Digestion of the sample: The most time-consuming step in the analysis, this is designed to break down the bonds that hold the polypeptides together and convert them to simpler chemicals such as water, carbon dioxide, and ammonia. Adding strong sulfuric acid and heating the mixture to about 370ºC to 400ºC for 60 to 90 minutes oxidizes the organic material and releases ammonium ions.
Distillation: This separates the ammonia from the digestion mixture by raising the pH with sodium hydroxide, which changes the ammonium ions into ammonia gas. The ammonia is collected through boiling and distillation of the gas into a trapping solution of hydrochloric acid.
Titration: As the ammonia dissolves into the trapping solution, it is back-titrated so that the quantity of distilled-off ammonia can be calculated and the amount of nitrogen in the protein determined.