Protein analysis by the Kjeldahl method

Materials and reagents

:

• Mineralization block
• Automatic distillation device
• Pure concentrated H₂SO₄ free of nitrogen compounds
• Sodium hydroxide at 30% (w/v)
• Catalyst (Kjeldahl tablets): 95.4%  K₂SO₄ - 2.8%  TiO₂ - 1.8%  CuSO₄
• Methyl red
• HCl 0.1 mol/L
• NaOH 0.1 mol/L
• Antifoam tablet

It is VERY IMPORTANT to follow the safety instructions for performing this procedure (laboratory coat, safety glasses and gloves) and to read at every step the instructions for using the equipment.
See the theme: All you need to know about safety in a chemistry laboratory

Step 1: Mineralization

(duration: 5 hours)

• Accurately weigh a given mass of the product (from a few mg to a g depending on the expected protein content of the product) and insert it into the tube to be mineralized.
• Add a Kjeldahl catalyst tablet, an antifoam tablet and 10 mL of sulfuric acid with the help of an automatic dispenser. Beware of the risk of foaming at the beginning of heating.
• Place the tube in the mineralization block reserved for this purpose. Make sure that the fume extractor is hermetically sealed (all the tubes or plugs must be present), and that the pump is running. Set the device to 150°C using the temperature dial and program the mineralization time (5 hours). Set the regulator to ON. After 10 minutes, and if no foam has formed in the tube, increase the temperature to 250°C, then to 350°C after another 10 minutes. Once the mineralization is finished, allow the tube to cool down, then add about 20 mL of water (under the fume hood).

Step 2: Distillation

(duration: about 15 minutes)

• Check that the water and sodium hydroxide tanks are sufficiently filled and open the water tap.
• Put the device under tension and set the sodium hydroxide volume to be dispensed (3 consecutive beeps).
• Position the tube containing the solution to be distilled in the tube holder and ensure that it is firmly fixed.
• Place an Erlenmeyer flask containing 25 mL of HCl 0.1 mol/L, 6 drops of methyl red and 10 mL of water on the rack, under the cooling coil. Ensure that the tube is immersed in the solution.
• Close the door, the contactor is then switched on. The device will only work if the door is closed.
• Press the STEAM button. The sodium hydroxide is now dispensed, and the distillation begins. Allow to distill for 8 minutes (a beeper will sound to indicate the end of the distillation), then lower the Erlenmeyer flask so that the tube is no longer immersed in the solution.
• Allow to cool down and retrieve the Erlenmeyer flask.

Step 3: Determination

(duration: about 15 minutes)

• Titrate with the sodium hydroxide 0.1 mol/L the amount of unreacted HCl (excess) and from it deduce the percentage of nitrogen in the sample.
• Make a control or blank sample under the same conditions.