Kjeldahl Method

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This method was specifically developed by Danish chemist, Johan G.C.T. Kjeldahl, in 1883 to determine the nitrogen contents in organic and inorganic substances  (like foodstuffs, fertilizers, wastewater, soil, feed, grain, and other substances). This method is also used for estimating the protein content in food.

Kjeldahl Method Principle

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The Kjeldahl method is mainly divided into three main steps. This method needs to be carried out in proper sequence. The sequences are as follows:

1. Digestion: 

In this process, a certain substance or sample is heated in the presence of concentrated sulphuric acid. The acid breaks down the organic substance by the process of oxidation and reduced nitrogen is liberated in the form of ammonium sulfate. Catalysts like copper, mercury, selenium, or ions of copper or mercury are also used in the process of digestion. The sample is said to be fully decomposed when we obtain a clear and colorless solution. (Note: Potassium sulfate is added to increase the boiling point of the medium).

2. Distillation

In this process, a very small quantity of sodium hydroxide is added to convert the ammonium salt to ammonia. The distilled vapors are then trapped in a special trapping solution of HCl (hydrochloric acid) and water.

3. Titration: 

The third step is the quantification of Ammonia. The amount of ammonia or the amount of nitrogen present in the sample is determined by the process of titration. 

Kjeldahl Method Reaction


Organic (C, H, N) + H₂SO₄ → [digest] Cu²⁺ + (NH₄)₂SO₄


(NH₄)₂SO₄ + 2NaOH → Na₂SO₄ + 2H₂O + 2NH₃

And, NH₃ + HCl → NH₄Cl


B(OH)₂ + H₂O + Na₂CO₃ → NaHCO₃ + CO₂ + H₂O 

Note: Same steps are involved for protein estimation by the Kjeldahl method.

Kjeldahl Method Formula

The equivalent weight of NH₃ is 17g/eq. And, 14 gm of Nitrogen is contained one equivalent weight of NH₃. So, the percentage of nitrogen can be determined using the following formula:

Kjeldahl method of nitrogen estimation =

\[\frac{1.4 V \times N}{W}\]


W = Weight of the sample used (in grams)

V = Acid used in titration (in ml)

N = Normality of standard acid

Limitations of Kjeldahl Method

This method measures only nitrogen bound to organic components (proteins, amino acids, nucleic acids) and ammonium in the sample. This method is not suitable for compounds containing nitrogen in azo and nitro groups or in rings (quinoline, pyridine, nitrate, and nitrite, etc). In these compounds, the nitrogen cannot be converted to ammonium sulfate following the Kjeldahl method.

Did you know?

Protein quantification (total protein content in a sample) is necessary to understand the total protein content in a formulated product. Accurate protein quantification is important because a range of other evaluations require precise total protein content results in order to generate accurate data.

FAQ (Frequently Asked Questions)

Question: What Does the Kjeldahl Method Measure?

Answer: Kjeldahl method is used for the quantitative determination of nitrogen contained in organic substances and the nitrogen contained in inorganic compounds like ammonium, ammonia.

Question: What is the Kjeldahl Flask?

Answer:  It is a round bottom flask with long wide necks that are used in the Kjeldahl method for the quantitative determination of a sample of nitrogen content. They are manufactured from borosilicate glass, which is resistant to heat and chemicals.

Question: During the Estimation of Nitrogen Present in a Compound by Kjeldahl Method Calculation, the Ammonia Evolved from 0.5 g of the Compound in Kjeldahl's Estimation of Nitrogen Neutralized 10 mL of 1M H₂SO₄. Find Out the Percentage of Nitrogen Present in the Compound.

Answer: 10 ml, 1M H₂SO₄ corresponds to 20 ml, 1M NH₃.

⇒1000 ml of 1 M ammonia contains 14 g of nitrogen.

Hence, 20 ml of 1 M ammonia will contain (14 х 20)/1000 g of Nitrogen

⇒0.5 g of organic compound contains 14 х 20)/1000 g of Nitrogen.

Hence, 100 g of the compound will contain

(14 х 100 х 20) / (0.5 х 1000) = 56% Nitrogen.