As stated by international labeling regulations in the food & feed industry as well as research facilities, the calculation of the total protein content is a vital means of quality control and protein declaration.
Protein content can be directly related to product features and categorizations, for example, dough properties, foam development, the flavor of the beer, or the variation between starch and gluten-free starch. In types of applications, it is vital to have matrix-independent protein analyses. Theoretically, protein quantification is realized through a sequence of specific and non-specific physical and chemical reactions, with subsequent appropriate detection.
As a result of the need for fully automated methodologies that meet industry standards, two distinct methods have been broadly accepted within modern laboratory operations for the calculation of total protein content. These are Kjeldahl’s wet chemical method and Dumas’ high-temperature combustion method. Near-infrared spectroscopy (NIRS) can be employed as a secondary means of analysis, but this process necessitates a primary method for calibration.
For over a century, the Kjeldahl method was the most frequently applied method and the industry standard for the determination of the total protein content of food products. As an analysis involving wet chemicals, it necessitates excessive time and labor costs, as well as involving toxic and hazardous chemicals.
In the modern era, this is undesirable for both safety-based and economic motives. It is for this reason that in recent times, the Kjeldahl method is increasingly being replaced by the Dumas principle.
Combustion versus Wet Chemistry
The Dumas principle is dependent on the quantitative conversion of the sample into distinct gaseous species at 950 °C in an oxygenated environment. Throughout the combustion phase, the total quantity of nitrogen in the sample is converted to nitrogen oxides, which are subsequently condensed to nitrogen gas and measured with a thermal conductivity sensor.
Ahead of this quantification, all other combustion gases, including surplus oxygen, are confined or absorbed. The analysis can be carried out in as little as four minutes. Figure 1 demonstrates the functional principle of Elementar’s state-of-the-art N/protein analyzer rapid N exceed, which offers a great reduction in cost per analysis through the employment of the EAS REGAINER® technology.
Figure 1. The functional principle of the rapid N exceed utilizing the EAS REGAINER technology.
In the initial stage of the Kjeldahl principle, the sample is digested with the application of concentrated sulfuric acid and a catalyst at 420 °C for 90 minutes. Throughout this procedure, the nitrogen in the sample is transformed into ammonium sulfate.
For the following stage, the ammonium solution is supplemented with sodium hydroxide. The released ammonia is distilled off and sealed in a solution of boric acid. To finish, the ammonium borate complex is titrated with sulfuric acid or hydrochloric acid. These two steps take around five minutes in total. A complete analysis requires around 100 minutes to account for the initial digestion step with the required heating and cooling time.
Comparability of Methods
While the key methodologies of the Kjeldahl and Dumas methods are quite distinct from one another, they share the same end goal of calculating the total nitrogen content of the sample. For food samples, a linear relationship between nitrogen and protein content has been clearly established.
The nitrogen concentration can thus be converted to protein content, through the use of an appropriate factor. The factor differs and is contingent on the relative amount of diverse proteins and their amino acid structure (see Table 1). Neither method distinguishes protein and non-protein nitrogen.
Table 1. Selected factors for different sample matrices.
In the majority of instances, the results achieved through the Dumas method are marginally higher than those of the Kjeldahl method. This is the result of a superior nitrogen recovery through the Dumas method against Kjeldahl.
There are a great number of scientific studies available that offer comparisons between the Kjeldahl and Dumas methods for a broad variety of distinct sample types. When considering that the calculated protein content in both approaches is based solely on estimates, choosing the calculation factor with great care is vital.
As suggested by Simonne et al.1 for specific food groups, a conversion between Kjeldahl and Dumas values using suitable coefficients can be carried out, however, as Željko et al.2 demonstrated, for the majority of cases, there is no statistically significant difference between the two methods.
Throughput and Amount of Work
The preparation of samples for Dumas analyses is rapid and easy. Samples are measured out into tin cups, covered and positioned in the autosampler of the rapid N exceed. For the rapid MAX N exceed, the samples are measured out into refillable stainless steel crucibles before being positioned in the autosampler.
The initial measurement can be instigated with under 60 seconds of sample preparation for both cases. As the rapid MAX N exceed offers up to 90 sample positions, it is possible to carry out runs longer than 7 hours without operator supervision. As such, up to 200 samples can be determined in a standard daily routine.
Conversely, in order to prepare samples for Kjeldahl analysis, it is necessary not only to weigh the sample but also to add a precise quantity of sulfuric acid solution and catalyst tablets. As previously noted, the total analysis time is, therefore, a minimum of 100 minutes.
The daily output of a Kjeldahl analyzer can be improved through batch processing. This approach can allow for up to 20 samples per batch, as the time required to carry out the distillation/titration stage for the entire batch matches the time needed for the digestion step. The ultimate daily sample limit for a Kjeldahl analyzer is therefore set at 100 per day (Figure 1).
While both steps are undertaken automatically, a manual sample transfer between the two modules is unavoidable, and thus, the analyzer can only run without operator support for a total of 100 minutes.
Figure 2. Comparison of typically daily operation with Dumas and Kjeldahl analyzers.
Cost Per Analysis
Combustion instruments conventionally use heated metals such as copper or tungsten to bind surplus oxygen and condense formed nitrogen oxides to N2. In this instance, the reduction metal suffers corrosion, usually turning it into an inactive metal oxide, following around 200 - 300 samples when considering whole gas analysis. The reduction metal along with the carrier gas is therefore the main cost factors.
Elementar's exclusive EAS REGAINER technology offers a reduction metal lifespan of up to five times higher while lessening maintenance requirements by a factor of up to five. Taking into account suggested end-user prices, the price per analysis totals around 0.25€ in the case of the rapid N exceed and 0.49€ for the rapid MAX N exceed, accounting for all necessary reagents and gases.
The main elements driving up costs in Kjeldahl analyses are the expenses of purchasing chemicals, along with staff costs to process them, and their safe disposal. This leads to a total cost per analysis of around 6.00€.
As seen in Table 2, a typical Kjeldahl methodology for 2000 samples results in chemical waste of quantities up to 560 liters, in comparison to a trivial amount of only 430 g solids for the rapid N exceed.
Table 2. Comparison of reagent usage for 2000 samples.
The EAS REGAINER reagent is entirely used up in the course of the analysis and therefore does not leave behind any solid waste. It should be noted that labor costs have not so far been incorporated into the calculations. The Dumas method allows for the analysis of two times as many samples each day, in 1/3 less working time than the Kjeldahl method.
Standards Using the Dumas Method
The shift away from the Kjeldahl method towards the Dumas method also accounts for the growing number of valid standards globally which classify the determination of total protein content in accordance with the Dumas principle.
Table 3. List of the most important international and national standards.
It has been demonstrated that in sample processing, quantities of chemical waste, laboratory safety, labor intensity, and cost-per-analysis, the Dumas method possesses a number of clear benefits over the Kjeldahl method. Moreover, Dumas’ high degree of automation decreases the risk of errors.
With Elementar’s state-of-the-art N/Protein analyzers, the cost-per-analysis was radically lowered in comparison to traditional Dumas analyzers. Even with just ten daily samples, a rapid N exceed is an economically sound investment. It also offers security and confidence in potential future growths of samples.
With all this in mind, the Kjeldahl analyzer’s low initial investment cost for entry-level machines can no longer be considered a key reason to buy.
Sources and Further Reading
- Simonne, A.H., Simonne, E.H., Eitenmiller, R.R., Mills, H.A., Cresman III, C.P. 1997. Could the Dumas method replace the Kjeldahl digestion for nitrogen and crude protein determinations in foods? Journal of the Science of Food and Agriculture, 73:39-45.
- Željko, A.M., Sandra M.J., Nadežda, B.P., Željko, N.Ć., Milica, M.Ž., Comparison of the Kjeldahl method, Dumas method and NIR method for total nitrogen determination in meat and meat products Journal of Agroalimentary Processes and Technologies 2015, 21(4), 365-370.
This information has been sourced, reviewed and adapted from materials provided by Elementar Analysensysteme GmbH.
For more information on this source, please visit Elementar Analysensysteme GmbH.