How to estimate protein sample by Micro – Kjeldahl's method

In analyzing the materials for their protein content the Kjeldahl’s method is employed. In the Kjeldahl’s method nitrogen and not protein is determined most protein contain about 6% nitrogen by we so that 16 mg of nitrogen
= 100 mg of protein. The nitrogen value is therefore multisided by 6.25 (100/16) to get the wt. of protein.
In kjddahl’s method the sample to be analyzed is treated with conc. H2SO4 and sodium sulphate and small amount of sodium dioxide. The conc. Sulfuric acid acts as and oxiding agent as follows.

Sodium sulphate is added in order to raise the boiling point of the mixture. The higher the temperature of the solution the more rapidly the oxidation tubes place on boiling the soln, clears up because of oxidation of carbonaceous material after. After digestion is complete. The nitrogen of the sample is in the form of ammonium sulfate. This ammonium salt is estimated using Nestlé’s reagent co lour development is measured in a photoelectric colorimeter

Requirements:-
1) Protein sample 2) 2N NaOH 3) COaH2SO4, 4) Ammonium sulfate
5) Sodium sulfate 6) Nestles reagent.


Procedure:- For sample: Weigh accurately about 50 mg of sample and transfer to a kjedhhl’s flask digest with 4 ml of digestion mix 14 ml conc. H2SO4 + 0.5 gm potassium sulfate. Over a micro-burner, fill the mixture becomes dear faded few porcelain pieces to avoid bumping quantitatively transfer the content of the flask to a 250 ml volumetric flask. Neutralize the soln, to litmus and make up the volume to 250 ml use 0.5 ml of this solution for co lour development.

Standard Curve:- Weigh 100 mg of (NH4)2SO4 and dissolve in 250 ml water take 10 ml of this soln dilute to 100 ml 196 ug / ml prepare a series of test tubes with varying amount of co-100 ug, (NH4)S2O4 soln make up the volume to 2 ml. Add 2 ml of Nesters reagent and 3 ml of NaOH. Mix properly and observe the absorbance at 420 nm in a photoelectric colouri meter after 15 min and before 30 min on addition of reagent

Calculation: - 132 ug (NH4)2 SO4 = 2 NH3 =2 N = 28 ugN
wt of protein = 6.25 * wt of nitrogen.

Result: - The given protein sample contains 100% w/w protein.

Calculation: - 132 ug of (NH4)2 SO4 = 2(NH4) = 2 N = 28 ug N.
wt of protein = 6.25 * wt of nitrogen.
132 ug of (NH4)2SO4 = 28 ug N
30.4 ug of (NH4)2SO4 = 6.44 N
wt of protein = 40.30 N
wt of protein = 6.25 * 6.44 = 40.30


0.5 ml of diluted soln = 40.30 ug of protein
therefore 250 ml of diluted soln = 20.150 mg of protein
therefore 20 mg of sample = 20.150 mg of protein.

The given protein sample contains 100% w/w protein.

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