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Plant Chemical Analyses

ASL offers the following analysis for plant

  1. Nitrogen by Kjeldahl Method
  2. Modified ASL Procedure (MAP) Nitric/Perchloric Acid Digestion for ICP analysis of Na, K, Ca, Mg, P, Fe, Mn, S, Cu, Zn, Al, and Mo


Nitrogen by Kjeldahl Method

The determination of total organic nitrogen is carried out by the Kjeldahl digestion method. The ammonia produced in the reaction is determined colorimetrically as indo-phenol blue on the Technicon Autoanalyzer.
This procedure involves the digestion of plant material with a mixture of potassium sulphate and concentrated sulphuric acid, with finely powdered selenium metal as the catalyst.

Potassium sulphate
This is present in order to raise the boiling point of concentrated sulphuric acid from 330OC to around 380OC, making the reaction proceed faster. If too much potassium sulphate (K2SO4) is added, the boiling point may be raised above 410OC, resulting in the loss of nitrogen.

The amount of K2SO4 normally used is 1g per 2mL (3.70g) of concentrated sulphuric acid (H2SO4). At this concentration the mixture boils at around 380OC.

Sulphuric acid
This is used in two ways. Firstly, it absorbs the ammonia gas produced, forming ammonium sulphate. Secondly, it acts as a source of oxygen for oxidation of organic matter.

The following reaction takes place:

C + 2H2SO4 --> CO2 + 2H2O + 2SO2

For every 12 mg of carbon oxidized 98 mg of sulphuric acid are used up. In any normal reaction involving 50mg of plant material containing an average 40% carbon, then the amount of sulphuric acid used up will be:

50 x 40/100 x 98/12 = 163 mg

Since 2 mL of concentrated sulphuric acid of specific gravity 1.85 g/mL are used in a normal digest, then the oxidation of carbon will reduce this from 3.70 g to around 3.54 g. This reduction in the amount of sulphuric acid by 4% is not serious and simply causes the temperature of the reaction to rise a little as it nears completion, due to the increase in potassium sulphate to acid ratio.

Where samples are low in nitrogen so that much material has to be digested, the above fact must be borne in mind and sufficient additional sulphuric acid added to allow for the complete oxidation of all the carbonaceous matter, leaving at the end of the reaction a salt to acid ratio of 1:2 w/v.


  1. Heating blocks or open coil heaters (King type) suitable for digestion
  2. Thick walled Pyrex test-tubes, 150x19mm or 10mL Kjeldahl flasks calibrated at 20mL.
  3. Vortex cyclone mixer


  • Concentrated sulphuric acid
  • Potassium sulfate/selenium mixture

Colorimetric Determination of Nitrogen by Salicylate-Nitroprusside Method using AutoAnalyzer

  • Reagents

      To 600 mL distilled water, add 200 g of sodium hydroxide solution, 50% w/w. Cool to room temperature and dilute to one liter with distilled water.


      Dissolve 200 g of sodium potassium tartrate in about 600 ml of distilled water. Dilute to one liter with distilled water and mix thoroughly.


      Dissolve 134 g of sodium phosphate, dibasic, crystal in about 800 mL of distilled water. Add 40 g of 50% w/w sodium hydroxide solution, dilute to one liter with distilled water and mix thoroughly.


      Combine the reagents in the stated order: add 250 mL of stock 20% sodium potassium tartrate solution, to 200 mL of 0.5 M stock buffer solution with swirling. Slowly, with swirling, add 250 mL of 20% sodium hydroxide solution. Dilute to one liter with distilled water, add 1.0 mL of 30% Brij-35 solution and mix thoroughly.


      Dissolve 100 g of sodium chloride in about 600 ml of distilled water. Add 7.5 mL of sulfuric acid and dilute to one liter with distilled water. Add 1.0 mL of 30% Brij-35 (about 20 drops) and mix thoroughly.


      Dissolve 150 g of sodium salicylate and 0.30 g of sodium nitroprusside in about 600 ml of distilled water. Filter through fast filter paper into a one liter volumetric flask and dilute to volume with distilled water. Add 1.0 mL of 30% Brij-35 and mix thoroughly. Store in a light-resistant container.


      Dilute 6.0 mL of 5.25% sodium hypochlorite solution to 100 mL with distilled water. Add 0.1 mL (2 drops) of 30% Brij-35 and mix thoroughly. Prepare fresh daily. [Any commercial bleach solution (e.g. Clorox) containing 5.25% available chlorine is satisfactory.

  • Standardization

    • Standard nitrogen solution - 1000 ppm N

      Dry "Analar" grade ammonium sulfate at 105oC for 2 hr. Cool in a desiccator. Accurately weigh 4.7159 g and dissolve in distilled water contained in a 1000 mL volumetric flask. Add a few drops of concentrated sulfuric acid to preserve the solution. Make to volume with distilled water and shake well to mix.

    • Nitrogen working standards

      All standards are made up in 10% v/v sulfuric acid and 5% w/v potassium sulfate. Certain standards are used much more frequently than others and are therefore made up in greater volume. The following method should be used when making up working standards.

      Add the potassium sulfate into the required flask through a funnel. Wash any solid in the funnel into the flask with distilled water until the flask is about half full. Carefully add the required amount of concentrated sulfuric acid to the flask from the acid dispenser used for the nitrogen analyses, (1 shot = 2 mL acid). Set aside to cool. When cool add the correct volume of standard nitrogen solution, taking care that the correct standard is used. Use a bulb pipette throughout.

  1. Varley, J. A. (1966) The determination of nitrogen, phosphorus and potassium ion plant materials. Analyst. 91:119-126
  2. Technicon AutoAnalyzer II Industrial Method No. 334-74W/B. Release January 1976/Revised March 1977. Individual/Simultaneous Determination of Nitrogen and/or Phosphorus in BD Acid Digest.

Modified ASL Procedure (MAP) Nitric/Perchloric Acid Digestion for ICP analysis of Na, K, Ca, Mg, P, Fe, Mn, S, Cu, Zn, Al, and Mo

The combination of nitric acid and perchloric acid is used for the oxidation of plant samples. Nitric acid acts as primary oxidant for the destruction of organic matter. It reacts readily with both aromatic and aliphatic organic materials, giving rise to oxidation, esterification and nitration reactions which rapidly degrade to simple carboxylic acids. The normal concentrated nitric acid boils at ~120oC which limits its effectiveness in degrading more resistant material. With perchloric acid, it continues the oxidation after nitric acid has been removed and more resistant materials are degraded. Reagent grade perchloric acid (60-72%) when heated loses water and the oxidation potential increases as the temperature rises to 203oC, the boiling point of perchloric acid water azeotrope. The danger with perchloric acid arises when it is heated with hydroxyl compounds which give rise to the formation of unstable perchlorate esters that may cause explosion. Predigestion with nitric acid in addition to perchloric acid will give protection against explosion during the digestion process, provided that sufficient nitric acid is present, and the reaction is allowed to proceed for an adequate period.


  1. Analytical balance
  2. Prototype digestor with plastic baffle or AIM 500 Digestion Block System
  3. Culture tubes, 25 x 250mm
  4. Vortex mixer


  • 1:10 of HClO4:HNO3 --> mix acid for digestion (final volume = 1100 ml )

    Measure 100ml of 70% HClO4 and transfer to a 2.5L bottle. Add 1000ml of 70% HNO3. Mix well and transfer to the bottle labeled 1:10 of HClO4:HNO3 located under the perchloric hood (right side).

  • 1% HNO3--> acid for final dilution (final volume = 2000 ml )

    Measure 22.22 ml of 90% HNO3, transfer in a 2L volumetric flask and dilute to mark with deionized water. Mix well and transfer the solution to the teflon bottle labeled 1% HNO3.

  • 5% HNO3 --> for acid-washing of glasswares (final volume = 5000 ml)

    Measure 385 ml of 65% HNO3. Transfer to a pre-calibrated 5L container. Dilute to 5L mark with deionized H2O. This solution must be changed every 2 months.

  1. Fowles, Teresa (resource person). ICP Procedure in Waite Analytical Service Lab, Adelaide University
  2. Rutzke, Mike (resource person). SOP for HNO3/HClO4 Digestion in Cornell University (March 1999)
  3. Vaughan, Byron (resource person).SOP for HNO3/H2O2 Digestion in MDS Harris Laboratory Services (January 2002)
  4. Wet Ashing, Manual of Procedures, ASL-IRRI.
  5. Gorsuch,T.T. 1970. The destruction of organic matter, pp.21-23.Pergamon Press Ltd.,1st edition.UK

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