🍎 Total nitrogen and protein in agricultural food products — the Kjeldahl method (mineralisation, distillation, titration)

Food chemistry PN-75/A-04018

Overview

The Kjeldahl method, known since 1883 and little modified since then, is the most frequently used and the reference method of determining nitrogen in foodstuffs. It consists in the mineralisation of the product with concentrated sulfuric(VI) acid („wet"), the making alkaline of the solution, the distilling off and the quantitative determination of the ammonia formed. In this way total nitrogen is determined, in which, besides the protein nitrogen, there is nitrogen from the products of the decomposition of proteins and of other organic compounds (ammonium ions, amide, amine and imine groups); the nitrogen of nitrites and nitrates and the nitrogen of aromatic heterocyclic rings are not determined. The standard PN-A-04018:1975 with the amendment Az3:2002 is cited in scientific publications on the composition of meat (protein = N × 6.25, a Büchi set) and in the list of methodologies of the Provincial Sanitary and Epidemiological Station; its text was not available to us — the description is based on the instructions of the Gdańsk University of Technology and of the University of Gdańsk and before implementation it has to be checked against the standard.

The average content of nitrogen in proteins is about 16 %, and for that reason for so-called crude protein the factor 6.25 (100 : 16) is used; because the proteins of individual products differ in their nitrogen content, specific factors are used: the protein of a hen’s egg 6.67, of milk 6.38, of meat 6.25, of rye, wheat and oats 5.70 — the factor is given next to the result (for example for milk N × 6.38). Indirect methods may be used only where the product contains no other nitrogen compounds or contains very few of them. For the determination a sample is taken such that it contains about 0.02 g of nitrogen (as a rule about 0.5–1.5 g of product).

The principle of the method has been used in automatic sets (a mineralisation apparatus, a unit for distillation or for distillation and automatic titration), allowing more than a hundred analyses a day to be carried out.

SEARCH KEY CORRECTED (23.09.2026). The key field had the form "PN-A-04018" in the new scheme, while the accreditation scopes use both conventions — the old "PN-75/A-04018" and the new "PN-A-04018:1975". The old one stands in 22 entries at 16 laboratories, the new one in 19 at 9; matching on the beginning of the notation covers only one of them, because they differ already in the first character after "PN-". The key has been set to the more numerous form: the "Laboratories" tab now shows 22 entries at 16 laboratories instead of 19 at 9. CHECKED BEFORE THE CHANGE: no other standard stands under the prefix "PN-75/A-04018" — all records concern the same document, most of them with the amendment "+Az3:2002" attached.

Method principle

Mineralisation: in a Kjeldahl flask or tube the organic compounds are oxidised in concentrated sulfuric acid in the presence of a catalyst (copper(II) sulfate(VI), mercury(II) sulfate, selenium or a selenium-copper mixture), often with the addition of potassium or sodium sulfate(VI) raising the boiling point of the acid and possibly of an oxidant (hydrogen peroxide); the nitrogen passes into ammonium sulfate (in the instructions of the Gdańsk University of Technology: ammonium hydrogen sulfate, the mineralisation at 380–410 °C, a mineralised sample has a dark brown colour). Distillation: after the addition of a solution of a strong base (30–33 % NaOH) the ammonia released is distilled off with steam into a receiver with an excess of boric acid (25 cm³ of a saturated solution with a mixed indicator: methyl red + bromocresol green), where it is bound into the ammonium salt of boric acid; alternatively into a measured amount of sulfuric acid 0.1 N, the excess of which is back-titrated with NaOH. Titration: the ammonia bound is titrated with a standardised solution of hydrochloric acid (for example 0.01 M) or of sulfuric acid until the colour of the indicator returns to what it was before the absorption of the ammonia. Calculation: %N = 14·V·c·100/(1000·m) (V — the volume of acid in cm³, c — the concentration in mol/dm³, m — the mass of the dry test portion in g), protein = %N × the factor.

Applications

• Determination of total protein in meat and meat products, milk, cereals, feeds and other agricultural food products
• The declaration of the nutritional value (protein = N × the factor) and control of conformity with the specification
• The reference method for calibrating indirect analysers (NIR, infrared spectrometry)
• The basic chemical composition of raw materials in scientific studies (protein N × 6.25)
• Exercises in food analysis and analytical chemistry (elemental analysis of nitrogen)

Key parameters

Standard

Standard number

PN-A-04018:1975 (+Az3:2002)

Title (PL)

Produkty rolniczo-żywnościowe — Oznaczanie azotu metodą Kjeldahla i przeliczanie na białko

Title (EN)

Agricultural food products — Determination of nitrogen by the Kjeldahl method and conversion to protein (working translation)

Step-by-step procedure

1. Test portion

   Weigh into a digestion tube a sample containing about 0.02 g of nitrogen (usually 0.5–1.5 g); for dry products determine the moisture in parallel, so as to convert to dry mass.

2. Mineralisation

   Add the catalyst (Se or CuSO4 with K2SO4) and 5 cm³ of concentrated sulfuric acid; place the tubes in the mineraliser, close the empty openings, put on the extraction head connected to a water pump and heat (the thermostat at full power). After about 10 min check whether the sample has been mineralised; when it is finished take the tubes out with the head and cool them for about 10 min, then rinse the walls with about 10 cm³ of water.

   ⏱ Time: about 10 min + cooling • 🌡 Temperature: 380–410 °C

3. Check of the distillation apparatus

   Fill the tanks of water and of lye, turn on the cooling water, put in an empty tube and a receiver, distil about 100 cm³ of water (a rinse), switch the steam off.

4. Distillation

   Put in the tube with the sample and the receiver with 25 cm³ of saturated boric acid with the indicator; dose about 20 cm³ of 30 % NaOH lye, switch the steam on and distil until about 100 cm³ of distillate have been collected; rinse the end of the condenser with water.

   ⏱ Time: to about 100 cm³ of distillate

5. Titration

   Titrate the contents of the receiver with hydrochloric acid 0.01 M until the colour of the indicator returns to what it was before the absorption of the ammonia (a comparison solution helps). In the variant with sulfuric acid in the receiver — back-titrate the unbound acid with 0.1 N NaOH to a green colour of Tashiro’s indicator.

6. Control test

   Carry out a blank with all the reagents without the sample and take it into account in the calculations.

7. Calculations

   %N = 14·V·c·100/(1000·m) (V — the volume of acid in cm³, c — the concentration in mol/dm³, m — the mass of the dry test portion in g; 1 cm³ of HCl 0.1 M corresponds to 0.0014 g N); in the reverse variant %N = (M1 − M2)·n·0.014·100/m. Protein = %N × the factor (6.25 general; 6.38 milk; 5.70 cereals; 6.67 egg). Give the uncertainty from the repetitions (RSD, k = 2).

8. Test report

   Give the content of nitrogen and of protein with the factor used, the mass of the test portion, the catalyst and the variant of the method (manual/automatic).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Concentrated sulfuric acid at 380–410 °C — the mineralisation only under a fume hood with extraction of the vapours (SO2, acid mist), goggles, a laboratory coat, acid-resistant gloves
• Selenium and the compounds of copper/mercury as catalysts are toxic — weigh them out in gloves, the waste as chemical waste
• Concentrated NaOH 30–33 % and the hot steam distillation — a risk of burns and of ejection; dose the lye automatically, do not open the apparatus under the pressure of steam
• Hydrochloric acid and boric acid — the standard protective measures

Frequently asked questions