Estimation of crude protein

Aim

         To estimate the percentage of crude protein present in the plant material or food staff or related  agricultural produces.

Principle

         The organic form of nitrogen is converted into NH4SO4 by digestion with concentrated H2SO4.  The digested material containing NH4SO4 is distilled with excess of alkali and the ammonia liberated is collected in a known quantity of a standard acid using methyl red as indicator.  The unreacted acid is then determined by back titration with standard alkali. The nitrogen content in the sample is  multiplied with the factor 6.25 to derive the crude protein content of the plant sample.

Reagents required

1. Concentrated  H2SO4

2. Copper sulphate

3. Potassium sulphate

4. 40% NaOH

5. 10% Na2S

6. 0.1N H2SO4

7. 0.1 N KOH

8. Methyl red indicator.

Procedure

Two process            1.  Digestion  2.Distillation

Digestion

         Weigh exactly 1 g of plant sample and transfer into a kjeldahl flask carefully without the sample adhering to the side walls of the flask.  To this add 10 g of K2SO4 and CuSO4 mixture.  Then add 30 ml of Concentrated H2SO4 using measuring cylinder with care. Mix the content well and digest over a bunsen burner till a light green or apple green colour is obtained.

Distillation

         Allow the kjeldahl flask to cool and dilute the contents with little distilled water taken in a squeeze bottle. Mix the content well and complete the transfer of the digested material into cleanly washed distillation flask. Repeat the washing for about 5-6 times, and ensure the complete transfer of digested material into the distillation flask.

         Pipette out 25 ml of 0.1N H2SO4 in to a cleanly washed 250 ml tall form beaker. To this add few drops of the methyl red indicator. Then keep this tall form beaker with the standard H2SO4 at the delivery end of the condenser of the distillation assembly. Add few bits of porcelain (for uniform boiling and uniform distribution of heat) into the distillation flask. Then add 10 ml of 10% Na­2S through measuring cylinder. Sodium sulphide prevents the formation of cupra ammonium complex, instead it forms sulphides of copper. Finally add 120 ml of 40% NaOH into the distillation flask and immediately close the distillation flask to avoid the loss of ammonia.  Put on the burner and continue the distillation.

         Test the completion of distillation with a moistened red litmus paper introduced at the tip of the condenser of distillation assembly.  Absence of blue colour indicates that the ammonia is already distilled.  Then stop the distillation and remove the distillate collected in the tall form beaker and titrate against 0.1N KOH to know the quantity of unreacted excess acid.  The end point is the change of color from pinkish red to straw yellow.

Calculation

Weight of the plant sample taken

=

W g

Volume of 0.1 N H2SO4 taken in excess

=

A ml

Volume of 0.1 N KOH used for back titration

=

B ml

Volume of 0.1 N H2SO4 actually used for neutralizing the ammonia

=

(A-B) ml

1 ml of 0.1N H2SO4

=

0.0014g of N

Hence, (A-B) ml of 0.1 N H2SO4

=

0.0014 x (A-B)

This is present in W g of the sample

Therefore percentage of N on moisture free basis

=

Percentage of crude protein     

=

Percentage of N x 6.25

Result:

Percentage of crude protein present in the plant sample  =