The method consists of heating a sample at 360–410°C with concentrated sulfuric acid, which decomposes the organic sample by oxidation to liberate the reduced nitrogen as ammonium sulfate. Catalysts like selenium, Hg2SO4 or CuSO4 are often added to make the digestion go faster. Na2SO4 is also added to increase the boiling point of H2SO4. Digestion is complete when the liquor clarifies with the release of fumes. A distillation system depicted below is built. The end of the condenser is dipped into a known volume of standard acid. A weak acid like boric acid in excess of ammonia is often used. Standardized HCl, H2SO4 or some other strong acid can be used instead, but this is less commonplace. The sample solution is then distilled with a small amount of sodium hydroxide. NaOH can also be added with a dropping funnel. NaOH reacts the ammonium to ammonia, which boils off the sample solution. Ammonia bubbles through the standard acid solution and reacts back to ammonium salts with the weak or strong acid. Ammonium ion concentration in the acid solution, and thus the amount of nitrogen in the sample, is measured via titration. If boric acid was used, direct acid-base titration is done with a strong acid of known concentration. HCl or H2SO4 can be used. Indirect back titration is used instead if strong acids were used to make the standard acid solution: strong base of known concentration is used to neutralize the solution. In this case, the amount of ammonia is calculated as the difference between the amount of HCl and NaOH. In the case of direct titration, it is not necessary to know the exact amount of weak acid because it does not interfere with the titration. Thus, one standard solution is needed in the direct titration, while two are needed in the back-titration. One of the suitable indicators for these titration reactions is Tashiro's indicator. In practice, this analysis is largely automated; specific catalysts accelerate the decomposition. Originally, the catalyst of choice was mercuric oxide. However, while it was very effective, health concerns resulted in it being replaced by cupric sulfate. Cupric sulfate was not as efficient as mercuric oxide, and yielded lower protein results. It was soon supplemented with titanium dioxide, which is currently the approved catalyst in all of the methods of analysis for protein in the Official Methods and Recommended Practices of AOAC International.
Applications
The Kjeldahl method's universality, precision and reproducibility have made it the internationally recognized method for estimating the protein content in foods and it is the standard method against which all other methods are judged. It is also used to assay soils, waste waters, fertilizers and other materials. It does not, however, give a measure of true protein content, as it measures nonprotein nitrogen in addition to the nitrogen in proteins. This is evidenced by the 2007 pet food incident and the 2008 Chinese milk powder scandal, when melamine, a nitrogen-rich chemical, was added to raw materials to fake high protein contents. Also, different correction factors are needed for different proteins to account for different amino acid sequences. Additional disadvantages, such as the need to use concentrated sulfuric acid at high temperature and the relatively long testing time, compare unfavorably with the Dumas method for measuring crude protein content.
Total Kjeldahl nitrogen
Total Kjeldahl nitrogen or TKN is the sum of nitrogen bound in organic substances, nitrogen in ammonia and in ammonium in the chemical analysis of soil, water, or waste water. Today, TKN is a required parameter for regulatory reporting at many treatment plants, and as a means of monitoring plant operations.
Conversion factors
TKN is often used as a surrogate for protein in food samples. The conversion from TKN to protein depends on the type of protein present in the sample and what fraction of the protein is composed of nitrogenous amino acids, like arginine and histidine. However, the range of conversion factors is relatively narrow. Example conversion factors, known as N factors, for foods range from 6.38 for dairy and 6.25 for meat, eggs, maize and sorghum to 5.83 for most grains; 5.95 for rice, 5.70 for wheat flour, and 5.46 for peanuts. In practice, 6.25 is used for almost all food and feed regardless of applicability. The factor 6.25 is specifically required by US Nutrition Label regulations in the absence of another published factor.
Kjeldahl method is not applicable to compounds containing nitrogen in nitro and azo groups and nitrogen present in rings as nitrogen of these compounds does not convert to ammonium sulfate under the conditions of this method.
