Nitrogen Determination by Kjeldahl Method

I. Introduction

The Kjeldahl method is the standard method for determining protein content of foods. The
protein content in a foodstuff is estimated by multiplying the Kjeldahl nitrogen content by
a nitrogen-to-protein conversion factor, usually set at 6.25, which assumes the nitrogen
content of proteins to be 16%. It would be important to note that this method measures the
total content of nitrogen exhibiting an oxidation number of -3, and therefore do not
distinguish protein-based nitrogen from nonprotein nitrogen. Consequently, the presence
of non-protein nitrogen compounds in foods (amino acids, ammonia, urea…) overestimates
their protein content. Separation of non-protein nitrogen compounds from proteins may be
performed by adding a protein precipitating agent (trichloroacetic acid, perchloric acid) or
by dialysis and gel filtration techniques.

The Kjeldahl method consists of three main steps: sample digestion, distillation, and
titration. In the digestion step, organic nitrogen exhibiting an oxidation number of -3 in the
samples is converted into ammonium sulfate by using sulfuric acid, salts, and a variety of
catalysts. During the distillation process, the digestate is treated with NaOH, which
converts the ammonium sulfate to ammonia. Ammonia is distilled off the mixture and
collected in a receiving flask of excess boric acid, forming ammonium borate. The
ammonium is then titrated with a standard acid solution to determine the Kjeldahl nitrogen
content of the samples.

In this practical experiment, you will be given a digestate solution and you will perform
the distillation (by Micro Kjeldahl apparatus or UDK 139 Semi-automatic Distillation unit)
and titration steps to determine the protein content of the sample.
 1. Preparation

- Check the schematic diagram of Micro Kjeldahl apparatus in the following Figure

Figure 1. Micro Kjeldahl apparatus.

- Check water level in steam generator.
- Check the valves of distillation unit.
- Open the valve to let water flow through the condenser slowly.
- Heat to boil the acidified water in steam generator.
- Wash 3 times the distillation unit (consender, the Kjeldahl’s flask).

2. Distillation
Test sample
- Add 20 mL of 3% boric acid (w/v) to an Erlenmeyer (conical) flask.
- Add 2-3 drops of Tashiro indicator to the boric acid solution.
- Install the conical flask into the Micro Kjeldahl apparatus (see Figure 1). Make sure
that the end of condenser is submerged in the boric acid solution.
- To the reaction chamber, add respectively the following solution:
 + 5 mL of digestate (using a pipette).
+ 2 mL distilled water (using a pipette).
+ 5 mL of 40% NaOH (using measuring cylinder)
Note: close immediately the stopple after adding each solution to the
reaction chamber

- After the solution in conical flask turns from red violet to green, wait for five
minutes.
- Check the pH of the distillate by lowering the conical flask and use purple litmus
paper to measure the pH of the solution from the condenser. If the color changes to
blue, put the receiver flask in the system and wait for five minutes. If no color
change is observed, remove the receiving flask from the distillation unit, and wash
the tip of condenser with distilled water.

Blank sample
- Add 20 mL of 3% boric acid (w/v) to an Erlenmeyer (conical) flask.
- Add 2-3 drops of Tashiro indicator to the boric acid solution.
- Install the conical flask into the Micro Kjeldahl apparatus (see Figure 1). Make sure
that the end of condenser is submerged in the boric acid solution.
- To the reaction chamber, add respectively the following solution:
+ 7 mL distilled water (using a pipette).
+ 5 mL of 40% NaOH (using measuring cylinder)
Note: close immediately the stopple after adding each solution to the
reaction chamber

- After the solution in conical flask turns from red violet to green, wait for five
minutes.
3. Titration
- Titrate the amount of (NH4)2B4O7 in test and blank samples with 0,1 N H2SO4.
The end point is reached when the Tashiro indicator permanently changes color
from green to red violet.
- Record the volume of H2SO4 used.

4. Calculation
Calculate the Kjeldahl nitrogen content (g/100 mL) of the digestate knowing that 1 mL
of H2SO4 0,1 N corresponds to 1,4 mg of nitrogen.
Assuming that the digestate was prepared from the precipitate of 5 g of sample by
trichloroacetic acid and the total volume of the digestate was 100 mL, calculate the
protein content of the sample.
 UDK 139 SEMI-AUTOMATIC DISTILLATION UNIT

1. Preparation
Step 1:
- Check the liquid levels of NaOH and H2O tanks.
- Put the Kjeldahl tube (without sample) into the
machine, carefully pull down the lever for insertion of
the tube and check that the spash head is in place in
the tube. Close the safety guard.

Step 2:
- Open the valve of tap water.
- Turn on the instrument by pressing the main power
switch on the right of the instrument. The steam generator needs 3 minutes to
start working.
Step 3:
- Wash the system by pressing at the same time the NaOH key and the (-) key to let
the system draw distilled water from the distilled H2O flask through the entire
system to the Kjeldahl tube (no sample at this time), then take this tube out, discard
the water.

2. Test sample
Step 4: Add 5 mL of digestate to a Kjendahl tube, install the tube into the machine.
Step 5: Add 20 mL of 3% H3BO3 into a 250 ml conical flask, add 3 drops of Tashiro
indicator, and place the reiceiving flask on the holder.
Step 6: NaOH addition: Press the NaOH key and the (+) key at the same time
Step 7: Set the distillation time. Time can be set for 3-10 minutes by using (+) or (-) key
(currently 7 minutes)
Step 8: Press the Start key to initiate the distillation process. The device will display: “d02”
then “d01”. The distillation time countdown is shown on the display during analysis. At
the end of the distillation process, the device will automatically stop. Remove the reiceiving
flask from the distillation unit.
Step 9: Wash the machine with distilled water as in Step 3.

3. Blank sample
Repeat the distillation process from Step 4 to Step 9 by using 5 mL of distilled water
instead of 5 mL of digestate.

Titration of test and blank samples by 0,1 N H2SO4 will be carried out as described above.