🥩 Protein — Kjeldahl method
In short
Classical method for determining total protein in food based on nitrogen content (N × conversion factor). The test is performed according to PN-EN ISO 8968-1:2014 / AOAC Official Method 2001.11; Measurement range: 0.1–100% protein (depending on matrix). The procedure comprises 10 steps (about 3 h 23 min–4 h 21 min in total); it is used for: Determination of protein in milk and dairy products (ISO 8968-1), Quality control of meat and meat products, Protein analysis in cereals, flours, and cereal products.
At a glance
- Standard: PN-EN ISO 8968-1:2014 / AOAC Official Method 2001.11
- Category: Food Chemistry
- Procedure steps: 10
- Total time of stages: 3 h 23 min–4 h 21 min
- Measurement range: 0.1–100% protein (depending on matrix)
- Conversion factors: 6.25 (general), 6.38 (milk), 5.70 (cereals), 6.25 (meat)
- Repeatability: ±0.038% protein (according to ISO 8968-1)
Overview
The Kjeldahl method is the world reference standard for determining nitrogen and crude protein content in food, feed, soil, and environmental samples. Developed in 1883 by Danish chemist Johan Kjeldahl, it remains the most widely used method for quantitative protein determination in food quality control laboratories worldwide.
The principle of the method involves mineralization of the sample in concentrated sulfuric acid with a catalyst (CuSO₄, K₂SO₄), during which organic nitrogen is converted to ammonium sulfate. Then, after alkalizing the solution with sodium hydroxide, the released ammonia is distilled with steam and absorbed in boric acid solution. The amount of ammonia is determined by titration with hydrochloric acid. Protein content is calculated by multiplying nitrogen content by a conversion factor (e.g., 6.25 — general; 6.38 — milk; 5.70 — cereals; 6.25 — meat).
The Kjeldahl method is required by EU and Polish regulations concerning food labeling, feed control, and certification of food raw materials. The repeatability of the method for protein content defined in AOAC 991.20 and ISO 8968-1 standards is 0.038% protein, making it a highly precise reference technique.
Modern Kjeldahl systems (BÜCHI, Gerhardt, VELP, FOSS) automate block mineralization, steam distillation, and titration, enabling analysis of 20–40 samples per day. The method is universal — suitable for both homogeneous and heterogeneous samples (meat, cereals, milk, feed).
Method principle
The method consists of three stages: (1) Mineralization — the sample is digested in concentrated H₂SO₄ (temp. 380–420°C) with selenium or copper catalyst and K₂SO₄ raising the boiling point, which causes decomposition of organic compounds and conversion of nitrogen to (NH₄)₂SO₄. (2) Distillation — after cooling and adding excess NaOH, released NH₃ is distilled with steam to a receiver with boric acid (H₃BO₃). (3) Titration — the resulting ammonium borate is titrated with standardized HCl solution to the endpoint (color change of mixed Tashiro indicator). Nitrogen content is converted to crude protein: protein [%] = N [%] × F, where F is the conversion factor depending on the product type.
Applications
- Determination of protein in milk and dairy products (ISO 8968-1)
- Quality control of meat and meat products
- Protein analysis in cereals, flours, and cereal products
- Testing of feed and feed mixtures (AOAC 2001.11)
- Food labeling control (EU Reg. 1169/2011)
- Protein analysis in special-purpose food (infant formula)
- Certification of food raw materials (soy, peas, whey)
Key parameters
| Parameter | Value |
|---|---|
| Measurement range | 0.1–100% protein (depending on matrix) |
| Conversion factors | 6.25 (general), 6.38 (milk), 5.70 (cereals), 6.25 (meat) |
| Repeatability | ±0.038% protein (according to ISO 8968-1) |
| Mineralization temperature | 380–420°C |
| Mineralization time | 45–90 min (depending on matrix) |
| Sample size | 0.5–2.0 g |
Standard
- Standard number
- PN-EN ISO 8968-1:2014 / AOAC Official Method 2001.11
- Title (PL)
- Oznaczanie zawartości azotu — Część 1: Metoda Kjeldahla i obliczanie białka surowego
- Title (EN)
- Determination of nitrogen content — Part 1: Kjeldahl principle and crude protein calculation
Step-by-step procedure
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Sample preparation
Homogenize the sample. Weigh 0.5–2.0 g on an analytical balance into a Kjeldahl flask. Record mass with 0.001 g accuracy.
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Addition of reagents
Add 2 catalyst tablets (K₂SO₄ + CuSO₄) and 20 mL conc. H₂SO₄. Mix carefully.
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Mineralization
Place flasks in block digestor. Heat gradually to 420°C. Mineralize until solution becomes clear (light green/colorless).
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Cooling
Turn off heating. Leave flasks to cool to room temperature. Carefully add 50 mL deionized water.
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Distillation preparation
Place flask in steam distillation unit. Prepare receiver with 50 mL 4% boric acid with Tashiro indicator (pink color).
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Alkalization and distillation
Distillation unit automatically doses 40% NaOH. Steam releases NH₃, which is absorbed in H₃BO₃ solution (color change to green).
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Titration
Titrate distillate with standardized 0.1 mol/L HCl to endpoint (color change from green to violet). Record HCl consumption.
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Blank test
Conduct full procedure without sample (with reagents only). Subtract HCl consumption for blank from result.
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Calculations
N [%] = (V_sample − V_blank) × c_HCl × 14.007 × 100 / (m_sample × 1000). Protein [%] = N [%] × conversion factor.
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Quality control
Analyze CRM every series (10–20 samples). Nitrogen recovery 98.5–101.5%. Maintain control chart.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Kjeldahl block digestor | BÜCHI SpeedDigester K-436 (20 positions), Gerhardt Kjeldatherm | 35 000–65 000 PLN |
| Kjeldahl steam distillation unit | BÜCHI KjelFlex K-360, VELP UDK 169 | 40 000–75 000 PLN |
| Gas scrubber | BÜCHI Scrubber B-414, Gerhardt Turbosog | 8 000–15 000 PLN |
| Analytical balance 0.1 mg | Mettler Toledo MS205DU, Sartorius Quintix 224 | 8 000–20 000 PLN |
| Automatic burette / Titrator | Metrohm 888 Titrando, SI Analytics TitroLine 7000 | 15 000–35 000 PLN |
| Kjeldahl flasks 250–500 mL | DURAN / Simax borosilicate, set of 20 pcs. | 600–1 200 PLN |
| Catalyst tablets | BÜCHI Kjeldahl Tablets (K₂SO₄ + CuSO₄), 1000 pcs. | 400–800 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Concentrated sulfuric acid 96% p.a. | 7664-93-9 | For mineralization, 2.5 L for ~50 determinations |
| Sodium hydroxide 40% | 1310-73-2 | For alkalization before distillation, 2.5 L |
| Boric acid 4% | 10043-35-3 | Ammonia absorbing solution, with mixed Tashiro indicator |
| Hydrochloric acid 0.1 mol/L standardized | 7647-01-0 | For titration, 1 L bottle with certificate |
| Potassium sulfate K₂SO₄ | 7778-80-5 | Catalyst — raises boiling point |
| Copper(II) sulfate CuSO₄·5H₂O | 7758-99-8 | Mineralization catalyst, in tablets or powder |
| Mixed Tashiro indicator | — | Methyl red + methylene blue, color change green→violet |
| Reference material CRM (protein) | — | E.g., NIST SRM 1849a (milk) or BIPEA, for validation and quality control |
Health and safety (OHS)
- Concentrated H₂SO₄ — highly corrosive (H314), risk of severe burns. Use acid-resistant gloves, apron, goggles.
- 40% NaOH — highly corrosive (H314). Reaction with H₂SO₄ highly exothermic.
- High-temperature mineralization — risk of splashing. Use shield and fume hood.
- SO₂ and NH₃ fumes — work under fume hood with scrubber.
- Hot flasks — use heat-insulated holders.
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 8968-1:2014 / AOAC Official Method 2001.11 — “Determination of nitrogen content — Part 1: Kjeldahl principle and crude protein calculation”.
How does this method work?
The method consists of three stages: (1) Mineralization — the sample is digested in concentrated H₂SO₄ (temp. 380–420°C) with selenium or copper catalyst and K₂SO₄ raising the boiling point, which causes decomposition of organic compounds and conversion of nitrogen to (NH₄)₂SO₄.
What is the measuring range and accuracy?
Measurement range: 0.1–100% protein (depending on matrix); Conversion factors: 6.25 (general), 6.38 (milk), 5.70 (cereals), 6.25 (meat); Repeatability: ±0.038% protein (according to ISO 8968-1); Mineralization temperature: 380–420°C.
How long does the test take?
The times given for the individual stages add up to approximately 3 h 23 min–4 h 21 min. The procedure comprises 10 steps.
What equipment is required?
Kjeldahl block digestor, Kjeldahl steam distillation unit, Gas scrubber, Analytical balance 0.1 mg, Automatic burette / Titrator, Kjeldahl flasks 250–500 mL, Catalyst tablets. Indicative cost of the core instrument (Kjeldahl block digestor): 35 000–65 000 PLN.
Where is this test used?
Determination of protein in milk and dairy products (ISO 8968-1); Quality control of meat and meat products; Protein analysis in cereals, flours, and cereal products; Testing of feed and feed mixtures (AOAC 2001.11); Food labeling control (EU Reg. 1169/2011); Protein analysis in special-purpose food (infant formula); Certification of food raw materials (soy, peas, whey).
What safety precautions apply?
Concentrated H₂SO₄ — highly corrosive (H314), risk of severe burns. Use acid-resistant gloves, apron, goggles.; 40% NaOH — highly corrosive (H314). Reaction with H₂SO₄ highly exothermic.; High-temperature mineralization — risk of splashing. Use shield and fume hood.; SO₂ and NH₃ fumes — work under fume hood with scrubber.; Hot flasks — use heat-insulated holders.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 8968-1 / AOAC 2001.11.