How Do They Calculate Protein? Understanding the Methods in Food and Science

October 15, 2025 •

5 min read

The Kjeldahl method for determining nitrogen—from which protein content is often estimated—was first developed in 1883 and remains a standard for comparison. This highlights that when we ask, 'How do they calculate protein?', we are delving into a long history of chemical analysis used for nutrition and science.

Quick Summary

This article explores the various analytical methods used to quantify protein in food and scientific samples, including the nitrogen-based Kjeldahl and Dumas techniques and the more modern colorimetric assays. It explains the principles, applications, and limitations of each approach.

Key Points

Nitrogen-based methods: The traditional Kjeldahl method and the modern Dumas method calculate "crude protein" by measuring total nitrogen and multiplying by a conversion factor, most commonly 6.25.
Crude vs. True Protein: Nitrogen-based methods measure total nitrogen, including non-protein nitrogen (NPN), which can lead to an overestimation of actual protein content.
Colorimetric Assays: Laboratory techniques like the Bradford, Lowry, and BCA assays quantify protein concentration by measuring a color change that occurs when reagents bind to proteins.
Bradford Assay Mechanics: The Bradford assay relies on the binding of Coomassie dye to basic amino acid residues, causing a color shift from red to blue that is measured by a spectrophotometer.
Amino Acid Analysis: The most accurate method for determining "true protein" involves hydrolyzing the sample and quantifying individual amino acids using sophisticated techniques like HPLC.
Method Choice Matters: The best protein calculation method depends on the context, balancing speed, cost, and the required level of accuracy. No single method is perfect for all applications.
Specific Conversion Factors: While 6.25 is a common factor, different foods and proteins require specific conversion factors for more accurate calculation due to varying amino acid compositions.

The Core Principle: From Nitrogen to Crude Protein

For over a century, the most common way to calculate protein content in food has been an indirect method based on measuring the nitrogen content. This approach relies on the fact that proteins are polymers of amino acids, which contain nitrogen. On average, proteins contain about 16% nitrogen by mass. This observation led to the adoption of a standard conversion factor, N x 6.25 (1/0.16), to estimate protein content from the measured nitrogen levels.

This method, however, measures what is known as "crude protein," not true protein. The value is considered 'crude' because it includes all nitrogenous compounds in the sample, not just those from protein. Non-protein nitrogen (NPN) sources like free amino acids, nucleotides, and urea can contribute to the final reading, leading to an overestimation of the actual protein content.

Nitrogen-Based Methods: The Kjeldahl and Dumas Techniques

The Kjeldahl Method

Developed in 1883, the Kjeldahl method was for decades the international reference for estimating protein in many materials, including dairy products and cereals. It is a reliable but lengthy and labor-intensive wet chemistry process involving three main steps: digestion, distillation, and titration.

The Kjeldahl process involves these steps:

Digestion: A food sample is heated with concentrated sulfuric acid and a catalyst, which breaks down organic matter and converts all organic nitrogen into ammonium sulfate.
Distillation: The solution is made alkaline with sodium hydroxide, converting the ammonium ions to ammonia gas, which is then distilled and captured in a boric acid solution.
Titration: The amount of trapped ammonia is determined by titrating the boric acid solution with a standard acid.

The Dumas Method

The Dumas method is a more modern, automated alternative to the Kjeldahl method that also measures total nitrogen. It overcomes some of the limitations of the Kjeldahl process, such as the use of hazardous chemicals. It is faster, taking only a few minutes per sample.

The Dumas process involves:

Combustion: A small, weighed sample is burned in a high-temperature furnace (approx. 900°C) with pure oxygen.
Gas Analysis: The combustion products, including N₂ gas, are passed through columns to remove interfering gases like CO₂ and water vapor.
Nitrogen Detection: The remaining N₂ is measured by a thermal conductivity detector.

Colorimetric and Dye-Binding Methods

In laboratory settings, especially for research involving isolated proteins, colorimetric assays are frequently used. These methods are fast and sensitive, relying on a color change that is directly proportional to protein concentration. They are particularly useful for quantifying proteins in small biological samples, such as cell lysates or serum.

Bradford Assay: This assay uses Coomassie Brilliant Blue G-250 dye, which binds to basic amino acid residues (arginine, lysine, and histidine) under acidic conditions. When the dye binds to protein, it shifts from a reddish-brown color to blue, and the change is measured by a spectrophotometer at 595 nm. A standard curve, typically made with known concentrations of bovine serum albumin (BSA), is used to determine the concentration of unknown samples.
Bicinchoninic Acid (BCA) Assay: A two-step assay, the BCA method involves the reduction of copper ions by protein in an alkaline medium. The cuprous ions formed then react with BCA to produce a purple-colored product that is measured at 562 nm. BCA is less susceptible to interference from detergents than the Bradford assay.
Lowry Assay: This is a more sensitive, but also more time-consuming, variation of the biuret method. It uses the Folin-Ciocalteu reagent in addition to copper chelation to produce an intense blue color. It is more sensitive than the biuret method but prone to interference from various compounds.

Specialized and Modern Techniques

Amino Acid Analysis

For the most accurate determination of 'true protein,' analysts can use methods that quantify the individual amino acid content of a food sample. This involves hydrolyzing the protein with strong acid and then using high-performance liquid chromatography (HPLC) to separate and quantify the individual amino acids. This approach is complex and expensive, but it bypasses the inaccuracies associated with the nitrogen-based conversion factors.

Near-Infrared (NIR) Spectroscopy

In the food industry, especially for quality control, rapid and non-destructive methods are often preferred. NIR spectroscopy analyzes the protein content by measuring the absorption of light waves in the near-infrared spectrum. It is extremely fast and requires minimal sample preparation once calibrated against a reference method like Kjeldahl.

Comparison of Major Protein Calculation Methods


Feature	Kjeldahl Method	Dumas Method	Bradford Assay	Amino Acid Analysis
Principle	Measures total nitrogen; uses conversion factor.	Measures total nitrogen via combustion.	Dye binds to basic amino acids, causing a color change.	Quantifies individual amino acids after hydrolysis.
Accuracy	Provides a "crude" estimate; overestimates if NPN is present.	Also provides a "crude" estimate; subject to same NPN limitations.	Dependent on amino acid composition; can vary between proteins.	Considered the most accurate method for "true" protein.
Speed	Time-consuming; 30 minutes to 2 hours or more.	Fast and automated; under 5 minutes per sample.	Very fast; results in under 10 minutes.	Very slow; requires extensive sample preparation.
Cost	Relatively low cost per sample, but requires trained personnel and reagents.	High initial cost for equipment, but lower running cost per sample.	Inexpensive and simple reagents.	Requires very expensive and specialized HPLC equipment.
Hazard	Uses concentrated sulfuric acid and other hazardous reagents.	Generally safer, as it relies on combustion rather than corrosive chemicals.	Uses acidic reagent, but generally safer than Kjeldahl.	Involves strong acid for hydrolysis, but is highly controlled.
Interferences	Affected by non-protein nitrogen (NPN) sources.	Also affected by NPN sources.	Interfered with by detergents.	Minimal interference if sample preparation is done correctly.

Conclusion

There is no single universal way to calculate protein, but rather a collection of methods chosen based on the specific application, desired accuracy, and available resources. For official food labeling, nitrogen-based methods like Kjeldahl and Dumas are still common, though their "crude protein" results can sometimes be misleading due to non-protein nitrogen. In research labs, fast and sensitive colorimetric assays like the Bradford method are staples for quantifying protein in purified samples. However, the gold standard for precision, particularly for evaluating protein quality and dietary requirements, remains the costly and time-consuming amino acid analysis. The choice of method, therefore, is a careful balancing act between speed, cost, and analytical rigor.

Authoritative Reference on Protein Analysis Methods

Frequently Asked Questions

The 6.25 conversion factor is a historic and common multiplier used to estimate protein content from a sample's total nitrogen content. It's based on the assumption that proteins contain approximately 16% nitrogen on average.

Crude protein is an estimation based on total nitrogen content, including non-protein nitrogen sources. True protein is a more accurate measurement derived from quantifying the actual amino acid content of a sample, excluding any non-protein nitrogen.

The Kjeldahl method has historically been the standard and is highly reproducible. Many regulations and databases are based on Kjeldahl results. Modern variants or alternatives like the Dumas method offer speed improvements while still relying on the same nitrogen-to-protein conversion principle.

The Bradford assay can be inaccurate if the protein has an unusual amino acid composition (especially a high or low number of basic residues). It is also highly susceptible to interference from detergents.

In laboratory settings, scientists often use fast and sensitive colorimetric assays like the Bradford or BCA (Bicinchoninic Acid) assays to quantify total protein concentration in cell lysates or other biological fluids.

Many food manufacturers use automated methods like the Dumas technique or rapid spectroscopic techniques such as Near-Infrared (NIR) spectroscopy for fast and efficient quality control checks on protein content.

Food fraud incidents have occurred where nitrogen-rich, non-protein compounds (like melamine) were added to products to artificially inflate the apparent protein content as measured by nitrogen-based methods like Kjeldahl. This highlights a key limitation of these indirect measurement techniques.