Nitrogen and calculation of crude protein
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1 Nitrogen and calculation of crude protein Ing. Petrová Jaroslava Workshop on Official Controls of Feed AGR 46230, , Ankara. Turkey ÚKZÚZ - NRL RO Praha 1
2 Content of this presentation 1. Methods of determination 2. Kjedahl 3. Dumas 4. Conclusion ÚKZÚZ - NRL RO Praha 2
3 Propojený obrázek nelze zobrazit. Příslušný soubor byl pravděpodobně přesunut, přejmenován nebo odstraněn. Ověřte, zda propojení odkazuje na správný soubor a umístění. 1. Methods of determination REGULATION (EC) No 152/2009, Annex III, C. Determination of content of crude protein (CSN) EN ISO Animal feeding stuffs - Determination of nitrogen content and calculation of crude protein content -Part 1: Kjeldahl method (CSN) EN ISO Animal feeding stuffs - Determination of nitrogen content and calculation of crude protein content -Part 2: Block digestion and steam distillation method (CSN) EN ISO Food products - Determination of the total nitrogen content by combustion according to the Dumas principle and calculation of the crude protein content - Part 1: Oilseeds and animal feeding stuffs ÚKZÚZ - NRL RO Praha 3
4 2. Kjeldahl method Consisted of : a) Digestion b) distillation and c) back titration. a) sulphuric acid reacted with organic compounds and ammonium salts were produced b) Ammonium salts reacted with lye and ammonia was released by distillation into. a standardized solution of hydrochloric acid or sulphuric acid. c) This solution was back titrated with caustic soda to indirectly measure nitrogen. During the 1880s, Kjeldahl used potassium sulphate to raise the boiling point of the acid and mercury as a catalyst to speed the decomposition. For the back titration process of the released ammonia, he used boric acid buffer solution. The method does not measure oxidized forms of nitrogen nor heterocyclic nitrogen compounds. The method does not distinguish between protein nitrogen and non-protein nitrogen ÚKZÚZ - NRL RO Praha 4
5 EN ISO Kjeldahl - ISO EN method Digestion of organic matter by sulphuric acid in the presence of a catalyst. Rendering of the reaction product in alkaline, then distillation and titration of the liberated ammonia. Calculation of the nitrogen content. Multiplication of the result by the conventional factor to obtain the crude protein content. Digestion - Sulphuric acid Catalyst - Copper(II), potassium sulphate Releasing agent - 33 % Sodium hydroxide solution. Collecting liquid - Sulphuric acid or Boric acid Solution for titration - Sodium hydroxide or Sulphuric acid ÚKZÚZ - NRL RO Praha 5
6 EN ISO Kjeldahl - ISO EN method Digestion in a block-digestion or equivalent apparatus. Distillation on manual, semi-automatic or fully automatic steam distillation unit. Manual or semi-automatic distillation - collection of ammonia in excess of boric acid. Titration with hydrochloric acid solution to a colorimetric endpoint. Automatic distillation - collection of ammonia in excess of boric acid with simultaneously titration. NOTE 1 Detection of endpoint by potentiometric ph system is also possible ÚKZÚZ - NRL RO Praha 6
7 2. Kjeldahl EC REGULATION method The sample is digested by sulphuric acid in the presence of a catalyst. The ammonia is distilled and collected in sulphuric acid standard solution, the excess of which is titrated with sodium hydroxide standard solution. Alternatively, the liberated ammonia is distilled into an excess of boric acid solution, followed by titration with hydrochloric acid or sulphuric acid solution ÚKZÚZ - NRL RO Praha 7
8 2. Kjeldahl Principle of all method is the same, Digestion - Sulphuric acid Catalyst - Copper(II), potassium sulphate Releasing agent - 33 % Sodium hydroxide solution. Collecting liquid - Sulphuric acid or Boric acid Solution for titration - Sodium hydroxide, Sulphuric acid or Hydrochloric acid ISO Scope: animal feedingstuffs Repeatability, Reproducibility, Annex differences in validation ISO Scope: animal feeding stuffs, pet foods and their raw materials, LOQ 0,5 % nitrogen Repeatability, Reproducibility, Annex EU method Scope: feed Repeatability ÚKZÚZ - NRL RO Praha 8
9 3. Dumas method In 1833, Jean-Babtiste Dumas developed a method for estimating the amount of elementary nitrogen in an organic compounds. Combustion (oxygen atmosphere, about 900 C ) Separation of releasing gases (CO2, H2O and N2) and removing all interfering components Measurement of nitrogen content by a thermal conductivity detector External calibration, e.g. EDTA (= 9.59%N) ÚKZÚZ - NRL RO Praha 9
10 3. Dumas method EN ISO Cereals, pulses, milled cereal products, oilseeds, oilseed residues and animal feeding stuffs Determination of total nitrogen and crude protein contents by combustion according to the Dumas principle Scope: Cereals, pulses, milled cereal products, oilseeds, oilseed residues and animal feeding stuffs Validation: Repeatability Reproducibility Annex ÚKZÚZ - NRL RO Praha 10
11 4. Conclusion Comparison between Kjedahl and Dumas method Kjeldahl method Advantages: universal, highly precise and has good reproducibility. Disadvantages: The use of concentrated sulphuric acid at high temperatures poses a considerable hazard. The technique is time consuming. Dumas method Advantages: much faster, doesn't need toxic chemicals or catalysts, automatic measurement, easy to use. Disadvantages: High initial cost, small sample size makes it difficult to obtain a representative sample, external calibration by pure material with a known nitrogen content, e.g. as EDTA (= 9.59%N) ÚKZÚZ - NRL RO Praha 11
12 4. Conclusion Comparison results Kjedahl and Dumas method 28, , , , ÚKZÚZ - NRL RO Praha 12
13 Thank you for your attention ÚKZÚZ - NRL RO Praha 13
Amino acids. Ing. Petrová Jaroslava. Workshop on Official Controls of Feed AGR 46230, , Ankara. Turkey ÚKZÚZ - NRL RO Praha 1
Amino acids Ing. Petrová Jaroslava Workshop on Official Controls of Feed AGR 46230, 6. 7. 12. 2011, Ankara. Turkey 6.12.2011 ÚKZÚZ - NRL RO Praha 1 Content of this presentation 1. Function of amino acids
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