How They Measure the Amount of Protein in Food

December 11, 2025 •

3 min read

Food manufacturers use standardized methods to determine protein content for nutritional labeling and quality control. For over a century, these methods have centered on measuring nitrogen content, as proteins are the main nitrogen-containing compounds in food. However, the precision of measurement can vary significantly depending on the technique employed.

Quick Summary

Several analytical methods exist to quantify protein in food, with the nitrogen-based Kjeldahl and Dumas methods being the most common. Other techniques include spectrophotometric assays and highly accurate amino acid analysis. Each method has distinct principles, advantages, and limitations.

Key Points

Crude Protein vs. True Protein: Crude protein is an estimate based on a food's total nitrogen content, while true protein is a more accurate measurement of actual protein and its component amino acids.
Kjeldahl Method: A classic, but slow, wet-chemistry technique that measures total nitrogen via digestion, distillation, and titration using hazardous chemicals.
Dumas Method: A rapid, automated combustion method that measures total nitrogen and is gaining favor over Kjeldahl due to its speed and safety.
Amino Acid Analysis: Considered the most accurate method, it involves hydrolyzing proteins and quantifying each amino acid individually, providing a true protein value.
Spectrophotometric Assays: Fast, colorimetric methods like the Bradford and Lowry assays measure relative protein concentrations but can be affected by interfering substances.
Modern Spectroscopy: Techniques like Near-Infrared (NIR) spectroscopy and ultrasonic analysis offer rapid, non-destructive protein measurement, particularly useful for quality control in manufacturing.
Conversion Factor Caveats: Nitrogen-based methods use a conversion factor (typically 6.25), but this can lead to protein overestimation as it doesn't account for non-protein nitrogen.

Nitrogen-Based Methods: Measuring Crude Protein

Protein measurement often relies on quantifying the total nitrogen in food, assuming proteins are about 16% nitrogen. This total nitrogen is multiplied by a factor (usually 6.25) to estimate 'crude protein'. The two primary nitrogen-based methods are Kjeldahl and Dumas.

The Kjeldahl Method

Introduced in 1883, the Kjeldahl method remains a standard wet-chemistry technique. It involves three steps: Digestion (heating the sample in sulfuric acid with a catalyst to convert organic nitrogen to ammonium sulfate), Distillation (releasing ammonia gas with alkali and capturing it), and Titration (quantifying ammonia to calculate nitrogen, then crude protein). While reliable, it's slow and uses hazardous chemicals. It also measures non-protein nitrogen (NPN), potentially overestimating true protein, as seen in the melamine scandal.

The Dumas Method

A faster, automated combustion technique, the Dumas method, is gaining popularity. It involves combusting the sample at high temperatures to convert nitrogen to nitrogen oxides, reducing these to elemental nitrogen gas ($N_2$), and measuring the $N_2$ with a thermal conductivity detector. This nitrogen value is then converted to crude protein. Like Kjeldahl, it measures all nitrogen but is faster and avoids toxic chemicals.

Spectrophotometric and Chromatography Methods: Targeting True Protein

To measure true protein more accurately, methods that directly target protein or amino acids are used.

Amino Acid Analysis

Considered the most accurate method for true protein, this involves hydrolyzing protein into amino acids, separating them using high-performance liquid chromatography (HPLC), and quantifying each one. The sum represents the true protein content. This method requires complex equipment and time but is highly accurate.

Spectrophotometric Assays

These rapid colorimetric assays react protein with a reagent to produce a colored product, the intensity of which is measured with a spectrophotometer. Examples include the Biuret, Lowry, and Bradford assays. While fast, they can be affected by other substances and protein composition.

Comparison of Protein Measurement Methods


Feature	Kjeldahl Method	Dumas Method	Amino Acid Analysis	Spectrophotometric Assays
Basis	Measures total nitrogen	Measures total nitrogen	Measures individual amino acids	Colorimetric reaction with proteins
Result	Crude protein	Crude protein	True protein	Relative protein concentration
Speed	Slow, labor-intensive	Fast, automated	Slow, complex	Fast, routine
Accuracy	Good, but can be inaccurate if NPN is present	Good, but can be inaccurate if NPN is present	Highly accurate for true protein	Can be unreliable due to interfering substances
Cost	Relatively low initial cost	High initial equipment cost	High initial and running costs	Low cost per test, but susceptible to errors
Safety	Uses hazardous chemicals (sulfuric acid)	Safe, uses no hazardous chemicals	Uses strong acid for hydrolysis	Generally safe, but uses specific reagents

Modern Spectroscopic Approaches

Rapid, non-destructive methods are also used, especially for quality control. Near-Infrared (NIR) Spectroscopy analyzes chemical composition using light waves and is fast with no sample prep. Ultrasonic Analysis measures protein in liquids like milk by analyzing wave passage.

Conclusion

The Kjeldahl and Dumas methods remain common for estimating crude protein based on nitrogen, suitable for nutritional labels. However, more precise techniques like amino acid analysis provide true protein content. Spectrophotometric assays are quick but less accurate, while modern methods like NIR and ultrasonic analysis offer rapid, non-destructive options for quality control. The best method depends on the required accuracy, food type, and practical considerations. Careful method selection is crucial for regulatory compliance and clear consumer information.

Frequently Asked Questions

Crude protein is an estimation of protein content based on the total nitrogen in a food sample, including nitrogen from non-protein sources. True protein is a more accurate measurement that quantifies only the actual protein by summing its constituent amino acids.

The Kjeldahl method involves digesting a food sample with sulfuric acid to convert all nitrogen into ammonium sulfate. The nitrogen is then converted to ammonia, distilled, and titrated to measure its quantity, which is then converted into a crude protein estimate.

The Dumas method involves combusting a food sample at high temperatures to convert all nitrogen into nitrogen gas. A detector then measures the amount of nitrogen gas, and this value is converted into a crude protein estimate. It is faster and more automated than the Kjeldahl method.

Nitrogen-based methods can overestimate protein content because they measure all nitrogen in the sample, including non-protein nitrogen (NPN) from compounds like urea, free amino acids, and creatine. Since the calculation assumes all nitrogen comes from protein, the final value may be higher than the true protein amount.

Spectrophotometric assays, such as the Bradford and Lowry methods, use a colorimetric reaction to measure protein. A reagent is added to the sample, and the resulting color intensity is measured by a spectrophotometer. The color intensity is proportional to the amount of protein.

Direct amino acid analysis using techniques like high-performance liquid chromatography (HPLC) is considered the most accurate method. It quantifies the sum of individual amino acids, providing a true protein content value without reliance on conversion factors or assumptions about non-protein nitrogen.

Yes, modern spectroscopic methods like Near-Infrared (NIR) spectroscopy and ultrasonic analysis are used for rapid, non-destructive protein testing. NIR is often used for in-line quality control of food products, while ultrasonic analysis is common for liquid dairy products.