IMPROVING PROTEIN

ANALYSIS RESULTS BY
KJELDAHL PROCEDURE
Pinky Pe Tobiano
16th Annual ASA-IM Sea Feed
Technology and Nutrition Workshop
May 28, 2008
Singapore

Introduction
Importance of crude protein values for
feed ingredients

Diet formulation

Monitoring quality of raw

materials and mixed feeds

Government regulation and

control

What is Kjeldahl analysis?

It is an analytical method to determine
quantitatively nitrogen in the
trinegative state in certain organic
compounds.
The method was developed in 1833 by
Johan Kjeldahl, a Danish Chemist.
This involves 3 main steps:
Digestion,
Distillation, and
Titration.

Principle of Kjeldahl Nitrogen

Analysis
The sample is digested to convert the
protein nitrogen into ammonium sulfate
The ammonium sulfate is treated with
an alkali, thus liberating ammonia
The ammonia is received by an acid and
quantified through titration

3 Different Steps of Kjeldahl

Method
Digestion conversion of nitrogen
in the sample to ammonium
sulfate
ProteinN + H2SO4

(NH4)2SO4

Distillation- separation of ammonia

from digestate using alkali (NaOH)
and collection of ammonia using a
receiver
(NH4)2SO4 + 2NaOH

2NH3 + H2O+ Na2SO4

NH3 + H3BO3 (Boric Acid)

NH4 Borate

Or, if the receiver used is hydrochloric acid:

NH3 + HCl

NH4Cl
+ excess HCl

Titration - quantification of ammonia and

calculation of the initial protein
concentration
NH4 Borate + H2SO4

(NH4)2SO4 + Boric Acid

The amount of NH3 evolved is directly

proportional to the amount of H2SO4 used
as titrant.
Or: NH4Cl
+ excess HCl + NaOH

NaCl + H2O

Table 1. Crude protein content based on 6.25

factor vs specific factor
Sample

%N

Skimmed
5.23
Milk
Rice
1.57
Oats
1.95
Soya Bean
7.43
Meal
Wheat Bran 2.57
Safflower
3.35
Sunflower
3.30

CP
Specific
using 6.25
factor

% Crude
Protein

32.69

6.38

33.38

9.78
12.19
46.44

5.95
5.83
5.71

9.31
11.37
42.42

16.04
20.94
20.60

5.70
5.30
5.30

14.63
17.75
17.47

FACTORS AFFECTING KJELDAHL

TEST RESULTS
1. Sample Preparation
2. Sample Amount
3. Catalyst
4. Digestion Time

Sample Preparation
Sample should be carefully prepared
to avoid errors in the final result.
This procedure may involve one or
more treatments to homogenize the
sample.
Example: The particle size of the sample
must be reduced to a size < 1mm.

Homogeneity of the analytical

sample improves the
reproducibility of the method
and also offers the possibility to
reduce the sample size used,
without sacrificing the quality of
the final results.

Physical treatments used to

homogenize samples:

shaking

stirring

mortaring

riffling

coning and quartering

grinding

blending

homogenizing

milling

Sample Weight
The actual weight of sample
required for analysis depends
primarily on the homogeneity
of the sample.

For non-homogeneous
samples, high precision of the
measurements cannot be
obtained using small sample
sizes.
For homogeneous samples,
amount is not as critical and
can be optimized to give a
suitable final titration volume.

Table 2. Rough Guide for

selecting the sample size
Protein Content

mg sample

Up to 5%
5-30%
more than 30%

1000- 5000
500- 1500
200- 1000

Note: the analytical sample should ideally

contain 10 -100mg N

Catalyst
The speed and efficiency of the digestion
is not only influenced by the
temperature used but can also be
improved by the addition of a suitable
catalyst.
Catalyst is used to hasten the chemical
reaction (oxidation) by increasing
boiling point
Selenium
Mercury
Copper
Titanium

Factors in choosing a suitable

catalyst:
1. Cost
2. Availability
3. Disposal

Usually, catalysts are composed of a

mixture of salts:
Example:
Potassium Sulfate plus Copper Sulfate
Sodium Sulfate plus Copper Sulfate

Digestion Time
Digestion time - the time it takes until the
digestate has cleared or become colorless.

Factors that affect the total

digestion time:
1.
2.
3.
4.
5.

Type of sample
Volume of acid
Amount of catalyst used
Oxidizing agent ( Sulfuric Acid)
Temperature of block digestor

Titration
Indicator- included in the receiver. Examples:
1. Methyl red (from light green to light pink)
2. Bromcresol green (from light green to
light blue or light gray to very light pink)
Titrant exact concentration and volume (titration reading) is
necessary
End point- achieved when the color of digestate changes
ex. The end point is achieved when the light
green clear digestate changes to light gray to very
light pink.
The digestate is over titrated when the color
becomes reddish pink.

20 placer Kjeldahl Unit

6 placer Kjeldahl unit

Conventional Kjeldahl Apparatus

Digested at 4200C for 1 hr

Distilled with 50ml 40% NaOH

End-point- light gray to very light pink

Over titrated- pink color

Verification of Kjeldahl Test

Using standard sample to check
accuracy of result
ex. glycine or tryptophan
urea
Using natural ingredient (ex. soya or
corn) for the reproducibility of test result

PER SYSTEM VERIFICATION

1. Digestion System
In order to ensure high quality of the
analytical results produced in routine
work, it is essential to include check
sample in all batches digested and
analyzed.

The following may be used for verifying

accuracy of the digestion procedure. A

standard substance with known

nitrogen content
1. Glycine pure, use 500mg
Nitrogen content= 18.66%
2. Tryptophan pure, use 500mg
Nitrogen content = 13.72 %

Table 3. Results of Kjeldahl

Verification Test
Standard Sample

%, N

%,N

%N

%N

Glycine

18.69

18.66

18.64 8.66

Urea

45.10

45.23

45.16 45.05

Note : % N= Nitrogen Content

2. Distillation System
Distillation principle is to convert ammonium
(NH4) into ammonia (NH3) by adding alkali,
and steam distilling into a receiver flask
containing boric acid with mixed
indicators.
Since all nitrogen in the samples after
digestion form ammonium sulfate, it can
be used as a standard to check the
recovery of the distilling unit.

The following procedures can be used:

1. Use ammonium sulfate > 99.5%
% Nitrogen (NH4)2SO4 = 21.09
weight to use = 0.15g
75-100 ml distilled water, 50ml
NaOH (40%)
2. Ammonium Iron (II) Sulfate
% Nitrogen = 7.145
weight to use = 0.5g
75-100ml distilled water, 50ml
NaOH 40%

3. Inter-laboratory Verification
Participate in round robin

sample tests with other testing

laboratories
Prepare reference sample within
the laboratory and verify versus
other laboratories.
Note: Always use this sample as internal
reference in daily routine use.

When comparing results with other

laboratories, it is important to know the
moisture content of the sample.
This can either be done by correcting for
the moisture content and reporting
results on dry basis, or by always
analyzing predried samples.
Content on dry basis = Ax100 /(100-B)
A= % Protein of sample
B= % Moisture of sample when
analyzed for protein

Table 4A. Common ingredients and their

observed values (CP%, meanstd) as
analyzed by Kjeldahl Procedure
Ingredient

As Analyzed
% (mean) Std. D

As Dry Basis
% (mean) Std.D

Fish Meal
Rice Bran
Soya Bean
Meal
Yellow Corn

15
6
12

62.67
12.65
45.47

1.91
0.23
1.09

68.98
13.99
51.75

2.55
0.33
1.55

7.56

0.36

8.83

0.23

Table 4B. Different Qualities of Soya Bean Meal

and their observed Crude Protein
value as analyzed by Kjeldahl Procedure
Ingredient

N
(No. of
samples)

Soya Bean Meal

Low Protein
(44.0% to 46.0%)

57

Soya Bean Meal

High Protein
(47.0 to 48.0 %)

As Analyzed
% (mean)
SD

45.45

47.41

0.87

0.40

SUMMARY/ CONCLUSION
The key to successful Kjeldahl analysis
can be found:
1. In the sample preparation step;
2. By validating the test procedure that
includes use of catalyst, digestion time,
amount of acid;
3. By verifying the digestion procedure;
4. By monitoring regularly the
performance of distillation system.

A SUCCESSFUL KJELDAHL
ANALYSIS WILL HAVE
THE RIGHT
KJELDAHL TEST RESULT.

THANK YOU!

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