Kjeldahl Method for Determining Nitrogen Digestion Titration 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. Various scientific associations approve and have refined the Kjeldahl method, including the AOAC International (formerly the Association of Official Analytical Chemists), Association of American Cereal Chemists, American Oil Chemists Society, Environmental Protection Agency, International Standards Organization, and United States Department of Agriculture. The Kjeldahl method may be broken down into three main steps: digestion, distillation, and titration. Digestion
Digestion is accomplished by boiling a homogen eous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution. The general equ ation for the digestion of an organic sample is shown below: Organic N + H 2SO4 → (NH4)SO4 + H2O + CO4 + other sample matrix byproducts
Distillation: Excess base is added to the digestion product to convert NH4 to NH3 as indicated in the following equation. The NH3 is recovered by distilling the reaction product. ammonium ammonia heat sulfate gas (NH4)2SO4 + 2NaOH → 2NH3 + Na2SO4 + 2H2O Go to Top Titration
Titration quantifies the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution. There are two types of titration—back titration and direct titration. Both methods indicate the ammonia present in the distillate with a color change.
In back titration (commonly used in macro Kjeldahl), the ammonia is captured by a carefully measured excess of a standardized acid solution in the receiving flask. The excess of acid in the receiving solution keeps the pH low, and the indicator does not change until the solution is "back titrated" with base.
ammonia 2NH3 +
ammonia sulfate (NH4)2SO4 +
standard sulfuric acid acid 2H2SO4
measured excess acid H2SO4 +
excess ammonium sulfate → (NH4)2SO4 + (no color change)
sulfuric acid H2SO4
measured ammonium sodium sulfate hydroxide 2NaOH → Na2SO4 + (NH4)2SO4 + 2H2O (color change occurs)
In direct titration, if boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical reaction is: ammonia gas NH3 +
boric ammoniumacid borate complex H3BO3 → NH4 + H2BO-3 + (color change occurs)
excess boric acid H3BO3
The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the color of the receiving solutions changes. ammoniumsulfuric ammonium borate acid sulfate complex 2NH4 + H2BO-3 + H2SO4 (NH4)2SO4 (color change occurs in reverse)
boric acid + 2H3BO3
The boric acid method has the advantages that only one standard solution is necessary for the determination and that the solution has a long shelf life.
NITROGEN ANALYSIS On March 7, 1883, Johan Kjeldahl presented his method of nitrogen analysis to the Danish Chemical Society. Since then, his method has been extensively studied, modified, and improved upon. Today, the Kjeldahl method for the determination of organic nitrogen is the worldwide standard for the purpose of calculating the
protein content in both human food and animal food. Additionally, Kjeldahl has been adapted as a standard method of nitrogen analysis in water, wastewater, fertilizer, and fossil fuels, to name a few. The Kjeldahl method for nitrogen analysis is composed of three distinct steps. These are digestion, distillation, and titration. DIGESTION STEP The purpose of the digestion step is to break the intricate structure and chemical bonds that hold a chemical substance (piece of meat, cup of flour or quart of oil) down to simple chemicals and ionic structures. Specifically, proteins and other forms of nitrogen are broken down and converted to ammonia. To accomplish this, one to two grams of the sample are placed on a digestion tube with 12-15 ml of concentrated sulfuric acid (H2SO4). Seven grams of potassium sulfate (K2SO4) and a metallic catalyst, usually copper, are then added. (The latter two chemicals are commercially available from Rose Scientific as the proper Kelmate NT™.) The digestion tube is laced into a digestion block where it is heated to the boiling temperature of the mixture. Digestion is usually completed after one hour at 370ºC to 400ºC. The Distillation Step 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). Now it is possible to separate the nitrogen by distilling the ammonia and collecting the distillate in a suitable trapping medium. With Foss Tecator’s Kjeltec Systems, distillation takes less than five minutes. Today collection of ammonia is usually done by absorption into a solution of four percent boric acid. The ammonia is bound to the boric acid in the form of ammonium borate. The Titration Step Determination of the amount of nitrogen on the condensate 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 mixed indicator. The mixed indicators (bromocresol green and methyl red) are available in the four percent boric acid solution. Calculation After all this chemistry it is now time to calculate the amount of nitrogen present in the sample. This calculation can either be performed as percent nitrogen or percent protein. For percent nitrogen: % N= 14.01 x (ml titrant – ml blank) – (N of titrant) x 100 Sample Wt. (grams) x 1000 It has been shown that protein is 16% nitrogen. (Wheat and dairy products are some exceptions.) By dividing 100 by 16, we get the conversion factor for nitrogen to protein of 6.25. Hence, the percent protein is calculated as follows: % Protein = 6.25 x %N
