Determination of Protein content in Foods

1. P R O T E I N
D E T E R M I N AT I O N
L E C T U R E R : A Y E S H A S I D D I Q A
P H D S C H O L A R

2. PROTEIN DETERMINATION BY
KJELDAHL METHOD
HISTORY OF KJELDAHL METHOD
In the year of 1883, march 7th Johan Gustav Christoffer Thorsager kjeldahl, a danish chemist
developed this method.
OBJECTIVE
“ Quantitative determination of nitrogen (N2) in the substances .”

3. PROCEDURE

4. 1. DIGESTION
• The purpose of this step is to break down the bonds that hold the polypeptides
together, and convert them to simpler chemicals such as water, carbon dioxide
and ammonia.
• The food sample to be analyzed is weighed into a digestion flask and then
digested by heating it in the presence of sulfuric acid (an oxidizing agent which
digests the food), potassium sulfate (K2SO4) (to speed up the reaction by raising
the boiling point of the digesting acid).
• Ammonia gas is not liberated in an acid solution because the ammonia is in
the form of the ammonium ion (NH +) which binds to the sulfate ion (SO 2-) and
thus remains in solution:
• The general equation for the digestion of an organic sample is shown below:
Protein + H2SO4 → (NH4)2SO4 + H2O + CO2

6. 2. DISTILLATION
 The purpose of the distillation step, is to separate the ammonia (that is, the
nitrogen) from the digestion mixture.
• This is done by adding NaOH that will raise the pH of the mixture. This has
the effect of changing the ammonium(NH4
+) ions (which are dissolved in the
liquid) to ammonia (NH3), which is a gas as indicated in the following
equation.
(NH4)2SO4+ 2NaOH → 2NH3 + Na2SO4 +2H2O
 Reaction with Boric Acid (H3BO3):
• If Boric Acid is receiving solution then the Ammonia produce in distillation is
react with H3BO3 and produce AMMONIUM BORATE (NH4+H2BO3-).
NH3 + H3BO3 =NH4+H2BO3-

7. TITRATION
• Ammonium borate complex can directly titrated against standard acid by
using METHYL RED as an indicator
• Toquantify the amount of ammonia in the receiving solution.

8. DETERMINATION OF PROTEIN CONTENT IN WHEAT
FLOUR SAMPLE BY KJELDAHL APPARATUS
Apparatus/Reagents Required:
0.1N H2SO4, 40% NaOH, 4% H3BO3, Methyl Red, Digestion Mixture/Tablets, Burette, Flask,
Kjeldahl Apparatus, Distilled Water
Principle:
The food sample is digested with the help of strong acid (H2SO4) to release nitrogen in the form of
ammonia ion. This ammonia ion would combine with sulphate ion to form ammonium sulphate.
Furthermore, this ammonium sulphate would decompose by sodium hydroxide to release
ammonia gas which is absorbed by boric acid to form ammonium borate. The amount of nitrogen
is calculated by titrating it against 0.1N H2SO4.

9. PROCEDURE
1- Digestion
1. Take 2g of sample in digestion tube and then add digestion tablet in it.
2. Then add 30ml of 0.1 N H2SO4 and digest the sample until light green color is obtained.
3. At last dilute that digested sample up to volume of 250ml with distilled water.
2- Distillation
1. Take 10ml of diluted sample and add it in flask.
2. Then add 10ml of 40% NaOH solution in that flask and take 10ml of 4% H3BO3 in Kjeldahl
flask and turn on distillation assembly.
3. Distilled the sample until silver yellow color is obtained as end point.

10. 3- Titration
1. Take 0.1N H2SO4 solution in burette for titration.
2. Add 1-2 drops of methyl red as indicator in distilled sample and titrate it against 0.1N H2SO4
until red color have appeared.
3. The protein content can be calculated by following expressions;
𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (%) = 𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 (%) 𝑥 6.25

11.  Advantages:
 Applicable to all types of foods
 Accurate; an official method for crude protein content
 Disadvantages
 It does not give a measure of the true protein, since all nitrogen in food is not
in the form of protein.
 Time consuming
 Different proteins need different correction factors because they have
different amino acid sequence