What is the Kjeldahl method?

2026-07-30 — Equipo JP Selecta

The Kjeldahl method is a standard quantitative chemical technique used worldwide to determine the organic and inorganic nitrogen content of biological, food and industrial substances.

Developed in 1883 by the Danish chemist Johan Kjeldahl, this analytical procedure has established itself as the official reference method for estimating crude protein concentration across a wide variety of organic matrices.

The fundamentals of the Kjeldahl method

The fundamentals of the Kjeldahl method consist of transforming the organic nitrogen present in the sample into ammonium through acid digestion. This is then converted into ammonia, distilled and quantified by acid-base titration.

First, the organic matter is broken down by digestion through heating at high temperature in the presence of concentrated sulphuric acid and metal catalysts. During this decomposition reaction, the organic nitrogen is reduced and trapped in the solution in the form of ammonium sulphate. The simplified chemical reaction of this process is expressed as follows:

Organic N + H2SO4 (heat, catalyst) -> (NH4)2SO4 + CO2 + SO2 + H2O

Next, the resulting mixture is made alkaline with a strong base to convert the ammonium ions into gaseous ammonia, which is carried over by steam and collected in a receiving acid to be finally quantified by volumetry.

Stages of the Kjeldahl method

The complete analytical protocol is rigorously structured into three consecutive steps. Understanding the stages of the Kjeldahl method in detail is essential to guarantee full nitrogen recovery and to avoid losses through volatilisation or incomplete digestion.

1. Digestion

The first stage of the Kjeldahl method consists of breaking down the organic matter of the sample using concentrated sulphuric acid and a mixture of catalysts, applying high temperatures.

During this process, carbon and hydrogen are transformed into carbon dioxide and water, while the organic nitrogen is retained in the form of ammonium sulphate. When digestion has finished, the solution takes on a transparent or slightly greenish appearance, a sign that the sample has been correctly broken down.

2. Distillation

Once digestion is complete, the solution is made alkaline with sodium hydroxide to transform the ammonium into gaseous ammonia. By means of a steam current, this ammonia is carried over to a vessel containing a boric acid solution, where it is retained for subsequent quantification.

3. Titration

In the final stage of the Kjeldahl method, the collected ammonia is determined by acid-base titration using a solution of known concentration. The volume of acid consumed makes it possible to calculate the nitrogen present in the sample and then to estimate its protein content using the corresponding conversion factor.

Calculating nitrogen and protein content

Once titration is complete, the volume of acid consumed is used to calculate the percentage of nitrogen present in the sample, correcting the result by means of a blank test. From this value, the crude protein content is estimated by applying a conversion factor, usually 6.25 for most foods, although specific factors exist for products such as milk, cereals or soya.

This calculation makes the Kjeldahl method one of the reference techniques for determining protein content in food, animal feed and other biological samples.

%N = [(V - Vb) x N x 1.4007] / m

Where:

• V: Volume of acid titration solution consumed by the sample (mL).
• Vb: Volume of acid solution consumed in the blank test (mL).
• N: Exact normality of the titrating acid (eq/L).
• 1.4007: Equivalent weight of nitrogen (14.007 g/mol) adjusted to give the result as a percentage.
• m: Mass of the analysed sample expressed in grams (g).

However, because of variations in amino acid composition between raw materials, standardisation bodies set specific factors by matrix:

• Meat and meat products: 6.25
• Milk and dairy products: 6.38
• Wheat and cereals: 5.70
• Nuts and seeds: 5.30 – 5.40
• Soya and pulses: 5.71

What materials are needed to carry out the Kjeldahl method?

Performing the full Kjeldahl analytical technique requires suitable instrumental infrastructure and analytical grade chemical reagents:

• Kjeldahl digestion tubes: high thermal resistance borosilicate glass vessels (Micro 100 mL or Macro 250 mL formats).
• Thermostatic digestion block: aluminium heating module capable of reaching 400 °C, coupled to a fume manifold and an acid gas scrubber.
• Steam distillation unit: programmable distiller for the safe addition of caustic reagents and steam generation.
• Digital burette or automatic titrator: for the exact dosing of the standard volumetric solution.
• Analytical reagents: concentrated sulphuric acid (H2SO4), sodium hydroxide (NaOH at 30–40%), boric acid (H3BO3), catalyst mixtures (CuSO4/K2SO4) and pH indicators.

Applications of this analytical technique

The Kjeldahl procedure is the official methodology required by health inspection and quality control regulations across several sectors:

• Human food and animal feed: control of nutritional labelling for protein content in flours, meats, whey products and animal feed.
• Agriculture and environment: determination of organic and ammoniacal nitrogen in agricultural soils, fertilisers, manures and sewage sludge.
• Pharmaceutical industry: quantification of nitrogen in raw materials, amino acid preparations and biotechnology products.
• International reference standards: governed by consolidated standards such as AOAC 2001.11 (food), ISO 8968-1 (dairy) and ISO 5983-2 (animal feed).

The advantages and limitations of Kjeldahl

Like any laboratory test procedure, the Kjeldahl method has significant analytical strengths, but also operational constraints that must be assessed:

Advantages

• Maximum precision and reproducibility: it achieves nitrogen recovery rates above 99.5%.
• Official reference method: it is the undisputed legal standard against which indirect rapid methods (such as NIR spectroscopy) are calibrated.
• High applicability: it can process liquid, solid or viscous samples regardless of their biological origin.

Limitations

• Lack of specificity (non-protein nitrogen): it does not distinguish between truly protein nitrogen and non-protein nitrogen (NPN) from urea, nitrates, nitrites or synthetic contaminants.
• Handling of corrosive reagents: it requires handling concentrated acids and boiling caustic soda, which demands appropriate protection systems.
• Time per test: although distillation is completed in minutes, the prior digestion stage takes between 1 and 2 hours.

To significantly reduce working times and minimise the risks of chemical handling, modern laboratories turn to automatic distillers such as the Kjeldahl Distillers catalogue with the Pro-Nitro range from J.P. Selecta.

Models such as the Pro-Nitro M (with automatic NaOH dosing), the Pro-Nitro S (semi-automatic with automatic sample emptying) or the Pro-Nitro A (which performs "on-line" colorimetric titration in real time simultaneously with distillation) optimise laboratory performance, guaranteeing maximum nitrogen recovery under strict traceability protocols and reports compatible with IQ/OQ standards and GLP inspections.