🍎 Determination of nitrogen content by the Kjeldahl method and calculation of crude protein in animal feeding stuffs — 0.3–1.0 g of sample is digested in a tube in a block heated to 420 °C with 12 ml of sulfuric acid and two catalyst tablets (potassium sulfate and copper sulfate), the ammonia released after addition of sodium hydroxide is steam-distilled into boric acid and titrated with 0.1 mol/l hydrochloric acid to the first pink colour or to pH 4.6; crude protein = nitrogen × 6.25; nitrogen content above 0.5 %, PN-EN ISO 5983-2

Food chemistry PN-EN ISO 5983-2

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN ISO 5983-2:2009 (Polish version), in full (our translation): “This part of ISO 5983 specifies a method for the determination of the nitrogen content of animal feeding stuffs according to the Kjeldahl method, and a method for the calculation of the crude protein content. It is suitable for use as a rapid routine semi-micro method using block digestion, copper catalyst and steam distillation into boric acid. The method is applicable to the determination of a Kjeldahl nitrogen content, expressed as a mass fraction greater than 0.5 %, in animal feeding stuffs, pet foods and their raw materials. The method does not measure oxidized forms of nitrogen or heterocyclic nitrogen compounds. The method does not distinguish between protein and non-protein nitrogen. NOTE If it is important to determine the content of non-protein nitrogen, an appropriate method can be applied”. We read the text of the standard in the sample of ISO 5983-2:2009 in the Russian version (text identical to the English one introduced by EN ISO 5983-2:2009) from the table of contents through the foreword and Clauses 1–8 up to and including 9.5.1. One sentence on the total digestion time (9.3.1) is cut off in the sample — we do not give that figure. Outside the sample remain 9.5.2–9.5.4 (recovery tests), Clause 10 (calculation), 11 (precision), 12 (test report) and Annexes A–B (results of interlaboratory tests, comparison of the colorimetric and potentiometric end point).

ACCORDING TO THE TEXT OF ISO 5983-2:2009 (Clauses 1–8, 9.1–9.5.1). The standard was prepared by ISO/TC 34 “Food products”, subcommittee SC 10 “Animal feeding stuffs”; it is the second edition, which after technical revision replaced ISO 5983-2:2005. Part 1 of the series is the Kjeldahl method, Part 2 — the method with block digestion and steam distillation. Warning: the standard may involve hazardous materials, operations and equipment; safety practices are established by the user. Scope (1): determination of nitrogen in animal feeding stuffs by the Kjeldahl method and calculation of crude protein; rapid routine semi-micro method with digestion block, copper catalyst and steam distillation into boric acid; for Kjeldahl nitrogen contents above 0.5 % (by mass) in feeding stuffs for farm and pet animals and their raw materials; it does not determine oxidized forms of nitrogen or heterocyclic nitrogen compounds and does not distinguish protein from non-protein nitrogen. References (2): ISO 1871 (general guidelines for the Kjeldahl method in food and feed), ISO 6498 (sample preparation). Definitions (3): nitrogen content — mass fraction of nitrogen determined according to this part of the standard, in percent or g/kg; crude protein — nitrogen content multiplied by 6.25, in percent or g/kg. Principle (4): the sample is decomposed in a digestion block; concentrated sulfuric acid converts protein nitrogen into ammonium sulfate at an elevated boiling point obtained by adding potassium sulfate, and the copper catalyst accelerates the reaction; excess sodium hydroxide is added to the cooled digest, and the released ammonia is steam-distilled in a manual, semi-automatic or automatic apparatus; in manual or semi-automatic distillation the ammonia is collected in excess boric acid and titrated with hydrochloric acid to a colorimetric end point, and in a fully automatic system titration runs simultaneously with distillation, also with a potentiometric end point; sulfuric acid may also be used for titration. Reagents (5): distilled or deionized water; catalyst tablets — 3.5 g of potassium sulfate and 0.4 g of copper(II) sulfate pentahydrate per tablet (other tablets permitted if the total number of tablets introduces 7 g of potassium sulfate and 0.8 g of copper sulfate and they contain no salts of toxic metals, e.g. selenium or mercury); sulfuric acid at least 98 %, nitrogen-free (approx. 1.84 g/ml); hydrogen peroxide approx. 30 g per 100 ml; antifoaming agent, e.g. 30 % silicone emulsion; sodium hydroxide approx. 40 %, nitrogen-free (below 5 µg N/g); indicators — methyl red and bromocresol green, 100 mg each in 100 ml of ethanol or methanol; concentrated boric acid 40.0 g/l (400 g per 10 l, with 70 ml of methyl red solution and 100 ml of bromocresol green, adjusted with alkali so that the blank consumes 0.05–0.15 ml of titrant and 25 ml of the solution after adding 100 ml of water turns green; stored at room temperature, away from light and ammonia vapours) and diluted 10.0 g/l (for titrators that start titration together with distillation); standard hydrochloric acid 0.1000 mol/l (other concentrations of HCl or of sulfuric acid permitted after correcting the calculations; concentration always to the fourth decimal place); ammonium sulfate min. 99.5 %, dried 4 h at (102 ± 2) °C — 21.09 % nitrogen; ammonium iron(II) sulfate — 7.145 % nitrogen; reference substances: tryptophan (nitrogen 137.2 g/kg) or acetanilide (min. 99 %, nitrogen 103.6 g/kg, without drying), and where possible reference materials with certified Kjeldahl nitrogen and crude protein; sucrose with nitrogen not more than 0.002 % — for the blank. Apparatus (6): balance accurate to 0.1 mg; digestion block (e.g. of aluminium alloy) with temperature control, maintaining (420 ± 5) °C; 250 ml tubes; exhaust manifold; centrifugal scrubber, water aspirator pump or exhaust of acid-resistant material; dispensers up to 25 ml; 50 ml cylinders; steam distillation apparatus (manual, semi-automatic or with automatic titration); 250 ml conical flasks; 25 ml burette readable to at least 0.05 ml (class A) or automatic burette; titrator with pH meter calibrated in the range pH 4–7. Sampling (7) is not part of the standard (ISO 6497 recommended); sample preparation (8) — according to ISO 6498. Test portion (9.2): to the nearest 0.1 mg — approx. 1.0 g at 3–30 % protein, approx. 0.5 g at 30–80 %, approx. 0.3 g above 80 %, not more than 1.2 g; in each series a check of the reference substances and a reagent blank. Digestion (9.3.1): test portion into the tube, two catalyst tablets, 12 ml of sulfuric acid (15 ml for products with more than 10 % fat) — at this point work may be interrupted until the next day; on foaming slowly 3–5 ml of hydrogen peroxide or a few drops of antifoaming agent; heat shields, exhaust manifold at full power, rack into the block heated to 420 °C; after 10 min reduce the suction so that the acid fumes stay in the manifold and the condensation zone in the tube; after the main fumes of sulfur oxides cease reduce the vacuum so as not to lose sulfuric acid; digest for approx. another 60 min; then 10–20 min of cooling with the manifold; carefully (gloves, eye protection) add water a few ml at a time — spattering means the tubes are too hot — and make up to approx. 80 ml; heat a solidified sample carefully until the salts dissolve or distil 30–60 s longer; crystallization during digestion may cause nitrogen losses. Distillation (9.3.2): flask with 25–30 ml of concentrated boric acid under the condenser, tube immersed in the solution; 50 ml of sodium hydroxide; distillation according to the manufacturer’s instructions — the amount of distillate depends on the nitrogen content. Titration (9.3.3): colorimetrically with hydrochloric acid from the burette to the first trace of pink colour, reading to the nearest 0.05 ml (an illuminated stirrer or a photometric detector helps); potentiometrically — with a titrator to pH 4.6, at the inflection point of the curve; with automatic titration from the start of distillation — diluted boric acid 10 g/l. Blank (9.4): the entire procedure with 2 ml of water and approx. 0.7 g of sucrose instead of the sample; titrant consumption always greater than 0.0 ml, the blank values in the laboratory should be constant over time, and changes are investigated. Recovery tests (9.5.1) are carried out regularly to check the correctness of the method and apparatus.

STATUS (catalogue cards as of 03.10.2026). The card of PN-EN ISO 5983-2:2009 (Polish version, published 17-06-2013, 22 pages, price group L) has no “Withdrawn” header: Food, Agriculture and Forestry Sector, KT 40 Animal Feeding Stuffs, ICS 07.100.30, 65.120, “Introduces: EN ISO 5983-2:2009 [IDT], ISO 5983-2:2009 [IDT]”, “Supersedes: PN-EN ISO 5983-2:2006 — Polish version”. The English version PN-EN ISO 5983-2:2009 (published 23-07-2009, 18 pages) also has no “Withdrawn” header — in the PKN search (query “PN-EN ISO 5983-2”, 03.10.2026) both versions are listed side by side, and the earlier editions 2005 (English version) and 2006 (Polish version) — as withdrawn. In the iTeh catalogue (read 03.10.2026) ISO 5983-2:2009 has the status “Published” and stage 90.93 “International Standard confirmed” — the ISO document was confirmed at systematic review.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 5983-2 appears in 6 records at 5 laboratories: AB 527, AB 546, AB 823, AB 1616, AB 1837 — everywhere as “PN-EN ISO 5983-2:2009”. In the current scopes on the PCA website (read 03.10.2026) the standard appears at only four: AB 546 (issue no. 24 of 14.10.2025), AB 823 (no. 18 of 06.07.2026), AB 1616 (no. 13 of 14.10.2025) and AB 1837 (no. 5 of 10.06.2026). In the document of AB 527 (issue no. 26 of 02.07.2026) the number 5983-2 does not appear even once — for feeding stuffs there are only moisture, homogeneity and other characteristics, and Kjeldahl nitrogen appears only for meat according to PN-75/A-04018+Az3:2002; in our copy AB 527 still has the item “Feeding stuffs — Kjeldahl nitrogen content (0.05–14.50) %N”. The “Laboratories” tab (“PN-EN ISO 5983-2”) on the production server labcoda.pl (03.10.2026) shows 5 laboratories, including AB 527 — because it is based on the same copy.

WHAT THE LABORATORIES TEST (PCA items). Feeding stuffs, animal feed, feed with feed materials and additives and their raw materials (AB 1837). Items: “Kjeldahl nitrogen content” with crude protein “by calculation” (AB 823, AB 1616, AB 1837) or “Nitrogen content” with protein “by calculation” (AB 546); technique — titrimetric method. Nitrogen ranges: (1.0–13.0) % (AB 546), (0.06–9.60) % (AB 823), (0.500–12.600) % (AB 1616), (0.50–16.00) %, i.e. (5.00–160.00) g/kg (AB 1837). The lower limit of AB 823 — 0.06 % — lies below 0.5 %, from which the standard applies (1). Just above in the scope of AB 823 is Kjeldahl nitrogen in plant material (1.00–8.00) % according to in-house procedure PB 35 — a different item and a different test object.

WHERE A SEEMINGLY CORRECT RESULT IS EASY. At the test portion: a portion above 1.2 g or 12 ml of acid instead of 15 ml for feed with more than 10 % fat is a deviation from 9.2 and 9.3.1. At digestion: too strong suction after the first phase carries away sulfuric acid, and crystallization in the tube causes nitrogen losses (9.3.1, note 2). At the reagents: sodium hydroxide and sulfuric acid must be nitrogen-free; boric acid without proper adjustment gives a blank equal to zero — the standard requires the titrant consumption in the blank to be always greater than 0.0 ml (5.7, 9.4). At the result: the method does not distinguish protein from non-protein nitrogen (1), so “crude protein” = N × 6.25 is conventional and counts non-protein nitrogen as protein too; nitrogen in oxidized (nitrates) and heterocyclic forms is not determined by the method (1). Protein is a calculated quantity: the only measurement of this method is nitrogen, and 6.25 is a fixed factor from definition 3.2.

WHAT WE DO NOT GIVE. We do not give the total digestion time (sentence cut off in the sample), recovery tests 9.5.2–9.5.4, the formulas of Clause 10, precision data or test report requirements, because we did not read these parts. We also do not give requirements for protein in feeding stuffs, because we did not read the texts of regulations for this entry.

Method principle

The organic nitrogen of the feed sample is converted into ammonium sulfate by digesting it in concentrated sulfuric acid at 420 °C; potassium sulfate raises the boiling point, and copper catalyses the decomposition. After making alkaline with excess sodium hydroxide, the ammonia is steam-distilled into boric acid and titrated with standard hydrochloric acid; the amount of ammonia gives the nitrogen content, and multiplied by 6.25 — the crude protein.

Applications

• Feeding stuffs for farm and pet animals and their raw materials — Kjeldahl nitrogen above 0.5 % and crude protein (Clause 1)
• Rapid routine semi-micro method with block digestion and steam distillation — manual, semi-automatic or with automatic titration (4, 6.8)
• Feeding stuffs, feed materials and additives — in the current accreditation scopes of 4 laboratories at PCA (5 in the database copy)

Key parameters

Standard

Standard number

PN-EN ISO 5983-2:2009

Title (PL)

Pasze — Oznaczanie zawartości azotu i obliczanie zawartości białka ogólnego — Część 2: Metoda mineralizacji w bloku i destylacji z parą wodną

Title (EN)

Animal feeding stuffs — Determination of nitrogen content and calculation of crude protein content — Part 2: Block digestion and steam distillation method

Step-by-step procedure

1. Preparation and test portion

   Sample according to ISO 6498; portion 0.3–1.0 g (≤ 1.2 g) depending on protein content; in the series a reference substance and a blank. PN-EN ISO 5983-2:2009 current (PKN catalogue card as of 03.10.2026).

2. Digestion

   2 catalyst tablets + 12 ml H2SO4 (15 ml at fat > 10 %); block 420 °C with manifold; after 10 min reduce suction; then approx. 60 min; cooling 10–20 min.

3. Dilution

   Carefully a few ml of water at a time (gloves, eye protection), to approx. 80 ml; dissolve solidified salts by heating.

4. Distillation

   Receiver 25–30 ml boric acid 40 g/l with immersed tube; 50 ml NaOH 40 %; steam distillation according to the manufacturer.

5. Titration and protein

   HCl 0.1000 mol/l to the first pink (reading 0.05 ml) or to pH 4.6; protein = N × 6.25. Formulas (Clause 10) outside the sample read.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard may involve hazardous materials, operations and equipment — safety practices are established by the user (warning at the beginning of the standard)
• Digestion at 420 °C releases fumes of sulfur oxides — exhaust manifold with scrubber and work in a fume hood (9.3.1)
• Water is added to hot tubes carefully, with gloves and eye protection — spattering means too high a temperature (9.3.1); concentrated sulfuric acid and 40 % NaOH are corrosive

Frequently asked questions