Nitrogen and protein determination

Paul Boughton

Cereals and seeds are significant components of the human diet and the principal part of feeding stock for domestic animals. One of the most important nutrients of cereals and seeds is protein. The monitoring of protein amount, through the estimation of nitrogen, must be accurate in order to determine the nutritional quality of cereals and seeds. If the amount of nitrogen is multiplied by a factor depending on the kinds of protein expected to be present in the food, then the total protein content can be determined. In addition to its dietary importance, the protein content has also become a guideline for some cereal trade transactions.

This article discusses current legislation governing the analysis of nitrogen content for protein determination in cereals and seeds and examines the use of an advanced combustion technique against the traditional Kjeldahl method. The advantages of the combustion method are further highlighted with an application example.

The Kjeldahl method

This is a method for the quantitative determination of nitrogen in chemical substances. For many years, the Kjeldahl method has been the internationally-recognised method for estimating protein concentration in food, due to its universality, precision and reproducibility.

Currently, it is the standard method against which all other methods are judged. Nevertheless, the Kjeldahl method is associated with many major downfalls. It is not capable of providing measurements of true protein content and different correction factors are required for different proteins to account for different amino acid sequences.

The Kjeldahl method is a multi-stepped process, including sample digestion in boiling sulphuric acid, neutralisation with sodium hydroxide solution, distillation of the resulting ammonia gas into a trapping solution, titration with an acid solution and determination of the amount of nitrogen and protein by calculation. The entire process may require over four hours to be completed while all steps, excluding digestion, require continuous technician contribution.

In addition, handling boiling sulphuric acid, especially with the addition of concentrated caustic solution, is a particularly hazardous task. Kjeldahl analyses also generate toxic waste since they involve the use of mercury or selenium catalysts during the digestion step.

All these disadvantages have ended the dominance of the Kjedahl method as the preferred technique for nitrogen/protein determination in food. On the contrary, the capabilities of the combustion method have been greatly improved to make it faster, safer and more reliable than the traditional Kjeldahl method.

Designed to offer an alternative to the Kjeldahl methodology for dedicated nitrogen and nitrogen/protein analysis in food, the upgraded combustion method features a large sample load of 2g, ensuring maximum accuracy and reliability of results as well as improved productivity capabilities, reducing sample handling time and minimising matrix effects. The technique reduces the need for sample preparation and is capable of processing more samples than the Kjeldahl method in only a few minutes. In addition, consumable costs associated with sample preparation are minimised and safety in the laboratory is increased.

The advanced combustion method features a unique automatic carbon dioxide (CO2) adsorber regenerating technology, which is automatically activated to adsorb the CO2 generated during the combustion.

Unlike traditional technologies, this self-cleaning filter never needs to be changed, saving time and money. This feature improves the autonomy of the system and reduces instrument downtime and maintenance. The benefits of the combustion method are further strengthened by an inherent water condensation drainage device (Peliter) and the combination of a safe, sensitive and reliable thermal conductivity detector (TCD), featuring an extensive working range to cover nitrogen/protein applications from low ppm to high percentage concentrations.


As a direct consequence of these key advantages, the combustion method has been approved and adopted by the Association of Official Analytical Chemists (AOAC) and the American Association of Cereal Chemists (AACC).

The official methodology requirements for protein determination by combustion include AOAC Method 992.23 and AACC Method 46-30, 1999. These methods indicate that the suitable fineness of grind must be determined for each different material analysed to achieve precision that gives RSD (equal or less) of two per cent for ten successive determinations of nitrogen.

The AACC 46-30 crude protein combustion method specifies that nitrogen, freed by pyrolysis and subsequent combustion at high temperature in pure oxygen, is quantified by thermal conductivity detection and converted to equivalent protein by appropriate numerical factor. This generic combustion method is applicable to all flours, cereal grains, oilseeds and animal feeds. Different conversion factors are used for various cereal grains and oilseeds. The AOAC 990.03 protein in animal feed combustion method is applicable to the determination of nitrogen in all types of forages.

Providing nutrition information on food labels is important to public health as it helps consumers to adopt a balanced diet. Current European legislation mandates that only foods claiming a specific nutrition value, for example ‘low fat’ or ‘high in fibre’, such as cereals, should carry nutritional information. These products must label energy content, seven core nutrients including protein, carbohydrates, total fat, saturated fat, trans fat, sodium and sugars and any nutrient for which a claim is made. This regulatory requirement further stresses the need for a powerful protein monitoring method.

A Thermo Scientific FLASH 4000 nitrogen/protein analyser was used to validate the combustion method in terms of accuracy and reproducibility according with the pre-treatment of the sample (Fig. 1). Different cereals and seeds were chosen for this analysis.

Protein content was calculated automatically using the Thermo Scientific Eager Xperience software with default protein factor of 6.25 (5.70 for rice). The protein factor can be changed in accordance with the food type.

Temperature of the left reactor was set at 950°C and 840°C for the right reactor. Temperature of the oven was 50°C. A 500 mg EDTA (9.59 per cent nitrogen) standard was used and a sample weight of 600mg-1.6g. The oxygen amount necessary for the complete combustion of samples was calculated automatically by the OxyTune function present in the Eager Xperience software.

Table 1 shows the nitrogen and protein determination in cereals samples. Barley and rice were homogenised to 1mm and 2mm particle sizes. Data obtained through ten consecutive determinations demonstrated excellent reproducibility. In all cases, the relative standard deviation was less than two per cent according to the official methods. No memory effect was observed when changing the type of sample, indicating the complete detection of nitrogen present in the sample. No significant differences in the results were observed changing the sample particle size from 2 to 1mm.

Table 2 shows the nitrogen and protein determination in sunflower seeds. Due to their sample nature and high content of fat, oil-seeds require a proper optimisation of the oxygen amount needed for combustion to obtain accurate data.

The sample was homogenised at 2mm particle size. Data were reproducible with a RSD percentage as per official method requirements. The left part shows the reproducibility of ten determinations using a weight of around 500mg, while the right part indicates the reproducibility of ten determinations in a range from 700-1000mg of the same sunflower seed sample. No memory effect was observed when changing sample weight.


Food safety is a growing and important market that requires superior analytical accuracy in order to ensure optimum productivity. The conventional Kjeldahl method for nitrogen determination is extremely time consuming, requiring extensive sample preparation and handling and using various unsafe chemicals and solutions. The Kjeldahl method is also associated with high waste costs and is incapable of operating continuously while being strongly dependent on the user’s experience. As a consequence, productivity is considerably jeopardised.

However, recent advances of the combustion method have made this technique perfectly suitable to cope effortlessly with the wide array of food safety laboratory requirements such as accuracy, day-to-day reproducibility and high sample throughput.

The Authors work for Thermo Fisher Scientific in Milan, Italy. Dr Liliana Krotz is OEA Product Specialist, Dr Elena Ciceri is OEA Analytical Product Development and Dr Guido Giazzi is OEA Product Manager. Tel: +1 800-532-4752 email: