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Kjeldahl: step 1: Digestion

The purpose of the digestion step is to break the intricate structure and chemical bonds in a sample down to simple chemicals and ionic structures. Specifically, proteins and other forms of nitrogen are broken down and converted to ammonia.

To accomplish this, the sample is heated in the presence of concentrated sulfuric acid (H 2 SO 4), an effective oxidizing agent.

Organic N + H 2 SO 4 + heat + catalyst  (NH 4)2 SO 4 + CO 2 + H 2 O

Ammonia gas is not liberated but remains in solution as ammonium sulfate ((NH4)2 SO4).

This step is accelerated by using catalysts such as selenium, copper, mercury etc. Most of these catalyst are toxic and the waste disposes problems. Potassium sulfate is added to increase the temperature.

Kjeldahl: step 2: Destillation

Stream distillation involves separation of ammonia–nitrogen from the digestate. This is accomplished by raising the pH with sodium hydroxide (NaOH). This changes the ammonium (NH4+) ion to ammonia (NH3). Then steam is purged through the sample tube. Now it is possible to separate the nitrogen by distilling the ammonia and collecting the distillate in a suitable trapping medium such as boric acid.

(NH 4)2 SO 4 + 2NaOH  NH 3 (g) + 2H 2 O + Na 2 SO 4

Kjeldahl: step 3 Titration

In a frequently used procedure, the liberated ammonia is adsorbed in an excess of boric acid solution (H3BO3) containing bromocresol green and methyl red as acid/base indicators. The low pH of this solution converts the ammonia intro ammonium ions and the boric acid to borate ions.

NH 3 (l) + H 3 BO 3  NH 4+ + H 2 BO 3-

Determination of the amount of nitrogen in the erlenmeyer flask can be accomplished by several methods. The most common is titration of the ammonia with a standard solution of one-tenth normal hydrochloric acid (0.1 H HCl) in the presence of indicators.

NH 4+ + H 2 BO 3- + HCl  H 3 BO 3 + NH 4 Cl

Sometimes spectroscopic methods are used for the direct determination of ammonia, making the time consuming distillation and titration steps unnecessary.

The application of Kjeldahl methods can be manual, or semi- or fully automated. Fully unattended operations allow the analysis of 60 samples within 4 hours. Modern types of Kjeldahl apparatus allow the analysis of N in the range of 0.2 - 160 mg per sample. Modified Kjeldahl micro methods can detect N concentration in the range of 0.3 - 1.3 mg/ml. However. if a sample is small, be aware that the sample could be less representative for the food, because of heterogeneity of foods.

Advantages:

• Applicable to all foods.

• Not sensitive for interference with other food components (except non protein nitrogen).

• Internationally used standard method (e.g. ISO standards). Different modifications based on the Kjeldahl method are available for meat, milk and cereals.

The Kjeldahl method

The Kjeldahl method (or Kjeldahl-Wilforth-Gunning method) is the most universal technique for the determination of organic N in foods and it is internationally recognised for official and statutory purposes. The Kjeldahl method consists of three distinct steps.

1. Digestion: to break the intricate structure and chemical bonds in a food down to simple chemicals and ionic structures. Specifically, proteins and other forms of nitrogen are broken down and converted to ammonia.

2. Destillation: to separate ammonia–nitrogen from the digestate.

3. Titration: to determine released ammonia (NH3) by titration.

Finally, nitrogen values are converted to protein values using conversion factors.

Play this slide show to learn more.