KJELDAHL METHOD OF PROTEIN ANALYSIS

KJELDAHL METHOD OF PROTEIN ANALYSIS

The Kjedahl method of protein analysis is a general method of calculating the concentration of protein in substances. The protein analysed from a sample through Kjedahl method are said to be crude and commonly called Total Crude Protein. This is because this method does not account for the presence of nitrogen from non-protein substances e.g. secondary metabolites, vitamins and nucleic acids in the sample.

Protein is one of the four major macro molecules (others are Fat, Carbohydrate, and Nucleic Acid). Proteins are composed of α-L- amino acids that are joined by peptide bonds to form linear chains or branched chain through formation of disulphide bonds.

In addition to carbon, hydrogen, and oxygen, proteins contain approximately 16% nitrogen by weight.

The digestive process breaks down proteins to their constituent amino acids, which enter the blood. The complete oxidation of proteins to CO₂, H₂O, and NH₄⁺ in the body yields approximately 4kcal/g.

The official method for analysis of crude protein in solid and powdered samples is the Kjeldahl method.

AIM: To determine the percentage protein through percentage Nitrogen

APPARATUS: Kjeldahl flask, ash less filter paper, Kjeldahl instrument, 250ml conical flask.

REAGENT: Concentrated H₂SO₄, 50% NaOH, 4% Boric Acid, Screened Methyl Red, Kjeldahl Tablet (Se₂SO₄) and distilled water

PRINCIPLE: The heating of the sample with sulfuric acid, decomposes the organic nitrogen present to ammonium sulphate. In this step there is also addition of  mercury oxide or copper sulphate which serves as catalyst to speed up the rate of the decomposition. Chemical decomposition of the sample is complete when the medium has become clear and colorless solution (initially black).

The solution is then distilled with sodium hydroxide which converts the ammonium salt to ammonia which is trapped with boric acid solution. The concentration of ammonia present (hence the amount of nitrogen present in the sample) is determined by titration, a type of titration known as back titration.

Equations for various Steps

1.      DIGESTION OF THE SAMPLE

Protein + H₂SO₄           (NH₄)₂SO_4(l) + SO_2(g) + CO₂ + H₂O_(g)

2.      DISTILLATION

(NH₄)₂SO_4(l) + NaOH           Na₂SO_4(s) + NH_3(g) + H₂O_(l)

Capturing of ammonia by boric acid

B(OH)_3(l) + NH_3(l) + H₂O_(l)         NH₄⁺_(l) + B(OH)₄^-_(l) (greenish colouration)

3.      TITRATION

 This is the final stage of the reaction, this involve the titration of the solution obtained from distillation against 0.1M HCl or 0.05M H₂SO₄.

NH₄⁺ + B(OH)₄^- + HCl         NH₄Cl + B(OH)_3(l) + H₂O_(l)

NH₄⁺ + B(OH)₄^- + H₂SO₄         (NH₄)₂SO_4(l) + B(OH)_3(l) + H₂O_(l)

PROCEDURE:

i) Weigh 0.2g of the sample in an ash less filter paper and transfer into the Kjeldahl flask

ii) Add 25ml concentrated H₂SO₄

iii) Add half Kjeldah tablet (catalyst)

iv) Placed in the Kjeldahl Digestion compartment and turned on the Heater. Heat till solution turn colourless, then turn off the heater and allow the solution to cool to room temperature.

v) Add 200ml of distilled water to the cool dilute the acidic solution

vi) Measure 50ml of 4% Boric acid (B(OH)3) into 250ml conical flask and add 3 drops of screened methyl red and place the solution in the ammonia outlet of the Kjedahl Apparatus.

vii) Add 75ml of 50% NaOH gently, glass beads, granulated zinc (anti-bumping agent) and 50ml of distilled water to the solution in step (v).

vii) Transfer immediately to the Kjeldahl distillation compartment and set up the apparatus, turn on the heater and distil to the 250ml mark in step (vi).

The distillate (whole 250ml) was titrated against 0.1M HCl (or 0.005M H₂SO₄) in the burette to a light reddish colour end point.

CALCULATIONS:

Percentage Nitrogen = Titre value *0.0014 * 100%

Weight taken

Percentage Protein = % Nitrogen * Factor

NB: Factors are derived from the compositional amino acids in the sample. Therefore different samples have different factors. The factors are 5.7 for flour, 6.38 for milk and 6.25 for other foods. Most time 6.25 is generally used.