Malaysian Institute of Chemical

& Bioengineering Technology

Food Technology Department Document SOP /TP/005 Version 03

Title Crude Protein determination –Kjeldahl Method Date 07/01/09
Page 1 of 4
1. Introduction
This document will be used for determine Crude Protein content in food product. A Kjeldahl method is a
universal method no consideration is given to optimizing the procedure describe for a specific type of sample.
Analyzing the protein results is throughout by the digestion, distillation and titration step.

2. Scope
For homogeneous sample

3. Procedure
3.1 Equipment and Supplies:
a. Kjeltec 2100 and its apparatus; ashless filter paper
b. Analytical balance
c. Chemical solution: H 2 SO4 , deionized water, HCl, boric acid. (Refers SOP/GP/002 –
Solution for Protein analysis (Kjeldahl Method)
d. Conical flaks 250ml

3.2 Test Procedure

NOTE: All samples and blanks should be prepared in triplicate. Those samples should be homogeneous.

 Sample preparation
Sample should be homogeneous.

 Digestion
1. Prepare the representative sample and weight 2 gram into a digestion tube. Use filter paper to weight
sample.
2. Prepare empty digestion tube for blank.
3. Add 2 kjeltabs Cu 3.5 and carefully add 12 ml of H 2 SO4 into each of ready tube. Gently shake to
“wet” the sample with acid.
NOTE: Sample contain high fat (> 10% fat)/ carbohydrate use 15ml H 2 SO4 )
4. Attach the exhaust system to the digestion tubes in the rack and set the water aspirator to full effect.
5. Load the rack with exhaust into a preheated digestion block (420°C). Press START button.
6. Continue to digest until all samples are clear with blue/green solution. Normally after 1 hour and 15
minutes.
7. Remove the rack of the tubes with exhaust still in the tray and put in the stand to cool for 10 - 20
minutes.
8. During digestion, protein nitrogen is liberated to form ammonium ions; sulfuric acid oxidizes organic
mater and combines with ammonium formed; carbon and hydrogen elements are converted to
carbon dioxide and water.

H 2 SO4
PROTEIN + > ( NH 4 2 SO4  CO 2  H 2 O  SO4
Heat , Catalyst
 Malaysian Institute of Chemical
& Bioengineering Technology

Food Technology Department Document SOP /TP/005 Version 03

Title Crude Protein determination –Kjeldahl Method Date 07/01/09
Page 2 of 4

 Distillation
1. Add 80ml deionized water to each cooled tube carefully. Hot acid may cause explosion when meet cool
water.
2. Dispense 25ml of 4% boric acid (receiver solution); containing the indicator methyl blue and methyl red
to a 250ml conical flask and place it into the distillation unit and raise the platform so that the distillation
outlet is submerged in the receiver solution.
NOTE: The tip must be below the surface of the boric acid solution to avoid loss of ammonia.
3. Connect the digestion tube in the distillation unit and close the safety door.
4. Press Analyze button. 50ml of 40% NaOH will dispense into the tube.
5. The receiver solution in the distillation flask will now be light green indicating the presence of an alkali –
ammonia.

( NH 4 ) 2 SO4  2 NaOH          2 NH 3 ( g )  2 H 2 O  Na 2 SO4


NH 3 ( I )  H 3 BO3            NH 4  H 2 BO3

 Titration
1. Titrate the distillate with standardizes HCl (usually 0.1000N or 0.2000N) until the color of distillate
change, from light green to light grey color. Note the volume of acid consumed in the titration
as V sample .

H 2 BO3  H               H 3 BO3

 Calculation

i. Moles of HCl = moles NH 3 = moles N in the sample

ii. %N = [(Normality of HCl) X (Volume sample – Volume blank) X 0.014 X 100]/ weight
sample
0.014 is atomic weight of nitrogen
iii. % Protein = %N X conversion factor, f
f = 6.25 because most protein contain 16% N, so the conversion factor is 6.25.
(100/16 = 6.25)
iv. Conversion factor for various foods ,f

Type of Food Factor

Egg or Meat 6.254
Dairy products 6.38
Wheat 5.70
Other cereal grains and oilseeds 6.25
Almond 5.18
Peanuts and Brazil nuts 5.46
Other tree nuts and coconut 5.30
 Malaysian Institute of Chemical
& Bioengineering Technology

Food Technology Department Document SOP /TP/005 Version 03

Title Crude Protein determination –Kjeldahl Method Date 07/01/09
Page 3 of 4

4. Precautions
1. Performed acid digestion in the fume hood with adequate ventilation.
2. Read carefully the Operation Procedure for Kjeltech 2100 unit.
3. Careful handling the solution because requires the digestion with strong acid at high temperature.
4. Take care when handling hot digestion tube.

5. Distribution List
1. Original – Document controller
2. Copy - Analysis Laboratory Food Technology Department

6. Appendix
 F/SOP/TP/005 – Crude Protein Test Form
 * refers SOP OP 001- Kjeltech 2100 to operate the equipment
 Refers SOP/GP/002 –Solution for Protein analysis (Kjeldahl Method)

7. Document History
Document No & Description Description Version Date
Issued
SOP/TP/005 Crude Protein Determination – New document 01 160807
Kjeldahl method
SOP/TP/005 Crude Protein Determination – Update procedure and format 02 060308
Kjeldahl Method
SOP/TP/005 Crude Protein Determination – Update procedure, calculation and format 03 070109
Kjeldahl Method

Document End
 Malaysian Institute of Chemical
& Bioengineering Technology

Food Technology Department Document Appendix SOP /TP/005 Version 03

Originating
Title CrudeDocument No: SOP/TP/005
Protein determination CrudeMethod
–Kjeldahl Protein Determination Date 07/01/09
Title: Crude Protein Determination Test Form Page 4 of 4

Form No: F/SOP/TP/005 Page: 4 of 4

Test Information
Date & Time Equipment Operator
Test Parameter :

Sample Information
Sample Name
Company
Sample Description

Test Data
Sample Data Replicate 1 Replicate 2 Replicate 3
Sample Weight, A (g)
Amount HCl used, V sample (ml)
Amount HCl used, V blank (ml)
Normality HCl, N acid (N)

Test Result
Test Data Replicate 1 Replicate 2 Replicate 3
%N
vsample  vblank  1.4007 N acid
A
% Protein = ( f x %N)
Mean Of Protein

Done By, Date Approved By, Date