  • \(Percent\;P\;=\;{((1.594*0.01*31) \over ((0.0359*1000)}*100 = 1.376\;Percent\)
Last update: October 20, 2022