Determination of Nitrogen Content Percentage by Kjeldahl Method

The Kjeldahl method of nitrogen determination is the worldwide standard for calculating the protein content in a wide variety of materials ranging from human and animal food, fertilizer, and fossil fuels.

Definition: The protein content as determined by the procedure described multiplied by 6.38 and expressed as a percentage by mass.

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Kjeldahl Method Principle

The sample is digested in concentrated sulphuric acid and a digestion mixture, using a digestion block system. Salt and catalysts are added to speed up the conversion of organic nitrogen to ammonia sulfate. 

An excess of sodium hydroxide is added to the cooled digest to liberate ammonia. The liberated ammonia is trapped in a weak boric acid solution and titrated with standard hydrochloric acid. The nitrogen content is calculated from the amount of determined ammonia.

Reagents and Apparatus

  •  Potassium sulfate, K2SO4+Se tablets (2 for each digestion tube)
  •  Concentrated H2SO4 (in the hood)
  •  35 % (w/v) NaOH(99.9%) purity for each digestion tube (already prepared).
  •  4.0 % (w/v) Boric acid, H3BO3 (already prepared)
  •  0.1 M HCl (already prepared, exact concentration will be given)
  •  Methyl orange (in droppers)
  •  Erlenmeyer flasks
  •  Burette
  •  Digestion tubes
  •  Milk: Do not forget to bring any brand of milk or casein power.

Nitrogen Content Procedure

Digestion is the decomposition of nitrogen in organic samples utilizing a concentrated acid solution. This is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution. The general equation for the digestion of an organic sample is shown below:

Protein + H2SO4 → (NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g) 

Sulfuric acid has been used alone for the digestion of organic samples. The amount of acid required is influenced by sample size and the relative amount of carbon and hydrogen in the sample, as well as the amount of nitrogen. A very fatty sample consumes more acid. 

1) Sample: 5.0 mL fresh cow’s milk in a digestion tube. Weight exactly 0.2 to 0.5 gm of WPC powder sample into the protein digestion tube. Add 10gm digestion mixture ( potassium sulfate and Copper sulfate). Add 25 ml conc. sulphuric acid. Place the digestion tube in the digester assembly and select heating programs so that the powder sample is digested completely.

2) Reagents for digestion: to each milk sample and also to an empty digestion tube (blank) add the following:

  • 2 tablets of K2SO4 + Se catalyst
  • 10.0 mL of concentrated H2SO4 (98%)

3) Digestion: heat for ca. 30 minutes at 420ºC.

ATTENTION: Do not inhale the gases that evolve in reaction 1.

4) Cooling and diluting: let the digestion tubes cool to 50-60 ºC and add to each 50 mL of distilled water.

ATTENTION: Let the tubes stand in the air to cool, cold water may break the tubes.


Distillation is adding excess base to the acid digestion mixture to convert NH4+ to NH3, followed by boiling and condensation of the NH3 gas in a receiving solution. This is accomplished by.

Raise the pH of the mixture using sodium hydroxide (NaOH solution). This has the effect of changing the ammonium (NH4+) ions (which are dissolved in the liquid) to ammonia (NH3), which is a gas. 

(NH4)2SO4(aq) + 2NaOH → Na2SO4(aq) + 2H2O(l) + 2NH3(g) (2)

Separating the nitrogen away from the digestion mixture by distilling the ammonia (converting it to a volatile gas, by raising the temperature to boiling point) and then trapping the distilled vapors in a special trapping solution of boric acid (H3BO3). The ammonia is bound to the boric acid in the form of the ammonium borate complex.

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protein content by kjeldahl method

Titration Procedure

Place the digested samples in digestion tubes to the distilling unit and add 50.0 mL of 35% (w/v) NaOH.

Sample: The sample is distilled until 100 mL of distillate is collected in 25.0 mL of 4.0 % (w/v) boric acid. Add 2-3 drops of mixed indicator to the Erlenmeyer flask and titrate it with 0.1 M HCl. Calculate the amount of protein (% protein) and compare the result with the value given on the milk or WPC powder.

Blank: Carry out blank determination with sucrose (0.5gm) instead of the test protein using the material in the same quantities.

protein determination


                                     Burette sample reading - Burette blank reading X 0.8932X100
Protein % (ODB) =

                                            Weight of sample (100 - Moisture/100)

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