FOSS Digestor Solution Sample Types

The FOSS Digestor is a heating block designed for the sample The method described in this
digestion process during protein analysis following the Kjeldahl Application Note covers the
method. To match different lab capacities, manual digestion, semi- following sample types:
automated digestion and fully automated digestion solutions can • Animal feed
be selected by choosing between different Digestor models with
• Forage (plant tissue)
various accessories.
• Grain
FOSS Kjeltec™ 9 Solution • Oilseedsds
Kjeltec 9 is an automated distillation unit designed primarily for • Fish feed
protein analysis following the Kjeldahl method but can be widely
• Pet food
used for many different distillation processes. Optional built-in
colourimetric titration and autosampler can fully automate the
Kjeldahl analysis.
 AN 5515
Rev. 1

1 Application Information
This Application Note should be used in conjunction with the Application Note AN 5511 [3]
“Determination of Nitrogen According to the Kjeldahl Method - General Application”.

1.1 Introduction
This application note provides guidance for using Kjeltec 9 series to analyse Kjeldahl nitrogen in
animal feed and forage. The method is applicable from 0.5-50% nitrogen and to the same matrices
as AOAC Official Method 2001.11[2]. This method does not measure oxidized forms of nitrogen and
does not fully recover heterocyclic nitrogen compounds.

1.2 Principle
A homogenous sample portion material is digested in a mixture of concentrated sulphuric acid and
suitable catalyst at a high temperature, thereby any present organic nitrogen will be converted to
ammonium sulphate. Excess sodium hydroxide is added to the cooled digest to liberate ammonia,
using steam distillation.
The liberated ammonia is trapped in a weak boric acid solution and titrated with a standardised
titrant. The nitrogen content in the sample is calculated from the amount of ammonia produced
and the volume of the titrant.
The nitrogen can be converted into Kjeldahl protein by applying a protein factor.

1.3 Safety Precautions

The Kjeldahl method requires the digestion of the sample using strong acid at high temperatures.
Carefully handling the solutions is mandatory for laboratory safety. Refer to the appropriate
material safety sheet for reagent handling instructions.
For added protection, acid digestions should be performed in a fume hood with adequate
ventilation. Personal protection equipment (PPE) such as gloves, lab coat and eye protection should
always be worn, and care should be taken when handling hot digestion tubes.
Follow safety instructions of instrument User Manuals and Material Safety Data Sheets.

8000190a

Fig. 1 Personal protection equipment

Application Note 5515 / Rev. 1 1(6)

1.4 Apparatus
A. Milling or grinding equipment, e.g., CT 293 Cyclotec™, CM 290 Cemotec™ or KN 295 Knifetec™
B. Analytical balance, accurate to 0.1 mg
C. Digestion block, e.g., FOSS Tecator™ Digestor 2520/2508 or Labtec™ Digestor 220/208
including accessories
D. Distillation, titrations and autosampler
• For steam distillation: Kjeltec 9 Distillator
• For steam distillation and auto titration: Kjeltec 9 Analyser
• Autosampler: Kjeltec 9 Auto Sampler 20 or 60 units in combination with Kjeltec 9 Analyser
E. 250 ml digestion tube
F. Titration burette, 50 ml or equivalent - for manual titration
G. Erlenmeyer flask (Receiver flask, up to 500 ml) - for manual titration
H. Pipette

1.5 Reagents
During the analysis, use only reagents of recognized analytical grade.
See AN 5511 for further information regarding to the preparation of reagents.
A. 98% sulfuric acid
B. Kjeltab Cu 3.5 (3.5 g K2SO4 + 0.4 g CuSO45H2O per tablet)
C. Antifoaming agent (optional)
D. 40% w/v sodium hydroxide
E. Titrant (Standardised)
• 0.1 N Hydrochloric acid
Note: The normality of the titrant is required to be four decimal places.
F. Receiver solution
• Boric acid 1% with bromocresol green/methyl red indicator solution - for auto titration.
• Boric acid 4% with bromocresol green/methyl red indicator solution - for manual titration
Note: Recommended blank value of the receiver solution is 0.05-0.15 ml using a 0.1 N
HCl.

1.6 Analytical Details

1.6.1 Digestion Procedure
Sample Sample Antifoaming Digestion Digestion
H2SO4 Kjeltab Cu 3.5
Type Size Tablet Temperature Time

Solid 1g 12 ml 2 pcs 0 pc 420 oC 60 min

General Procedure for Digestion

Application Note 5515 / Rev. 1 2(6)

1.6.2 Distillation Procedure
Model Dilution Alkali Steam Power Delay/SAfE Receiver solution Distillation End

KT9 Distillator 80 ml 50 ml 100% 12 s / SAfE 30ml 4% Boric acid Time: 4 min

KT9 Analyser 80 ml 50 ml 100% 12 s / SAfE 30ml 1% Boric acid Auto

General Procedure for Distillation

Note: The total volume of mixed solution inside the 250 ml digestion tube must not exceed
170 ml during distillation.

1.6.3 Titrant
Selection of titrant depends on the content of nitrogen. The table below describes the
recommended concentration of titrant based on the nitrogen content of samples. Titrant
concentration is required to 4 decimal places, e.g. 0.1002 mol/L.
0.01 N HCl (range of Nitrogen, e.g., 0.1-2 mg of N)

0.1 N HCl (range of Nitrogen, e.g., 2-100 mg of N)

0.2 N HCl (range of Nitrogen, e.g., 50-200 mg of N)

Recommended titrant concentration

1.7 Procedure
1.7.1 Sample Preparation
1. Grind the samples by using a suitable laboratory mill (e.g., FOSS Cyclotec). For results on a dry
basis perform a separate moisture determination of the ground sample.
2. Weigh an appropriate amount of homogenised sample into a 250 ml digestion tube and record
the weight nearest to 0.1 mg.
If the approximate protein content of the sample is known, the test portion weight can be selected
according to:
Protein Content Sample Weight (g)

<5% 1-5

5-30% 0.5-1.5

>30% 0.2-1

Sample Amount for Samples with Different Protein Content

Note: The analytical sample should ideally contain 30-140 mg N

1.7.2 Digestion
Prepare at least three reagents blank digestion tubes for each batch of samples, the blanks should
be prepared and treated as analytical samples, but without sample material.
1. Add two Kjeltabs Cu 3.5 (or 7 g K2SO4 + 0.8 g CuSO45 H2O) to each digestion tube.
2. Using a pipette, add 12 ml of concentrated H2SO4 to each tube (15 ml for high-fat materials (>
10% fat)). It is possible to stop at this point and proceed to work the following day.
Note: Adding two tabs of antifoaming tablets to each digestion tube can prevent
foaming issues during digestion.
3. Place the fume manifold tightly on the tubes and turn the H2O aspirator or Scrubber on
completely. Place the rack of tubes in the pre-heated digestion block.

Application Note 5515 / Rev. 1 3(6)

 Note: Steps 3-6 can be done automatically with the Tecator Digestor equipped with an
auto lift system and Tecator Scrubber 2501.
4. After 10 minutes, turn the H2O aspirator or Scrubber down until the acid fumes are just
contained within the exhaust hood. A condensation zone should be maintained within the
tube. After the bulk of the sulphur oxide fumes is produced during the initial stages of
digestion, the vacuum source must be reduced to prevent loss of H2SO4.
5. Digest an additional 50 minutes. Total digestion time is approximately 60 minutes.
Note: If 15 ml sulfuric acid is used for digestion, please extend the total digestion time to
75 min. Besides, 60 ml alkali solution should be used for distillation later.
6. Turn the digestion block off. Remove the rack of tubes with exhaust still in place and put in the
stand to cool for 10-20 minutes. Cooling can be increased by using a commercial air blower or
by placing it in a hood with the hood sash pulled down to increase airflow cross tubes. When
fuming has stopped, remove the manifold and shut off the aspirator.

1.7.3 Distillation
Note: If Kjeltec 9 Analyser is used, the distillation, titration and calculation is performed
automatically.
1. Turn on the instrument and complete a successful self-test.
2. Select the program profile.
3. Select the Product (Sample Type, only for Kjeltec 9 Analyser).
4. Select created batch (only for Kjeltec 9 Analyser).
5. Load digestion tube into instrument or autosampler.
6. Start to run a sample.
7. Remove sample from the distillation unit and titrate distillate with standardised HCl.

1.8 Expression of Results

The results are reported as %Nitrogen, the calculations are as follows:

1.8.1 Calculation

% Nitrogen =
(T − B )  N  14,007  100
weight sample (mg )

T = Titration volume for sample (ml)

B = Titration volume for blank (ml)
N = Normality of acid, 4 places of decimal

%𝑃𝑟𝑜𝑡𝑒𝑖𝑛 = %𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 × 𝐹

F = Conversion factor for Nitrogen to Protein.

Application Note 5515 / Rev. 1 4(6)

 Sample / Commodity Factor

Animal feeding stuff and most food products 6.25

Milk and milk products 6.38

Meat and meat products 6.25

Common wheat 5.7

Durum wheat 5.7

Wheat milling products 5.7 or 6.25

Wheat for feed 6.25

Barley 6.25

Oats 5.7 or 6.25

Rye 5.7

Triticale 6.25

Corn 6.25

Pulses 6.25

Oilseeds and oilseed meal 5.3

Soybeans, seeds, meal or products 5.71

Protein factor for different sample types

1.8.2 Method Performance

Samples
Eight different kinds of feed and forage samples are analysed to check the performance of Kjeltec 9
series. Below results are based on these test conditions. The steam power used for distillation is
100%. The concentration of standard acid used for titration is 0.1 N with four decimal places.
Sample Sample Catalyst H2SO4 Digestion Dilution Alkali Receiver *Distillation
Type Weight and Salt (mL) (°C/Min.) (mL) (mL). Solution End
(g) (mL)

Corn silage 1.2 2 Kjeltab 12 420 / 60 80 50 30 4 min/ Auto

Cu 3.5

Sunflower 1.2 2 Kjeltab 15 420 / 75 80 60 30 4 min/ Auto

seeds Cu 3.5

Swine 1.2 2 Kjeltab 12 420 / 60 80 50 30 4 min/ Auto

pellet Cu 3.5

Fish meal 0.5 2 Kjeltab 15 420 / 75 80 60 30 4 min/ Auto

Cu 3.5

Meat and 0.5 2 Kjeltab 15 420 / 75 80 60 30 4 min/ Auto

bone Cu 3.5

Pig feed 1.2 2 Kjeltab 12 420 / 60 80 50 30 4 min/ Auto

Cu 3.5

Barley 1.2 2 Kjeltab 12 420 / 60 80 50 30 4 min/Auto

Cu 3.5

Application Note 5515 / Rev. 1 5(6)

 Sample Sample Catalyst H2SO4 Digestion Dilution Alkali Receiver *Distillation
Type Weight and Salt (mL) (°C/Min.) (mL) (mL). Solution End
(g) (mL)

Wheat 1.2 2 Kjeltab 12 420 / 60 80 50 30 4 min/ Auto

Cu 3.5

Analytical conditions for sample types used for the Method Performance data. *Auto mode
was applied for the Analyser distillation and titration program

1.8.3 Results for Distillator and Analyser

The study was performed at FOSS using block digestor, Kjeltec 9 distillation unit and auto titrator.
The study was performed using the procedure described within this application note and AN 5511.
Sample Average n SD RSD *Reference Result Obtained Result Obtained
Type Obtained Result (%) Value from Kjetec 8200 from Dumatec
(%Protein) (%Protein) (%Protein) n=3 (%Protein) n=2

Corn silage 2.28 10 0.01 0.66 - 2.28 2.255

Sunflower 17.01 10 0.15 0.86 - 16.78 21.378

seeds

Swine 14.93 10 0.09 0.61 15.3 14.79 15.305

pellet

Fish meal 73.09 10 0.34 0.47 - 72.93 74.94

Meat and 65.40 10 0.56 0.85 - 65.56 67.045

bone

Pig feed 13.60 10 0.12 0.88 12.6-13.97 13.62 -

Barley 9.34 10 0.02 0.26 10 9.30 9.325

Wheat 11.11 10 0.06 0.53 12 11.10 11.26

Performance Data from Kjeltec 9 Distillator *Reference value provided on label

Sample Average n SD RSD *Reference Result Obtained Result Obtained

Type Obtained Result (%) Value from Kjeltec 8400 from Dumatec
(%Protein) (%Protein) (%Protein) n=3 (%Protein) n=2

Corn silage 2.15 10 0.02 0.79 - 2.16 2.255

Sunflower 16.97 10 0.22 1.32 - 16.75 21.378

seeds

Swine 15.13 10 0.05 0.34 15.3 15.04 15.305

pellet

Fish meal 73.22 10 0.24 0.32 - 72.38 74.94

Meat & 65.81 10 0.58 0.88 - 64.67 67.045

bone

Pig feed 13.66 10 0.07 0.51 12.6-13.97 13.55 -

Barley 9.31 10 0.04 0.40 10 9.27 9.325

Wheat 11.20 10 0.02 0.17 12 11.11 11.26

Performance Data of Kjeltec 9 Analyser *Reference value provided on label

Application Note 5515 / Rev. 1 6(6)

1.9 References
EN ISO 5983-2: 2005 Animal feeding stuff - Determination of nitrogen content and calculation of
[1]

crude protein content - Part 2: Block digestion/steam distillation method

AOAC Official Method 2001.11, Crude Protein in Animal Feed, Forage, Grain and Oilseeds, Block
[2]

Digestion Using Copper Catalyst, System Distillation into Boric Acid, AOAC INTERNATIONAL,
Gaithersburg, MD.
[3]
Application Note AN 5511
[4]
Application Note AN 3001

2 Questions and Feedback

If you have questions or feedback concerning this Application Note, please contact your FOSS
support team.

Joi Chen
Product Specialist, Service

3 Revision History
Rev. Date of Issue Revised Material Approved by

1 2022-06-10 First issue. JICH

Application Note 5515 / Rev. 1 7(6)