Determination of the mass fraction of the nitrogen according to Kjeldahl method and the mass fraction of protein

The Kjeldahl method is used for the quantitative analysis of nitrogen-containing organic substances and drugs containing amine, amide and heterocyclic nitrogen.

For the first time the Kjeldahl method was applied for the determination of nitrogen in meat and various tissues without preliminary drying and degreasing of samples by the prominent Russian physiologist I. P. Pavlov together with the D. P. Pavlov.
 
The main advantage of this method is that it allows for determination of the total nitrogen with high accuracy and reproducibility and is currently the most popular official method for determining of nitrogen and protein content.
Despite a number of modifications that the method has undergone during its history while retaining its specificity it has become the standard method for determining nitrogen which is based on a combination of mineralization of organic substances with the subsequent application of acid-base titration.

However, due to the fact that some method’s stages are difficult to automate the method is currently still quite laborious and time consuming.
 
The method includes the following several steps performed successively:
 
Stage 1: Mineralization 

Mineralization is carried out by heating a sample with concentrated sulfuric acid. The purpose of the procedure of wet combustion/ wet ashing is the complete destruction of chemical bonds in a sample of nitrogen and conversion of the total nitrogen to the ammonia ions.
 
For accelerating the process various catalysts are introduced: copper oxide, selenium, lead, etc., the boiling point of the sulfuric acid is increased by adding salts, potassium sulfate or sodium sulfate, besides adding of a catalyst and salts is combined during the wet combustion of the sample.
 
Based on the amount of acid it is possible to assess the amount of nitrogen.
 
R-NH₂ + [O] + H₂SO₄ → CO₂↑ + H₂O + NH₄HSO_4.

 
Stage 2: Distillation
 
Hydrosulfate ammonium is treated with hydroxide sodium and the ammonia evolved is distilled into the receiver containing boric acid solution.
 
NH₄HSO₄ + 2NaOH → NH₃↑ + 2H₂O + Na₂SO_4.
 
Since the boric acid reacts with ammonia to form salts of tetraboric and metaboric acid, ammonium metaborate and tetraborate is formed in a receiver:

NH₃ + Н₃ВО₃ → NH₄ВO₂ + Н₂O,
2NH₃ + 4Н₃ВO₃ → (NH₄)₂В₄O₇ + 5Н₂O.

Stage 3: Titration
The collected distillate containing all the resulting ammonia in the form of meta- and tetraborate ammonium is titrated with 0,1 M solution of hydrochloric acid:
NH₄ВO₂ + НС1 + H₂O → NH₄С1 + Н₃ВО₃,

The Kjeldahl method allows for the determination of the amount of the total nitrogen in any modification.
Mass fraction of protein is calculated by multiplying the obtained value of total nitrogen by the conversion coefficient/ factor of 6.25 assuming that the average protein contains 16% nitrogen.

It is obvious that the results obtained as a result of this conversion are conventional as not the total nitrogen contained in the foodstuffs is in the form of protein, besides the percentage of nitrogen in the proteins is subject to fluctuations in upward and downward by 16%.

In some foodstuffs the nitrogenous substances of nonprotein nature achieve significant amounts (muscle tissue of fish - 15%, animal meats - 10-16% of the total quantity of nitrogenous compounds).

Therefore to obtain more accurate results it is necessary to use different coefficients for recalculation of the total nitrogen to protein depending on the percentage content of nitrogen in the proteins of different foodstuffs/ products: meat and vegetables - 6.25; wheat, rye, peas, etc. - 5.7.; buckwheat, rice - 6.0; milk - 6.37, etc.

To improve the accuracy of the analysis the control test is performed/ carried out simultaneously with the main test/ experiment. The difference between the amount (the number of milliliters) of titrated solution of hydrochloric acid in the main and control test/ experiments, multiplied by 0,0014 corresponds to the amount of nitrogen (g), contained in the test substance.

Since this method is used for a long time, it is constantly improved in terms of energy consumption required space and weight of the control samples.

Analysis of protein by the Kjeldahl method is the most common method, but unfortunately it has some significant drawbacks such as the low speed of analysis, a large number of possible losses/ big quantity of waste in the process of analysis, bulky equipment and the need to use aggressive/ corrosive reagents (concentrated sulfuric acid and alkali solution).

The use of KELTUN semi-automated complex for the determination of nitrogen/ protein eliminates or minimizes the above disadvantages of the Kjeldahl method.

The maximum operating temperature of the digester of KELTRUN is 450°C that significantly reduces the time required for the sample preparation.
 
Optional system for removing and neutralization of the vapors and the vacuum pump are highly efficient.
The operating conditions required for work with this equipment are fully reproducible and safe, allow for the use of the minimum reagents and working volume.
 
The second important step in the analysis is a steam distillation.

The transparent solution obtained at the stage of decomposing cannot be used for the direct determination of ammonium nitrogen due to the high content of interfering components.
For the separation of ammonia nitrogen, it is converted/ transferred to the ammonium form by adding of alkali and is distilled with the steam in special devices called distillers.
 
The use of automated devices helps not only speed up the analysis itself significantly, but also improve the reproducibility of the results. The distiller uses steam generator working on distilled water that does not require maintenance in the process of operation.
 
The performance of the distiller is up to 3 kg water vapor per hour, the sample distillation is carried out within a few minutes.
 
The final stage of the analysis is the determination of the nitrogen ammonium/ ammonia nitrogen in a distilled sample by titration.
 
The ammonia distilled with steam is collected in the flask which is placed preliminarily in the solution of boric acid or sulfuric acid with the known normality. The obtained transparent solution of borate or ammonium sulfate can be easily titrated using the direct or reverse method.

KELTRUN is the set of equipment for conducting of the complete analysis cycle in accordance with the Kjeldahl method in foodstuffs, dairy products, beverages, water, soil, fodder, medical preparations, chemicals, atmospheric precipitation and many other substances.

KELTRUN Complex for the analysis of nitrogen and protein includes:
-       KELTRUN Digestor and Scrubber (Stage 1: Ignition/ wet combustion)
-       KELTRUN Distiller/ (KELTRUN Distillation unit for steam distillation) (Stage2: Distillation)
-       KELTRUN Titration unit: manual or automatic (Stage 3: Titration)

KELTRUN allows for determination nitrogen/ protein content in accordance with the Kjeldahl method in the wide range of products:
* Foodstuffs: dairy products, grain-crops, cereals, meat;
* Feeding stuffs, fodder;
* Fertilizers;
* Water, beverages, beer;
* Soil, bottom sediments, atmospheric precipitation, activated sledge;
* Crude oil, lubricants;
* Different types of chemical substances;
* Alcohol content in wine, wort and spirits;
* Etc.

The analysis allows for determination of nitrogen in the range from 0,02 – to 99,0% and is conducted in accordance with the GOST (State Standards of the Russian Federation).

KELTRUN - Сomplex for the analysis of nitrogen and protein according to the Kjeldahl method is an affordable solution which can be used to minimize and eliminate the drawbacks of the Kjeldahl method and providing convenience and ease of operation.