Determination of Crude Protein

Determination of Crude
Protein

B.K.K.K.Jinadasa
12/13/2009

(GS/M.Sc./FOOD/3608/08)
Determination of Crude Protein 2009

3.1. Introduction

In Kjeldahl method a food is digested with a strong acid so that it releases nitrogen which can be
determined by a suitable titration technique. The amount of protein present is then calculated
from the nitrogen concentration of the food. It is usually considered to be the standard method of
determining protein concentration. Because the Kjeldahl method does not measure the protein
content directly a conversion factor (F) is needed to convert the measured nitrogen concentration
to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of
protein) is used for many applications, however, this is only an average value, and each protein
has a different conversion factor depending on its amino-acid composition. The Kjeldahl method
can conveniently be divided into three steps: digestion, neutralization and titration. In the
digestion step nitrogen in protein is oxidized to ammonium sulphate by using sulphuric acid and
catalyst.

2
Determination of Crude Protein 2009

3.2. Equipment s & reagents:

Kjeldhal digestion kit (Kjeldhal digestion flasks, Kjeldhal heater, fume trap, distillation unit
(Pranas Wagner still)
Titration flasks
Weighing balance
Sodium sulphate solution
(Dissolve 50 g of NaOH pellets and 8g of sodiumthiosulphate in 100 mL distilled water)
100 mL distilled water
4% Boric acid solution
0.02 M HCl standard hydrochloric acid solutions
Kjeldhal catalyst tablets
Indicators – Methyl red
Methylene blue
(Prepare a solution by mixing two parts of 2% methyl red (alcoholic) solution with one part of
0.2% (alcoholic) methylene blue solution.)
conc. H2SO4

3.2. Methods

Weigh 0.05 g of prepared sample was transferred on a tissue paper to the kjeldhal digestion flask.
Catalyst powder and 2 mL of conc. H2SO4 were added to the flask. Then the flask was connected
to fume trap and attached to the pump. Sample was allowed to digest for four hours, until a clear
solution without black particles was obtained. Then the sample was cooled for one hour. Blank
digestion was also carried out.
Sample was dissolved in a minimum amount of ammonia free distilled water and transferred to a
semi micro kjeldhal distillation apparatus which has been previously conditioned by passing
steam for several minutes.
Add 8 mL of NaOH/Na2SO3 solution to the kjeldhal apparatus.
5mL of 4% boric acid solution and 3 drops of indicator were added into a titration flask and kept
at the end of the apparatus to trap the ammonia liberated.
Steam was passed through the flask until about 15 mL of distillate was received.
This solution was collected at the titration flask and titrated with standard HCl solution. The end
point is pink colour.
Same procedure was applied to a blank sample as well.

3
Determination of Crude Protein 2009

3.3. Results and calculation

Weight of sample (g) Sample titre (mL)

01 0.0000 _
02 0.0557 5.60
03 0.0506 5.30
04 0.0540 5.40
05 0.0525 5.90

Sample No. Nitrogen % Protein % AVG protein %

Sample 2 2.8 17.50

Sample 3 2.9 18.12

Sample 4 2.8 17.50 18.13

Sample 5 3.1 19.38

3.4. Discussion

In the digestion step sample was weighed into a digestion flask and then digested by heating it in
the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium
sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper,
selenium, titanium, or mercury (to speed up the reaction). Here we used a catalyst powder and it
contained anhydrous sodium sulphate and selenium. Since the selenium is very efficient
digestion was very fast. Digestion converts any nitrogen in the food (other than that which is in
the form of nitrates or nitrites) into ammonia, and other organic matter to C02 and H20. Ammonia
gas is not liberated in an acid solution because ammonia is in the form of ammonium ion (NH4+)
which binds to the sulfate ion (SO42-) and thus remains in solution:

Protein + H2SO4 (conc.) (NH4)2SO4

In the neutralization step the solution in the digestion flask is made alkaline by addition of
sodium hydroxide, which converts the ammonium sulfate into ammonia gas. Sodium
thiosulphate is used to break the ammonium sulphate complex.

(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

4
Determination of Crude Protein 2009

NH3 + H3BO3 NH4+ + H2BO3-

The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium
ion, and simultaneously converts the boric acid to the borate ion:

H2BO3- + H+ H3BO3

First sample didn’t give a proper result since all the ammonia gas has been evolved before
trapping it into boric acid solution.

Advantages
High precision and good reproducibility have made this the major method for the estimation of
protein in foods.

Disadvantages
It does not give a measure of the true protein, since all nitrogen in foods is not in the form of
protein.
Different proteins need different correction factors because they have different amino acid
sequences.
The use of concentrated sulfuric acid at high temperatures poses a considerable hazard.

3.5. References:
AOAC, “official methods of analysis”, method 981.10
Tacon, A.G.T. (1975). The nutrition and feeding of farmed fish- a training manual 2- Nutrient
sources and composition
UDK 132, Semi automatic Kjeltec system manuals