Index >> Rhizobium and Legume Root Nodulation >>Determination of Total Nitrogen by Kjeldahl Method

The biological material (about 1 g) is placed in a 250 ml Kjeldahl flask and digestion mixture is added to it. The digestion mixture consists of the following: 1 g of a well-ground mixture of 20 g CUSO₄ 5H₂0 and 1 g Se. To one part of this mixture 20 parts of anhydrous Na₂S0₄ or K2S0₄ are added. Five g of this catalyst mixture is added to the digestion flask along with 5 ml of mercuric sulphate solution (12 ml concentrated H₂SO₄ in 100 ml water in which 10 g red ,mercuric oxide is dissolved). The Na₂S0₄ raises the boiling temperature of H₂SO₄, CUSO₄ and Se, accelerates the rate of digestion while the mercuric salt helps in the digestion of methylamines and prevents loss of N₂ which may occur if only Se is present. Fifteen ml concentrated H₂SO₄ and a few glass beads are then added. The digestion flasks heated at first gently until all the water is removed and charring is completed. The heat is then gradually increased so that the solution is brought to constant boiling with slight bubbling.

Hoskins steam distillation apparatus is commonly used for distillation. Measured quantity of the digested material (10-25 ml, depending on the N₂ content of the material) is taken in the distillation flask and 10-15 ml of 40% NaOH added to the sample. The flask containing sulphuric acid and indicator solution is kept under the condenser of the distillation apparatus. Toshiro's indicator (0.25 g of methylene blue, 0.375 g of methyl red and 300 ml of 95% ethanol) or a mixed indicator (0.099 g bromocresol green and 0.066 g of methyl red in 100 ml of ethanol) could be used advantageously. Heating should be carefully regulated to prevent sucking back of the sulphuric acid.

After sufficient distillate has been collected, the sulphuric acid is back titrated with standard 0.05 N NaOH (1 ml of 0.05 N H₂S0₂ = 0.7 mg ammonium N). The colour changes from green to pink or purple depending on the indicator.