Plant Phosphorus Protocol – Sulfuric acid digestion




Christopher Buyarski, Sandra Brovold

Author affiliations

University of Minnesota, Hobbie Lab


Method for Phosphorous determination using a sulfuric acid digestion. NB. Treat this entire procedure as you would any hazardous waste creating procedure (see Health and safety notice below).


For phosphorus in a dried sample


  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

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


  1. Use acid soaked glassware for all steps.
  2. Pre-weigh samples and standards.
    • APPLE STANDARD (NIST 1515) can be placed in an aluminum foil boat and dried in 60C 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 0.1 to 6 mg and place it in the bottom of test tubes. For samples, weigh out between 1-4 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 300C for 30 minutes then at 550C 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 100C 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 121C 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 Shimadzu UV1600A spectrophotometer with 1 cm cells.


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

Water: 99.27NaV P dried weight calculations: (concentration of sample (abs) x final vol. of sample in L x mass of P (31)) / (dried weight of sample x 1000)) x 100

i.e. 0.0359 mg sample gives 1.594 uM conc. when filled to 10 mL.

% P = ((1.594*0.01*31) / ((0.0359*1000)*100 = 1.376

Notes and troubleshooting tips

Reference materials:

  1. NIST #8414- bovine muscle powder (0.836 % P). Used when running insects.
  2. NIST #1515- apple leaves (0.159%P). Used when running leaves.
  • Standard methods for the examination of water and wastewater. 20th edition. Prepared and Published jointly by American Public Health Association, American Water Works Association, Water Environment Federation. 1998.
  • Franson, M.A.H. managing editor. Persulfate Digestion Method and Ascorbic Acid Method for Phosphorus determination. APHA.

Note: All reagents should be kept in the fridge except for Ascorbic acid which should be made fresh daily.

Health, safety & hazardous waste disposal considerations

Treat this entire procedure as you would any hazardous waste creating procedure. Persulfate, Sulfuric Acid, Phenolphthalein, Sodium Hydroxide, and the color reagent are all hazardous.

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