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Scientists use the Kjeldahl method to analyze the percent of organic nitrogen in a substance. The nitrogen levels can then be used to determine the amount of protein. The full name of the method is the Kjeldahl method of nitrogen analysis — sometimes protein analysis is used in place of nitrogen analysis, but the terms refer to the same method.
Chemist Johan Kjeldahl first presented his method to the Danish Chemical Society in 1883. He determined that, since nitrogen is a major element in protein, nitrogen analysis could be used to determine the amount of protein in a substance. His findings have been improved upon since that time, but the basic method remains in place.
The Kjeldahl method consists of three steps, commonly termed digestion, distillation, and titration. Digestion breaks down the nitrogen into ammonia and distillation separates the ammonia from other components. The amount of ammonia is calculated using titration, then the amounts of nitrogen and protein can be calculated based on the amount of ammonia.
During the digestion step, a small sample of the substance to be analyzed is mixed with sulfuric acid, potassium sulfate, and a catalyst that speeds up the reaction. This mixture is heated to a very high temperature — up to 750°F (about 400°C) — for about an hour, then cooled. The reactions that take place in the heated mixture break down large molecules into smaller components, including ammonium ions.
The distillation step converts the ammonium ions to ammonia gas by adding sodium hydroxide to the mixture. Then the temperature of the solution is raised, converting the ammonia to a volatile gas that rises in a vapor. The vapors are trapped in a solution, such as hydrochloric acid or boric acid.
Ammonia trapped in an acid neutralizes some of the acid, meaning that it lowers the pH. The amount of acid left after this neutralization is titrated with a base, such as sodium hydroxide. A dye is added to the acid and ammonia solution, which changes color when the pH changes. Then small amounts of the base are added to the acid just until the solution changes color. The amount of base needed to reach this endpoint can be used to calculate the amount of ammonia in the original solution.
To calculate the amount of nitrogen, a scientist must first know the number of moles of acid and base that were present in the final solution. Subtracting the moles of base from the moles of acid gives the moles of ammonia. The moles of ammonia in the final solution is the same as the moles of nitrogen, so this number is multiplied by 14 — the atomic mass of nitrogen — to find the grams of nitrogen.
Percent nitrogen is found by dividing the grams of nitrogen by the total grams in the original sample and multiplying by 100. The percent protein the Kjeldahl method is found by multiplying the percent nitrogen by a conversion factor. This conversion factor is usually 6.25, with the exception of a few substances such as wheat and dairy products.