1. Lab Activity 4 Determination of Nitrogen & Crude Protein
IUG,
Dr. Tarek Zaida

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

3. 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

4. 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)

5. The solution is then distilled with NaOH (added in small quantities) which converts the (NH4)2SO4 + 2NaOH NH3 + Na2SO4 + 2H2O
Amount of NH3 present (hence the amount of nitrogen present in sample) is distilled into boric acid
NH4OH + H3BO NH4H2BO3 + H2O

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

7. Summary Vs –Vb of 0.01 N HCl x 0.14 = mg of N in the sample
1. Digestion:
Protein H2SO (NH4)2SO4 + CO2 + SO2 + H2O
2. Liberation of NH3
(NH4)2SO NaOH NH3 + Na2SO H2O
3. Capturing NH3
NH3 + H2O + H3BO NH4 H2BO3 + H2O
4. Titration of NH3
NH4 H2BO HCl NH4Cl H3BO3
1 ml 0.01N HCl = mg Nitrogen
Vs –Vb of 0.01 N HCl x = 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

8. 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.

9. Experiment
Look at the lab manual for more details