Lab Activity 4 Determination of Nitrogen Crude Protein

Lab Activity 4 Determination of Nitrogen & Crude Protein IUG, 2016 -2017 Dr. Tarek Zaida 1

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

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 3

Principle of the Kjeldahl Method • The method is based on heating a substance with H 2 SO 4, • H 2 SO 4 decomposes the organic substance by oxidation • The reduced nitrogen will liberate as ammonium sulfate (NH 4)2 SO 4 Note: Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark) 4

• The solution is then distilled with Na. OH (added in small quantities) which converts the (NH 4)2 SO 4 + 2 Na. OH 2 NH 3 + Na 2 SO 4 + 2 H 2 O Amount of NH 3 present (hence the amount of nitrogen present in sample) is distilled into boric acid NH 4 OH + H 3 BO 3 5 NH 4 H 2 BO 3 + H 2 O

• The ammonia present in the boric acid is then titrated with standardized hydrochloric acid HCl NH 4 H 2 BO 3 + HCl 6 NH 4 Cl + H 3 BO 3

Summary • 1. Digestion: Protein + H 2 SO 4 2. Liberation of NH 3 (NH 4)2 SO 4 + 2 Na. OH 3. Capturing NH 3 + H 2 O + H 3 BO 3 4. Titration of NH 3 NH 4 H 2 BO 3 + HCl (NH 4)2 SO 4 + CO 2 + SO 2 + H 2 O NH 3 + Na 2 SO 4 + H 2 O NH 4 H 2 BO 3 + H 2 O NH 4 Cl + H 3 BO 3 1 ml 0. 01 N 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 7

• 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. Ø 8 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 9