Determination of Crude Protein Content - Kjeldahl Method: Principle

Determination of crude protein content – Kjeldahl method
Principle
Digestion of organic matter with sulfuric acid in the presence of a catalyst, Rendering the
reaction product alkaline then distillation and titration of the liberated ammonia, Calculation
of the nitrogen content, Multiplication of the result by the conventional factor 6.25 to obtain
the crude protein content.
Reagents and materials

• Potassium sulfate
• Copper (II) oxide (CuO)
• Copper (II) sulfate pentahydrate (CuSO 4.5H 2O)
• Sulfuric acid - 18 mol/l, = 1.84 g/ml
• Paraffin wax
• Saccharose
• Acetanilide, with melting point 114ºC, nitrogen content 103.6 g/kg.
• Tryptophan, with melting point 282ºC, nitrogen content 137.2 g/kg. Before use, dry
the tryptophan.
• Sodium hydroxide solution, (NaOH) = 33% (m/m).
• Boric acid = 40 g/l.
• Sodium hydroxide - 0.1 mol/l or (NaOH) = 0.25 mol/l.
• Sulfuric acid - 0.05 mol/l or (H 2SO 4) = 0.125 mol/l.
• Mixed indicator, neutral point at pH 4.4 to 5.8.
• Dissolve 2 g of methyl red and 1 g of methylene blue in 1000 ml of ethanol (w(C H
2 OH)
2
= 95% (v/v).
Apparatus
• Analytical balance (Fig. 7.3)
• Digestion, distillation and titration apparatus
Procedure
Weigh, to the nearest 1 mg, a mass of the test sample chosen according to the expected
nitrogen content so that the test portion contains between
0.005 g and 0.2 g of nitrogen and, preferably more than
0.02 g.
Note: The mass of the test portion of homogeneous air-
dry samples should be between 0.5 g and 2.0 g. The
mass of the test portion of wet and/ or inhomogeneous
samples should be between 2.5 g and 5.0 g.
Digestion of organic matter

Transfer the test portion quantitatively into a Kjeldahl
digestion flask of suitable size (usually 800 ml).
Add 15 g of potassium sulfate.
Add an appropriate quantity of catalyst as follows: 0.3 g
of copper (II) oxide or 0.9 g to 1.2 g of copper (II) sulfate
pentahydrate.
Fig. 7.3 Analytical balance

Add 25 ml of sulfuric acid (18 mol/l) for the first gram of dry matter of the test portion and 6
to 12 ml for each additional gram of dry matter. Mix thoroughly, ensuring complete wetting
of the test portion.
Heat the flask moderately at first to prevent foam from rising into the neck of the flask or
escaping from the flask.
Heat moderately, swirling from time to time, until the mass has carbonized and the foam
has disappeared. Then heat more intensively until the liquid is boiling steadily.
Avoid overheating of the walls of the flask not in contact with liquid.
After the liquid has become clear with a light green-blue colour, heat for 2 h.
Leave to cool. If the digest starts to solidify, add some water and mix by swirling.
Distillation of ammonia
Carefully add 250 to 350 ml of water to dissolve the sulfates completely. If necessary,
facilitate dissolving by heating the flask in warm water. Mix by swirling and allow cooling.
Add a few boiling aids.
Pipette into the collecting flask of the distillation apparatus 25 ml of the sulfuric acid (0.05
mol/l), choosing the concentration according to the expected nitrogen content of the test
portion. Add 100 to 150 ml of water. Add a few drops of the mixed indicator. Proceed as
follows:
Immerse the end of the condenser in the liquid contained in the collecting flask, to a depth
of at least 1 cm.
Slowly pour 100 ml of sodium hydroxide solution (33 per cent) into the digestion flask along
the wall.
Immediately connect the flask to the distillation apparatus.
Heat the flask in such a manner that approximately 150 ml of distillate is collected in 30 min.
At the end of this time, check the pH of the distillate at the tip of the condenser using litmus
paper. If the reaction is alkaline, continue distillation.
Alternatively, transfer into the collecting flask 100
to 250 ml of boric acid. Add a few drops of mixed
indicator.
Titration
If sulfuric acid is used as the collecting liquid, titrate
in the collecting flask, the excess sulfuric acid with
sodium hydroxide solution 0.1 mol/l or 0.25 mol/l
as appropriate, until the endpoint is indicated by the
pH meter or until the color changes from violet to
green.
If boric acid is used as the collecting liquid, titrate the
ammonia with sulfuric acid 0.05 mol/l or 0.125 mol/l
as appropriate, until the endpoint is indicated by the
pH-meter or the color changes from green to violet
(Fig. 7.4).
Blank test
Perform a blank test using about 1 g of saccharose in
place of the test portion. Fig. 7.4 Titration
Check test
Perform a check test by determining the nitrogen content of acetanilide or tryptophan after
addition of 1 g of saccharose.
Calculation and expression of results

Distillate collected in sulfuric acid:
(V0 – V1) x C1 x M
WN1 = -----------------------
m
Where,
WN1 is the nitrogen content, in grams per kilogram, of the test sample.
V is the volume, in milliliters, of the sodium hydroxide solution required for the
blank test.
V1 is the volume, in ml, of the sodium hydroxide solution required for the
determination.
C1 is the concentration, in moles per litre, of the sodium hydroxide solution used
for the titrations;
M is the molar mass, in grams per mole, of nitrogen (M = 14 g/mol);
C2 is the concentration, in moles per litre, of the sulfuric acid (4.9.2) used for the
titrations;
m is the mass, in grams, of the test portion.
Distillate collected in boric acid:
2(V3 – V2) x C2 x M
WN2 = -----------------------
---- m
WN2 is the nitrogen content, in grams per kilogram, of the test sample.
V2 is the volume, in ml, of the sulfuric acid required for the blank test.
V3 is the volume, in ml, of the sulfuric acid required for the determination.
M is the molar mass, in grams per mole, of nitrogen (M = 14 g/mol);
m is the mass, in grams, of the test
portion. Calculation of crude protein content
Calculate the crude protein content of the test sample by the equation.
Wp = 6.25 x wN
Where,
Wp is the crude protein content, in grams per kilogram, of the test sample;
Wn is the nitrogen content, in grams per kilogram, of the test sample (either wN1
or
N2
Reference: IS 14825 : 2000, ISO 5983 : 1997

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Determination of crude protein content – Kjeldahl method

Reagents and materials

Digestion of organic matter

Fig. 7.3 Analytical balance

Calculation and expression of results

Reference: IS 14825 : 2000, ISO 5983 : 1997

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