Protein Determination

Total nitrogen in the samples may be estimated by conventional Micro – Kjeldahl method. Multiply nitrogen contents with suitable factor to calculate crude protein content.  Generally, 16% nitrogen is present in proteins. So 100/16 = 6.25 factor may be used to convert nitrogen content to protein. If nitrogen content in protein in a particular crop is more, value of the factor will be low. So, factor for conversion of nitrogen to crude protein varies from crop to crop. Nitrogen present in proteins or other organic compounds reacts with sulphuric acid during digestion and release ammonia, which may be trapped in boric acid or dilute sulphuric acid. Thus nitrogen content may be quantified by titrating it with standard acid or alkali.

Digestion of protein

• 1g of the pretreated sample, 3g of cupric sulphate, 10g of sodium sulphate, and 20ml of sulphuric acid is turned into a flask. Then few grains of antibombing granules is added.
• The sample is heated gently and then   strongly in a fume hood until it changes colour from brown to colourless.
• It is then allowed to cool followed by dilution with 100ml of distilled and filtration using filter paper.

Distillation of protein digest

• 10ml of the protein digest plus few grains of antibombing granules and 30ml of 10%
• NaOH using a syringe is turned into the round bottom flask.
• At the receiving end 10ml of 2% Boric acid is added and a drop of double indicator.
• Distill until the volume of the distillate doubles the initial volume at the receiving end.

Titration

• 0.1M HCl is prepared  in 250ml and turned into 50ml burette
• Then titrate against the distillate in the beaker. The titre value of the colour change is taken.
• The colour change will be from light blue colour to deep blue.