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Plant Phosphorus Protocol - Sulfuric acid digestion

Megan Ogdahl , Christopher Buyarski
Contributors : Jennifer.Funk262 points 

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Author: Christopher Buyarski

Co-Author:  Sandra Brovold

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

Units, terms, definitions


  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 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 0.1 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 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 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

Other resources

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.

Links to resources and suppliers

Literature references

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.

Contributors to this page: Admin36802 points  , Admin26202 points  and Jennifer.Funk262 points  .
Page last modified on Sunday 05 of May, 2013 22:42:18 EST by Admin36802 points . (Version 11)