How do scientists know how much protein is in food?: The Analytical Methods Behind Nutrition Labels

October 30, 2025 •

5 min read

On average, proteins contain about 16% nitrogen by weight. This fundamental chemical property is the key to understanding how do scientists know how much protein is in food, using a variety of sophisticated methods for nutritional analysis.

Quick Summary

Scientists quantify protein content in food using several methods, including traditional Kjeldahl and modern Dumas techniques that measure total nitrogen, and more precise amino acid analysis.

Key Points

Indirect Measurement: Most common methods like Kjeldahl and Dumas measure total nitrogen content, which is then multiplied by a conversion factor to estimate protein.
The NPN Problem: Both Kjeldahl and Dumas methods can overestimate protein because they also detect non-protein nitrogen compounds (NPN) like free amino acids and creatine.
Direct Measurement (Gold Standard): The most accurate method is amino acid analysis, which breaks down the protein and quantifies each individual amino acid, thereby measuring true protein.
Speed vs. Accuracy: The modern Dumas method is fast and automated, ideal for routine quality control, while the traditional Kjeldahl method is slower but still widely accepted as a reference standard.
Reference Databases: Much of the data on nutrition labels comes from extensive food composition databases, which compile nutritional averages based on standardized laboratory analysis.
Purpose-Driven Methods: The choice of method depends on the application, with high-accuracy, complex methods used for specific research and quality assurance, and faster, cost-effective methods for routine production checks.

Determining the protein content of food is a fundamental process in nutrition research, quality control, and for creating accurate nutritional labels. However, it's not as simple as weighing the protein itself. Food scientists and analysts must use specialized, and sometimes complex, analytical techniques to accurately measure protein levels. The primary challenge lies in the fact that not all nitrogen in food comes from protein—a problem that has led to the evolution of different measurement methods over time.

Indirect Methods: Measuring Nitrogen Content

For over a century, the standard approach has been to measure the total nitrogen in a food sample and then convert it to a protein value. This approach is based on the assumption that protein contains a relatively constant percentage of nitrogen.

The Kjeldahl Method: The Gold Standard

Developed in 1883, the Kjeldahl method is a wet-chemistry technique that has long been the international reference standard for protein determination. The process involves three main steps:

Digestion: A food sample is heated with concentrated sulfuric acid and a catalyst, which breaks down the organic matter and converts all organic nitrogen into ammonium sulfate.
Distillation: The solution is neutralized with sodium hydroxide, releasing ammonia gas, which is then captured in a boric acid solution.
Titration: The captured ammonia is quantified by titration with a standard acid, allowing the total nitrogen content to be calculated.

Finally, this nitrogen content is multiplied by a conversion factor (most commonly 6.25, reflecting the average 16% nitrogen content of proteins) to estimate the 'crude protein' amount. This method is highly precise and reproducible but has some notable limitations, including its use of hazardous chemicals and a time-consuming procedure.

The Dumas Method: A Modern Alternative

As a faster, safer, and more automated alternative, the Dumas method has gained significant traction since the 1990s. It is based on the principle of combustion analysis:

A food sample is burned at a high temperature (around 900°C) in a pure oxygen environment.
This combustion releases all nitrogen from the sample as nitrogen gas ($N_2$) and other gases.
The gas mixture is passed through a series of columns to remove other products like carbon dioxide and water.
The remaining nitrogen gas is measured by a thermal conductivity detector, and the total nitrogen is then calculated and converted to protein using a factor.

Compared to Kjeldahl, the Dumas method is much faster (a few minutes vs. an hour or more), uses no toxic reagents, and is well-suited for high-throughput analysis. However, similar to Kjeldahl, it does not distinguish between protein and non-protein nitrogen (NPN), which can lead to overestimation.

Direct Methods: Measuring Amino Acid Content

For the most accurate measurement of 'true protein'—the nitrogen that is actually bound within amino acid chains—scientists can perform a direct amino acid analysis. This method bypasses the reliance on conversion factors and the NPN problem.

High-Performance Liquid Chromatography (HPLC)

This method involves the following steps:

Hydrolysis: The protein is first broken down into its constituent amino acids using strong acid, typically hydrochloric acid.
Derivatization: The individual amino acids are then modified with a chemical reagent to make them more easily detectable.
Chromatographic Separation: The sample is injected into an HPLC machine, which separates the amino acids based on their chemical properties.
Detection and Quantification: As each amino acid emerges, a detector measures its quantity, and the total protein is calculated by summing the mass of all individual amino acids.

The major advantage of amino acid analysis is its high accuracy, making it the reference method recommended by the Food and Agriculture Organization (FAO) for determining true protein content. The main drawbacks are the high cost of equipment and reagents, plus the complexity and time-consuming nature of the procedure, which is why it's not used for routine analysis in many labs.

Other Spectrophotometric and Physical Methods

In addition to the primary methods, other techniques exist for specific applications, particularly for research and quality control, though they may have limitations for general food analysis:

Bradford, Lowry, and Biuret Assays: These are colorimetric assays that measure protein concentration based on the reaction of reagents with specific chemical groups on proteins. They are less accurate for complex food matrices due to interference from other compounds.
Infrared (IR) Spectroscopy: This rapid, non-destructive method measures protein content based on characteristic absorption patterns of the peptide bonds. It is primarily used for rapid quality control but requires extensive calibration against a standard method like Kjeldahl or Dumas.
Nuclear Magnetic Resonance (NMR) Spectroscopy: Similar to IR, NMR can quantify protein by measuring signals from the protein fraction. It's fast and non-destructive but very expensive and requires robust calibration.

Comparison of Protein Analysis Methods


Feature	Kjeldahl Method	Dumas Method	Amino Acid Analysis (HPLC)
Principle	Measures total nitrogen via wet digestion, distillation, and titration.	Measures total nitrogen via high-temperature combustion.	Measures true protein by quantifying individual amino acids after hydrolysis.
Speed	Slow (approx. 1-2 hours per sample).	Fast (approx. 3-5 minutes per sample).	Slow and complex (involves multiple steps).
Accuracy	Good, but overestimates protein by including non-protein nitrogen (NPN).	Good, but also overestimates protein due to NPN and can be affected by certain matrices.	High; measures only true protein, avoiding the NPN issue.
Cost	Relatively low initial equipment cost, but includes chemical disposal.	High initial equipment cost, but low cost per sample and no hazardous chemical waste.	High equipment and operational costs.
Chemicals	Uses hazardous sulfuric acid and other corrosive reagents.	No hazardous wet chemicals used.	Uses hydrochloric acid for hydrolysis and other reagents.
Primary Use	Traditional regulatory standard and reference method.	High-throughput labs, routine quality control.	High-accuracy research, verification, and determining specific protein quality.

The Role of Food Composition Databases

Beyond lab testing, much of the nutritional data found on food labels is compiled from comprehensive food composition databases (FCDBs). These databases, maintained by organizations like the USDA and the FAO, contain average nutritional information for a vast range of food items based on extensive laboratory analysis. Food manufacturers and analysts use these standardized data sets to calculate nutritional information for their products, rather than conducting new tests for every batch.

Conclusion: More Than Meets the Label

Determining the protein content of food is a sophisticated process involving several analytical techniques, each with distinct advantages and drawbacks. Indirect methods like Kjeldahl and Dumas are efficient for measuring total nitrogen but may slightly overestimate true protein due to the presence of non-protein nitrogen compounds. Direct amino acid analysis provides the most accurate measurement of true protein but is more complex and costly. Ultimately, the choice of method depends on the required level of accuracy and the analytical context. When you read a nutrition label, it's the result of this rigorous scientific effort, whether through direct testing or referencing authoritative food composition databases. For example, a manufacturer might choose the rapid, high-throughput Dumas method for routine quality control of their protein powder production, while a research lab developing a novel infant formula would rely on the higher precision of amino acid analysis to ensure complete nutritional adequacy.

For a deeper dive into how food standards are set, visit the Food and Agriculture Organization website, an authority on global food composition data and analysis methods. https://www.fao.org/home/en/

Frequently Asked Questions

Crude protein is an estimate based on a food's total nitrogen content, which includes both protein and non-protein nitrogen (NPN). True protein, measured by amino acid analysis, is the actual mass of the protein, excluding NPN compounds.

The Kjeldahl method has a long history as a globally recognized and highly precise reference method. Its results are often used to validate newer, faster methods like the Dumas technique, and it is still required by some regulatory bodies for specific applications.

The Dumas method is significantly faster because it relies on high-temperature combustion and automated gas analysis, which takes minutes, compared to the hours required for the multi-stage wet-chemistry process of the Kjeldahl method.

A nitrogen-to-protein conversion factor is a multiplier used to convert measured total nitrogen into an estimate of crude protein. The standard factor is 6.25, though more specific factors ('Jones factors') exist for different food types based on their typical amino acid composition.

Yes, methods that measure total nitrogen (Kjeldahl and Dumas) can be susceptible to food adulteration. In some past food scandals, nitrogen-rich compounds like melamine were added to products to artificially inflate the protein content based on these tests.

While amino acid analysis provides the most accurate measure of 'true protein,' its high cost, complexity, and slow processing time make it impractical for routine use. It is typically reserved for specialized applications where high precision is critical.

Most manufacturers use standardized food composition databases (FCDBs) or conduct tests using validated methods like Dumas or Kjeldahl. The results from these analyses are then used to calculate the average protein content shown on the Nutrition Facts panel.