TheKjeldahl method orKjeldahl digestion (Danish pronunciation:[ˈkʰelˌtɛˀl]) inanalytical chemistry is a method for the quantitative determination of a sample'sorganicnitrogen plusammonia/ammonium (NH3/NH4+). Without modification, other forms of inorganic nitrogen, for instancenitrate, are not included in this measurement. Using an empirical relation between Kjeldahl nitrogen and protein, it is an important method for indirectly quantifyingprotein content of a sample. This method was developed by theDanishchemistJohan Kjeldahl in 1883.[1][2]
The method consists of heating a sample to 360–410 °C with concentratedsulfuric acid (H2SO4), which decomposes, or digests, the organic sample byoxidation to liberate the reduced nitrogen as stableammonium sulfate:(NH4)2SO4.[3]
Hot concentrated sulfuric acid oxidizescarbon (asbituminous coal) andsulfur (seesulfuric acid's reactions with carbon):
Most oforganic carbon and sulfur are decomposed and eliminated as gaseousCO2 andSO2.
In contrast to organic carbon and sulfur, the digested organicnitrogen remains preserved in the concentrated sulfuric acid as stableammoniumcation (NH+4). Ammonium does not further oxidize to gaseousN2, or a higher oxidized form of nitrogen, such as, e.g.,N2O, NO, NO−2, NO2, or NO−3. If it was the case, the Kjeldahl method would not work.
Catalysts likeselenium,Hg2SO4 orCuSO4 are often added to accelerate the digestion.Na2SO4 orK2SO4 is also added to increase theboiling point of H2SO4. Digestion is complete when the liquor clarifies with the release of fumes.[3]
After complete digestion of the sample, to recoverammonia (NH3) from the ammonium sulfate,sodium hydroxide (NaOH) is first added to the residual sulfuric acid to neutralize it and to convert the soluble ammonium ion into volatileammonia:
Then, ammonia is recovered bydistillation using the system below (right side of the figure).
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The end of thecondenser is dipped into a known volume of standard acid (i.e. acid of known concentration). Aweak acid likeboric acid (H3BO3) in excess of ammonia is often used. StandardizedHCl, H2SO4 or some other strong acid can be used instead, but this is less commonplace. The sample solution is then distilled with a small excess ofsodium hydroxide (NaOH).[3] NaOH can also be added with adropping funnel.[4] NaOH converts dissolvedammonium (NH4+) to gaseousammonia (NH3), 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.[3]
Ammonium ion concentration in the acid solution, and thus the amount of nitrogen in the sample, is measured via titration. If boric acid (or some other weak acid) was used, directacid–base titration is done with a strong acid of known concentration. HCl or H2SO4 can be used. Indirectback titration is used instead if strong acids were used to make the standard acid solution: a strong base of known concentration (like NaOH) 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 (e.g. boric acid) because it does not interfere with the titration (it does have to be in excess of ammonia to trap it efficiently). Thus, one standard solution (e.g., HCl) is needed in the direct titration, while two are needed (e.g., HCl and NaOH) in the back-titration. One of the suitable indicators for these titration reactions isTashiro's indicator.[3]
In practice, this analysis is largely automated; specificcatalysts accelerate the decomposition. Originally, the catalyst of choice wasmercuric oxide. However, while it was very effective, health concerns resulted in its replacement withcupric sulfate. Cupric sulfate was less efficient than mercuric oxide and yielded lower protein results. It was soon supplemented withtitanium dioxide, the approved catalyst in all protein analysis methods in the Official Methods and Recommended Practices of AOAC International.[5]
The Kjeldahl method's universality, precision and reproducibility have made it the internationally recognized method for estimating the protein content in foods. 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. However, it does not allow for determining the true protein content, as it measures non-protein nitrogen in addition to the nitrogen in proteins. This is evidenced by the2007 pet food incident and the2008 Chinese milk powder scandal, whenmelamine, 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 (an hour or more), compare unfavorably with theDumas method for measuring crude protein content.[6]
Total Kjeldahl nitrogen or TKN is the sum ofnitrogen bound in organic substances, nitrogen inammonia (NH3-N) and inammonium (NH4+-N) in the chemical analysis of soil, water, or waste water (e.g. sewage treatment plant effluent).
Today, TKN is a required parameter for regulatory reporting at many treatment plants and for monitoring plant operations.
TKN is often used as a surrogate forprotein infood 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 nitrogenousamino acids, likearginine andhistidine. 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 (corn) and sorghum to 5.83 for most grains; 5.95 for rice, 5.70 for wheat flour, and 5.46 for peanuts.[7] 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.[8]
| Animal origin | Factor | Grass seeds | Factor | Beans and peanuts | Factor |
|---|---|---|---|---|---|
| Eggs | 6.25 | Barley | 5.83 | Castor bean | 5.30 |
| Meat | 6.25 | Corn (maize) | 6.25 | Jack bean | 6.25 |
| Milk | 6.38 | Millets | 5.83 | Lima bean | 6.25 |
| Oats | 5.83 | Navy bean | 6.25 | ||
| Rice | 5.95 | Mung bean | 6.25 | ||
| Rye | 5.83 | Soybean | 5.71 | ||
| Sorghum | 6.25 | Velvet bean | 6.25 | ||
| Wheat: Whole kernel | 5.83 | Peanuts | 5.46 | ||
| Wheat: Bran | 6.31 | ||||
| Wheat: Endosperm | 5.70 |
The Kjeldahl method is poorly sensitive in the original version. Other detection methods have been used to quantify NH4+ after mineralisation and distillation, achieving improved sensitivity: in-line generator of hydride coupled to aplasma atomic emission spectrometer (ICP-AES-HG, 10–25 mg/L),[10] potentiometric titration (> 0.1 mg of nitrogen), zone capillary electrophoresis (1.5 μg/mL of nitrogen),[11] andion chromatography (0.5 μg/mL).[12]
Kjeldahl method does not apply to compounds containing nitrogen innitro andazo groups andnitrogen present in rings (e.g.pyridine,quinoline,isoquinoline) as nitrogen of these compounds does not convert toammonium sulfate under the conditions of this method.