Estimation of Nitrogen by Kjeldahl Method | Plants

In this article we will discuss about the Kjeldahl method for the estimation of nitrogen in plants.

Principle:

Digestion of nitrogenous compounds by concentrated sulphuric acid in presence of catalysts (reduced iron powder, and a mixture of CuSO₄ and K₂SO₄) produces ammonium compound. Further, such ammo­nium compound when distilled by steam in presence of strong alkali (40% NaOH soln.) produces ammonia which can be condensed in liquid form (NH₄OH) and absorbed by N/50 HCl.

The titrimetric estimation of residual N/50 HCl by N/50 NaOH soln. gives the data about the amount of N/50 HCl consumed by liquid ammonia produced during distillation of desired amount of digested nitrogenous materials. The percentage of nitrogen can thus be obtained by the known data i.e. 1 ml of N/50 HCl = 0.28 mg of nitrogen.

The reactions are:

Requirements:

(a) Reagent:

1. Reduced iron powder.

2. Mixture of K₂SO₄ and CuSO₄ (1: 3).

3. Cone. H₂SO₄ and 50% H₂SO₄.

4. NaOH Soln. 40% (400 gms. NaOH in 1,000 ml dist. water).

5. N/50 NaOH Soln. (0.8 gms NaOH/lit).

6. N/50 HCl (1.8 ml conc. HCl/lit).

7. Weslow’s indicator (prepared by mixture of methyl red and methylene blue).

(b) Apparatus:

Microkjeldahl’s flask, distillating apparatus, conical flask, pipette, burette etc.

(c) Sample solution preparation:

Nitrogenous salts are soluble in water, thus soln. of respective salts can be used as sample solution for digestion.

Soil or plant materials (ash or dry materials in the form of powder) can be directly used for digestion to estimate the total nitrogen content.

Test Procedure:

(a) Digestion of material:

Sample solution or soil or powdered materials are digested by dilute H₂SO₄ and reduced iron powder over low flame for about an hour in digestion flask (1st step of digestion).

Then the digestion flasks are cooled and 2 ml of conc. H₂SO₄ along with a pinch of K₂SO₄: CuSO₄ mixture (1: 3) is added. The flasks are heated over high flame for about 3-4 hours until the sample becomes clear. 3-5 sets of each sample are digested in this manner. Soil samples, however, require pro­longed high temp, digestion for a period of about 4-6 hour duration.

This digestion method is applicable to amino and amide containing nitrogenous compounds and also to many heterocyclic nitrogenous compounds, but it fails in case of compounds containing N — N, NO and NO₂ groups. Thus, it is necessary to reduce such compounds by reducing agents before digestion. Satisfactory results can often be obtained by adding glucose soln. to the digestion mixture.

(b) Distillation:

After digestion the flasks are cooled and then transferred entirely to the distillation flask separately. The digestion flask is also washed by distilled water at least thrice and the contents transferred to the distillation flask. Then 20 ml of 40% NaOH soln. is added to each distillation flask. Thereafter, a continuous steam is passed through the distillation flask for about 15 mins.

The distilled material is passed through condenser and finally absorbed in 10 ml of N/50 HC1 solution. After distilla­tion is over, the HC1 containing flask is removed and 2-3 drops of Weslow’s indicator added before titration against N/50 NaOH soln. to estimate the residual acid content.

Meanwhile the distillation flask is cooled by ice water and the residual content is refluxed back to the collecting chamber after cutting off the steam source. The distillation flask is also washed twice by water in the same manner.

(c) Titration:

The residual acid of the N/50 HCl containing flask which receives the distilled content of nitrogenous material is estimated by N/50 NaOH soln. using Weslow’s indicator. The volume of N/50 NaOH soln. required to titrate 10 ml N/50 HC1 is also estimated in the control set.

(d) Finally, nitrogen contents are calculated on the basis of titration values and nitrogen equiva­lence of HCl as stated earlier.