🌱 Determination of the nitrogen content by the Kjeldahl method and calculation of crude protein in cereals, pulses and derived products — sample ground through a 0.8 mm sieve, digestion with sulfuric acid and catalyst (potassium sulfate, copper sulfate, titanium oxide) for at least 2 h at (420 ± 10) °C, alkalisation, distillation of ammonia into boric acid, titration with 0.05 mol/l sulfuric acid; recovery from acetanilide at least 99.5 %, from tryptophan 99.0 %; result on dry matter, PN-EN ISO 20483

Physicochemistry PN-EN ISO 20483

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN ISO 20483:2014-02 (Polish version), in full (our translation): “This International Standard specifies a method for the determination of the nitrogen content of cereals, pulses and derived products according to the Kjeldahl method and a way of conversion to protein content. The method does not distinguish protein nitrogen from non-protein nitrogen. If it is important to determine the non-protein nitrogen content, an appropriate method should be applied. NOTE In certain cases full recovery of the nitrogen from nitrates(V) and nitrates(III) is not possible with this method”. We read the text of the standard in two iTeh samples: ISO 20483:2013 (English) — from the contents through the foreword and Clauses 1–10 up to and including 11.1; SIST EN ISO 20483:2014 (text of EN ISO 20483:2013) — title pages, European foreword and endorsement notice. Outside the samples remain 11.2 (calculation of crude protein), Clauses 12 (precision — interlaboratory test, repeatability, reproducibility, critical difference) and 13 (test report) and Annexes A (results of interlaboratory tests), B (critical difference and limits for different protein contents) and C (factors for converting nitrogen to protein).

ACCORDING TO THE TEXT OF ISO 20483:2013 (Clauses 1–10, 11.1). The standard was prepared by ISO/TC 34 “Food products”, subcommittee SC 4 “Cereals and pulses”; it is the second edition, which replaced ISO 20483:2006 after technical revision; CEN approved EN ISO 20483:2013 on 7 September 2013 without modification, and it replaced EN ISO 20483:2006. Scope (1): nitrogen in cereals, pulses and derived products by the Kjeldahl method and calculation of crude protein; the method does not distinguish protein from non-protein nitrogen; in some cases nitrogen of nitrates and nitrites is not fully recovered. Definitions (3): nitrogen content and crude protein content — both as a mass fraction of the dry product in percent; crude protein is nitrogen multiplied by a factor depending on the type of cereal or pulse. Principle (4): digestion with sulfuric acid and catalyst, alkalisation, distillation, absorption of ammonia in boric acid and titration with sulfuric acid. Reagents (5): nitrogen-free; Kjeldahl tablets — CuSO4·5H2O 2.8 %, TiO2 2.8 %, K2SO4 94.3 % (or a mixture in this ratio); sulfuric acid 18 mol/l (ρ = 1.84 g/ml); antifoaming agent (paraffin oil, silicone, tablets); acetanilide or tryptophan of at least 99 % purity as reference materials; boric acid 40 g/l (or the concentration recommended for the apparatus); mixed indicator — solution A: bromocresol green 200 mg in 95 % ethanol to 100 ml, solution B: methyl red 200 mg in ethanol to 100 ml, e.g. 5 volumes A and 1 volume B (a ready-made boric acid solution with indicator or potentiometric titration with a pH electrode checked daily is allowed); sodium hydroxide 30–40 % with nitrogen not more than 0.001 %; standard sulfuric acid 0.05 mol/l — recommended instead of HCl because it does not produce bubbles in the tubes; standard ammonium sulfate 0.05 mol/l (or e.g. Mohr’s salt); pumice or glass rods against bumping; nitrogen-free sucrose (optional); phosphorus pentoxide. The warning concerns sulfuric acid, sodium hydroxide, the standard acid and phosphorus pentoxide. Apparatus (6): mill, 0.8 mm sieve, balance to 0.001 g, digestion, distillation and titration set; the temperature homogeneity of the block is checked by a full determination of a reference material, and the distillation unit — by distilling a known amount of ammonium salt (e.g. 10 ml of ammonium sulfate solution) with a recovery of at least 99.8 %. Sampling (7) is not part of the standard (ISO 24333); the laboratory should receive a representative sample, undamaged and unchanged during transport and storage. Preparation (8): if necessary grinding so that the whole passes through a 0.8 mm sieve; grains — at least 200 g. Moisture (9): according to ISO 712 (cereals), ISO 6540 (maize), ISO 24557 (pulses). Procedure (10): two determinations when repeatability is to be checked; portion weighed to 0.001 g, containing 0.005–0.2 g of nitrogen, preferably more than 0.02 g; into the flask tablets with 10 g potassium sulfate, 0.30 g copper sulfate and 0.30 g titanium oxide and 20 ml sulfuric acid (the amount of acid may be changed only after confirming recovery of 99.5 % from acetanilide and 99.0 % from tryptophan); block preheated to (420 ± 10) °C, digestion at least 2 h from the time the temperature is reached again — the minimum time is checked on the reference material with which recovery is hardest to obtain; too strong suction of fumes risks loss of nitrogen; after cooling 50 ml water, into the receiver 50 ml boric acid and at least 10 drops of indicator, sodium hydroxide with an excess of 5 ml over the amount needed to neutralise the acid; titration during or after distillation, with a visual end point, optical probe or potentiometrically. Blank (10.4) — without sample, possibly with 1 g sucrose. Check (10.5) — reference material dried at 60–80 °C under vacuum over phosphorus pentoxide, portion of at least 0.15 g; recovery at least 99.5 % (acetanilide) and 99.0 % (tryptophan). Result (11.1): as a percentage of dry matter, to two decimal places, from a formula containing the acid volumes for the sample and blank, the normality T of the acid, the constant 0.014, the portion mass and the moisture.

STATUS (card as of 03.10.2026). The card of PN-EN ISO 20483:2014-02 (Polish version, published 14-09-2015, 20 pages, price group K) has no “Withdrawn” header: Food, Agriculture and Forestry Sector, KT 36 Cereals and Cereal Products, ICS 67.060, “Introduces: EN ISO 20483:2013 [IDT], ISO 20483:2013 [IDT]”, “Replaces: PN-EN ISO 20483:2007 — Polish version”. The card of the English version (published 03-02-2014) lists the additional element PN-EN ISO 20483:2014-02/Ap1:2015-08 and the note “title changed by amendment”; we have not read the content of the amendment. In the iTeh catalogue (read on 03.10.2026) ISO 20483:2013 has the status “Published” and stage 90.93 “International Standard confirmed” (completed 29.08.2026). The same Kjeldahl scheme for feed is described in a separate entry “Determination of nitrogen content by the Kjeldahl method and calculation of crude protein in animal feeding stuffs” (PN-EN ISO 5983-2).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read on 03.10.2026). The number 20483 appears in 14 records at 4 laboratories: AB 452, AB 1179, AB 1275, AB 1616. In the current PCA documents (read on 03.10.2026, issues from 14.10.2025 to 25.09.2026) the number appears in 13 items of the same 4 laboratories (AB 1179 — 10, the others 1 each; at AB 452 the copy splits the item into two records — nitrogen and protein). Notation everywhere “PN-EN ISO 20483:2014-02”. The “Laboratories” tab (“PN-EN ISO 20483”) shows the same 4 laboratories — checked with the tab query on the production server labcoda.pl on 03.10.2026.

WHAT THE LABORATORIES TEST (items in PCA). In line with the subject of the standard: AB 452 — cereal grain (nitrogen (1.120–2.982) % d.m.), cereal products ((1.053–3.200) % d.m.) and pulse seeds ((4.000–6.400) %), with protein by calculation; AB 1275 — wheat grain, nitrogen (1.2–2.6) % d.m. and protein by calculation; AB 1616 — cereal grain, nitrogen (1.00–14.10) % d.m. and protein on dry matter by calculation. More broadly: AB 1179 cites the standard in 10 items — tea, coffee, food concentrates, vegetable products, fruit products, cereals and cereal products, bread and pulse seeds, confectionery, bakery products and salty snacks, foods for special nutritional purposes — mostly with nitrogen (0.03–14.27) % and protein (0.19–90) % by calculation; the standard covers only cereals, pulses and derived products, and the conversion factors (Annex C) refer to types of cereals and pulses. In the AB 1179 document the footnotes on suspension of part of the scope from 24.04.2026 to 21.10.2026 concern other items.

WHERE A SEEMINGLY CORRECT RESULT IS EASY. In interpretation: the result includes non-protein nitrogen, so the calculated “protein” is raised by every non-protein form of nitrogen, and nitrates and nitrites may not be recovered (1). In digestion: too short a time or uneven block temperature leave no visible trace — that is why the standard requires setting the minimum time on the more difficult reference material and checking the recovery of acetanilide (99.5 %) or tryptophan (99.0 %) (10.3.1, 10.5); too strong suction of fumes carries nitrogen away (10.3.1). In distillation: recovery from ammonium salt below 99.8 % indicates a leak or fault in the distillation unit (6.4). In calculation: Formula 11.1 uses the normality T of the acid, while reagent 5.9 has its concentration given in mol/l (0.05 mol/l sulfuric acid); the explanation of the constant 0.014 in the text refers to 1 ml of 0.5 mol/l acid — anyone who puts the molar concentration into the formula instead of the normality gets a result twice too low (for sulfuric acid the normality is twice the molar concentration — our conversion, not a statement of the standard). On dry matter: the result on dry matter requires moisture determined by the method appropriate to the product (ISO 712, ISO 6540 or ISO 24557) — another moisture method shifts the result.

WHAT WE DO NOT GIVE. We have not read 11.2, Clauses 12–13 or the annexes, so we do not give the factors for converting nitrogen to protein for individual cereals and pulses, the repeatability and reproducibility limits or the critical difference. Nor do we give the content of amendment Ap1:2015-08 or requirements of regulations on cereal quality.

Method principle

A portion of the ground sample (0.005–0.2 g nitrogen) is digested with sulfuric acid with potassium sulfate and a copper–titanium catalyst for at least 2 h at (420 ± 10) °C; organic nitrogen is converted to ammonium sulfate. After dilution with water and addition of excess sodium hydroxide, ammonia is distilled into boric acid with indicator and titrated with standard sulfuric acid (visually, with an optical probe or potentiometrically). From the acid volumes for the sample and blank, nitrogen is calculated as a percentage of dry matter, and crude protein — by multiplying nitrogen by a factor specific to the cereal or pulse.

Applications

• Cereal grain, pulse seeds and derived products — nitrogen and crude protein (1)
• Does not distinguish protein from non-protein nitrogen; nitrogen of nitrates and nitrites not always fully recovered (1)
• Cereal grain, cereal products, pulse seeds, and at one laboratory also tea, coffee, fruit and vegetable products, confectionery — in the accreditation scopes of 4 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 20483:2014-02

Title (PL)

Ziarno zbóż i nasiona roślin strączkowych — Oznaczanie zawartości azotu i przeliczanie na zawartość białka — Metoda Kjeldahla

Title (EN)

Cereals and pulses — Determination of the nitrogen content and calculation of the crude protein content — Kjeldahl method

Step-by-step procedure

1. Sample preparation

   Grinding through a 0.8 mm sieve (grains — at least 200 g), mixing; moisture by the method appropriate to the product. PN-EN ISO 20483:2014-02 current (PKN card as of 03.10.2026).

2. Digestion

   Portion with 0.005–0.2 g nitrogen, catalyst tablets, 20 ml H2SO4; at least 2 h at (420 ± 10) °C; care with fume suction.

3. Distillation

   50 ml water; into the receiver 50 ml boric acid and at least 10 drops of indicator; NaOH with 5 ml excess; distillation.

4. Titration and checks

   H2SO4 0.05 mol/l; blank (possibly with 1 g sucrose); check with acetanilide (99.5 %) or tryptophan (99.0 %).

5. Result

   Nitrogen as a percentage of dry matter to two decimal places; crude protein — nitrogen times the factor from Annex C (which we do not give).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Concentrated sulfuric acid, sodium hydroxide solution and phosphorus pentoxide require caution (warning in Clause 5)
• Digestion at 420 °C releases sulfuric acid fumes — work under an efficient hood or with fume extraction (10.3.1)
• Adding water and alkali to the flask after digestion — carefully, after cooling (10.3.2)

Frequently asked questions