Advanced Biochemistry Lab : Determination of Nitrogen and Crude protein Ms. Nadia Amara

Description of the method: The Kjeldahl method was developed over 100 years ago for determining the nitrogen contents in organic and inorganic substances. Although the technique and apparatus have been modified over the years, the basic principles introduced by Johan Kjeldahl still endure today. Kjeldahl nitrogen determinations are performed on a variety of substances such as meat, feed, grain, waste water, soil, and many other samples. Description of the method: The method consists of heating a substance with sulfuric acid, which decomposes the organic substance by oxidation . As the organic material is oxidized, the carbon it contains is converted to carbon dioxide and the hydrogen is converted into water. While the nitrogen, from the amine groups found in the peptide bonds of the polypeptide chains, is converted to ammonium ion, which dissolves in the oxidizing solution, and can later be converted to ammonia gas. In this step potassium sulfate is added to increase the boiling point of the medium (169°C to 189°C) and a catalyst, which helps the conversion of the amine nitrogen to ammonium ions.

Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark). The solution is then distilled with sodium hydroxide which converts the ammonium salt to ammonia. Finally The amount of ammonia present ( the amount of nitrogen present in the sample) is determined by titration. ** Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 is used .

The Kjeldahl method consists of three steps, which have to be carefully carried out in sequence: Digestion , Distillation & Titration 1. Digestion : This is the most time-consuming step in the analysis. The purpose of this step is to break down the bonds that hold the polypeptides together, and convert them to simpler chemicals such as water, carbon dioxide and ammonia. The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), potassium sulfate (K2SO4) (to speed up the reaction by raising the boiling point of the digesting acid) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction).

Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution: The general equation for the digestion of an organic sample is shown below: Organic N + H2SO4 → (NH4)SO4 + H2O + CO2 2. Distillation: The purpose of the distillation step, is to separate the ammonia (that is, the nitrogen) from the digestion mixture. This is done by, raising the pH of the mixture using (NaOH ). This has the effect of changing the ammonium(NH4+) ions (which are dissolved in the liquid) to ammonia (NH3), which is a gas as indicated in the following equation. (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

3. Titration: The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution (Boric acid ). The boric acid captures the ammonia gas, forming an ammonium-borate complex. NH3 +H3BO3 NH4 H2BO3 +H2O (color change occurs ) (Red color) (Green color complex ) Ammonium-borate complex titrates with HCl and using Tashiro’s indicator to quantify the amount of ammonia in the receiving solution. NH4 H2BO3 + HCl NH4Cl + H3BO3 (Green color complex ) (Red color) (color change reverse )

Calculations: One mole of ammonia coming from the digestion mixture (and hence from the original protein) will neutralize exactly one mole of the acid . 1 mole NH3=1 mole N =1mole HCl gm N/14 = Vs * 0.1N Where vs is the titration volume of the sample , and 14g is the molecular weight of nitrogen N. %nitrogen = (gm nitrogen / gm sample) x 100 %N = (gN / gS) x 100 Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor: Crude protein (CP) = %N x 6.25

Advantages and Disadvantages: Advantages: The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods. Disadvantages : 1. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. 2. Different proteins need different correction factors because they have different amino acid sequences. 3. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts. 4. The technique is time consuming to carry-out.