Lab Activity 4 Determination of Nitrogen & Crude Protein IUG, 2016-2017 Dr. Tarek Zaida

Methods used for protein determination 1- Total Nitrogen 2- Kjeldahl 3- UV 4- Refractometry 5- Biuret 6- Dye- binding

Introduction The percentage composition of a protein in nitrogen is 16% or 1/6 The quantitative determination of nitrogen in chemical substances was developed by Johan Kjeldahl in 1883

Principle of the Kjeldahl Method The method is based on heating a substance with H2SO4, H2SO4 decomposes the organic substance by oxidation The reduced nitrogen will liberate as ammonium sulfate (NH4)2SO4 Note: Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark)

The solution is then distilled with NaOH (added in small quantities) which converts the (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O Amount of NH3 present (hence the amount of nitrogen present in sample) is distilled into boric acid NH4OH + H3BO3 NH4H2BO3 + H2O

The ammonia present in the boric acid is then titrated with standardized hydrochloric acid HCl NH4H2BO3 + HCl NH4Cl + H3BO3

Summary Vs –Vb of 0.01 N HCl x 0.14 = mg of N in the sample 1. Digestion: Protein + H2SO4 (NH4)2SO4 + CO2 + SO2 + H2O 2. Liberation of NH3 (NH4)2SO4 + 2NaOH NH3 + Na2SO4 + H2O 3. Capturing NH3 NH3 + H2O + H3BO3 NH4 H2BO3 + H2O 4. Titration of NH3 NH4 H2BO3 + HCl NH4Cl + H3BO3 1 ml 0.01N HCl = 0.14 mg Nitrogen Vs –Vb of 0.01 N HCl x 0.14 = mg of N in the sample Where Vs : ml of 0.01 N used in sample titration Vb : ml of 0.01 N HCl used in blank titration Crude protein = total N% x 6.25

kjeldahl method is a universe method, precise, reproducible, therefore it is recognized as a good method for estimating the protein content in food It does not however give a measure of true protein content, as it measures non protein nitrogen in addition to the nitrogen in proteins. In 2007 there was a pet food incident, and in 2008 another incident recorded in the Chinese milk powder scandal due to the use of melamine; a nitrogen-rich chemical added to raw material to fake high protein contents.

Experiment Look at the lab manual for more details