Kjeldahl Analysis

The Kjeldahl method is an analytical method for the determination of Nitrogen in the trinegative state in certain organic compounds. The method was developed in 1883 by Johan Kjeldahl, a Danish chemist, and is used extensively in the determination of protein in foods, since protein is a macromolecule made up of Nitrogen-containing amino acids linked together. When used for protein determination, the percent Nitrogen measured is converted to the equivalent protein content by use of an appropriate numerical factor. For meat samples, this factor is 6.25, since meat protein is approximately 16% Nitrogen. The Kjeldahl method does not directly detect N-N and N-O linkages (e.g., azides, nitrates, nitrites, nitro groups, etc.). Samples containing these groups must be pretreated or subjected to reducing conditions before Kjeldahl analysis. The amino Nitrogen in the sample is converted to Ammonium Bisulfate as the organic material in the sample is destroyed by digestion with boiling, concentrated Sulfuric Acid. Potassium Sulfate is added to raise the boiling point of the mixture, thus speeding the decomposition. The amounts of Sulfuric Acid and Potassium Sulfate used must be controlled, depending on the amount of organic material present in the sample, to insure that a proper digestion temperature range of 370 - 400°C is maintained. Too low a temperature may lead to long digestion times and/or incomplete digestion, while too high a ratio of Potassium Sulfate to Sulfuric Acid may raise the temperature above 400°C, resulting in pyrolytic loss of Nitrogen and low results. A metal catalyst is also added to accelerate the digestion. In the past, Mercury was the catalyst of choice for this purpose, but Copper and Selenium catalysts are now more commonly used for safety and environmental reasons.

After digestion of the sample is complete, excess Sodium Hydroxide is added to the digestion mixture to neutralize the remaining Sulfuric Acid and release the Ammonia formed as the Nitrogen-containing molecules were broken down. The Ammonia is then distilled over into a measured excess of a standard acid, and the excess acid is back-titrated with a standard base. As an alternative, the Ammonia can be distilled over into a Boric Acid solution, and the Ammonia then can be determined by direct titration with a standard acid. The Ammonia can also be determined colorimetrically (e.g., via reaction with Phenate ion) or by using an Ammonia Selective Electrode. In these cases, a standard mineral acid is used as the Ammonia absorbent. If Mercury or a Mercuric salt is used as the catalyst, Thiosulfate or Sulfide is added with the Sodium Hydroxide to decompose any Mercuric-Ammonium complex, thus releasing the Ammonia and precipitating the Mercuric compound which may interfere with the distillation of the Ammonia. Zinc granules, pumice, or other suitable boiling stones are added to the distillation flask to prevent "bumping" which may lead to erroneous results due to carry-over of some of the caustic in addition to the Ammonia. If the Ammonia, after absorption in Boric Acid solution, is titrated directly with a standardized acid, Methyl Red, Methyl Purple, or Bromocresol Green - Methyl Red Mixed Indicator can be used as the indicator. These indicators can also be used in a back-titration using standard Sodium Hydroxide, if an excess standard acid is used as the absorbent for the Ammonia.

The reactions involved may be summarized as follows:

  Organic N + H2SO4 + Heat + Catalyst → CO2 + H2O + NH4HSO4 Sample Digestion
  NH4HSO4 + 2 NaOH → NH3 + Na2SO4 + H2O Neutralization of Digestion Mixture and Release of Ammonia
  NH3 + HCl [or H2SO4] → NH4Cl [or (NH4)2SO4] Direct Titration of Ammonia
  NaOH + HCl [or H2SO4] → NaCl [or Na2SO4] + H2O Back Titration of Standardized Acid

RICCA CHEMICAL COMPANY has all of the absorbents, neutralizing agents, titrants, and indicators required for Kjeldahl analysis, as well as the reagents required for the Phenate colorimetric determination or for use in the Ammonia Selective Electrode method.

for digestion of water samples Digestion Reagents, Group Nos. 2550 - 2551
for neutralization of digestion mixture and release of Ammonia Sodium Hydroxide Solutions, Group Nos. 7280-7290
  Sodium Hydroxide - Sulfide Solution, Group No. 7480
  Sodium Hydroxide - Thiosulfate Solutions, Group Nos. 7490 - 7495.
for absorption of released Ammonia after distillation Boric Acid Solutions, Group Nos. 1064 - 1070
  Standardized Hydrochloric Acid or Sulfuric Acid (as appropriate)
for direct titration of released Ammonia Standardized Hydrochloric Acid or Sulfuric Acid (as appropriate)
for back titration of excess standardized acid    Standardized Sodium Hydroxide solution (as appropriate)
for pH indicators and color development reagents see appropriate chemical name or list in the back of this catalog
Ammonia-selective electrode reagents and standards see appropriate chemical name or list in the back of this catalog