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Quantiative analysis
Kjeldahl Method

Nitrogen is one of the five major elements found in organic materials such as protein. This fact was recognized by a Danish chemist, Johan Kjeldahl, who used it as a method of determining the amount of protein in samples taken from a wide variety of organisms. In 1883 Kjeldahl presented to the Danish Chemical Society a method (much revised since his day) for determining the amount of nitrogen in mixtures of substances containing ammonium salts, nitrate, or organic nitrogen compounds.

The central basis used in this procedure is the oxidation of the organic compound using strong sulfuric acid. As the organic material is oxidized the carbon it contains is converted to carbon dioxide and the hydrogen is converted into water.

The nitrogen, from the amine groups found in the peptide bonds of the polypeptide chains, is converted to ammonium ion, which dissolves in the oxidizing solution, and can later be converted to ammonia gas.

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

A three step procedure

The Kjeldahl method consists of three steps, which have to be carefully carried out in sequence:

  1. the sample is first digested in strong sulfuric acid in the presence of a catalyst, which helps in the conversion of the amine nitrogen to ammonium ions,

  2. the ammonium ions are then converted into ammonia gas, heated and distilled. The ammonia gas is led into a trapping solution where it dissolves and becomes an ammonium ion once again,

  3. finally the amount of the ammonia that has been trapped is determined by titration with a standard solution, and a calculation made.

Step One: Digestion of the Sample

This is the most time-consuming step in the analysis. The purpose of this step is to break down the bonds that hold the polypeptides together, and convert them to simpler chemicals such as water, carbon dioxide and, of course, ammonia.

Such reactions can be considerably speeded up by the presence of a catalyst and by a neutral substance, such as potassium sulfate (K2SO4), which raises the boiling point of the digesting acid and thus the temperature of the reaction.

Catalysts are also used to help in the digestion process; many different one have been tried including selenium, mercury, copper, or ions of mercury or copper.

Digestion is accomplished by:

  1. Weighing out approximately 1 gm of the sample containing protein, making a note of the weight, and placing the sample into a digestion flask, along with 12-15 ml of concentrated sulfuric acid (H2SO4).

  2. Adding seven grams of potassium sulfate and a catalyst, usually copper.

  3. Bringing the digestion tube/flask and mixture to a "rolling boil" (about 370oC to 400oC) using a heating a block.

  4. Heating the mixture in the tube/flask until white fumes can be seen, and then continuing the heating for about 60-90 mins.

  5. Cooling the tube/flask and cautiously adding 250 mls of water.

Step Two: Distillation

The purpose of the next step, distillation, is to separate the ammonia (that is, the nitrogen) from the digestion mixture. This is done by,

  1. raising the pH of the mixture using sodium hydroxide (45% 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.

  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 about 15 ml HCl (hydrochloric acid) in 70 ml of water.

  3. removing the trapping flask and rinsing the condenser with water so as to make sure that all the ammonia has been dissolved.

Step Three: Titration

As the ammonia dissolves in the acid trapping solution, it neutralizes some of the HCl it finds there. What acid is left can then be "back titrated", that is titrated with a standard, known solution of base (usually NaOH). In this way the amount of ammonia distilled off from the digestive solution can be calculated, and hence the amount of nitrogen in the protein determined.

The quantities of acid, and hence ammonia are determined by,

  1. adding an indicator dye to the acid/ammonia trapping solution. This dye should turn a strong color, indicating that a significant amount of the original trapping acid is still present.

  2. putting a standard solution of NaOH (sodium hydroxide) into the buret (a long tube with a tap at the end), and slowly, slowly adding small amounts of the sodium hydroxide solution to the acid solution with the dye.

  3. watching for the point at which the dye turns orange, indicating that the "endpoint" has been reached and that now all the acid has been neutralized by the base.

  4. recording the volume of the neutralizing base (sodium hydroxide solution) that was necessary to reach the endoint.

  5. performing a calculation to find the amount of ammonia, and thus nitrogen, that came from the original sample.


One mole of ammonia coming from the digestion mixture (and hence from the original protein) will neutralize exactly one mole of the acid in the trapping flask.

The first calculation, therefore, is to find the number of moles of ammonia that have been produced and then trapped from your sample(s).

This is done by,

  • calculating the number of moles of acid in the trapping flask originally (before any ammonia was trapped) by multiplying the molarity of the acid solution by the volume of the trapping solution
    moles of acid =
    molarity of acid x volume used in flask
    (molesA = M x V)

  • calculating the number of moles of base (NaOH) that were added from the buret to neutralize the remaining acid (that NOT neutralized by the ammonia).
    moles of base =
    molarity of base x volume added from buret
    (molesB = M x V)

  • subtracting the "moles of base" added from the "moles of acid" present at the beginning, to get,
  • the number of "moles of ammonia" coming from the protein,
  • the number of "moles of ammonia" is the same as the "moles of nitrogen",
  • so ... to calculate the number of grams of nitrogen in the original sample of protein, multiply the "moles of nitrogen" by the atomic mass of nitrogen (mass of atoms of nitrogen),
    gms nitrogen =
    moles nitrogen x atomic mass

    (gN = molesN x 14.0067)

It is also possible to calculate the amount of crude protein in the sample. Although there are differences between different samples, the amount of "crude protein" (CP) can be found by multipling the percent Nitrogen by a factor (usually 6.25).

CP = %N x 6.25

percent Nitrogen

The percentage of nitrogen found in the orginal sample can now be calculated by:

%nitrogen =
(gms nitrogen / gms sample) x 100

%N = (gN / gS) x 100

Science@a Distance
© 2003, Professor John Blamire