EXPERIMENT-2

Determination of protein content in food products by Kjeldahl method

2.1 Introduction
The Kjeldahl method for the determination of protein in food products are
based on the nitrogen determination. The protein content of foods is usually
calculated from total nitrogen by multiplying with a suitable conversion
factor that is based upon the nitrogen % present in a particular protein.
2.2 Principle
The Kjeldahl procedure can be basically divided into three parts: (1)
digestion, (2) distillation, (3) titration. In the digestion step, organic nitrogen
is converted to an ammonium in the presence of a catalyst at approximately
370°C. In the distillation step, the digested sample is made alkaline with
NaOH and the nitrogen is distilled off as NH 3. This NH3 is “trapped” in a
boric acid solution. The amount of ammonia nitrogen in this solution is
quantified by titration with a standard H 2SO4 solution. A reagent blank is
carried through the analysis and the volume of H2SO4 titrant required for
this blank is subtracted from each determination.

Equations

Carbohydrate + Protein + Fat (NH 4)2SO4 + CO2+ SO2 + H2O

(NH4)2SO4 + 2NaOH 2NH4OH + Na2SO4
2NH4OH + H2SO4 Neutralization (NH4)2SO4 + 2H2O

2.3 Requirements
1. Kjedahl catalyst: Mix 5 part of potassium sulphate with 1 part of cupric
sulphate
2. Conc Sulfuric acid (H2SO4)
3. 40% NaOH solution
4. 4% Boric acid
5. 0.1 N H2SO4 solution
Sl no. Sample name Sample weight (g) 0.1N H2SO4 used
(ml)

Table 2.1: Observation

Calculation
6. Indicator solution: Mix 100 ml of 0.1% methyl red (in 95% ethanol)
with 200 ml of 0.2% bromocresol green (in 95% ethanol)

2.4 Procedure

Digestion

 Place sample (0.2-0.5 g) in digestion flask.

 Add 3 g Kjedahl catalyst and 10 ml of conc. H2SO4

 Prepare a tube containing the above chemical except sample as blank.

Place flasks in inclined position and heat gently unit frothing ceases.
Boil briskly until solution clears. Ensure any frothing of samples are
there, if frothing is not there then increase the temp to 420° C

Distillation

• Cool and add 50 ml of distilled water cautiously.

• Immediately connect flask to digestion bulb on condenser and with tip

of condenser immersed in standard acid and 5-7 drops of mix
indicator in receiver. Rotate flask to mix content thoroughly; then heat
until all NH3 is distilled.

Titration

• Remove receiver, wash tip of condenser and titrate excess standard

acid distilled with standard 0.1 N H2SO4 solutions.

Formula
14  N  (S - B) x 100

NITROGEN (%) = -------------------------

W x 1000

Where,
14 = Atomic weight of Nitrogen

B = volume (ml) 0.1N H2SO4 used titration for blank,

S= volume (ml) 0.1N H2SO4 used titration for sample
N= normality of acid used for titration,
W= weight, in g, of sample taken for test.

PROTEIN (%) = Kjeldhal Factor X Nitrogen %

2.5 Results
The protein content (%) in the given sample is______________

2.6 Inference
Duplicate determinations of the nitrogen should agree within 0.05%
nitrogen. Appropriate conversion factor for protein from nitrogen should be
used for specific food samples. The following Kjeldahl factors are used for
different food products in converting nitrogen to protein.

S. No. Types of food material Kjeldahl

factor

1. Rye, oat meal, whole wheat 5.83

2. Wheat flour & its products viz. bread, macaroni, 5.70

spaghetti,

3. Maize, rice polish, pulses, tea, cocoa, coffee, 6.25

malt, beer, etc.

4. Groundnut, brazil nut 5.46

5. Cashew, coconut & other tree nuts, sesame, 5.30

safflower, sunflower, castor, cottonseed, linseed

6. Milk & milk products, margarine 6.38

7. Egg whole, egg powder, 6.68

2.7 Precautions
 Sample to be analyzed should be homogeneous.
 Determine the strength of H2SO4 before use.
 Sample should be checked for complete digestion through colour and
there should not be any presence of carbon particles adhering to the
neck of Kjeldahl flask.