Kjeldahl's nitrogen determination

Scheme of determination

The Kjeldahl nitrogen determination , sometimes also referred to as Kjeldahlometry in the clinical field , is a quantitative , frequently used determination method of the nitrogen content developed in 1883 . The Danish chemist Johan Kjeldahl developed them at the research center of the Danish brewery Carlsberg . It quickly replaced the previously widespread nitrogen determination according to Will-Varrentrapp . With the Kjeldahl method, nitrogen can be determined in many nitrogen-containing substances, which is reflected in their broad field of application:

• Food industry
• Environmental analysis
• Pharmaceutical and chemical industry
• Agricultural analysis ( manure , soil samples )

In the classic procedure, a precisely weighed amount of sample (0.5 to 3 g, depending on its nitrogen content) is digested with sulfuric acid in a Kjeldahl flask . The organic components of the sample are removed and the nitrogen is converted into ammonium sulfate. The addition of a strong base releases ammonia from the digestion solution, which is collected in acid and determined titrimetrically. The specification is made as Total Kjeldahl Nitrogen .

There are also micro-methods for samples from 5 to 15 mg.

In addition, the entire determination can be automated using fully automatic laboratory devices - even with several samples at the same time - so that the analyzes can be carried out overnight without staff present.

Exposure

During the digestion, the sample is boiled in an open flask with an excess of sulfuric acid. The carbon in the organic material is oxidized to carbon dioxide (CO ₂ ) and sulfuric acid is reduced to sulfur dioxide (SO ₂ ) :

${\displaystyle \mathrm {(CH{\color {Blue}N}O)_{\ (Probe,\ s)}{\xrightarrow {\Delta ,\ H_{2}SO_{4\ (l)}}}\ CO_{2\ (g)}+SO_{2\ (g)}+H_{2}O_{\ (g)}+({\color {Blue}N}H_{4})_{2}SO_{4\ (solv)}} }$

Kjeldahl flask with a sample of slurry in boiling sulfuric acid

A catalyst consisting of Hg, Se, Cu or Ti compounds is used to improve the implementation of the reaction. The digestion temperature in an open flask is limited by the boiling point of the sulfuric acid. In order to achieve a higher temperature, a compound that is difficult to volatilize can be added. To raise the boiling point of this kind , sodium or potassium sulfate (Na ₂ SO ₄ or K ₂ SO ₄ ) are used in the Kjeldah digestion .

Not all substances can be digested directly. If the nitrogen is present in a nitro, nitroso or azo compound, a reduction (for example with zinc or Devard's alloy ) must first be carried out.

In addition to the determination of nitrogen, the sulfuric acid digestion can also be used to determine phosphorus, arsenic and metals in organic material.

Steam distillation

After the digestion, the nitrogen is in the form of ammonium sulfate (NH ₄ ) ₂ SO _{4 dissolved} in sulfuric acid. When a strong base (for example NaOH) is added, the sulfuric acid is neutralized and ammonia is expelled, which can be quantitatively introduced into an acid reservoir by means of steam distillation .

${\displaystyle \mathrm {({\color {Blue}N}H_{4})_{2}SO_{4\ (solv)}+2\ NaOH_{\ (aq)}\longrightarrow Na_{2}SO_{4\ (aq)}+2\ {\color {Blue}N}H_{3}+2\ H_{2}O_{\ (l)}} }$

In principle, any acid can be used to collect it. If a strong acid is used, it must be measured precisely because the acid remaining after the introduction of ammonia is titrated back with a base . If, on the other hand, a weak acid is used, such as boric acid with a p K _s value of 9.25, then the strong base formed during the collection can be titrated with an acid without excess collection acid being recorded. This has the advantage that the weak acid does not have to be measured precisely. Boric acid does not react directly as a proton donor , but as an OH ^- acceptor in the sense of a Lewis acid :

${\displaystyle \mathrm {B(OH)_{3\ (aq)}+H_{2}O+{\color {Blue}N}H_{3\ (g)}\longrightarrow B(OH)_{4\ (aq)}^{-}+{\color {Blue}N}H_{4\ (aq)}^{+}} }$

Titration

For the direct titration of borate, an indicator mixture of methyl red and methylene blue ( Tashiro ) is used, which turns acidic. The volume of standard solution used can be converted into the amount of nitrogen in the sample.

${\displaystyle \mathrm {{\color {Blue}N}H_{4\ (aq)}^{+}+B(OH)_{4\ (aq)}^{-}+HCl_{\ (aq)}\longrightarrow {\color {Blue}N}H_{4}Cl_{\ (aq)}+B(OH)_{3\ (aq)}+H_{2}O} }$

Protein content

The nitrogen content determined using this method is related to the protein content of a biological sample. In most foods, one can assume that the Kjeldahl nitrogen comes mainly from proteins. When converting nitrogen content to protein content, it must be taken into account that the individual amino acids have a different nitrogen content and thus a protein composition that differs far from the average leads to a different conversion factor. This factor must therefore be determined beforehand for various protein sources using another method, such as cleavage of the proteins , followed by determination of the individual amino acids. The nitrogen proportions found in this way in the protein are on average 16%. The analytically determined nitrogen content of a sample must be multiplied by a factor of 6.25 to calculate the protein content. With wheat flour, semolina and haze , the factor is 5.7.

Further deviations can occur if there are other nitrogen sources in addition to the proteins. For example, the melamine scandal in China in 2008 shows the limits of this process: stretching of dairy products in order to simulate a higher protein content with the usual measuring processes.

literature

• Johan Kjeldahl: New method for determining nitrogen in organic bodies. In: Journal for Analytical Chemistry. 1883, pp. 366-382.
• R. Hoegger: Training Papers Nitrogen determination according to Kjeldahl. Büchi Labortechnik AG, Flawil 1998, pp. 1-18.
• R. Bock: Digestion methods in inorganic and organic chemistry. Wiley-VCH, Weinheim 1972, ISBN 978-3527254033 , pp. 142-145.