Fish Nutrition and Feed Technology (2+1)

Practical 1-Proximate composition analysis of feed ingredients and prepared feeds

(b) Determination of Crude Protein (Kjeldahl Method)

Principle

A known weight of powdered sample is decomposed by digesting with concentrated sulphuric acid in the presence of catalyst mixture to accelerate the process, which results in the formation of ammonium sulphate on addition of known quantity of 40% NaOH and subsequent distillation, ammonia is liberated, which is absorbed in a known volume of 4% boric acid solution. Then formed ammonium borate is titrated against 0.1N HCl using indicator.

Reagents

• Sulphuric acid (98%)
• Catalyst mixture: Add 35g of Potassium sulphate (K₂SO₄) and 4g of Copper sulphate (CuSO₄).
• 0.1 N of Std. HCl: Dilute 4ml of Conc. HCl in 450ml distilled water. Standardize against 1.9% Borax as given bellow

• Take 10ml of 1.9% Borax in a conical flask and add 3 drops of methyl orange
• Take 0.1N of Std. HCl in burette and titrate against the borax solution till the colour changes to pink.
• Note the titre value in the buratte

Normality of the borax solution is calculated as in bellow

• 4% Boric acid solution: Dissolve 40mg of boric acid in 900ml of hot distilled water. Cool and add 10ml of Bromocresol green and 7ml of methyl red solution. Mix the solution thoroughly and make the volume upto 1litre with the distilled water. Add a drop of alkali (40% NaOH).
  

• Sodium hydroxide, 40% solution

Apparatus

• Micro Kjeldahl digestion and distillation units,
• Kjeldahl glass digestion tubes (300 ml capacity), and
• Conical flasks, 250 ml.
• Burette

Method

Accurately weigh 1 g of sample into a digestion tube. Add 10 g of catalyst mixture and 10 ml of Conc. sulphuric acid. Heat the tube gently at a temperature of about 350ºC and gradually increase the temperature upto 450ºC and boil the solution until it turns light green colour.

On cooling, add about 35 ml distilled water, recool, and add 40 ml of NaOH and initiate distillation. Ammonia liberated is traped in 25ml of 4% Boric acid kept in a conical flask. When the volume of the solution in the flask reaches nearly 150ml stop distillation, titrate the solution against 0.1N HCl till the green colour turns to pink. Note down the the burette reading. Calculate the protein level by using following equation.

Calculation