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Short answer: the Kjeldahl method measures nitrogen in a sample by three stages: digesting it in boiling concentrated sulfuric acid to turn organic nitrogen into ammonium, releasing that ammonia with strong base and distilling it into an absorbing solution, then determining the ammonia, usually by titration. In food labs the nitrogen result is multiplied by a conversion factor to estimate protein; in water labs it is reported as total Kjeldahl nitrogen (TKN).
Definition and scope
The method is named for Johan Kjeldahl, a Danish chemist who introduced it in 1883 (Ricca Chemical technical tip, read 2026-11-08). It is a wet-chemistry procedure for total nitrogen of the amine/amide type, and it is still written into published methods. The US EPA’s Method 351.2 (Revision 2.0, August 1993) applies it to drinking, ground and surface waters and to domestic and industrial wastes, with a stated applicable range of 0.1-20 mg/L TKN that can be extended by dilution (EPA, read 2026-11-08).
The same chemistry therefore appears under two names that are easy to confuse. A food laboratory usually says “Kjeldahl nitrogen” or “crude protein”; an environmental laboratory says “total Kjeldahl nitrogen”. They describe the same digestion step, applied to different matrices and reported in different units. This is an analytical-chemistry technique, not a clinical test; see what analytical chemistry is for the wider context and the analytical process from sampling to validated result for where a method like this sits.
How the Kjeldahl method works in practice
1. Digestion
The sample is heated in boiling concentrated sulfuric acid, which converts organic nitrogen into ammonium bisulfate. Potassium sulfate is added to raise the boiling point of the acid. Copper and selenium catalysts are now common; mercury was historically used but has been replaced because of safety and environmental concerns (Ricca). Boiling stones or zinc granules are used to prevent bumping. Ricca states that the digestion temperature should stay between 370 and 400 °C; one published lab protocol (Prometheus Protocols, read 2026-11-08) digests at about 420 °C with selenium catalyst tablets, so the working temperature is a property of the specific validated procedure rather than a universal constant.
2. Neutralization and distillation
Excess sodium hydroxide neutralizes the acid and releases the ammonia, which is distilled into an absorbing solution. The protocol cited above uses about 40% (w/v) NaOH and about 2% boric acid as the receiving solution; other procedures absorb the ammonia in a measured excess of standard acid instead (Ricca).
3. Determination
The ammonia can be determined by titration, by colorimetry or with an ammonia-selective electrode (Ricca). EPA Method 351.2 is a semi-automated colorimetric version in which the digest, diluted to 25 mL, is analyzed for ammonia after a 2.5-hour heating step (EPA).
Worked calculation
In the titration version described by Prometheus Protocols, nitrogen is calculated as (mL acid – mL blank) × normality of the acid × 1.4007 ÷ sample mass in g. With volumes in mL and mass in g this gives nitrogen as a percentage of the sample mass (editorial arithmetic: 14.007 mg N per milliequivalent, divided by the sample mass, expressed as a percentage). Multiplying by a protein conversion factor gives estimated protein. Ricca gives 6.25 for meat, which corresponds to assuming that protein is about 16% nitrogen. An ammonium sulfate standard is used for calibration in that protocol. For other uses of volumetric endpoints, see sources of error in titration.
Direct versus back titration
| Feature | Direct titration | Back titration |
|---|---|---|
| Receiving solution | Boric acid | Measured excess of standard acid |
| What is titrated | The borate formed, with standard acid | The unreacted acid, with standard sodium hydroxide |
| Indicators named by Ricca | Methyl red, methyl purple, or bromocresol green-methyl red mixed indicator | |
Source: Ricca Chemical technical tip, read 2026-11-08.
Where the method fails: temperature and ammonia loss
| Condition | Effect | Source |
|---|---|---|
| Digestion too cool | Digestion takes long and may be incomplete | Ricca |
| Digestion too hot | Nitrogen is lost and results are low | Ricca |
| Leaks in the distillation train, or too little boric acid | Ammonia is lost and results read falsely low | Prometheus Protocols |
| No boiling stones or zinc granules | Bumping during digestion | Ricca |
Where it matters for labs
What the nitrogen number does and does not mean
- Nitrate, nitrite and N-N / N-O bonds are not captured. Ricca notes that azides, nitrates, nitrites and nitro compounds are not detected directly without pretreatment under reducing conditions; Prometheus Protocols likewise says total nitrogen is underestimated unless nitrate and nitrite are reduced first.
- Some nitrogen in wastes is not converted. EPA Method 351.2 says the digestion may not convert amines, nitro compounds, hydrazones, oximes, semicarbazones and some refractory tertiary amines in industrial wastes.
- TKN is ammonia plus organic nitrogen. The method defines total Kjeldahl nitrogen as free ammonia plus organic nitrogen converted to ammonium sulfate, and organic Kjeldahl nitrogen as TKN minus free ammonia.
- The protein figure depends on the conversion factor. It is an estimate of protein from nitrogen, not a direct protein measurement. For choosing a food or nutrition testing provider, see how to evaluate a nutritional analysis lab.
- Water samples need preservation. EPA Method 351.2 requires samples to be preserved with H2SO4 to pH below 2 and cooled to 4 °C.
Safety
The procedure uses boiling concentrated acid and concentrated sodium hydroxide. The protocol cited above specifies trained staff, a fume hood, a face shield and acid-resistant gloves for handling hot digestion tubes, and reading the safety data sheets first. See what a fume hood is and how to read a safety data sheet. Local procedures, your chemical hygiene plan and the validated method govern in your lab.
Common misunderstandings
- “Kjeldahl measures protein.” It measures nitrogen; protein is calculated from it with an assumed factor.
- “Kjeldahl measures all nitrogen.” It does not, as the list above shows.
- “The method is obsolete.” Prometheus Protocols states that combustion analysis has largely replaced Kjeldahl over the last 25 years in their experience; EPA Method 351.2 (1993) still describes a Kjeldahl-based procedure for water and waste.
- “Any digestion temperature will do.” Too low and too high both give wrong results, as the table above shows.
FAQ
What is the Kjeldahl method used for?
Determining nitrogen, most often to estimate protein in foods and to report total Kjeldahl nitrogen in water and wastewater samples.
What are the three steps of the Kjeldahl method?
Digestion in concentrated sulfuric acid, neutralization with sodium hydroxide followed by distillation of ammonia, and determination of the ammonia, typically by titration.
Why is potassium sulfate added?
It raises the boiling point of the sulfuric acid, which allows the digestion to run at the required temperature (Ricca).
Why was mercury replaced as a catalyst?
For safety and environmental reasons; copper and selenium catalysts are now common (Ricca).
Sources
- Ricca Chemical, “Kjeldahl Analysis” technical tip: riccachemical.com/pages/tech-tips/kjeldahl-analysis (read 2026-11-08).
- Prometheus Protocols, “Kjeldahl Nitrogen Analysis”: prometheusprotocols.net/kjeldahl-nitrogen-analysis (read 2026-11-08).
- US EPA, Method 351.2, Revision 2.0 (August 1993), Determination of Total Kjeldahl Nitrogen by Semi-Automated Colorimetry: EPA Method 351.2 (PDF) (read 2026-11-08).
